Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases Related to the GIT

*Luís Cláudio Lima de Jesus, Fernanda Alvarenga Lima, Nina Dias Coelho-Rocha, Tales Fernando da Silva, Júlia Paz, Vasco Azevedo, Pamela Mancha-Agresti and Mariana Martins Drumond*

## **Abstract**

Many diseases that affect the gastrointestinal tract (GIT) have great influence on the quality of life of the majority of patients. Many probiotic strains are being highly studied as a promising candidate due to their beneficial effect reported in the GIT. With the purpose of increasing the beneficial characteristics of some probiotics strains and, consequently, to improve further the reported results, many probiotic strains expressing or encoding different proteins, with anti-inflammatory activities, have been developed. These recombinant strains have been reported as good candidates for the treatment of different pathological conditions, especially colitis and mucositis disease since they have been shown to have positive results and good perspectives for GIT inflammation. Thus, this chapter will first address the aspects of the gastrointestinal tract in humans as well as its microbiota. In a second moment, it will discuss about chronic diseases, mainly the intestinal ones. Finally, it will discuss about probiotics, especially concerning on lactic acid bacteria (LAB), and its action in the prevention and treatment of these diseases. At the final part, we will point out aspects on the development of recombinant strains and the results found in the literature on disease models.

**Keywords:** *L. lactis*, *Lactobacillus*, DNA vaccine, heterologous protein

## **1. The human gastrointestinal tract**

The human gastrointestinal tract is formed by a complex ecosystem which includes the gastrointestinal epithelium, immune cells, and resident microbiota [1] and comprehends one of the biggest existent interfaces between the host, environmental factors, and antigens in the human body.

The intestine encompasses a broad variety of microorganisms (bacteria, archaea, eukarya, and viruses) [2] from more than 3500 different species [3, 4] that coevolved with the host in a mutually beneficial relationship [5, 6]. The composition and density of bacterial populations in adult individuals differ considerably over the GIT. The area of the GIT that has highest microorganism abundance is the colon (1014) followed by dental plaque (1012), ileum (1011), saliva (1011), and skin (1011) [7]. However, low concentrations (up to 102 –107 cells/mL) and bacterial diversity are found in the upper GIT (stomach, duodenum, jejunum) [3, 4], since the presence of acid, bile salts, and pancreatic secretions hinders the bacterial colonization [8], so that there is no nutritional competition between the microbiota and the host [9]. Thus, both function and structure of microbial communities are significant and are closely related. However, function could be the more important measure of microbiome health, since bacterial ecology suggests that analogous ecosystems have similar function although they have moderately diverse composition [10, 11].

### **2. Gut microbiota**

The importance and the specific functions that gut microbiota has in human nutrition and health are well settled. The attributed functions can be classified in three classes: metabolic, protective, and trophic [12]. The gene diversity of the microbial community provides a variety of enzymes and biochemical pathways, specific to the host, able to contribute to short-chain fatty acid (SCFA) production by carbohydrate fermentation and production of some vitamins such as K, B12, biotin, folic acid, and pantothenate. These factors added to synthesis of amino acids from ammonia or urea contributing to the metabolic function of the microbiota [13, 14].

The gut microbiota's protective function is related to barrier effect, once the resident bacteria generate a resistance line which avoid pathogens/opportunistic bacteria and maintain normal mucosal function. The activity of some bacteria to secrete antimicrobial substances, such as bacteriocins, is able to inhibit the growth of other bacteria and nutrient competition [15, 16].

Regarding trophic functions of gut microbiota, the interaction between resident microorganisms has influence in differentiation and proliferation of epithelial cells [17], as well as in the development and regulation of the immune system by numerous and varied interactions between microbes, epithelium, and gut lymphoid tissues [18].

It is important to highlight that the interactions between the gut microbiota and the host immune system are required to preserve the gut homeostasis [19–21]. When this relationship is affected, alterations in bacterial function and diversity lead to the imbalance in the composition of the resident microbiota, favoring either the growing of pathogenic bacteria or the decreasing in beneficial bacteria in a process known as dysbiosis [22], which appoint a great threat to gut integrity and is intrinsically related to the development and progression of several diseases, such as inflammatory bowel diseases.

#### **3. Chronic inflammatory diseases**

One of the most well-characterized chronic inflammatory diseases that mainly affect the digestive tract is inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD). The exact etiology of IBD is still unclear, but the strict relation between genetic and the environmental factors, such as enteric immune dysregulation and alterations in the intestinal microbiome [23, 24], is broadly known. Besides, these diseases generate substantial morbidity and have a high prevalence in developed countries (5 in 1000 individual are affected) they remain to increase in developing nations [25].

Both diseases, UC and CD, present different pathogenesis, symptomatology, inflammatory profiles, and gut microbiota composition. CD is characterized by the irregular transmural inflammation (extending deeply into the submucosal regions) which can

**51**

*Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases...*

**4. IBD complications and microbiota manipulation**

tumorigenesis associated with inflammation [31].

most frequently identified cancer in females and the third in males.

affect any portion of the GIT and often made difficult by strictures, abscesses, and fistulae. On the other hand, the inflammation presented in UC is restricted to the superficial layers of the intestinal mucosa characterized by mucosa erosion and/or ulcer, generally localized in the region of the gut most colonized by bacteria, the colon [26, 27]. In addition, regarding the immune response associated with these diseases, it is possible to relate CD with an increased IL-12, IL-23, IL-27, interferon γ (IFN-γ), and tumor necrosis factor-α (TNF-α) production, all associated with Th1 and Th17 immune responses, different from UC which is correlated with a Th2 immune response, with high levels of IL-5 and transforming growth factor-β (TGF-β) production [28].

It is important to highlight that the principal cause of death in IBD patients is colorectal cancer (CRC) [29]. Frequent episodes of inflammatory process in the intestinal mucosa are related to development of this disease, which is the second

There are increased evidences that environmental factors such as lifestyle and diet alterations have effect in CRC incidence [30]. This effect has been documented

because there is evidence showing an essential relationship between dietary antigens and antigens of commensal bacteria with the regulatory T cells (Tregs), which maintain the immune tolerance and, consequently, reduce the risk of

In this context, it was reported that the higher consumption of diet rich in grains and vegetables decreases the incidence of CRC. This effect involves different mechanisms such as the diminution in the fecal transit time due to the increase in the stool bulk, and consequently, it reduces the contact of carcinogen with colon cells and the fermentation of these fibers of colonic components [14, 32]. In addition, significant reduction in concentration of acetate, propionate, and butyrate with increase in fecal pH [33] and the decrease in the number of obligate anaerobe microorganisms have been reported in individuals with colon cancer [34] when compared with healthy people. Thus, intestinal environmental alterations are the keys to evolution toward adenoma and afterward to CRC progression [35].

It has been also reported that up to 30% of patients with UC need surgical management such as the restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) [36]. This procedure removes the entire colon and rectum while preserving the anal sphincter and, hence, normal bowel function and fecal continence, therefore acting as an internal pelvic place for intestinal contents [37]. Around 50–60% of UC patients with following IPAA develop inflammation in the ileal pouch, generating the condition called "pouchitis." The reported incidence of pouchitis is variable, generally because of the diagnostic criteria that have been used to define this syndrome [38, 39]. In addition, although its pathogenesis is uncertain, the main hypothesis for the mechanism by which the disease occurs is the break in the mucosal barrier generated by dysbiotic microbiome in susceptible patients, generating an unusual mucosal immune

Corresponding to the increased attention given to the role of the intestinal microbiota in a variety of diseases, there has been an intense exploration of potential means to manipulate the intestinal microbiome either by probiotic administra-

In this context, a randomized clinical trial based on a 1-week treatment with anaerobically prepared donor FMT, compared with autologous FMT, resulted in a higher probability of remission in 8 weeks for patients with UC, revealing that stool administration from healthy donors to UC or CD patients is an intervention that seeks to restore a healthier balance of gut microbes and control IBD [42]. Data on FMT for

activation [40]; still the disease typically responds to antibiotics.

tion or fecal microbiota transplant (FMT) for therapeutic effect [41].

*DOI: http://dx.doi.org/10.5772/intechopen.88325*

*Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases... DOI: http://dx.doi.org/10.5772/intechopen.88325*

affect any portion of the GIT and often made difficult by strictures, abscesses, and fistulae. On the other hand, the inflammation presented in UC is restricted to the superficial layers of the intestinal mucosa characterized by mucosa erosion and/or ulcer, generally localized in the region of the gut most colonized by bacteria, the colon [26, 27]. In addition, regarding the immune response associated with these diseases, it is possible to relate CD with an increased IL-12, IL-23, IL-27, interferon γ (IFN-γ), and tumor necrosis factor-α (TNF-α) production, all associated with Th1 and Th17 immune responses, different from UC which is correlated with a Th2 immune response, with high levels of IL-5 and transforming growth factor-β (TGF-β) production [28].

## **4. IBD complications and microbiota manipulation**

It is important to highlight that the principal cause of death in IBD patients is colorectal cancer (CRC) [29]. Frequent episodes of inflammatory process in the intestinal mucosa are related to development of this disease, which is the second most frequently identified cancer in females and the third in males.

There are increased evidences that environmental factors such as lifestyle and diet alterations have effect in CRC incidence [30]. This effect has been documented because there is evidence showing an essential relationship between dietary antigens and antigens of commensal bacteria with the regulatory T cells (Tregs), which maintain the immune tolerance and, consequently, reduce the risk of tumorigenesis associated with inflammation [31].

In this context, it was reported that the higher consumption of diet rich in grains and vegetables decreases the incidence of CRC. This effect involves different mechanisms such as the diminution in the fecal transit time due to the increase in the stool bulk, and consequently, it reduces the contact of carcinogen with colon cells and the fermentation of these fibers of colonic components [14, 32]. In addition, significant reduction in concentration of acetate, propionate, and butyrate with increase in fecal pH [33] and the decrease in the number of obligate anaerobe microorganisms have been reported in individuals with colon cancer [34] when compared with healthy people. Thus, intestinal environmental alterations are the keys to evolution toward adenoma and afterward to CRC progression [35].

It has been also reported that up to 30% of patients with UC need surgical management such as the restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) [36]. This procedure removes the entire colon and rectum while preserving the anal sphincter and, hence, normal bowel function and fecal continence, therefore acting as an internal pelvic place for intestinal contents [37]. Around 50–60% of UC patients with following IPAA develop inflammation in the ileal pouch, generating the condition called "pouchitis." The reported incidence of pouchitis is variable, generally because of the diagnostic criteria that have been used to define this syndrome [38, 39]. In addition, although its pathogenesis is uncertain, the main hypothesis for the mechanism by which the disease occurs is the break in the mucosal barrier generated by dysbiotic microbiome in susceptible patients, generating an unusual mucosal immune activation [40]; still the disease typically responds to antibiotics.

Corresponding to the increased attention given to the role of the intestinal microbiota in a variety of diseases, there has been an intense exploration of potential means to manipulate the intestinal microbiome either by probiotic administration or fecal microbiota transplant (FMT) for therapeutic effect [41].

In this context, a randomized clinical trial based on a 1-week treatment with anaerobically prepared donor FMT, compared with autologous FMT, resulted in a higher probability of remission in 8 weeks for patients with UC, revealing that stool administration from healthy donors to UC or CD patients is an intervention that seeks to restore a healthier balance of gut microbes and control IBD [42]. Data on FMT for

*The Health Benefits of Foods - Current Knowledge and Further Development*

–107

of other bacteria and nutrient competition [15, 16].

inflammatory bowel diseases.

**3. Chronic inflammatory diseases**

moderately diverse composition [10, 11].

concentrations (up to 102

**2. Gut microbiota**

of the GIT that has highest microorganism abundance is the colon (1014) followed by dental plaque (1012), ileum (1011), saliva (1011), and skin (1011) [7]. However, low

GIT (stomach, duodenum, jejunum) [3, 4], since the presence of acid, bile salts, and pancreatic secretions hinders the bacterial colonization [8], so that there is no nutritional competition between the microbiota and the host [9]. Thus, both function and structure of microbial communities are significant and are closely related. However, function could be the more important measure of microbiome health, since bacterial ecology suggests that analogous ecosystems have similar function although they have

The importance and the specific functions that gut microbiota has in human nutrition and health are well settled. The attributed functions can be classified in three classes: metabolic, protective, and trophic [12]. The gene diversity of the microbial community provides a variety of enzymes and biochemical pathways, specific to the host, able to contribute to short-chain fatty acid (SCFA) production by carbohydrate fermentation and production of some vitamins such as K, B12, biotin, folic acid, and pantothenate. These factors added to synthesis of amino acids from ammonia or urea contributing to the metabolic function of the microbiota [13, 14]. The gut microbiota's protective function is related to barrier effect, once the resident bacteria generate a resistance line which avoid pathogens/opportunistic bacteria and maintain normal mucosal function. The activity of some bacteria to secrete antimicrobial substances, such as bacteriocins, is able to inhibit the growth

Regarding trophic functions of gut microbiota, the interaction between resident microorganisms has influence in differentiation and proliferation of epithelial cells [17], as well as in the development and regulation of the immune system by numerous and varied interactions between microbes, epithelium, and gut lymphoid tissues [18]. It is important to highlight that the interactions between the gut microbiota and the host immune system are required to preserve the gut homeostasis [19–21]. When this relationship is affected, alterations in bacterial function and diversity lead to the imbalance in the composition of the resident microbiota, favoring either the growing of pathogenic bacteria or the decreasing in beneficial bacteria in a process known as dysbiosis [22], which appoint a great threat to gut integrity and is intrinsically related to the development and progression of several diseases, such as

One of the most well-characterized chronic inflammatory diseases that mainly affect the digestive tract is inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD). The exact etiology of IBD is still unclear, but the strict relation between genetic and the environmental factors, such as enteric immune dysregulation and alterations in the intestinal microbiome [23, 24], is broadly known. Besides, these diseases generate substantial morbidity and have a high prevalence in developed countries (5 in 1000 individual are affected) they remain to increase in developing nations [25].

Both diseases, UC and CD, present different pathogenesis, symptomatology, inflammatory profiles, and gut microbiota composition. CD is characterized by the irregular transmural inflammation (extending deeply into the submucosal regions) which can

cells/mL) and bacterial diversity are found in the upper

**50**

Crohn's disease is rather more limited than for UC, but it has been shown that single standardized FMT resulted in a clinical remission sustained for more than 9 months in CD patients [43]. However, the authors suggest that further studies are needed to enhance the knowledge about the use of stool transplantation for IBD treatment.

Alteration in the gut microbiome composition with increase in some groups of microorganisms, such as *Clostridium* and *Fusobacterium*, was also reported in patients with pouchitis [44, 45]. In this context, literature evidences indicate that the probiotic administration such as VSL#3 is effective in the chronic pouchitis prevention [46]. On the other hand, FMT to pouchitis treatment did not report the same beneficial results. Only three reports with this approach [47–49] exposed that neither clinical remission nor any adequate response was observed in the evaluated patients suggesting that the efficacy of FMT for pouchitis after proctocolectomy is limited [49]. The importance of standardization of this procedure needs to be highlighted to improve its efficacy, since frequency, route of administration (e.g., endoscopy, nasogastric tube, colonoscopy), and the criteria of choice of healthy donor are very important parameters to be considered.

## **5. Intestinal mucositis**

Different chemotherapy regimens such as FOLFOX (5-fluorouracil and oxaliplatin), FOLFIRI (5-fluorouracil and irinotecan), and triple FOLFOXIRI regimen (5-fluorouracil, oxaliplatin, and irinotecan) [50, 51] are adopted for different types of cancer but with a broad range of collateral effects.

Mucositis is the most common side effect in patients undergoing chemotherapy/ radiotherapy treatments, which consist in an inflammation and/or ulcers in the gastrointestinal tract [52] with consequent loss of cells from the epithelial barrier of the GIT. Many symptoms are related to gastrointestinal mucositis, such as diarrhea, severe abdominal pain, bleeding, fatigue, malnutrition, dehydration, electrolyte imbalance, and infections, with potential fatal complications which can conduce to reduction or interruption of antitumor treatment [53] and consequently leads to longer hospitalization.

This pathology occurs due to cytotoxic effects of anticancer drugs/radiotherapy that cause damage at the DNA of stem cell (epithelial cell progenitors) with intense oxidative stress and consequent cell death. This apoptotic process is exacerbated affecting the absorption by shortening the villi structure of enterocytes and causing the loss of epithelial barrier with an invasion of inflammatory cells (neutrophils, eosinophils, and macrophages) leading to an increased production of inflammatory mediators at the mucosal area with consequent epithelial erosion and ulceration. The progressive destruction of mucosal integrity causes the rupture of the *tight junctions* proteins, leading to an increase in the intestinal permeability with subsequent penetration of commensal microbiota to the submucosal layer generating bacteria translocation which exacerbates the inflammatory process and intensifies the symptoms [53–57]. Besides, the intestinal microbiota composition is also modified by the chemotherapeutic drugs and radiotherapy action [54, 58, 59] resulting in dysbiosis. After the end of treatment, recovery and restoration of the GIT structure occur [60].

### **6. Metabolic syndrome**

Besides IBD and mucositis, it has been reported that intestinal microbiota has an intrinsic effect on metabolism, potentially contributing to several features of the pathophysiology of metabolic syndrome [61, 62]. The metabolic syndrome is an accumulation of various risk factors (glucose intolerance, hyperinsulinemia,

**53**

**7. Functional foods**

markers.

*Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases...*

hypertension, as well as dyslipidemia) which can often be associated with insulin resistance, hypertension with abdominal fat accumulation, and obesity [63–65]. The etiology of metabolic syndrome is not well-defined; however there are evident characteristics and life habits that could contribute to its development such as unbalanced diet, smoking, lack of physical activity, and the genetic predisposition [66]. These factors directly increase the risk of cardiovascular disease and chronic diseases as type 2 diabetes mellitus and obesity, and the interaction between components of both the clinical and biological phenotypes of the syndrome contributes to the development of a pro-inflammatory state [67]. The inflammatory process observed in MS is directly associated with increased

oxidative stress. The reactive oxygen species (ROS) are capable of mediating symptoms of diabetes mellitus, such as insulin resistance and decrease in insulin secretion, and attend as precursors for the formation of LDLox (oxidized lowdensity lipoproteins), responsible for a large part of the development of atherosclerotic lesions, and the increase in circulating cholesterol fractions and glucose [68, 69]. In addition, chronic diseases are directly related to changes in the intestinal microbiome [70, 71], and they are also associated with elevated circulating levels of

The probiotic use in attenuating symptoms of different inflammatory diseases is widely reported in the literature. Among the commercial probiotics studied for treatment of these diseases, only a few products have been extensively tested in clinical trials in patients with MS, in order to demonstrate an effective result on weight loss, lipid metabolism, and reduction of inflammatory

Studies performed with *Lactobacillus* strains have shown the ability of these probiotics in reducing the lipid accumulation in adipose tissues, as well as in inducing the subexpression of lipogenic genes [73, 74]. Animals that received diets with high concentrations of lipids and then treated with *L. gasseri* SBT2050 had shown lower intestinal permeability and bacterial translocation, as well as reduction of inflammatory parameters, suggesting that this strain improves the intestinal barrier function [75–78]. In addition, *L gasseri* BRN17 was studied to treat animals with MS caused by the carbohydrate-rich diet consumption. This strain reduced the accumulation of adipose tissue in mice, and it has a beneficial effect on weight loss [79–81]. Another important approach with associated probiotics (*Bifidobacterium*, *Lactobacillus*, and *S. thermophilus*) for treatment of overweight patients has shown an improvement in lipid profile, as well as insulin sensitivity [82]. Besides, recently Hsieh e collaborators [83] demonstrated that administration of live *Lactobacillus reuteri* ADR-1 and killed *Lactobacillus reuteri* ADR-3 strain ameliorated type 2 diabetes mellitus in a clinical trial. The results indicated that the consumption of ADR-1 displayed a reduction effect on serum glycated hemoglobin (HbA1c), triglyceride, and cholesterol levels. On the other hand, the intake of ADR-3 showed a beneficial effect on blood pressure reduction. Besides, a reduction in the levels of pro-inflammatory cytokines (IL-1β), increase in antioxidant enzyme (superoxide dismutase), and the changes in intestinal microflora composition (increase in intestinal level of *Lactobacillus* spp. and *Bifidobacterium* spp. and decrease in *Bacteroidetes*) were observed. Thus, these strategies highlight the beneficial and potential effect of interventions targeting gut microbiota modulation by the use of probiotic strains to treat components or complications of metabolic syndrome.

The human being for more than 4000 years has been consuming fermented products, by the fermentation process. At the beginning this practice was done

pro-inflammatory cytokines such as TNF and IL-6 [72].

*DOI: http://dx.doi.org/10.5772/intechopen.88325*

#### *Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases... DOI: http://dx.doi.org/10.5772/intechopen.88325*

hypertension, as well as dyslipidemia) which can often be associated with insulin resistance, hypertension with abdominal fat accumulation, and obesity [63–65].

The etiology of metabolic syndrome is not well-defined; however there are evident characteristics and life habits that could contribute to its development such as unbalanced diet, smoking, lack of physical activity, and the genetic predisposition [66]. These factors directly increase the risk of cardiovascular disease and chronic diseases as type 2 diabetes mellitus and obesity, and the interaction between components of both the clinical and biological phenotypes of the syndrome contributes to the development of a pro-inflammatory state [67].

The inflammatory process observed in MS is directly associated with increased oxidative stress. The reactive oxygen species (ROS) are capable of mediating symptoms of diabetes mellitus, such as insulin resistance and decrease in insulin secretion, and attend as precursors for the formation of LDLox (oxidized lowdensity lipoproteins), responsible for a large part of the development of atherosclerotic lesions, and the increase in circulating cholesterol fractions and glucose [68, 69]. In addition, chronic diseases are directly related to changes in the intestinal microbiome [70, 71], and they are also associated with elevated circulating levels of pro-inflammatory cytokines such as TNF and IL-6 [72].

The probiotic use in attenuating symptoms of different inflammatory diseases is widely reported in the literature. Among the commercial probiotics studied for treatment of these diseases, only a few products have been extensively tested in clinical trials in patients with MS, in order to demonstrate an effective result on weight loss, lipid metabolism, and reduction of inflammatory markers.

Studies performed with *Lactobacillus* strains have shown the ability of these probiotics in reducing the lipid accumulation in adipose tissues, as well as in inducing the subexpression of lipogenic genes [73, 74]. Animals that received diets with high concentrations of lipids and then treated with *L. gasseri* SBT2050 had shown lower intestinal permeability and bacterial translocation, as well as reduction of inflammatory parameters, suggesting that this strain improves the intestinal barrier function [75–78]. In addition, *L gasseri* BRN17 was studied to treat animals with MS caused by the carbohydrate-rich diet consumption. This strain reduced the accumulation of adipose tissue in mice, and it has a beneficial effect on weight loss [79–81]. Another important approach with associated probiotics (*Bifidobacterium*, *Lactobacillus*, and *S. thermophilus*) for treatment of overweight patients has shown an improvement in lipid profile, as well as insulin sensitivity [82]. Besides, recently Hsieh e collaborators [83] demonstrated that administration of live *Lactobacillus reuteri* ADR-1 and killed *Lactobacillus reuteri* ADR-3 strain ameliorated type 2 diabetes mellitus in a clinical trial. The results indicated that the consumption of ADR-1 displayed a reduction effect on serum glycated hemoglobin (HbA1c), triglyceride, and cholesterol levels. On the other hand, the intake of ADR-3 showed a beneficial effect on blood pressure reduction. Besides, a reduction in the levels of pro-inflammatory cytokines (IL-1β), increase in antioxidant enzyme (superoxide dismutase), and the changes in intestinal microflora composition (increase in intestinal level of *Lactobacillus* spp. and *Bifidobacterium* spp. and decrease in *Bacteroidetes*) were observed. Thus, these strategies highlight the beneficial and potential effect of interventions targeting gut microbiota modulation by the use of probiotic strains to treat components or complications of metabolic syndrome.

## **7. Functional foods**

The human being for more than 4000 years has been consuming fermented products, by the fermentation process. At the beginning this practice was done

*The Health Benefits of Foods - Current Knowledge and Further Development*

donor are very important parameters to be considered.

**5. Intestinal mucositis**

longer hospitalization.

**6. Metabolic syndrome**

Crohn's disease is rather more limited than for UC, but it has been shown that single standardized FMT resulted in a clinical remission sustained for more than 9 months in CD patients [43]. However, the authors suggest that further studies are needed to enhance the knowledge about the use of stool transplantation for IBD treatment. Alteration in the gut microbiome composition with increase in some groups of microorganisms, such as *Clostridium* and *Fusobacterium*, was also reported in patients with pouchitis [44, 45]. In this context, literature evidences indicate that the probiotic administration such as VSL#3 is effective in the chronic pouchitis prevention [46]. On the other hand, FMT to pouchitis treatment did not report the same beneficial results. Only three reports with this approach [47–49] exposed that neither clinical remission nor any adequate response was observed in the evaluated patients suggesting that the efficacy of FMT for pouchitis after proctocolectomy is limited [49]. The importance of standardization of this procedure needs to be highlighted to improve its efficacy, since frequency, route of administration (e.g., endoscopy, nasogastric tube, colonoscopy), and the criteria of choice of healthy

Different chemotherapy regimens such as FOLFOX (5-fluorouracil and oxaliplatin), FOLFIRI (5-fluorouracil and irinotecan), and triple FOLFOXIRI regimen (5-fluorouracil, oxaliplatin, and irinotecan) [50, 51] are adopted for

Mucositis is the most common side effect in patients undergoing chemotherapy/ radiotherapy treatments, which consist in an inflammation and/or ulcers in the gastrointestinal tract [52] with consequent loss of cells from the epithelial barrier of the GIT. Many symptoms are related to gastrointestinal mucositis, such as diarrhea, severe abdominal pain, bleeding, fatigue, malnutrition, dehydration, electrolyte imbalance, and infections, with potential fatal complications which can conduce to reduction or interruption of antitumor treatment [53] and consequently leads to

This pathology occurs due to cytotoxic effects of anticancer drugs/radiotherapy that cause damage at the DNA of stem cell (epithelial cell progenitors) with intense oxidative stress and consequent cell death. This apoptotic process is exacerbated affecting the absorption by shortening the villi structure of enterocytes and causing the loss of epithelial barrier with an invasion of inflammatory cells (neutrophils, eosinophils, and macrophages) leading to an increased production of inflammatory mediators at the mucosal area with consequent epithelial erosion and ulceration. The progressive destruction of mucosal integrity causes the rupture of the *tight junctions* proteins, leading to an increase in the intestinal permeability with subsequent penetration of commensal microbiota to the submucosal layer generating bacteria translocation which exacerbates the inflammatory process and intensifies the symptoms [53–57]. Besides, the intestinal microbiota composition is also modified by the chemotherapeutic drugs and radiotherapy action [54, 58, 59] resulting in dysbiosis. After the end of treatment, recovery and restoration of the GIT structure occur [60].

Besides IBD and mucositis, it has been reported that intestinal microbiota has an intrinsic effect on metabolism, potentially contributing to several features of the pathophysiology of metabolic syndrome [61, 62]. The metabolic syndrome is an accumulation of various risk factors (glucose intolerance, hyperinsulinemia,

different types of cancer but with a broad range of collateral effects.

**52**

to preserve foods from either physical, chemical, or microbial alterations. The microorganisms participating in this process are the lactic acid bacteria, extensively widespread in nature and also belong to the GIT communities, able to convert the sugar in lactic acid as well as produce other metabolites which contribute to food modifications, either sensorial or nutritional value. Thus, the terminology "functional food" was attributed to food with health benefits to the consumer including nutritional and physiological function [84–86].

During the fermentation, these bacteria can contribute to improving the digestion of nutrients (lactose, proteins, small peptides, and polysaccharides); providing essential micronutrients (vitamins) as well as bioactive compounds (metabolites) with potential health benefits to the host, such as prevention against enteric inflammation [87, 88]; providing antimicrobial, antihypertensive, hypocholesterolemic, immunomodulatory, antioxidant, and anticancer effects [46, 85, 89–92]; showing ability to regulate the immunity; and, consequently, improving host quality of life [93].

Therefore, the gut communities and the microbial-derived molecules present in the gut lumen have been strongly influenced, either qualitatively or quantitatively, by consumption of dairy products [94] such as yogurts, cheeses, and fermented milk, among other fermented products using probiotic bacteria. Thus, the microbiota manipulation by functional food, probiotics, and prebiotics are evaluated as a beneficial option for treatment of GIT diseases [95].

#### **8. Lactic acid bacteria: the largest group of probiotic bacteria**

There is a constant interaction between the host and the bowel commensal bacterial community in order to maintain the homeostasis [3, 96–98]. However, when this mutualist relationship is compromised, the intestinal microbiota may cause and/or contribute to either the establishment or the progression of inflammatory diseases [96–99]. In this context, the search for therapeutic strategies that minimize the development and progression of pathologies caused directly and indirectly by the unbalance of the commensal microbiota has grown. The consumption of probiotic bacteria is one of these strategies, as they present several effects, such as ability to improve the intestinal barrier, stimulate the systemic and mucosal immune system, regulate the composition of the intestinal microbiota, and provide essential micronutrients (such as vitamins and SCFAs) and other bioactive compounds (metabolites) with potential health benefits for the host [100–103].

Probiotics are defined as "live microorganisms that offer host health benefits when administered in adequate amounts" [104, 105]. The majority of the studied probiotics belongs to the group of lactic acid bacteria. However, other microorganisms with probiotic properties also deserve attention, such as yeasts (*Saccharomyces* spp.) and bacteria of the genus *Bifidobacterium* and *Faecalibacterium*, among others [106–108].

LAB, which include, mainly, species from the genus *Lactobacillus*, *Leuconostoc*, *Lactococcus*, *Pediococcus*, and *Streptococcus*, constitute a group of Gram-positive, anaerobic or aerotolerant, nonspore-forming, nonmobile, and highly low pH-tolerant microorganisms. However, the main characteristic of this group is its ability to produce lactic acid as the final product of the fermentation of carbohydrates [109–111].

#### **9. Probiotic effects in gastrointestinal inflammation**

LAB are often present in the human gut but also can be introduced by the ingestion of fermented foods, such as yogurt and other fermented milk products and fermented cured meat by-products [103], having the generally recognized as

**55**

*Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases...*

safe (GRAS) status by the Food and Drug Administration (FDA). *Lactobacillus* spp., *Streptococcus* spp., and *Lactococcus* spp. are the major LAB species with probiotic effects, and they have been used in therapeutic applications for treatment and

Scientific evidence reveals that the mechanisms by which probiotic bacteria ameliorate inflammatory bowel damage are heterogeneous, strain specific, and dependent on the number of available bacteria. Thus, administration of probiotic bacteria, specially LAB, improves intestinal inflammatory responses by (i) modulation and normalization of perturbed intestinal microbial communities; (ii) competitive exclusion of pathogens such as *Staphylococcus aureus* and *Salmonella typhimurium*, among others; (iii) bacteriocin and SCFA production; (iv) enzymatic activities related to metabolization of a number of carcinogens and other toxic substances; (v) adhesion to mucosal cells, cell antagonism, and mucin production; (vi) intestinal permeability reduction by tight junctions protein modulation (e.g., zonulin, claudin, occludin, junctional adhesion molecule); (vii) modulation of the immune system by stimulating Tregs cells, IgA production by B cells, and NF-kβ signaling pathway inhibition; and (viii) interaction with the brain-gut axis via the

In order to potentialize the beneficial effects of probiotic strains, research has been conducted over the last decades, based on genetic engineering techniques, especially those related to DNA manipulation. Thus, modern methods of genetic engineering open the new opportunities to design and create genetically modified probiotic strains with the desired characteristics or to exclusively target a specific pathogen or toxin to be used either as a vaccine or for drug delivery [119, 120]. Since most of the probiotic strains are part of the LAB group, most of the genetic manipulation studies are carried out with species that belong to this group, such as *Lactococcus* and *Lactobacillus* genera. Consequently, recombinant probiotics have been created for mucosal delivery of therapeutic and/or prophylactic molecules comprising DNA, peptides, single-chain variable fragments, cytokines, enzymes, and allergens [121, 122], leading to the concept of "biodrug" for the prevention and treatment of various diseases [123]. Thus, researches have emphasized the use of species of these genera in two different approaches: the first as producers of heterologous protein and the second as vehicle for delivery of DNA vaccines [124].

*DOI: http://dx.doi.org/10.5772/intechopen.88325*

**Figure 1.**

prevention of various intestinal disorders [112, 113].

*A schematic diagram about potential action mechanisms of probiotic bacteria.*

generation of bacterial metabolites (**Figure 1**) [103, 114–118].

**10. Recombinant LAB probiotics**

*Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases... DOI: http://dx.doi.org/10.5772/intechopen.88325*

**Figure 1.**

*The Health Benefits of Foods - Current Knowledge and Further Development*

nutritional and physiological function [84–86].

beneficial option for treatment of GIT diseases [95].

**8. Lactic acid bacteria: the largest group of probiotic bacteria**

**9. Probiotic effects in gastrointestinal inflammation**

LAB are often present in the human gut but also can be introduced by the ingestion of fermented foods, such as yogurt and other fermented milk products and fermented cured meat by-products [103], having the generally recognized as

There is a constant interaction between the host and the bowel commensal bacterial community in order to maintain the homeostasis [3, 96–98]. However, when this mutualist relationship is compromised, the intestinal microbiota may cause and/or contribute to either the establishment or the progression of inflammatory diseases [96–99]. In this context, the search for therapeutic strategies that minimize the development and progression of pathologies caused directly and indirectly by the unbalance of the commensal microbiota has grown. The consumption of probiotic bacteria is one of these strategies, as they present several effects, such as ability to improve the intestinal barrier, stimulate the systemic and mucosal immune system, regulate the composition of the intestinal microbiota, and provide essential micronutrients (such as vitamins and SCFAs) and other bioactive compounds (metabolites) with potential health benefits for the host [100–103].

Probiotics are defined as "live microorganisms that offer host health benefits when administered in adequate amounts" [104, 105]. The majority of the studied probiotics belongs to the group of lactic acid bacteria. However, other microorganisms with probiotic properties also deserve attention, such as yeasts (*Saccharomyces* spp.) and bacteria of the genus *Bifidobacterium* and *Faecalibacterium*, among others [106–108]. LAB, which include, mainly, species from the genus *Lactobacillus*, *Leuconostoc*, *Lactococcus*, *Pediococcus*, and *Streptococcus*, constitute a group of Gram-positive, anaerobic or aerotolerant, nonspore-forming, nonmobile, and highly low pH-tolerant microorganisms. However, the main characteristic of this group is its ability to produce lactic acid as the final product of the fermentation of carbohydrates [109–111].

to preserve foods from either physical, chemical, or microbial alterations. The microorganisms participating in this process are the lactic acid bacteria, extensively widespread in nature and also belong to the GIT communities, able to convert the sugar in lactic acid as well as produce other metabolites which contribute to food modifications, either sensorial or nutritional value. Thus, the terminology "functional food" was attributed to food with health benefits to the consumer including

During the fermentation, these bacteria can contribute to improving the digestion of nutrients (lactose, proteins, small peptides, and polysaccharides); providing essential micronutrients (vitamins) as well as bioactive compounds (metabolites) with potential health benefits to the host, such as prevention against enteric inflammation [87, 88]; providing antimicrobial, antihypertensive, hypocholesterolemic, immunomodulatory, antioxidant, and anticancer effects [46, 85, 89–92]; showing ability to regulate the immunity; and, consequently, improving host quality of life [93].

Therefore, the gut communities and the microbial-derived molecules present in the gut lumen have been strongly influenced, either qualitatively or quantitatively, by consumption of dairy products [94] such as yogurts, cheeses, and fermented milk, among other fermented products using probiotic bacteria. Thus, the microbiota manipulation by functional food, probiotics, and prebiotics are evaluated as a

**54**

*A schematic diagram about potential action mechanisms of probiotic bacteria.*

safe (GRAS) status by the Food and Drug Administration (FDA). *Lactobacillus* spp., *Streptococcus* spp., and *Lactococcus* spp. are the major LAB species with probiotic effects, and they have been used in therapeutic applications for treatment and prevention of various intestinal disorders [112, 113].

Scientific evidence reveals that the mechanisms by which probiotic bacteria ameliorate inflammatory bowel damage are heterogeneous, strain specific, and dependent on the number of available bacteria. Thus, administration of probiotic bacteria, specially LAB, improves intestinal inflammatory responses by (i) modulation and normalization of perturbed intestinal microbial communities; (ii) competitive exclusion of pathogens such as *Staphylococcus aureus* and *Salmonella typhimurium*, among others; (iii) bacteriocin and SCFA production; (iv) enzymatic activities related to metabolization of a number of carcinogens and other toxic substances; (v) adhesion to mucosal cells, cell antagonism, and mucin production; (vi) intestinal permeability reduction by tight junctions protein modulation (e.g., zonulin, claudin, occludin, junctional adhesion molecule); (vii) modulation of the immune system by stimulating Tregs cells, IgA production by B cells, and NF-kβ signaling pathway inhibition; and (viii) interaction with the brain-gut axis via the generation of bacterial metabolites (**Figure 1**) [103, 114–118].

#### **10. Recombinant LAB probiotics**

In order to potentialize the beneficial effects of probiotic strains, research has been conducted over the last decades, based on genetic engineering techniques, especially those related to DNA manipulation. Thus, modern methods of genetic engineering open the new opportunities to design and create genetically modified probiotic strains with the desired characteristics or to exclusively target a specific pathogen or toxin to be used either as a vaccine or for drug delivery [119, 120]. Since most of the probiotic strains are part of the LAB group, most of the genetic manipulation studies are carried out with species that belong to this group, such as *Lactococcus* and *Lactobacillus* genera. Consequently, recombinant probiotics have been created for mucosal delivery of therapeutic and/or prophylactic molecules comprising DNA, peptides, single-chain variable fragments, cytokines, enzymes, and allergens [121, 122], leading to the concept of "biodrug" for the prevention and treatment of various diseases [123]. Thus, researches have emphasized the use of species of these genera in two different approaches: the first as producers of heterologous protein and the second as vehicle for delivery of DNA vaccines [124].

#### **10.1 LAB as producers of heterologous protein**

Many studies are carried out with *Lactococcus lactis* due to its economic importance in the production of cheese and its easy growth and manipulation. In addition, it was the first species of LAB to have its genome completely sequenced, which allowed a greater understanding of its genetic and physiological mechanisms, aiding in the development of technological packages for its genetic manipulation in a laboratory environment [124–128].

There are several ways to make LAB produce heterologous proteins, and the most used form is through the insertion of a plasmid into its cytoplasm. Plasmids are elements of extrachromosomal DNA that are naturally found in prokaryotes. With the advent of the recombinant DNA technique, these elements have been manipulated to act as molecular vehicles that allow the production of proteins of interest by the bacterium [129].

The first heterologous protein production system based on plasmid insertion in LAB was developed for *L. lactis.* These systems included both inducible and constitutive promoters, which ensure efficient expression of the antigen of interest under different conditions [130, 131]. Although it is possible to choose the type of promoter to be used in the vector, the vast majority of expression vectors present inducible promoters that allow controlled expression of the protein of interest by protecting against aggregation and protein degradation in the bacterial cytoplasm. On the other hand, these vectors present safety issues that need to be analyzed since it is necessary to introduce chemical compounds into the culture medium to induce protein expression prior to animal administration [132–134].

With the improvement of cloning and expression techniques, several production systems were developed, specifically for LAB, allowing the production of different molecules of interest, including pathogen antigens, by a large number of LAB species [135–139]. The most commonly used regulation systems in LAB are the following:

#### *10.1.1 Nisin-controlled gene expression (NICE)*

Among the heterologous production systems, the most widely studied is the nisin-controlled gene expression system. This system is based on the expression of three genes (nisA, nisF, and nisR) that are involved in the production and regulation of the antimicrobial peptide nisin, which is naturally secreted by different strains of *L. lactis*. In this system the membrane-located histidine kinase NisK senses the signal inducer nisin and autophosphorylates and then transfers the phosphorous group to the intracellular response regulator protein NisR which acts as a transcription activator of nisA/nisF and induces gene expression under pNis promoter. Depending on the presence or absence of the corresponding targeting signals, the protein is either expressed into the cytoplasm or the cell envelope or secreted into the external medium [140]. Thus, it has already been successfully used for the expression of different proteins of medical and biotechnological interest [141, 142].

#### *10.1.2 Xylose-inducible expression system (XIES)*

In 2004, Miyoshi and colleagues [143] developed the xylose-inducible expression system whose promoter is the xylose permease gene (pxylT) found in *L. lactis* NCDO2118. This system produces either cytoplasmic or secreted proteins being activated in the presence of xylose and strongly repressed in the presence of glucose, fructose, or mannose [143].

**57**

*Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases...*

using the *L. lactis* groESL promoter [134]. This system induces expression of proteins of interest via stress stimuli such as those found in the GIT (e.g., bile salt, acid pH, antimicrobial peptide, and heat shock proteins) [134, 144]. This system does not require the induction of bacterial culture or the presence of regulatory genes, being a good alternative in the delivery and production of therapeutic

**10.2 LAB as a live vehicle to deliver DNA vaccine plasmids to eukaryotic cells**

Among the available approaches to stimulate efficient mucosal responses, the use of bacterial system for DNA delivery and its expression using the eukaryotic cell machinery have been extensively explored. Unlike the production of heterologous protein, in which the bacterium is responsible for the synthesis of the protein of interest, in the DNA vaccine platform, the bacteria only act as a delivery vehicle for

New vectors had been developed to approach the DNA vaccine using LAB as live delivery vehicles [146–150]. These vectors present a series of common characteristics such as the presence of a eukaryotic promoter, which allows protein expression by eukaryotic cells; a prokaryotic region, which has a selection marker (usually antibiotic resistance); a multiple cloning site, where the open reading frame (ORF) of interest will be inserted; and a prokaryotic origin of replication, which ensures that the plasmid replicates only in prokaryotic cells [151]. Some molecules (IL-10, IL-4, and HSP65) have been cloned in these vectors to evaluate their effect, espe-

[152, 153], as well as reporters (GFP and Cherry) which allowed the understanding of this platform in the mammalian body [148, 154]. Although further studies need to be conducted in order to elucidate whether the cloning of ORFs of interest in these vectors is really effective pointing to disease prevention and treatment, this approach is undoubtedly an important tool for the development of new techniques

Among the different techniques used to construct recombinant LAB strains, the most recent is associated with the use of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, based on the use of a system present in several bacterial strains that works as part of the adaptive immune system of bacteria and archaea against the presence of external DNA, such as plasmids and

Although this system has been studied for more than 30 years [160], it was only in 2013 that the first experiments were carried out emphasizing its use as a tool for genome editing [161, 162]. Evaluating the CRISPR databases, it is possible to observe that about 46% of all bacterial genomes presents the CRISPR-Cas system, and this percentage reaches approximately 63% of the sequenced *Lactobacillus* genomes [163]. The natural presence of this system in most of the LAB strains expands the possibilities of genetic manipulation of microorganisms of this group,

The first gene editing experiment in LAB based on the CRISPR-Cas system was conducted by Oh and van Pijkeren [165] where they were able to edit three different

More recently, the stress-inducible controlled expression system was developed

*DOI: http://dx.doi.org/10.5772/intechopen.88325*

proteins at mucosal surfaces.

*10.1.3 Stress-inducible controlled expression system (SICE)*

prophylactic and therapeutic purposes [109, 145].

with potential in the medical clinic.

bacteriophages [155–159].

including probiotic ones [164].

cially as a treatment approach in diseases related to the bowel

**11. Next-generation recombinants: using CRISPR-Cas system**

*Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases... DOI: http://dx.doi.org/10.5772/intechopen.88325*

#### *10.1.3 Stress-inducible controlled expression system (SICE)*

*The Health Benefits of Foods - Current Knowledge and Further Development*

Many studies are carried out with *Lactococcus lactis* due to its economic importance in the production of cheese and its easy growth and manipulation. In addition, it was the first species of LAB to have its genome completely sequenced, which allowed a greater understanding of its genetic and physiological mechanisms, aiding in the development of technological packages for its genetic manipulation in

There are several ways to make LAB produce heterologous proteins, and the most used form is through the insertion of a plasmid into its cytoplasm. Plasmids are elements of extrachromosomal DNA that are naturally found in prokaryotes. With the advent of the recombinant DNA technique, these elements have been manipulated to act as molecular vehicles that allow the production of proteins of

The first heterologous protein production system based on plasmid insertion in LAB was developed for *L. lactis.* These systems included both inducible and constitutive promoters, which ensure efficient expression of the antigen of interest under different conditions [130, 131]. Although it is possible to choose the type of promoter to be used in the vector, the vast majority of expression vectors present inducible promoters that allow controlled expression of the protein of interest by protecting against aggregation and protein degradation in the bacterial cytoplasm. On the other hand, these vectors present safety issues that need to be analyzed since it is necessary to introduce chemical compounds into the culture medium to induce protein expression prior to animal administration

With the improvement of cloning and expression techniques, several production systems were developed, specifically for LAB, allowing the production of different molecules of interest, including pathogen antigens, by a large number of LAB species [135–139]. The most commonly used regulation systems in LAB are the following:

Among the heterologous production systems, the most widely studied is the nisin-controlled gene expression system. This system is based on the expression of three genes (nisA, nisF, and nisR) that are involved in the production and regulation of the antimicrobial peptide nisin, which is naturally secreted by different strains of *L. lactis*. In this system the membrane-located histidine kinase NisK senses the signal inducer nisin and autophosphorylates and then transfers the phosphorous group to the intracellular response regulator protein NisR which acts as a transcription activator of nisA/nisF and induces gene expression under pNis promoter. Depending on the presence or absence of the corresponding targeting signals, the protein is either expressed into the cytoplasm or the cell envelope or secreted into the external medium [140]. Thus, it has already been successfully used for the expression of different proteins of medical and biotechnological

In 2004, Miyoshi and colleagues [143] developed the xylose-inducible expression system whose promoter is the xylose permease gene (pxylT) found in *L. lactis* NCDO2118. This system produces either cytoplasmic or secreted proteins being activated in the presence of xylose and strongly repressed in the presence of

**10.1 LAB as producers of heterologous protein**

a laboratory environment [124–128].

interest by the bacterium [129].

*10.1.1 Nisin-controlled gene expression (NICE)*

*10.1.2 Xylose-inducible expression system (XIES)*

glucose, fructose, or mannose [143].

[132–134].

**56**

interest [141, 142].

More recently, the stress-inducible controlled expression system was developed using the *L. lactis* groESL promoter [134]. This system induces expression of proteins of interest via stress stimuli such as those found in the GIT (e.g., bile salt, acid pH, antimicrobial peptide, and heat shock proteins) [134, 144]. This system does not require the induction of bacterial culture or the presence of regulatory genes, being a good alternative in the delivery and production of therapeutic proteins at mucosal surfaces.

#### **10.2 LAB as a live vehicle to deliver DNA vaccine plasmids to eukaryotic cells**

Among the available approaches to stimulate efficient mucosal responses, the use of bacterial system for DNA delivery and its expression using the eukaryotic cell machinery have been extensively explored. Unlike the production of heterologous protein, in which the bacterium is responsible for the synthesis of the protein of interest, in the DNA vaccine platform, the bacteria only act as a delivery vehicle for prophylactic and therapeutic purposes [109, 145].

New vectors had been developed to approach the DNA vaccine using LAB as live delivery vehicles [146–150]. These vectors present a series of common characteristics such as the presence of a eukaryotic promoter, which allows protein expression by eukaryotic cells; a prokaryotic region, which has a selection marker (usually antibiotic resistance); a multiple cloning site, where the open reading frame (ORF) of interest will be inserted; and a prokaryotic origin of replication, which ensures that the plasmid replicates only in prokaryotic cells [151]. Some molecules (IL-10, IL-4, and HSP65) have been cloned in these vectors to evaluate their effect, especially as a treatment approach in diseases related to the bowel [152, 153], as well as reporters (GFP and Cherry) which allowed the understanding of this platform in the mammalian body [148, 154]. Although further studies need

to be conducted in order to elucidate whether the cloning of ORFs of interest in these vectors is really effective pointing to disease prevention and treatment, this approach is undoubtedly an important tool for the development of new techniques with potential in the medical clinic.

## **11. Next-generation recombinants: using CRISPR-Cas system**

Among the different techniques used to construct recombinant LAB strains, the most recent is associated with the use of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, based on the use of a system present in several bacterial strains that works as part of the adaptive immune system of bacteria and archaea against the presence of external DNA, such as plasmids and bacteriophages [155–159].

Although this system has been studied for more than 30 years [160], it was only in 2013 that the first experiments were carried out emphasizing its use as a tool for genome editing [161, 162]. Evaluating the CRISPR databases, it is possible to observe that about 46% of all bacterial genomes presents the CRISPR-Cas system, and this percentage reaches approximately 63% of the sequenced *Lactobacillus* genomes [163]. The natural presence of this system in most of the LAB strains expands the possibilities of genetic manipulation of microorganisms of this group, including probiotic ones [164].

The first gene editing experiment in LAB based on the CRISPR-Cas system was conducted by Oh and van Pijkeren [165] where they were able to edit three different regions of the genome, with efficiency up to 100% in the selected clones. After this pioneering work, few others were published focusing on LAB gene editing [166–168].

Therefore, the use of this technology is presented as a widely viable strategy to be applied in LAB, enabling the development of food-grade recombinant strains in order to allow their future use in the clinic [169].

## **12. Use of recombinant LAB to treat GIT-related disorders**

The use of recombinant *L. lactis* strains, as well as others recombinant LAB strains, using different systems has shown promising results in many studies as an alternative therapy to treat, especially, GIT inflammation and other diseases (**Table 1**).

To arrive at mucosa in sufficient quantities to exert their therapeutic effects, many LAB strains must survive, during their passage through the GIT, stressor factors such as pH, temperature, bile salt concentration, and the presence of antimicrobial peptides [170–172]. In this context, an interest approach was recently developed by Coelho-Rocha and colleagues [154] using an encapsulated recombinant strain (*L. lactis* pExu:*mcherry*) and tested it through the GIT at different times post-administration. They have shown that the microencapsulation process is an effective method to improve DNA delivery, guaranteeing a greater number of viable bacteria able to reach different sections of the bowel [154].

The use of recombinant probiotics to improve therapeutic approaches has been widely studied using different systems with different molecules. As IBDs are a serious clinical topic, many strategies have been tested trying to improve previous results found with wild type strains.

*L. lactis* MG1363 strain carrying the pTREX1 vector expressing the mouse IL-27 protected mice against the inflammatory effects of dextran sulfate sodium (DSS) induced colitis. This recombinant strain was able to reduce disease activity scores and pathology features of the large and small bowels and also led to reduced levels of inflammatory cytokines IL-1β, TNF-α, and IFN-γ in colonic tissue. In addition, reduction in the number of CD4+ and IL-17<sup>+</sup> T cells in gut-associated lymphoid tissue and increase in IL-10 production were observed [173].

Besides, it was also demonstrated in a DSS-induced colitis mouse model that the oral administration of *L. lactis* NZ900 strain harboring the NICE system expressing either the anti-inflammatory cytokine IL-10, TGF-β1, secretory leukocyte protease inhibitor (SLPI), or elafin was able to ameliorate some clinical parameters in inflamed mice. Even though it was possible to observe the reduction of weight loss and diarrhea, microscopic colonic damage scores, colon thickness, and myeloperoxidase (MPO) activity, the authors reported that treatments with recombinant *L. lactis* strain delivering either SLPI or elafin were more efficient to reduce signs of colitis than treatments with anti-inflammatory cytokines. Altogether these recombinant strains display anti-inflammatory effects in inflamed mice [174].

Approaches using the invasive *L. lactis* MG1363 FnBPA+ , by expressing the FnBpA protein at their surface and carrying the pValac eukaryotic expression vector coding either the IL-10 cytokine [r*L. lactis* FnPBA+ (pValac:*il-10*)] or the IL-4 cytokine [r*L. lactis* FnPBA+ (pValac:*il-4*)] in DSS or trinitrobenzenesulfonic acid (TNBS)-induced acute model of colitis, respectively, were also investigated. The administration of *L. lactis* FnPBA+ (pValac:*il-10*) recombinant strain was capable to reduce the intestinal inflammation by increasing IL-10 levels and sIgA production, accompanied by decreasing IL-6, as well as the restoration of intestinal architecture of mice colon [153]. Besides, the engineered *L. lactis* FnPBA+ (pValac:*il-4*) was able to slump the level of pro-inflammatory cytokine (IL-12, IL-6) and myeloperoxidase activity and increase levels of IL-4 and IL-10, consequently decreasing the colitis harshness [153].

**59**

*Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases...*

**Inflamation Condition**

DNBS-induced colitis

Mouse model of DSS/TNBSinduced colitis

of DSS-induced colitis

of DSS-induced colitis

of DSS-induced colitis

DNBS-induced colitis

Mouse model of TNBS-induced Crohn's disease

Mouse model of TNBS-induced colitis

Hamster model of radiation-induced oral mucositis

5-FU-induced intestinal mucositis

NICE Mouse model of

NICE Mouse model

XIES Mouse model

XIES Mouse model

**Anti-Inflamatory Properties**

Restoration of intestinal architecture; IgA production and IL-6 reduction; Reduced tissue damage

Decreased IL-6, IL-12 and MPO activity Reduced tissue damage

Reduced tissue damage Decreased proinflammatory cytokines

Reduced tissue damage

Restoration of intestinal architecture CD4+Foxp3+ and CD4+LAP+ regulatory T cells production

Reduced tissue damage

Reduced tissue damage Reduced microbial translocation Increase IL-10/ INF-γ reduction

Reduced tissue damage Reduced microbial translocation IL-17 reduction

Reduced clicnical scores of oral mucositis

Microbiota Regulation Villus architecture preservation Increased Paneth cells activity

**References**

[134]

[152, 153]

[174]

[175]

[176]

[177]

[180]

[181]

[186]

[185, 187]

**System**

pValac vector

*L. lactis* MG1363 Mouse IL-10 SICE Mouse model of

*DOI: http://dx.doi.org/10.5772/intechopen.88325*

**Microorganism Gene Expression** 

*L. lactis* MG1363 Mouse IL-10 and IL-4

*L. lactis* NZ9000 Mouse TGF-β1; IL-10 and leukocyte protease inhibitor Human Elafin

*L. casei* BL23 Superoxide

*L.lactis* AG013 Human Trefoil

*L. lactis* NZ9000 Human

Human 15-lipoxygenase-1

*M. leprae* Hsp65 protein

dismutase A from *L.lactis* MG1363 Catalase from *L.plantarum* ATCC

Superoxide dismutase A from *L.lactis* MG1363 Catalase from *L.plantarum* ATCC

Factor 1 (Htff-1)

pancreatitis associated protein (Reg3A)

*B. bifidum* BS42 Mouse IL-10 BEST Mouse model of

pLEM415 vector

pIL253 vector

ThyA native promoter of *L.lactis*

*L. lactis* NCDO

*L. lactis* NCDO

*S. thermophilus* CLR807

2118

2118


*Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases... DOI: http://dx.doi.org/10.5772/intechopen.88325*

*The Health Benefits of Foods - Current Knowledge and Further Development*

**12. Use of recombinant LAB to treat GIT-related disorders**

bacteria able to reach different sections of the bowel [154].

tissue and increase in IL-10 production were observed [173].

Approaches using the invasive *L. lactis* MG1363 FnBPA+

either the IL-10 cytokine [r*L. lactis* FnPBA+

[153]. Besides, the engineered *L. lactis* FnPBA+

results found with wild type strains.

reduction in the number of CD4+

therapy to treat, especially, GIT inflammation and other diseases (**Table 1**).

order to allow their future use in the clinic [169].

regions of the genome, with efficiency up to 100% in the selected clones. After this pioneering work, few others were published focusing on LAB gene editing [166–168]. Therefore, the use of this technology is presented as a widely viable strategy to be applied in LAB, enabling the development of food-grade recombinant strains in

The use of recombinant *L. lactis* strains, as well as others recombinant LAB strains, using different systems has shown promising results in many studies as an alternative

To arrive at mucosa in sufficient quantities to exert their therapeutic effects, many LAB strains must survive, during their passage through the GIT, stressor factors such as pH, temperature, bile salt concentration, and the presence of antimicrobial peptides [170–172]. In this context, an interest approach was recently developed by Coelho-Rocha and colleagues [154] using an encapsulated recombinant strain (*L. lactis* pExu:*mcherry*) and tested it through the GIT at different times post-administration. They have shown that the microencapsulation process is an effective method to improve DNA delivery, guaranteeing a greater number of viable

The use of recombinant probiotics to improve therapeutic approaches has been

*L. lactis* MG1363 strain carrying the pTREX1 vector expressing the mouse IL-27 protected mice against the inflammatory effects of dextran sulfate sodium (DSS) induced colitis. This recombinant strain was able to reduce disease activity scores and pathology features of the large and small bowels and also led to reduced levels of inflammatory cytokines IL-1β, TNF-α, and IFN-γ in colonic tissue. In addition,

Besides, it was also demonstrated in a DSS-induced colitis mouse model that the oral administration of *L. lactis* NZ900 strain harboring the NICE system expressing either the anti-inflammatory cytokine IL-10, TGF-β1, secretory leukocyte protease inhibitor (SLPI), or elafin was able to ameliorate some clinical parameters in inflamed mice. Even though it was possible to observe the reduction of weight loss and diarrhea, microscopic colonic damage scores, colon thickness, and myeloperoxidase (MPO) activity, the authors reported that treatments with recombinant *L. lactis* strain delivering either SLPI or elafin were more efficient to reduce signs of colitis than treatments with anti-inflammatory cytokines. Altogether these recombinant strains display anti-inflammatory effects in inflamed mice [174].

protein at their surface and carrying the pValac eukaryotic expression vector coding

acute model of colitis, respectively, were also investigated. The administration of *L.* 

level of pro-inflammatory cytokine (IL-12, IL-6) and myeloperoxidase activity and increase levels of IL-4 and IL-10, consequently decreasing the colitis harshness [153].

inflammation by increasing IL-10 levels and sIgA production, accompanied by decreasing IL-6, as well as the restoration of intestinal architecture of mice colon

(pValac:*il-4*)] in DSS or trinitrobenzenesulfonic acid (TNBS)-induced

(pValac:*il-10*) recombinant strain was capable to reduce the intestinal

T cells in gut-associated lymphoid

(pValac:*il-10*)] or the IL-4 cytokine [r*L.* 

(pValac:*il-4*) was able to slump the

, by expressing the FnBpA

widely studied using different systems with different molecules. As IBDs are a serious clinical topic, many strategies have been tested trying to improve previous

and IL-17<sup>+</sup>

**58**

*lactis* FnPBA+

*lactis* FnPBA+


#### **Table 1.**

*Protein with anti-inflammatory properties produced in different strains of bacteria.*

The human 15-lipoxygenase-1-producing *L. lactis* NCDO2118 harboring the xylose-inducible expression system (pXylt:CYT:*15-LOX-1*) was also effective in attenuating the symptoms of DSS-induced colitis in a murine model [175]. Its oral administration improved the body weight, decreased pro-inflammatory cytokines (IFN-γ and IL-4) while increasing the anti-inflammatory cytokine IL-10, and, consequently, ameliorated the macroscopic damage scores associated with the inflammation.

The oral pretreatment with genetically modified *L. lactis* NCDO2118 able to secrete HSP65 protein from *Mycobacterium leprae*, using XIES system (pXylt:SEC:*hsp65*), prevented DSS-induced colitis in C57BL/6 mice [176]. This protection was associated with reduced pro-inflammatory cytokines, such as IFN-γ, IL-6, and TNF-α; it also increased IL-10 production in colonic tissue and expansion of CD4+ FoxP3<sup>+</sup> and CD4+ latency-associated peptide (LAP+ ) regulatory T cells in the spleen and mesenteric lymph nodes. Besides, the authors showed that this effect was dependent on IL-10 and toll-like receptor 2 (TLR-2) [176].

Although *L. lactis* represents an excellent candidate for a live mucosal vector delivery system, other bacteria have also been explored as promising live vehicles for molecule expression with therapeutic properties, such as *Lactobacillus*, *Bifidobacterium*, and *Streptococcus*. In this context, Mauras et al. [177] using the new *Bifidobacteria* Expression SysTem (BEST) allowing the production of IL-10 in *Bifidobacterium bifidum* BS42(pBESTExp4:*il-10* and pBESTBL1181:*il-10*) demonstrated that the use of these recombinant strains in a DNBS-induced colitis model showed its ability to decrease local inflammation and confirmed therefore its potential for delivery of therapeutic molecules in the colon.

It is well known that IBD is associated with oxidative stress by the increase in concentration of reactive oxygen species in the GIT and impaired antioxidant defenses [178, 179]. In this context, it has been shown that some probiotic LAB strains may play a protective role in IBD by expressing antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) [180, 181].

LeBlanc et al. and Del Carmen et al. [180, 181] showed, respectively, that *L. casei* BL23 and *S. thermophilus* CRL807 transformed with two different plasmids (pLEM415:*mnkat*; pLEM415:*sodA*) (pIL253:*sodA* and pIL253:*mnkat*) harboring

**61**

[134, 182].

and CD4+

development.

and diabetes [182–185].

*Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases...*

with either CAT- or SOD-producing lactobacilli/streptococci.

the genes encoding catalase (CAT) or superoxide dismutase (SOD) antioxidant enzymes exhibited anti-inflammatory activities in a mouse model of Crohn's and colitis disease induced by trinitrobenzenesulfonic acid (TNBS). The authors observed a reduction in weight loss, fewer liver microbial translocation, lower macroscopic and microscopic damage scores, and modulation of the IFN-γ/IL-10 [180] and IL-10/IL-17 [181] cytokine production in the large intestines of mice treated

The stress-inducible controlled expression (SICE) system represented by *L. lactis* MG1363 strain harboring the pLB333 plasmid was developed to avoid the external induction of culture before the host administration [134]. Several interesting molecules were cloned in this system such as IL-10 [134] and IL-17 [182], and the effect of *L. lactis* secreting them was evaluated in mice models. *L. lactis* (pSICE:*il-10*) was tested in a DNBS-induced colitis mice model, resulting in a significant reduction in colitis parameters with improvement in weight loss and a decrease in macroscopic scores [134]. The intranasal administration with *L. lactis* secreting IL-17A (pSICE:*il-17*), in a mice model of human papilloma virus (HPV)-induced cancer, was able to reduce tumor size and induce IL-6 and IL-17 secretion in reactivated splenocytes from mice challenged with the tumoral cell line [182]. Both works confirmed the potential use of *L. lactis* harboring the SICE system to deliver interesting molecules either to colitis or colon cancer patients

Although many studies have focused on the use of recombinant bacteria for the treatment of IBDs, as was previously discussed, the use of recombinant probiotic strains expressing/delivering therapeutic molecules has been explored for treatment or prevention of other diseases such as mucositis, cancer, obesity, multiple sclerosis,

An in vivo study reported by Caluwaerts et al. [186] showed that recombinant *L. lactis* AG013 secreting human trefoil factor 1(hTFF-1) was able to reduce the severity and course of radiation-induced oral mucositis. Carvalho et al. [187] also demonstrated that a recombinant strain of *L. lactis* NZ9000 using the inducible NICE system to express the human pancreatitis-associated protein (PAP) was able to prevent 5-FU-induced intestinal mucositis in a murine model. It was observed that this protein preserved villous architecture, increased Paneth cell activity [187],

FoxP3<sup>+</sup>

) regulatory T cells in the spleen,

T cells to the spinal cord

and suppressed the growth of *Enterobacteriaceae* during inflammation [185]. It also has been shown that oral administration of a recombinant *L. lactis* NCDO2118 strain (pXylT:SEC:*hsp65*) prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice [184]. Mice fed daily with this recombinant strain increased the number of natural and inducible CD4+

inguinal and mesenteric lymph nodes, as well as in the spinal cord. In addition, a

was observed, which decreased IgG response against HSP65 and induced an antiinflammatory cytokine profile (IL-17 reduction and IL-10 increase) during EAE

The oral administration of recombinant *L. lactis* expressing HSP65 and tandemly repeated P277 (pCYT:*HSP65-6P277*) was also analyzed in a model of type 1 diabetes mellitus (DM1) [183]. The authors observed that oral administration of recombinant *L. Lactis* resulted in the prevention of hyperglycemia, improved glucose tolerance and reduced insulitis, and induced HSP65- and P277-specific T-cell immunotolerance, as well as antigen-specific proliferation of splenocytes, demonstrating to be an effective therapeutic approach in preventing DM1 [183]. Another study using the *E. coli* Nissle 1917 strain engineered to secrete N-acylphosphatidylethanolamines (NAPEs) (pDEST-At1g78690 expression

latency-associated peptide (LAP+

reduction in the recruitment of encephalitogenic CD4+

*DOI: http://dx.doi.org/10.5772/intechopen.88325*

*Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases... DOI: http://dx.doi.org/10.5772/intechopen.88325*

the genes encoding catalase (CAT) or superoxide dismutase (SOD) antioxidant enzymes exhibited anti-inflammatory activities in a mouse model of Crohn's and colitis disease induced by trinitrobenzenesulfonic acid (TNBS). The authors observed a reduction in weight loss, fewer liver microbial translocation, lower macroscopic and microscopic damage scores, and modulation of the IFN-γ/IL-10 [180] and IL-10/IL-17 [181] cytokine production in the large intestines of mice treated with either CAT- or SOD-producing lactobacilli/streptococci.

The stress-inducible controlled expression (SICE) system represented by *L. lactis* MG1363 strain harboring the pLB333 plasmid was developed to avoid the external induction of culture before the host administration [134]. Several interesting molecules were cloned in this system such as IL-10 [134] and IL-17 [182], and the effect of *L. lactis* secreting them was evaluated in mice models. *L. lactis* (pSICE:*il-10*) was tested in a DNBS-induced colitis mice model, resulting in a significant reduction in colitis parameters with improvement in weight loss and a decrease in macroscopic scores [134]. The intranasal administration with *L. lactis* secreting IL-17A (pSICE:*il-17*), in a mice model of human papilloma virus (HPV)-induced cancer, was able to reduce tumor size and induce IL-6 and IL-17 secretion in reactivated splenocytes from mice challenged with the tumoral cell line [182]. Both works confirmed the potential use of *L. lactis* harboring the SICE system to deliver interesting molecules either to colitis or colon cancer patients [134, 182].

Although many studies have focused on the use of recombinant bacteria for the treatment of IBDs, as was previously discussed, the use of recombinant probiotic strains expressing/delivering therapeutic molecules has been explored for treatment or prevention of other diseases such as mucositis, cancer, obesity, multiple sclerosis, and diabetes [182–185].

An in vivo study reported by Caluwaerts et al. [186] showed that recombinant *L. lactis* AG013 secreting human trefoil factor 1(hTFF-1) was able to reduce the severity and course of radiation-induced oral mucositis. Carvalho et al. [187] also demonstrated that a recombinant strain of *L. lactis* NZ9000 using the inducible NICE system to express the human pancreatitis-associated protein (PAP) was able to prevent 5-FU-induced intestinal mucositis in a murine model. It was observed that this protein preserved villous architecture, increased Paneth cell activity [187], and suppressed the growth of *Enterobacteriaceae* during inflammation [185].

It also has been shown that oral administration of a recombinant *L. lactis* NCDO2118 strain (pXylT:SEC:*hsp65*) prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice [184]. Mice fed daily with this recombinant strain increased the number of natural and inducible CD4<sup>+</sup> FoxP3<sup>+</sup> and CD4+ latency-associated peptide (LAP+ ) regulatory T cells in the spleen, inguinal and mesenteric lymph nodes, as well as in the spinal cord. In addition, a reduction in the recruitment of encephalitogenic CD4+ T cells to the spinal cord was observed, which decreased IgG response against HSP65 and induced an antiinflammatory cytokine profile (IL-17 reduction and IL-10 increase) during EAE development.

The oral administration of recombinant *L. lactis* expressing HSP65 and tandemly repeated P277 (pCYT:*HSP65-6P277*) was also analyzed in a model of type 1 diabetes mellitus (DM1) [183]. The authors observed that oral administration of recombinant *L. Lactis* resulted in the prevention of hyperglycemia, improved glucose tolerance and reduced insulitis, and induced HSP65- and P277-specific T-cell immunotolerance, as well as antigen-specific proliferation of splenocytes, demonstrating to be an effective therapeutic approach in preventing DM1 [183].

Another study using the *E. coli* Nissle 1917 strain engineered to secrete N-acylphosphatidylethanolamines (NAPEs) (pDEST-At1g78690 expression

*The Health Benefits of Foods - Current Knowledge and Further Development*

*L.lactis* MG1363 Mouse IL-17 SICE Mice model HPV-

*Protein with anti-inflammatory properties produced in different strains of bacteria.*

**System**

**Inflamation Condition**

experimental encephalomyelitis

induced cancer

diabetes type 1

XIES Mice model of

NICE Mice model of

**Anti-Inflamatory Properties**

Increased CD4+Foxp3+ regulatory T cells Reduced encephalytogenic CD4+ T cells

Reduced tumor

Induced IL-6 and IL-17 secretion

Inhibition of T cell proliferation

Reduction of insulitis

size

**References**

[184]

[182]

[183]

**Microorganism Gene Expression** 

*M. leprae* Hsp65 protein

*L.lactis* NZ9000 *M. leprae* Hsp65

protein and peptide derived of human Hsp60 protein

The human 15-lipoxygenase-1-producing *L. lactis* NCDO2118 harboring the xylose-inducible expression system (pXylt:CYT:*15-LOX-1*) was also effective in attenuating the symptoms of DSS-induced colitis in a murine model [175]. Its oral administration improved the body weight, decreased pro-inflammatory cytokines (IFN-γ and IL-4) while increasing the anti-inflammatory cytokine IL-10, and, consequently, ameliorated the macroscopic damage scores associated with the

The oral pretreatment with genetically modified *L. lactis* NCDO2118 able to secrete HSP65 protein from *Mycobacterium leprae*, using XIES system (pXylt:SEC:*hsp65*), prevented DSS-induced colitis in C57BL/6 mice [176]. This protection was associated with reduced pro-inflammatory cytokines, such as IFN-γ, IL-6, and TNF-α; it also increased IL-10 production in colonic tissue and expansion

was dependent on IL-10 and toll-like receptor 2 (TLR-2) [176].

potential for delivery of therapeutic molecules in the colon.

superoxide dismutase (SOD) and catalase (CAT) [180, 181].

latency-associated peptide (LAP+

the spleen and mesenteric lymph nodes. Besides, the authors showed that this effect

Although *L. lactis* represents an excellent candidate for a live mucosal vector delivery system, other bacteria have also been explored as promising live vehicles for molecule expression with therapeutic properties, such as *Lactobacillus*, *Bifidobacterium*, and *Streptococcus*. In this context, Mauras et al. [177] using the new *Bifidobacteria* Expression SysTem (BEST) allowing the production of IL-10 in *Bifidobacterium bifidum* BS42(pBESTExp4:*il-10* and pBESTBL1181:*il-10*) demonstrated that the use of these recombinant strains in a DNBS-induced colitis model showed its ability to decrease local inflammation and confirmed therefore its

It is well known that IBD is associated with oxidative stress by the increase in concentration of reactive oxygen species in the GIT and impaired antioxidant defenses [178, 179]. In this context, it has been shown that some probiotic LAB strains may play a protective role in IBD by expressing antioxidant enzymes such as

LeBlanc et al. and Del Carmen et al. [180, 181] showed, respectively, that *L. casei* BL23 and *S. thermophilus* CRL807 transformed with two different plasmids (pLEM415:*mnkat*; pLEM415:*sodA*) (pIL253:*sodA* and pIL253:*mnkat*) harboring

) regulatory T cells in

**60**

inflammation.

*L.lactis* NCDO2118

**Table 1.**

of CD4+

FoxP3<sup>+</sup>

and CD4+

plasmid) demonstrated that this strain was able to reduce the obesity of mice fed with a high-fat diet when added to drinking water. N-acyl phosphatidylethanolamines are precursors to the N-acylethanolamine (NAE) family of lipids, which are synthesized in the small intestine in response to feeding and reducing food intake and obesity. Mice that received modified bacteria had dramatically lower food intake, adiposity, insulin resistance, and hepatosteatosis than mice receiving standard water or control bacteria [188]. In addition, it was observed that changes on intestinal microbiota significantly decreased the abundance of *Firmicutes* and increased the abundance of *Proteobacteria*. Thus, these results provide evidence of the potential efficacy of this approach to inhibit the development of metabolic disorders and related diseases.

## **13. Conclusion**

Currently the association between disease progression, especially chronic inflammatory diseases, and intestinal dysbiosis has been more frequently observed. As a clinical strategy, the use of probiotic bacteria, which naturally benefit the host, has been increasingly used on the treatment of diseases related to the GIT. In view of the good results obtained with this approach, researchers have sought through bacterial genetic modification to increase the beneficial potential of probiotics, either through their use for heterologous protein production or as a vehicle for vaccinal plasmid delivery, by developing recombinant bacterial strains and by testing their action in different disease models. And while there are still a number of questions that need to be answered about the use of genetically modified organisms for health care, especially in human, the use of these strains has proven to be a potentially effective therapeutic alternative, so much so that clinical trials using recombinant lineages have already been authorized and conducted in humans.

## **Author details**

Luís Cláudio Lima de Jesus1 , Fernanda Alvarenga Lima1 , Nina Dias Coelho-Rocha1 , Tales Fernando da Silva1 , Júlia Paz1 , Vasco Azevedo1 , Pamela Mancha-Agresti1 \* and Mariana Martins Drumond1,2

1 Laboratório de Genética Celular e Molecular (LGCM), Instituto de Ciências Biológicas, Departamento de Genética, Ecologia e Evolução, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil

2 Departamento de Ciências Biológicas, Centro Federal de Educação Tecnológica de Minas Gerais (CEFET/MG), Belo Horizonte, Minas Gerais, Brazil

\*Address all correspondence to: p.mancha.agresti@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**63**

*Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases...*

[9] Walter J, Ley R. The human gut microbiome: Ecology and recent evolutionary changes. Annual Review of Microbiology [Internet]. 2011;**65**(1):411-429. Available from: http://www.ncbi.nlm.nih.gov/

[10] Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature [Internet]. 2012;**486**(7402):207-214. Available from: http://www.ncbi.nlm.nih.gov/

[11] Bäckhed F, Fraser CM, Ringel Y, Sanders ME, Sartor RB, Sherman PM, et al. Defining a healthy human gut microbiome: Current concepts, future directions, and clinical applications. Cell Host & Microbe [Internet]. 2012;**12**(5):611-622. Available from: http://www.ncbi.nlm.nih.gov/

[12] Guarner F, Malagelada J-R. Gut flora in health and disease. The Lancet (London, England) [Internet]. 2003;**361**(9356):512-519. Available from: http://www.ncbi.nlm.nih.gov/

[13] Hooper LV, Midtvedt T, Gordon JI. How host-microbial interactions shape the nutrient environment of the mammalian intestine. Annual Review of Nutrition [Internet]. 2002;**22**(1):283- 307. Available from: http://www.ncbi. nlm.nih.gov/pubmed/12055347

[14] den Besten G, van Eunen K, Groen AK, Venema K, Reijngoud D-J, Bakker BM. The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism. Journal of Lipid Research [Internet]. 2013;**54**(9):2325-2340. Available from: http://www.ncbi.nlm.

nih.gov/pubmed/23821742

pubmed/21682646

pubmed/22699609

pubmed/23159051

pubmed/12583961

*DOI: http://dx.doi.org/10.5772/intechopen.88325*

[1] Thursby E, Juge N. Introduction to the human gut microbiota. Biochemical

[2] Peterson J, Garges S, Giovanni M, McInnes P, Wang L, Schloss JA, et al. The NIH Human Microbiome Project. Genome Research [Internet]. 2009;**19**(12):2317-2323. Available from: http://www.ncbi.nlm.nih.gov/

[3] Mowat AM, Agace WW. Regional specialization within the intestinal immune system. Nature Reviews. Immunology. 2014;**14**(10):667-685

Subramanyam C, Vuyyuru H, Sasikala M, Nageshwar Reddy D. Role of the normal

[4] Jandhyala SM, Talukdar R,

gut microbiota. World Journal of Gastroenterology [Internet]. 2015;**21**(29):8787-8803. Available from: http://www.ncbi.nlm.nih.gov/

Journal. 2017;**474**(11):1823-1836

pubmed/19819907

**References**

pubmed/26269668

pubmed/19026645

pubmed/27541692

pubmed/16498248

[5] Neish AS. Microbes in

gastrointestinal health and disease. Gastroenterology [Internet]. 2009;**136**(1):65-80. Available from: http://www.ncbi.nlm.nih.gov/

[6] Bäckhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science [Internet]. 2005;**307**(5717): 1915-1920. Available from: http://www. ncbi.nlm.nih.gov/pubmed/15790844

[7] Sender R, Fuchs S, Milo R. Revised estimates for the number of human and bacteria cells in the body. PLoS Biology [Internet]. 2016;**14**(8):1-14. Available from: http://www.ncbi.nlm.nih.gov/

[8] Guarner F. Enteric flora in health and disease. Digestion [Internet]. 2006;**73**(1):5-12. Available from: http://www.ncbi.nlm.nih.gov/

*Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases... DOI: http://dx.doi.org/10.5772/intechopen.88325*

## **References**

*The Health Benefits of Foods - Current Knowledge and Further Development*

disorders and related diseases.

**13. Conclusion**

**Author details**

Luís Cláudio Lima de Jesus1

and Mariana Martins Drumond1,2

provided the original work is properly cited.

Tales Fernando da Silva1

plasmid) demonstrated that this strain was able to reduce the obesity of mice fed with a high-fat diet when added to drinking water. N-acyl phosphatidylethanolamines are precursors to the N-acylethanolamine (NAE) family of lipids, which are synthesized in the small intestine in response to feeding and reducing food intake and obesity. Mice that received modified bacteria had dramatically lower food intake, adiposity, insulin resistance, and hepatosteatosis than mice receiving standard water or control bacteria [188]. In addition, it was observed that changes on intestinal microbiota significantly decreased the abundance of *Firmicutes* and increased the abundance of *Proteobacteria*. Thus, these results provide evidence of the potential efficacy of this approach to inhibit the development of metabolic

Currently the association between disease progression, especially chronic inflammatory diseases, and intestinal dysbiosis has been more frequently observed. As a clinical strategy, the use of probiotic bacteria, which naturally benefit the host, has been increasingly used on the treatment of diseases related to the GIT. In view of the good results obtained with this approach, researchers have sought through bacterial genetic modification to increase the beneficial potential of probiotics, either through their use for heterologous protein production or as a vehicle for vaccinal plasmid delivery, by developing recombinant bacterial strains and by testing their action in different disease models. And while there are still a number of questions that need to be answered about the use of genetically modified organisms for health care, especially in human, the use of these strains has proven to be a potentially effective therapeutic alternative, so much so that clinical trials using recombinant lineages have already been authorized and conducted in humans.

, Fernanda Alvarenga Lima1

2 Departamento de Ciências Biológicas, Centro Federal de Educação Tecnológica de

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Laboratório de Genética Celular e Molecular (LGCM), Instituto de Ciências Biológicas, Departamento de Genética, Ecologia e Evolução, Universidade Federal

, Vasco Azevedo1

, Júlia Paz1

de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil

Minas Gerais (CEFET/MG), Belo Horizonte, Minas Gerais, Brazil

\*Address all correspondence to: p.mancha.agresti@gmail.com

, Nina Dias Coelho-Rocha1

, Pamela Mancha-Agresti1

,

\*

**62**

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[2] Peterson J, Garges S, Giovanni M, McInnes P, Wang L, Schloss JA, et al. The NIH Human Microbiome Project. Genome Research [Internet]. 2009;**19**(12):2317-2323. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/19819907

[3] Mowat AM, Agace WW. Regional specialization within the intestinal immune system. Nature Reviews. Immunology. 2014;**14**(10):667-685

[4] Jandhyala SM, Talukdar R, Subramanyam C, Vuyyuru H, Sasikala M, Nageshwar Reddy D. Role of the normal gut microbiota. World Journal of Gastroenterology [Internet]. 2015;**21**(29):8787-8803. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/26269668

[5] Neish AS. Microbes in gastrointestinal health and disease. Gastroenterology [Internet]. 2009;**136**(1):65-80. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/19026645

[6] Bäckhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science [Internet]. 2005;**307**(5717): 1915-1920. Available from: http://www. ncbi.nlm.nih.gov/pubmed/15790844

[7] Sender R, Fuchs S, Milo R. Revised estimates for the number of human and bacteria cells in the body. PLoS Biology [Internet]. 2016;**14**(8):1-14. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/27541692

[8] Guarner F. Enteric flora in health and disease. Digestion [Internet]. 2006;**73**(1):5-12. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/16498248

[9] Walter J, Ley R. The human gut microbiome: Ecology and recent evolutionary changes. Annual Review of Microbiology [Internet]. 2011;**65**(1):411-429. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/21682646

[10] Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature [Internet]. 2012;**486**(7402):207-214. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/22699609

[11] Bäckhed F, Fraser CM, Ringel Y, Sanders ME, Sartor RB, Sherman PM, et al. Defining a healthy human gut microbiome: Current concepts, future directions, and clinical applications. Cell Host & Microbe [Internet]. 2012;**12**(5):611-622. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/23159051

[12] Guarner F, Malagelada J-R. Gut flora in health and disease. The Lancet (London, England) [Internet]. 2003;**361**(9356):512-519. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/12583961

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[185] Carvalho R, Vaz A, Pereira FL, Dorella F, Aguiar E, Chatel J-M, et al. Gut microbiome modulation during treatment of mucositis with the dairy bacterium *Lactococcus lactis* and recombinant strain secreting human antimicrobial PAP. Scientific Reports [Internet]. 2018;**8**(1):15072. Available from: http://www.ncbi.nlm.nih.gov/

Medeiros SR, Gomes-Santos AC, Alves AC, Loli FG, et al. Hsp65-producing *Lactococcus lactis* prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells. Journal of Autoimmunity [Internet]. 2013;**40**(1):45- 57. Available from: http://www.ncbi.nlm.

Lu Y, Jin L, et al. Oral administration of recombinant *Lactococcus lactis* expressing HSP65 and tandemly repeated P277 reduces the incidence of type I diabetes in non-obese diabetic mice. PLoS One [Internet]. 2014;**9**(8):e105701. Available from: http://www.ncbi.nlm.nih.gov/

antioxidant enzymes exhibit enhanced anti-inflammatory activities. Applied and Environmental Microbiology [Internet]. 2014;**80**(3):869-877. Available from: http://www.ncbi.nlm.

[182] Jacouton E, Torres Maravilla E, Boucard A-S, Pouderous N, Pessoa Vilela AP, Naas I, et al. Anti-tumoral effects of recombinant *Lactococcus lactis* strain secreting IL-17A cytokine. Frontiers in Microbiology [Internet]. 2018;**9**(1):3355. Available from: http://www.ncbi.nlm.nih.gov/

*DOI: http://dx.doi.org/10.5772/intechopen.88325*

Available from: http://journals.ke-i.org/

[176] Gomes-Santos AC, de Oliveira RP,

[177] Mauras A, Chain F, Faucheux A, Ruffié P, Gontier S, Ryffel B, et al. A new bifidobacteria expression system (BEST) to produce and deliver interleukin-10 in *Bifidobacterium bifidum*. Frontiers in Microbiology [Internet]. 2018;**9**(1):3075. Available from: http://www.ncbi.nlm.nih.gov/

index.php/mra/article/view/612

Moreira TG, Castro-Junior AB, Horta BC, Lemos L, et al. Hsp65 producing *Lactococcus lactis* prevents inflammatory intestinal disease in mice by IL-10- and TLR2-dependent pathways. Frontiers in Immunology [Internet]. 2017;**8**(1):30. Available from: http://www.ncbi.nlm.nih.gov/

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[178] Tian T, Wang Z, Zhang J. Pathomechanisms of oxidative stress in inflammatory bowel disease and potential antioxidant therapies. Oxidative Medicine and Cellular Longevity [Internet]. 2017;**2017**(1):4535194. Available from: http://www.ncbi.nlm.nih.gov/

[179] Bourgonje AR, von Martels JZH, Bulthuis MLC, van Londen M, Faber KN, Dijkstra G, et al. Crohn's disease in clinical remission is marked by systemic oxidative stress. Frontiers in Physiology [Internet]. 2019;**10**(1):499. Available from: http://www.ncbi.nlm.nih.gov/

[180] LeBlanc JG, del Carmen S, Miyoshi A, Azevedo V, Sesma F, Langella P, et al. Use of superoxide dismutase and catalase producing lactic acid bacteria in TNBS induced Crohn's disease in mice. Journal of Biotechnology [Internet]. 2011;**151**(3):287-293. Available from: http://www.ncbi.nlm.nih.gov/

*Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases... DOI: http://dx.doi.org/10.5772/intechopen.88325*

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*The Health Benefits of Foods - Current Knowledge and Further Development*

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[174] Bermúdez-Humarán LG, Motta J-P, Aubry C, Kharrat P, Rous-Martin L, Sallenave J-M, et al. Serine protease inhibitors protect better than IL-10 and TGF-β anti-inflammatory cytokines against mouse colitis when delivered by recombinant Lactococci. Microbial Cell Factories [Internet]. 2015;**14**(1):26. Available from: http://www.ncbi.nlm.

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[168] Berlec A, Škrlec K, Kocjan J, Olenic M, Štrukelj B. Single plasmid systems for inducible dual protein expression and for CRISPR-Cas9/ CRISPRi gene regulation in lactic acid bacterium *Lactococcus lactis*. Scientific Reports [Internet]. 2018;**8**(1):1009. Available from: http://www.ncbi.nlm.

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[166] Sanozky-Dawes R, Selle K, O'Flaherty S, Klaenhammer T, Barrangou R. Occurrence and activity of a type II CRISPR-Cas system in *Lactobacillus gasseri*. Microbiology [Internet]. 2015;**161**(9):1752-1761. Available from: http://www.ncbi.nlm.

**76**

[176] Gomes-Santos AC, de Oliveira RP, Moreira TG, Castro-Junior AB, Horta BC, Lemos L, et al. Hsp65 producing *Lactococcus lactis* prevents inflammatory intestinal disease in mice by IL-10- and TLR2-dependent pathways. Frontiers in Immunology [Internet]. 2017;**8**(1):30. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/28194152

[177] Mauras A, Chain F, Faucheux A, Ruffié P, Gontier S, Ryffel B, et al. A new bifidobacteria expression system (BEST) to produce and deliver interleukin-10 in *Bifidobacterium bifidum*. Frontiers in Microbiology [Internet]. 2018;**9**(1):3075. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/30622516

[178] Tian T, Wang Z, Zhang J. Pathomechanisms of oxidative stress in inflammatory bowel disease and potential antioxidant therapies. Oxidative Medicine and Cellular Longevity [Internet]. 2017;**2017**(1):4535194. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/28744337

[179] Bourgonje AR, von Martels JZH, Bulthuis MLC, van Londen M, Faber KN, Dijkstra G, et al. Crohn's disease in clinical remission is marked by systemic oxidative stress. Frontiers in Physiology [Internet]. 2019;**10**(1):499. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/31080419

[180] LeBlanc JG, del Carmen S, Miyoshi A, Azevedo V, Sesma F, Langella P, et al. Use of superoxide dismutase and catalase producing lactic acid bacteria in TNBS induced Crohn's disease in mice. Journal of Biotechnology [Internet]. 2011;**151**(3):287-293. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/21167883

[181] Del Carmen S, de Moreno de LeBlanc A, Martin R, Chain F, Langella P, Bermúdez-Humarán LG, et al. Genetically engineered immunomodulatory *Streptococcus thermophilus* strains producing antioxidant enzymes exhibit enhanced anti-inflammatory activities. Applied and Environmental Microbiology [Internet]. 2014;**80**(3):869-877. Available from: http://www.ncbi.nlm. nih.gov/pubmed/24242245

[182] Jacouton E, Torres Maravilla E, Boucard A-S, Pouderous N, Pessoa Vilela AP, Naas I, et al. Anti-tumoral effects of recombinant *Lactococcus lactis* strain secreting IL-17A cytokine. Frontiers in Microbiology [Internet]. 2018;**9**(1):3355. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/30728820

[183] Ma Y, Liu J, Hou J, Dong Y, Lu Y, Jin L, et al. Oral administration of recombinant *Lactococcus lactis* expressing HSP65 and tandemly repeated P277 reduces the incidence of type I diabetes in non-obese diabetic mice. PLoS One [Internet]. 2014;**9**(8):e105701. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/25157497

[184] Rezende RM, Oliveira RP, Medeiros SR, Gomes-Santos AC, Alves AC, Loli FG, et al. Hsp65-producing *Lactococcus lactis* prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells. Journal of Autoimmunity [Internet]. 2013;**40**(1):45- 57. Available from: http://www.ncbi.nlm. nih.gov/pubmed/22939403

[185] Carvalho R, Vaz A, Pereira FL, Dorella F, Aguiar E, Chatel J-M, et al. Gut microbiome modulation during treatment of mucositis with the dairy bacterium *Lactococcus lactis* and recombinant strain secreting human antimicrobial PAP. Scientific Reports [Internet]. 2018;**8**(1):15072. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/30305667

[186] Caluwaerts S, Vandenbroucke K, Steidler L, Neirynck S, Vanhoenacker P, Corveleyn S, et al. AG013, a mouth rinse formulation of *Lactococcus lactis* secreting human Trefoil Factor 1, provides a safe and efficacious therapeutic tool for treating oral mucositis. Oral Oncology [Internet]. 2010;**46**(7):564-570. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/20542722

[187] Carvalho RD, Breyner N, Menezes-Garcia Z, Rodrigues NM, Lemos L, Maioli TU, et al. Secretion of biologically active pancreatitisassociated protein I (PAP) by genetically modified dairy *Lactococcus lactis* NZ9000 in the prevention of intestinal mucositis. Microbial Cell Factories [Internet]. 2017;**16**(1):27. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/28193209

[188] Chen Z, Guo L, Zhang Y, Walzem RL, Pendergast JS, Printz RL, et al. Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity. The Journal of Clinical Investigation [Internet]. 2014;**124**(8):3391-3406. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/24960158

**79**

**Chapter 3**

**Abstract**

**1. Introduction**

Nutritional Profile and Medicinal

Properties of Pumpkin Fruit Pulp

Having high nutritional value and low cultivation costs, pumpkin fruit makes a great candidate to be used by the food industry as a functional ingredient. To prolong its shelf life and widen the array of its potential uses in food products, drying and powdering have been applied, producing pumpkin flour. Several studies have been done to optimize the drying method of pumpkin in order to preserve or reduce the loss of its nutritional constituents and color changes during drying and storage. As vacuum freeze drying produces great quality pumpkin powder and best preserves the β-carotene and phenolic contents of the fruit, it is considered an expensive technique that could be inconvenient to be used in developing countries or for cost-reduction purposes. Air drying is a cheaper technique but results in less nutrient preservation than vacuum drying. This highlights the role of pretreatments in order to reduce the loss of nutrients and produce better quality pumpkin flour. Hot water blanching followed by metabisulfite pretreatment results in the best carotenoid stability and preservation of phenolic compounds in the produced powder. Incorporation of pumpkin powder in wheat bread could increase its nutritional value by increasing the levels of dietary fiber, pro-vitamin A β-carotene, calcium,

iron, and zinc and by decreasing the carbohydrate and caloric contents.

Pumpkin belongs to the family *Cucurbitaceae*, genus *Cucurbita*. It is extensively grown throughout the tropical and subtropical countries, with the most common types worldwide being *Cucurbita maxima*, *Cucurbita moschata*, and *Cucurbita pepo* (**Figure 1**) [1]. The giant type pumpkins tend to be *C. maxima* varieties ('Boston Marrow' and 'Mammoth'), and the miniature pumpkins tend to be *C. pepo* ('Jack-O-Lantern'). *C. moschata* is the most commonly cultivated species in Asia and United States [2]. The characteristic yellow-orange color of pumpkins is due to the presence of carotenoids that have major roles in nutrition as pro-vitamin A [3]. Pumpkins have an abundance of macro- and micro-nutrients and antioxidants that promote the human body immunity against cancer and other diseases; "*It has such* 

Pumpkins are high-yield fruits and their cultivation is inexpensive [5]. They are stable for 1–3 months after their harvest, but they become susceptible to microbial spoilage, moisture loss, and color changes after peeling. Thus, in order to prolong their shelf life, drying and powdering techniques have been applied. This also allows pumpkin to be used as an ingredient in manufacturing foods such as bakery

**Keywords:** pumpkin fruit, pumpkin flour, artisanal food

*nutritional potential unequal to any other single crop*" [4].

*Sami El Khatib and Mariam Muhieddine*

## **Chapter 3**

*The Health Benefits of Foods - Current Knowledge and Further Development*

[186] Caluwaerts S, Vandenbroucke K, Steidler L, Neirynck S, Vanhoenacker P, Corveleyn S, et al. AG013, a mouth rinse formulation of *Lactococcus lactis* secreting human Trefoil Factor 1, provides a safe and efficacious therapeutic tool for treating oral mucositis. Oral Oncology [Internet]. 2010;**46**(7):564-570. Available from: http://www.ncbi.nlm.nih.gov/

pubmed/20542722

pubmed/28193209

pubmed/24960158

[187] Carvalho RD, Breyner N, Menezes-Garcia Z, Rodrigues NM, Lemos L, Maioli TU, et al. Secretion of biologically active pancreatitisassociated protein I (PAP) by genetically

modified dairy *Lactococcus lactis* NZ9000 in the prevention of intestinal mucositis. Microbial Cell Factories [Internet]. 2017;**16**(1):27. Available from: http://www.ncbi.nlm.nih.gov/

[188] Chen Z, Guo L, Zhang Y,

Walzem RL, Pendergast JS, Printz RL, et al. Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity. The Journal of Clinical Investigation [Internet]. 2014;**124**(8):3391-3406. Available from: http://www.ncbi.nlm.nih.gov/

**78**

## Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp

*Sami El Khatib and Mariam Muhieddine*

## **Abstract**

Having high nutritional value and low cultivation costs, pumpkin fruit makes a great candidate to be used by the food industry as a functional ingredient. To prolong its shelf life and widen the array of its potential uses in food products, drying and powdering have been applied, producing pumpkin flour. Several studies have been done to optimize the drying method of pumpkin in order to preserve or reduce the loss of its nutritional constituents and color changes during drying and storage. As vacuum freeze drying produces great quality pumpkin powder and best preserves the β-carotene and phenolic contents of the fruit, it is considered an expensive technique that could be inconvenient to be used in developing countries or for cost-reduction purposes. Air drying is a cheaper technique but results in less nutrient preservation than vacuum drying. This highlights the role of pretreatments in order to reduce the loss of nutrients and produce better quality pumpkin flour. Hot water blanching followed by metabisulfite pretreatment results in the best carotenoid stability and preservation of phenolic compounds in the produced powder. Incorporation of pumpkin powder in wheat bread could increase its nutritional value by increasing the levels of dietary fiber, pro-vitamin A β-carotene, calcium, iron, and zinc and by decreasing the carbohydrate and caloric contents.

**Keywords:** pumpkin fruit, pumpkin flour, artisanal food

## **1. Introduction**

Pumpkin belongs to the family *Cucurbitaceae*, genus *Cucurbita*. It is extensively grown throughout the tropical and subtropical countries, with the most common types worldwide being *Cucurbita maxima*, *Cucurbita moschata*, and *Cucurbita pepo* (**Figure 1**) [1]. The giant type pumpkins tend to be *C. maxima* varieties ('Boston Marrow' and 'Mammoth'), and the miniature pumpkins tend to be *C. pepo* ('Jack-O-Lantern'). *C. moschata* is the most commonly cultivated species in Asia and United States [2]. The characteristic yellow-orange color of pumpkins is due to the presence of carotenoids that have major roles in nutrition as pro-vitamin A [3]. Pumpkins have an abundance of macro- and micro-nutrients and antioxidants that promote the human body immunity against cancer and other diseases; "*It has such nutritional potential unequal to any other single crop*" [4].

Pumpkins are high-yield fruits and their cultivation is inexpensive [5]. They are stable for 1–3 months after their harvest, but they become susceptible to microbial spoilage, moisture loss, and color changes after peeling. Thus, in order to prolong their shelf life, drying and powdering techniques have been applied. This also allows pumpkin to be used as an ingredient in manufacturing foods such as bakery

**Figure 1.** *(A) Cucurbita moschata Duchesne; (B) Cucurbita pepo (variety ovifera) [8].*

products for quality addition [6], as the rich nutrient base of this vegetable increases the nutritional quality of baked products [7]. Pumpkin wheat composite bread has been found to have good nutritional value and sensory characteristics that could make it acceptable and well-appreciated to consumers [3].

## **2. Nutritional profile and medicinal properties of pumpkin fruit pulp**

A growing interest in pumpkin fruit and its derived products has been taken by agriculture, pharmaceuticals, and food-processing due to its nutritional and health promoting values [9]. Many countries, such as India, China, Brazil and Argentina have been using different species of this fruit as a medicine. The Traditional Chinese Medicine considers pumpkin as being *immensely valuable for human health* [8]. The various health benefits of pumpkin nutritional components include anti-diabetic, anti-carcinogenic, antioxidant [10] and possible antifatigue effects [11].

The composition of fresh pumpkin is shown in **Table 1**. Additional physical and chemical characteristics of ripe pumpkin fruit are shown in **Table 2**. It must be noted though that differences in the chemical components are found between different species of pumpkin, and among cultivars grown in different regions [5]. Pumpkin fruit is composed of pulp and seeds. Pumpkin pulp contains polysaccharides, pigments, amino acids, active proteins, and minerals. Pumpkin seeds are high in lipids and proteins, and they are a good source of many elements such as potassium, phosphorus and magnesium [8]. This chapter aims at characterizing the main nutritional components of pumpkin fruit pulp and its medicinal properties.


**81**

**Table 2.**

**2.1 Pumpkin pulp polysaccharides**

*Physical and chemical characteristics of ripe pumpkin [2].*

*(n = 4), YGY: Yellow to golden yellow.*

**Minerals**, **mg/100 g edible portion**

Many studies have been done on the anti-diabetic effect of pumpkin polysaccharides. They have been shown to decrease blood glucose and lipid levels in diabetic rats. *C. moschata* polysaccharides, which include soluble and insoluble dietary fiber, had a clear effect on reduction of serum glucose in diabetic rats. Clinical trials have also demonstrated significant reduction of post-prandial serum glucose and fasting glucose in non insulin dependent diabetes mellitus (NIDDM) subjects, after oral administration of pumpkin polysaccharides liquid and granules; and they have also shown that a daily supplement of 30 g pumpkin powder can significantly reduce blood glucose

*Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp*

Weight, g 3730.0 ± 67.71 Length, cm 32.6 ± 2.32 Diameter, cm 69.1 ± 2.05 Color YGY Pulp recovery, % 76.7 ± 0.006 Pulp:Skin:Seed 23:6:1 Firmness, lb./in2 21.3 ± 0.11 Seed oil recovery, % 35.7 ± 0.003 Moisture, % 6.2 ± 0.07 Total soluble solids, °B 9.2 ± 0.06 Total sugars, % 3.9 ± 0.01 Reducing sugars, % 2.1 ± 0.02 Titratable acidity, % 0.07 ± 0.003 pH 4.5 ± 0.003 β-carotene, mg/100 g 11.2 ± 0.007 Ascorbic acid, mg/100 g 14.5 ± 0.03 Pectin, % 1.2 ± 0.01 Fiber, % 0.66 ± 0.003 Ash, % 0.52 ± 0.003

Ca 10 P 30 Fe 0.44 Mg 38 Na 5.6 K 139 Cu 0.05 Mn 0.05 Zn 0.26 S 16 Cl 4

*DOI: http://dx.doi.org/10.5772/intechopen.89274*

#### **Table 1.** *Proximate composition of fresh pumpkin [9].*


#### *Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp DOI: http://dx.doi.org/10.5772/intechopen.89274*

**Table 2.**

*The Health Benefits of Foods - Current Knowledge and Further Development*

make it acceptable and well-appreciated to consumers [3].

*(A) Cucurbita moschata Duchesne; (B) Cucurbita pepo (variety ovifera) [8].*

products for quality addition [6], as the rich nutrient base of this vegetable increases the nutritional quality of baked products [7]. Pumpkin wheat composite bread has been found to have good nutritional value and sensory characteristics that could

**2. Nutritional profile and medicinal properties of pumpkin fruit pulp**

by agriculture, pharmaceuticals, and food-processing due to its nutritional and health promoting values [9]. Many countries, such as India, China, Brazil and Argentina have been using different species of this fruit as a medicine. The Traditional Chinese Medicine considers pumpkin as being *immensely valuable for human health* [8]. The various health benefits of pumpkin nutritional components include anti-diabetic, anti-carcinogenic, antioxidant [10] and possible anti-

A growing interest in pumpkin fruit and its derived products has been taken

The composition of fresh pumpkin is shown in **Table 1**. Additional physical and chemical characteristics of ripe pumpkin fruit are shown in **Table 2**. It must be noted though that differences in the chemical components are found between different species of pumpkin, and among cultivars grown in different regions [5]. Pumpkin fruit is composed of pulp and seeds. Pumpkin pulp contains polysaccharides, pigments, amino acids, active proteins, and minerals. Pumpkin seeds are high in lipids and proteins, and they are a good source of many elements such as potassium, phosphorus and magnesium [8]. This chapter aims at characterizing the main nutritional components of pumpkin fruit pulp and its

Moisture 92.24 Fat 0.15 Protein 0.98 Ash 0.76 Crude fiber 0.56 Carbohydrate 5.31

**80**

**Table 1.**

fatigue effects [11].

**Figure 1.**

medicinal properties.

**Composition of fresh pumpkin (%)**

*Proximate composition of fresh pumpkin [9].*

*Physical and chemical characteristics of ripe pumpkin [2].*

## **2.1 Pumpkin pulp polysaccharides**

Many studies have been done on the anti-diabetic effect of pumpkin polysaccharides. They have been shown to decrease blood glucose and lipid levels in diabetic rats. *C. moschata* polysaccharides, which include soluble and insoluble dietary fiber, had a clear effect on reduction of serum glucose in diabetic rats. Clinical trials have also demonstrated significant reduction of post-prandial serum glucose and fasting glucose in non insulin dependent diabetes mellitus (NIDDM) subjects, after oral administration of pumpkin polysaccharides liquid and granules; and they have also shown that a daily supplement of 30 g pumpkin powder can significantly reduce blood glucose

concentrations in NIDDM patients [8]. A Protein-bound polysaccharide isolated from water-soluble substances of pumpkin fruits, was also proven to improve tolerance to glucose by reducing blood glucose levels and increasing the levels of serum insulin in alloxan diabetic rats [5].

Pectin, a complex polysaccharide, is an important structural component of the cell wall of plants. It is mainly found in the peels of pumpkin, but the pressed pulp also contains a promising amount of it. Pumpkin pectin has been reported to have remarkable effects on lowering cholesterol levels in blood plasma and reducing triacylglycerols in liver, and thus reducing fatty acids in blood. It also simultaneously decreases the rate of fat assimilation and causes quick dissimilation of fat. In addition to their hypoglycemic and hypolipidemic activities, pumpkin polysaccharides antitumor effects were investigated and observed [8].

#### **2.2 Pumpkin pulp pigments**

Pumpkin pulp pigments are widely used as additives in food products, in medicine and in cosmetics. Pumpkin pigments include carotenoids, lutein and zeaxanthin. The carotenoids are responsible for the characteristic yellow-orange color of pumpkins [8]. In fact, the yellow color of pumpkin at its young stage develops to orange in its ripened stage due to a dramatic increase by 11 fold in the carotenoid content of the fruit [12].

The high carotenoid content is one of the reasons why pumpkin is such a nutritionally valuable fruit [13]. Carotenoids are considered a major source of vitamin A which is necessary for embryonic development, growth, and normal eyesight. Pumpkin is an excellent source of pro-vitamin A carotenoids. The major carotenoid in pumpkin is β-carotene, followed by small amounts of α-carotene, lutein and lycopene [8]. β-carotene content of pumpkin varies from 1.6 to 45.6 mg/100 g [14]. Indian cultivars have 132 to 527 mg/100 g of β-carotene content [1]. Research has indicated that pumpkin could be a primary vegetable to satisfy children's needs for carotenoids [8]. Moreover, β-carotene can protect against certain cancers and is considered a *powerful ally against degeneration aspect of aging*. Analysis of β-carotene content of pumpkin fruit has also been done for its possible use in combating eye diseases [2].

## **2.3 Pumpkin pulp minerals, amino acids, and active proteins**

The human body acquires its needed minerals from the daily diet. Minerals have key roles in several body functions. Pumpkin is considered as an eminent source of many minerals important for human health [8]. Pumpkin pulp is rich in K, Fe, Mn, Mg, P, vitamin C, vitamin E and phytosterols [2]. The pulp of *C. moschata* contains high amounts of calcium (205.45 μg/g) and potassium (1840.30 μg/g) and a low amount of sodium (28.70 μg/g), making it a suitable food for the prevention of osteoporosis and hypertension. Chrome is another mineral that is found in pumpkin in an amount higher than any other vegetable. Chrome is part of glucose tolerance factor which is essential for the activity of insulin and improves tolerance of blood glucose. Cobalt is also an essential microelement present in pumpkin. It is essential for islet cells of the pancreas and improves the body's metabolic capacity and participates in the synthetic action of vitamin B12 [8].

Protein content of pumpkin is less than 2.0% of dry matter weight. Yet, there are some essential amino acids present in pumpkin pulp. *C. moschata*, for example, contains 0.609% valine, 0.700% leucine, and 0.508% lysine, which are relatively

**83**

**Table 3.**

*Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp*

high amounts. Several studies of purified pumpkin extracts including proteins and polysaccharides have shown anticancer activity against melanoma, ehrlich ascites and leukemia. In addition, enzyme preparations of pumpkin have been found to

Processing of fruits or vegetables can transform these perishable foods into more stable foods that can be advantageous to both consumers and food industries. Pumpkins are consumed in various ways, whether fresh, canned, frozen, or dried. Preservation of pumpkin by drying is an important way to prevent postharvest losses. Though they keep longer than other fruits and vegetables, they can only do so if the fruits are free from any bruise. However, this is sometimes not possible because of insect bites or bruises acquired by harvesting, or by transportation after

Pumpkin fruit can be processed into flour which has a longer shelf-life, highly desirable -sweet- flavor, and deep yellow-orange color [2]. The rich nutrient potential of dried pumpkin makes utilization of pumpkin flour or pumpkin flour based products a good source of vitamin A from the β-carotene content, protein [4] and

Analyses on the composition of pumpkin (*Cucurbita moschata* Decne) flour, for example (**Table 3**), show that it contains high levels of carbohydrates, starch, dietary fiber, protein, total ash, and low levels of lipids and crude fiber. Authors proposed that it is an ideal food for diabetes patients, cardiovascular disease patients, and elderly [9]. Moreover, the functional properties of the flour such as water solubility and absorption indices and the pasting properties suggest that it may have suitable applications in the food industry such as a thickener in soup, gravy, and as an

ingredient in bakery products such as bread, cake and fried noodles [9].

**3.2 Effect of different convective drying methods on selected characteristics** 

Production of powders from vegetables and fruits have been done by various drying techniques such as hot air-drying, freeze drying, spray drying, vacuum

Moisture content (%) 3.73 ± 0.01 Fat (%) 3.60 ± 0.12 Crude fiber (%) 3.65 ± 0.14 Protein (%) 7.81 ± 0.18 Ash (%) 5.29 ± 0.01 Carbohydrate (%) 79.57 ± 0.01 Dietary fiber (g/100 g) 12.1 ± 0.00 Starch (%) 48.30 ± 0.54 Vitamin A (μg/100 g) 262 ± 0.32

*DOI: http://dx.doi.org/10.5772/intechopen.89274*

possess antitumor potential [8].

**3.1 Characteristics of pumpkin flour**

**3. Pumpkin flour**

harvest [4].

dietary fiber [15].

**Parameter**

**of pumpkin fruit flour**

*Proximate composition of pumpkin powder [9].*

high amounts. Several studies of purified pumpkin extracts including proteins and polysaccharides have shown anticancer activity against melanoma, ehrlich ascites and leukemia. In addition, enzyme preparations of pumpkin have been found to possess antitumor potential [8].

## **3. Pumpkin flour**

*The Health Benefits of Foods - Current Knowledge and Further Development*

alloxan diabetic rats [5].

observed [8].

diseases [2].

**2.2 Pumpkin pulp pigments**

content of the fruit [12].

concentrations in NIDDM patients [8]. A Protein-bound polysaccharide isolated from water-soluble substances of pumpkin fruits, was also proven to improve tolerance to glucose by reducing blood glucose levels and increasing the levels of serum insulin in

Pectin, a complex polysaccharide, is an important structural component of the cell wall of plants. It is mainly found in the peels of pumpkin, but the pressed pulp also contains a promising amount of it. Pumpkin pectin has been reported to have remarkable effects on lowering cholesterol levels in blood plasma and reducing triacylglycerols in liver, and thus reducing fatty acids in blood. It also simultaneously decreases the rate of fat assimilation and causes quick dissimilation of fat. In addition to their hypoglycemic and hypolipidemic activities, pumpkin polysaccharides antitumor effects were investigated and

Pumpkin pulp pigments are widely used as additives in food products, in medicine and in cosmetics. Pumpkin pigments include carotenoids, lutein and zeaxanthin. The carotenoids are responsible for the characteristic yellow-orange color of pumpkins [8]. In fact, the yellow color of pumpkin at its young stage develops to orange in its ripened stage due to a dramatic increase by 11 fold in the carotenoid

The high carotenoid content is one of the reasons why pumpkin is such a nutritionally valuable fruit [13]. Carotenoids are considered a major source of vitamin A which is necessary for embryonic development, growth, and normal eyesight. Pumpkin is an excellent source of pro-vitamin A carotenoids. The major carotenoid in pumpkin is β-carotene, followed by small amounts of α-carotene, lutein and lycopene [8]. β-carotene content of pumpkin varies from 1.6 to 45.6 mg/100 g [14]. Indian cultivars have 132 to 527 mg/100 g of β-carotene content [1]. Research has indicated that pumpkin could be a primary vegetable to satisfy children's needs for carotenoids [8]. Moreover, β-carotene can protect against certain cancers and is considered a *powerful ally against degeneration aspect of aging*. Analysis of β-carotene content of pumpkin fruit has also been done for its possible use in combating eye

The human body acquires its needed minerals from the daily diet. Minerals have key roles in several body functions. Pumpkin is considered as an eminent source of many minerals important for human health [8]. Pumpkin pulp is rich in K, Fe, Mn, Mg, P, vitamin C, vitamin E and phytosterols [2]. The pulp of *C. moschata* contains high amounts of calcium (205.45 μg/g) and potassium (1840.30 μg/g) and a low amount of sodium (28.70 μg/g), making it a suitable food for the prevention of osteoporosis and hypertension. Chrome is another mineral that is found in pumpkin in an amount higher than any other vegetable. Chrome is part of glucose tolerance factor which is essential for the activity of insulin and improves tolerance of blood glucose. Cobalt is also an essential microelement present in pumpkin. It is essential for islet cells of the pancreas and improves the body's metabolic capacity and participates in the synthetic action of

Protein content of pumpkin is less than 2.0% of dry matter weight. Yet, there are some essential amino acids present in pumpkin pulp. *C. moschata*, for example, contains 0.609% valine, 0.700% leucine, and 0.508% lysine, which are relatively

**2.3 Pumpkin pulp minerals, amino acids, and active proteins**

**82**

vitamin B12 [8].

## **3.1 Characteristics of pumpkin flour**

Processing of fruits or vegetables can transform these perishable foods into more stable foods that can be advantageous to both consumers and food industries. Pumpkins are consumed in various ways, whether fresh, canned, frozen, or dried. Preservation of pumpkin by drying is an important way to prevent postharvest losses. Though they keep longer than other fruits and vegetables, they can only do so if the fruits are free from any bruise. However, this is sometimes not possible because of insect bites or bruises acquired by harvesting, or by transportation after harvest [4].

Pumpkin fruit can be processed into flour which has a longer shelf-life, highly desirable -sweet- flavor, and deep yellow-orange color [2]. The rich nutrient potential of dried pumpkin makes utilization of pumpkin flour or pumpkin flour based products a good source of vitamin A from the β-carotene content, protein [4] and dietary fiber [15].

Analyses on the composition of pumpkin (*Cucurbita moschata* Decne) flour, for example (**Table 3**), show that it contains high levels of carbohydrates, starch, dietary fiber, protein, total ash, and low levels of lipids and crude fiber. Authors proposed that it is an ideal food for diabetes patients, cardiovascular disease patients, and elderly [9]. Moreover, the functional properties of the flour such as water solubility and absorption indices and the pasting properties suggest that it may have suitable applications in the food industry such as a thickener in soup, gravy, and as an ingredient in bakery products such as bread, cake and fried noodles [9].

## **3.2 Effect of different convective drying methods on selected characteristics of pumpkin fruit flour**

Production of powders from vegetables and fruits have been done by various drying techniques such as hot air-drying, freeze drying, spray drying, vacuum


## **Table 3.**

*Proximate composition of pumpkin powder [9].*

drying, and microwave vacuum drying. Spray and freeze drying have been reported to produce a good quality product, but are too expensive. On the other hand, hot air-drying *could result in a quality product that is characterized by uniform, hygienic, and attractive color of dried fruit and vegetable powder* in a condition that it is not done in a rapid manner which might result in an inferior product quality [6].

A study performed by Kiharason et al. [4] aimed to determine the effect of three drying methods of pumpkin fruit slices on the nutrient integrity of certain components of pumpkin: open solar (OSD), oven electric (OED), and enhanced solar (ESD) drying methods. The drying methods were applied, then dry fruit slices were milled and analyzed to determine their nutritive value.

### *3.2.1 Drying of pumpkin slices*

After washing, peeling and deseeding mature fruits, the fruit pulps were sliced and then cut at 2.55 cm length by 0.5 cm width. Next, they were blanched by dipping fast in boiling water for 1 min, cooled with running tap water for 1 min as well, and then wiped with absorbent paper. Afterwards, they were subjected to drying while weighing every 3 h until constant weight was recorded. The dried pumpkin slices obtained were ground, sieved and analyzed to determine their nutritive value [4].

## *3.2.2 The effect of the three drying methods on the moisture content and nutritive value of pumpkin powder*

#### *3.2.2.1 Moisture content*

In ESD and OED (temperature set at 50°C), different shelves at which pumpkin slices were put had great variations in terms of drying time, whereas the drying time in OSD, where tables used to dry pumpkin slices were at the same height, did not have much variation. Generally, OED took the shortest time and ESD took the longest time to dry the pumpkin slices (**Table 4**). As for the moisture content (MC), milled pumpkin slices that were dried by the OED exhibited the highest MC while ESD had the lowest (**Table 6**) [4].

Drying in a solar drier occurs in a closed environment whereas open sun drying happens in the open without any barrier, leading to quicker drying. A high evaporation rate during drying leads to a high possibility of nutrient losses. In addition, open sun drying has the poorest protection against insects, dust, microbes, and is inconvenient due to certain weather conditions such as rain where samples become subject to spoilage. Both oven drying and open solar drying showed a moisture content above the acceptable safe level, which is 14%. Moisture levels of 14% and above make the food susceptible to attack by microbes and promote fungal growth, while lower


#### **Table 4.**

*The mean drying time of pumpkin fruit slices and the average moisture content obtained by three drying methods [4].*

**85**

**Temperature (°C)**

*ppm parts-per-million, 10−6.*

Enhanced solar

*\**

**Table 5.**

*aw Water Activity.*

*different temperatures [6].*

**Table 6.**

*MCdb Moisture Content – dry basis matter.*

**MCdb of fresh pumpkin**

*Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp*

levels slow down the growth of microbes and prolong the shelf-life. These results make enhanced solar drying a better method for longer preservation of pumpkin

Pumpkin flour obtained by oven drying OED retained the highest amount of β-carotene, followed by ESD (**Table 5**). The fast rate of drying caused less nutrients to be lost, and samples at the core were still intact by the time constant weight was achieved. OSD had the least amount of β-carotene, most likely since unprotected exposure to the sun's UV rays caused photo-degradation of the carotenoids. Protein content showed no significant differences between the three drying methods but it showed a significant difference between the flours and the fresh fruits for almost

As for mineral, drying generally was found to reduce their levels compared to fresh fruits. Zinc was significantly lower in enhanced solar drying than oven and open sun drying, yet the fresh fruit exhibited the highest amount. Calcium levels were reduced greatly after drying showing 200% loss from fresh fruit. Whereas for iron, calcium and energy levels, no significant difference was noted between all treatments. It is therefore concluded, in this study, that enhanced solar drying is the best method for drying pumpkin and obtaining better quality pumpkin flour [4].

> **Zinc (ppm)**

62.9875ab 16.4875a 9.058b 49.5400a 539.08a 3.76350a

Fresh fruit 16.6150c 2.6175b 44.075c 94.5000a 1116.82a 4.26575a Oven dried 74.8425a 13.7850a 24.948a 66.3225a 830.23a 3.84675a

Open sun 27.1750bc 16.4900a 20.995ba 94.7975a 525.43a 3.62875a *F-value* 8.497 58.832 17.616 1.595 1.705 2.376 *P-value* 0.003 0.000 0.000 0.242 0.219 0.121

> **MCdb of dried pumpkin powder**

50 82.10 10.21a 0.98 0.65a 60 82.58 7.46b 0.95 0.42b

*Data are expressed as mean values. Mean values with different superscripts in the same column differ significantly at* 

*Mean values for moisture content and water activity of dried pumpkin powders prepared by hot air-drying at* 

70 84.09 5.47c 0.97

*Means followed by the same letter within a column are not significantly different at P = 0.05.*

*Nutrient levels of fresh pumpkin fruit and pumpkin flour obtained from three drying methods [4].*

**Protein (%)**

*P* ≤ *0.05. The symbol ns means that the mean values are not significantly different.*

**aw of fresh pumpkin**

**Iron (ppm) Calcium** 

**(ppm)**

ns

**aw of dried pumpkin powder**

**Energy (kcal/g)**

0.30c

*DOI: http://dx.doi.org/10.5772/intechopen.89274*

*3.2.2.2 Analysis of nutritive value*

800% increase in the flour [4].

**Treatment β-carotene** 

**(μg/g)\***

flour [4].

*Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp DOI: http://dx.doi.org/10.5772/intechopen.89274*

levels slow down the growth of microbes and prolong the shelf-life. These results make enhanced solar drying a better method for longer preservation of pumpkin flour [4].

### *3.2.2.2 Analysis of nutritive value*

*The Health Benefits of Foods - Current Knowledge and Further Development*

milled and analyzed to determine their nutritive value.

*3.2.1 Drying of pumpkin slices*

*value of pumpkin powder*

ESD had the lowest (**Table 6**) [4].

nutritive value [4].

*3.2.2.1 Moisture content*

drying, and microwave vacuum drying. Spray and freeze drying have been reported to produce a good quality product, but are too expensive. On the other hand, hot air-drying *could result in a quality product that is characterized by uniform, hygienic, and attractive color of dried fruit and vegetable powder* in a condition that it is not done in a rapid manner which might result in an inferior product quality [6].

A study performed by Kiharason et al. [4] aimed to determine the effect of three drying methods of pumpkin fruit slices on the nutrient integrity of certain components of pumpkin: open solar (OSD), oven electric (OED), and enhanced solar (ESD) drying methods. The drying methods were applied, then dry fruit slices were

After washing, peeling and deseeding mature fruits, the fruit pulps were sliced

In ESD and OED (temperature set at 50°C), different shelves at which pumpkin slices were put had great variations in terms of drying time, whereas the drying time in OSD, where tables used to dry pumpkin slices were at the same height, did not have much variation. Generally, OED took the shortest time and ESD took the longest time to dry the pumpkin slices (**Table 4**). As for the moisture content (MC), milled pumpkin slices that were dried by the OED exhibited the highest MC while

Drying in a solar drier occurs in a closed environment whereas open sun drying happens in the open without any barrier, leading to quicker drying. A high evaporation rate during drying leads to a high possibility of nutrient losses. In addition, open sun drying has the poorest protection against insects, dust, microbes, and is inconvenient due to certain weather conditions such as rain where samples become subject to spoilage. Both oven drying and open solar drying showed a moisture content above the acceptable safe level, which is 14%. Moisture levels of 14% and above make the food susceptible to attack by microbes and promote fungal growth, while lower

**Drying method Average drying time (hours)\* Average moisture content (%)**

Enhanced solar drying 13.27 <sup>a</sup> 12.82 Open solar drying 9.50b 14.91 Open drying 7.25c 15.15

*The mean drying time of pumpkin fruit slices and the average moisture content obtained by three drying* 

*Means followed by the same letter within a column are not significantly different at P = 0.05.*

and then cut at 2.55 cm length by 0.5 cm width. Next, they were blanched by dipping fast in boiling water for 1 min, cooled with running tap water for 1 min as well, and then wiped with absorbent paper. Afterwards, they were subjected to drying while weighing every 3 h until constant weight was recorded. The dried pumpkin slices obtained were ground, sieved and analyzed to determine their

*3.2.2 The effect of the three drying methods on the moisture content and nutritive* 

**84**

*\**

**Table 4.**

*methods [4].*

Pumpkin flour obtained by oven drying OED retained the highest amount of β-carotene, followed by ESD (**Table 5**). The fast rate of drying caused less nutrients to be lost, and samples at the core were still intact by the time constant weight was achieved. OSD had the least amount of β-carotene, most likely since unprotected exposure to the sun's UV rays caused photo-degradation of the carotenoids. Protein content showed no significant differences between the three drying methods but it showed a significant difference between the flours and the fresh fruits for almost 800% increase in the flour [4].

As for mineral, drying generally was found to reduce their levels compared to fresh fruits. Zinc was significantly lower in enhanced solar drying than oven and open sun drying, yet the fresh fruit exhibited the highest amount. Calcium levels were reduced greatly after drying showing 200% loss from fresh fruit. Whereas for iron, calcium and energy levels, no significant difference was noted between all treatments. It is therefore concluded, in this study, that enhanced solar drying is the best method for drying pumpkin and obtaining better quality pumpkin flour [4].


*\* Means followed by the same letter within a column are not significantly different at P = 0.05. ppm parts-per-million, 10−6.*

#### **Table 5.**

*Nutrient levels of fresh pumpkin fruit and pumpkin flour obtained from three drying methods [4].*


*Data are expressed as mean values. Mean values with different superscripts in the same column differ significantly at P* ≤ *0.05. The symbol ns means that the mean values are not significantly different. MCdb Moisture Content – dry basis matter.*

*aw Water Activity.*

#### **Table 6.**

*Mean values for moisture content and water activity of dried pumpkin powders prepared by hot air-drying at different temperatures [6].*

## **3.3 Hot air-drying of pumpkin: the effects of using different temperatures on physico-chemical characteristics of pumpkin flour**

## *3.3.1 Drying of pumpkin slices by three different temperatures*

A study performed by Roongruangsri and Bronlund [6] examined the effect of three hot-air drying temperatures (50, 60, and 70°C) on physico-chemical properties and sorption characteristics of pumpkin powder after the drying process. *Cucurbita maxima* Duch., also called buttercup pumpkin, was cleaned, peeled, and deseeded. The pulp was cut into slabs with a 5 mm thickness, 40 mm length and 20 mm width. Pumpkin slices were then blanched by immersing in hot water at 95°C for 5 minutes, then cooled to room temperature. Hot-air drying was then performed in a cross-flow cabinet hot-air tray dryer at three different temperatures of 50, 60 and 70°C. Afterwards, samples were weighed to calculate the moisture content (MCdb), and then ground in a blender and sieved.

## *3.3.2 The effect of drying temperatures on the characteristics of pumpkin powder*

### *3.3.2.1 Moisture content and water activity*

Results of MCdb and water activity analyses showed that dried pumpkin powder produced at 70°C exhibited the lowest MCdb and water activity levels compared to those produced at drying temperatures 50 and 60°C, as shown in **Table 6**. The low MCdb and water activity levels of pumpkin powders produced at 60 and 70°C suggest a better keeping quality than those produced at 50°C, since the occurrence of most unfavorable changes of food during storage is less when water activity drops below 0.4 [6].

## *3.3.2.2 Color of pumpkin powder*

Color of food is one of the important quality parameters since it may indicate changes in food quality due to processing, storage or other conditions. As mentioned earlier, the yellowish color of dried pumpkin powder is due to the carotenoid pigments naturally found in the pumpkin fruit. Powders produced at drying temperatures of 50 and 60°C showed lighter color retention than those produced at 70°C. Pumpkin powder produced at 50°C had the lightest color compared to that produced at 60 and 70°C, indicating that increase of drying temperature causes increase in the darkening of the color [6].

#### *3.3.2.3 Carotenoid content*

Dried pumpkin powder produced at 70°C showed the highest percentage decrease in carotenoid content (56%) compared to the decrease in those produced at 50 and 60°C (18% and 33% respectively). Decrease in total carotenoid content may be attributed to the degradation of β-carotene and other carotenoids due to auto-oxidation, since the highly unsaturated chemical structure of carotenoids makes them very sensitive to thermal degradation and oxidation [6].

#### *3.3.2.4 Powder properties*

**Table 7** shows the effects of drying temperatures on bulk density, solubility, water adsorption and oil adsorption capacities of the pumpkin powder. These

**87**

**Table 8.**

*Different types of pretreatments [1].*

*Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp*

**Water solubility (%)**

 *a, b, cMean values with different superscripts in the same column differ significantly at P* ≤ *0.05.*

**Water adsorption (g water/g dry sample)** **Oil adsorption capacity (g oil/g dry sample)**

ing properties that are important in bakery products [6].

**3.4 Freeze-dried pumpkin powder**

**Samples Pretreatment** T1 Control

T2 Dipping in 0.1% Citric Acid (CA) for 15 minutes T3 Hot water blanching at 95°C for 3 minutes

T5 Blanching at 95°C in 1%NaCl for 3 minutes

(KMS) for 45 minutes

T6 Dipping in 0.2% potassium metabisulfite (KMS) for 45 minutes

T7 Hot water blanching for 2 minutes followed by dipping in potassium metabisulfite

T4 Steam blanching for 5 minutes

properties affect the functional characteristics of the powder and are critical parameters for controlling quality; fruit and vegetable powders that have high water adsorption and oil adsorption capacities can convey water-retention and fat-bind-

*Physical characteristics of dried pumpkin powders obtained by hot air-drying at different temperatures [6].*

50 0.62c 54a 3.50a 4.42a 60 0.86b 50b 3.00b 3.97<sup>b</sup> 70 0.91a 43c 2.33c 3.87<sup>b</sup>

have more potential for baking purposes than those produced at 70°C [6].

The results implied that higher drying temperatures have an effect of decreasing water solubility and water and oil adsorption capacities of pumpkin powder: the dried pumpkin powder produced at 50 and 60°C had a water solubility above 50%, and higher water and oil adsorption capacities compared to that obtained at 70°C. These results indicate that dried pumpkin powders produced at 50 and 60°C

Freeze drying is a dehydration process employing two steps: freezing the food material, and sublimation of ice from the frozen material. Freeze drying is generally recommended for drying foods that have heat sensitive components such as tocopherols, carotenoids, and phenolics. It is considered a great method for drying foods of high quality where color, flavor, texture, nutrient content, taste, chemical composition and biological activity of the fresh sample only undergo minimal changes [16]. In a study performed by Dirim and Caliskan [16], it was observed that the chemical compositions such as vitamin C and total phenolics contents of dried pumpkin powder obtained by freeze-drying were not significantly different from that of fresh pumpkin. Freeze drying only reduced the total phenolic content by 3% in this study, but in the study performed by Aydin and Gocmen [17], pumpkin powder that was produced by hot-air oven scored higher than that produced by

*DOI: http://dx.doi.org/10.5772/intechopen.89274*

**Bulk density (g/ml)**

**Temperature (°C)**

**Table 7.**

*Data are expressed as mean values.*


*Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp DOI: http://dx.doi.org/10.5772/intechopen.89274*

 *a, b, cMean values with different superscripts in the same column differ significantly at P* ≤ *0.05.*

#### **Table 7.**

*The Health Benefits of Foods - Current Knowledge and Further Development*

**on physico-chemical characteristics of pumpkin flour**

*3.3.1 Drying of pumpkin slices by three different temperatures*

content (MCdb), and then ground in a blender and sieved.

*3.3.2.1 Moisture content and water activity*

below 0.4 [6].

*3.3.2.2 Color of pumpkin powder*

increase in the darkening of the color [6].

*3.3.2.3 Carotenoid content*

*3.3.2.4 Powder properties*

**3.3 Hot air-drying of pumpkin: the effects of using different temperatures** 

*3.3.2 The effect of drying temperatures on the characteristics of pumpkin powder*

Results of MCdb and water activity analyses showed that dried pumpkin powder produced at 70°C exhibited the lowest MCdb and water activity levels compared to those produced at drying temperatures 50 and 60°C, as shown in **Table 6**. The low MCdb and water activity levels of pumpkin powders produced at 60 and 70°C suggest a better keeping quality than those produced at 50°C, since the occurrence of most unfavorable changes of food during storage is less when water activity drops

Color of food is one of the important quality parameters since it may indicate changes in food quality due to processing, storage or other conditions. As mentioned earlier, the yellowish color of dried pumpkin powder is due to the carotenoid pigments naturally found in the pumpkin fruit. Powders produced at drying temperatures of 50 and 60°C showed lighter color retention than those produced at 70°C. Pumpkin powder produced at 50°C had the lightest color compared to that produced at 60 and 70°C, indicating that increase of drying temperature causes

Dried pumpkin powder produced at 70°C showed the highest percentage decrease in carotenoid content (56%) compared to the decrease in those produced at 50 and 60°C (18% and 33% respectively). Decrease in total carotenoid content may be attributed to the degradation of β-carotene and other carotenoids due to auto-oxidation, since the highly unsaturated chemical structure of carotenoids

**Table 7** shows the effects of drying temperatures on bulk density, solubility, water adsorption and oil adsorption capacities of the pumpkin powder. These

makes them very sensitive to thermal degradation and oxidation [6].

A study performed by Roongruangsri and Bronlund [6] examined the effect of three hot-air drying temperatures (50, 60, and 70°C) on physico-chemical properties and sorption characteristics of pumpkin powder after the drying process. *Cucurbita maxima* Duch., also called buttercup pumpkin, was cleaned, peeled, and deseeded. The pulp was cut into slabs with a 5 mm thickness, 40 mm length and 20 mm width. Pumpkin slices were then blanched by immersing in hot water at 95°C for 5 minutes, then cooled to room temperature. Hot-air drying was then performed in a cross-flow cabinet hot-air tray dryer at three different temperatures of 50, 60 and 70°C. Afterwards, samples were weighed to calculate the moisture

**86**

*Physical characteristics of dried pumpkin powders obtained by hot air-drying at different temperatures [6].*

properties affect the functional characteristics of the powder and are critical parameters for controlling quality; fruit and vegetable powders that have high water adsorption and oil adsorption capacities can convey water-retention and fat-binding properties that are important in bakery products [6].

The results implied that higher drying temperatures have an effect of decreasing water solubility and water and oil adsorption capacities of pumpkin powder: the dried pumpkin powder produced at 50 and 60°C had a water solubility above 50%, and higher water and oil adsorption capacities compared to that obtained at 70°C. These results indicate that dried pumpkin powders produced at 50 and 60°C have more potential for baking purposes than those produced at 70°C [6].

#### **3.4 Freeze-dried pumpkin powder**

Freeze drying is a dehydration process employing two steps: freezing the food material, and sublimation of ice from the frozen material. Freeze drying is generally recommended for drying foods that have heat sensitive components such as tocopherols, carotenoids, and phenolics. It is considered a great method for drying foods of high quality where color, flavor, texture, nutrient content, taste, chemical composition and biological activity of the fresh sample only undergo minimal changes [16].

In a study performed by Dirim and Caliskan [16], it was observed that the chemical compositions such as vitamin C and total phenolics contents of dried pumpkin powder obtained by freeze-drying were not significantly different from that of fresh pumpkin. Freeze drying only reduced the total phenolic content by 3% in this study, but in the study performed by Aydin and Gocmen [17], pumpkin powder that was produced by hot-air oven scored higher than that produced by


#### **Table 8.**

*Different types of pretreatments [1].*


*The Health Benefits of Foods - Current Knowledge and Further Development*

**Table 9.**

**89**

*Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp*

less carotenoid degradation [16] than that of hot-air oven drying.

in developing countries or for cost-reduction purposes.

the final powder product produced by drying [17].

leaching out during the blanching process [1].

browning compounds during storage [1].

**4. Pumpkin wheat composite bread**

**4.1 Nutritive value of pumpkin wheat composite flour**

freeze drying in terms of phenolic contents and antioxidant activity. The latter study, however, showed that freeze drying reduced browning, preserved redness, resulted in a lighter color, higher water holding capacity, oil binding capacity, emulsion stability, and the highest total dietary fiber compared to oven produced powders. Color values obtained by Aydin and Gocmen [17] and Mujaffar et al. [18] supported the overall results that freeze drying was able to preserve a closer color of powder to that of fresh pumpkin, producing pumpkin powder of high quality color. Moreover, freeze drying was reported to produce higher yields of powder [18] and

Although freeze drying preserved the deep-orange color of fresh pumpkin and produced better physico-chemical properties of pumpkin flour, the cost of freeze drying application is very high [17], making oven drying a more suitable technique

**3.5 The effect of pre-treatments on selected properties of pumpkin flour**

Fruits and vegetables are often pretreated in order to extend their shelf life, preserve their color and flavor, decrease the loss of nutrients and reduce activity of enzymes. In the production of dried products, pretreatments can lead to improvement of product quality and help in the inhibition of enzymatic browning (Kripanand et al., 2016). Since conventional air drying can adversely affect the color, flavor and nutritional value of the dried products, pretreatments prior to air drying are considered one of the most important factors that affect the quality of

In order to optimize different pretreatments to obtain good quality pumpkin powder and β-carotene retention during storage, Kripanand et al. [1] performed a study employing six different pretreatments for the production of pumpkin flour from fresh *Cucurbita maxima*. The different types of pretreatments are presented in

Results of this study (**Table 9**) showed that pretreated flour samples retained a higher moisture content compared to the control sample. Blanching was found to significantly affect the protein content, where cold pre-treated samples (T1, T2 and T3) had higher protein values compared to hot pre-treated samples. Blanching was also found to reduce starch, ash, fiber, phosphorus and iron quantities due to

As for the carotenoid content, it was observed that chemical pretreatments lead to improvement in the amount of total carotene in pumpkin flour. But the use of blanching and sulfiting together (T7) showed a most favorable effect on total carotenoid stability T7 pretreatment also attained the highest score for color and overall acceptability, followed by T6. In addition, less browning was observed in all T7 samples during storage indicating that metabisulfite reduced the formation of

Consumers are becoming more aware of healthy eating and high quality foods that contain additional health benefits. Yet, the modern consumers rely on the food industry to provide such high quality food products as they purchase more processed foods and ready meals [7]. Development of healthy products with the incorporation of fruits and vegetables represent one strategy for the production

**Table 8**, where the control sample (T1) represents no pretreatment.

*DOI: http://dx.doi.org/10.5772/intechopen.89274*

*Effect of pretreatments on the proximate parameters of pumpkin flour [1].*

#### *Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp DOI: http://dx.doi.org/10.5772/intechopen.89274*

*The Health Benefits of Foods - Current Knowledge and Further Development*

**88**

**Composition** Moisture (%)

Protein (%)

Ash (%) Crude fiber (%)

**Minerals (mg/100**

Phosphorus

Iron Total carotene (mg/100 g)

Starch (%) SO2 (mg/kg)

**Table 9.** *Effect of pretreatments on the proximate parameters of pumpkin flour [1].*

241.977 ± 0.02

22.54 ± 0.05 2.816 ± 0.01 30.16 ± 0.05

—

—

—

—

—

—

317.545 ± 0.02

16.01 ± 0.03 5.492 ± 0.03 40.77 ± 0.01

19.8 ± 0.01

23.7 ± 0.05

22.68 ± 0.02

30.22 ± 0.03

9.196 ± 0.01

10.35 ± 0.01

2.17 ± 0.01

7.581 ± 0.00

5.078 ± 0.00

10.075 ± 0.01

11.629 ± 0.00

18.61 ± 0.04

177.449 ± 0.06

167.514 ± 0.01

28.35 ± 0.05

269.451 ± 0.02

142.988 ± 0.04

21.794 ± 0.01

17.769 ± 0.00

32.14 ± 0.04

1279.14 ± 0.03

 **g)**

**T1** 6.40 ± 0.005

8.51 ± 0.01 6.52 ± 0.02 6.58 ± 0.02

**T2** 7.38 ± 0.01 7.25 ± 0.01 5.70 ± 0.15 6.41 ± 0.03

6.9 ± 0.2

7.04 ± 0.02

7.5 ± 0.05

8.36 ± 0.01

5.17 ± 0.02 4.02 ± 0.01

6.61 ± 0.02

6.59 ± 0.03

6.04 ± 0.01

**T3** 12.78 ± 0.02

**Pumpkin flour (0 days)**

**T4** 12.62 ± 0.01

6.16 ± 0.01

6.68 ± 0.01

9.54 ± 0.01

**T5** 13.8 ± 0.05

11.44 ± 0.01

**T6**

**T7**

10.99 ± 0.01

5.45 ± 0.02

6.54 ± 0.01

12.011 ± 0.02

freeze drying in terms of phenolic contents and antioxidant activity. The latter study, however, showed that freeze drying reduced browning, preserved redness, resulted in a lighter color, higher water holding capacity, oil binding capacity, emulsion stability, and the highest total dietary fiber compared to oven produced powders. Color values obtained by Aydin and Gocmen [17] and Mujaffar et al. [18] supported the overall results that freeze drying was able to preserve a closer color of powder to that of fresh pumpkin, producing pumpkin powder of high quality color. Moreover, freeze drying was reported to produce higher yields of powder [18] and less carotenoid degradation [16] than that of hot-air oven drying.

Although freeze drying preserved the deep-orange color of fresh pumpkin and produced better physico-chemical properties of pumpkin flour, the cost of freeze drying application is very high [17], making oven drying a more suitable technique in developing countries or for cost-reduction purposes.

## **3.5 The effect of pre-treatments on selected properties of pumpkin flour**

Fruits and vegetables are often pretreated in order to extend their shelf life, preserve their color and flavor, decrease the loss of nutrients and reduce activity of enzymes. In the production of dried products, pretreatments can lead to improvement of product quality and help in the inhibition of enzymatic browning (Kripanand et al., 2016). Since conventional air drying can adversely affect the color, flavor and nutritional value of the dried products, pretreatments prior to air drying are considered one of the most important factors that affect the quality of the final powder product produced by drying [17].

In order to optimize different pretreatments to obtain good quality pumpkin powder and β-carotene retention during storage, Kripanand et al. [1] performed a study employing six different pretreatments for the production of pumpkin flour from fresh *Cucurbita maxima*. The different types of pretreatments are presented in **Table 8**, where the control sample (T1) represents no pretreatment.

Results of this study (**Table 9**) showed that pretreated flour samples retained a higher moisture content compared to the control sample. Blanching was found to significantly affect the protein content, where cold pre-treated samples (T1, T2 and T3) had higher protein values compared to hot pre-treated samples. Blanching was also found to reduce starch, ash, fiber, phosphorus and iron quantities due to leaching out during the blanching process [1].

As for the carotenoid content, it was observed that chemical pretreatments lead to improvement in the amount of total carotene in pumpkin flour. But the use of blanching and sulfiting together (T7) showed a most favorable effect on total carotenoid stability T7 pretreatment also attained the highest score for color and overall acceptability, followed by T6. In addition, less browning was observed in all T7 samples during storage indicating that metabisulfite reduced the formation of browning compounds during storage [1].

## **4. Pumpkin wheat composite bread**

#### **4.1 Nutritive value of pumpkin wheat composite flour**

Consumers are becoming more aware of healthy eating and high quality foods that contain additional health benefits. Yet, the modern consumers rely on the food industry to provide such high quality food products as they purchase more processed foods and ready meals [7]. Development of healthy products with the incorporation of fruits and vegetables represent one strategy for the production

#### *The Health Benefits of Foods - Current Knowledge and Further Development*


*\* This study does not mention whether pumpkin seeds were removed or not before drying and powdering, which might explain the higher fat content in pumpkin flour compared to wheat flour if the seeds were kept.*

#### **Table 10.**

*Proximate composition of wheat flour and pumpkin flour [19].*


*\* Means followed by the same letter within a column are not significantly different at P = 0.05. PF = pumpkin flour. g/d = grams per day. mg/d = milligrams per day. Kcal/d = kilocalories per day.*

*\*\*Applies to retinol: 1 μg retinol = 12 μg β-carotene, hence RDI values should be multiplied by 12 to relate to table values.*

*RDI Reference Daily Intake*

#### **Table 11.**

*Mean of nutrient content in pumpkin bread at five blending levels [7].*

of these 'functional foods' [19]. Use of functional ingredients in bakery products for the aim of nutrient enrichment is increasingly becoming important in bakery industries [7]. Pumpkin flour has been used to supplement cereal flours in bakery products, soups, sauces, instant noodles and spices [3].

*Pumpkin wheat composite flour improves the texture, nutritional value, and color of different bakery products* and thus, it is likely to produce bread with improved nutritional value and good sensory characteristics by using pumpkin wheat composite flour [20]. **Table 10** compares the proximate composition of wheat flour and pumpkin flour. Pumpkin flour was shown to have higher amounts of calcium, iron, zinc, β-carotene, ash and total dietary fiber. This indicates that pumpkin flour could be used to supplement wheat flour with these nutrients for the production of higher quality bread [19].

**Table 11** shows the contents of various nutrients in wheat bread supplemented with different levels of pumpkin flour. Incorporation of pumpkin flour resulted

**91**

*Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp*

in a uniform trend of increase in protein, β-carotene, calcium, iron and zinc, and uniform decrease in energy content with increasing levels of pumpkin flour. Reduction of calories with increasing pumpkin flour levels is attributed to increased fiber content and lower carbohydrate content in the composite flour, which is a

**Composition % Control 5% 10% 15%** Moisture 32.02 ± 0.54bc 32.63 ± 0.50c 34.25 ± 0.08ab 35.32 ± 0.06a Fat 2.59 ± 0.01a 2.55 ± 0.01a 2.48 ± 0.01b 2.44 ± 0.01b Protein 15.72 ± 0.04a 15.17 ± 0.09b 14.71 ± 0.02c 14.47 ± 0.06c Ash 1.83 ± 0.07d 2.09 ± 0.01c 2.26 ± 0.02b 2.43 ± 0.03a Crude fiber 1.56 ± 0.02d 2.46 ± 0.03c 2.62 ± 0.01b 2.90 ± 0.04a Carbohydrate 46.28 ± 0.14a 45.10 ± 0.21b 43.68 ± 0.05c 42.44 ± 0.05d

271.31a 264.03b 255.88c 249.60d

**4.2 Physico-chemical properties of pumpkin wheat composite bread**

The effects of adding different levels of pumpkin flour on the physico-chemical properties of bread have been studied. Substitution of higher levels of pumpkin powder in bread have been shown to decrease the fat content of the bread. This might be attributed to the lower content of fat in pumpkin flour compared to wheat flour. The same effect was observed for the carbohydrate content, as increasing the level of pumpkin flour resulted in decreased total carbohydrate content of the bread [3]. Protein content has been also shown to decrease with increased incorporation of pumpkin flour (**Table 12**) [3, 20], which opposes the results obtained by Kiharason et al. [7] (**Table 11**) that shows increased protein content with increased pumpkin flour content. This might be attributed -as mentioned in chapter I- to the different nutritional profile of different species and cultivars of pumpkin, or to the pumpkin powder preparation methods in which seeds were removed or kept. Pumpkin seeds are rich in protein and lipids [2], and thus keeping them as part of the pulp in the flour preparation process would increase the amount of these constituents in the produced powder. Ash, total fiber and reducing sugar levels increased with increasing substitution of pumpkin flour in bread [3, 21]. Increasing the level of pumpkin flour also resulted in increase of the moisture content of the composite bread which could be explained by the higher water absorption capac-

In the study conducted by See et al. [3], incorporation of 5% pumpkin flour resulted in the highest loaf volume and specific volume compared to the other samples giving more significant softness in bread. The weight of the loaf significantly increased as increasing levels of pumpkin flour were incorporated, which was attributed to the increased water absorption capacity of pumpkin flour. Opposite results were obtained by Kundu et al. [19] where supplementation of increased levels of pumpkin flour lead to decreased water absorption (**Table 13**).

Dough development time, defined as *the time to the nearest half minute from the first addition of the water to the development of the maximum consistency of the dough*

good approach in the direction of health promotion [7].

*Proximate composition of bread for different levels of pumpkin flour [3].*

*a, b, c Means in a row with similar superscripts are not significantly different at α = 0.05.*

ity of the composite flour compared to wheat flour [3].

The result was related to the dilution of gluten.

*DOI: http://dx.doi.org/10.5772/intechopen.89274*

*Values are the Means ± SD and n = 3 for each group.*

Calorie (kcal/100 g)

**Table 12.**

*Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp DOI: http://dx.doi.org/10.5772/intechopen.89274*


*a, b, c Means in a row with similar superscripts are not significantly different at α = 0.05. Values are the Means ± SD and n = 3 for each group.*

#### **Table 12.**

*The Health Benefits of Foods - Current Knowledge and Further Development*

Moisture 11.1% 4.8% Protein 12.4% 11.6% Fat 1.4% 2.4%\* Dietary fiber 10.1% 28.3% Crude fiber 1.2% 16.9% Ash 0.63% 6.7% Calcium 17.0 mg/100 g 121.7 mg/100 g Iron 5.3 mg/100 g 7.1 mg/100 g Zinc 2.8 mg/100 g 3.1 mg/100 g β-carotene — 1.8 mg/100 g

of these 'functional foods' [19]. Use of functional ingredients in bakery products for the aim of nutrient enrichment is increasingly becoming important in bakery industries [7]. Pumpkin flour has been used to supplement cereal flours in bakery

*Pumpkin wheat composite flour improves the texture, nutritional value, and color of different bakery products* and thus, it is likely to produce bread with improved nutritional value and good sensory characteristics by using pumpkin wheat composite flour [20]. **Table 10** compares the proximate composition of wheat flour and pumpkin flour. Pumpkin flour was shown to have higher amounts of calcium, iron, zinc, β-carotene, ash and total dietary fiber. This indicates that pumpkin flour could be used to supplement wheat flour with these nutrients for the production of higher quality bread [19]. **Table 11** shows the contents of various nutrients in wheat bread supplemented with different levels of pumpkin flour. Incorporation of pumpkin flour resulted

products, soups, sauces, instant noodles and spices [3].

*Mean of nutrient content in pumpkin bread at five blending levels [7].*

**90**

**Level (%PF)**

**Table 10.**

*\**

RDI (adult)

RDI (child)

*values.*

**Table 11.**

*\**

1 (0%) 0.1108b

*RDI Reference Daily Intake*

**Protein (g/mg)**

**β-carotene (μg/g)**

*Proximate composition of wheat flour and pumpkin flour [19].*

*explain the higher fat content in pumpkin flour compared to wheat flour if the seeds were kept.*

34–71 g/d 600–1300 μg/d\*\* 1000–

*g/d = grams per day. mg/d = milligrams per day. Kcal/d = kilocalories per day.*

**Calcium (mg/g)**

*This study does not mention whether pumpkin seeds were removed or not before drying and powdering, which might* 

2 (5%) 0.1284ab 3.583ab 0.2850b 0.0739c 0.0407ab 2.4494b 3 (20%) 0.1298ab 3.768ab 0.4549ab 0.0164bc 0.0512ab 2.3141bc 4 (50%) 0.1350a 5.125a 0.8063ab 0.1175ab 0.0551ab 2.2147bc 5 (95%) 0.1378a 5.128a 1.0113a 0.1495a 0.0631a 2.1104c

1300 mg/d

*Means followed by the same letter within a column are not significantly different at P = 0.05. PF = pumpkin flour.* 

*\*\*Applies to retinol: 1 μg retinol = 12 μg β-carotene, hence RDI values should be multiplied by 12 to relate to table* 

13–19 g/d 300–400 μg/d\*\* 500–800 mg/d 7–10 mg/d 3–5 mg/d 1046–

**Iron (mg/g)**

**Wheat Flour Pumpkin Flour**

\* 1.433b 0.2736b 0.0216 0.0344b 2.6792a

**Zinc (mg/g)**

8–18 mg/d 8–13 mg/d 2403–

**Energy (kcal/g)**

3067 kcal/d

1742 kcal/d

*Proximate composition of bread for different levels of pumpkin flour [3].*

in a uniform trend of increase in protein, β-carotene, calcium, iron and zinc, and uniform decrease in energy content with increasing levels of pumpkin flour. Reduction of calories with increasing pumpkin flour levels is attributed to increased fiber content and lower carbohydrate content in the composite flour, which is a good approach in the direction of health promotion [7].

#### **4.2 Physico-chemical properties of pumpkin wheat composite bread**

The effects of adding different levels of pumpkin flour on the physico-chemical properties of bread have been studied. Substitution of higher levels of pumpkin powder in bread have been shown to decrease the fat content of the bread. This might be attributed to the lower content of fat in pumpkin flour compared to wheat flour. The same effect was observed for the carbohydrate content, as increasing the level of pumpkin flour resulted in decreased total carbohydrate content of the bread [3]. Protein content has been also shown to decrease with increased incorporation of pumpkin flour (**Table 12**) [3, 20], which opposes the results obtained by Kiharason et al. [7] (**Table 11**) that shows increased protein content with increased pumpkin flour content. This might be attributed -as mentioned in chapter I- to the different nutritional profile of different species and cultivars of pumpkin, or to the pumpkin powder preparation methods in which seeds were removed or kept. Pumpkin seeds are rich in protein and lipids [2], and thus keeping them as part of the pulp in the flour preparation process would increase the amount of these constituents in the produced powder. Ash, total fiber and reducing sugar levels increased with increasing substitution of pumpkin flour in bread [3, 21]. Increasing the level of pumpkin flour also resulted in increase of the moisture content of the composite bread which could be explained by the higher water absorption capacity of the composite flour compared to wheat flour [3].

In the study conducted by See et al. [3], incorporation of 5% pumpkin flour resulted in the highest loaf volume and specific volume compared to the other samples giving more significant softness in bread. The weight of the loaf significantly increased as increasing levels of pumpkin flour were incorporated, which was attributed to the increased water absorption capacity of pumpkin flour. Opposite results were obtained by Kundu et al. [19] where supplementation of increased levels of pumpkin flour lead to decreased water absorption (**Table 13**). The result was related to the dilution of gluten.

Dough development time, defined as *the time to the nearest half minute from the first addition of the water to the development of the maximum consistency of the dough*


#### **Table 13.**

*Effect of incorporation of various levels of pumpkin powder on farinographic characteristics of wheat flour [19].*

#### **Figure 2.**

*The changes in the value of dry off and baking loss in bread samples [21].*

increased with the addition of pumpkin flour, which was related to the difference in the physiochemical properties between the constituents of pumpkin flour and wheat flour. Dough consistency was also maintained almost at the same level after increased levels of pumpkin flour indicating that the dough was stable and had more resistance against mechanical mixing. Increased concentration of pumpkin flour also lead to decrease in mixing tolerance index indicating stronger flour, since the lower the mixing tolerance index, the stronger the flour. Extensibility and resistance to extensibility were also shown to significantly increase with increased incorporation of pumpkin flour, resulting in rubber-like properties [19].

Rakcejeva et al. [21] studied the effect of incorporating 10% pumpkin flour in wheat bread on the bread baking loss which *forms the biggest loss in technological processes*. Results showed an insignificant decrease by 0.95% compared to 100% wheat flour bread (**Figure 2**). Thus, technological bread weight loss during the addition of 10% pumpkin flour is considered insignificant. These results show that pumpkin powder supplemented bread can be used for making good quality bread.

#### **4.3 Sensory evaluation of pumpkin wheat composite bread**

Conducting tests that determine consumer acceptance, liking, preference and opinions is among the key activities that relay important information for consumer

**93**

bread (higher for 5% pumpkin flour bread).

*Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp*

*a, b, c Means in a row with similar superscripts are not significant different at α = 0.05.*

*Mean value of sensory attributes of bread incorporated with different levels of pumpkin flour [3].*

T0 7.00a 7.00a 2.80a 2.90a 2.90a T1 8.00b 7.00a 2.80a 2.80a 2.80a T2 7.10bc 6.90b 2.60a 2.60a 2.60a T3 6.90c 6.62a 2.20a 2.20a 2.20a *T0 = control (0% pumpkin flour), T1 = 5% pumpkin flour, T2 = 10% pumpkin flour, T3 = 15% pumpkin flour.*

> **Aroma of bread**

T0 7.50a 8.10a 8.00a 12.60a 8.00a 12.20a T1 7.50a 8.00a 7.70a 12.60a 7.60a 12.20a T2 7.40ab 7.60a 7.50ab 11.00b 7.20a 11.90a T3 6.80b 6.90b 6.90b 10.80b 6.40b 10.80b *T0 = control (0% pumpkin flour), T1 = 5% pumpkin flour, T2 = 10% pumpkin flour, T3 = 15% pumpkin flour.*

**Symmetry of form**

> **Taste of bread**

**Evenness of bake**

**Mastication of bread**

**Character of crust**

> **Texture of bread**

**Crust color**

**Parameter Control 5% 10% 15%** Crust color 6.00 ± 1.67 <sup>a</sup> 6.07 ± 0.88a 5.67 ± 0.81a 5.33 ± 0.90a Crumb color 6.13 ± 0.99ab 7.67 ± 0.49c 6.67 ± 0.49b 5.73 ± 0.70a Moistness 5.60 ± 0.51ab 6.07 ± 0.80a 5.33 ± 0.49bc 5.00 ± 0.38c Softness 5.93 ± 0.80ab 6.47 ± 0.83a 5.53 ± 0.64bc 5.20 ± 0.41c Aftertaste 5.73 ± 0.59a 6.13 ± 0.52a 5.20 ± 0.41b 4.87 ± 0.35b Overall acceptability 6.60 ± 0.74ab 6.93 ± 0.59a 6.13 ± 0.35bc 5.73 ± 0.46c

goods companies. The results of these tests help companies make product decisions concerning marketing, development of new products, reformulation of existing products, etc.. Sensory evaluation performed to assess pumpkin wheat composite bread showed the highest acceptability and preference for 5% pumpkin flour supplemented bread in the studies conducted by See et al. [3] and Pasha et al. [20]. **Table 14** shows the data of the sensory evaluation obtained by See et al. [3]. The data indicated that consumers preferred the crust color, moistness, softness and aftertaste of the 5% pumpkin flour bread and the control sample that were not significantly different. Similar results were obtained by Pasha et al. [20] where external and internal characteristics (**Tables 15** and **16**) of control bread and 5% pumpkin flour supplemented bread were only significantly different by volume of

*DOI: http://dx.doi.org/10.5772/intechopen.89274*

*Values are the Means ± SD and n = 15 for each group.*

**bread**

*a, b, c Means in a row with similar superscripts are not significantly different.*

**Crumb color**

*a, b, c Means in a row with similar superscripts are not significantly different.*

**Treatments Volume of** 

*External characteristics of bread [20].*

*Internal characteristics of bread [20].*

**bread**

**Treatments Grain of** 

**Table 14.**

**Table 15.**

**Table 16.**

### *Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp DOI: http://dx.doi.org/10.5772/intechopen.89274*


*a, b, c Means in a row with similar superscripts are not significant different at α = 0.05. Values are the Means ± SD and n = 15 for each group.*

#### **Table 14.**

*The Health Benefits of Foods - Current Knowledge and Further Development*

**5% pumpkin powder**

Water absorption (%) 67.0 ± 0.0 65.0 ± 0.0 62.5 ± 0.16

Dough stability 2.0 ± 0.83 3.0 ± 0.08 3.5 ± 0.08

*Effect of incorporation of various levels of pumpkin powder on farinographic characteristics of wheat flour [19].*

**Flour supplemented with 10% pumpkin powder**

2.5 ± 0.0 2.7 ± 0.0 4.1 ± 0.08

70.0 ± 1.6 60.0 ± 1.6 50 ± 1.6

5.1 ± 0.0 6.0 ± 0.08 7.5 ± 1.6

51.4 ± 0.0 60 ± 0.83 75 ± 1.6

**Flour supplemented with 15% pumpkin powder**

**Parameter Flour supplemented with** 

Dough development time

Mixing tolerance index

Time to break down

Farinographic quality

(min.)

(BU)

(min.)

number

**Table 13.**

**Figure 2.**

increased with the addition of pumpkin flour, which was related to the difference in the physiochemical properties between the constituents of pumpkin flour and wheat flour. Dough consistency was also maintained almost at the same level after increased levels of pumpkin flour indicating that the dough was stable and had more resistance against mechanical mixing. Increased concentration of pumpkin flour also lead to decrease in mixing tolerance index indicating stronger flour, since the lower the mixing tolerance index, the stronger the flour. Extensibility and resistance to extensibility were also shown to significantly increase with increased

Rakcejeva et al. [21] studied the effect of incorporating 10% pumpkin flour in wheat bread on the bread baking loss which *forms the biggest loss in technological processes*. Results showed an insignificant decrease by 0.95% compared to 100% wheat flour bread (**Figure 2**). Thus, technological bread weight loss during the addition of 10% pumpkin flour is considered insignificant. These results show that pumpkin

Conducting tests that determine consumer acceptance, liking, preference and opinions is among the key activities that relay important information for consumer

incorporation of pumpkin flour, resulting in rubber-like properties [19].

powder supplemented bread can be used for making good quality bread.

**4.3 Sensory evaluation of pumpkin wheat composite bread**

*The changes in the value of dry off and baking loss in bread samples [21].*

**92**

*Mean value of sensory attributes of bread incorporated with different levels of pumpkin flour [3].*


*T0 = control (0% pumpkin flour), T1 = 5% pumpkin flour, T2 = 10% pumpkin flour, T3 = 15% pumpkin flour. a, b, c Means in a row with similar superscripts are not significantly different.*

#### **Table 15.**

*External characteristics of bread [20].*


*T0 = control (0% pumpkin flour), T1 = 5% pumpkin flour, T2 = 10% pumpkin flour, T3 = 15% pumpkin flour. a, b, c Means in a row with similar superscripts are not significantly different.*

#### **Table 16.**

*Internal characteristics of bread [20].*

goods companies. The results of these tests help companies make product decisions concerning marketing, development of new products, reformulation of existing products, etc.. Sensory evaluation performed to assess pumpkin wheat composite bread showed the highest acceptability and preference for 5% pumpkin flour supplemented bread in the studies conducted by See et al. [3] and Pasha et al. [20]. **Table 14** shows the data of the sensory evaluation obtained by See et al. [3]. The data indicated that consumers preferred the crust color, moistness, softness and aftertaste of the 5% pumpkin flour bread and the control sample that were not significantly different. Similar results were obtained by Pasha et al. [20] where external and internal characteristics (**Tables 15** and **16**) of control bread and 5% pumpkin flour supplemented bread were only significantly different by volume of bread (higher for 5% pumpkin flour bread).

In the study performed by Rakcejeva et al. [21], the highest assessment after expert sensory evaluation was scored for 10% pumpkin flour bread, and elevated levels of pumpkin flour (over 10%) became unacceptable due to worse porosity, stickier bread soft part and unpleasantly sweet taste of bread. A higher degree of liking was also scored for 10% pumpkin flour bread over control bread: bread sample with pumpkin additive was shown to be tastier than the control bread sample.

## **5. Conclusion and discussion**

The nutritional value of pumpkin fruit is high and exquisite, which calls for its exploitation by the food industry as a functional food. Studies have found antioxidant, anti-diabetic, anti-carcinogenic and anti-fatigue effects of pumpkin pulp nutritional components. Being a perishable fruit, means for prolonging its shelf life had to be employed. Drying is one of the methods that prolong the shelf life of food products by reducing the moisture content to inhibit the growth of microbes and thus prevent spoilage of the food material.

To preserve the nutritional value of the dried pumpkin, several drying methods were studied in an attempt to reduce the degradation of nutritive components during drying and during storage. Vacuum freeze drying was shown to be a great method to preserve the β-carotene and phenolic acid contents of dried pumpkin but is an expensive drying technique. Convective drying methods are common methods to dry food materials and are cheaper but could result in a greater loss of nutrients. To reduce this loss, the appropriate drying conditions such as drying temperature and pretreatments had to be optimized. It was found that a drying temperature of 60°C resulted in good quality pumpkin powder with acceptable water activity, β-carotene content retention, color quality and good potential for baking purposes. Drying temperatures of 50 and 70°C lead to unacceptable water activity level and greater degradation of β-carotene, respectively. Metabisulfite pretreatment of pumpkin slices preceded by hot water blanching was found to have the most favorable effect on total carotenoid stability, color, phenolic content and overall acceptability compared to several other pretreatments in the production of hot air dried pumpkin powder.

Production of pumpkin powder from dried pumpkin slices allows its supplementation into baking products –among others- to enhance their nutritional value. Development of pumpkin wheat composite bread was studied using different levels of pumpkin flour. Increasing the level of pumpkin flour incorporation into wheat flour led to increased contents of total fiber, β-carotene, calcium, iron and zinc, and it led to decrease in carbohydrate and caloric contents which is a good approach for health promotion. Incorporation of 5 and 10% pumpkin flour were found to have good dough and bread physical characteristics and the best sensory evaluation of pumpkin wheat composite bread.

**95**

**Author details**

Beqaa Valley, Lebanon

University, Beqaa Valley, Lebanon

provided the original work is properly cited.

Sami El Khatib1,2\* and Mariam Muhieddine2

\*Address all correspondence to: sami.khatib@liu.edu.lb

1 Department of Biological Sciences, Lebanese International University,

2 Department of Food Sciences and Technologies, Lebanese International

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp*

*DOI: http://dx.doi.org/10.5772/intechopen.89274*

*Nutritional Profile and Medicinal Properties of Pumpkin Fruit Pulp DOI: http://dx.doi.org/10.5772/intechopen.89274*

*The Health Benefits of Foods - Current Knowledge and Further Development*

**5. Conclusion and discussion**

thus prevent spoilage of the food material.

dried pumpkin powder.

pumpkin wheat composite bread.

In the study performed by Rakcejeva et al. [21], the highest assessment after expert sensory evaluation was scored for 10% pumpkin flour bread, and elevated levels of pumpkin flour (over 10%) became unacceptable due to worse porosity, stickier bread soft part and unpleasantly sweet taste of bread. A higher degree of liking was also scored for 10% pumpkin flour bread over control bread: bread sample

with pumpkin additive was shown to be tastier than the control bread sample.

The nutritional value of pumpkin fruit is high and exquisite, which calls for its exploitation by the food industry as a functional food. Studies have found antioxidant, anti-diabetic, anti-carcinogenic and anti-fatigue effects of pumpkin pulp nutritional components. Being a perishable fruit, means for prolonging its shelf life had to be employed. Drying is one of the methods that prolong the shelf life of food products by reducing the moisture content to inhibit the growth of microbes and

To preserve the nutritional value of the dried pumpkin, several drying methods

Production of pumpkin powder from dried pumpkin slices allows its supplementation into baking products –among others- to enhance their nutritional value. Development of pumpkin wheat composite bread was studied using different levels of pumpkin flour. Increasing the level of pumpkin flour incorporation into wheat flour led to increased contents of total fiber, β-carotene, calcium, iron and zinc, and it led to decrease in carbohydrate and caloric contents which is a good approach for health promotion. Incorporation of 5 and 10% pumpkin flour were found to have good dough and bread physical characteristics and the best sensory evaluation of

were studied in an attempt to reduce the degradation of nutritive components during drying and during storage. Vacuum freeze drying was shown to be a great method to preserve the β-carotene and phenolic acid contents of dried pumpkin but is an expensive drying technique. Convective drying methods are common methods to dry food materials and are cheaper but could result in a greater loss of nutrients. To reduce this loss, the appropriate drying conditions such as drying temperature and pretreatments had to be optimized. It was found that a drying temperature of 60°C resulted in good quality pumpkin powder with acceptable water activity, β-carotene content retention, color quality and good potential for baking purposes. Drying temperatures of 50 and 70°C lead to unacceptable water activity level and greater degradation of β-carotene, respectively. Metabisulfite pretreatment of pumpkin slices preceded by hot water blanching was found to have the most favorable effect on total carotenoid stability, color, phenolic content and overall acceptability compared to several other pretreatments in the production of hot air

**94**

## **Author details**

Sami El Khatib1,2\* and Mariam Muhieddine2

1 Department of Biological Sciences, Lebanese International University, Beqaa Valley, Lebanon

2 Department of Food Sciences and Technologies, Lebanese International University, Beqaa Valley, Lebanon

\*Address all correspondence to: sami.khatib@liu.edu.lb

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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**Chapter 4**

**Abstract**

**1. Introduction**

**1.1 Tree-borne oilseeds**

Tree-Borne Edible Oilseeds as

Acids for Human Health

*Bithika Chaliha, Debajit Saikia* 

*and Siddhartha Proteem Saikia*

Sources of Essential Omega Fatty

Certain positional isomers of polyunsaturated omega-3 and omega-6 fatty acids are the essential fatty acids that the human body needs for metabolic functioning but cannot produce themselves and therefore must be acquired from the diet. The beneficial effects of omega-3 fatty acids are related to brain development, coronary heart disease (CHD), cancer, inflammatory bowel disease, rheumatoid arthritis, psoriasis, mental health, and neurodegenerative disorders. The essential omega-3 fatty acid is α-linolenic acid (ALA; 18:3ω3), found in green leafy vegetables and in the seeds of flax, rape, chia, and walnuts. The essential omega-6 fatty acid, linoleic acid (LA; 18:2ω6), is plentiful in nature and being found in the seeds of many edible plants. There are at least hundred species of plants occurring in wild or cultivated from forest areas that may be a source of vegetable oil. These vegetable oils are rich in polyunsaturated fatty acids, which are highly beneficial for human health.

**Keywords:** fatty acids, oilseeds, tree-borne, PUFA, prostaglandins, human health

Triglycerides constitute a vital part of human nutrition, and 90% of the global

production from plant, animal, and aquatic sources is used as edibles or as an ingredient in edible products. A major portion of the dietary energy comes from triacylgycerols which contain more than twice the value of identical amount of carbohydrate. Tree-borne seed oil can be defined as a vegetable oil that is obtained

from the seed (endosperm) of some trees, rather than the fruit (pericarp). Vegetable oil production and bioenergy generation from high oil-yielding tree-borne oilseeds have been a topic of interest [1]. The popular tree-borne oilseed (TBO) species include *Azadirachta indica* (neem), *Calophyllum inophyllum* (Undi), *Garcinia indica* (Kokum), *Jatropha curcas* (Ratanjot), *Madhuca longifolia* and *M. indica* (mahua), *Pongamia pinnata* (Karanj), and *Simarouba glauca* (*Simarouba*). Tree-borne seeds rich in non-edible oils, mostly produced by perennial species, are referred to as tree-borne oilseed species. *Simarouba*, which is not a familiar species in India, was studied to standardize various aspects of its cultivation [2]. *Simarouba glauca*, an exotic species belonging to family Simaroubaceae, is indigenous to North

## **Chapter 4**
