Selection of useful probiotic lactic acid bacteria from the Lactobacillus acidophilus group and their applications to functional foods

In the present review, a new mass screening system for selecting probiotic strains from Lactobacillus (L) acidophilus group lactic acid bacteria (LAB) with strong adhesion to the human intestinal tract is described. Characteristics of antimicrobial peptides (bacteriocin), lactose‐hydrolyzing enzymes and immunostimulative oligo DNA motifs in L. gasseri strains are also described. Finally, the use of L. acidophilus LAB, selected by our screening method, that have strong adhesion to the human colonic mucosa in functional yogurt products is described. Adhesiveness to the human intestine is one of the most important characteristics of probiotic LAB. A new screening system that involves a combination of three methods is proposed: rat colonic mucin (RCM)‐micro plate assay, Carnoy's histochemical staining method and carbohydrate probe binding assay. By using an RCM‐coated poly‐vinylidene‐diflouride membrane that mimics the human colonic mucous layer, a new lectin was isolated and its structure was clarified by gene cloning. Furthermore, the structures and functions of a new cyclic bacteriocin (gassericin A), new lactose‐hydrolyzing enzymes, and new immunostimulating oligo DNA motifs from Lactobacillus gasseri (B₁ subgroup) were clarified. A new functional yogurt ‘Fit down’ is proposed, that is fermented by an adhesive strain of L. acidophilus LA67 selected by our screening and contains antihypertensive peptides derived from whey proteins by protease digestion. In the future, superior functional foods containing more effective probiotic LAB are expected to be developed by the use of the proposed mass screening system.     

Oral administration of Lactobacillus plantarum Lq80 and Megasphaera elsdenii iNP‐001 induces efficient recovery from mucosal atrophy in the small and the large intestines of weaning piglets

Weaning causes atrophy of intestinal mucosa and a drop of IgA protection in piglets which increases vulnerability to pathogenic infections. Probiotic lactobacilli may support recovery from such weaning stresses. Butyrate‐produce bacteria may support the growth of colonic mucosa. Megasphaera elsdenii, a lactate‐utilizing butyrate producer, may help butyrate production particularly when combined with lactobacilli. Weaned piglets (Experiment 1: 20 days old, Experiment 2: 28 days old) were orally dosed once a day with either (L) 10¹⁰ (cell/dose) L. plantarum Lq80, or (LM) 10¹⁰ (cell/dose) Lq80 with 10⁹ (cell/dose) M. elsdenii iNP‐001. Lq80 was contained in capsules resistant to gastric digestion. M. elsdenii was contained in capsules resistant to gastric and intestinal digestion. An untreated control (C) was also prepared. After 2 weeks of administration, L. plantarum enhanced the recovery from the villous atrophy in both experiments. The rectal and colonic IgA tended to be higher in L and LM than in C in Experiment 1. Colonic butyrate was higher in LM than in the others in Experiment 1. The thickness of the colonic mucosa was greater in LM than in the others in Experiment 1. In early weaned piglets, the effects of L. plantarum and M. elsdenii were clear.     

Construction of the Shuttle Vector Expressing Green Fluorescent Protein and its Application in Lactobacillus acidophilus

In order to further study the mechanism of Lactobacillus acidophilus,GFP labeling shuttle expression vector pSET4s-P1-GFP-P2 was constructed by inserting green fluorescence protein (GFP),upstream and downstream homology arm of Lactobacillus acidophilus based on temperature shuttle expression vector pSET4s.Recombinant Lactobacillus acidophilus expressing green fluorescence gene was constructed by transforming pSET4s-P1-GFP-P2 into Lactobacillus acidophilus using electrotransformation.We could obtain the recombinant Lactobacillus acidophilus by resistance screening and temperature screening,and named it as ΔMG6243(GFP) which could expressed GFP gene stably by fluorescence microscopy observation and Western blotting analysis.We could analyse the genetic stability and biological characteristics with fluorescence microscopy observation and plate count.Green fluorescent of recombinant Lactobacillus acidophilus could be observed in the blue light,and also be observed after 10 generations.There were significant differences neither growth characteristics nor pH and salt tolerance compared with the wild mushrooms.The results showed that GFP labeling recombinant Lactobacillus acidophilus was constructed successfully.Exogenous gene could be non-resistant and integrative to express in the Lactobacillus acidophilus with the vector.It established the foundation for construction of recombinant Lactobacillus acidophilus.At the same time,the role of the GFP as marker laid the foundation for distribution,adhesion of probiotic in animal gastrointestinal tract and the mechanism of Lactobacillus acidophilus.     

绿色荧光蛋白穿梭载体的构建及其在嗜酸乳杆菌中的应用

为深入研究嗜酸乳杆菌的作用机理,以温敏型穿梭载体pSET4s为基本骨架,将绿色荧光蛋白(GFP)和嗜酸乳杆菌上、下游同源臂插入该载体,构建GFP标记的穿梭表达载体pSET4s-P1-GFP-P2。利用电转化方法将穿梭表达载体pSET4s-P1-GFP-P2转化入嗜酸乳杆菌,通过抗性和温度双重筛选获得能稳定表达GFP的重组嗜酸乳杆菌,将Western blotting和荧光显微镜观察验证的重组菌命名为ΔMG6243(GFP)。利用荧光显微镜观察和平板计数的方法对重组菌进行遗传稳定性和生物学特性研究。结果显示,重组菌在蓝光激发下可见明显绿色荧光,连传10代仍可见明显荧光,与野生菌相比,其生长特性、酸碱盐耐受性均无显著差别。结果表明,本试验已成功构建稳定表达GFP基因的重组嗜酸乳杆菌,利用温敏型穿梭载体pSET4s实现了外源基因在嗜酸乳杆菌中非抗性、整合型表达,为嗜酸乳杆菌基因重组菌的构建奠定了基础,同时GFP的标记作用也为嗜酸乳杆菌在动物体内分布、定植规律和作用机理创造了可行的条件。     

嗜酸乳杆菌对黄羽肉鸡生长性能、肠道微生物及经济效益的影响

为探究饲料中嗜酸乳杆菌对肉鸡生产的效果,试验选取11日龄健康的黄羽肉鸡200羽,随机分为5组。分别为对照组(饲喂基础日粮),试验1、2、3、4组在基础日粮中分别添加5、10、15g/kg的嗜酸乳杆菌和150mg/kg的抗菌肽。结果表明:(1)与对照组相比,嗜酸乳杆菌组显著提高了肉鸡的日增重与采食量(P<0.05);但与抗菌肽组相比差异不显著(P>0.05)。(2)添加嗜酸乳杆菌15g/kg时,试验组中盲肠段内的双歧杆菌数量显著提高(P<0.05)。与对照组相比,4个试验组中的盲肠、回肠乳酸杆菌与双歧杆菌均有所提高,但只有试验3、4组呈现显著性差异(P<0.05),除此之外,各分组中回肠大肠杆菌数量无明显差异(P>0.05)。(3)与对照组相比,饲料中添加不同嗜酸乳杆菌能明显提高肉鸡生长性能和饲料利用率,增加经济效益。     

ORIGINAL ARTICLE: Inulin‐coated butyrate increases ileal MCT1 expression and affects mucosal morphology in the porcine ileum by reduced apoptosis

Carbohydrates, which were not digested in the jejunum, will be fermented by micro‐organisms to short chain fatty acids. These are transported by the monocarboxylate transporter 1 (MCT1) through the gut wall and serve as fuels for colonic cells. To deliver butyrate to the distal part of the intestine, inulin with a low precaecal digestibility was chosen as a coating material. Approximately 150 g of inulin‐coated butyrate (containing 81 g butyrate) per day was fed to pigs (mean weight: 97 kg) over a period of 6 days after an adaptation period of 6 days with linear increasing amounts of butyrate. The following observations compared to controls were observed: (1) coating was digested microbially in the ileum; (2) MCT1‐mRNA showed a higher expression in the ileum; (3) apoptosis was reduced in the ileum but mitosis was not changed; and (4) length of villi increased by approximately 25% in the ileum. Feeding inulin‐coated butyrate resulted in an increased ileal surface. Delivery of butyrate to the colon requires a more resistant inulin‐coating.     

Effects of fermentable carbohydrates and low dietary phosphorus supply on the chemical composition of faecal bacteria and microbial metabolites in the gastrointestinal tract of pigs

The objective was to determine the effects of fermentable carbohydrates on phosphorus (P) metabolism, the chemical composition of the faecal mixed bacterial mass (MBM) and the microbial activity in the large intestine (LI) of pigs. Eight barrows (mean BW 35.9 ± 0.9 kg), fitted with simple T‐cannulas at the terminal ileum, were either fed a low‐P corn–soybean meal‐based control diet or 75% of the control diet supplemented with 25% cellulose, starch or pectin according to a 4 × 3 Latin Square design. Both pectin and cellulose caused higher faecal than ileal P recoveries. Ileal volatile fatty acids (VFA) levels were more pronounced for the starch (p < 0.05) rather than the cellulose and pectin treatments, whereas pectin resulted in a higher faecal VFA concentration in comparison to starch and cellulose (p < 0.05). The differences in faecal VFA concentrations corresponded to the pH values obtained in faeces. The N content of MBM was higher (p < 0.05) when cellulose was supplemented. Pectin caused a decrease in the P content of the MBM compared to the control (p < 0.05). As a result, the N:P ratio was significantly higher for the pectin (N:P = 4.33) than for the control treatment (N:P = 2.63), while the Ca:P ratio remained constant for all treatments, suggesting changes in the accumulation of N, P and Ca in MBM, probably due to changes in the species composition and activity of the microflora.     

Effect of Megasphaera elsdenii NCIMB 41125 dosing on rumen development,volatile fatty acid production and blood β‐hydroxybutyrate in neonatal dairy calves

Thirty calves were randomly assigned to two treatments and fed until weaning [42 days (d) of age]. Treatments were a control group (n = 15), which did not receive Megasphaera elsdenii (Me0) and a M. elsdenii group, which received a 50‐ml oral dose of M. elsdenii NCIMB 41125 (10⁸ CFU/ml) at day 14 day of age (Me14). Calves were given colostrum for the first 3 day followed by limited whole milk feeding. A commercial calf starter was offered ad libitum starting at day 4 until the end of the study. Fresh water was available throughout the study. Feed intake and growth were measured. Blood samples were collected via jugular venipuncture to determine β‐hydroxybutyrate (BHBA) concentrations. Fourteen male calves (seven per group) were euthanised on day 42 and digestive tracts harvested. Reticulo‐rumen weight was determined and rumen tissue samples collected from the cranial and caudal sacs of the ventral and dorsal portions of the rumen for measurements of papillae length, papillae width and rumen wall thickness. Dosing with M. elsdenii NCIMB 41125 improved starter dry matter intake (DMI), weaning body weight (BW) and tended to improve average daily gain. Calves in Me14 group had greater plasma BHBA concentration than Me0‐calves during the last 3 weeks of the trial and had at day 42 greater reticulo‐rumen weight, papillae width and papillae density compared to Me0. No differences in rumen wall thickness or papillae length were observed between the two groups. Total volatile fatty acids, acetate and propionate production did not differ between treatments, but butyrate production was greater in Me14 than Me0. Dosing M. elsdenii NCIMB 41125 showed benefit for calves with improved feed intake and rumen development suggesting increased epithelium metabolism and improved absorption of digestive end products.     

Intravaginal administration of lactic acid bacteria modulated the incidence of purulent vaginal discharges,plasma haptoglobin concentrations,and milk production in dairy cows

This investigation studied the effects of intravaginal administration of a mixture of lactic acid bacteria (LAB) on the incidence of purulent vaginal discharges (PVD), plasma haptoglobin concentrations, and milk production in dairy cows. A total of 82 pregnant primiparous and multiparous Holstein dairy cows were used in this study. Half of the cows received intravaginally 1 mL of LAB at 10¹⁰–10¹² cfu/mL and the other half 1 mL of reconstituted skim milk (i.e., carrier) (controls). Administration of LAB was conducted once per wk during 2 and 1 wk before the expected day of calving and at 1, 2, 3, and 4 wk postpartum. Data demonstrated that intravaginal administration of LAB decreased the occurrence of PVD at 3 wk postpartum (P < 0.05). Concentrations of plasma haptoglobin, an acute phase protein often associated with uterine infections, was lower in cows treated with the LAB mixture at 2 wk (P < 0.001) and 3 wk (P < 0.05) postpartum. Treatment with LAB did not improve overall pregnancy rate, but the treated multiparous cows produced more milk than their control counterparts (P < 0.05), whereas no difference was observed in primiparous cows regarding milk yield (P > 0.05). Overall, this is the first study demonstrating that intravaginal LAB administration lowers the incidence of PVD and enhances milk production in dairy cows. Further research is warranted to evaluate the effects of LAB on reproductive performance in a larger cohort of cows.     

Associations of the variation in the porcine myogenin gene with muscle fibre characteristics, lean meat production and meat quality traits

Pig breeding is aimed at improving lean meat production ability as well as meat quality, and muscle fibre characteristics may be important for enhancing these traits. Therefore, new molecular markers have been demanded for selecting lean meat production ability and meat quality in live animals. Myogenin belongs to the MyoD gene family, and is a candidate gene responsible for muscle fibre characteristics. We identified a new single nucleotide polymorphism (SNP) site in the 5' upstream region of the myogenin gene (nucleotides C and T). A total of 252 pigs of three breeds were genotyped by polymerase chain reaction–restriction fragment length polymorphism using BspCNI . Additionally, they were genotyped for the previously detected Msp I site in the 3'-flanking region (alleles A and B). The CCBB diplotype had the highest frequency over breeds, followed by TCBB and CCAB. The other diplotypes were not found in studied pigs. Association analysis performed for the markers found that the TCBB diplotype has desirable effects on the total number of fibres (p   < 0.002), fibre cross-sectional area (p   < 0.06), and loin eye area (p   < 0.001) than the other diplotypes. Moreover, the diplotype had the highest muscle pH value (p   < 0.07) and all meat quality traits were near the upper limit of the normal range as a reddish pink, firm and non-exudative (RFN) pork. Therefore, we suggest that selection for the myogenin diplotypes could improve total muscle fibre number, size and lean meat production ability with good meat quality.     

不同酸度的乳酸菌饲料添加剂对6月龄梅花鹿消化率及小肠微生物氮流量的影响

试验通过研究不同酸度的乳酸菌添加剂对6月龄梅花鹿的采食量、氮平衡、消化率以及小肠微生物氮流量的影响,确定乳酸菌添加剂在梅花鹿饲喂中的适宜酸度。结果表明:添加不同酸度的乳酸菌可明显提高梅花鹿对DM、OM、CF、CP以及氮采食量(P<0.05),其中以未调酸的乳酸菌组各种营养成分采食量最高;DM、OM、CF采食量,各组间差异显著(P<0.05),氮的采食量,除调酸组与未调酸组间差异不显著外(P>0.05),其余各组间差异显著(P<0.05)。不同酸度的乳酸菌饲料添加剂可提高梅花鹿DOM、DCF、氮沉积以及小肠微生物氮流量,未调酸组的DOM、DCF、氮沉积以及小肠微生物氮流量显著高于对照组(P<0.05)。     

Effect of crushing unhulled rice and the addition of fermented juice of epiphytic lactic acid bacteria on the fermentation quality of whole crop rice silage, and its digestibility and rumen fermentation status in sheep

This experiment was carried out to examine the effect of crushing unhulled rice and the addition of fermented juice of epiphytic lactic acid bacteria (FJLB) on the fermentation quality of whole crop rice silage, and its digestibility and rumen fermentation in sheep. After whole rice plants (Oryza sativa L. cv. Haenuki) were harvested between the yellow and full‐ripened period of maturity, and cut at a length of 2 cm, four silage treatments were set: (i) non‐treatment; (ii) crushing treatment of unhulled rice division (crushing); (iii) addition of 1% FJLB; and (iv) crushing + addition of 1% FJLB. Lactic acid content increased with the addition of FJLB, and was highest in the crushing + FJLB treatment. Digestibilities of crude fiber, acid detergent fiber and neutral detergent fiber were significantly higher during the crushing + FJLB treatment. There were no differences among treatments in the volatile fatty acid concentrations before and after feeding. At 4 h after feeding, however, the ratio of acetic acid : propionic acid was significantly lower with the addition of FJLB. It was proven that the fermented quality of silage was improved when FJLB was added to rice plants and then ensilaged, and the digestibility of the fibrous component was raised when unhulled rice division was crushed and FJLB was supplemented.