Effects of cow's and goat's milk as fermentation media on the microbial ecology of sugary kefir grains

In the present study, we have investigated the importance of fermentation media on grain formation and the microbial characteristics of sugary kefir. The sugary kefir grains were fermented in brown sugar, cow's milk or goat's milk. Using culture-dependent and culture-independent methods, we identified the microorganisms present in both the grains and filtrate and then evaluated their distribution. The structure of the grains was also observed by scanning electronic microscopy (SEM). The identification results indicated that there were remarkable changes in microbial ecological profiles of the sugary kefir grains and their filtrates when brown sugar and milk were compared as fermentation media. Three lactic acid bacteria (LAB) species (Leuconostoc mesenteroides, Lactobacillus mali and Lactobacillus hordei) were found in the grains fermented using brown sugar. However, four species, named Leu. mesenteroides, Lactococcus lactis, Bifidobacterium psychraerophilum and Enterococcus faecalis, were identified in the grains fermented using either cow's or goat's milk. The size and structure of the kefir grains were also significantly influenced by the culture medium. We hypothesize that the grains originally may contain many different microorganisms and the identified changes are an adaption to each specific medium during grain formation and growth. The distribution of strains thus may vary depending on the carbon and energy sources available for grain fermentation and these microbial changes will further affect the granulation and growth of the grains. This study is important to our understanding of the mechanism of kefir grain formation and growth because it explores the relationship between fermentation media and kefir microorganisms.     

两歧双歧杆菌发酵橙汁酸豆奶的研究

本文尝试研究豆奶中两歧双歧杆菌的生长情况,以得到风味良好的发酵产品.以大豆与水1∶8的比例制取豆奶,添加一定量的橙汁,并加入两歧双歧杆菌进行发酵.考察了培养温度、橙汁添加量、接种量等因素对产品pH值和活菌教的影响,并在此基础上进行正交试验优化.正交试验结果表明,培养温度为39℃、接种量为5％、橙汁添加量为10％,18 ...     

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.     

Growth of probiotic bacteria and bifidobacteria in a soy yogurt formulation

Soy beverage and cows' milk yogurts were produced with Steptococcus thermophilus (ATCC 4356) and Lactobacillus delbrueckii subsp. bulgaricus (IM 025). The drop in pH during fermentation was faster in the soy beverage than in cows' milk, but the final pH values were similar. Yogurts were prepared with a yogurt starter in conjunction with either the probiotic bacteria Lactobacillus johnsonii NCC533 (La-1), Lactobacillus rhamnosus ATCC 53103 (GG) or human derived bifidobacteria. The presence of the probiotic bacteria did not affect the growth of the yogurt strains. Approximately 2 log increases in both L. rhamnosus GG and L. johnsonii La-1 were observed when each was added with the yogurt strains in both cows' milk and the soy beverage. Two of the five bifidobacteria strains grew well in the cows' milk and soy beverage during fermentation with the yogurt bacteria. High pressure liquid chromatography (HPLC) analyses showed that the probiotic bacteria and the bifidobacteria were using different sugars to support their growth, depending on whether the bacteria were growing in cows' milk or soy beverage.     

HYDROLYSIS OF ISOFLAVONE PHYTOESTROGENS IN SOYMILK FERMENTED BY LACTOBACILLUS AND BIFIDOBACTERIUM COCULTURES

This study evaluated the hydrolysis of isoflavones in soymilk fermented at 37C for 48 h by four different Lactobacillus and Bifidobacterium cocultures. The hydrolysis of isoflavone β-glucosides significantly increased ( P <  0.05) the bioactive aglycones from 36 to over 90% of total isoflavones in soymilk fermented with any of the four Lactobacillus and Bifidobacterium cocultures as compared with unfermented soymilk. Compared with three other cocultures of Lactobacillus and Bifidobacterium, fermentation of soymilk with the Lactobacillus paracasei/Bifidobacterium longum cocultures yielded better isoflavone hydrolytic potential (Otieno-Shah index) and the highest β-glucosidase activity after 12 h of incubation.

PRACTICAL APPLICATIONS

Isoflavones are known as phytoestrogens because they are present in soy products and have estrogen-like activity. During fermentation, the majority of glucoside isoflavones in soymilk are converted to bioactive aglycones via microorganism-derived β-glucosidase. In human intestines, aglycone isoflavones are absorbed faster and in greater amounts than their glucosides. Using probiotic Lactobacillus and Bifidobacterium cocultures to ferment soymilk efficiently increases the bioactive aglycone concentrations. Hence, fermenting soymilk with this coculture could enhance the nutritional value of the product.     

The mixed coculture effect of primary rat hepatocytes and bone marrow cells is caused by soluble factors derived from bone marrow cells

Heterospheroids consisting of hepatocytes and bone marrow cells (BMCs) are formed by the mixed coculture of these cells and enhance the expression and maintenance of the liver-specific functions of hepatocytes. Not only the soluble factors derived from these cells, but also functional organoid (heterospheroid) formation, are considered to underlie this coculture effect. Therefore, in the present study, we aimed to clarify the mechanism of this co-culture effect. We performed hepatocyte monoculture with conditioned media prepared from hepatocyte cultures, BMC cultures and a coculture of hepatocytes and BMCs. When using any type of conditioned medium, no hepatocyte spheroids formed, and the hepatocytes formed a monolayer. In addition, an effect for these conditioned media was shown in terms of the albumin production and ammonia metabolism activities of the hepatocytes; conditioned medium from BMCs showed the strongest effect. The monocultured hepatocytes in the conditioned medium derived from BMCs showed equivalent albumin production and ammonia metabolism activities to the cocultured spheroids of hepatocytes and BMCs. Therefore, it was determined that the effect of the coculture of hepatocytes and BMCs was caused by soluble factors derived from BMCs.     

EFFECT OF COLD STORAGE ON CULTURE VIABILITY AND SOME RHEOLOGICAL PROPERTIES OF FERMENTED MILK PREPARED WITH YOGURT AND PROBIOTIC BACTERIA

ABSTRACT

We examined the effect of storage time on culture viability and some rheological properties (yield stress, storage modulus, loss modulus, linear viscoelastic region, structural recuperation and firmness) of fermented milk made with Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus (LA) and Bifidobacterium animalis ssp. lactis in coculture with Streptococcus thermophilus (ST). Acidification profiles and factors that affect viability (postfermentation acidification, acidity and dissolved oxygen) were also studied during 35 days at 4C. Fermented milk prepared with a coculture of ST and Bifidobacterium lactis gave the most constant rheological behavior and the best cell viability during cold storage; it was superior to ST plus LA for probiotic fermented milk production.

PRACTICAL APPLICATIONS

Probiotic cultures should grow quickly in milk, provide adequate sensory and rheological properties to the product, and remain viable during storage. Commercially, it is very common to use yogurt starter culture (i.e. Streptococcus thermophilus[ST] and Lactobacillus delbrueckii ssp. bulgaricus) in combination with the probiotic bacteria in order to reduce fermentation time. However, LB tends to post acidify fermented milk, which reduces the viability of the probiotic bacteria; thus, it is recommended to use starter cultures devoid of this species. We found that the technological properties and the viability of the probiotic bacterium Bifidobacterium animalis ssp. lactis BL O4 in coculture with ST make it suitable for probiotic fermented milk production; it produces rheological characteristics similar to those of yogurt.     

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.     

Utilization of soy yoghurt in tarhana production

Tarhana, a traditional fermented food made from a mixture of white wheat flour and yoghurt, is widely consumed in Turkey. Tarhana samples with cow's milk yoghurt, soy milk yoghurt, and a blend of cow's and soy milk yoghurt were produced and evaluated for pH, viscosity, color, and sensory properties. Acid production was slower in soy milk-added samples, and increasing yoghurt amount in tarhana significantly contributed to acidity of the samples (P<0.05). Soy milk-added samples had higher viscosity compared with cow's milk yoghurt-added samples (P<0.05). Soy milk yoghurt bleached the wheat flour and therefore these samples appeared whiter than the cow's milk yoghurt-added samples (P<0.05). Panelists could not detect any beany-off flavor in soy milk yoghurt-added samples and gave similar scores to soy milk yoghurt-added tarhanas for all sensory properties     

Growth Medium for Culturing Probiotic Bacteria for Applications in Vegetarian Food Products

Vegetarian probiotic foods by definition, must be free from all animal-derived ingredients. This not only includes the product ingredients but the probiotic inoculum as well. Probiotic starter cultures are traditionally grown and stored in media containing milk or meat-derived ingredients. The presence of these ingredients makes the probiotic cell concentrates unsuitable for use in vegetarian products and thus creates the need for a growth medium free from animal-derived ingredients. This study investigated the growth of two strains of Lactobacillus acidophilus (MJLA1 and La-5), L. paracasei ssp. paracasei (LCSH1 and 01) and Bifidobacterium lactis (BDBB2 and Bb-12) in five media. Growth parameters such as maximum cell concentrations, lag phase and growth rate, were calculated using the growth curves generated by DModel, Institute of Food Research, U.K. Results showed that several media were suitable, with results comparable or better than the commonly used laboratory media MRS and RCM. Media containing 25 g/L soy peptone, yeast extract and glucose monohydrate gave the most desirable results with the strains examined.     

Immunomodulatory Properties of Fermented Soy and Dairy Milks Prepared with Lactic Acid Bacteria

ABSTRACT:  Fermented soy and dairy milk preparations provide a means for delivering lactic acid bacteria and their fermentation products into the diet. Our aims were to test immunomodulatory bioactivity of fermented soy beverage (SB) and dairy milk blend (MB) preparations on human intestinal epithelial cells (IEC) and to determine the impact of freezing medium on culture survival prior to bioactivity analyses. Fermented SB and MB were prepared using pure or mixed cultures of  Streptococcus thermophilus  ST5,  Bifidobacterium longum  R0175, and  Lactobacillus helveticus  R0052. Immunomodulatory bioactivity was assessed by testing selected SB and MB ferments on tumor necrosis factor α (TNFα)-treated IEC and measuring effects on Interleukin-8 (IL-8) production. Impact of timing of ferment administration relative to this pro-inflammatory challenge was investigated. The most pronounced reductions in IEC IL-8 production were observed when IEC were treated with either SB or MB ferment preparations prior to TNFα challenge. These results indicate that freezing-stable MB and SB ferments prepared with selected strains can modulate IEC IL-8 production  in vitro , and suggest that yogurt-like fermented soy formulations could provide a functional food alternative to milk-based fermented products.     

Food Microorganisms that Effectively Hydrolyze O-Glycoside but Not C-Glycoside Isoflavones in Puerariae Radix

ABSTRACT: Puerariae radix (PR) is known to contain abundant glucoside conjugates of daidzein isoflavones. This study was performed to determine the daidzin hydrolysis activity of the various strains of probiotic bacteria and edible fungi during fermentation of PR. Among the strains tested, Bifidobacterium sp. Int-57 showed the greatest β-glucosidase activity and daidzin hydrolysis activity. When yeast extract (2.5%) was added to the PR medium during fermentation with Bifidobacterium sp. Int-57, all of the daidzin was hydrolyzed to near completion. The addition of skim milk and whole milk also improved the conversion of daidzin into daidzein. Puerarin, a C-glucoside of daidzein in PR, was not hydrolyzed into daidzin by any of the experimental strains during fermentation. This study demonstrates that daidzin can be efficiently converted into daidzein by the proper combination of probiotic strains and fermentation conditions.     

Fermented soymilk with a monoculture of Lactococcus lactis

Lactococcus lactis strain (LL3) isolated from mothers' milk was used to produce fermented soymilk. The strain survived at levels of over 7 log cfu/ml for 3 weeks in the fermented soymilk. A consumer survey was carried out to compare the acceptability of the fermented product with a similar product made with L. lactis ATCC11545 originally isolated from cow's milk. Blind samples produced by fermentation with the two strains were rated equally attractive, whereas information on the origin of the strains significantly enhanced the pleasantness of the fermented soymilk.     

Incorporation of citrus fibers in fermented milk containing probiotic bacteria

The incorporation of Lactobacillus acidophilus CECT 903, Lactobacillus casei CECT 475 and Bifidobacterium bifidum CECT 870 together with lemon (LF) and orange (OF) fibers obtained from juice by-products were tested in (i) a model system: fiber enriched with de Man Rogosa Sharp (MRS) broth cultured with each probiotic bacteria and (ii) evaluation of populations of probiotic bacteria in fermented milks formulated with citrus fibers. Citrus fibers enhanced L. acidophilus CECT 903, and L. casei CECT 475 survival in MRS during refrigerated storage, whereas erratic results were obtained for B. bifidum CECT 870, OF enhanced its growth and LF had inhibitory effect. Populations of probiotic bacteria decreased with storage time in MRS broth. The presence of yogurt starter bacteria in probiotic fermented milks favored the growth and survival of L. acidophilus and B. bifidum. Citrus fiber presence in fermented milks also enhanced bacterial growth and survival of the tested probiotic bacteria. This study indicates that citrus fiber enriched fermented milk have good acceptability and are good vehicles for a variety of commercial probiotics but survival of B. bifidum will need to be improved.     

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.     

Preparation of the bifidus milk powder

Malt extract and skim milk were used as the culture medium for bifidobacteria. Bifidobacterium longum E194b (ATCC 15707) selected from among four strains grew well in a nitrogen atmosphere regardless of the initial dissolved oxygen level of the culture medium. The optimum temperature range for growth was 34 to 37 degrees C. The viable cell numbers of Bifidobacterium in freeze-dried powder from neutralized bifidus milk were 20 times those found in powder from nonneutralized milk after storage for 30 d at 35 degrees C. The optimum residual water content in the powder for bacterial stability was about 3.5% or less. Bifidus milk powder prepared under these optimum conditions contained at least one billion viable cells even after storage for 30 d at 35 degrees C. The powder must be sealed with material made of aluminium for storage.     

Bifidobacterium longum Survival During Frozen and Refrigerated Storage as Related to pH during Growth

Four strains of Bifidobacterium longum were grown at pH 5.5, 6.0, 6.5 and 7.0 and evaluated for survival and bile tolerance during frozen and subsequent refrigerated storage in milk. There were no reductions in cell numbers following initial freezing. There were effects for strain, pH and storage for three of the four strains of B. longum du ring storage at 5°C in milk. Bifidobacterium longum S9 was more stable than other strains in that no losses occurred, regardless of pH during growth. Results were variable for strains II, III, and ATCC 15707 grown at the various pH levels. Bifidobacterium longum S9 did not lose bile resistance during refrigerated storage as the other three strains did.     

成人粪便中长双歧杆菌长亚种的分离鉴定及多位点序列分型分析

为了对成人粪便中分离的长双歧杆菌长亚种进行多位点序列分型分析,采用改良MRS培养基从健康成人粪便中分离的长双歧杆菌长亚种,通过生理生化试验结合16S rDNA和热应激蛋白60(heat-shock protein,hsp60)同源性分析鉴定分离株,利用同源性分析及多位点序列分型(multilocus sequence typing,MLST)技术进一步分析长双歧杆菌长亚种的基因多态性。从12个健康成人体内分离得到24株厌氧的细菌菌株,经形态学观察、生理生化试验、16S rDNA及hsp60同源性分析发现,其中14株分离株为长双歧杆菌长亚种(Bifidobacterium longum subsp. longum)。MLST结果表明:14株长双歧杆菌长亚种可分为10种基因型,且均与Bifidobacterium longum subsp. longum ATCC 15707为不同基因型。健康成人粪便中分离的长双歧杆菌长亚种具有较大的基因多态性。     

Fermentation of rice using amylolytic Bifidobacterium

Twenty-two amylolytic bifidobacteria grown in BHI-starch media were compared for the amylase activity of the intra- and extra-cellular enzymes. The activity of the cells grown in the liquid medium differed considerably. Among the strains tested B. adolescentis Int57 and B. adolescentis ZS8 exhibited higher activities than others. In rice medium containing 0.05% -cysteine·HCl and 0.2% yeast extract, the amylolytic Bifidobacterium strains grew considerably better than non-amylolytic Bifidobacterium strains. B. adolescentis Int57, which showed highest growth and amylase activity in the rice medium, was chosen and rice fermentation was investigated. The titratable acidity (TA) reached 2.43 and pH decreased to 4.4 after 24 h fermentation. The relative ratio of acetic acid to lactic acid gradually decreased during fermentation. The concentration of reducing sugar and amylase activity gradually increased and reached 14 mg maltose equivalent/ml and 35 mU/ml min, respectively, in 24 h. The accumulated reducing sugar was mostly maltotriose. The layer-separation of fermented product was stabilized by the addition of 1% gelatin. It was suggested that amylolytic bifidobacteria may be used for the production of fermented rice products.     

Protocols for the isolation and detection of lactic acid bacteria with bacteriocinogenic potential

The objective of this study was to evaluate culture media and methodologies for isolation and detection of lactic acid bacteria (LAB) capable to produce bacteriocin-like substances. Samples of milk and cheese were pour plated on de Mann-Rogosa-Sharpe agar (MRS) and Kang-Fung-Sol agar (KFS) (both at 35 °C/48 h, under anaerobiosis), from which 389 and 256 LAB cultures were selected. The antagonistic activity of them was evaluated using the spot-on-the-lawn and two culture media: brain-heart infusion agar with catalase (BHI + C) and M17 (both at 35 °C/24 h). The proteinaceous nature of the antagonistic cultures was verified using: spot-on-the-lawn (MRS, 25 °C/24 h, under anaerobiosis) and well-diffusion (cultures amplified on modified MRS broth at 25 °C/24 h, and then neutralized using NaOH). Listeria monocytogenes ATCC 7644 was used as indicator. A larger number of antagonist cultures were isolated from MRS (83 by M17 and 65 by BHI + C) in comparison to KFS (24 by M17 and 15 by BHI + C). The spot-on-the-lawn identified a higher frequency of LAB capable of producing bacteriocin-like substances. MRS was considered to be the best culture media for the isolation of LAB capable to produce bacteriocin-like substances, activity that was better identified using the spot-on-the-lawn methodology.