Heterologous Expression of Pediocin/Papa in Bacillus Subtilis

Listeria moniocytogenes is food-borne pathogen. Pediocin is group II α bacteriocin with anti-listeria activity, naturally produced by Pediococuus acidilactic and Lactobacillus plantarum. Gene pepA/papA encode for pediocin. Expression and secretion of active papA was relayed on transporter papC and accessary protein papD on the same operon in native host. The excretion machines were also necessary for pediocin protein expression in heterologous host of E. coli, Lactobacillus lactis, and Corynebacterium glutamicum. In this study, two vector carrying codon sequence of papA mature peptide was constructed, with or without His tag. Both fragments were inserted into plasmid pHT43 and transformed Bacillus subtilis WB800N. The strains were induced with IPTG to secrete recombination protein PA1 and PA2 respectively. Supernatant from both recombination strains can inhibit Listeria monocytogenes ATCC54003 directly. The fused protein possesses inhibition activity as a whole, exempting from cleavage of leading peptide. Protein PA1 can be purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography. This is the first report for active pediocin expression without assistance of papCD in heterogenous host.

BAKTERI Lactobacillus spp DAN PERANANNYA BAGI KEHIDUPAN

Kelompok BAL (Bakteri Asam Laktat) yaitu berasal dari genus Lactobacillus terdiri dari hampir 80 spesies berbeda. Jenis Lactobacillus terdiri dari dua kelompok yang bersifat heterofermentatif dan homofermentatif. Spesies dari bakteri Lactobacillus yang tergolong homofermentatif adalah Lactobacillus bulgaricus, Lactobacillus thermophiles, Lactobacillus lactis, dan Lactobacillus acidophilu. Spesies bakteri yang bersifat heterofermentatif adalah Lactobacillus Fermentum. Bakteri Asam Laktat (BAL) adalah bakteri yang digunakan sebagai probiotik. Genus Lactobacillus terdiri atas banyak spesies yang digunakan untuk fermentasi dan pengawet makanan. Ada 106 spesies Lactobacillus, namun hanya 56 spesies berpotensi sebagai probiotik. Penelitian ini menggunakan metode adalah deskriptif restrospektif. Sampel penelitian berupa hasil literature dari jurnal dan penelitian yang telah diterbitkan. Tujuan penelitian ini adalah mereview berbagai jenis bakteri Lactobacillus spp dan perananya untuk kehidupan.

DISERTASI (FERMENTASI BIJI KAKAO KERING TERKENDALI MENGGUNAKAN INOKULUM MIKROBIA)

Biji kakao kering ditingkat petani sebagian besar dihasilkan tanpa fermentasiyang mempunyai beberapa kelemahan diantaranya tidak menghasilkan prekursorflavour khas kakao. Upaya untuk mendapatkan biji kakao kering yang memilikiprekursor flavour khas kakao dapat dilakukan, apabila masih terdapat subtrat yangdapat difermentasi oleh mikrobia yang terlibat dalam fermentasi biji kakao segardengan kondisi proses yang sesuai.Tujuan penelitian adalah: 1) mengetahui komposisi pulp biji kakao keringsebagai subtrat untuk fermentasi; 2) mengevaluasi pengaruh variasi teknikfermentasi biji kakao kering terhadap parameter mutu biji kakao kering hasilfermentasi; dan 3) mengevaluasi prekursor flavour dan senyawa volatil yangdihasilkan biji kakao hasil fermentasi pasca sangrai. Tahapan penelitian yangdilakukan adalah sebagai berikut: 1) pengujian komposisi dan kadar air pulp bijikakao kering sebagai subtrat fermentasi; 2) fermentasi biji kakao kering dengan 3variasi teknik fermentasi; 3) Analisis senyawa volatil biji kakao hasil fermentasidari tiga perlakuan tersebut. Ketiga variasi teknik fermentasi yaitu tanpapenambahan inokulum (kontrol), perlakuan kedua menggunakan inokulumSacharomyces cerevisiae (FNCC 3056), Lactobacillus lactis (FNC 0086) danAcetobacter aceti (FNCC 0016), masing–masing sekitar 108cfu/g diberikanserentak diawal fermentasi (IA) dan perlakuan ketiga, pemberian inokulum secarabertahap yaitu yeast di awal fermentasi, bakteri asam laktat pada jam ke 24 dan bakteriasam asetat pada jam ke 48 dengan populasi mikrobia sama dengan perlakuan kedua(IB). Fermentasi dilaksanakan selama 120 jam. Suhu diatur selama fermentasi,berturut-turut 35 oC (24 jam pertama), 45 oC (24 jam kedua), 55 oC (24 jam ketiga)dan 35 oC (48 jam terakhir)..Hasil penelitian menunjukan bahwa setelah direhidrasi sebanyak 50% dariberat bahan menghasilkan komposisi pulp biji kakao kering dapat digunakansebagai subtrat fermentasi. Selama fermentasi, biji kakao kering yang telahdirehidrasi mengalami penurunan kadar gula total, pH dan total polifenol padaketiga perlakuan. Cut test keping biji kakao kering menunjukkan persentase warnacoklat naik pada ketiga perlakuan. Kadar gula reduksi dan indeks fermentasi bijikakao menunjukan kenaikan pada seluruh perlakuan. Konsentrasi etanol, asamlaktat dan asam asetat pada semua perlakuan mencapai jumlah puncaknya berturut– turut pada jam ke 24, 60 dan 108 fermentasi. S. cerevisiae, L. lactis dan A. acetidari ketiga perlakuan mencapai populasi tertinggi berturut –turut pada jam ke 24,48 dan 72 fermentasi. Biji kakao kering pasca fermentasi menghasilkan asam aminohidrofobik yaitu alanin, tirosin, valin, phenilalanin, isoleusin dan methionin sebagaiprekursor flavor dengan total asam amino hidrofobik dimiliki oleh perlakuanpenambahan inokulum secara serentak. Setelah penyangraian biji kakao keringhasil fermentasi menghasilkan senyawa volatil antara lain aldehid, asam, alkoholdan ester.Seluruh pengujian parameter tersebut diatas menunjukan perlakuanpenambahan inokulum secara bertahap mencapai nilai tertinggi dibandingkankedua perlakuan yang lain. Dari hasil penelitian ini dapat disimpulkan bahwa xxiirehidrasi pulp biji kakao kering dapat memperbaiki komposisi pulp sebagai subtratfermentasi. Parameter pengujian, prekursor aroma dan senyawa volatil tertinggiditunjukan pada perlakuan penambahan inokulum secara bertahap.

Molecular Response in Intestinal and Immune Tissues to in Ovo Administration of Inulin and the Combination of Inulin and Lactobacillus lactis Subsp. cremoris

Intestinal microbiota are a key factor in maintaining good health and production results in chickens. They play an important role in the stimulation of immune responses, as well as in metabolic processes and nutrient digestion. Bioactive substances such as prebiotics, probiotics, or a combination of the two (synbiotic) can effectively stimulate intestinal microbiota and therefore replace antibiotic growth promoters. Intestinal microbiota might be stimulated at the early stage of embryo development in ovo. The aim of the study was to analyze the expression of genes related to energy metabolism and immune response after the administration of inulin and a synbiotic, in which lactic acid bacteria were combined with inulin in the intestines and immune tissues of chicken broilers. The experiment was performed on male broiler chickens. Eggs were incubated for 21 days in a commercial hatchery. On day 12 of egg incubation, inulin as a prebiotic and inulin with Lactobacillus lactis subsp. cremoris as a synbiotic were delivered to the egg chamber. The control group was injected with physiological saline. On day 35 post-hatching, birds from each group were randomly selected and sacrificed. Tissues (spleen, cecal tonsils, and large intestine) were collected and intended for RNA isolation. The gene panel (ABCG8, HNF4A, ACOX2, APBB1IP, BRSK2, APOA1, and IRS2) was selected based on the microarray dataset and biological functions of genes related to the energy metabolism and immune responses. Isolated RNA was analyzed using the RT-qPCR method, and the relative gene expression was calculated. In our experiment, distinct effects of prebiotics and synbiotics following in ovo delivery were manifested in all analyzed tissues, with the lowest number of genes with altered expression shown in the large intestines of broilers. The results demonstrated that prebiotics or synbiotics provide a potent stimulation of gene expression in the spleen and cecal tonsils of broiler chickens. The overall number of gene expression levels and the magnitude of their changes in the spleen and cecal tonsils were higher in the group of synbiotic chickens compared to the prebiotic group.

Effects of Fermentation on the Antioxidant and Antinutritional Compositions of Green Pea

Green peas are known to contain anti-nutritional factors like enzymes inhibitors, phytates, oxalates, saponins and polyphenolic compounds, all of which limit their utilization hence, the study evaluate the effect of fermentation on the antioxidant and antinutrients content of green pea. Fermentation of green pea was done using both submerged and solid state fermentation for 7days. Isolation and identification of microorganism from the fermented sample was done on daily basis using standard microbiological and molecular techniques. The type of organism isolated from the submerged fermentation of Green pea included the bacteria (Bacillus subtilis, Lactobacillus Plantarum, Micrococcus roseus, Lactobacillus lactis, and Lactobacillus fermentum) and the fungi Rhizopus oryzae, Penicillium chrysogenum and Rhizopus stolonifer. While the type of organism isolated from the solid state fermentation of Green pea included some bacteria (Bacillus subtilis, Lactobacillus Plantarum and Lactobacillus lactis) and fungi (Penicillium notatum, Rhizopus oryzae, Penicillium chrysogenum, Candida albicans, Alternaria alternate and Rhizopus stolonifer). Fermentation reduced the antinutritional content of the fermented sample with submerged fermentation resulting in the highest reduction from 32.18 mg/g, 4.14 mg/g, 1.62 mg/g, 51.08 mg/g and 36.37 mg/g in the raw sample to 26.27 mg/g, 0.48 mg/g, 0.27 mg/g, 7.82 mg/g and 24.07 mg/g in submerged fermented green pea for saponin, tannin, oxalate, phytate and alkaloid respectively. However, Fermentation significantly p ≤ 0.05 increased the phenol, flavonoid and FRAP content of the fermented green pea with the solid state fermentation resulting in the highest increase from 3.50, 0.03 and 1.41 in the raw sample to 9.32, 0.12 and 9.66 in the solid state fermented green pea for phenol, flavonoid and FRAP content respectively. This study revealed that fermentation had significant effect on the antioxidant and antinutritional compositions of Green pea thereby reducing the antinutrient composition of Green pea in which will improve the nutrient value of Green pea.

ANALISIS VIABILITAS LACTOBACILLUS LACTIS PADA INOVASI MEDIA DASAR PERTUMBUHAN ALTERNATIF DAN MEDIA DASAR PENEPUNGAN BAKTERI ASAM LAKTAT

The limited shelf life in a food requires a natural preservative so that the food used is not easily damaged and has a longer shelf life, namely by using lactic acid bacteria (BAL) using alternative media. By using lactic acid bacteria, the time in the storage period food products can be extended. The purpose of this study was to determine the viability of the Lactobacillus lactis bacteria on an alternative growth base media and a media on the media of bacteria. Lactic acid bacteria were rejuvenated and culture propagation of 5 ?l was inoculated into 5 mL of MRSB media. Formulation media used for bacterial growth such as whey tofu + 5% sucrose + 1% urea. The alternative media was incubated for 24 hours. Bacterial growth was observed at 0, 4, 8 and 16 hours using the TPC (Total Plate count) method. Various media Lactobacillus lactis bacterial deposition was grown on MRSB media and dried with freeze dry for 48 hours and the viability of Lactobacillus lactis was tested. The basic growth media that can be used are Lactobacillus lactis bacteria, namely whey tofu + sucrose 5% + urea 1% as well as Lactobacillus lactis viability results in various media which are grown on MRS media and various alternative media shows that the media has a 100% carrageenan composition able to maintain the viability of Lactobacillus lactis cells.