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.     

Differences between Lactobacillus casei subsp. casei 2206 and Citrate-Positive Lactococcus lactis subsp. lactis 3022 in the Characteristics of Diacetyl Production

Lactobacillus casei subsp. casei 2206 exhibited much lower levels of diacetyl reductase activity than Citr⁺Lactococcus lactis subsp. lactis 3022 but two-, three-, and more than eightfold-higher levels of diacetyl synthase, lactate dehydrogenase, and NADH oxidase activities, respectively. A requirement for metal ions by the diacetyl synthases in both species was observed. The extracts of strain 2206 but not strain 3022 produced more diacetyl from pyruvate when the reaction for diacetyl synthase was aerated than when it was conducted statically.     

Determining the Suitability of Lactobacilli Antifungal Metabolites for Inhibiting Mould Growth

Summary In recent years, public concern about indoor mould growth has increased dramatically in the United States. In this study, lactic acid bacteria (LAB), which are known to produce antimicrobial compounds important in the biopreservation of food, were evaluated to determine if the same antimicrobial properties can be used to inhibit mould fungi that typically colonize wood. Based on biomass measurement, cell-free supernatants from Lactobacillus casei subsp. rhamnosus and Lactobacillus acidophilus grown in deMan Rogosa Sharpe (MRS) broth inhibited 95–100% growth of three mould fungi and one stain fungus associated with wood-based building materials. Lactic acid and four unknown compounds ⩽ kDa molecular weight were fractionated from the culture supernatant by thin layer chromatography and high-performance liquid chromatography. Antifungal activity, which was attributed to one or more unknown metabolites, was retained during heating and neutralization. A 1:2 dilution of L. casei supernatant inhibited 100% growth of all test fungi.     

Localization of Lactococcus lactis ssp lactis bv diacetylactis in alginate gel beads affects biomass density and synthesis of several enzymes involved in lactose and citrate metabolism

Summary Lactococcus lactis ssp lactis bv diacetylactis, immobilized in calcium alginate beads, was grown in synthetic medium in a continuous flow reactor. Cell distribution inside the gel, as well as the activity of various enzymes, was measured after 30 h of operation. The included biomass tended to concentrate at the periphery of the bead along a section of radius about 100 m long. ATPase activity was maximal in this zone. The activity of NADH oxidase, alcohol dehydrogenase, diacetyl reductase and acetoin reductase, which are repressed in the presence of citrate, were higher in the deeper zones than at the surface of the beads. This result shows that only the peripheral zone of the bead is responsible for the bioconversion of citrate into flavour compounds (diacetyl and acetoin).     

Production of lactic acid from date juice extract with free cells of single and mixed cultures of Lactobacillus casei and Lactococcus lactis

The production of lactic acid from date juice by single and mixed cultures of Lactobacillus casei and Lactococcus lactis was investigated. In the present conditions, the highest concentration of lactic acid (60.3 g l⁻¹) was obtained in the mixed culture system while in single culture fermentations of Lactobacillus casei or Lactococcus lactis, the maximum concentration of lactic acid was 53 and 46 g l⁻¹, respectively. In the case of single Lactobacillus casei or Lactococcus lactis, the total percentage of glucose and fructose utilized were 82.2; 94.4% and 93.8; 60.3%, respectively, whereas in the case of mixed culture, the total percentage of glucose and fructose were 96 and 100%, respectively. These results showed that the mixed culture system gave better results than single cultures regarding lactic acid concentration, and sugar consumption.     

Diacetyl and acetoin production from whey permeate using engineered <Emphasis Type="Italic">Lactobacillus casei</Emphasis>

The capability of Lactobacillus casei to produce the flavor-related compounds diacetyl and acetoin from whey permeate has been examined by a metabolic engineering approach. An L. casei strain in which the ilvBN genes from Lactococcus lactis, encoding acetohydroxyacid synthase, were expressed from the lactose operon was mutated in the lactate dehydrogenase gene (ldh) and in the pdhC gene, which codes for the E2 subunit of the pyruvate dehydrogenase complex. The introduction of these mutations resulted in an increased capacity to synthesize diacetyl/acetoin from lactose in whey permeate (1,400 mg/l at pH 5.5). The results showed that L. casei can be manipulated to synthesize added-value metabolites from dairy industry by-products.     

Effect of citrate on growth of Lactococcus lactis subsp. lactis in milk

The effect of citrate on the growth of Lactococcus lactis subsp. lactis var. diacetylactis in milk has been investigated. Five strains of Lactococcus lactis subsp. lactis var. diacetylactis were compared to their citrate-negative variants, which lack the plasmid coding for citrate permease. In most cases, acidification kinetics and the final bacterial concentration of pure cultures of parental and variant strains did not differ significantly. Co-cultures of parental and variant strains, however, systematically tended towards the predominance of parental strains. Citrate metabolism is responsible for this change, since the predominance of citrate-positive strains was not observed in the absence of citrate. Continuous culture in milk enabled the difference in growth rates between the parental strain Lactococcus lactis subsp. lactis var. diacetylactis CDI1 and its citrate-negative variant to be quantified by following changes in the populations of the two co-cultured strains. At 26 °C, the growth rate of the parental strain was 7% higher than that of its citrate-negative variant. These results show that citrate metabolism slightly stimulates the growth of lactococci in milk. Received: 18 February 1997 / Received revision: 2 May 1997 / Accepted: 4 May 1997     

A descriptive model for citrate utilization by Lactococcus lactis ssp lactis bv diacetylactis

A model for the use of citrate by Lactococcus lactis ssp lactis bv diacetylactis CNRZ 125 is proposed. Citrate metabolism by this strain leads to the production of acetate, CO₂ and C4 compounds (diacetyl, acetoin, 2,3-butylene glycol). The model furnishes correct simulations, consistent with published results on the pathways used and on lactose-citrate co-metabolism. Citric acid is incorporated independently of growth. The production of flavoring compounds is a complex process, depending on the rate of citrate utilization, on the proportion of pyruvate arising from citrate and which condenses to form -acetolactate and CO₂, on the rate of transformation of -acetolactate to diacetyl and acetoin, as well as on the rate of reduction of these compounds to 2,3-butylene glycol.     

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.     

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.     

Effect of ilvBN-encoded α-acetolactate synthase expression on diacetyl production in Lactococcus lactis

 Conversion of pyruvate to α-acetolactate, which is broken down to diacetyl and acetoin, can be catalysed by two α-acetolactate synthases in Lactococcus lactis. The enzyme encoded by the als gene (Als) has previously been shown to have a low affinity for pyruvate, which limits the formation of diacetyl. In this study we have expressed from a plasmid the ilvBN genes, which encode the other α-acetolactate synthase (IlvBN). This plasmid-directed enzyme expression provided up to 3.6-fold increased product formation in the L. lactis MG1363 and IL1403 backgrounds. Plasmid-based expression of the ilvBN genes, in an IL1403 derivative from which the leu.ilv.ald and flanking genes had been deleted, yielded up to 0.1 mM diacetyl whereas the host strain produced none. In addition, IlvBN, with a K _m value of 8.3 mM, was shown to have a greater affinity for pyruvate than does Als. Received: 28 June 1995 / Received revision: 26 September 1995 / Accepted: 29 September 1995     

pH Homeostasis and Citric Acid Utilization: Differences Between Leuconostoc mesenteroides and Lactococcus lactis

This study presents the effects of citric acid and extracellular pH (pH_e) on the intracellular pH (pH_i) of wild-type and citrate negative variants (cit⁻) Leuconostoc mesenteroides subsp. mesenteroides (Ln. mesenteroides M) and Lactococcus lactis subsp. lactis bv. diacetylactis (L. lactis LD). A recent method using a pH-sensitive fluorescent indicator carboxyfluorescein succinimidyl ester (cFSE) was adapted to measure the pH_i of these two lactic acid bacteria in resting cells. Energized cells with 10 mM lactose of Ln. mesenteroides M and L. lactis LD modified their pH gradient (ΔpH) in the same manner; when the pH_e was decreased from 7 to 4, the pH_i decreased from 7 to about 5. The adjunction of 10 mM citric acid had no effect on the pH_i of wild-type and cit(−) variant of L. lactis LD, nor on the pH_i of Ln. mesenteroides cit(−) variant. Nevertheless, in Ln. mesenteroides M wild-type, citric acid utilization increased the pH_i, which was maintained at about 6.5–7.0 when the pH_e was decreased from 7 to 4. It could be concluded that citric acid allows the maintenance of pH homeostasis in Leuconostoc mesenteroides. Received: 7 March 1997 / Accepted: 14 April 1997     

Nisin-selectable food-grade secretion vector for Lactococcus lactis

A nisin-resistant Lactococcus lactis strain TML01 was isolated from crude milk. A gene with 99% homology to the nisin-resistance gene, nsr, was identified. The food-grade secretion plasmid, pLEB690 (3746 bp), was constructed based on this novel nsr gene enabling primary selection with up to 5 μg nisin/ml. The functionality of pLEB690 as a secretion vector was shown by expressing and secreting the pediocin AcH gene papA in L. lactis. pLEB690 is therefore, a functional food-grade secretion vector potentially useful for the food industry.     

Diacetyl production by different strains of Lactococcus lactis subsp. lactis var. diacetylactis and Leuconostoc spp.

Summary Several strains of Lactococcus lactis subsp. lactis var. diacetylactis and Leuconostoc spp. were compared for product formation from citrate in milk cultures. Most strains produced acetoin and butanediol. Some strains derived from buffer starter cultures produced, in addition, -acetolactate. Lactococcus lactis strain C17, which produced acetoin and butanediol but no -acetolactate in culture, was compared physiologically with L. lactis strain Ru4, which produced only -acetolactate. Activities of enzymes involved in citrate metabolism were almost identical in both strains, with the exception of -acetolactate decarboxylase, which was missing in strain Ru4. The formation of -acetolactate, acetoin and diacetyl was further analysed in cell-free extracts. -Acetolactate synthase activity saturated at a high pyruvate concentration (100 mm). This is in agreement with the observed accumulation of pyruvate externally, and probably internally, during -acetolactate, acetoin and butanediol production by L. lactis cells.Correspondence to: J. Hugenholtz     

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.     

Growth and aflatoxin production by Aspergillus parasiticus when in the presence of Streptococcus lactis

Streptococcus lactis was grown with Aspergillus parasiticus in modified APT broth. Three inoculation procedures were used: (a) S. lactis was grown 3 days, then conidia of A. parasiticus were added (SLAP), (b) both organisms were added simultaneously (ST) and (c) A. parasiticus was grown 3 days, then S. lactis was added (APSL). At 3, 6 and 10 days of incubation, contents of flasks were analyzed for growth of each organism, pH of broth and aflatoxin content. S. lactis did not survive past 3 days when grown alone. In ST cultures, S. lactis grew to the same extent as in the control at 3 days; it remained viable at a low level through 10 days. In APSL cultures, S. lactis growth was inhibited at 3 days but the bacterium survived through 7 days (10 days of mold growth) at reduced numbers. At 3 days there were no appreciable differences in growth of A. parasiticus. At 6 days, in ST and SLAP cultures, growth of the mold was inhibited, while in the APSL culture growth increased over that in the control. At 10 days, growth of mold was somewhat increased over the control in all test conditions. The pH of broth in the A. parasiticus control and APSL culture was 6 at 3 days, dropped to 4.5–4.6 at 6 days and rose to 7 by 10 days. In ST and SLAP cultures, the pH was at 4.1 at 3 days and rose to pH 7 by 10 days. Aflatoxin (B₁ plus G₁) content was lowest at 3 days and increased at 6 days. Between 6 and 10 days two patterns were observed. In APSL and SLAP cultures, aflatoxin content decreased, while it increased in the ST culture. These patterns occurred when aflatoxin content was expressed on a total or per gram of dried mycelium basis. At 3 days the amounts of aflatoxin B₁ and G₁ were approximately equal. Between 3–6 days the amount of G₁ increased more rapidly than that of B₁. Between 6 and 10 days in the ST culture, the amount of G₁ increased at a slower rate than that of B₁ while in SLAP and APSL cultures, the amount of G₁ decreased more rapidly than that of B₁. When a different lot of the same medium was used, aflatoxin production was greatly reduced. The pH of broth at all test conditions rose through the incubation period.     

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.     

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

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

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.