利用16S rDNA序列及tuf-RFLP鉴定蒙古国发酵乳中的乳酸菌

运用16S rDNA序列分析和tuf-RFLP技术对采于蒙古国扎布汗省的25份发酵乳样中分离出的110株乳酸菌进行鉴定。首先将分离的110株乳酸菌的16S rRNA基因进行扩增,测序并构建系统发育树,初步鉴定为41株嗜热链球菌,40株瑞士乳杆菌,11株德氏乳杆菌保加利亚亚种,2株发酵乳杆菌,1株乳明串珠菌,2株肠膜明串肠膜亚种,1株乳酸乳球乳酸亚种和12株属于干酪族的菌株。由于干酪乳杆菌族的16S rDNA序列差异很小,故采用tuf-RFLP技术对这12株进行了进一步的验证,通过分离菌株与模式菌株tuf-RFLP图谱的比较分析,结果表明这12株菌均为干酪乳杆菌。     

Chemical and microbiological characterisation of kefir grains

Chemical and microbiological composition of four Argentinean kefir grains from different sources as well as characteristics of the corresponding fermented milk were studied. Kefir grains CIDCA AGK1, AGK2 and AGK4 did not show significant differences in their chemical and microbiological composition. In contrast, protein and yeast content of AGK3 was higher than in the other grains. Although grain microflora comprised lactobacilli, lactococcus, acetic acid bacteria and yeast, we found an important difference regarding species. Lactococcus lactis subsp. lactis, Lactobacillus kefir, Lactobacillus plantarum, Acetobacter and Saccharomyces were present in all types of kefir grain. While Leuconostoc mesenteroides was only isolated from grains CIDCA AGK1 and Lactococcus lactis subsp. lactis biovar diacetylactis, Lactobacillus parakefir and Kluyveromyces marxianus were only isolated from CIDCA AGK2 grains. All grains produced acid products with pH between 3.5 and 4.0. The apparent viscosity of AGK1 fermented milk was greater than the product obtained with AGK4. All fermented milks had inhibitory power towards Escherichia coli but AGK1 and AGK2 supernatants were able to halt the bacterial growth for at least 25 h. Grain weight increment in AGK1, AGK2 and AGK3 during growth in milk did not show significant differences. Despite their fermenting activity, AGK4 grains did not increase their weight.     

保加利亚乳杆菌诱导产亚油酸异构酶条件研究

亚油酸异构酶可由保加利亚乳杆菌经诱导产生,可以将亚油酸(LA)转化为共轭亚油酸(CLA)。本实验对诱导保加利亚乳杆菌产亚油酸异构酶的条件进行研究,利用紫外和气质联用仪(GC-MS)检测所生成的CLA。结果表明：在培养基中添加1.5‰(V/V) LA 时所产酶的共轭亚油酸转化率最高;温度为36℃,培养36h 为较适的培养条件;单独添加0.1%(m/V)的乳糖或0.1%(m/V)的氯化钠有利于诱导产酶;在培养基中直接添加LA 的效果优于培养3至12h 后再进行添加。诱导所产酶可将LA 转化为CLA,且含有9c,11t-CLA 异构体。     

Conjugated linoleic acid concentration as affected by lactic cultures and added linoleic acid

Six lactic cultures: Lactobacillus acidophilus (CCRC14079), L. delbrueckii subsp. bulgaricus (CCRC14009), L. delbrueckii subsp. lactis (CCRC14078), Lactococcus lactis subsp. cremoris (CCRC12586), Lc. lactis subsp. lactis (CCRC10791), and Streptococcus salivarius subsp. thermophlius (CCRC12257) were tested for the effects of addition of 1000 and 5000 μg/ml linoleic acid, and incubation time from 0 to 48 h. The levels of conjugated linoleic acid (CLA) formation were determined by gas chromatography. A sharp increase in CLA level was observed in linoleic acid added cultures. Incubation of L. acidophilus in 1000 μg/ml linoleic acid added-skim milk medium for 24 h was most effective in promoting CLA formation. Increasing linoleic acid addition from 1000 to 5000 μg/ml and prolonging incubation from 24 to 48 h did not appear to enhance CLA formation.     

The microbiology of South African traditional fermented milks

A total of 15 samples of traditional fermented milk were collected from individual households in South Africa and Namibia. Lactic acid bacteria dominated the microflora of these samples, especially the genera Leuconostoc, Lactococcus and Lactobacillus. Other groups identified included pyogenic streptococci and enterococci. The dominant lactococci species was Lactococcus lactis subsp. lactis. Eighty-three percent of the leuconostoc isolates were identified as Leuconostoc mesenteroides subsp. dextranicum. Other species identified included Leuconostoc citreum, Leuconostoc lactis, Lactobacillus delbrueckii subsp. lactis and Lactobacillus plantarum.     

Increase of Conjugated Linoleic Acid Content in Milk by Fermentation with Lactic Acid Bacteria

ABSTRACT: The objectives of this study were to identify the factors and procedures responsible for increasing the conjugated linoleic acid (CLA) content in fermented milk. Fourteen lactic acid bacteria were screened for CLA-producing ability using sunflower oil (containing 70% linoleic acid) as a substrate. Among the screened strains, Lactococcus lactis I-01 showed the highest CLA-producing ability. The optimal concentration of sunflower oil for CLA production was 0.1 g/L in whole milk, which accounted for 0.25% of total milk fat. Our results demonstrated that CLA formation in fermented milk could be affected by numerous factors such as bacterial strain, cell number, optimal substrate concentration, and the period of incubation at neutral pH.     

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.     

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.     

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     

Growth inhibition of Listeria monocytogenes by a bacteriocin of Pediococcus acidilactici M during fermentation of kimchi

Four lactic acid bacteria known to produce bacteriocins were evaluated for the inhibitory effect against Listeria monocytogenes strain Scott A. Lactococcus lactis subsp. lactis biovar diacetylactis 26-2 was the least antilisterial, whereas Pediococcus acidilactici M resulted in the largest zone of inhibition on an agar medium. Leuconostoc paramesenteroides OX and Lactobacillus sake Lb 706 were intermediate. Aqueous solutions of crude bacteriocin powders derived from supernatant fluids of L. sake Lb 706 and P. acidilactici M. cultures inhibited the growth of Lactobacillus plantarum UGA8 in MRS agar. The addition of 10 mg of crude bacteriocin powder from L. sake to 150 g of kimchi fermented at 14 ± 1°C for 16 days had no effect on the growth of L. monocytogenes. The same amount of crude bacteriocin derived from P. acidilactici, however, had an initial lethal effect on L. monocytogenes and controlled growth of the pathogen throughout the 16-day fermentation. The successful application of bacteriocins to control the growth of L. monocytogenes in lightly fermented foods such as kimchi would appear to be achievable.     

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.     

Inhibitory and stimulatory effects of cumin, oregano and their essential oils on growth and acid production of Lactobacillus plantarum and Leuconostoc mesenteroides

Cumin at concentrations of 0.5, 1.0 and 2.0% (w/w) stimulated growth and acid production of Lactobacillus plantarum and Leuconostoc mesenteroides in a liquid medium. Essential oil from cumin at high concentrations (300 and 600 ppm) inhibited growth and acid production of Lactobacillus plantarum. After a certain period, growth of Leuconostoc mesenteroides was observed at all concentrations (150, 300 and 600 ppm) and its acid production was stimulated at 600 ppm. Oregano and its essential oil at all concentrations inhibited growth of both cultures. Acid production by Lactobacillus plantarum was stimulated by oregano spice.     

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.     

Lactococcus Genus: A selective and Differential Agar Medium

An agar medium (Alsan), was selective for Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. lactis biovar. diacetylactis. The former developed white or yellow colonies; the latter gave blue-green or blue colonies. Eleven strains of L. lactis subsp. cremoris did not grow in this medium. The medium contained phenylethanol agar, lactose, glycine anhydride, lithium chloride and trimethoprim. In comparison with M17 medium, Alsan was not significantly different (P > 0.05) in recovering either bacterium. Furthermore, the medium inhibited the common food genera Pseudomonas, Escherichia, Leuconostoc, Enterococcus and Lactobacillus. Bacteriophage plaque sizes of 0.4–0.5 cm were demonstrated on the medium using a lactococcal bacteriophage.     

盐渍藠头发酵过程中乳酸菌的分离及特性研究

对盐渍藠头发酵过程中不同时期的乳酸菌进行了分离、鉴定及特性研究,从中分离出9种菌株。通过菌落形态、培养特征和生化试验等鉴定表明,这9种菌株分别为植物乳球菌、发酵乳杆菌、短乳杆菌、菊糖芽孢杆菌、乳酸链球菌、肠膜明串珠菌葡聚糖亚种、植物乳杆菌、皱膜假丝酵母、膜醭毕赤氏酵母菌。并观察了发酵过程中各个时期的主导菌相,最后得出短乳杆菌、发酵乳杆菌可作为盐渍藠头发酵过程中人工接种的试验菌株。     

Response of milk fatty acid composition to dietary supplementation of soy oil, conjugated linoleic acid, or both

Thirty-six Holstein cows were blocked by parity and allotted by stage of lactation to 6 treatments to evaluate the effects of dietary soy oil, conjugated linoleic acid (CLA; free acid or calcium salt), or both, on CLA content of milk. Diets were fed for 4 wk and are as follows: (1) control, (2) control + 5% soy oil, (3) control + 1% CLA, (4) control + 1% Ca(CLA)₂, (5) control + 1% CLA + 4% soy oil, and (6) control + 1% Ca(CLA)₂ + 4% soy oil. Rumen volatile fatty acid -concentrations, blood fatty acid concentrations, milk yield, and milk composition were measured weekly or biweekly. Dry matter intake and milk yield were recorded daily. Dietary supplementation of soy oil or CLA had no effect on daily milk yield, milk protein concentration and production, or milk lactose concentration and production. Supplementation of unsaturated fatty acids as soy oil, CLA, or Ca(CLA)₂ increased total fatty acid concentration in plasma, decreased milk fat concentration and production, and had no effect on rumen volatile fatty acid concentrations. The weight percentage of CLA in milk was increased from 0.4 to 0.7% with supplementation of 1% CLA, to 1.2% with supplementation of soy oil, and to 1.3% with supplementation of 1% CLA plus soy oil. Supplementation with Ca(CLA)₂ or Ca(CLA)₂ + soy oil increased the CLA content of milk fat to 0.9 and 1.4%, respectively. In summary, adding 5% soy oil was as effective as supplementing CLA, Ca(CLA)₂, or a combination of 1% CLA (free acid or calcium salt) + 4% soy oil at increasing CLA concentrations in milk fat. Feeding CLA as the calcium salt resulted in greater concentrations of CLA in milk fat than did feeding CLA as the free acid. Dietary supplementation of 5% soy oil or 4% soy oil + 1% CLA as the free acid or the calcium salt increased the yield of CLA in milk.     

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.     

Isolation of halotolerant Lactococcus lactis subsp. lactis from intestinal tract of coastal fish

We isolated lactic acid bacteria from the intestinal tract of the pufferfish Takifugu niphobles caught in Shimoda, Shizuoka, Japan by using MRS broth prepared with 50% seawater. Additional screening was carried out using phenotypic tests such as Gram staining, cell morphology, catalase, oxidase and fermentation of glucose. Subsequently 227 isolates screened by the phenotypic tests were subjected to species-specific PCR for Lactococcus lactis, resulting in four positive isolates. The 16S rRNA gene sequences from three isolates were highly similar to that of L. lactis subsp. lactis (DNA database accession number M58837), while that of one isolate was identical to that of Leuconostoc mesenteroides (AB023246). These isolates were characterized by API 50 CH for carbohydrate fermentation and other phenotypic criteria for salt tolerance, and the characteristics were compared with those of L. lactis subsp. lactis from a cheese starter culture. The carbohydrate fermentation profiles of these isolates were characteristic of L. lactis subsp. lactis strains, whereas the tolerance of these isolates to salt was higher than that of L. lactis subsp. lactis from the cheese starter culture: the new L. lactis isolates showed high salt tolerance in MRS-agar plates containing 200% seawater or 6% sodium chloride. This is the first report of the isolation of halotolerant strains of L. lactis subsp. lactis from a marine environment.     

Production of free conjugated linoleic acid by Lactobacillus acidophilus and Lactobacillus casei of human intestinal origin

A gas chromatographic procedure was used for analysis of conjugated linoleic acid (CLA) isomers cis-9, trans-11-octadecadienoic; trans-10, cis-12 octadecadienoic; and trans-9, trans-11-octadecadienoic (c9t11, t10c12, t9t11) produced by lactobacilli. Four different cultures, two strains each of Lactobacillus acidophilus and Lactobacillus casei were tested for their ability to produce CLA from free linoleic acid in MRS broth supplemented with linoleic acid. Different concentrations of linoleic acid (0, 0.05, 0.1, 0.2 and 0.5 mg/ml) were added to MRS broth, inoculated with the lactobacilli, and incubated at 37 degrees C. Viable counts and amounts of individual isomers of CLA (c9t11, t10c12, t9t11) were measured at 0, 24, 48, and 72 h. All the cultures were able to produce free CLA in media supplemented with linoleic acid. Maximum production of CLA (80.14 to 131.63 microg/ml) was observed at 24 h of incubation in broth containing 0.02% of free linoleic acid. No significant (P > 0.05) increases in total CLA levels were observed after 24 h of incubation. The ability of the cultures to produce CLA in skim milk supplemented with 0.02% free linoleic acid also was studied. In this medium, the total amounts of free CLA after 24 h of incubation ranged from 54.31 to 116.53 microg/ml. The use of lactic acid bacteria able to form free CLA in cultured dairy products may have potential health or nutritional benefits. Free CLA in the products likely would be more readily available for absorption from the digestive tract than if it were incorporated into the cells of the starter culture.