Analisis Metagenomik Komunitas Bakteri yang Berasosiasi dengan Larva Attacus atlas L.

Ulat Attacus atlas merupakan ulat sutera liar yang berpotensi sebagai penghasil serat sutera. Kultur spesies serangga ini sangat diperlukan untuk menjamin kontinuitas produk sutera dan mencegah eksploitasi berlebihan spesies ini di alam. Penelitian ini bertujuan untuk mengetahui struktur komunitas dan keanekaragaman bakteri pada larva A. atlas liar dan larva A. atlas yang dipelihara. Penelitian ini telah dilaksanakan pada bulan Juni 2010- Juni 2011 di laboratorium Biologi FMIPA UNS. Metode penelitian ini terdiri kultur bakteri, ekstraksi DNA, amplifikasi gen 16S rRNA dan sekuensing gen 16S rRNA. Hasil kultur bakteri dari larva A. atlas liar diperoleh 6 isolat, sedangkan dari larva A. atlas yang dipelihara diperoleh 16 isolat. Hasil penelitian ini menunjukkan bahwa larva A. atlas yang dipelihara memiliki struktur komunitas bakteri yang didominasi oleh isolat LA-1, berdasarkan hasil sekuensing gen 16S rRNA isolat tersebut memiliki kesamaan 97 % dengan uncultured bacteria, sedangkan pada larva A. atlas liar didominasi oleh isolat us2-s, yang memiliki kesamaaan 98 % dengan Providencia sp. Keanekaragaman bakteri berdasarkan indeks Shannon- Wiener/H’ dan Evenness pada larva A. atlas yang dipelihara lebih tinggi daripada larva A. atlas liar. Kata kunci: komunitas bakteri, larva A. atlas, sekuensing gen 16S rRN

Isolasi, Karakterisasi, dan Kloning Gen Penyandi α α α α α-Amilase Bakteri Halofil Moderat asal Bledug Kuwu

A moderately halophilic bacteria, BK-05, was isolated from Bledug Kuwu, a saline terrestrial area at Central Java. Based on partial sequence of 16S-rRNA gene, the isolate was closely related to Halobacillus litoralis. This isolate showed amylolytic activity when grown on saline media [15% (w/v) NaCl] suplemented with starch. A pair of primer was designed based on the sequence of amyH gene from Halomonas meridiana and Pseudoalteromonas haloplanktis. PCR amplification using these primers showed three DNA bands with each size approximately 1.50, 1.00, and 0.75 kb. Partial DNA sequencing analysis based on its deduced protein sequence revealed that the 1.50 kb band was closely related to the sequence of metalloprotease from Bacillus subtilis (approximately 54.3% identity in 184 amino acid overlap). Southern hybridization analysis showed that the 1.50 kb fragment was located within a 4.0 kb fragment of BamHI, 4.8 kb of EcoRI, 4.3 kb of HindIII, and 4.0 kb of XhoI digestion of BK-05 genomic DNA, respectively

Produksi Gula Reduksi oleh Rhizopus oryzae dari Substrat Bekatul

Rice bran was produced from rice-hulling process. Rice bran was contain high starch, therefore it was used to saccharification process. The aim of this research was to study the optimum concentration of rice bran to produce reducing sugar on saccharification process by Rhizopus oryzae. The 100 mL-rice bran medium (5, 10, 15, 20, 25, and 30%) were sterilized at 1210C, for 15 minutes and inoculated with R. oryzae (± 2x106 cfu) and incubated for 3 days. The concentration of reducing sugar, starch, pH value, and biomass were analyzed everyday. Concentration of starch was decreasing during saccharification process. However, the concentration of reducing sugar was maximum at first day saccharification. The optimum concentration of rice bran for saccharification process was showed by maximum production of reducing sugar (15,347 mg/mL); it was in 20% concentration of medium. The pH value was decreasing during saccharification process, and the biomass was reversed. Keywords: rice brans, Rhizopus oryzae, saccharification, reducing sugar

Use of Edible Film Incorporated with Parijoto Fruit Extract (Medinilla speciosa Blume) to Inhibit Microbiological and Oxidative Damages of Sausages

Background and Objective: Edible film is one of the solutions for food packaging that carry antimicrobial and antioxidant compounds, usually found in Medinilla speciosa fruits. The objective of this study was to assess effects of Medinilla speciosa fruit extract on physical, chemical and bioactivity of edible films as well as effects of coating on sausage quality during storage. Material and Methods: The edible film included 2% w w-1 chitosan, 2% w w-1 sorbitol and Medinilla speciosa fruit extract. Variations of Medinilla speciosa extract included 0, 2.5, 5 and 10% (w w-1), while the storage temperature included 4 and 27 oC. Seven parameters of edible film characteristics were assessed, including tensile strength, elongation, water vapor permeability, antimicrobial and antioxidant activities, surface microstructure and Fourier-transform infrared response. Parameters assessed in storage treatment included total plate count, yeast mold count and thiobarbituric acid reactive substances. Data were analyzed using Kruskal-Wallis test. Organoleptic characteristics were analyzed using Friedman test and SPSS Software. Results and Conclusion: Results showed that the higher the concentration of Medinilla speciosa extract was, the higher the value of tensile strength, water vapor permeability and antioxidant activity and lower the elongation value were. The film control with 0% Medinilla speciosa extract was the only film that met Japanese standard for the water vapor permeability value, including 6.74 gm-2 h-1. Furthermore, shelf life of sausages coated with edible films revealed that the higher the concentration of Medinilla speciosa extract was, the lower the total plate count, yeast mold count and thiobarbituric acid reactive substances values were. Study demonstrated that the Medinilla speciosa extract edible film inhibited microbiological and oxidative damages. ​Oon Day 15 of storage, sausages coated with edible films with 10% Medinilla speciosa extract included lower total plate count, yeast mold count and thiobarbituric acid reactive substances values, respectively including 2.4 ±0.02 log CFU g-1, 1.3 ±0.08 log CFU g-1 and 13.38 ±0.22 mg malonaldehyde kg-1 sausage, compared to the control film. Organoleptic assessment showed no major differences in consumer acceptance. In conclusion, edible film with 10% Medinilla speciosa extract is the best physical, chemical and bioactivity film. Moreover, this film extends the sausage shelf life. Conflict of interest: The authors declare no conflict of interest

AKTIVITAS PERTUMBUHAN DAN KELANGSUNGAN HIDUP IKAN SIDAT Anguilla bicolor DENGAN PAKAN Wolffia arrhiza

Budidaya ikan sidat (Anguilla bicolor) salah satu peluang usaha yang sangat potensial untuk dikembangkan. Kendala pada pengembangan budidaya pembesaran ikan ini adalah kualitas pakan. Wolffia arrhiza memiliki kesempatan besar sebagai bahan pangan, mengingat berbagai sifat-sifatnya: kandungan protein relatif tinggi, karbohidrat dan lemak yang baik, memiliki pertumbuhan yang cepat dan beradaptasi dengan baik di danau dan kolam. Penelitian ini bertujuan untuk mengetahui pengaruh penggunaan tepung  W. arrhiza sebagai bahan penyusun pelet terhadap pertumbuhan dan kelangsungan hidup ikan sidat (A. bicolor). Percobaan yang digunakan dalam penelitian adalah Rancangan Acak Lengkap (RAL) dengan 5 perlakuan. Ikan sidat dibagi menjadi 5 kelompok, tiap akuarium perlakuan berisi 3 ekor, dengan perlakuan pemberian subtitusi pakan 0% (kontrol), 20%, 40%, 60% dan 80% dari berat total ikan sidat. Data yang diperoleh subtitusi tepung W. arrhiza  ada pengaruh nyata (P>0,05) terhadap pertumbuhan dan kelangsungan hidup ikan sidat (A. bicolor) akan tetapi berpengaruh ke arah hasil negatif. Nilai terbaik laju pertumbuhan pada perlakuan P2 (0,6 cm) dan terendah pada perlakuan P4 (0,1 cm) dan perubahan berat yaitu pada perlakuan P0 (kontrol) 10,6 – 10,7 dan penurunan berat ikan 10.4 – 6 g. Dari hasil penelitian ini dapat disimpulkan bahwa penggunaan tanaman W. Arrhiza mempunyai potensi sebagai bahan pakan budidaya ikan sidat. Akan tetapi perlu adanya uji lebih lanjut bagaimana tekhnik pemberian pakan yang tepat

Pemanfaatan potensi Azolla microphylla sebagai pakan untuk ikan sidat (Anguilla bicolor)

Azolla microphylla termasuk dalam famili Azollaceae yang sangat kaya protein, asam amino esensial, vitamin dan mineral. Tanaman azolla dapat menjadi sumber nutrien potensial untuk ikan. Anguilla bicolor merupakan salah satu ikan yang memiliki potensial ekspor dengan permintaan pasar dunia yang meningkat dari tahun ke tahun.. Tujuan penelitian ini adalah mengetahui pemanfaatan potensi A. microphylla  sebagai pakan untuk ikan sidat (A. bicolor). Penelitian ini menggunakan Rancangan Acak Lengkap (RAL), dengan lima perlakuan dan tiga kali ulangan. Sekitar 15 ekor A. bicolor dengan rata-rata panjang tubuh 20-25 cm dan berat ± 10 g dibagi menjadi lima kelompok, ke dalam akuarium masing-masing berisi tiga ikan untuk diberi pakan uji dengan substitusi A. microphylla (0%, 10%, 20%, 30%, 40 %) selama 30 hari. Parameter penelitian ini adalah pertambahan berat mutlak, laju pertumbuhan spesifik, rasio konversi pakan dan kelangsungan hidup A. bicolor. Hasil analisis ANOVA dilanjutkan uji Tukey dengan taraf kepercayaan 95%, menunjukkan hasil beda nyata (P<0,05) pada perlakuan kontrol (0% A. microphylla) dengan pertambahan berat mutlak: 0,13±0,07g, SGR: 0,55±0,11(%/hari), FCR: 13,25±2,62 dan SR: 100%. Seluruh perlakuan dengan substitusi A. microphylla belum menunjukkan hasil positif untuk ikan sidat. Dari hasil penelitian dapat disimpulkan bahwa pemberian A. microphylla dengan substitusi konsentrasi (10%, 20%, 30%, 40%) selama 30 hari, belum menunjukkan potensi sebagai pakan untuk A. bicolor

Investigating Untapped Potentials: Velvet Beans as Novel Prebiotic Sources and Their Effects on Gut Microbiota and Short-Chain Fatty Acid Level

  Abstract   Background and Objective: Prebiotics are non-digestible carbohydrates that selectively facilitate growth of beneficial microorganisms in the gut. Legumes naturally contain carbohydrates with potentials as prebiotic sources. However, numerous legume species remain uninvestigated in this context. The aim of this study was to identify such uninvestigated legumes as potential sources of prebiotics. Material and Methods: Nine legume samples collected from Central Java and East Java, Indonesia, were extracted using maceration method. Digestion with HCl buffer and α-amylase followed by analysis with dinitrosalicylic acid and phenol-sulfuric acid methods assessed quantities of non-digestible carbohydrates. Three legumes with the highest non-digestible carbohydrates levels were assessed in vitro to investigate their abilities to promote the probiotics growth. Then, the most promising extract was assessed on mice to assess its effects on short-chain fatty acid levels using GC-2010 Plus and gut microbiota composition using metagenomic 16S rRNA markers. Results and Conclusion: From the nine legumes assessed, bambara groundnut (23,51 mg.g-1), velvet beans (22.36 mg.g-1) and chickpeas (12.1 mg.g-1) included the highest non-digestible carbohydrates levels. Velvet beans showed a greater ability to stimulate growth of Lactobacillus plantarum and Bifidobacterium bifidum, compared to bambara groundnut and chickpeas. Administration of velvet beans to mice increased short-chain fatty acid levels in forms of acetate (12.6 mM) and propionate (3.28 mM). Significantly, velvet beans could modify composition of the gut bacteria by increasing diversity, decreasing dominance levels, increasing abundance of Bacteroides, Helicobacter, Mucispirillum, Bifidobacterium and Lawsonia genera and decreasing abundance of Lachnospiraceae NK4A136 group, Blautia and Lachnoclostridium species. Conflict of interest: The authors declare no conflict of interest. Introduction   Prebiotics consist of non-digestible carbohydrates (NDCs) and are resistant to stomach acid and digestive enzymes; therefore, they selectively promote growth of beneficial bacteria in the large intestine [1,2]This fermentation process by gut bacteria produces short-chain fatty acids (SCFAs) such as acetate, propionate and butyrate, which can lower pH of the gastrointestinal tract (GIT) and affect gut microbiota compositions [3]. Decreases in pH can decrease number of pathogenic microorganisms and increase growth of beneficial microorganisms, which is linked to the tolerance of the latter microorganisms to acidic conditions. Previous studies have shown that specific prebiotics such as inulin can promote growth of beneficial gut bacteria such as Lactobacillus and Bifidobacterium. [4,5]. Similar effects were observed with fructo-oligosaccharides, increasing Bifidobacterium while decreasing harmful bacteria [6]. Legumes are renowned for their nutritional values, particularly as protein sources. However, they contain NDCs in the form of oligosaccharides and polysaccharides, which include potentials as prebiotics [7]. These components pass through the small intestine undigested and reach the large intestine, where they can promote growth of beneficial gut bacteria [8]. Studies have demonstrated prebiotic effects of common legumes such as cowpeas and black beans, including decreased pH levels, increased growth of Bifidobacterium and Lactobacillus and increased SCFA levels [9,10] however, a vast majority of legume species are grouped under the minor category, remaining virtually uninvestigated. Much legumes are consumed only by local communities in Java, Indonesia. Additionally, food industries ignore legumes due to a limited knowledge of their compositions and potential benefits. Investigating prebiotic potentials of the minor legumes presents a compelling opportunity to enhance their economic values. No reports have been published on the prebiotic potentials of specific minor legume varieties used in the current study. While most studies on prebiotics have focused on familiar major legumes, the current study investigated the lesser-known varieties. Significantly, one minor legume, Mucuna pruriens (velvet bean), has shown promises to combat obesity in mice, but its prebiotic potentials must be investigated [11]. In the current study, minor legume samples were selected based on their NDCs content and ability to promote growth of common gut bacteria of Bifidobacterium bifidum and Lactobacillus plantarum [12,13]. Legumes with the most promising prebiotic potentials were further assessed in mice to assess their effects on SCFA levels and gut microbiota compositions. This study targeted caecum, the major fermentation site in mice (pH 4.4-4.6), and used metagenomic analysis of 16S rRNA gene sequences to characterize the microbial communities. The aim of this study was to investigate novel potential prebiotic sources from several assessed legume candidates. Materials and Methods 2.1. Selection of Legumes A total of nine types of legumes that were not investigated as prebiotics were species grown and consumed by local people in Java, Indonesia. The minor legumes, including velvet beans (M. pruriens), bambara groundnut (Vigna subterranea), chickpea (Phaseolus vulgaris), calopo beans (Calopogonium mucunoides), snow pea (Pisum sativum var. saccharatum), winged beans (Psophocarpus tetragonolobus), sword beans (Canavalia ensiformis), red beans or senerek beans (P. vulgaris) and turi beans (Sesbania grandiflora). 2.2 Extraction of Carbohydrates The prebiotic components in the legume samples were extracted using maceration method. Ethanol, a polar solvent known for its attraction to polar carbohydrates, was chosen as the extraction solvent. Then, 70% ethanol (v v-1) was used for maceration with a 1:10 sample:solvent ratio [14,15]. The mixture was set for 4 d and then filtered and the solvent was evaporated at 60 °C using rotary evaporator.     2.3. Assessment of non-digestible carbohydrate contents Carbohydrate resistance of nine minor legume extracts was assessed using simulated stomach acid and digestive enzymes. Each extract was prepared as a 10% stock solution (w v-1) in distilled water (DW). Resistance was assessed using acidic and enzymatic digestions. Acidic digestion involved incubating 200 µl of 1% extract solution (v v-1) with 200 µl of HCl buffer (pH 1) at 37 °C for 4 h (16). The reaction was stopped with 1 N NaOH. Enzymatic digestion was followed by further incubation of 200 µl of acid-digested solutions with 200 µl of α-amylase enzyme (2 U.ml-1) in sodium phosphate buffer at 37 °C for 6 h. Heating at 80 °C for 10 min stopped the reaction. Each digestion was carried out in triplicate [15].    The NDCs content was assessed using dinitrosalicylic acid (DNS) method for reducing sugars before digestion and the phenol sulfuric acid method for total sugars after acid-enzymatic digestion. Acid and enzyme analyses were carried out respectively to simulate digestion in the stomach and small intestine. The DNS method involved preparation of a reagent by dissolving 10 g of 3,5-dinitrosalicylic acid, 2 g of phenol, 0.5 g of sodium sulfite and 10 g of sodium hydroxide in 1 l of DW. Assay involved adding 100 µl of 1% legume extract solution (v v-1) and 100 µl of DNS reagent, followed by vortexing for 30 s, heating at 95 ℃ for 10 min (until color change), adding 33 µl of 40% sodium potassium tartrate (w v-1) and diluting 10× with DW. Absorbance was measured at 540 nm using ELISA reader [17] For the phenol sulfuric acid method, 50 µl of digested solution were mixed with 50 µl of phenol solution and vortexed for 30 s; followed by mixing with 250 µl of H2SO4 (sample:phenol:H2SO4 ratio of 1:1:5), vortexing for 30 s, diluting 10× with DW and measuring the absorbance at 490 nm using ELISA reader (18). The NDC content in the extracts was calculated using Eq. 1 (15):                                                      Eq.1 2.4. Probiotics Growth Stimulation This study used the probiotic B. bifidum and L. plantarum. Bacterial growth was estimated using standard growth curve based on optical density at 600 nm and colony counts (CFU.ml-1)19. Bifidobacterium bifidum was incubated anaerobically at 37 °C, while L. plantarum was incubated aerobically at 37 °C using shaker incubators. The two bacteria were first propagated on MRS agar media for 24 h at 37 °C before inoculation into MRS broth media. After 24 h, inoculum was used to count cells and colonies. The OD 600 measurement at each dilution (100-10-10) estimated the number of cells. Colony counts for dilutions of 10-7, 10-8 and 10-9 were carried out using total plate count (TPC) method with triplicate plating. Bifidobacterium bifidum and L. plantarum were propa-gated in each modified MRS broth growth medium. The MRS broth was prepared using 10 g of peptone, 4 g of yeast extract, 8 g of meat extract, 20 g of carbon sources (glucose, inulin and three types of legume extracts with the highest NDCs), 1 g of Tween 80, 2 g of potassium phosphate dibasic (K2HPO4), 5 g sodium acetate (CH3COONa.3H2O), 2 g of tri-ammonium citrate (C6H17N3O7), 0,2 g magnesium sulfate (MgSO4·7H2O) and 0,04 g manganese (II) sulfate (MnSO4·H2O), which were dissolved in DW up to 1 l [20]. The OD 600 was measured at 0, 24 and 48 h using ELISA reader. Then, number of the bacterial colonies was estimated from the standard curve. 2.5. Experiments on Animal Models Promising prebiotic function legumes, based on their NDC levels and probiotic-stimulating abilities, were assessed in mice to investigate their effects on SCFA levels and gut microbiota compositions. Fifteen mice were equally divided into three groups of a negative control group fed with a standard diet, a positive control group fed with a standard diet supplemented with 5% inulin (w w-1) and a treatment group fed with a standard diet supplemented with 5% of the selected legume extract (w w-1). Each mice was housed individually, fed at 5 g per min rate with ad libitum water. Body weight and food intake of each mice were assessed daily for 28 d. After 28 d, mice were euthanized and their cecum were collected for further analysis. 2.6. Assessment of Short-Chain Fatty Acid Levels    Analysis of SCFA levels in caecum contents was carried out using gas chromatography (Shimadzu GC-2010 Plus, Japan). First, 0.15 g of the sample was mixed with 1 ml of DW and centrifuged at 1008 × g for 10 min. Then, supernatant was analyzed using GC-202 Plus, Japan, provided by the Food and Agricultural Product Technology Testing Laboratory, Faculty of Agricultural Technology, UGM, Indonesia [21]. 2.7. Metagenomic Analysis    Genomic DNA was extracted from the caecum using ZymoBIOMICS DNA mini kit, following the manufacturer’s instructions. The extracted DNA concentration was assessed using BioPhotometer Plus (Eppendorf, Germany). Poly-merase chain reaction (PCR) amplified the V3 and V4 regions of the 16S rRNA gene using primers of 341F (CCTACGGGRGGCAGCAG) and 806R (GGACTACC-AGGGTTTCTA) [22]. Then, DNA sequencing was carried out using next-generation sequencing (NGS) method and MGISeq platform. All PCR and NGS studies were carried out at PT. Genetic Sciences Jakarta, Indonesia. 2.8. Data Analysis Data were analyzed using SPSS software v.24.0 and one-way ANOVA with a significance level of α = 0.05. For significant differences, Duncan's multiple range test (DMRT) was used for post-hoc analysis. FASTA-formatted sequence data were analyzed with UPARSE v7.0.1001 to group sequences into OTUs (operational taxonomic units). Sequences with ≥97% similarity were assigned to the same OTU. Taxonomic profiling was carried out using QIIME v.1.7.0 and SILVA database. Multiple sequence alignment was carried out using MUSCLE v.3.8.31. Then, OTUs with abundance less than 0.005% were removed. Normalized OTU abundance was used to assess alpha and beta diversities. Alpha diversity analysis was carried out using QIIME v.1.7.0 and visualized using R v.2.15.3. This captured the within-habitat bacterial diversity, including the number of OTUs, Shannon-Wiener diversity index and Simpson index. Beta diversity, reflecting bacterial diversity between the habitats, was estimated using PCoA-based index calculated using FactoMineR, ggplot2 and R v.2.15.3. Results and Discussion 3.1. Quantity of Non-digestible Carbohydrates in Legumes To qualify a compound as a prebiotic agent, its carbohydrates must resist digestion in the stomach and small intestine. Table 1 shows that velvet beans and bambara groundnut included significantly higher quantities of NDC, compared to other beans (p&lt;0.05). Chickpeas demonstrated a relatively high quantities of NDC, compared to other legumes. In addition to the high quantity of NDCs, the low hydrolysis proportions of velvet beans (4.57%), bambara groundnut (8%) and chickpea (24%) indicated that most of the carbohydrates in these three beans were resistant to acid and enzyme digestions. These results suggested that these three beans could reach the large intestine for selective fermentation by the beneficial bacteria. Based on the results, these three beans were further assessed in vitro to assess their abilities to stimulate beneficial bacteria. Velvet beans (M. pruriens) are beans with high carbohydrates contents. Previous studies reported that raw velvet bean seeds contained up to 49.22% of carbohydrates [23] Total dietary fiber content of the velvet beans is known to reach 86.6 mg.g-1, which is higher than that of Canavalia gladiata and Vigna unguiculata [24]. Bambara groundnuts (V. subterranean) are known as food sources with high carbohydrate contents, reaching up to 64.4%. Previous studies have shown that most of the carbohydrates contained in bambara groundnut are dominated by oligosaccharides and polysaccharides [25]. Chickpeas (P. vulgaris) are known as sources of carbohydrates that consist of starch, fibers and oligosaccharides. Previous studies have reported that the total polysaccharide contents in chickpeas reach to 300-370 mg.g-1, while the indigestible carbohydrate contents in form of oligosaccharides reach to 41.8-85.3 mg.g-1 [26]. 3.2. Propagation of Probiotics on Various Carbon Sources The NDCs are qualified as prebiotics if they selectively encourage propagation of beneficial bacteria in the large intestine. This study investigated several prebiotic candidates known for their acid and enzymatic resistances, based on their high quantity of NDCs. The candidates included velvet beans, bambara groundnuts and chickpeas. The study assessed their abilities to stimulate propagation of the representative probiotics of B. bifidum and L. plantarum, commonly detected in GIT of humans and rodents [27,13]. Bacteria were cultured in MRS broth media with various carbon sources. Each medium contained 2% extracts (w v-1) of velvet beans (MRS-VB), bambara groundnuts (MRS-BR) and chickpeas (MRS-CP). Propagation of B. bifidum and L. plantarum on these sources was compared with those on cultures using 2% of prebiotic inulin (w v-1) (MRS-INU), 2% of glucose (w.v-1) (MRS+) and no carbon sources (MRS-). Figure 1a,b showed that the growth of B. bifidum and L. plantarum in MRS- media did not show significant increases after incubation for 48 h. Carbon source in the media is a substrate that is utilized by bacteria to form amino acids and other cell components, making it important for the multiplication of bacterial cells [28]. Figure 1a reveals diverse growth patterns for B. bifidum in various MRS media. Significantly, MRS-VB and MRS-CP media stimulated B. bifidum propagation after 24 h, compared to MRS-BR. However, the number of B. bifidum colonies in MRS-VB and MRS-CP media was lower than that in MRS-INU at 24 h (p&lt;0.05). At 48 h, B. bifidum in media with the prebiotic candidate (MRS-VB, MRS-BR and MRS-CP) included a lower number than that it did in MRS+ and MRS-INU media (p&lt;0.05). These results indicated that the three prebiotic candidates were not able to stimulate propagation of B. bifidum as well as inulin prebiotics. However, B. bifidum in MRS+ media included a better propagation rate, compared to that in MRS-INU. Despite its positive effects, glucose failed to control harmful bacteria such as Escherichia coli and Salmonella spp. This suggests that specific media and prebiotics might be needed to support particular beneficial bacteria while limiting harmful ones [29]. Figure 1b reveals that L. plantarum in MRS-VB, MRS-BR and MRS-CP media included a lower number of colonies at 24 h than that it did MRS-INU (p&lt;0.05). At 48 h, L. plantarum in MRS-VB media included the highest number of colonies, compared to that it did in MRS-VB, MRS-CP and MRS-INU media. A carbon source of 2% velvet bean extract could stimulate propagation of L. plantarum to reach to 99.83 ±1.45 × 107 CFU.ml-1, which was significantly higher than inulin prebiotics and the other two prebiotic candidates (p&lt;0.05). These results indicated that velvet beans included a better ability to stimulate L. plantarum during 48 h of incubation, compared to that inulin prebiotics did. Proliferative Index was reported from log cell number at 48 h minus log cell number at 0 h. Table 2 shows proliferative index of the probiotic bacteria during 48 h of incubation in media containing various prebiotic carbon sources. Significantly, B. bifidum in MRS-VB media included a higher proliferative index and was significantly different, compared to that it did in MRS-BR and MRS-CP (p&lt;0.05). However, the proliferative index of B. bifidum in MRS-VB media included a lower value, compared to that it did in MRS-INU and MRS+ (p&lt;0.05). These results indicated that B. bifidum utilized inulin and glucose better than that the prebiotics in velvet beans did. These results were similar to previous results, which stated that propagation of B. bifidum could only be stimulated by inulin [30]. This was because B. bifidum could not utilize oligosaccharides such as fructooligosaccharides (FOS) and galactooligosaccharides (GOS) in legumes. Previous studies have reported that FOS-type prebiotics could stimulate propagation of all Bifidobacterium spp., except B. bifidum [30]. This was because B. bifidum did not include genes encoding β-fructofuranosidase enzyme, functioning to hydrolyze FOS [31].    The proliferative index of L. plantarum (Table 2) in MRS-VB media showed the highest value, compared to that the other groups did (p&lt;0.05). These results indicated that L. plantarum could better utilize carbohydrates in velvet beans better, compared to that inulin prebiotics and the other two prebiotic candidates did. Velvet beans (M. pruriens) are known to contain FOS prebiotics, which consist of glucose and fructose units linked by β(2-1) glycosidic bonds [32,33]. Previous studies have shown that L. plantarum can utilize FOS as a selective carbon source because it includes β-fructofuranosidase enzyme, which plays roles in FOS degradation [34,33]. Nucleotide sequence of the L. plantarum genome has demonstrated presence of the sacA gene, which expresses an enzyme that can hydrolyze FOS internally. Unlike extracellular enzymes in L. pentosus and L. paracasei, intracellular enzymes in L. plantarum can maximally utilize FOS prebiotics because there are no hydrolysis products that are consumed by other bacterial species in the large intestine. This reveals that L. plantarum utilizes FOS to compete with other microorganisms in the large intestine. Fermentation of FOS by L. plantarum produces secondary metabolites that inhibit propagation of pathogenic bacteria such as E. coli that produce β-glucuronidase enzyme [34]. 3.3. Assessment of the prebiotic effects with prebiotic index A prebiotic index value greater than 1 (&gt;1) shows that a carbohydrate includes positive effects on propagation of the probiotic bacteria. Figure 2 shows that the prebiotic index of velvet beans and inulin in L. plantarum culture includes values greater than 1 (&gt;1). Velvet beans included the highest value, compared to that inulin and other prebiotic candidates did (p&lt;0.05). In B. bifidum culture, the three legume candidates included a lower proliferative index, compared to that inulin prebiotics did (p&lt;0.05). These results demonstrated that prebiotics in velvet beans included good abilities to stimulate L. plantarum, compared to that the inulin did. Moreover, propagation of B. bifidum could only be stimulated by the inulin prebiotics. 3.4. Experiments on Animal Models: Changes in Body Weight and Food Consumption Identified as promising prebiotic candidates, velvet beans were assessed in vivo using mice models. Mice in all three groups included similar body weights at baseline (Day 0) (Table 3). On Day 7, all groups gained weight with the treatment group (13.3%) showed the highest increase (not statistically significant), compared to the negative (12,3%) and positive controls (12,67%). By Days 21 and 28, all groups gained weight steadily with no significant differences between them (Table 4). However, all groups consumed more than 78% of the provided foods, demonstrating that prebiotics reached digestive system of all mice. Although significantly indifferent, treatment and positive control groups included lower body weights than that the negative control group did. A relatively high feed consumption in the treatment group did not cause significant increases in body weight of the mice. Previous studies have reported that velvet beans include anti-obesity effects by decreasing body weight in obese groups [11]. 3.5. Short-chain fatty acid levels in animal models Anaerobic bacteria primarily produce SCFA such as acetic, propionic and butyric acids via fermenting NDCs [35]. Significantly, positive control and treatment groups demonstrated higher leve

Production, Characterization, and Molecular Phylogenetic Analysis of Phytase from Aspergillus niger Isolates of an Indonesia Origin

This research aimed at analyzing the phytase from fungal isolates SB1, SB2, BS, and WF produced in cornstarch with glucose medium (CS+Glu) as carbon sources and Potatoes dextrose broth (PDB). The activity of phytase from isolates SB1, SB2, BS, and WF produced in CS+Glu medium was 2.97 UmL-1, 2.87 UmL-1, 3.18 UmL-1, and 4.37 UmL-1, respectively, while the activity of phytases was 2.07 UmL-1, 2.17 UmL-1, 2.22 UmL-1, and 2.78 UmL-1 respectively in PDB medium. The optimal temperature of SB1 and WF phytase was 40°C, while SB2 and BS were 50°C and 60°C, respectively. The optimal pH of SB1 and WF phytase was 5.0, while SB2 and BS phytase were 6.0, and 4.0 respectively. 18S rRNA gene analysis revealed that SB1 was 99% identical to Aspergillus niger ANTS (KY825168.1), SB2, BS, and WF were 99% identical to A. niger Moriga leaf (MG889596.1). Multiple sequences and phylogenetic analysis of phytase gene showed that phyA_SB1 and phyA_SB2 were 98% homology with A. ficuum (AAB26466), 97% with A. niger (ADP05107) while phyA_WF was 99% with A. ochraceoroseus (PLB29348), 98% with A. niger (ADP05105). The deduced proteins contain conserved motifs RHGARYPTD at N-terminal while lacking HD motif at C-terminal. These phytases were in the same cluster with Aspergillus sp. phytase A indicating that they belong to Histidine Acid Phosphatases (HAP) family

Formulasi dan Pengujian Aktivitas Antibakteri Sediaan Gel Hand Sanitizer Berbahan Aktif Kitooligosakarida

Kitooligosakarida merupakan derivat dari kitosan yang memiliki sifat antimikroba dan dijadikan bahan aktif dalam cairan hand sanitizer. Hand sanitizer bebas alkohol dapat menghindari terjadinya toleransi bakteri terhadap bahan kimia seperti alkohol dan mengurangi efek buruk alkohol bagi kulit. Penelitian bertujuan untuk mengetahui karakteristik sediaan hand sanitizer kitooligosakarida dan efektivitasnya dalam membunuh mikroba. Serbuk kitooligosakarida ditambahkan dalam formulasi hand sanitizer dengan variasi berat 1 g (formula 1), 1,5 g (formula 2), dan 2 g (formula 3) menggunakan basis NaCMC, propilenglikol, essens, dan metil paraben. Evaluasi stabilitas fisik hand sanitizer berdasarkan warna, tekstur, aroma, pH, homogenitas, dan daya sebar. Uji daya hambat hand sanitizer diuji melalui metode disc diffusion dilanjutkan dengan perbandingan daya hambat melalui pengukuran diameter zona penghambatan dan uji one-way ANOVA. Uji penurunan angka koloni mikroba dengan metode swabbing dan dilanjutkan perbandingan rerata penurunan koloni mikroba melalui uji T berpasangan. Hasil evaluasi stabilitas fisik hand sanitizer memenuhi kriteria gel yang baik. Hasil uji formula 1 memiliki kriteria angka diameter zona hambat yang lemah pada S. aureus (14,33 cm) dan daya hambat sedang pada E. coli (17,33 cm), Formula 2 memiliki angka diameter zona hambat kuat sebesar 21,33 cm pada S. aureus dan 20,33 cm pada E. coli. Formula 3 menghasilkan zona hambat kuat, yaitu 20,67 cm pada S. aureus dan 22,33 cm pada E. coli. Hasil penurunan angka koloni mikroba sediaan hand sanitizer paling besar yaitu formula 3 sebesar 80,22%. Hand sanitizer kitooligosakarida menunjukkan kemampuan penurunan angka koloni mikroba dan dapat digunakan sebagai formulasi alternatif hand sanitizer

PEMANFAATAN Azolla microphylla SEBAGAI PAKAN SUBSTITUSI UNTUK IKAN SIDAT (Anguilla bicolor)

Anguilla bicolor merupakan salah satu spesies ikan sidat yang hidup di perairan Indonesia. Permintaan komoditas ikan sidat ini semakin meningkat, namun lambatnya laju pertumbuhan perlu disediakan pakan berprotein tinggi untuk memacu pertumbuhan ikan. Azolla microphylla merupakan salah satu alternatif bahan baku pakan untuk memenuhi kebutuhan nutrisi ikan. Penelitian ini bertujuan untuk mengetahui pengaruh tepung A. microphylla sebagai bahan substitusi pada pakan terhadap pertumbuhan ikan sidat (A. bicolor). Rancangan percobaan yang digunakan adalah Rancangan Acak Lengkap (RAL) dengan 5 perlakuan. Ikan sidat dengan berat ± 10 gr dibagi menjadi 5 kelompok, kemudian masing-masing perlakuan diberi tambahan 0%, 10%, 20%, 30%, 40% tepung A. microphylla dari total berat pakan uji. Hasil uji pakan dengan substitusi tepung A. microphylla pada berat ikan menunjukkan penurunan bobot berkisar 10-10,9 g mejadi 0-9 g, sedangkan panjang tubuh ikan mengalami pertambahan berkisar 22,3-28,2 cm menjadi 23,4-28,4 cm. Dari hasil penelitian dapat disimpulkan bahwa A. microphylla tidak cocok untuk bahan substitusi pakan, namun melihat pengaruhnya terhadap pertambahan panjang tubuh ikan dapat dimanfaatkan sebagai bahan tambahan untuk membuat pakan ikan