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1 Mold control in cheese using metabolites from lactic acid bacteria July 2013 By: Aubrey Mendonca Iowa State University, Dept of Food Science and Human Nutrition David Manu, graduate student, ISU Terri Boylston, Co Pl, ISU Joseph Sebranek, ISU Aura Daraba, visiting scientist, Univeristy Dunarea De Jos of Galati, Romani Partners: Midwest Dairy Association

2 TABLE OF CONTENTS ABSTRACT... 2 BACKGROUND INFORMATION... 2 OBJECTIVES AND MATERIALS AND METHODS... 3 RESULTS AND DISCUSSION... 5 CONCLUSIONS... 7 BENEFITS TO MINNESOTA ECONOMIC DEVELOPMENT... 7 REFERENCES

3 Abstract The antifungal efficacy of selected metabolites produced by certain Lactobacillus species against molds (mainly Aspergillus spp and Penicillum spp) that spoil cheddar cheese was evaluated. Various concentrations of metabolites were screened for their antifungal ability in laboratory media. The metabolite that proved to strongly inhibit mold growth was further evaluated for control of mold growth in shredded cheddar cheese inoculated with mold (~10 3 CFU/g) and held at 7 C and 25 C. Compared to other metabolites tested, DL 3 phenyllactic acid (PLA) exhibited the strongest antifungal effect. In laboratory broth (ph 5.5) growth of all molds tested were completely inhibited by PLA at 10 or 15 mg/ml. Penicillium glabrum and Penicillium roqueforti were most sensitive to PLA; their growth was strongly inhibited by PLA at 7.5 mg/ml. Generally, decreasing the ph of broth containing PLA from ph 5.5 to 5.3 or 5.0 increased the antifungal effectiveness of PLA. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of PLA decreased significantly (P<0.05) at ph 5.0 indicating increased sensitivity of molds to PLA at that ph. In shredded cheddar cheese without added PLA visible mold growth occurred within 7 to 10 days and 3 to 5 days, respectively in cheese held at 7 C and 25 C. In contrast, cheese treated with PLA at 10 mg/ml exhibited visible mold within 24 to 35 days (at 7 C) and 7 to 13 days (at 25 C). Of the 7 mold species evaluated none grew within 42 days in cheese treated with PLA at 12.5 mg/ml. These results indicate that PLA can inactivate molds in shredded cheddar cheese and offer new perspectives for use of this natural antimicrobial to control mold growth in this important dairy product. Background Information A major economic loss that continues to plague shredded cheese manufacturers is product return because of mold growth. Currently used antifungal agents such as potassium sorbate and natamycin are not considered ideal due to unique problems linked to use of these preservatives. Although sorbate effectively inhibits mold growth in cheese, certain species of Aspergillus and Penicillum can degrade sorbate via decarboxylation and produce unpleasant odors (Marth et al., 1966; Sofos and Busta, 1981; Kinderlerer and Hatton, 1990). Therefore, the dairy industry currently depends on natamycin for control of mold in cheese. The relatively poor solubility, distribution and stability of natamycin in cheese limit its effectiveness in preventing mold growth. Certain substances (produced by lactic acid bacteria) that have antifungal efficacy and good stability in cheese may provide may provide an attractive alternative to currently used antifungal agents. Some lactic acid bacteria (LAB) can produce low molecular weight substances that exhibit antifungal properties (Lavermicocca et al., 2000; Niku Paavola et al., 1999; Strom et al., 2002). Magnusson et al (2003) isolated several LAB strains (with antifungal properties) from plant materials stored anaerobically. A strain of Lactobacillus plantarum (ITM21B) used as a starter culture in sourdough bread, inhibited growth of Aspergillus niger and Penicillium roqueforti for one week and significantly extended the shelf life of the bread (Lavermicocca et al., 2000; Lavermicocca et al., 2003). In the culture filtrate of the starter, those researchers detected phenyllactic acid (PLA) and 4 hydroxyphenyllactic acid (OH PLA). The PLA was identified as the main product responsible for the antifungal activity of the starter culture (Lavermicocca et al., 2000). 2

4 The antifungal activity of PLA has been reported against several species of molds including Aspergillus ochraceus, Penicillium verrucosum, and Penicillium citrinum, isolated bakery products, cereals, and flour (Lavermicocca et al., 2000; Lavermicocca et al., 2003). Phenyllactic acid and hydroxyl phenyllactic acid are metabolites that are associated with LAB strains involved in formation of cheese flavor. Phenyllactic acid and hydroxyl phenyllactic acid are formed by degradation of phenylalanine and tyrosine, respectively, by certain LAB strains (Kieronczyk, et al., 2003). A proteinaceous antifungal compound and antifungal cyclic dipeptides are also produced from certain lactobacillus species including Lactobacillus plantarium (certain strains) and Lactobacillus coryniformis subsp. coryniformis (Magnusson and Schnurer, 2001; Strom et al., 2002). Considering the potential of specific LAB metabolites as antifungal agents, the objectives of the present research were: Objectives 1. Evaluate the antifungal activity of selected metabolic products of lactic acid bacteria (LAB) against several species of molds in laboratory broth. 2. Demonstrate the efficacy of selected antifungal LAB metabolites for preventing mold growth on shredded cheddar cheese Materials and Methods Objective 1. Evaluate the antifungal activity of selected metabolic products of lactic acid bacteria (LAB) against several species of molds in laboratory broth Experimental design: Molds ( Penicillium and Aspergillus species) and four LAB metabolites at five concentrations (mg/ml) each were used in evaluating the antifungal activity of the LAB metabolites in brain heart infusion broth (BHI) in wells of microtiter plates. Treatments (LAB metabolites) that exhibited strong antifungal activity were selected and evaluated against several mold species at 3 different ph conditions (ph 5.0, 5.3, and 5.5). Broth at each ph without added LAB metabolite served as control. Three independent evaluations were performed for each treatment (concentration of LAB metabolite) within each of two replicate experiments. Metabolites of lactic acid bacteria. The following metabolites were used: 4 hydroxy phenyllactic acid, DL 3 phenyllactic acid, 3 hydroxy fatty acids, and a cyclic dipeptide, all products of Lactobacillus spp. Microdilution tests. Microdilution tests were carried out using sterile disposable, microtiter plates (each with 100 wells) designed for use in the Bioscreen C turbidometer. Brain heart infusion broth with added chloramphenicol (100 µg/ml) and various concentrations of LAB metabolites were dispensed into wells (190 ul per well). Each well was inoculated with a 10 ul conidial suspension containing approximately 5 x10 5 mold conidia. Inoculated microtiter plates were incubated in the Bioscreen C at 25 C for 5 days. Fungal growth was monitored by optical density at 600 nm every 12 hours. Non inoculated control (BHI broth plus antifungal 3

5 metabolite) and a non treated control (broth without antifungal metabolite) were included in each experiment. Determination of minimum inhibitory and fungicidal concentration. Optical density measurements of mold cultures were taken every 12 hours for 120 hours and used to prepare growth curves for each mold species. Results were used to determine the minimum inhibitory concentration (MIC) of the selected LAB metabolite for each mold culture tested. The lowest concentration of LAB metabolite that inhibited growth of a mold culture for 120 hours in BHI under specified conditions (ph and temperature) was reported as the MIC for that specific mold. To determine minimum fungicidal concentration (MFC) 10 µl aliquots of mold cultures from wells with LAB metabolite were plated on dichloran rose bengal chloramphenicol agar (DRBC). Plate counts of molds were performed on cultures with LAB metabolite concentration higher than the MIC, at the MIC and just below the MIC. The lowest concentration of LAB metabolite at which no conidial germination was detected in BHI (25 C) after 120 hours at a specified ph was reported as the MFC for a specified mold. Objective 2. Demonstrate the efficacy of selected antifungal LAB metabolite for preventing mold growth on shredded cheddar cheese Experimental design: The experimental design consisted of 9 treatments (concentrations of LAB antifungal metabolite including control), 2 storage temperatures (7 C and 25 C), 42 storage days (for cheese samples held at 7 C), and 14 storage days (for cheese samples held at 25 C). Methods Cheese production and inoculation. Blocks of cheddar cheese (approximately 1.5 lb each) manufactured without added antifungal preservatives, were purchased from a local organic food store in Ames, Iowa. The cheese was aseptically shredded in 4.5 lb batches using a sterile stainless steel hand shredder in a laminar flow hood. The shredded cheese was manually mixed in a sanitized plastic tub and duplicate 10 g samples were aseptically weighed in appropriatelylabeled sterile Petrie dishes (each 56 cm 2 ). For each Petrie dish a sterile spatula was used to evenly spread the shreds of cheese to form a monolayer that covered the bottom of the dish. Each dish of shredded cheese was inoculated with 20 µl of mold spore suspension dispensed as four droplets on the surface of the layer of cheese to give approximately 10 4 CFU of mold spores per 10 g sample. Samples were stored at 7 C and at 25 C in laboratory incubators for 42 and 14 days, respectively. Observation of mold growth and microbiological analysis. All samples of shredded cheese were observed daily for mold growth. At 10 day intervals microbiological analysis was performed on cheese samples (7 C) inoculated 4 molds that exhibited the highest resistance to the LAB metabolite based on MIC results at ph 5.30 to 5.5. For microbial analysis, samples (10 g) of cheese were aseptically transferred to separate sterile two compartment filter stomacher bags. Sterile buffered peptone water (BPW; 90 ml) was added to each bag. Each mixture was pummeled for 1.0 minute using a laboratory stomacher at medium speed. The resulting cheese homogenates were serially diluted (10 fold) in BPW and diluted samples were surface plated on DRBC. All inoculated plates were incubated at 25 C for 5 days before counting fungal colonies. 4

6 Data analysis. Each experiment was repeated three times. A least significant difference analysis, adjusted by the Tukey Kramer procedure for treatment means was performed. Statistical differences were determined at a significance level of P<0.05. Statistical analysis was performed using SAS software (Version 7.0, SAS Institute Inc., Cary, NC). Results and Discussion Objective 1. Evaluate the antifungal activity of selected metabolic products of lactic acid bacteria (LAB) against several species of molds in laboratory broth Of the lactic acid bacterial products tested (4 hydroxy phenyllactic acid, DL 3 phenyllactic acid, 3 hydroxy fatty acids, and a cyclic dipeptide), the one that was most inhibitory to growth of molds was DL 3 phenyllactic acid (PLA). On this basis PLA was selected for use in subsequent experiments to characterize its effect against molds in a model system (BHI broth) and in shredded cheddar cheese. Figures 1, 2, and 3 show the growth of six mold species in BHI broth (25 C; ph 5.5) with or without (control) added PLA. Molds in control (0 mg/ml) grew rapidly within 100 to 120 hours. PLA at 2.5 or 3.75 mg/ml failed to inhibit growth irrespective of the mold species tested. Higher concentrations of 5.0 and 7.5 mg/ml extended the lag phase and decreased the growth rate of the molds. Growth of P. glabrum and P. roqueforti was strongly inhibited compared to that of the other molds tested. Similar results were obtained in BHI broth at ph 5.0 and ph 5.3; however, all molds tests exhibited a greater sensitivity to PLA at those lower ph values (Table 1A). Tables 1A and 1B show the MIC and MFC of PLA against 7 mold species in BHI broth (25 C) at ph 5.0, 5.3 and 5.5. Generally, the MIC of PLA against the molds decreased with decreasing ph values. Interestingly, for Penicillium roqueforti, Penicillium citrinum, and Aspergillus niger, the concentration of PLA that represented the MIC of PLA for these molds at ph 5.0 or 5.3 also caused death of their conidia (fruiting bodies that give rise to new mold). In fact no conidial germination was observed when aliquots (10 µl) of conidial suspension with added PLA (5.0 to 10 mg/ml) were sub cultured on DRBC plates and incubated for 120 hours, confirming the MFC of PLA for these molds. Our results indicate that PLA can inhibit growth of the molds tested in the present study and that the antifungal effect of PLA increases with decreased ph values. This increased antimicrobial activity of PLA at lowered ph is similar to that of other weak organic acid preservatives such as propionic acid, benzoic acid and sorbic acid. This ph dependent activity of PLA indicates that its antimicrobial mechanism of action is likely linked to its lipophilic properties which facilitate passage of the undissociated form of PLA across microbial membranes (Gould, 1996). In this regard PLA should be more effective against molds in cheddar cheese at ph 5.1 or 5.2 compared to ph 5.3 or 5.4. Apart from its strong growth inhibitory effect PLA exhibited fungicidal activity against all mold species tested (Table 1B). This suggests that application of PLA as an antifungal agent in cheese not only delays mold growth but also kill molds thus giving PLA an advantage over more traditional antifungal preservatives 5

7 such as sorbic acid, benzoic acid, and propionic acid. These preservatives are fungistatic and only temporarily delay mold growth (Lacey et al., 1991). Objective 2. Efficacy of PLA for preventing mold growth on shredded cheddar cheese Tables 2A and 2B show days when growth of molds became visible on shredded cheddar cheese treated with PLA and held at 7 C and 25 C, respectively. In control shredded cheddar cheese (without added PLA) held aerobically at 7 C, growth of molds became visible within 7 to 11 days. Cheese treated with PLA at 7.5 mg/ml and 10 mg/ml exhibited mold growth within 19 to 25 days and 24 to 35 days, respectively (Table 2A). In contrast, no mold growth was observed for 42 days in any of the shredded cheese treated with PLA at 12.5 mg/ml. In control cheese held at 25 C, mold became visible within 3 to 5 days. Mold growth in cheese treated with PLA at 7.5 and 10 mg/ml was visible within 6 to 11 days and 11 to 14 days, respectively. No mold growth was visible for 14 days in any of the shredded cheese treated with PLA at mg/ml (Table 2B). Growth of molds in shredded cheddar cheese with or without added PLA and stored aerobically at 7 C for 42 days are shown in Figures 4 and 5. The 4 mold species used in these viability studies were those that exhibited the highest resistance to the PLA based on MIC results at ph 5.30 to 5.5. In shredded cheese with no added PLA all molds grew rapidly; initial viable count increased from about 3 Log 10 CFU/g to more than 7 Log 10 CFU/g at 14 and ~ 21 days for Penicillium spp and Aspergillus spp, respectively. Growth inhibition increased with increasing concentrations of PLA treatments. For all molds the PLA treatment of 12.5 mg/ml completely suppressed growth. The observed inhibition of mold growth in cheddar cheese under aerobic conditions at 7 C is significant because cheeses are refrigerated and can exhibit mold growth if not protected by an antifungal agent. Also molds are aerobic and if present, they will grow on shredded cheddar cheese when this product is exposed to air. In the present study, the marked delay in appearance of mold on cheese treated PLA at 10 or 12.5 mg/ml and stored aerobically at 7 C or 25 C attests to the antifungal ability of PLA. These results indicate that PLA has good potential for practical use as a fungicidal agent in cheese due to its broad inhibitory effect against several species of fungi especially Penicillium species which represent a major group of fungi that spoil cheese. In fact, the most common molds isolated from hard cheeses are Penicillium species (>80%) with Aspergillus spp representing less than 8% (Committee on Toxicity of Chemicals in Foods, Consumer Products and the Environment, 2006). While natamycin and sorbates are the only approved antifungal preservatives for preventing mold growth in cheese, several problems with use of those preservatives warrant their replacement. For example, some cheese spoilage fungi including Penicillium and Aspergillus can degrade sorbate to produce an unpleasant hydrocarbon like odor in cheese (Marth et al., 1966; Sofos and Busta, 1981; Kinderlerer and Hatton, 1990). Also, the effectiveness of natamycin as an antifungal agent in cheese is limited by its relative insolubility and uneven distribution on the cheese surface. These problems can be circumvented by use of PLA which exhibits potent antifungal effect, good water solubility and ability to be evenly distributed in cheese without wastage as occurs in application of natamycin. 6

8 Conclusions Based on the results of the present study, it can be concluded that PLA has good potential for use as a natural fungicidal preservative in shredded cheddar cheese. The natural occurrence of various amounts of PLA in samples of honey from several countries around the world (Weston et al., 1999) and non toxicity of PLA for animal and human cells (Oberdoerster et al., 2000) might permit its safe use in cheese. Benefit to Minnesota Economic Development The two types of cheese that are associated with the highest consumption in the United States are cheddar and mozzarella with an estimated per capita consumption of lb and lb, respectively. In fact, cheddar is among four cheese types (Cheddar, Mozzarella, American and Colby Jack) with the largest volume of sales at retail supermarkets (International Dairy Foods Association, 2011). Considering the fact that health conscious consumers are demanding more natural food additives as replacements for synthetic or some traditional food preservatives, the use of PLA as a replacement for Natamycin in cheddar cheese can further increase sales of this nutritious dairy product. This in turn would produce positive economic benefits due to increased sales of milk for dairy producers. References Committee on Toxicity of Chemicals in Foods, Consumer Products and the Environment Mycotoxins in cheese. TOX/2006/43. Available at: Accessed December 12, Gould, G. W Methods for preservation and extension of shelf life. Int. J. Food Microbiol. 33: International Dairy Foods Association Cheese Sales and Trends. Available at: views/media kits/cheese/cheese sales and trends/ Accessed January 15, Kieronczyk, A., S. Skeie, T. Langsrud, and M. Yvon Cooperation between Lactococcus lactis and nonstarter lactobacilli in the formation of cheese aroma from amino acids. Appl. Environ. Microbiol. 69: Kinderlerer, J. L., and P. V. Hatton Fungal metabolites of sorbic acid. Food Addit. Contam. 7: Lacey, J., N. Ramakrishna, A. Haner, N. Magan, and I. C. Marfleet Grain fungi, p In D. K. Arora, K. G. Murkerji, and E. H. Marth (ed.) Handbook of Applied mycology, Vol 3. Foods and Feeds, marcel Dekker, Inc, New York, NY. 7

9 Niku Paavola, M. L., A. Laitila, T. Mattila Sandholm, and A. Haikara New types of antimicrobial compounds produced by Lactobacillus plantarum. J. Appl. Microbiol. 86: Lavermicocca, P. F., A. Valerio, A. Evidente, S. Lazzaroni, A. Corsetti, and M. Gobbetti Purification and characterization of novel antifungal compounds from the sourdough Lactobacillus plantarum strain 21B. Appl. Environ. Microbiol. 66: Lavermicocca, P. F., A. Valerio, and A. Visconti Antifungal activity of phenyllactic acid against molds isolated from bakery products. Appl. Environ. Microbiol. 69: Magnusson, J., and J. Schnurer Lactobacillus coryniformis subsp. Coryniformis strain Si3 produces a broad spectrum proteinaceous antifungal compound. Appl. Environ. Microbiol. 67:1 5. Magnusson, J., K. Strom, S. Roos, J. Sjogren and J. Schnurer Broad and complex antifungal activity among environmental isolates of lactic acid bacteria. FEMS Microbiol. Lett. 219: Marth, E. H., C. M. Capp, L. Hasenzahl, H. W. Jackson, and R. V. Hussong Degradation of potassium sorbate by Penicillium species. J. Dairy Sci. 49: Oberdoerster, j., M. Guizzetti, and L. G. Costa Effect of phenylalanine and its metabolites on the proliferation and viability of neuronal and astroglial cells: possible relevance in maternal phenylketonuria. J. Pharmacol. Exp. Ther. 295; Sofos, J. N., and F. F. Busta Antimicrobial activity of sorbate. J. Food Prot. 44: Strom, K., J. Sjogren, A. Broberg, and J. Schnurer Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo (L Phe L Pro) and cyclo (L Phe Trans 4 OH L Prp) and 3 phenyllactic acid. Appl. Environ. Microbiol. 68: Weston, R. J., k. R. Mitchell, and K. L. Allen Antibacterial phenolic components of New Zealand manuka honey. Food Chem. 64:

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