Citrate metabolism in Lactococcus lactis subsp. lactis var. diacetylactis strains

The formation of diacetyl, acetoin, 2,3-butylene glycol, acetaldehyde, ethanol and lactic acid during 24 h of cultivation in milk with 0.19 and 0.5 % of citrate has been studied. Depending on the strain, bacteria produced 1.5 - 1.9 mg of diacetyl, 212 - 311 mg of acetoin and 137 - 156 mg of butylene glycol in 1 1 milk. An increase of the citrate concentration in milk to 0.5 % resulted in an increase in the production of diacetyl from 58 to 74 % and of acetoin by 2.8 - 3.7 times. The strains of distinct activity of acetoin reductase produced in these conditions 2.3 - 2.7 times as much as 2,3-butylene glycol. The recovery of citrate in the from of C₄-compounds ranged from 76 to 98 %, yet barely 0.18 - 0.44 % in the from of diacetyl. Increased concentration of citrate in milk stimulated the production of diacetyl and acetaldehyde to the similar extent, thereby it did not result in the deterioration of organoleptic qualities of starters and milk products. Within the doses used citrate did not significantly affect growth and acidifying activity of the bacteria.     

Influence of acetaldehyde on growth and acetoin production by Leuconostoc citrovorum

Addition of acetaldehyde (100 μg/ml) to glucose-citrate broth stimulated growth and approximately doubled the production of acetoin plus diacetyl by Leuconostoc citrovorum. Radioactive acetaldehyde was converted to ethanol; the acetoin, diacetyl and acetic acid produced were not radioactive. A discussion of the mechanisms by which species of Leuconostoc utilize citrate and carbohydrate suggests that the added acetaldehyde enhanced production of acetoin and diacetyl by increasing the availability of hydroxyethylthiamine pyrophosphate and acetyl-coenzyme A, and that it stimulated growth by permitting greater conversion of acetyl-coenzyme A and acetyl-phosphate to acetate and adenosine 5′-triphosphate.     

Production of acetoin and diacetyl by lactic acid bacteria in skimmed milk with added citrate and pyruvate

Summary Thirty-two strains of starter cultures and lactic acid bacteria belonging to the genera Streptococcus, Leuconostoc and Lactobacillus were studied for their ability to produce acetoin (A) and diacetyl (D) from citrate and pyruvate added separately to skim milk and from pyruvate added to peptone-yeast extract-glucose broth (PYG).The largest amount of A + D was produced from citrate and by the citrate fermenters Streptococcus cremoris AM₂, Str. faecalis, Leuconostoc citrovorum ATCC 8082, L. Gasei ATCC 393, L. Gasei ssp. alactosus and L. xylosus. Some strains of Str. cremoris, Str. thermophilus, some strains of L. Gasei, L. plantarum and L. coryniformis on the contrary produced more A + D from pyruvate than from citrate. Str. cremoris AM₂ should be designated as Str. diaceticremoris like Str. diacetilactis.PYG was more favourable than skim milk for production of A + D from pyruvate by Str. lactis, some strains of Str. cremoris, Str. faecalis and lactobacilli. Pyruvate as a sole source of carbon gave a poor yield of A + D compared to itself in the presence of glucose in case of most of the streptococci and all of the lactobacilli.The largest amount of diacetyl was produced by 70% of the streptococci from citrate added to skim milk, and by the great most of the lactobacilli from pyruvate in PYG medium.

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Produktion von Acetoin durch Milchsäurebakterien in mit Zitrat und Pyruvat angereicherter Magermilch

Zusammenfassung Insgesamt 32 Säureweckerkulturen der Gattungen Streptococcus, Leuconostoc und Lactobacillus wurden unter Verwendung unterschiedlicher Nährsubstrate auf die Bildung von Acetoin und Diacetyl untersucht. Es zeigte sich, daß je nach Nährbodenzusammensetzung und Bakterienstamm unterschiedliche Mengen der genannten Aromastoffe gebildet wurden.

     

Effect of alpha-acetolactate decarboxylase inactivation on alpha-acetolactate and diacetyl production by Lactococcus lactis subsp. lactis biovar diacetylactis

Strains of Lactococcus lactis subsp. lactis biovar diacetylactis deficient in alpha-acetolactate decarboxylase produce alpha-acetolactate. This unstable compound is a precursor of acetoin and an aromatic compound, diacetyl. Following random mutagenesis of strain CNRZ 483, alpha-acetolactate decarboxylase-negative mutant 483 M1 was selected. When grown in milk, its growth and acidification characteristics were similar to those of the parental strain. In anaerobic conditions, the parental strain produced 2.10 mM acetoin and less than 0.05 mM diacetyl. The mutant accumulated up to 2.11 mM alpha-acetolactate, which spontaneously degraded to acetoin and diacetyl. After 24 h of culture, the alpha-acetolactate concentration was only 0.49 mM and the acetoin and diacetyl concentrations reached 1.50 mM and 0.26 mM, respectively. Diacetyl production by both strains increased in aerobic conditions, as well as when citrate was added. In contrast to cultures of the parental strain, however, diacetyl and acetoin concentrations in mutant cultures continued to increase without reaching a plateau. The results also showed that diacetyl production by wild type L. lactis subsp. lactis biovar diacetylactis strains cannot be explained uniquely by the spontaneous decarboxylation of the alpha-acetolactate produced in the culture medium.     

Determination of several flavours in beer with headspace sampling–gas chromatography

In order to determine flavours (diacetyl, pentanedione, acetoin and acetaldehyde) in beer, gas chromatography coupled with headspace sampling technique was developed in our laboratory. The calibrations were accomplished by six level standard addition methods, and relative coefficients were ?0.999. Repeatability experiments were accomplished by detecting 20 successive same standard samples, which showed that detection precision was <2% relative standard deviation (RSD) for three flavours (diacetyl, pentanedione and acetaldehyde), and detection precision of acetoin was <2.5% (RSD). Four flavours gradually increased and then decreased during the process of fermentation, and they leveled off when the fermentation was over. The ratio (diacetyl/pentanedione) reflected the degree of contamination: normal beer had a ratio of approximate 1; the ratio (diacetyl/pentanedione) was higher than 1, displaying that beer was contaminated by microbes. Our data suggested that gas chromatography coupled with headspace sampling technique could be used successfully to determine flavours (diacetyl, pentanedione, acetoin and acetaldehyde) in beer.     

雪里蕻腌菜风味物质的研究

利用GC-MS技术对雪里蕻腌菜卤汁乙醚萃取液中组分进行了鉴定分析,结果鉴定出有机酸组分8个,醇类组分4个,腈类组分2个,酯类组分1个,芳烃类组分2个,并对卤汁中的醇类组分乙醇、丙醇、丁醇及2,3-丁二醇进行了气相色谱检测,卤汁中的乙醛、乙偶姻、双乙酰经2,4*二硝基苯肼衍生化后进行了高效液相色谱分析,还对其中重要风味组分的乳酸发酵机理及其对雪里蕻腌菜风味的影响进行了分析讨论。     

Volatile flavour compounds of yoghurt

The volatile flavour compounds of three samples of Egyptian yoghurt were analysed over a two-week period at 8°C using a simple headspace gas chromatography technique in order to study the changes and relate them to flavour acceptability.
Volatile compounds present were acetaldehyde, diacetyl, acetoin, acetone, butanone, and acetic acid. Acetone and butanone disappeared within the first week of storage, whereas acetaldehyde, diacetyl and acetoin declined steadily but were still present after two weeks. Acetic acid increased to about twice its original level after 10d of storage.
The decreases in acetaldehyde, diacetyl, acetoin, and the increase of acetic acid were closely related to the rapid decrease in product acceptability after 8–10d storage.     

Characterization of the lactic acid bacteria in ewe's milk and cheese from northwest Argentina

Indigenous lactic acid bacteria in ewe's milk and artisanal cheese were studied in four samples of fresh raw milk and four 1-month-old cheeses from the provinces of northwest Argentina. Mean growth counts on M17, MRS, and MSE agar media did not show significant differences (P < 0.05) in raw milk and cheeses. Isolates of lactic acid bacteria from milk were identified as Enterococcus (48%), lactococci (14%), leuconostocs (8%), and lactobacilli (30%). All lactococci were identified as Lactococcus lactis (subsp. lactis and subsp. cremoris). Lactobacilli were identified as Lactobacillus plantarum (92%) and Lactobacillus acidophilus (8%). Enterococci (59%) and lactobacilli (41%) were isolated from cheeses. L. plantarum (93%), L. acidophilus (5%), and Lactobacillus casei (2%) were most frequently isolated. L. lactis subsp. lactis biovar diacetylactis strains were considered as fast acid producers. L. lactis subsp. cremoris strains were slow acid producers. L. plantarum and L. casei strains identified from the cheeses showed slow acid production. The majority of the lactobacilli and Lactococcus lactis strains utilized citrate and produced diacetyl and acetoin in milk. Enzyme activities (API-ZYM tests) of lactococci were low, but activities of L. plantarum strains were considerably higher. The predominance of L. plantarum in artisanal cheese is probably important in the ripening of these cheeses due to their physiological and biochemical characteristics.     

Factors influencing the production of acetoin and diacetyl by lactic acid bacteria in skim milk

Nine strains of starter cultures belonging to the genera Streptococcus, Leuconostoc and Lactobacillus were studied for the effect of pyruvate concentration, incubation period and the associative growth on the production of acetoin and diacetyl in skim milk. The absolute amount of acetoin and diacetyl and the amount pro μmol pyruvate increased with increasing the concentration of pyruvate. At the low concentration of pyruvate the maximum amount of these neutral carbonyl compounds was first reached after 34 days, whereas at the high concentration it was readily achieved after 6 days and then decreased. Production of acetoin and diacetyl by mixed-species cultures from added citrate was either increased or markedly decreased compared to the single-species culture.     

Microorganisms and characteristics of laban.

Laban had a titratable acidity of about 1.0%, a pH of 4.25, an ethanol content of 1.25%, and contained 4.2 mug acetaldehyde and 34 mug acetoin/ml. There was no diacetyl. Five microorganisms, classified as Streptococcus thermophilus, Lactobacillus acidophilus, Leuconostoc lactis, Kluyveromyces fragilis, and Saccharomyces cerevisiae, were responsible for the fermentation. Streptococcus thermophilus and L. acidophilus were responsible for acid production with S. thermophilus producing acid more rapidly. Most of the acetaldehyde was produced by K. fragilis, little ethanol was found in absence of S. cerevisiae, and the acetoin was producted by S. thermophilus.     

SOME EFFECTS OF AERATION ON THE GROWTH AND METABOLISM OF SACCHAROMYCES CEREVISIAE IN CONTINUOUS CULTURE

In continuous fermentation of a complex, synthetic medium by Saccharomyces cerevisiae, a steady state was maintained for prolonged periods in the absence of oxygen. Growth reached a maximum at a low rate of aeration and under these conditions ester formation was markedly inhibited. At higher levels of aeration growth was reduced, the cells formed chains and aggregated, the rate of fermentation decreased and large quantities of acetoin and acetaldehyde were produced. Under fully aerobic conditions, ethanol production continued to account for the major part of the glucose consumed. The amount of diacetyl produced was insignificant at all rates of aeration. The extent of growth and the nitrogen content of the yeast were inversely related.     

Effect of Lactate on the Growth and Production of Diacetyl and Acetoin by Lactobacilli

Diacetyl and acetoin production and cell growth in Lactobacillus acidophilus CNRZ 232, Lactobacillus casei ssp. rhamnosus ATCC 7469, Lactobacillus brevis ATCC 14869, and Lactobacillus fermentum ATCC 9338 were studied. The first three species produced large amounts of diacetyl and acetoin when pyruvate was included in a medium with yeast extract, peptone, tryptone, and glucose. Lactobacillus fermentum failed to produce the compounds under consideration.Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus brevis utilized lactate as a carbon source. When lactate was added to the medium containing pyruvate, the diacetyl, acetoin, and biomass decreased in Lactobacillus casei and Lactobacillus brevis. In Lactobacillus acidophilus both compounds decreased somewhat, and bacterial growth increased, although it decreased again at high lactate concentrations. There was no production of diacetyl and acetoin at pH lower then 4.25 and only little at 4.25.     

Dissimilation of organic acids by dairy lactic acid bacteria

One hundred and eleven different strains of lactic acid bacteria, belonging to the genera Leuconostoc, Streptococcus and Lactobacillus, were examined for their ability to degrade 10 organic acids by detecting CO₂ production, using the conventional Durham tube method. All the strains did not break down succinate, glutarate, 2-oxoglutarate, or mucate. Malate, fumarate, citrate, gluconate, tartrate and pyruvate were variably attacked. A malo-lactic fermentation was brought about by two thirds of S. cremoris and S. lactis strains as well as L. casei but not by L. buchneri or L. brevis. One third of the lactic streptococci examined, i.e. S. cremoris AM₂, ML₈ and SK₁₁ were capable of diacetyl production. They required glucose for cleavage of citrate, whereas Leuconostoc citrovorum and S.faecalis did not. S. cremoris differed from S. lactis in not producing CO₂ from gluconate. From all the lactic acid bacteria examined, only L. plantarum dissimilated tartrate. Production of acetoin and diacetyl is a more reliable evidence for assessing the degradation of pyruvate, compared to detection of evolved CO₂. Facultatively heterofermentative lactobacilli and Leuconostoc citrovorum produced acetoin and diacetyl from pyruvate, whereas the obligately heterofermentative lactobacilli did not, a character that would be of taxonomic value. Glucose fermented by both the facultatively and obligately heterofermentative lactobacilli did not prove to be a metabolic source of acetoin and diacetyl. The facultative heterofermenters degraded pyruvate in the presence of glucose with lactate as the major product together with a mean of acetate of 4.1%, ethanol of 7.9%, acetoin of 1.7% and diacetyl of 2.6% yield on a molar basis after 60 days at 30°C. L. brevis produced acetate and lactate. The role of S. cremoris and the facultatively and obligately heterofermentative lactobacilli in flavour development during the ripening of many cheese varieties has been confirmed.     

Effects of the volatile organic compounds produced by Enterococcus spp. strains isolated from maize grain silos on Fusarium verticillioides growth and fumonisin B1 production

Fusarium verticillioides is a phytopathogenic fungus that can contaminate maize grain silos and result in important losses in the post-harvest product. The objective of this work was to investigate the effects of volatile organic compounds produced by four lactic acid bacterial strains isolated from maize grain silos on F. verticillioides M3125 growth and fumonisin B₁ (FB₁) production. The bacterial isolates 55 and 49 were identified as Enterococcus faecium and M4A and M4G as Enterococcus casseliflavus. The fungal growth was inhibited by 33.33% by the volatiles released by the M4A strain and by approximately 10% by the volatiles emitted by the 55 and 49 strains. The volatiles produced by the M4A strain also significantly reduced (88.75%) FB₁ biosynthesis. The gas chromatography–mass spectrometer analysis identified 21 volatile organic compounds, with diacetyl, acetic acid and acetoin being the main volatiles emitted by the four bacterial strains. Acetoin was the volatile produced in the highest proportion by the four strains, with M4A generating the highest proportion of diacetyl (35.11%). Diacetyl and acetic acid completely inhibited fungal growth at concentrations of 0.3 and 1 mM, respectively, while acetoin promoted fungal growth. Only acetoin significantly reduced FB₁ production. These results showed that diacetyl was the main compound involved in fungal inhibition, while the effect on FB₁ production could have been due to the combination of the volatile organic compounds produced by the M4A strain. In conclusion, the volatiles emitted by the E. casseliflavus M4A strain could be a promising tool for the biocontrol of F. verticillioides in storage maize grain silos.     

Solvent Desorption Dynamic Headspace Method for Diacetyl and Acetoin in Buttermilk

Major buttermilk volatiles recovered included diacetyl, acetic acid, and acetoin. Detection limits were diacetyl 0.2 and acetoin 2.0 μg/g. Mean percent recoveries were 112% for diacetyl and 8.08% for acetoin. Normalized detector responses were linear over the range of concentrations tested (R² > 0.999) for diacetyl and acetoin. Percent relative standard deviations from quadruplicate analysis of 7-day-old buttermilk were <8% for diacetyl, acetoin, and acetic acid. The method enabled quantitative estimation of diacetyl and acetoin in <30 min, including sample preparation time.     

Growth and metabolism of selected strains of probiotic bacteria, in maize porridge with added malted barley

A fermented probiotic maize porridge with high energy density and low viscosity was prepared, using maize flour and barley malt. The porridge was fermented with four probiotic strains (grown separately): Lactobacillus reuteri, Lb. acidophilus (LA5 and 1748) and Lb. rhamnosus GG. These strains were inoculated at two levels; to obtain approx. 7 or 6 log cfu g(-1) in the porridge at 0 h. The porridge was fermented for 24 h at 37 degrees C, and analysed for viable cell count, pH, organic acids, volatile aromatic compounds and sugar content. The inoculated cell concentration was shown to be particularly important during the first hours of the fermentation period, showing a delayed production of most metabolites in porridge inoculated with approx. 6 log cfu g(-1). Most strains reached maximum cell count after 12-h fermentation (7.2-8.2 log cfu g(-1)), with a pH below 4.0. Depending on the strain, lactic acid was produced in amounts ranging from 1360 to 4000 mg kg(-1). Lb. reuteri metabolised succinate, while pyruvate and small amounts of diacetyl were detected in porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748. High amounts of diacetyl (6 mg kg(-1)) and acetoin (27 mg kg(-1)) were detected in porridge inoculated with Lb. rhamnosus GG. Porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748, contained acetaldehyde, while both Lb. reuteri and Lb. rhamnosus GG reduced the acetaldehyde to ethanol. Lb. reuteri utilised both maltose and glucose as carbohydrate sources, while Lb. acidophilus LA5, Lb. acidophilus 1748 and Lb. rhamnosus GG utilised only glucose.     

Diacetyl levels and volatile profiles of commercial starter distillates and selected dairy foods

Starter distillates (SDL) are used as ingredients in the formulation of many food products such as cottage cheese, margarine, vegetable oil spreads, processed cheese, and sour cream to increase the levels of naturally occurring buttery aroma associated with fermentation. This buttery aroma results, in part, from the presence of the vicinal dicarbonyl, diacetyl, which imparts a high level of buttery flavor notes and is a key component of SDL. Diacetyl (2,3-butanedione) is a volatile product of citrate metabolism produced by certain bacteria, including Lactococcus lactis ssp. diacetylactis and Leuconostoc citrovorum. In the United States, SDL are regarded as generally recognized as safe ingredients, whereby usage in food products is limited by good manufacturing practices. Recently, diacetyl has been implicated as a causative agent in certain lung ailments in plant workers; however, little is published about the volatile composition of SDL and the levels of diacetyl or other flavoring components in finished dairy products. The objective of this work was to characterize the volatile compounds of commercial SDL and to quantitate levels of diacetyl and other Flavor and Extract Manufacturers Association-designated high-priority flavoring components found in 18 SDL samples and 24 selected dairy products. Headspace volatiles were assessed using a solid-phase microextraction and analyzed by gas chromatography-mass spectrometry. In addition to diacetyl (ranging from 1.2 to 22,000 μg/g), 40 compounds including 8 organic acids, 4 alcohols, 3 aldehydes, 7 esters, 3 furans, 10 ketones, 2 lactones, 2 sulfur-containing compounds, and 1 terpene were detected in the SDL. A total of 22 food samples were found to contain diacetyl ranging from 4.5 to 2,700 μg/100g. Other volatile compounds, including acetaldehyde, acetic acid, acetoin, benzaldehyde, butyric acid, formic acid, furfural, 2,3-heptanedione, 2,3-pentanedione, and propanoic acid, were also identified and quantified in SDL or food samples, or both. The results obtained in this work summarize the volatile composition of commercial SDL and the approximate levels of diacetyl and other Flavor and Extract Manufacturers Association-designated high-priority flavoring components found in SDL and selected dairy foods.     

Volatile flavor compounds in yogurt: a review

Considerable knowledge has been accumulated on the volatile compounds contributing to the aroma and flavor of yogurt. This review outlines the production of the major flavor compounds in yogurt fermentation and the analysis techniques, both instrumental and sensory, for quantifying the volatile compounds in yogurt. The volatile compounds that have been identified in plain yogurt are summarized, with the few key aroma compounds described in detail. Most flavor compounds in yogurt are produced from lipolysis of milkfat and microbiological transformations of lactose and citrate. More than 100 volatiles, including carbonyl compounds, alcohols, acids, esters, hydrocarbons, aromatic compounds, sulfur-containing compounds, and heterocyclic compounds, are found in yogurt at low to trace concentrations. Besides lactic acid, acetaldehyde, diacetyl, acetoin, acetone, and 2-butanone contribute most to the typical aroma and flavor of yogurt. Extended storage of yogurt causes off-flavor development, which is mainly attributed to the production of undesired aldehydes and fatty acids during lipid oxidation. Further work on studying the volatile flavor compounds-matrix interactions, flavor release mechanisms, and the synergistic effect of flavor compounds, and on correlating the sensory properties of yogurt with the compositions of volatile flavor compounds are needed to fully elucidate yogurt aroma and flavor.     

Short communication: Enzymatic perspective of galactosidases reveals variations in lactose metabolism among Lactococcus lactis strains

To date, most studies of lactose utilization have focused on the genetic diversity of lactic acid bacteria or its influence on product quality, but phenotypic evaluation has rarely been based on metabolic characteristics. In the present study, we investigated the growth, acid production, β-galactosidase, and 6-phospho-β-galactosidase activities of 16 Lactococcus lactis strains obtained from various habitats with lactose as the sole carbon source. The 15 L. lactis strains obtained from various habitats exhibited significant differences in growth and acid production characteristics in the de Man, Rogosa, and Sharpe-lactose broth, and 4 strains consumed more lactose when cultured in skim milk than the type strain ATCC 19435. Among these strains, DQHXNQ38–12 mainly produced acetoin and diacetyl when cultured in skim milk, whereas the strains 15M2 and 5G2 produced high levels of acid and formed curd rapidly. We concluded that the use of lactose is necessary for strain adaptation to the dairy niche. Comprehensive studies of lactose use and the fermentation characteristics of L. lactis are of significant importance.     

Method development for the determination of diacetyl and acetoin at a microwave popcorn plant

Separate sampling and analytical methods for the determination of diacetyl and acetoin have been developed to assess workplace exposures at a popcorn processing facility have been described. Diacetyl (NMAM 2557) is efficiently recovered from an Anasorb CMS sampler tube when the composition of methanol in the desorption solvent is 1%, and acetoin (NMAM 2558) is efficiently recovered when the concentration of methanol is increased to 5%. Desorption efficiencies for diacetyl and acetoin were acceptable, 89.9% (RSD = 0.018) and 94.9% (RSD = 0.019), respectively. Recoveries for nonanone, methyl ethyl ketone, and ethyl acetate were not optimized because they were present in very low concentrations in the popcorn processing facility and not considered to be major occupational health hazards. Samples were collected on Anasorb CMS solid sorbent tubes. All analytes were separated using a 30-m Stabilwax-DA fused silica capillary column, followed by analysis using gas chromatography with flame ionization detection. These methods were acceptable for monitoring and identifying exposures to diacetyl and acetoin present in the butter flavoring mixture used at popcorn processing facilities. For example, in the initial site visit the method was used to determine that maximum workers exposures to diacetyl (462.6 mg/m3), acetoin (59.1 mg/m3), and nonanone (0.45 mg/m3) occurred as the butter flavoring was added to the mixing kettle. When protective measures were recommended by NIOSH personnel and implemented bythe popcorn processing facility, the methods were then used to determine the effectiveness of these changes, which showed that diacetyl and acetoin concentrations had been reduced significantly to 0.97 and 2.3 mg/m3, respectively, while the concentration of nonanone fell to levels below the detection limit (LOD).