Yoghurt: an unlikely source of Campylobacter jejuni/coli

Knowledge of the relative insensitivity of Campylobacter jejuni to moderately acid environments prompted us to study its survival in different batches of yoghurt of pH range 4.2-5.3 and the role of organic or inorganic acid in the die-off of this pathogen. None of the 11 strains of C. jejuni or C. coli survived more than 25 min in yoghurt. Suspecting that this rapid die-off cannot be accounted for by the pH of the yoghurt we compared the survival rates of C. jejuni in milk, whose pH had been adjusted by lactic, propionic and hydrochloric acid respectively, with that of yoghurt. Even for an inoculum of 10(5)-10(8) cfu/ml propionic acid was bactericidal in minutes. Lactic acid reduced the bacterial populations by 3-5 logs in 30 min. Strong inorganic acid HCl, by contrast, had little or no effect on the populations. Although lactic acid is quite bactericidal for C. jejuni, it is apparently not the only factor to which the prompt elimination of this pathogen from yoghurt could be attributed.     

The survival of Staphylococcus aureus during the fermentation and storage of yoghurt

The effect of yoghurt culture R _x on the survival of Staphylococcus aureus CCM 5984 added to milk in various concentrations was observed during the fermentation and storage of yoghurt. The end of the fermentation process (3.5 h) was only accompanied by a slight reduction. During the storage of yoghurt at 4°C a 1-2 log reduction was observed. No Staph. aureus was detected in yoghurt produced from milk contaminated by 10³ Staph. aureus cells 1^-1 after 48 h of cold storage. When a concentration of 10² Staph. aureus cells was used for milk contamination, the pathogen was not recovered from yoghurt during the fermentation and storage. The fermentation and storage of yoghurt was accompanied by increases in lactic acid and titrimetric acidity, as well as by a decrease in pH value.     

The immediate bactericidal effect of lactic acid on meat-borne pathogens

P. VAN NETTEN, J.H.J. HUIS IN'T VELD AND D.A.A. MOSSEL. 1994. The kinetics of the bactericidal effect of lactic acid decontamination (LAD) on meat-borne pathogens ( Salmonella spp., Campylobacter jejuni and Listeria monocytogenes ) was studied in an in-vitro model. The bactericidal effect was greatest on organisms in the lactic acid film that replaced the natural fluid on the meat surface during LAD. A stepwise increase in pH from 2.6 to 3.5 and 4.0 progressively reduced the bactericidal effect of decontamination. For treatment with 2% lactic acid for 30–90 s at 21C, the immediate death of Salmonella spp. decreased from about 0.5–2 log₁₀ cfu at pH 2.6 to an insignificant level at pH 4.0. The immediate death for Camp. jejuni decreased from 2.6 to >5.3 at pH 2.6 to 0.3–1.0 at pH 4.0. The decrease in bactericidal effect with increasing pH could, however, be countered by an increase in the temperature from 21C to 37C. It is suggested that 2% LAD at 37C for 30–90 s is suitable for elimination of salmonellas on meat but not for L. monocytogenes. Decontamination with 1% lactic acid at pH 3.0 and 21C for at least 30 s was effective for Camp. jejuni. Mesophilic Enterobacteriaceae were reliable indicators of the LAD-induced bactericidal effect on Salmonella spp. and Camp. jejuni.     

Protective effect of casein toward Salmonella typhimurium in acid-milk

Lactic acid is the inhibitory agent in yoghurt responsible for the inhibition of Salmonella typhimurium. Casein, however, may exert a protective effect toward the survival of the salmonella in acid-milk products. Salmonella typhimurium was found to die-off 21.2% more rapidly in 18-h yoghurt-whey than in 18-h yoghurt at 37 degrees C with a pH of 3.85 and 1.42% lactic acid. When casein was added to yoghurt-whey, the die-off rate of the salmonellas was reduced to that found in yoghurt. The rate remained unchanged when 4.8% sodium caseinate was added to the whey. When 0 to 14% casein was added to the acid-whey the die-off rate changed from 9.7 to 24.0 min/log reduction of cells, respectively. There was a direct correlation between the increase in casein concentration and length of survival of the salmonellas. At a pH of 3.85, 4.2 or 4.5, the die-off rate was 6.5, 13.0 or 40 min/log reduction of cells in milk containing 1.42% lactic acid, and was 4.0, 10.0 or 33.3 min/log reduction, respectively, in whey with 1.42% lactic acid. Thus, the protective effect of casein toward Salm. typhimurium increased as the pH increased. This indicated that casein exerts a protective effect on Salm. typhimurium in acid dairy products and the degree of protection depends on the casein concentration, the form of the casein molecule and the pH.     

Protective effect of casein toward Salmonella typhimurium in acid-milk

Lactic acid is the inhibitory agent in yoghurt responsible for the inhibition of Salmonella typhimurium. Casein, however, may exert a protective effect toward the survival of the salmonella in acid-milk products. Salmonella typhimurium was found to die-off 21.2% more rapidly in 18-h yoghurt-whey than in 18-h yoghurt at 37 C with a pH of 3.85 and 1.42% lactic acid. When casein was added to yoghurt-whey, the die-off rate of the salmonellas was reduced to that found in yoghurt. The rate remained unchanged when 4.8% sodium caseinate was added to the whey. When 0 to 14% casein was added to the acid-whey the die-off rate changed from 9.7 to 24.0 min/log reduction of cells, respectively. There was a direct correlation between the increase in casein concentration and length of survival of the salmonellas. At a pH of 3.85, 4.2 or 4.5, the die-off rate was 6.5, 13.0 or 40 min/log reduction of cells in milk containing 1.42% lactic acid, and was 4.0, 10.0 or 33.3 min/log reduction, respectively, in whey with 1.42% lactic acid. Thus, the protective effect of casein toward Salm. typhimurium incieased as the pH increased. This indicated that casein exerts a protective effect on Salm. typhimurium in acid dairy products and the degree of protection depends on the casein concentration, the form of the casein molecule and the pH.     

Survival of Escherichia coli O157 : H7 in yoghurt during preparation and storage at 4°C

Cow's milk was inoculated with ca 10³ and 10⁷ cfu ml⁻¹ Escherichia coli O157 : H7. After fermentation at 42°C for 0–5 h, the yoghurt was stored at 4°C. Two kinds of yoghurt were used : traditional yoghurt (TY), made with Streptococcus thermophilus and Lactobacillus bulgaricus starter cultures, and 'bifido' yoghurt (BY), made with the two starter cultures plus Bifidobacterium bifidum . After 7 d E. coli O157 : H7 decreased from 3·52 to 2·72 log₁₀ cfu ml⁻¹ and from 7·08 to 5·32 log₁₀ cfu ml⁻¹ in TY, and from 3·49 to 2·73 log₁₀ cfu ml⁻¹ and from 7·38 to 5·41 log₁₀ cfu ml⁻¹ in BY. The pH values of yoghurt dropped from 6·6 to 4·5 and 4·4 in TY (for low and high pathogen inocula, respectively), and from 6·6 to 4·6 and 4·5 in BY (for low and high pathogen inocula, respectively).     

Viability of Cryptosporidium parvum during ensilage of perennial ryegrass

The survival of Cryptosporidium parvum during ensilage of perennial ryegrass was examined in laboratory silos with herbage prepared in one of three different ways; either untreated, inoculated with a strain of Lactobacillus plantarum or by direct acidification with formic acid. The pH values of all silages initially fell below 4.5, but only formic acid-treated silage remained stable at less than pH 4 after 106 d, with the pH of the untreated and inoculant-treated silages rising to above 6. The formic acid-treated silage had a high lactic acid concentration (109 g kg^-1 dry matter (DM)) and low concentrations of propionic and butyric acids after 106 d. However, the untreated and inoculant-treated silages showed an inverse relationship, with low lactic acid concentrations and high concentrations of acetic, propionic and butyric acids. These silages also contained ammonia-N concentrations in excess of 9 g kg^-1 DM. In terms of the viability of Cryptosporidium parvum oocysts very few differences were seen after 14 d of ensilage with ca 50% remaining viable, irrespective of treatment and total numbers had declined from the initial level of 5.9 × 10⁴ to 1 x 10⁴ g^-1 fresh matter. Total oocyst numbers remained approximately the same until the end of the ensiling period, with the percentage of viable oocysts declining to 46, 41 and 32% respectively for formic acid, inoculant and untreated silages. The results are discussed in terms of changes occurring during the silage fermentation, in particular the products which may influence the survival of Cryptosporidium and implications for agricultural practice and the health of silage fed livestock.     

A note on the antibacterial properties of 3-(3'-isocyanocyclopent-2'-enylidene)propionic acid in the ovine rumen

B rewer , D., P arkinson , V.O. & T aylor , A. 1990. A note on the antibacterial properties of 3-{3'-isocyanocyclopent-2'-enylidene)propionic acid in the ovine rumen. Journal of Applied Bacteriology 69 , 701–704.
The concentration of 3-(3'-isocyanocyclopent-2'-enylidene)propionic acid after introduction into the ovine rumen declined by 50% during 30 min. Its antibacterial activity in the rumen could be demonstrated by the decline in numbers of cellulo-lytic rumen bacteria from about 10⁶/ml to about 10²/ml.     

Antimicrobial activity of Leuconostoc gelidum against closely related species and Listeria monocytogenes

H arding , C.D. & S haw , E.G. 1990. Antimicrobial activity of Leuconostoc gelidum against closely related species and Listeria monocytogenes. Journal of Applied Bacteriology 69 , 648–654.
A newly isolated strain of Leuconostoc gelidum was evaluated for its ability to inhibit a wide spectrum of lactic acid bacteria, meat spoilage bacteria and food-related human pathogens, including Listeria monocytogenes . It was inhibitory to most of the lactobacilli, all the leuconostocs, and three strains of L. monocytogenes when tested both by direct and well diffusion methods. Cell-free extract retained activity after 60 min at 100C but was sensitive to protease. Dialysis suggested a molecular weight in excess of 10⁴ daltons. The inhibitory effect was bactericidal and rapid.     

The use of the bacteriocin, nisin, as a preservative in ricotta-type cheeses to control the food-borne pathogen Listeria monocytogenes

The efficacy of nisin to control the food-borne pathogen Listeria monocytogenes in ricotta-type cheeses over long storage (70 d) at 6–8°C was determined. Cheeses were prepared from unpasteurized milk by direct acidification with acetic acid (final pH 5·9) and/or calcium chloride addition during heat treatment. Nisin was added in the commercial form of Nisaplin^® pre-production to the milk. Each batch of cheese was inoculated with 10²–10³ cfu g⁻¹ of a five-strain cocktail of L. monocytogenes before storage. Shelf-life analysis demonstrated that incorporation of nisin at a level of 2·5 mg l⁻¹ could effectively inhibit the growth of L. monocytogenes for a period of 8 weeks or more (dependent on cheese type). Cheese made without the addition of nisin contained unsafe levels of the organism within 1–2 weeks of incubation. Measurement of initial and residual nisin indicated a high level of retention over the 10-week incubation period at 6–8°C, with only 10–32% nisin loss.     

The effect of pH, acidulant and temperature on the survival of Yersinia enterocolitica

The survival of Yersinia enterocolitica at sub-optimal temperatures (0–23°C) and growth inhibitory pH values, achieved using a range of acidulants, was investigated. At a given pH, survival was greater the lower the temperature. Sulphuric and citric acids had lower bactericidal activity than acetic and lactic acids and in nearly all cases where the four acids could be compared at the same pH the order of bactericidal activity was acetic > lactic > citric > sulphuric. Attempts to model this behaviour by a negative square root relationship gave good correlation coefficients for plots of the square root of death rate against temperature at different combinations of pH and acidulant but so too did several other functions of death rate. The high coefficient of variation for T ₀ determined from square root plots prevented construction of a combined temperature/pH model similar to that already described for growth.     

The effects of temperature and pH on the growth of yeast species during the fermentation of grape juice

The effects of temperature and pH on the survival and growth of Saccharomyces cerevisiae, Kloeckera apiculata, Candida stellata, Candida krusei, Candida pulcherrima and Hansenula anomala were examined during mixed culture in grape juice. At 25°C, pH 3.0 and pH 3.5, S. cerevisiae dominated the fermentation and the other species died off before fermentation was completed. Saccharomyces cerevisiae also dominated the fermentation at 20°C but there was increased growth and survival of the other species. At 10°C the fermentation was dominated by the growth of both S. cerevisiae and K. apiculata and there was extended growth and survival of C. stellata and C. krusei. Juices fermented at 10°C exhibited ethanol concentrations between 7.4 and 13.4% and populations of K. apiculata, C. stellata and C. krusei in the range 10⁶-10⁸ cells/ml. However, these species produced maximum ethanol concentrations in the range 2.7–6.6% when grown as single cultures in grape juice.     

Different inhibition of Lactobacillus delbrueckii subsp. bulgaricus by D- and L-lactic acid: effects on lag phase, growth rate and cell yield

Inhibition of Lactobacillus bulgaricus by lactic acids at constant pH is governed by two factors: the total concentration of lactic acids and the D/L ratio of lactic acid stereoisomers. L-Lactic acid is more inhibitory than the D-isomer, the latter being the only isomer produced by Lact. bulgaricus. The major inhibitory effect is an extremely prolonged lag phase (2–39 h for the range 0–25 g 1^-1 added lactic acids). Added lactic acids reduce the maximum cell concentration obtained in pH controlled fermentations, but have virtually no effect on maximum specific growth rate and on the yield of biomass on produced lactic acid. The added lactic acids have no effect on the growth-related ATP consumption nor on the non-growth related ATP consumption. The culture is heterogeneous since a part of the culture is unable to grow in the presence of L-lactic acid. A different inhibitory effect of D- and L-lactic acid has implications for yoghurt manufacture and potential for improved food preservation.     

Fate of low temperature and acid-adapted Yersinia enterocolitica and Listeria monocytogenes that contaminate lactic acid decontaminated meat during chill storage

Pathogens found in the environment of abattoirs may become adapted to lactic acid used to decontaminate meat. Such organisms are more acid tolerant than non-adapted parents and can contaminate meat after lactic acid decontamination (LAD). The fate of acid-adapted Yersinia enterocolitica and Listeria monocytogenes, inoculated on skin surface of pork bellies 2 h after LAD, was examined during chilled storage. LAD included dipping in 1%, 2% or 5% lactic acid solutions at 55°C for 120 s. LAD brought about sharp reductions in meat surface pH, but these recovered with time after LAD at ≈1–1·5 pH units below that of water-treated controls. Growth permitting pH at 4·8–5·2 was reached after 1% LAD in less than 0·5 d (pH 4·8–5·0), 2% LAD within 1·5 d (pH 4·9–5·1) and after 5% LAD (pH 5·0–5·2) within 4 d. During the lag on 2% LAD meat Y. enterocolitica counts decreased by 0·9 log₁₀ cfu per cm² and on 5% LAD the reduction was more than 1·4 log₁₀ cfu per cm². The reductions in L. monocytogenes were about a third of those in Y. enterocolitica . On 1% LAD the counts of both pathogens did not decrease significantly. The generation times of Y. enterocolitica and L. monocytogenes on 2–5% LAD meats were by up to twofold longer than on water-treated controls and on 1% LAD-treated meat they were similar to those on water-treated controls. Low temperature and acid-adapted L. monocytogenes and Y. enterocolitica that contaminate skin surface after hot 2–5% LAD did not cause an increased health hazard, although the number of Gram-negative spoilage organisms were drastically reduced by hot 2–5% LAD and intrinsic (lactic acid content, pH) conditions were created that may benefit the survival and the growth of acid-adapted organisms.     

APPARENT IN VIVO ACETYLCHOLINE TURNOVER RATE IN WHOLE MOUSE BRAIN: EVIDENCE FOR A TWO COMPARTMENT MODEL BY TWO INDEPENDENT KINETIC ANALYSES

Abstract— Acetylcholine turnover has been determined in whole mouse brain using a newly available high specific activity [³H]choline (70 Ci/mmol). Animals were killed at various time points (0.25–10 min) after pulse adminstration of [³H]choline (Ch) by microwave irradiation of the head. Steady-state levels of ACh were determined by radioenzymatic analysis as described by G oldberg & M c C aman (1973) as modified by M c C aman & S tetzer , 1977. Ch levels were determined by a modification of the method of M c C aman & S tetzer (1977). Radiolabelled metabolites of [³H]Ch were separated by selective extraction of [³H]Ch and [³H]ACh inio tetraphenylboron in 3-heptanone (C arroll et al. , 1977) coupled with an enzymatic separation of [³H]Ch from [³H]ACh. A precursor-product relationship was verified for Ch and ACh specific activities. Acetylcholine turnover rate was determined by the biosynthesis ratio method (S chuberth et al. , 1969, Method 1) and by the finite-differences method (N eff et al. , 1971, Method 2). Both methods of kinetic analysis revealed two distinct turnover rates for acetylcholine. In the first phase (0.25–1.5 min post-[³H]Ch), the ACh turnover rate averaged 22nmol/g/min (both methods). During the second phase, (2–10 min) acetylcholine turnover rates were significantly ( P < 0.05 and P < 0.01) lower; i.e. 7nmol/g/min (Method 1) and 5.9 nmol/g/min (Method 2). The data are consistent with a 2-compartment model for ACh turnover in whole mouse brain. Additionally, the method described for the separation of radiolabelled metabolites of [³H]Ch allows an accurate determination of ACh turnover in as little as 2 mg of tissue.     

Influence of pH and lactic acid concentration on Clostridium tyrobutyricum during continuous growth in a pH-auxostat

The aim of this project was to establish the minimal inhibitory concentration (MIC) of lactic acid for growth of Clostridium tyrobutyricum. A pH-auxostat was used to maintain a constant pH and to allow continuous growth at the highest possible rates at fixed, but adjustable concentrations of lactate. By raising the concentration of lactic acid and keeping the pH constant, the growth rate was shown to decrease linearly with increasing lactic acid concentration. The p K _a of lactic acid, measured in the actual growth medium at 37°C, was 3.40 (±0.03). Based on this value, the MIC_undiss values for each pH were estimated. The MIC of total lactic acid (MIC_tot) ranged from 150 mmol l⁻¹ to 1510 mmol l⁻¹ at pH 4.6–6.25, respectively. The corresponding MIC values of undissociated lactic acid (MIC_undiss) ranged from 8.9 to 2.1 mmol l⁻¹ at the same pH values. These results emphasize the importance of a rapid pH decrease and an equally rapid initial lactic acid fermentation of the ensilage, in order to sufficiently suppress clostridial growth.     

ADSORPTION OF FULVIC ACID ON ALGAL SURFACES AND ITS EFFECT ON CARBON UPTAKE

Adsorption of Suwannee River fulvic acid (SRFA) to algal surfaces of three green algae was studied at environmentally relevant pH values (4 –7) and SRFA concentrations (5–100 mg·L ^{− 1}). The influence of adsorbed SRFA on carbon uptake of Scenedesmus subspicatus Chodat was also examined. Although no adsorption was observed at neutral pH values (pH 6 and 7), at pH 4 up to 31 mg SRFA·m ^{− 2} and at pH 5 up to 4 mg SRFA·m ^{− 2} was adsorbed to the algal surfaces. Electrophoretic mobility measurements of S. subspicatus demonstrated an increase in the negative surface charge of the alga in the presence of SRFA at pH 4. The adsorbed SRFA also influenced ¹⁴C uptake in S. subspicatus; in this case, enhanced carbon uptake could be related to the amount of adsorbed SRFA. The binding of humic substances by algal surfaces was interpreted as the result of hydrogen bonding and hydrophobic interactions.     

In vitro susceptibility of Erwinia amylovora (Burrill) Winslow et al. to geraniol and citronellol

The in vitro antimicrobial activity of geraniol and citronellol towards seven strains of Erwinia amylovora , the causal agent of 'fire blight'of Rosaceous plants, was assessed in tube cultures. All of the strains tested at 1 × 10⁵ cfu/ml were inhibited for 24 h by geraniol in the range 600–1500 mg/1, whereas its minimum bactericidal concentration was 800–1700 mg/1. Citronellol was less effective, being bactericidal for only two of seven strains. RIF-NY, isolated from apple orchards, was relatively resistant to geraniol; 1700 mg/1 of the chemical only reduced the growth of an inoculum of 1 × 10⁷ cfu/ml. In general, such terpenoids commenced exerting a bactericidal effect 6 h after addition to the suspensions, even if geraniol added at 1700 mg/1 to 1 × 10³ cfu/ml of five strains, commenced its bactericidal activity earlier than 6 h.     

Sterilization of soybean cotyledons by boiling in lactic acid solution

F. RUÍZ-TERÁN AND J.D. OWENS. 1996. The effect of pH on the heat resistance of Bacillus stearothermophilus spores at 100°C in the presence of 0.11 mol 1^-1 lactic acid and 0.2 mol 1^-1 sodium phosphate buffer was examined. At pH values of 7.0 and 6.0 spores survived 60 min exposure unharmed but at pH 4.3 and 3.0 they died with decimal reduction times (DRTs) of 27 min and 2.8 min, respectively. Death rates were similar in the presence or absence of hydrated soybean cotyledons. In the presence of phosphate buffer and cotyledons at mean pH 3.6 the DRT was 118 min but in the presence, in addition, of lactic acid it was 11 min. It is suggested that the enhanced death rate was due to toxic effects of undissociated lactic acid. Rhizopus oligosporus NRRL 2710 grew well on cotyledons, having pH values from 7.0 to 3.7, prepared by boiling for 60 min in the presence of 0.11 mol 1^-1 lactic acid and 0.2 mol 1^-1 phosphate buffer.     

Some effects on the growth of brown trout from exposure to aluminium at different pH levels

Yearling brown trout, Salmo trutta L., were exposed to various concentrations of inorganic aluminium (0–3.7 μM1⁻¹) over a pH range of 4.3–6.5 in a flow-through bioassay apparatus using synthetic test media. Low pH, in the absence of aluminium, produced little effect on growth or survival except at the lowest pH tested (4.3). At pH less than 5.5, concentrations of total aluminium in excess of 1 μM 1⁻¹ (27μg 1⁻¹) were found to retard growth. The effects of a given aluminium concentration were markedly reduced at pH above 5.5.
The change in aluminium toxicity with pH must be related to changes in aluminium chemistry. When growth rates are correlated with the different aluminium species, calculated using thermodynamic equilibrium constants given in the literature, it appears that the Al(OH)^{2 +} species is the most toxic, with a small contribution also coming from polymeric complexes.