Ability of thermophilic lactic acid bacteria to produce aroma compounds from amino acids

Although a large number of key odorants of Swiss-type cheese result from amino acid catabolism, the amino acid catabolic pathways in the bacteria present in these cheeses are not well known. In this study, we compared the in vitro abilities of Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, and Streptococcus thermophilus to produce aroma compounds from three amino acids, leucine, phenylalanine, and methionine, under mid-pH conditions of cheese ripening (pH 5.5), and we investigated the catabolic pathways used by these bacteria. In the three lactic acid bacterial species, amino acid catabolism was initiated by a transamination step, which requires the presence of an alpha-keto acid such as alpha-ketoglutarate (alpha-KG) as the amino group acceptor, and produced alpha-keto acids. Only S. thermophilus exhibited glutamate dehydrogenase activity, which produces alpha-KG from glutamate, and consequently only S. thermophilus was capable of catabolizing amino acids in the reaction medium without alpha-KG addition. In the presence of alpha-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than S. thermophilus, which mainly produced alpha-keto acids and a small amount of hydroxy acids and acids. L. helveticus mainly produced acids from phenylalanine and leucine, while L. delbrueckii subsp. lactis produced larger amounts of alcohols and/or aldehydes. Formation of aldehydes, alcohols, and acids from alpha-keto acids by L. delbrueckii subsp. lactis mainly results from the action of an alpha-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols. In contrast, the enzyme involved in the alpha-keto acid conversion to acids in L. helveticus and S. thermophilus is an alpha-keto acid dehydrogenase that produces acyl coenzymes A.     

Hydrolysis of sequenced beta-casein peptides provides new insight into peptidase activity from thermophilic lactic acid bacteria and highlights intrinsic resistance of phosphopeptides

The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified beta-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the beta-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp. lactis ITG LL14 and ITG LL51, L. delbrueckii subsp. bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the beta-casein hydrolysate as the substrate at pH 5.5 and 24 degrees C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry. Regardless of strain, L. helveticus was the most efficient in hydrolyzing beta-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the beta-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species.     

Co-culturing of Lactobacillus paracasei subsp. paracasei with a Lactobacillus delbrueckii subsp. delbrueckii Mutant to Make High Cell Density for Increased Lactate Productivity from Cassava Bagasse Hydrolysate

To increase the productivity of lactic acid, a co-culture of lactobacilli was made by mixing 1:1 ratio of Lactobacillus paracasei subsp. paracasei and a fast growing L. delbrueckii subsp. delbrueckii mutant. The culture was embedded on to polyurethane foam (PUF) cubes as a biofilm and used for fermentation. In order to prevent the cell leakage, the PUF cubes were further entrapped in calcium cross-linked alginate. The maximum lactic acid production using a high cell density free culture was >38 g l(-1) from ~40 g l(-1) of reducing sugar within 12 h of fermentation. Using PUF biofilms, the same yield of lactic acid attained after 24 h. When the cubes were further coated with alginate it took 36 h for the maximum yield. Even though, the productivity is slightly lesser with the alginate coating, cell leakage was decreased and cubes were reused without much decrease in production in repeated batches. Using a conventional control inoculum (3%, w/v), it took 120 h to yield same amount of lactic acid.     

Growth Response and Modifications of Organic Nitrogen Compounds in Pure and Mixed Cultures of Lactic Acid Bacteria from Wine

The interactions between the proteolytic X₂L strain of Oenococcus oeni and the non-proteolytic 12p strain of Pediococcus pentosaceus were assayed. The characteristics of cell growth, protein degradation, and amino acid production of both strains were determined in pure and mixed cultures. O. oeni showed poor cell growth and greater ability in the release of amino acids to the extracellular medium, whereas P. pentosaceus showed a higher yield in cell production with a decrease in the amino acid concentration in the medium. P. pentosaceus especially consumed essential amino acids for growth, and O. oeni released several of the essential amino acids important for growth of P. pentosaceus. In the mixed culture, mutualism was observed. The higher activity of the proteolytic system of O. oeni in mixed culture produced an increase in cell growth and in the amount of essential amino acids released. These findings provide new knowledge about the metabolic interactions between lactic acid bacteria isolated from wine when proteins are degraded in mixed bacterial populations.     

Lactic acid production by Lactobacillus delbreuckii in a hollow fiber fermenter

Summary Lactic acid was produced by viable Lactobacillus delbreuckii NRRL-B445 in a hollow fiber fermenter. Final cell densities in the fluid surrounding the fibers in the fermenter were apparently as high as 480 gms DW/L, and volumetric productivities reached 100 gms/L-hr lactic acid. The observed cell yields were appreciably lower than batch cell yields.     

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.     

乳杆菌代谢产物对化脓性链球菌的抑制作用

【目的】分析乳杆菌代谢产物对化脓性链球菌的抑制作用。【方法】基于双层平板打孔法,通过测量抑菌圈大小来检测乳杆菌代谢产物对化脓性链球菌的抑菌作用;然后分别采用高效液相色谱法和4-氨酰安替比林法检测乳杆菌代谢产物中的有机酸和H2O2含量;最后,检测乳酸、乙酸和H2O2对化脓性链球菌的最小抑菌浓度(MIC)、最小杀菌浓度(MBC)。【结果】对化脓性链球菌的抑菌效果以植物乳杆菌KLDS1.0667最好,副干酪乳杆菌KLDS1.0342-1次之,瑞士乳杆菌KLDS1.0203抑菌效果最差;乳酸和乙酸产量KLDS1.0667>KLDS1.0342-1>KLDS1.0203;H2O2产量KLDS1.0203>KLDS1.0667>KLDS1.0342-1。在抑菌试验中,乳杆菌的发酵上清液经去除H2O2处理后抑菌圈直径都减小;将发酵上清液的p H调至7.0后均检测不到抑菌圈。结果表明,乳杆菌代谢产物中对化脓性链球菌起抑制作用的主要物质为有机酸和H2O2,其中乳酸是产生抑菌作用的最主要物质。乳酸、乙酸和H2O2对化脓性链球菌的最小抑菌浓度(MIC)分别为1.28、0.64和0.008 g/L,对化脓性链球菌的最小杀菌浓度(MBC)分别为5.12、2.56和0.032 g/L。【结论】乳杆菌可利用其代谢产物对化脓性链球菌产生抑制作用,主要抑菌物质为有机酸和H2O2。     

Sugar Utilization and Acid Production by Free and Entrapped Cells of Streptococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactococcus lactis subsp. lactis in a Whey Permeate Medium

Cells of Streptococcus salivarius subsp. thermophilus and Lactococcus lactis subsp. lactis entrapped in k-carrageenan-locust bean gum gel performed similarly to free cells in the conversion of lactose to lactic acid. Bead diameter influenced the fermentation rate. Cells entrapped in smaller beads (0.5 to 1.0 mm) showed higher release rates, higher lactose, glucose, and formic acid utilization, higher galactose accumulation, and higher lactic acid production than did cells entrapped in larger beads (1.0 to 2.0 mm). Values for smaller beads were comparable with those for free cells. Immobilization affected the fermentation rate of lactic acid bacteria, especially Lactobacillus delbrueckii subsp. bulgaricus. Entrapped cells of L. delbrueckii subsp. bulgaricus demonstrated a lower lactic acid production than did free cells in batch fermentation. The kinetics of the production of formic and pyruvic acids by L. lactis subsp. lactis and S. salivarius subsp. thermophilus are presented.     

Pure Culture Fermentation of Brined Cucumbers

The relative abilities of Pediococcus cerevisiae, Lactobacillus plantarum, L. brevis, and several other species of lactic acid bacteria to grow and produce acid in brined cucumbers were evaluated in pure culture fermentations. Such fermentations were made possibly by the use of two techniques, gamma radiation (0.83 to 1.00 Mrad) and hot-water blanching (66 to 80 C for 5 min), designed first to rid the cucumbers of naturally occurring, interfering, and competitive microbial groups prior to brining, followed by inoculation with the desired lactic acid bacteria. Of the nine species tested, strains of the three common to cucumber fermentations, P. cerevisiae, L. plantarum, and L. brevis, grew to the highest populations, and produced the highest levels of brine acidity and the lowest pH values in fermentations at 5.4 to 5.6% NaCl by weight; also, their sequence of active development in fermentations, with the use of a three-species mixture for inoculation, was in the species order just named. This sequence of occurrence was similar to that estimated by others for natural fermentations. The rates of growth and acid production in fermentations with a mixture of P. cerevisiae, L. plantarum, and L. brevis increased as the incubation temperature was increased from 21 to 27 to 32 C; however, the maximal populations and acidities attained were essentially the same for fermentations at each temperature. Further, these same three species were found to be the most salt tolerant of those tested; their upper limit for appreciable growth and measurable acid production was about 8% salt, whereas thermophilic species such as L. thermophilus, L. lactis, L. helveticus, L. fermenti, and L. delbrueckii exhibited a much lower salt tolerance, ranging from about 2.5 to 4.0%. However, certain strains of L. delbrueckii grew very rapidly in cucumbers brined at 2.5 to 3.0% salt, and produced sufficient acid in about 30 hr at 48 C to reduce the brine pH from above 7.0 to below 4.0. An inexpensive, pure culture fermentor which was suitable for gamma radiation, resistant to salt and acid, and which permitted repeated aseptic sampling of the fermenting brine, is illustrated and the specifications are given.     

Utilization of amino acids to enhance glutathione production in Saccharomyces cerevisiae

The effects of amino acids on glutathione (GSH) production by Saccharomyces cerevisiae T65 were investigated in this paper. Cysteine was the most important amino acids, which increased intracellular GSH content greatly but inhibited cell growth at the same time. The suitable amino acids addition strategy was two-step addition: in the first step, cysteine was added after two hours culture to 2 mM and then, the three amino acids (glutamic acid, glycine, and serine) were added after seven hours culture. The optimum concentration of those three key amino acids (10 mM glutamic acid, 10 mM glycine, and 10 mM serine) was obtained by orthogonal matrix method. With the optimum amino acids addition strategy a 1.63% intracellular GSH content was obtained in shake flask culture. Intracellular GSH content was 55.2% higher than the experiments without three amino acids addition. The cell biomass and GSH yield were 9.4 g/L and 153.2 mg/L, respectively. Using this amino acids addition strategy in the fed-batch culture of S. cerevisiae T65, GSH content, the biomass, and GSH yield reached 1.41%, 133 g/L, and 1875 mg/L, respectively, after 44 hours fermentation. GSH yield was about 2.67 times as that of amino acids free.     

Production of D-lactic acid from defatted rice bran by simultaneous saccharification and fermentation

Production of d-lactic acid from rice bran, one of the most abundant agricultural by-products in Japan, is studied. Lactobacillus delbrueckii subsp. delbrueckii IFO 3202 and defatted rice bran powder after squeezing rice oil were used for the production. Since the rice bran contains polysaccharides as starch and cellulose, we coupled saccharification with amylase and cellulase to lactic acid fermentation. The indigenous bacteria in the rice bran produced racemic lactic acid in the saccharification at pH 6.0-6.8. Thus the pH was controlled at 5.0 to suppress the growth of the indigenous bacteria. L. delbrueckii IFO 3202 produced 28 kgm(-3) lactic acid from 100 kgm(-3) rice bran after 36 h at 37 degrees C. The yield based on the amount of sugars soluble after 36-h hydrolysis of the bran by amylase and cellulase (36 kgm(-3) from 100 kgm(-3) of the bran) was 78%. The optical purity of produced d-lactic acid was 95% e.e.     

Differentiation of Lactobacillus helveticus,Lactobacillus delbrueckii subsp bulgaricus,subsp lactis and subsp delbrueckii using physiological and genetic tools and reclassification of some strains from the ATCC collection

Several physiological tests of glucose metabolism and genetic tools including species specific probes and 16S rDNA sequences were used to identify strains of L. helveticus and the group of L. delbrueckii with its three subspecies lactis, bulgaricus, and delbrueckii. These species are important for the milk industry as fermenting lactic acid bacteria. The identification procedure was applied to the different strains of these species available from the ATCC collection and allowed to reclassify part of them.     

Plasmids, Lactic Acid Production, and N-Acetyl-d-Glucosamine Fermentation in Lactobacillus helveticus subsp. jugurti

Two lactobacillus strains, Lactobacillus helveticus subsp. jugurti S13-8 and L. helveticus subsp. jugurti S36-2, were examined for the presence of plasmids. Plasmids of 16.45, 13.03, and 11.83 kilobases (kb) were found in the first, low lactic acid-producing strain; their function is not presently known. A single plasmid species of 13.17 kb was found in the second, high lactic acid-producing strain. This plasmid was found to be associated with lactic acid production and N-acetyl-d-glucosamine fermentation.     

Genetic Characterization of a Cell Envelope-Associated Proteinase from Lactobacillus helveticus CNRZ32

A cell envelope-associated proteinase gene (prtH) was identified in Lactobacillus helveticus CNRZ32. The prtH gene encodes a protein of 1,849 amino acids and with a predicted molecular mass of 204 kDa. The deduced amino acid sequence of the prtH product has significant identity (45%) to that of the lactococcal PrtP proteinases. Southern blot analysis indicates that prtH is not broadly distributed within L. helveticus. A prtH deletion mutant of CNRZ32 was constructed to evaluate the physiological role of PrtH. PrtH is not required for rapid growth or fast acid production in milk by CNRZ32. Cell surface proteinase activity and specificity were determined by hydrolysis of alpha(s1)-casein fragment 1-23 by whole cells. A comparison of CNRZ32 and its prtH deletion mutant indicates that CNRZ32 has at least two cell surface proteinases that differ in substrate specificity.     

Production of lactic acid and fructose from media with cane sugar using mutant of Lactobacillus delbrueckii NCIM 2365

AIMS: To examine the potential of Lactobacillus delbrueckii mutant, Uc-3 to produce lactic acid and fructose from sucrose-based media. METHODS AND RESULTS: The mutant of L. delbrueckii NCIM 2365 was cultivated in shake flask containing hydrolysed cane sugar (sucrose)-based medium. The lactic acid yield and volumetric productivity with hydrolysed cane concentration up to 200 g l(-1) were in the range of 92-97% of the theoretical value and between 2.7 and 3.8 g l(-1) h(-1), respectively. The fructose fraction of the syrup produced was more than 95% when the total initial sugar concentration in the medium was higher (150-200 g l(-1)). There are no unwanted byproducts detected in the fermentation broth. CONCLUSIONS: We demonstrated that L. delbrueckii mutant Uc-3 was able to utilize glucose preferentially to produce lactic acid and fructose from hydrolysed cane sugar in batch fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings will be useful in the production of lactic acid and high fructose syrups using media with high concentrations of sucrose-based raw materials. This approach can lead to modification of the traditional fermentation processes to obtain value-added byproducts, attaining better process economics.     

Extractive bioconversion for lactic acid production using solid sorbent and organic solvent

Extraction of lactic acid from fermentation broths have been studied. The extractant selected for this research was paraffin oil which, in addition to having satisfactory physical properties, is non-toxic and does not affect the fermentability of the broth. Several amine compounds and a polymeric resin (Bonopore) have been tested to increase the extraction efficiency of the paraffin oil. Addition of these compounds gave higher K _D values. However, all of the amine compounds tested are highly toxic to the free cells of L. delbrueckii (NRRL B-445). Bonopore, the polymer giving good absorption pattern and no deleterious effects was tested with paraffin oil in repeated batch culture of L. delbrueckii.     

Optimization of nutritional supplements for enhanced lactic acid production utilizing sugar refinery by-products

The present study investigated the synergistic effect of nutritional supplements (amino acid and Tween 80) on lactic acid production by Lactobacillus delbruckii utilizing a sugar refinery by product (cane molasses) in a submerged fermentation process. Initially, the effect of individual factors on lactic acid yield was studied by supplementing amino acids and their combinations, Tween 80 and cane molasses at varying concentrations in production medium. A combination of l-phenylalanine and l-lysine gave a maximum lactic acid yield of 47.89?±?0.1 g/L on a dry cell weight basis at individual factor level. Similarly, maximum lactic acid yield was obtained by supplementing the production medium with 40.0 g/L and 2.0 g/L Tween 80 and cane molasses, respectively, at individual factor level. In order to further improve the lactic acid yield, nutritional supplements were optimized by central composite rotatable design (CCRD) using Minitab 15 software. Shake flask cultivation under optimized conditions, i.e., cane molasses (32.40 g/L), Tween 80 (2.0 g/L) and l-phenylalanine and l-lysine (34.0 mg/L) gave a lactic acid yield of 64.86?±?0.2 g/L, corresponding to 95.0 % of the predicted yield of 67.78?±?0.3 g/L. Batch cultivation performed in 7.5 L bioreactor (working volume: 3.0 L) under optimized conditions gave maximum lactic acid yield and productivity of 79.12?±?0.2 g/L and 3.40 g/L·h, which is higher than previous studies with reduced fermentation time. Screening of lactic acid producing bacteria and characterization of lactic acid was also done.     

Lacticin, a bacteriocin produced by Lactobacillus delbrueckii subsp. lactis

Twenty-one strains of Lactobacillus delbrueckii and L. helveticus were tested for bacteriocin production against each other. Lactobacillus delbrueckii subsp. lactis JCM 1106 and 1107 produced an inhibitory agent active against L. delbrueckii subsp. bulgaricus JCM 1002 and NIAI yB-62, L. delbrueckii subsp. lactis JCM 1248 and L. delbrueckii subsp. delbrueckii JCM 1012. Lactobacillus delbrueckii subsp. lactis JCM 1248 inhibited only the growth of L. delbrueckii subsp. bulgaricus NIAI yB-62. These agents were sensitive to proteolytic enzymes and heating (at 60°C for 10min). These agents were considered to be bacteriocins and designated lacticin A and B.     

Use of whey lactose from dairy industry for economical kefiran production by Lactobacillus kefiranofaciens in mixed cultures with yeasts

To evaluate the feasibility of producing kefiran industrially, whey lactose, a by-product from dairy industry, was used as a low cost carbon source. Because the accumulation of lactic acid as a by-product of Lactobacillus kefiranofaciens inhibited cell growth and kefiran production, the kefir grain derived and non-derived yeasts were screened for their abilities to reduce lactic acid and promote kefiran production in a mixed culture. Six species of yeasts were examined: Torulaspora delbrueckii IFO 1626; Saccharomyces cerevisiae IFO 0216; Debaryomyces hansenii TISTR 5155; Saccharomyces exiguus TISTR 5081; Zygosaccharomyces rouxii TISTR 5044; and Saccharomyces carlsbergensis TISTR 5018. The mixed culture of L. kefiranofaciens with S. cerevisiae IFO 0216 enhanced the kefiran production best from 568 mg/L in the pure culture up to 807 and 938 mg/L in the mixed cultures under anaerobic and microaerobic conditions, respectively. The optimal conditions for kefiran production by the mixed culture were: whey lactose 4%; yeast extract 4%; initial pH of 5.5; and initial amounts of L. kefiranofaciens and S. cerevisiae IFO 0216 of 2.1×10(7) and 4.0×10(6)CFU/mL, respectively. Scaling up the mixed culture in a 2L bioreactor with dissolved oxygen control at 5% and pH control at 5.5 gave the maximum kefiran production of 2,580 mg/L in batch culture and 3,250 mg/L in fed-batch culture.