Incorporation of selected nutraceuticals and probiotic bacteria into a fermented milk

Yoghurts were produced from a base milk containing three important nutraceuticals, namely ω-3-fatty acids, isoflavones and phytosterols. The cultures employed to make the yoghurts were single probiotic strains of Lactobacillus gasseri or Bifidobacterium infantis and, to achieve a short production time, a two-stage fermentation procedure was used with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus providing the rapid acidification. Yoghurts containing counts of >1.0×10⁸ cfu mL⁻¹ of the individual probiotics and high counts of the traditional species from yoghurt were awarded overall scores for sensory acceptability >4.0 out of 5.0; the nutraceuticals appeared to have no adverse effect on flavour. Storage trials at 5 °C showed that the viability of the probiotic cultures was retained over 15 days.     

Identification of peptides in traditional and probiotic sheep milk yoghurt with angiotensin I-converting enzyme (ACE)-inhibitory activity

Two sets of traditional Greek sheep milk yoghurt were produced: the first one (YC) using normal yoghurt culture (Lactobacillus delbrueckii subsp. bulgaricus ?10.13 and Streptococcus thermophilus ?10.7) and the second (PR) with the same normal culture mixed with Lactobacillus paracasei subsp. paracasei DC412. YC and PR had similar physicochemical properties and proteolysis patterns throughout storage. Both products showed similar peptide profiles by RP-HPLC but quantitative differences were observed in respect to storage time. Single-strain cultures of the microorganisms used showed similar peptide profiles for both lactobacilli, yet L. delbrueckii subsp. bulgaricus was the most proteolytic of all three microorganisms. The peptide content and the ACE-inhibitory activity of the water-soluble extracts of yoghurts, YC and PR, increased throughout storage. Major peptides were identified from yoghurt PR and from the separate cultures of L. delbrueckii subsp. bulgaricus and L. paracasei subsp. paracasei. Most of these peptides were derived from β-casein. A peptide, β-CN f114-121, with well-established ACE-inhibitory and opiate-like activity was identified in yoghurt PR. Further identified peptides were regarded as potential ACE-inhibitors according to their sequence.     

Effect of acidification on the activity of probiotics in yoghurt during cold storage

The viability of Lactobacillus acidophilus LAFTI^® L10, Bifidobacterium lactis LAFTI^® B94, and L. paracasei LAFTI^® L26 and their proteolytic activities were assessed in yoghurt at different termination pH of 4.45, 4.50, 4.55, and 4.60 in the presence of L. delbrueckii ssp. bulgaricus Lb1466 and Streptococcus thermophilus St1342 during 28 days of storage at 4 °C. All strains achieved the recommended level of 6.00 log cfu g⁻¹ of the product with L. acidophilus LAFTI^® L10 and L. paracasei LAFTI^® L26 exceeding the number to 8.00 and 7.00 log cfu g⁻¹, respectively. Lactobacilli strains showed a good cellular stability maintaining constant concentration throughout storage period regardless of termination pH. On the other hand, the cell counts of B. lactis LAFTI^® B94 decreased by one log cycle at the end of storage. The presence of probiotic organisms enhanced proteolysis significantly in comparison with the control batch containing L. delbrueckii ssp. bulgaricus Lb1466 and S. thermophilus St1342 only. The proteolytic activity varied due to termination pH, but also appeared to be strain related. The increased proteolysis improved survival of L. delbrueckii ssp. bulgaricus Lb1466 during storage resulting in lowering of pH and production of higher levels of organic acids, which might have caused the low cell counts for B. lactis LAFTI^® B94.     

Production of traditional Greek yoghurt using Lactobacillus strains with probiotic potential as starter adjuncts

Lactobacillus plantarum ACA-DC 146 and L. paracasei subsp. tolerans ACA-DC 4037 were examined for their potential application as adjuncts in the production of traditional Greek set-type yoghurt. Both strains displayed low milk acidification activity, while no inhibition was observed towards or from the yoghurt starters used. Yoghurt produced with L. paracasei subsp. tolerans ACA-DC 4037 exhibited the best sensory properties, with a rich traditional smooth taste, and the strain was selected for further trials. Yoghurt produced with this strain as an adjunct had good physicochemical properties. After 2 weeks of refrigerated storage, microbial loads (>7.0 log cfu g⁻¹) were in accordance with international recommendations and guidelines for probiotic and starter cultures in milk products. Increasing the microbial load further, using concentrated and encapsulated inocula (10–11 log cfu g⁻¹), gave yoghurt with long fermentation times and poor organoleptic properties.     

Supercritical CO<Subscript>2</Subscript> extraction of <Emphasis Type="Italic">Alkanna</Emphasis> species and investigating functional characteristics of alkannin-enriched yoghurt during storage

Alkannin is a potent pharmaceutical substance with a wide spectrum of biological activities. In the scope of this study, supercritical CO₂ extraction and sonication with hexane were applied to various Alkanna species, which were then subjected to hydrolysis. Total alkannins were quantified by HPLC/DAD and incorporated into yoghurt. Viscosities, pH values and microbial analyses were reported at 7 days of intervals for 21 days of storage. A. tinctoria possessed the highest amounts of alkannins and total phenols (686.3 mg GAE/g extract). The results revealed no significant changes in pH values (4.1–4.0), viable counts of Streptococcus salivarius ssp. thermophilus (80–150 × 10⁶ cfu g⁻¹) and slightly lower viscosities of enriched yoghurts (8,250–6,750 cPs) compared with the control (4.15–4.0; 110–105 × 10⁶ cfu g⁻¹; 12,600–11,310 cPs) during storage. However, viable counts of Lactobacillus delbrueckii ssp. bulgaricus of enriched yoghurts (87 × 10³ cfu g⁻¹) were much better than the control (191 × 10³ cfu g⁻¹), indicating a significant decrease in post acidification and generation of bitter peptides. Among the species investigated, A. tinctoria is the most promising source, obtained at higher yields via supercritical fluid extraction technology as a green alternative to solvent extraction and thus can be utilized at industrial scale in order to develop yoghurt products with improved health benefits.     

Survival and activity of selected probiotic organisms in set-type yoghurt during cold storage

The growth and metabolism of two probiotic organisms (L. acidophilus LAFTI^® L10 and Lactobacillus casei LAFTI^® L26) and a regular yoghurt culture (L. delbrueckii ssp. bulgaricus Lb1466 and Streptococcus thermophilus St1342) were studied in yoghurt containing 0.5%, 1.0%, and 1.5% (w/v) of high amylose corn starch powder (Hi-maize^®) or inulin. Viable cell counts of probiotic organisms, their metabolites and proteolytic activities, and viscosity of the yoghurts were determined during refrigerated storage for 28 d at 4 ^oC. In the presence of inulin, cultures showed better retention of viability (8.0 log cfu g⁻¹) in comparison with that of Hi-maize, which had a reduction by one log cycle. Lower concentrations of 0.5–1.0% Hi-maize improved (P<0.05) the production of propionic acid and also increased proteolytic activity of probiotic organisms substantially. A greater release of free amino acids may have sustained better growth of the organisms in yoghurts. Supplementation with either Hi-maize or inulin increased the viscosity of probiotic yoghurts significantly (P<0.05).     

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.     

Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt--a review

Yoghurt is increasingly being used as a carrier of probiotic bacteria for their potential health benefits. To meet with a recommended level of ≥ 10⁶ viable cells/g of a product, assessment of viability of probiotic bacteria in market preparations is crucial. This requires a working method for selective enumeration of these probiotic bacteria and lactic acid bacteria in yoghurt such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lb. acidophilus, Lb. casei and Bifidobacterium. This chapter presents an overview of media that could be used for differential and selective enumerations of yoghurt bacteria. De Man Rogosa Sharpe agar containing fructose (MRSF), MRS agar pH 5.2 (MRS 5.2), reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) or reinforced clostridial agar at pH 5.3 (RCA 5.3) are suitable for enumeration of Lb. delbrueckii subsp. bulgaricus when the incubation is carried out at 45 °C for 72 h. S. thermophilus (ST) agar and M17 are recommended for selective enumeration of S. thermophilus. Selective enumeration of Lb. acidophilus in mixed culture could be made in Rogosa agar added with 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-Glu) or MRS containing maltose (MRSM) and incubation in a 20% CO₂ atmosphere. Lb. casei could be selectively enumerated on specially formulated Lb. casei (LC) agar from products containing yoghurt starter bacteria (S. thermophilus and Lb. delbrueckii subsp. bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei. Bifidobacterium could be enumerated on MRS agar supplemented with nalidixic acid, paromomycin, neomycin sulphate and lithium chloride (MRS-NPNL) under anaerobic incubation at 37 °C for 72 h.     

Probiotic strains: survival under simulated gastrointestinal conditions,in vitro adhesion to Caco-2 cells and effect on cytokine secretion

This study evaluated three probiotic strains (Lactobacillus paracasei subsp. paracasei LC-01, L. acidophilus LA-5, Bifidobacterium lactis Bb-12) and two yoghurt strains (L. delbrueckii subsp. bulgaricus LBY-27 and Streptococcus thermophilus STY-31) with regard to their resistance to simulated gastrointestinal stress, and their ability to interact with human intestinal epithelial cells. The viability of strains was analyzed by measurements of fluorescence-stained cells and their growth by plate colony-counts. The results reveal that for all tested strains, gastric emptying (above pH 3.0) would release a large number of viable cells ranging from 91% for L. paracasei to 53% for S. thermophilus into the intestinal tract, and that between 12 and 23% of them subsequently survive intestinal stress. Among them L. paracasei showed the highest resistance to gastric stress. All the bacteria adhered to the Caco-2 cell line, with the highest adhesions being observed for L. delbrueckii subsp. bulgaricus (9%) and L. acidophilus (7%). Binding of all strains to Caco-2 cells did not result in a significant increase in the production of IL-6 and IL-8 cytokines, suggesting that these bacteria do not trigger an overt inflammatory response in human intestine epithelial cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production of volatile aroma compounds by kefir starter cultures

Production of carbonyl compounds by single-strain cultures, kefir starter (Lactobacillus delbrueckii subsp. bulgaricus HP1+Lb. helveticus MP12+Lactococcus lactis subsp. lactis C15+Streptococcus thermophilus T15+Saccharomyces cerevisiae A13) and kefir grains during fermentation and storage of kefir was studied. The content of carbonyl compounds produced by kefir starter was greater than that produced by kefir grains. The maximum acetaldehyde concentration (18.3 μg g⁻¹) in kefir with starter culture was mainly due to the metabolic activity of Lb. delbrueckii subsp. bulgaricus HP1 isolated from kefir grains. The highest diacetyl production activity was recorded in the starter culture (1.87 μg g⁻¹) and the single-strain culture St. thermophilus T15 (1.62 μg g⁻¹), followed by Lb. helveticus MP12 (0.85 μg g⁻¹) and Lc. lactis subsp. lactis C15 (0.42 μg g⁻¹). The lactobacilli Lb. delbrueckii subsp. bulgaricus HP1 and Lb. helveticus MP12 produced acetone, which was not found in the cocci cultures. The presence of 2-butanone was related to the production ability of Lb. helveticus MP12. In comparison, Lc. lactis subsp. lactis C15 synthesized ethyl acetate more actively than the other single-strain cultures included in the starter. S. cerevisiae A13 produced ethanol and CO₂ in amounts (3975 μg g⁻¹; 1.80 g L⁻¹) that lent cultured kefir distinctive flavour and aroma characteristic of authentic kefir.     

Effects of glutathione on acid stress resistance and symbiosis between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus

Streptococcus thermophilus SDMCC18 contains a novel bi-functional glutathione synthetase gene gshF, and it can produce glutathione (GSH). In the present study, we demonstrated that the produced GSH by S. thermophilus SDMCC18 could protect cells against acid challenge and be secreted into the medium. Moreover, using a gshF-defective mutant strain as the control, we found that the GSH conferred a protective role against acid stress to Lactobacillus delbrueckii subsp. bulgaricus ATCC11842. In addition, L. bulgaricus ATCC11842 could import the exogenous GSH into the cytoplasm, leading to an improved growth of strain ATCC11842. Taken together, our study reported a novel proto-cooperation relationship between S. thermophilus and L. bulgaricus in yoghurt fermentation.     

Effect of probiotic cultures on the stability of anthocyanins in blueberry yoghurts

Dependent on anthocyanin pigments, colour is one of the main quality factors of berry products. In this paper we assess the influence of probiotic bacteria on the degradation rate of anthocyanins in yoghurts with a highbush blueberry preparation during storage. Four types of yoghurts were prepared: first with the yoghurt starter culture YC-X16 only (Steptococcus thermophilus and Lactobacilllus delbrueckii subsp. bulgaricus) and three more, each with the starter culture YC-X16 and one of the three probiotic cultures: Bb-12, La-5 and LCP, respectively (Bif. animalis subsp. lactis – Bb-12; Lactobacillus. acidophilus – La-5 and Lactobacillus. paracasei subsp. paracasei – LCP).Derivatives of malvidin were the predominant anthocyanins in the tested yoghurts. Degradation of pigments occurred in accordance with the first-order reaction and its half-life time depended on the bacterial cultures. Anthocyanins in the probiotic yoghurt made with LCP culture were characterized by lower stability than those made with the other bacterial cultures. The impact of lactic bacteria or their metabolic products on the stability of anthocyanins in berry yoghurts shows that an appropriate selection of culture for production of yoghurt is recommended.     

Evolution of β-lactoglobulin and α-lactalbumin content during yoghurt fermentation

The evolution of the concentrations of β-lactoglobulin (BLG) and α-lactalbumin (ALAC) was studied during the early stages of yoghurt fermentation by YC 191, a mixed strain culture from Chr Hansen containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Radial immunodiffusion of samples taken at different times indicated that the concentration of both proteins remained constant during fermentation. Electrophoresis performed on 12% polyacrylamide slab gels confirmed the results obtained with radial immunodiffusion. In model experiments, the strains were incubated either separately or in combination with both whey proteins, one by one or together. BLG proteolysis required a longer time than that used during yoghurt fermentation. ALAC was susceptible to proteolysis, especially by Streptococcus thermophilus. Despite evident possession of adequate proteolytic system, the strains used for yoghurt production did not cleave detectable amounts of the whey proteins during yoghurt fermentation.     

Isolation and characterisation of a Lactobacillus delbrueckii subsp. bulgaricus mutant with low H+‐ATPase activity

Lactobacillus delbrueckii subsp. bulgaricus is an acid‐tolerant species used in yoghurt production and considered important in postacidification reductions in palatability of yoghurt during storage. In this study, a variant (ND06‐2) of L. delbrueckii subsp. bulgaricus ND06 was selected using neomycin sulphate. ND06‐2 exhibited sensitivity to acids and lower H⁺‐ATPase activity compared with ND06. High‐pressure liquid chromatography (HPLC) was used to demonstrate that the capacity of ND06‐2 to produce lactic acid was significantly lower than for ND06 during fermentation or storage. These results suggest that the L. delbrueckii subsp. bulgaricus variant could be used in yoghurt starters and have a lower tendency to cause postacidification.     

Comparative analysis of physicochemical,rheological, sensory and flavour properties of yoghurts using a new probiotic Bacillus coagulans 13002 with traditional yoghurt starter

Bacillus coagulans 13002 (13002) as a new potential starter was incorporated into the yoghurts for fermentation in this study. We compared three groups of yoghurt, including Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (SL), 13002 (BCS), and 13002 and SL (BCS + SL). Compared with the combination of SL and BCS, the group of BCS + SL exhibited better flavour, higher titratable acidity, cohesiveness and richer volatile compounds, suggesting that the combination of BCS and SL was more efficient to produce the best sensory properties of yoghurt (P < 0.05). The production of volatile compounds in fermentation with and without the addition of BCS was mainly affected by aldehydes, ketones, esters and hydrocarbons, respectively. Furthermore, BCS additions into traditional starters resulted in increasing organic acids and speeding up the usage of sucrose and lactose during the fermentation period of yoghurt. Our study suggested that BCS is a favourable starter supplement for improving the quality of probiotic yoghurts.     

Rheology,spontaneous whey separation,microstructure and sensorial characteristics of probiotic yoghurts enriched with passion fruit fiber

Beyond demonstrated beneficial health attributes, passion fruit rinds are a by-product of the fruit pulp industry, rich in total dietary fiber, particularly pectin. The aim of this study was to evaluate the influence of the addition of passion fruit fiber on the whey formation, rheological parameters, microstructure and sensorial characteristics of probiotic yoghurts. Skim milk bases enriched with 1% of passion fruit fiber or not were heat treated and inoculated with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and divided into four groups according to the probiotic strain added — Lactobacillus acidophilus strains L10 and NCFM and Bifidobacterium animalis subsp. lactis strains Bl04 and B94. Fermentations were performed until the pH reached 4.5. Rheological characteristics of yoghurts were determined by a rotational rheometer in two cycles of shear rate ranging from 0 to 15 s^? 1 in both upward and downward curves. Sensorial analysis of passion fruit fiber yoghurts, either without any probiotic or co-fermented by L. acidophilus L10 or B. animalis subsp. lactis Bl04, was evaluated against a control yoghurt without fiber. Photomicrographs of freeze-dried yoghurts were made by field-emission scanning electron microscope (SEM). Thixotropy of enriched yoghurts was higher than that of their respective controls in the two cycles of shear rate. Apparent viscosity was significantly higher in fiber yoghurts co-fermented by the lactobacilli than in their controls at the end of cold storage. Photomicrographs demonstrated that in passion fruit fiber yoghurts the casein gel was more compact and overlaid the fiber, while filaments of exopolysaccharides were more frequent in control yoghurts. Appearance, odor and color of the passion fruit fiber yoghurts received scores as ‘good’, and the intensity of the passion fruit flavor was considered weak by the sensory assessors. Results indicate that the passion fruit fiber is an almost neutral ingredient for the design of new high value-added yoghurt.     

Microbial and physiochemical changes in yoghurts containing different Lactobacillus delbrueckii subsp. bulgaricus strains in association with Lactobacillus plantarum as an adjunct culture

Two strains of slow and normal acid‐producing Lactobacillus delbrueckii subsp. bulgaricus, mixed separately with Streptococcus thermophilus, were used in the preparation of yoghurt with or without Lactobacillus plantarum A7. Viable cell counts of lactic bacteria, pH, lactic acid and acetic acid concentrations, soluble nitrogen content, spontaneous syneresis, viscosity and firmness were studied at selected points during the refrigerated storage period. Results showed that survival of L. plantarum A7 in yoghurt was independent of the acidification capability of L. delbrueckii subsp. bulgaricus strains which had already been characterised in their pure culture. Thus, in the preparation of probiotic yoghurt, using slow acid‐producing L. delbrueckii subsp. bulgaricus strain in combination with nondairy lactobacilli postfermentation acidification is not restricted but instability in other physicochemical properties of the yoghurt may be expected.     

High genetic and phenotypic variability of Streptococcus thermophilus strains isolated from artisanal Yuruk yoghurts

Streptococcus thermophilus is a commonly used starter bacterium in dairy industry. It reduces the pH of milk rapidly and equilibrates the medium for the growth of Lactobacillus delbrueckii subsp. bulgaricus during yoghurt fermentation. Efforts to increase the diversity of artisanal yoghurt starters are not only important to bring new strains with novel and desirable characteristics, but also for the preservation of natural diversity which diminishes with the overuse and spread of industrial starters to natural resources. In the present study, 14 artisanal yoghurt samples were processed for the isolation of promising strains for yoghurt starter culture production and 66 strains were subsequently characterized. They were all identified as S. thermophilus using species-specific PCR and 16S rRNA gene sequencing. Genotypic diversity at the strain level was investigated by pulsed field gel electrophoresis (PFGE), and 22 homology groups were obtained. Further phenotypic characterization unearthed a significant phenotypic heterogeneity within homology groups, mostly with atypical novel character. Only 7 out of 66 strains showed S. thermophilus type-strain like phenotypic traits. Majority of the isolates were determined to be protease positive and fast milk acidifier to be used as yoghurt starter culture.     

Effect of Greek-style yoghurt manufacturing processes on starter and probiotic bacteria populations during storage

This study compared viable counts of Streptococcus thermophilus (starter) and Lactobacillus helveticus R0052 (probiotic), as a function of two manufacturing processes of Greek-style (GS) yoghurt: centrifugation of a curd (GS-CF process) or ultrafiltration of milk prior to fermentation (GS-UF process). Fresh GS-UF and GS-CF yoghurts had between 3 and 7 times higher counts of Lb. helveticus R0052 and S. thermophilus than the regular stirred yoghurt (Control). Strain R0052 was three times more stable in the GS-CF yoghurt than in the Control over the 44 d storage period. The highest lactose content (44 g L⁻¹) was obtained in the Control, while 60% less was obtained in the GS-UF yoghurt; the opposite trend was observed for lactic acid and galactose. Free amino acid levels were 33% higher in the GS products. GS yoghurt can be 10 times better than traditional yoghurt to deliver probiotic bacteria, as a function of the process used.     

Viability and activity of bifidobacteria in yoghurt containing fructooligosaccharide during refrigerated storage

The viability of yoghurt bacteria and two commercial strains of bifidobacteria was assessed in either yoghurt containing chicory fructooligosaccharide (FOS) or without any prebiotic, during 28 days storage at 4 °C. All the products showed a decrease in the viable count of yoghurt bacteria and bifidobacteria during storage. Numbers of Lactobacillus delbrueckii ssp. bulgaricus decreased faster than those for Streptococcus thermophilus. The viability of bifidobacteria in yoghurt was affected by the strain type and the presence of FOS. Bifidobacterium animalis exhibited better stability in the yoghurt than B. longum. The recommended level of 1 million cells was exceeded for B. animalis throughout storage. The highest viable number of bifidobacteria (3.59–2.25 × 10⁷ CFU g^?1) was obtained in the product containing B. animalis and FOS. Viability of B. longum in yoghurt containing FOS remained above 10⁶ CFU g^?1 for up to 21 days, whereas this level was maintained for only 7 days for that organism in yoghurt without any prebiotic.