South Indian Journal of Biological Sciences 2015; 1(1); 1-6. Online ISSN: Introduction

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1 South Indian Journal of iological Sciences 2015; 1(1); 1-6 Online ISSN: Growth and metabolite profile of Pediococcus pentosaceus and Lactobacillus plantarum in different juice Soundarrajan Ilavenil, Srisesharam Srigopalram, Hyung Soo Park, Ki hoon hoi* Grassland and forage division, National Institute of Animal Science, RA, Seonghwan-Eup, heonan-si, hungnam, , Republic of Korea orresponding Author Ki hoon hoi Tel: Fax: manuscript details article History: Received 1 April 2015 Revised 5 May 2015 Accepted 4 June 2015 Published 25 June 2015 Keywords: Lactic acid bacteria K-23 and K-24 growth and metabolites silage abstract The lactic acid bacteria (LA) are much important for food processing industry and used as inoculants for silage preparation. The aim of the study was to isolate LA from Italian ryegrass and investigates their growth and metabolite profiles in barley, corn, and Italian rye grass juice. We isolated number of the LA, among these two isolates exhibited potent antifungal activity, and named as K-23 and K-24. iochemical and 16S rrna results revealed that the K-23 and K-24 as a Pediococcus pentosaceus and Lactobacillus plantarum respectively. The K-23 and K-24 showed significant growth in barley, corn and Italian ryegrass and MRS broth as compared with standard strains (KA and KA-91067). Also, K- 23 and K-24 produced the significant amount of lactic acid, succinic acid, and acetic acid. From the results, we concluded that the K-23 and K-24 could be useful for making quality silage from different grass and plants. 1. Introduction The lactic acid bacteria (LA) are industrially important microbes used in worldwide especially in industrial food fermentations (Arasu et al., 2013a). Primary responsible for this fermentation is due the production lactic acid from available carbon sources. It also stimulates rapid acidification of the food-raw materials. It is a key parameter in the preservation of these products. Also, LA has the ability to contribute the other characteristics such as flavor, texture and nutrition. Silage is a commonly preserved forage crop and grass in worldwide. The silage preservation is based on the production of sufficient acid to inhibit the undesirable microbial activity under the anaerobic conditions (ai 1999). Lactic acid bacteria utilize the carbohydrate to produce lactic acid (Arasu et al., 2013b). It is primary acid responsible for reducing the ph in the silage. Plants are having number of lactic acid bacteria, but it is hetro-fermentative bacteria (ai et al., 1998; ai 1999). The concept of using microbial inoculants for silage preparation involves adding fast growing homofermentative LA to dominate the fermentation, which provides high-quality silage (Weinberg 1996; Weinberg et al., 2003). Some of the commonly used homofermentative LA in silage preparations were Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Pediococcus acidilactici and Enterococcus faecium. Also, hetero-fermentative LA species, such as Lactobacillus brevis, Lactobacillus fermentum and Lactobacillus reuteri were developed as silage additives (Georgieva et al., 2008; Ávila et al., 2010). For silage preparation, the lactic acid bacteria must be growing fast and need to produce higher amount of lactic acid (Arasu et al., 2014a, Arasu et al., 2014b; Arasu et al., 2014c; Arasu et al., 2014d). Therefore, we plan to isolate the suitable lactic acid bacteria from Italian ryegrass and investigate their growth and metabolites profile in the barley, corn Italian rye-grass and MRS broth under different conditions. 2. Material and methods 2.1. Sample collection The Italian rye-grass forage crop was collected from heonan, South Korea in sterile propylene bags. One gram of sample was mixed with 10 ml of sterile water and made a serial dilution up to Then, the plates 1

2 A Fig. 1. Growth profile of K-23 and K-24 in barley, corn, Italian rye-grass and MRS broth under the aerobic condition. A-arley, -orn, -Italian rye-grass, - MRS. A Fig. 2. Growth profile of K-23 and K-24 in barley, corn, Italian rye-grass and MRS broth under the micro-aerobic condition. A-arley, -orn, -Italian rye-grass, - MRS. 2

3 A Fig. 3. Growth profile of K-23 and K-24 in barley, corn, Italian rye-grass and MRS broth under the anaerobic condition. A-arley, -orn, -Italian rye-grass, - MRS. A Fig. 4. Lactic acid productions by P. pentosaceus K-23 and L. plantarum K-24 in different juice. A- aerobic-k-23, -micro-aerobic-k-23, - aerobic-k-23, - micro-aerobic-k-24. 3

4 A Fig. 5. Succinic acid production by P. pentosaceus K-23 and L. plantarum K-24 in different juice. A- aerobic-k-23, -micro-aerobic-k-23, - aerobic-k-23, - micro-aerobic-k-24. A Fig. 6. Acetic acid productions by P. pentosaceus K-23 and L. plantarum K-24 in different juice. A- aerobic-k-23, -micro-aerobic-k-23, - aerobic-k-23, - micro-aerobic-k-24. 4

5 were incubated at 30 ± 2 for 24 to 48 h. Maximum biological potentials were displayed in K-23 and K- 24. Therefore, we selected for further analysis iochemical and physiological characterization of - K-23 and K-24 Gram stains, catalase activity, and motility were determined (Kozaki et al., 1992) Molecular Identification of K-23 and K-24 The 16S rrna gene was amplified by PR using universal primers (27F AGA GTT TGA TM TGG T AG and 1492R GTA TTA G GG TG TG G) with genomic NA as a template and it s sequenced by Genetic Analyzer 3130 in Solgent o. Ltd. The aligned 16S rrna of the isolate was subjected into LAST with the non redundant database of NI Gene ank Juice preparations The barley, corn, and Italian rye-grass were collected from grassland and forage form, South Korea. These samples were dried under the shadow and powdered. Taken 100 gram of each sample and 1L of sterile distilled water in the 5L bottle and kept at room temperature for 72 h with mild shaking. Then extracted samples were filtered through Whatman no-4 filter paper and stored at 4 for further experiment Growth profile analysis Fresh K-23 and K-24 was inoculated in different Juice such as barley, corn and Italian rye-grass and MRS broth and kept at 30 ± 2 in the shaker. From that aliquots were withdrawn at different time intervals and absorbance was measured at 600 nm until 48 h Metabolites profile analysis Fresh K-23 and K-24 was inoculated in different juice such as barley, corn and Italian rye-grass and MRS broth and kept at 30 ± 2 in the shaker for 48 h. Then supernatant was collected by centrifuging at 8000 rpm for 10 min. The lactic acid, succinic acid and acetic acid were quantified using the megazyme assay kit Statistical analysis Each sample was carried out in three replications. Excel 2007 was used for the calculation of the mean and standard deviation. 3. Result and discussion The LA plays an important role in the various food processing industries for making quality products (Leroy and e Vuyst 2004). The genetically modified organisms used to improve the properties of starter cultures. However, for fermented foods remains to be limited for number of reasons. The discovery of wild strains from natural sources must be considered as a most suitable approach for identifying the novel lactic acid (Ruas-Madiedo et al., 2005; Arasu et al., 2013a). For that, we isolated the number of the lactic acid bacteria from Italian rye-grass. Among these, two isolates showed strong antifungal activity. We named as these strain are K-23 and K- 24. Further, it was characterized by biochemical and 16S rrna sequence. iochemical results revealed that the K-23 was Gram-positive, non-motile, circular shape, catalase-negative and mesophilic in nature. The molecular identification of K-23 was done by the basis of 16S rrna sequence analysis, and it was revealed that the K-23 as a strain of Pediococcus pentosaceus (accession number: KM396463). K-24 was a creamy color, rod-shaped, anaerobic, Gram positive, and nonspore forming bacteria. The 16S rrna results revealed that the K-24 as Lactobacillus plantarum that displayed a similarity of 99 % to other L. plantarum strains (accession number KM396462). We previously investigated their antifungal and probiotics characteristics of K-23 and K-24. It showed potent antifungal and probiotic nature (Ilavenil et al., 2015; Vijayakumar et al., 2015). The growth and metabolite profiles of K-23 and K-24 in different types of juice such as barley, corn, IRG and MRS broth were determined by cultivating under the different condition such as aerobic, micro aerobic and microaerobic. In aerobic condition, K-24 growth was higher in barley than the other strains, but K-23 growth was higher in corn juice as compared with other strains. The growth profile of K-23 and K-24 was little lesser in IRG and MRS broth as compared with KA and KA (Fig. 1). Micro aerobic condition, K-23 and K-24 growth was higher in IRG and MRS broth than the KA and KA-91067, whereas in lesser in the barley and corn (Fig. 2). In anaerobic condition, K-23 and K- 24 growth was almost similar in IRG, whereas, K-24 growth was higher in barley as compared with other strains (Fig. 3). Plants contain number of the native lactic acid bacteria which produced lactic acid under the aerobic conditions. It is very useful to make quality silage because lactic acid reduces the ph of the silage that helps to prevent the undesired microbial growth. However, LA is not the only microorganisms on plants. acteria like lostridia and Enterobacteria, as well as yeast and molds, also are present on plants and compete with LA for sugars (Pahlow et al., 2003; Arasu et al., 2014d; Arasu et al., 2014f). The lactic acid and acetic acid together with low residual butyric acid content constitute the most important factors to reach aerobic stability in silages (Weinberg and Muck, 1996; Weinberg et al., 2003). Lactobacillus buchneri is a hetero-fermentative lactic acid bacteria used as inoculants for silage preparations. These bacteria could convert the lactic acid to acetic acid and other compounds. Generally, acetic acid is a potent inhibitor for yeast and molds and it improves the bunk of life or aerobic stability (Georgieva et al., 2008). The present study, K-23 produced the higher amount of lactic acid in MRS broth under the aerobic condition followed by corn, barley, and IRG. In micro aerobic condition, K-23 produced the higher amount of lactic acid in MRS broth followed by barley, IRG, and corn. K-24 5

6 produced higher amount of lactic acid in MRS and corn followed by corn, barley and IRG in aerobic and microaerobic conditions (Fig. 4). K-23 and K-24 produced the higher amount of succinic acid in corn soup in both aerobic and micro-aerobic conditions than the other Juice (Fig. 5). For acetic acid, K-23 was higher in IRG and corn in aerobic and micro-aerobic conditions respectively than the other juice. Whereas, K-24 produced comparatively higher amounts of acetic acid in IRG in both aerobic and micro-aerobic conditions than the other juice (Fig. 6). Generally, lactic acid, acetic acid, and succinic acid could play an important role in the preventing undesired microbial growth through decreases of ph of the surrounding places. 4. onclusion The P. pentosaceus-k-23 and L. plantarum-k-24 showed significant growth in the different juice as compared with standard strains. Also it produced the significant amount of extra cellular metabolites such as lactic acid, succinic acid, and acetic acid. Therefore, we suggest that these strains could be useful for making quality silage in future. onflict of interest statement We declare that we have no conflict of interest. Acknowledgement This work was carried out with the support of ooperative Research Program for Agriculture Science & Technology evelopment (Project title: Isolation of lactic acid bacteria for the manufacture of high quality silage and utilization technology; Project No. PJ ) Rural evelopment Administration, Republic of Korea. This study was supported by (2014) Postdoctoral Fellowship Program of (National Institute of Animal Science), Rural evelopment Administration, Republic of Korea. References 1. Arasu VM, Jung MW, Ilavenil S, Jane M, Kim H, Lee K, Park HS, Hur TY, hoi GJ, Lim Y, Al-habi NA, hoi K. (2013a). 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