**8. Effect of the addition of LAB on bread quality**

### **8.1. Shelf life**

Traditionally, chemical preservatives and fungicides are used to inhibit fungal growth but concerns about environmental pollution and consumer health, along with problems of microbial resistance, favour the demand for alternative methods in controlling the growth of fungi (Druvefors et al., 2005). The shelf life of bread has been reported to be extended when certain LAB strains were added to bread formulations (Muhialdin et al., 2011a; Ogunbanwo et al., 2008; Rizzello et al., 2010; Ryan et al., 2011) (Table 2). The use of safe microbes in bread to extend the shelf life of the product is a great research area. Since LAB isolates are safe for use in foods, they are a significant alternative to chemical preservatives. Several researchers in the area of the bakery industry have successfully added LAB to dough and these strains grew well, producing the desired antifungal compounds in the dough.

Various fungi isolated from bakeries were inhibited by *L. plantarum* (LB1) and *L. rossiae* (LB5) isolated from raw wheat germ. Organic acids and peptides synthesized during fermentation were responsible for the antifungal activity; formic acid had the highest inhibition activity (Rizzello et al., 2011). However, the inhibitory compounds characterized were different, depending upon the LAB strains and flour type used. Dal Bello et al., (2007) characterized lactic acid, phenyllactic acid (PLA), cyclic dipeptides cyclo (L-Leu–L-Pro) and cyclo (L-Phe–L-Pro) produced by *L. plantarum* FST 1.7 and found them to inhibit the growth of *Fusarium* spp. in wheat bread. Ryan et al., (2008) reduced the use of calcium propionate from 3000 ppm to 1000 ppm when using sourdough fermented with *L. plantarum* FST 1.7 (LP 1.7) and *L. plantarum* FST 1.9 (LP 1.9), in which the growth of *A. niger*, *F. culmorum* and *P. expansum* was delayed for over six days while the growth of *P. roqueforti* appeared after three days of incubation at 30 °C. *L. plantarum* VTT E-78076. *Pediococcus pentosaceus* VTT E-90390 was reported to inhibit the growth of rope-forming *Bacillus subtilis* and *Bacillus licheniformis* in laboratory conditions and in the bread when the selected strains were inoculated to sourdough and subsequently 20-30 g of the inoculated sourdough was added to 100 g of wheat dough (Katina et al., 2002). Lavermicocca et al. (2000) found that *L.*


Lactic Acid Bacteria in Biopreservation and the Enhancement of the Functional Quality of Bread 163

(Ryan et al., 2011; Muhialdin et al., 2011a). The growth of fungi is responsible for the formation of off-flavours and the production of mycotoxins; adding LAB to dough can prevent the growth of fungi and enhance the flavour of bread. The produced compound plays an important role for any technological application to enhance the flavour, such as diacetyl which gives a buttery flavour. Sourness in white bread indicates spoilage in contrast to the sourness of sourdough bread; for this reason, the search for new LAB for application in white bread becomes essential. Finding a new LAB strain that produces less acid and does not drop the pH below 4 will mark a good strategy for resolving such an issue. The addition of *L. paracasi* D5 and *L. fermentum* Te007 in the production of white bread resulted in an improved aroma and a pleasant caramel-like flavour in the baked bread itself

The quality of bread produced with LAB as a starter culture was reported to improve the texture and the quality of bread by increasing the air cells (Coda et al., 2008; Katina et al., 2002; Lavermicocca et al., 2000). Baker's yeast - also referred to as 'baking yeast' (*Saccharomyces cerevisiae*) - has the ability to ferment different carbohydrates and produce CO2; the most important factor involving baking yeast in bread manufacturing is to leaven the dough during the bread's preparation. The presence of antimicrobials in the dough is used to inhibit the growth of spoilage microorganisms that can affect the growth of the baker's yeast and delay the fermentation of dough, thereby resulting in economic losses to the bakery industry (Pattison & von Holy, 2001). Baking yeast is a excellent producer of the necessary flavour and aroma compounds from the products of secondary metabolism

Pattison & von Holy (2001) found that the presence of propionic salts reduced the baking yeast activity by up to 34.4% in an *in vitro* study carried out using several natural antimicrobials with positive control calcium propionate. In comparison, lactic acid and acetic acid displayed slight effects on the activity reduction of the yeast compared with the positive control. Baking yeast and lactic acid bacteria commonly have live symbiotically in the natural ecosystem of fermenting food and beverages (Kenns et al., 1991). The volume of the dough was increased by adding sourdough containing *L. amylovorus* DSM 19280 when compared with chemical acidification (Ryan et al., 2011). Rizzello et al. (2010) reported the improvement of bread texture properties and the delaying of the staling of the bread because of the anti-staling effect produced by LAB and the synthesis of antifungal compounds. As mentioned previously, *S. cerevisiae* is responsible of leaving the dough and

The key role in achieving the optimum growth and activity of the bakery yeast is played by selecting a LAB that does not exhibit inhibition activity against the bakery yeast. Before choosing the LAB to be added to the dough as a co-starter, a simple experiment can be conducted in order to examine the tolerance of the bread yeast to the selected LAB strain. In a test tube mix of 10 ml water, 5 g of white flour, the LAB strain and baking yeast, we

(Muhialdin et al., 2011a).

(Evans 1990).

**8.3. Quality and acceptability** 

giving the most desirable texture to the bread.

**Table 2.** Delay of the appearance of fungal growth on bread with added lactic acid bacteria cells

*plantarum* 21B inhibited the bread spoilage fungi *Aspergillus, Fusarium, Penicillium* and *Eurotium*; the active compounds were phenyllactic and 4-hydroxyphenyllactic acids. The growth of *Aspergillus niger* appeared after two days in the control sample while *L. plantarum* 21B delayed the growth of the stated fungi for seven days at 20 °C.

### **8.2. Flavour**

Flavour is one of the most valued sensory attributes in bread - volatile and non-volatile compounds produced during the fermentation of dough contribute to bread's flavour. Reports show that the fermentation of dough with LAB can enhance the aroma and flavour (Ryan et al., 2011; Muhialdin et al., 2011a). The growth of fungi is responsible for the formation of off-flavours and the production of mycotoxins; adding LAB to dough can prevent the growth of fungi and enhance the flavour of bread. The produced compound plays an important role for any technological application to enhance the flavour, such as diacetyl which gives a buttery flavour. Sourness in white bread indicates spoilage in contrast to the sourness of sourdough bread; for this reason, the search for new LAB for application in white bread becomes essential. Finding a new LAB strain that produces less acid and does not drop the pH below 4 will mark a good strategy for resolving such an issue. The addition of *L. paracasi* D5 and *L. fermentum* Te007 in the production of white bread resulted in an improved aroma and a pleasant caramel-like flavour in the baked bread itself (Muhialdin et al., 2011a).

### **8.3. Quality and acceptability**

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

**days** 

*L. plantarum* 12 *Rhizopus oryzae A.* 

*L. brevis* AM7 21 *P. roqueforti*

*L. plantarum* 10 *A. niger, F.* 

**Target fungi Storage** 

*L. plantarum* 21B 7 Broad spectrum 20 Lavermicocca et

*niger A. flavus Penicillium sp. F. oxysporum* 

DPPMAF1

*expansum*

8 *Aspergillus,* 

28 *P. roqueforti* 

*culmorum,* and *P.* 

*Fusarium,* and *Penicillium*

DPPMAF1

14 *F. culmorum* FST 4.05, *A. niger*  FST4.21, *P. expansum* FST 4.22, *P. roqueforti* FST 4.11

9-12 *A. niger* and *A. oryzae*

21B delayed the growth of the stated fungi for seven days at 20 °C.

**Table 2.** Delay of the appearance of fungal growth on bread with added lactic acid bacteria cells

*plantarum* 21B inhibited the bread spoilage fungi *Aspergillus, Fusarium, Penicillium* and *Eurotium*; the active compounds were phenyllactic and 4-hydroxyphenyllactic acids. The growth of *Aspergillus niger* appeared after two days in the control sample while *L. plantarum*

Flavour is one of the most valued sensory attributes in bread - volatile and non-volatile compounds produced during the fermentation of dough contribute to bread's flavour. Reports show that the fermentation of dough with LAB can enhance the aroma and flavour

**temperature °C** 

**Reference** 

al., (2000)

al., (2008)

(2008)

(2008)

(2009)

(2011)

(2011)

(2011)

27 Ogunbanwo et

25 Coda et al.,

25 Ryan et al.,

30 Gerez et al.,

25 Coda et al.,

25 Ryan et al.,

30 Muhialdin et al.,

**Strains No. of** 

*L. plantarum* CRL 778, *L. reuteri* CRL 1100, and *L. brevis*  CRL 772 and CRL

*L. plantarum* 1A7

*L. amylovorus* DSM

*L. fermentum* Te007, *P. pentosaceus*  Te010, *L. pentosus* G004, and *L. paracasi* D5

**8.2. Flavour** 

796

(S1A7)

19280

The quality of bread produced with LAB as a starter culture was reported to improve the texture and the quality of bread by increasing the air cells (Coda et al., 2008; Katina et al., 2002; Lavermicocca et al., 2000). Baker's yeast - also referred to as 'baking yeast' (*Saccharomyces cerevisiae*) - has the ability to ferment different carbohydrates and produce CO2; the most important factor involving baking yeast in bread manufacturing is to leaven the dough during the bread's preparation. The presence of antimicrobials in the dough is used to inhibit the growth of spoilage microorganisms that can affect the growth of the baker's yeast and delay the fermentation of dough, thereby resulting in economic losses to the bakery industry (Pattison & von Holy, 2001). Baking yeast is a excellent producer of the necessary flavour and aroma compounds from the products of secondary metabolism (Evans 1990).

Pattison & von Holy (2001) found that the presence of propionic salts reduced the baking yeast activity by up to 34.4% in an *in vitro* study carried out using several natural antimicrobials with positive control calcium propionate. In comparison, lactic acid and acetic acid displayed slight effects on the activity reduction of the yeast compared with the positive control. Baking yeast and lactic acid bacteria commonly have live symbiotically in the natural ecosystem of fermenting food and beverages (Kenns et al., 1991). The volume of the dough was increased by adding sourdough containing *L. amylovorus* DSM 19280 when compared with chemical acidification (Ryan et al., 2011). Rizzello et al. (2010) reported the improvement of bread texture properties and the delaying of the staling of the bread because of the anti-staling effect produced by LAB and the synthesis of antifungal compounds. As mentioned previously, *S. cerevisiae* is responsible of leaving the dough and giving the most desirable texture to the bread.

The key role in achieving the optimum growth and activity of the bakery yeast is played by selecting a LAB that does not exhibit inhibition activity against the bakery yeast. Before choosing the LAB to be added to the dough as a co-starter, a simple experiment can be conducted in order to examine the tolerance of the bread yeast to the selected LAB strain. In a test tube mix of 10 ml water, 5 g of white flour, the LAB strain and baking yeast, we

incubate and observe the production of gas at the top of the tube, which is a good indicator of the yeast activity. Ogunbanwo et al. (2008) isolated LAB from retted cassava and studied the effects of lactic acid bacteria as a starter co-culture in combination with *S. cerevisiae* in order to produce cassava-wheat bread. The improvement in the nutritional contents, physical properties and the extension of the shelf life were reported. Bread produced using *L. acidophilus* and *L. brevis* had the highest acceptability on average in relation to the bread produced with other strains of LAB. The use of LAB in bread in terms of improving the quality of wheat bread, bread volume and crumb structure has been reported (Clarke et al., 2002; Zannini et al., 2009).

Lactic Acid Bacteria in Biopreservation and the Enhancement of the Functional Quality of Bread 165

Rezzillo et al., 2011). The use of lactic acid bacteria as an antifungal agent or as a starter culture for bakery and processed foods can solve two global issues; firstly, it can extend the shelf life of the food products, which will reduce their cost and the need for low temperatures, secondly, it will satisfy the high demand of modern consumers for high quality food that is free of chemicals. Above all, the product must be safe with an extended

The growth of LAB and the production of antifungal compounds are largely affected by the food matrix itself (Helander, 1997). Most of the studies regarding the antifungal activity of LAB were done using the universal MRS agar. As demonstrated earlier, there are few studies that evaluate the ability of LAB isolates to produce the active compounds in non-defined media as well as few *in situ* studies. The challenge for the food industry is the need for the high production of biomass and the bioactive compounds using an inexpensive fermentation growth medium. A defined medium is all well and necessary for laboratory screening purposes but it is not suitable for heavy industrial plant. The question here is whether the selected LAB can produce the biomass and maintain the antifungal activity. In our laboratory, *L. fermentum* Te007, *Pediococcus pentosaceus* Te010, *L. pentosus* G004 and *L. paracasi* D5 were used to ferment white bread dough and they maintained the antifungal activity, as detected using MRS agar, indicating that these isolates produced the antifungal compounds in the bread dough (Muhialdin et al., 2011a). *Pediococcus pentosaceus* Te010 was further investigated for its ability to grow in formulated media from plant extracts supplemented with the basic growth needs of LAB, such as vitamins, carbohydrates, nitrogen sources and salts. The results indicated that the selected isolate was able to grow in the formulated media and maintain the production of the antifungal activity but, unfortunately, the compounds have not yet been characterized

The growth conditions of any microbe are the key to success during the fermentation process. As for LAB, the generally optimum temperature for growth is 37 °C for 48 h in anaerobic conditions. This is not exactly what can be applied for the production of antagonistic fungal inhibitor compounds. Some of the LAB are psychrophilic and prefer low temperatures for their growth while others are thermophilic and prefer high temperatures for their growth. This should be considered as a significant factor because the optimum growth temperature has a significant impact on the production of antifungal compounds. As well as temperature, the incubation time has a significant effect on the production of antifungal compounds with respect to the availability of nutrients in the growth medium

**10. Production of LAB cells and inhibitory compounds** 

shelf life and good sensory properties.

**10.1. Growth medium** 

(unpublished data).

**10.2. Growth conditions** 

and the production of primary or secondary metabolites.

### **8.4. Enhancement of a specific nutrient**

LAB fermentation in dough has been approved for enhancing the nutritional value and digestibility of bread. Vitamin B, organic acids and the free amino acids produced through the fermentation of LAB can enhance the nutrients' presence in bread. The human body cannot synthesize B-group vitamins and this is why the body needs an external source of the vitamins. Certain LAB has been proven to synthesize B-group vitamins during the fermentation of foods; at the same time, LAB are considered to be the perfect vehicle for delivering the vitamins to the human body.

There are reports about the production of B-group vitamins by LAB isolates. Keuth and Bisping (1993) described the production of Riboflavin (Vitamin B 2) by *Streptococcus* and *Enterococcus* isolated from tempeh (Indonesian fermented food). Folates were observed to be produced by *L. plantarum* in low amounts (Sybesma et al., 2003). Vitamin B 12 (Cobalamin) was also produced by *L. reuteri* as well as the other groups of vitamin B (Santos et al., 2008). LAB enzymatic activity by proteases that take place during dough fermentation will release small peptides and free amino acids, which are considered to be important nutrients that should be present in bread in high quantities (Thiele et al., 2002). Essential amino acids, including lysine, threonine, phenylalanine and valine were reported to be produced by LAB (Gerez et al., 2006). The enzymes produced by LAB including amylases, proteases, phytases and lipases improve the food quality through the hydrolysis of polysaccharides, proteins, phytates and lipids. Anti-nutrients such as phytic acid and tannins can be reduced by LAB fermentation in food, leading to increased sensory properties of the bread (Chelule et al., 2010). The growth of fungi in food materials can cause the synthesis of allergenic spores and hazardous mycotoxins, which will lead to the reduction of the nutritional value of food stuffs. Adding 4% of fermented sourdough to the white wheat flour improved the texture and physical sensation of the bread. Furthermore, it enhanced the free amino acids, protein digestibility, phytase and antioxidant activities (Rizzello et al., 2010).

## **9. Starter cultures for the bread industry**

Lactic acid bacteria were reported as being used as a starter culture or co-culture in the bread industry with success in terms of survivability in dough (Lavermicocca et al., 2000; Rezzillo et al., 2011). The use of lactic acid bacteria as an antifungal agent or as a starter culture for bakery and processed foods can solve two global issues; firstly, it can extend the shelf life of the food products, which will reduce their cost and the need for low temperatures, secondly, it will satisfy the high demand of modern consumers for high quality food that is free of chemicals. Above all, the product must be safe with an extended shelf life and good sensory properties.
