Acetoin Fermentation by Citrate-Positive Lactococcus lactis subsp. lactis 3022 Grown Aerobically in the Presence of Hemin or Cu2+

Citr⁺Lactococcus lactis subsp. lactis 3022 produced more biomass and converted most of the glucose substrate to diacetyl and acetoin when grown aerobically with hemin and Cu²⁺. The activity of diacetyl synthase was greatly stimulated by the addition of hemin or Cu²⁺, and the activity of NAD-dependent diacetyl reductase was very high. Hemin did not affect the activities of NADH oxidase and lactate dehydrogenase. These results indicated that the pyruvate formed via glycolysis would be rapidly converted to diacetyl and that the diacetyl would then be converted to acetoin by the NAD-dependent diacetyl reductase to reoxidize NADH when the cells were grown aerobically with hemin or Cu²⁺. On the other hand, the Y_Glu value for the hemincontaining culture was lower than for the culture without hemin, because acetate production was repressed when an excess of glucose was present. However, in the presence of lipoic acid, an essential cofactor of the dihydrolipoamide acetyltransferase part of the pyruvate dehydrogenase complex, hemin or Cu²⁺ enhanced acetate production and then repressed diacetyl and acetoin production. The activity of diacetyl synthase was lowered by the addition of lipoic acid. These results indicate that hemin or Cu²⁺ stimulates acetyl coenzyme A (acetyl-CoA) formation from pyruvate and that lipoic acid inhibits the condensation of acetyl-CoA with hydroxyethylthiamine PP_i. In addition, it appears that acetyl-CoA not used for diacetyl synthesis is converted to acetate.     

Diacetyl production by immobilized citrate-positiveLactococcus lactis subsp.lactis 3022 in the fibrous Ca-alginate gel

Summary Diacetyl production by (Citr^*)Lactococcus lactis subsp.lactis 3022 was found to be an oxygen-dependent reaction. The diacetyl production by the cells immobilized in conventional Ca-alginate gel beads (Diameter: 3 mm) was lower than that of the cells immobilized in Ca-alginate gel fibers (Diameter: 0.2 mm), probably because oxygen transfer to the immobilized cells is better in gel fibers than in gel beads.     

Expression of citrate permease gene of plasmid pCM1 isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7 in Lactobacillus casei L-49-4

Recombinant vector pJLECit (8,232 bp) was constructed using citrate permease gene contained in the 3,919-bp fragment of plasmid pCM1 (8,280 bp) isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7, repA and ori from pLU1, and pMB1 ori and the erythromycin resistance gene from pJIR418. Lactobacillus casei L-49-4 (plasmid-free mutant of strain L-49) harboring the constructed pJLECit converted citrate into diacetyl/acetoin. Citrate uptake rate of resting cells was the highest at pH 5.5 and 10 mM citrate concentration. Diacetyl formation activity by the cell-free extracts of Lb. casei L-49-4 (pJLECit) grown in de Man–Rogosa–Sharpe (MRS) broth was higher than that of cells grown in MRS broth without citrate. On the other hand, diacetyl reductase activity of cells grown in MRS broth was lower than that of cells grown in MRS broth without citrate.     

Diacetyl production by co-immobilized citrate-positiveLactococcus lactis subsp.lactis 3022 and homogenized bovin liver in alginate fibers with double gel layers

Summary Diacetyl production by (Citr⁺)Lactococcus lactis subsp.lactis 3022 cells immobilized in Ca-alginate fine fibers with single layer in the presence of catalase was three times higher than that in the absence of catalase. A co-immobilized culture system of the lactic acid bacterial cells (outer) and the homogenized bovine liver (inner layer) in Ca-alginate fibers with double gel layers was developed. The culture system gave high diacetyl productivity (30 mg/l) for ten repeated batch cultures.     

Properties of 2,3-Butanediol Dehydrogenases from Lactococcus lactis subsp. lactis in Relation to Citrate Fermentation

Two 2,3-butanediol dehydrogenases (enzymes 1 and 2; molecular weight of each, 170,000) have been partially purified from Lactococcus lactis subsp. lactis (Streptococcus diacetylactis) D10 and shown to have reductase activity with either diacetyl or acetoin as the substrate. However, the reductase activity with 10 mM diacetyl was far greater for both enzymes (7.0- and 4.7-fold for enzymes 1 and 2, respectively) than with 10 mM acetoin as the substrate. In contrast, when acetoin and diacetyl were present together, acetoin was the preferred substrate for both enzymes, with enzyme 1 showing the more marked preference for acetoin. meso-2,3-Butanediol was the only isomeric product, with enzyme 1 independent of the substrate combinations. For enzyme 2, both the meso and optical isomers of 2,3-butanediol were formed with acetoin as the substrate, but only the optical isomers were produced with diacetyl as the substrate. With batch cultures of strain D10 at or near the point of citrate exhaustion, the main isomers of 2,3-butanediol present were the optical forms. If the pH was sufficiently high (>pH 5), acetoin reduction occurred over time and was followed by diacetyl reduction, and meso-2,3-butanediol became the predominant isomer. Interconversion of the optical isomers into the meso isomer did occur. The properties of 2,3-butanediol dehydrogenases are consistent with diacetyl and acetoin removal and the appearance of the isomers of 2,3-butanediol.     

Influence of citrate on the diacetyl and acetoin production by fully grown cells ofStreptococcus lactis subsp.diacetylactis

The effects of citrate on diacetyl and acetoin level by fully grown cells ofStreptococcus lactis subsp.diacetylactis CNRZ 124 were studied. In the absence of citrate, diacetyl synthase as well as acetolactate synthase and acetoin and diacetyl reductases exhibited a basal activity confirming their constitutive nature. However, when initial citrate concentration ranged from 8.8 to 59 mM, the enzyme levels increased in the same way, indicating no saturation rate of citrate metabolism. These results were reflected by a similar enhancement in acetoin and diacetyl production. When citrate was added in fed-batch conditions, its utilization by the fully grown cells led to a twofold increase in diacetyl yield over batch conditions.     

Characterization of the Heat Shock Response in Lactococcus lactis subsp. lactis

The heat shock response in Lactococcus lactis subsp. lactis was characterized with respect to synthesis of a unique set of proteins induced by thermal stress. A shift in temperature from 30 to 42°C was sufficient to arrest the growth of L. lactis subsp. lactis, but growth resumed after a shift back to 30°C. Heat shock at 50°C reduced the viable cell population by 10³; however, pretreatment of the cells at 42°C made them more thermoresistant to exposure at 50°C. The enhanced synthesis of approximately 13 proteins was observed in cells labeled with ³⁵S upon heat shock at 42°C. Of these heat shock-induced proteins, two appeared to be homologs of GroEL and DnaK, based on their molecular weights and reactivity with antiserum against the corresponding Escherichia coli proteins. Therefore, we conclude that L. lactis subsp. lactis displays a heat shock response similar to that observed in other mesophilic bacteria.     

Acetoin Fermentation by Citrate-Positive Lactococcus lactis subsp. lactis 3022 Grown Aerobically in the Presence of Hemin or Cu

CitrLactococcus lactis subsp. lactis 3022 produced more biomass and converted most of the glucose substrate to diacetyl and acetoin when grown aerobically with hemin and Cu. The activity of diacetyl synthase was greatly stimulated by the addition of hemin or Cu, and the activity of NAD-dependent diacetyl reductase was very high. Hemin did not affect the activities of NADH oxidase and lactate dehydrogenase. These results indicated that the pyruvate formed via glycolysis would be rapidly converted to diacetyl and that the diacetyl would then be converted to acetoin by the NAD-dependent diacetyl reductase to reoxidize NADH when the cells were grown aerobically with hemin or Cu. On the other hand, the Y(Glu) value for the hemincontaining culture was lower than for the culture without hemin, because acetate production was repressed when an excess of glucose was present. However, in the presence of lipoic acid, an essential cofactor of the dihydrolipoamide acetyltransferase part of the pyruvate dehydrogenase complex, hemin or Cu enhanced acetate production and then repressed diacetyl and acetoin production. The activity of diacetyl synthase was lowered by the addition of lipoic acid. These results indicate that hemin or Cu stimulates acetyl coenzyme A (acetyl-CoA) formation from pyruvate and that lipoic acid inhibits the condensation of acetyl-CoA with hydroxyethylthiamine PP(i). In addition, it appears that acetyl-CoA not used for diacetyl synthesis is converted to acetate.     

Starvation-Induced Stress Resistance in Lactococcus lactis subsp. lactis IL1403

Carbohydrate-starved cultures of Lactococcus lactis subsp. lactis IL1403 showed enhanced resistance to heat, ethanol, acid, osmotic, and oxidative stresses. This cross-protection seems to be established progressively during the transitional growth phase, with maximum resistance occurring when cells enter the stationary phase. Chloramphenicol or rifamycin treatment does not abolish the development of a tolerant cell state but, on the contrary, seems to provoke this response in L. lactis subsp. lactis.     

Insertion of Transposon Tn917 Derivatives into the Lactococcus lactis subsp. lactis Chromosome

Two transposition vectors, pTV32 and pLTV1, containing transposon Tn917 derivatives TV32 and LTV1, respectively, were introduced into Lactococcus lactis subsp. lactis MG1614. It was found that pTV32 and pLTV1 replicate and that TV32 and LTV1 transpose in this strain. A protocol for production of a collection of Tn917 insertions in L. lactis subsp. lactis was developed. The physical locations of TV32 on the chromosomal SmaI fragments of 62 independent transpositions were established by pulsed-field gel electrophoresis. These transpositions could be divided into at least 38 different groups that exhibited no Tn917-dominating hot spots on the L. lactis subsp. lactis chromosome. A total of 10 of the 62 transpositions resulted in strains that express β-galactosidase. This indicates that there was fusion of the promoterless lacZ of the Tn917 derivatives to a chromosomal promoter. Thus, the Tn917-derived transposons should be powerful genetic tools for studying L. lactis subsp. lactis.     

Genetic Evidence for Plasmid-Linked Lactose Metabolism in Streptococcus lactis subsp. diacetylactis

It has been previously observed that loss of plasmid pGK4101 occurred concomitantly with loss of lactose-fermenting ability in Streptococcus lactis subsp. diacetylactis 18-16. Transfer of this 41-megadalton plasmid to LM0230, a lactosenegative (Lac⁻) strain of S. lactis, required cell-to-cell contact and resulted in a conversion of LM0230 to the Lac⁺ phenotype. This confirms the linkage of lactose-fermenting ability to the 41-megadalton plasmid in S. lactis subsp. diacetylactis and, in addition, demonstrates transfer by a process resembling conjugation in the group N streptococci.     

Study of the Citrate Metabolism of Lactococcus lactis subsp. lactis Biovar Diacetylactis by Means of 13C Nuclear Magnetic Resonance

The metabolic fate of citrate and pyruvate in four strains of Lactococcus lactis subsp. lactis biovar diacetylactis has been studied by means of ¹³C nuclear magnetic resonance, using as a substrate either [3-¹³C]pyruvic acid or custom-synthesized citric acid that is ¹³C labeled either at carbons 2 and 4 or at carbon 3. The fermentations were carried out batchwise in modified M17 broth. For the actual conversions of the ¹³C-labeled substrates, cells at the end of their logarithmic growth phase were used to minimize the conversion to lactic acid. A mass balance of the main citric acid metabolites was obtained; the four strains produced from 50 to 70% (on a molar basis) lactic acid from either citrate or pyruvate. The remaining 50 to 30% was converted mainly to either α-acetolactic acid (for one strain) or acetoin (for the other three strains). One of the strains produced an exceptionally high concentration of the diacetyl precursor α-acetolactic acid. Another strain (SDC6) also produced α-acetolactic acid, but this was decarboxylated to acetoin at a high rate. The ¹³C nuclear magnetic resonance method confirmed that the biosynthesis of α-acetolactic acid occurs via condensation of pyruvate and “active” acetaldehyde. Diacetyl was not found as a direct metabolite of citrate or pyruvate metabolism.     

Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor

Summary The transfer of plasmids was studied in a stirred fermentor in the course of mixed batch cultures combining recombinant strains of Lactococcus lactis subsp. lactis (donor strains) with L. lactis subsp. lactis CNRZ 268M3 (recipient strain). Donor strains contained one or two of the following plasmids (coding for erythromycin or chloramphenicol resistance): pIL205 (self-transmissible), pIL252, pIL253 (non-transmissible but mobilizable by pIL205, respectively small and large copy number) and pE194 (inserted in the chromosome). Only self-transmissible plasmid pIL205 was transferred, with frequencies ranging from 10^–7 to 10^–8 after 12 h of fermentation. These frequencies were 60–400 times lower than in unstirred M17 broth and 100 000 times lower than on agar medium. In the latter case, non-transmissible plasmids pIL252 and pIL253 were mobilized by pIL205 with a frequency of about 10^–5–10^–6. Correspondence to: C.-Y. Boquien     

Characterization of Nitrite Degradation by Lactobacillus casei subsp. rhamnosus LCR 6013

Nitrites are potential carcinogens. Therefore, limiting nitrites in food is critically important for food safety. The nitrite degradation capacity of Lactobacillus casei subsp. rhamnosus LCR 6013 was investigated in pickle fermentation. After LCR 6013 fermentation for 120 h at 37°C, the nitrite concentration in the fermentation system was significantly lower than that in the control sample without the LCR 6013 strain. The effects of NaCl and Vc on nitrite degradation by LCR 6013 in the De Man, Rogosa and Sharpe (MRS) medium were also investigated. The highest nitrite degradations, 9.29 mg/L and 9.89 mg/L, were observed when NaCl and Vc concentrations were 0.75% and 0.02%, respectively in the MRS medium, which was significantly higher than the control group (p ≤ 0.01). Electron capture/gas chromatography and indophenol blue staining were used to study the nitrite degradation pathway of LCR 6013. The nitrite degradation products contained N₂O, but no NH₄ ⁺The LCR 6013 strain completely degraded all NaNO₂ (50.00 mg/L) after 16 h of fermentation. The enzyme activity of NiR in the periplasmic space was 2.5 times of that in the cytoplasm. Our results demonstrated that L. casei subsp. rhamnosus LCR 6013 can effectively degrade nitrites in both the pickle fermentation system and in MRS medium by NiR. Nitrites are degraded by the LCR 6013 strain, likely via the nitrate respiration pathway (NO₂ ⁻>NO⁻>N₂O⁻>N₂), rather than the aammonium formation pathway (dissimilatory nitrate reduction to ammonium, DNRA), because the degradation products contain N₂O, but not NH₄ ⁺.     

Diacetyl and α-Acetolactate Overproduction by Lactococcus lactis subsp. lactis Biovar Diacetylactis Mutants That Are Deficient in α-Acetolactate Decarboxylase and Have a Low Lactate Dehydrogenase Activity

Lactococcus lactis subsp. lactis biovar diacetylactis strains are utilized in several industrial processes for producing the flavoring compound diacetyl or its precursor α-acetolactate. Using random mutagenesis with nitrosoguanidine, we selected mutants that were deficient in α-acetolactate decarboxylase and had low lactate dehydrogenase activity. The mutants produced large amounts of α-acetolactate in anaerobic milk cultures but not in aerobic cultures, except when the medium was supplemented with catalase, yeast extract, or hemoglobin.     

PCR Amplification of the Gene acmA Differentiates Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris

The occurrence of the acmA gene, encoding the lactococcal N-acetylmuramidase in new lactococcal isolates from raw milk cheeses, has been determined. Isolates were genotypically identified to the subspecies level with a PCR technique. On the basis of PCR amplification of the acmA gene, the presence or absence of an additional amplicon of approximately 700 bp correlated with Lactococcus lactis subspecies. L. lactis subsp. lactis exhibits both the expected 1,131-bp product and the additional amplicon, whereas L. lactis subsp. cremoris exhibits a single 1,131-bp fragment.     

Adaptation and Response of Bifidobacterium animalis subsp. lactis to Bile: a Proteomic and Physiological Approach

Bile salts are natural detergents that facilitate the digestion and absorption of the hydrophobic components of the diet. However, their amphiphilic nature makes them very inhibitory for bacteria and strongly influences bacterial survival in the gastrointestinal tract. Adaptation to and tolerance of bile stress is therefore crucial for the persistence of bacteria in the human colonic niche. Bifidobacterium animalis subsp. lactis, a probiotic bacterium with documented health benefits, is applied largely in fermented dairy products. In this study, the effect of bile salts on proteomes of B. animalis subsp. lactis IPLA 4549 and its bile-resistant derivative B. animalis subsp. lactis 4549dOx was analyzed, leading to the identification of proteins which may represent the targets of bile salt response and adaptation in B. animalis subsp. lactis. The comparison of the wild-type and the bile-resistant strain responses allowed us to hypothesize about the resistance mechanisms acquired by the derivative resistant strain and about the bile salt response in B. animalis subsp. lactis. In addition, significant differences in the levels of metabolic end products of the bifid shunt and in the redox status of the cells were also detected, which correlate with some differences observed between the proteomes. These results indicate that adaptation and response to bile in B. animalis subsp. lactis involve several physiological mechanisms that are jointly dedicated to reduce the deleterious impact of bile on the cell's physiology.     

Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412^T, which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.     

Effect of lactic acid on diacetyl and acetoin production byLactobacillus casei subsp.rhamnosus ATCC 7469

Lactic acid or its acidity apparently play an important role in the regulation of the biosynthesis of flavor compounds inLactobacillus casei subsp.rhamnosus ATCC 7469. In pyruvate-containing media,L. casei produces lactic acid, acetoin, and diacetyl. A specific pH-dependent system is necessary for both the use of pyruvate and the induction of acetoin and diacetyl production. In cell extracts ofL. casei, lactic acid inhibits the enzymatic activity of acetolactate decarboxylase (ALD) and acetolactate synthetase (ALS); this effect does not occur in whole cells under standard physiological conditions. Lactic acid prevents the use of pyruvate, and the induction of acetoin and diacetyl production. When pyruvate-containing media are used, the pH must be kept close to 6.0 in order to obtain the best production of acetoin and diacetyl.     

Effect of Initial Oxygen Concentration on Diacetyl and Acetoin Production by Lactococcus lactis subsp. lactis biovar diacetylactis

The production of aroma compounds (acetoin and diacetyl) in fresh unripened cheese by Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 was studied at 30°C at different initial oxygen concentrations (0, 21, 50, and 100% of the medium saturation by oxygen). Regardless of the initial O₂ concentration, maximal production of these compounds was reached only after all the citrate was consumed. Diacetyl and acetoin production was 0.01 and 2.4 mM, respectively, at 0% oxygen. Maximum acetoin concentration reached 5.4 mM at 100% oxygen. Diacetyl production was increased by factors of 2, 6, and 18 at initial oxygen concentrations of 21, 50, and 100%, respectively. The diacetyl/acetoin concentration ratio increased linearly with initial oxygen concentration: it was eight times higher at 100% (3.3%) than at 0% oxygen (0.4%). The effect of oxygen on diacetyl and acetoin production was also shown with other lactococci. At 0% oxygen, specific activity of α-acetolactate synthetase (0.15 U/mg) and NADH oxidase (0.04 U/mg) was 3.6 and 5.4 times lower, respectively, than at 100% oxygen. The increasing α-acetolactate synthetase activity in the presence of oxygen would explain the higher production of diacetyl and acetoin. The NADH oxidase activity would replace the role of the lactate dehydrogenase, diacetyl reductase, and acetoin reductase in the reoxidation of NADH, allowing accumulation of these two aroma compounds.