Survival of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in the Terminal Ileum of Fistulated Göttingen Minipigs

The ability of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus administered in yogurt to survive the passage through the upper gastrointestinal tract was investigated with Göttingen minipigs that were fitted with ileum T-cannulas. After ingestion of yogurt containing viable microorganisms, ileostomy samples were collected nearly every hour beginning 3 h after food uptake. Living L. delbrueckii subsp. bulgaricus and S. thermophilus were detected in the magnitude of 10⁶ to 10⁷ per gram of intestinal contents (wet weight) in all animals under investigation. A calculation of the minimum amount of surviving bacteria that had been administered is presented. Total DNA extracted from ileostomy samples was subjected to PCR, which was species specific for L. delbrueckii and S. thermophilus and subspecies specific for L. delbrueckii subsp. bulgaricus. All three bacterial groups could be detected by PCR after yogurt uptake but not after uptake of a semisynthetic diet. One pig apparently had developed an endogenous L. delbrueckii flora. When heat-treated yogurt was administered, L. delbrueckii was detected in all animals. S. thermophilus or L. delbrueckii subsp. bulgaricus was not detected, indicating that heat-inactivated cells and their DNAs had already been digested and their own L. delbrueckii flora had been stimulated for growth.     

Rapid detection of Escherichia coli in waters using fluorescent in situ hybridization,direct viable counting and solid phase cytometry

Aims: We developed an improved Fluorescent In Situ Hybridization FISH‐based method to detect viable Escherichia coli cells by solid phase cytometry (SPC), and results were compared to those obtained by the standard culture method. Methods and Results: The method includes a direct viable count (DVC) assay, multi‐probes labelled and unlabelled (helpers) to detect specifically viable E. coli cells and to enhance SPC cell counts. We demonstrate that helpers increase the fluorescence intensity of hybridized E. coli cells as detected by SPC and assess the high specificity of the DVC–FISH procedure on a large panel of cultured strains. Application to seawater, freshwater and wastewater samples showed a good correlation between SPC cells counts and standard plate counts. Conclusion: The high specificity of the procedure was demonstrated as well as its accuracy for detecting and counting viable E. coli cells in environmental samples. Significance and Impact of the Study: The developed approach may be used to monitor faecal contamination sources and to investigate the occurrence of viable E. coli in natural environments.     

Lowering of ochratoxin A level in milk by yoghurt bacteria and bifidobacteria

A decrease in the content of ochratoxin A (OA) was observed in milk samples fermented by yoghurt bacteria and bifidobacteria. OA was added to the milk before fermentation at a rate of 0.05–1.5 mg/L. No residues of OA were found in samples containing 0.05 and 0.1 mg/L of OA, fermented byS. salivarius subsp.thermophilus, L. delbrueckii subsp.bulgaricus andB. bifidum. Yoghurt bacteria (S. salivarius subsp.thermophilus andL. delbrueckii subsp.bulgaricus) were the most effective since no residues were detected even in fermented samples containing originally 0.5 mg/L OA.     

Pediocin production in milk by Pediococcus acidilactici in co-culture with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus

The production of pediocin in milk by Pediococcus acidilactici was evaluated in co-culture with the dairy fermentation cultures Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. The cultures were tested singly and in different combinations in milk (0 or 2% fat content) during incubation at 40°C for up to 10 h. Cell-free milk samples taken every 60 min were tested for bacteriocin activity against Listeria monocytogenes. Pediocin activity was not detectable when P. acidilactici was inoculated into milk as a monoculture. When P. acidilactici was grown in combination with the yogurt starter cultures S. thermophilus and Lb. delbrueckii ssp. bulgaricus, pediocin concentration reached 3,200–6,400 units ml⁻¹ after 8 h of incubation. The results showed that pediocin producing pediococci may be useful adjunct components in mixed cultures of S. thermophilus and Lb. delbrueckii ssp. bulgaricus to amplify the bioprotective properties of fermented dairy foods against Listeria contamination.     

Demonstration of viable but nonculturable Vibrio cholerae O1 in fresh water environment of India using ciprofloxacin DFA-DVC method

Aim: To demonstrate the presence of culturable and nonculturable viable pathogenic Vibrio cholerae O1 in fresh water environments of a cholera‐endemic region in India. Methods and Results: Conventional culture and ciprofloxacin DFA–DVC were utilized to investigate the existence of V. cholerae O1. We isolated pathogenic culturable V. cholerae O1 from water samples collected from cholera‐affected areas. No culturable V. cholerae O1 was isolated from water and plankton samples from natural fresh water bodies. Ciprofloxacin was used for DFA–DVC as V. cholerae O1 are 100% resistant to nalidixic acid in our region. The viable but nonculturable O1 cells were demonstrated in 2·21 and 40·69% samples from natural water bodies and cholera‐affected areas, respectively. Conclusion: Vibrio cholerae O1 VBNC could be demonstrated using modified DFA–DVC technique. Ciprofloxacin is preferable to nalidixic acid for DVC in view of existing high‐level resistance to nalidixic acid in cholera‐endemic areas. Significance and Impact of the study: We endorse that for public health surveillance, cholera outbreak investigation and disease control water samples in addition to culture should be tested for V. cholerae using DFA–DVC.     

Effect of oxygen on batch yogurt cultures

A yogurt culture (Streptococcus thermophilus 15HA + Lactobacillus delbrueckii subsp. bulgaricus 2-11) was studied in conditions of aerobic batch fermentation (10–40% dissolved oxygen in milk). The growth and acidification of S. thermophilus 15HA were stimulated at 20% oxygen concentration and the lactic acid process in a mixed culture was shortened by 1 h (2.5 h for the aerobic culture and 3.5 h for the anaerobic mixed culture). Streptococcus thermophilus 15HA oxygen tolerance was significantly impaired at oxygen concentrations in the milk above 30%. Though S. thermophilus 15HA was able to overcome to some extent the impact of high oxygen concentration (40%), the lactic acid produced was insufficient to coagulate the milk casein (4.0 g lactic acid l⁻¹ in the mixed culture and 3.8 g lactic acid l⁻¹ in the pure culture). A dramatic decrease in the viable cell count of L. delbrueckii subsp. bulgaricus 2-11 in the pure and mixed cultures was recorded at 30% dissolved oxygen. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative Sequence Analysis of Plasmids from Lactobacillus delbrueckii and Construction of a Shuttle Cloning Vector

While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with Lactobacillus delbrueckii, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of Lactobacillus delbrueckii subsp. bulgaricus was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic ori and encoded six open reading frames (ORFs), of which functions could be predicted for three—a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available L. delbrueckii plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for L. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from Streptococcus thermophilus. This observation and the ability of the replicon to function in S. thermophilus support the idea that the origin of plasmids in L. delbrueckii was likely from S. thermophilus. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from L. delbrueckii has previously been constructed, an Escherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A ori, the chloramphenicol resistance gene of pCI372, and the lacZ polylinker from pUC18. This cloning vector was successfully introduced into E. coli, L. delbrueckii subsp. bulgaricus, S. thermophilus, and Lactococcus lactis. This shuttle cloning vector provides a new tool for molecular analysis of Lactobacillus delbrueckii and other lactic acid bacteria.     

Comparison of Campylobacter jejuni isolates from human, food, veterinary and environmental sources in Iceland using PFGE, MLST and fla-SVR sequencing

Aims: To evaluate the probiotic properties of strains isolated from smoked salmon and previously identified as bacteriocin producers. Methods and Results: Strains Lactobacillus curvatus ET06, ET30 and ET31, Lactobacillus fermentum ET35, Lactobacillus delbrueckii ET32, Pediococcus acidilactici ET34 and Enterococcus faecium ET05, ET12 and ET88 survived conditions simulating the gastrointestinal tract (GIT) and produced bacteriocins active against several strains of Listeria monocytogenes, but presented very low activity against other lactic acid bacteria (LAB). Cell‐free supernatants containing bacteriocins, added to 3‐h‐old cultures of L. monocytogenes 603, suppressed growth over 12 h. Auto‐aggregation was strain‐specific, and values ranged from 7·2% for ET35 to 12·1% for ET05. Various degrees of co‐aggregation with L. monocytogenes 603, Lactobacillus sakei ATCC 15521 and Enterococcus faecalis ATCC 19443 were observed. Adherence of the bacteriocinogenic strains to Caco‐2 cells was within the range reported for Lactobacillus rhamnosus GG, a well‐known probiotic. The highest levels of hydrophobicity were recorded for Lact. curvatus (61·9–64·6%), Lact. fermentum (78·9%), Lact. delbrueckii (43·7%) and Ped. acidilactici (51·3%), which are higher than the one recorded for Lact. rhamnosus GG (53·3%). These strains were highly sensitive to several antibiotics and affected by several drugs from different generic groups in a strain‐dependent manner. Conclusions: Smoked salmon is a rich source of probiotic LAB. All strains survived conditions simulating the GIT and produced bacteriocins active against various pathogens. Adherence to Caco‐2 cells was within the range reported for Lact. rhamnosus GG, a well‐known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics. Significance and Impact of the Study: Smoked salmon contains a number of different probiotic LAB and could be marketed as having a potential beneficial effect.     

Effect of frozen storage and aging on the Kashkaval cheese starter culture

Summary The changes in the number of the starter microorganisms Lb. delbrueckii subsp. bulgaricus and Str. thermophiluswere followed in frozen-stored Kashkaval cheese made from cow’s milk. Kashkaval samples of various aging times were produced industrially, frozen at T=−16 °C and stored at T=−10 to −12 °C for 12 months. It was found that the number of Lb. delbrueckiisubsp. bulgaricus and Str. thermophilusdecreased considerably during frozen storage. The decrease was more substantial for Lb. delbrueckiisubsp. bulgaricus, which was evidence for its greater sensitivity to the impact of low temperatures. The aging time of Kashkaval did not influence the changes in the starter culture during frozen storage but is important for its amount in the product aged after defrosting. There was an increase in the Str. thermophilus: Lb. delbrueckiisubsp. bulgaricus ratio in samples with shorter aging time subjected to frozen storage and aged after defrosting. The changes in the starter culture in frozen stored Kashkaval cheese can be controlled by an appropriate combination of the two factors: aging time and period of frozen storage.     

Growth and Proteolytic Activity of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus in Reduced Sodium Kashkaval Cheese

Summary Reduced sodium Kashkaval cheese was produced from cow’s milk. Mixtures of NaCl, NaCl:KCl (1:1, 2:1) and NaCl:KH₂PO₄ (1:1, 2:1) were used for hot brining and salting of the cheddarized cheese curd. There were no significant differences (P < 0.05) in the count of Lb. delbrueckii ssp. bulgaricus after aging of Kashkaval samples. At the end of the ripening process the counts of Lb. delbrueckii ssp. bulgaricus reached 10⁶ c.f.u./g and the counts of Streptococcus thermophilus varied from 10⁴ to 10⁵ c.f.u./g. Proteolysis during ripening of reduced sodium Kashkaval cheese, initiated by the starter microorganisms Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, was studied through the changes in the levels of non-casein and non-protein nitrogen. It was observed that non-casein and non-protein nitrogen increased significantly (P < 0.05) during ripening. The amounts of non-casein and non-protein nitrogen accumulated in the studied Kashkaval samples were similar. That indicates that the partial replacement of NaCl with KCl or KH₂PO₄ does not cause significant changes in the course of proteolysis of Kashkaval cheese by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus.     

Genotyping of Streptococcus thermophilus strains isolated from traditional Egyptian dairy products by sequence analysis of the phosphoserine phosphatase (serB) gene with phenotypic characterizations of the strains

Aims: To develop a new, simplified genotyping method for examining the genetic diversity of Streptococcus thermophilus strains isolated from traditional Egyptian fermented dairy products and to characterize phenotypic traits of those strains related to their potential use in bioprocessing applications. Methods and Results: A novel, simplified approach was developed for genotyping Strep. thermophilus involving the analysis of nucleotide sequence variations within a housekeeping gene encoding the phosphoserine phosphatase (SerB). Using this method, it was possible to identify ten genotypes involving diverse serB alleles among 54 Strep. thermophilus isolates cultured from Egyptian dairy products. These isolates harboured five de novo serB alleles that have not been detected in other Strep. thermophilus strains, deposited in a multilocus sequence typing (MLST) database. To assess distinct genotypes of the organism with phenotypic traits relevant to their potential use in industry, Strep. thermophilus strains were all subjected to a series of phenotypic characterizations. The strains were found to exhibit phenotypic diversity in terms of their ability to ferment lactose and galactose, express urease activity, produce exopolysaccharides and develop acidity. Conclusions: The analysis of nucleotide sequence variations within the serB gene could serve as a suitable tool for probing diverse genotypes of Strep. thermophilus. Streptococcus thermophilus isolates associated with traditional Egyptian dairy products show high degree of genetic and phenotypic diversity. Significance and Impact of the Study: This study presents a novel, simplified procedure based on serB nucleotide sequencing for genotyping Strep. thermophilus. It also provides a pool of phenotypically diverse Strep. thermophilus cultures, from which certain strains could be selected for use in bioprocessing applications including the preparation of fermented dairy products.     

The application of viable count procedures for measuring viable cells in the various marine environments

Aims: To investigate whether the use of direct viable count (DVC), quantitative viable count (qDVC), colony‐forming units and the contribution of capsule‐bearing bacteria to the total number of bacteria and esterase‐active bacteria could be used to clearly differentiate viable cells in various trophic status of seawater. Methods and Results: Hundred and four marine isolates from various marine environments in Turkey (Western Black Sea, northern part of the Sea of Marmara, Northern Aegean Sea and eastern part of the Sea of Marmara) were screened. Seawater samples were taken from the surface (the upper 0–30 cm) and deeper layers (from 5 to 500 m) of the sea at different time periods between February 2002 and June 2007. For the assessment of cell elongation, minor modifications were made on DVC procedure in order to optimize the concentration of yeast extract and incubation time for enumeration of bacteria in response to nutrient addition. The best results were obtained when the yeast extract was used at a final concentration of 250 mg l^?1 (at 35°C 24 h incubation) for bacteria isolated from eutrophic areas and a final concentration of 50 mg l^?1 for those selected from oligotrophic areas. A positive correlation was found between the trophic level and the level of metabolically active bacteria. Among these methods, the bacterial number obtained by qDVC is higher than those gained by other methods. Conclusions: The results indicate that the qDVC procedure could easily differentiate between viable cells and dormant or dead cells. We suggest that this method may be applicable to detecting the level of metabolic potential of bacterial communities in marine environments. Significance and Impact of the Study: The study resulted in increased knowledge on the applicability of the qDVC method that arranges the substrate amount and incubation time as well as on the comparison of various viable bacteria count procedures related to trophic situation of seawater samples.     

Genetically Engineered Immunomodulatory Streptococcus thermophilus Strains Producing Antioxidant Enzymes Exhibit Enhanced Anti-Inflammatory Activities

The aims of this study were to develop strains of lactic acid bacteria (LAB) having both immunomodulatory and antioxidant properties and to evaluate their anti-inflammatory effects both in vitro, in different cellular models, and in vivo, in a mouse model of colitis. Different Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were cocultured with primary cultures of mononuclear cells. Analysis of the pro- and anti-inflammatory cytokines secreted by these cells after coincubation with candidate bacteria revealed that L. delbrueckii subsp. bulgaricus CRL 864 and S. thermophilus CRL 807 display the highest anti-inflammatory profiles in vitro. Moreover, these results were confirmed in vivo by the determination of the cytokine profiles in large intestine samples of mice fed with these strains. S. thermophilus CRL 807 was then transformed with two different plasmids harboring the genes encoding catalase (CAT) or superoxide dismutase (SOD) antioxidant enzymes, and the anti-inflammatory effects of recombinant streptococci were evaluated in a mouse model of colitis induced by trinitrobenzenesulfonic acid (TNBS). Our results showed a decrease in weight loss, lower liver microbial translocation, lower macroscopic and microscopic damage scores, and modulation of the cytokine production in the large intestines of mice treated with either CAT- or SOD-producing streptococci compared to those in mice treated with the wild-type strain or control mice without any treatment. Furthermore, the greatest anti-inflammatory activity was observed in mice receiving a mixture of both CAT- and SOD-producing streptococci. The addition of L. delbrueckii subsp. bulgaricus CRL 864 to this mixture did not improve their beneficial effects. These findings show that genetically engineering a candidate bacterium (e.g., S. thermophilus CRL 807) with intrinsic immunomodulatory properties by introducing a gene expressing an antioxidant enzyme enhances its anti-inflammatory activities.     

Nisin‐induced expression of pediocin in dairy lactic acid bacteria

Aims: To test whether a single vector, nisin‐controlled expression (NICE) system could be used to regulate expression of the pediocin operon in Streptococcus thermophilus, Lactococcus lactis subsp. lactis and Lactobacillus casei. Methods and Results: The intact pediocin operon was cloned immediately into pMSP3535 downstream of the nisA promoter (PnisA). The resulting vector, pRSNPed, was electrotransformed into Strep. thermophilus ST128, L. lactis subsp. lactis ML3 and Lact. casei C2. Presence of the intact vector was confirmed by PCR, resulting in the amplification of a 0·8‐kb DNA fragment, and inhibition zones were observed for all lactic acid bacteria (LAB) transformants following induction with 50 ng ml^?1 nisin, when Listeria monocytogenes Scott A was used as the target bacterium. Using L. monocytogenes NR30 as target, the L. lactis transformants produced hazy zones of inhibition, while the Lact. casei transformants produced clear zones of inhibition. Zones of inhibition were not observed when the Strep. thermophilus transformants were tested against NR30. Conclusions: The LAB hosts were able to produce enough pediocin to inhibit the growth of L. monocytogenes Scott A; the growth of L. monocytogenes NR30 was effectively inhibited only by the Lact. casei transformants. Significance and Impact of the Study: This is the first time that the NICE system has been used to express the intact pediocin operon in these LAB hosts. This system could allow for the in situ production of pediocin in fermented dairy foods supplemented with nisin to prevent listeria contamination.     

Dynamic metagenome-scale metabolic modeling of a yogurt bacterial community

Genome-scale metabolic models and flux balance analysis (FBA) have been extensively used for modeling and designing bacterial fermentation. However, FBA-based metabolic models that accurately simulate the dynamics of coculture are still rare, especially for lactic acid bacteria used in yogurt fermentation. To investigate metabolic interactions in yogurt starter culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, this study built a dynamic metagenome-scale metabolic model which integrated constrained proteome allocation. The accuracy of the model was evaluated by comparing predicted bacterial growth, consumption of lactose and production of lactic acid with reference experimental data. The model was then used to predict the impact of different initial bacterial inoculation ratios on acidification. The dynamic simulation demonstrated the mutual dependence of S. thermophilus and L. d. bulgaricus during the yogurt fermentation process. As the first dynamic metabolic model of the yogurt bacterial community, it provided a foundation for the computer-aided process design and control of the production of fermented dairy products.     

Assimilation of cholesterol by some cultures of lactic acid bacteria and bifidobacteria

Summary Cultures ofStr. thermophilus assimilated less cholesterol than those ofLactobacillus delbrueckii subsp.bulgaricus. A significant difference was found between strains ofL. delbrueckii subsp.bulgaricus — LB1 and LB2 and LB3 (p<0.001).Bif. bifidum actively assimilated cholesterol, but no significant difference was observed between their two strains (p>0.05). Cultures ofL. asidophilus assimilated significantly more cholesterol than those ofStr. thermophilus and a commercial yoghurt culture.     

Comparison of growth,acidification and productivity of pure and mixed cultures of Streptococcus salivarius subsp. thermophilus 404 and Lactobacillus delbrueckii subsp. bulgaricus 398

Pure and mixed controlled-pH batch cultures of Streptococcus salivarius subsp. thermophilus 404 and Lactobacillus delbrueckii subsp. bulgaricus 398 have been conducted. The characteristics of growth and acidification and the productivity of the cultures were compared. During the mixed cultures, the growth characteristics revealed a pronounced stimulation of S. thermophilus whereas L. bulgaricus metabolism was not significantly improved. The final total population was 1.4 to 4.9 higher than in pure cultures. The acidification characteristics were not enhanced by the mixed culture conditions. The productivity of mixed cultures was 1.7 to 2.4 times higher as compared to an equivalent mixing of pure cultures.Correspondence to: C. Béal     

Survival of Yogurt Bacteria in the Human Gut

Whether Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus can be recovered after passage through the human gut was tested by feeding 20 healthy volunteers commercial yogurt. Yogurt bacteria were found in human feces, suggesting that they can survive transit in the gastrointestinal tract.     

Lactobacillus delbrueckii subsp lactis (strain CIDCA 133) resists the antimicrobial activity triggered by molecules derived from enterocyte‐like Caco‐2 cells

Aims: The aim of the present study was to assess the ability of a potentially probiotic strain to resist, in vitro, the effect of intestinal antimicrobial molecules. Methods and results: Strain CIDCA 133 of Lactobacillus delbrueckii subsp lactis was studied. Lactobacillus delbrueckii subsp bulgaricus as well as other gram‐positive and gram‐negative bacteria were used for comparison purposes. The effect of different antimicrobial extracts was determined by diffusion assays, viable counts and growth kinetics. Human‐defensins (hβD1 and hβD2) were also included in the study. Two types of cellular fractions from Caco‐2 cells were tested: (i) cytosolic fractions, obtained by sonication of cultured human enterocytes and (ii) cationic fraction, obtained by batch extraction of the cytosolic fraction with a weak cation exchange resin. In addition, the effect of Caco‐2‐secreted factors was studied. Strain CIDCA 133 was neither inhibited by Caco‐2 secreted, cytosolic nor cationic fractions. Of note, human‐defensins were inactive against strain CIDCA 133. In contrast, a related lactobacilli: Lactobacilli delbrueckii subsp bulgaricus (strain CIDCA 331) and other species of gram‐positive or gram‐negative bacteria were strongly inhibited. Conclusions: Strain CIDCA 133 is able to survive and grow in the presence of enterocyte‐derived antimicrobial molecules. This ability is not a general property of lactobacilli. Significance and Impact of the Study: Results could provide a new insight into the mechanisms of the probiotic effect and encourage further studies on this field. Resistance to antimicrobial peptides can be relevant to understand the interaction of potentially probiotic strains with the host′s immune system. This ability can be also relevant as a selection criterion for new probiotic strains.     

Enhanced production of lactic acid by an adapted strain of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis>

Lactobacillus delbrueckii subsp. lactis strains were developed having increased activity, by gradually acclimatizing the bacteria to acidic conditions over repeated batch culture. Cells from one batch culture were used as the inoculum for the subsequent batch culture and thereby an adapted strain of Lactobacillus was obtained showing improved lactic acid productivity, cell growth and total glucose utilization. Furthermore, the acclimatized cells used significantly less nitrogen for a given level of lactic acid production, which is significant from an industrial point of view. The developed procedure decreases fermentation time and nutrient use, leading to reduced operation costs, while providing a lactic acid yield superior to previously reported methods.