Use of tuf gene-based primers for the PCR detection of probiotic Bifidobacterium species and enumeration of bifidobacteria in fermented milk by cultural and quantitative real-time PCR methods

Due to the increasing use of bifidobacteria in probiotic products, it is essential to establish a rapid method for the qualitative and quantitative assay of the bifidobacteria in commercial products. In this study, partial sequences of the tuf gene for 18 Bifidobacterium strains belonging to 14 species were determined. Alignment of these sequences showed that the similarities among these Bifidobacterium species were 82.24% to 99.72%. Based on these tuf gene sequences, 6 primer sets were designed for the polymerase chain reaction (PCR) assay of B. animalis subsp. animalis, B. animalis subsp. lactis, B. bifidum, B. breve, B. longum subsp. infantis, B. longum subsp. longum, and the genus of Bifidobacterium, respectively. These Bifidobacterium species are common probiotic species present in dairy and probiotic products. When each target Bifidobacterium spp. was assayed with the designed primers, PCR product with expected size was generated. In addition, for each target species, more than 70 bacterial strains other than the target species, including strains of other Bifidobacterium species, strains of Lactobacillus spp., Enterococcus spp., and other bacterial species, all generated negative results. PCR assay with primers specific to B. animalis subsp. lactis and B. longum subsp. longum confirmed the presence of these Bifidobacterium species in commercial yogurt products. In addition, for each product, enumeration of the bifidobacteria cells by culture method with BIM-25 agar and the quantitative real-time PCR showed similar cell counts. Such results indicated that within 15-d storage (4 °C) after manufacture, all the bifidobacteria cells originally present in yogurt products were viable and culturable during the storage.     

Viability of lactic acid bacteria and bifidobacteria in fermented soymilk after drying, subsequent rehydration and storage

To develop a probiotic dietary adjunct, soymilk fermented with various combinations of lactic acid bacteria (Streptococcus thermophilus and Lactobacillus acidophilus) and bifidobacteria (Bifidobacterium longum and Bifidobacterium infantis) was subjected to freeze-drying and spray-drying. Survival of the starter organisms during the drying process, subsequent rehydration at different temperatures and during a 4-month period of storage under different storage conditions was examined. After freeze-drying, lactic acid bacteria and bifidobacteria exhibited a survival percent of 46.2-75.1% and 43.2-51.9%, respectively, higher than that noted after spray-drying. Regardless of the drying condition, S. thermophilus showed a higher percentage of survival than L. acidophilus, while B. longum survived better than B. infantis. Further study with soymilk fermented with S. thermophilus and B. longum revealed that the freeze-dried and spray-dried fermented soymilk rehydrated at 35-50 degrees C and 20 degrees C, respectively, was optimum for the recovery of the starter organisms. Both S. thermophilus and B. longum survived better in the freeze-dried than the spray-dried fermented soymilk during storage. A higher percent of survival was also noted for both the starter organisms when the dried fermented soymilk was stored at 4 degrees C than 25 degrees C. Holding the dried fermented soymilk in the laminated pouch enabled S. thermophilus and B. longum to exhibit a higher percentage of survival than in the deoxidant- and desiccant-containing glass or polyester (PET) bottle. Among all the packaging materials and storage temperatures tested, starter organisms were most stable in the dried fermented soymilk held in laminated pouch and stored at 4 degrees C. Under this storage condition, S. thermophilus and B. longum showed a survival percentage of 51.1% and 68.8%, respectively, in the freeze-dried fermented soymilk after 4 months of storage. Meanwhile, S. thermophilus and B. infantis in the spray-dried fermented soymilk showed a survival percent of 29.5% and 57.7%, respectively.     

Application of Conductimetry for Evaluation of Lactic Starter Cultures in Soymilk

ABSTRACT: :
Soymilk (SM), a potential culture medium for applying conductimetric techniques to evaluate the behavior of probiotic lactobacilli and bifidobacteria was examined. Media LAPTg, LM, and SM was standardized for the growth of Lactobacillus fermentum and Bifidobacterium longum. A dilution of 1/100 for L. fermentum and 1/1 for B. longum were considered optimal to obtain a detection time (DT) between 5 and 10 min. The relationship between the initial number of cells and the DT was linear throughout the incubation period considered (40 h). The effect of temperature (30, 37, and 42 °C) on the metabolic activity of the cells was also determined after 40 h of fermentation. The higher metabolic changes were observed at 42 °C with conductance values from 600 to 800 μS and DT of 5-7 min. Results obtained were confirmed by viable counts.     

Viability of bifidobacteria in commercial yogurt products in North Carolina during refrigerated storage

Fifty-eight commercial yogurt products of seven brands (which claimed to include bifidobacteria) were obtained from local stores in Greensboro, North Carolina, USA. These products were examined at 0, 1, 2, 3 and 4 weeks for the viability of bifidobacteria and yogurt starter culture during refrigerated storage at 4°C. Our results showed that bifidobacteria counts were variable, ranging from 0 to 5.5 log cfu/mL. The average yogurt starter culture counts ranged from 5.20 to 8.87 log cfu/mL and 7.51–8.94 log cfu/mL for Lactobacillus delbrueckii spp. bulgaricus and Streptococcus thermophilus , respectively. Of the 58 products tested, only 44 products (76%) contained viable cultures. Viability of bifidobacteria in yogurt samples remained within the same levels during 3 weeks of storage at 4°C; however, the bacterial count started to decline during the fourth week. These results suggest optimal beneficial consumption of yogurt foods with live bifidobacteria should occur within 3 weeks of production. Results obtained from this research could be used by the industry to develop new technologies to ensure consumers receive high-quality products.     

Effect of yogurt cultures on the survival of bifidobacteria in fermented milks

Three species of Bifidobacterium, Bif bifidum, Bif longum and Bif adolescentis were grown in reconstituted skim milk (12% total solids) either individually or in combination with one of three commercial yogurt cultures. Total colony counts were recorded for the bifidobacteria over a growth period of 24 hours and, for samples with the fermentation halted at pH 4.6, during storage at 5°C for up to 21 days. The results indicated that, while co-inoculation with the yogurt organisms tended to suppress the growth of the bifidobacteria, subsequent storage in the presence of the yogurt cultures did not lead to any significant decline in numbers.     

Physicochemical Properties of Pacific Whiting Surimi as Affected by Various Freezing and Storage Conditions

ABSTRACT: Effects of various freezing methods of surimi on the biochemical and physical properties, were examined. Stress values increased up to 3 mo and then decreased. Strain values significantly decreased over time, except freeze-dried surimi stored at -18 °C. Yellowness (b*) of the freeze-dried surimi stored at 22 °C increased significantly during storage. In addition, salt-extractable proteins (SEP) decreased while dimethylamine (DMA) increased. Freeze-dried surimi showed the highest SEP and the lowest DMA values after 9 mo storage. Electrophoretic patterns did not show any apparent damages to the MHC until 6 mo. At 6 and 9 mo, development of proteins with smaller molecular weights was observed, indicating proteolytic degradation during frozen 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.     

Microbiological and Color Quality Changes of Channel Catfish Frame Mince During Chilled and Frozen Storage

Microbial counts and color values of channel catfish frame mince were determined during storage at -20, 0, and 5 °C. Aerobic plate counts increased from 5.5 to greater than 8 log10 CFU/g by 3 d at 0 and 5 °C. Total coliform counts increased 1 log during refrigerated storage for 5 d. Both microbial populations declined during 3 mo frozen storage. Washed mince was lighter in color, less red, and less yellow than unwashed mince. Color of both mince types did not change during storage. Microbial results suggested that prepared catfish frame mince should be stored no longer than 3 d at 0 to 5 °C. Frozen mince remained acceptable for at least 3 mo at 20 °C. Key Words: catfish, mince, quality, microorganisms, color     

The influence of Bifidobacterium Bb-12 and lactic acid incorporation on the properties of Minas Frescal cheese

The effects of a probiotic bacterium (Bifidobacterium Bb-12) and lactic acid on the microbiological, physicochemical, rheological and microstructural proprieties of Minas Frescal cheese were evaluated after 1 day and after 28 days of storage (5 ± 1 °C). The cheese was produced with four different treatments: with no lactic acid (C1 and C2), with lactic acid (C3 and C4) and with bifidobacteria (C2 and C3). The cheese formulations containing bifidobacteria were classified as probiotic. The addition of bifidobacteria to the cheese did not influence its yield, protein or lipid levels one day after production. Moreover, after 28 days of storage, the cheese samples with no lactic acid showed lower moisture levels compared to the samples containing lactic acid (P < 0.05). The absence of lactic acid had an influence on the rheological and microstructural behavior, making them more elastic, firm and compact, however all the cheese samples evaluated showed a higher tendency to viscosity than to elasticity. The Minas Frescal cheese with Bifidobacterium Bb-12 and lactic acid showed great potential as a functional food, with possible industrial and commercial application.     

Suitability of whey and buttermilk for the growth and frozen storage of probiotic lactobacilli

The growth of six probiotic commercial strains of lactobacilli was assessed in reconstituted dried whey and buttermilk supplemented with yeast extract, meat peptone, soy peptone, tryptone or casein acid hydrolysate at 0.3%, 0.6% or 1%. The addition of 1% glucose was also tested. Growth and acidification kinetics were determined at 37°C using MRS broth and a commercial culture medium as references. The suitability of whey and buttermilk as cryoprotectants at –20°C and –70°C was also assessed. Whey and buttermilk with 0.3% yeast extract were chosen for the growth of probiotic lactobacilli, since no satisfactory growth was observed without an external nitrogen source, whereas glucose did not improve the growth of any of the strains assayed. In general, buttermilk performed as satisfactorily as the reference media. The effectiveness of these media as cryoprotectants was strain dependent: skimmed milk and whey were the most suitable ones, especially for long-term storage at –20°C. However, at –70°C, no significant differences were observed between the culture media assessed. The use of whey or buttermilk as culture media for the production of probiotic lactic acid bacteria and for their cryopreservation implies a novel use of these low-cost products, offering an alternative way of utilizing the by-products of the dairy industry, helping to minimize their negative impact on the environment.     

Viability assessment of lactic acid bacteria in commercial dairy products stored at 4 °C using LIVE/DEAD® BacLightTM staining and conventional plate counts

The viability of lactic acid bacteria (LAB) from commercial fermented milks was studied during storage at 4 °C. The enumeration of total viable bacteria was assessed using fluorescence microscopy. Plate counting on selective media was used to enumerate LAB. Using LIVE/DEAD® BacLight^TM viability staining, it was observed that bacterial counts decreased gradually after expiry dates, the number of viable bacteria remaining above 10⁶ bacteria g^?1 for all of the products. Viable cell counts estimated by plating onto selective media were lower than those obtained by direct microscopic counting. The viability of LAB contained in acid products decreased during their storage period at 4 °C. All products contain viable LAB ranging from 10⁸ to 10⁹ bacteria g^?1 and could be considered as probiotic, given that the recommended minimum number of probiotic bacteria in such food products is approximately 10⁷ bacteria mL^?1 product. The number of bifidobacteria in commercial fermented milks declared to contain bifidobacteria varied from 10⁴ to 10⁷ bacteria mL^?1. This study confirms the usefulness of fluorescent techniques for a rapid and accurate evaluation of bacterial viability in probiotic products.     

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

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

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.     

Acceleration of Thai fish sauce fermentation using proteinases and bacterial starter cultures

ABSTRACT:  A means to accelerate fish sauce fermentation without adversely affecting fish sauce quality was investigated. Starter cultures prepared from Virgibacillus sp. SK33, Virgibacillus sp. SK37, and Staphylococcus sp. SK1-1-5 were added separately to anchovy that was hydrolyzed by 0.25% Alcalase at 60 °C for 2 h followed by 0.5% Flavourzyme at 50 °C for 4 h. The mixtures were then adjusted to contain 25% solar salt and incubated at 35 °C for 4 mo. α-Amino contents of all inoculated samples were higher than the control (without the addition of starter culture) during the course of fermentation. After 4-mo fermentation, the samples inoculated with Staphylococcus sp. SK1-1-5 contained the highest α-amino content of 733.37 ± 13.89 mM while that of the control was 682.67 ± 3.33 mM. Amino acid profiles of inoculated samples showed similar patterns to that of commercial product fermented for 12 mo, with glutamic, aspartic, and lysine being predominant amino acids. Virgibacillus sp. SK33 appeared to decrease histamine content of fish sauce by 50% when compared to the control. Volatile compounds analyzed by GC–MS of all inoculated samples fermented for 4 mo exhibited a similar pattern to those of the 12-mo-old commercial product. Samples inoculated with Staphylococcus sp. SK1-1-5 produced higher levels of volatile fatty acids and showed similar sensory characteristics to the commercial fish sauce fermented for 12 mo. Staphylococcus sp. SK1-1-5 is a potential strain that can be applied to produce fish sauce with overall sensory characteristics of traditional fish sauce in shorter time.     

Functional, Nutritional, Mycological, and Akara-making Properties of Stored Cowpea Meal

ABSTRACT: Cowpea meal was packaged in high-density polyethylene bags, stored at -18 °C, 21 °C/55% RH, 37 °C/55% RH, and evaluated at 0, 4, 8, 12, 18, and 24 mo for functional, nutritional, mycological, and akara-making properties. Moisture content of meal varied no more than 0.5% and there were no marked changes in yeast and mold populations. Meal stored at -18 and 21 °C retained color, foam capacity, and akara-making quality for 24 mo. Overall, storage of cowpea meal at 37 °C was detrimental to its foaming characteristics, a key measure of performance in akara-making. A decline in protein solubility was also observed in the 37 °C samples.     

Effect of refrigerated storage temperature on the viability of probiotic micro-organisms in yogurt

The effect of refrigerated storage temperature was studied at 2, 5 and 8°C on the viability of probiotics in ABY ( Lactobacillus acidophilus, Bifidobacterium lactis BB-12 and yogurt bacteria. Bulgaricus , i.e. Streptococcus thermophilus and Lactobacillus delbrueckii ssp. Bulgaricus ) probiotic yogurt. The study was carried out during a 20-day refrigerated storage period to identify the best storage temperature(s). Also, the viability change of the probiotic micro-organisms was analysed at 5-day intervals throughout the refrigerated storage period. After 20 days, storage at 2°C resulted in the highest viability of L. acidophilus , whereas for Bifidobacterium lactis the highest viability was obtained when yogurt was stored at 8°C.     

Viability During Storage of Selected Probiotic Lactobacilli and Bifidobacteria in a Yogurt-like Product

ABSTRACT: Multiple species cultures, including 2 strains of Streptococcus thermophilus and Lactobacillus acidophilus NCFM plus 1 strain each of Bifidobacterium longum and Lactobacillus casei , were used to make yogurt-like products. The lactobacilli and bifidobacteria were tested for growth in the products and subsequent viability during refrigerated storage. During fermentation, L. casei Com-5 actually declined in numbers, while L. casei E5 and E10 increased about 2 fold. Numbers of B. longum S9 increased about 3 fold while B. longum Com-4 did not increase. During storage, L. acidophilus NCFM appeared stable in all mixtures and both strains of bifidobacteria decreased. Lactobacillus casei E5 and E10 were more stable than was L. casei Com-5.     

Fecal Microbiota Changes with the Consumption of Follow-up Formulas Containing Bifidobacterium spp. and/or Galactooligosaccharides by Rats and a Follow-up Infant Formula Containing Bifidobacterium spp. by Human Infants

ABSTRACT Seven groups of rats were fed during 1 mo using 1 infant formula containing Bifidobacterium bifidum and Bifidobacterium longum, 3 infant formulas containing 4‐galactosyllactose at 1.2%, 5.0%, and 10.0%, and 3 infant formulas containing both ingredients. During 3 periods, corresponding to day 8 to 10, 18 to 20, and 28 to 30, fecal samples were collected for total aerobes, total anaerobes, and bifidobacteria counts. Results showed that bifidobacteria represented an important proportion out of the anaerobe group in the 1st period. However, in the 2nd period bifidobacteria decreased significantly, and in the 3rd period bifidobacteria counts increased, especially in the group fed diet containing galactooligosaccharides 1.2%. In a 2nd study, 12 human infants were fed with the infant formula containing B. bifidum and B. longum, whereas the other 12 were fed using a control infant formula. Fecal samples were collected at the age of 1, 3, 5, 7, 9, and 12 mo for total anaerobes, bacteroides, bifidobacteria, clostridia counts, as well as for fecal pH determination. Infants fed infant formula containing bifidobacteria in samples corresponding to 7th and 9th mo of age had significantly (P< 0.05) higher bifidobacteria counts and a lower fecal pH than those fed control infant formula.     

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。     

Comparison of Sample Storage Methods for Vitamin B6, Assay in Broiler Meats

Five methods were compared for storage stability of vitamin B₆ in broiler meat samples. Maximum retention of vitamin B₆ was observed in the N₂-packed freeze-dried samples at -34°C (101–103% up to 20 mo). Good retentions occurred in the intact tissues of half birds at -34°C (90% in 16 mo) and in ground meat samples (200g portions) at -34°C (91% up to 12 mo). Significantly lower retentions were found in freeze-dried samples stored at room temperature (85–77% in 1–5 mo, respectively) and for ground meat small portion samples (5g) at -34°C (84–72% in 3–5 mo, respectively).