Effects of lactobacilli on yeast-catalyzed ethanol fermentations.

Normal-gravity (22 to 24 degrees Plato) wheat mashes were inoculated with five industrially important strains of lactobacilli at approximately 10(5), approximately 10(6), approximately 10(7), approximately 10(8), and approximately 10(9) CFU/ml in order to study the effects of the lactobacilli on yeast growth and ethanol productivity. Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus #3, Lactobacillus rhamnosus, and Lactobacillus fermentum were used. Controls with yeast cells but no bacterial inoculation and additional treatments with bacteria alone inoculated at approximately 10(7) CFU/ml of mash were included. Decreased ethanol yields were due to the diversion of carbohydrates for bacterial growth and the production of lactic acid. As higher numbers of the bacteria were produced (depending on the strain), 1 to 1.5% (wt/vol) lactic acid resulted in the case of homofermentative organisms. L. fermentum, a heterofermentative organism, produced only 0.5% (wt/vol) lactic acid. When L. plantarum, L. rhamnosus, and L. fermentum were inoculated at approximately 10(6) CFU/ml, an approximately 2% decrease in the final ethanol concentration was observed. Smaller initial numbers (only 10(5) CFU/ml) of L. paracasei or Lactobacillus #3 were sufficient to cause more than 2% decreases in the final ethanol concentrations measured compared to the control. Such effects after an inoculation of only 10(5) CFU/ml may have been due to the higher tolerance to ethanol of the latter two bacteria, to the more rapid adaptation (shorter lag phase) of these two industrial organisms to fermentation conditions, and/or to their more rapid growth and metabolism. When up to 10(9) CFU of bacteria/ml was present in mash, approximately 3.8 to 7.6% reductions in ethanol concentration occurred depending on the strain. Production of lactic acid and a suspected competition with yeast cells for essential growth factors in the fermenting medium were the major reasons for reductions in yeast growth and final ethanol yield when lactic acid bacteria were present.     

Pressure measurement to evaluate ethanol or lactic acid production during glucose fermentation by yeast or heterofermentative bacteria in pure and mixed culture

A rapid and simple technique to follow CO2 release during fermentation of glucose by heterofermentative bacteria or yeasts was used in order to evaluate ethanol and lactate production in pure and mixed cultures of yeast and bacteria. In pure cultures, good correlations were found between gas pressure variations (deltaP) and ethanol or lactate production by yeasts or heterofermentative bacteria, and ratios between deltaP and ethanol or lactate produced could be established. In mixed cultures, ratios between maximal deltaP and total amount of glucose consumed were determined. It was thus possible to evaluate the amount of glucose that was consumed by each strain and then deduce the bacterial lactate production. Good results were obtained for mixed cultures of yeast and homofermentative bacteria. This technique may be useful to evaluate the activity of strains in mixed cultures of yeast and lactic acid bacteria.     

Distribution of the phosphoenolpyruvate:glucose phosphotransferase system in fermentative bacteria.

A number of selected fermentative bacteria were surveyed for the presence of the phosphoenolpyruvate:glucose phosphotransferase system, with particular attention to those organisms which ferment glucose by pathways other than the Embden-Meyerhof-Parnas pathway. The phosphoenolpyruvate:glusoe phosphotransferase system was found in all homofermentative lactic acid bacteria tested that ferment glucose via the Embden-Meyerhof-Parnas pathway, but in none of a group of heterofermentative species of Lactobacillus or Leuconostoc, which ferment glucose via the phosphoketolase pathway. A phosphoenolpyruvate:glucose phosphotransferase system was also absent in Zymomonas mobilis, which ferments glucose via an anaerobic Entner-Doudoroff pathway. It thus appears that the phosphotransferase mode of glucose transport is limited to bacteria with the Embden-Meyerhof-Parnas mode of glucose fermentation.     

Population Dynamics and Metabolite Target Analysis of Lactic Acid Bacteria during Laboratory Fermentations of Wheat and Spelt Sourdoughs

Four laboratory sourdough fermentations, initiated with wheat or spelt flour and without the addition of a starter culture, were prepared over a period of 10 days with daily back-slopping. Samples taken at all refreshment steps were used for determination of the present microbiota. Furthermore, an extensive metabolite target analysis of more than 100 different compounds was performed through a combination of various chromatographic methods including liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. The establishment of a stable microbial ecosystem occurred through a three-phase evolution within a week, as revealed by both microbiological and metabolite analyses. Strains of Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus rossiae, Lactobacillus brevis, and Lactobacillus paraplantarum were dominating some of the sourdough ecosystems. Although the heterofermentative L. fermentum was dominating one of the wheat sourdoughs, all other sourdoughs were dominated by a combination of obligate and facultative heterofermentative taxa. Strains of homofermentative species were not retrieved in the stable sourdough ecosystems. Concentrations of sugar and amino acid metabolites hardly changed during the last days of fermentation. Besides lactic acid, ethanol, and mannitol, the production of succinic acid, erythritol, and various amino acid metabolites, such as phenyllactic acid, hydroxyphenyllactic acid, and indolelactic acid, was shown during fermentation. Physiologically, they contributed to the equilibration of the redox balance. The biphasic approach of the present study allowed us to map some of the interactions taking place during sourdough fermentation and helped us to understand the fine-tuned metabolism of lactic acid bacteria, which allows them to dominate a food ecosystem.     

Ethanol addition enhances acid treatment to eliminate Lactobacillus fermentum from the fermentation process for fuel ethanol production

Fermentation is one of the most critical steps of the fuel ethanol production and it is directly influenced by the fermentation system, selected yeast, and bacterial contamination, especially from the genus Lactobacillus. To control the contamination, the industry applies antibiotics and biocides; however, these substances can result in an increased cost and environmental problems. The use of the acid treatment of cells (water‐diluted sulphuric acid, adjusted to pH 2·0–2·5) between the fermentation cycles is not always effective to combat the bacterial contamination. In this context, this study aimed to evaluate the effect of ethanol addition to the acid treatment to control the bacterial growth in a fed‐batch system with cell recycling, using the industrial yeast strain Saccharomyces cerevisiae PE–2. When only the acid treatment was used, the population of Lactobacillus fermentum had a 3‐log reduction at the end of the sixth fermentation cycle; however, when 5% of ethanol was added to the acid solution, the viability of the bacterium was completely lost even after the first round of cell treatment. The acid treatment +5% ethanol was able to kill L. fermentum cells without affecting the ethanol yield and with a low residual sugar concentration in the fermented must.

Significance and Impact of the Study

In Brazilian ethanol‐producing industry, water‐diluted sulphuric acid is used to treat the cell mass at low pH (2·0) between the fermentative cycles. This procedure reduces the number of Lactobacillus fermentum from 10⁷ to 10⁴ CFU per ml. However, the addition of 5% ethanol to the acid treatment causes the complete loss of bacterial cell viability in fed‐batch fermentation with six cell recycles. The ethanol yield and yeast cell viability are not affected. These data indicate the feasibility of adding ethanol to the acid solution replacing the antibiotic use, offering a low cost and a low amount of residue in the biomass.     

A survey of lactic acid bacteria in Italian silage

G razia , L. & S uzzi , G. 1984. A survey of lactic acid bacteria in Italian silage. Journal of Applied Bacteriology 56 , 373–379.
Lactic acid bacteria, isolated from Italian ensiled products, were represented by strains of the genera Lactobacillus and Leuconostoc . The predominant strains were heterofermentative lactobacilli, with Lactobacillus buchneri being the most frequent. Among homofermentative lactic acid bacteria, strains of Lact. plantarum and Lact. casei were recovered. Almost all strains utilized malic acid and showed good acid-tolerance, but only some of them were able to metabolize malic acid at extremely low pH; these were five homofermentative lactobacilli (4 Lact. plantarum and 1 Lacr. casei var. casei ) and two heterofermentative lactobacilli ( Lact. cellobiosus and Lactobacillus sp.).     

Interaction between lactic acid bacteria and yeasts in sour-dough using a rheofermentometer

Rheofermentometer assays were used to characterize the leavening of sour-doughs produced using species of lactic acid bacteria (LAB) and yeasts, alone or in combination. Saccharomyces cerevisiae 141 produced the most CO₂ and ethanol whereas S. exiguus M14 and Lactobacillus brevis subsp. lindneri CB1 contributed poorly to leavening and gave sour-doughs without porosity. In comparison with that seen in sour-dough produced with yeast alone, yeast fermentation with heterofermentative LAB present was faster whereas that with homofermentative LAB (L. plantarum DC400, L. farciminis CF3) present was slower and produced more CO₂. Combining L. brevis subsp. lindneri CB1 with S. cerevisiae 141 decreased bacterial cell numbers and souring activity. However, addition of fructose to the sour-dough overcame these problems as well as activating S. cerevisiae 141.The authors are with the Institute of Dairy Microbiology, Faculty of Agriculture, University of Perugia, S. Costanzo, 06126 Perugia, Italy     

Regulation of Lactobacillus plantarum contamination on the carbohydrate and energy related metabolisms of Saccharomyces cerevisiae during bioethanol fermentation

During the industrial bioethanol fermentation, Saccharomyces cerevisiae cells are often stressed by bacterial contaminants, especially lactic acid bacteria. Generally, lactic acid bacteria contamination can inhibit S. cerevisiae cell growth through secreting lactic acid and competing with yeast cells for micronutrients and living space. However, whether are there still any other influences of lactic acid bacteria on yeast or not? In this study, Lactobacillus plantarum ATCC 8014 was co-cultivated with S. cerevisiae S288c to mimic the L. plantarum contamination in industrial bioethanol fermentation. The contaminative L. plantarum-associated expression changes of genes involved in carbohydrate and energy related metabolisms in S. cerevisiae cells were determined by quantitative real-time polymerase chain reaction to evaluate the influence of L. plantarum on carbon source utilization and energy related metabolism in yeast cells during bioethanol fermentation. Contaminative L. plantarum influenced the expression of most of genes which are responsible for encoding key enzymes involved in glucose related metabolisms in S. cerevisiae. Specific for, contaminated L. plantarum inhibited EMP pathway but promoted TCA cycle, glyoxylate cycle, HMP, glycerol synthesis pathway, and redox pathway in S. cerevisiae cells. In the presence of L. plantarum, the carbon flux in S. cerevisiae cells was redistributed from fermentation to respiratory and more reducing power was produced to deal with the excess NADH. Moreover, L. plantarum contamination might confer higher ethanol tolerance to yeast cells through promoting accumulation of glycerol. These results also highlighted our knowledge about relationship between contaminative lactic acid bacteria and S. cerevisiae during bioethanol fermentation.     

The biodiversity of lactic acid bacteria in Greek traditional wheat sourdoughs is reflected in both composition and metabolite formation

Lactic acid bacteria (LAB) were isolated from Greek traditional wheat sourdoughs manufactured without the addition of baker's yeast. Application of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell protein, randomly amplified polymorphic DNA-PCR, DNA-DNA hybridization, and 16S ribosomal DNA sequence analysis, in combination with physiological traits such as fructose fermentation and mannitol production, allowed us to classify the isolated bacteria into the species Lactobacillus sanfranciscensis, Lactobacillus brevis, Lactobacillus paralimentarius, and Weissella cibaria. This consortium seems to be unique for the Greek traditional wheat sourdoughs studied. Strains of the species W. cibaria have not been isolated from sourdoughs previously. No Lactobacillus pontis or Lactobacillus panis strains were found. An L. brevis-like isolate (ACA-DC 3411 t1) could not be identified properly and might be a new sourdough LAB species. In addition, fermentation capabilities associated with the LAB detected have been studied. During laboratory fermentations, all heterofermentative sourdough LAB strains produced lactic acid, acetic acid, and ethanol. Mannitol was produced from fructose that served as an additional electron acceptor. In addition to glucose, almost all of the LAB isolates fermented maltose, while fructose as the sole carbohydrate source was fermented by all sourdough LAB tested except L. sanfranciscensis. Two of the L. paralimentarius isolates tested did not ferment maltose; all strains were homofermentative. In the presence of both maltose and fructose in the medium, induction of hexokinase activity occurred in all sourdough LAB species mentioned above, explaining why no glucose accumulation was found extracellularly. No maltose phosphorylase activity was found either. These data produced a variable fermentation coefficient and a unique sourdough metabolite composition.     

Use of sulfite and hydrogen peroxide to control bacterial contamination in ethanol fermentation.

Lactic acid bacteria isolated from an industrial-scale ethanol fermentation process were used to evaluate sulfite as a bacterial-contamination control agent in a cell-recycled continuous ethanol fermentation process. The viabilities of bacteria were decreased by sulfite at concentrations of 100 to 400 mg liter-1, while sulfite at the same concentrations did not change the viability of the Saccharomyces cerevisiae strain used in this process. Sulfite was effective only in the presence of oxygen. Bacteria showed differences in their susceptibilities to sulfite. Facultatively heterofermentative Lactobacillus casei 4-3 was more susceptible than was obligatory heterofermentative Lactobacillus fermentum 7-1. The former showed higher enzyme activities involved in the production and consumption of hydrogen peroxide than did the latter. The viability of L. fermentum 7-1 could be selectively controlled by hydrogen peroxide at concentrations of 1 to 10 mM. Based on these findings, it is hypothesized that the sulfur trioxide radical anions formed by peroxidase in the presence of hydrogen peroxide are responsible for the control of contaminating bacteria. Sulfite did not kill the yeast strain, which has catalase to degrade hydrogen peroxide. A cell-recycled continuous ethanol fermentation process was run successfully with sulfite treatments.     

Population dynamics and metabolite target analysis of lactic acid bacteria during laboratory fermentations of wheat and spelt sourdoughs

Four laboratory sourdough fermentations, initiated with wheat or spelt flour and without the addition of a starter culture, were prepared over a period of 10 days with daily back-slopping. Samples taken at all refreshment steps were used for determination of the present microbiota. Furthermore, an extensive metabolite target analysis of more than 100 different compounds was performed through a combination of various chromatographic methods including liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. The establishment of a stable microbial ecosystem occurred through a three-phase evolution within a week, as revealed by both microbiological and metabolite analyses. Strains of Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus rossiae, Lactobacillus brevis, and Lactobacillus paraplantarum were dominating some of the sourdough ecosystems. Although the heterofermentative L. fermentum was dominating one of the wheat sourdoughs, all other sourdoughs were dominated by a combination of obligate and facultative heterofermentative taxa. Strains of homofermentative species were not retrieved in the stable sourdough ecosystems. Concentrations of sugar and amino acid metabolites hardly changed during the last days of fermentation. Besides lactic acid, ethanol, and mannitol, the production of succinic acid, erythritol, and various amino acid metabolites, such as phenyllactic acid, hydroxyphenyllactic acid, and indolelactic acid, was shown during fermentation. Physiologically, they contributed to the equilibration of the redox balance. The biphasic approach of the present study allowed us to map some of the interactions taking place during sourdough fermentation and helped us to understand the fine-tuned metabolism of lactic acid bacteria, which allows them to dominate a food ecosystem.     

Use of Gas-Liquid Chromatography to Determine the End Products of Growth of Lactic Acid Bacteria

A simple gas-liquid chromatographic procedure for analyzing ethanol, acetic acid, acetoin, and racemic and meso-2,3-butylene glycol in broth media is described. Overnight broth cultures were filtered or centrifuged, and the filtrate or supernatant was treated with formic acid to aid separation of volatile fatty acids. Samples were then directly analyzed by gas-liquid chromatography on a 20% Tween 80-Chromosorb W-AW column and propionic acid as an internal standard. A complete analysis took ca. 8 min. The method can be used to distinguish homofermentative from heterofermentative lactic acid bacteria based on the level of ethanol produced and citrate-utilizing from non-citrate-utilizing lactic acid bacteria based on the levels of acetic acid produced. The method also has potential in distinguishing other bacterial fermentations. Of the 13 species of lactic acid bacteria tested, Streptococcus lactis subsp. diacetylactis was the major producer of 2,3-butylene glycol (total range, 0.3 to 3.5 mM), and, except for strain DRC1, both the racemic and meso isomers were produced in approximately equal amounts.     

Contaminant occurrence, identification and control in a pilot-scale corn fiber to ethanol conversion process

While interest in bioethanol production from lignocellulosic feedstocks is increasing, there is still relatively little pilot-plant data and operating experience available for this emerging industry. A series of batch and continuous fermentation runs were performed in a pilot-plant, some lasting up to six weeks, in which corn fiber-derived sugars were fermented to ethanol using glucose-fermenting and recombinant glucose/xylose-fermenting yeasts. However, contamination by Lactobacillus bacteria was a common occurrence during these runs. These contaminating microorganisms were found to readily consume arabinose, a sugar not utilized by the yeast, producing acetic and lactic acids that had a detrimental effect on fermentation performance. The infections were ultimately controlled with the antibiotic virginiamycin, but routine use of antibiotics is cost prohibitive. The severity of the problem encountered during this work is probably due to use of a highly contaminated feedstock. Lignocellulosic conversion facilities will not employ aseptic designs. Instead, techniques similar to those employed in the corn-based fuel ethanol industry to control infections will be used. Effective control may also be possible by using fermentative microorganisms that consume all biomass-derived sugars.     

Acetic Acid Increases Stability of Silage under Aerobic Conditions

The effects of various compounds on the aerobic stability of silages were evaluated. It has been observed that inoculation of whole-crop maize with homofermentative lactic acid bacteria leads to silages which have low stability against aerobic deterioration, while inoculation with heterofermentative lactic acid bacteria, such as Lactobacillus brevis or Lactobacillus buchneri, increases stability. Acetic acid has been proven to be the sole substance responsible for the increased aerobic stability, and this acid acts as an inhibitor of spoilage organisms. Therefore, stability increases exponentially with acetic acid concentration. Only butyric acid has a similar effect. Other compounds, like lactic acid, 1,2-propanediol, and 1-propanol, have been shown to have no effect, while fructose and mannitol reduce stability.     

A differential medium for the enumeration of homofermentative and heterofermentative lactic Acid bacteria

A medium was developed for the differential enumeration of homofermentative and heterofermentative lactic acid bacteria. Essential components of the medium included fructose (14 mM), KH(2)PO(4) (18 mM), bromcresol green (as a pH indicator), and other nutrients to support growth. In agar medium, homofermentative colonies were blue to green, while heterofermentative colonies remained white. A total of 21 Lactobacillus, Pediococcus, Leuconostoc, and Streptococcus species were correctly classified with the medium.     

Evolution of the lactic acid bacterial community during malt whisky fermentation: a polyphasic study.

The development of the lactic acid bacterial community in a commercial malt whisky fermentation occurred in three broad phases. Initially, bacteria were inhibited by strong yeast growth. Fluorescence microscopy and environmental scanning electron microscopy revealed, in this early stage, both cocci and rods that were at least partly derived from the wort and yeast but also stemmed from the distillery plant. Denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA genes and sequence analysis revealed cocci related to Streptococcus thermophilus or Saccharococcus thermophilus, Lactobacillus brevis, and Lactobacillus fermentum. The middle phase began 35 to 40 h after yeast inoculation and was characterized by exponential growth of lactobacilli and residual yeast metabolism. Lactobacillus casei or Lactobacillus paracasei, L. fermentum, and Lactobacillus ferintoshensis were detected in samples of fermenting wort examined by DGGE during this stage. Bacterial growth was accompanied by the accumulation of acetic and lactic acids and the metabolism of residual maltooligosaccharides. By 70 h, two new PCR bands were detected on DGGE gels, and the associated bacteria were largely responsible for the final phase of the fermentation. The bacteria were phylogenetically related to Lactobacillus acidophilus and Lactobacillus delbrueckii, and strains similar to the former had previously been recovered from malt whisky fermentations in Japan. These were probably obligately homofermentative bacteria, required malt wort for growth, and could not be cultured on normal laboratory media, such as MRS. Their metabolism during the last 20 to 30 h of fermentation was associated with yeast death and autolysis and further accumulation of lactate but no additional acetate.     

Bacterial community associated with ensilage process of wilted guinea grass

Aims: To determine the effects of wilting, storage period and bacterial inoculant on the bacterial community and ensiling fermentation of guinea grass silage. Methods and Results: Fermentation products, colony counts and denaturing gradient gel electrophoresis (DGGE) profiles were determined. There was more lactic acid than acetic acid in all silages, but the lactic acid to acetic acid ratio decreased with storage time. This shift from lactic to acetic acid was not prevented even with a combination of wilting and bacterial inoculant. The DGGE analyses suggest that facultatively heterofermentative lactic acid bacteria (Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus pentosus) were involved in the shift to acetic acid fermentation. Conclusions: Lactic acid can dominate the fermentation in tropical grass silage with sufficient wilting prior to ensiling. Prolonged storage may lead to high levels of acetic acid without distinctive changes in the bacterial community. Significance and Impact of the Study: The bacterial community looks stable compared to fermentation products over the course of long storage periods in tropical grass silage. Acetic acid fermentation in tropical grass silage can be a result of the changes in bacterial metabolism rather than community structure.     

Microbial Community Dynamics during Production of the Mexican Fermented Maize Dough Pozol

The dynamics of the microbial community responsible for the traditional fermentation of maize in the production of Mexican pozol was investigated by using a polyphasic approach combining (i) microbial enumerations with culture media, (ii) denaturing gradient gel electrophoresis (DGGE) fingerprinting of total community DNA with bacterial and eukaryotic primers and sequencing of partial 16S ribosomal DNA (rDNA) genes, (iii) quantification of rRNAs from dominant microbial taxa by using phylogenetic oligonucleotide probes, and (iv) analysis of sugars and fermentation products. A Streptococcus species dominated the fermentation and accounted for between 25 and 75% of the total flora throughout the process. Results also showed that the initial epiphytic aerobic microflora was replaced in the first 2 days by heterofermentative lactic acid bacteria (LAB), including a close relative of Lactobacillus fermentum, producing lactic acid and ethanol; this heterolactic flora was then progressively replaced by homofermentative LAB (mainly close relatives of L. plantarum, L. casei, and L. delbrueckii) which continued acidification of the maize dough. At the same time, a very diverse community of yeasts and fungi developed, mainly at the periphery of the dough. The analysis of the DGGE patterns obtained with bacterial and eukaryotic primers targeting the 16S and 18S rDNA genes clearly demonstrated that there was a major shift in the community structure after 24 h and that high biodiversity—according to the Shannon-Weaver index—was maintained throughout the process. These results proved that a relatively high number of species, at least six to eight, are needed to perform this traditional lactic acid fermentation. The presence of Bifidobacterium, Enterococcus, and enterobacteria suggests a fecal origin of some important pozol microorganisms. Overall, the results obtained with different culture-dependent or -independent techniques clearly confirmed the importance of developing a polyphasic approach to study the ecology of fermented foods.     

The bacterial community and metabolome dynamics and their interactions modulate fermentation process of whole crop corn silage prepared with or without inoculants

Multi-omics approach was adopted to investigate the modulation of bacterial microbiota and metabolome as well as their interactions in whole crop corn ensiling systems by inoculating homofermentative Lactobacillus plantarum or heterofermentative Lactobacillus buchneri. Inoculations of the two different inoculants resulted in substantial differences in microbial community and metabolic composition as well as their dynamics in ensiled corn. Inoculants also altered the correlations of microbiota in different manners, and various keystone species were identified in corn silages with different treatments. Many metabolites with biofunctional activities like bacteriostatic, antioxidant, central nervous system inhibitory and anti-inflammatory were found in the present silage. A constitutive difference in microbiota dynamics was found for several pathways, which were upregulated by specific taxa in middle stage of fermentation, and widespread associations between metabolites with biofunctions and the species of lactic acid bacteria dominated in silage were observed. Multiple microbial and metabolic structures and dynamics were correlated and affected the fermentation process of the corn ensiling systems. Results of the current study improve our understanding of the complicated biological process underlying silage fermentation and provide a framework to re-evaluate silages with biofunctions, which may contribute to target-based regulation methods to produce functional silage for animal production.     

Use of mixed cultures for the fermentation of cereal-based substrates with potential probiotic properties

The production of a new cereal-based probiotic foods with suitable aroma, flavor and pH using mixed culture fermentation has been investigated. This required the selection of suitable types of cereal grains and probiotic microorganisms. In a medium of 5% (w/v) malt suspension the effects of yeast presence on the fermentation of a lactic acid bacterium (LAB), Lactobacillus reuteri, was studied. With different inoculum ratios between the yeast and the LAB, the characteristics of the fermentation broth including pH and the contents of free amino nitrogen (FAN), reducing sugar, lactic acid and ethanol were investigated. It was found that LAB growth was enhanced by the introduction of the yeast. In mixed culture broth pH was lowered and the production of lactic acid and ethanol were increased in comparison against pure LAB culture.