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Evaluation of the efficacy of commercial protective cultures to inhibit mold and yeast in cottage cheese

Open ArchivePublished:January 14, 2021DOI:https://doi.org/10.3168/jds.2020-19136

      ABSTRACT

      Biopreservation is defined as using microbes, their constituents, or both to control spoilage while satisfying consumer demand for clean-label products. The study objective was to investigate the efficacy of bacterial cultures in biopreserving cottage cheese against postprocessing fungal contamination. Cottage cheese curd and dressing were sourced from a manufacturer in New York State. Dressing was inoculated with 3 different commercial protective cultures—PC1 (mix of Lacticaseibacillus spp. and Lactiplantibacillus spp.), PC2 (Lacticaseibacillus rhamnosus), and PC3 (Lactic. rhamnosus)—following the manufacturer recommended dosage and then mixed with curd. A control with no protective culture was included. Nine species of yeast (Candida zeylanoides, Clavispora lusitaniae, Debaryomyces hansenii, Debaryomyces prosopidis, Kluyveromyces marxianus, Meyerozyma guilliermondii, Pichia fermentans, Rhodotorula mucilaginosa, and Torulaspora delbrueckii) and 11 species of mold (Aspergillus cibarius, Aureobasidium pullulans, Penicillium chrysogenum, Penicillium citrinum, Penicillium commune, Penicillium decumbens, Penicillium roqueforti, Mucor genevensis, Mucor racemosus, Phoma dimorpha, and Trichoderma amazonicum) were included in the study. Fungi strains were previously isolated from dairy processing environments and were inoculated onto the cheese surface at a rate of 20 cfu/g. Cheese was stored at 6 ± 2°C. Yeast levels were enumerated at 0, 7, 14, and 21 d postinoculation. Mold growth was visually observed on a weekly basis through d 42 of storage and imaged. Overall, the protective cultures were limited in their ability to delay the outgrowth in cottage cheese, with only 8 of the 20 fungal strains showing an effect of the cultures compared with the control. The protective cultures were not very effective against yeast, with only PC1 able to delay the outgrowth of 3 strains: D. hansenii, Tor. delbrueckii, and Mey. guilliermondii. The efficacy of these protective cultures against molds in cottage cheese was more promising, with all protective cultures showing the ability to delay spoilage of at least 1 mold strain. Both PC1 and PC2 were able to delay Pen. chrysogenum and Pho. dimorpha outgrowth, and PC1 also delayed Pen. commune, Pen. decumbens, and Pen. roqueforti to different extents compared with the controls. This study demonstrates that commercial lactic acid bacteria cultures vary in their performance to delay mold and yeast outgrowth, and thus each protective culture should be evaluated against the specific strains of fungi of concern within each specific dairy facility.

      Key words

      INTRODUCTION

      In 2017, average annual consumption of cottage cheese in the United States was estimated at 675 million pounds (∼307 million kg;
      • Statistica
      U.S. domestic consumption of cottage cheese 2018.
      ). Cottage cheese is a soft, fresh cheese formed by mixing dry curd with a creaming mixture (
      • FDA (Food and Drug Administration)
      Code of Federal Regulations Title 21 Food and Drugs. Vol. 2, Part 133.
      ). For regular cottage cheese with 4% fat, moisture content is around 80% with pH values as high as 5.2 (
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      ). These attributes make cottage cheese highly susceptible to microbial spoilage (
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      ).
      Fungi are ubiquitous in nature. Fresh cheeses are nutrient rich, and fungal contamination can occur at different points of the value chain, including at the farm level, at the processing plant, and once it has reached consumers. Fungal strains commonly associated with fresh cheese spoilage include Candida spp., Debaryomyces spp., Pichia spp., Penicillium spp., Aspergillus spp., and Mucor spp. (
      ;
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      Antifungal activity of lactic and propionic acid bacteria and their potential as protective culture in cottage cheese.
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      ).
      To control potential spoilage, the food industry has adopted hurdle technology, which involves creating barriers (hurdles) to microbial growth, including heat treatment, modified atmospheric packaging, food-grade preservatives, and biopreservation (
      • Fernandez B.
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      Antifungal activity of lactic and propionic acid bacteria and their potential as protective culture in cottage cheese.
      ). These are being explored and used in the dairy industry as postpasteurization contamination represents a continued challenge. However, even with the use of hurdle technology, technologies may create additional challenges in regard to consumer acceptability. For example, the ability of carbon dioxide to dissolve in water and fat has been shown to result in packaging collapse and negatively affect consumer acceptability of modified atmospheric packaging (
      • Ho T.M.
      • Howes T.
      • Bhandari B.R.
      Methods to extend the shelf-life of cottage cheese—A review.
      ). Some fungal strains are able to degrade sorbate, creating a “kerosene-like” flavor and decreasing the effectiveness of sorbate as a preservative (
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      • Fliss I.
      Antifungal activity of lactic and propionic acid bacteria and their potential as protective culture in cottage cheese.
      ). Exploration into other technologies is necessary to prevent spoilage while maintaining consumer acceptance levels.
      Increased consumer demand for clean-labels products has necessitated the exploration of natural alternatives to protect against fungal spoilage. Biopreservation is gaining interest as a way to naturally preserve food, enhance food safety, and extend shelf life using agents of animal, plant, or bacterial origin. Bacteriocins, propionic bacteria, and lactic acid bacteria (LAB) are examples of biopreservatives of bacterial origin (
      • Garnier L.
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      • Mounier J.
      Diversity and control of spoilage fungi in dairy products: An update.
      ). Lactic acid bacteria are non-spore-forming, facultative, aerobic, gram-positive bacteria that have a long history of being used in food fermentations. Due to this history and their Generally Recognized as Safe status by the US Food and Drug Administration, there is great interest in expanding the application of LAB as a method of biopreservation (
      • Crowley S.
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      Current perspectives on antifungal lactic acid bacteria as natural bio-preservatives.
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      The antifungal activity of LAB can be attributed to a range of mechanisms. First, the production of a broad range of acids such as lactic acid, phenyllactic, and 4-hydroxyphenyllactic reduces the medium pH (
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      Isolation of lactic acid bacteria with antifungal activity against the common cheese spoilage mould Penicillium commune and their potential as biopreservatives in cheese.
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      Food Biopreservation. SpringerBriefs in Food, Health, and Nutrition.
      ). Another proposed mechanism of action is the secretion of metabolites such as reuterin by Lactobacillus spp., which may disrupt DNA synthesis (
      • Galvez A.
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      • Pérez Pulido R.
      Food Biopreservation. SpringerBriefs in Food, Health, and Nutrition.
      ). Dipeptides, diacetyl, bacteriocins, and fatty acids exhibiting antifungal activity can also contribute to the antifungal properties of LAB (
      • Crowley S.
      • Mahony J.
      • van Sinderen D.
      Current perspectives on antifungal lactic acid bacteria as natural bio-preservatives.
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      • Aunsbjerg S.D.
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      • Knøchel S.
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      ). Effective inhibition of yeast and mold thought to be competition for essential resources, such as manganese, by LAB has also been demonstrated (
      • Siedler S.
      • Rau M.H.
      • Bidstrup S.
      • Vento J.M.
      • Aunsbjerg S.D.
      • Bosma E.F.
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      • Beisel C.L.
      • Neves A.R.
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      ).
      Several commercially available LAB cultures are marketed for their antifungal efficacy, but little published data exist for cheese manufactures to gauge how these blends compare in antifungal spectrum and their potential for shelf-life improvement in fresh cheeses. In this study, we investigated the efficacy of LAB cultures in biopreservation against spoilage fungi in cottage cheese. The study objectives were to evaluate the efficacy of 3 commercially available LAB cultures advertised as providing protection against fungal spoilage in fresh cheese and to provide a benchmark for cottage cheese producers to determine which cultures may be most effective for their products. We hypothesized that commercial LAB cultures vary in performance of mold and yeast inhibition at both genus and species level, and thus each may be ideal against specific strains depending on the food matrix.

      MATERIALS AND METHODS

      Lactic Acid Bacteria and Fungi Strains

      Three commercial bioprotective cultures—PC1 (mix of Lacticaseibacillus spp. and Lactiplantibacillus spp.), PC2 (Lacticaseibacillus rhamnosus), and PC3 (Lactic. rhamnosus)—were used throughout the study to challenge fungal spoilage organisms in cottage cheese. The manufacturer and brand name of the protective cultures were not disclosed for proprietary purposes, but further information is available upon written request to the authors. However, cultures were confirmed as a mix of Lacticaseibacillus spp. and Lactiplantibacillus spp., Lactic. rhamnosus, and Lactic. rhamnosus, respectively, through 16s rDNA sequencing (
      • Makki G.M.
      • Kozak S.M.
      • Jencarelli K.G.
      • Alcaine S.D.
      Evaluation of the efficacy of commercial protective cultures against mold and yeast in queso fresco.
      ). All protective cultures were kept at −80 ± 2°C until use.
      Fungal spoilage strains used throughout the study were previously isolated from dairy processing plants and were sourced from the Cornell University Food Safety Laboratory isolate collection and the Alcaine Research Group isolate collection (Ithaca, NY). Nine species of yeast (Candida zeylanoides, Clavispora lusitaniae, Debaryomyces hansenii, Debaryomyces prosopidis, Kluyveromyces marxianus, Meyerozyma guilliermondii, Pichia fermentans, Rhodotorula mucilaginosa, and Torulaspora delbrueckii) and 11 species of mold spanning 6 genera (Aspergillus cibarius, Aureobasidium pullulans, Penicillium chrysogenum, Penicillium citrinum, Penicillium commune, Penicillium decumbens, Penicillium roqueforti, Mucor genevensis, Mucor racemosus, Phoma dimorpha, and Trichoderma amazonicum) were used throughout the study. Fungal stocks were kept frozen at −80 ± 2°C. Table 1 shows genus and species identification, type of microorganism, and isolation source for spoilage organisms used throughout the study.
      Table 1Genus and species identification, type, location, and isolation source for spoilage organisms used throughout the study
      Strain
      All strains were from the Food Safety Laboratory at Cornell University (Ithaca, NY) expect for Mucor genevensis,which was from the Alcaine Research Group collection at Cornell University.
      TypeID
      References identification of isolate in Food Microbe Tracker. Additional information can be found at www.foodmicrobetracker.com.
      Isolate source
      Candida zeylanoidesYeastB90031Cheese
      Clavispora lusitaniaeYeastB90007Raw milk
      Debaryomyces hanseniiYeastB90013Cheese
      Debaryomyces prosopidisYeastB90028Cheese
      Kluyveromyces marxianusYeastB90008Raw milk
      Meyerozyma guilliermondiiYeastE20377Yogurt
      Pichia fermentansYeastB90001Raw milk
      Rhodotorula mucilaginosaYeastE20331Dairy processing environment
      Torulaspora delbrueckiiYeastE20442Yogurt
      Aspergillus cibariusMoldE20323Dairy processing environment
      Aureobasidium pullulansMoldE20290Yogurt
      Mucor genevensisMoldTD0021Yogurt
      Mucor racemosusMoldE20368Yogurt
      Penicillium chrysogenumMoldE20332Dairy processing environment
      Penicillium citrinumMoldE20297Yogurt
      Penicillium communeMoldB90026Cheese
      Penicillium decumbensMoldE20320Dairy processing environment
      Penicillium roquefortiMoldE20329Dairy processing environment
      Phoma dimorphaMoldE20369Yogurt
      Trichoderma amazonicumMoldE20387Yogurt
      1 All strains were from the Food Safety Laboratory at Cornell University (Ithaca, NY) expect for Mucor genevensis,which was from the Alcaine Research Group collection at Cornell University.
      2 References identification of isolate in Food Microbe Tracker. Additional information can be found at www.foodmicrobetracker.com.

      Preparation of Yeast Inoculum

      For each respective yeast strain, frozen yeast stocks were allowed to thaw on ice and propagated on the surface of potato dextrose agar plates (PDA; Hardy Diagnostics, Santa Maria, CA) followed by incubation at 25°C for 48 h. A single colony was isolated from each plate and inoculated in 5 mL of potato dextrose broth (HiMedia Laboratories Pvt. Ltd., Mumbai, India), followed by incubation at 25°C for 16 h. Cultures were transferred into sterile 2-mL cryovials (Simport, Beloeil, QC, Canada) and supplemented with glycerol at 50% (vol/vol) to form yeast stock suspensions. Yeast stocks were stored at −80 ± 2°C until use. To enumerate yeast stocks, each stock was serially diluted in PBS and plated on PDA, followed by incubation at 25°C for 48 h. Yeast colonies were enumerated using a Q-Count colony counter (Advanced Instruments, Norwood, MA).

      Preparation of Mold Spore Suspensions

      Frozen isolates of each mold strain were allowed to thaw on ice and spotted onto the surface of malt extract agar plates (Difco, Franklin Lakes, NJ), followed by incubation at 25°C for 30 d until spore formation. Plates were flooded with PBS containing 0.1% Tween 80 (Tokyo Chemical Industry Co. Ltd., Tokyo, Japan), and then gently scraped using a sterile cell spreader to release spores and mycelia. Mycelia were filtered from the mold suspensions by passing through 4 layers of sterile cheesecloth. Spore formation was confirmed microscopically (Reichert microscope, Reichert Technologies, Depew, NY). In sterile 50-mL conical tubes (VWR, Radnor, PA), spore suspensions were supplemented with glycerol at 50% (vol/vol). Spore suspensions were stored at −80 ± 2°C until use.
      For each mold strain, spore suspension concentrations were determined by microscopy with a Neubauer's improved counting chamber hemocytometer (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany). Spore counts were confirmed by plating in duplicate on dichloran rose bengal chloramphenicol agar (Becton, Dickinson and Co., Sparks, MD), followed by incubation at 25°C for 5 d.

      Cottage Cheese Sourcing and Inoculation with Protective Cultures

      Separate dry curd and cream dressing of freshly made, preservative-free, regular cottage cheese (4% fat) were sourced from a cheese manufacturer in upstate New York. Immediately before inoculation, each commercial protective culture was resuspended in UHT processed fat-free milk (Parmalat, Buffalo, NY) and used to inoculate cream dressing. For each treatment, cream dressing was inoculated with the commercial protective cultures (PC1, PC2, and PC3) to achieve the manufacturer recommended dosage in the cottage cheese. Curd was added to cream dressing and mixed well to ensure homogeneity. A positive control with no protective culture was included. All steps were carried out under aseptic conditions. Cheese was stored under refrigeration conditions (6 ± 2°C) overnight.

      Efficacy of Bioprotective Cultures Against Yeast on Cottage Cheese

      Following overnight refrigeration of creamed cottage cheese, four 6-well plates (3.5 cm in diameter; Falcon, Corning, NY) were prepared for each yeast strain, where a single plate was assigned for each of the 3 treatments (PC1, PC2, PC3) and the positive control. In each well, 5 ± 0.5 g of cottage cheese was added aseptically. In each well, yeast strains were inoculated on the surface of cheese samples with a target rate of 20 cfu/g. Throughout the study, plates were stored at refrigeration temperature (6 ± 2°C).
      Yeast enumeration was conducted at d 0, 7, 14, and 21 postinoculation. For sampling, the contents of one well were aseptically transferred to a stomacher bag (Whirl-Pak, Nasco, Fort Atkinson, WI) and 45 mL of PBS was added to realize a 1:9 ratio followed by homogenization at 230 rpm for 60 s using a Seward Stomacher 400 Circulator blender (Seward Ltd., Worthing, UK). Samples were serially diluted and plated in duplicate on PDA supplemented with 25 mg of chloramphenicol/L of medium (Sigma-Aldrich, St. Louis, MO). Plates were incubated at 25°C for 5 d. Figure 1 shows a diagram outlining yeast inoculation and enumeration in cottage cheese.
      Figure thumbnail gr1
      Figure 1Schematic diagram of yeast challenge study in cottage cheese. C = control cheese without protective culture; PC1 = mix of Lacticaseibacillus spp. and Lactiplantibacillus spp.; PC2 = Lacticaseibacillus rhamnosus; PC3 = Lactic. rhamnosus.

      Efficacy of Bioprotective Cultures Against Mold on Cottage Cheese

      Following overnight refrigeration of cottage cheese, two 6-well plates were prepared for each challenged mold strain, where the positive control and PC1 represent the first and second rows of wells, respectively, of the first 6-well plate, and PC2 and PC3 represent the first and second rows of wells, respectively, of the second 6-well plate. In each well, 5 ± 0.5 g of cottage cheese was aseptically added. Mold strains were surface-inoculated on cottage cheese samples in each well at a target rate of 20 cfu/g. Plates were stored at refrigeration temperature (6 ± 2°C) throughout the study.
      To assess mold outgrowth on cottage cheese, plates were visually observed at d 0, 7, 14, 21, 28, 35, and 42 postinoculation and imaged using a PowerShot SX530 160-MP HS digital camera (Canon, Tokyo, Japan). After 42 d, fungal spoilage occurred in the negative control cheeses. The following scale was used to report results: (−) no visible mold growth, (+*) cheeses transitioning into matte appearance in some replicates, (+) matte appearance with no colored mold growth in all replicates, (++*) cheeses transitioning into colored mold growth in some replicates, and (++) mold growth with change in color across replicates. Figure 2 shows a schematic diagram of mold inoculation and visual examination in cottage cheese stored at 6 ± 2°C.
      Figure thumbnail gr2
      Figure 2Schematic diagram of mold challenge study on cottage cheese. C = control cheese without protective culture; PC1 = mix of Lacticaseibacillus spp. and Lactiplantibacillus spp.; PC2 = Lacticaseibacillus rhamnosus; PC3 = Lactic. rhamnosus.

      Cheese Physical Properties

      Moisture content and pH values were measured for 2 samples per treatment using a microwave oven (CEM Inc., Matthews, NC) and edge pH meter (Hanna Instruments, Smithfield, RI), respectively.

      Statistical Analysis

      Experiments were performed in triplicate for both yeast and mold studies. Significant differences (P < 0.05) between the counts of each yeast strain were determined by comparing yeast counts (log cfu/g) for each protective culture against the control cheese with no protective culture using a 1-way ANOVA with Bonferroni correction performed individually at time points 7, 14, and 21 d postinoculation. For these studies, a biological difference was defined as having both a significant difference (P < 0.05) and at least a 1 log cfu/g difference between the counts on the treatment and control cheeses. Statistical analysis was performed using JMP Pro software version 14 (SAS Institute Inc., Cary, NC).

      RESULTS AND DISCUSSION

      The average pH values of the control cheese and the PC1-, PC2-, and PC3-inoculated cheeses were 4.97 ± 0.09, 4.75 ± 0.16, 4.95 ± 0.04, and 4.90 ± 0.06, respectively. Average moisture levels of the control cheese and the PC1-, PC2-, and PC3-inoculated cheeses were 78.32 ± 1.32, 78.51 ± 1.44, 78.50 ± 1.43, and 79.10 ± 1.28%, respectively. For the yeast challenge study, negative controls of each treatment showed no growth through 21 d of incubation at 6 ± 2°C. In the context of this study, the yeast count that results in spoilage was selected as >5 log cfu/g. At this level, consumers can detect flavor changes, visible defects, and textural changes (
      • Zantar S.
      • Yedri F.
      • Mrabet R.
      • Laglaoui A.
      • Bakkali M.
      • Zerrouk M.H.
      Effect of Thymus vulgaris and Origanum compactum essential oils on the shelf life of fresh goat cheese.
      ). For yeast counts below the detection limit, a value of 1.3 log cfu/g was used to express counts.
      Overall, the protective cultures were mostly ineffective at controlling the growth of yeast in cottage cheese (Figure 3). Of the 9 strains used in experiments, 6 strains (Cla. lusitaniae, Can. zeylanoides, D. prosopidis, Klu. marxianus, Pic. fermentans, and R. mucilaginosa) all readily grew in cottage cheeses, with no significant difference in outgrowth between the control and the treatments with protective cultures (Figure 3). Two strains, Can. zeylanoides and D. prosopidis, rapidly surpassed the spoilage limit of 5 log cfu/g by d 7, whereas the other 4 strains reached the spoilage limit by d 14.
      Figure thumbnail gr3
      Figure 3Growth of yeast strains on cottage cheese at 6 ± 2°C. C = control without protective culture (black squares and dotted line); PC1 = mix of Lacticaseibacillus spp. and Lactiplantibacillus spp. (gray squares and solid line); PC2 = Lacticaseibacillus rhamnosus (gray circles and dotted line); PC3 = Lactic. rhamnosus (gray triangles and dashed line). (A) Clavispora lusitaniae, (B) Candida zeylanoides, (C) Debaryomyces hansenii, (D) Debaryomyces prosopidis, (E) Kluyveromyces marxianus, (F) Meyerozyma guilliermondii, (G) Pichia fermentans, (H) Rhodotorula mucilaginosa, and (I) Torulaspora delbrueckii. For counts below the detection limit, a value of 1.3 log cfu/g was used. The horizontal dashed line represents the threshold (cfu/g) for spoilage. Error bars reflect SD. Asterisk indicates that counts were significantly different from C within a sampling day (P < 0.05).
      Only 1 of the protective cultures in this study, PC1, had an effect on the outgrowth of D. hansenii, Mey. guilliermondii, and Tor. delbrueckii compared with the control (Figure 3). In the case of D. hansenii, at d 14, PC1 had significantly slowed the outgrowth of this strain compared with the control and other protective cultures (Figure 3) to below the spoilage limit. However, by d 21 D. hansenii was able to overcome the inhibitory effect of PC1, surpassing the spoilage limit and reaching a final level that was not significantly different from that of the control. In regard to Mey. guilliermondii, cheeses treated with PC1 resulted in significantly lower counts compared with the control cheese and the PC2 and PC3 treatments on d 14 and 21 (P < 0.0001; Figure 3F), although Mey. guilliermondii counts still exceeded spoilage limits by d 14. Our results indicate that PC1 does inhibit Mey. guilliermondii and may delay time to spoilage if the load of Mey. guilliermondii in a cottage cheese facility is lower than the inoculation level used in this study, although more research is needed. Torulaspora delbrueckii was the only yeast strain that was significantly inhibited by a protective culture, again PC1, compared with the control and that was kept from exceeding the spoilage limit over the 21-d study. Recent studies on Lactic. rhamnosus and Lacticaseibacillus paracasei have demonstrated the ability of several strains to inhibit the outgrowth of both D. hansenii and Tor. delbrueckii in fermented milk through competition for manganese (
      • Siedler S.
      • Rau M.H.
      • Bidstrup S.
      • Vento J.M.
      • Aunsbjerg S.D.
      • Bosma E.F.
      • McNair L.M.
      • Beisel C.L.
      • Neves A.R.
      Competitive exclusion is a major bioprotective mechanism of Lactobacilli against fungal spoilage in fermented milk products.
      ). In this study, manganese levels in the cottage cheese were not measured, so it is unclear whether such trace mineral competition accounts for the ability of PC1 to affect the growth of these spoilage organisms in cottage cheese; however, PC2 and PC3, which are Lactic. rhamnosus strains, did not show any inhibitory effect. In a similar challenge study that we performed with these protective cultures in queso fresco, all 3 cultures were able to inhibit D. hansenii outgrowth, and 2 were able inhibit Tor. delbrueckii (
      • Makki G.M.
      • Kozak S.M.
      • Jencarelli K.G.
      • Alcaine S.D.
      Evaluation of the efficacy of commercial protective cultures against mold and yeast in queso fresco.
      ), but it is not uncommon to see LAB, including Lactic. rhamnosus strains, perform differently in different dairy substrates (
      • Leyva Salas M.L.
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      • Mounier J.
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      • Coton E.
      Antifungal activity of lactic acid bacteria combinations in dairy mimicking models and their potential as bioprotective cultures in pilot scale applications.
      ,
      • Leyva Salas M.L.
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      • Maillard M.-B.
      • Valence F.
      • Coton E.
      • Thierry A.
      Identification and quantification of natural compounds produced by antifungal bioprotective cultures in dairy products.
      ). The difference in matrix attributes, such as pH (which was around 4.9 for the cottage cheese vs. 6.3 for the queso fresco in our study) as well as moisture (which was around 78% for the cottage cheese vs. 60% in the queso fresco), potentially affects the activity of both the protective cultures and the challenge fungi, although more research is need to understand in what manner (
      • Makki G.M.
      • Kozak S.M.
      • Jencarelli K.G.
      • Alcaine S.D.
      Evaluation of the efficacy of commercial protective cultures against mold and yeast in queso fresco.
      ).
      In this study, the antifungal activity of protective cultures against mold in cottage cheese was evaluated by comparing visible mold growth on control cheese with no protective culture and on cheeses with protective cultures inoculated with the same mold strain. Examination was performed on a weekly basis over 42 d of refrigerated storage (6 ± 2°C). Day 42 was selected as the cutoff point of the study because beyond that negative control cheeses, thus not inoculated with mold, started showing visual growth. Table 2 shows the results of mold growth over 42 d on cottage cheese at 6 ± 2°C.
      Table 2Mold growth over 42 d on cottage cheese at 6 ± 2°C
      Mold growth rating scale: (−) no visible mold growth, (+*) transitioning to matte appearance, (+) matte appearance with no colored mold growth, (++*) transitioning to colored mold growth, (++) mold growth with change in color. At d 0 and 7 no growth was observed for any mold strain across treatments.
      Visual examination dayStrain
      PC1 = mix of Lacticaseibacillus spp. and Lactiplantibacillus spp.; PC2 = Lacticaseibacillus rhamnosus; PC3 = Lactic. rhamnosus; C = control with no protective culture.
      Aspergillus cibariusAureobasidium pullulansMucor genevensisMucor racemosusPenicillium chrysogenumPenicillium citrinumPenicillium communePenicillium decumbensPenicillium roquefortiPhoma dimorphaTrichoderma amazonicumNegative control
      Day 42PC3++++++++*
      PC2++++++++*
      PC1+*++*
      C++++++++*
      Day 35PC3++++++++*
      PC2++++++++*
      PC1+*
      C++++++++*
      Day 28PC3++++++*++*
      PC2++++++*++*
      PC1+*
      C++++++*++*
      Day 21PC3++++++++
      PC2++++*++++
      PC1+++*++
      C++++++++++*
      Day 14PC3++*+++++*+++*
      PC2++*+++++++*
      PC1++*++++
      C++*+++++*+++*
      1 Mold growth rating scale: (−) no visible mold growth, (+*) transitioning to matte appearance, (+) matte appearance with no colored mold growth, (++*) transitioning to colored mold growth, (++) mold growth with change in color. At d 0 and 7 no growth was observed for any mold strain across treatments.
      2 PC1 = mix of Lacticaseibacillus spp. and Lactiplantibacillus spp.; PC2 = Lacticaseibacillus rhamnosus; PC3 = Lactic. rhamnosus; C = control with no protective culture.
      For 3 mold species (Asp. cibarius, Tri. amazonicum, and Pen. citrinum), no visual growth was observed throughout d 42 on any treatment, including positive control with no protective culture (Table 2). The viability of the stocks was confirmed (data not shown), and although the isolates were from dairy products and the dairy environment, these results suggest that they do not grow well in cottage cheese.
      None of the protective cultures inhibited the outgrowth of strains of Aur. pullulans, Muc. genevensis, or Muc. racemosus compared with the control (Table 2). Figure 4 shows Muc. racemosus outgrowth on cottage cheese at d 7 and 14 after incubation at 6 ± 2°C. At d 14, all cheeses inoculated with protective cultures as well as control cheese showed “cat hair” growth characteristic for Mucor spp. on their surface. This aligns with our queso fresco study, where none of the protective cultures delayed visible outgrowth of the Mucor spp. strains compared with the control (
      • Makki G.M.
      • Kozak S.M.
      • Jencarelli K.G.
      • Alcaine S.D.
      Evaluation of the efficacy of commercial protective cultures against mold and yeast in queso fresco.
      ). A study screening antifungal LAB in both yogurt and cheese systems also found only slight inhibition of Muc. racemosus by Lactic. rhamnosus strains (
      • Leyva Salas M.L.
      • Thierry A.
      • Lemaître M.
      • Garric G.
      • Harel-Oger M.
      • Chatel M.
      • Lê S.
      • Mounier J.
      • Valence F.
      • Coton E.
      Antifungal activity of lactic acid bacteria combinations in dairy mimicking models and their potential as bioprotective cultures in pilot scale applications.
      ). Another study screening LAB strains, including Lactic. rhamnosus, did show evidence of inhibition of Muc. racemosus in an overlay assay; however, its performance in a dairy matrix was not evaluated (
      • Fernandez B.
      • Vimont A.
      • Desfossés-Foucault É.
      • Daga M.
      • Arora G.
      • Fliss I.
      Antifungal activity of lactic and propionic acid bacteria and their potential as protective culture in cottage cheese.
      ).
      Figure thumbnail gr4
      Figure 4Mucor racemosus outgrowth on cottage cheese at d 7 and 14 of incubation at 6 ± 2°C. C = control cheese without protective culture; PC1 = mix of Lacticaseibacillus spp. and Lactiplantibacillus spp.; PC2 = Lacticaseibacillus rhamnosus; PC3 = Lactic. rhamnosus.
      The 4 Penicillium spp. strains that grew on cottage cheese were all inhibited differently by each protective culture (Table 2). Visible outgrowth of Pen. decumbens was inhibited over the 42-d challenge study by PC1, whereas the other 2 protective controls did not delay outgrowth compared with the control. A similar pattern was observed with Pen. chrysogenum, which was inhibited over the study by PC1, and but in this case PC2 was also able to delay visible outgrowth of the mold by 1 wk over the control to d 21; PC3 did not inhibit the outgrowth of Pen. chrysogenum. Figure 5 shows Pen. chrysogenum growth on cottage cheese at d 21 and 42 of storage at 6 ± 2°C.
      • Fernandez B.
      • Vimont A.
      • Desfossés-Foucault É.
      • Daga M.
      • Arora G.
      • Fliss I.
      Antifungal activity of lactic and propionic acid bacteria and their potential as protective culture in cottage cheese.
      demonstrated the ability of Lactic. rhamnosus strain A238, both alone and when used with Bifidobacterium animalis ssp. lactis A026, to inhibit Pen. chrysogenum outgrowth on cottage cheese by at least 21 d under refrigerated conditions. In our queso fresco study, 2 of the protective cultures were able to inhibit the outgrowth of Pen. chrysogenum over the 42-d study (
      • Makki G.M.
      • Kozak S.M.
      • Jencarelli K.G.
      • Alcaine S.D.
      Evaluation of the efficacy of commercial protective cultures against mold and yeast in queso fresco.
      ).
      Figure thumbnail gr5
      Figure 5Penicillium chrysogenum outgrowth on cottage cheese at d 21 and 42 of incubation at 6 ± 2°C. C = control cheese without protective culture; PC1 = mix of Lacticaseibacillus spp. and Lactiplantibacillus spp.; PC2 = Lacticaseibacillus rhamnosus; PC3 = Lactic. rhamnosus.
      Both Pen. roqueforti and Pen. commune grew well in cottage cheese, with only PC1 able to delay visible outgrowth by 1 wk compared with the control (Table 2). Partial inhibition of the level of Pen. commune by PC1 continued through d 42. In contrast, another study by
      • Cheong E.Y.L.
      • Sandhu A.
      • Jayabalan J.
      • Kieu Le T.T.
      • Nhiep N.T.
      • My Ho H.T.
      • Zwielehner J.
      • Bansal N.
      • Turner M.S.
      Isolation of lactic acid bacteria with antifungal activity against the common cheese spoilage mould Penicillium commune and their potential as biopreservatives in cheese.
      screening the antifungal activity of 800 LAB isolates in cottage cheese found 12 isolates that exhibited antifungal activity against Pen. commune, although all were strains of Lactip. plantarum. In our queso fresco study, visible outgrowth of Pen. roqueforti and Pen. commune was delayed by varying degrees by all 3 protective cultures (
      • Makki G.M.
      • Kozak S.M.
      • Jencarelli K.G.
      • Alcaine S.D.
      Evaluation of the efficacy of commercial protective cultures against mold and yeast in queso fresco.
      ). A study evaluating the effect of commercially available protective cultures to improve Greek yogurt quality also found Pen. commune readily inhibited over 60 d, even with culture levels used at rates below the manufacturer recommendations (
      • Buehler A.J.
      • Martin N.
      • Boor K.
      • Wiedmann M.
      Evaluation of biopreservatives in Greek yogurt to inhibit yeast and mold spoilage and development of a yogurt spoilage predictive model.
      ).
      Interestingly, all 3 protective cultures demonstrated some level of efficacy against Pho. dimorpha (Table 2). All 3 were able to delay visible outgrowth by 21 wk, to d 28, and PC1 was able to delay outgrowth for an additional 2 wk until d 42. All 3 cultures were also effective at delaying Pho. dimorpha outgrowth in queso fresco (
      • Makki G.M.
      • Kozak S.M.
      • Jencarelli K.G.
      • Alcaine S.D.
      Evaluation of the efficacy of commercial protective cultures against mold and yeast in queso fresco.
      ). This suggests potential efficacy of Lactobacillus spp. in protecting against spoilage caused by Pho. dimorpha in several cheese matrices under refrigerated conditions.
      Several other studies have also examined the interaction of LAB and molds in dairy products and found it to be quite varied. As with yeast, this competition may be due to direct competition for trace nutrients. For example, outgrowth of strains of Penicillium brevicompactum, Penicillium crustosum, and Penicillium solitum was shown to be inhibited by Lactic. rhamnosus strains but was restored when provided with excess manganese. Another study found that diacetyl production by Lactic. paracasei was able to inhibit the outgrowth of Pen. solitum (
      • Aunsbjerg S.D.
      • Honoré A.H.
      • Marcussen J.
      • Ebrahimi P.
      • Vogensen F.K.
      • Benfeldt C.
      • Skov T.
      • Knøchel S.
      Contribution of volatiles to the antifungal effect of Lactobacillus paracasei in defined medium and yogurt.
      ). A study by
      • Sedaghat H.
      • Eskandari M.H.
      • Moosavi-Nasab M.
      • Shekarforoush S.S.
      Application of non-starter lactic acid bacteria as biopreservative agents to control fungal spoilage of fresh cheese.
      demonstrated the ability of Lactip. plantarum PIN, Lactip. plantarum CAG23, Lactic. casei D31, Lactip. plantarum NBRC107151, and Lactip. pentosus H39 to delay mycelial growth of Aspergillus flavus and Aspergillus parasiticus on cheese surfaces under different storage conditions. A study by
      • Leyva Salas M.L.
      • Thierry A.
      • Lemaître M.
      • Garric G.
      • Harel-Oger M.
      • Chatel M.
      • Lê S.
      • Mounier J.
      • Valence F.
      • Coton E.
      Antifungal activity of lactic acid bacteria combinations in dairy mimicking models and their potential as bioprotective cultures in pilot scale applications.
      also demonstrated the variability in inhibition of other mold species not included in this study, such as Penicillium bialiwiezense and Galactomyces geotrichum, by LAB including Lactic. rhamnosus stains that are commonly found in commercial protective cultures, and the variability across dairy matrices such as cheese and yogurt. This highlights the importance of understanding both the resident spoilage organisms within a dairy facility and the application of protective cultures in different dairy products.
      In this study, protective cultures were incorporated into the cottage cheese dressing before being mixed with the cheese curd and subsequently packaged. Adding protective cultures together with cheese starter culture could theoretically allow the protective culture more time to multiply and hence enhance the antifungal activity against selected mold and yeast strains. Conversely, these cultures could potentially compete with starter culture over nutrients, or either culture could secrete metabolites compromising medium quality for optimum growth. It is likely that adding protective cultures to cottage cheese at a different processing step would result in a different inhibitory scheme against the fungal challenge strains, and thus cheese processors should validate the step at which protective culture is added to maximize antifungal activity.
      In summary, the protective cultures evaluated in this study were mostly ineffective at controlling the growth of yeast in cottage cheese. Only 1 of the protective cultures, PC1, was able to delay the outgrowth of 3 strains, and only Tor. delbrueckii was kept below the detectable spoilage level over the 21-d period. The outgrowth of the other 6 yeast strains was unimpeded by any of the protective cultures in this study. The efficacy of these protective cultures against molds in cottage cheese was more promising, with all protective cultures showing the ability to delay visible spoilage of at least 1 mold strain in the study. In 5 of the 8 mold strains that grew in cottage cheese, PC1 was able to delay outgrowth compared with the control. This study was not an exhaustive study on all commercially available protective cultures, and new LAB strains are being constantly identified and used to replace or augment strains in currently available products. It is highly recommended that cottage cheese producers thoroughly evaluate any protective culture against the spoilage species and strains of interest before adoption to ensure their products get the extended shelf life they expect.
      Overall, the findings show that commercial LAB cultures vary in performance against various yeast and molds, and thus each protective culture may exhibit a broad range of antifungal activity against different fungal strains taking into consideration food matrix factors. Cheese processors considering protective LAB cultures as a clean-label alterative should investigate the efficacy of these cultures against fungal strains of concern within their production system.

      ACKNOWLEDGMENTS

      This project was funded by the National Dairy Council (Rosemont, IL). We are especially grateful for the support received from the Food Safety Laboratory at Cornell University (Ithaca, NY). The authors have not stated any conflicts of interest.

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