Culture conditions affect the chemical composition of the exopolysaccharide synthesized by the fungus Aureobasidium pullulans

Aims:  To identify if culture conditions affect the chemical composition of exopolysaccharide (EPS) produced by Aureobasidium pullulans .
Methods and Results:  In batch airlift and continuously stirred tank (CSTR) reactors the EPS produced with low (0·13 g l⁻¹ N) initial NaNO₃ or (NH₄)₂SO₄ levels contained pullulan, with maltotriose as its major component, similar to that synthesized in the airlift reactor with high (0·78 g l⁻¹ N) initial NaNO₃ levels. EPS produced by CSTR grown cultures with high (NH₄)₂SO₄ levels contained little pullulan, possibly because of a population shift from unicells to mycelium. This chemical difference may explain why total EPS yields did not fall as they did with cultures grown under identical conditions with high NaNO₃ levels, where the pullulan component of the EPS disappeared. EPS synthesized in N-limiting chemostat cultures of A. pullulans changed little with growth rate or N source, being predominantly pullulan consisting of maltotriose units.
Conclusions:  While the EPS chemical composition changed little under N-limiting conditions, high initial medium N levels determined maltotriose content and/or pullulan content possibly by dictating culture morphology.
Significance and Impact of the Study:  These results emphasize the requirement of all studies to determine EPS chemical composition when examining the influence of culture conditions on EPS yields.     

Modelling the growth of Weissella cibaria as a function of fermentation conditions

Aims:  To investigate the effect of pH, water activity ( a _w) and temperature on the growth of Weissella cibaria DBPZ1006, a lactic acid bacterium isolated from sourdoughs.
Methods and Results:  The kinetics of growth of W. cibaria DBPZ1006 was investigated during batch fermentations as a function of pH (4·0–8·0), a _w (0·935–0·994) and temperature (10–45°C) in a rich medium. The growth curve parameters (lag time, growth rate and asymptote) were estimated using the dynamic model of Baranyi and Roberts (1994. A dynamic approach to predicting bacterial growth in food. Int J Food Microbiol 23, 277–294). The effect of pH, a _w and temperature on maximum specific growth rate (μ_max) were estimated by fitting a cardinal model. μ_max under optimal conditions (pH = 6·6, a _w = 0·994, T  = 36·3°C) was estimated to be 0·93 h⁻¹. Minimum and maximum estimated pH and temperature for growth were 3·6 and 8·15, and 9·0°C and 47·8°C, respectively, while minimum a _w was 0·918 (equivalent to 12·2% w/v NaCl).
Conclusions:  Weissella cibaria DBPZ1006 is a fast-growing heterofermentative strain, which could be used in a mixed starter culture for making bread.
Significance and Impact of the Study:  This is the first study reporting the modelling of the growth of W. cibaria , a species that is increasingly being used as a starter in sourdough and vegetable fermentations.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5·2 with HCl and incubated at 30°C, inocula containing < 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give > 10⁸ bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10–20 weeks. Citric acid concentrations of 10–50 mmol/l at pH 5·2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10⁶. The citric acid also reduced the concentration of free Ca²⁺ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5·2 could be prevented by the addition of Ca²⁺ or Mg²⁺ and greatly reduced by Fe²⁺ and Mn²⁺. The addition of Ca²⁺, but not of the remaining divalent metal ions, restored the concentration of free Ca²⁺ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca²⁺. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5·2. The effect of citric acid and Ca²⁺ at pH 5·2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.     

Production and stability of pediocin N5p in grape juice medium

The production and stability of pediocin N5p from Pediococcus pentosaceus , isolated from wine, were examined in grape juice medium. Maximum growth and higher titre (4000 U ml^-1) were observed at a initial pH of 7·5 and 30°C. The activity of the inhibitory substance was stable between pH values from 2·0 to 5·0 at 4° and 30°C. At pH 10·0 it was completely inactivated. When submitted to 30 min at 80°, 100° and 115°C, maximal stability was observed at pH 2·0. Ethanol up to 10% did not affect pediocin activity at acid pH, nor did 40–80 mg 1^-1 SO₂, independently or combined with different ethanol concentrations, affect inhibitory activity.     

Research note : Unsuitability of the MRS medium for the screening of hydrogen peroxide-producing lactic acid bacteria

The effect of MRS broth on the stability of hydrogen peroxide (H₂O₂) has been studied. Known concentrations (1–100 μg ml⁻¹) of H₂O₂ were prepared in distilled water, phosphate buffer (pH 7·0) and MRS broth (pH 6·2 and 3·9). H₂O₂ was very stable in aqueous and buffer solutions but it was rapidly degraded in MRS broth (pH 3·9). The presence of H₂O₂ in MRS broth (pH 6·2) could not be detected.     

Note: Purification of amylase secreted from Bifidobacterium adolescentis

Bifidobacterium adolescentis Int-57 isolated from human faeces produced extracellular amylase. The enzyme was purified from the culture supernatant fluids by ammonium sulphate precipitation, gel-filtration chromatography (Sephadex-G-75), ion-exchange chromatography (CM-cellulose) and FPLC. SDS-PAGE of the purified enzyme revealed a major band with an apparent molecular weight of 66 kDa. The pI was 5·2. Enzyme activity was optimal at 50°C, and at pH 5·5. The enzyme was stable at 20–40°C, and at pH 5–6 with a K _m value of 2·4 g l⁻¹ soluble starch. The activation energy was 42·3 kJ mol⁻¹. The enzyme was significantly inhibited by maltose (10%), glucose (10%), Cu²+ (5 mmol l⁻¹), Zn²+ (5 mmol l⁻¹), N- bromosuccinimide (5 mmol l⁻¹), EDTA (5 mmol l⁻¹), I₂ (1 mmol l⁻¹) and activated by β-mercaptoethanol (10 mmol l⁻¹).     

Fermentation characteristics of Clostridium pasteurianum LMG 3285 grown on glucose and mannitol

Clostridium pasteurianum fermented glucose to acetate, butyrate, CO₂ and H₂. In batch cultures the fermentation pattern was only slightly affected by culture pH over the range 8·0 to 5·5. The acetate/butyrate ratio was always higher than or equal to one. Between 2·14 and 2·33 mol H₂ was produced per mol glucose fermented. At unregulated pH, more butanol and less butyrate was formed. In a carbon-limited chemostat, the steady-state acetate/butyrate ratio was always lower than one. H₂ production was approximately 1·70 mol per mol glucose consumed. Substantial amounts of extracellular protein were formed. With decreasing pH, acetate and formate production decreased, while H₂ production was highest at pH 6.0. With increasing dilution rate ( D ), the product spectrum hardly changed, but more biomass was formed. Y _glucose^max and Y _ATP^max were 55·97 and 31·48 g dry weight per mol glucose or ATP respectively. With increasing glucose input the formation of fatty acids and H₂ slightly decreased.
Continuous cultures fermented mannitol to acetate, butyrate, butanol, CO₂ and H₂. With acetate as co-substrate, butanol production and molar growth yields, Y _mannitol and Y _ATP, markedly decreased, while the butyrate and H₂ production increased. The latter reached a value of 2·21 mol H₂ per mol mannitol consumed.     

Growth, diet and metabolism of common wolf–fish in the North Sea, a fast–growing population

The von Bertalanffy growth parameters for common wolf–fish Anarhichas lupus in the North Sea were: male: L ∞=111·2 cm, t ₀=–0·43 and K =0·12; and female: L ∞=115·1 cm, t ₀=–0·39 and K =0·11, making this the fastest growing stock reported. Resting metabolic rates (RMR±S.E.) and maximum metabolic rates (MMR±S.E.) for six adult common wolf–fish (mean weight, 1·39 kg) at 5° C were 12·18±1·6 mg O₂ kg^–1 h^–1 and 70·65±7·63 mg O₂ kg^–1 h^–1 respectively, and at 10° C were 25·43±1·31 mg O₂ kg^–1 h^–1 and 113·84±16·26 mg O₂ kg^–1 h^–1. Absolute metabolic scope was 53% greater at 10° C than at 5° C. The diet was dominated by Decapoda (39% overall by relative occurrence), Bivalvia (20%) and Gastropoda (12%). Sea urchins, typically of low energy value, occupied only 7% of the diet. The fast growth probably resulted from summer temperatures approximating to the optimum for food processing and growth, but may have been influenced by diet, and reduced competition following high fishing intensity.     

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.     

SULPHIDE PRODUCTION FROM SULPHATE-ENRICHED SEWAGE SLUDGES

SUMMARY: Sterilized raw sewage sludge enriched with sulphate and inoculated with pure strains of Desulphovibrio desulphuricans produced negligible sulphide. Unsterilized sludge supplemented with 7% (w/v) CaSO₄.2H₂O and inoculated with crude cultures of sulphate-reducing bacteria obtained from sewage yielded 1·0% S^2- (wt S^2- produced as H₂S/vol. of raw sludge) in 6 months at 30°. By repeated subculture more active cultures developed which produced 1% S^2- in 7 days and 1·2–1·9% in 28 days. Digested sludge yielded only 0·1% S^2-. In semicontinuous fermentations at 30°, raw sludge without added sulphate produced 20 times its own volume of gas containing 70% CH₄ and 30% CO₂. When 5% CaSO₄.2H₂O and an active crude culture of sulphate reducers were added, gas production decreased steadily to zero. There were no differences in pH, temperature and redox potential in sludges producing methane or sulphide. The chief cause of inhibition appeared to be the action of sulphide: 0·02% soluble sulphide (S^2-) totally inhibited methane formation; 0·01% S^2- initially decreased gas production by one-quarter but there was a slow recovery to normal, suggesting acclimatization of the methane-producing organisms to sulphide.
Linked fermentations, in which gas from a methane fermentation swept H₂S from a sulphide fermentation, gave a final gas mixture of about 60% CH₄, 30% CO₂ and 5–10% H₂S. The yield of sulphide depended on the rate of sweeping.     

Effects of gaseous environment and temperature on the storage behaviour of Listeria monocytogenes on chicken breast meat

Portions of skinless chicken breast meat (pH 5·8) were inoculated with a strain of Listeria monocytogenes and stored at 1, 6 or 15°C in (1) aerobic conditions; (2) 30% CO₂+ air; (3) 30% CO₂+ N₂; and (4) 100% CO₂. When samples were held at 1°C the organism failed to grow under any of the test conditions, despite marked differences between treatments in spoilage rate and ultimate microflora. At 6°C counts of L. monocytogenes increased ca 10-fold in aerobic conditions before spoilage of the meat, but only when the inoculum culture was incubated at 1°C rather than 37°C. In CO₂ atmospheres growth of L. monocytogenes was inhibited on meat held at 6°C, especially under 100% CO₂. By contrast, storage at 15°C led to spoilage of the meat within 2 d, in all gaseous environments, and listeria levels increased up to 100-fold. Differences in the behaviour of L. monocytogenes on poultry and red meats are discussed.     

The content of prostaglandins and their precursors in Mortierella and Cunninghamella species

Five strains of filamentous fungi belonging to the genera Mortierella and Cunninghamella were examined for the content of dihomo-γ-linolenic, arachidonic, eicosapentaenoic acids and prostaglandins (type E₂ and F _2α). Prostaglandins were detected using an ELISA method in mycelia of all tested strains (range 50–4800 ng g⁻¹ of PGE₂ and 6–30 ng g⁻¹ of PG F _2α). Several micro-organisms also produced prostaglandins in the culture medium (2·2–137·6 μg l⁻¹ for PGE₂ and 0·4–7·8 μg l⁻¹ for PG F _2α).     

The combined effect of sub-optimal temperature and sub-optimal pH on growth and toxin formation from spores of Clostridium botulinum

Low-acid foods (pH ≥ 4.5) are not sufficiently acidic to prevent growth of Clostridium botulinum in otherwise optimal conditions. The combination of sub-optimal pH and sub-optimal temperature may, however, result in a very significant reduction in the risk of growth of this bacterium compared with the risk in optimal conditions. The combined effect of incubation temperatures of 12° and 16°C and pH values between 5·2 and 5·5 on growth and toxin production from spores of Cl. botulinum during incubation for 28 d has been investigated. Growth and formation of toxin (type B) were detected only in medium at pH 5·5 and incubated at 16°C, corresponding to a probability of growth from a single spore within 14 d of 1·6 × 10^-5. The probability of growth in 28 d in the remaining conditions was <9 × 10^-6. After transfer of inoculated media from 12° to 30°C growth occurred at pH 5·2–5·5 within 19 d. After transfer of inoculated media from 12° to 20°C growth occurred at pH 5·5 and 5·4 but not at pH 5·3 or 5·2 in 40 d. Growth at pH 5·2–5·5 was accompanied by formation of toxin, in most cases of types A or B. In addition to the effect of sub-optimal temperature and pH, chelation of divalent metal ions by citrate may have contributed to inhibition.     

The effect of temperature and fish size on growth and feed efficiency ratio of juvenile spotted wolffish Anarhichas minor

The growth properties of juvenile spotted wolffish Anarhichas minor reared at 4, 6, 8 and 12° C, and a group reared under 'temperature steps', (T‐step) i.e . with temperature reduced successively from 12 to 9 and 6° C were investigated. Growth rate and feed efficiency ration was significantly influenced by temperature and fish size. Overall growth rate was highest at 6° C (0·68% day⁻¹) and lowest at 12° C (0·48% day⁻¹), while the 4 and 8° C, and the T‐step groups had similar overall growth rates, i.e . 0·59, 0·62 and 0·51% day⁻¹ respectively. Optimal temperature for growth ( T _{opt G} ) and feed efficiency ratio (T_{opt FCE}) decreased as fish size increased, indicating an ontogenetic reduction in T _{opt G} and T _{opt FCE}. The results suggest a T _{opt G} of juvenile spotted wolffish in the size range 135–380 g, dropping from 7·9° C for 130–135 g to 6·6° C for 360–380 g juveniles. The T _{opt FCE} dropped from 7·4° C for 120–150 g to 6·5° C for 300–380 g juveniles. A wider parabolic regression curve between growth, feed efficiency ratio and temperature as fish size increased, may indicate increased temperature tolerance with size. Individual growth rates varied greatly at all time periods within the experimental temperatures, but at the same time significant size rank correlations were maintained and this may indicate stable size hierarchies in juvenile spotted wolffish.     

EXOCELLULAR PRODUCT FORMATION BY MICROORGANISMS IN CONTINUOUS CULTURE. I. PRODUCTION OF 2:3-BUTANEDIOL BY AEROBACTER AEROGENES IN A SINGLE STAGE PROCESS

SUMMARY: A quantitative study of the growth and carbohydrate metabolism of Aerobacter aerogenes in continuous culture with particular reference to 2:3-butanediol production is described. The effects of varying dilution rate, oxygen uptake rate, pH value, temperature and CO₂ pressure were studied.
The optimum pH value was 5·0°6·0. The optimum temperature was 35–37° with a dilution rate of 0·2 hr⁻¹. The rate of sugar metabolism was decreased by raising the CO₂ pressure.
In continuous culture the maximum diol output rate/1. of working volume exceeded the greatest rate reported for batch culture by a factor of three. The maximum diol concentration and yield as a percentage of the theoretical maximum were lower in continuous culture than in batch culture. Reasons for this are given.
Some variants of the parent organism were found in continuous cultures but they caused no change in the yield of butanediol. There was no sign of loss of butanediol-producing ability by any of the cultures, some of which were maintained for 2,000 hr. For the first few generations the rate of production of butanediol actually increased.     

Nisin, temperature and pH effects on growth and viability of Pectinatus frisingensis, a Gram-negative, strictly anaerobic beer-spoilage bacterium

Pectinatus frisingensis , a Gram-negative and strictly anaerobic beer spoilage bacterium is sensitive to nisin. An increase in nisin concentration (0 to 1100 IU ml⁻¹) added to the culture medium prolonged the lag phase, and decreased the growth rate of the bacterium. In addition, late exponential cells of P. frisingensis exposed to low concentrations of nisin lost immediately a part of their intracellular K⁺. Presence of Mg²⁺ up to 15 mmol l⁻¹ did not protect P. frisingensis from nisin-induced loss of viability and K⁺ efflux. Potassium leaks were also measured in P. frisingensis late exponential phase cells exposed to combined effects of nisin addition (100–500 IU ml⁻¹), 10 min mild heat-treatment (50 °C) or rapid cooling (2 °C), and pH (4·0 and 6·2). Net K⁺ efflux from both starving and glucose-metabolizing cells, was more important at pH 6·2, whatever the temperature treatment and nisin addition. Reincubation at 30 °C of P. frisingensis glucose-metabolizing cells exposed to a preliminary combination of nisin addition and mild heat or cooling down treatment, showed that cells exposed to rapid cooling reaccumulated more K₊ than heat-treated cells, whatever the pH conditions. A combination of nisin and mild heat-treatment could thus be of interest to prevent P. frisingensis growth in beers.     

Effects of oxygen, pH and nitrate concentration on denitrification by Pseudomonas species

Abstract The production of nitrogen-containing gases by denitrification in three organisms was examined using membrane inlet mass spectrometry. The effects of O₂ (during both growth and maintenance) and of pH, nitrate concentration and carbon source were tested in non-proliferating cell suspensions. Two strains of Pseudomonas aeruginosa were capable of co-respiration of NO₃⁻ and O₂ and, under controlled O₂ supply, gave oscillatory denitrification. Variations in culture and assay conditions affected both the rate of denitrification and the ratio of end products (N₂O:N₂). Higher rates were seen following anaerobic growth. Optimum values of pH and nitrate concentration for denitrification are given. Generally, the optimum pH was 7.0–7.5, approximately that of the growth medium. Optimum nitrate concentration was generally 20 mM.     

Studies on the Production of Extracellular Proteinases by a Non-pigmented Strain of Chromobacterium lividum Isolated from Abattoir Effluent

A highly proteolytic bacterium isolated from abattoir effluent was identified as a non-pigmented strain of Chromobacterium lividum. Ferrous or ferric ions at concentrations between 1·8 × 10^-5 and 9 × 10^-4 g ions/1, which is 2–3 orders of magnitude greater than that required for growth, were essential for extracellular proteinase production in aerated but not in static culture. Co²⁺, Ni²⁺, Mn²⁺, Cu²⁺ or Zn²⁺ ions could not replace iron. Four proteinases (I-IV) were produced in static culture, but only proteinase I was formed in significant quantities in aerated culture. With both forms of culture amino nitrogen was essential for proteinase production; glucose inhibited formation in aerated, but not static, cultures. Growth occurred over the range 1–33 °C, whereas proteinase production ceased at 27 °C, with maximum activity at 13 °C. Proteinase production appeared to be controlled by an interaction between iron, oxygen tension and glucose.     

Survival and growth of Cronobacter species (Enterobacter sakazakii) in wheat-based infant follow-on formulas

Aims:  To determine the survival and growth characteristics of Cronobacter species ( Enterobacter sakazakii ) in infant wheat-based formulas reconstituted with water, milk, grape juice or apple juice during storage.
Methods and Results:  Infant wheat-based formulas were reconstituted with water, ultra high temperature milk, pasteurized grape or apple juices. The reconstituted formulas were inoculated with Cronobacter sakazakii and Cronobacter muytjensii and stored at 4, 25 or 37°C for up to 24 h. At 25 and 37°C, Cronobacter grew more (>5 log₁₀) in formulas reconstituted with water or milk than those prepared with grape or apple juices ( c. 2–3 log₁₀). The organism persisted, but did not grow in any formulas stored at 4°C. Formulas reconstituted with water and milk decreased from pH 6·0 to 4·8–5·0 after 24 h, whereas the pH of the formulas reconstituted with fruit juices remained at their initial pH values, c. pH 4·8–5·0.
Conclusions:  Cronobacter sakazakii and C. muytjensii can grow in reconstituted wheat-based formulas. If not immediately consumed, these formulas should be stored at refrigeration temperatures to reduce the risk of infant infection.
Significance and Impact of the Study:  The results of this study will be of use to regulatory agencies and infant formula producers to recommend storage conditions that reduce the growth of Cronobacter in infant wheat-based formulas.     

Growth and physiological changes during metamorphosis of Senegal sole reared in the laboratory

The metamorphosis of Solea senegalensis was studied in larvae reared at 20° C and fed four different feeding regimes. A, Artemia (4 nauplii ml⁻¹); B, Artemia (2 nauplii ml⁻¹); C, mixed diet (2 nauplii ml⁻¹ and 3 mg ml⁻¹ microencapsulated diet); and D, microencapsulated diet (3·7 mg ml⁻¹). Rotifers were also supplied in all cases during the first days of feeding. These feeding regimes supported different growth rates during the pre-metamorphosis period (regime A, G=0·376 day⁻¹; regime B, G=0·253 day⁻¹; regime C, G=0·254 day⁻¹; regime D, G=0·162 day⁻¹). Larvae started metamorphosis 9 days after hatching (DAH) when fed the regime A, 13 DAH with regime B, 11 DAH with regime C and 15 DAH with regime D. A minimum 5·6–5·9 mm L_T was required under all feeding regimes to initiate the metamorphosis. Eye translocation was completed when the larvae reached 8·6–8·7 mm L_T (regimes A, B and C), but only 7·3 mm L_T with regime D. 4·4–6·2 days were required to complete eye migration under the regimes A, B and C, and 18·3 days under the regime D. This transformation is concomitant with changes in body reserves, and with the pattern of some digestive enzymes.