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The potential of vancomycin-resistant enterococci to persist in fermented and pasteurised meat products.

Experiments with 148 isolates of vancomycin-resistant enterococci (VRE) were performed to assess their potential to persist and grow in fermented sausages and pasteurised meat products. All strains were meat isolates and Van-type A, except a single VanC1 strain. In total, 143 strains of Enterococcus faecium were involved. Eight selected strains were examined for their potential to grow at high salt and nitrite levels and at reduced pH. The same isolates were used in experiments with fermented sausages. All available strains were subjected to heating tests in meat suspensions with added curing ingredients. All but one of the eight tested isolates grew at pH 4.0 in tryptone soya broth (TSB). With the combination of 8% w/w NaCl, 400 ppm NaNO2 and 0.5% w/w glucose in the meat suspension, all isolates grew at 37 degrees C, whereas none grew at 7 degrees C even after 56 days. With the addition of 10% w/w NaCl, 200 ppm NaNO2 and 0.5% w/w glucose, still one E. faecium isolate grew at 37 degrees C, although very slowly. Overall, the strains tolerated high salt and nitrite concentrations and reduced pH very well, even beyond levels applied in the regular production of fermented and/or pasteurised meat products. The tested strains could be isolated after the fermentation and further ripening of "boerenmetworst" and "snijworst". Overall, their colony counts decreased on average about 1 log-unit over a period of 60 days after batter manufacture. All 148 isolates demonstrated a relatively weak thermal resistance compared to results for selected vancomycin-sensitive enterococci strains reported in the literature and to results collected under identical experimental conditions in this laboratory. None of the strains (log inoculation level about 5-6 ml(-1) for each isolate) could be cultured after heating at 70 degrees C for 10 min.

Animals↗

Isolation and characterization of a Lactobacillus plantarum bacteriophage, phiJL-1, from a cucumber fermentation.

A virulent Lactobacillus plantarum bacteriophage, PhiJL-1, was isolated from a commercial cucumber fermentation. The phage was specific for two related strains of L. plantarum, BI7 and its mutant (deficient in malolactate fermenting ability) MU45, which have been evaluated as starter cultures for controlled cucumber fermentation and as biocontrol microorganisms for minimally processed vegetable products. The phage genome of PhiJL-1 was sequenced to reveal a linear, double-stranded DNA (36.7 kbp). Sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) profiles indicated that PhiJL-1 contains six structural proteins (28, 34, 45, 50, 61, and 76 kDa). Electron microscopy revealed that the phage has an isometric head (59 nm in diameter), a long non-contractile tail (182 nm in length and 11 nm in width), and a complex base plate. The phage belongs to the Bradley group B1 or Siphoviridae family. One-step growth kinetics of the phage showed that the latent period was 35 min, the rise period was 40 min, and the average burst size was 22 phage particles/infected cell. Phage particles (90%) adsorbed to the host cells 20 min after infection. Calcium supplementation (up to 30 mM CaCl(2)) in MRS media did not affect the first cycle of phage adsorption, but promoted rapid phage propagation and cell lysis in the infection cycle subsequent to adsorption. The D values of PhiJL-1 at pH 6.5 were estimated to be 2.7 min at 70 degrees C and 0.2 min at 80 degrees C by a thermal inactivation experiment. Knowledge of the properties of L. plantarum bacteriophage PhiJL-1 may be important for the development of controlled vegetable fermentations.

Adsorption↗

The role of interaction between yeasts and lactic acid bacteria in African fermented milks: a review.

Yeasts are present in indigenous African fermented milks in numbers up to log 8 cfu g(-1), together with a varied lactic acid bacteria (LAB) flora, and therefore potentially contribute to product characteristics. However, interaction between yeasts and LAB in these products has received little notice. In studies of indigenous fermented milk in Zimbabwe and Uganda, many samples contained more than one species of yeast, but Saccharomyces cerevisiae was most commonly isolated. Other frequent isolates were other species of Saccharomyces and several species of Candida. Most yeast isolates were lactose-negative but usually galactose-positive. Some strains assimilated lactate and citrate. The growth in milk of strains of yeasts and LAB, isolated from naturally soured milk in Zimbabwe, and their interaction when selected pairs of strains were grown together has been studied. Interactions were shown by the significantly different amounts of certain metabolites produced, such as acetaldehyde and malty aldehydes, when co-cultures were compared to pure cultures. Preliminary sensory acceptance tests did not show, however, that milks made from a co-culture with Candida kefyr and LAB were preferable to the pure LAB culture. Further work is still needed to elucidate the reactions that may be taking place in fermented milk between varying LAB and yeast populations. The potential for use as starter cultures depends on various aspects, including the final product being prepared. The role of other microorganisms in naturally fermented milk also needs to be studied.

Animals↗

Munkoyo beverage, a traditional Zambian fermented maize gruel using Rhynchosia root as amylase source.

A typical munkoyo beverage was made by fermenting Rhynchosia heterophylla root extract-cooked maize meal mixture with Lactobacillus confusus LZI and Saccharomyces cerevisiae YZ20. The fermented munkoyo beverage had a pH of 3.3, lactic acid content of 60 mmol/l, ethanol 320-410 mmol/l and a characteristic 'munkoyo' aroma. L. confusus, used alone, produced a beverage with a faint munkoyo flavour note whilst beverage produced with S. cerevisiae alone seemed not to have a typical munkoyo note. R. heterophylla root extract converted 75% of the starch in sterile cooked maize meat to maltose (80% of total sugars), maltotriose (17%) and glucose (3%) in I h at 45 degrees C. During fermentation by the mixed culture or the yeast alone most of the maltose was utilised but little or none of the maltotriose. The ratio of yeast to lactic acid bacteria in the starter culture affected the rate of production of ethanol but had no effect on the growth or acid production by the bacterium. To obtain a munkoyo beverage with the desired low alcohol concentration (< 100 mmol/l), the ratio of yeast concentration (cfu/ml) to Lactobacillus concentration in the starter culture should be 1:1000 or less and the beverage should be fermented for 24 h only.

Amylases↗

Ecology of inoculated and spontaneous fermentations in Rioja (Spain) musts, examined by mitochondrial DNA restriction analysis.

Analysis of mitochondrial DNA restriction patterns was used to study the introduction of a selected strain of Saccharomyces cerevisiae for fermentation of non-sterile musts of La Rioja (Spain). All of the isolates from the inoculated musts showed the restriction pattern of the selected strain. The same technique was used to study the spontaneous fermentation of musts, showing that a few strains were responsible for the fermentations. One of the strains identified from the spontaneous fermentations had been identified in a previous vintage.

Animals↗

Differentiation of lactate-fermenting, gas-producing Clostridium spp. isolated from milk.

Endospores of Clostridium spp. capable of producing gas in a lactate-containing medium were enumerated from 14 pasteurized milk samples from Wisconsin cheese plants. Concentrations of endospores of lactate-fermenting, gas-producing Clostridium spp. were between 5.0 x 10(-2) and 1.7 x 10(0) MPN ml(-1). Concentrations of presumptive C. tyrobutyricum endospores (defined by subterminal endospore position and lactate dehydrogenase activity) were lower, not exceeding 2.0 x 10(-2) MPN ml(-1). Based on subterminal endospore position, lactate dehydrogenase activity, and a carbohydrate fermentation profile identical to C. tyrobutyricum strain ATCC 25755, five isolates (Ct) were initially characterized as C. tyrobutyricum, a known cause of late-blowing in high-pH cheeses. Twenty-eight other isolates (Cx) produced gas from lactate, but differed from ATCC 25755 in either endospore position, lactate dehydrogenase activity or carbohydrate fermentation profile. When inoculated at high concentrations in Gouda cheese, strain ATCC 25755, two Ct isolates and 18 Cx isolates tested produced gas during ripening. Among the five Ct isolates obtained and two reference strains confirmed as C. tyrobutyricum, there were four qualitatively different volatile organic acid byproduct profiles. Each of the two confirmed C. tyrobutyricum reference strains and five Ct isolates had distinct quantitative cell membrane fatty acid (CMFA) profiles. The Cx isolates represented 14 different volatile organic acid byproduct profiles and each isolate had a unique CMFA profile. Pulsed field gel electrophoresis (PFGE) of DNA from the two confirmed reference C. tyrobutyricum strains, four Ct and three Cx isolates, showed a low degree of relatedness. The results of this study suggest that a heterogeneous group of lactate-fermenting, gas-producing Clostridium spp. may be found in milk. Gas chromatographic analysis of volatile organic acid byproducts or CMFA, and PFGE of DNA are highly discriminating methods for differentiating Clostridium spp. that may cause late blowing in high-pH cheeses.

Animals↗

Oat bran beta-gluco- and xylo-oligosaccharides as fermentative substrates for lactic acid bacteria.

The influence of oat bran oligosaccharides on carbohydrate utilization and fermentation end-products was studied with reference to three different lactic acid bacteria (LAB: Lactobacillus rhamnosus, Lactobacillus plantarum and Lactococcus lactis). The main results were that all three LAB utilized oat beta-gluco-oligosaccharides, while only L. plantarum utilized xylo-oligosaccharides. The main products of LAB metabolism were lactic acid, acetic acid, formic acid and ethanol. The results indicated that oat beta-gluco-oligosaccharides and xylo-oligosaccharides induce LAB to form the end-products of a typical mixed-acid fermentation. The formation of mixed-acid production from xylo-oligosaccharides was mainly due to the starvation of cells. This study indicates that oat bran oligosaccharides affect both qualitatively and quantitatively the fermentation end-products of LAB grown on these substrates. This should be taken into account when selecting strains for new fermented cereal based food products.

Acetic Acid↗

Effect of proteolytic starter cultures of Staphylococcus spp. on biogenic amine formation during the ripening of dry fermented sausages.

The effect of proteolytic starter cultures of Staphylococcus carnosus and Staphylococcus xylosus on biogenic amine production was examined during the fermentation process of dry sausages. Microbial counts (lactic acid bacteria, Micrococcaceae and Enterobactenaceae), pH, moisture and proteolysis-related parameters were also studied. The polyamines spermine and spermidine were the main amines found in the raw material and they only showed slight fluctuations during the fermentation. The four elaborated batches presented a significant (P < 0.001) formation of tyramine and putrescine. The main rate of amine production was during the first three days, when a sharp pH decrease and the development of lactic acid bacteria occurred. Sausages fermented with starters had lower amounts of tyramine than naturally fermented sausages (control), but differences in the Micrococcaceae counts were only significant during the first week of the ripening process. A slight formation of diaminopropane, cadaverine, agmatine, tryptamine and phenylethylamine was observed. The amounts of histamine were constant and remained below 0.5 mg/kg of dry matter, while serotonin, octopamine and dopamine were not detected. The sausages with Staphylococcus as starter culture showed strong proteolysis that was correlated with higher pH values than those of the control sausages. However, no positive correlation was found between the proteolysis index and biogenic amine production. Since proteolysis was stronger during the second half of the ripening process, the release of free amino acids as amine precursors occurred later than the early amine production.

Animals↗

Antimicrobial activity of tea as affected by the degree of fermentation and manufacturing season.

Bacillus subtilis, Escherichia coli, Proteus vulgaris, Pseudomonas fluorescens, Salmonella sp. and Staphylococcus aureus were used to test the antimicrobial activity of tea flush extract and extracts of various tea products. Among the six test organisms, P. fluorescens was the most sensitive to the extracts, while B. subtilis was the least sensitive. In general, antimicrobial activity decreased when the extents of tea fermentation increased. The antimicrobial activities of tea flush extract and extracts of tea products with different extents of fermentation varied with test organisms. Tea flush and Green tea, the unfermented tea, exerted the strongest antimicrobial activity followed by the partially fermented tea products such as Longjing, Tieh-Kuan-Ying, Paochung, and Oolong teas. On the other hand, Black tea, the completely fermented tea, showed the least antimicrobial activity. It was also noted that extracts of Oolong tea prepared in summer exhibited the strongest antimicrobial activity, followed by those prepared in spring, winter and fall.

Anti-Bacterial Agents↗

Scale-up of dialysis fermentation for high cell density cultivation of Escherichia coli.

In dialysis fermentations inhibiting metabolites can be removed from cell suspensions resulting in a prolonged exponential growth phase and higher production yields. Because of successful high cell density cultivations of Escherichia coli in a laboratory dialysis reactor, a scale-up of the process was investigated. To provide sufficient membrane area for dialysis in a technical scale fermenter, an external membrane module was used, that was also applied for oxygen supply to the culture in the external loop. Cultivations with recombinant E. coli K12, with and without induction, in 2- and 300-l reactors were carried out using external modules. Cell densities exceeding 190 g l(-1), previously obtained in laboratory dialysis fermentation, were also produced with external dialysis modules. Protein concentration in a 300-l reactor was increased to the 3.8-fold of industrial fed-batch-fermentations.

Culture Media↗

Evaluation of a recombinant Klebsiella oxytoca strain for ethanol production from cellulose by simultaneous saccharification and fermentation: comparison with native cellobiose-utilising yeast strains and performance in co-culture with thermotolerant yeast and Zymomonas mobilis.

In the simultaneous saccharification and fermentation to ethanol of 100 g l(-1) microcrystalline cellulose, the cellobiose-fermenting recombinant Klebsiella oxytoca P2 outperformed a range of cellobiose-fermenting yeasts used in earlier work, despite producing less ethanol than reported earlier for this organism under similar conditions. The time taken by K. oxytoca P2 to produce up to about 33 g l(-1) ethanol was much less than for any other organism investigated, including ethanol-tolerant strains of Saccharomyces pastorianus, Kluyveromyces marxianus and Zymomonas mobilis. Ultimately, it produced slightly less ethanol (maximum 36 g l(-1)) than these organisms, reflecting its lower ethanol tolerance. Significant advantages were obtained by co-culturing K. oxytoca P2 with S. pastorianus, K. marxianus or Z. mobilis, either isothermally, or in conjunction with temperature-profiling to raise the cellulase activity. Co-cultures produced significantly more ethanol, more rapidly, than either of the constituent strains in pure culture at the same inoculum density. K. oxytoca P2 dominated the early stages of the co-cultures, with ethanol production in the later stages due principally to the more ethanol tolerant strain. The usefulness of K. oxytoca P2 in cellulose simultaneous saccharification and fermentation should be improved by mutation of the strain to increase its ethanol tolerance.

Carbohydrate Metabolism↗

Anaerobic thermophilic fermentation for acetic acid production from milk permeate.

Fermentation of milk permeate to produce acetic acid under anaerobic thermophilic conditions (approximately 60 degrees C) was studied. Although none of the known thermophilic acetogenic bacteria can ferment lactose, it has been found that one strain can use galactose and two strains can use lactate. Moorella thermoautotrophica DSM 7417 and M. thermoacetica DSM 2955 were able to convert lactate to acetate at thermophilic temperatures with a yield of approximately 0.93 g g(-1). Among the strains screened for their abilities to produce acetate and lactate from lactose, Clostridium thermolacticum DSM 2910 was found precisely to produce large amounts of lactate and acetate. However, it also produced significant amounts of ethanol, CO2 and H2. The lactate yield was affected by cell growth. During the exponential phase, acetate, ethanol, CO2 and H2 were the main products of fermentation with an equimolar acetate/ethanol ratio, whereas during the stationary phase, only lactic acid was produced with a yield of 4 mol per mol lactose, thus reaching the maximal theoretical value. When this bacterium was co-cultured with M. thermoautotrophica, lactose was first converted mainly to lactic acid, then to acetic acid, with a zero residual lactic acid concentration and an overall yield of acetate around 80%. Under such conditions, only 13% of the fermented lactose was converted to ethanol by C. thermolacticum.

Acetic Acid↗

Degradation of organochlorine pesticides by meat starter in liquid media and fermented sausage.

The effect of meat starter on the degradation of DDT and lindane was investigated. The insignificant role of Lactobacillus plantarum in degrading p,p'-DDT and lindane presented in tryptone soya broth (TSB) and mineral salt medium (MSM) with or without 120 ppm nitrite was observed.The degradation of DDT and lindane by Micrococcus varians in TSB and MSM with or without nitrite were studied. The results indicated that DDT or lindane were degraded during the incubation period. The reduction in DDT at the end of the incubation period (15 days) was about 24.1 and 32.5% in TSB and MSM without nitrite, respectively. Corresponding values in the same media with nitrite were 37.5 and 46.4%. Regarding the reduction in lindane, it was recorded as 27.9 and 40.0% in TSB and MSM without nitrite, respectively and 38.4 and 48.4% in the same media with nitrite. The results indicated that culture media M. varians metabolized DDT mainly to DDD and lindane mainly to 2,4-, 2,5-, 2,6- and 3,4-dichlorophenol; 2,3,4- and 2,3,5-trichlorophenol; hexachlorobenzene; and pentachlorophenol. The effect of pesticides on the growth rate of meat starter was also investigated. The addition of DDT or lindane resulted in a slight decrease in counts of the strains during the initial incubation in TSB or MSM. Then the microorganisms recovered and began to grow logarithmically, but not as well as in a normal situation. The effect of fermentation stage by meat starter on DDT and lindane in fermented sausage was recorded. The results indicated that during the 72 h of fermentation, the reduction was 10 and 18% of DDT and lindane, respectively. These results confirmed that the fermentation process in meat products reduced pesticide residues and these reductions were due to the activity of meat starter.

Animal Feed↗

Determination of beta-lactams and their biosynthetic intermediates in fermentation media by pre-column derivatisation followed by fluorescence detection.

This paper describes a novel and sensitive pre-column derivatisation method for the detection and quantitation of beta-lactams and their biosynthetic precursors at trace levels in fermentation media. Filtered broths from fermentations of strains of Penicillium chrysogenum and Cephalosporium acremonium, after deproteination and centrifugation, were incubated with 9-fluorenylmethylchloroformate for 5 min at 20 degrees C in 0.2 M borate buffer at pH 7.7. Following two-fold pentane extraction of the reagent hydrolysis product, the aqueous layer was injected directly onto a C18 reversed-phase column, and products were detected spectrofluorimetrically with excitation and emission wavelengths of 260 and 313 nm, respectively. Detection limits of 0.01 and 0.05 micrograms ml-1 were achieved for both 6-aminopenicillanic acid (6-APA) and isopenicillin N in borate buffer and filtered fermentation broths, respectively, using a 10-microliter injection volume. A linear calibration for 6-APA in fermentation broth was obtained for a very wide concentration range (0.05-100 micrograms ml-1). Detection limits for solutions of cephalosporin C, deacetylcephalosporin C and deacetoxycephalosporin C in broth were all 0.25 micrograms ml-1. The detection limit for the beta-lactam precursor delta-(L-aminoadipyl)-L-alpha-cysteinyl-D-valine (ACV) dimer in borate buffer was 0.5 microgram ml-1. The cephalosporins and ACV dimer gave linear plots in the ranges 3-25 and 1-100 micrograms ml-1, respectively. Repeated analysis of 6-APA at a concentration of 10 micrograms ml-1 in filtered broth gave a mean peak area of 2.5.10(6) with a standard deviation of 2.6.10(5) using a 10-microliter injection volume. Ampicillin spiked into deproteinated blood serum gave a linear calibration in the concentration range 2-100 micrograms ml-1.

Acremonium↗

High-performance capillary electrophoresis of a fermentation-derived cyclic peptide analog, animal growth promoter.

We have developed HPCE methods for the analysis of sulfomycin (trivial name) and related compounds (code name, crude material = U82127 = I), which is an animal growth promoter derived from a fermentation beer. The crude material, I, isolated from the fermentation consisted of more than 40 components which were not completely separated by conventional HPLC. Thus, as a complementary analysis method, we have optimized HPCE conditions for I using various capillaries including uncoated, coated, and packed using various buffers. The optimized HPCE conditions were obtained with an uncoated fused-silica capillary and a buffer that consisted of 30 mM Tris-tricine, 10 mM SDS, 10 mM NaCl and 20% MeOH, pH 8.0. Using these HPCE conditions, we were able to separate the one main component collected from the HPLC effluent into two or three components. In order to identify the main components of the fermentation product, an off-line HPLC-HPCE-MS analysis for I was performed. From the MALDI-TOF-MS results, the separated components collected from HPCE had different molecular masses. Four lots of I samples having different characteristics were also analyzed by HPCE to investigate lot-to-lot differences in peak profiles. The four lots of I were found to have very similar peak profiles. In this paper, I refers to the crude fermentation product and sulfomycin to the purified, major HPLC component of I.

Animals↗

Expression levels and patterns of glycolytic yeast genes during wine fermentation.

Promoters from glycolytic genes are widely used for gene overexpression in the yeast Saccharomyces cerevisiae. Wine strains are not an exception, and genes of enological interest have been expressed in this way. However, the transcriptional pattern of glycolytic genes has never been studied during wine fermentation. In this work we describe the levels and expression patterns of glycolytic genes for a wine yeast strain during the alcoholic fermentation of three different musts. Results show similar transcriptional patterns for all genes studied: maximal levels of mRNA at the exponential growth stage, and a gradual decrease during the stationary phase, in accordance with the fermentation rates. The glyceraldehyde 3-phosphate dehydrogenase genes reach the highest transcriptional levels during wine fermentation, similarly as previously described for laboratory strains and conditions.

Fermentation↗

[Alcoholic fermentation of inulin by various strains of yeasts].

Strains of fourteen species of yeasts able to ferment inulin without previous chemical or physical hydrolysis were studied on semi-synthetic medium by evaluation of CO2 production under anaerobic conditions. Among them, Kluyveromyces cicerisporus, Candida macedoniensis and Candida utilis showed the best kinetic characteristics of fermentation. Experiments were carried out to specify the action of different parameters such as temperature, pH and exogenous ethanol concentration. The results obtained on semi-synthetic medium were confirmed on Jerusalem artichoke juice. The optimal temperature for fermentation was 35 degrees C for the three strains, but K. cicerisporus alone conserved its fermentative capacity at 40 degrees C, at low pH (3.5) and with 6% (v/v) exogenous ethanol concentration. This strain appears to be a good yeast for industrial production of ethanol from inulin substrate.

Culture Media↗

Clostridium lentocellum SG6--a potential organism for fermentation of cellulose to acetic acid.

A cellulolytic, acetic acid producing anaerobic bacterial isolate, Gram negative, rod-shaped, motile, terminal oval shaped endospore forming bacterium identified as Clostridium lentocellum SG6 based on physiological and biochemical characteristics. It produced acetic acid as a major end product from cellulose fermentation at 37 degrees C and pH 7.2. Acetic acid production was 0.67 g/g cellulose substrate utilized in cellulose mineral salt (CMS) medium. Yeast extract (0.4%) was the best nitrogen source among the various nitrogenous nutrients tested in production medium containing 0.8% cellulose as substrate. No additional vitamins or trace elemental solution were required for acetic acid fermentation. This is the highest acetic acid fermentation yield in monoculture fermentation for direct conversion of cellulose to acetic acid.

Acetic Acid↗