As shown in Table 4, after 60 d of storage, all LAB-inoculated samples showed significantly lower pH values than no-additive groups, reflecting higher lactic acid content (P < 0.05). The number of LAB were measured by plate count on lactobacilli de Man, Rogosa, Sharpe (MRS) agar incubated at 30°C for 48 h under anaerobic conditions (DG 250/min MACS; Don Whitley Science; England). In contrast to cocci, lactobacilli are important promoters of lactic acid fermentation for a longer time, particularly during the latter stage of ensiling [21,22]. lactis subsp. Data Availability: All relevant data are within the paper. The numbers of LAB in LAB-treated samples showed significantly higher than the control (P < 0.05). The organic acid contents were measured by high performance liquid chromatography (HPLC, 1200series; Agilent; American). Henan Provincial Key Laboratory of Ion Beam Bio-engineering, Zhengzhou University, Henan, China, Affiliation The Group A to H strains were identified as Lactobacillus (L.) plantarum subsp. Three repetitions of each sample were analyzed. Morphological, physiological and biochemical tests of LAB morphology and Gram-staining response were examined after 24 h of incubation on MRS agar. Performed the experiments: DL. Although they are part of the normal human gastrointestinal and vaginal flora, they can also be occasional human pathogens. Thus, other phylogenetic analysis methods were required, such as an analysis of the recA gene, which is more variable and has been proposed as a phylogenetic marker for distantly related species [12]. In this study, the selection was performed at the strain and not the species level. The Group C and F strains were tolerant to low pH 3.5. https://doi.org/10.1371/journal.pone.0121967.t001, https://doi.org/10.1371/journal.pone.0121967.t002. This metabolism takes place in an aerotolerant environment, in which oxygen is not present (11). In a study performed using the SIV model of AIDS (a version of the virus that is found only in primates and not humans), inoculation of intestinal loops with Lactobacillus plantarum showed a rapid anti inflammatory response to SIV infected macaques suggesting a reversal of the responses cause by a disruption in the  IL-1β pathway that is disrupted as a result of the virus.(12). garvieae (15.2%), E. faecium (9.1%) and Lc. The comparison of the fermentation quality with respect to the control demonstrated that the LAB-treated silages presented lower (P < 0.05) pH values and a higher (P < 0.05) content of lactic acid; in addition, among the four groups, the R 465-treated silages had the lowest pH values and the highest content of lactic acid, and propionic and butyric acids were not detected in any of the treated silages. Strain R421 in Group F can be assigned to Lc. No, Is the Subject Area "Phylogenetic analysis" applicable to this article? From this investigation, eight LAB species were found to be present in rice silages, and most of these species belong to cocci. HIV is an immunodeficiency virus that targets the CD4+ T cells that are an important contributor to immune response especially in the intestinal epithelial layer. plantarum JCM 1149T and L. plantarum subsp. plantarum. During the process of silage fermentation, LAB involved in adequate acidification play an important role in the production of higher-quality silage, and different types of LAB exhibit different effects on the silage quality [7]. lactis was the most closely related species. plantarum), R 427 (L. plantarum subsp. Yes The PCR mixture and amplifications were performed as described by Torriani et al [13]. A selected strain, namely L. casei R 465, was found to be able to grow under low pH conditions and to improve the silage quality with low pH and a relatively high content of lactic acid. In this study, after 60 days of storage, the three inoculated strains exerted different effects on the fermentation quality of the paddy rice silages. As shown in Figs. This drops the pH and makes a desirable environment for acetic acid bacteria to grow and continue the fermentation process. Wrote the paper: KN. Sequence similarity searches were performed using the DNA Database of Japan (DDBJ) and the Basic Local Alignment Search Tool (BLAST). The samples (10g) were blended with 90ml of sterilized water, and serially diluted from 10-1 to 10-5 in sterilized water. Wet silage (10g) was homogenized with 90ml sterilized distilled water. Lactic acid is usually the main reason for low pH in high quality silage. [27] found that inoculation homofermentative LAB may decrease the amount of gas production and DM loss by inhibiting the clostridia and aerobic bacteria, and Carvalho et al. Growth at pH 3.0, 3.5, 4.0, 4.5 and 7.0 was observed in MRS broth after incubation at 30°C for 7 days. AB921218, AB921219, AB921220, AB921221, AB921222, AB921223, AB921224 and AB921225 for the strains R 413, R 419, R 420, R 421, R 422, R 423, R 442 and R 465, respectively. DM loss during the fermentation constitutes the main problems in the ensilage of the forage and the majority of these losses were most likely due to the fermentation process with remainder due to plant and microbial respiration. Punctiform (pointy), convex, entire margin colony morphology. garvieae, E. faecium and Lc. The Group C strain R 413 was closely related to the Ln. In this regard, the present results suggest that it is necessary to inoculate paddy rice silage with excellent LAB, particularly lactobacilli, to increase the initial load of the inoculated LAB. Animal Physiology and Nutrition Division, National Institute of Livestock and Grassland Science (NILGS), Ibaraki, Japan. The lactic acid content and acetic acid content in the uninoculated samples were higher in the current study compared with the results reported by Tohno et al [3]. here. However, the other strains showed homofermentative products. No, Is the Subject Area "Yeast" applicable to this article? Silages were prepared using a small scale system, approximately 100g portions of forge material chopped into about 20-mm length and packed into plastic bags (N-9, Asahi Kasei Co., Ltd., Japan). garvieae, and this finding was supported with a bootstrap value of 100%. plantarum (species ratio: 8.1%), L. casei (5.1%), Leuconostoc (Ln.) Salt tolerance of LAB was tested in MRS broth containing 3.0 and 6.5% NaCl at 30°C for 2 days. Lactobacillus plantarum  is incredibly diverse in its microbial habitat. plantarum, L. plantarum subsp. lactis subsp. Lactobacillus plantarum can perform these transformations because it can bind DNA and uptake that DNA. Lactobacillus plantarum are heterofermentative bacteria, meaning they produce both lactic acid and ethanol as metabolic byproducts. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Edit them in the Widget section of the, How microbes create our favorite delicacies, Kluyveromyces marxianus in Chocolate Fermentation, Lactobacillus acidophilus’s Duty in Sourdough Production, Chocolate Fermentation: Lactobacillus Fermentum, Pseudomonas fluorescens in Coffee Fermentation, La Colombe Executive Baker Talks Sourdough. Physiological and biochemical test results of Lactobacillus The physiological and biochemical test results of Lactobacillus were showed in Tables 1-3 , the stain was identified as Lactobacillus plantarum . With an increase in the population of China and improvements in the living standards, the resulting growth in the demand for animal products has resulted in increased utilization of forage crops, such as paddy rice. Competing interests: The authors have declared that no competing interests exist. For more information about PLOS Subject Areas, click L. = Lactobacillus. [10] evaluated the effect of different LAB species on silage quality and observed that the effect of the inoculant is more related to the strain than the species, and Tohno et al. Growth at different temperatures was observed in MRS broth after incubation at 5 and 10°C for 14 days, and at 45 and 50°C for 7 days. The bar indicates 1% sequence divergence. Among them, 27 (R 381-R 407), 21 (R 408–428), 21 (R 429–449) and 30 (R 450–479) LAB strains were isolated at the 3d, 7d, 30d and 60d of ensiling, respectively. Change ), You are commenting using your Google account. Pure cultures were grown on MRS agar at 30°C for 24 h, and then the purified strains were stored at -80°C in nutrient broth (Difco) for further examination. No, Is the Subject Area "Fermentation" applicable to this article? This analysis resulted in the delineation of eight groups of isolates. Ninety-nine of the presumptive LAB strains were characterized using phenotypic characteristics (Table 1) and sugar fermentation assays with API 50 CH strips (Table 2). First, the ninety-nine LAB strains were cultivated in MRS broth for 24h at 30°C. Change ), You are commenting using your Twitter account. Silages were prepared using a small scale system, approximately 100g portions of forage material chopped into about 20-mm length and packed into plastic bags (N-9, Asahi Kasei Co., Ltd., Japan). plantarum subsp. The bags were sealed with Sharp Vacuum Seal/Package (SQ-202, Sharp Co., Ltd., Japan), and the plastic bags were stored in a room at ambient temperature. PLoS ONE 10(3): Contributed reagents/materials/analysis tools: YC.