DNA fingerprinting of thermophilic lactic acid bacteria using repetitive sequence-based polymerase chain reaction

DNA fingerprints of lactic acid bacteria were generated by polymerase chain reaction using a primer based on the repetitive elements found in the genome of Streptococcus pneumoniae (BOX-PCR). The method made it possible to identify 37 isolates from raw milk. industrial starters and yogurt. Differentiation at species, subspecies and strain level was possible for Lactobacillus delbrueckii subsp. lactis, Lb. delbrueckii subsp bulgaricus and Str. thermophilus. BOX-PCR was also applied to studying the strain composition of a starter culture and the direct detection of strains in commercial fermented milk.     

Growth of Probiotic and Traditional Yogurt Cultures in Milk Supplemented with Whey Protein Hydrolysate

ABSTRACT: Growth of some probiotic bacteria was significantly improved in milk supplemented with whey protein hydrolysate (WPH). However, WPH had no effect on the growth of Lactobacillus delbrueckii ssp. bulgaricus 18, L. delbrueckii ssp. bulgaricus 10442, and Streptococcus thermophilus 1. When the probiotic bacteria were grown in combination with different yogurt cultures in milk, WPH caused significant increases in growth of Bifidobacterium longum S9, L. acidophilus O16, and L. acidophilus L-1. However, by day 28 of refrigerated storage, the populations of the probiotic cultures that had been grown in samples supplemented with WPH were similar or below those in the control samples.     

Effect of Versagel on the growth and metabolic activities of selected lactic acid bacteria

ABSTRACT:  In this study, the influence of a protein-based fat replacer, Versagel ^® added at 1% and 2% (w/v) to reconstituted skim milk (RSM), on the growth and metabolic activities of selected strains of yogurt starters ( Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus ) as well as probiotic organisms ( Lactobacillus casei , Lactobacillus acidophilus , and Bifidobacterium longum ) was examined. Addition of Versagel resulted in significantly improved growth of S. thermophilus and B. longum but inhibited that of L. casei , L. acidophilus , and L. delbrueckii ssp. bulgaricus . This is also reflected in the extent of reduction of pH in RSM with added Versagel by these organisms. Among the biochemical activities, proteolytic activity of all the organisms except B. longum was adversely affected by the presence of Versagel, although the angiotensin-I converting enzyme (ACE) inhibitory and α-glucosidase (α-glu) inhibitory activities were improved. Versagel at 1% level influenced the growth, while ACE inhibitory and α-glu inhibitory activities of the organisms were better at 2% level.     

Effect of thermophilic lactic acid bacteria on the viability of Salmonella serovar Typhimurium PT8 during milk fermentation and preparation of buffalo's yogurt

Viability of dairy-borne Salmonella enterica ssp. enterica serovar Typhimurium PT8 was studied during the fermentation of skim milk by thermophilic lactic acid bacteria (LAB). Longer generation times of Salmonella were found in mixed cultures of skim milk containing Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus or a mixture of them (1:1), as compared with single cultures of the pathogen. Salmonella was less able to survive in mixed cultures with these LAB during prolonged incubation at 41°C and also during cold storage of the fermented milk. L actobacillus ssp. bulgaricus and its mixture with S. thermophilus were more inhibitory to the growth and survival of Salmonella than was S. thermophilus . This was associated with higher ability of L . ssp. bulgaricus and the mixture to develop acidity in milk than S. thermophilus . Examining the antibacterial activity of these LAB towards Salmonella showed that other factors including heat-resistant and heat-labile compounds were involved in inhibiting the pathogen by these cultures. The viability of the same Salmonella strain during the preparation and cold storage of buffalo's yogurt was also examined. Salmonella was found to survive longer in yogurt made with starter containing probiotic bacteria than in that prepared with the traditional starter. This was ascribed to the development of lower pH by the traditional starter.     

5L立瓶发酵制备益生菌直投式发酵剂及应用试验

对长双歧杆菌、保加利亚乳杆菌、嗜热乳酸链球菌在5 L立瓶中分别进行培养,确定了各自的发酵最适收获期,菌体收集后经制备所获得冷冻浓缩直投式发酵剂活菌数大于1010mL-1,冷冻干燥直投式发酵剂活菌数大于1011g-1。以制备的冷冻浓缩、冷冻干燥直投式发酵剂,进行牛乳发酵应用试验,生产的益生菌酸奶风味纯正、品质良好。     

The viability of three probiotic organisms grown with yoghurt starter cultures during storage for 21 days at 4°C

This study investigated the viability of probiotic ( Lactobacillus acidophilus LA5, Lactobacillus rhamnosus LBA and Bifidobacterium animalis subsp . lactis BL-04) in milk fermented with Lactobacillus delbrueckii subsp . bulgaricus LB340 and Streptococcus thermophilus TAO (yoghurt – Y). Each probiotic strain was grown separately in co-culture with Y and in blends of different combinations. Blends affected fermentation time(s), pH and firmness during storage at 4°C. The product made with Y plus B. animalis subsp . lactis and L. rhamnosus had counts of viable cells at the end of shelf life that met the minimum required to achieve probiotic effect. However, L. acidophilus and L. delbrueckii subsp . bulgaricus were inhibited.     

Capsule formation by nonropy starter cultures affects the viscoelastic properties of yogurt during structure formation

The aim of this work was to study the structure formation of yogurt made with cultures containing ropy Lactobacillus delbrueckii ssp. bulgaricus (R), capsule-forming nonropy Streptococcus thermophilus (CNR), and noncapsule-forming nonropy cultures (NCNR). Similar gelation profiles were shown for milk fermented by ropy and nonropy lactic cultures. The gelation point occurred at lower pH values in milk fermented with R or NCNR compared to that of milk fermented with CNR culture. Differences between capsule forming ropy and nonropy cultures were observed in the aggregation behavior of the caseins. Gels made with R culture had the highest maximum in loss tangent (tan delta). However, this maximum occurred at the highest pH value when CNR culture was used. The earlier gelation of milk fermented by the encapsulated nonropy strain of Strep. thermophilus resulted in increased structure rearrangement as the pH dropped, interfering with the formation of a more compact structure.     

优选保加利亚乳杆菌发酵特性的测定

对实验室研究较为成熟的4株保加利亚乳杆菌进行发酵产酸、后酸化测定,同时对单菌株发酵、与嗜热链球菌组合发酵制备的酸奶进行质构测定和感官评价,旨为菌株开发成直投式酸奶发酵提供基础。     

Use of chemical mutagenesis for the isolation of food grade beta-galactosidase overproducing mutants of bifidobacteria, lactobacilli and Streptococcus thermophilus

A classical chemical mutagenesis protocol was evaluated for increasing beta-galactosidase production by probiotic bacteria to improve their potential to treat symptoms of lactose malabsorption in humans. Two Bifidobacterium species (B. breve and B. longum) and one strain each of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus were tested by a single exposure to two chemical mutagens, ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). To screen for beta-galactosidase (beta-gal) overproducing mutants, optimized EMS and MNNG mutant pots for each strain were plated on BHI agar containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal). Colonies that exhibited a blue color were selected for quantitative beta-gal activities using the o-nitrophenyl-beta-galactoside (ONPG) assay. Seventy-five mutants were obtained out of more than 2 million colonies screened and showed increased beta-galactosidase activities compared with the wild-type strains. EMS gave a higher frequency of beta-gal overproducing mutants than MNNG for three of the four strains, S. thermophilus, B. breve, and B. longum, whereas the frequency of L. delbrueckii ssp. bulgaricus beta-gal mutants was similar with both mutagens. The highest beta-gal increases, when induced during growth in lactose, for mutants of each culture were 137% for L. delbrueckii ssp. bulgaricus; 104% for S. thermophilus; 70% for B. breve; and 222% for B. longum mutants. This food-grade classical approach has the ability to moderately increase beta-gal concentrations in probiotic cultures to improve their potential for treating the symptoms of lactose malabsorption in humans.     

Proteolytic profiles and angiotensin-I converting enzyme and alpha-glucosidase inhibitory activities of selected lactic acid bacteria

ABSTRACT:  This study was conducted to examine the growth, proteolytic profiles as well as angiotensin-I converting enzyme (ACE) and α-glucosidase (α-glu) inhibitory potentials of selected strains of lactic acid bacteria (LAB). Two strains each of yogurt bacteria ( Streptococcus thermophilus —1275 and 285, and Lactobacillus delbrueckii ssp. bulgaricus —1092 and 1368), and probiotics ( L. acidophilus —4461 and 33200, and L. casei —2607 and 15286, and 1 strain of Bifidobacterium longum 5022), were cultivated in reconstituted skim milk (RSM) at 37 °C and their proteolytic profiles and ACE as well as α-glu inhibitory activities were determined. Among all the strains of lactic acid bacteria studied, yogurt bacteria grew very well, with the exception of L. delbrueckii ssp. bulgaricus 1368 which showed a slower growth during the initial 3 h of incubation. The growth pattern corresponded well with the decrease in pH for the organisms. All the organisms showed an increase in proteolysis with time. The variations in proteolytic capabilities translated into corresponding variations in ACE inhibitory potential of these organisms. Bifidobacterium longum 5022 showed the highest ACE inhibitory potential followed by L. delbrueckii ssp. bulgaricus 1368, L. casei 15286, S. thermophilus 1275, and L. acidophilus 4461. Organisms with high intracellular enzymatic activities grew well. Also, aminopeptidases of strains of L. acidophilus 4461 and S. thermophilus 1275 that could better utilize proline containing substrates showed enhanced ACE inhibitory potential. Lactic acid bacteria possessed the ability to inhibit α-glu activity, which endowed them an antidiabetic property as well.     

Contribution of Streptococcus thermophilus to growth-stimulating effect of yogurt on rats

The origin of the growth-stimulating factor in yogurt was studied in rats fed liquid or freeze-dried diets of milk, yogurt, milks fermented individually by Streptococcus thermophilus and Lactobacillus bulgaricus, milks to which cells of Streptococcus thermophilus and Lactobacillus bulgaricus were added. Diets containing sonicated cells, cell supernatant, and cell fractions also were fed. Milk fermented by Streptococcus thermophilus and milk plus Streptococcus thermophilus cells stimulated growth as effectively as did yogurt. That finding and the absence of stimulation in rats fed Lactobacillus bulgaricus showed that Streptococcus thermophilus is responsible for stimulation of growth by yogurt. Growth was stimulated by an intracellular factor and not by fermentative changes in the milk.     

EFFECT OF COLD STORAGE ON CULTURE VIABILITY AND SOME RHEOLOGICAL PROPERTIES OF FERMENTED MILK PREPARED WITH YOGURT AND PROBIOTIC BACTERIA

ABSTRACT

We examined the effect of storage time on culture viability and some rheological properties (yield stress, storage modulus, loss modulus, linear viscoelastic region, structural recuperation and firmness) of fermented milk made with Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus (LA) and Bifidobacterium animalis ssp. lactis in coculture with Streptococcus thermophilus (ST). Acidification profiles and factors that affect viability (postfermentation acidification, acidity and dissolved oxygen) were also studied during 35 days at 4C. Fermented milk prepared with a coculture of ST and Bifidobacterium lactis gave the most constant rheological behavior and the best cell viability during cold storage; it was superior to ST plus LA for probiotic fermented milk production.

PRACTICAL APPLICATIONS

Probiotic cultures should grow quickly in milk, provide adequate sensory and rheological properties to the product, and remain viable during storage. Commercially, it is very common to use yogurt starter culture (i.e. Streptococcus thermophilus[ST] and Lactobacillus delbrueckii ssp. bulgaricus) in combination with the probiotic bacteria in order to reduce fermentation time. However, LB tends to post acidify fermented milk, which reduces the viability of the probiotic bacteria; thus, it is recommended to use starter cultures devoid of this species. We found that the technological properties and the viability of the probiotic bacterium Bifidobacterium animalis ssp. lactis BL O4 in coculture with ST make it suitable for probiotic fermented milk production; it produces rheological characteristics similar to those of yogurt.     

Improved media for differentiation of rods and cocci in yogurt

Selected yogurt starters and commercial samples grew on Elliker's lactic agar supplemented with .1% Tween 80 and 50 micrograms/ml of 2,3,5-triphenyltetrazolium chloride to produce small, red Streptococcus thermophilus colonies and larger, white Lactobacillus bulgaricus colonies. The distinction was somewhat strain dependent but was satisfactory in most cases. Addition of 7% skim milk (11% solids) to lactic agar in place of 2,3,5-triphenyltetrazolium chloride allowed good rod-coccus differentiation, regardless of strain or yogurt brand. On this medium, called yogurt lactic agar, L. bulgaricus appeared as large white colonies surrounded by a cloudy zone and S. thermophilus as smaller white colonies devoid of a surrounding halo. Casein precipitation was responsible for the halo effect around the more acidogenic L. bulgaricus colonies. Yogurt lactic agar compared favorably with S. thermophilus and Lactobacillus agar media for the recovery of S. thermophilus and L. bulgaricus in single and mixed cultures.     

Sensory Acceptability and Stability of Probiotic Microorganisms and Vitamin C in Fermented Acerola (Malpighia emarginata DC.) Ice Cream

ABSTRACT:  Six fermented acerola ice creams were produced, containing different starter cultures ( Bifidobacterium longum , Bi.lactis , and traditional yogurt starter culture— Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus ) and final pH (5 and 4.5). The ice creams were evaluated for probiotic culture viability, vitamin C stability, and sensory acceptance. Mix fermentations were stopped when pH 5.0 and 5.5 were attained. However, after the addition of acerola pulp the determined pH were 4.5 and 5, respectively. Mixes were frozen and stored for 15 wk at −18 °C. The viable counts for probiotic cultures remained above the recommended minimum limit of 10⁶ cfu/g during 15 wk storage even in products with pH 4.5. Vitamin C concentration remained around 140 mg/100 g of product. The attributes of aroma, taste, texture, and overall acceptance obtained scores in the range of 5.15 to 7.22. The fermented acerola ice cream was a suitable food for the delivery of vitamin C and Bifidobacterium strains with excellent viability and acceptable sensory characteristics.     

Direct observation of bacterial exopolysaccharides in dairy products using confocal scanning laser microscopy

The objective of this work was to develop a methodology for direct visualization of bacterial exopolysaccharides (EPS) in fully hydrated dairy products. The new method involved staining EPS with wheat germ agglutinin labeled with Alexa fluor 488 or staining with concanavalin A 488. Samples were observed using confocal scanning laser microscopy. Distribution of EPS produced by Lactococcus lactis (CHCC 3367), a combination of Streptococcus thermophilus (CHCC 3534) and Lactobacillus delbrueckii ssp. bulgaricus (CHCC 769) and Lactobacillus delbrueckii ssp. bulgaricus RR in milk was compared in stirred and unstirred fermented milk. The EPS and proteins were observed as distinct entities, with EPS present in the protein network pores. EPS was observed in greater amounts in milk fermented by the ropy L. lactis culture than in milk fermented by the less ropy strain of S. thermophilus. Stirring the fermented milk caused aggregation of EPS into more extended structures. The more ropy the culture, the longer and larger the strands formed during stirring. The method was also applied to Feta cheese made with an EPS-producing strain of S. thermophilus. EPS was observed in the cheese as thick sheets filling pores in the protein network.     

Evaluation of the possible use of potentially probiotic Lactobacillus strains in dairy products

In the study, the ability of two potentially probiotic strains Lactobacillus plantarum 14 and Lactobacillus fermentum 4a to milk fermentation and the possibility to use them in yogurt production were investigated. The strains did not acidify milk during 24 h and 72 h fermentation at 37C, but grew well and remained at the level of 10⁸ colony-forming units (CFU)/mL during 21 days of cold storage. Their application to yogurt production along with commercial starter culture consisted on L. delbrueckii ssp. bulgaricus and S. thermophilus allowed to obtain products with typical sensory properties, pH values and numbers of potentially probiotic bacteria at desired level 10⁷ CFU/mL.     

Growth of probiotic bacteria and bifidobacteria in a soy yogurt formulation

Soy beverage and cows' milk yogurts were produced with Steptococcus thermophilus (ATCC 4356) and Lactobacillus delbrueckii subsp. bulgaricus (IM 025). The drop in pH during fermentation was faster in the soy beverage than in cows' milk, but the final pH values were similar. Yogurts were prepared with a yogurt starter in conjunction with either the probiotic bacteria Lactobacillus johnsonii NCC533 (La-1), Lactobacillus rhamnosus ATCC 53103 (GG) or human derived bifidobacteria. The presence of the probiotic bacteria did not affect the growth of the yogurt strains. Approximately 2 log increases in both L. rhamnosus GG and L. johnsonii La-1 were observed when each was added with the yogurt strains in both cows' milk and the soy beverage. Two of the five bifidobacteria strains grew well in the cows' milk and soy beverage during fermentation with the yogurt bacteria. High pressure liquid chromatography (HPLC) analyses showed that the probiotic bacteria and the bifidobacteria were using different sugars to support their growth, depending on whether the bacteria were growing in cows' milk or soy beverage.     

Use of isolated autochthonous starter cultures in yogurt production

The aim of this study was to produce yogurt from Iranian autochthonous starters. Strains of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus were isolated from autochthonous yogurts. The strains were used as starter for yogurt production. Chemical, microbial and organoleptic characteristics of products were examined. There were significant increases in acidity and acetaldehyde content, and significant decreases in pH and microbial population of samples during cold storage. There was no significant difference in organoleptic characteristics (taste, smell and texture) during cold storage. We conclude that autochthonous starters in production of yogurt can be satisfactorily used and the starters can introduce to national microbial collection.     

Commercially important characteristics of Turkish origin domestic strains of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus

Thirty native (Turkish origin) strains of Streptococcus thermophilus and 24 strains of Lactobacillus delbrueckii ssp. bulgaricus were isolated and identified. These strains were examined for their technologically important properties such as acidification, proteolysis, acetaldehyde production and antimicrobial activity. By taking into consideration their phage resistant properties, two strains of S. thermophilus (St.7.7, and St.26.2) and one of L. bulgaricus, numbered as 2004, were found to have the potential to be starters for yogurt production.     

Characterisation of lactic acid bacteria isolated from fermented milk "laban"

The technological properties of 96 lactic acid bacteria isolated from Lebanese traditional fermented milk "laban" were characterised. They were classified by phenotypic and biochemical analyses as Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, thus indicating that laban is a fermented milk similar to yogurt. Most strains of L. bulgaricus (87.5%) exhibited a high acidification activity, whereas strains of streptococci showed low acidification ability. 33.3% of streptococci strains and 25% of lactobacilli strains displayed similar acidification performances as European strains. Results obtained for syneresis, texture and rheological parameters led us to consider that isolated strains were not low polymer-producing strains. Some of them displayed interesting characteristics such as low syneresis and high values for rheological parameters. The major flavour compounds found in pure cultures were acetaldehyde, acetone, 2-butanone, dimethyl disulfide, acetoin, 2,3-butanedione, 2,3-pentanedione, and acetic, hexanoic and butanoic acids. Acetaldehyde (7.4%) and organic acids (48.3%) were mainly produced by L. bulgaricus strains, whereas streptococci cultures contained high relative levels of 2,3-butanedione and acetoin, which represented around 82% of the total flavour compounds. Finally, strains isolated from laban samples exhibited different technological properties than those used in yogurt production, thus conferring specific characteristics to this product.