pH and acidity in lactic-fermenting cereal gruels: effects on viability of enteropathogenic microorganisms

Survival of Bacillus cereus, Campylobacter jejuni, enterotoxigenic Escherichia coli, Salmonella typhimurium and Shigella flexneri during lactic acid fermentation of cereal gruels prepared from low-tannin (white) and high-tannin (red) sorghum varieties was studied. A previously fermented gruel (starter culture, SC) recycled daily or stored for 7, 14 or 28 days, germinated cereal flour (power flour, PF), or a combination of PF and SC (PF+SC) were used as starters. At 24 h, the pH of all gruels with added starter was 4; the pH in control gruels without starter was 5.2. pH decrease was significantly faster in gruels made with PF+SC than with either PF or SC alone (P<0.05). A daily recycled SC resulted in a significantly faster decrease in pH (P<0.05) than SC stored for more than 7 days. Acid production was correlated with pH decrease (r=–0.94; P<0.01). In control gruels, the enteropathogens remained at the inoculation level or increased in number. Their growth was inhibited within 24 to 48 h in the fermented gruels, in the order: Bacillus > Campylobacter > Escherichia coli > Salmonella > Shigella. The inhibition rate was significantly faster in fermenting gruel with PF+SC (P<0.05) than in gruel with PF or SC alone and correlated with pH development (r=0.71; P<0.01). Both white and red sorghum gruels gave similar results. Using PF+SC as a starter resulted in a faster decrease in pH as well as a more rapid inhibition of enteropathogenic microorganisms. The effect is optimal if the SC is transferred daily.     

Inhibition of enterotoxin production by, and growth of enteropathogens in a lactic acid-fermenting cereal gruel

Growth and enterotoxin production of three strains of Campylobacter jejuni or Escherichia coli known to produce heat labile enterotoxins were evaluated when added into a lactic acid-fermenting cereal gruel, togwa. A single strain of each of the enteropathogens was simultaneously inoculated with a lactic acid starter culture (sc) to reach a level of about 107 c.f.u./ml and was left to ferment for 48h. Gruels without sc (control gruel), pure cholera toxin in fermenting or control gruel and the test bacteria inoculated into nutrient broth were used as positive toxin controls; gruel without inoculated test bacteria was the toxin-negative control. Viable colonies were counted by spread plating 0.1ml of gruel subsamples collected at intervals during the fermentation period onto different selective media and the heat labile enterotoxin production was evaluated using an assay on Chinese hamster ovary (CHO) cells. In the fermenting gruels, no viable cells of the C. jejuni and E. coli strains were detected after 8 and 24h incubation, respectively, but inocula increased in number or remained at the initial level in the control gruel and in the nutrient broth. After 24h incubation, all supernatants of control gruels with inoculated bacteria showed enterotoxicity to the CHO cells (indicated by elongation of 20–50% of the cells). No toxin activity was observed in the fermenting gruels with or without added bacteria or in control gruel alone. Pure cholera toxin added to control gruel caused the enterotoxigenic effect in 70–100% of the CHO cells, but no activity was detected when it was added to the fermenting gruel. The CHO cells were affected instead by a low pH level but were not elongated. Adjusting the pH of fermented gruels to approximately neutral levels restored the toxin effect when cholera toxin was added but not in the presence of added test bacteria. We conclude that lactic acid fermented cereal gruels with pH 4 have a high potential to inhibit the growth of enteropathogenic bacteria of the genera C. jejuni and E. coli and to inhibit production of heat labile enterotoxins. Regular consumption of fermented cereal weaning foods will therefore reduce transmission of enterotoxin-producing bacteria, and ingestion of enterotoxins.     

The effect of fermentation on the growth and survival ofSalmonella typhimurium,Staphylococcus aureus,Bacillus cereus andPseudomonas aeruginosa in fermenting tef (Eragrostis tef)

Summary Growth ofSalmonella typhimurium, Staphylococcus aureus, Bacillus cereus andPseudomonas aeruginosa was inhibited when the pH of fermenting tef approached 5.0, 5.0, 5.5 and 5.0, respectively. However the test organisms grew in far more acidic conditions in broth than in fermenting tef and this is due to antimicrobial substance(s) being produced by some of the lactic acid bacteria. Except forBacillus cereus spores, all the test organisms were heat-inactivated during the baking process of the final tef injera.

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Effet de la fermentation sur la croissance et la survie de Salmonella typhimurium, Staphylococcus aureus, Bacillus cereus et Pseudomonas aeruginosa dans le tef (Eragrostis tef) en fermentation

Résumé La croissance deSalmonella typhimurium, deStaphylococcus aureus, deBacillus cereus et dePseudomonas aeruginosa est inhibée lorsque les pH du tef en fermentation approchent respectivement 5:0, 5.0, 5.5 et 5.0. Toutefois, les organismes tests croissent dans des conditiones bien plus acides que dans le tef en fermentation. Ceci est dû à des substances antimicrobiennes produites par certaines bactéries lactiques. A l'exception des spores deBacillus cereus, tous les organismes tests sont inactivés par la chaleur durant le processus de cuisson.

     

Lytic effect ofVibrio cholerae elastase on Gram-positive and-negative bacteria

Elastase ofVibrio cholerae caused the lysis of freshly grown cells of Gram-negative (Pseudomonas aeruginosa, Proteus vulgaris, Salmonella paratyphi A andKlebsiella pneumoniae) bacteria. Gram-positive (Staphylococcus aureus andS. epidermidis) organisms were resistant to this enzyme. Heat killed and lyophilized Gram-positive and-negative bacteria (exceptS. aureus andS. epidermidis) showed higher sensitivity to elastase. Both Gram-negative and-positive bacteria were lyzed maximally by elastase at pH 8.0. At this pH, lytic activity of elastase was maximum in Tris-HCl and glycine-NaOH buffers followed by Tris-maleate and cacodylate buffers.     

Absence or presence of glucose in growth medium and its effect on heat injury inStaphylococcus aureus

Summary Staphylococcus aureus 196E, when grown in a glucose (0.25% wt./vol.)-containing medium, produced cells that would undergo injury when subjected to sublethal heat conditions (45 min at 50°C); however, if glucose was omitted from the growth medium, the extent of injury was greatly reduced. Media containing glucose sterilized by filtration or by separate autoclaving produced cells equal in injury susceptibility to medium in which glucose was autoclaved as part of the medium components. Injury also occurred when other sugars such as fructose, mannose, maltose, or lactose were substituted for glucose. Sugar-containing media that producedStaphylococcus aureus of maximal susceptibility to heat injury reached a pH of approximately 6 or lower during growth of the cells. Incubation of staphylococci in growth medium acidified with acetic or lactic acids or HCl did not lead to cells that would undergo injury under the stated conditions. The stimulatory effect of glucose on injury appears to be related to the metabolism of the sugar byStaphylococcus aureus.Agricultural Research Service, U.S. Department of Agriculture. Reference to brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.     

Photodynamic effects of deuteroporphyrin on Gram-positive bacteria

The photodynamic effects of deuteroporphyrin (DT) on the growth and viability ofStaphylococcus aureus, Streptococcus faecalis, andBacillus cereus are described. By exposure to light and DT, the growth ofSta. aureus andStr. Faecalis strains was markedly inhibited, whereasB. cereus was undergoing lysis. Counting the viable bacteria during the DT treatment revealed that more than 99% of the initial bacterial population was killed within the first 30 min of treatment. The pH dependence of the photodynamic inhibitory effect shows that it is best obtained at pH 6.5. No temperature dependence in the range of 37°–44°C could be detected. The best photodynamic effect was achieved when the culture was in the mid-log phase. DT-treated bacteria, when grown in the dark or in the presence of horse serum albumin, were unaffected by the porphyrin action. Electron microscopic examinations ofSta. aureus andStr. faecalis showed the appearance of well-developed mesosomes within the cell cytoplasm.B. cereus preparations have not shown any mesosome formation but showed some lysed cells as well as some spores. None of the mentioned effects by DT and light could be observed on Gramnegative bacteria such asEscherchia coli orPseudomonas aeruginosa.     

Acetic acid production from whey lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum

Summary Acetic acid was produced from anaerobic fermentation of lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum at 35° C and pHs between 7.0 and 7.6. Lactose was converted to lactic acid, and then to acetic acid in this mixed culture fermentation. The overall acetic acid yield from lactose was about 95% at pH 7.6 and 90% at pH 7.0. The fermentation rate was also higher at pH 7.6 than at pH 7.0. In batch fermentation of whey permeate containing about 5% lactose at pH 7.6, the concentration of acetic acid reached 20 g/l within 20 h. The production rate then became very slow due to end-product inhibition and high Na⁺ concentration. About 30 g/l acetate and 20 g/l lactate were obtained at a fermentation time of 80 h. However, when diluted whey permeate containing 2.5% lactose was used, all the whey lactose was converted to acetic acid within 30 h by this mixed culture.     

Microbiome precision editing: Using PEG as a selective fermentation initiator against methicillin‐resistant Staphylococcus aureus

Recent creation of a Unified Microbiome Initiative (UMI) has the aim of understanding how microbes interact with each other and with us. When pathogenic Staphylococcus aureus infects the skin, the interplay between S. aureus and skin commensal bacteria occurs. Our previous data revealed that skin commensal bacteria can mediate fermentation against the growth of USA300, a community‐acquired methicillin‐resistant S. aureus MRSA. By using a fermentation process with solid media on a small scale, we define poly(ethylene glycol) dimethacrylate (PEG‐DMA) as a selective fermentation initiator which can specifically intensify the probiotic ability of skin commensal Staphylococcus epidermidis bacteria. At least five short‐chain fatty acids including acetic, butyric and propionic acids with anti‐USA300 activities are produced by PEG‐DMA fermentation of S. epidermidis. Furthermore, the S. epidermidis‐laden PEG‐DMA hydrogels effectively decolonized USA300 in skin wounds in mice. The PEG‐DMA and its derivatives may become novel biomaterials to specifically tailor the human skin microbiome against invading pathogens.     

Growth and survival of Bacillus cereus in mageu,a sour maize beverage

The growth and survival of Bacillus cereus, a known pathogen commonly found in cereals, during lactic acid fermentation of mageu, a sour maize beverage, was studied. In the mageu base inoculated with both the starter culture and B. cereus, the acidity developed to pH 4.00 and 0.10% titratable acidity after 24 h; the growth of B. cereus was reduced from 10⁶ c.f.u./ml to 10² c.f.u./ml within 24 h; after the first 6 h of fermentation, the rate of inhibition of B. cereus was correlated to the rate of decrease in pH (r = 0.85, p < 0.05); the redox potential (E_h) decreased from 463 to 149 mV within the first 12 h. The control mageu base to which neither starter nor lactic acid was added, had a pH of 6.50, titratable acidity of 0.015% and lowest E_h of 244 mV. In the mageu base to which lactic acid and B. cereus were added, the pathogen was inhibited to < 10¹ c.f.u./ml. The B. cereus in the mageu base to which no starter culture nor lactic acid was added, grew to over 10⁷ c.f.u./ml after 12 h. The decrease in E_h seemed to have no inhibitory effect on the growth and survival of B. cereus. No strains of lactic acid bacteria were found to produce bacteriocins antagonistic to B. cereus. Low pH and acidity were found to be the major factors inhibiting growth of B. cereus in mageu.     

Lactic acid bacteria population dynamics during spontaneous fermentation of radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.) roots in brine

The aim of the present study was to assess the microecosystem development and the dynamics of the lactic acid bacteria population during spontaneous fermentation of radish (Raphanus sativus L.) roots in brine at 20 and 30?°C. In both temperatures, lactic acid bacteria prevailed the fermentation; as a result, the pH value was reduced to ca. 3.6 and total titrable acidity increased to ca. 0.4% lactic acid. Enterococci population increased and formed a secondary microbiota while pseudomonads, Enterobacteriaceae and yeasts/molds populations were below enumeration limit already before the middle of fermentation. Pediococcus pentosaceus dominated during the first days, followed by Lactobacillus plantarum that prevailed the fermentation until the end. Lactobacillus brevis was also detected during the final days of fermentation. A succession at sub-species level was revealed by the combination of RAPD-PCR and rep-PCR analyses. Glucose and fructose were the main carbohydrates detected in brine and were metabolized into lactic acid, acetic acid and ethanol.     

Seasonal variation in allelopathic potential of<Emphasis Type="Italic">Artemisia princeps</Emphasis> var.<Emphasis Type="Italic">orientalis</Emphasis> on plants and microbes

We investigated seasonal variations in allelopathic potential ofArtemisia princeps var.orientalis. Aqueous and meth-anol extracts and volatile substances were prepared in the laboratory from samples collected monthly (April through October). Their impacts were then assessed on the germination and seedling growth ofLactuca sativa andAchyranthes japonica. The allelopathic potential varied with the time of sample collection and the concentration tested. For example, germination ofL. sativa was not inhibited by the aqueous extract but seedling growth (shoots and roots) was, with its seasonal effect being significant. ForA. japonica, seed germination was not inhibited at lower concentrations (except for August samples). However, at higher concentrations and in certain months (especially July), germination was more negatively affected. The degree of seedling growth inhibition also differed by month and by extract concentration, with roots being impacted more than shoots. Volatile substances also had a time-dependent influence on the germination and seedling elongation ofA. japonica. In a separate experiment, the ethyl-acetate and water fractions of a crude methanol extract were prepared monthly fromA. princeps var.orientalis. Here, we examined their antimicrobial activities against three gram-positive bacteria (Bacillus cereus, Bacillus subtilis, andStaphylococcus aureus), two gramnegative bacteria (Escherichia coli andPseudomonas fluorescens), and one lactic acid bacterium,Lactobacillus plantar urn. The ethyl-acetate fraction that was sampled in September was remarkably potent againstB. cereus andB. subtilis, whereas the water fraction collected in August and September showed great antimicrobial activity against the grampositive and -negative bacteria. In contrast,L. plantarum was not inhibited by the water fraction, regardless of the sampling month. Likewise, the ethyl-acetate and water fractions collected in April and October had the lowest levels of antimicrobial activity.     

Effect of γ radiation on physiological parameters of the ethanolic fermentation

This work examines the efficacy of radiation in reducing the viability of certain contaminating bacteria of sugar-cane must and the consequential beneficial effect of lethal doses of radiation on some physiological parameters of the yeast-based ethanolic fermentation. The must from sugar-cane juice was inoculated with different bacteria that usually contaminate the must in the production facilities: Bacillus and Lactobacillus. The contaminated must was irradiated at 2.0, 4.0, 6.0, 8.0 and 10.0 kGy of gamma radiation. The population density of the bacteria in the irradiated must was recorded. Ethanolic fermentation by yeast (Saccharomyces cerevisiae) was carried out and the total acidity, the volatile acidity and the organic acids (lactic and acetic) produced during the fermentation were determined. The ethanol yield was also recorded. The treatment with radiation reduced the population of the contaminating microorganisms of the sugar-cane must. The acidity and the organic acids (lactic and acetic) produced during the fermentation decreased as the dose of radiation applied to the must increased. It is concluded that irradiation was efficient in decontaminating the sugar-cane must and improved the biochemical parameters of the ethanolic fermentation, including the ethanol yield by 2%.     

Genetic organization ofAcetobacter for acetic acid fermentation

Plasmid vectors for the acetic acid-producing strains ofAcetobacter andGluconobacter were constructed from their cryptic plasmids and the efficient transformation conditions were established. The systems allowed to reveal the genetic background of the strains used in the acetic acid fermentation. Genes encoding indispensable components in the acetic acid fermentation, such as alcohol dehydrogenase, aldehyde dehydrogenase and terminal oxidase, were cloned and characterized. Spontaneous mutations at high frequencies in the acetic acid bacteria to cause the deficiency in ethanol oxidation were analyzed. A new insertion sequence element, IS1380, was identified as a major factor of the genetic instability, which causes insertional inactivation of the gene encoding cytochromec, an essential component of the functional alcohol dehydrogenase complex. Several genes including the citrate synthase gene ofA. aceti were identified to confer acetic acid resistance, and the histidinolphosphate aminotransferase gene was cloned as a multicopy suppressor of an ethanol sensitive mutant. Improvement of the acetic acid productivity of anA. aceti strain was achieved through amplification of the aldehyde dehydrogenase gene with a multicopy vector. In addition, spheroplast fusion of theAcetobacter strains was developed and applied to improve their properties. ADH membrane-bound alcohol dehydrogenase - ALDH membrane-bound aldehyde dehydrogenase - IS insertion sequence - NTG N-methyl-N-nitro-N-nitrosoguanidine - PQQ pyrroloquinoline quinone     

Effect of pH on<Emphasis Type="Italic"> Lactobacillus fermentum</Emphasis> growth,raffinose removal, α-galactosidase activity and fermentation products

In this study, the behaviour of Lactobacillus fermentum CRL 722 and CRL 251 were evaluated under different pH conditions (pH 6.0, 5.5, 5.0, 4.5) and without pH control. Growth was similar under all conditions assayed except at pH 4.5. These microorganisms were able to eliminate raffinose, a nondigestible -oligosaccharide (NDO) found in soy products, showing a consumption rate of 0.25 g l^–1 h^–1 (pH 6.0–5.0). The removal of raffinose was due to the high -galactosidase (-gal) activities of these lactic acid bacteria, which was highest at pH 5.5 (5.0 U/ml). The yield of organic acids produced during raffinose consumption was also highest at this pH. The results of this study will allow selection of the optimum growth conditions of L. fermentum with elevated levels of -gal to be used in the reduction of NDO in soy products when used as starter cultures.     

Effect of photoactivated hematoporphyrin derivative on the viability ofStaphylococcus aureus

The photodynamic effect of hematoporphyrin derivative (HPD) on the viability of penicillin-resistantStaphylococcus aureus is described. Growth rate of the bacteria was markedly reduced by exposure to light and HPD.Staphylococcus viability was decreased by 80% in 3 h of growth even at low HPD concentration (12 g/ml). A synergistic killing effect of HPD, light, and penicillin (10 g/ml) onStaphylococcus aureus was demonstrated, although the bacteria were originally resistant to 100 g/ml penicillin. A residual viability of only 3% was found by growth in medium containing this drug combination. The surviving bacteria after 3 h of treatment were sensitive even to 1 g penicillin/ml. The mechanism of HPD action onStaphylococcus is composed of two steps: i) penetration of HPD into the bacterial cell, which may be accomplished in the dark with no harm to the cells; ii) damaging of the bacterial cell upon photoactivation. The photoactivated HPD completely inhibited thymidine incorporation into the bacterial DNA. This effect was accompanied by inhibition of RNA and protein synthesis, which was parallel in extent to the reduction in growth rate. Electron microscopic examination ofS. aureus exposed for 3 h to HPD and light showed the appearance of well-developed mesosomes in the bacterial cells. None of these effects was observed on Gram-negative bacteria.     

Antimicrobial activity of naphthoquinones from Fusaria

Twenty-two naphthoquinone compounds isolated or derived synthetically from culture extracts ofFusarium solani andF. oxysporum were examined for antimicrobial activity. Fifteen exhibited antibiotic activity againstStaphylococcus aureus, and 12 were active againstStreptococcus pyogenes, but none were active at the highest rate of 128 g/ml againstEscherichia coli, Klebsiella pneumoniae, Salmonella typhi, Proteus vulgaris, Serratia marcescens, orPseudomonas aeruginosa. Of 8 plant pathogenic bacteria tested against 11 naphthoquinones,Corynebacterium poinsettiae was inhibited by 6 compounds, andPseudomonas viridiflava was weakly inhibited by one. Only one of a group of 6 fluorescent soil pseudomonads was inhibited by one naphthoquinone. Antifungal activity of 10 compounds against 8 fungal plant pathogens was limited to inhibition ofPhytophthora parasitica by one naphthopyran.South Atlantic, Agricultural Research Service, U.S. Department of Agriculture. Mention of a trademark or proprietary product is for identification only and does not imply a warranty or guarantee of the product by the U.S. Department of Agriculture over other products which may also be suitable.     

Microbiological and biochemical changes during the fermentation of maize (Zea mays) grains formasa production

Microorganisms associated with fermentation ofmasa wereLactobacillus plantarum, Pediococcus acidilactici, Lactobacillus fermentum andSaccharomyces cerevisiae. During the fermentation of maize grains, the temperature and moisture increased and the pH decreased. Most amino acids decreased in concentration during fermentation, with glutamic acid being the most abundant amino acid, but he protein content of the masa increased during fermentation. The bacteria played the major role in the fermentation.

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Résumé Les microorganismes associés avec la fermentation demasa sontLactobacillus plantarum, pediococcus acidilactici, Lactobacillus fermentum etSaccharomyces cerevisiae. Durant la fermentation de grains de mais, la température et l'humidité augmentent, et le pH diminue. La concentration de la plupart des acides aminés diminue durant la fermentation. L'acide aminé le plus abondant est l'acide glutamique. Toutefois, le contenu en protéines dumasa augmente au cours de la fermentation. Les bactéries jouent le rôle majeur dans la fermentation.

     

Critical factors in chitin production by fermentation of shrimp biowaste

Factors affecting Lactobacillus fermentation of shrimp waste for chitin and protein liquor production were determined. The objective of the fermentation is medium conditioning by Lactobacillus through production of proteases and lowering of the pH. The efficiency was tested by conducting fermentation of biowaste in 1-l beakers with or without pH adjustment using different acids. Addition of 5% glucose to the biowaste supported the growth of lactic acid bacteria and led to better fermentation. Among four acids tested to control pH at the start and during fermentation, acetic acid and citric acid proved to be the most effective. In biowaste fermented with 6.7% L. plantarum inoculum, 5% glucose, and pH 6.0 adjusted with acetic acid, 75% deproteination and 86% demineralization was achieved. Replacement of acetic acid by citric acid gave 88% deproteination and 90% demineralization. The fermentation carried out in the presence of acetic acid resulted in a protein fraction that smelled good and a clean chitin fraction. Received: 4 April 2000 / Received revision: 9 June 2000 / Accepted: 9 June 2000     

Resistance to cadmium salts and metal absorption by different microbial species

The present study evaluates the inhibitory activity and the absorption of cadmium (Cd) salts by different microbial species, including Gram-positive (Staphylococcus aureus andS. epidermidis), Gram-negative (Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, andProteus mirabilis) bacteria and one yeast (Candida albicans). The metal absorption by growing cells was considered both in liquid and in solid medium. For one strain ofP. aeruginosa the presence of Cd deposits inside the cell was investigated by transmission electron microscopy. Generally, the Gram-negative species tested proved to be highly resistant to Cd ions and accumulated great amounts of Cd during growth. Two strains ofP. aeruginosa showed a high degree of resistance to Cd and were particularly efficient in removing the metal from solutions. The Gram-positive bacteria showed a heterogeneous behavior: anS. aureus strain susceptible to Cd absorbed, at low metal concentrations, higher amounts of metal than a Cd-resistant one. The metal absorption for Gram-negative species was dose dependent, while for the Cd-resistant staphylococci it reached a plateau. Our results suggest that microorganisms can represent a good model to study the interactions between heavy metals and living organisms.     

Absence of a role for lytic microorganisms in the decline of bacteria andSaccharomyces introduced into soil

The populations ofKlebsieila pneumoniae, Escherichia coli, Enterobacter aerogenes, andPseudomonas sp. fell following their addition to soil, but species lysing these gram-negative bacteria were not detected. The numbers ofStaphylococcus aureus andMicrococcus flavus fell by more than four orders of magnitude and ofSaccharomyces cerevisiae by more than two orders after their addition to soil. Organisms lysing these gram-positive bacteria were present in soil, but their numbers did not increase as a result of the additions. Lytic activity againstS. aureus was detected in soil filtrates, but this activity was not enhanced by inoculation of soil with the bacterium. Addition of cycloheximide to soil suspensions delayed the fall in abundance ofM. flavus but did not suppress the lytic populations. We conclude that lysis is not responsible for the decline of bacteria orS. cerevisiae added to soil.