Growth of yeasts, lactic and acetic acid bacteria in palm wine during tapping and fermentation from felled oil palm (Elaeis guineensis) in Ghana

AIM: To investigate the microbiological and biochemical changes which occur in palm wine during the tapping of felled oil palm trees. METHODS AND RESUlts: Microbiological and biochemical contents of palm wine were determined during the tapping of felled oil palm trees for 5 weeks and also during the storage. Saccharomyces cerevisiae dominated the yeast biota and was the only species isolated in the mature samples. Lactobacillus plantarum and Leuconostoc mesenteroides were the dominated lactic acid bacteria, whilst acetic acid bacteria were isolated only after the third day when levels of alcohol had become substantial. The pH, lactic and acetic acid concentrations during the tapping were among 3.5-4.0%, 0.1-0.3% and 0.2-0.4% respectively, whilst the alcohol contents of samples collected within the day were between 1.4% and 2.82%; palm wine which had accumulated over night, 3.24% to 4.75%; and palm wine held for 24 h, over 7.0%. CONCLUSION: Accumulation of alcohol in palm wine occurs in three stages during the tapping and marketing with the concurrent lactic and acetic acid fermentation taking place as well. SIGNIFICANCE AND IMPACT OF THE STUDY: Yeasts, lactic and acetic acid bacteria are all important in the fermentation of palm wine and influence the composition of the product.     

Interaction effects of lactic acid and acetic acid at different temperatures on ethanol production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in corn mash

The combined effects of lactic acid and acetic acid on ethanol production by S. cerevisiae in corn mash, as influenced by temperature, were examined. Duplicate full factorial experiments (three lactic acid concentrations × three acetic acid concentrations) were performed to evaluate the interaction between lactic and acetic acids on the ethanol production of yeast at each of the three temperatures, 30, 34, and 37°C. Corn mash at 30% dry solids adjusted to pH 4 after lactic and acetic acid addition was used as the substrate. Ethanol production rates and final ethanol concentrations decreased (P<0.001) progressively as the concentration of combined lactic and acetic acids in the corn mash increased and the temperature was raised from 30 to 37°C. At 30°C, essentially no ethanol was produced after 96 h when 0.5% w/v acetic acid was present in the mash (with 0.5, 2, and 4% w/v lactic acid). At 34 and 37°C, the final concentrations of ethanol produced by the yeast were noticeably reduced by the presence of 0.3% w/v acetic acid and ≥2% w/v lactic acid. It can be concluded that, as in previous studies with defined media, lactic acid and acetic acid act synergistically to reduce ethanol production by yeast in corn mash. In addition, the inhibitory effects of combined lactic and acetic acid in corn mash were more apparent at elevated temperatures.     

Inhibition of the growth of acid tolerant yeasts by acetate, lactate and propionate and their synergistic mixtures

Saccharomyces uvarum, Geotrichum candidum, Endomycopsis burtonii and Hansemula canadensis have been isolated from deteriorated silages and/or spoiled lactic acid fermented foods. All yeasts could grow in relatively high (100 mmol/l) concentrations of acetate or lactate in Wickerham's assimilation broth but were inhibited by increasing concentrations of these acids. Propionate was much more inhibitory than acetate or lactate. Growth rate, but not final cell yield, was adversely affected by increasing concentrations of these acids. The three acids used in combination were synergistically inhibitory to H. canadensis and G. candidum. Saccharomyces uvarum was inhibited by synergistic mixtures of acetate/propionate. Endomycopsis burtonii and H. canadensis were inhibited by synergistic mixtures of acetate/lactate, acetate/propionate and lactate/propionate. Lactate and propionate had a synergistic inhibitory effect on G. candidum. The mechanism of inhibition of growth of S. uvarum by acetate and propionate was further investigated. Acetate and propionate each caused a reduction in cellular efficiency (Y, μg cells/μmole glucose consumed). Mixtures caused a drastic reduction in growth rate and Y. These results suggest that the cells may expend energy in reducing intracellular concentrations of acetate while propionate probably acts in a different manner.     

Cocoa Fermentations Conducted with a Defined Microbial Cocktail Inoculum

Cocoa fermentations were performed in wooden boxes under the following four experimental regimens: beans naturally fermented with wild microflora; aseptically prepared beans with no inoculum; and beans inoculated with a defined cocktail containing microorganisms at a suitable concentration either at zero time or by using phased additions at appropriate times. The cocktail used consisted of a yeast, Saccharomyces cerevisiae var. chevalieri, two lactic acid bacterial species, Lactobacillus lactis and Lactobacillus plantarum, and two acetic acid bacterial species, Acetobacter aceti and Gluconobacter oxydans subsp. suboxydans. The parameters measured were cell counts (for yeasts, filamentous fungi, lactic acid bacteria, acetic acid bacteria, and spore formers, including reisolation and identification of all residual cell types), sugar, ethanol, acetic acid, and lactic acid contents (and contents of other organic acids), pH, and temperature. A cut test for bean quality and a sensorial analysis of chocolate made from the beans were also performed. The natural fermentation mimicked exactly the conditions in 800-kg boxes on farms. The aseptic box remained largely free of microflora throughout the study, and no significant biochemical changes occurred. With the zero-time inoculum the fermentation was almost identical to the natural fermentation. The fermentation with the phased-addition inoculum was similar, but many changes in parameters were slower and less pronounced, which led to a slightly poorer end product. The data show that the nearly 50 common species of microorganisms found in natural fermentations can be replaced by a judicious selection and concentration of members of each physiological group. This is the first report of successful use of a defined, mixed starter culture in such a complex fermentation, and it should lead to chocolate of more reliable and better quality.     

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.     

Effects of acetic acid and lactic acid on the growth of Saccharomyces cerevisiae in a minimal medium

Specific growth rates (μ) of two strains of Saccharomyces cerevisiae decreased exponentially (R ²>0.9) as the concentrations of acetic acid or lactic acid were increased in minimal media at 30°C. Moreover, the length of the lag phase of each growth curve (h) increased exponentially as increasing concentrations of acetic or lactic acid were added to the media. The minimum inhibitory concentration (MIC) of acetic acid for yeast growth was 0.6% w/v (100 mM) and that of lactic acid was 2.5% w/v (278 mM) for both strains of yeast. However, acetic acid at concentrations as low as 0.05–0.1% w/v and lactic acid at concentrations of 0.2–0.8% w/v begin to stress the yeasts as seen by reduced growth rates and decreased rates of glucose consumption and ethanol production as the concentration of acetic or lactic acid in the media was raised. In the presence of increasing acetic acid, all the glucose in the medium was eventually consumed even though the rates of consumption differed. However, this was not observed in the presence of increasing lactic acid where glucose consumption was extremely protracted even at a concentration of 0.6% w/v (66 mM). A response surface central composite design was used to evaluate the interaction between acetic and lactic acids on the specific growth rate of both yeast strains at 30C. The data were analysed using the General Linear Models (GLM) procedure. From the analysis, the interaction between acetic acid and lactic acid was statistically significant (P≤0.001), i.e., the inhibitory effect of the two acids present together in a medium is highly synergistic. Journal of Industrial Microbiology & Biotechnology (2001) 26, 171–177. Received 06 June 2000/ Accepted in revised form 21 September 2000     

Comparative metabolic profiling to investigate the contribution of <Emphasis Type="Italic">O. oeni</Emphasis> MLF starter cultures to red wine composition

In this research work we investigated changes in volatile aroma composition associated with four commercial Oenococcus oeni malolactic fermentation (MLF) starter cultures in South African Shiraz and Pinotage red wines. A control wine in which MLF was suppressed was included. The MLF progress was monitored by use of infrared spectroscopy. Gas chromatographic analysis and capillary electrophoresis were used to evaluate the volatile aroma composition and organic acid profiles, respectively. Significant strain-specific variations were observed in the degradation of citric acid and production of lactic acid during MLF. Subsequently, compounds directly and indirectly resulting from citric acid metabolism, namely diacetyl, acetic acid, acetoin, and ethyl lactate, were also affected depending on the bacterial strain used for MLF. Bacterial metabolic activity increased concentrations of the higher alcohols, fatty acids, and total esters, with a larger increase in ethyl esters than in acetate esters. Ethyl lactate, diethyl succinate, ethyl octanoate, ethyl 2-methylpropanoate, and ethyl propionate concentrations were increased by MLF. In contrast, levels of hexyl acetate, isoamyl acetate, 2-phenylethyl acetate, and ethyl acetate were reduced or remained unchanged, depending on the strain and cultivar evaluated. Formation of ethyl butyrate, ethyl propionate, ethyl 2-methylbutryate, and ethyl isovalerate was related to specific bacterial strains used, indicating possible differences in esterase activity. A strain-specific tendency to reduce total aldehyde concentrations was found at the completion of MLF, although further investigation is needed in this regard. This study provided insight into metabolism in O. oeni starter cultures during MLF in red wine.     

Characterization of interactions between Lactobacillus hilgardii and Saccharomyces florentinus isolated from sugary kefir grains

Stimulation of the growth of Lactobacillus hilgardii and production of lactic acid in the presence of Saccharomyces florentinus have already been observed in a poor glucose medium. In this study we showed that CO₂, pyruvate, propionate, acetate and succinate excreted by the yeast were responsible for this phenomenon, whereas ethanol, fumarate and a cocktail of vitamins had no effect. Fermentation by the yeast did not enrich the total nitrogen or free amino acid content of the medium. The use of proteases confirmed that it was improbable that any nitrogenous compound could be responsible for the bacterial activation. Between values of 0.1 and 5%, the initial yeast: bacteria ratio had no effect on the stimulation of Lact. hilgardii, even though yeast fermentation was drastically affected by a low initial value. The interactions differed greatly according to the sugar source. With monosaccharides, stimulation began after 48 h of culture, and was higher on glucose than on fructose. With sucrose, stimulation appeared at the beginning of fermentation. Different mechanisms seemed to be involved.     

Novel Characteristics of Bifidobacterium bifidum in Solid State Fermentation System

Summary The characteristics of Bifidobacterium bifidum grown in solid state fermentation (SSF) system (water content of media 54.5 and 68.8%) was compared with the submerged fermentation (SmF) system (water content of medium: 89.8%). Besides lactic acid (lactate) and acetic acid (acetate), the bacterium was able to secrete propionic acid (propionate) and butyric acid (butyrate) under SSF conditions. However, it only produced lactate and acetate under SmF conditions. The ratio of lactate to acetate was 1.26–1.62:1 in SSF but it was 1:2 in SmF. A higher content of C16:0 and C18:1 as well as a lower content of C18:0 cell membrane fatty acids were observed in SSF than in SmF. There was a lower growth rate, a lower viable count and a longer logarithmic growth phase for B. bifidum cultivated in SSF than in SmF.     

代谢工程改造野生耐酸酵母生产L-乳酸

以选育低pH条件下高产L-乳酸的酵母菌为目的,从自然样品中筛选分离得到一株能在pH 2.5 (乳酸调节) 的培养基中生长且不利用乳酸的酵母 (初步鉴定为木兰假丝酵母Candida magnolia);进一步将来源于米根霉As3.819的乳酸脱氢酶编码基因 (ldhA) 插入含有G418抗性基因的酵母穿梭载体,构建了重组质粒pYX212-kanMX-ldhA,电转化入野生型C. magnolia中,筛选获得了一株具有产L-乳酸能力的重组菌株C. magnolia-2;通过发酵实验表明,该重组菌产L-乳酸的最     

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.     

发酵抑制物对絮凝酵母戊糖发酵的影响

将絮凝剂加入酵母溶液中,使酵母絮凝成颗粒以此作为固定化酵母进行戊糖发酵。研究了常见发酵抑制物(甲酸、乙酸、糠醛和乳酸等)对絮凝酵母发酵木糖的影响。结果表明:在60.0g/L木糖发酵液中,经过24h发酵,木糖利用率达94.6%,当分别添加抑制物甲酸、乙酸、糠醛、乙醇和乳酸时,聚氧乙烯絮凝酵母分别对其的耐受浓度为0.5、0.5、1.0、30.0和8.0g/L。当抑制物添加量超过各自的耐受浓度后,对絮凝酵母发酵会产生明显的抑制作用。     

High production system of the antibacterial peptide PLG-1

Propionibacterium thoenii P-127 produces and releases to the growth medium antibacterial agents that can be used as natural preservatives. The concentrations of these antibacterial agents in the growth medium are very low, and their activity can be detected only in concentrated medium, even in a bioreactor. A simple and efficient system to produce propionicin PLG-1 without the use of a bioreactor was investigated. Fermentation in screw-cap bottles without shaking produced antibacterial activity similar to that of fermentation in plates, but in a shorter time. Sodium lactate medium (NaLa) was found to be the most supportive for PLG-1 production compared to lactic acid bacteria media such as M-17 or beet molasses/corn. The initial concentration of the carbon source, sodium lactate, agar concentration, and the initial pH of the medium affected the synthesis of PLG-1. Additions of NaCl up to 1% showed no effect on the antibacterial agent production. The optimal conditions for production of the antibacterial agent were fermentation for 9 days in screw-cap bottles in modified NaLa medium (M-NaLa) containing 1% yeast extract, 1% tryptic soy broth, 0.9% lactic acid, and 0.6% agar, adjusted to pH of 9.     

Microbiological and physicochemical characterization of small-scale cocoa fermentations and screening of yeast and bacterial strains to develop a defined starter culture

Spontaneous cocoa bean fermentations performed under bench- and pilot-scale conditions were studied using an integrated microbiological approach with culture-dependent and culture-independent techniques, as well as analyses of target metabolites from both cocoa pulp and cotyledons. Both fermentation ecosystems reached equilibrium through a two-phase process, starting with the simultaneous growth of the yeasts (with Saccharomyces cerevisiae as the dominant species) and lactic acid bacteria (LAB) (Lactobacillus fermentum and Lactobacillus plantarum were the dominant species), which were gradually replaced by the acetic acid bacteria (AAB) (Acetobacter tropicalis was the dominant species). In both processes, a sequence of substrate consumption (sucrose, glucose, fructose, and citric acid) and metabolite production kinetics (ethanol, lactic acid, and acetic acid) similar to that of previous, larger-scale fermentation experiments was observed. The technological potential of yeast, LAB, and AAB isolates was evaluated using a polyphasic study that included the measurement of stress-tolerant growth and fermentation kinetic parameters in cocoa pulp media. Overall, strains L. fermentum UFLA CHBE8.12 (citric acid fermenting, lactic acid producing, and tolerant to heat, acid, lactic acid, and ethanol), S. cerevisiae UFLA CHYC7.04 (ethanol producing and tolerant to acid, heat, and ethanol), and Acetobacter tropicalis UFLA CHBE16.01 (ethanol and lactic acid oxidizing, acetic acid producing, and tolerant to acid, heat, acetic acid, and ethanol) were selected to form a cocktail starter culture that should lead to better-controlled and more-reliable cocoa bean fermentation processes.     

Effect of pH and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash

The effects of lactic and acetic acids on ethanol production by Saccharomyces cerevisiae in corn mash, as influenced by pH and dissolved solids concentration, were examined. The lactic and acetic acid concentrations utilized were 0, 0.5, 1.0, 2.0, 3.0 and 4.0% w/v, and 0, 0.1, 0.2, 0.4, 0.8 and 1.6% w/v, respectively. Corn mashes (20, 25 and 30% dry solids) were adjusted to the following pH levels after lactic or acetic acid addition: 4.0, 4.5, 5.0 or 5.5 prior to yeast inoculation. Lactic acid did not completely inhibit ethanol production by the yeast. However, lactic acid at 4% w/v decreased (P<0.05) final ethanol concentration in all mashes at all pH levels. In 30% solids mash set at pH ≤5, lactic acid at 3% w/v reduced (P<0.05) ethanol production. In contrast, inhibition by acetic acid increased as the concentration of solids in the mash increased and the pH of the medium declined. Ethanol production was completely inhibited in all mashes set at pH 4 in the presence of acetic acid at concentrations ≥0.8% w/v. In 30% solids mash set at pH 4, final ethanol levels decreased (P<0.01) with only 0.1% w/v acetic acid. These results suggest that the inhibitory effects of lactic acid and acetic acid on ethanol production in corn mash fermentation when set at a pH of 5.0–5.5 are not as great as that reported thus far using laboratory media.     

Fed-batch and continuous fermentation ofSelenomonas ruminantium for natural propionic,acetic and succinic acids

An anaerobic fermentation process was developed for production of natural propionic, acetic and succinic acids froml-lactic acid usingSelenomonas ruminantium. Thel-lactic acid was quickly converted to a racemic mixture and there was no enantiomeric preference for further metabolism. The lactic acid was metabolized to propionic, acetic and succinic acids typically in a molar ratio of about 531. However, the ratio of propionate: succinate started high (as much as 221), before declining to as low as 51 after the first 48 h. Nutrients in corn steep liquor and yeast extract were necessary for optimal production of propionic acid. The corn steep liquor and yeast extract were heat stable at neutral pH, but some nutritional qualities were lost when heated at pH 2.4. In fed-batch fermentation on lactic acid 2.0% propionic acid was produced in 48 h and 2.3% in 68 h. A continuous culture operated at a dilution rate of 0.055 h^–1 and a lactic acid feed concentration of 30 gL^–1 had a propionic acid productivity of 0.59 gL^–1h^–1. The steady state results were: lactic acid 0.6%, propionic acid 1.1%, acetic acid 0.50%, and succinic acid 0.33%.     

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.     

Antifungal activity of Leuconostoc citreum and Weissella confusa in rice cakes

The antifungal activity of organic acids greatly improves the shelf life of bread and bakery products. However, little is known about the effect of lactic acid fermentation on fungal contamination in rice cakes. Here, we show that lactic acid fermentation in rice dough can greatly retard the growth of three fungal species when present in rice cakes, namely Cladosporium sp. YS1, Neurospora sp. YS3, and Penicillium crustosum YS2. The antifungal activity of the lactic acid bacteria against these fungi was much better than that of 0.3% calcium propionate. We found that organic acids including lactic and acetic acid, which are byproducts of lactic fermentation or can be artificially added, were the main antifungal substances. We also found that some Leuconostoc citreum and Weissella confusa strains could be good starter species for rice dough fermentation. These results imply that these lactic acid bacteria can be applicable to improve the preservation of rice cakes.     

Whey fermentation by immobilized cells of Propionibacterium shermanii

Growth of Propionibacterium shermanii B-123 was faster on media containing lactate than on that containing lactose. Cheese whey was therefore fermented with Lactobacillus helveticus and neutralized with NaOH or Ca(OH)₂, before inoculation with B-123. Fermentation rate by immobilized propionibacteria was best in Ca(OH)₂-neutralized whey, and at lactate concentrations between 1 and 2%. Calcium propionate concentrations of 1 and 3% reduced fermentation rates by 40% and 55% respectively. Optimum temperature for propionate fermentation by immobilized cells was 37°C. Ratios of propionic acid to acetic acid increased as incubation temperature was increased. Agitation increased propionic acid fermentation rates but lowered the ratio of propionic acid to acetic acid. Beads containing immobilized propionibacteria were re-utilized for ten consecutive fermentations. Fermentation rates increased upon re-utilization. Escherichia coli and Staphylococcus aureus were inhibited by the propionic fermentation but did not die; they kept growing at a reduced rate.