Isolation and Characterization of Yeasts from Rumen Fluids for Potential Use as Additives in Ruminant Feeding.

Saccharomyces cerevisiae is a yeast strain often used to improve the feed quality of ruminants. However, S. cerevisiae has limited capacity to provide biomass when inoculated with carbon sources and a low ability to produce cellulase enzymes. Here, we hypothesized that yeast in the rumen produces a large amount of biomass and could release cellulase enzymes to break down fiber content. Therefore, the aim of this study was to screen, isolate and identify yeast from the rumen fluids of Holstein Friesian steers and measure the efficiency of biomass production and cellulase activity. A fermentation medium containing sugarcane molasses as a carbon source and urea as a nitrogen source was optimized. Two fistulated-crossbred Holstein Friesian steers averaging 350 ± 20 kg body weight were used to screen and isolate the ruminal yeast. Two experiments were designed: First, a 12 × 3 × 3 factorial was used in a completely randomized design to determine biomass and carboxymethyl cellulase activity. Factor A was the isolated yeast and S. cerevisiae. Factor B was sugarcane molasses (M) concentration. Factor C was urea (U) concentration. In the second experiment, potential yeasts were selected, identified, and analyzed for 7 × 4 factorial use in a completely randomized design. Factor A was the incubation times. Factor B was the isolated yeast strains, including codes H-Khon Kaen University (KKU) 20 (as P. kudriavzevii-KKU20), I-KKU20 (C. tropicalis-KKU20), and C-KKU20 (as Galactomyces sp.-KKU20). Isolation was imposed under aerobic conditions, resulting in a total of 11 different colonies. Two appearances of colonies including asymmetric colonies of isolated yeast (indicated as A, B, C, E, and J) and ovoid colonies (coded as D, F, G, H, I, and K) were noted. Isolated yeast from the rumen capable of providing a high amount of biomass when inoculant consisted of the molasses 15% + urea 3% (M15 + U3), molasses 25% + urea 1% (M25 + U1), molasses 25% + urea 3% (M25 + U3), and molasses 25% + urea 5% (M25 + U5) when compared to the other media solution (p < 0.01). In addition, 11 isolated biomass-producing yeasts were found in the media solution of M25 + U1. There were 4 isolates cellulase producing yeasts discovered in the media solution of M25 + U1 and M25 + U5 whereas molasses 5% + urea 1% (M5 + U1), molasses 5% + urea 3% (M5 + U3), molasses 5% + urea 5% (M5 + U5), molasses 15% + urea 1% (M15 + U1), molasses 15% + urea 3% (M5 + U3), and M25 + U3 were found with 2, 3, 1, 2, 1, and 2 isolates, respectively. Ruminal yeast strains H-KKU20, I-KKU20, and C-KKU20 were selected for their ability to produce biomass. Identification of isolates H-KKU20 and I-KKU20 revealed that those isolates belonged to Pichia kudriavzevii-KKU20 and Candida tropicalis-KKU20 while C-KKU20 was identified as Galactomyces sp.-KKU20. Two strains provided maximum cell growth: P. kudriavzevii-KKU20 (9.78 and 10.02 Log cell/mL) and C. tropicalis-KKU20 (9.53 and 9.6 Log cells/mL) at 60 and 72 h of incubation time, respectively. The highest ethanol production was observed in S. cerevisiae at 76.4, 77.8, 78.5, and 78.6 g/L at 36, 48, 60, and 72 h of incubation time, respectively (p < 0.01). The P. kudriavzevii-KKU20 yielded the least reducing sugar at about 30.6 and 29.8 g/L at 60 and 72 h of incubation time, respectively. The screening and isolation of yeasts from rumen fluids resulted in 11 different yeasts being obtained. The potential yeasts discovered in the rumen fluid of cattle were Pichia kudriavzevii-KKU20, Candida tropicalis-KKU20, and Galactomyces sp.-KKU20. P. kudriavzevii-KKU20 had higher results than the other yeasts in terms of biomass production, cellulase enzyme activity, and cell number.

Author Correction: Diversity in gut bacterial community of school-age children in Asia.

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Thermotolerant Yeast Kluyveromyces marxianus Reveals More Tolerance to Heat Shock than the Brewery Yeast Saccharomyces cerevisiae.

The thermotolerant yeast Kluyveromyces marxianus, growing at high temperature (45℃) , showed stronger survival under heat shock at 50℃ than the brewing yeast Saccharomyces cerevisiae, which was unable to grow at 45℃. The survival rate of K. marxianus decreased to 10% during heat shock at 50℃ for 20 min, and to less than 0.01% at 60℃ for 20 min. Cells with damaged cellular membranes were infrequently observed at 50℃ and had decreased significantly from heat shock at 60℃. The metabolic activity of K. marxianus was retained at 50℃, whereas that of S. cerevisiae was not. The trehalose content of K. marxianus was approximately two times that of S. cerevisiae. These results suggest that K. marxianus protects itself from heat shock-induced damage through the use of trehalose (a protective molecule in S. cerevisiae) as well as other different factors.

Heterophil Phagocytic Activity Stimulated by Lactobacillus salivarius L61 and L55 Supplementation in Broilers with Salmonella Infection.

Newborn chicks are susceptible to Salmonella enterica serovar Enteritidis (SE). The objective of this study was to evaluate the effect of Lactobacillus probiotic isolated from chicken feces on heterophil phagocytosis in broiler chicks. A total of 150 newborn broiler chicks were divided into 5 groups (30 chicks per group) as follows: group 1 (normal control), given feed and water only, group 2 (positive control) given feed, water and SE infection, group 3 (L61 treated) given feed, water, SE infection followed by Lactobacillus salivarius L61 treatment, group 4 (L55 treated) given feed, water, SE infection followed by L. salivarius L55 treatment, and group 5 given feed, water, SE infection followed by L. salivarius L61 + L55 combination treatment. After SE infection, L. salivarius treatment lasted for 7 days. The results showed that L. salivarius L61 and L. salivarius L55 treatment, either alone or combination of both, increased the survival rate after SE infection, and upregulated heterophil phagocytosis and phagocytic index (PI). Conversely, chick groups treated with Lactobacillus showed lower SE recovery rate from cecal tonsils than that of the positive control group. The PI values of the chicken group with SE infection, followed by the combination of L. salivarius L61 and L. salivarius L55 were the highest as compared to either positive control or normal control group. Two Lactobacillus strains supplementation group showed significantly (p<0.05) higher PI value at 48 h than 24 h after treatment.

Distinct gut microbiota of healthy children from two different geographic regions of Thailand.

In Thailand, food consumption by people from each region is different. This can be an important environmental factor which shapes the gut microbiota further affecting their health. This study aimed to use quantitative PCR (qPCR) to investigate the intestinal microbial community in 60 healthy children (aged 8-11 years) living in specific areas, namely central (CT) and northeastern (NE) Thailand where each region has its own typical food consumption. The children from NE had significantly higher consumption frequency of meat (chicken and beef), a wide variety of carbohydrate sources (noodle, fermented rice and sweet potato) including vegetables and fruit, while in CT, there was a significant preference for rice, breakfast cereal and cow milk. The qPCR analysis resulted in significantly higher abundance of lactobacilli, Clostridium coccoides-Eubacterium rectale, Clostridium leptum, Prevotella and Bacteroides fragilis in children from the NE region. However, no significant difference in the count of Bifidobacterium spp., Enterobacteriaceae and methanogens was observed. Considering the correlation of food sources and microbial groups, the consumption frequency of vegetables showed a moderately positive correlation coefficient of 0.42 and 0.34 to the Lactobacillus group (P = 0.001) and the Prevotella group (P = 0.008), respectively, while a diet of fish and beef showed a moderately negative correlation coefficient of -0.41 (P = 0.001) and -0.33 (P = 0.09) to Bifidobacterium spp., respectively. Our results suggested that high frequency consumption of varieties of carbohydrates, protein sources, fruits and vegetables by the NE children promoted a high abundance of bacterial species in the phyla Firmicutes and Bacteroidetes.

Diversity in gut bacterial community of school-age children in Asia.

Asia differs substantially among and within its regions populated by diverse ethnic groups, which maintain their own respective cultures and dietary habits. To address the diversity in their gut microbiota, we characterized the bacterial community in fecal samples obtained from 303 school-age children living in urban or rural regions in five countries spanning temperate and tropical areas of Asia. The microbiota profiled for the 303 subjects were classified into two enterotype-like clusters, each driven by Prevotella (P-type) or Bifidobacterium/Bacteroides (BB-type), respectively. Majority in China, Japan and Taiwan harbored BB-type, whereas those from Indonesia and Khon Kaen in Thailand mainly harbored P-type. The P-type microbiota was characterized by a more conserved bacterial community sharing a greater number of type-specific phylotypes. Predictive metagenomics suggests higher and lower activity of carbohydrate digestion and bile acid biosynthesis, respectively, in P-type subjects, reflecting their high intake of diets rich in resistant starch. Random-forest analysis classified their fecal species community as mirroring location of resident country, suggesting eco-geographical factors shaping gut microbiota. In particular, children living in Japan harbored a less diversified microbiota with high abundance of Bifidobacterium and less number of potentially pathogenic bacteria, which may reflect their living environment and unique diet.

Low cost medium for spore production of Bacillus KKU02 and KKU03 and the effects of the produced spores on growth of giant freshwater prawn (Macrobrachium rosenbergii de Man).

In order to extend the shelf life of 2 high potential Bacillus probiotic isolates which were Bacillus KKU02 and Bacillus KKU03, the spore forms of these 2 Bacillus isolates were studied for using as probiotic instead. The low cost medium for spore production of these 2 Bacillus isolates was examined in order to produce probiotic spores for feeding the shrimps. It was found that cassava at 100 g L(-1) and supplemented with 20.0 g L(-1) dextrose, 0.1 g L(-1) MgSO4 and 2.0 g L(-1) (NH4)2SO4 showed the highest spore concentration at about 1 x 10(8) CFU mL(-1). The effects of feeding these 2 Bacillus spores on the growth of giant freshwater prawns were further examined. The spores of Bacillus KKU02 and Bacillus KKU03 (-10(7) spore mL(-1)) in pure and mixed culture forms were mixed with commercial prawn feed (200 mL kg(-1)) to give six feed treatments. Body length and weight of the prawns in mixed spore culture tanks after rearing for 90 days (13.5 cm and 59.8 g, respectively) were significantly higher (p = 0.05) than other treatments. The treated prawns were further challenged with Aeromonas hydrophila for 7 days. The percentages of survival after the challenge in the prawns fed with the mixed spores (46.8%) were also found significantly higher (p = 0.05) than others groups, except the mixed live cell treatment (60%). These results indicated that the spores of Bacillus KKU02 and Bacillus KKU03 had a high potential for using as commercial probiotics.

Thermotolerant bacteriocin-producing lactic acid bacteria isolated from thai local fermented foods and their bacteriocin productivity.

Twenty-one samples of Thai local fermented foods were screened for thermotolerant bacteriocin-producing lactic acid bacteria. From 529 isolates of lactic acid bacteria, 121 isolates were able to inhibit the growth of certain bacterial strains. Of these 121 isolates, only 11 produced antibacterial agents that were capable of inhibiting the growth of multiple bacterial strains in a liquid medium. One strain (KKU 170) of these 11 isolates produced an antibacterial agent that could strongly inhibit the growth of selected strains of gram-positive bacteria including Listeria sp. The antibacterial agent produced by the strain KKU 170 was identified as a bacteriocin since it was inactivated by proteinase K treatment. The strain KKU 170 was identified as Pediococcus acidilactici by both biochemical tests and molecular biological techniques. Optimal production of bacteriocin by the strain KKU 170 was found in culture medium containing 0.2% glucose, at an initial culture pH of 6.5, and temperature of 45 ºC. The maximum bacteriocin activity (1600 AU ml(-1)) was reached at the late exponential phase of growth and displayed primary metabolite production. The partially purified bacteriocin of the strain KKU 170 was tolerant to heat treatment at 121 ºC for 30 min.

Effect of feeding Bacillus sp. as probiotic bacteria on growth of giant freshwater prawn (Macrobrachium rosenbergii de Man).

The effects of feeding two Bacillus spp. isolated from the intestine of the giant freshwater prawn on the growth of Giant Freshwater Prawns (Macrobrachium rosenbergii de Man) was examined. The isolated Bacillus KKU02 and Bacillus KKU03 (approximately 10(7) CFU mL(-1)) were mixed into commercial prawn feed (200 mL kg(-1)). After rearing shrimp with the bacteria in four feed treatments, (Bacillus KKU03, Bacillus KKU02, mixed culture and control groups) for 120 days, body length and weight of the prawns in mixed culture tanks were significantly higher (p = 0.05) than in the control tanks (7.48 cm and 3.32 g, vs 6.6 cm and 2.1 g, respectively). Both isolates were found to produce amylase and protease. The stabilities of the single Bacillus sp., mixed culture and commercial probiotic in the feeds were examined during storage at 4 degrees C and room temperature. The percentage viability of Bacillus KKU02, Bacillus KKU03 and mixed culture stored at room temperature declined dramatically to 2.54, 21.88 and 10.92% within 2 weeks, respectively. At 4 degrees C however, the percentage viability of the tested probiotics reduced slowly. The survival of the commercial probiotics was the same at both temperatures about 50% after 70 days' storage.

Immunological adjuvant activities of saponin extracts from the pods of Acacia concinna.

Pods of Acacia concinna (Leguminosae) contain several saponins. In this study, four saponin fractions which were acetone fraction (AAC), aqueous fraction (WAC), hydromethanolic fraction (HAC) and methanolic fraction (MAC) were generated and their haemolytic activities and surface activities were determined in comparison with quillaja saponin (QS). There were no significant differences between the haemolytic activities of MAC and QS. However, the surface tensions of MAC was significantly lower than QS (p < 0.001). Furthermore, the immunomodulatory effect and the adjuvant potential of MAC on the cellular and humoral immune response of BALB/c mice against ovalbumin were investigated. The splenocyte proliferations induced by MAC were significantly higher than QS at the concentrations of 200, 400, 800 and 1000 microg/ml (p < 0.05). BALB/c mice were immunized subcutaneously either with OVA 20 microg alone or with OVA 20 microg combining with QS (10 microg) or MAC (10 and 40 microg). Ten days after the second immunization, concanavalin A (Con A)-, pokeweed mitogen (PWM)-, and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. The results suggested that MAC (40 microg) could activate T and B cells. In addition, OVA-specific IgG, IgG1 IgG2a and IgG2b antibody levels in serum were significantly enhanced by MAC (40 microg) as compared with OVA control group (p < 0.001). This finding suggested that MAC might be effect on Th1 and Th2 helper T cells. In conclusion, the results indicated that MAC at a dose of 40 microg could be used as vaccine adjuvant to increase immune responses.

Growth characteristics of fusants by protoplast fusion between the thermotolerant yeast, Kluyveromyces marxianus, and the starch-assimilating yeast, Schwanniomyces occidentalis.

Intergeneric fusants were obtained by protoplast fusion between the thermotolerant yeast, Kluyveromyces marxianus, and the starch-assimilating yeast, Schwanniomyces occidentalis. Two thermotolerant fusants growing at 40 degrees C were screened on the medium containing soluble starch. These fusants showed weak growth in a soluble starch medium and the production of a little amylase. The carbon source assimilation and the chromosome composition of the fusants were similar to those of the K. marxianus parent. However, a chromosomal difference from K. marxianus was recognized in the fusants. These results show the possibility that the fusants are amylase-producing strains rearranged from K. marxianus.