Advertisement

Antimicrobial effects of a bioactive glycolipid on spore-forming spoilage bacteria in milk

Open ArchivePublished:February 12, 2021DOI:https://doi.org/10.3168/jds.2020-19769

      ABSTRACT

      The growth of psychrotolerant aerobic spore-forming bacteria during refrigerated storage often results in the spoilage of fluid milk, leading to off-flavors and curdling. Because of their low toxicity, biodegradability, selectivity, and antimicrobial activity over a range of conditions, glycolipids are a novel and promising intervention to control undesirable microbes. The objective of this study was to determine the efficacy of a commercial glycolipid product to inhibit spore germination, spore outgrowth, and the growth of vegetative cells of Paenibacillus odorifer, Bacillus weihenstephanensis, and Viridibacillus arenosi, which are the predominant spore-forming spoilage bacteria in milk. For spore germination and outgrowth assays, varying concentrations (25–400 mg/L) of the glycolipid product were added to commercial UHT whole and skim milk inoculated with ∼4 log10 spores/mL of each bacteria and incubated at 30°C for 5 d. Inhibition of spore germination in inoculated UHT whole milk was only observed for V. arenosi, and only when glycolipid was added at 400 mg/L. However, concentrations of 400 and 200 mg/L markedly inhibited the outgrowth of vegetative cells from spores of P. odorifer and B. weihenstephanensis, respectively. No inhibition of spore germination or outgrowth was observed in inoculated UHT skim milk for any strain at the concentrations tested (25 and 50 mg/L). The effect of glycolipid addition on vegetative cell growth in UHT whole and skim milk when inoculated with ∼4 log10 cfu/mL of each bacteria was also determined over 21 d of storage at 7°C. Glycolipid addition at 50 mg/L was bactericidal against P. odorifer and B. weihenstephanensis in inoculated UHT skim milk through 21 d of storage, whereas 100 mg/L was needed for similar control of V. arenosi. Concentrations of 100 and 200 mg/L inhibited the growth of vegetative cells of B. weihenstephanensis and P. odorifer, respectively, in inoculated UHT whole milk, whereas 200 mg/L was also bactericidal to B. weihenstephanensis. Additional studies are necessary to identify effective concentrations for the inhibition of Viridibacillus spp. growth in whole milk beyond 7 d. Findings from this study demonstrate that natural glycolipids have the potential to inhibit the growth of dairy-spoilage bacteria and extend the shelf life of milk.

      Key words

      INTRODUCTION

      The Economic Research Service of the United States Department of Agriculture estimates that 17 billion pounds of fluid milk, or 32% of the total supply, is lost at the retail and consumer levels every year in the United States (
      • Buzby J.C.
      • Farah-Wells H.
      • Hyman J.
      The estimated amount, value, and calories of postharvest food losses at the retail and consumer levels in the United States. USDA-ERS Economic Information Bulletin Number 121.
      ), with microbial spoilage as a leading cause of this loss (
      • Buzby J.C.
      • Hyman J.
      Total and per capita value of food loss in the United States.
      ;
      • Ivy R.A.
      • Ranieri M.L.
      • Martin N.H.
      • den Bakker H.C.
      • Xavier B.M.
      • Wiedmann M.
      • Boor K.J.
      Identification and characterization of psychrotolerant sporeformers associated with fluid milk production and processing.
      ;
      • Rawat S.
      Food spoilage: Microorganisms and their prevention.
      ). Psychrotolerant aerobic spore-forming bacteria present in raw milk can survive pasteurization and subsequent growth during refrigerated storage, ultimately causing off-flavors and curdling of milk (
      • Ranieri M.L.
      • Ivy R.A.
      • Mitchell W.R.
      • Call E.
      • Masiello S.N.
      • Wiedmann M.
      • Boor K.J.
      Real-time PCR detection of Paenibacillus spp. in raw milk to predict shelf life performance of pasteurized fluid milk products.
      ;
      • Trmčić A.
      • Martin N.H.
      • Boor K.J.
      • Wiedmann M.
      A standard bacterial isolate set for research on contemporary dairy spoilage.
      ). The predominant spore-forming bacteria in fluid milk are members of the genera Paenibacillus, Viridibacillus, and Bacillus (
      • Ranieri M.L.
      • Boor K.J.
      Short communication: Bacterial ecology of high-temperature, short-time pasteurized milk processed in the United States.
      ;
      • Ivy R.A.
      • Ranieri M.L.
      • Martin N.H.
      • den Bakker H.C.
      • Xavier B.M.
      • Wiedmann M.
      • Boor K.J.
      Identification and characterization of psychrotolerant sporeformers associated with fluid milk production and processing.
      ;
      • Ranieri M.L.
      • Ivy R.A.
      • Mitchell W.R.
      • Call E.
      • Masiello S.N.
      • Wiedmann M.
      • Boor K.J.
      Real-time PCR detection of Paenibacillus spp. in raw milk to predict shelf life performance of pasteurized fluid milk products.
      ;
      • Buehler A.J.
      • Martin N.H.
      • Boor K.J.
      • Wiedmann M.
      Psychrotolerant spore-former growth characterization for the development of a dairy spoilage predictive model.
      ). Many Bacillus spp. strains are capable of degrading milk proteins, fat, and phospholipids (
      • Mehta D.S.
      • Metzger L.E.
      • Hassan A.N.
      • Nelson B.K.
      • Patel H.A.
      The ability of spore formers to degrade milk proteins, fat, phospholipids, common stabilizers, and exopolysaccharides.
      ). Bacillus spp. and to a lesser extent Paenibacillus spp. are also prevalent in dairy environments, including bedding, water, feed, and air (
      • Huck J.R.
      • Sonnen M.
      • Boor K.J.
      Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product.
      ;
      • Martin N.H.
      • Kent D.J.
      • Evanowski R.L.
      • Zuber Hrobuchak T.J.
      • Wiedmann M.
      Bacterial spore levels in bulk tank raw milk are influenced by environmental and cow hygiene factors.
      ). They have also been associated with postpasteurization contamination of milk (
      • Huck J.R.
      • Hammond B.H.
      • Murphy S.C.
      • Woodcock N.H.
      • Boor K.J.
      Tracking spore-forming bacterial contaminants in fluid milk-processing systems.
      ), in which case the onset of spoilage is more rapid in the absence of the spore germination process. Strategies to impede spoilage have focused on reducing spore levels in raw milk by controlling the entry of spores into milk on the farm (
      • Evanowski R.L.
      • Kent D.J.
      • Wiedmann M.
      • Martin N.H.
      Milking time hygiene interventions on dairy farms reduce spore counts in raw milk.
      ) or by mechanically removing them (
      • Doll E.V.
      • Scherer S.
      • Wenning M.
      Spoilage of microfiltered and pasteurized extended shelf life milk is mainly induced by psychrotolerant spore-forming bacteria that often originate from recontamination.
      ).
      Antimicrobial approaches for the inhibition of spore germination and the growth of vegetative cells in milk have also been investigated, including the addition of monolaurin (
      • Mansour M.
      • Amri D.
      • Bouttefroy A.
      • Linder M.
      • Milliere J.B.
      Inhibition of Bacillus licheniformis spore growth in milk by nisin, monolaurin, and pH combinations.
      ;
      • Mansour M.
      • Millière J.-B.
      An inhibitory synergistic effect of a nisin–monolaurin combination on Bacillus sp. vegetative cells in milk.
      ) and potassium sorbate (
      • Aouadhi C.
      • Mejri S.
      • Maaroufi A.
      Inhibitory effects of nisin and potassium sorbate alone or in combination on vegetative cells growth and spore germination of Bacillus sporothermodurans in milk.
      ). The antimicrobial effects of the bacteriocin nisin against Bacillus spp. has been studied extensively as a single application and in combination with other antimicrobial treatments, including pulsed electric fields (
      • Bermúdez-Aguirre D.
      • Dunne C.P.
      • Barbosa-Cánovas G.V.
      Effect of processing parameters on inactivation of Bacillus cereus spores in milk using pulsed electric fields.
      ) and heat and hydrostatic pressure (
      • Aouadhi C.
      • Simonin H.
      • Mejri S.
      • Maaroufi A.
      The combined effect of nisin, moderate heating and high hydrostatic pressure on the inactivation of Bacillus sporothermodurans spores.
      ). Although the applications of single antimicrobial treatments were typically insufficient to inhibit spore germination, combination treatments have shown synergistic effects against both spores and vegetative cells. However, these studies focused on a single Bacillus species, and their effects on other predominant spore-forming bacteria such as Paenibacillus spp. and Viridibacillus spp. are not known. Moreover, repetitive exposure to nisin increases the potential for resistance to this bacteriocin (
      • Zhou H.
      • Fang J.
      • Tian Y.
      • Lu X.Y.
      Mechanisms of nisin resistance in Gram-positive bacteria.
      ). This possibility, coupled with some technologies (e.g., hydrostatic pressure) not being readily available to producers, necessitates the investigation of alternative interventions.
      The use of glycolipids, which have demonstrated antibacterial, antifungal, and antiviral activities (
      • Singh P.
      • Cameotra S.S.
      Potential applications of microbial surfactants in biomedical sciences.
      ) including activity against Bacillus subtilis (
      • Lang S.
      • Katsiwela E.
      • Wagner F.
      Antimicrobial effects of biosurfactants.
      ), presents a novel approach. The antimicrobial activity of glycolipids has been attributed to the disruption of membranes, resulting in their increased permeability, the leakage of intracellular material, and cell lysis (
      • Sánchez M.
      • Teruel J.A.
      • Espuny M.J.
      • Marqués A.
      • Aranda F.J.
      • Manresa A.
      • Ortiz A.
      Modulation of the physical properties of dielaidoylphosphatidylethanolamine membranes by a dirhamnolipid biosurfactant produced by Pseudomonas aeruginosa..
      ;
      • Sotirova A.V.
      • Spasova D.I.
      • Galabova D.N.
      • Karpenko E.
      • Shulga A.
      Rhamnolipid-biosurfactant permeabilizing effects on gram-positive and gram-negative bacterial strains.
      ;
      • de Freitas Ferreira J.
      • Vieira E.
      • Nitschke M.
      The antibacterial activity of rhamnolipid biosurfactant is pH dependent.
      ). Use of glycolipids is appealing because of their low toxicity, biodegradability, selectivity, and activity at a wide range of temperatures, pH, and salinity (
      • Nitschke M.
      • Costa S.G.V.A.O.
      Biosurfactants in food industry.
      ). Studies on their application in food are currently limited. Recent research in our laboratory has demonstrated the potential of a novel, natural glycolipid to control Listeria monocytogenes in milk during refrigerated storage (
      • Sun L.
      • Forauer E.C.
      • Brown S.R.B.
      • D'Amico D.J.
      Application of bioactive glycolipids to control Listeria monocytogenes biofilms and as post-lethality contaminants in milk and cheese.
      ). Therefore, we hypothesized that the addition of the same glycolipid product to inoculated UHT whole and skim milk would inhibit spore germination and the growth of vegetative cells of spore-forming bacteria associated with milk spoilage. We additionally hypothesized that treatments would be more efficacious in skim milk. The objectives of this study were to determine the efficacy of a novel natural glycolipid product to control (1) spore germination and (2) the growth of vegetative cells of the predominant milk spoilers Paenibacillus odorifer, Bacillus weihenstephanensis, and Viridibacillus arenosi in whole and skim milk.

      MATERIALS AND METHODS

      Bacterial Strains, Growth Conditions, and Inoculum Preparation

      Paenibacillus odorifer FSL H8-237, Bacillus weihenstephanensis FSL M7-669, and Viridibacillus arenosi FSL R5-213 were selected from a standard bacterial isolate set for research on contemporary dairy spoilage (
      • Trmčić A.
      • Martin N.H.
      • Boor K.J.
      • Wiedmann M.
      A standard bacterial isolate set for research on contemporary dairy spoilage.
      ;
      • Buehler A.J.
      • Martin N.H.
      • Boor K.J.
      • Wiedmann M.
      Psychrotolerant spore-former growth characterization for the development of a dairy spoilage predictive model.
      ); the isolates were kindly provided by the Food Safety Laboratory at Cornell University. Frozen (−80°C) stock cultures were streaked on tryptic soy agar with 0.6% yeast extract added (TSAYE) and incubated at 30°C overnight prior to use as described below.
      Working stock solutions (2% wt/vol) of the glycolipid product Nagardo (Lanxess Corp., Pittsburgh, PA) were prepared before each experiment by dissolving the powdered product in sterile deionized water (SDW). Nagardo is a natural glycolipid obtained via fermentation of glucose by the edible jelly fungus Dacryopinax spathularia (

      Stadler, M., J. Bitzer, B. Köpcke, K. Reinhardt, and J. Moldenhauer. 2012. Long chain glycolipids useful to avoid perishing or microbial contamination of materials. Patent WO2012/167920A1.

      ). It is currently affirmed as GRAS (generally recognized as safe) for use in nonalcoholic beverages (
      • United States Food and Drug Administration (FDA)
      GRAS Notices: GRN No. 740 Glycolipids from Dacryopinax spathularia..
      ) and is currently in commercial use in this market segment at 2 to 25 ppm. Antimicrobial activity of the glycolipid was initially screened by growing vegetative cells of each microbe individually in UHT whole and skim milk (Natrel, Agropur Inc., St. Paul, MN) for 24 h at 30°C in the presence of 2-fold dilutions of glycolipid from 4,000 to 31.25 mg/L. Surviving populations were enumerated as described below, and final experimental concentrations, as shown in the resulting figures, were selected based on these results. Commercial UHT milk was used in the present study to reduce the potential for confounding effects of native spores and vegetative cells of the P. odorifer, B. weihenstephanensis, and V. arenosi in the milk.

      Spore Preparation

      Although data related to Paenibacillus spp. and Viridibacillus spp. are scant, the optimum heat treatment for the activation of spores of Bacillus spp. varies depending on the species (
      • Ghosh S.
      • Zhang P.
      • Li Y.
      • Setlow P.
      Superdormant spores of Bacillus species have elevated wet-heat resistance and temperature requirements for heat activation.
      ). Preliminary studies determined that the standard spore activation conditions of 80°C for 12 min (
      • Frank J.F.
      • Yousef A.E.
      Tests for groups of microorganisms.
      ) did not allow for spores of any bacteria used in the present study to germinate. Such heat activation conditions have been shown to damage and inactivate spores (
      • Turnbull P.C.
      • Frawley D.A.
      • Bull R.L.
      Heat activation/shock temperatures for Bacillus anthracis spores and the issue of spore plate counts versus true numbers of spores.
      ), and optimal heat activation conditions ranging between 60 to 75°C for 15 to 30 min have been reported for several species of Bacillus (
      • Ghosh S.
      • Zhang P.
      • Li Y.
      • Setlow P.
      Superdormant spores of Bacillus species have elevated wet-heat resistance and temperature requirements for heat activation.
      ). Therefore, sporulation was conducted as described by
      • Baril E.
      • Coroller L.
      • Postollec F.
      • Leguerinel I.
      • Boulais C.
      • Carlin F.
      • Mafart P.
      The wet-heat resistance of Bacillus weihenstephanensis KBAB4 spores produced in a two-step sporulation process depends on sporulation temperature but not on previous cell history.
      and heat activation was performed at 63°C for 30 min, which allowed for the germination of all 3 bacteria. Isolated colonies from TSAYE plates were first individually inoculated into nutrient broth (BD, Franklin Lakes, NJ) and then incubated at 30°C for 18 h. Following 2 successive transfers, cultures were diluted 1:9 in sporulation mineral buffer and incubated at 30°C for 5 d. Sporulation mineral buffer was composed of phosphate buffer (K2HPO4·3H2O: 4.5 g/L; anhydrous KH2PO4: 1.8 g/L; Sigma-Aldrich, Saint Quentin Fallavier, France), supplemented with CaCl2·2H2O (8.0 mg/L; Sigma-Aldrich) and MnSO4·H2O (1.5 mg/L; ThermoFisher Scientific, Waltham, MA). Cultures were centrifuged (10 min, 4,000 × g at 4°C; Thermo Scientific Sorvall Legend X1R, ThermoFisher Scientific), and the resulting pellets were resuspended in SDW and heated on a heating block at 63°C for 30 min. Spores were washed again, resuspended in SDW, and stored at 4°C. Resulting spore suspensions were enumerated on brain heart infusion agar supplemented with vitamin B12 (BHI-B12, 1 mg/L; Sigma-Aldrich) following incubation at 30°C for 24 h (
      • Aouadhi C.
      • Mejri S.
      • Maaroufi A.
      Inhibitory effects of nisin and potassium sorbate alone or in combination on vegetative cells growth and spore germination of Bacillus sporothermodurans in milk.
      ). The sporulation and heat activation process yielded 5 to 6 log10 cfu/mL. Because the heat treatment was sufficient to eliminate vegetative cells (5–6 log cfu/mL reduction to levels below limit of enumeration) as confirmed through preliminary studies, resulting cells were confidently enumerated as spores.

      Control of Spores in Milk

      The effect of glycolipid addition to milk on the inactivation of spores, spore germination, spore outgrowth, and vegetative growth was determined over 5 d based on previously published methods (
      • Mansour M.
      • Amri D.
      • Bouttefroy A.
      • Linder M.
      • Milliere J.B.
      Inhibition of Bacillus licheniformis spore growth in milk by nisin, monolaurin, and pH combinations.
      ;
      • Aouadhi C.
      • Mejri S.
      • Maaroufi A.
      Inhibitory effects of nisin and potassium sorbate alone or in combination on vegetative cells growth and spore germination of Bacillus sporothermodurans in milk.
      ). Spore outgrowth refers to the period following germination during which the germinated spore transitions into a growing vegetative cell (
      • Paidhungat M.
      • Setlow P.
      Spore germination and outgrowth.
      ;
      • Setlow P.
      Spore germination.
      ). Spores from refrigerated storage were heat activated at 63°C for 30 min, pelleted through centrifugation (3 min, 13,000 × g at 4°C; Sorvall Legend Micro 21 Microcentrifuge, ThermoFisher Scientific), washed with 0.1% peptone water, and resuspended in UHT whole or skim milk (Natrel) supplemented with l-alanine (1 g/L; Sigma-Aldrich) as a germination promoter to achieve 4 log10 spores/mL. Glycolipid treatments for each strain were achieved by adding 22 μL of glycolipid stocks of varying concentrations to 198 μL of spore-inoculated UHT skim or whole milk in 96-well polystyrene plates (Corning Costar, Cambridge, MA). Samples were incubated at 30°C for 5 d, and aliquots were removed for enumeration after 4, 8, 24, 48, 72, 96, and 120 h of incubation. Aliquots for the enumeration of total counts (vegetative cells + spores) were serially diluted in peptone water and plated on BHI-B12, whereas aliquots for the enumeration of spores were first heated at 63°C for 30 min before serial dilution and plating on BHI-B12. These heat treatment conditions may allow for the enumeration of injured spores (
      • Frank J.F.
      • Yousef A.E.
      Tests for groups of microorganisms.
      ) resulting from the glycolipid treatment. Colonies were enumerated after incubation at 30°C for 24 h. Spore germination rates (%) were calculated using the formula [1 − (cfu/mL of spores at sampling)/(cfu/mL spores at inoculation)] × 100 (
      • Johnson K.M.
      • Nelson C.L.
      • Busta F.F.
      Germination and heat resistance of Bacillus cereus spores from strains associated with diarrheal and emetic food-borne illnesses.
      ).

      Control of Vegetative Cells in Milk During Refrigerated Storage

      To determine the efficacy of the glycolipid to control the growth of vegetative cells during refrigerated storage, a single colony from each plate was inoculated into tryptic soy broth with 0.6% yeast extract added and incubated at 30°C for 18 h. Following 2 subsequent transfers, cultures were harvested by centrifugation (3 min, 13,000 × g at 4°C) and washed with peptone water. Washed pellets of P. odorifer, B. weihenstephanensis, or V. arenosi were resuspended individually in 4.5 mL of either UHT whole or skim milk to achieve 4 log10 cfu/mL, to which 0.5 mL of SDW (control) or glycolipid stocks of varying concentrations were added and vortexed to mix. Samples were incubated at 7°C for 3 wk. Aliquots were removed at d 1, 3, 5, 7, 14, and 21 of incubation; serially diluted in 0.1% peptone water; and plated on TSAYE for enumeration following overnight incubation at 30°C. Treatments were considered bacteriostatic at a given time point when counts were < 1 log10 cfu/mL higher than initial inoculation and bactericidal when >3 log10 cfu/mL lower than initial inoculation.

      Statistical Analysis

      Data were analyzed using R (version 3.6.1; RStudio, PBC, Boston, MA). The 3-phase linear model of Buchanan (
      • Baranyi J.
      • Roberts T.A.
      A dynamic approach to predicting bacterial growth in food.
      ) was fit to the data (R package: “nlsMicrobio,” version 0.0.1) to calculate growth parameters. Cell counts below the limit of enumeration (10 cfu/mL) were scored as 5 cfu/mL (0.5× limit of enumeration) before log-transformation and analysis. Spore germination experiments were analyzed by 2-way ANOVA followed by 1-way ANOVA at each time point. Post hoc Tukey's honest significant difference tests were performed. Repeated-measures ANOVA including an autocorrelation structure in the mixed effects model was used for experiments of vegetative cell growth in milk (R package: “nmle,” version 3.1.140). Post hoc Tukey's honest significant difference tests were performed. A significance level of the confidence intervals was set at P < 0.05 for all analyses.

      RESULTS AND DISCUSSION

      Effect of Glycolipid on Spore Germination and Outgrowth of P. odorifer Inoculated in UHT Milk

      An approximate lag time of 8 h was observed for P. odorifer spores to germinate into multiplying vegetative cells in whole milk without glycolipid. Mean spore counts in the control decreased to 2.68 log10 cfu/mL with a germination rate of 95.5% in the first 48 h (Figure 1A). Spore counts started to increase at 48 h, which could have been due to the sporulation of vegetative cells as they reached the end of exponential phase at 48 h (vegetative cell count of 6.38 log10 cfu/mL). Spore counts in the control continued to increase, reaching 3.73 log10 cfu/mL after 5 d. Addition of glycolipid at 200 and 400 mg/L did not inhibit germination but did inactivate emerging vegetative cells, with total cell counts overlapping spore counts at 8 h. Continued vegetative cell growth was observed in the 200 mg/L treatment, whereas spore outgrowth was inhibited in the 400 mg/L treatment through the following 5 d. A previous study by
      • Aouadhi C.
      • Mejri S.
      • Maaroufi A.
      Inhibitory effects of nisin and potassium sorbate alone or in combination on vegetative cells growth and spore germination of Bacillus sporothermodurans in milk.
      also reported that the additions of the antimicrobials nisin and potassium sorbate did not affect spore germination but were able to control spore outgrowth under similar experimental conditions. Because vegetative cells are more susceptible to stresses and are more readily inactivated compared with spores, allowing spores to germinate but inhibiting spore outgrowth is a common strategy to control spore-forming bacteria in foods (
      • Paredes-Sabja D.
      • Torres J.A.
      • Setlow P.
      • Sarker M.R.
      Clostridium perfringens spore germination: Characterization of germinants and their receptors.
      ;
      • Aouadhi C.
      • Simonin H.
      • Maaroufi A.
      • Mejri S.
      Optimization of nutrient-induced germination of Bacillus sporothermodurans spores using response surface methodology.
      ). The addition of glycolipid to whole milk at 400 mg/L may be applied to prevent milk spoilage by spore-forming bacteria.
      Figure thumbnail gr1
      Figure 1Change in spore and total cell counts (mean ± SD) of Paenibacillus odorifer (A), Bacillus weihenstephanensis (B), and Viridibacillus arenosi (C) over time in inoculated UHT whole milk without (control) or with glycolipid (200 or 400 mg/L for P. odorifer and V. arenosi; 100 or 200 mg/mL for B. weihenstephanensis). Filled symbols represent total cell counts, and open symbols represent spore counts. Dashed line indicates initial inoculation level (4.02–4.3 log10 cfu/mL). ^Counts below the limit of enumeration (10 cfu/mL). #Data points for 200_total cell treatment at 24, 48, and 72 h represents 2 trials following removal of significant outliers in a third trial at these time points.
      Spores of P. odorifer did not germinate under the described conditions in skim milk (Figure 2A), which may be attributed to several factors including conditions during spore activation (e.g., temperature and medium composition) and environmental conditions during incubation (
      • Griffiths M.W.
      • Phillips J.D.
      Incidence, source and some properties of psychrotrophic Bacillus spp. found in raw and pasteurized milk.
      ;
      • Ghosh S.
      • Zhang P.
      • Li Y.
      • Setlow P.
      Superdormant spores of Bacillus species have elevated wet-heat resistance and temperature requirements for heat activation.
      ;
      • Buehler A.J.
      • Martin N.H.
      • Boor K.J.
      • Wiedmann M.
      Psychrotolerant spore-former growth characterization for the development of a dairy spoilage predictive model.
      ). The optimal temperature for P. odorifer growth is 6°C, with limited or no growth when incubated at 30°C and 37°C, respectively (
      • Trmčić A.
      • Martin N.H.
      • Boor K.J.
      • Wiedmann M.
      A standard bacterial isolate set for research on contemporary dairy spoilage.
      ). Germination of the same strain has been previously observed when incubated at 6°C in skim milk broth medium (
      • Buehler A.J.
      • Martin N.H.
      • Boor K.J.
      • Wiedmann M.
      Psychrotolerant spore-former growth characterization for the development of a dairy spoilage predictive model.
      ). Therefore, incubating spores at 30°C may not be optimized for germination and growth of P. odorifer in UHT skim milk.
      Figure thumbnail gr2
      Figure 2Change in spore and total cell counts (mean ± SD) of Paenibacillus odorifer (A), Bacillus weihenstephanensis (B), and Viridibacillus arenosi (C) over time in inoculated UHT skim milk without (control) or with glycolipid (25 or 50 mg/L for P. odorifer and B. weihenstephanensis; 50 or 100 mg/L for V. arenosi). Filled symbols represent total cell counts, and open symbols represent spore counts. Dashed line indicates initial inoculation level (4.02–4.3 log10 cfu/mL). ^Counts below the limit of enumeration (10 cfu/mL).

      Effect of Glycolipid on Spore Germination and Outgrowth of B. weihenstephanensis Inoculated in UHT Milk

      In the absence of glycolipid, spores of B. weihenstephanensis rapidly decreased in whole milk with a germination rate of 99.6% after 4 h (Figure 1B). Similar germination rates were observed with glycolipid added at 100 and 200 mg/L in whole milk. Total cell counts of B. weihenstephanensis in the 100 mg/L treatment increased to levels similar to the control after 24 h (∼8 log10 cfu/mL). Interestingly, the presence of glycolipid in the 100 mg/L treatment in whole milk induced sporulation with higher spore counts compared with the control from 48 h through 5 d (Figure 1B). Cell membrane movement is critical for bacterial sporulation because it enables the mother cell to engulf the forespore to form endospores (
      • Ojkic N.
      • López-Garrido J.
      • Pogliano K.
      • Endres R.G.
      Cell-wall remodeling drives engulfment during Bacillus subtilis sporulation.
      ). It is possible that glycolipids, which are major components of Bacillus membranes, can affect cell membrane function and the sporulation process (
      • Griffiths K.K.
      • Setlow P.
      Effects of modification of membrane lipid composition on Bacillus subtilis sporulation and spore properties.
      ). In contrast, total counts in the 200 mg/L treatment were >6 log10 cfu/mL lower than control at 8 h through the remainder of storage when significant outliers in a single trial at 24, 48, and 72 h were removed (Figure 1B). With these outliers included, counts in this treatment were >4 log10 cfu/mL lower than the control from 8 to 72 h and >6 log10 cfu/mL lower thereafter.
      In the absence of glycolipid, B. weihenstephanensis spores germinated rapidly in skim milk, and the resulting vegetative cells reached a stationary phase within 24 h, while spore counts remained low over 5 d (Figure 2B). Addition of glycolipid at 25 and 50 mg/L reduced total counts through 4 and 8 h, respectively, but growth followed thereafter, with treatments matching the control at 24 and 48 h, respectively. Similar to observations made in whole milk, glycolipid addition in skim milk also enhanced sporulation of B. weihenstephanensis when added at 25 mg/L, with mean spore counts reaching 2.65 log10 cfu/mL at d 5. The germination rate of B. weihenstephanensis was highest among the 3 bacteria in both whole and skim milk. This outcome is in agreement with the observation that Bacillus spp. are typically isolated from fluid milk in the first week of shelf life, whereas Paenibacillus spp. are more frequently isolated close to the end of shelf life (>17 d) (
      • Ranieri M.L.
      • Boor K.J.
      Short communication: Bacterial ecology of high-temperature, short-time pasteurized milk processed in the United States.
      ). The comparatively early identification of Bacillus spp. may be associated with its rapid germination, whereas the rapid outgrowth of other species dominates at later stages in milk.

      Effect of Glycolipid on Spore Germination and Outgrowth of V. arenosi Inoculated in UHT Milk

      Spore counts of V. arenosi in whole milk decreased in the first 8 h (germination rate: 55.3%) along with rapid growth of vegetative cells (Figure 1C). The population reached stationary phase by 24 h (7.75 log10 cfu/mL) with a concomitant increase in V. arenosi spore counts at 24 h (3.78 log10 cfu/mL). Addition of glycolipid to whole milk at 200 mg/L did not inhibit germination, but it inactivated emerging vegetative cells of V. arenosi within the first 8 h, followed by increases in both total cell and spore counts from 8 to 24 h. Mean spore counts on d 5 reached 6.33 and 6.54 log10 cfu/mL in the control and 200 mg/L treatment, respectively. Glycolipid at 400 mg/L was not sporicidal, but it inhibited germination in whole milk, with mean spore counts remaining near 2.3 log10 cfu/mL over 5 d. Counts of V. arenosi spores in skim milk started to increase at 8 h following the rapid growth of vegetative cells, reaching a stationary phase between 8 to 24 h (Figure 2C). Although glycolipid addition at 50 mg/L slowed vegetative cell growth and 100 mg/L inhibited spore outgrowth through 8 h, growth was observed thereafter. Addition of glycolipid at 50 and 100 mg/L delayed sporulation through 24 h, but spore counts increased to >6 log10 cfu/mL on d 5 in both treatments.
      Sporulation of V. arenosi, previously classified as Bacillus arenosi (
      • Heyrman J.
      • Rodríguez-Díaz M.
      • Devos J.
      • Felske A.
      • Logan N.A.
      • De Vos P.
      Bacillus arenosi sp. nov., Bacillus arvi sp. nov. and Bacillus humi sp. nov., isolated from soil.
      ;
      • Ivy R.A.
      • Ranieri M.L.
      • Martin N.H.
      • den Bakker H.C.
      • Xavier B.M.
      • Wiedmann M.
      • Boor K.J.
      Identification and characterization of psychrotolerant sporeformers associated with fluid milk production and processing.
      ), was highest among the 3 strains tested in both UHT whole and skim milk. Although sporulation of this species has been shown to be limited on nutrient-rich media, abundant spores were formed in nutrient-poor media after ∼3 d of incubation at 30°C, with no identification of vegetative cells after 5 d (
      • Heyrman J.
      • Rodríguez-Díaz M.
      • Devos J.
      • Felske A.
      • Logan N.A.
      • De Vos P.
      Bacillus arenosi sp. nov., Bacillus arvi sp. nov. and Bacillus humi sp. nov., isolated from soil.
      ). Although research into the control of this species in milk is limited, the need is increasing due to its high sporulation frequency when nutrition is depleted and its prevalence in fluid milk (
      • Ivy R.A.
      • Ranieri M.L.
      • Martin N.H.
      • den Bakker H.C.
      • Xavier B.M.
      • Wiedmann M.
      • Boor K.J.
      Identification and characterization of psychrotolerant sporeformers associated with fluid milk production and processing.
      ;
      • Buehler A.J.
      • Martin N.H.
      • Boor K.J.
      • Wiedmann M.
      Psychrotolerant spore-former growth characterization for the development of a dairy spoilage predictive model.
      ).

      Growth of Vegetative Cells of Spore-Forming Spoilage Bacteria in UHT Milk During Cold Storage

      Because milk can be contaminated post pasteurization with vegetative cells of spore-forming dairy spoilers, the efficacy of the glycolipid product to control the growth of vegetative cells was also determined over 3 wk of refrigerated storage (Figures 3 and 4). We first identified the growth patterns of each bacteria in the absence of glycolipid in skim and whole milk during refrigerated storage (Table 1). Bacillus weihenstephanensis had the longest lag phase among the 3 strains (∼4 d), as the others entered exponential phase around the first day of incubation. Bacillus weihenstephanensis also had the lowest growth rate (<1 log10 cfu/mL per day) and maximum cell density (∼6.4 log10 cfu/mL). Counts of each bacterium increased in whole and skim milk and reached a stationary phase around d 14, except for P. odorifer in skim milk. The growth pattern of P. odorifer in skim milk differed from that in whole milk, whereby mean counts increased initially over the first 7 d to 5.64 log10 cfu/mL, followed by a gradual decrease to 4.09 log10 cfu/mL on d 21 (Figure 4A).
      Table 1Growth parameters for vegetative cells of Paenibacillus odorifer, Bacillus weihenstephanensis, and Viridibacillus arenosi in inoculated UHT whole and skim milk
      SpeciesWhole milkSkim milk
      Lag (d)μmax
      Maximum growth rate at 7°C (log10 cfu/mL per day).
      Nmax
      Maximum cell density at 7°C (log10 cfu/mL).
      Lag (d)μmax
      Maximum growth rate at 7°C (log10 cfu/mL per day).
      Nmax
      Maximum cell density at 7°C (log10 cfu/mL).
      P. odorifer
      Growth parameters for skim milk were calculated based on data through d 7 because P. odorifer populations reached the death phase on d 14.
      0.951.017.271.461.755.59
      B. weihenstephanensis4.230.796.364.440.906.43
      V. arenosi0.401.087.390.861.227.67
      1 Maximum growth rate at 7°C (log10 cfu/mL per day).
      2 Maximum cell density at 7°C (log10 cfu/mL).
      3 Growth parameters for skim milk were calculated based on data through d 7 because P. odorifer populations reached the death phase on d 14.

      Effect of Glycolipid on Vegetative Cells of Sporeformers Inoculated in UHT Milk

      The addition of glycolipid to whole milk at 50 and 100 mg/L did not affect P. odorifer growth at any time point. Application at 200 mg/L resulted in initial reductions with counts that were 2.29 to 5.85 log10 cfu/mL lower than the control between d 1 and 7 of storage (Figure 3A). Growth was further inhibited to <1 log10 cfu/mL higher than the initial inoculation through d 21 (4.46 log10 cfu/mL) (Figure 3A) and was therefore considered bacteriostatic. Glycolipid addition at 1 mg/L was ineffective against P. odorifer in skim milk, but total counts were 5.18 log10 cfu/mL lower than the control at d 7 when the glycolipid was added at 25 mg/L (Figure 4A). Mean counts increased slightly thereafter, reaching 1.49 log10 cfu/mL at 21 d. Addition of the glycolipid at 50 mg/L was bactericidal, with mean counts below the limit of enumeration at d 5 with no observable growth through d 21.
      Figure thumbnail gr3
      Figure 3Change in vegetative cell counts (mean ± SD) of Paenibacillus odorifer (A), Bacillus weihenstephanensis (B), and Viridibacillus arenosi (C) over time in inoculated UHT whole milk without (control) or with glycolipid (50 to 200 mg/L). Dashed line indicates initial inoculation level (3.78–4.26 log10 cfu/mL). ^Counts below the limit of enumeration (10 cfu/mL). Different relative to control: **P < 0.01; ***P < 0.001.
      Growth of B. weihenstephanensis in whole milk was not affected by the addition of glycolipid at 50 mg/L (Figure 3B). However, 100 mg/L reduced mean counts to 3.07 log10 cfu/mL lower than the control at d 7 and held mean counts to <1 log10 cfu/mL higher than the initial inoculation (4.71 log10 cfu/mL) through 21 d of storage, and it was thus considered bacteriostatic. Glycolipid addition at 200 mg/L was bactericidal to B. weihenstephanensis and reduced counts to levels below the limit of enumeration at d 1, where they remained through 21 d of storage at 7°C, save for a slight increase at d 5. Glycolipid concentrations as low as 10 mg/L were bacteriostatic and inhibited the growth of vegetative cells of B. weihenstephanensis in skim milk to mean counts below inoculation levels through 14 d (3.30 log10 cfu/mL, Figure 4B). Bactericidal treatments at 50 and 100 mg/L reduced counts in skim milk to levels below the limit of enumeration at d 1, where they remained throughout the 21-d storage period.
      The growth of V. arenosi in whole milk was not affected at any time point by the addition of glycolipid at 50 and 100 mg/L. Application at 200 mg/L resulted in an initial reduction in mean counts of 2.82 log10 cfu/mL at d 1 (Figure 3C), but mean counts rapidly increased in this treatment, reaching inoculation levels by d 7 (4.40 log10 cfu/mL) and counts similar to control at d 14 and 21 (∼7.5 log10 cfu/mL). The addition of glycolipid at 10 mg/L to skim milk did not affect V. arenosi counts at any time point (Figure 4C). Glycolipid at 50 mg/L resulted in an initial 2.5 log10 cfu/mL reduction in mean counts. Counts in this treatment remained below inoculation levels through d 7 (3.58 log10 cfu/mL) but did not differ from the control at d 14 and 21 (6.96 log10 cfu/mL, P = 0.18; and 7.45 log10 cfu/mL, P = 0.99, respectively). The 100 mg/L treatment was bactericidal, with mean counts below the limit of enumeration on d 3, 7, 14, and 21.
      Figure thumbnail gr4
      Figure 4Change in vegetative cell counts (mean ± SD) of Paenibacillus odorifer (A), Bacillus weihenstephanensis (B), and Viridibacillus arenosi (C) over time in inoculated UHT skim milk without (control) or with glycolipid (1 to 100 mg/L). Dashed line indicates initial inoculation level (3.74–4.29 log10 cfu/mL). ^Counts below limit of enumeration (10 cfu/mL). Different relative to control: ***P < 0.001.
      Similar to previous studies with Bacillus spp., antimicrobials added to milk initially decreased counts of spore-forming bacteria, but subsequent increases followed over prolonged incubation (
      • Mansour M.
      • Millière J.-B.
      An inhibitory synergistic effect of a nisin–monolaurin combination on Bacillus sp. vegetative cells in milk.
      ;
      • Aouadhi C.
      • Mejri S.
      • Maaroufi A.
      Inhibitory effects of nisin and potassium sorbate alone or in combination on vegetative cells growth and spore germination of Bacillus sporothermodurans in milk.
      ). In addition, the decreased efficacy of glycolipids in milk in the presence of fat (i.e., whole milk) reported here was also observed for the control of L. monocytogenes in milk (
      • Sun L.
      • Forauer E.C.
      • Brown S.R.B.
      • D'Amico D.J.
      Application of bioactive glycolipids to control Listeria monocytogenes biofilms and as post-lethality contaminants in milk and cheese.
      ). This outcome has also been reported for other antimicrobial treatments including essential oils, lysozyme, and nisin (
      • Dabbah R.
      • Edwards V.M.
      • Moats W.A.
      Antimicrobial action of some citrus fruit oils on selected food-borne bacteria.
      ;
      • García-Graells C.
      • Masschalck B.
      • Michiels C.W.
      Inactivation of Escherichia coli in milk by high-hydrostatic-pressure treatment in combination with antimicrobial peptides.
      ;
      • Cava-Roda R.M.
      • Taboada-Rodríguez A.
      • Valverde-Franco M.T.
      • Marín-Iniesta F.
      Antimicrobial activity of vanillin and mixtures with cinnamon and clove essential oils in controlling Listeria monocytogenes and Escherichia coli O157:H7 in milk.
      ) and has been attributed to the solubility of antimicrobial compounds in fat, which can limit the interactions with bacteria in the aqueous phase (
      • Tassou C.C.
      • Drosinos E.H.
      • Nychas G.J.E.
      Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4° and 10°C.
      ;
      • Mejlholm O.
      • Dalgaard P.
      Antimicrobial effect of essential oils on the seafood spoilage micro-organism Photobacterium phosphoreum in liquid media and fish products.
      ). In contrast to B. weihenstephanensis and V. arenosi, counts of P. odorifer in skim milk increased in the first 7 d but started to decrease in the following days to 4.12 log10 cfu/mL on d 21.
      • Trmčić A.
      • Martin N.H.
      • Boor K.J.
      • Wiedmann M.
      A standard bacterial isolate set for research on contemporary dairy spoilage.
      reported >4 log10 cfu/mL growth of this strain in skim milk broth at 21 d of storage at 6°C. Aside from differences in culture preparation and starting inoculation levels, the results of the present study suggest that growth in skim milk broth medium may not be a good predictor of growth in commercial UHT skim milk. Likewise, it is not known if the results observed for UHT milk in the present study would be similar in commercial HTST pasteurized milk.
      Overall, data in the present study indicate that the glycolipid product was more effective in UHT skim compared with UHT whole milk for the control of spores, spore outgrowth, and vegetative cells of inoculated spore-forming dairy spoilers. Addition of the glycolipid product at 400 mg/L to inoculated UHT whole milk was sufficient to inhibit V. arenosi spore germination and inhibited outgrowth of P. odorifer and B. weihenstephanensis from spores. Although effective concentrations were not identified for the control of V. arenosi spore germination and outgrowth in skim milk, the effective concentration is expected to be between 100 and 400 mg/L because the glycolipid product was more effective in skim compared with whole milk. Lower concentrations were needed for the control of inoculated vegetative cells, whereby 100 mg/L was bactericidal against P. odorifer, B. weihenstephanensis, and V. arenosi inoculated in UHT skim milk through 21 d of storage. Concentrations of 200 mg/L were bactericidal and bacteriostatic to vegetative cells of B. weihenstephanensis and P. odorifer inoculated in UHT whole milk, respectively, but higher concentrations would be needed to inhibit Viridibacillus spp. growth beyond 7 d.

      CONCLUSIONS

      Together, the findings from this proof-of-concept study demonstrate that glycolipids have the potential to control spore-forming dairy-spoilage bacteria and extend the shelf life of milk, thereby reducing food waste. Future studies should evaluate the effect of glycolipid addition on the native spores in commercial HTST milk because spore levels are expected to be lower in real-life conditions. With consideration of the results from previous studies, future studies could also address the potential for synergistic combinations of antimicrobial interventions to enhance efficacy against both spores and vegetative cells of spore-forming dairy-spoilage bacteria. Although the glycolipid product is currently used in the nonalcoholic beverage sector, future studies will be needed to evaluate potential changes to the sensory properties of milk containing glycolipids at effective concentrations to ensure consumer acceptance. However, addition to milk is restricted by the current US Code of the Federal Regulations pertaining to the standard of identity for milk (21CFR131) that place limitations on added ingredients. Revisions to these standards to allow for antimicrobial applications or other interventions with demonstrated efficacy and safety could help to improve food quality and to reduce food waste.

      ACKNOWLEDGMENTS

      The authors thank the Lanxess Corporation (Pittsburgh, PA; project no. 3059) for their support of the project and for providing the Nagardo glycolipid product. The authors have not stated any conflict of interest.

      REFERENCES

        • Aouadhi C.
        • Mejri S.
        • Maaroufi A.
        Inhibitory effects of nisin and potassium sorbate alone or in combination on vegetative cells growth and spore germination of Bacillus sporothermodurans in milk.
        Food Microbiol. 2015; 46 (25475264): 40-45
        • Aouadhi C.
        • Simonin H.
        • Maaroufi A.
        • Mejri S.
        Optimization of nutrient-induced germination of Bacillus sporothermodurans spores using response surface methodology.
        Food Microbiol. 2013; 36 (24010613): 320-326
        • Aouadhi C.
        • Simonin H.
        • Mejri S.
        • Maaroufi A.
        The combined effect of nisin, moderate heating and high hydrostatic pressure on the inactivation of Bacillus sporothermodurans spores.
        J. Appl. Microbiol. 2013; 115 (23611251): 147-155
        • Baranyi J.
        • Roberts T.A.
        A dynamic approach to predicting bacterial growth in food.
        Int. J. Food Microbiol. 1994; 23 (7873331): 277-294
        • Baril E.
        • Coroller L.
        • Postollec F.
        • Leguerinel I.
        • Boulais C.
        • Carlin F.
        • Mafart P.
        The wet-heat resistance of Bacillus weihenstephanensis KBAB4 spores produced in a two-step sporulation process depends on sporulation temperature but not on previous cell history.
        Int. J. Food Microbiol. 2011; 146 (21354646): 57-62
        • Bermúdez-Aguirre D.
        • Dunne C.P.
        • Barbosa-Cánovas G.V.
        Effect of processing parameters on inactivation of Bacillus cereus spores in milk using pulsed electric fields.
        Int. Dairy J. 2012; 24: 13-21
        • Buehler A.J.
        • Martin N.H.
        • Boor K.J.
        • Wiedmann M.
        Psychrotolerant spore-former growth characterization for the development of a dairy spoilage predictive model.
        J. Dairy Sci. 2018; 101 (29803413): 6964-6981
        • Buzby J.C.
        • Farah-Wells H.
        • Hyman J.
        The estimated amount, value, and calories of postharvest food losses at the retail and consumer levels in the United States. USDA-ERS Economic Information Bulletin Number 121.
        • Buzby J.C.
        • Hyman J.
        Total and per capita value of food loss in the United States.
        Food Policy. 2012; 37: 561-570
        • Cava-Roda R.M.
        • Taboada-Rodríguez A.
        • Valverde-Franco M.T.
        • Marín-Iniesta F.
        Antimicrobial activity of vanillin and mixtures with cinnamon and clove essential oils in controlling Listeria monocytogenes and Escherichia coli O157:H7 in milk.
        Food Bioprocess Technol. 2012; 5: 2120-2131
        • Dabbah R.
        • Edwards V.M.
        • Moats W.A.
        Antimicrobial action of some citrus fruit oils on selected food-borne bacteria.
        Appl. Microbiol. 1970; 19 (4905947): 27-31
        • Doll E.V.
        • Scherer S.
        • Wenning M.
        Spoilage of microfiltered and pasteurized extended shelf life milk is mainly induced by psychrotolerant spore-forming bacteria that often originate from recontamination.
        Front. Microbiol. 2017; 8 (28197147): 135
        • Evanowski R.L.
        • Kent D.J.
        • Wiedmann M.
        • Martin N.H.
        Milking time hygiene interventions on dairy farms reduce spore counts in raw milk.
        J. Dairy Sci. 2020; 103 (32197847): 4088-4099
        • de Freitas Ferreira J.
        • Vieira E.
        • Nitschke M.
        The antibacterial activity of rhamnolipid biosurfactant is pH dependent.
        Food Res. Int. 2019; 116 (30717003): 737-744
        • Frank J.F.
        • Yousef A.E.
        Tests for groups of microorganisms.
        in: Wehr H.M. Frank J.F. Standard Methods for the Examination of Dairy Products. 17th ed. American Public Health Association, Washington, DC2004: 227-248
        • García-Graells C.
        • Masschalck B.
        • Michiels C.W.
        Inactivation of Escherichia coli in milk by high-hydrostatic-pressure treatment in combination with antimicrobial peptides.
        J. Food Prot. 1999; 62 (10571312): 1248-1254
        • Ghosh S.
        • Zhang P.
        • Li Y.
        • Setlow P.
        Superdormant spores of Bacillus species have elevated wet-heat resistance and temperature requirements for heat activation.
        J. Bacteriol. 2009; 191 (19592590): 5584-5591
        • Griffiths K.K.
        • Setlow P.
        Effects of modification of membrane lipid composition on Bacillus subtilis sporulation and spore properties.
        J. Appl. Microbiol. 2009; 106 (19291241): 2064-2078
        • Griffiths M.W.
        • Phillips J.D.
        Incidence, source and some properties of psychrotrophic Bacillus spp. found in raw and pasteurized milk.
        Int. J. Dairy Technol. 1990; 43: 62-66
        • Heyrman J.
        • Rodríguez-Díaz M.
        • Devos J.
        • Felske A.
        • Logan N.A.
        • De Vos P.
        Bacillus arenosi sp. nov., Bacillus arvi sp. nov. and Bacillus humi sp. nov., isolated from soil.
        Int. J. Syst. Evol. Microbiol. 2005; 55 (15653863): 111-117
        • Huck J.R.
        • Hammond B.H.
        • Murphy S.C.
        • Woodcock N.H.
        • Boor K.J.
        Tracking spore-forming bacterial contaminants in fluid milk-processing systems.
        J. Dairy Sci. 2007; 90 (17881711): 4872-4883
        • Huck J.R.
        • Sonnen M.
        • Boor K.J.
        Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product.
        J. Dairy Sci. 2008; 91 (18292280): 1218-1228
        • Ivy R.A.
        • Ranieri M.L.
        • Martin N.H.
        • den Bakker H.C.
        • Xavier B.M.
        • Wiedmann M.
        • Boor K.J.
        Identification and characterization of psychrotolerant sporeformers associated with fluid milk production and processing.
        Appl. Environ. Microbiol. 2012; 78 (22247129): 1853-1864
        • Johnson K.M.
        • Nelson C.L.
        • Busta F.F.
        Germination and heat resistance of Bacillus cereus spores from strains associated with diarrheal and emetic food-borne illnesses.
        J. Food Sci. 1982; 47: 1268-1271
        • Lang S.
        • Katsiwela E.
        • Wagner F.
        Antimicrobial effects of biosurfactants.
        Eur. J. Lipid Sci. Technol. 1989; 91: 363-366
        • Mansour M.
        • Amri D.
        • Bouttefroy A.
        • Linder M.
        • Milliere J.B.
        Inhibition of Bacillus licheniformis spore growth in milk by nisin, monolaurin, and pH combinations.
        J. Appl. Microbiol. 1999; 86 (10063630): 311-324
        • Mansour M.
        • Millière J.-B.
        An inhibitory synergistic effect of a nisin–monolaurin combination on Bacillus sp. vegetative cells in milk.
        Food Microbiol. 2001; 18: 87-94
        • Martin N.H.
        • Kent D.J.
        • Evanowski R.L.
        • Zuber Hrobuchak T.J.
        • Wiedmann M.
        Bacterial spore levels in bulk tank raw milk are influenced by environmental and cow hygiene factors.
        J. Dairy Sci. 2019; 102 (31447152): 9689-9701
        • Mehta D.S.
        • Metzger L.E.
        • Hassan A.N.
        • Nelson B.K.
        • Patel H.A.
        The ability of spore formers to degrade milk proteins, fat, phospholipids, common stabilizers, and exopolysaccharides.
        J. Dairy Sci. 2019; 102 (31521346): 10799-10813
        • Mejlholm O.
        • Dalgaard P.
        Antimicrobial effect of essential oils on the seafood spoilage micro-organism Photobacterium phosphoreum in liquid media and fish products.
        Lett. Appl. Microbiol. 2002; 34 (11849488): 27-31
        • Nitschke M.
        • Costa S.G.V.A.O.
        Biosurfactants in food industry.
        Trends Food Sci. Technol. 2007; 18: 252-259
        • Ojkic N.
        • López-Garrido J.
        • Pogliano K.
        • Endres R.G.
        Cell-wall remodeling drives engulfment during Bacillus subtilis sporulation.
        eLife. 2016; 5 (27852437)e18657
        • Paidhungat M.
        • Setlow P.
        Spore germination and outgrowth.
        in: Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Its Closest Relatives: From Genes to Cells. ASM Press, Washington, DC2001: 537-548
        • Paredes-Sabja D.
        • Torres J.A.
        • Setlow P.
        • Sarker M.R.
        Clostridium perfringens spore germination: Characterization of germinants and their receptors.
        J. Bacteriol. 2008; 190 (18083820): 1190-1201
        • Ranieri M.L.
        • Ivy R.A.
        • Mitchell W.R.
        • Call E.
        • Masiello S.N.
        • Wiedmann M.
        • Boor K.J.
        Real-time PCR detection of Paenibacillus spp. in raw milk to predict shelf life performance of pasteurized fluid milk products.
        Appl. Environ. Microbiol. 2012; 78 (22685148): 5855-5863
        • Ranieri M.L.
        • Boor K.J.
        Short communication: Bacterial ecology of high-temperature, short-time pasteurized milk processed in the United States.
        J. Dairy Sci. 2009; 92 (19762798): 4833-4840
        • Rawat S.
        Food spoilage: Microorganisms and their prevention.
        Asian J. Plant Sci. Res. 2015; 5: 47-56
        • Sánchez M.
        • Teruel J.A.
        • Espuny M.J.
        • Marqués A.
        • Aranda F.J.
        • Manresa A.
        • Ortiz A.
        Modulation of the physical properties of dielaidoylphosphatidylethanolamine membranes by a dirhamnolipid biosurfactant produced by Pseudomonas aeruginosa..
        Chem. Phys. Lipids. 2006; 142 (16678142): 118-127
        • Setlow P.
        Spore germination.
        Curr. Opin. Microbiol. 2003; 6 (14662349): 550-556
        • Singh P.
        • Cameotra S.S.
        Potential applications of microbial surfactants in biomedical sciences.
        Trends Biotechnol. 2004; 22 (15036865): 142-146
        • Sotirova A.V.
        • Spasova D.I.
        • Galabova D.N.
        • Karpenko E.
        • Shulga A.
        Rhamnolipid-biosurfactant permeabilizing effects on gram-positive and gram-negative bacterial strains.
        Curr. Microbiol. 2008; 56 (18330632): 639-644
      1. Stadler, M., J. Bitzer, B. Köpcke, K. Reinhardt, and J. Moldenhauer. 2012. Long chain glycolipids useful to avoid perishing or microbial contamination of materials. Patent WO2012/167920A1.

        • Sun L.
        • Forauer E.C.
        • Brown S.R.B.
        • D'Amico D.J.
        Application of bioactive glycolipids to control Listeria monocytogenes biofilms and as post-lethality contaminants in milk and cheese.
        Food Microbiol. 2020; 95103683
        • Tassou C.C.
        • Drosinos E.H.
        • Nychas G.J.E.
        Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4° and 10°C.
        J. Appl. Bacteriol. 1995; 78 (7615414): 593-600
        • Trmčić A.
        • Martin N.H.
        • Boor K.J.
        • Wiedmann M.
        A standard bacterial isolate set for research on contemporary dairy spoilage.
        J. Dairy Sci. 2015; 98 (26026752): 5806-5817
        • Turnbull P.C.
        • Frawley D.A.
        • Bull R.L.
        Heat activation/shock temperatures for Bacillus anthracis spores and the issue of spore plate counts versus true numbers of spores.
        J. Microbiol. Methods. 2007; 68 (17055602): 353-357
        • United States Food and Drug Administration (FDA)
        GRAS Notices: GRN No. 740 Glycolipids from Dacryopinax spathularia..
        • Zhou H.
        • Fang J.
        • Tian Y.
        • Lu X.Y.
        Mechanisms of nisin resistance in Gram-positive bacteria.
        Ann. Microbiol. 2014; 64: 413-420