Trophic relationships between Saccharomyces cerevisiae and Lactobacillus plantarum and their metabolism of glucose and citrate

Glucose and citrate are two major carbon sources in fruits or fruit juices such as orange juice. Their metabolism and the microorganisms involved in their degradation were studied by inoculating with an aliquot of fermented orange juice a synthetic model medium containing glucose and citrate. At pH 3.6, their degradation led, first, to the formation of ethanol due to the activity of yeasts fermenting glucose and, eventually, to the formation of acetate resulting from the activity of lactobacilli. The yeast population always outcompeted the lactobacilli even when the fermented orange juice used as inoculum was mixed with fermented beet leaves containing a wider variety of lactic acid bacteria. The evolution of the medium remained similar between pH 3.3 and 5.0. At pH 3.0 or below, the fermentation of citrate was totally inhibited. Saccharomyces cerevisiae and Lactobacillus plantarum were identified as the only dominant microorganisms. The evolution of the model medium with the complex microbial community was successfully reconstituted with a defined coculture of S. cerevisiae and L. plantarum. The study of the fermentation of the defined model medium with a reconstituted microbial community allows us to better understand the behavior not only of fermented orange juice but also of many other fruit fermentations utilized for the production of alcoholic beverages.     

Citrate metabolism by Lactobacillus plantarum isolated from orange juice

The behaviour of Strains of Lactobacillus plantarum isolated from fermented orange juice and Lact. plantarum DSM 20174 was studied in the presence of citrate. When used as sole carbon source, citrate scarcely supported the growth of the bacteria. It was shown to enhance the growth of Lact. plantarum in glucose media. Under acid conditions (pH 4·0–5·0), 1 mol of citrate yielded 1·7 mol of acetate as sole major final metabolite with release of CO₂ in the gas phase.     

Fermentation of citrate by Lactobacillus plantarum in the presence of a yeast under acid conditions

Summary At pH 3.6, Lactobacillus plantarum is unable to grow on citrate or to ferment it in the absence of another carbon source such as glucose. In a defined medium containing glucose and citrate, with a higher concentration of the former than the latter, as in many fermented alcoholic beverages, L. plantarum will first ferment the sugar. The production of lactate from glucose degradation increases the acidity of the medium and inhibits the fermentation of citrate. In co-culture with Saccharomyces cerevisiae, part of the glucose is fermented by the yeast, partly avoiding the pH drop and the inhibition of citrate fermentation by L. plantarum. Fermentation was still possible at pH values around 3.0. Offprint requests to: C. Kennes     

Citrate utilization by homo- and heterofermentative lactobacilli

Citrate utilization by several homo- and heterofermentative lactobacilli was determined in Kempler and McKay and in calcium citrate media. The last medium with glucose permitted best to distinguish citrate-fermenting lactobacilli. Lactobacillus rhamnosus ATCC 11443, Lactobacillus zeae ATCC 15820 and Lactobacillus plantarum ATCC 8014 used citrate as sole energy source, whereas in the other strains, glucose and citrate were cometabolized. Some lactobacilli strains produced aroma compounds from citrate. Citrate transport experiments suggested that all strains studied presented a citrate transport system inducible by citrate. The levels of induction were variable between several strains. Dot blot experiment showed that lactobacilli do not present an equivalent plasmid coding for citrate permease.     

液质发酵食品中微生物群落及与乳酸菌间相互作用研究进展

液质发酵食品发酵过程中微生物组成复杂,复杂的微生物发酵体系会影响微生物的生长和代谢,继而改变微生物的群落结构与功能,最终影响液质发酵食品的品质.乳酸菌在食品发酵中对形成风味物质、提高营养价值、抑制腐败菌生长具有重要的作用.本文主要对近年来食醋、酱油和饮料酒等液质发酵食品中微生物群落及与乳酸菌间相互作用关系进行综述,了解...     

Effects of Orange Juice Formulation on Prebiotic Functionality Using an In Vitro Colonic Model System

A three-stage continuous fermentative colonic model system was used to monitor in vitro the effect of different orange juice formulations on prebiotic activity. Three different juices with and without Bimuno, a GOS mixture containing galactooligosaccharides (B-GOS) were assessed in terms of their ability to induce a bifidogenic microbiota. The recipe development was based on incorporating 2.75g B-GOS into a 250 ml serving of juice (65°Brix of concentrate juice). Alongside the production of B-GOS juice, a control juice – orange juice without any additional Bimuno and a positive control juice, containing all the components of Bimuno (glucose, galactose and lactose) in the same relative proportions with the exception of B-GOS were developed. Ion Exchange Chromotography analysis was used to test the maintenance of bimuno components after the production process. Data showed that sterilisation had no significant effect on concentration of B-GOS and simple sugars. The three juice formulations were digested under conditions resembling the gastric and small intestinal environments. Main bacterial groups of the faecal microbiota were evaluated throughout the colonic model study using 16S rRNA-based fluorescence in situ hybridization (FISH). Potential effects of supplementation of the juices on microbial metabolism were studied measuring short chain fatty acids (SCFAs) using gas chromatography. Furthermore, B-GOS juices showed positive modulations of the microbiota composition and metabolic activity. In particular, numbers of faecal bifidobacteria and lactobacilli were significantly higher when B-GOS juice was fermented compared to controls. Furthermore, fermentation of B-GOS juice resulted in an increase in Roseburia subcluster and concomitantly increased butyrate production, which is of potential benefit to the host. In conclusion, this study has shown B-GOS within orange juice can have a beneficial effect on the fecal microbiota.     

水稻秸秆低温复合菌系多样性及发酵动态

【目的】为解决中国寒冷地区水稻秸秆大面积废弃问题,加快低温地区水稻秸秆饲料转化,本文筛选了可以低温下加速秸秆发酵过程的微生物复合菌系,研究其微生物组成并跟踪其发酵动态。【方法】通过5℃下连续定向富集筛选,获得低温复合菌系。采用克隆文库方法分析复合菌系的组成。将复合菌系和商业接种剂(由Lactobacillus plantarum,Enterococcus faecium,L.salivarilus,Pediococcus acidilactici组成)分别接入稻秸进行10℃发酵。气质联机(GC-MS)测定发酵产物的同时,通过变性梯度凝胶电泳检测微生物在发酵体系的定殖情况。采用定量PCR方法追踪复合菌系组成菌在发酵过程中的动态。【结果】16S rDNA克隆文库分析结果表明复合系主要由两种微生物组成,一种属乳酸杆菌(Lactobacillus),一种属乳酸球菌(Leuconostoc)。10℃稻秸发酵结果表明,在发酵第6天接种复合菌系处理的pH已经下降到4.3,乳酸菌菌落形成单位为2.9×109CFU/g鲜样,而接种商业接种剂的处理pH为5.3,乳酸菌菌落形成单位为3.6×108 CFU/g鲜样;在发酵30 d时,接种复合菌系处理的乳酸含量为8.1 g/kg鲜样,接种商业接种剂处理的乳酸含量为2.0 g/kg鲜样。变性梯度凝胶电泳结果表明,在接种复合菌系的稻秸中,从发酵的第6天开始,检测到的微生物主要为L.sakei和Leuconostoc inhae,在整个发酵过程中,两菌一直存在;在商业接种剂处理中,发酵第6天检测到的微生物除其四种组成菌外,还包括Uncultured bacterium;而在发酵第16天和第30天,只检测到组成菌中的L.plantarum和E.faecium。定量PCR结果显示,接种复合菌系处理中,L.sakeiDNA在发酵第6天达到41.0%,在发酵第16天已达到65%,Le inhae在发酵的第6天达到整个发酵过程中的最大值(5.5%)。【结论】接种复合菌系,可以有效促进水稻秸秆的低温发酵进程。复合菌系组成菌可以定殖在发酵体系中,并占据优势。复合菌系的关键菌为L.sakei。     

Citrate metabolism in Lactobacillus casei and Lactobacillus plantarum

Information on the factors influencing citrate metabolism in lactobacilli is limited and could be useful in understanding the growth of lactobacilli in ripening cheese. Citrate was not used as an energy source by either Lactobacillus casei ATCC 393 or Lact. plantarum 1919 and did not affect the growth rate when co-metabolized with glucose or galactose. In growing cells, metabolism of citrate was minimal at pH 6 but significant at pH 4·5 and was greater in cells co-metabolizing galactose than in those co-metabolizing glucose or lactose. In non-growing cells, optimum utilization of citrate also occurred at pH 4·5 and was not increased substantially by the presence of fermentable sugars. In both growing and non-growing cells, acetate and acetoin were the major products of citrate metabolism; pyruvate was also produced by non-growing cells and was transformed to acetoin once the citrate was exhausted. Citrate was metabolized more rapidly than sugar by non-growing cells; the reverse was true of growing cells. Citrate metabolism by Lact. plantarum 1919 and Lact. casei ATCC 393 increased six- and 22-fold, respectively, when the cells were pre-grown on galactose plus citrate than when pre-grown on galactose only. This was probably due to induction of citrate lyase by growth on citrate plus sugar. These results imply that lactobacilli, if present in large enough numbers, can metabolize citrate in ripening cheese in the absence of an energy source.     

益生乳酸菌Lactobacillus casei Zhang和Lactobacillus plantarum P8对全价饲料pH及微生物类群变化的研究

研究了益生乳酸菌干酪乳杆菌Zhang(Lactobacillus casei Zhang)和植物乳杆菌P8(Lactobacillus planta-rum P8)对全价饲料pH及微生物类群变化的影响。分别将L.casei Zhang、L.plantarum P8单一菌种及复合菌种(11)以6.30 lg cfu/g的接种总量发酵全价饲料,测定25℃10 d发酵期间全价饲料pH和微生物类群的变化,应用选择培养基测定发酵饲料中的乳酸菌及杂菌(酵母菌、霉菌、大肠菌群、芽胞杆菌和梭状芽胞杆菌)的动态变化,应用RT-PCR技术测定试验组中的L.casei Zhang和L.plantarum P8的动态变化。结果显示,试验组pH下降显著,发酵10 d时,L.casei Zhang、L.plantarum P8单一菌种和复合菌种发酵饲料的pH分别为4.23、4.24和4.22,显著低于对照组(P0.05);L.casei Zhang、L.plantarum P8单一菌种和复合菌种发酵饲料中的L.casei Zhang、L.plantarum P8活菌数分别为8.91、8.89、6.58和8.69 lg cfu/g。发酵期间,试验组中酵母菌、霉菌、大肠菌群、芽胞杆菌及梭状芽胞杆菌活菌数显著低于对照组(P0.05),其中L.plantarum P8单一菌种发酵和复合菌种发酵对杂菌抑制效果显著优于L.casei Zhang单一菌种发酵(P0.05)。结果表明,全价饲料经L.casei Zhang、L.plantarum P8发酵可以显著降低其pH,抑制其中杂菌的生长,同时L.casei Zhang、L.plantarum P8在饲料中具有良好的稳定性。     

A microbiological study of Hussuwa: a traditional Sudanese fermented food from germinated Sorghum bicolor c.v. feterita

Hussuwa is a traditional Sudanese fermented food. Hussuwa made from Sorghum bicolor variety feterita exists in northern, central and eastern Sudan. The microbiological study indicated that the fermentation was primarily a lactic acid fermentation. The changes in microbial population, acetic acid bacteria, lactic acid bacteria and yeasts during all stages of hussuwa preparation and ripening were studied. The identification of fermented hussuwa microorganisms revealed that the main microorganisms were Lactobacillus saccharolyticum, Gluconobacter oxydans, Acetobacter xylinum and Saccharomyces cerevisiae. The metabolic products were studied in all stages of preparation and the period of ripening of hussuwa. The values of pH decreased as fermentation proceeded, and titratable acidity and volatile fatty acids increased.     

Changes in the Lactobacillus community during Ricotta forte cheese natural fermentation

The loss of microbial biodiversity due to the increase in large-scale industrial processes led to the study of the natural microflora present in a typical little known dairy product. The community of lactobacilli was studied in order to understand the natural fermentation of Ricotta forte cheese. The combined use of RAPD analysis, 16S rDNA sequencing and physiological tests allowed 33 different strains belonging to 10 species of Lactobacillus to be characterized. RAPD analysis revealed the heterogeneity of both the Lact. kefiri and Lact. paracasei species. The sequence analysis of the large 16S/23S rRNA spacer region enabled Lact. plantarum to be distinguished from Lact. paraplantarum, two closely related species belonging to the Lact. plantarum group. The recovery of strains endowed with interesting physiological characteristics, such as strong stress resistance, could improve technological and/or organoleptic characteristics of Ricotta forte cheese and other fermented foods.     

Effect of process parameters on the production and drying of Leuconostoc mesenteroides cultures

Leuconostoc mesenteroides BLAC was grown on MRS broth or on a carrot juice medium, and the effects of sugar concentration, type of pH control, aeration and fermentor size on viable counts were examined. The effect on viability of the type of centrifuge used to concentrate the bacterial culture was also examined. When the MRS broth had the traditional 110 mM glucose, pH control did not increase the final population. However, using a zone pH control mode, increasing the glucose content of MRS both from 110 to 220 mM almost doubled the population. In MRS broth, the amount of acetic acid produced was the same for all treatments, and was proportional to the amount of citrate consumed. There was a significantly lower cell yield in the carrot juice medium when the pH was not regulated. In the carrot juice medium, pH had a more pronounced effect on the final population level than did aeration, even though the quantity of viable cells was greater when the culture was aerated. In MRS broth, glucose was completely consumed during fermentation, but this was not the case in carrot juice medium. Aeration resulted in increased acetic acid content of the fermented medium. Viable counts were not affected by scaling the volume of the fermentation from 2 to 15 l ,or by the type of centrifuge used to concentrate the cells. Cells were concentrated by a factor of 10, but in both centrifuge types, viable counts showed only an eightfold average increase. However, freeze-dried powders obtained from the continuous pilot-plant-centrifuged cultures had, on the average, 33% lower populations than those obtained from the laboratory unit. Journal of Industrial Microbiology & Biotechnology (2002) 28, 291–296 DOI: 10.1038/sj/jim/7000245 Received 09 July 2001/ Accepted in revised form 25 January 2002     

Identification of prebiotic fructooligosaccharide metabolism in Lactobacillus plantarum WCFS1 through microarrays

Short-chain fructooligosaccharides (scFOS) and other prebiotics are used to selectively stimulate the growth and activity of lactobacilli and bifidobacteria in the colon. However, there is little information on the mechanisms whereby prebiotics exert their specific effects upon such microorganisms. To study the genomic basis of scFOS metabolism in Lactobacillus plantarum WCFS1, two-color microarrays were used to screen for differentially expressed genes when grown on scFOS compared to glucose (control). A significant up-regulation (8- to 60-fold) was observed with a set of only five genes located in a single locus and predicted to encode a sucrose phosphoenolpyruvate transport system (PTS), a beta-fructofuranosidase, a fructokinase, an alpha-glucosidase, and a sucrose operon repressor. Several other genes were slightly overexpressed, including pyruvate dehydrogenase. For the latter, no detectable activity in L. plantarum under various growth conditions has been previously reported. A mannose-PTS likely to encode glucose uptake was 50-fold down-regulated as well as, to a lower extent, other PTSs. Chemical analysis of the different moieties of scFOS that were depleted in the growth medium revealed that the trisaccharide 1-kestose present in scFOS was preferentially utilized, in comparison with the tetrasaccharide nystose and the pentasaccharide fructofuranosylnystose. The main end products of scFOS fermentation were lactate and acetate. This is the first example in lactobacilli of the association of a sucrose PTS and a beta-fructofuranosidase that could be used for scFOS degradation.     

Monitoring the bacterial community during fermentation of sunki, an unsalted, fermented vegetable traditional to the Kiso area of Japan

Aims:  To investigate the microbial community in sunki , an indigenous, unsalted, fermented vegetable, made from the leaves of red beet.
Methods and Results:  Fermenting samples were collected at 1- to 2-day intervals from four houses and investigated by culture-dependent and culture-independent techniques. PCR-Denaturing-Gradient-Gel-Electrophoresis profiles indicated that the bacterial community was stable and Lactobacillus delbrueckii , Lact. fermentum and Lact. plantarum were dominant during the fermentation. This result agreed well with that obtained by the culturing technique. Moulds, yeasts or bacteria other than lactic acid bacteria (LAB) were not detected.
Conclusions:  The bacterial community was stable throughout the fermentation, and Lact. delbrueckii , Lact. fermentum and Lact. plantarum were dominant. The acidic pH and lactic acid produced by LAB probably preserve the sunki from spoilage.
Significance and Impact of the Study:  This is the first report on the use of both culture-dependent and culture-independent techniques to study the bacterial community in sunki . A combination of culture-dependent and culture-independent techniques is necessary for the analysis of complex microbial communities.     

Production of probiotic cabbage juice by lactic acid bacteria

Research was undertaken to determine the suitability of cabbage as a raw material for production of probiotic cabbage juice by lactic acid bacteria (Lactobacillus plantarum C3, Lactobacillus casei A4, and Lactobacillus delbrueckii D7). Cabbage juice was inoculated with a 24-h-old lactic culture and incubated at 30 degrees C. Changes in pH, acidity, sugar content, and viable cell counts during fermentation under controlled conditions were monitored. L. casei, L. delbrueckii, and L. plantarum grew well on cabbage juice and reached nearly 10x10(8) CFU/mL after 48 h of fermentation at 30 degrees C. L. casei, however, produced a smaller amount of titratable acidity expressed as lactic acid than L. delbrueckii or L. plantarum. After 4 weeks of cold storage at 4 degrees C, the viable cell counts of L. plantarum and L. delbrueckii were still 4.1x10(7) and 4.5x10(5) mL(-1), respectively. L. casei did not survive the low pH and high acidity conditions in fermented cabbage juice and lost cell viability completely after 2 weeks of cold storage at 4 degrees C. Fermented cabbage juice could serve as a healthy beverage for vegetarians and lactose-allergic consumers.     

Development of food grade media for the preparation of Lactobacillus plantarum starter culture

Based on MRS medium, two types of food grade (FG) culture media (FG medium I and FG medium II) for the preparation of a concentrated starter culture of Lactobacillus plantarum NRIC 0380 to manufacture a new type of instant Chinese noodle, the fermented instant Chinese noodle, were developed using FG materials. FG medium I, which is for normal static culture, contains table sugar (sucrose), Yeast peptone standard type F, Sunsoft Q-17S (emulsifier), sodium acetate, trisodium citrate and MnSO(4).4-5H(2)O. FG medium II was designed to be used for the pH-controlled jar fermentor culture conditions. Therefore, sodium acetate and trisodium citrate as a buffer to prevent acidification of medium were omitted from FG medium I. When L. plantarum NRIC 0380 was cultured under the pH-controlled jar fermentor culture conditions, the kinetics of growth, sugar consumption and lactic acid production in FG medium II were quite similar to those observed in the Difco Lactobacilli MRS Broth. Furthermore, growths of many lactobacilli strains isolated from various fermented foods in FG medium I were also quite similar to those observed in MRS medium. Therefore, simple and practical FG media for the culture of lactobacilli were successfully established.     

The effect of acetic acid bacteria upon the growth and metabolism of yeasts during the fermentation of grape juice

The influence of species of Acetobacter and Gluconobacter upon growth of the wine yeasts Saccharomyces cerevisiae, Kloeckera apiculata and Candida stellata was examined during mixed culture in grape juice. Acetobacter pasteurianus, A. aceti and Gluconobacter oxydans grew in conjunction with yeasts during juice fermentation. As determined by viable counts, yeast growth was only slightly impaired by the presence of bacteria. However, as judged by the concentrations of glucose, fructose, ethanol, glycerol, acetaldehyde, ethyl acetate, iso -amyl alcohol and organic acids in the fermented juice, acetic acid bacteria significantly influenced the alcoholic fermentation by yeasts.     

Anaerobic l-lactate degradation by Lactobacillus plantarum

Abstract Lactobacillus plantarum strains used as silage inoculants were investigated for their ability to metabolize lactic acid anaerobically after prolonged incubation (7–30 days) when glucose was absent from the medium. When citrate was present in the medium together with glucose during the initial fermentation, the lactic acid produced was degraded. Citrate was concomitantly degraded, resulting in accumulation of formic, acetic and succinic acids along with CO₂. The anaerobic degradation was confirmed by the use of l ¹⁴C(U) labelled lactate. The existence of pyruvate formate lyase in L. plantarum was indicated by using ¹⁴C-labelled pyruvate and HPLC identification of end-products. The 1-¹⁴C-carboxylic acid group of pyruvate was converted to formic acid, and the 3-¹⁴C was found in acetic acid. The key enzyme(s) in this metabolic pathway appears to require anaerobic conditions and induction by citrate.     

A pH Control System Based on Malate Decarboxylation for the Cultivation of Lactic Acid Bacteria

Most species of lactic acid bacteria decarboxylate l-malate to lactate and CO(2) if an energy source such as glucose is present. A proton is taken up in the reaction, which prevents pH decreases in the growth medium caused by lactic acid production from glucose fermentation. MRS broth (pH 7.0) (Difco Laboratories) containing 10 mM glucose and various concentrations of l-malate (0, 25, 50, 75, and 100 mM) was used to cultivate Lactobacillus plantarum. After 72 h at 37 degrees C, all malate was decarboxylated and all glucose was fermented, with resultant final pH values of 4.5, 6.3, 6.9, 7.3, and 7.5, respectively. When d-malate (which cannot be decarboxylated) was substituted for l-malate, the final pH values were 4.5, 5.2, 5.6, 5.8, and 5.9. By varying the ratios of glucose to l-malate in the growth medium, it was possible to obtain pH values which were lower, the same, or higher than the initial pH values. In contrast, buffers such as phosphate only retard decreases in pH. l-Malate, when compared with K(2)PO(4) on an equal molar basis, provided greater resistance to decreases in pH. Higher specific growth rates were observed for L. plantarum and Leuconostoc mesenteroides when l-malate rather than K(2)PO(4) was incorporated into the growth medium.     

Simultaneous and sequential fermentations with yeast and lactic acid bacteria in apple juice

A complex substrate, reconstituted concentrated apple juice, was used for testing the principal processes during yeast and malolactic bacteria fermentations. Interactions between microorganisms were studied based on two controlled inoculation procedures, and at different fermentation temperatures. Temperature had a more important effect on yeast growth than the presence of malolactic bacteria in the medium. Acceleration of the death phase of the bacterial population was detected at increased temperatures. In all cases, malic acid degradation was affected by the fermentation temperature. When experiments were carried out with simultaneous inoculation, acidification of the medium took place at both temperatures tested (15°C and 22°C), that was not observed when the malolactic bacteria were inoculated after completion of alcoholic fermentation by yeasts. Received 4 August 1998/ Accepted in revised form 9 December 1998