Inhibition of Listeria monocytogenes by fatty acids and monoglycerides.

Fatty acids and monoglycerides were evaluated in brain heart infusion broth and in milk for antimicrobial activity against the Scott A strain of Listeria monocytogenes. C12:0, C18:3, and glyceryl monolaurate (monolaurin) had the strongest activity in brain heart infusion broth and were bactericidal at 10 to 20 micrograms/ml, whereas potassium (K)-conjugated linoleic acids and C18:2 were bactericidal at 50 to 200 micrograms/ml. C14:0, C16:0, C18:0, C18:1, glyceryl monomyristate, and glyceryl monopalmitate were not inhibitory at 200 micrograms/ml. The bactericidal activity in brain heart infusion broth was higher at pH 5 than at pH 6. In whole milk and skim milk, K-conjugated linoleic acid was bacteriostatic and prolonged the lag phase especially at 4 degrees C. Monolaurin inactivated L. monocytogenes in skim milk at 4 degrees C, but was less inhibitory at 23 degrees C. Monolaurin did not inhibit L. monocytogenes in whole milk because of the higher fat content. Other fatty acids tested were not effective in whole or skim milk. Our results suggest that K-conjugated linoleic acids or monolaurin could be used as an inhibitory agent against L. monocytogenes in dairy foods.     

Natural milk fatty acids affect survival and invasiveness of Listeria monocytogenes

The effects of 12 fatty acids, naturally occurring in milk from several mammalian species, on the survival and invasion ability of Listeria monocytogenes , a food-borne pathogen, were determined. The survival was tested in the presence of 200 μg ml⁻¹ fatty acids suspended in brain hearth infusion broth or in storage conditioning solution (NaCl 1%) of Mozzarella cheese, an Italian soft unripened cheese, at pH 7·0 or 5·0. Lauric (C12:0), linoleic (C18:2) and linolenic (C18:3) acids exerted the strongest bactericidal activity. The invasive efficiency of L. monocytogenes , determined in the Caco-2 enterocyte-like cell line, was strongly decreased in the presence of the fatty acids tested (from about 20 to 500-fold). This research suggests that naturally occurring fatty acids of milk, supplemented in milk derivatives, could affect both bacterial growth and invasiveness and consequently, could serve as barriers towards L. monocytogenes infection.     

Comparison of media and methods for detecting and enumerating Listeria monocytogenes in refrigerated cabbage.

Direct plating, selective enrichment, and cold enrichment followed by secondary selective enrichment procedures were compared for detecting and enumerating Listeria monocytogenes in chopped cabbage stored at 5 degrees C for up to 64 days. Addition of Fe3+ to solid media enhanced detection of the organism. Cold enrichment (5 degrees C) in nutrient broth and brain heart infusion broth followed by secondary enrichment (48 h, 30 degrees C) in Trypticase soy-yeast extract-antibiotic broth and thiocyanate-nalidixic acid broth and plating on selective agar media (Doyle and Schoeni selective enrichment agar [minus acriflavin hydrochloride, supplemented with 5 micrograms of Fe3+/ml] and McBride Listeria agar) resulted in the detection of highest populations.     

Comparison of media and methods for detecting and enumerating Listeria monocytogenes in refrigerated cabbage

Direct plating, selective enrichment, and cold enrichment followed by secondary selective enrichment procedures were compared for detecting and enumerating Listeria monocytogenes in chopped cabbage stored at 5 degrees C for up to 64 days. Addition of Fe3+ to solid media enhanced detection of the organism. Cold enrichment (5 degrees C) in nutrient broth and brain heart infusion broth followed by secondary enrichment (48 h, 30 degrees C) in Trypticase soy-yeast extract-antibiotic broth and thiocyanate-nalidixic acid broth and plating on selective agar media (Doyle and Schoeni selective enrichment agar [minus acriflavin hydrochloride, supplemented with 5 micrograms of Fe3+/ml] and McBride Listeria agar) resulted in the detection of highest populations.     

Inhibition of Bacillus licheniformis spore growth in milk by nisin, monolaurin, and pH combinations

The effects of nisin and monolaurin, alone and in combination, were investigated on Bacillus licheniformis spores in milk at 37 degrees C. In the absence of inhibitors, germinated spores developed into growing vegetative cells and started sporulation at the end of the exponential phase. In the presence of nisin (25 IU ml-1), spore outgrowth was inhibited (4 log10 reduction at 10 h). Regrowth appeared between 10 and 24 h and reached a high population level (1.25 x 10(8) cfu ml-1) after 7 d. Monolaurin (250 micrograms ml-1) had a bacteriostatic effect during the first 10 h but thereafter, regrowth occurred slowly with a population level after 7 d (4 x 10(5) cfu ml-1) lower than that of nisin. Different combined effects of nisin (between 0 and 42 IU ml-1), monolaurin (ranging from 0 to 300 micrograms ml-1), pH values (between 5.0 and 7.0) and spore loads (10(3), 10(4), 10(5) spores ml-1) were investigated using a Doehlert matrix in order to study the main effects of these factors and the different interactions. Results were analysed using the Response Surface Methodology (RSM) and indicated that nisin and monolaurin had no action on spores before germination; only pH values had a significant effect (P < or = 0.001), i.e. spore count decreased as the pH value increased in relation to germination. Sublethal concentrations of nisin (30 IU ml-1) and monolaurin (100 micrograms ml-1) in combination acted synergistically on outgrown spores and vegetative cells, showing total inhibition at pH 6.0, without regrowth, within 7 d at 37 degrees C.     

Control of Listeria monocytogenes in a biofilm by competitive-exclusion microorganisms

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37 degrees C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10(3) CFU of L. monocytogenes/ml and 10(5) CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37 degrees C for 24 h, 15 degrees C for 14 days, 8 degrees C for 21 days, and 4 degrees C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37 degrees C, two at 15 and 8 degrees C, and three at 4 degrees C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4 degrees C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log(10) CFU of L. monocytogenes/cm(2)). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37 degrees C.     

Inhibition of Listeria monocytogenes by using bacteriocin PA-1 produced by Pediococcus acidilactici PAC 1.0

The bacteriocin produced by Pediococcus acidilactici PAC 1.0, previously designated PA-1 bacteriocin, was found to be inhibitory and bactericidal for Listeria monocytogenes. A dried powder prepared from PAC 1.0 culture supernatant fortified with 10% milk powder was found to contain bacteriocin activity. An MIC against L. monocytogenes and lytic effects in broth cultures were determined. Inhibition by PA-1 powder occurred over the pH range 5.5 to 7.0 and at both 4 and 32 degrees C. In addition, inhibition of L. monocytogenes was demonstrated in several food systems including dressed cottage cheese, half-and-half cream, and cheese sauce.     

Inhibition of Listeria monocytogenes by using bacteriocin PA-1 produced by Pediococcus acidilactici PAC 1.0.

The bacteriocin produced by Pediococcus acidilactici PAC 1.0, previously designated PA-1 bacteriocin, was found to be inhibitory and bactericidal for Listeria monocytogenes. A dried powder prepared from PAC 1.0 culture supernatant fortified with 10% milk powder was found to contain bacteriocin activity. An MIC against L. monocytogenes and lytic effects in broth cultures were determined. Inhibition by PA-1 powder occurred over the pH range 5.5 to 7.0 and at both 4 and 32 degrees C. In addition, inhibition of L. monocytogenes was demonstrated in several food systems including dressed cottage cheese, half-and-half cream, and cheese sauce.     

Inhibition of Listeria monocytogenes growth by the lactoperoxidase-thiocyanate-H2O2 antimicrobial system.

The lactoperoxidase-thiocyanate-H2O2 system (LP system), consisting of lactoperoxidase (0.37 U/ml), KSCN (0.3 mM), and H2O2 (0.3 mM), delayed but did not prevent growth of L. monocytogenes Scott A at 5, 10, 20, and 30 degrees C in broth and at 20 degrees C in milk. The net lag periods determined spectrophotometrically varied inversely with temperature and were shorter at 5 and 10 degrees C for cultures from shaken versus from statically grown inocula. Lag periods for cultures from shaken and statically grown inocula, respectively, were 73 and 98 h at 5 degrees C, 22 and 32 h at 10 degrees C, both 8.9 h at 20 degrees C, and both 2.8 h at 30 degrees C. After the lag periods, the maximum specific growth rates were similar for each of the three treatments (complete LP system, H2O2 alone, or control broth) at 5, 10, and 20 degrees C and were 0.06 to 0.08, 0.09 to 0.1, and 0.32 to 0.36/h, respectively. At 20 degrees C in sterile reconstituted skim milk, the LP system restricted growth of Scott A, with log CFU counts per ml at 0, 36, and 68 h being 5.7, 6.4 and 7.9 (versus 5.7, 9.8, and 11.2 for controls). Possible explanations for the decreased lag times observed for cultures from aerobically grown inocula are discussed.     

Inhibition of Listeria monocytogenes growth by the lactoperoxidase-thiocyanate-H2O2 antimicrobial system

The lactoperoxidase-thiocyanate-H2O2 system (LP system), consisting of lactoperoxidase (0.37 U/ml), KSCN (0.3 mM), and H2O2 (0.3 mM), delayed but did not prevent growth of L. monocytogenes Scott A at 5, 10, 20, and 30 degrees C in broth and at 20 degrees C in milk. The net lag periods determined spectrophotometrically varied inversely with temperature and were shorter at 5 and 10 degrees C for cultures from shaken versus from statically grown inocula. Lag periods for cultures from shaken and statically grown inocula, respectively, were 73 and 98 h at 5 degrees C, 22 and 32 h at 10 degrees C, both 8.9 h at 20 degrees C, and both 2.8 h at 30 degrees C. After the lag periods, the maximum specific growth rates were similar for each of the three treatments (complete LP system, H2O2 alone, or control broth) at 5, 10, and 20 degrees C and were 0.06 to 0.08, 0.09 to 0.1, and 0.32 to 0.36/h, respectively. At 20 degrees C in sterile reconstituted skim milk, the LP system restricted growth of Scott A, with log CFU counts per ml at 0, 36, and 68 h being 5.7, 6.4 and 7.9 (versus 5.7, 9.8, and 11.2 for controls). Possible explanations for the decreased lag times observed for cultures from aerobically grown inocula are discussed.     

Mechanism of Synergistic Inhibition of Listeria monocytogenes Growth by Lactic Acid, Monolaurin, and Nisin

The combined lactic acid, monolaurin, and nisin effects on time-to-detection (optical density at 600 nm) extension were greater (P < 0.05) than any single or paired combination effect, which demonstrates a synergistic interaction among the antimicrobials. Monolaurin exposure caused C12:0 cell membrane incorporation. Lactic acid caused increased monolaurin C12:0 membrane incorporation, while nisin had no influence. We postulate that lactic acid-enhanced monolaurin C12:0 incorporation into the cell membrane increased membrane fluidity resulting in increased nisin activity.     

Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy.

The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.     

Effects of sample handling temperatures on bovine skim milk progesterone concentrations

The effects of incubation of whole milk at various temperatures and times on the concentration of progesterone in the skim milk fraction was determined. For the study, milk samples were collected from 10 pregnant Holstein cows. The milk from each cow was transferred to culture tubes to provide 32 replicates of 3 ml volume. To begin the incubation study, all samples were placed in a 37 degrees C water bath for 4 h. The end of this incubation was designated as time 0 and a sample from each cow was centrifuged to harvest skim milk. At time 0, samples from each cow were divided among incubation temperatures of 0 degrees, 4 degrees, 20 degrees and 37 degrees C. Samples were removed from each incubation group at 30, 60, 90 and 120 min. After 120 min, all remaining samples were returned to the 37 degrees C incubation and skim milk was collected at 30, 60 and 90 min. Progesterone was measured in skim milk by radioimmunoassay. The mean +/- SE concentration of progesterone in skim milk at time 0 was 10.9 +/- 1.1 nmol/L. The mean concentration of progesterone in skim milk was higher (P < 0.05) in all samples incubated at 0 degrees and 4 degrees C, with incremental increases ranging from 34% to 67% above time 0. Progesterone in skim milk returned to time 0 concentrations in milk samples transferred from 0 degrees or 4 degrees C to 37 degrees C. There was no change in skim milk progesterone in whole milk samples incubated at 20 degrees or 37 degrees C. From this study, it can be concluded that the concentration of progesterone in skim milk is temperature dependent. Inconsistency in handling whole milk samples can have a profound effect in the concentration of progesterone on skim milk. The temperature-dependent effect was reversible and may be related to solubility of progesterone in milk fat.     

Factors affecting sensitivity of Staphylococcus aureus 196E to polyphosphates.

The effect of polyphosphates (eight compounds) on growth of Staphylococcus aureus 196E in brain heart infusion broth was studied. The organism was sensitive (in decreasing order) to chain polyphosphates with 21, 3, 13, and 15 PO4 groups, and bactericidal effects were observed with 0.5% of these compounds. No inhibition was effected by PPi or a metaphosphate. The inhibitory effects were pH dependent, and bacterial sensitivity was highest at pH greater than 7.4. Initial populations affected the number of survivors. No growth was observed after 24 h at 35 degrees C when the initial cell population was less than 10(4) CFU/ml, and a 100- to 1,000-fold decline in cell numbers occurred when initial populations were higher than 10(4) CFU/ml. Sodium tripolyphosphate produced less inhibition after heat sterilization (15 min, 121 degrees C) than after filter sterilization, whereas sodium hexametaphosphate (n = 21) retained most of its antimicrobial activity after heat sterilization. Supplementation of broth with Mg2+ was effective in overcoming inhibition by 0.5% sodium tripolyphosphate, and an addition of 0.25 to 1.0 mM cation restored most of the growth. Inhibition was partially eliminated by Ca2+ and Fe2+, but not by Zn2+ or Mn2+.     

The effect of various acidulants on the growth of Listeria monocytogenes

The ability of four Listeria monocytogenes strains to initiate growth in brain heart infusion broth adjusted to various pH values with either acetic, lactic, citric or hydrochloric acid was investigated. Acetic acid was the most effective inhibitor tested, since in broth adjusted with this acid a higher minimum pH was required for growth of the various strains at both 4 and 30°C, as compared with broth adjusted with the other acidulants. The minimum pH value required for the initiation of growth of L. monocytogenes ranged from 5·0 to 5·7 at 4°C, and from 4·3 to 5·2 at 30°C, depending upon the acidulant used.     

Factors affecting sensitivity of Staphylococcus aureus 196E to polyphosphates

The effect of polyphosphates (eight compounds) on growth of Staphylococcus aureus 196E in brain heart infusion broth was studied. The organism was sensitive (in decreasing order) to chain polyphosphates with 21, 3, 13, and 15 PO4 groups, and bactericidal effects were observed with 0.5% of these compounds. No inhibition was effected by PPi or a metaphosphate. The inhibitory effects were pH dependent, and bacterial sensitivity was highest at pH greater than 7.4. Initial populations affected the number of survivors. No growth was observed after 24 h at 35 degrees C when the initial cell population was less than 10(4) CFU/ml, and a 100- to 1,000-fold decline in cell numbers occurred when initial populations were higher than 10(4) CFU/ml. Sodium tripolyphosphate produced less inhibition after heat sterilization (15 min, 121 degrees C) than after filter sterilization, whereas sodium hexametaphosphate (n = 21) retained most of its antimicrobial activity after heat sterilization. Supplementation of broth with Mg2+ was effective in overcoming inhibition by 0.5% sodium tripolyphosphate, and an addition of 0.25 to 1.0 mM cation restored most of the growth. Inhibition was partially eliminated by Ca2+ and Fe2+, but not by Zn2+ or Mn2+.     

Properties of Bac W42, a bacteriocin produced by Bacillus subtilis W42 isolated from Cheonggukjang

Ten Bacillus strains with antimicrobial activities were isolated from Cheonggukjang produced at different parts in Korea. They all inhibited Listeria monocytogenes ATCC 19111 and nine inhibited Bacillus cereus ATCC 14579. Four isolates (W42, H27, SKE 12, and K21) showing strong inhibiting activities were identified as B. subtilis. B. subtilis W42 was the most inhibiting strain. The antimicrobial activity of culture supernatant from B. subtilis W42 was destroyed completely by proteinase K treatment, indicating that a bacteriocin was the responsible agent. The bacteriocin, Bac W42, was most stable at pH 7 and stable between pH 3-6 and 8-9. Bac W42 was stable up to 80°C. BHI (brain heart infusion) and TSB (tryptic soy broth) were the best media for the activity (320 AU/ml) followed by LB (160 AU/ml). Bac W42 was partially purified by column chromatographies. The specific activity was increased from 1,151.2 AU/ml to 9,043.5 AU/ml and the final yield was 26.3%. Bac W42 was 5.4 kDa in size as determined by SDS-PAGE. Bac W42 showed bactericidal activity against L. monocytogenes ATCC 19111.     

Occurence of lactose-negative mutants in chemostat cultures of lactic streptococci.

Batch and chemostat cultures of Streptococcus cremoris HP and Streptococcus lactis 829 were examined for lactose-hegative (lac-)mutants on indicator agar. In batch cultures, S. cremoris HP gave less than 1% of the total count as lac- colonies while S. lactis 829 consistently contained about 15% of the total as lac- colonies. In chemostat cultures of S. cremoris HP in 2% skim milk containing casamino acids and yeast extract (0.1% each), the percentage of lac- colonies increased markedly when the temperature of growth was 18 degrees C but not when the temperature of growth was 25 degrees C. The percentage of lac- colonies in chemostat cultures in the skim milk medium at 25 degrees C was about the same as that in batch cultures. On the other hand, when chemostat cultures of S. lactis 829 in the skim milk medium were grown at several temperatures between 18 and 33 degrees C, the percentage of lac- colonies was markedly lower than that found in batch cultures of this organism. Cultivation of S. cremoris HP in chemostats with yeast extract-glucose broth at low temperature (18 degrees C) resulted in a selection of cells giving lac- colonies and atypical (small) lac+ colonies. The results show that cultivation of S. cremoris HP and S. lactis 829 in chemostats sometimes gave rise to altered populations. Conditions causing a change in one organism did not necessarily cause a similar change in the other. The results indicate that the successful propagation of lactic streptococci in chemostats for use as starter cultures in the dairy industry will require the careful establishment of optimum conditions for every strain so as to minimize the possible selection of undesirable populations.     

Identification of the conjugated linoleic acid isomer that inhibits milk fat synthesis

Conjugated linoleic acids (CLA) are octadecadienoic fatty acids that have profound effects on lipid metabolism. Our previous work showed that CLA (mixture of isomers) markedly reduced milk fat synthesis. In this study, our objective was to evaluate the effects of specific CLA isomers. Multiparous Holstein cows were used in a 3x3 Latin square design, and treatments were 4-day abomasal infusions of 1) skim milk (control), 2) 9,11 CLA supplement, and 3) 10,12 CLA supplement. CLA supplements provided 10 g/day of the specific CLA isomer (cis-9,trans-11 or trans-10,cis-12). Treatments had no effect on intake, milk yield, or milk protein yield. Only the 10,12 CLA supplement affected milk fat, causing a 42 and 44% reduction in milk fat percentage and yield, respectively. Milk fat composition revealed that de novo synthesized fatty acids were extensively reduced. Increases in ratios of C(14:0) to C(14:1) and C(18:0) to C(18:1) indicated the 10,12 CLA supplement also altered Delta(9)-desaturase. Treatments had minimal effects on plasma concentrations of glucose, nonesterified fatty acids, insulin, or insulin-like growth factor-I. Overall, results demonstrate that trans-10,cis-12 CLA is the isomer responsible for inhibition of milk fat synthesis.     

A comparative study of preservation and storage of Haemophilus influenzae

The aim of this study was to compare the efficacy of conservation by freezing the strains of Haemophilus influenzae at -20 degrees C and -70 degrees C. Skim milk supplemented with glucose, yeast extract and glycerol allowed highest viability of H. influenzae both at -20 degrees C and -70 degrees C from the media analyzed. Trypticase soy broth and brain heart infusion broth supplemented with glycerol, allowed excellent recovery. Use of cotton swaps as supporting material, with or without addition of cryoprotective agents, did not modify H. influenzae viability after six months of storage. Concentration of the initial inoculum positively affected viability when stored at -20 degrees C. Initial concentration did not influence survival after storage at -70 degrees C. Thawing at room temperature should not exceed 3 h as to get highest survival percentage.