Modelling the effect of pH, acidulant and temperature on the growth rate of Yersinia enterocolitica

Growth of two pathogenic and one environmental serotype of Yersinia enterocolitica under acidic conditions and at 4 and 25°C was investigated. At both temperatures the maximum growth inhibitory pH depended on the acidulant used and was in the order acetic > lactic > citric > sulphuric. At the lower temperature the maximum growth inhibitory pH was 0.3-0.5 pH units higher than at 25°C. No difference was observed between the behaviour of pathogenic and environmental serotypes in this respect. Measurement of growth at a number of sub-optimal temperatures and pH values showed that the variation of growth rate with temperature could be represented by a square root plot. The effect of different pH values could be incorporated into the model by replacing the regression coefficient b by its relationship with pH. Values of maximum growth inhibitory pH derived from the model were in good agreement with experimental values with the exception of acetic acid.     

The influence of pH, temperature and organic acids on the initiation of growth of Yersinia enterocolitica

The influence of incubation temperature, and of acetic, lactic and citric acids on the minimum pH for the initiation of growth of six strains of Yersinia enterocolitica was determined. The strains included two of serotype O : 9, two of serotype O : 3, and one each of serotypes O : 8 and O : 5, 27. In a culture medium acidified with HC1 to pH values between 4.0 and 6.0 at intervals of approximately 0.1 unit the minimum pH at which growth was detected after incubation at 20°, 10°, 7° and 4°C for 21 d was in the ranges 4.18–4.36, 4.26–4.50, 4.36–4.83 and 4.42–4.80, respectively. The minimum pH for growth was also determined in media that contained 17, 33 and 50 mmol/1 acetic acid adjusted to pH values between 5.1 and 5.9 at intervals of approximately 0.2 unit, 24, 48 and 95 mmol/1 citric acid adjusted to pH values between 41 and 4.9 at intervals of approximately 0.2 unit, and 22, 44, and 111 mmol/1 lactic acid adjusted to pH values between 4.3 and 5.7 at intervals of approximately 0.4 or 0.5 unit. The effect of these concentrations of organic acids was, in most cases, to increase the minimum pH that allowed growth. The order of effectiveness of the organic acids in raising the minimum pH for growth was acetic > lactic > citric and the minimum inhibitory concentrations were greater at higher temperatures.     

The influence of pH, temperature and organic acids on the initiation of growth of Yersinia enterocolitica

The influence of incubation temperature, and of acetic, lactic and citric acids on the minimum pH for the initiation of growth of six strains of Yersinia enterocolitica was determined. The strains included two of serotype O : 9, two of serotype O : 3, and one each of serotypes O : 8 and O : 5, 27. In a culture medium acidified with HCl to pH values between 4.0 and 6.0 at intervals of approximately 0.1 unit the minimum pH at which growth was detected after incubation at 20 degrees, 10 degrees, 7 degrees and 4 degrees C for 21 d was in the ranges 4.18-4.36, 4.26-4.50, 4.36-4.83 and 4.42-4.80, respectively. The minimum pH for growth was also determined in media that contained 17, 33 and 50 mmol/l acetic acid adjusted to pH values between 5.1 and 5.9 at intervals of approximately 0.2 unit, 24, 48 and 95 mmol/l citric acid adjusted to pH values between 4.1 and 4.9 at intervals of approximately 0.2 unit, and 22, 44, and 111 mmol/l lactic acid adjusted to pH values between 4.3 and 5.7 at intervals of approximately 0.4 or 0.5 unit. The effect of these concentrations of organic acids was, in most cases, to increase the minimum pH that allowed growth. The order of effectiveness of the organic acids in raising the minimum pH for growth was acetic greater than lactic greater than citric and the minimum inhibitory concentrations were greater at higher temperatures.     

Modelling the effect of pH, acidulant and temperature on the growth rate of Yersinia enterocolitica

Growth of two pathogenic and one environmental serotype of Yersinia enterocolitica under acidic conditions and at 4 and 25 degrees C was investigated. At both temperatures the maximum growth inhibitory pH depended on the acidulant used and was in the order acetic greater than lactic greater than citric greater than sulphuric. At the lower temperature the maximum growth inhibitory pH was 0.3-0.5 pH units higher than at 25 degrees C. No difference was observed between the behaviour of pathogenic and environmental serotypes in this respect. Measurement of growth at a number of sub-optimal temperatures and pH values showed that the variation of growth rate with temperature could be represented by a square root plot. The effect of different pH values could be incorporated into the model by replacing the regression coefficient b by its relationship with pH. Values of maximum growth inhibitory pH derived from the model were in good agreement with experimental values with the exception of acetic acid.     

Influence of pH on antimicrobial activity of organic acids against rabbit enteropathogenic strain of<Emphasis Type="Italic">Escherichia coli</Emphasis>

Susceptibility of the rabbit enteropathogenic strain Escherichia coli C6 (O128 serogroup) to C6-C14 fatty acids, oleic, citric, lactic and fumaric acid at 5 mg/mL was determined by the plating technique in the near-neutral pH region (pH approximately 6.5), and in a weakly acid and acid environment (pH 5.4 +/- 0.1 and 2.2-2.5, respectively). In the near-neutral pH region caproic and caprylic acid reduced the concentration of viable cells by 3 and 6 orders, respectively. At lower pH the bactericidal effect of caproic acid remained similar, but caprylic acid decreased the concentration of viable cells to < 100/mL. The bactericidal activity of capric acid was low at pH 6.5 but increased at pH 5.3. High environmental acidity was intrinsically bactericidal and at very low pH the effects of fatty acids were thus less pronounced. Citric acid reduced the counts of viable cells to 1/10. Antimicrobial activity of other acids examined was marginal or absent. Medium-chain fatty acids, caprylic and, to a lesser extent, also caproic and capric acid were better antimicrobials than other organic acids examined; the antimicrobial activity of fatty acids toward the C6 strain was pH-dependent. Beneficial effects of citric, lactic and fumaric acid reported by animal nutritionists are thus probably related to factors other than their direct antimicrobial action.     

Augmenting effect of acetic acid for acidification on bactericidal activity of hypochlorite solution

AIMS: Bactericidal activity of chlorine solution is enhanced by weak acidification. We compared the effects of various acids on the bactericidal activity of hypochlorite solution to establish a method for safe and effective use of an acidic hypochlorite solution. METHODS AND RESULTS: The bactericidal activities of acidic hypochlorite solutions that had been adjusted to pH 5.0 with hydrochloric acid, acetic acid, citric acid, lactic acid, formic acid, phosphoric acid or sulphuric acid against Bacillus subtilis spores were compared. The acidic solutions prepared with hydrochloric acid and acetic acid showed the highest bactericidal activity, and all of the spores (5 x 106 cfu ml(-1)) were killed within 10 min. On the other hand, the solutions prepared with citric acid and lactic acid showed no bactericidal activity against any bacterial strains tested in this study despite the low pH. The amount of chlorine gas produced by the preparation using acetic acid was sixfold less than that produced from the preparation using hydrochloric acid. CONCLUSIONS: Acetic acid is the most suitable and safe acid for the preparation of an acidic hypochlorite solution. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide useful information for establishing a method for safe and effective use of an acidic hypochlorite solution.     

Influence of acidity and initial substrate temperature on germination of Rhizopus oligosporus sporangiospores during tempe manufacture

J.C. DE REU, F.M. ROMBOUTS AND M.J.R. NOUT. 1995. During the soaking of soya beans according to an accelerated acidification method organic acids were formed, resulting in a pH decrease from 6·0 to 3·9. After 24 h of fermentation at 30°C, lactic acid was the major organic acid (2·1% w/v soak water), while acetic acid (0·3% w/v soak water) and citric acid (0·5% w/v soak water) were also found. During cooking with fresh water (ratio raw beans: water, 1: 6·5) the concentrations of lactate/lactic acid and acetate/acetic acid in the beans were reduced by 45% and 51%, respectively.
The effect of organic acids on the germination of Rhizopus olgosporus sporangiospores was studied in liquid media and on soya beans. Germination in aqueous suspensions was delayed by acetic acid: within 6 h no germination occurred at concentrations higher than 0·05% (w/v incubation medium), at pH 4·0. When soya beans were soaked in the presence of acetic acid, the inhibitory concentration depended on the pH after soaking. Lactic acid and citric acid enhanced germination in liquid medium, but not in tempe.
Inoculation of soya beans with R. oligosporus at various temperatures followed by incubation at 30°C resulted in both increased and decreased periods for the lag phase of fungal growth. A maximum difference of 3 h lag phase was found between initial bean temperatures of 25 and 37°C.
When pure cultures of homofermentative lactic acid bacteria were used in the initial soaking process, less lactic acid and acetic acid was formed during soaking than when the accelerated acidification method was used. This resulted in a reduction of the lag phase before growth of R. oligosporus by up to 4·7 h.     

The influence of temperature and organic matter on the bactericidal activity of short-chain organic acids on salmonellas

CHRISTINA A. CHERRINGTON, VIVIEN ALLEN AND M. HINTON. 1992. Acetic and lactic acids and BioAdd, a commercial preparation of formic and propionic acid, were tested at a concentration of 0.1% (w/w) at 20, 30, 40 and 50°C and in the presence of organic material for bactericidal activity against Salmonella serotype Kedougou. BioAdd was the most active of the solutions at all temperatures, followed by lactic acid and acetic acid. The presence of horse blood at all four temperatures, and milk and serum at 50°C, did not greatly affect the antibacterial activity of the acids although yeast extract (50°C) provided some protection for the salmonella. Acid activity was related to low pH values although the bactericidal activity of acetic acid with blood and milk was greater than the unadulterated acid even though the pH was 0.4 units higher.     

The influence of temperature and organic matter on the bactericidal activity of short-chain organic acids on salmonellas.

Acetic and lactic acids and BioAdd, a commercial preparation of formic and propionic acid, were tested at a concentration of 0.1% (w/w) at 20, 30, 40 and 50 degrees C and in the presence of organic material for bactericidal activity against Salmonella serotype Kedougou. BioAdd was the most active of the solutions at all temperatures, followed by lactic acid and acetic acid. The presence of horse blood at all four temperatures, and milk and serum at 50 degrees C, did not greatly affect the antibacterial activity of the acids although yeast extract (50 degrees C) provided some protection for the salmonella. Acid activity was related to low pH values although the bactericidal activity of acetic acid with blood and milk was greater than the unadulterated acid even though the pH was 0.4 units higher.     

Sugar and organic acid metabolism in mixed cultures of Pediococcus pentosaceus and Leuconostoc oenos isolated from wine

Pediococcus pentosaceus 12p and Leuconostoc oenos X₂L isolated from Argentinian wine were examined for growth and changes in the concentrations of glucose, fructose, sucrose and mannitol and malic, citric, acetic and lactic acids in pure and mixed cultures. In mixed cultures a mutualistic growth response and a change in the balance of end-products of sugar and organic acid metabolism were observed. The production of mannitol and acetic acid was lower while D(-) and L(+) lactic acids were detected in higher levels than in pure cultures. Malic and citric acids were metabolized simultaneously, but the amount of citric acid consumed was lower than in pure culture of Leuc. oenos.     

Growth and survival of Escherichia coli O157:H7 under acidic conditions.

The effect of pH reduction with acetic (pH 5.2), citric (pH 4.0), lactic (pH 4.7), malic (pH 4.0), mandelic (pH 5.0), or tartaric (pH 4.1) acid on growth and survival of Escherichia coli O157:H7 in tryptic soy broth with 0.6% yeast extract held at 25, 10, or 4 degrees C for 56 days was determined. Triplicate flasks were prepared for each acid treatment at each temperature. At 25 degrees C, populations increased 2 to 4 log10 CFU/ml in all treatments except that with mandelic acid, whereas no growth occurred at 10 or 4 degrees C in any treatments except the control. However, at all sampling times, higher (P < 0.05) populations were recovered from treatments held at 4 degrees C than from those held at 10 degrees C. At 10 degrees C, E. coli O157:H7 was inactivated at higher rates in citric, malic, and mandelic acid treatments than in the other treatments. At the pH values tested, the presence of the organic acids enhanced survival of the pathogen at 4 degrees C compared with the unacidified control. E. coli O157:H7 has the ability to survive in acidic conditions (pH, > or = 4.0) for up to 56 days, but survival is affected by type of acidulant and temperature.     

Survival and growth of Escherichia coli O157:H7 in ground, roasted beef as affected by pH, acidulants, and temperature.

A study was undertaken to determine the fate of Escherichia coli O157:H7 in ground, roasted beef as influenced by the combined effects of pH, acidulants, temperature, and time. There was essentially no change in the viable population of E. coli O157:H7 when beef salads (pH 5.40 to 6.07) containing up to 40% mayonnaise were incubated at 5 degrees C for up to 72 h. At 21 and 30 degrees C, significant (P < or = 0.05) increases in populations of the organism occurred in salads containing 16 to 32% mayonnaise (pH 5.94 to 5.55) between 10 and 24 h of incubation. Death was more rapid as the pH of acidified beef slurries incubated at 5 degrees C was decreased from 5.98 to 4.70. E. coli O157:H7 grew in control slurries (pH 5.98) and in slurries containing citric and lactic acids (pHs 5.00 and 5.40) incubated at 21 degrees C for 24 h; decreases occurred in slurries acidified to pHs 4.70, 5.00, and 5.40 with acetic acid or pH 4.70 with citric or lactic acid. At 30 degrees C, populations decreased in slurries acidified to pHs 4.70 and 5.00 with acetic acid. Citric and lactic acids failed to prevent significant increases in populations in slurries at pH 4.70 to 5.40 between 10 and 24 h of incubation. The order of effectiveness of acidulants in inhibiting growth was acetic acid > lactic acid > or = citric acid. The same order was observed for inactivation of E. coli O157:H7 in acidified (pH 5.00) beef slurry heated at 54 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tolerance of acid-adapted and non-adapted Escherichia coli O157:H7 cells to reduced pH as affected by type of acidulant

A study was carried out to determine if three strains of Escherichia coli O157:H7 grown (18 h) in Tryptic Soy Broth (TSB) and TSB supplemented with 1.25% glucose (TSBG), i.e. unadapted and acid-adapted cells, respectively, exhibited changes in tolerance to reduced pH when plated on Tryptic Soy Agar (TSA) acidified (pH 3.9, 4.2, 4.5, 4.8, 5.1 and 5.4) with acetic, citric or malic acids. All test strains grew well on TSA acidified with acetic acid at pH > or = 5.4 or malic acid at pH > or = 4.5; two strains grew on TSA acidified with citric acid at pH > or = 4.5, while the third strain grew at pH > or = 4.8. Acid-adapted and control (unadapted) cells differed little in their ability to form visible colonies on TSA containing the same acid at the same pH. However, on plates not showing visible colonies, acid-adapted cells retained higher viability than unadapted cells when plated on acidified TSA. Growth of acid-adapted and control cells of E. coli O157:H7 inoculated into TSB containing acetic acid (pH 5.4 and 5.7) and citric or malic acids (pH 4.2 and 4.5) was also studied. There was essentially no difference in growth characteristics of the two types of cells in TSB acidified at the same pH with a given acid. Tolerance of acid-adapted and control cells on subsequent exposure to low pH is influenced by the type of acidulant. The order of sensitivity at a given pH is acetic > citric > malic acid. When performing acid challenge studies to determine survival and growth characteristics of E. coli O157:H7 in foods, consideration should be given to the type of acid to which cells have been exposed previously, the procedure used to achieve acidic environments and possible differences in response among strains. The use of strains less affected by pH than type of acidulant or vice versa could result in an underestimation of the potential for survival and growth of E. coli O157:H7 in acid foods.     

Inhibited growth of common enteropathogenic bacteria in lactic-fermented cereal gruels

A natural lactic fermentation of mixtures of water and whole flour of either maize or high-tannin sorghum was obtained either before or after cooking to a weaning gruel: The preparations had a final pH of about 3.8 (range 3.67 to 4.00) and a ratio of lactic acid to acetic acid of 91 (w/w). The growth of added (about 10⁷ c.f.u./g gruel) Gram-negative intestinal pathogenic bacteria, enterotoxigenicEscherichia coli, Campylobacter jejuni, Shigella flexneri andSalmonella typhimurium, was strongly inhibited in the sour gruels, and the effect could primarily be explained by the low pH caused by the formation of lactic and acetic acids during the fermentation process. Of the added Gram-positive bacteria,Bacillus cereus andStaphylococcus aureus showed similar inhibited growth up to 7h after inoculation in the sour gruels. The strain ofStaphylococcus, however, showed only a continued reduction in growth in the fermented gruel samples, which had a viable lactic bacteria culture indicating the presence of a bacteriocin. This implies that a low pH (< 4.0) alone is not sufficient to sustain the inhibition of the growth ofStaphylococcus aureus. The survival studies were carried out at optimal temperatures for each respective enteropathogen.     

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.     

Effect of acetic and trans-aconitic acids on citric acid production by Aspergillus niger.

The effect of acetic and trans-aconitic acids on citric acid production by A. niger at different pH values was studied. The presence of acetic acid at pH 2 prevented spore germination, while it decreased the fungal growth and citric acid production at other pH values. In the presence of trans-aconitic acid the inhibition was less marked at lower than at higher pH values.     

Individual and combined effects of ph and lactic acid concentration on Listeria innocua inactivation: development of a predictive model and assessment of experimental variability

In food technology, organic acids (e.g., lactic acid, acetic acid, and citric acid) are popular preservatives. The purpose of this study was to separate the individual effects of the influencing factors pH and undissociated lactic acid on Listeria innocua inactivation. Therefore, the inactivation process was investigated under controlled, initial conditions of pH (pH0) and undissociated lactic acid ([LaH]0). The resulting inactivation curves consisted of a (sometimes negligible) shoulder period followed by a descent phase. In a few cases, a tailing phase was observed. Depending on the conditions, the descent phase contained one or two log-linear parts or had a convex or concave shape. In addition, the inactivation process was characterized by a certain variability, dependent on the severity of the conditions. Furthermore, in the neighborhood of the growth/no growth interface sometimes contradictory observations occurred. Overall, the individual effects of the influencing factors pH and undissociated lactic acid could clearly be distinguished and were also apparent based on fluorescence microscopy. Appropriate model types were developed and enabled prediction of which conditions of pH0 and [LaH]0 are necessary to obtain a predetermined inactivation (number of decimal reductions) within a predetermined time range.     

Inhibition of enterobacteria and Listeria growth by lactic, acetic and formic acids

Minimum inhibitory concentrations (MIC) of undissociated lactic, acetic and formic acids were evaluated for 23 strains of enterobacteria and two of Listeria monocytogenes. The evaluation was performed aerobically and anaerobically in a liquid test system at pH intervals of between 4.2 and 5.4. Growth of the enterobacteria was inhibited at 2–11 mmol 1⁻¹, 0.5–14 mmol 1⁻¹ and 0.1–1.5 mmol 1⁻¹ of undissociated lactic, acetic and formic acids, respectively. The MIC value was slightly lower with anaerobic conditions compared with aerobic conditions. The influence of protons on the inhibition was observed for acetic acid at the low pH values. Undissociated lactic acid was 2 to 5 times more efficient in inhibiting L. monocytogenes than enterobacteria. Acetic acid had a similar inhibitory action on L. monocytogenes compared with enterobacteria. Inorganic acid (HCl) inhibited most enterobacteria at pH 4.0; some strains, however, were able to initiate growth to pH 3.8. The results indicate that the values of undissociated acid which occur in a silage of pH 4.1–4.5 are about 10–100 times higher than required in order to protect the forage from the growth of enterobacteria and L. monocytogenes.     

Growth and toxin formation by Clostridium botulinum at low pH values

Spores of Clostridium botulinum were found to initiate growth and to produce toxin in aqueous suspensions of soya protein at pH values as low as 4-2 and in skimmed milk at pH 4.4. Most of the experiments were done with mixed cultures of CI. botulinum types A and B in the presence of two strains of Bacillus subtilis. The role of the latter organism was concluded to be to lower the oxygen content and the E_h of the suspensions. Toxin was produced at pH 4-4 after 4 weeks of incubation at 30^oC when either hydrochloric or citric acids were used as the acidulant and after 12 and 14 weeks when, respectively, lactic and acetic acids were used. Thus, amongst other factors the nature of the acid and not solely the pH value is an important factor in controlling the growth of Cl. botulinum at low pH. Pure cultures of Cl botulinum type A grew at 30^oC under strictly anaerobic conditions and produced toxin at pH 4-3 in the presence of hydrochloric acid.     

Individual and Combined Effects of pH and Lactic Acid Concentration on Listeria innocua Inactivation: Development of a Predictive Model and Assessment of Experimental Variability

In food technology, organic acids (e.g., lactic acid, acetic acid, and citric acid) are popular preservatives. The purpose of this study was to separate the individual effects of the influencing factors pH and undissociated lactic acid on Listeria innocua inactivation. Therefore, the inactivation process was investigated under controlled, initial conditions of pH (pH₀) and undissociated lactic acid ([LaH]₀). The resulting inactivation curves consisted of a (sometimes negligible) shoulder period followed by a descent phase. In a few cases, a tailing phase was observed. Depending on the conditions, the descent phase contained one or two log-linear parts or had a convex or concave shape. In addition, the inactivation process was characterized by a certain variability, dependent on the severity of the conditions. Furthermore, in the neighborhood of the growth/no growth interface sometimes contradictory observations occurred. Overall, the individual effects of the influencing factors pH and undissociated lactic acid could clearly be distinguished and were also apparent based on fluorescence microscopy. Appropriate model types were developed and enabled prediction of which conditions of pH₀ and [LaH]₀ are necessary to obtain a predetermined inactivation (number of decimal reductions) within a predetermined time range.