**4. Probiotics and functional foods**

An offshoot of the initial research on bacteriocins of LAB isolated from indigenous fermented foods is the emergence of probiotic research towards developing functional foods for biomedical applications. Probiotics refer to microorganisms that, when administered in adequate amounts, confers health benefits to the host. Although there are many microorganisms that can be considered as probiotics, LAB are the most common types because they produce antimicrobial compounds that inhibit other harmful microorganisms, they are able to tolerate acids and bile present in the digestive system, and they are able to adhere and establish themselves in the gut surfaces.

Many benefits have been ascribed to probiotics. For example, *Lactobacillus casei* (Shirota strain in Yakult®) have been shown effective in preventing diarrhea due to enterotoxigenic *Escherichia coli* (ETEC) and choleragenic vibrios (*V. cholerae* biotype E1 Tor and classical *V. cholerae*) using rats (Jacalne et al., 1990). This may be accounted for by its ability to kill the pathogens and inhibit further growth (Consignado et al., 1994). Because the probiotic used in the two studies mentioned is a commercial strain, current research on probiotics progressed to the search for indigenous LAB for use in the development of locally-produced functional food and investigation of their utility for biomedical applications. Metagenomic approaches to investigating LAB present in fermented foods have shown the diversity of potentially beneficial species present other than those that are readily detected by conventional culturebased methods. The development of functional food products shows potential in disease management. Research using metagenomic analysis in searching for microbial markers for use in functional foods to address certain lifestyle diseases as well as malnutrition is on the way.

### **4.1. Metagenomic and diversity studies**

Traditional culture-based methods have been used for isolating LAB from fermented foods. These studies form the basis for the starter cultures used in food fermentation technologies employed for commercial production. Sanchez (2008) gives detailed information on the different technologies and cultures used for the production of some traditional as well as developed technologies that have arisen from the culture-based studies conducted in earlier years.

In recent years culture-based approaches in LAB isolation have become more targeted for detection of bacteriocin-producers and those that have potential as probiotics. In one initiative, LAB isolates from fermented foods were screened for bacteriocin production and a PCR-based assay was used to detect specific bacteriocin-encoding genes. Acid and bile tolerance were also determined. Among all the isolates tested, *Lactobacillus fermentum* 4B1 and *Lactobacillus pentosus* 3G3 (later identified as *Pediococcus acidilactici*) have been identified as most promising for the development of new probiotic food products, hence they were chosen for subsequent biomedical application assays (Lim and Gervacio, 2007). In another study, LAB from traditionally fermented wine and vinegar from Visayas and Mindanao were isolated, identified, and tested for inhibitory activity against *Enterococcus faecium*, *Listeria innocua*, and *Staphylococcus aureus*. Five *Lactobacillus paracasei* and one *Lactobacillus brevis* showed antimicrobial properties against the tester strains (Licaros and Bautista, 2009).

With the advent of molecular techniques, the existence of non-culturable microorganisms has been acknowledged especially since the occurrence of culture-bias is already wellaccepted. Culture-independent approaches, therefore, have been gaining popularity in microbial diversity studies and this includes researches on microorganisms found in fermented foods. The microbial populations in selected Philippine fermented foods were assessed through Polymerase Chain Reaction followed by Denaturing Gradient Gel Electrophoresis (PCR-DGGE) in two recent studies (Dalmacio et al., 2011; Larcia, 2010). Food samples tested include *burong mustasa* (fermented mustard), *alamang* (fermented shrimp paste), *burong isda* (fermented rice-fish mixture), *balao-balao/burong hipon* (fermented riceshrimp mixture), *tuba* (sugar cane wine), and *sinamak* (spiced vinegar). Analysis of the 16S rRNA gene sequences revealed the presence of several LAB that have not been reported in these food products before. *Weissella cibaria*, *Lactobacillus plantarum*, *Lactobacillus pontis*, *Lactobacillus panis*, and *Lactobacilus fermentum* were detected in *burong mustasa* (Larcia, 2010). *L. panis* and *L. fermentum* were present in *alamang*; *L. pontis* and *L. plantarum* in *burong isda*; *L. panis*, *L. pontis*, and *L. fermentum* in burong hipon; and *W. cibaria*, *L. pontis*, *L. panis*, *L. fermentum* and *L. plantarum* in *burong mustasa* (Dalmacio et al., 2011).

The results of the two studies using molecular approaches in defining diversity of LAB in Philippine fermented foods show that culture-independent approaches are efficient tools for the analysis of microbial populations in fermented foods. Majority of the identified bacteria (LAB and other bacterial groups) have not been reported in culture-dependent studies. As such, the isolated bacterial 16S rRNA genes were cloned to have an initial partial 16S rRNA gene library for Philippine fermented foods (Dalmacio et al., 2011).

### **4.2. Biomedical applications**

### 1. Anti-Obesity

580 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

**4. Probiotics and functional foods** 

adhere and establish themselves in the gut surfaces.

**4.1. Metagenomic and diversity studies** 

inocula in fermented food products (Gervasio and Lim, 2007).

and plantaricins show promise, their applications are limited at present because it is a wellknown fact that other bacteriocins aside from nisin are not yet approved for food use. For pediocins and plantaricins, the most practical use for now would be dermatological and animal health care use. But since the bacteriocin-producing LAB are of GRAS status, those with probiotic properties such as tolerance to acidic pH (2.0 -3.0) and bile (0.3%) and adhesion properties to intestinal mucosa would be an advantage when used as adjunct

Perhaps another importance of bacteriocin-producing LAB is their effectiveness in biomedical applications. In one study, for example, partially-purified pediocin K2a2-3, through pHmediated bacteriocin extraction method, was found cytotoxic against human colon adenocarcinoma (HT29) and human cervical carcinoma (HeLa) cells *in vitro* as determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Villarante et al., 2011). Other potential biomedical applications will be discussed in the succeeding section.

An offshoot of the initial research on bacteriocins of LAB isolated from indigenous fermented foods is the emergence of probiotic research towards developing functional foods for biomedical applications. Probiotics refer to microorganisms that, when administered in adequate amounts, confers health benefits to the host. Although there are many microorganisms that can be considered as probiotics, LAB are the most common types because they produce antimicrobial compounds that inhibit other harmful microorganisms, they are able to tolerate acids and bile present in the digestive system, and they are able to

Many benefits have been ascribed to probiotics. For example, *Lactobacillus casei* (Shirota strain in Yakult®) have been shown effective in preventing diarrhea due to enterotoxigenic *Escherichia coli* (ETEC) and choleragenic vibrios (*V. cholerae* biotype E1 Tor and classical *V. cholerae*) using rats (Jacalne et al., 1990). This may be accounted for by its ability to kill the pathogens and inhibit further growth (Consignado et al., 1994). Because the probiotic used in the two studies mentioned is a commercial strain, current research on probiotics progressed to the search for indigenous LAB for use in the development of locally-produced functional food and investigation of their utility for biomedical applications. Metagenomic approaches to investigating LAB present in fermented foods have shown the diversity of potentially beneficial species present other than those that are readily detected by conventional culturebased methods. The development of functional food products shows potential in disease management. Research using metagenomic analysis in searching for microbial markers for use in functional foods to address certain lifestyle diseases as well as malnutrition is on the way.

Traditional culture-based methods have been used for isolating LAB from fermented foods. These studies form the basis for the starter cultures used in food fermentation technologies employed for commercial production. Sanchez (2008) gives detailed information on the different

Obesity is defined as an abnormal or excessive fat accumulation that presents risks to health. Probiotics can help in fighting obesity by reducing lipid absorption through its action on bile

acid metabolism, and by assimilation of cholesterol thus eliminating it from the host's system. Several studies were conducted to examine anti-obesity properties of different probiotic strains.

Lactic Acid Bacteria in Philippine Traditional Fermented Foods 583

The study also showed that isolate 4B1 exhibited better immune-enhancing effect than the probiotic species found in a commercial probiotic drink. T-cell proliferation, however, was not observed in any of the treatments in this study and was attributed to the delayed stimulation in cells responding to a first-time exposure to the different probiotic strain preparations used.

A study by Ngo et al. (2008) showed that oral administration of kefir, a common fermented food consumed by the elderly, significantly decreased blood glucose levels and body weight of diabetic obese male Sprague Dawley rats. The results of the study showed lower blood glucose levels (from 198.5 to 105.6 mg/dL) and clinically lower body weights (from 342.9 to 311.5 g) of the treated diabetic-obese rats than the untreated diabetic-obese control group.

The effect of *P. acidilactici* 3G3 administration on hypercholesterolemic Swiss Albino mice was determined (Parungao et al., 2009). This strain was able to assimilate cholesterol in the *in vitro* plate assay and decrease HDL, LDL, and total cholesterol in the *in vivo* assay using mice. Strain 3G3 was also shown to adhere well to the duodenum and middle colon. Results

The development of functional foods containing known probiotic strains stems from earlier researches on bacteriocins and isolation of potential probiotics from traditional fermented foods. The beneficial effects of probiotic-supplemented chocolate bars (Arroyo et al., 2010; Arroyo and Fabiculana, 2011), fermented mustard leaves (Calapardo et al., 2006), and coffee wine (Parungao, 2007) have been investigated. Initial studies on mango-milk and carrot juice drinks supplemented with probiotic strains have also been conducted (Bugarin et al., 2010; Elegado et al., 2005). These potential functional foods contain probiotic strains, previously isolated from traditional fermented foods such as *P. acidilactici* AA5a (Elegado et al., 2003), *L. plantarum* BS25 (Banaay et al., 2004), and *P. acidilactici* 3G3 (Lim and Gervacio, 2007). Research on functional foods is still in its infancy but this food category shows promise in disease management as well as in contributing to food security in the country. Commercial interest in probiotic food products is increasing due to the growing understanding of its health benefits. This growing industry can derive benefits from the

Aside from the research works presented earlier in this paper as well as on-going follow-up studies related to them, future goals may include research on a variety of other possible biomedical applications of LAB with potential probiotic properties. The effect of probiotics on *Helicobacter pylori* infections (that may cause peptic ulcers) may be determined. Their ability to modulate inflammatory and hypersensitivity responses as well as their effect on

suggest the potential of *P. acidilactici* 3G3 in preventing hypercholesterolemia.

3. Reduction of blood glucose levels

4. Prevention of hypercholesterolemia

**4.3. Development of functional foods** 

researches conducted on this emerging food category.

**5. Future perspectives** 

In one study, oral administration of *Lactobacillus paracasei* K3-4C, isolated from a locally fermented food had significant effect on lowering blood glucose levels (by 46%) and body weight (by 13%) in female BALB/c mice induced to be diabetic and obese through a 28-day high-fat diet (Parungao et al., 2006). In another study, orally administered *L. fermentum* 4B1 reduced adipose cell size, and decreased adipose tissue weight and overall body weight of mice fed with a high-fat diet for 49 days (Bautista et al., 2008). Likewise, oral administration of *P. acidilactici* 3G3 reduced body weight in diet-induced obese female Swiss mice (Parungao et al., 2009). In the last two studies described, the effects of the probiotics were determined to be comparable with the effects of the commercial anti-obesity drug Orlistat based on the parameters measured.

Recently, it has been postulated that the development of obesity may be caused by a shift in the composition of the gut microbiota towards the Firmicutes population (Ley et al., 2005). Firmicutes characterize obese versus lean/non-obese individuals together with a drop or no change in Bacteroidetes (Delzenne and Cani, 2010). Interestingly, Ley et al. (2006) found that a low fat diet had an effect to reverse the shift of Firmicutes/Bacteroidetes proportion. Because of this, dietary manipulation has been seen as a potential means of changing bacterial populations in the colonic microbiota and perhaps treating or at least preventing diseases like obesity. Although the root cause of obesity is excessive caloric intake coupled with a sedentary lifestyle (Blaut and Bischoff, 2010), Ley et al. (2005) proposed in their findings that alteration in the populations of mice gut microflora may have caused or may have been an effect of obesity. Because of this, current researches aim in using probiotics in the treatment of diseases such as obesity.

In two related studies (Arroyo and Fabiculana, 2011; Parungao et al., 2012), the effect of a functional food containing *P. acidilactici* 3G3 on microbial community changes in the gut of obese and non-obese mice was determined through PCR-DGGE. Results of these two preliminary studies showed that obese and non-obese mice had different baseline colonic microbiota. There were also indications that treatment with probiotics shifts the microbiota of obese mice towards the normal non-obese type. As these are preliminary studies, more research is warranted to elucidate the nature of the changes in gut microbiota and how it is related to obesity and the anti-obesity effects of probiotics.

2. Immuno-enhancement

A preliminary *in vitro* study to examine the immune-enhancing properties of viable and heat-killed preparations of two LAB previously isolated from traditional fermented foods (*L. fermentum* 4B1 and *P. acidilactici* 3G3) on murine peritoneal macrophage cells and spleenic Tcells showed that isolate 4B1 was able to induce NO production in murine macrophages but, like 3G3, was unable to stimulate murine T-cell proliferation (Tan et al., 2008). Furthermore, this study showed that preparations of *L. fermentum* 4B1 have the ability to induce NO production in murine macrophage cells and its effects were more potent when it was alive. The study also showed that isolate 4B1 exhibited better immune-enhancing effect than the probiotic species found in a commercial probiotic drink. T-cell proliferation, however, was not observed in any of the treatments in this study and was attributed to the delayed stimulation in cells responding to a first-time exposure to the different probiotic strain preparations used.

3. Reduction of blood glucose levels

582 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

probiotic strains.

based on the parameters measured.

the treatment of diseases such as obesity.

2. Immuno-enhancement

related to obesity and the anti-obesity effects of probiotics.

acid metabolism, and by assimilation of cholesterol thus eliminating it from the host's system. Several studies were conducted to examine anti-obesity properties of different

In one study, oral administration of *Lactobacillus paracasei* K3-4C, isolated from a locally fermented food had significant effect on lowering blood glucose levels (by 46%) and body weight (by 13%) in female BALB/c mice induced to be diabetic and obese through a 28-day high-fat diet (Parungao et al., 2006). In another study, orally administered *L. fermentum* 4B1 reduced adipose cell size, and decreased adipose tissue weight and overall body weight of mice fed with a high-fat diet for 49 days (Bautista et al., 2008). Likewise, oral administration of *P. acidilactici* 3G3 reduced body weight in diet-induced obese female Swiss mice (Parungao et al., 2009). In the last two studies described, the effects of the probiotics were determined to be comparable with the effects of the commercial anti-obesity drug Orlistat

Recently, it has been postulated that the development of obesity may be caused by a shift in the composition of the gut microbiota towards the Firmicutes population (Ley et al., 2005). Firmicutes characterize obese versus lean/non-obese individuals together with a drop or no change in Bacteroidetes (Delzenne and Cani, 2010). Interestingly, Ley et al. (2006) found that a low fat diet had an effect to reverse the shift of Firmicutes/Bacteroidetes proportion. Because of this, dietary manipulation has been seen as a potential means of changing bacterial populations in the colonic microbiota and perhaps treating or at least preventing diseases like obesity. Although the root cause of obesity is excessive caloric intake coupled with a sedentary lifestyle (Blaut and Bischoff, 2010), Ley et al. (2005) proposed in their findings that alteration in the populations of mice gut microflora may have caused or may have been an effect of obesity. Because of this, current researches aim in using probiotics in

In two related studies (Arroyo and Fabiculana, 2011; Parungao et al., 2012), the effect of a functional food containing *P. acidilactici* 3G3 on microbial community changes in the gut of obese and non-obese mice was determined through PCR-DGGE. Results of these two preliminary studies showed that obese and non-obese mice had different baseline colonic microbiota. There were also indications that treatment with probiotics shifts the microbiota of obese mice towards the normal non-obese type. As these are preliminary studies, more research is warranted to elucidate the nature of the changes in gut microbiota and how it is

A preliminary *in vitro* study to examine the immune-enhancing properties of viable and heat-killed preparations of two LAB previously isolated from traditional fermented foods (*L. fermentum* 4B1 and *P. acidilactici* 3G3) on murine peritoneal macrophage cells and spleenic Tcells showed that isolate 4B1 was able to induce NO production in murine macrophages but, like 3G3, was unable to stimulate murine T-cell proliferation (Tan et al., 2008). Furthermore, this study showed that preparations of *L. fermentum* 4B1 have the ability to induce NO production in murine macrophage cells and its effects were more potent when it was alive.

A study by Ngo et al. (2008) showed that oral administration of kefir, a common fermented food consumed by the elderly, significantly decreased blood glucose levels and body weight of diabetic obese male Sprague Dawley rats. The results of the study showed lower blood glucose levels (from 198.5 to 105.6 mg/dL) and clinically lower body weights (from 342.9 to 311.5 g) of the treated diabetic-obese rats than the untreated diabetic-obese control group.

4. Prevention of hypercholesterolemia

The effect of *P. acidilactici* 3G3 administration on hypercholesterolemic Swiss Albino mice was determined (Parungao et al., 2009). This strain was able to assimilate cholesterol in the *in vitro* plate assay and decrease HDL, LDL, and total cholesterol in the *in vivo* assay using mice. Strain 3G3 was also shown to adhere well to the duodenum and middle colon. Results suggest the potential of *P. acidilactici* 3G3 in preventing hypercholesterolemia.

### **4.3. Development of functional foods**

The development of functional foods containing known probiotic strains stems from earlier researches on bacteriocins and isolation of potential probiotics from traditional fermented foods. The beneficial effects of probiotic-supplemented chocolate bars (Arroyo et al., 2010; Arroyo and Fabiculana, 2011), fermented mustard leaves (Calapardo et al., 2006), and coffee wine (Parungao, 2007) have been investigated. Initial studies on mango-milk and carrot juice drinks supplemented with probiotic strains have also been conducted (Bugarin et al., 2010; Elegado et al., 2005). These potential functional foods contain probiotic strains, previously isolated from traditional fermented foods such as *P. acidilactici* AA5a (Elegado et al., 2003), *L. plantarum* BS25 (Banaay et al., 2004), and *P. acidilactici* 3G3 (Lim and Gervacio, 2007). Research on functional foods is still in its infancy but this food category shows promise in disease management as well as in contributing to food security in the country. Commercial interest in probiotic food products is increasing due to the growing understanding of its health benefits. This growing industry can derive benefits from the researches conducted on this emerging food category.

## **5. Future perspectives**

Aside from the research works presented earlier in this paper as well as on-going follow-up studies related to them, future goals may include research on a variety of other possible biomedical applications of LAB with potential probiotic properties. The effect of probiotics on *Helicobacter pylori* infections (that may cause peptic ulcers) may be determined. Their ability to modulate inflammatory and hypersensitivity responses as well as their effect on

irritable bowel syndrome and colitis may be investigated. Further research on possible anticancer properties of probiotics is warranted as follow-up studies on the work done by Villarante et al. (2011). These studies are very important as these have the potential to address some of the more serious health concerns of our society.

Lactic Acid Bacteria in Philippine Traditional Fermented Foods 585

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Bautista RLS, Ecarma NCA, Balolong ECJr, Hallare AV, Parungao MM (2008) The Effects of Orally-Administered *Lactobacillus* sp. 4B1 on the Adipose Tissues of Diet-Induced Obese Mice (*Mus musculus* L.). International Symposium on Probiotic from Asian Traditional Fermented Foods for Healthy Gut Function. August 19-20, 2008. Sari-Pan Pacific Hotel,

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Bugarin MA, Sison AAD, Elegado FB, Calapardo M, Parungao MM (2010) Survival of *Lactobacillus plantarum* BS25 on Varying Ratio of Mango-Milk Substrates and Storage Temperature. 39th Annual Convention and Scientific Meeting, PSM, Inc. April 29-30,

Calapardo MR, Bueno MOV, Guillermo MKB, Saguibo JD, Parungao MM, Elegado FB (2006) Bile and Acid Tolerance of Bacteriocinogenic *Lactobacillus plantarum* and its Use as Adjunct Inoculum in Pickled Mustard Leaves. Proceedings of the 5th Asia-Pacific Biotechnology Congress and 35th Annual Convention of the PhilippineSociety for Microbiology Inc. (PSM). "Microbiology and Biotechnology: Roadmaps and Milestones for Enhancing Sustainable Productivity in the Asia-Pacific Region." Bohol Tropics

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Dalmacio LMM, Angeles AKJ, Larcia LLH, Parungao-Balolong MM, Estacio RC (2011) Assessment of microbial diversity in Philippine fermented food products through polymerase chain reaction- denaturing gradient gel electrophoresis (PCR-DGGE).

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23.

Much is still to be learned about the existing probiotic strains. The molecular biology and genomics of these isolates may be pursued in order to further elucidate their properties and mechanisms of action.

Determination of factors affecting probiotic viability in foods is also important as these will determine if their survival in the food, and therefore their delivery into the host, is maintained. This will constitute a quality control for functional foods.

The potential physiological effects of multiple prebiotic strains, as opposed to a single strain, are also interesting areas of research. The delivery of multiple probiotic strains may help ensure its effectiveness in an environment that contains high diversity of resident microflora. The potential benefits of synbiotics, (combination of probiotic and prebiotic) which have synergistic interaction, may also be investigated. A good combination will greatly enhance the health benefits to humans.
