Detection of Plantaricin-Encoding Gene and Its Partial Purification in Lactobacillus plantarum BP102

Lactobacillus plantarum BP102 isolated from garlic bulb tissue has probiotic properties, including producing bacteriocin called plantaricin. This study aimed to detect the gene encoding bacteriocin produced by Lactobacillus plantarum BP102, and to evaluate the bacteriocin activity at each stage of partial purification. After the end of the log phase of L. plantarum BP102 was determined, and the bacteriocin-encoding genes were checked by PCR technique. Partial purification of bacteriocin was elucidated including pH-neutralized cell-free-supernatant (CFS), precipitation using 80% of ammonium sulfate, and dialysis (cut-off 10 kDa), then the bacteriocin activity in every partial purification stage was evaluated. The molecular weight of plantaricin was estimated using SDS-PAGE analysis. Lactobacillus plantarum BP102 harbored the gene encoding plantaricin (pln) biosynthesis, namely plnEF and plnK genes. The activity of crude bacteriocin was inactivated by the presence of proteinase-K enzyme. The protein concentration was gradually decreased along with the purification process. The bacteriocin activity was demonstrated at each step of the purification process (CFS, precipitation, and dialysis) against Bacillus cereus by 9.23 ± 0.20 mm, 7.86 ± 0.15 mm, and 7.6 ± 0.10 mm, respectively; while, Escherichia coli by 10.3 ± 0.55 mm, 7.4 ± 0.1 mm, and 6.86 ± 0.45, respectively. The molecular weight of partially purified bacteriocin BP102 was found to be approximately 15.9 kDa. The overlaid part of the gel showed a slight inhibition against E. coli due to a low protein concentration. This bacteriocin purification process should be further optimized to improve the bacteriocin activity that could be useful for food preservation

Screening and identification of potential indigenous yeasts isolated during fermentation of wine coffee

Wine coffee is a fermented coffee product that involves yeast as the fermentative agent which has potency as probiotics. This study aimed to determine the potency of yeast isolated from wine coffee fermentation and to identify the yeast species with the best probiotic properties. This study comprised three main steps: coffee fermentation, yeast isolation, and probiotic characterization. A series of probiotic tests were carried out, including resistance tests at low pH (pH 2, 3, & 4) and bile salts (0.5% & 2%), antimicrobial activity tests, antibiotic resistance tests, hemolytic activity tests, and species identification based on the ITS rDNA sequence. The data obtained were analyzed using One-way ANOVA (p≤0.05) and continued with the Tukey test. A total of 25 yeast isolates were isolated and purified. Nine isolates (A2, B1, B3, C3, D4, D5, E2, E3 & E5) had the highest tolerance to pH 2 and 2% bile salts with survival rates were more than 100% and 90%, respectively. Nine isolates were resistant to all tested antibiotics, and only isolate A2 exhibited a pathogenic characteristic (β-hemolysis). Three isolates (B3, E3 & E5) could inhibit the five indicator pathogens, with the highest inhibitory activity shown by isolating E3 against Bacillus cereus by 68 AU/mL. The isolate E3 was selected as the best yeast with probiotic properties identified as Pichia kudriavzevii with 100% similarities towards strain iwate20191107

Uji Aktivitas Bakteriofage Litik dari Limbah Rumah Tangga Terhadap Salmonella Typhi

Salmonella Typhi merupakan salah satu bakteri yang menjadi agen penyakit bawaan makanan. Bakteriofage sebagai alternatif penggunaan antibiotika telah digunakan untuk mengendalikan bakteri tersebut. Penelitian ini dilakukan untuk mendapatkan isolat bakteriofage litik yang mampu melisis beberapa bakteri patogen yang diujikan dan mengetahui pengaruh aktivitas bakteriofage litik terhadap pertumbuhan SalmonellaTyphi. Bakteriofage diisolasi dari limbah rumah tangga. Selanjutnya penentuan host range bakteriofage terhadap bakteri patogen lain dilakukan dengan metode spot test. Uji aktivitas bakteriofage terhadap SalmonellaTyphi dilakukan menggunakan metode bacterial challenge test. Berdasarkan hasil isolasi, didapat enam isolat bakteriofage, yaitu B2-St, B3-St, S1-St, S2-St, SL1-St, dan SL3-St. Semua isolat bakteriofage mampu melisiskan sel bakteri Escherichia coli dan Salmonella Typhimurium namun tidak mampu melisiskan Bacillus cereus, Staphylococcus aureus dan Shigella disentriae.Tiga isolat bakteriofagetelah terpilih berdasarkan densitas plaque terbanyak yaitu B2-St, SL3-St dan S2-St. Kemampuan isolat bakteriofage B2-St dalam melisiskan sel Salmonella Typhi lebih tinggi (6,81 ± 0,35 log sel/mL) daripada isolat bakteriofage SL3-St (7,39 ± 0,31 log sel/mL) dan S2-St (7,60 ± 0,27 log sel/mL). Penurunan densitas sel inang terendah oleh ketiga isolat bakteriofage terjadi pada jam ke-4. Bakteriofage B2-St merupakan bakteriofage terbaik dan berpotensi sebagai agen biokontrol Salmonella  Typhi.

Next-Generation Sequencing of the Microbial Community Profile In Free-Range Chicken (Gallus gallus domesticus) Cecum from East Nusa Tenggara Province

Free-range chicken is livestock reared to support the humans' need for protein alongside its ritualistic use in traditional medicine to treat diseases. This study investigates the diversity of bacterial communities in the free-range chicken cecum reared in different East Nusa Tenggara Province localities comprising Sambi 1, Sambi 2 villages, Labuan Bajo, and Kupang City. The extracted chromosomal DNA was subjected to next-generation sequencing using the V3-V4 region primers. Results revealed that the Kupang chicken cecum had the highest total tags, while the Sambi 2 village recorded the lowest. Similarly, Sambi 2 chicken cecum exhibited the highest unique tags (6662) and OTUs number (1261), while the Kupang samples gave the lowest at 2550 and 745, respectively. The Shannon diversity index for bacterial diversity demonstrated that cecum samples from Labuan Bajo (5.679) were more diverse than Sambi 1 (5.378), Sambi 2 (5.653), and Kupang samples (3.77). The bacteria with the highest dominance index (0.935) was found in Sambi 2, while the lowest was observed in the Kupang samples (0.082). The three bacterial phyla showing the highest relative abundance were those from Sambi 1, Sambi 2, and Labuan Bajo cecum samples, comprising Firmicutes, Bacteroidota, and Actinobacteriota.Conversely, the Kupang samples showed an abundance of Firmicutes, Bacteroidota, and Campilobacterota, compared to the Lactobacillus-dominated Kupang, Sambi 1, and Sambi 2 chicken cecum samples. The highest relative abundance for Bifidobacterium occurred in Sambi 1 and Sambi 2 chicken cecum samples, the Kupang samples were Campylobacter dominated, and Olsenella was abundant in the Labuan Bajo samples. Intriguingly, the bacterial composition in the tested chicken cecum samples largely comprised beneficial bacteria such as the lactic acid bacteria group. This bacterial group can be further characterized for obtaining probiotic cultures that could improve the health of free-range chickens

Prevalence of multidrug-resistant and extended-spectrum β-lactamase producing Escherichia coli from local and broiler chickens at Cibinong market, West Java, Indonesia

Background and Aim: Antimicrobial resistance (AMR) is becoming a public health concern. Foodborne pathogens are infectious agents that can be transmitted from animals to humans through food and can become resistant due to misuse and overuse of antibiotics, especially in poultry. This study aimed to detect the prevalence of multidrug-resistant and extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolated from local and broiler chickens at the Cibinong market, West Java, Indonesia. Materials and Methods: A total of 60 cloacal swab samples from 30 local and broiler chickens sold at the Cibinong market in West Java were obtained by random sampling. From these samples, 39 E. coli isolates were obtained after being cultured on eosin methylene blue agar and molecularly identified using polymerase chain reaction (PCR). Six antibiotic disks were used for the antibiotic sensitivity test against E. coli isolates cultured on Mueller-Hinton agar. PCR was performed to detect ESBL genes (blaTEM, blaSHV, and blaCTX-M). Results: A total of 76.47% (39/51) cloacal swab samples were positive for E. coli. All E. coli isolates were sensitive to imipenem (100%), and 38 isolates were sensitive to cefoxitin (FOX) (97.4%). On average, the isolates were sensitive to amoxicillin-clavulanic acid (AMC) (69.2%) and ceftriaxone (CRO) (89.7%). E. coli isolates were occasionally resistant to enrofloxacin (25.64%), followed by gentamicin (20.51%), CRO (10.25%), AMC (7.69%), and FOX (2.56%). The prevalence of E. coli AMR was 10.25% (4/39). All four multidrug-resistant E. coli isolates (blaTEM and blaCTX-M) were confirmed to have the ESBL gene based on PCR. Conclusion: The prevalence of multidrug-resistant and ESBL-producing E. coli is still found, proving that there is still inappropriate use of antibiotics and a need for strict supervision of their use, especially around Cibinong market, West Java

Benefits of Coriandrum sativum L. seed extract in maintaining immunocompetent cell homeostasis

Diabetes mellitus (DM) is a metabolic disease followed by an increase in blood glucose and impaired metabolism of proteins, lipids and carbohydrates. In general, these conditions are caused by impaired insulin secretion and/or defects of insulin receptors. As yet, there is no effective drug capable of treating DM. Manifestations of DM worsen when accompanied by severe inflammation. Therefore, the avoiding strategy and management of inflammation in DM are the primary courses of action in preventing further damage. In this study, we had evidence that Coriandrum sativum L. extract (CSE) could be used as an alternative to relieve symptoms in DM mice model. Previously, CSE has been shown to be able to inhibit the synthesis of the pro-inflammatory molecule interleukin (IL)-6 by both B220 and CD11b cells. Here, we showed that CSE prevents over-activation of CD4 and CD8 T lymphocytes. The predominance of T cells expressing CD62L on both CD4 and CD8 T cells after administration of CSE indicated that there were obstacles to activation. CSE also suppressed excessive CD25 expression, causing CD25 expression in CD4 T cells to return to normal levels. Thus, this study showed the therapeutic activity of CSE in DM mice model by suppressing the pro-inflammatory cytokines, modulate the activation of naïve T cells, and maintain the population of CD4+ CD25+ cells

The effect of Phyllanthus niruri and Catharanthus roseus on Macrophage Polarization in Breast Cancer Mice Model

Cancer death cases have increased yearly, and there are estimated to be 21.6 million cancer cases in 2030. Studies of herbal compounds for cancer treatment alternatives are essential because cancer treatment is relatively expensive and has adverse effects. Phyllanthus niruri (Pn) and Catharanthus roseus (Cr) are plants that are known as herbal medicines. Combining the two plants is expected to prevent and enhance the immune system in breast cancer cases. This study aims to analyze the anti-cancer and immunomodulatory effects of P. niruri and C. roseus extract (PCE) in modulating macrophage polarization in breast cancer mice. Experimental animals are divided into six groups and there is healthy control (normal mice), cancer (DMBA-induced mice), cancer mice with cisplatin administration, cancer mice with PCE administration with three different doses, including dose 1 (500 mg/kg Pn + 15 mg/kg Cr), dose 2 (1000 mg/kg Pn + 75 mg/kg Cr), and dose 3 (2000 mg/kg Pn + 375 mg/kg Cr). The mice were injected with DMBA once a week for six weeks to induce cancer in mice. The breast cancer mice model was administered with PCE orally for 14 days. The expression of CD11b+IL-10+ and CD11b+IFN-γ+ demonstrated macrophage polarization. The results showed that breast cancer induction using DMBA increased the level of IL-10 and decreased the level of IFN-γ significantly compared to the normal group (p < 0.05). In specific doses, administration of PCE could reduce IL-10 levels and increase the level of IFN-γ significantly (p < 0.05). PCE can modulate the polarization of macrophages by suppressing the M2-like macrophage and increasing the M1-like macrophage. The ability of PCE to modulate macrophage polarization indicates that the combination of P. niruri and C. roseus has activity as an anti-cancer

Naturally fermented milk from Indonesia: a study of microbial diversity and probiotic potency for the potential treatment of intestinal mucositis

Naturally fermented milk (NFM) is prepared from fresh milk which is fermented spontaneously, without any inoculation of starter cultures. With increasing interest in novel dairy products, naturally fermented milks have become of interest to food microbiologists as a result of their potential as technologically important microorganisms. Dadih is a well-known naturally fermented milk product developed by local people in West Sumatra, Indonesia. This product is manufactured using unpasteurized buffalo milk which is then fermented spontaneously at ambient temperature. Dangke is prepared from heat-treated buffalo milk, and then processed enzymatically utilizing papain from papaya latex. Identification and characterization of the indigenous microbiota is essential for understanding how the microbial ecology impacts on the organoleptic, safety and potential health benefits of dadih and dangke. In addition, the presence of probiotic microorganisms in these products was also evaluated by investigating their potential to reduce the severity of chemotherapy-induced intestinal mucositis in rats. Probiotics have been defined as ‘live microorganisms that, when administered in adequate amounts, confer a health benefit on the host’ (Hill et al. 2014). Lactic acid bacterial (LAB) groups detected using culture-dependent techniques in dadih were Lactobacillus plantarum, Lactococcus lactis subsp. lactis, and Enterococcus faecium (Chapter 2). Only one species of acetic acid bacteria was found, namely Acetobacter orientalis, while yeasts isolates were identified as Saccharomyces cerevisiae, Candida metapsilosis and Kluyveromyces marxianus, with C. metapsilosis as the principal yeast (Chapter 3). Other bacteria detected included Klebsiella oxytoca, Klebsiella sp. and Bacillus pumilus. Among these bacteria, L. plantarum was the most frequently isolated LAB from dadih, followed by L. lactis subsp. lactis. Indigenous microbiota detected in dangke were relatively similar to dadih (Chapter 2). However, E. faecium and B. pumilus were not found in dangke. Lactococcus lactis subsp. lactis was the most predominant LAB, while S. cerevisiae was the most frequently isolated yeast (Chapter 3). Moreover, based on a culture-independent method (pyrosequencing), genus Lactococcus had the greatest relative abundance in dadih. Based on pyrosequencing results, a more diverse population of mesophilic LAB was found in dangke sourced from cow’s milk; while family Enterobacteriaceae dominated dangke samples from buffalo milk. Lactobacillus plantarum S1.30 isolated from dadih demonstrated probiotic properties which included tolerance to low pH and bile salts, antimicrobial activity and the presence of a bacteriocin regulating gene (plantaricin A) and msa and bsh genes, susceptibility to antibiotics and ability to adhere to Caco-2 cells (Chapter 4). From these probiotic features, only antimicrobial activity and the presence of msa and bsh genes were not demonstrated by L. lactis subsp. lactis SL3.34. However, from the pyrosequencing results, this strain was selected as the representative of the dominant genus/species in dadih. The efficacy of probiotics evaluated in the present study was variable at treating 5-fluorouracil (5-FU)-induced intestinal damage in vivo (Chapter 5). The results suggested that L. plantarum S1.30 and L. lactis subsp. lactis SL3.34 could have beneficial effects through partially improving metabolic parameters such as water intake, urine output, food intake, and fecal output in 5-FU challenged rats. The severity of damage in the jejunum and ileum was also reduced following probiotic culture treatment. In conclusion, this insight into the microbial composition of dadih and dangke will assist in the development of sustainable and technologically feasible starter cultures with probiotic properties. This information has the potential to enhance human health, food safety and food security from locally produced traditional fermented milk products.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Animal and Veterinary Sciences, 2017

Isolation and Detection of Bacteriocin-Like Inhibitory Substances-Producing Bacteria from Fermented Mare’s Milk Sumbawa

  Bacteriocin-like inhibitory substances (BLIS) produced by bacteria is a promising future food preservative agent. This study aimed to obtain bacterial strains that can produce broad-spectrum antibacterial agents and identify the best BLIS producer species based on 16S rDNA sequences. The bacterial strains were isolated from fer-mented mare’s milk using MRS and M17 agar medium. The isolates then were initially screened based on its antibacterial activity of crude cells against Staphylococcus aureus ATCC 6538. The selected strains were cultured and harvested for its cell-free supernatant (CFS). The pH of CFS was adjusted to 6.5 then used for antibacterial activity as-says against ten pathogenic bacteria. Also, the proteinaceous nature of BLIS compound was confirmed by testing with proteinase K. The gDNA of selected isolates was extracted and the 16S rDNA was am-plified using the polymerase chain reaction method then sequenced. The 16S rDNA sequences of the selected strains were used to identify the species using BLAST nucleotides from NCBI then the phylogenetic trees were constructed. 32 isolates was obtained, but only three iso-lates (BC9, SB7, and DC12) were selected as a result of antibacterial screening for further assays. The neutralized-CFS (N-CFS) of these isolates exhibited broad-spectrum antibacterial activity. The N-CFS could be assumed as BLIS. The isolate of BC9 was identified as Ba-cillus amyloliquefaciens strain BC9 that has 99.99 % similarity with B. amyloliquefaciens KC-1, SB7 was Lactobacillus plantarum strain SB7 that has 99.99 % similarity with Lb. plantarum JMC 1149T, and DC12 was Lactobacillus rhamnosus strain DC12 that has 100 % sim-ilarity with Lb. rhamnosus DSM 20021T. Thus, the BLIS produced by those strains is potential for future food and beverages preservations.

Selection of Pectinolytic Yeast from Liberica Green Coffee Beans (Coffea liberica)

Liberica coffee has an inferior quality when compared to arabica and robusta coffee. This flavour can be improved through a fermentation process involving indigenous microbes, one of which is a group of pectinolytic yeasts. Therefore, this study aimed to obtain yeast isolates with pectinolytic activity. Green bean samples were used for yeast isolation using YEPD agar media. Selected yeast isolates were screened for their pectinolytic activity semi-quantitatively using the well-diffusion method and quantitatively using the dinitrosalicylic acid (DNS) method. A total of six yeast isolates with different colony morphological characters were obtained and referred to as isolates Y1, Y2, Y3, Y4, Y5, and Y6. The isolates suspected to produce pectinase enzyme due to a clear zone around the well are Y2, Y3, Y4, and Y5. Y5 had the highest pectinolytic index, 022 and 0,73 on the second and fifth days of incubation, respectively. The four selected isolates were then screened quantitatively and showed that Y2 had the highest pectinase enzyme activity with a value of 12,66 ± 0,17 U/mL. The results showed that four yeast isolates from Liberica green coffee beans had pectinolytic activity with the highest enzyme activity found in Y2