Microbiota of 'airag', 'tarag' and other kinds of fermented dairy products from nomad in Mongolia

The traditional fermented dairy products were collected from three nomadic families in Donto‐Govi prefecture in Mongolia (central Mongolia), and those microbiota were analyzed. These samples consist three of ‘airag’, two of ‘tarag’, two of ‘isgelen tarag’ and ‘qoormog’, and some cheeses. In airag, Lactobacillus (L.) helveticus, L. kefiri, and Saccharomyces (S.) dairensis were common, and L. paracasei, L. plantarum, L. farciminis, S. cerevisiae, Issachenkia (I.) orientalis, Kluyveromyces (K.) wickerhamii were also found. In tarag, isgelen tarag and qoormog, L. helveticus, L. kefiri, L. fermentum, L. paracasei and L. acetotolerance were found. L. delbrueckii subsp. bulgaricus was also found in one tarag and one qoormog samples. Randomly amplified polymorphic DNA (RAPD) analysis showed that there were diversity in each L. helveticus family and products, and there were common strains found in airag and tarag in the same family.     

Microflora in traditional starter cultures for fermented milk, hurunge, from Inner Mongolia, China

Microflora were investigated in traditional starter cultures for fermented milk, hurunge, which are used for fermented dairy products by nomadic families in the Inner Mongolia Autonomic Region, China. The acid‐forming bacteria and yeast counts ranged from 1.8 × 10⁵ to 5.3 × 10⁸ c.f.u./g and from 6.1 × 10⁵ to 3.2 × 10⁶ c.f.u./g, respectively. Sixty‐six strains of lactic acid bacteria and 30 strains of yeasts were isolated and identified from three hurunge samples collected from the nomadic families. Lactococcus raffinolactis was the most predominant lactococci isolated from these samples. The other lactococci were Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, and Leuconostoc mesenteroides ssp. cremoris. Two major lactobacilli strains, Lactobacillus plantarum and Lactobacillus casei, were identified. In addition, Lactobacillus kefiranofaciens, Lactobacillus acetotolerans, which grew in 11% acetic acid culture medium, and Lactobacillus homohiochii, which grew in the culture medium containing 16% ethanol, were also identified. The isolated yeast strains were identified as Candida kefyr, Saccharomyces cerevisiae, Kluyveromyces marxianus var. lactis, Candida krusei and Candida valida.     

Genotypic and phenotypic characterization of lactic acid bacteria isolated from Italian ryegrass silage

Twenty-three lactic acid bacteria (LAB) isolated from three cultivars (Akiaoba, Nagahahikari and Tachiwase) of Italian ryegrass (Lolium multiflorum Lam.) silage were precisely characterized by a combination of phenotypic tests, genotypic 16S ribosomal DNA sequencing and rapid PCR-based analyses, focusing on their useful phenotypes for silage preparation as inoculants. We successfully identified both at the species and subspecies levels: phenotypically novel Lactococcus lactis subsp. lactis, Lactobacillus brevis, Lactobacillus coryniformis subsp. torquens, Lactobacillus curvatus, Lactobacillus plantarum subsp. plantarum, Lactobacillus sakei subsp. carnosus, Leuconostoc mesenteroides subsp. dextranicum and Pediococcus parvulus. This is the first report to elucidate the presence of Lactobacillus coryniformis ssp. torquens and Leuconostoc mesenteroides subsp. dextranicum in Italian ryegrass silages. Physiological and biochemical tests revealed that phenotypic characteristics are different among the different strains of the same species and subspecies, and that the isolates show unique and diverse phenotypes related to fermentation factors, such as available carbohydrates, optimal growth pH and temperature. These results suggest that, for various well-preserved silage preparations, the isolates may be useful in producing novel inoculants corresponding to their optimally climatic and ecological niches.     

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.     

The effect of selected microbial strains on internal milieu of broiler chickens after peroral administration

The aim of this work was to investigate the effect of probiotic preparation with multi-strains composition (Lactobacillus delbrueckii ssp. bulgaricus LAT 187, Lactobacillus acidophilus LAT 180, Lactobacillus helveticus LAT 179, Lactobacillus delbrueckii ssp. lactis LAT 182, Streptococcus thermophilus LAT 205 and Enterococcus faecium E 253) on internal milieu, antioxidant status and body weight of broiler chickens. Chickens were divided into three groups (C, control; E1 and E2, experimental groups). Experimental chickens received the probiotic preparation in drinking water. Probiotic preparation caused a significant increase of serum calcium and potassium content. In the group with higher dose of probiotic strains serum triglycerides level decreased. Total antioxidant status in groups with addition of probiotic strains showed higher values in comparison to control group. Serum albumin level was found to increase after consumption of probiotic preparation in group with higher dose of probiotic strains. Probiotic strains improved body weight in last observed weeks of feeding.     

New lactic acid bacterial strains from traditional Mongolian fermented milk products have altered adhesion to porcine gastric mucin depending on the carbon source

Attachment of lactic acid bacteria to the mucosal surface of the gastrointestinal tract is a major property of probiotics. Here, we examined the ability of 21 lactic acid bacterial strains isolated from traditional fermented milk products in Mongolia to adhere to porcine gastric mucin in vitro. Higher attachment was observed with Lactobacillus delbrueckii subsp. bulgaricus strains 6‐8 and 8‐1 than with Lactobacillus rhamnosus GG (positive control). Lactococcus lactis subsp. cremoris strain 7‐1 adhered to mucin as effectively as did strain GG. Heat inactivation decreased the adhesive ability of strains 6‐8 and 8‐1 but did not affect strain 7‐1. The adhesion of strains 6‐8, 7‐1 and 8‐1 was significantly inhibited when the cells were pretreated with periodate and trypsin, indicating that proteinaceous and carbohydrate‐like cell surface compounds are involved in the adhesion of these strains. The adhesion of strain 7‐1 was affected by the type of carbohydrate present in the growth medium, being higher with fructose than with lactose, galactose or xylose as the carbon source. The sugar content of 7‐1 cells grown on various carbohydrates was negatively correlated with its adhesive ability. We provide new probiotic candidate strains and new information regarding carbohydrate preference that influences lactic acid bacterial adhesion to mucin.     

Investigation of lactic acid bacterial strains for meat fermentation and the product's antioxidant and angiotensin‐I‐converting‐enzyme inhibitory activities

In the lactic acid bacteria (LAB) strains screened from our LAB collection, Lactobacillus (L.) sakei strain no. 23 and L. curvatus strain no. 28 degraded meat protein and tolerated salt and nitrite in vitro. Fermented sausages inoculated strains no. 23 and no. 28 showed not only favorable increases in viable LAB counts and reduced pH, but also the degradation of meat protein. The sausages fermented with these strains showed significantly higher antioxidant activity than those without LAB or fermented by each LAB type strain. Angiotensin‐I‐converting‐enzyme (ACE) inhibitory activity was also significantly higher in the sausages fermented with strain no. 23 than in those fermented with the type strain. Higher ACE inhibitory activity was also observed in the sausages fermented with strain no. 28, but did not differ significantly from those with the type strain. An analysis of the proteolysis and degradation products formed by each LAB in sausages suggested that those bioactivities yielded fermentation products such as peptides. Therefore, LAB starters that can adequately ferment meat, such as strains no. 23 and no. 28, should contribute to the production of bioactive compounds in meat products.     

Interaction of Campylobacter spp. and Human Probiotics in Chicken Intestinal Mucus

Campylobacter is the most common cause of bacterial food‐borne diarrhoeal disease throughout the world. The principal risk of human contamination is handling and consumption of contaminated poultry meat. To colonize poultry, Campylobacter adheres to and persists in the mucus layer that covers the intestinal epithelium. Inhibiting adhesion to the mucus could prevent colonization of the intestine. The aim of this study was to investigate in vitro the protective effect of defined commercial human probiotic strains on the adhesion of Campylobacter spp. to chicken intestinal mucus, in a search for alternatives to antibiotics to control this food‐borne pathogen. The probiotic strains Lactobacillus rhamnosus GG and Propionibacterium freudenreichii ssp. shermanii JS and a starter culture strain Lactococcus lactis ssp. lactis adhered well to chicken intestinal mucus and were able to reduce the binding of Campylobacter spp. when the mucus was colonized with the probiotic strain before contacting the pathogen. Human‐intended probiotics could be useful as prophylactics in poultry feeding for controlling Campylobacter spp. colonization.     

Identification of lactic acid bacteria isolated from corn stovers

One hundred and twenty‐six strains were isolated from corn stover in Henan Province, China, of which 105 isolates were considered to be lactic acid bacteria (LAB) according to Gram‐positive, catalase‐negative and mainly metabolic lactic acid product. Analysis of the 16S ribosomal DNA sequence of 21 representative strains was used to confirm the presence of the predominant groups and to determine the phylogenetic affiliation of isolates. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank type strains between 99.4% and 100%. The prevalent LAB, predominantly Lactobacillus (85.6%), consisted of L. plantarum (33.3%), L. pentosus (28.6%) and L. brevis (23.7%). Other LAB species as Leuconostoc lactis (4.8%), Weissella cibaria (4.8%) and Enterococcus mundtii (4.8%) also presented in corn stover. The present study is the first to fully document corn stover‐associated LAB involved in the silage fermentation. The identification results revealed LAB composition inhabiting corn stover and enabling the future design of appropriate inoculants aimed at improving the fermentation quality of silage.     

Identification and characterization of lactic acid bacteria isolated from mixed pasture of timothy and orchardgrass,and its badly preserved silage

In order to understand the relationship between lactic acid bacteria (LAB) species and silage fermentation, a total of 65 LAB strains isolated from mixed pasture of timothy (Phleum pratense L.) and orchardgrass (Dactylis glomerata L.), and its badly preserved silages were subjected to phenotypic and genetic analysis. According to these analyses, the isolates were divided into 13 groups, including Enterococcus gallinarum, Lactobacillus acidipiscis, L. coryniformis subsp. coryniformis, L. coryniformis subsp. torquens, L. curvatus, L. paraplantarum, L. plantarum subsp. argentoratensis, L. plantarum subsp. plantarum, L. sakei subsp. carnosus, Lactococcus garvieae, Lactococcus lactis subsp. cremoris, Leuconostoc pseudomesenteroides, Pediococcus acidilactici, Pediococcus pentosaceus, Weissella hellenica, Weissella paramesenteroides and Carnobacterium divergens. This is the first report to document that C. divergens, L. acidipiscis, L. sakei subsp. carnosus, L. garvieae, phenotypically novel L. lactis subsp. cremoris, E. gallinarum and W. hellenica are present in vegetative forage crops. L. plantarum group strains were most frequently isolated from the badly preserved silages. Some isolates showed a wide range of growth preferences for carbohydrate utilization, optimal growth pH and temperature in vitro, indicating that they have a high growth potential. These results are useful in understanding the diversity of LAB associated with decayed silage of timothy and orchardgrass.     

New lactic acid bacteria for skin health via oral intake of heat‐killed or live cells

Lactic acid bacteria play an essential role in the food industry in the manufacture of many fermented products (cheese, yogurt, fermented vegetables, etc.). Application of these organisms is now being extended to the area of health improvement, as their probiotic activities become known. Probiotics are defined as viable microorganisms that exert a beneficial effect on the health of the host when they are ingested in sufficient quantity. Lactic acid bacteria and bifidobacteria isolated from the human intestine are the most common probiotics used for human consumption. The development of new probiotics with new beneficial effects is eagerly awaited in the food industry. This review introduces Lactococcus, which are one of the genera of lactic acid bacteria and are mainly isolated from dairy products and fermented vegetables, as new probiotics, focusing especially on Lactococcus lactis H61, which improves skin status in Japanese women with oral intake of heat‐killed or live cells. The deduced mechanisms associated with the beneficial effects of strain H61 are also discussed.     

Characterization and application of lactic acid bacteria for tropical silage preparation

Strains TH 14, TH 21 and TH 64 were isolated from tropical silages, namely corn stover, sugar cane top and rice straw, respectively, prepared in Thailand. These strains were selected by low pH growth range and high lactic acid‐producing ability, similar to some commercial inoculants. Based on the analysis of 16S ribosomal RNA gene sequence and DNA‐DNA relatedness, strain TH 14 was identified as Lactobacillus casei, and strains TH 21 and TH 64 were identified as L. plantarum. Strains TH 14, TH 21, TH 64 and two commercial inoculants, CH (L. plantarum) and SN (L. rhamnosus), were used as additives to fresh and wilted purple Guinea and sorghum silages prepared using a small‐scale fermentation method. The number of epiphytic lactic acid bacteria (LAB) in the forages before ensilage was relatively low but the numbers of coliform and aerobic bacteria were higher. Sorghum silages at 30 days of fermentation were all well preserved with low pH (3.56) and high lactic acid production (72.86 g/kg dry matter). Purple Guinea silage inoculated with LAB exhibited reduced count levels of aerobic and coliform bacteria, lower pH, butyric acid and ammonia nitrogen and increased lactic acid concentration, compared with the control. Strain TH 14 more effectively improved lactic acid production compared with inoculants and other strains. © 2016 Japanese Society of Animal Science     

Fermentation quality and aerobic stability of mulberry silage prepared with lactic acid bacteria and propionic acid

The objective of this study was to examine the fermentation quality and aerobic stability of mulberry (Morus alba L.) silage prepared with lactic acid bacteria (LAB) and propionic acid (PA). The selected LAB strains Lactobacillus (L.) plantarum LC365281 (L1) and L. brevis LC365282 (L2), and commercial inoculant strains L. plantarum Gaofuji (GF) and L. buchneri Fresh (FR), and PA were used as additives for silage preparation. Silage treatments were designed as control, L1, L2, GF, FR, PA, PA + L1, PA + L2, PA + GF, or PA + FR. After 30 days of ensiling, the fermentation quality of silages treated with PA + L1 was improved, with a lower (p < 0.05) pH and NH₃‐N content than those of other treatments. During the aerobic exposure, the PA + LAB‐treated silages displayed an aerobic stability with stable pH value and lactic acid content. The results confirm that L. plantarum L1 and PA were the best additive combination for ensiling mulberry.     

In vitro safety assessments and antimicrobial activities of Lactobacillus rhamnosus strains isolated from a fermented mare's milk

Safety and probiotic characteristics such as antimicrobial activities of three Lactobacillus rhamnosus strains, FSMM15, FSMM22 and FSMM26, previously isolated as potential probiotics from fermented mare's milk were investigated. The three FSMM strains were susceptible to ampicillin, gentamycin, kanamycin, streptomycin, tetracycline and chloramphenicol, whereas they were resistant to erythromycin (minimal inhibitory concentration (MIC) = 4?8 µg/mL) and clindamycin (MIC = 4 µg/mL); bioconversion of bile salts, hemolytic activity and mucin degradation activity were negative; enzymatic activities of α‐chymotrypsin and β‐glucosidase were detected, but those of α‐galactosidase, β‐glucuronidase and N‐acetyl‐β‐glucosaminidase, were undetectable. Among the strains, strain FSMM15 was chosen as a safer probiotic candidate due mainly to the lack of plasminogen binding ability. Despite lower acid production of strain FSMM15 than others, its cell‐free culture supernatant inhibited growths of Salmonella Typhimurium LT‐2, Shigella sonnei , Listeria monocytogenes , and Escherichia coli O157 with comparable levels of ampicillin, suggesting a favorable aspect of strain FSMM15 as a probiotic strain.     

Major microbiota of lactic acid bacteria from Matsoni, a traditional Georgian fermented milk

A total of 26 samples of Matsoni were collected in Georgia. From these samples 80 strains of lactic acid cocci and 173 strains of lactobacilli were isolated. The number of lactic acid bacteria varied between 10⁵ and 10¹⁰ colony forming unit (CFU)/mL. All the isolated lactic acid bacteria were thermophilic bacteria that could grow at 45°C. The predominant lactic acid bacteria were Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus in 25 samples of Matsoni, while Lactobacillus helveticus was also a predominant species, together with the two previous species in one sample of Matsoni. We showed there was diversity in both S. thermophilus and L. delbrueckii spp. bulgaricus at the strain level by randomly amplified polymorphic DNA analysis.     

Effects of a probiotic intervention in acute canine gastroenteritis – a controlled clinical trial

Objectives : To evaluate the effect of a probiotic product in acute self-limiting gastroenteritis in dogs. Methods : Thirty-six dogs suffering from acute diarrhoea or acute diarrhoea and vomiting were included in the study. The trial was performed as a randomised, double blind and single centre study with stratified parallel group design. The animals were allocated to equal looking probiotic or placebo treatment by block randomisation with a fixed block size of six. The probiotic cocktail consisted of thermo-stabilised Lactobacillus acidophilus and live strains of Pediococcus acidilactici, Bacillus subtilis, Bacillus licheniformis and Lactobacillus farciminis. Results : The time from initiation of treatment to the last abnormal stools was found to be significantly shorter (P = 0·04) in the probiotic group compared to placebo group, the mean time was 1·3 days and 2·2 days, respectively. The two groups were found nearly equal with regard to time from start of treatment to the last vomiting episode. Clinical Significance : The probiotic tested may reduce the convalescence time in acute self-limiting diarrhoea in dogs.     

A novel strategy to select Bifidobacterium strains and prebiotics as natural growth promoters in newly weaned pigs

The study addresses the first two weeks of piglet weaning and is aimed at i) identifying new probiotic strains of Bifidobacterium spp. and non-digestible oligosaccharides (NDO) able to increase the level of autochthonous bifidobacteria in the gut, and ii) finding the best probiotic doses, and probiotic-NDO combinations for piglet growth and gut health.Initially, on a total of 60 pigs we administered 12 strains of 4 bifidobacteria species taken from our collection of strains of different ecological origin. We found the most promising probiotic candidates to be two strains of B. animalis subsp. lactis (M 354 and Ra 18) and one strain of B. choerinum (Su 891), as these led to an increase in the number of viable bifidobacteria in the caecum. In a second trial, a different set of 64 piglets were fed a diet containing a galacto-oligosaccharide (1%) from milk whey, and two fructo-oligosaccharides (4%) from chicory inulin and sugar beet (SbFOS). There was virtually no effect on the hindgut microbiota, except for SbFOS at 4% which tended to increase the endogenous bifidobacteria. Then, in a third trial employing 128 weaning pigs, the probiotic strains Ra 18 and Su 891 were tested individually, at different doses (0; 10⁷; 10⁹; 10¹¹ per pig per day) and combined with 0% or 2% SbFOS supplementation. It was found that Ra 18 improved the live weight growth linearly, while the Su 891 did not. The FOS supplementation showed no interaction with the Ra 18 or Su 891 probiotics for growth performance and no effect on this response criterion.Our selection procedure has highlighted that B. animalis subsp. lactis Ra 18, at 10¹¹ CFU per pig per day, appears to be the best probiotic choice for improved growth performance in weaning piglets.     

Microstructure of the small intestine in broiler chickens fed a diet with probiotic or synbiotic supplementation

The aim of the study was to determine the effect of dietary supplementation of a probiotic and a synbiotic on the morphometric parameters of the small intestine of broiler chickens. The experiment was conducted on three hundred sixty, one‐day‐old female Ross 308 chicks, which were randomly selected from 20,000 birds and divided into three treatment groups (n = 120) with ten replicates per treatment. The control group (C) was fed a commercial diet, the probiotic group (PRO) was fed the same diet with an added 1% of the probiotic Lavipan® (Lactococcus lactis, Carnobacterium divergens, Lactobacillus casei, Lactobacillus plantarum and Saccharomyces cerevisiae), and the synbiotic group (SYN) was fed the commercial diet with an added synbiotic: 0.8% of the prebiotic RFO (extracted from lupin seeds) and 1% Lavipan®. According to the manufacturer's data, apart from the typical probiotic action,microorganisms contained in the preparation release anti‐bacterial substances (hydrogen peroxide and bacteriocins) and, therefore, are antagonistic towards pathogenic bacteria present in the gut of animals. Supplementation took place in the first seven days of rearing, and all birds had ad libitum access to water and feed during the 42 days of the experiment. On the last day, all birds were slaughtered and samples from three segments of the small intestine were taken. Villi area, height, width and crypt depth ratios were read using Multiscan software. Synbiotic supplementation increased the BWG of broilers from first to tenth day of rearing, compared to the control group. The PRO group had improved villi morphometric parameters of the duodenum. In the jejunum and ileum, both bioactive substances improved villus width and villus surface area. Crypts were deeper in the small intestine of birds supplemented with bioactive substances, which allows greater renewal of the villi. As expected, the intestinal morphometric parameters of broiler chickens benefited from bioactive substance supplementation.     

Effects of probiotic lactic acid bacteria on growth performance,carcass characteristics,hematological indices,humoral immunity,and IGF-I gene expression in broiler chicken

The effects of screened lactic acid bacteria strains were evaluated on growth performance, humoral immunity, and IGF-1 gene expression in broiler chickens. The three dietary groups of negative control fed basal diet, the native LAB probiotic group (NP), and PrimaLac commercial LAB probiotic (PC) were studied. The results revealed that NP and PC diets significantly improved feed conversion ratio and increased body weight, as well as relative weight of carcass compared with group fed NC diet (P?<?0.05). Lymphocyte level was significantly increased in birds fed NP and PC (P?<?0.01), while serum triglycerides and total cholesterol levels were significantly decreased compared with the NC (P?<?0.05). Significant increases were observed in antibody titers against Newcastle disease virus of vaccinated birds (P?<?0.03), and morphological analysis of ileum revealed significant increases (P?<?0.05) in the villus height and villus height/crypt depth in birds fed NP and PC compared with the NC. The dietary significantly increased Lactobacillus spp. (P?<?0.05), while Escherichia coli (P?<?0.04) populations were significantly decreased, and also, the expression of IGF-1 gene in liver tissue of broilers fed NP and PC was significantly increased compared with the NC (P?<?0.05). These results indicated that the identified native LAB strains can be used commercially as a low-cost probiotic in poultry industry of Iran.

     

Classification of lactic acid bacteria isolated from chigee and mare milk collected in Inner Mongolia

One hundred and seventeen isolates from chigee and 191 isolates from mare milk collected in Inner Mongolia were classified into six and 10 groups, respectively, based on the protein profiles obtained by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). A dendrogram on the basis of the molecular sizes of the protein bands was drawn and the similarity among the groups ranged from 96% to 44%. Representative isolates from all groups were identified by 16S rDNA sequencing and a phylogenetic tree was drawn. Furthermore, the results of carbohydrate fermentation tests of all groups were compared with those obtained by 16S rDNA sequencing and SDS‐PAGE analyzes. The isolates from chigee consisted of mainly Lactobacillus sp., Lactobacillus (Lb.) plantarum, Lb. pentosus and Lactococcus (Lc.) lactis ssp. cremoris were isolated at the rates of 48, 33 and 19%, respectively. The isolates from mare milk consisted of coccus, namely, Leuconostoc (Leuc.) mesenteroides, Leuc. pseudomesenteroides, Lc. garvieae, Lc. lactis ssp. lactis, Streptococcus (Sc.) parauberis and Enterococcus (Ec.) faecium. The isolation rates were 45, 19, 7, 8, 16 and 6%, respectively. The results demonstrated that the species isolated from chigee were different from those in mare milk.