Culture-dependent and culture-independent qualitative analysis of probiotic products claimed to contain bifidobacteria

A total of 58 probiotic products obtained worldwide, which were claimed to contain Bifidobacterium strains (including 22 yoghurts, 5 dairy fruit drinks, 28 food supplements and 3 pharmaceutical preparations) were investigated in parallel using a culture-dependent and a culture-independent approach. Three isolation media previously reported as selective for Bifidobacterium were evaluated for their suitability in the quality analysis of these products. Subsequently, possible bifidobacterial colonies were picked from the best medium and identified by means of rep-PCR fingerprinting using the BOX primer (BOX-PCR). Bifidobacterium animalis subsp. lactis, formerly classified as Bifidobacterium lactis, was most frequently found, but strains belonging to Bifidobacterium longum biotypes longum and infantis, Bifidobacterium bifidum and Bifidobacterium breve were recovered also. In parallel, all products were also subjected to culture-independent analysis which involved a nested-PCR step on total bacterial DNA extracted directly from the product, followed by separation of the amplicons by Denaturing Gradient Gel Electrophoresis (DGGE) and subsequent identification of species from the band patterns. By conventional cultivation, 70.7% of the products analysed were found to contain culturable bifidobacteria whereas by culture-independent DGGE analysis members of the genus Bifidobacterium could be detected in 96.5% of the analysed products. Genotypic characterization of a number of bifidobacterial isolates at the strain level by means of Pulsed-Field Gel Electrophoresis (PFGE) revealed a relatively high degree of genomic homogeneity among the Bifidobacterium strains currently used in the probiotic industry.     

Evaluation of real-time PCR targeting the 16S rRNA and recA genes for the enumeration of bifidobacteria in probiotic products

The application of real-time PCR targeting the multicopy 16S rRNA gene and the single copy recA gene was evaluated for the enumeration of bifidobacteria in 29 probiotic products claimed to contain these organisms. Both assays relied on the use of genus-specific primers and the non-specific SYBR Green I chemistry. For both applications, the calibration curve was constructed using the type strain of Bifidobacterium animalis subsp. lactis. Upon correction with a factor corresponding to the 16S rRNA gene copy number, both assays generally produced comparable enumeration results. Only in exceptional cases, differences between both gene targets were found in probiotic products containing low amounts of bifidobacteria in which case the quantification of the multicopy 16S rRNA gene turned out to be more sensitive than the recA-based assay. On the other hand, the use of the latter single copy gene in real-time PCR quantification offers the advantage that no prior knowledge of bacterial content is required when using genus-specific primers, since no correction for multiple gene copies has to be performed. Only 11 of the analysed products (38%), including one dairy based product and ten dried products, contained a minimal Bifidobacterium concentration of 10(6) CFU per ml or g of product. Depending on the application, both assays proved to be rapid and reproducible alternatives for culture-based detection and quantification of bifidobacteria in probiotic products.     

Antibiotic resistance of lactic acid bacteria and Bifidobacterium spp. isolated from dairy and pharmaceutical products

The outlines of antibiotic resistance of some probiotic microorganisms were studied. This study was conducted with the double purpose of verifying their ability to survive if they are taken simultaneously with an antibiotic therapy and to increase the selective properties of suitable media for the isolation of samples containing mixed bacterial populations. We isolated from commercial dairy and pharmaceutical products, 34 strains declared as probiotics, belonging to the genera Bifidobacterium and Lactobacillus, and 21 strains of starter culture bacteria. All the microorganisms have been compared by electrophoresis of the soluble proteins for the purpose of identifying them. A Multiplex-PCR with genus- and species-specific primers was used to detect for Bifidobacterium animalis subsp. lactis presence. All bifidobacteria were B. animalis subsp. lactis except one Bifidobacterium longum. Sometimes the identification showed that the used strain was not the one indicated on the label. The lactobacilli were Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus delbrueckii subsp. bulgaricus. The streptococci were all Streptococcus thermophilus. The minimal inhibitory concentration (MIC) of 24 common antibiotic substances has been valued by the broth microdilution method. All tested strains were susceptible to ampicillin, bacitracin, clindamycin, dicloxacillin, erytromycin, novobiocin, penicillin G, rifampicin (MIC(90) ranging from 0.01 to 4 microg/ml); resistant to aztreonam, cycloserin, kanamycin, nalidixic acid, polymyxin B and spectinomycin (MIC(90) ranging from 64 to >1000 microg/ml). The susceptibility to cephalothin, chloramphenicol, gentamicin, lincomycin, metronidazole, neomycin, paromomycin, streptomycin, tetracycline and vancomycin was variable and depending on the species.     

Rapid discrimination of Bifidobacterium animalis subspecies by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Currently, the species Bifidobacterium animalis consists of two subspecies, B. animalis subsp. lactis and B. animalis subsp. animalis. Among these two subspecies, B. animalis subsp. lactis is especially important because it is widely used in the manufacture of probiotic dairy products. The application of these microbes in the food industry demands fast, accurate and low cost methods to differentiate between species and strains. Although various genotypic methods have been employed to discriminate between these two subspecies, they are not easily adapted for rapid identification in the industry. The purpose of this study was to evaluate the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to differentiate between the two subspecies of B. animalis, and for discrimination at strain level. We identified twenty-three strains of B. animalis at subspecies and strain level by genotypic methods and by proteomics using MALDI-TOF MS. The proteomics identification by MALDI-TOF was nearly identical to that obtained by genotypic identification using comparison of tuf and atpD gene sequences, and single-nucleotide polymorphisms (SNPs), insertions, and deletions (INDELs). We identified four protein markers, L1, L2, A1, and A2, which are useful for discriminating between both subspecies. Proteomics identification using MALDI-TOF MS was therefore an accurate method for discriminating and identifying these bacteria. Given the speed in which this method is achieved (~20 min including sample preparation), MALDI-TOF MS is promising as a tool for rapid discrimination of starter cultures and probiotics.     

婴儿源益生性双歧杆菌的筛选及肠道定殖性研究

本研究旨在从婴儿粪便中筛选出具有潜在益生特性的双歧杆菌,并探究其肠道定殖情况,为双歧杆菌的产品开发提供优良的菌株。采用MRS培养基对样品进行分离纯化,菌株经F6PPK检测及16S r DNA测序鉴定,之后进行模拟胃肠液、胆盐耐受性、对食源性致病菌(大肠杆菌、沙门氏菌、单增李斯特菌等)的抑制及对HT-29细胞的粘附能力测定,将筛选出的菌株进行动物实验,测定其肠道定殖能力。分离到的27株双歧杆菌,经分子生物学鉴定为7个不同的种:Bifidobacterium longum、Bifidobacterium breve、Bifidobacterium bifidum、Bifidobacterium pseudocatenulatum、Bifidobacterium infantis、Bifidobacterium animalis和Bifidobacterium adolescentis。体外实验表明,B.longum A9、B.breve A4、B.bifidum B6、B.longum C6、B.adolescentis F8和B.infantis H6等具有较强的潜在益生特性;动物实验表明,B.infantis H6和B.longum C6具有较强的肠道定殖能力。B.longum C6和B.infantis H6有望作为优良的益生性菌株,应用于双歧杆菌的产品开发。     

Effects of administration of Bifidobacterium animalis subsp. lactis GCL2505 on defecation frequency and bifidobacterial microbiota composition in humans

The aim of this study was to evaluate the changes in endogenous bifidobacteria and administered Bifidobacterium animalis subsp. lactis (B. lactis) GCL2505 (GCL2505) in the intestine after administration of GCL2505 by means of a randomized, placebo-controlled double-blind, cross-over study. An increase in the number of total bifidobacteria (the sum of B. bifidum, B. breve, B. longum subsp. longum, B. adolescentis, B. anglatum, B. catenulatum, B. pseudocatenulatum, B. dentium, B. longum subsp. infantis and B. lactis) in the feces were observed after administration of GCL2505 using species- and subspecies-specific real-time polymerase chain reaction analysis. However, the number of endogenous bifidobacteria species (excluding B. lactis) remained unchanged. B. lactis also became the predominant bifidobacterial species. Taking into account the number of GCL2505 administered, the findings further suggested that GCL2505 proliferated in the intestine. In addition, the defecation frequency increased during GCL2505 administration compared with the placebo. Moreover, a single administration study (n=17) clearly demonstrated that GCL2505 successfully reached the intestine before proliferating at least 10-fold. This is the first report to show an increase in intestinal bifidobacteria, with no changes to the endogenous species, and improvements in constipation following proliferation of administered bifidobacteria.     

Short communication: Identification and differentiation of bifidobacteria obtained from Ukraine

Ten freeze-dried bifidobacterial strains used as probiotics in Ukrainian dairy foods, identified by the supplier as Bifidobacterium adolescentis (2), Bifidobacterium bifidum (2), Bifidobacterium longum (4), Bifidobacterium animalis (1), and Bifidobacterium infantis (1), were characterized. Following rehydration and anaerobic growth on de Man, Rogosa, and Sharpe-cysteine medium at 37°C for 72 h, single-colony isolates were picked and evaluated using PCR primers specific for the Bifidobacterium genus, for the supplier-identified species, and for B. animalis ssp. lactis. All isolates were identified as members of the genus Bifidobacterium; however, species-specific PCR revealed all 10 isolates were actually strains of B. animalis ssp. lactis. Further evaluation using pulsed-field gel electrophoresis was only able to separate a single strain (RT 09) from the other 9 strains evaluated. Application of genome-wide allelic profiling to the Ukrainian bifidobacterial strains revealed 4 distinct groups. Interestingly, 6 (60%) of the isolates fell into the same cluster as that containing the common commercial probiotic strain BB-12. Two of the strains (RT 02 and RT 09) were found to be in the same group as ATCC 27536 and one strain (RT 08) was in the same group as the RB 7239 (a previously evaluated commercial strain). One strain, RT 04, was placed on a unique branch. These results highlight the importance of employing routine typing of bifidobacterial isolates, demonstrate the utility of single nucleotide polymorphism/insertion-deletion polymorphism-based allelic typing in B. animalis ssp. lactis strain differentiation and further point to the limited genetic variability of B. animalis ssp. lactis strains and the worldwide distribution of a small number of commercial strains.     

实时荧光定量PCR法鉴定婴幼儿配方乳粉中动物双歧杆菌乳亚种

该研究通过系统发育树分析确定目标基因,并设计引物和探针,建立一种婴幼儿配方乳粉中动物双歧杆菌乳亚种（Bifidobacterium animalis subsp. lactis）的实时荧光定量聚合酶链式反应（RT-fqPCR）鉴定方法。通过特异性、灵敏性和抗干扰实验对该方法进行验证,并对市售的64份标识含有动物双歧杆菌乳亚种的婴幼儿配方乳粉样品进行检测。结果表明,atpD基因在动物双歧杆菌乳亚种间具有较高的种间特异性,种间差异率＞10%,确定其为目标基因。基于该基因建立的RT-fqPCR方法能够特异性的检测动物双歧杆菌乳亚种,检测绝对灵敏度可以达到1 pg/μL,相对灵敏度可以达到103 CFU/mL;基因水平和培养物水平抗干扰能力良好。采用该方法从64份标识含有动物双歧杆菌乳亚种的乳粉样品中均能检测出动物双歧杆菌乳亚种,表明基于atpD基因建立的RT-fqPCR方法能够快速准确的对动物双歧杆菌乳亚种进行检测。     

Comparison of four methods to enumerate probiotic bifidobacteria in a fermented food product

Four methods of enumeration were compared by monitoring levels of probiotic bifidobacteria in fermented oat drink during storage. Strains of Bifidobacterium longum and B. lactis were quantified by plate counts, fluorescent in situ hybridization (FISH), quantitative real-time PCR and commercial LIVE/DEAD BacLight bacterial viability kit, and the methods were further developed to suit the enumeration of bifidobacteria in fermented foods. Plate counts of both B. lactis and B. longum were lower than the PCR and FISH counts. The LIVE/DEAD counts of B. lactis were comparable to PCR and FISH counts. The plate counts of B. lactis were slightly but significantly lower than LIVE/DEAD counts, suggesting that the cells that were not able to grow on plates may have become dormant. The plate counts of B. longum were several log units lower than LIVE/DEAD counts, suggesting that a remarkable part of the cells were dormant. Real-time PCR and FISH were shown to suit the quantification of the total amount of probiotic bifidobacteria in foods. Plate counts and LIVE/DEAD counts provided conflicting information on viability especially in the case of B. longum. We conclude that the choice of enumeration method for probiotic bacteria may have significant effect on the results of the analysis. The strain-specific properties and the objects of the analysis should be taken into account when enumeration methods for different probiotic strains are chosen.     

Production of oligosaccharides in yogurt containing bifidobacteria and yogurt cultures

Yogurts were prepared by using yogurt cultures combined to mixed cultures of bifidobacteria (Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum) and by adding a preincubation step (1.5 h at 50 degrees C) with bifidobacteria to the conventional method of manufacture in order to produce oligosaccharides. The survival of bifidobacteria was drastically affected during storage of yogurts, except for products containing B. animalis, in which viable counts remained at >10(6) cfu/g after 28 d of storage at 4 degrees C. Oligosaccharides with a degree of polymerization of 3 were produced during the preincubation step (0.31 to 0.68%), and the amount in the final products varied according to the species of bifidobacteria inoculated during the preincubation step or the concentration of bifidobacteria used as second inoculum during the fermentation process. In fact, the higher concentration of oligosaccharides measured at the end of the fermentation process (0.72%) and the 28 d-storage period (0.67%) was obtained for yogurts containing B. infantis. However, yogurts containing B. breve showed higher beta-galactosidase activities and had lower lactose concentrations after the fermentation process and the storage period than the other yogurts. The use of a mixed cultures of bifidobacteria (B. animalis, B. infantis, or B. breve) thus allows the production of yogurts in which bifidobacteria can survive in relatively high cell numbers and contain appreciable amount of oligosaccharides.     

H+-ATPase activity in Bifidobacterium with special reference to acid tolerance

The acid tolerance of 17 strains of nine species of bifidobacteria was compared using brief exposures to acidic conditions (pH 2-5). In addition, because it has been hypothesized that the acid tolerance of bifidobacteria depends on H+-ATPase activity, the activity of this enzyme in various strains and species was compared. In general, the acid tolerance of bifidobacteria was found to be weak, with the exception of Bifidobacterium lactis and Bifidobacterium animalis. High numbers of all strains of B. lactis and B. animalis survived exposure to pH 3-5 for 3 h. The H+-ATPase activity of the acid-tolerant strains B. lactis LKM512 and JCM 10602T, and B. animalis JCM 1190T, JCM 1253, JCM 7117, and JCM 7124 was higher at pH 4 than at pH 5. In contrast, the H+-ATPase activity of nonacid-tolerant strains was lower at pH 4 than at pH 5.     

Probiotic viability and physico-chemical and sensory properties of plain and stirred fruit yogurts made from goat's milk

Probiotic plain and stirred fruit yogurts were made from goat's milk using bacterial cultures comprising, Lactobacillus acidophilus LA-5, Bifidobacterium animalis subsp. lactis BB-12 and Propionibacterium jensenii 702. The products were stored at 4°C for 4weeks, during which time the viability of the yogurt starter culture and probiotic bacteria was analysed weekly. P. jensenii 702 demonstrated the highest viability (10(8)cfu/g) in all types of yogurt throughout the storage period, while the viability of the bifidobacteria (～10(7)cfu/g) also remained above the minimum therapeutic level. The viability of L. acidophilus LA-5 fell below 10(6)cfu/g in yogurts, however, the addition of fruit juice appeared to support the viability of lactobacilli, with higher microorganism numbers observed in fruit yogurts than in plain yogurt throughout the shelf life. Addition of fruit juice significantly increased the syneresis, and decreased viscosity and water holding capacity of yogurts (p<0.05), and also enhanced their sensory acceptability.     

Rapid and accurate bacterial identification in probiotics and yoghurts by MALDI-TOF mass spectrometry

Probiotic food is manufactured by adding probiotic strains simultaneously with starter cultures in fermentation tanks. Here, we investigate the accuracy and feasibility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for bacterial identification at the species level in probiotic food and yoghurts. Probiotic food and yoghurts were cultured in Columbia and Lactobacillus specific agar and tested by quantitative real-time PCR (qPCR) for the detection and quantification of Lactobacillus sp. Bacterial identification was performed by MALDI-TOF analysis and by amplification and sequencing of tuf and 16S rDNA genes. We tested 13 probiotic food and yoghurts and we identified by qPCR that they presented 10(6) to 10(7) copies of Lactobacillus spp. DNA/g. All products contained very large numbers of living bacteria varying from 10(6) to 10(9) colony forming units/g. These bacteria were identified as Lactobacillus casei, Lactococcus lactis, Bifidobacterium animalis, Lactobacillus delbrueckii, and Streptococcus thermophilus. MALDI-TOF MS presented 92% specificity compared to the molecular assays. In one product we found L. lactis, instead of Bifidus spp. which was mentioned on the label and for another L. delbrueckii and S. thermophilus instead of Bifidus spp. MALDI-TOF MS allows a rapid and accurate bacterial identification at the species level in probiotic food and yoghurts. Although the safety and functionality of probiotics are species and strain dependent, we found a discrepancy between the bacterial strain announced on the label and the strain identified. Practical Application: MALDI-TOF MS is rapid and specific for the identification of bacteria in probiotic food and yoghurts. Although the safety and functionality of probiotics are species and strain dependent, we found a discrepancy between the bacterial strain announced on the label and the strain identified.     

双歧杆菌胞外多糖对酸豆乳品质的影响

为探索益生菌及其胞外多糖对酸豆乳的影响,利用产胞外多糖双歧杆菌及其活性多糖与传统发酵剂共同发酵豆浆,研究其对酸豆乳理化、感官及质构特性的作用。结果表明：添加双歧杆菌（107 CFU/mL）和其活性胞外多糖（质量分数0.3%,下同）均对酸豆乳凝乳速度及后酸化无显著影响（P＞0.05）。添加0.3%胞外多糖能显著提高酸豆乳的黏度、持水力和质构特性（P＜0.05）。添加产胞外多糖双歧杆菌107 CFU/mL,对发酵期间酸豆乳发酵速率及凝胶特性影响不大;在后酵期间产生86 mg/L 胞外多糖,显著提高酸豆乳的黏度和质构特性（P＜0.05）,但对产品持水力的影响并不显著（P＞0.05）。双歧杆菌胞外多糖可有效改善酸豆乳凝胶特性,提高产品品质。     

Preservation of functionality of Bifidobacterium animalis subsp. lactis INL1 after incorporation of freeze-dried cells into different food matrices

The aim of this work was to investigate how production and freeze-drying conditions of Bifidobacterium animalis subsp. lactis INL1, a probiotic strain isolated from breast milk, affected its survival and resistance to simulated gastric digestion during storage in food matrices. The determination of the resistance of bifidobacteria to simulated gastric digestion was useful for unveiling differences in cell sensitivity to varying conditions during biomass production, freeze-drying and incorporation of the strain into food products. These findings show that bifidobacteria can become sensitive to technological variables (biomass production, freeze-drying and the food matrix) without this fact being evidenced by plate counts.     

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.     

Viability of microencapsulated bifidobacteria in set yogurt during refrigerated storage

Bifidobacteria are probiotic organisms that improve the microbial balance in the human gut. They can be incorporated as live cultures in fermented dairy foods, including yogurt, for transmission to humans. Because bifidobacteria are sensitive to high acidity, their viability in yogurt is limited. The objective of the present study was to investigate the effect of microencapsulation on the viability of bifidobacteria in yogurt during refrigerated storage for 30 d. Live bifidobacterial cells were encapsulated in kappa-carrageenan. Cell enumeration, determination of titratable acidity and pH, quantitation of lactic and acetic acids, and sensory evaluation (consumer test) were carried out on the yogurt samples. Microbiological results showed a decline of 78 and 70.5% in the population of Bifidobacterium longum B6 and B. longum ATCC 15708, respectively, for the treatments containing nonencapsulated cells. No difference in bifidobacterial population was observed in the encapsulated treatments. The acetic acid content in the yogurt with nonencapsulated bifidobacteria was higher than that in the plain yogurt (control) and encapsulated treatments. The increase in lactic acid content during storage was not different among the various treatments for B. longum B6, but was greater for nonencapsulated than encapsulated B. longum 15708 and the control. Consumers judged the nonencapsulated treatment as the most sour, which was likely due to the higher acetic acid content. Consumers preferred the control and nonencapsulated treatments over the encapsulated treatment. Microencapsulation appears to increase the viability of bifidobacteria in yogurt. This technique can be used to transmit bifidobacteria via fermented products provided that sensory characteristics of the product are improved or maintained.     

益生菌混合菌种在发酵豆乳中的优化

采用益生菌混合物AST和TLB分别在42℃进行豆乳的发酵,在发酵豆乳的发酵和贮存过程中(4℃下28d),观察pH值和活菌数的变化情况。在AST益生菌混合物(嗜酸性乳杆菌,双歧杆菌和嗜热链球菌)的培育下,42℃下发酵时间减少到8h。但嗜酸性乳杆菌在冷藏的过程中生存状态较差,其活菌数在冷藏后一周后未达到标准。将豆乳在42℃下用TLB益生菌(嗜热链球菌,保加利亚乳杆菌和动物双歧杆菌乳酸亚种Bb12)进行发酵,结果发酵时间缩短到4h,双歧杆菌活菌数目的对数增加了约一半,而且经过28d的冷藏,细菌数仍然维持在107 CFU/mL以上。     

Interactions among lactic acid starter and probiotic bacteria used for fermented dairy products

Interactions among lactic acid starter and probiotic bacteria were investigated to establish adequate combinations of strains to manufacture probiotic dairy products. For this aim, a total of 48 strains of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium spp. (eight of each) were used. The detection of bacterial interactions was carried out using the well-diffusion agar assay, and the interactions found were further characterized by growth kinetics. A variety of interactions was demonstrated. Lb. delbrueckii subsp. bulgaricus was found to be able to inhibit S. thermophilus strains. Among probiotic cultures, Lb. acidophilus was the sole species that was inhibited by the others (Lb. casei and Bifidobacterium). In general, probiotic bacteria proved to be more inhibitory towards lactic acid bacteria than vice versa since the latter did not exert any effect on the growth of the former, with some exceptions. The study of interactions by growth kinetics allowed the setting of four different kinds of behaviors between species of lactic acid starter and probiotic bacteria (stimulation, delay, complete inhibition of growth, and no effects among them). The possible interactions among the strains selected to manufacture a probiotic fermented dairy product should be taken into account when choosing the best combination/s to optimize their performance in the process and their survival in the products during cold storage.     

Viability of probiotic (Bifidobacterium, Lactobacillus acidophilus and Lactobacillus casei) and nonprobiotic microflora in Argentinian Fresco cheese

We evaluated the suitability of Argentinian Fresco cheese as a food carrier of probiotic cultures. We used cultures of Bifidobacterium bifidum (two strains), Bifidobacterium longum (two strains), Bifidobacterium sp. (one strain), Lactobacillus acidophilus (two strains), and Lactobacillus casei (two strains) in different combinations, as probiotic adjuncts. Probiotic, lactic starter (Lactococcus lactis and Streptococcus thermophilus), and contaminant (coliforms, yeasts, and molds) organisms were counted at 0, 30, and 60 d of refrigerated storage. Furthermore, the acid resistance of probiotic and starter bacteria was determined from hydrochloric solutions (pH 2 and 3) of Fresco cheese. The results showed that nine different combinations of bifidobacteria and L. acidophilus had a satisfactory viability (count decreases in 60 d <1 log order) in the cheese. Both combinations of bifidobacteria and L. casei cultures assayed also showed a satisfactory survival (counts decreased <1 log order for bifidobacteria but no decrease was detected for L. casei). On the other hand, the three combinations of bifidobacteria, L. acidophilus, and L. casei tested adapted well to the Fresco cheese environment. When a cheese homogenate at pH 3 was used to partially simulate the acidic conditions in the stomach, the probiotic cultures had an excellent ability to remain viable up to 3 h. At pH 2, the cell viability was more affected; B. bifidum was the most resistant organism. This study showed that the Argentinian Fresco cheese could be used as an adequate carrier of probiotic bacteria.