**7. Method for determining antifungal activity**

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

**6. Bioactive compounds as antifungal agents** 

19280 and produced seventeen antifungal compounds.

CB1

E78076

393

*L. plantarum* MiLAB14

*Pediococcus acidilactici* 

*L. lactis* subsp. *Lactis* CHD 28.3

*L. sanfranciscencis*

*L. plantarum* VTT

*L. plantarum* MiLAB

Possibly proteinaceous

Possibly proteinaceous

acid

Caproic acid, propionic acid, butyric acid, valeric

Benzoic acid, methylhydantoin, mevalonolactone,

hydroxy-

phenyllactic and 4-

phenyllactic acids

3-Phenyllactic acid, cyclo (Phe-OH-Pro), cyclo (Phe-Pro).

Hydroxy fatty acids, phenyllactic acid, cyclo(Phe-Pro), cyclo(Phe-OH-Pro),

antifungal activity against several food spoilage and mycotoxin-producing fungi while

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

**Compound Producer Inhibited fungi References** 

*L. plantarum* 21B Broad spectrum

*Saccharomyces cerevisiae*

*A. flavus*, *A. parasiticus*, *Fusarium* spp.

*Fusarium* spp., *Penicillium* spp., *Aspergillus* spp., *Monilia* spp.

against bakery spoilage fungi

*F. sporotrichioides*  and *A. fumigatus*

Vandenbergh & Kanka (1989)

Roy et al. (1996)

*F. avenaceum* Niku-Paavola et al.

Broad spectrum Magnusson et al.

(1999)

(200)

(2003)

Corsetti et al. (1998)

Lavermicocca et al.

Ström et al. (2002)

commercial preservatives are usually used to control only one or few fungi.

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.

Lactic Acid Bacteria in Biopreservation and the Enhancement of the Functional Quality of Bread 161

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

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

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.*

**7.4. Micro-titter 96 well plate** 

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

grew well, producing the desired antifungal compounds in the dough.

supernatant.

**8.1. Shelf life** 

### **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**

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

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.

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

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

**7.1. Dual agar overlay method** 

**7.2. Agar well diffusion method** 

**7.3. Dry weight of biomass** 

measuring the clear zones' diameters around the wells.

contains the fungi and the growth medium with no supernatant.

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.
