Molecular detection of Bifidobacterium animalis DN-173010 in human feces during fermented milk administration

We tested different techniques to detect exogenous bifidobacteria (DN-173010) in feces; genus- and species-specific PCR technique; amplified ribosomal DNA restriction analysis (ARDRA) and fluorescent in situ hybridization (FISH) technique. A significant increase in the number of bifidobacteria in feces was observed during ingestion of fermented milk, and also, we detected a decrease in this number when the ingestion stopped. The number of bifidobacteria enumerated by culturing was 10–100-fold lower than by FISH technique. Bifidobacterium animalis DN-173010 can survive passage through the gastrointestinal tract and was detected viable in human feces. Combination of ARDRA and FISH was a powerful tool to detect exogenous bifidobacteria. The aim of this study is to demonstrate that Bifidobacterium DN-173010 can survive passage through the gastrointestinal tract and be recovered live in human feces. For this purpose, we have assessed different techniques to detect and quantify the number of bifidobacteria following fermented milk supplemented with B. animalis subsp. lactis DN-173010 administration and to confirm that this strain can survive passage through the gastrointestinal tract by recovering viable cells in human feces.     

Molecular discrimination of new isolates of Bifidobacterium animalis subsp. lactis from reference strains and commercial probiotic strains

Fifteen new isolates from pig faeces were identified as Bifidobacterium animalis subsp. lactis by polymerase chain reaction (PCR) using primers specific for this subspecies. Ten of the isolates could be differentiated at strain level by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). These new strains were different from the type strain and other reference strains of this subspecies, and from all commercial dairy strains with probiotic functionality. Thus, possibly some of the isolates are potential candidates for new probiotic Bifidobacterium strains that can be unambiguously identified by RAPD-PCR and PFGE, which could be of interest for the food industry. In contrast, reference strains and commercial dairy strains revealed great genetic homogeneity, showing almost identical DNA fingerprints using RAPD-PCR and PFGE. Some reference strains and all commercial probiotic strains that had been originally designated as B. animalis or B. lactis have to be assigned to B. animalis subsp. lactis to be correctly labelled.     

Survival of three Bifidobacterium animalis subsp. lactis strains is related to trans-vaccenic and α-linolenic acids contents in organic fermented milks

This study aims at relating the survival at 4 °C for 28 days in organic and conventional fermented milks of three strains of Bifidobacterium animalis subsp. lactis (BB12, B94 and BL04), in co-culture with Streptococcus thermophilus TA040 and Lactobacillus bulgaricus LB340, to milk fatty acids profile. Cultivability after 28 days of cold storage was improved in organic fermented milks as compared to conventional products, with slight differences among strains. In addition, the poly-unsaturated fatty acids fraction was higher in organic products, as well as the relative trans-vaccenic (TVA) and α-linolenic (ALA) fatty acids contents that were respectively 1.7 and 2.4 times higher in organic than in conventional fermented milks. From these results, it was concluded that elevated levels of TVA and ALA, together with a lower ratio between linoleic acid and α-linolenic acid, as found in organic products, improved the survival of the bifidobacteria during chilled storage.     

Intestinal survival and persistence of probiotic Lactobacillus and Bifidobacterium strains administered in triple-strain yoghurt

The aim of the present study was to investigate the intestinal survival and persistence of probiotic strains Lactobacillus F19, Lactobacillus acidophilus NCFB 1748, and Bifidobacterium animalis subsp. lactis Bb-12 consumed in a yoghurt (ABC product), and also their effect on the intestinal microbiota. Based on the results of culture studies and strain-level analysis by randomly amplified polymorphic DNA (RAPD) fingerprinting Lactobacillus F19 and B. animalis subsp. lactis Bb-12 survived well through the human gastrointestinal tract; they were detected in reasonable numbers in the faeces of 100% and 79% of the study subjects, respectively. Ingestion of the probiotic yoghurt increased transiently the numbers of bifidobacteria and lactobacilli. For lactobacilli the increase was due to the detection of the ingested probiotic strains in faeces, while in bifidobacteria the increase was likely caused by the increase of indigenous bifidobacteria since the ingested Bifidobacterium strain did not comprise the predominant part of bifidobacterial population during the intervention. Probiotic strains were infrequently detected in mucosal biopsy samples. The present study indicates that developing probiotic food products with multiple probiotic strains is feasible.     

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.     

Short communication: Survival of the characteristic microbiota in probiotic fermented camel,cow, goat,and sheep milks during refrigerated storage

The objective of this study was to monitor the viability during storage of Lactobacillus acidophilus LA-5 (A), Bifidobacterium animalis ssp. lactis BB-12 (B), and Streptococcus thermophilus CHCC 742/2130 (T) in probiotic cultured dairy foods made from pasteurized camel, cow, goat, and sheep milks fermented by an ABT-type culture. The products manufactured were stored at 4°C for 42 d. Microbiological analyses were performed at weekly intervals. Streptococcus thermophilus CHCC 742/2130 was the most numerous culture component in all 4 products both at the beginning and at the end of storage. The viable counts of streptococci showed no significant decline in fermented camel milk throughout the entire storage period. The initial numbers of Lb. acidophilus LA-5 were over 2 orders of magnitude lower than those of Strep. thermophilus CHCC 742/2130. With the progress of time, a slow and constant decrease was observed in lactobacilli counts; however, the final viability percentages of this organism did not differ significantly in the probiotic fermented milks tested. The cultured dairy foods made from cow, sheep, and goat milks had comparable B. animalis ssp. lactis BB-12 counts on d 0, exceeding by approximately 0.5 log₁₀ cycle those in the camel milk-based product. No significant losses occurred in viability of bifidobacteria in fermented camel, cow, and sheep milks during 6 wk of refrigerated storage. In conclusion, all 4 varieties of milk proved to be suitable raw materials for the manufacture of ABT-type fermented dairy products that were microbiologically safe and beneficial for human consumption. It was suggested that milk from small ruminants be increasingly used to produce probiotic fermented dairy foods. The development of camel milk-based probiotic cultured milks appears to be even more promising because new markets could thus be conquered. It must be emphasized, however, that further microbiological and sensory studies, technology development activities, and market research are needed before such food products can be successfully commercialized.     

Survival of Bifidobacterium strains in organic fermented milk is improved as a result of membrane fatty acid composition

Survival of three Bifidobacterium strains in organic and conventional fermented milks in relation to their membrane fatty acid composition was studied during chilled storage. Survival of Bifidobacterium lactis BB12 and BL04 over 21 days at 4 °C was improved in organic fermented milks, whereas these parameters were less effective for Bifidobacterium infantis ATCC15697. These different behaviours were linked to acidification activity, oxidoreduction potential and relative fatty acid composition, which differed among the strains and the types of milk used. The higher relative unsaturated fatty acid content in organic products, including trans-vaccenic, conjugated linoleic and α-linolenic acids, resulted in an increase in the linoleic and α-linolenic acid content in the cell membranes of B. lactis BB12 and BL04. The study showed that the membrane fatty acid composition, which depended on the strain and on the milk fatty acid composition, affected the survival of bifidobacteria during chilled storage in fermented milk products.     

Short communication: the complete genome sequence of Bifidobacterium animalis subspecies animalis ATCC 25527(T) and comparative analysis of growth in milk with B. animalis subspecies lactis DSM 10140(T)

The objective of this work was to sequence the genome of Bifidobacterium animalis ssp. animalis ATCC 25527^T, the subspecies most closely related to B. animalis ssp. lactis, some strains of which are widely added to dairy foods as probiotics. The complete 1,932,963-bp genome was determined by a combination of 454-shotgun sequencing and PCR gap closing, and the completed assembly was verified by comparison with a KpnI optical map. Comparative analysis of the B. animalis ssp. animalis ATCC 25527^T and B. animalis ssp. lactis DSM 10140^T genomes revealed high degrees of synteny and sequence homology. Comparative genomic analysis revealed 156 and 182 genes that were unique to and absent in the B. animalis ssp. animalis genome, respectively. Among these was a set of unique clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes and a novel CRISPR locus containing 30 spacers in the genome of B. animalis ssp. animalis. Although previous researchers have suggested that one of the defining phenotypic differences between B. animalis ssp. animalis and B. animalis ssp. lactis is the ability of the latter to grow in milk and milk-based media, the differential gene content did not provide insights to explain these differences. Furthermore, growth and acid production in milk and milk-based media did not differ significantly between B. animalis ssp. lactis (DSM 10140^T and Bl04) and B. animalis ssp. animalis (ATCC 25527^T). Growth of these strains in supplemented milk suggested that growth was limited by a lack of available low-molecular-weight nitrogen in the 3 strains examined.     

Effect of Aqueous Extract of the Seaweed Gracilaria domingensis on the Physicochemical,Microbiological, and Textural Features of Fermented Milks

The effects of the Gracilaria domingensis seaweed aqueous extract in comparison with gelatin on the physicochemical, microbial, and textural characteristics of fermented milks processed with the mixed culture SAB 440 A, composed of Streptococcus thermophilus, Lactobacillus acidophilus, and Bifidobacterium animalis ssp. lactis, were investigated. The addition of G. domingensis aqueous extract did not affect pH, titratable acidity, and microbial viability of fermented milks when compared with the control (with no texture modifier) and the products with added gelatin. Fermented milk with added the seaweed aqueous extract showed firmness, consistency, cohesiveness, and viscosity index at least 10% higher than those observed for the control product (P < 0.05). At 4 h of fermentation, the fermented milks with only G. domingensis extract showed a texture comparable to that observed for products containing only gelatin. At 5 h of fermentation, firmness and consistency increased significantly (P < 0.05) in products with only seaweed extract added, a behavior not observed in products with the full amount of gelatin, probably due to the differences between the interactions of these ingredients with casein during the development of the gel network throughout the acidification of milk. The G. domingensis aqueous extract appears as a promising gelatin alternative to be used as texture modifier in fermented milks and related dairy products.     

Safety and intestinal microbiota modulation by the exopolysaccharide-producing strains Bifidobacterium animalis IPLA R1 and Bifidobacterium longum IPLA E44 orally administered to Wistar rats

Bifidobacterium animalis subsp. lactis IPLA R1 and Bifidobacterium longum IPLA E44 strains were tested for their safety and ability to modulate the intestinal microbiota in vivo. Chemically simulated gastrointestinal digestion showed considerably lower survival of E44 than R1 strain, the first microorganism also being more sensitive to refrigerated storage in 10% skimmed milk at 4 °C. Harmful glycosidic activities were absent, or at low levels, in the strains R1 and E44. Both strains were sensitive to most antibiotics and resistant to aminoglycosides, a common feature in bifidobacteria. Similar to several other bifidobacteria strains, B. animalis subsp. lactis IPLA R1 displayed a moderate resistance against tetracycline which correlated with the presence of tet(W) gene in its genome. The general parameters indicating well-being status, as well as translocation to different organs and histological examination of the gut tissues, revealed no changes induced by the administration of bifidobacteria to rats. Twelve-week-old male Wistar rats were distributed into three groups, eight rats in each. Two groups were administered daily over 10⁸ cfu of the corresponding strain suspended in 10% skimmed milk for 24 days, whereas rats in the placebo group received skimmed milk without microorganisms added. The microbiota and short chain fatty acids (SCFA) were monitored in faeces at different time points during treatment and in caecum content at the end of the assay. Quantitative PCR (qPCR) showed that faecal and caecal Bifidobacterium levels were higher in bifidobacteria-fed rats than in the placebo rats at the end of the intervention, whereas total anaerobic plate counts did not show significant differences. Quantification of B. animalis and B. longum by qPCR showed that, independent of the microorganism administered, treatment with bifidobacteria resulted in higher levels of B. animalis in the caecum. PCR-DGGE analysis of microbial populations revealed a higher diversity of bands in caecum content of rats fed B. animalis IPLA R1 than in the placebo group and rats fed B. longum IPLA E44. Remarkably, although no variations in the proportion of acetate, propionate and butyrate were found, at the end of the assay the total SCFA concentration in the faeces of rats fed bifidobacteria was significantly higher and those in caecum content significantly lower, than that of the placebo group. This suggests a displacement of the SCFA production to parts of the colon beyond the caecum in rats receiving bifidobacteria. Therefore, the oral administration of B. animalis IPLA R1 and B. longum E44 can be considered safe, these microorganisms having the ability to modulate the intestinal microbiota of rats by influencing SCFA and the bifidobacterial population levels.     

Physicochemical,textural and volatile characteristics of fermented milk co-cultured with Streptococcus thermophilus,Bifidobacterium animalis or Lactobacillus plantarum

Fermented milks were prepared with various culture combinations of Lactobacillus plantarum or Bifidobacterium animalis with Streptococcus thermophilus, and the impact of microbes combinations on acidification, proteolysis, lipolysis, texture, volatiles and sensory quality of fermented milk was investigated during 21-day storage at 4 °C. The results showed that products from the co-cultures displayed higher titratable acidity, more peptides, higher proteolytic activity and lipolysis capacity compared with that from the pure strains of S. thermophilus. When L. plantarum and B. animalis combined in a ratio of 2:1 with S. thermophilus, the products maintained relatively more viable cell counts of the 3 strains, strong proteolysis ability and lipolysis capacity during the storage. Milk fermented by co-cultures of the 3 strains exhibited higher cohesiveness, more volatiles and better sensory quality compared with these fermented by S. thermophilus only or in co-cultures with either B. animalis or L. plantarum based on principal component analysis.     

Short communication: Effect of the addition of Bifidobacterium monocultures on the physical,chemical, and sensory characteristics of fermented goat milk

The aim of the study was to use 3 monocultures of Bifidobacterium (Bifidobacterium animalis ssp. lactis AD600, Bifidobacterium animalis ssp. lactis BB-12, and Bifidobacterium longum AD50) in fermented goat milk to assess the microbial, physicochemical, rheological, and sensory quality of beverages during a 3-wk storage period at 5°C. The results indicated that selected bifidobacteria may be used for production of fermented goat milk because they comply with the minimum standards specified by the Food and Agriculture Organization of the United Nations and the World Health Organization during the entire period of storage. However, goat milk fermented by Bif. longum AD50 had less than 10⁶ cfu/g after 21 d of storage. The acidity, acetaldehyde content, viscosity, and hardness of fermented goat milk beverages depended on the strain and the storage period. Sensory properties were similar and acceptable, with a tendency for the quality to be reduced with an extended storage time. Depending on the monoculture of bifidobacteria used to manufacture fermented goat milk, the product had a different pH value. Titratable acidity in all fermented goat milk increased significantly along with the time of storage. Our study has shown that monocultures of bifidobacteria had a significant effect on the content of acetaldehyde, but the lowest effect over the entire storage period was observed in goat milk fermented by Bif. animalis ssp. lactis BB-12. This sample also had the lowest viscosity values compared with other samples and the best organoleptic properties during a 3-wk storage period.     

The residual occurrences of Bacillus thuringiensis biopesticides in food and beverages

In 2006, 54 pasteurized full fat milk samples, 40 ice-cream samples, and two green-tea beverage samples were analyzed and a total of 19 Bacillus thuringiensis-like strains were isolated, nine from seven pasteurized milks, one from an ice-cream with peach pulp and juice, and nine from two green-tea beverages. These strains were classified as B. thuringiensis, contained the cry1A gene and produced crystal inclusions during sporulation. All strains were characterized by a serotyping test, SDS-PAGE, random amplified polymorphic DNA, and enterotoxic gene PCR analysis. Most isolates produced bipyramidal crystals and belonged to serotypes H_3a3b, H_5a5b, or H₇. Furthermore, two strains from pasteurized full fat milks and three strains from green-tea beverages were indistinguishable from the B. thuringiensis subsp. kurstaki strains isolated from commercial biopesticides (Kaiyan®, Qiangdi®, Lvpuan® and Sutai®), suggesting the residual occurrences of B. thuringiensis from biopesticides in food and beverages.     

Distribution of Penicillium commune isolates in cheese dairies mapped using secondary metabolite profiles, morphotypes, RAPD and AFLP fingerprinting

In an 8-year study of the diversity and distribution of Penicillium commune contaminants in two different cheese dairies, swab and air samples were taken from the production plants, the processing environment and contaminated cheeses. A total of 321 Penicillium commune isolates were characterized using morphotypes (colony morphology and colours) and secondary metabolite profiles. Based on production of secondary metabolites the P. commune isolates were classified into 6 groups. The genetic diversity of the P. commune isolates was assessed using randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). For a sub-set of 272 P. commune isolates RAPD analysis generated 33 RAPD groups whereas AFLP profiling revealed 55 AFLP groups. This study conclusively showed that the discriminatory power of AFLP was high compared to RAPD and that AFLP fingerprinting matched morphotyping. P. commune isolates with identical profiles using all four typing techniques were interpreted as closely related isolates with a common origin and the distribution of these isolates in the processing environment indicated possible contamination points in the cheese dairies. The coating process and unpacking of cheeses with growth of P. commune seemed to cause the contamination problems. Several identical P. commune isolates remained present in the processing environment for more than 7 years in both dairies.     

Probiotic monocultures in fermented goat milk beverages – sensory quality of final product

The aim of our study was to conduct a selection of the monocultures capable of providing the most attractive sensory features of the final product. Four fermented goat's milk beverages were produced with probiotic monocultures containing Lactobacillus (Lb. acidophilus La‐5, Lb. rhamnosus K3 and Lb. plantarum O20) and Bifidobacterium (Bif. animalis subsp. lactisBB‐12). A sensory analysis and microbiological assessment of fermented goat's milk beverages were made at the beginning of the study and after 3, 7, 10 and 14 days of refrigerated storage (5 ± 1 °C). We found that samples including monocultures Lb. plantarum O20 and Bif. animalis subsp. lactisBB‐12 were differentiated from other goat's milk beverages.     

Influence of cofermentation by amylolytic Lactobacillus strains and probiotic bacteria on the fermentation process,viscosity and microstructure of gruels made of rice,soy milk and passion fruit fiber

Gruels tailored to school-age children and made of soy milk and rice flour with or without total dietary fiber from passion fruit by-product were fermented by amylolytic lactic acid bacteria strains (Lactobacillus fermentum Ogi E1 and Lactobacillus plantarum A6), by commercial probiotic bacteria strains (Lactobacillus acidophilus L10, Lactobacillus casei L26 and Bifidobacterium animalis subsp. lactis B94) and by co-cultures made of one amylolytic and one probiotic strain. The influence of ingredient composition and bacterial cultures on kinetics of acidification, α-amylase activity of the bacteria, apparent viscosity and microstructure of the fermented products was investigated. During fermentation of the gruels, α-amylase activity was determined through the Ceralpha method and apparent viscosity, flux behavior and thixotropy were determined in a rotational viscometer. Rheological data were fitted to Power Law model. The combination of amylolytic and probiotic bacteria strains reduced the fermentation time of the gruels as well as increased the α-amylase activity. The addition of passion fruit fiber exerted less influence on the apparent viscosity of the fermented products than the composition of the bacterial cultures. Scanning electron microscopy provided evidence of exopolysaccharide production by amylolytic bacteria strains in the food matrices tested. The co-cultures made of amylolytic and probiotic bacteria strains are suitable to reduce the fermentation time of a soy milk/rice matrix and to obtain a final product with pH and viscosity similar to yoghurt.     

Improved viability of bifidobacteria in fermented milk by cocultivation with Lactococcus lactis subspecies lactis

The poor survival of probiotic bacteria in commercial yogurts may limit their potential to exert health benefits in humans. The objective was to improve the survival of bifidobacteria in fermented milk. Cocultivation with some strains of Lactococcus lactis ssp. lactis improved the survival of bifidobacteria in fermented milk during refrigerated storage. Studies on one strain, Lc. lactis ssp. lactis MCC866, showed that the concentrations of dissolved oxygen were kept lower in the cocultivated fermented milk during storage compared with monocultured Bifidobacterium longum BB536 or samples cocultured with another noneffective Lc. lactis ssp. lactis strain. Degradation of genomic DNA was suppressed in the cocultivating system with Lc. lactis ssp. lactis MCC866. Several genes that participated in protection from active oxygen species (e.g., genes coding for alkyl hydroperoxide reductase and Fe²⁺ transport system) were expressed at higher levels during refrigerated storage in Lc. lactis ssp. lactis MCC 866 compared with another noneffective Lc. lactis ssp. lactis strain. Concentration of free iron ion was also lower in supernatants of fermented milk cocultivated with B. longum BB536 and Lc. lactis ssp. lactis MCC866. These results suggest that Lc. lactis ssp. lactis MCC 866 is potentially superior in reducing oxygen damage and consequently improves the survival of bifidobacteria in the cocultivating system. This cocultivation system is of industrial interest for producing fermented milk containing viable bifidobacteria with long shelf life.     

Influence of adjunct cultures on angiotensin‐converting enzyme (ACE)‐inhibitory activity,organic acid content and peptide profile of kefir

The angiotensin‐converting enzyme (ACE)‐inhibitory activities, peptide profiles and organic acid contents in kefir produced by kefir grains plus lactic acid bacteria as adjunct cultures were determined. All the kefir samples showed almost similar peptide profiles as detected by RP‐HPLC, but quantitative differences were observed during storage. The ACE‐inhibitory activities of different lactic cultures did not exhibit a linear tendency during storage period. After 7 days of storage, there was a significant increase in ACE‐inhibitory activity of the sample fermented with Lactobacillus helveticus. However, a kefir sample containing Streptococcus thermophilus, Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis exhibited a higher ACE‐inhibitory activity (92.23%) compared to the other samples.     

Suitability of Bifidobacterium spp. and Lactobacillus plantarum as Probiotics Intended for Fruit Juices Containing Citrus Extracts

A strain of Lactobacillus plantarum and 4 strains of bifidobacteria were inoculated in apple juice and in a commercial beverage labeled as “red‐fruit juice,” containing citrus extracts as natural preservatives; the suitability of the probiotics was evaluated in relation to their resistance to 2 kinds of citrus extracts (biocitro and lemon extract), survival in juices at 4 and 37 °C, and inhibition of Zygosaccharomyces bailii. Cell count of L. plantarum and bifidobacteria over time was fitted through the Weibull equation, for the evaluation of the first reduction time (δ), death time, and microbiological shelf life (the break‐point was set to 7 log cfu/mL). Bifidobacterium animalis subsp. lactis experienced the highest δ‐value (23.21 d) and death time (96.59 d) in the red‐fruit juice at 4 °C, whereas L. plantarum was the most promising strain in apple juice at 37 °C. Biocitro and lemon extract did not exert a biocidal effect toward probiotics; moreover, the probiotics controlled the growth of Z. bailii and the combination of L. plantarum with 40 ppm of biocitro reduced the level of the yeast after 18 d by 2 log cfu/mL.     

Comparative effect of probiotic and paraprobiotic addition on rheological and sensory properties of yoghurt

Yoghurt was produced using killed cells of Lactobacillus acidophilus ATCCSD 5221 and Bifidobacterium animalis subsp. lactis BB‐12. The rheological and sensory properties of the yoghurt were investigated at the end of the fermentation. The results revealed that the samples made with killed cells gave the lowest storage modulus, loss modulus, stress crossover point and loss tangent. The frequency‐sweep of yoghurt samples containing paraprobiotics added before fermentation showed the highest correlation coefficient. In the case of sensory assessment, significant differences were indicated among treatments. Adding killed cells of B. animalis subsp. lactis BB‐12 before fermentation produced yoghurt with the highest total score, whereas samples containing killed cells of L. acidophilus ATCCSD 5221 and B. animalis subsp. lactis BB‐12 added before fermentation exhibited the lowest total score.