Growth and Acid Production by Lactic Acid Bacteria and Bifidobacteria Grown in Skim Milk Containing Honey

ABSTRACT: Twelve percent nonfat dry milk containing 5% (w/w) honey, fructose, or sucrose were pasteurized and inoculated with Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus delbrukeii subsp bulgaricus , or Bifidobacterium bifidum. Inoculated tubes were incubated at 37 °C, 24 h. Samples were collected at 0 and 24 h and examined for (a) viability of bacteria, and (b) levels of fermentation end products (lactic and acetic acids) as measured by HPLC. Honey supported growth of all 4 organisms similar to other sweeteners and was not inhibitory. Lactic acid production was similar for all, except for bifidobacteria and was not influenced by sweetener type. Although lactic acid production was enhanced (p < 0.05) when bifidobacteria were grown in the presence of honey, acetic acid production was not affected. Various oligosaccharides found in honey may be responsible for enhanced lactic acid production by bifidobacteria.     

Growth and Viability of Commercial Bifidobacterium spp in Skim Milk Containing Oligosaccharides and Inulin

Two commercial strains of Bifidobacterium spp (Bf ‐1 and Bf ‐6) were cultured in 12% (w/w) reconstituted nonfat dry milk (NDM) containing 0, 0.5, 1.0, 3.0, or 5.0% (w/v) fructooligosaccharide (FOS), galactooligosaccharide (GOS), and inulin. Inoculated samples were incubated anaerobically at 37° for 48 h. Growth and activity of the cultures in the presence of FOS, GOS, and inulin were determined. Viability of each strain was assessed after 4 weeks of refrigerated storage at 4°. Growth promotion, enhancement of activity and retention of viability were greatest when Bifidobacterium Bf‐1 and Bf‐6 were grown in the presence of FOS followed in a descending order by GOS and inulin. The effects of oligosaccharides and inulin increased with increasing carbohy drate concentration and was maximal at 5% (w/v).     

A selective medium for the enumeration and differentiation of Lactobacillus delbrueckii ssp. bulgaricus

Modified reinforced clostridial medium (mRCM) was developed and evaluated for the differential enumeration of Lactobacillus delbrueckii ssp. bulgaricus. Lactobacillus bulgaricus, an important species of lactic acid bacteria with health benefits, is used in the production of yogurt and other fermented foods. Our results showed that supplementing reinforced clostridial medium with 0.025% CaCl₂, 0.01% uracil, and 0.2% Tween 80 (mRCM) significantly enhanced the growth rate of L. bulgaricus RR and ATCC 11842 strains as measured by the optical densities of these strains after 12 h of incubation at 42°C. The bacterial populations (plate count) of the RR and ATCC 11842 strains were 0.76 and 0.77 log cfu/g higher in mRCM than in de Man, Rogosa, and Sharpe and reinforced clostridial medium media, respectively. Conversely, the population counts for other bacterial species (Bifidobacterium, Lactobacillus rhamnosus, and Lactobacillus reuteri) were significantly inhibited in the mRCM medium. The addition of aniline blue dye to mRCM (mRCM-blue) improved the selectivity of L. bulgaricus in mixed lactic bacterial cultures compared with de Man, Rogosa, and Sharpe medium and lactic agar with regard to colony appearance and morphology. The mRCM-blue performed better than the conventional medium in culturing, enumerating, and differentiating L. bulgaricus. Therefore, mRCM-blue could be used as a selective medium to enhance the growth and differentiation of L. bulgaricus in order to meet the increasing demand for this beneficial species of bacteria.     

Carbohydrate composition of honey from different floral sources and their influence on growth of selected intestinal bacteria: An in vitro comparison

Five human intestinal Bifidobacterium spp. (B. longum, B. adolescentis, B. breve, B. bifidum, and B. infantis) and intestinal microorganism (Bacteriodes thetaiotaomicron, Clostridium perfringens, Eubacterium aerofaciens, and Enterococcus faecalis) were cultured in De Man Rogosa Sharpe (MRS) medium or thioglycollate medium supplemented (5% w/v) with different unifloral honeys (sourwood, alfalfa, or sage). Inoculated samples were incubated anaerobically at 37 °C for 48 h. Samples were collected at 12 h intervals and examined for specific growth rate. Levels of fermentation end products (lactic and acetic acids) produced by Bifidobacterium spp. were measured by high-pressure liquid chromatography. Growth of intestinal microorganisms co-cultured with Bifidobacterium spp. in the presence of different unifloral honeys were also examined. All three honeys enhanced (p < 0.05) growth and activity of the five intestinal Bifidobacterium spp. Their effect on other organisms of the intestinal microflora was selective. Growth of C. perfringens and E. aerofaciens was inhibited (p < 0.05) in the presence of honey and further inhibited when co-cultured with Bifidobacterium spp. Bifidobacterium spp. was not affected.     

Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp. lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures

Exopolysaccharides (EPS) isolated from two Bifidobacterium strains, one of human intestinal origin (Bifidobacterium longum subsp. longum IPLA E44) and the other from dairy origin (Bifidobacterium animalis subsp. lactis IPLA R1), were subjected to in vitro chemically simulated gastrointestinal digestion, which showed the absence of degradation of both polymers in these conditions. Polymers were then used as carbon sources in pH-controlled faecal batch cultures and compared with the non-prebiotic carbohydrate glucose and the prebiotic inulin to determine changes in the composition of faecal bacteria. A set of eight fluorescent in situ hybridisation oligonucleotide probes targeting 16S rRNA sequences was used to quantify specific groups of microorganisms. Growth of the opportunistic pathogen Clostridium histolyticum occurred with all carbohydrates tested similarly to that found in negative control cultures without added carbohydrate and was mainly attributed to the culture conditions used rather than enhancement of growth by these substrates. Polymers E44 and R1 stimulated growth of Lactobacillus/Enterococcus, Bifidobacterium, and Bacteroides/Prevotella in a similar way to that seen with inulin. The EPS R1 also promoted growth of the Atopobium cluster during the first 24 h of fermentation. An increase in acetic and lactic acids was found during early stages of fermentation (first 10–24 h) correlating with increases of Lactobacillus, Bifidobacterium, and Atopobium. Propionic acid concentrations increased in old cultures, which was coincident with the enrichment of Clostridium cluster IX in cultures with EPS R1 and with the increases in Bacteroides in cultures with both microbial EPS (R1 and E44) and inulin. The lowest acetic to propionic acid ratio was obtained for EPS E44. None of the carbohydrates tested supported the growth of microorganisms from Clostridium clusters XIVa+b and IV, results that correlate with the poor butyrate production in the presence of EPS. Thus, EPS synthesized by bifidobacteria from dairy and intestinal origins can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of short chain fatty acids.     

Growth and viability of commercial Bifidobacterium spp. in honey-sweetened skim milk.

Two commercial Bifidobacterium bifidum (Bf-1 and Bf-6) were cultured in 12% (wt/vol) reconstituted nonfat dry milk (NDM) containing 5% (wt/wt) honey, sucrose, fructose, or glucose. Inoculated samples were incubated anaerobically at 37 degrees C for 48 h. Samples were collected at 12-h intervals and examined for (i) specific growth rate, (ii) pH, and (iii) levels of fermentation end products (lactic and acetic acids) as measured by high-performance liquid chromatography (HPLC). Viability of the organisms during 28 days of refrigerated storage at 4 degrees C was also assessed at 7-day intervals. Growth promotion and acid production were greatest when Bf-1 and Bf-6 were grown in the presence of honey. For both Bf-1 and Bf-6, retention of viability was greatest up to 14 days of refrigerated storageat 4 degrees C when they were grown and stored in the presence of honey compared to other sweeteners.     

Influence of sucrose,high fructose corn syrup and honey from different floral sources on growth and acid production by lactic acid bacteria and bifidobacteria

Sage, alfalfa and sourwood honey, which vary in their oligosaccharide contents, were compared with sucrose, high fructose corn syrup and inulin in their ability to support growth, activity and viability of lactic acid bacteria and bifidobacteria typically used in yoghurt manufacturing. Growth and the end products of fermentation (lactic and acetic acids) were determined. Growth and acid production by organisms studied in the presence of different sweeteners were dependent on the specific organism investigated; however, it was not influenced by sweetener type, oligosaccharide content or the floral source of the honeys. All the sweeteners studied supported the growth, activity and viability of the organisms studied.     

Effect of prebiotics on viability and growth characteristics of probiotics in soymilk

BACKGROUND: Soy products have attracted much attention lately as carriers for probiotics. This study was aimed at enhancing the growth of probiotics in soymilk via supplementation with prebiotics. RESULTS: Lactobacillus sp. FTDC 2113, Lactobacillus acidophilus FTDC 8033, Lactobacillus acidophilus ATCC 4356, Lactobacillus casei ATCC 393, Bifidobacterium FTDC 8943 and Bifidobacterium longum FTDC 8643 were evaluated for their viability and growth characteristics in prebiotic‐supplemented soymilk. In the presence of fructooligosaccharides (FOS), inulin, mannitol, maltodextrin and pectin, all strains showed viability exceeding 7 log₁₀ colony‐forming units mL^?1 after 24 h. Their growth was significantly (P < 0.05) increased on supplementation with maltodextrin, pectin, mannitol and FOS. Additionally, supplementation with FOS, mannitol and maltodextrin increased (P < 0.05) the production of lactic acid. Supplementation with FOS and maltodextrin also increased the α‐galactosidase activity of probiotics, leading to enhanced hydrolysis and utilisation of soy oligosaccharides. Finally, prebiotic supplementation enhanced the utilisation of simpler sugars such as fructose and glucose in soymilk. CONCLUSION: Supplementation with prebiotics enhances the potential of soymilk as a carrier for probiotics. Copyright © 2009 Society of Chemical Industry     

Effect of inulin on growth and bacteriocin production by Lactobacillus plantarum in stationary and shaken cultures

The prebiotic effect of inulin added to MRS medium on growth and bacteriocin production by L. plantarum ST16 Pa was investigated in stationary cultures in anaerobic jars with medium containing 0.025% sodium thioglycolate or in flasks shaken at 100 rpm. In the presence of 1% inulin in anaerobic stationary cultures, this strain produced lactic acid at a level that was 36.5% higher than in the absence of the polysaccharide. In shaken cultures without inulin, cell count was 54% higher than in the stationary ones. Under stationary conditions in anaerobic jars, the addition of inulin increased the maximum specific growth rate from 0.37 to 0.49 h^?1 and reduced the generation time from 1.85 h to 1.40 h. Consequently, the exponential phase was shortened from 12 to 9 h when the cells were grown in stationary cultures with the oxygen scavenger. Despite this effect of inulin on growth rate, stationary cultures without inulin displayed higher antimicrobial activity against Listeria monocytogenes L104 (3200 AU/mL) than cultures with inulin (1600 AU/mL); therefore, inulin behaved as a compound able to accelerate growth rather than to stimulate bacteriocin production. The results presented in this study are very promising, as L. monocytogenes is a well‐known foodborne pathogenic microorganism. Moreover, L. plantarum ST16 Pa has proven to be a potential producer of a natural food preservative at an industrial level.     

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.     

Lipolysis in probiotic and synbiotic cheese: The influence of probiotic bacteria, prebiotic compounds and ripening time on free fatty acid profiles

The influence of probiotic bacteria (Lactobacillus casei-01, Bifidobacterium lactis B94), prebiotic compounds (FOS and inulin) and ripening time (0-60 days) on the free fatty acid (FFA) profile of cheese, with special emphasis on the conjugated linoleic acid (CLA) content, was investigated. After 60 days of ripening, 10⁹-10¹⁰ cfu g⁻¹ cheese were recorded in both probiotic and synbiotic cheeses, despite harsh conditions of low pH values (4.1-5.1) and low moisture content (<30%, w/w). Increases in total FFA and CLA were observed throughout the ripening period, especially in synbiotic cheeses containing FOS and inulin (50:50) inoculated with B. lactis B94. The addition of FOS alone or combined with inulin did not significantly affect probiotic strain growth and viability during the ripening period; however, the advantage of the addition of prebiotic compounds in probiotic cheese manufacture is that it may allow the production of cheeses with improved performance as far as functional CLA compounds are concerned, as well as an improved nutritional quality reflected in a lower atherogenicity index.     

Rapid enumeration of Bifidobacterium lactis in milk powders using impedance

An impedance method was evaluated to enumerate Bifidobacterium lactis, a probiotic, added to milk powder. Impedance changes were measured at 40 °C and recorded using the BacTrac™ 4100 microorganism growth analyser. Five different media were compared for the optimum impedance response. A raffinose-based medium (B. lactis medium) produced the fastest and most reproducible results. Good correlations were obtained between cell numbers from pure cultures of B. lactis (DR 10™) on reinforced clostridial agar plates and the impedance changes in the B. lactis medium. Enumeration of these bacteria in milk powder using the BacTrac™ 4100 impedance system showed no significant difference when compared with agar plate count results. The impedance counts estimated the cell counts of 10⁶ cells g⁻¹ within 15 h and was faster than the 3 days required to obtain a result using the agar plate count method.     

响应面法优化长双歧杆菌增殖培养基

为了提高长双歧杆菌发酵液中的活菌数,对其增殖培养基进行响应面优化。通过单因素试验筛选出长双歧杆菌的最佳碳源为乳糖,并发现低聚木糖、菊糖、低聚异麦芽糖、低聚果糖、苯丙氨酸、蛋氨酸、脯氨酸、谷氨酸及赖氨酸均能显著促进长双歧杆菌的生长。利用Design Expert 8.06软件设计Plackett-Burman 试验筛选出影响长双歧杆菌生长的3个最重要因子,通过Box-Behnken试验及响应面分析确定3个因子的最佳添加量为：低聚木糖1.7g/L、菊糖3.6g/L、脯氨酸0.4g/L,用优化后的增殖培养基培养长双歧杆菌,18h后其活菌数达(1.75±0.02)×109CFU/mL,比优化前提高了95.64%。     

Viability and Activity of Bifidobacteria During Refrigerated Storage of Yoghurt Containing Mangifera pajang Fibrous Polysaccharides

Abstract: The viability and activity of Bifidobacterium pseudocatenulatum G4, B. longum BB 536 and yoghurt cultures (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) were studied in yoghurt containing 0.75%Mangefira pajang fibrous polysaccharides (MPFP) and inulin. Growth of probiotic organisms, their proteolytic activities, the production of short chain fatty acids (lactic, acetic and propionic) and the pH of the yoghurt samples were determined during refrigerated storage at 4 °C for 28 d. B. pseudocatenulatum G4 and B. longum BB 536 showed better growth and activity in the presence of MPFP and inulin, which significantly increased the production of short chain fatty acids as well as the proteolytic activity of these organisms. Practical Application: This is the first study reported on produce synbiotic yoghurt as a functional food for specified health uses contains bifidobacteria and M. pajang fibrous polysaccharides. M. pajang fibrous polysaccharides can be used as a prebiotic particularly in dairy products to increase the viability and activity of bifidobacteria which can be used as probiotic to exert health benefit to the human by yoghurt that is considered common use in society; thus, the benefits of synbiotic yoghurt are readily accessible to the member of society.     

Effect of inulin polymerization degree on various properties of synbiotic fermented milk including Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12

The effects of inulin degree of polymerization (DP) on the viabilities of Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 and on some parameters of fermented milk, such as microbiological, rheological, biochemical, and sensory properties, were investigated during 30 d of storage. Samples were produced using L. acidophilus La-5 and B. animalis Bb-12, along with inulin having different DP as prebiotic, and the effects of high-DP (DP ≥ 23) and low-DP (DP ≤ 10) inulin on fermented milk, were determined. The viability of both strains increased when they were used with inulin having any DP. The addition of inulin increased the consistency index of all samples. During storage, we observed an increase in lactic and acetic acid contents of samples in which high-DP inulin was used, for both strains of bacteria. Of the combinations we tested, the sample produced with L. acidophilus La-5 and high-DP inulin demonstrated the highest rheological and sensory performance as well as the best viability of probiotics.     

主要代谢产物对双歧杆菌生长的影响

通过对双歧杆菌F 3 5在液体培养基中的生长特性、基质消耗和主要代谢产物的研究 ,揭示了代谢产物H+ ,乙酸根和乳酸根对两歧双歧杆菌F 3 5生长影响。结果表明 :基质中碳源和氮源不是造成双歧杆菌死亡的主要原因。H+ 对菌体生长有一定影响 ,但不是主要的因素。代谢产物中酸根 ,主要是乙酸根和乳酸根是抑制双歧杆菌生长的主要因素 ,乳酸的影响比乙酸大 ,乳酸达到 2 0mg/mL时有明显的抑制作用 ,而乙酸的耐受浓度可达 2 5mg/mL。 2者的比例也有一定的影响。     

Acid production by bifidobacteria and yoghurt bacteria during fermentation and storage of milk

Three species of bifidobacteria, namelyBifidobacterium bifidum, Bifidobacterium longumandBifidobacterium adolescentiswere used in pure culture and in combination with yoghurt bacteria (B3 and SBI cultures) for the production of fermented milks. The number of bacteria during fermentation and the level of acid produced during fermentation and storage were assessed using Rogosa's modified selective agar and high performance liquid chromatography (HPLC). It was found that during fermentation all bifidobacteria exhibited growth uncoupled from acid production. Two of the species examined produced only low levels of acids when grown individually and onlyB. adolescentisproduced appreciable amounts. In mixed cultures, the level of acid was a reflection of the combination of yoghurt culture and species ofBifidobacterium, and this, observation suggests that there is a degree of influence between the cultures. During storage, the acid concentration remained quite stable in most samples. The prevention of post-production acidification that normally occurs during storage of yoghurt can be attributed to the presence of bifidobacteria, and it could be that acetic acid has a marginally inhibitory effect on theLactobacillusandStreptococcusspp.     

Competition mechanisms of lactic acid bacteria and bifidobacteria: Fermentative metabolism and colonization

Enzyme activities (α- and β-glucosidases, α- and β-galactosidases and β-fructofuranosidase) and organic acid production of four strains of lactic acid bacteria (LAB; Streptococcus thermophilus STY-31, Lactobacillus delbrueckii subsp. bulgaricus LBY-27, Lactobacillus casei LC-01 and Lactobacillus acidophilus LA-5) and Bifidobacterium lactis BB-12 were tested on milk and MRS fermentation broth with glucose, lactose or fructooligosaccharides (FOS) as carbon source. The highest β-galactosidase activity was found in L. acidophilus growing on milk. As compared to milk, α-glucosidase activity was increased with FOS by B. lactis, L. acidophilus and L. casei. The analysis of organic acids and short-chain fatty acids in the medium growth showed that lactate and acetate were the major fermentation metabolites produced by LAB and bifidobacteria, respectively. However, a metabolic shift towards more acetate and formate production, at the expense of lactate production, was observed during growth of L. casei on FOS. When grown on FOS as sole carbon source, L. acidophilus showed the highest production of lactate among the species tested. In addition, L. acidophilus demonstrated resistance to colonization against the intestinal pathogens Escherichia coli and Salmonella enterica in competition assays.     

乳酸菌代谢低聚果糖/菊粉途径及机理的研究进展

合生元又称合生素,指益生元与益生菌结合使用的生物制剂（微生态制剂）,近年来,越来越多研究表明合生元具有多种益处,益生元和益生菌的选择对其最终效果起着至关重要的作用。低聚果糖和菊粉是合生元中常见的益生元,可被肠道内的乳酸菌选择性吸收,通过促进益生菌增殖的方式来改善机体健康。本文综述了乳酸菌对低聚果糖和菊粉的代谢差异及代谢途径,并从分子水平阐述水解酶、转运系统、调控蛋白的作用机制,以期为探明乳酸菌调控低聚果糖和菊粉的代谢网络提供依据;同时总结了近年来低聚果糖/菊粉合生元的应用,为益生元与益生菌的进一步联用提供参考。     

Growth, Viability and Activity of Bifidobacterium spp. in Skim Milk Containing Prebiotics

Growth, activity and mean doubling time (T_d) of five Bifidobacterium species in the presence of four types of prebiotics, and concentrations of acetic and lactic acids were determinted during fermentation and after 4 weeks of refrigerated storage. The T_d was lowest for B. animalis with raftilose and inulin. Retention of viability of bifidobacteria was greatest with hi‐amylose corn starch (hi‐maize). The average pH of skim milk at the end of 4 wks storage averaged 4.34 (for B. animalis with raftilose) to 4.07 (for B. longum with inulin). The highest levels of acetic acid and lactic acid were produced by B. pseudolongum with lactulose and B. infantis with lactulose, respectively.