Lactis Biovar Research Articles

Fatty acid profile of sour dairy products produced by different starter cultures

In this research we have investigated the effect of various cultures (Lactobacillus lactis subsp. lactis, Lactobacillus lactis subsp. cremoris, Streptococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus lactis subsp. lactis biovar, Lactobacillus diacetilactis, Lactobacillus acidophilus, Bifidobacterium lactis) on fatty acid composition of soured dairy products (Sana, yoghurt) manufactured using different technologies, with special regard to conjugated linoleic acid (CLA). It was established that the cultures we used and which are also commonly used in the dairy industry, had only a minor effect on fatty acid composition of milk. Although minimal differences were found in case of the individual fatty acids, however, due to the small differences it can be established, that the cultures have no influence on nutritional value of milk fat.

Utilization of UF-Permeate for Production of β-galactosidase by Lactic Acid Bacteria

Four lactobacilli strains (Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacilus casei and Lactobacillus reuteri) were grown in MRS broth and three lactococci strains (Streptococcus thermophilus, Lactococcus lactis subsp. Lactis and Lactococcus lactis subsp. lactis biovar. diacetilactis) were grown in M17 broth. L. reuteri and S. thermophilus were chosen on the basis of the best mean beta-galactosidase activity of 10.44 and 10.01 U/ml respectively, for further studies on permeate-based medium. The maximum production of beta-galactosidase by L. reuteri was achieved at lactose concentration of 6%, initial pH 5.0-7.5, ammonium phosphate as nitrogen source at a concentration of 0.66 g N/L and incubation temperature at 30 degrees C/24 hrs to give 6.31 U/ml. While in case of S. thermophilus, maximum beta-galactosidase production was achieved at 10% lactose concentration of permeate medium, supplemented with phosphate buffer ratio of 0.5:0.5 (KH2PO4:K2HPO4, g/L), at initial pH 6.0-6.5, ammonium phosphate (0.66g N/L) as nitrogen source and incubation temperature 35 degrees C for 24 hrs to give 7.85 U/ml.

Cell surface properties affect colonisation of raw milk by lactic acid bacteria at the microstructure level

Cell localisation in the matrix microstructure of fermented food is important for microbial activity and ripening. To evaluate the importance of physicochemical factors in these phenomena, the physicochemical behaviour of four strains of lactic acid bacteria were investigated after inoculation of raw milk or milk fractions. To discriminate between physicochemical and metabolic properties, media were (i) rapidly acidified to avoid cell division or (ii) bacteria were allowed to ferment milk. To evaluate the impact of milk components, cells were dispersed in protein or fatty globule fractions. From two Lactococcus lactis subsp. lactis biovar. diacetylactis strains selected from their different surface properties investigated in previous studies, after rapid acidification one was encountered in fatty globules and protein gels whereas the other was found in serum and on the surface of globules. After fermentation, the first one formed colonies in the lipid phase or next to it in the whey and the second formed colonies in the protein gel. Strains forming long chains (Lactobacillus bulgaricus and Streptococcus thermophilus) were always found in contact with fatty globules, where they developed. Our results suggest that localisation and subsequent spatial colonisation depend on the physicochemical properties of cell surfaces, on the structure in chains as well as on the metabolic ability to develop on proteins or lipids.

Inhibition of 1,2-dimethylhydrazine induced colon genotoxicity in rats by the administration of probiotic curd

Epidemiologic and experimental studies suggest that the probiotic organisms are effective in preventing colon carcinogenesis, which is the major cause of mortality and morbidity in western countries. Keeping this in view, a curd (a common Indian fermented milk product) was prepared by the addition of probiotic cultures Lactobacillus acidophilus, Lactobacillus casei and curd culture Lactococcus lactis biovar. diacetylactis. In present study, we have evaluated the anti tumor effect of probiotic curd by monitoring the DNA damage through comet assay. The rats were allocated to four groups, first group was DMH control group, second group was probiotic curd group in which probiotic curd was given along with DMH (1,2-dimethylhydrazine) injection, third group was normal curd group in which normal curd was given along with DMH injection and fourth group was normal control group. Animals received subcutaneous injection of DMH dissolved in normal saline at a dose rate of 20 mg/kg body weight, once weekly for 15 weeks. The rats were dissected at 40th week of experiment and comet assay was done in colonic cells to assess the DNA damage. A significant reduction in DNA damage (54.7%) was observed in probiotic curd group as compared to DMH control group (88.1%). The probiotic curd was effective to significantly reduce the L:W ratio in comparison to DMH control group and normal curd. The results of present study show the protective effects of probiotic curd against DMH induced genotoxicity in colonic cells.

Identification of Lactococcus lactis subsp. Isolates Based on Their Cell Wall Protein Profiles and Plasmid Contents

In this study, among the total 50 lactococci isolates, 44% L. lactis subsp. lactis, 28% L. lactis subsp. lactis biovar. diacetylactis and 28% L. lactis subsp. cremoris strains were identified by their phenotypic characterization. According to the cell wall protein patterns, 8.6, 12.5, 14.7, 20.3, 23.4, 25.4, and 31.8 kDa proteins were found identical within 82% of isolates. Moreover 93% of L. lactis subsp. cremoris strains contained a unique 157.8 kDa protein; however, L. lactis subsp. lactis and L. lactis subsp. lactis biovar. diacetylactis had no unique protein in their groups. Also a key plasmid profile, which would be used to determine the isolates at subspecies level, could not be obtained.

Behavior of Lactococcus lactis subsp. lactis biovar. diacetylactis in a Four Lactococcus Strain Starter during Successive Milk Cultures

Cheeses are frequently made with natural whey starters (NWS). The whey from the previous cheese making is cultured and used for the next day. This practice, although essential for the development of typical sensory characteristics, can sometimes lead to acidification defects. In this work, the ability of Lactococcus lactis subsp. lactis biovar. diacetylactis to dominate over the other lactic acid bacteria (LAB) was tested in a controlled system as a possible explanation for these acidification breakdowns. A starter made of two Lc lactis subsp. lactis strains (LL), one Lc lactis subsp. cremoris strain (LC), and one Lc lactis subsp. lactis biovar. diacetylactis (LD) was added to sterilized milk. After incubation, the whey was removed and used to re-seed sterilized milk, the next day. This process was made during a five and twelve days' period. During the eight first days, the proportion of LD population increased, while the other LAB remained rather stable. Thereafter, LD strains dominated. At the same time, the diversity of LD population diminished considerably. If acidification ability of these LAB is not altered, this simplification was particularly hazardous in case of phages attack. LC and LL behavior was tested in milk containing increasing diacetyl concentrations. As long as diacetyl did not exceed a 5 ppm level — frequently measured in dairy products — its influence was impossible to detect. The selective advantage conferred by the citrate metabolism was proposed as a possible explanation for the LD population dominance. Other potential factors were also examined.

Interaction between starter bacteria and adjunct Lactobacillus plantarum INF15D on the degradation of citrate, asparagine and aspartate in a washed-curd cheese

Washed-curd brine-salted cheeses were made with four different starters: citrate degrading (Cit +) heterofermentative mesophilic DL-starters (CHN-11 and CHN-19), a citrate negative (Cit −) mesophilic homofermentative O-starter (R-704) and a thermophilic starter TCC-20, all with the addition of an adjunct Lactobacillus plantarum INF15D to study the interaction between the starter and adjunct on the degradation of citrate to aspartate (Asp), diacetyl and acetoin in cheese. Citrate degradation by Lb. plantarum INF15D in cheese varied according to the type of starter used. In cheese made with Cit + DL-starters, the degradation of citrate and Asp by the adjunct Lb. plantarum INF15D was less extensive as diacetyl and acetoin were produced from citrate via α-acetolactate by Cit + Lactococcus lactis subsp. lactis biovar. diacetylactis and/or Leuconostoc mesenteroides subsp. cremoris. In the presence of a Cit − O-starter, Lb. plantarum INF15D degraded citrate mainly to Asp which was further converted to acetoin and diacetyl. However, in the presence of a thermophilic starter, Lb. plantarum INF15D degraded citrate mainly to succinic acid. The highest degradation of asparagine (Asn) was found in cheese made with the Cit − O-starter (R-704) and adjunct Lb. plantarum INF15D. Both Lb. plantarum INF15D and the starter cultures were capable of deaminating Asn to Asp.

Selective enumeration and viability of Bifidobacterium animalis subsp. lactis in a new fermented milk product

One of the key focuses of today’s dairy industry worldwide is the continued development of new products, especially probiotic-based products. Buttermilk is originally a by-product of butter making fermented by Mesophilic Aromatic Cultures (MAC). It can also be made by fermentation of pasteurized whole milk or skimmed milk. This product is not marketed in Brazil. The objectives of this work were: (1) to develop a selective medium for Bifidobacterium animalis subsp. lactis enumeration and (2) to determine the viability of this microorganism during the shelf life of the buttermilk. Skim milk added with 10% sucrose or 0.03% sucralose was pasteurized and inoculated with a composite starter culture consisting of 1% MAC (containing Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc mesenteroides subsp. cremoris) and 2% Bifidobacterium animalis subsp. lactis. To attain selective counts of Bif. animalis subsp. lactis the MRS agar supplemented with 0.5% L-cysteine hydrochloride at 10%, 1% lithium chloride at 10%, 0.01% aniline blue and 0.5% dicloxacillin at 0.1% was modified by increasing the antibiotic concentration, addition of NaCl, adjusting pH to 4.8 or increasing the incubation temperature (from 37 to 45oC). Raising the incubation temperature to 45oC was found to be efficient in inhibiting the MAC cultures, even in media not added with dicloxacillin. Bif. animalis subsp. lactis exhibited high viability in the product. The buttermilk product prepared with sucrose and sweetener contained in excess of 10 8 cfu.ml -1 bifidobacteria throughout the shelf life of the product (28 days).

Selection and properties of α-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 α-acetolactate decarboxylase are used in some food processes for their ability to produce large amounts of diacetyl. However, up until now, the use of α-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 α-acetolactate decarboxylase-deficient mutants of S. thermophilus. We observed that in a chemically defined medium (CDM) containing α-ketobutyrate plus leucine, or α-ketobutyrate plus leucine plus isoleucine, the α-acetolactate decarboxylase-deficient mutant TIL865, obtained by directed mutagenesis, grew faster than its parent strain. This property was used for selecting spontaneous α-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.

Effect of addition of native cultures on characteristics of Armada cheese manufactured with pasteurized milk: A preliminary study

Six batches of Armada cheese were produced, one from raw milk with no added starter, another from pasteurized milk with a commercial mesophilic starter and four from pasteurized milk with experimental starters. These starters included: Lactococcus lactis subsp. lactis and L. lactis subsp. lactis biovar. diacetylactis; or the same strains plus either Enterococcus raffinosus, Leuconostoc mesenteroides subsp. dextranicum or Lactobacillus plantarum, all isolated from Armada cheese made from raw milk. The highest counts of aerobic mesophilic bacteria and populations of lactic acid bacteria for all batches were reached in the first week of ripening. These counts declined later throughout the ripening, although not at identical rates in every batch. Counts of lactobacilli were significantly higher in cheeses made from raw milk and those inoculated with the lactobacilli strain than in the other batches, over the whole ripening period. Added native starters minimized growth of Enterobacteriaceae. Cheeses made with the starter containing the Enterococcus strain had the most favourable sensory attributes throughout ripening.

Milk fermentation by functional mixed culture producing nisin Z and exopolysaccharides in a fresh cheese model

A nisin Z-producing strain, Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 and two nisin-sensitive cultures, Lactobacillus rhamnosus RW-9595 M producing exopolysaccharide (EPS), and Lc. lactis subsp. cremoris for acidification, were tested in pure and mixed cultures during milk fermentation. The mixed culture of the three strains showed a higher acidifying capacity at 34°C and 38°C, even though populations of Lc. cremoris were largely reduced compared with pure cultures. Bacteriocin production was 3.1–4.6-fold higher in mixed cultures than for pure cultures of Lc. diacetylactis UL719. These data can be explained by commensalism behavior relying on high proteolytic activity of Lc. cremoris and autolysis and nisin Z-induced lysis. In mixed culture, EPS production was 3-fold lower than for Lb. rhamnosus RW-9595 M pure culture. Our data showed that this strain combination, with nisin-producing and sensitive strains, can be used in mixed cultures for manufacture of fresh cheese with improved functional properties.

A novel plasmid-encoded phage abortive infection system from Lactococcus lactis biovar. diacetylactis.

A 16-kb plasmid (pND859) was identified from Lactococcus lactis biovar. diacetylactis UK12922 which encodes phage resistance to the small isometric phage 712 when tested in L. lactis LM0230. The gene encoding phage abortive infection, designated abi-859, was localized on a 1.2-kb region which consists of an open reading frame (ORF) of 846 bp preceded by a potential ribosome-binding site and a putative promoter region. A helix-turn-helix region typical of DNA-binding motifs was identified near the N-terminal of the abi-859 product, suggesting a possible interaction with the phage DNA.

Antimicrobial properties of lactic acid bacteria and yeast-LAB cultures isolated from traditional fermented milk against pathogenic Escherichia coli and Salmonella enteritidis strains

The survival and growth of Escherichia coli 3339 and Salmonella enteritidis 949575 isolated from human clinical samples, in milk fermented with lactic acid bacteria (LAB) and yeast strains previously isolated from Zimbabwean naturally fermented milk (NFM) was studied. The LAB starter cultures used were Lactococcus lactis subsp. lactis biovar. diacetylactis C1 alone (C1) or in combination with Candida kefyr 23 (C1/23), L. lactis subsp. lactis Lc261 alone (LC261) or in combination with C. kefyr 23 (Lc261/23). The growth of the same pathogens in milk fermented with a commercial DL culture (CH-N 22) and spontaneously fermented raw milk was also monitored. The C1 and C1/23 cultures significantly ( P < 0.05) inhibited the growth of both pathogens. When inoculated at the beginning of the fermentation, both E. coli 3339 and S. enteritidis 949575 counts were significantly ( P < 0.05) reduced by about two log cycles in C1 and C1/23 cultured milk. However, in naturally fermented milk and the DL cultured milk, both E. coli 3339 and S. enteritidis 949575 grew and reached high populations of about 9 and 8.8 log cfu ml − 1 , respectively, after 18 h. When E. coli 3339 was inoculated into previously fermented milk, the viable counts were significantly ( P < 0.05) reduced in the presence of C1 and C1/23 from 7 log cfu ml − 1 to 3 log cfu ml − 1 after 48 h. S. enteritidis 949575 could not be recovered from these cultures after 48 h. The addition of the yeast did not enhance or diminish the inhibitory capacity of the LAB cultures. The pathogens survived in high numbers when inoculated into pre-fermented NFM and the commercial DL- (CH-N 22) cultured milk. The C1 strain, therefore, offered the best protection against the pathogens. Its inhibitory effect was mainly related to fast acid production.

Increased stress tolerance of Bifidobacterium longum and Lactococcus lactis produced during continuous mixed-strain immobilized-cell fermentation.

The effect of immobilization and long-term continuous culture was studied on probiotic and technological characteristics of lactic acid and probiotic bacteria. A continuous culture in a two-stage system was carried out for 17 days at different temperatures ranging from 32 to 37 degrees C, with a first reactor containing Bifidobacterium longum ATCC 15707 and Lactococcus lactis subsp. lactis biovar. diacetylactis MD immobilized separately in gel beads, and a second reactor operated with free cells released from the first reactor. The tolerance of free cells from both strains produced in the effluent medium of both reactors to hydrogen peroxide, simulated gastric and intestinal juices, antibiotics and nisin, and freeze-drying markedly increased with culture time and was generally higher after 6 days than that of stationary-phase cells produced during free-cell batch fermentations. The reversibility of the acquired tolerance of B. longum, but not L. diacetylactis, to antibiotics was shown during successive free-cell batch cultures. Free cells produced from continuous immobilized-cell culture exhibited altered physiology and increased tolerance to various chemical and physico-chemical stresses. Continuous culture with immobilized cells could be used to produce probiotic and lactic acid bacteria with enhanced technological and probiotic characteristics.

Microorganisms Involved in Fulani Traditional Fermented Milk in Burkina Faso

Thirty samples of traditional fermented milk were collected in northern Burkina from Fulani individual household. Microbial analysis was done by standard methods and with API 50 CH identification system. The predominant microflora was lactic acid bacteria, belonging to the genus Lactobacillus (32%), following by Leuconostoc (30%), Lactococcus (20%), Leuconostoc/s-bacterium (10%), Streptococcus (6%) and Enteroccoccus (2%) genus. Yeasts, molds and Enterobacteria were also isolated. Twenty representative lactic acid bacteria strains were identified to species level belonging to species Lactococcus lactis ssp. lactis, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactobacillus confusus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus plantarum, Leuconostoc citreum, Leuconostoc lactis.

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.

Co-fermentation of glucose and citrate by Lactococcus lactis diacetylactis: quantification of the relative metabolic rates by isotopic analysis at natural abundance.

The simultaneous catabolism of citrate and glucose by growing Lactococcus lactis subsp. lactis biovar. diacetylactis to obtain energy was followed quantitatively, using a non-enrichment isotopic technique. Both citrate and glucose are precursors of pyruvate, which may either be reduced to lactate, the principle product that accumulates, or be converted to diacetyl and acetoin. Under suitable conditions, both routes regenerate NAD+. Until recently, however, the quantitative relationships between the two substrates and these three products were poorly defined. It was recently shown, by exploiting differences in natural abundance 13C/12C ratios in the two substrates, that there is no metabolic separation of the catabolism of these two carbon sources. In this study, it is shown that the relative consumption rates change throughout the growth phase, citrate being preferentially metabolised at the onset of a culture of energy-depleted cells, with a subsequent evolution towards a metabolism dominated by glucose consumption. Additionally, it is shown that the relative consumption rates are influenced by environmental factors, notably initial pH and temperature.

Technological characterization of lactic acid bacteria isolated from Armada cheese (a Spanish goats’ milk cheese)

Thirty-one strains of lactic acid bacteria (LAB) isolated from Armada cheese, Sobado variety, (eight strains of Lactococcus lactis subsp. lactis, four strains of Lactococcus lactis subsp. cremoris, two strains of L. lactis subsp. lactis biovar. diacetylactis, two strains of Leuconostoc mesenteroides subsp. mesenteroides, two strains of Leuconostoc mesenteroides subsp. dextranicum, five strains of Lactobacillus plantarum, six strains of Lactobacillus casei subsp. casei and two strains of Lactobacillus brevis) were screened for their acidifying capacity and enzymatic activity, that included the rapid API-ZYM system, the proteolytic activity, the amino-, di-, and carboxypeptidase activity and the caseinolytic activity. The strains of L. lactis subsp. lactis exhibited the highest acidifying and proteolytic activity. Lipase and esterase activity was practically non-existent for lactococci and lactobacilli; a certain esterase activity was observed among leuconostoc. The highest aminopeptidase activity was demonstrated by the cell-free extract (CFE) of some strains of L. plantarum, L. casei subsp. casei and L. mesenteroides subsp. dextranicum. The CFEs of L. lactis subsp. cremoris and L. lactis subsp. lactis possessed carboxypeptidase and dipeptidase activities, at levels depending on the strain. Appreciable caseinolytic activity was detected for the CFE of L. plantarum and those some lactococci.

Molecular Characterization of a Lactococcal Plasmid Reducing the Growth Rate of Host Cells

Lactococcus Lactis subsp. lactis biovar. diacetylactis DRC1 carries more than 6 plasmids, including a 7.4 kb cryptic plasmid, which was designated as pDR1-1. The pDR1-1 plasmid was found to significantly affect the maximum specific growth rate (μ m a x ) of the host cells because of its limiting effect on growth. To investigate the properties of the limiting effect, the entire nucleotide sequence of pDR1-1 was determined. It consisted of 7412 bp, and 6 open reading frames (ORFs) were identified. The first ORF showed a high degree of similarity to a family of replication genes (rep) that are commonly found in lactococcal strains. The rep gene in pDR1-1was followed by a second ORF of unknown function. Directly downstream of the second ORF, a third ORF was found, that showed homology to the S subunit from type 1 restriction/modification systems. No significant similarity to the contents of the database was found for the other ORFs. PCR analysis was carried out in order to detect pDR1-1 in the other L lactis strains. The μ m a x of the pDR1-1-positive strain was the same as that of DRC1. These results suggest that the load of pDR1-1 (or pDR1-1-like plasmid) is a major factor influencing the μ m a x of DRC1 because of its limiting effect on growth, an effect which is much more pronounced than that produced by the overall load of other coexisting plasmids.

Quantitative determination of the spatial distribution of pure- and mixed-strain immobilized cells in gel beads by immunofluorescence.

A new method was developed to detect and quantify two strains, Lactococcus lactis subsp. lactis biovar. diacetylactis MD and Bifidobacterium longum ATCC 15707, immobilized separately and co-immobilized in gel beads, using specific polyclonal antibodies and confocal laser-scanning microscopy. The establishment of biomass concentration profiles for each strain was measured during colonization of beads using successive pH-controlled batch fermentations. Growth occurred preferentially in 200- and 300-microm peripheral layers of the beads for L. diacetylactis and B. longum, respectively. Repeated-batch cultures with immobilized cells permitted the production of a mixed culture containing a non-competitive strain of bifidobacteria, as a result of immobilized-cell growth and high cell-release activity from the beads. During co-immobilized fermentations, there were no apparent interactions between the strains.