Conjugated linoleic acid production by immobilized cells of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus acidophilus

Lactobacillus delbrueckii ssp. bulgaricus (CCRC14009) and L. acidophilus (CCRC14079), immobilized with chitosan and polyacrylamide, were tested for CLA production. A 10-ml aliquot of L. delbrueckii ssp. bulgaricus cell suspension (3.59 × 10⁷ CFU/ml) was adsorbed to 0.5 g chitosan and polyacrylamide, mixed with 0.2 ml linoleic acid (0.9 g/ml), and incubated at 37 °C for 24 h at pH 5, 6, 7, and 8 for CLA production. CLA levels, produced by immobilized cells of L. delbrueckii ssp. bulgaricus and L. acidophilus with increasing cell counts to 1.08 and 1.28 × 10¹⁰ CFU/ml, respectively, at optimal reaction pHs were evaluated. More CLA was formed at pH 8 of chitosan and pH 7 of polyacrylamide-immobilized L. delbrueckii ssp. bulgaricus cell treatments. Increase in cell count resulted in higher CLA production. The adsorption of L. delbrueckii ssp. bulgaricus cells onto polyacrylamide at pH 7 showed significant improvement in total CLA level. Results demonstrated a potential for enhancing CLA production through immobilization.     

Some low homogenization pressures improve certain probiotic characteristics of yogurt culture bacteria and Lactobacillus acidophilus LA-K

Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus salivarius ssp. thermophilus, and Lactobacillus acidophilus are dairy cultures widely used in the manufacture of cultured dairy products. Commonly used homogenization pressures in the dairy industry are 13.80 MPa or less. It is not known whether low homogenization pressures can stimulate bacteria to improve their probiotic characteristics. Objectives were to determine the effect of homogenization at 0, 3.45, 6.90, 10.34, and 13.80 MPa on acid tolerance, bile tolerance, protease activity, and growth of L. delbrueckii ssp. bulgaricus LB-12, S. salivarius ssp. thermophilus ST-M5, and L. acidophilus LA-K. The cultures were individually inoculated in cool autoclaved skim milk (4°C) and homogenized for 5 continuous passes. Growth and bile tolerance of samples were determined hourly for 10 h of incubation. Acid tolerance was determined every 20 min for 120 min of incubation. Protease activity was determined at 0, 12, and 24 h of incubation. All homogenization pressures studied improved acid tolerance of L. delbrueckii ssp. bulgaricus LB-12 but had no beneficial effect on protease activity and had negative effects on growth and bile tolerance. A pressure of 6.90 MPa improved acid tolerance, bile tolerance, and protease activity of S. salivarius ssp. thermophilus ST-M5, but none of the homogenization pressures studied had an effect on its growth. Homogenization pressures of 13.80 and 6.90 MPa improved acid tolerance and bile tolerance, respectively, of L. acidophilus LA-K but had no effect on protease activity and its growth. Some low homogenization pressures positively influenced some characteristics of yogurt culture bacteria and L. acidophilus LA-K. Culture pretreatment with some low homogenization pressures can be recommended for improvement of certain probiotic characteristics.     

Effects of rare sugar D-allulose on acid production and probiotic activities of dairy lactic acid bacteria

It has recently been reported that the rare sugar d-allulose has beneficial effects, including the suppression of postprandial blood glucose elevation in humans, and can be substituted for sucrose as a low-calorie food ingredient. To examine the applications of d-allulose in the dairy industry, we investigated the effects of d-allulose on the acid production of 8 strains of yogurt starter (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) and 4 strains of lactococci, including potential probiotic candidates derived from dairy products. Acid production by 2 L. delbrueckii ssp. bulgaricus yogurt starter strains in milk was suppressed by d-allulose, but this phenomenon was also observed in some strains with another sugar (xylose), a sugar alcohol (sorbitol), or both. In contrast, among the dairy probiotic candidates, Lactococcus lactis H61, which has beneficial effects for human skin when drunk as part of fermented milk, was the only strain that showed suppression of acid production in the presence of d-allulose. Strain H61 did not metabolize d-allulose. We did not observe suppression of acid production by strain H61 with the addition of xylose or sorbitol, and xylose and sorbitol were not metabolized by strain H61. The acid production of strain H61 after culture in a constituted medium (tryptone–yeast extract–glucose broth) was also suppressed with the addition of d-allulose, but growth efficiency and sugar fermentation style were not altered. Probiotic activities—such as the angiotensin-converting enzyme inhibitory activity of H61-fermented milk and the superoxide dismutase activity of H61 cells grown in tryptone–yeast extract–glucose broth—were not affected by d-allulose. d-Allulose may suppress acid production in certain lactic acid bacteria without altering their probiotic activity. It may be useful for developing new probiotic dairy products from probiotic strains such as Lactococcus lactis H61.     

Potential of LAB starter culture isolated from Chinese traditional fermented foods for yoghurt production

A total of 32 indigenous lactic acid bacteria (LAB) isolates were previously obtained from 7 samples of traditional dairy products in specific ecological niches throughout the northwestern area of China. Among the 32 isolates, 9 strains of LAB were selected for their potential in fermented dairy products and identified by 16S rDNA sequence analysis. In this study, these 9 isolates were combined each other and 8 combinations were selected according to their fermentation characteristics in yoghurt production. The effects of these 8 combinations on acidification, post acidification, texture and sensory quality of yoghurt were analysed. Results indicated that Streptococcus thermophilus SP1.1 combined with Lactobacillus delbrueckii ssp. bulgaricus 3 4.5 was the best combination and better than the commercial starter culture in terms of yoghurt fermentation time and sensory quality, suggesting that the combination has potential as a commercial starter to improve the quality of yoghurt.     

Conjugated linoleic acid production by cells and enzyme extract of Lactobacillus delbrueckii ssp. bulgaricus with additions of different fatty acids

The objective of the study was to determine the effect of fatty acid additions to the cells and enzyme extract of Lactobacillus delbrueckii ssp. bulgaricus (CCRC14009) on CLA production. Washed cells of L. delbrueckii ssp. bulgaricus, obtained by cultivation in a MRS broth, were mixed with BSA and each of the three fatty acids: linoleic, oleic, and linolenic acids in sodium phosphate buffer at pH 6.5. After incubation at 37 °C for 108 h, CLA concentration was analyzed by HPLC. Enzyme extract from the culture was also reacted with each fatty acid at 50 °C for 10 min at pH 5 to test for CLA production. Results showed that linoleic acid addition to the culture improved CLA production, indicating the presence of linoleic acid isomerase activity in the culture. The crude enzyme extract from the culture was observed to be capable of oleic and linolenic acid conversions into CLA, demonstrating the possible presence of desaturase activity in the enzyme extract.     

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.     

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.     

Effects of yogurt starter cultures on the survival of Lactobacillus acidophilus

Recognized to confer health benefits to consumers, probiotics such as Lactobacillus acidophilus are commonly incorporated into fermented dairy products worldwide; among which yogurt is a popular delivery vehicle. To materialize most of the putative health benefits associated with probiotics, an adequate amount of viable cells must be delivered at the time of consumption. However, the loss in their viabilities during refrigerated storage has been demonstrated previously. This study focused on the effects of yogurt starter cultures on the survival of five strains of L. acidophilus, with emphases on low pH and acid production. Differential survival behavior between L. acidophilus strains was further analyzed. To this end, viable cell counts of L. acidophilus were determined weekly during 4 °C storage in various types of yogurts made with Streptococcus thermophilus alone, L. delbrueckii ssp. bulgaricus alone, both species of the starter cultures, or glucono-delta-lactone (GDL). All yogurt types, except for pasteurized yogurts, were co-fermented with L. acidophilus. Yogurt filtrate was analyzed for the presence of any inhibitory substance and for the amount of hydrogen peroxide. Multiplication of L. acidophilus was not affected by the starter cultures as all strains reached high level on day 0 of the storage period. Throughout the 28-day storage period, cell counts of L. acidophilus PIM703 and SBT2062 remained steady (~ 6 × 10⁷ CFU/g) in yogurts made with both starter cultures, whereas those of ATCC 700396 and NCFM were reduced by a maximum of 3 and 4.6 logs, respectively. When starter cultures were replaced by GDL, all strains survived well, suggesting that a low pH was not a critical factor dictating their survival. In addition, the filtrate collected from yogurts made with starter cultures appeared to have higher inhibitory activities against L. acidophilus than that made with GDL. The presence of viable starter cultures was necessary to adversely affect the survival of some strains, as pasteurized yogurts had no effect on their survival. In particular, the inhibitory effect exerted by L. delbrueckii ssp. bulgaricus on L. acidophilus NCFM was highly pronounced than by S. thermophilus, nevertheless, the same effect was not observed on SBT2062. The inhibition against stationary-phase NCFM cells might be caused by the elevated level of hydrogen peroxide produced by L. delbrueckii ssp. bulgaricus. Delineating factors driving the differences in survival trait among probiotic strains will lead to a more efficacious delivery of health benefits in fermented dairy products through targeted technological interventions.     

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.     

Characterization of volatile compounds in fermented milk using solid-phase microextraction methods coupled with gas chromatography-mass spectrometry

Lactic acid bacteria (LAB) are industrially important bacteria that are widely used in the fermented food industry, especially in the manufacture of yogurt. Characteristic flavors are produced by LAB during fermentation and storage that affect the quality and acceptability of fermented milk products. In this study, the volatile compounds in milk fermented by Streptococcus thermophilus IMAU80842 alone, Lactobacillus delbrueckii ssp. bulgaricus IMAU20401 alone, or both species together were identified using solid-phase microextraction methods coupled with gas chromatography-mass spectrometry. A total of 53, 43, and 32 volatile compounds were identified in milk fermented by S. thermophilus alone, L. delbrueckii ssp. bulgaricus alone, or both species together, respectively. The presence of some important flavor compounds was confirmed: acetic acid, acetaldehyde, acetoin, 2,3-butanedione, ethanol, and 1-heptanol. Our results demonstrate that the composition of the volatile compounds in fermented milk depends on the species of LAB used and whether they are used alone or in combination. This is important for the selection of appropriate starter cultures for the production of different types of fermented milk product with particular flavors.     

Application of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and L. helveticus for sourdough bread making

The application of Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) as starter cultures for sourdough bread making was examined. Production of lactic and acetic acids, bread rising, volatile composition, shelf-life and organoleptic quality of the sourdough breads were evaluated. The amount of starter culture added to the flour, the dough fermentation temperature and the amount of sourdough used were examined in order to optimise the bread making process. The use of mixed cultures led to higher total titratable acidities and lactic acid concentrations compared to traditionally made breads. Highest acidity (3.41 g lactic acid/kg of bread) and highest resistance to mould spoilage were observed when bread was made using 50% sourdough containing 1% K. marxianus and 4% L. delbrueckii ssp. bulgaricus. The use of these cultures also improved the aroma of sourdough breads, as shown by sensory evaluations and as revealed by GC–MS analysis.     

Proteolytic profiles of yogurt and probiotic bacteria

Nine strains of Streptococcus thermophilus, 6 strains of Lactobacillus delbrueckii ssp. bulgaricus, 14 strains of Lactobacillus acidophilus and 13 strains of Bifidobacterium spp. were screened for proteolytic, amino-, di-, tri- and endopeptidase activity by using the o-pthaldialdehyde-based spectrophotometric assay. Strains showing the highest and lowest proteolytic activity were further studied for their peptidase activities at the extracellular and intracellular levels. The amounts of free amino groups released by S. thermophilus, L. delbrueckii ssp. bulgaricus and L. acidophilus strains were higher than that by Bifidobacterium strains. Aminopeptidase activity was detected for all bacterial strains both at the extracellular and intracellular levels. The specific activity towards the six substrates studied was higher at the intracellular level for all strains. High dipeptidase activity was demonstrated by all bacterial strains for L. delbrueckii ssp. bulgaricus, L. acidophilus, and Bifidobacterium spp. whereas S. thermophilus had greater dipeptidase activity at the extracellular level. All bacterial cultures tested were able to hydrolyse large biologically active peptides, bradykinin, Ala–Ala–Ala–Ala–Ala and the tripeptide substrate Gly–Ala–Tyr at both the extracellular and intracellular levels. However, with the substrates ending with a C-terminal of phenylalanine, the hydrolysis only occurred at the intracellular level.     

The impact of alternative nitrogen sources on the growth and viability of Lactobacillus delbrueckii ssp. bulgaricus

In this study, we developed and optimized a growth medium using various nitrogen sources for the cultivation of Lactobacillus delbrueckii ssp. bulgaricus, a probiotic and essential dairy starter culture. The composition of de Man, Rogosa, and Sharpe (MRS) culture medium was modified, and the nitrogen content was replaced by alternative nitrogen sources X-Seed Nucleo Max, X-Seed KAT, and X-Seed Carbo Max (Ohly GmbH) in various blends of 5 and 10 g/L. Results showed that bacterial growth was significantly higher when the nitrogen source blend of 10 g/L of KAT and 10 g/L of Carbo Max [KCMax (10/10)] was used. The optical densities of the Lb. bulgaricus strains were significantly higher in the KCMax (10/10) medium than in the MRS medium. There was no significance in bacterial counts for both the MRS and the KCMax (10/10) medium, and all bacterial counts were estimated at 8 log cfu/mL. The buffering capacity of the KCMax (10/10) medium was also tested and supplemented with l-histidine and was significantly higher than that of the MRS control medium. KCMax (10/10) also supported the freeze-stability and viability of the Lb. bulgaricus cells during freezing and freeze-drying operations. Our results suggest that the alternative nitrogen sources X-Seed Nucleo Max, X-Seed KAT and X-Seed Carbo Max can substantially support the growth of lactic acid bacteria as demonstrated with Lb. bulgaricus. These alternative nitrogen sources could thus be recommended for lactic acid bacteria fermentation and for the cultivation of dairy starter cultures.     

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.     

Proteolytic action of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 reduces antigenic response to bovine β-lactoglobulin

The whey protein β-lactoglobulin (BLG) is highly allergenic. Lactic acid bacteria can degrade milk proteins. The capacity of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 to hydrolyse the major BLG epitopes (V41–K60; Y102–R124; L149–I162) and decrease their recognition by IgE of allergic patients was evaluated. The intensity of BLG degradation was analysed by Tricine SDS–PAGE and RP-HPLC. Peptides released were identified by LC–MS/MS and the hydrolysates were tested for their capacity to inhibit IgE binding by ELISA test. L. delbrueckii subsp. bulgaricus CRL 656 degraded BLG (35%, 8 h). The sequence analysis of the released peptides indicated that this strain degraded three main BLG epitopes. BLG-positive sera (3–5 year old children) were used for testing IgE binding inhibition of BLG hydrolysates from the Lactobacillus strain. The hydrolysates were less immuno-reactive (32%) than the heated BLG. L. delbrueckii subsp. bulgaricus CRL 656 could be used for developing hypoallergenic dairy products.     

Influence of addition of Raftiline HP on the growth, proteolytic, ACE- and α-glucosidase inhibitory activities of selected lactic acid bacteria and Bifidobacterium

This study examined the influence of Raftiline HP^®, added at the rate of 1, 2 and 3 g 100 mL⁻¹ to reconstituted skim milk, on the growth and biochemical activities of selected strains of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus casei, Lactobacillus acidophilus and Bifidobacterium longum. The growth of B. longum and S. thermophilus was improved in reconstituted skim milk (RSM) containing 1 g 100 mL⁻¹ Raftiline HP^®. All the organisms except for S. thermophilus produced more lactic acid and acetic acid in the presence of Raftiline HP^® than in the control. L. acidophilus and B. longum showed improvement in the proteolytic capabilities at all the three levels of Raftiline HP^® addition. L. delbrueckii ssp. bulgaricus showed maximum percent ACE inhibition in RSM containing 2 g 100 mL⁻¹ Raftiline HP^® while B. longum exhibited this potential in RSM containing 3 g 100 mL⁻¹ Raftiline HP^®. All organisms, except L. delbrueckii ssp. bulgaricus, however, showed improvement in the α-glucosidase inhibitory activity in RSM containing Raftiline HP^®.     

Immunomodulatory effects of polysaccharides produced by Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1

The extracellular polysaccharides (EPS) produced by lactic acid bacteria (LAB) are associated with the rheology, texture, and mouthfeel of fermented milk products, including yogurt. This study investigated the immunomodulatory effects of EPS purified from the culture supernatant of Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) OLL1073R-1. The crude EPS were prepared from the culture supernatant of L. bulgaricus OLL1073R-1 by standard chromatographic methods, and were fractionated into neutral EPS and acidic EPS (APS). Acidic EPS were further fractionated into high molecular weight APS (H-APS) and low molecular weight APS (L-APS). High molecular weight APS were shown to be phosphopolysaccharides containing D-glucose, D-galactose, and phosphorus. Stimulation of mouse splenocytes by H-APS significantly increased interferon-γ production, and, moreover, orally administered H-APS augmented natural killer cell activity. Oral administration of yogurt fermented with L. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059 to mice showed a similar level of immunomodulation as H-APS. However, these effects were not detected following administration of yogurt fermented with the starter combination of L. bulgaricus OLL1256 and S. thermophilus OLS3295. We conclude from these findings that yogurt fermented with L. bulgaricus OLL1073R-1, containing immunostimulative EPS, would have an immunomodulatory effect on the human body.     

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.