Partial biochemical characterization of tetrazolium red‐reactive Lactobacillus plantarum mutants

One thousand colonies derived from Lactobacillus plantarum ATCC 8014 cells that survived 34–43 × 10³ ergs cm^‐2 ultraviolet irradiation were screened on media containing tetrazolium red to detect fermentative mutants. Fermentation end‐products formed from pyruvate, glucose, or lactose catabolism were determined. All 37 stable tetrazolium red‐reactive mutants had increased pyruvate utilization compared to the wild‐type strain. Only two did not produce lactate from exogenous pyruvate. When glucose or lactose were substrates, these two mutants and six other representative mutants produced lactate at levels similar to the parent strain. Although the average lactate and acetoin production from pyruvate by the mutants and wild‐type strains were similar, 25% of the mutants had increased acetoin production.     

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.     

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.     

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.     

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.     

INFLUENCE OF SODIUM NITRITE ON THE AEROBIC CATABOLISM OF GLUCOSE BY STAPHYLOCOCCUS AUREUS1

The inhibitory effect of sodium nitrite upon glucose catabolism by Staphylococcus aureus was investigated using [U–^{1 4}C] glucose, liquid chromatography, and gas-liquid chromatography. Acetate and acetoin are the end-products of glucose metabolism by S. aureus at 37°C and pH 6.3. In the presence of inhibitory levels of sodium nitrite, acetate and lactate with traces of pyruvate and acetoin are the end products. Acetate production per unit of growth is significantly lower in the sodium nitrite inhibited cultures. The decreased acetoin accumulation was not due to inhibition of diacetyl reduction. The production of acetoin was induced by the addition of acetate to the sodium nitrite containing medium.     

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.     

Effect of alsD deletion and overexpression of nox and alsS on diacetyl and acetoin production by Lacticaseibacillus casei during milk fermentation

Diacetyl and acetoin are key aroma components of fermented milk but are produced in low concentrations by starter cultures. In this study, we expressed NADH oxidase, acetolactate synthase, and inactivated acetolactate decarboxylase in Lacticaseibacillus casei TCS to generate recombinant L. casei strains, and investigated the effects of the genes encoding these enzymes on diacetyl and acetoin production during milk fermentation. In the single-gene recombinant strains tested, diacetyl concentrations were highest in milk fermented by L. casei TCSI-nox (nox gene overexpressed, 3.68 mg/kg), whereas acetoin concentrations were highest in milk fermented by L. casei TCS-ΔalsD (alsD gene deleted, 32.94 mg/kg). Moreover, diacetyl and acetoin concentrations were higher in the inducible strains than in the corresponding constitutive strains (e.g., TCSI-nox vs. TCSC-nox, and TCSI-ΔalsD-nox vs. TCSC-ΔalsD-nox). This phenomenon was also reflected in the protein expression levels and enzyme activities. In the double-gene recombinant strains tested, the highest concentrations of diacetyl and acetoin were produced by L. casei TCSI-ΔalsD-nox (nox overexpressed and alsD deleted, 4.66 mg/kg, 69.62 mg/kg, respectively). The triple-gene recombinant L. casei TCS-ΔalsD-nox-alsS produced the highest concentrations of diacetyl and acetoin, which were 2.38 and 11.19 times, respectively, the concentrations produced by the original strain. These results show that the nox, alsS, and alsD genes make key contributions to the biosynthesis of diacetyl and acetoin by L. casei. The modification of multiple genes had a synergistic effect, leading to greatly increased synthesis of diacetyl and acetoin by L. casei during its fermentation of milk.     

Physiological Activity of Deep-Frozen Concentrates of Leuconostoc Strains

11 strains of Leuconostoc cremoris and 1 strain of Leuconostoc lactis were the subject of investigations. Basing on the evaluation of growth parameters in the different cultivation media and on the diacetyl production ability in milk 2 strains of Leuconostoc cremoris and 1 strain of Leuconostoc lactis were chosen for preparing frozen concentrated biomass. Sterile milk or cream (18% fat) were used as protecting agents. Concentrated biomass was frozen at ?70 °C and stored at ?30 °C for 3 months. The prepared concentrates contained from 1.6 × 10¹¹ to 2.8 × 10¹¹ colony forming units (CFU)/g. Except for one (strain “S”) the strains retained 100% survival. All the strains showed high growth activity after their recovery in milk (3.3 × 10⁸ - 8.4 × 10⁸ CFU/ml). Both protecting agents applied proved to be equally good for preserving viability of the cells and for their acidifying activity. In spite of the protecting agent a decrease in the aroma activity by 25–50% was observed after one month of the biomass storage. Nevertheless, concentrated frozen multicomponent starters containing Leuconostoc strains retained the ability to produce diacetyl and acetoin on the unchanged level during the three months storage period.     

COLOUR REACTION SCREENING OF CLONES WITH DIACETYL AND ACETOIN REDUCTASE ACTIVITIES

A genomic library of the chromosomal DNA of a brewing strain (Saccharomyces carlsbergensis) has been made in a Escherichia coli HB101 strain as host. A coloured test allowing one to isolate recombined clones possessing diacetyl (DR) and acetoin (AR) reductase activities is described. This test allowed one to detect 3 Escherichia coli clones having both activities (DR and AR) from among 3000 E. coli strains constituting the genomic library of Saccharomyces carlsbergensis in E. coli.     

Oxygen uptake activity and aerobic metabolism of Streptococcus thermophilus STH450

Streptococcus thermophilus STH450 had a very high oxygen uptake. This strain was then compared with aerobic metabolism to S. thermophilus ATCC 19258, a reference strain for aerobic metabolism. Molecular oxygen, which was absorbed by S. thermophilus STH450 during aerobic glycolytic metabolism, was involved in the oxidation of NADH by the catalytic activity of NADH oxidase. The portion of pyruvate that corresponded to the oxidized NADH was committed to form alpha-acetolactate, acetoin, and diacetyl. Both strains were deficient in peroxidase and pyruvate oxidase activities; therefore, NADH oxidase was probably the terminal oxidase in aerobic glycolytic metabolism. Oxygen uptake and NADH oxidase activities were significantly higher in S. thermophilus STH450 than in S. thermophilus ATCC 19258. alpha-Acetolactate, acetoin, and diacetyl also accumulated during aerobic glycolytic metabolism of S. thermophilus STH450. However, when both strains were grown in the presence of pyruvate, these metabolites were equivalent. Hence, less oxygen might be needed for pyruvate metabolism.     

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.     

Citrate utilization in milk by Leuconostoc cremoris and Streptococcus diacetilactis.

Citrate utilization and diacetyl, acetoin and acetaldehyde production by 2 strains each of Leuconostoc cremoris and Streptococcus diacetilactis in milk were studied. With the leuconostoc bacteria no growth and little citrate utilization occurred unless a stimulant (yeast extract) was present, when complete utilization of citrate without concomitant production of diacetyl or acetoin was obtained. The additon of Mn2+ stimulated growth resulted in diacetyl and acetoin production. Destruction of diacetyl and acetoin occurred when the citric acid level fell to c.1000 and 600 mug/g in the case of Leuc. cremoris FR8-1 and CAF1, respectively. Only strain FR8-1 produced acetaldehyde. In contrast, Str. diacetilactis produced diacetyl, acetoin and acetaldehyde concomitant with citrate utilization.     

Growth and metabolic activity of a cheese starter in CO2-acidified and non-acidified refrigerated milk

 The growth and activity of two Lactococcus strains and one Leuconostoc strain in CO₂-acidified and non-acidified refrigerated milk were evaluated separately and as a mixed culture to determine their suitability for use as a starter in the manufacture of Afuega’l Pitu, an acid-coagulated Spanish cheese, from refrigerated CO₂-acidified milk. The growth of the strains studied and their production of organic acids were similar in CO₂-acidified and non-acidified refrigerated or fresh milk, indicating that CO₂ treatment does not affect the metabolic activity of the strains. However, refrigeration enhanced the production of acetaldehyde, ethanol and diacetyl in CO₂-acidified and non-acidified milk. The level of diacetyl was also greater in refrigerated CO₂-acidified milk than in refrigerated non-acidified milk. It was concluded that refrigerated milk acidified with CO₂ can be satisfactorily used in the manufacture of Afuega’l Pitu cheese, and that this technique can be also used in the production of other acid-coagulated cheeses. Received: 1 July 1997 / Revised version: 1 September 1997     

Growth and metabolic activity of a cheese starter in CO2-acidified and non-acidified refrigerated milk

 The growth and activity of two Lactococcus strains and one Leuconostoc strain in CO₂-acidified and non-acidified refrigerated milk were evaluated separately and as a mixed culture to determine their suitability for use as a starter in the manufacture of Afuega’l Pitu, an acid-coagulated Spanish cheese, from refrigerated CO₂-acidified milk. The growth of the strains studied and their production of organic acids were similar in CO₂-acidified and non-acidified refrigerated or fresh milk, indicating that CO₂ treatment does not affect the metabolic activity of the strains. However, refrigeration enhanced the production of acetaldehyde, ethanol and diacetyl in CO₂-acidified and non-acidified milk. The level of diacetyl was also greater in refrigerated CO₂-acidified milk than in refrigerated non-acidified milk. It was concluded that refrigerated milk acidified with CO₂ can be satisfactorily used in the manufacture of Afuega’l Pitu cheese, and that this technique can be also used in the production of other acid-coagulated cheeses. Received: 1 July 1997 / Revised version: 1 September 1997     

Sensorically and antimicrobially active metabolite production of Lactobacillus strains on Jerusalem artichoke juice

BACKGROUND: In the tubers of Jerusalem artichoke (Helianthus tuberosus L.) the main carbohydrate is the well‐known prebiotic inulin, which is a good growth substrate for gut microorganisms. Jerusalem artichoke tuber is traditionally consumed boiled or pickled rather than in fermented form. Lactic acid bacteria are traditionally used in the production of fermented foods; nevertheless their behavior and metabolite production are considerably influenced by the substrate. The purpose of this study was to investigate the growth and production of the most important sensorically and antimicrobially active metabolites of different Lactobacillus strains on Jerusalem artichoke juice. RESULTS: All investigated strains grew well (in the range 10⁹ cfu mL^?1) in the media. The organic acids (lactic acid, 110–337 mmol L^?1; acetic acid, 0–180 mmol L^?1; and succinic acid, 0–79 mmol L^?1), hydrogen peroxide (0.25–1.77 mg L^?1), mannitol (0.06–3.24 g L^?1), acetoin and diacetyl production of strains varies not only according to the species but also from strain to strain, which will be demonstrated and discussed in the paper. CONCLUSION: Our results showed that lactobacilli can be used for the fermentation of Jerusalem artichoke, which in this form could be used, alone or mixed with other raw food material, as a new synbiotic functional food. Copyright © 2011 Society of Chemical Industry     

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.     

The effects of incorporating wild‐type strains of Lactococcus lactis into Turkish white‐brined cheese (Beyaz peynir) on the fatty acid and volatile content

The development of free fatty acids (FFA) and volatile flavour compounds in the Turkish white‐brined cheese Beyaz peynir made by using three wild strains of Lactococcus lactis subsp. lactis was investigated over 90 days. Results showed that production of both FFA and flavour compounds in the control (PK1) and experimental cheeses (MBLL9, MBLL23 and MBL27) was strain dependent. The hydrolysis of milk fat was more evident in the cheese made using Lc. lactis subsp. lactis MBL27. Considering the production of fat breakdown compounds and acidification activities of the strains MBLL23 and MBL27, the combination of these strains could be proposed for the production of white‐brined cheese.     

Two strains of nonstarter lactobacilli increased the production of flavor compounds in soft cheeses

The contribution to flavor generation and secondary proteolysis of 2 strains of mesophilic lactobacilli isolated from cheese was studied. Miniature soft cheeses (200 g) were produced with or without the inclusion of a culture of Lactobacillus plantarum I91 or Lactobacillus casei I90 in the starter composed of Streptococcus thermophilus. During ripening, cheeses containing the added lactobacilli showed an increased content of total free amino acids, but this increase was only significant in cheeses with Lb. plantarum I91. In addition, free amino acid profiles were modified by selective increases of some amino acids, such as Asp, Ser, Arg, Leu, and Phe. Cheeses inoculated with Lb. plantarum I91 or Lb. casei I90 were also characterized by a significantly higher concentration of diacetyl, a key flavor compound, and an increased content of acetoin. Results suggest an increase in the catabolism of either citrate or aspartate, with the production of the derived aroma compounds. Overall, aspartate content increased in both lactobacilli-added cheeses, whereas citrate was more or less constant, suggesting that aspartate could be the source of increased diacetyl and acetoin. A triangle aroma test showed that the addition of the lactobacilli strains significantly changed the sensory attributes of cheeses. At least 11 of 12 panelists commented that the aroma of cheeses with adjuncts was more buttery than that of control cheeses, which is desirable in most soft cheeses. Both Lb. plantarum I91 and Lb. casei I90 performed well as adjunct cultures by influencing cheese aroma development and cheese proteolysis.     

Study of Cheese Associated Lactic Acid Bacteria Under Carbohydrate-Limited Conditions Using D-Stat Cultivation

The metabolic pathways alternative to glycolytic energy (ATP) during growth of starter and nonstarter lactic acid bacteria were studied simulating the depletion of carbohydrates during cheese ripening. D-stat cultivation strategy with the gradual decrease of galactose concentration in tryptone-arginine feeding medium was used. With the decrease of galactose feeding, the biomass yield calculated on carbohydrate consumption (Y_X/HEX) and acetate/lactate production ratio of all strains increased. We assume that ATP and biomass yields improved by directing the pyruvate flow from lactate to acetate and that metabolic energy could be obtained either by producing acetate from carbohydrates or from arginine metabolism in ADI-positive strains. Four LAB strains out of eight produced ornithine from arginine indicating active arginine-deiminase (ADI) pathway. These ADI-positive strains achieved 3–10 times higher Y_X/HEX than ADI-negative strains in tryptone-arginine medium. Lactobacillus plantarum also used serine as an energy source. Starters and NSLAB strains using the amino acids arginine and serine or limited amounts of carbohydrates therefore have the potential to influence flavor production in cheese more efficiently.