Open access

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

Written By

Belal J. Muhialdin, Zaiton Hassan and Nazamid Saari

Submitted: 15 April 2012 Published: 30 January 2013

DOI: 10.5772/51026

From the Edited Volume

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

Edited by Marcelino Kongo

Chapter metrics overview

5,454 Chapter Downloads

View Full Metrics

1. Introduction

LAB have a long history in preserving foods from spoilage microorganisms - they are commonly used in food fermentation, may produce several metabolites with beneficial health effects and, thus, are generally recognized as safe (GRAS). The increasing resistance of food spoilage microorganisms to current preservatives, the consumer’s high demand for safe, minimally processed foods and the hazards associated with the use of high doses of chemical preservatives has led to the need for finding safer alternatives in food preservation. The application of LAB with the simultaneous control of factors that affect fungal growth can help to minimize food spoilage. The selection and addition of novel isolates of LAB may be the key to reducing the use of chemicals, enhancing nutrients and extend the shelf life of bakery products. In this chapter, the focus will be on the use of LAB as biopreservative agents to extend the shelf life of bakery products and the inhibition of the common spoilage fungi of bread.

Advertisement

2. Sources of LAB

LAB are found in many habitats and occur naturally in a variety of food products, such as dairy, vegetables and meat products (Carr et al., 2002), all of which are rich in the nutrients required for the fastidious metabolism of LAB (Björkroth & Holzapfel, 2003; Hammes & Hertel, 2003). Some LAB are associated with the mouth flora, intestine and vagina of mammals (Whittenbury, 1964), while others are present in fermented seafood, such as Lactobacillus plantarum (IFRPD P15) and L. reuteri (IFRPD P17), which are reported to be associated with plaa-som fermented Thai fish (Saithong et al., 2010). LAB are the most important bacteria used in the fermentation industry of dairy products, such as yogurt, cheese, sour milk and butter, and in combination with yeast are commonly used to ferment cereal products such as dough (Lavermicocca et al., 2000; Muhialdin et al., 2011a; Ryan et al., 2008).

Advertisement

3. Spoilage fungi in food

The economic losses and the health hazards of the mycotoxins produced by spoilage fungi are the main concerns of the food industry (Gray & Bemiller, 2003). According to Gerez et al., (2009) the spoilage of bakery products by fungi is more common in countries with a high humidity and temperature. Pitt and Hocking (1999) estimated that about 5-10% of food production is spoiled by the growth of yeast and fungi in food materials. Similarly, in Western Europe, the growth of the spoilage fungi of bread is estimated to reach more than 200 million Euros per year (Legan, 1993; Schnürer & Magnusson, 2005). The history conditions of the food can be a major factor in determining any fungal spoilage - for example, stored and processed foods are more sensitive to spoilage when compared with fresh and prepared foods. Aspergillus and Penicillium species are the most common spoilage fungi for many foods and feeds while Fusarium species are reported to attack the cereal grains in the field (Samson et al., 2000).

The most widespread species of fungi that contaminate bakery products belong to the genera Aspergillus, Penicillium, Eurotium (Abellana et al., 1997; Guynot et al., 2005), Monilia, Mucor, Endomyces, Cladosporium, Fusarium and Rhizopus (Lavermicocca et al., 2000, 2003). In addition, fungi may be responsible for off-flavours, the production of mycotoxins and allergenic compounds. There are more than 400 known mycotoxins produced by different fungi (Filtenborg et al., 1996). Mycotoxigenic fungi such as Aspergillus, Fusarium and Penicillium are serious hazards for human health. The six classes of mycotoxins frequently encountered in different food systems are: aflatoxins, fumonisins, ochratoxins, patulin, trichothecenes and zearalenone (Dalié et al., 2009).

Advertisement

4. Common techniques to control spoilage fungi in bakery products

Two types of techniques/factors are commonly used to control spoilage fungi: physical ones such as drying, freeze drying, cold storage, modified atmosphere storage, irradiation, the pasteurization of packaged bread and heat treatment; and chemical ones, in general based on the use of organic acids such as propionic acid and its salts (Farkas, 2001; Legan, 1993). Heat treatment is one of the most important physical factors in controlling fungi growth and mycotoxin production, as mycotoxins are destroyed by heat, although the effectiveness of destruction is affected by the food matrix and the composition of the mycotoxin (Scott, 1984). Mycotoxins have different heat stability - for example, ochratoxin A is highly stable even at 200 ºC (Trivedi et al., 1992), aflatoxins are destroyed only at temperatures of approximately 250 ºC (Levi, 1980), while zearalenone and fumonisin require high temperatures between 150-200 ºC to be efficiently destroyed (Bennett et al., 1980). Microwaves are effective in destroying mycotoxins - the aflatoxin in peanuts is reported to be destroyed using microwaves at a power level of 1.6 kW for 16 min and at 3.2 kW for 5 min (Luter et al., 1982). Among the physical methods, a modified atmosphere and gamma irradiation are preferred to the chemical methods and they have been used successfully in grain storage (Shapira & Paster, 2000).

Chemical methods that use weak acids and salts such as propionic, sorbic and benzoic acids, are usually applied only to inhibit the growth of spoilage microorganisms. The allowable concentrations of sorbate, propionate and ethanol have a limit up to 0.2% (wt/wt), 0.3% (wt/wt) and 2% (wt/wt) respectively. The use of such low concentration may not be sufficient to prevent the growth of spoilage fungi (Dantigny et al., 2005; European Union, 1995). Propionic acid is inhibitory to fungi and Bacillus spores and has commonly been used to preserve bakery products. Its activity relies on the un-dissociated form which, at low pH, has optimum activity (Coda et al., 2008; Pattison et al., 2004). The use of propionic acid at a concentration of 4% led to the appearance of cancer-like tumours in rats and eventually led to the prohibition of the use of calcium propionate in some European countries (Pattison et al., 2004). There is a major concern with microorganisms that can develop resistance to chemical preservatives, namely food spoilage and human pathogen fungi resistant to antibiotics and chemicals additives, such as sorbic and benzoic acids (Brul & Coote, 1999; Lourens-Hattingh & Viljoen, 2001). Calcium propionate has been reported to inhibit the growth of many fungi but, after a lag phase, it stimulated the growth of resistant strains of Penicillium roqueforti (Suhr & Nielsen, 2004). Interest in natural bio-preservation from LAB has been on the rise as an alternative to chemical preservatives.

Advertisement

5. Significance of the metabolites of LAB

LAB are well known for their antifungal activity, which is related to the production of a variety of compounds including acids, alcohols, carbon dioxide, diacetyl, hydrogen peroxide, phenyllactic acid, bacteriocins and cycle peptides (Gerez et al., 2009; Lavermicocca et al., 2000; Magnusson et al., 2003; Prema et al., 2008). These compounds were added to several foods in order to conserve them from food-borne and spoilage microorganisms. Organic acids are the main product of LAB in the fermentation systems of the raw materials. The main acids produced by LAB are lactic acid and acetic acid, besides certain other acids depending upon the strain of LAB (El-Ziney, 1998). These acids will be diffused through the membrane of the target organisms in their hydrophobic un-dissociated form and then used to reduce the cytoplasmic pH and stop metabolic activities (Piard & Desmazeaud, 1991). Other factors that contribute to the preservative action of the acids are the sole effect of pH, the extent of the dissociation of the acid and the specific effect of the molecule itself on the microorganisms (Axelsson, 1998).

Bacteriocins exhibit good potential for use in the food industry and as bio-preservation agents (Ennahar et al., 1999). Bacteriocins are small, ribosomally synthesized, antimicrobial peptides or proteins that display inhibition activity toward related species, with no reports about fungal inhibition (Cotter & Ross, 2005). The notable property of LAB supernatant is the heat stability of the antifungal compounds present in it. This will promote the use of LAB supernatant and/or antifungal compounds in heat-treated foods. The supernatant of certain LAB observed to be active within a wide range of pH, starting from as low as 3 and up to 9 depending upon the strain (Muhialdin et al., 2011b). This could be considered as a major factor whereby LAB are used in food preservation when compared with the chemical preservative which are usually active at low pH between 3 and 4.5. Additionally, LAB have a broad spectrum of antifungal activity against several food spoilage and mycotoxin-producing fungi while commercial preservatives are usually used to control only one or few fungi.

Advertisement

6. Bioactive compounds as antifungal agents

Several lactobacilli species are reported to have antifungal activity (Gerez et al., 2009; Muhialdin et al., 2011b; Plockova et al., 2001; Stiles et al., 1999). The antifungal compounds consist of organic acids, reuterin, hydrogen peroxide and other peptides (Table 1). The organic acids are active at low pH and the activity relies on the un-dissociated form of the acids. Recently, interest has dramatically increased in the use of bioactive peptides produced by LAB as an antifungal agent. The use of protein-like compounds are preferred over the use of acids because their activity is present over a wide range of pH and they are heat stable compounds which are ideal for use in heat processed foods (Muhialdin et al., 2011a). Cyclic dipeptides cyclo (Phe-Pro) and cyclo (Phe-OH-Pro) were produced by the L. coryniformis subsp. coryniformis Si3 strain and were inhibitory to Aspergillus sp. (Magnusson, 2003; Ström et al., 2002). Ryan et al. (2011) observed that sourdough made with L. amylovorus DSM 19280 had a longer shelf life compared with bread produced with calcium propionate. The selected strain inhibited the growth of Fusarium culmorum FST4.05, Aspergillus niger FST4.21, Penicillium expansum FST4.22, Penicillium roqueforti FST4.11 and L. amylovorus DSM 19280 and produced seventeen antifungal compounds.

CompoundProducerInhibited fungiReferences
Possibly proteinaceousPediococcus acidilacticiSaccharomyces cerevisiaeVandenbergh & Kanka (1989)
Possibly proteinaceousL. lactis subsp. Lactis CHD 28.3A. flavus, A. parasiticus, Fusarium spp.Roy et al. (1996)
Caproic acid, propionic acid, butyric acid, valeric acidL. sanfranciscencis CB1Fusarium spp., Penicillium spp., Aspergillus spp., Monilia spp.Corsetti et al. (1998)
Benzoic acid, methylhydantoin, mevalonolactone,L. plantarum VTT E78076F. avenaceumNiku-Paavola et al. (1999)
phenyllactic and 4-hydroxy-phenyllactic acidsL. plantarum 21BBroad spectrum against bakery spoilage fungiLavermicocca et al. (200)
3-Phenyllactic acid, cyclo (Phe-OH-Pro), cyclo (Phe-Pro).L. plantarum MiLAB 393F. sporotrichioides and A. fumigatusStröm et al. (2002)
Hydroxy fatty acids, phenyllactic acid, cyclo(Phe-Pro), cyclo(Phe-OH-Pro),L. plantarum MiLAB14Broad spectrumMagnusson et al. (2003)
Possibly cyclic dipeptideP. pentosaceusP. expansumRouse et al. (2008)
diacetyl and hydrogen peroxideL. fermentum and Leuconostoc mesenteroidesRhizopus oryzae, A. niger, A. flavus, Penicillium sp and F. oxysporumOgunbanwo et al. (2008)
Acetic acid, phenyllactic acidL. reuteri 1100F. graminearumGerez et al. (2009)
(cyclo(Leu–Leu))L. plantarum AF1Aspergillus flavus ATCC 22546Yang & Chang (2010)
Four peptides and organic acid mixtureL. plantarum
LB1 and L. rossiae LB5
Penicillium roqueforti DPPMAF1Rizzello et al. (2011)
Mixture of peptidesL. plantarum 1A7 (S1A7)Broad spectrumCoda et al. (2011)
Possibly protein-likeL. fermentum Te007, P. pentosaceus Te010, L. pentosus G004, and L. paracasi D5A. niger and A. oryzaeMuhialdin et al. (2011a)
nine carboxylic acids, two nucleosides, sodium decanoate and five cyclic dipeptidesL. amylovorus DSM 19280A. niger FST 4.21, A. fumigatus J9, F. culmorum TMW 4.0754 P. expansum FST 4.22 and P. roqueforti FST 4.11Ryan et al. (2011)
3-phenyllactic acid and Benzene acetic acid, 2- propenyl esterL. plantarum IMAU10014Botrytis cinerea, Glomerella cingulate, Phytophthora drechsleri Tucker, P. citrinum, P. digitatum and F. oxysporumWang et al. (2012)

Table 1.

Antifungal compounds produced by lactic acid bacteria and their target fungi

Advertisement

7. Method for determining antifungal activity

Rapid, reliable and sensitive methods for the detection of the antifungal activity of LAB becomes essential in the search for new replacements for chemical preservatives with potential industrial applications.

7.1. Dual agar overlay method

This method has been described by several authors (Magnusson & Schnürer, 2001; Ström et al., 2002; Hassan & Bullerman, 2008) and it is accurate and simple for determining the antifungal activity of LAB isolates. The method consists of inoculating the LAB cells in two 2-cm-long lines and/or small circle spots on a MRS agar surface then incubating the plates at 30 °C for 24-48 h in anaerobic jars. The plates are overlaid with 10 ml of malt extract soft agar (2% malt extract, 0.7% agar; Oxoid) containing different concentrations of the spore inoculant of 104 and 105 spore/ml. The plates are then incubated aerobically at 30 °C for 48-72 h. The inhibition activity is indicated by the clear zones around the bacterial streaks. The scale for measuring the activity can be recorded as follows: -, no activity; +, no fungal growth on 0.1 to 3% of the plate area; ++, no fungal growth on 3 to 8% of the plate area; and+++, no fungal growth on 8% of plate area. Another way to measure the activity is by recording the clear zone diameter around the isolates streak, which refers to the inhibition of the fungi growth. The dual agar overlay method is also a good method for the screening of the antifungal activity of the supernatant of LAB isolates. The supernatant can be mixed with the de Man, Rogosa and Sharpe (MRS) agar or potato dextrose agar (PDA) and poured into Petri dishes followed by a similar step, mentioned previously. The supernatant can be added to the agar before it is autoclaved in order to determine the heat stability of the antifungal compounds present in the supernatants, which is a good indicator of whether the supernatant is used in heat processed foods.

7.2. Agar well diffusion method

The well diffusion method is another approach for determining the antifungal activity of LAB, described as a simple, accurate and flexible method. It is suitable to determine the inhibition activity of LAB supernatant. A fungi numbering 104 -105 spore/ml are mixed with the selected agar and allowed to solidify. The wells can be made on a variety of agar surfaces - for example, wells are made on potato dextrose agar if the target is a fungi or on a nutrient agar if the target is a bacteria; the wells are made by using a sterilized cork borer with a diameter of 3 or 5 mm. 50 µl of the same agar is added to each well in order to seal the base so as to avoid leakage. The cell-free supernatants are then added to wells in amounts of 30-80 µl and incubated at room temperature for 3-6 h in order to allow the supernatant to be diffused through the agar. The antifungal activity is recorded by measuring the clear zones' diameters around the wells.

7.3. Dry weight of biomass

The reduction of the biomass of the fungi can be a tool for determining the growth inhibition activity of the supernatant. 50 ml of the supernatant is inoculated into a 250 ml flask containing the growth medium for the target fungi and then the suspension of the fungi spores is added at a concentration of 105. The fungal mass is harvested on filter paper and dried in an oven at 50 °C for 2 days. The average of the fungal biomass inhibition can be calculated by comparing the weight of treated fungi with the positive control which contains the fungi and the growth medium with no supernatant.

7.4. Micro-titter 96 well plate

The method is simple, inexpensive and practical for determining antibacterial and antifungal activity. The supernatant of LAB is placed into the wells of 190 µl and inoculated with 10 µl of a conidial suspension containing about 104-105 spore/ml. The plates are then incubated at 25-30 °C. The control is a conidial suspension placed in the wells in equal amounts without the addition of the LAB supernatant. Fungal growth is observed by the naked eye and determined by measuring the optical density at 560-580 nm, starting from 0 h and repeated every 24 h with a spectrophotometer. The result can be obtained by comparing the OD readings of the control with the treated wells. The method is appropriate for evaluating the MIC, heat stability, enzyme activity and effects of pH for the LAB supernatant.

Advertisement

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.

StrainsNo. of daysTarget fungiStorage temperature °CReference
L. plantarum 21B7Broad spectrum20Lavermicocca et al., (2000)
L. plantarum12Rhizopus oryzae A. niger A. flavus Penicillium sp. F. oxysporum27Ogunbanwo et al., (2008)
L. brevis AM721P. roqueforti DPPMAF125Coda et al., (2008)
L. plantarum10A. niger, F. culmorum, and P. expansum25Ryan et al., (2008)
L. plantarum CRL 778, L. reuteri CRL 1100, and L. brevis CRL 772 and CRL 7968Aspergillus, Fusarium, and Penicillium30Gerez et al., (2009)
L. plantarum 1A7 (S1A7)28P. roqueforti DPPMAF125Coda et al., (2011)
L. amylovorus DSM 1928014F. culmorum FST 4.05, A. niger FST4.21, P. expansum
FST 4.22, P. roqueforti FST 4.11
25Ryan et al., (2011)
L. fermentum Te007, P. pentosaceus Te010, L. pentosus G004, and L. paracasi D59-12A. niger and A. oryzae30Muhialdin et al., (2011)

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

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).

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).

Advertisement

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.

Advertisement

10. Production of LAB cells and inhibitory compounds

10.1. Growth medium

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 (unpublished data).

10.2. Growth conditions

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 and the production of primary or secondary metabolites.

11. Future research

The high demand by consumers for foods free of chemical preservatives has led to increasing amounts of research to provide alternatives for these chemicals. LAB provides technologically practicable alternatives for the replacement of chemical preservatives. The achievement of selecting LAB as starter cultures or co-cultures in fermentation processes can improve the desired properties of bread, at the same time providing consumers with new chemical-free foods. There is a need to study the interaction between the food matrix and the kinetics of the starter culture of LAB in bread; such studies will contribute to the bread industry by increasing the yield of the antifungal and nutritional compounds produced by LAB. Besides using the LAB cells in bread formulations, the use of the supernatant of LAB should be considered, especially the supernatant of LAB that are grown in non-conventional media such as plant extract and other cheap materials. Additional studies on the contribution of bioactive molecules to the quality and shelf life of foods will surely widen the use of LAB strains as a novel bio-control strategy in bakery products.

12. Conclusion

LAB can be used as a starter culture or a co-culture in the bread industry to enhance the sensory properties of bread and extend the shelf life. The nutritional value of the bread is enhanced due to the production of free amino acids, organic acids and a variety of Group-B vitamins. The antifungal compounds produced by LAB are important for the food industry for replacing or reducing the use of chemical preservatives. Several methods have been developed to determine the antifungal activity of the cells and the free cell supernatant. Natural sources of food preservatives - especially LAB - are important and reflect one possibility for fulfilling the needs of modern consumers of bakery products that are free of chemicals. Challenges are evident in finding new and novel isolates of LAB that can be applied in bread and which do not affect the activity of the yeast or inhibit their growth. Future works should consider the use of the LAB supernatant as well as the cells because the active compounds can be present in the supernatant. Inexpensive media are also important for high-scale industry, especially the use of plant extracts that are rich in carbohydrates and which can be supplied in bulk over the course of the year.

References

  1. 1. AbellanaM. LTorresV. Sand A. JRamos1997Caracterizacio´n de diferentesproductos de bollería industrial. II. Estudio de la Microflora. Alimentaria, 2875156
  2. 2. AxelssonL1998Lactic acid bacteria: Classification and physiologyIn Lactic Acid Bacteria: Microbiology and functional aspects, 2nd Edition, Revised and Expanded. Edited by S. Salminen & A. von Wright. 172Marcel Dekker, Inc. New York.
  3. 3. BennettG. AShotwellO. Land HesseltineC. W1980Destruction of zearalenone in contaminated cornJournal of the American Oil Chemists’ Society572457
  4. 4. BjörkrothJand HolzapfelW2003Genera Leuconostoc, Oenococcus and Weissella, In M. Dworkin et al., eds., The Prokaryotes: An Evolving Electronic Resource for the Microbiological Community, 3rd edition, Springer-Verlag, New York, http://link.springer-ny.com/link/service/books/10125/
  5. 5. BrulSand CooteP1999Preservative agents in foods-mode of action and microbial resistance mechanisms.International Journal of Food Microbiology50117
  6. 6. CarrF. JChillDand MaidaN2002The lactic acid bacteria: a literature surveyCritical Reviews in Microbiology28281370
  7. 7. CheluleP. KMokoenaM. Pand GqaleniN2010Advantages of traditional lactic acid bacteria fermentation of food in Africa. RORMATEX, 11641167http://www.formatex.info/microbiology2/1160-1167.pdf
  8. 8. ClarkeCSchoberT. Jand ArendtE. K2002Effect of single strain and traditional mixed strain starter cultures in rheological properties of wheat dough and bread quality.Cereal Chemistry79640647
  9. 9. CodaRRizzelloC. GNigroFDe AngelisMArnaultPand GobbettiM2008Long-term fungi inhibitory activity of water-soluble extract from Phaseolus vulgaris cv Pinto and sourdough lactic acid bacteria during bread storage. Applied and Environmental Microbiology, 7473917398
  10. 10. CodaRCassoneARizzelloC. GNionelliLCardinaliGand GobbettiM2011Antifungal Activity of Wickerhamomyces anomalus and Lactobacillus plantarum during Sourdough Fermentation: Identification of Novel Compounds and Long-Term Effect during Storage of Wheat BreadApplied and Environmental Microbiology7734843492
  11. 11. CorsettiAGobettiMRossiJand DamianiP1998Antimould activity of sourdough lactic acid bacteria: identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1.Applied Microbiology and Biotechnology50253256
  12. 12. CotterP. DHillCand RossR. P2005Bacteriocins: developing innate immunity for food.Nature ReviewsMicrobiology, 3777788
  13. 13. Dal BelloF., Clarke, C. I., Ryan, L. A. M., Ulmer, H., Schober, T. J., Strom, K., Sjorgren, J., Van Sinderen, D., Schnurer, J. and Arendt, E. K. 2007Improvement of the quality and shelf life of wheat bread by fermentation with the antifungal strain Lactobacillus plantarum FST 1.7Journal of Cereal Science45309318
  14. 14. DaliéD. K. DDeschampsA. Mand Richard-forgetF2009Lactic acid bacteria- Potential for control of mould growth and mycotoxins.A review. Food Control, 21370380
  15. 15. DantignyPGuilmartARadoiFBensoussanMand ZwieteringM2005Modelling the effect of ethanol on growth rate of food spoilage mouldsInternational Journal of Food Microbiology98261269
  16. 16. DruveforsU. APassothVand SchnurerJ2005Nutrient effects on biocontrol of Penicillium roqueforti by Pichia anomala J121 during airtight storage of wheatApplied Environmental Microbiology, 7118651869
  17. 17. El-ZineyM. Gand DebevereJ. M1998The effect of reuterin on Listeria monocytogenes and Escherichia coli O157:H7 in milk and cottage cheese.Journal of Food Protection6112751280
  18. 18. EnnaharSSonomotoKand IshizakiA1999Class IIa bacteriocins from lactic acid bacteria: antibacterial activity and food preservationJournal of Bioscience and Bioengineering87705716
  19. 19. European Union1995European Parliament and Council directive 95EC of 20 February 1995 on food additives other than colours and sweeteners, 53. Office for Official Publications of the European Communities, Luxembourg. http://europa.eu.int/eur-lex/en/consleg/pdf/1995en_1995L0002_do_001.pdf.
  20. 20. EvansI. H1990Yeast strains for baking. In: Yeast Technology, eds., Spencer, J. F. T. and Spencer, D. M. 13Berlin, Germany: Springer-Verlag.
  21. 21. FarkasJ2001Physical methods for food preservation. In Food microbiology: Fundamentals and frontiers. Edited by M. P. Doyle, L. R. Beuchat & T. J. Montville. 567592ASM press. Washington, USA.
  22. 22. FiltenborgOFrisvadJ. Cand ThraneU1996Moulds in food spoilage.International Journal of Food Microbiology3385102
  23. 23. HammesW. Pand HertelC2003The Genera Lactobacillus and Carnobacterium. In The Prokaryotes: An Evolving Electronic Resource for the Microbiological Community, Edited by M. Dworkin. Springer-Verlag, New York. http://link.springer-ny.com/link/service/books/10125/.
  24. 24. HassanY. Iand BullermanL. B2008Antifungal activity of Lactobacillus paracasei ssp. tolerans isolated from a sourdough bread culture. International Journal of Food Microbiology, 121112115
  25. 25. HelanderI. MVon WrightAand Mattila-sandholmT-M1997Potential of lactic acid bacteria and novel antimicrobials against Gram-negative bacteria. Trends in food Science and Technology, 8146150
  26. 26. GerezC. LRollánGand Font de Valdez, G. 2006Gluten breakdown by lactobacilli and pediococci strains isolated from sourdough. Letters in Applied Microbiology, 42459464
  27. 27. GerezC. LTorinoM. IRollanGand Font de Valdez, G. 2009Prevention of bread mould spoilage by using lactic acid bacteria with antifungal properties. Food Control, 20144148
  28. 28. GrayJand BemillerJ2003Bread staling: Molecular basis and control. Comprehensive Reviews in Food Science and Safety, 2121
  29. 29. GuynotM. EMarÍn, S., Setu, L., Sanchis, V. and Ramos, A. J. 2005Screening for antifungal activity of some essential oils against common spoilage fungi of bakery products. Food Science and Technology International, 112532
  30. 30. JamesSand StratfordM2003Spoilage yeast with emphasis on the genus Zygosaccharomyces. In Yeasts in food. Edited by B. T & R. V. Behrs, Verlag, pp. 171191
  31. 31. KatinaKSauriMAlakomiH. Land Mattila-sandholmT2002Potential of Lactic Acid Bacteria to Inhibit Rope Spoilage in Wheat Sourdough Bread. Lebensm.-Wiss. u.-Technology, 353845
  32. 32. KennsCVeigaM. CDubourgularH. CTouzelJ. PAlbengaeGNaveanHand NynsE. J1991Tropic relationship between Saccharomyces cerevisiae and Lactobacillus plantarum and their metabolism of glucose and citrate. Journal of Applied Environmental Microbiology, 5710471051
  33. 33. KeuthSand BispingB1993Formation of vitamins by pure cultures of tempe moulds and bacteria during the tempe solid substrate fermentation. Journal of Applied Bacteriology, 75427434
  34. 34. LavermicoccaPValerioFEvidenteALazzaroniSCorsettiAand GobbettiM2000Purification and characterization of novel antifungal compounds by sourdough Lactobacillus plantarum 21B. Applied and Environmental Microbiology, 6640844090
  35. 35. LavermicoccaPValerioFand ViscontiA2003Antifungal activity of phenyllactic acid against molds isolated from bakery products. Applied Environmental Microbiology, 69634640
  36. 36. LeganJ. D1993Mould spoilage of bread: the problem and some solutions. International Biodeterioration and Biodegradation, 323353
  37. 37. LeviC1980Mycotoxins in coffee. Journal- Association of Official Analytical Chemists, 6312821285
  38. 38. Lourens-hattinghAand ViljoenB. C2001Yoghurt as probiotic carrier food. International Dairy Journal, 11117
  39. 39. LuterLWyslouzilWand KashyapS. C1982The destruction of aflatoxin in peanuts by microwave roasting. Canadian Institute of Food Science and Technology Journal, 15236238
  40. 40. MagnussonJand SchnurerJ2001Lactobacillus coryniformis subsp. coryniformis strain Si3 produces a broad-spectrum proteinaceous antifungal compound. Applied and Environmental Microbiology, 6715
  41. 41. MagnussonJ2003Antifungal activity of lactic acid bacteria. Ph.D. Thesis, Agraria 397, Swedish University of Agricultural Sciences, Uppsala, Sweden.
  42. 42. MagnussonJStrömKRoosSSjögrenJand SchnürerJ2003Broad and complex antifungal activity among environmental isolates of lactic acid bacteria. FEMS Microbiology Letters, 219129135
  43. 43. MuhialdinB. JHassanZand SadonS. K2011aAntifungal Activity of Lactobacillus fermentum Te007, Pediococcus pentosaceus Te010, Lactobacillus pentosus G004 and L. paracasi D5 on Selected Foods. Journal of Food Science, 76493499
  44. 44. MuhialdinB. JHassanZSadonS. KNurAqilah, Z. and AZFAR, A. A. 2011bEffect of pH and Heat Treatment on Antifungal Activity of Lactobacillus fermentum Te007, Lactobacillus pentosus G004 and Pediococcuspentosaceus Te010. Innovative Romanian Food Biotechnology, 84153
  45. 45. MuhialdinB. Jand HassanZ2011Screening of Lactic Acid Bacteria for Antifungal Activity against Aspergillus oryzae. American Journal of Applied Science, 8447451
  46. 46. Niku-paavolaM. LLaitilaAMattila-sandholmTand HaikaraA1999New types of antimicrobial compounds produced by Lactobacillus plantarum. Journal of Applied Microbiology, 862935
  47. 47. OgunbanwoS. TAdebayoA. AAyodeleM. AOkanlawonB. Mand EdemaM. O2008Effects of lactic acid bacteria and Saccharomyces cerevisiae co-cultures used as starters on the nutritional contents and shelf life of cassava-wheat bread. Journal of Applied Biosciences, 12612622
  48. 48. PattisonT. Land Von HolyA2001Effect of selected natural antimicrobials on Baker’s yeast activity. Letters in Applied Microbiology, 33211215
  49. 49. PattisonT. LLindsayDand Von HolyA2004Natural antimicrobial as potential replacements for calcium propionate in bread. South Africa Journal of Science, 100339342
  50. 50. PiardJ. Cand DesmazeaudM1991Inhibiting factors produced by lactic acid bacteria. 1. Oxygen metabolites and catabolism end-products. Le Lait, 71525541
  51. 51. PittJ. JHockingA. D1999Fungi and food spoilage Second ed. Aspen Publications. pp.
  52. 52. PlockovaMStilesJand ChumchalovaJ2001Control of mould growth by Lactobacillus rhamnosus VT1 and Lactobacillus reuteri CCM 3625 on milk agar plates. Czech Journal of Food Science, 194650
  53. 53. PremaPSmilaDPalavesamAand ImmanuelG2008Production and Characterization of an Antifungal Compound (3-Phenyllactic Acid) Produced by Lactobacillus plantarum Strain. Food Bioprocess Technology, 3379386
  54. 54. RizzelloC. GNionelliLCodaRDi Cagno, R. and Gobbetti, M. 2010Use of sourdough fermented wheat germ for enhancing the nutritional, texture and sensory characteristics of the white bread. European Food Research and Technology, 230645654
  55. 55. RizzelloC. GCassoneACodaRand GobbettiM2011Antifungal activity of sourdough fermented wheat germ used as an ingredient for bread making. Food Chemistry, 127952959
  56. 56. RouseSHarnettDVaughanAand Van SinderenD2008Lactic acid bacteria with potential to eliminate fungal spoilage in foods. Journal of Applied Microbiology, 104915923
  57. 57. RoyUBatishV. KGroverSand NeelakantanS1996Production of antifungal substance by Lactococcus lactis subsp. lactis CHD-28.3.International Journal Food Microbiology, 322734
  58. 58. RyanL. A. MDal Bello, F. and Arendt, E. K. 2008The use of sourdough fermented by antifungal LAB to reduce the amount of calcium propionate in bread. International Journal of Food Microbiology, 125274278
  59. 59. RyanL. A. MZanniniEDal Bello, F., Pawlowska, A., Koehler, P. and Arendt, E. K. 2011Lactobacillus amylovorus DSM 19280 as a novel food-grade antifungal agent for bakery products. International Journal of Food Microbiology, 146276283
  60. 60. SaithongPPanthaveeWBoonyaratanakornkitMand SikkhamondholC2010Use of a starter culture of lactic acid bacteria in plaa-som, a Thai fermented fish. Journal of Bioscience and Bioengineering, 110553557
  61. 61. SamsonR. ASeifertK. AKuijpersA. F. AHoubrakenJ. A. M. Pand FrisvadJ. C2004Phylogenetic analysis of Penicillium subgenus Penicillium using partial b-tubulin sequences. Studies in Mycology, 49175200
  62. 62. SantosFVeraJ. LVan Der HeijdenRValdezGDe VosW. MSesmaFand HugenholtzJ2008The complete coenzyme B 12 biosynthesis gene cluster of Lactobacillus reuteri CRL1098. Microbiology, 1548193
  63. 63. ScottP. M1984Effect of food processing on mycotoxins. Journal of Food Protection, 47489499
  64. 64. SchnürerJand MagnussonJ2005Antifungal lactic acid bacteria as biopreservatives. Trends in Food Science and Technology, 167078
  65. 65. ShapiraRand PasterN2004Control of mycotoxins in storage and techniques for their decontamination. Mycotoxins in food. Woodhead Publishing Limited, Abington Hall, Abington, Cambridge CB1 6AH, England. 190223
  66. 66. StilesJPlockovaMTothVand ChumchalovaJ1999Inhibition of Fusarium sp. DMF 0101 by Lactobacillus strains grown in MRS and Elliker broths. Advances in Food Science, 21117121
  67. 67. StrömKSjörgenJBrobergAand SchnürerJ2002Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3 phenyllactic acid. Applied and Environment Microbiology, 6843224327
  68. 68. SuhrK. Iand NielsenP. V2004Effect of weak acid preservatives on growth of bakery product spoilage fungi at different water activities and pH values. International Journal of Food Microbiology, 956778
  69. 69. SybesmaWStarrenburgMTijsselingLHoefnagelM. Hand HugenholtzJ2003Effects of cultivation conditions on folate production by lactic acid bacteria. Applied and Environmental Microbiology, 6945424548
  70. 70. ThieleCGänzleM. Gand VogelR. F2002Contribution of sourdough lactobacilli, yeast, and cereal enzymes to the generation of amino acids in dough relevant for bread flavor. Cereal Chemistry, 794551
  71. 71. TrivediA. BDoiEand KitabatakeN1992Detoxification of ochratoxin A on heating under acidic and alkaline conditions. Bioscience, Biotechnology and Biochemistry, 56741755
  72. 72. VandenberghP. Aand KankaB. S1989Antifungal product. United States Patent. 4,877,615.
  73. 73. WangHYanYWangJZhangHQiW2012Production and Characterization of Antifungal Compounds Produced by Lactobacillus plantarum IMAU10014. PLoS ONE, 7(1): e29452. doi:10.1371/journal.pone.0029452
  74. 74. WhittenburyR1964Hydrogen peroxide formation and catalase activity in the lactic acid bacteria. Journal of General Microbiology, 351326
  75. 75. YangE. Jand ChangH. C2010Purification of a new antifungal compound produced by Lactobacillus plantarum AF1 isolated from kimchi. International Journal of Food Microbiology, 1395663
  76. 76. ZanniniEGarofaloCAquilantiLSantarelliSSilvestriGand ClementiF2009Microbiological and technological characterization of sourdoughs destined for bread-making with barley flour. Food Microbiology, 26744753

Written By

Belal J. Muhialdin, Zaiton Hassan and Nazamid Saari

Submitted: 15 April 2012 Published: 30 January 2013