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.     

Effect of packaging conditions and temperature on viability of microencapsulated bifidobacteria during storage

In this study, Bifidobacterium longum B6 and B infantis CCRC 14633 were microencapsulated in various wall materials, including skim milk, gum arabic, gelatin and soluble starch. The stability of these microencapsulated bifidobacteria held at 25 or 4 °C in glass or polyester bottles with or without deoxidant and desiccant was determined. Microencapsulated cells of B longum B6 were generally more stable than the corresponding microencapsulated cells of B infantis CCRC 14633 under the various storage conditions tested. The presence of deoxidant and desiccant, especially at 25 °C, increased the survival of microencapsulated cells. Furthermore, the survival of bifidobacteria was enhanced when they were stored at 4 °C in glass bottles. It was also found that the wall material affected the survival of microencapsulated bifidobacteria. The viability of B longum B6 and B infantis CCRC 14633 was best when they were encapsulated in skim milk and held at 4 °C in glass bottles. Skim milk‐encapsulated B longum B6 cells showed a relatively low viability reduction of only 0.15–0.20 log (colony‐forming units (cfu g^?1)) after 42 days of storage at 4 °C in glass bottles, regardless of the presence of deoxidant and desiccant. A reduction of 0.38–0.76 log (cfu g^?1) was noted for skim milk‐encapsulated cells of B infantis CCRC 14633 under similar storage conditions. Copyright © 2004 Society of Chemical Industry     

Survival of Lactobacillus casei,Lactobacillus plantarum and Bifidobacterium bifidum in free and microencapsulated forms on Iranian white cheese produced by ultrafiltration

Survival of probiotic strains Lactobacillus casei ( ATCC 39392 ), Lactobacillus plantarum ( ATCC 8014 ) and Bifidobacterium bifidum ( ATCC 29521 ) was investigated either in microencapsulated or in free form in the Iranian white cheese produced by ultrafiltration technique. The results indicated that the survival of encapsulated probiotic bacteria was higher than free cells. Both free and microencapsulated forms were successful in keeping counts of L. casei, L. plantarum and B. bifidum in the cheese high enough for the therapeutic minimum (10⁶–10⁷ cfu/g) after 60 days. Addition of probiotic adjunct also did not alter the chemical composition, but pH was lower in probiotic cheeses.     

Survival and Metabolic Activity of Microencapsulated Bifidobacterium longum in Stirred Yogurt

ABSTRACT: Live cells of Bifidobacterium longum, microencapsulated in K‐carrageenan, were added to stirred yogurt after fermentation (pH 4.6) and stored at 4.4 °C for 30 d. Cell enumeration indicated no decline of encapsulated cell number in yogurt samples, while there was significant reduction in nonencapsulated cell population (89.3% for B. longum B6 and 91.8% for B. longum ATCC 15708). Ion‐exchange high‐performance liquid chromatography showed comparable amounts of lactic and acetic acids in all samples, indicating little metabolic activity by bifidobacteria in experimental yogurts. Consumer sensory analysis of blackberry‐flavored yogurts revealed that samples containing encapsulated bifidobacteria had a grainy texture. Results suggested that microencapsulation protected bifidobacteria from the low pH of yogurt.     

Fermentation Capabilities of Bifidobacteria Using Nondigestible Oligosaccharides, and Their Viability as Probiotics in Commercial Powder Infant Formula

The species Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum biotype infantis (Spanish type culture collection), and Bifidobacterium longum (Morinaga nutritional foods) were evaluated in vitro in the presence of 4 commercial nondigestible oligosaccharides (NDO) (short‐chain fructooligosaccharides [SCFOS] [degree of polymerization, DP: 2–3], inulin [DP: 10–0], oligofructose [DP: 2–0] and 4′‐galactosyllactose [4′‐GOS] [DP: 3–]). Each species was incubated anaerobically in tryptone phytone yeast (TPY) broth for 7 d with NDO. Every 24 h, bifidobacteria growth was evaluated by means of broth turbidity as optical density at 600 nm. Moreover, another sample was collected for pH culture measurement. Results showed that inulin was the substrate with the least effect on the stimulation of bifidobacteria growth and pH decrease. On the last day of incubation, the substrate 4′‐GOS stimulated bacterial growth more strongly and produced a larger decrease in culture broth pH than the other substrates. On the other hand, B. bifidum and B. longum showed a greater growth with 4′‐GOS. In a 2nd study, these 2 bifidobacteria species were added to a powder follow‐on probiotic infant formula. The viability of the bifidobacteria during the formula's period of consumption was evaluated in 2 studies of 6 and 14 d. Both corresponded to the minimum and maximum time of consumption of the formula according to the manufacturer's directions. It was found that, although in both studies bifidobacteria counts decreased significantly (P < 0.05) with time, they were always above the recommended addition level (10⁶ colony‐forming units [CFU]/g) at the time of sale for dairy products by the Intl. Standard of Fédération Internationale de Laiterie/International Dairy Federation (FIL/IDF). Moreover, because the pH of the reconstituted formula was always close to neutrality (from 6.74 to 7.06), the number of bacteria did not drop below the recommended level.     

Viability of Bifidobacteria Strains in Yogurt with Added Oat Beta-Glucan and Corn Starch during Cold Storage

Abstract: Probiotics must be consumed at a level of 10⁷ CFU/mL for successful colonization of the gut. In yogurts containing beneficial cultures, the survival of probiotic strains can quickly decline below this critical concentration during cold storage. We hypothesized that beta-glucan would increase the viability of bifidobacteria strains in yogurt during cold storage. Yogurts were produced containing 0.44% beta-glucan (concentrated or freeze-dried) extracted from whole oat flour and/or 1.33% modified corn starch, and bifidobacteria (B. breve or B. longum) at a concentration of at least 10⁹ CFU/mL. All yogurts were stored at 4 °C. Bifidobacteria and yogurt cultures, Streptococcus thermophilus and Lactobacillus delbureckii subsp. bulgaricus, were enumerated from undisturbed aliquots before fermentation, after fermentation, and once a week for 5 wk. S. thermophilus and L. bulgaricus maintained a concentration of at least 10⁸ CFU/mL in yogurts containing concentrated or freeze-dried beta-glucan regardless of starch addition, and in the control with no added beta-glucan or starch. Similarly, the probiotic, Bifidobacterium breve, survived above a therapeutic level in all treatments. The addition of beta-glucan prolonged the survival of Bifidobacterium longum at a concentration of at least 10⁷ CFU/mL by up to 2 wk on average beyond the control. Further, the inclusion of concentrated beta-glucan in yogurt improved survival of B. longum above 10⁷ CFU/mL by 1 wk longer than did freeze-dried beta-glucan. Study results suggest that beta-glucan has a protective effect on bifidobacteria in yogurt when stressed by low-temperature storage. Practical Application: This study suggests that beta-glucan (oat fiber) may improve bifidobacteria survival in yogurt during refrigerated storage.     

Human milk oligosaccharide consumption by probiotic and human-associated bifidobacteria and lactobacilli

Human milk contains high concentrations of nondigestible complex oligosaccharides (human milk oligosaccharides; HMO) that reach the colon and are subsequently fermented by the infant gut microbiota. Using a high-throughput, low-volume growth determination, we evaluated the ability of 12 lactobacilli and 12 bifidobacteria strains, including several commercial probiotics, to ferment HMO and their constituent monomers. Of the 24 strains tested, only Bifidobacterium longum ssp. infantis ATCC 15697 and Bifidobacterium infantis M-63 were able to ferment 3′-sialyllactose, 6′-sialyllactose, 2′-fucosyllactose, and 3′-fucosyllactose. Bifidobacterium infantis M-63 degraded almost 90% of the 2′-fucosyllactose but left most of the fucose in the supernatant, as detected by HPLC. Among bifidobacteria, only the B. infantis strains and Bifidobacterium breve ATCC 15700 were able to ferment lacto-N-neotetraose (LNnT). Among lactobacilli, Lactobacillus acidophilus NCFM was found to be the most efficient at utilizing LNnT. The extracellular β-galactosidase (lacL, LBA1467) of L. acidophilus NCFM cleaves the terminal galactose of LNnT for growth, leaving lacto-N-triose II in the media as detected by HPLC. Inactivation of lacL abolishes growth of L. acidophilus NCFM on LNnT. These results contribute to our knowledge of HMO–microbe interactions and demonstrate the potential for synbiotic combinations of pre- and probiotics.     

Sodium Acetate and Bifidobacteria Increase Shelf-Life of Refrigerated Catfish Fillets

Microbiological and sensory evaluations of catfish fillets treated with sodium acetate (SA) and bifidobacteria were studied at 4°C. Cultures of Bifidobacterium adolescentis, B. infantis, or B. longum (2.5% V/W) extended lag phase, but not generation time of aerobic bacteria. One-half percent (W/W) SA increased lag phase and generation time. SA and bifidobacteria reduced counts more than either alone, extending shelf-life by 3 days. Amount of culture (1.5–4.5%) or culture age (24–72 hr) had no effect on inhibition. Treated fillets odor and appearance resembled fresh fillets for up to 6 days, while untreated fillets were unacceptable aher 3 days. SA, either alone or combined with bifidobacteria, extended refrigerated shelf-life of catfish fillets.     

PROPERTIES OF SALTED YOLK SOLIDS AND THEIR FUNCTIONALITY IN MAYONNAISE 1

The apparent viscosity, emulsification capacity, and functionality in mayonnaise of plain, free-flow, and low viscosity yolk solids were studied. Liquid yolk was used for comparison. All three types of yolk solids had considerably higher apparent viscosities than liquid yolk and there was a siginficant (P > 0.05) difference in apparent viscosity between each yolk solids sample. Free-flow yolk solids had the highest apparent viscosity, followed by plain yolk solids and low viscosity yolk solids. There was no significant (P > 0.05) difference in emulsification capacity between plain and free-flow yolk solids. Low viscosity yolk solids had a significantly (P > 0.05) lower emulsification capacity. There was a significant (P > 0.05) difference in apparent viscosity mayonnaise made from each yolk solids sample. Mayonnaise made from liquid yolk had the highest apparent viscosity. Of the mayonnaise samples made from yolk solids, that made from low viscosity yolk solids had an apparent viscosity of 38,250 c.p.s., followed by plain yolk solids mayonnaise at 29,500 c.p.s. and free flow yolk solids mayonnaise at 23,000 c.p.s. Although mayonnaise made from liquid yolk had a slightly higher spread, there was no significant (P > 0.05) difference in spread among the mayonnaise samples made from the three types of yolk solids. Mayonnaise made from free flow yolk solids had the highest stability at 12 days. Mayonnaise samples made from plain and from low viscosity yolk solids had stabilities of 10 days.     

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.     

Evaluation of physiochemical,textural, microbiological and sensory characteristics in set yogurt reinforced by microencapsulated Bifidobacterium bifidum F‐35

The objectives of this study were to encapsulate the Bifidobacterium bifidum F‐35 into whey protein for the production of one‐layer microcapsules, and then the microcapsules were covered by sodium alginate to produce double‐layer microcapsules for examining the effectiveness of microcapsules in set yogurt. The reinforced treatment by double layer exhibited a significant increase (P < 0.05) in B. bifidum F‐35 count more than the treatments of free cells and one‐layer microcapsules. Microcapsules of double layer in yogurt led to record a value of titratable acidity that was 1.51 in comparison with the treatments of one layer and free cells that were 1.65 and 1.73, respectively. The hardness values were recorded as 206.88 at the treatment of double layer and 130.31 at the treatment of one layer after 7 days of storage. Microencapsulation of double layer caused a slight bitterness and creamy texture in yogurt, whereas the samples of free cells were described to have sour and bad texture.     

Microencapsulation of Bifidobacterium bifidum F‐35 in reinforced alginate microspheres prepared by emulsification/internal gelation

Alginate microspheres containing Bifidobacterium bifidum F‐35 prepared by emulsification/internal gelation were reinforced by blending with pectin or starch or coating with chitosan or poly‐L‐lysine to provide extra protection for the strain. The influence of these treatments on the size of microspheres, encapsulation yield (EY) and protective effect of microencapsulation on the cells was studied. No difference was detected in EY with different types of reinforcement, which was approximately 43–50%. The mean diameter of reinforced alginate microspheres ranged from 117 to 178 μm, reaching a maximum value when starch was incorporated in the alginate matrix. It was observed that the protective effects varied with the type of reinforcement. However, chitosan‐coated alginate microspheres provided the best protection for microencapsulated cells in simulated gastrointestinal tract and during 1 month of storage at 4 °C, and this system could be the comparatively effective vector of bifidobacteria for intestinal delivery.     

Survivability and β‐galactosidase activity of bifidobacteria stored at low temperatures

Abstract

This work evaluated the ability of strains representing six species of Bifidobacterium with probiotic potential to survive and maintain β‐galactosidase activity through a two‐step, low‐temperature storage period. Cultures were also evaluated for their ability to ferment skim milk and retain viability during storage at 4°C. Bifidobacterium longum ATCC 15707, B. breve 15700, and B. bifidum 29521 maintained the greatest viabilities at > 1 x 10⁷ CFU/mL, and B. infantis 15702 maintained the highest β‐galactosidase activity at > 1 U/ml (with < 1 × 10⁵ CFU/mL) after ‐60 to 4°C storage. In fermented skim milk, B. breve 15700, B. bifidum 29521, and B. animalis 25527 tolerated a final product pH of 4.75 with > 1 × 10⁸ CFU/mL remaining after 14 days of storage at 4°C. Overall, it was found that highest levels of β‐galactosidase activities did not necessarily correlate to the highest plate‐count populations.     

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.     

Survival of probiotics encapsulated in chitosan-coated alginate beads in yoghurt from UHT- and conventionally treated milk during storage

Survival of the microencapsulated probiotics, Lactobacillus acidophilus 547, Bifidobacterium bifidum ATCC 1994, and Lactobacillus casei 01, in stirred yoghurt from UHT- and conventionally treated milk during low temperature storage was investigated. The probiotic cells both as free cells and microencapsulated cells (in alginate beads coated with chitosan) were added into 20 g/100 g total solids stirred yoghurt from UHT-treated milk and 16 g/100 g total solids yoghurt from conventionally treated milk after 3.5 h of fermentation. The products were kept at 4 °C for 4 weeks. The survival of encapsulated probiotic bacteria was higher than free cells by approximately 1 log cycle. The number of probiotic bacteria was maintained above the recommended therapeutic minimum (10⁷ cfu g⁻¹) throughout the storage except for B. bifidum. The viabilities of probiotic bacteria in yoghurts from both UHT- and conventionally treated milks were not significantly (P>0.05) different.     

Stimulating fermentative activities of bifidobacteria in milk by highintensity ultrasound

The effect of ultrasonic processing at 20 kHz on the fermentative activities of four different strains of Bifidobacterium (i.e., Bifidobacterium breve ATCC 15700, Bifidobacterium infantis, Bifidobacterium animalis ssp. lactis (BB-12), and Bifidobacterium longum (BB-46)) in milk was investigated. Results showed that ultrasound under the applied conditions could reduce the fermentation time required to reach pH 4.7 for samples with B. infantis, B. breve ATCC 15700, and BB-12, but not for BB-46. The viability of each the first three strains at the end of the fermentation was comparable with that of the corresponding control. The probiotic bacteria cells were ruptured by ultrasound and released intracellular enzyme β-galactosidase that promoted the hydrolysis of lactose and trans-galactosylation, and subsequently enhanced the growth of the remaining bacterial cells in inoculated milk during fermentation. The lower the concentration of lactose, the higher the amount of oligosaccharides (degree of polymerization = 3) found in the fermented milk with ultrasound treatment.     

Viability of microencapsulated bifidobacteria in simulated gastric juice and bile solution

Microencapsulated cells of Bifidobacterium longum B6 and Bifidobacterium infantis CCRC 14633 were prepared by spray drying the cell suspension containing the test organism and 10% (w/w) of the carrier material of either gelatin, soluble starch, skim milk or gum arabic. Survival of these microencapsulated and free cells of bifidobacteria in simulated gastric juice (pH 2.0 and 3.0) and bile solution (0.5% and 2.0%) was then examined. B. infantis CCRC 14633 was more susceptible than B. longum B6 to the simulated gastric environment and bile solution tested. Microencapsulated bifidobacteria exhibited a lower population reduction than free cells during exposure to simulated gastric environment and bile solution. This phenomenon was most pronounced when the test organism was exposed to gastric juice at pH 2.0 or 2.0% bile solution. Moreover, it was also observed that the protective effect exerted by encapsulation with spray drying varied with the carriers used and the strains of bifidobacteria.     

Utilization of galactooligosaccharides by Bifidobacterium longum subsp. infantis isolates

Prebiotics are non-digestible substrates that stimulate the growth of beneficial microbial populations in the intestine, especially Bifidobacterium species. Among them, fructo- and galacto-oligosaccharides are commonly used in the food industry, especially as a supplement for infant formulas. Mechanistic details on the enrichment of bifidobacteria by these prebiotics are important to understand the effects of these dietary interventions. In this study the consumption of galactooligosaccharides was studied for 22 isolates of Bifidobacterium longum subsp. infantis, one of the most representative species in the infant gut microbiota. In general all isolates showed a vigorous growth on these oligosaccharides, but consumption of larger galactooligosaccharides was variable. Bifidobacterium infantis ATCC 15697 has five genes encoding β-galactosidases, and three of them were induced during bacterial growth on commercial galactooligosaccharides. Recombinant β-galactosidases from B. infantis ATCC 15697 displayed different preferences for β-galactosides such as 4′ and 6′-galactobiose, and four β-galactosidases in this strain released monosaccharides from galactooligosaccharides. Finally, we determined the amounts of short chain fatty acids produced by strain ATCC 15697 after growth on different prebiotics. We observed that biomass and product yields of substrate were higher for lactose and galactooligosaccharides, but the amount of acids produced per cell was larger after growth on human milk oligosaccharides. These results provide a molecular basis for galactooligosaccharide consumption in B. infantis, and also represent evidence for physiological differences in the metabolism of prebiotics that might have a differential impact on the host.     

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.     

Effect of high intensity ultrasound on carbohydrate metabolism of bifidobacteria in milk fermentation

This study investigated the effect of high intensity ultrasound (20 kHz) on carbohydrate metabolism in milk fermentation by Bifidobacterium breve ATCC 15700, Bifidobacterium infantis, Bifidobacterium animalis subsp. Bifidobacterium lactis (BB-12), and Bifidobacterium longum (BB-46). After ultrasonication, lactose hydrolysis and the trans-galactosylation reaction in all fermented milk were accelerated during 24 h of fermentation. Lactose consumption of BB-46, BB-12, B. breve and B. infantis increased up to 2, 4, 3 and 2.5 times, respectively, in comparison with those found in control samples. This resulted in remarkable changes in the acid profiles of the strains. The ultrasonication stimulated the production of major organic acids in the later stage of the milk fermentation by BB-12, B. breve, and B. infantis, whilst it decreased the ratio of acetic acid to lactic acid and the ratio of total acetic and propionic acids to lactic acid in BB-12 and BB-46 samples, respectively.