Diversity of lactic acid bacteria associated with traditional fermented dairy products in Mongolia

Spontaneous milk fermentation has a long history in Mongolia, and beneficial microorganisms have been handed down from one generation to the next for use in fermented dairy products. The objective of this study was to investigate the diversity of lactic acid bacteria (LAB) communities in fermented yak, mare, goat, and cow milk products by analyzing 189 samples collected from 13 different regions in Mongolia. The LAB counts in these samples varied from 3.41 to 9.03 log cfu/mL. Fermented yak and mare milks had almost identical mean numbers of LAB, which were significantly higher than those in fermented goat milk but slightly lower than those in fermented cow milk. In total, 668 isolates were obtained from these samples using de Man, Rogosa, and Sharpe agar and M17 agar. Each isolate was considered to be presumptive LAB based on gram-positive and catalase-negative properties, and was identified at the species level by 16S rRNA gene sequencing, multiplex PCR assay, and restriction fragment length polymorphism analysis. All isolates from Mongolian dairy products were accurately identified as Enterococcus faecalis (1 strain), Enterococcus durans (3 strains), Lactobacillus brevis (3 strains), Lactobacillus buchneri (2 strains), Lactobacillus casei (16 strains), Lactobacillus delbrueckii ssp. bulgaricus (142 strains), Lactobacillus diolivorans (17 strains), Lactobacillus fermentum (42 strains), Lactobacillus helveticus (183 strains), Lactobacillus kefiri (6 strains), Lactobacillus plantarum ssp. plantarum (7 strains), Lactococcus lactis ssp. lactis (7 strains), Leuconostoc lactis (22 strains), Leuconostoc mesenteroides (21 strains), Streptococcus thermophilus (195 strains), and Weissella cibaria (1 strain). The predominant LAB were Strep. thermophilus and Lb. helveticus, which were isolated from all sampling sites. The results demonstrate that traditional fermented dairy products from different regions of Mongolia have complex compositions of LAB species. Such diversity of LAB provides useful information for further studies of probiotic strain selection and starter culture design, with regard to the industrial production of traditional fermented milk.     

Probiotic potential and biochemical and technological properties of Lactococcus lactis ssp. lactis strains isolated from raw milk and kefir grains

Lactococcus lactis ssp. lactis is one of the most important starter bacteria used in dairy technology and it is of great economic importance because of its use in the production of dairy products, including cheese, butter, cream, and fermented milks. Numerous studies have evaluated the biochemical and probiotic properties of lactococci; however, limited studies on the probiotic characteristics of lactococci were conducted using strains originating from raw milk and dairy products. Characterizing the probiotic properties of strains isolated from raw milk and fermented milk products is important in terms of selecting starter culture strains for the production of functional dairy products. In this study, biochemical properties (including antibiotic sensitivity, lipolytic activity, amino acid decarboxylation, antioxidant activity) and probiotic properties (including antimicrobial activity, growth in the presence of bile salts, bile salts deconjugation, and hydrophobicity) of 14 Lactococcus lactis strains isolated from raw milk and kefir grains were investigated. Strains originating from kefir grains had better characteristics in terms of antimicrobial activity and bile salt deconjugation, whereas strains from raw milk had better hydrophobicity and antioxidant activity characteristics. None of the strains were able to grow in the presence of bile salt and did not show amino acid decarboxylation or lipolytic activities. Biochemical and probiotic properties of L. lactis strains varied depending on the strain and some of these strains could be used as functional cultures depending on their properties. However, these strains did not possess all of the properties required to meet the definition of a probiotic.     

Screening selected strains of probiotic lactic acid bacteria for their ability to produce biogenic amines (histamine and tyramine)

The aim of this study was to investigate the production of biogenic amines (BA), histamine and tyramine by some probiotic lactic acid bacteria (LAB). Fifteen strains representing six LAB species were screened qualitatively by growing them in a decarboxylase medium. Quantitative analysis was carried out by HPLC analysis with direct derivatization of acid extracts. Lactobacillus casei (TISTR 389) and Lactobacillus delbrueckii subsp. bulgaricus (TISTR 895) were found to produce BA. The highest levels of histamine (1820.9 ± 3.5 mg L^?1) and tyramine (5486.99 ± 47.6 mg L^?1) formation were observed for the TISTR 389 strain, while TISTR 895 produced only histamine (459.1 ± 0.63 mg L^?1) in the decarboxylase broth. Biogenic amine potential was not observed for the Lactobacillus acidophilus, Lactobacillus lactis subsp. lactis, Lactococcus lactis subsp. lactis, and Lactobacillus plantarum strains studied. This study confirmed that BA formation is strain dependent and not related to the species. Therefore, careful screening for amino acid decarboxylase activity is recommended before selecting LAB as appropriate starter or probiotic strains in food and dairy industry.     

Influence of compounds associated with fermented dairy products on the growth of lactic acid starter and probiotic bacteria

The growth of 24 strains of lactic acid starter bacteria (Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Lactococcus lactis) and 24 strains of probiotic bacteria (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus and bifidobacteria) in liquid media containing different substances was assessed. The substances used were salts (NaCl and KCl); sugars (sucrose and lactose); sweeteners (acesulfame and aspartame); aroma compounds (diacetyl, acetaldehyde and acetoin); natural colorings for fermented milk (red, yellow and orange colorings); flavoring agents (strawberry, vanilla, peach and banana essences); flavoring–coloring agents (strawberry, vanilla and peach); nisin, natamycin and lysozyme. Bacterial growth in the presence of natural fruit juices (green apple, kiwi, pineapple, peach and strawberry) with or without neutralization and cell viability in lactic acid acidified (pH 4 and 5) milk for 4 weeks at 5°C were also studied.Some compounds (KCl, sweeteners, aroma compounds, natamycin, flavoring agents and the peach flavoring–coloring agent) did not influence the growth of the strains in the concentrations commonly used in the dairy industry. The effect of other substances (especially flavoring–coloring agents) on the growth of lactic acid starters and probiotic bacteria was strain-dependent. Natural fruit juices weakly inhibited mainly S. thermophilus strains. Cell viability during cold storage in acidified milk was satisfactory for L. delbrueckii subsp. bulgaricus and L. casei group strains. For L. acidophilus and Bifidobacterium, the decreases in cell counts at pH 5 were negligible. Nevertheless, decreases from 1.6 to 6.2 and from 0.1 to 7.6 log orders, respectively were observed at pH 4.     

Exploring the industrial potential of Lactobacillus delbrueckii ssp. bulgaricus by population genomics and genome-wide association study analysis

Lactobacillus delbrueckii ssp. bulgaricus is one of the most commonly used starter cultures for yogurt production. However, how its genetic background affects acid production capacity is unclear. This study investigated the industrial potential of L. delbrueckii ssp. bulgaricus using population genomics and GWAS analysis. To meet our goal, population genetics and functional genomics analyses were performed on 188 newly sequenced L. delbrueckii ssp. bulgaricus strains isolated from naturally fermented dairy products together with 19 genome sequences retrieved from the NCBI database. Four distinct clusters were identified, and they were correlated with the geographical sites where the samples were collected. The GWAS analysis about acidification fermentation results with sucrose-fortified reconstituted skim milk revealed a significant association between l-lactate dehydrogenase (lldD; Ldb2036) and the bacterial acid production rate. Our study has broadened the understanding of the population structure and genetic diversity of L. delbrueckii ssp. bulgaricus by identifying potential targets for further research, development, and use of lactic acid bacteria in the dairy industry.     

Characterization of probiotic bacteria involved in fermented milk processing enriched with folic acid

Yogurt products fermented with probiotic bacteria are a consumer trend and a challenge for functional food development. So far, limited research has focused on the behavior of the various probiotic strains used in milk fermentation. In the present study, we characterized folic acid production and the sensory and textural characteristics of yogurt products fermented with probiotic bacteria. Yogurt fermented with Lactobacillus plantarum had improved nutrient content and sensory and textural characteristics, but the presence of L. plantarum significantly impaired the growth and survival of Lactobacillus delbrueckii ssp. bulgaricus during refrigerated storage. Overall, L. plantarum was a good candidate for probiotic yogurt fermentation; further studies are needed to understand the major metabolite path of lactic acid bacteria in complex fermentation.     

Fatty acid profile, trans-octadecenoic, α-linolenic and conjugated linoleic acid contents differing in certified organic and conventional probiotic fermented milks

Development of dairy organic probiotic fermented products is of great interest as they associate ecological practices and benefits of probiotic bacteria. As organic management practices of cow milk production allow modification of the fatty acid composition of milk (as compared to conventional milk), we studied the influence of the type of milk on some characteristics of fermented milks, such as acidification kinetics, bacterial counts and fatty acid content. Conventional and organic probiotic fermented milks were produced using Bifidobacterium animalis subsp. lactis HN019 in co-culture with Streptococcus thermophilus TA040 and Lactobacillus delbrueckii subsp. bulgaricus LB340. The use of organic milk led to a higher acidification rate and cultivability of Lactobacillus bulgaricus. Fatty acids profile of organic fermented milks showed higher amounts of trans-octadecenoic acid (C18:1, 1.6 times) and polyunsaturated fatty acids, including cis-9 trans-11, C18:2 conjugated linoleic (CLA-1.4 times), and α-linolenic acids (ALA-1.6 times), as compared to conventional fermented milks. These higher levels were the result of both initial percentage in the milk and increase during acidification, with no further modification during storage. Finally, use of bifidobacteria slightly increased CLA relative content in the conventional fermented milks, after 7 days of storage at 4 °C, whereas no difference was seen in organic fermented milks.     

Development of a pentaplex PCR assay for the simultaneous detection of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. helveticus, L. fermentum in whey starter for Grana Padano cheese

A pentaplex PCR assay for the rapid, selective and simultaneous detection of Lactobacillus helveticus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and L. fermentum, was developed. The target sequences were a group of genes coding for beta-galactosidase production (S. thermophilus and L. delbrueckii subsp. bulgaricus), for cell-enveloped associated proteinase synthesis (L. helveticus), for dipeptide transport system production (L. delbrueckii subsp. lactis) and for arginine-ornithine antiporter protein production (L. fermentum). The analytical specificity of the assay was evaluated with 5 reference strains and 140 lactic acid bacterial strains derived from raw milk cheeses and belonging to the Lactobacillus, Streptococcus, Lactococcus and Enterococcus genera. The identification limit for each target strain was 10³ CFU/ml. This new molecular assay was used to investigate the LAB population by direct extraction of DNA from the 12 whey cultures for Grana Padano. The pentaplex PCR assay revealed a good correspondence with microbiological analyses and allowed to identify even minor LAB community members which, can be out-competed in vitro by numerically more abundant microbial species.     

Effect of exopolysaccharides (EPSs) produced by Lactobacillus delbrueckii subsp. bulgaricus strains to bacteriophage and nisin sensitivity of the bacteria

Lactobacillus strains used in this study were isolated from village-type yogurt and raw milk. The isolates were identified as Lactobacillus delbrueckii subsp. bulgaricus by 16 s rDNA sequence analysis and API 50 CHL identification systems. The exopolysaccharide (EPS) production of the strains growth in skim milk were investigated. In addition sensitivity and insensitivity of these strains against domestic bacteriophages and nisin were examined. It was deduced that those strains which had relatively high EPS-producing capacity were insensitive against phages and nisin. Linear relationships were determined between EPS production of the bacteria and bacteriophage and nisin insensitivity of the bacteria.There was a negative correlation between EPS production quantity and phage and nisin sensitivity of the bacteria. Of all the strains, L. delbrueckii subsp bulgaricus B3 produced the highest EPS quantity, and it was insensitive against phages and nisin. Based on these results, it is suggested that L. delbrueckii subsp bulgaricus B3 can be used with the starter culture in dairy industry for stable and high-quality yogurt production.     

Use of gases to improve survival of Bifidobacterium bifidum by modifying redox potential in fermented milk

The aim of this work was to study the effect of the oxidoreduction potential, modified using gas, on the growth and survival of a probiotic strain, Bifidobacterium bifidum, and 2 yogurt strains, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. Three fermented milks were manufactured with an initial oxidoreduction potential value adjusted to +440 mV (control milk), +350 mV (milk gassed with N₂), and −300 mV [milk gassed with N₂ plus 4% (vol/vol) H₂ (N₂-H₂)]. Acidification profiles, growth during milk fermentation and survival during storage at 4°C for 28 d were determined. This study showed that fermented probiotic dairy products made from milk gassed with N₂ and, more particularly, those made from milk gassed with N₂H₂ were characterized by a significant increase in B. bifidum survival during storage without affecting the fermentation kinetics and the survival of Strep. thermophilus and L. delbrueckii ssp. bulgaricus.     

Effect of high hydrostatic pressure treatment on the viability and acidification ability of lactic acid bacteria

The effect of high pressures of 100–450 MPa combined with temperatures of 20–40 °C on Lactobacillus delbrueckii subsp. bulgaricus ACA-DC0105, Streptococcus thermophilus ACA-DC0022 and Lactococcus lactis ACA-DC0049 cell viability and acid production ability was studied. The rates of decrease in pH and cell viability were estimated for all the process combinations studied. The viability and acidification ability of the cells depended on the process conditions. More intense process conditions resulted in a lower number of viable cells and simultaneous reduction of lactic acid production correlated to lower rates of decrease of pH. Lb. bulgaricus appeared to be the microorganism most resistant to pressure, while Lc. lactis the most sensitive. Similar behaviour was observed for the acidification ability of these microorganisms, with Lc. lactis being least able to decrease the pH value of cheese. The HP-treated strains could be used as adjunct starters for cheese production.     

Optimization of the rheological properties of probiotic yoghurts supplemented with milk proteins

This study aimed to optimize the rheological properties of probiotic yoghurts supplemented with skimmed milk powder (SMP), whey protein concentrate (WPC) and sodium caseinate (Na-Cn) by using an experimental design type simplex-centroid for mixture modeling. It included seven batches/trials: three were supplemented with each type of the dairy protein used, three corresponding to the binary mixtures and one to the ternary one in order to increase protein concentration in 1 g 100 g⁻¹ of final product. A control experiment was prepared without supplementing the milk base. Processed milk bases were fermented at 42 °C until pH 4.5 by using a starter culture blend that consisted of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Bifidobacterium animalis subsp. lactis. The kinetics of acidification was followed during the fermentation period as well the physico-chemical analyses, enumeration of viable bacteria and rheological characteristics of the yoghurts. Models were adjusted to the results (kinetic responses, counts of viable bacteria and rheological parameters) through three regression models (linear, quadratic and cubic special) applied to mixtures. The results showed that the addition of milk proteins affected slightly acidification profile and counts of S. thermophilus and B. animalis subsp. lactis, but it was significant for L. delbrueckii subsp. bulgaricus. Partially-replacing SMP (45 g/100 g) with WPC or Na-Cn simultaneously enhanced the rheological properties of probiotic yoghurts taking into account the kinetics of acidification and enumeration of viable bacteria.     

Probiotic viability and storage stability of yogurts and fermented milks prepared with several mixtures of lactic acid bacteria

Currently, the food industry wants to expand the range of probiotic yogurts but each probiotic bacteria offers different and specific health benefits. Little information exists on the influence of probiotic strains on physicochemical properties and sensory characteristics of yogurts and fermented milks. Six probiotic yogurts or fermented milks and 1 control yogurt were prepared, and we evaluated several physicochemical properties (pH, titratable acidity, texture, color, and syneresis), microbial viability of starter cultures (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) and probiotics (Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus reuteri) during fermentation and storage (35 d at 5°C), as well as sensory preference among them. Decreases in pH (0.17 to 0.50 units) and increases in titratable acidity (0.09 to 0.29%) were observed during storage. Only the yogurt with S. thermophilus, L. delbrueckii ssp. bulgaricus, and L. reuteri differed in firmness. No differences in adhesiveness were determined among the tested yogurts, fermented milks, and the control. Syneresis was in the range of 45 to 58%. No changes in color during storage were observed and no color differences were detected among the evaluated fermented milk products. Counts of S. thermophilus decreased from 1.8 to 3.5 log during storage. Counts of L. delbrueckii ssp. bulgaricus also decreased in probiotic yogurts and varied from 30 to 50% of initial population. Probiotic bacteria also lost viability throughout storage, although the 3 probiotic fermented milks maintained counts ≥10⁷ cfu/mL for 3 wk. Probiotic bacteria had variable viability in yogurts, maintaining counts of L. acidophilus ≥10⁷ cfu/mL for 35 d, of L. casei for 7 d, and of L. reuteri for 14 d. We found no significant sensory preference among the 6 probiotic yogurts and fermented milks or the control. However, the yogurt and fermented milk made with L. casei were better accepted. This study presents relevant information on physicochemical, sensory, and microbial properties of probiotic yogurts and fermented milks, which could guide the dairy industry in developing new probiotic products.     

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.     

Gelation and resistance to shearing of fermented milk: Role of exopolysaccharides

Milk was fermented with the exopolysaccharide-producing (EPS⁺) strains Lactococcus lactis subsp. cremoris, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and with the non-EPS-producing strain (EPS⁻) L. lactis subsp. cremoris. The kinetics of gelation and the behaviour of set fermented milk during and after shearing were studied using rheometry and confocal scanning laser microscopy. The time of gelation of milk depended on the kinetics of acidification of strains whereas the pH of gelation depended mostly on the presence of exopolysaccharides (EPS). In set fermented milk with EPS⁺ strains, bacteria were observed in protein-free areas likely filled with EPS. Phase-separated EPS and caseins contributed to induce the gelation of fermented milk at pH 5.6. The high resistance to shearing of milk fermented with the EPS⁺ strain L. lactis subsp. cremoris might be due to the negative charge of the exopolysaccharide allowing an attractive interaction with caseins.     

In vitro evaluation of physiological probiotic properties of different lactic acid bacteria strains of dairy and human origin

Eleven lactic acid bacteria strains of importance to the dairy industry were subjected to in vitro analyses to determine their probiotic potential. Seven strains were isolated from ewe’s and cow’s milk (Enterococcus faecalis – five –, Lactococcus lactis and Lactobacillus paracasei). Four were obtained from American Type Culture Collection (ATCC), isolated from cheese (Lactobacillus casei 393), human feces (L. paracasei 27092 and Lactobacillus rhamnosus 53103) and used in cheese making (L. lactis 54104). Although none of the strains was able to degrade mucin, all E. faecalis showed, at least, one transferable antibiotic resistance, which excluded them as candidates for addition to foods. Of the remaining six safe strains, L. lactis strains were more tolerant to low pH than Lactobacillus spp.; all were tolerant to pancreatin and bile salts and showed antibacterial activity. The highest level of adhesion to Caco-2 cells was observed with L. lactis 660, even higher than L. rhamnosus ATCC 53103 (recognized probiotic and used as control). The physiological probiotic properties of these strains, mainly isolated from dairy sources, are interesting in view of their use in cheese productions as starter and non starter cultures. The five LAB safe strains studied may have potential as novel probiotics in the dairy foods.     

Effects of ingesting milk fermented by Lactococcus lactis H61 on skin health in young women: A randomized double-blind study

We conducted a randomized double-blind trial to evaluate the effects of fermented milk produced using only Lactococcus lactis strain H61 as a starter bacterium (H61-fermented milk) on the general health and various skin properties of young women. Healthy female volunteers (n = 23; age = 19–21 r) received H61-fermented milk (10¹⁰ cfu of strain H61/d) or conventional yogurt (10¹⁰ cfu of both Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus per day), as a reference food, daily for 4 wk. Before and at the end of 4 wk, blood samples were taken, and skin hydration (inner forearms and cheek) and melanin content, elasticity, and sebum content (cheek only) were measured. Skin hydration at the inner forearm was higher at wk 4 than at wk 0 in both groups. Sebum content in cheek rose significantly after intervention in the H61-fermented milk group, but not the conventional yogurt group. Other skin parameters did not differ in either group. Serum analysis showed that total protein concentration and platelet count were elevated and reactive oxygen species decreased in both groups after the intervention. Although H61-fermented milk and conventional yogurt had similar effects on skin status and some blood characteristics of participants, an increase of sebum content in cheek is preferable to H61-fermented milk. As skin lipids contribute to maintaining the skin barrier, H61-fermented milk would provide beneficial effects on skin for young women.     

Effect of lactose hydrolysis on the milk-fermenting properties of Lactobacillus delbrueckii ssp. bulgaricus 2038 and Streptococcus thermophilus 1131

Yogurt is a well-known nutritious and probiotic food and is traditionally fermented from milk using the symbiotic starter culture of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. However, yogurt consumption may cause health problems in lactose-intolerant individuals, and the demand for lactose-free yogurt has been increasing. The standard method to prepare lactose-free yogurt is to hydrolyze milk by lactase; however, this process has been reported to influence the fermentation properties of starter strains. This study aimed to investigate the fermentation properties of an industrial starter culture of L. bulgaricus 2038 and S. thermophilus 1131 in lactose-hydrolyzed milk and to examine the metabolic changes induced by glucose utilization. We found that the cell number of L. bulgaricus 2038, exopolysaccharide concentration, and viscosity in the coculture of L. bulgaricus 2038 and S. thermophilus 1131 was significantly increased in lactose-hydrolyzed milk compared with that in unhydrolyzed milk. Although the cell number of S. thermophilus 1131 showed no difference, production of formic acid and reduction of dissolved oxygen were enhanced in lactose-hydrolyzed milk. Further, in lactose-hydrolyzed milk, S. thermophilus 1131 was found to have increased the expression of NADH oxidase, which is responsible for oxygen reduction. These results indicated that glucose utilization promoted S. thermophilus 1131 to rapidly reduce the dissolved oxygen amount and produce a high concentration of formic acid, presumably resulting in the increased cell number of L. bulgaricus 2038 in the coculture. Our study provides basic information on the metabolic changes in starter strains in lactose-hydrolyzed milk, and demonstrates that lactose-free yogurt with increased cell number of L. bulgaricus can be prepared without delay in fermentation and decrease in the cell number of S. thermophilus.     

Comparison of Associative Growth and Proteolytic Activity of Yogurt Starters in Whole Milk from Camels and Cows

Growth and proteolytic activities were studied using yogurt starter cultures incubated in pasteurized whole milk from camels and cows at 42°C as single and mixed cultures. In general, the growth of four strains of Streptococcus thermophilus and three strains of Lactobacillus delbrueckii ssp. bulgaricus was higher in cow milk than in camel milk. However, proteolysis was higher in camel milk than in cow milk. Lactobacillus delbrueckii ssp. bulgaricus LB12 in combination with streptococci had lowered pH more than did the other lactobacilli. Mixed cultures released the same amount of free amino groups as the corresponding single cultures, except for L. delbrueckii ssp. bulgaricus LB12.