Leuconostoc Citrovorum Research Articles

THE INFLUENCE OF LACTIC CULTURES ON GROUND BEEF QUALITY

SUMMARY– The effects of adding 5 and 10% lactic culture (Streptococcus lactis and Leuconostoc citrovorum) grown in skim milk; 2.5, 5, 10, and 20% lactic cultures grown in 20% milk solids; 1 and 2% frozen concentrated lactic cultures; lactic acid to pH 5.0 and 4.5; and 2.5, 5, and 10% skim milk to ground beef stored at refrigeration temperature (7°C) were studied in the 6 preliminary trials. Results obtained were used to formulate the principal investigation. The influence of 10% lactic culture and 10% lactic culture plus 450 ppm of ascorbic acid were tested in 5 replicate trials for the principal investigation. Cultures used in the replicate trials were grown in 20% milk solids. CVT (crystal violet tetrazolium) counts for gram‐negative bacteria, pH, VNC (volatile nitrogen content), and organoleptic observations were evaluated. Data from the preliminary study indicated a profound inhibitory action of the lactic cultures on the growth of the inherent gram‐negative bacteria in ground beef. The addition of 10% lactic culture grown in 20% milk solids was effective in preventing aerobic growth. The addition of pure lactic acid inhibited microbial growth, but caused an undesirable color and aroma. Frozen concentrated cultures required 1% lactose to inhibit the growth. The CVT count in the uncultured meat significantly increased (P < .01) as the storage time progressed. Cultured meat did not exhibit a significant increase in CVT count until 7 days of storage. The pH of the cultured meat significantly declined (P <.01) whereas the uncultured meat indicated a significant increase in pH (P < .01) during storage. VNC in the uncultured meat was significantly higher (P <.01) than in the cultured meat. Cultured meat with 450 ppm of ascorbic acid was consistently preferred for flavor, aroma, and when color was compared with the uncultured meat and the meat with culture alone.

Growth stimulants in plant extracts for Leuconostoc citrovorum.

The growth of Leuconostoc citrovorum ML 34, an isolate associated with the malo-lactic fermentation of wine, was stimulated in part by grape, orange, cabbage, and tomato juices. The stimulatory activity of tomato serum was associated with the carbohydrate fraction. Further purification of the fraction showed that fructose was the factor responsible for initiating growth. In addition to fructose, the organism required CO(2) for establishing growth. Saturated CO(2) atmosphere and catalytic amounts of fructose served as substitutes for plant extracts in a complex glucose medium.

INHIBITION OF LACTIC STREPTOCOCCI AND SOME PATHOGENIC BACTERIA BY CERTAIN MILK-ASSOCIATED VOLATILE COMPOUNDS AS MEASURED BY THE DISC ASSAY

Twenty-seven milk-associated volatile compounds were tested with the disc assay procedure for their ability to inhibit Streptococcus cremoris, Streptococcus lactis, Streptococcus diacetilactis, Leuconostoc citrovorum, Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus. Anisaldehyde, butyraldehyde, formaldehyde, glyoxal, diacetyl, butyric acid, decanoic acid, formic acid, hexanoic acid, octanoic acid, hexylamine, and propylamine inhibited all test cultures although frequently only at concentrations of 10,000 and 100,000 ppm. In addition, isobutyraldehyde (100,000 ppm) inhibited L. citrovorum, E. coli, S. typhimurium, and S. aureus and acetaldehyde (100,000 ppm) inhibited S. aureus. Compounds most active (inhibition at 1,000 ppm) against S. lactis, L. citrovorum, and S. aureus were formaldehyde, glyoxal, and decanoic acid; against S. cremoris were formaldehyde, butyric acid, decanoic acid, formic acid, and hexanoic acid; against one strain of S. diacetilactis were glyoxal, diacetyl, and decanoic acid; against another strain of S. diacetilactis were formaldehyde, decanoic acid, and formic acid; against E. coli were formaldehyde and formic acid; and against S. typhimurium were formaldehyde, diacetyl, butyric acid, formic acid, and hexanoic acid. At a concentration of 1,000 ppm formaldehyde inhibited all test cultures except the fast acid-producing S. diacetilactis and decanoic acid all but E. coli and S. typhimurium. The disc assay procedure was unsatisfactory for evaluating highly volatile compounds since they evaporated before adequate diffusion into the agar occurred.

Inhibition of Salmonella gallinarum by Culture Filtrates of Leuconostoc citrovorum

Salmonella gallinarum was inhibited at an acidic pH by a metabolite produced by Leuconostoc citrovorum. The primary inhibitor was found to be acetic acid, although lactic acid was slightly inhibitory.

The Effect of Grape Skin Treatments on Induced Malo-Lactic Fermentation in Ohio Wines

Induced malo-lactic fermentation was attempted in grape musts prepared by cold pressing, hot pressing, and fermentation "on the skins" for 1, 3, and 5 days before pressing. The musts were inoculated with the malo-lactic bacterium <i>Leuconostoc citrovorum</i> (ML 34) and otherwise treated by a standard vinification procedure. Results revealed that fermentation "on the skins" had a profound effect on the occurrence and rate of malo-lactic fermentation. The grape musts prepared by cold pressing, hot pressing, and fermentation "on the skins" for one day supported only limited growth of the inoculated bacteria, and no malo-lactic fermentation was defected after one year. The wines that were fermented "on the skins" for 3 days underwent an incomplete malo-lactic fermentation. Wines that were fermented 5 days "on the skins" completed malo-lactic fermentation by 11.3 weeks after the bacterial inoculation. In addilion, model grape musts were prepared which simulated the grape musts prepared by hot and cold pressing as well as by fermentation "on the skins." Isolates prepared from these model musts were demonstrated to contain substance(s) stimulatory to the growth of <i>L. citrovorum</i> (ML 34). Microbiological assays indicated that the isolates from the skins caused a greater growth stimuation than the isolates from the model musts stimulating hot- and cold-pressed musts.

Concentrated Cultures of Leuconostoc citrovorum1

Two single-strain cultures of Leuconostoc citrovorum were grown in a broth medium with automatic pH control. Culture concentrates were prepared by centrifugally harvesting the cells and resuspending them in 1/50th the original volume in 10% nonfat milk solids. The concentrates were stored in liquid nitrogen until analyzed. The maximum population attainable was approximately equal when cultures were grown at pH 6.0, 6.5, or 7.0 with sodium hydroxide or ammonium hydroxide as the neutralizer. Citrate was required in the growth medium for the cultures to be able to produce diacetyl subsequently in milk. At pH 6.0, the cells reached maximum population and ability to produce diacetyl. Organoleptic analysis by an experienced flavor panel showed a preference for cottage cheese creamed with a creaming mixture prepared with a culture concentrate rather than a normal culture. The culture concentrates maintained their viability and ability to produce diacetyl for at least 30 days when stored in liquid nitrogen.

Growth Stimulants in Plant Extracts for Leuconostoc citrovorum1

The growth of Leuconostoc citrovorum ML 34, an isolate associated with the malo-lactic fermentation of wine, was stimulated in part by grape, orange, cabbage, and tomato juices. The stimulatory activity of tomato serum was associated with the carbohydrate fraction. Further purification of the fraction showed that fructose was the factor responsible for initiating growth. In addition to fructose, the organism required CO(2) for establishing growth. Saturated CO(2) atmosphere and catalytic amounts of fructose served as substitutes for plant extracts in a complex glucose medium.

Chain Length of Leuconostoc citrovorum Modified by Dilution and Shaking Procedures of the Plate Count

Strains of Leuconostoc citrovorum producing long (CAF-B; Da3) and short (9; 14) chains were grown in sterile skimmilk for 18 hr at 22 and 30C, then shaken before and after dilution in buffered distilled water with and without glass beads. Stained preparations were examined microscopically to enumerate cells in chains of L. citrovorum.In milk cultures: a) Chains of CAF-B grown at 22 instead of 30C resisted breakup most; the reverse was true of Da3, b) breakup of chains of Strains 9 and 14 was unrelated to incubation temperature, c) shaking three strains 50 or more times yielded chains most of which contained four or less cells, and d) chains of Strains 9 and 14 broke up most readily. Chains of some strains, when added to diluent, partially broke up before shaking. Additional shaking (50 or more times) yielded a preponderance of chains, each with four or less cells. Similar results were obtained when milk cultures were shaken, transferred to diluent, and shaken again. Glass beads failed to markedly enhance breakup of chains.Leuconostoc citrovorum always grew to form chains with even numbers of cells. When these chains broke up, they most commonly yielded two-, four-, or six-cell fragments.

Growth of Leuconostoc citrovorym in Skimmilk at 22 and 30 C

Strains of Leuconostoc citrovorum were compared for their growth characteristics in sterile skimmilk at 22 and 30C. Numbers of organisms were determined periodically during incubation using f count procedure and Elliker's lactic agar. Microscopic observations were made on all strains at intervals during the 46-hr incubation period used in most of these experiments. Maximum growth of all L. citrovorum strains tested occured after an incubation of 14-30 hr at 22 and 30C, depending on the strain; however, most strains examined attained maximum numbers after incubation for 18 hr at 30C or 22 hr at 22C. Four strains developed nearly equal maximum populations at both incubation temperatures, whereas four others tended toward higher numbers at 22 than 30C. Although strain variation existed, generally chains formed by the cultures tended to be longest after 10 to 14 hr of incubation, regardless of temperature. Chains formed by most strains tended to be slightly longer (average values seldom more than five cells greater) at 30C incubation than at 22C.Populations of nearly all cultures remained essentially stationary for at least 46 hr when held at either 22 or 30C. Tests on three strains indicated their maximum stationary phase extended beyond 100 hr at 22C. At 30C, numbers of two strains began to decline slightly after 62 and 78 hr of incubation. A third strain revealed an initial decrease in numbers between 26 and 46 hr of incubation, followed by a slow increase which continued for at least 106 hr. Overall, a somewhat more rapid rate of growth during the logarithmic phase was observed at 30C (average generation time, 3.8 hr). In comparison, average generation times of strains attaining nearly equal number at 22 and 30C were 3.4 hr at 30C and 4.3 hr at 22C incubation. Generation times for strains with highest populations at 22C were 2.4 hr at 30C and 3.1 hr at 22C incubation.

Request for an Opinion that the name Leuconostoc citrovorum be rejected and the name Leuconostoc cremoris be conserved

The names Streptococcus citrovorus, Leuconostoc citrovorus and Betacoccus cremoris have been regarded as synonyms. Evidence is given which suggests that Hammer used the name Streptococcus citrovorus for strains which in fact belonged to several species of bacteria. One of these strains which is a sucrose fermenting leuconostoc is indicated in the 1968 ATCC catalogue as the representative strain of Leuconostoc citrovorum. The strain indicated as representative under Leuconostoc cremoris is a sucrose nonfermenting leuconostoc. An Opinion is requested whether the specific epithet citrovorum should have been perpetuated in the species name as Leuconostoc citrovorum and also whether the name Leuconostoc cremoris should be conserved for the sucrose nonfermenting species of Leuconostoc. An Opinion on whether Leuconstoc citrovorum and Leuconostoc cremoris are synonyms is also requested.

Production of acetic acid and other volatile compounds by Leucoostoc citrovorum and Leuconostoc dextranicum.

Single-strain milk cultures of Leuconostoc dextranicum are capable of reducing added acetaldehyde, propionaldehyde, and butanone to the corresponding alcohols at 30 C. L. dextranicum and L. citrovorum reduced propionaldehyde to n-propanol quantitatively in 30 hr, and the reduction of this compound paralleled culture growth. Under unagitated conditions, these organisms produced large amounts of acetic acid and ethyl alcohol. The yield of acetic acid increased when cultures were agitated during growth. This increase in acetic acid production was accompanied by a 20- to 70-fold decrease in ethyl alcohol. The addition of acetaldehyde to the fermentation caused a reduction in the final concentration of acetic acid.

Effect of Temperature on Growth and Diacetyl Production by Aroma Bacteria in Single- and Mixed-Strain Lactic Cultures

By the use of a differential plating medium, the growth of aroma bacteria (Leuconostoc citrovorum and Streptococcus diacetilactis) in mixed-strain lactic starter cultures was followed. Populations were greater at 30C than at the temperature (21C) normally used in the dairy industry for incubation of cultures for manufacture of fermented dairy products. At 30C, peak level of diacetyl produced in mixed-strain lactic starters was lower than at 21C; however, at the higher temperature there was an earlier initiation of the synthesis and the attainment of the peak level. Prompt cooling of cultures to 2C inhibited diacetyl reductase activity and, consequently, loss of desirable flavor and aroma was prevented. The cooling, however, had to be carried out when the diacetyl level was high and before complete destruction of the flavor compound. By a careful combination of time and temperature of incubation and prompt cooling, the diacetyl flavor in milk cultures and Cottage cheese dressed with cultured cream could be stabilized and improved.

Production of Acetic Acid and Other Volatile Compounds by Leuconostoc citrovorum and Leuconostoc dextranicum1

Single-strain milk cultures of Leuconostoc dextranicum are capable of reducing added acetaldehyde, propionaldehyde, and butanone to the corresponding alcohols at 30 C. L. dextranicum and L. citrovorum reduced propionaldehyde to n-propanol quantitatively in 30 hr, and the reduction of this compound paralleled culture growth. Under unagitated conditions, these organisms produced large amounts of acetic acid and ethyl alcohol. The yield of acetic acid increased when cultures were agitated during growth. This increase in acetic acid production was accompanied by a 20- to 70-fold decrease in ethyl alcohol. The addition of acetaldehyde to the fermentation caused a reduction in the final concentration of acetic acid.

Diacetyl biosynthesis in Streptococcus diacetilactis and Leuconostoc citrovorum.

Pyruvate was shown to be the precursor of diacetyl and acetoin in Streptococcus diacetilactis, but dialyzed cell-free extracts of S. diacetilactis and Leuconostoc citrovorum that had been treated with anion-exchange resin to remove coenzyme A (CoA) formed only acetoin from pyruvate in the presence of thiamine pyrophosphate (TPP) and Mg(++) or Mn(++) ions. The ability to produce diacetyl was restored by the addition of acetyl-CoA. Acetyl-phosphate did not replace the acetyl-CoA. Neither diacetyl nor acetoin was formed when the otherwise complete reaction system was modified by using boiled extract or by omitting the extract, pyruvate, TPP, or the metal ions. Free acetaldehyde was not involved in the biosynthesis of diacetyl or acetoin from pyruvate, dialyzed cell-free extracts of the bacteria produced only acetoin (besides CO(2)) from alpha-acetolactate, and acetoin was not involved in the biosynthesis of diacetyl. Only one of the optical isomers present in racemic alpha-acetolactate was attacked by the extracts, and there was no appreciable spontaneous decarboxylation of the alpha-acetolactate at the pH (4.5) used in experiments.

Some Factors Influencing Carbon Dioxide Production by Leuconostoc citrovorum1

This study was undertaken to determine whether Leuconostoc citrovorum plays a role in carbon dioxide production in milk. The ability of L. citrovorum strains to produce gas was studied by two methods. A qualitative method, in which an agar plug was forced up the neck of a volumetric flask, measured gas visually. This method demonstrated that 0.25% yeast extract, in a milk medium at 30 C, stimulated the production of at least 10 ml of gas. Studies using a Gilson Differential Respirometer revealed that L. citrovorum produced 500 to 900 muliters of CO(2) in 6.5 hr, whereas 800 to 1,500 muliters of CO(2) was produced in nonfat milk which contained 0.33% yeast extract. Cell extracts of Streptococcus cremoris, S. lactis, Lactobacillus lactis, L. casei, and L. helveticus also enhanced gas production of L. citrovorum from 20 to 70%. Autolysates of these bacteria, present during the ripening or ageing of certain cheeses, may stimulate L. citrovorum, a common organism in starter cultures, to produce gas, causing, for example, the slit-open defect of cheddar cheese. Yeast extract caused an increase in acid and gas production per cell but did not cause an increase in growth. Experiments indicated that one metabolic source of carbon dioxide was the decarboxylation of pyruvate produced during catabolism of citric acid. Yeast extract stimulated this reaction by 16%.

Control of Malo-Lactic Fermentation Induced byLeuconostoc Citrovorum

Musts and new wines were treated in a variety of ways to determine methods of controlling malo-lactic fermentation induced by <i>Leuconostoc citrovorum</i> ML 34 under California winemaking conditions. Malo-lactic fermentation rate was stimulated by bacterial inoculation, the rate being greater at higher concentrations of inoculum, and by lowered sulfur dioxide concentration. Malo-lactic fermentation was inhibited by added acid or sulfur dioxide and by heavy fining, but it was prevented only by a combination of these treatments. Factors which seemed to have little influence on the malo-lactic fermentation rate under the conditions established were aeration, delayed racking, and date of grape harvest. Unexpectedly, alcoholic fermentation in the presence of grape skins had a profound stimulatory influence on rate of malo-lactic fermentation, in both red and white wines. The predominant bacterial species isolated from the wine fermented on the skins was the same as that inoculated. It was suggested that some material extracted from the skins was influential in promoting malo-lactic fermentation with <i>L. citrovorum</i>.

Chemical Characterization of Wines Fermented with Various Malo-lactic Bacteria

Six malo-lactic strains of lactic acid bacteria were isolated from California wines and identified as Lactobacillus delbrueckii, L. buchneri, L. brevis, Leuconostoc citrovorum , and two strains of Pediococcus cerevisiae . Malo-lactic fermentation was induced in separate lots of wine by inoculation of each lot with one of the strains of bacteria. Malo-lactic fermentation had occurred in each inoculated wine within 2 months. The resultant wines were subjected to chemical analysis, including gas chromatographic examination of concentrated extracts of the wines. Only a few differences in composition were found when the malo-lactic wines were compared one with another. The differences that were found were in volatile acidity and in concentrations of acetoin (plus diacetyl) and probably diethyl succinate.

Chemical Characterization of Wines Fermented with Various Malo-lactic Bacteria1

Six malo-lactic strains of lactic acid bacteria were isolated from California wines and identified as Lactobacillus delbrueckii, L. buchneri, L. brevis, Leuconostoc citrovorum, and two strains of Pediococcus cerevisiae. Malo-lactic fermentation was induced in separate lots of wine by inoculation of each lot with one of the strains of bacteria. Malo-lactic fermentation had occurred in each inoculated wine within 2 months. The resultant wines were subjected to chemical analysis, including gas chromatographic examination of concentrated extracts of the wines. Only a few differences in composition were found when the malo-lactic wines were compared one with another. The differences that were found were in volatile acidity and in concentrations of acetoin (plus diacetyl) and probably diethyl succinate.

Green Flavor Defect in Lactic Starter Cultures1,2

The diacetyl-to-acetaldehyde ratio was found to be approximately 4:1 in desirably flavored mixed-strain butter cultures. When the ratio of the two compounds was lower than 3:1, a green flavor was observed. Acetaldehyde utilization at 21C by Leuconostoc citrovorum 91404 was very rapid in both acidified (pH 4.5) and nonacidified (pH 6.5) milk cultures. The addition of 5ppm of acetaldehyde to nonacidified milk media prior to inoculation greatly enhanced growth of L. citrovorum 91404 during incubation at 21C. A proposed mechanism of acetaldehyde utilization and its effect on associative growth in mixed-strain butter cultures is discussed. Combinations of single-strain organisms demonstrated that the green flavor defect can result from excess numbers of Streptococcus lactis or Streptococcus diacetilactis in relation to the L. citrovorum population.

Turbidimetric Microbiological Assay of Amino Acids

To shorten the time necessary for the determination of l-glutamic acid a turbidimetric microbiological assay method with Lactobacillus arabinosus 17-5 has been studied. It was found that the assay can be completed within 30 hours including the incubation period for the inoculum, the assay and the time for medium preparation. Turbidimetric assay methods of other amino acids such as dl-alanine with Leuconostoc citrovorum 8081, l-aspartic acid and l-lysine with Leuc. mesenteroides P-60 have also been studied.