Selection and properties of alpha-acetolactate decarboxylase-deficient spontaneous mutants of Streptococcus thermophilus

Many lactic acid bacteria produce diacetyl, which is a desirable aroma compound in some fermented dairy products. Strains or mutants of Lactococcus lactis subsp. lactis biovar. diacetylactis that are deficient in alpha-acetolactate decarboxylase are used in some food processes for their ability to produce large amounts of diacetyl. However, up until now, the use of alpha-acetolactate decarboxylase-deficient mutants of Streptococcus thermophilus for increased diacetyl production has not been evaluated. The objective of the present study was to devise a procedure for selecting spontaneous alpha-acetolactate decarboxylase-deficient mutants of S. thermophilus. We observed that in a chemically defined medium (CDM) containing alpha-ketobutyrate plus leucine, or alpha-ketobutyrate plus leucine plus isoleucine, the alpha-acetolactate decarboxylase-deficient mutant TIL865, obtained by directed mutagenesis, grew faster than its parent strain. This property was used for selecting spontaneous alpha-acetolactate decarboxylase-deficient mutants on agar plates. The resulting mutants were able to grow in milk, and their acidifying activity was slightly lower than that of the parent strain. Under partial anaerobic or aerobic conditions, they produced approximately three times more diacetyl than the parent strain. Such spontaneous mutants may be useful for increasing the diacetyl content of fermented milks whose production involves S. thermophilus strains.     

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.     

Method for the simultaneous assay of diacetyl and acetoin in the presence of alpha-acetolactate: application in determining the kinetic parameters for the decomposition of alpha-acetolactate

A simultaneous assay method for diacetyl and acetoin was developed to investigate the formation of diacetyl during the brewing of alcoholic beverages. A GC-MS analysis after the extraction from neutralized sample by ethyl acetate gave accurate assay results. The detection limit was below 0.1 mg/l and the assay was quantitative from 0.1 to 100 mg/l for both compounds. Unlike other methods, the assay results were unaffected by the presence of alpha-acetolactate (up to 26 mg/l), which easily decomposes to diacetyl or acetoin, because the extraction condition prevents the decomposition and extraction of this acidic compound. Since our assay is compatible with samples that contain alpha-acetolactate, the kinetic parameters for decomposition of alpha-acetolactate to diacetyl and acetoin were determined. The decomposition rate constants were affected by the ethanol concentration. Overall kinetics for the decomposition of alpha-acetolactate was formulated as a function of ethanol concentration, pH and temperature. The kinetics can be applied to alcoholic beverages such as sake.     

Impact of Autolytic and Proteolytic Lactobacilli and Nisin-Producing Culture on Proteolysis and Sensory Characteristics in Cheddar Cheese

ABSTRACT: Cheddar cheeses were made using a nisin-tolerant starter culture with either Lactobacillus delbrueckii subsp. bulgaricus UL12 (autolytic strain), Lactobacillus casei subsp. casei L2A (proteolytic strain), Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 (nisin producer), or of Lb. bulgaricus UL12 and Lc. diacetylactis UL719. Lb. bulgaricus UL12 produced more trichloroacetic acid-soluble nitrogen than did Lb. casei L2A, which produced more phosphotungstic acid-soluble nitrogen than did Lc. diacetylactis UL719. High-performance liquid chromatography analyses showed that either lactobacilli or Lc. diacetylactis UL719 increased the hydrophilic and hydrophobic peptide contents. Cheeses containing both Lb. bulgaricus UL12 and Lc. diacetylactis UL719 had the most intense old Cheddar cheese flavor after 6 mo of ripening.     

Growth characteristics of Candida kefyr and two strains of Lactococcus lactis subsp. lactis isolated from Zimbabwean naturally fermented milk.

Lactic acid bacteria (LAB) and yeasts constitute part of the microflora in Zimbabwean traditional fermented cows' milk, amasi. The present study was carried out to investigate the growth characteristics of Candida kefyr 23, Lactococcus lactis subsp. lactis biovar. diacetylactis C1 and L. lactis subsp. lactis Lc261, previously isolated from amasi, in ultrahigh temperature (UHT)-treated cows' milk. The strains were inoculated into the UHT milk as both single and yeast     

Screening of some potentially probiotic lactic acid bacteria for their ability to synthesis conjugated linoleic acid

Strains of potentially probiotic lactobacilli, propionibacteria, leuconostoc, lactococcus, enterococcus, and pediococcus, were tested for their ability to convert linoleic acid to conjugated linoleic acid (CLA). Growth and CLA production were followed during incubation for 48 h in reconstituted skim milk containing 0.2% lipolysed sesame oil. Lactococcus lactis subsp. lactis biovar diacetylactis and Leuconostoc mesenteroides subsp. mesenteroides gave the highest CLA production. Also, the effect of lipolysed oil concentration on the growth and CLA production of six strains were studied in medium containing 0.0–1% lipolysed oil. Leuconostoc mesenteroides subsp. mesenteroides and Lac. lactis subsp. lactis biovar diacetylactis gave maximum dienes in medium containing 0.6% and 0.8% lipolysed oil respectively.     

Analysis of Volatile Compounds Produced by 2 Strains of Lactococcus lactis Isolated from Leben (Tunisian Fermented Milk) Using Solid-Phase Microextraction-Gas Chromatography

ABSTRACT:  The volatile compounds that characterize Leben during fermentation with 2 Lactococcus lactis strains (SLT6 and SLT10) in flasks, in a 100-L fermentor, and during storage at 4 °C, were investigated and compared to those from commercial Leben. Volatile compounds from Leben were concentrated by a Carboxen–PDMS fiber and analyzed by GC-MS. These compounds include acids, alcohols, aldehydes, ketones, sulfur compounds, and hydrocarbons. Commercial Leben presented a poor volatile profile compared to the laboratory-made Leben. The mixed culture of 2 Lactococcus lactis strains resulted in higher volatile compound formation than the single strain culture. The GC volatile profiles of Leben produced in flask and in the 100-L fermentor were similar. Changes in volatile compounds were observed during storage at 4 °C. The effect of culture conditions on production of volatiles by SLT6 strain was studied. Aeration (0.1 mL/min) and agitation enhanced the production of diacetyl, acetoin, 3-methylbutanal, and 3-methylbutanol. Fermentation at pH 5 had no effect on volatile production.     

Process for improving the quality of direct acidified Cottage cheese

The effects of plain and fermented curd dressing ripened by single (Lactococcus lactis subsp. lactis biovar. diacetylactis) as well as mixed‐strain starter cultures (L. lactis subsp. lactis; L. lactis subsp. cremoris; L. lactis subsp. lactis biovar. diacetylactis:: 1:1:1), different levels of fat (18–24%) in curd dressing and inoculation rate (1–5%) on direct acidified Cottage cheese were observed. Ripened curd dressing containing 22% fat and mixed‐strain starter cultures at 3% imparted a pleasant acidic note, delicate overtones of diacetyl, improved the body and texture, visual appearance and thereby enhanced the overall quality of the product.     

Analysis of hemin effect on lactate reduction in Lactococcus lactis

Lactococcus lactis is a facultative anaerobic microorganism that produces lactate as the major product, and acetate and acetoin as by-products; some strains of this species produce an antimicrobial compound, nisin. Lactate has a strong inhibitory effect on L. lactis growth. On the other hand, hemin has a suppressive effect on lactate production during L. lactis growth under aerobic condition. To achieve the optimum effect of hemin on lactate amount reduction in L. lactis ATCC11454, cultures entailing various conditions were performed with and without hemin. In the culture with hemin, L. lactis growth and lactate reduction improved compared with those in the culture without hemin; that is, lactate production was suppressed by 1.8- and 1.3-fold under batch and fed-batch cultures, respectively. In microaerobic fed-batch culture with hemin, lactate production was sufficiently suppressed. This result suggests that microaerobic fed-batch culture could be applied to the maintenance of the low lactate amount. Under this condition, metabolic shift was observed from lactate to acetoin and acetate. However, no increase in nisin production was observed even though lactate production could significantly decrease in L. lactis ATCC11454.     

Impact of autolytic, proteolytic, and nisin-producing adjunct cultures on biochemical and textural properties of cheddar cheese

The effect of incorporating a highly autolytic strain (Lactobacillus delbrueckii subsp. bulgaricus UL12) a proteolytic strain (Lactobacillus casei subsp. casei L2A), or a nisin Z-producing strain (Lactococcus lactis, subsp. lactis biovar diacetylactis UL719) into Cheddar cheese starter culture (Lactococcus lactis KB and Lactococcus cremoris KB) on physicochemical and rheological properties of the resultant cheeses was examined. Cheeses were ripened at 7 degrees C and analyzed over a 6-mo period for viable lactococcal and lactobacilli counts, pH, titratable acidity (TA), lipolysis, proteolysis, and textural characteristics. The combination of the nisin-producing strain and autolytic adjuncts significantly increased the production of water-soluble nitrogen, free amino acids, and free fatty acids. The effect of Lc. diacetylactis UL719 alone or of Lb. casei L2A on water-soluble nitrogen and free amino acid contents were also significant, whereas their effect on free fatty acids was not. Viable counts of Lb. bulgaricus UL12 were significantly reduced in the presence of Lc. diacetylactis UL719. Lactobacilli-containing cheeses showed significantly lower values for hardness, fracturability, and springiness. It could be concluded that the addition of Lb. bulgaricus UL12 together with a nisin-producing strain produces a greater increase in cheese proteolysis and an improvement in Cheddar cheese texture.     

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.     

Redirection of Pyruvate Pathway of Lactic Acid Bacteria to Improve Cheese Quality

Metabolic engineering in Lactic acid bacteria (LAB) has focused on changing of pyruvate metabolism to increase production of desired flavor compounds. A constructed mutant strain should contain no foreign DNA and antibiotic resistance genes. Therefore, food grade lactate dehydrogenase (ldh ^d) and diacetyl reductase (dar ^d) mutant strains were created using two plasmid system in this study. Metabolic end products (pyruvate, lactate, formate and acetoin) of these strains in glucose medium and in cheese were determined using HPLC. Created mutant and wild type strains were used as a starter culture in cheese. Compared to the wild type strain, different levels of metabolites were observed in cheese during three weeks of ripening. The ldh ^d strains produced less lactate but high acetoin as a result of gene deletion. Deletion of dar gene decreased the production of acetoin. The dar deficient strains have low diacetyl reductase activity and are able to reduce significant amounts of acetoin but not terminate it completely. Genetic modification made the shift from homolactic to mixed acid fermentation, but the desired compound production hardly improved. The basis of these results and techniques are promising for the further studies.     

Proteolysis and formation of volatile compounds in cheese manufactured with a bacteriocin-producing adjunct culture

Hispánico cheese, a semi-hard Spanish variety, was manufactured from a mixture of pasteurized cows' and ewes' milks (4:1) using a commercial mesophilic LD-type starter comprising Lactococcus lactis subsp. cremoris, Lc. lactis subsp. lactis, Lc. lactis subsp. lactis var diacetylactis and Leuconostoc mesenteroides subsp. cremoris. Varying amounts (0-1.0 g/kg) of an Enterococcus faecalis INIA 4 culture in milk were added as a bacteriocin-producing adjunct. Differences in pH between cheeses manufactured with and without the bacteriocin producer did not exceed 0.11 pH units. Starter lactococci lost viability more rapidly in cheeses made with the bacteriocin producer, which reached counts of up to 6 x 10(7) cfu/g during ripening. Aminopeptidase activity in 1-d-old cheese made from milk inoculated with 1.0 g bacteriocin-producing culture/kg was twice that in control cheese. Degrees of overall proteolysis and levels of total free amino acids in 45-d-old cheese made with 1.0 g bacteriocin-producing culture/kg were 1.80-fold and 2.17-fold those in control cheese of the same age. Inoculating milk with 1.0 g/kg bacteriocin-producing culture reduced the level of hydrophobic peptides in the resultant cheese, increased the concentrations of 3-methyl-1-butanal, diacetyl and acetoin, and resulted in the highest scores for flavour quality and flavour intensity throughout ripening.     

丁二酮乳酸乳球菌发酵对羊奶脂肪酸组成影响分析

为研究乳酸乳球菌乳酸亚种丁二酮变种(Lactococcus lactis ssp.Lactis biovar diacetylactis)在单菌发酵,或与嗜热链球菌(Streptococcus thermophilus)和保加利亚乳杆菌(Lactobacillus bulgaricus)混合发酵条件下对羊奶中脂肪酸含量影响情况,利用气相色谱法进行脂肪酸分析,结果表明:L.diacetylactis发酵显著提高了羊奶中、短链脂肪酸百分含量,降低了长链脂肪酸百分含量(p<0.05);L.diacetylactis接种量对发酵羊奶成品中脂肪酸组成影响不显著;L.diacetylactis与S.thermophilus、L.bulgaricus混合发酵羊奶中脂肪酸组成不受S.thermophilus、L.bulgaricus影响。因此,L.diacetylactis发酵适用于开发风味良好、营养合理的酮香型羊奶保健品。     

乳酸菌发酵产生丁二酮的初步研究

将Hansen公司D 1乳酸菌及英国NCIMB的乳酸乳球菌NCIMB8763分别在牛奶培养基和MRS培养基中进行发酵柠檬酸盐产生丁二酮的特性研究。结果表明 ,Hansen公司D 1乳酸菌株能够发酵柠檬酸盐产生丁二酮 ,最高产量为 98mg/L ,而乳酸乳球菌NCIMB8763则产生很少量的丁二酮     

Effects of mixed starter composition on nisin Z production by lactococcus lactis subsp. lactis biovar. diacetylactis UL 719 during production and ripening of Gouda cheese

A starter culture system that produced both acid and nisin at acceptable rates in milk for manufacture of Gouda cheese was developed using nisin Z-producing L. lactis subsp. lactis biovar. diacetylactis UL 719 (UL 719) and a commercial Flora Danica (FD) starter culture. Different compositions of mixed cultures (0, 0.2, 0.4, 0.6 or 0.8% UL 719 with 1.4% FD) were tested for acidification and nisin Z production in milk after 12 h incubation at 30 degrees C. The 0.6/1.4% combination, selected as the optimal mixture of starter cultures, acidified milk to a suitable pH and produced nisin Z at a high concentration of 512 IU/ml. With this optimal combination, FD numbers of citrate-fermenting and non-fermenting bacteria did not change compared with the control (1.4% FD). However, with 0.8% of L. lactis strain UL 719 and 1.4% of the FD starter culture, the numbers of citrate-fermenting and non-fermenting bacteria in fermented milk decreased compared with those obtained when milk was inoculated with 0.2, 0.4 or 0.6% of UL 719 added to 1.4% FD or control cultures (1.4% FD). Mixed starter culture ratios 0.6/1.4%, 0.4/1.4% and 0.5/1.4% (UL 719/FD) were used to manufacture nisin Z containing Gouda cheese which was ripened up to 45 weeks. The composition of control cheeses made with 1.4% FD, and nisin Z-containing Gouda cheeses were similar with respect to percent moisture, fat, salt and protein. During the ripening period, the cell counts observed were approximately two logs higher in cheese made with the 0.6/1.4% mixed starter culture than in control cheese. In experimental cheese produced with 0.6/1.4% (UL 719/FD) mixed starter culture, nisin activity increased from 256 IU/g at the end of manufacture to a maximum of 512 IU/g after 6 weeks of ripening; the levels then decreased to 128 and 32 IU/g after 27 and 45 weeks of ripening, respectively. In contrast, nisin Z was not detected in experimental cheeses made with 0.4/1.4% or 0.5/1.4% (UL 719/FD) mixed starters. Using an affinity purified anti-nisin polyclonal antibody, anti-rabbit gold-conjugate and transmission electron microscopy, nisin Z was found to be localized in the cheese matrix, in fat globules, in the casein phase and concentrated at the fat-casein interface. After 27 weeks of ripening, nisin Z was detected preferentially in the fat globules of the experimental cheese.     

Comparison of newly isolated strains of Lactobacillus delbrueckii subsp. lactis for hydrogen peroxide production at 5 degrees C

Isolates of Lactobacillus delbrueckii subsp. lactis obtained from raw milk samples were compared for the ability to produce hydrogen peroxide (H2O2) at 5 degrees C. Nineteen out of 101 lactobacilli isolated were identified as L. delbrueckii subsp. lactis. The isolates of L. delbrueckii subsp. lactis from most raw milk samples produced more H2O2 than did isolates of other species of lactobacilli from the same samples. Seven isolates of L. delbrueckii subsp. lactis, which produced the highest levels of H2O2 at 5 degrees C were selected for comparison with a laboratory strain, L. delbrueckii subsp. lactis I. In 24 h, isolate RM2-5 produced 7.0 microg/10(9) cfu in buffer containing 5 mM sodium lactate and 4.4 microg/10(9) cfu in buffer containing 5 mM glucose. Three other isolates also produced more H2O2 on sodium lactate than on glucose. However, three remaining new isolates produced more H2O2 on glucose than on sodium lactate. All seven of the most active new isolates of L. delbrueckii subsp. lactis produced significantly higher concentrations of H2O2 than did L. delbrueckii subsp. lactis I in both solutions. Strain RM2-5 produced more H2O2 than did the other six most active newly isolated strains of L. delbrueckii subsp. lactis in this comparison.     

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.     

微生物发酵与酶法结合制备天然奶味香基的研究

选用具有丁二酮合成能力的乳酸乳球菌NRRL B-2356发酵全脂乳与脂肪酶结合制备天然奶味香基。乳酸乳球菌NRRL B-2356发酵全脂乳丁二酮、3-羟基丁酮产量在27h时达最大(52.95μg/mL),与德氏乳杆菌混合发酵,两者接种量比值为5:1时,丁二酮、3-羟基丁酮含量达60.1μg/mL,香气评分最高。利用脂肪酶palatase2000L对发酵液进行酶解修饰以提高香气强度,酸值为6.73mgKOH/g时香气评分最高。同时蒸馏萃取装置与GC-MS结合对奶味香基中挥发性成分进行了分析,结果表明,游离脂肪酸为98.586%,酮类1.219%,内酯类0.142%。研究制备的奶味香基香气纯正浓郁,可用于调配高档奶味香精。     

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.