Complete genome sequence of Indonesian probiotic strain Lactiplantibacillus plantarum subsp. plantarum Dad-13.

Previous studies have investigated the probiotic properties of L. plantarum subsp. plantarum Dad-13. Nevertheless, genomic sequence data from previous studies were not yet available to support each probiotic characteristic. This study focused on the complete genome sequence of the strain to validate its role in specific probiotic properties.

Correlation between the chemical, microbiological and sensory characteristics of cream cheese using a mixed and single probiotic culture.

The use of mixed culture in the fermentation industry requires more complicated equipment, processes, and monitoring systems; therefore, a single culture may be preferable. This study aimed to investigate the correlation between chemical and microbiological properties and sensory characteristics. In addition, this study aimed to determine the different characteristics of cheese made using single probiotic cultures of Lactobacillus plantarum Dad-13 and Lactobacillus plantarum Kita-3 and mixed culture. The obtained results showed that the chemical characteristics of cream cheese made using single cultures were similar to those of cream cheese made using mixed cultures. The viability of the cells remained high after 35 days of storage for the single culture, which was not the case for the mixed culture. In terms of sensory analysis, cream cheese made using the single culture L. plantarum Kita-3 showed higher overall liking score among the samples, which might correlate with the high ester and ketone content. In addition, there was a strong relationship between the ethyl octanoate and methyl butanol contents and the overall liking score. The results of this study showed that the use of a single culture of L. plantarum Kita-3 could improve the sensory characteristics of cream cheese with probiotic properties. This study also contributed to the development of cream cheese production, particularly in the screening of potential starters.

Study of Viability, Storage Stability, and Shelf Life of Probiotic Instant Coffee Lactiplantibacillus plantarum Subsp. plantarum Dad-13 in Vacuum and Nonvacuum Packaging at Different Storage Temperatures.

Probiotic coffee is an alternative to processed coffee that is preferred and can improve the balance of intestinal microflora so that it has a positive impact on health. Cell viability of probiotics may decrease during storage. Factors that can affect viability during storage are storage temperature, packaging, oxygen, and water activity. This study is aimed at evaluating the viability, storage stability, and shelf life of the probiotic instant coffee Lactiplantibacillus plantarum subsp. plantarum Dad-13 in vacuum and nonvacuum aluminium foil packaging and different storage temperatures. This study used a complete randomised design with three replicates of treatments. They were packaged using 90 µm thick aluminium foil in a vacuum and nonvacuum and stored at 4°C and 30°C for 50 days and 37°C for 15 days. Based on the literature, a temperature of 4°C can maintain the viability of probiotics for more than one month, the temperature commonly used to store dry products is room temperature (30°C), so longer storage (50 days) is tried. Meanwhile, to accelerate the prediction of quality degradation, extreme temperatures were used based on the literature that the viability of probiotics decreased drastically after being stored at 37°C for 7 days, then tried for longer storage (15 days). The evaluation of product was carried by sensory testing by comparing commercial instant coffee. The product has been tested for cell viability, water activity, and shelf life. The result showed that the colour attribute was significantly different for all formulations. The bitterness of probiotic instant coffee differed significantly from other formulations. The commercial instant coffee was preferred by panellist in terms of colour and bitterness. The aroma, sweetness, and overall attributes of all formulations were not significantly different. The cell viability in vacuum was higher than nonvacuum treatment, and it was higher in 4°C. However, cell viability for all treatments and during storage was still above 107 log CFU/g. Water activity in probiotic instant coffee with vacuum packaging is lower than in nonvacuum and stored at 4°C lower than in other temperatures. However, all treatments were still below 0.60. The shelf life of products reaches two years when they are stored in vacuum packaging at 4°C while a temperature of 30°C reaches 3 months. So, the panellists accepted probiotic instant coffee, vacuum packaging, and low temperature could maintain viability, stability, and longer shelf life.

Whole-genome sequence data of cellulase-producing fungi Trichoderma asperellum PK1J2, isolated from palm empty fruit bunch in Riau, Indonesia.

Trichoderma asperellum PK1J2 is a promising cellulase-producing fungus isolated from a palm empty fruit bunch in Riau, Indonesia. Presented here is the genome assembly of T. asperellum PK1J2. The whole genome of the fungi was sequenced using Illumina NovaSeq PE150. The genome assembly was performed using SOAPdenovo, SPAdes, and Abyss software, and the assembly results of the three types of software were integrated with CISA software. T. asperellum PK1J2 has 6,835 protein-coding genes with a length of 9,233,597 bp. The final genome assembly was approximately 36 Mbp with a GC content of 48.45%. This whole genome shotgun project has been deposited at DDBJ/ENA/GenBank under accession JAGJIK000000000.

Gut Microbiota Composition in Undernourished Children Associated with Diet and Sociodemographic Factors: A Case-Control Study in Indonesia.

Malnutrition, which consists of undernutrition and overnutrition, is associated with gut microbiota composition, diet, and sociodemographic factors. Undernutrition is a nutrient deficiency that that should be identified to prevent other diseases. In this study, we evaluate the gut microbiota composition in undernourished children in association with diet and sociodemographic factors. We observed normal children (n= 20) and undernourished children (n= 20) for ten days in Lombok and Yogyakarta. Diet, sociodemographic factors, and medical records were recorded using food records, screening forms, and standard household questionnaires. Gut microbiota analysis was performed using 16S rRNA gene sequencing targeting the V3-V4 region. The result showed that the undernourished group had lower energy intake. In addition, the undernourished group had lower quality of medical records, parent knowledge, education, and exclusive breastfeeding. Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Verrucomicrobia were significantly different between normal and undernourished children. Based on LefSe, we determined that Akkermansia is a biomarker for undernourished children. In conclusion, diet and sociodemographic factors affect the gut microbiota composition of undernourished children.

Safety assessment of the indigenous probiotic strain Lactiplantibacillus plantarum subsp. plantarum Kita-3 using Sprague-Dawley rats as a model.

Lactiplantibacillus plantarum subsp. plantarum Kita-3 is a candidate probiotic from Halloumi cheese produced by Mazaraat Artisan Cheese, Yogyakarta, Indonesia. This study evaluated the safety of consuming a high dose of L. plantarum subsp. plantarum Kita-3 in Sprague-Dawley rats for 28 days. Eighteen male rats were randomly divided into three groups, such as the control group, the skim milk group, and the probiotic group. Feed intake and body weight were monitored, and blood samples, organs (kidneys, spleen, and liver), and the colon were dissected. Organ weight, hematological parameters, serum glutamic oxaloacetic transaminase (SGOT), and serum glutamic pyruvic transaminase (SGPT) concentrations, as well as intestinal morphology of the rats, were measured. Microbial analyses were carried out on the digesta, feces, blood, organs, and colon. The results showed that consumption of L. plantarum did not negatively affect general health, organ weight, hematological parameters, SGOT and SGPT activities, or intestinal morphology. The number of L. plantarum in the feces of rats increased significantly, indicating survival of the bacterium in the gastrointestinal tract. The bacteria in the blood, organs, and colon of all groups were identified using repetitive-polymerase chain reaction with the BOXA1R primers and further by 16S rRNA gene sequencing analysis, which revealed that they were not identical to L. plantarum subsp. plantarum Kita-3. Thus, this strain did not translocate to the blood or organs of rats. Therefore, L. plantarum subsp. plantarum Kita-3 is likely to be safe for human consumption.

Adhesion Properties of Lactobacillus plantarum Dad-13 and Lactobacillus plantarum Mut-7 on Sprague Dawley Rat Intestine.

Adhesion capacity is considered one of the selection criteria for probiotic strains. The purpose of this study was to determine the adhesion properties of two candidate probiotics, Lactobacillus plantarum Dad-13 and Lactobacillus plantarum Mut-7. The evaluation included the hydrophobicity of the cell surface using microbial adhesion to hydrocarbons (MATH), autoaggregation, and the adhesion of L. plantarum Dad-13 and L. plantarum Mut-7 to the intestinal mucosa of Sprague Dawley rat, followed by genomic analysis of the two L. plantarum strains. L. plantarum Dad-13 and L. plantarum Mut-7 showed a high surface hydrophobicity (78.9% and 83.5%) and medium autoaggregation ability (40.9% and 57.5%, respectively). The exposure of both isolates to the surface of the rat intestine increased the total number of lactic acid bacteria on the colon compartment, from 2.9 log CFU/cm2 to 4.4 log CFU/cm2 in L. plantarum Dad-13 treatment and to 3.86 log CFU/cm2 in L. plantarum Mut-7 treatment. The results indicate the ability of two L. plantarum to attach to the surface of the rat intestine. The number of indigenous E. coli in the colon also decreased when the compartment was exposed to L. plantarum Dad-13 and Mut-7, from 2.9 log CFU/cm2 to 1 log CFU/cm2. Genomic analysis revealed that both strains have genes related to adhesion properties that could play an important role in increasing the adherence of probiotics to the intestinal mucosa such as gene encoding fibronectin-binding protein, chaperonin heat shock protein 33 (Hsp33), and genes related to the capsule and cell wall biosynthesis. Based on these findings, we believe that L. plantarum Dad-13 and L. plantarum Mut-7 have adhesion properties to the intestinal mucosa in the rat intestine model system. The present research will be essential to elucidate the molecular mechanism associated with adhesion in our two probiotic strains.

Characterization of the biosynthetic gene cluster for cryptic phthoxazolin A in Streptomyces avermitilis.

Phthoxazolin A, an oxazole-containing polyketide, has a broad spectrum of anti-oomycete activity and herbicidal activity. We recently identified phthoxazolin A as a cryptic metabolite of Streptomyces avermitilis that produces the important anthelmintic agent avermectin. Even though genome data of S. avermitilis is publicly available, no plausible biosynthetic gene cluster for phthoxazolin A is apparent in the sequence data. Here, we identified and characterized the phthoxazolin A (ptx) biosynthetic gene cluster through genome sequencing, comparative genomic analysis, and gene disruption. Sequence analysis uncovered that the putative ptx biosynthetic genes are laid on an extra genomic region that is not found in the public database, and 8 open reading frames in the extra genomic region could be assigned roles in the biosynthesis of the oxazole ring, triene polyketide and carbamoyl moieties. Disruption of the ptxA gene encoding a discrete acyltransferase resulted in a complete loss of phthoxazolin A production, confirming that the trans-AT type I PKS system is responsible for the phthoxazolin A biosynthesis. Based on the predicted functional domains in the ptx assembly line, we propose the biosynthetic pathway of phthoxazolin A.

Activation of cryptic phthoxazolin A production in Streptomyces avermitilis by the disruption of autoregulator-receptor homologue AvaR3.

The genomes of actinomycetes encode many cryptic novel/useful bioactive compounds, but access to these cryptic secondary metabolites remains limited. Streptomyces avermitilis predominantly produces three polyketide antibiotics (avermectin, filipin, and oligomycin) but has the potential to produce more secondary metabolites based on the number of cryptic biosynthetic gene clusters. Here, we extensively investigated the metabolite profiles of a gene disruptant of AvaR3 (an autoregulator receptor homologue), which is involved in the pleiotropic regulation of antibiotic production and cell morphology. Unlike the wild-type strain, the avaR3 mutant accumulated compound 3 in the culture. The chemical structure of compound 3 was elucidated on the basis of various spectroscopic analyses, and was identified as phthoxazolin A, a cellulose synthesis inhibitor. Bioassays demonstrated that compound 3 exerts growth inhibitory activity against a broad range of plant pathogenic oomycetes. Moreover, unlike avermectin production, phthoxazolin A (3) production was negatively controlled by avenolide, a new type of autoregulator in streptomycetes, through the function of AvaR3. These results suggest that the genetic manipulation of autoregulator receptor homologues would be a valuable tool for the discovery of cryptic bioactive compounds.

Establishment of Agrobacterium tumefaciens-mediated transformation of an oleaginous fungus, Mortierella alpina 1S-4, and its application for eicosapentaenoic acid producer breeding.

Gene manipulation tools for an arachidonic-producing filamentous fungus, Mortierella alpina 1S-4, have not been sufficiently developed. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for M. alpina 1S-4 transformation, using the uracil-auxotrophic mutant (ura5(-) strain) of M. alpina 1S-4 as a host strain and the homologous ura5 gene as a selectable marker gene. Furthermore, the gene for omega3-desaturase, catalyzing the conversion of n-6 fatty acid to n-3 fatty acid, was overexpressed in M. alpina 1S-4 by employing the ATMT system. As a result, we revealed that the frequency of transformation surpassed 400 transformants/10(8) spores, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and most of the transformants (60 to 80%) showed mitotic stability. Moreover, the accumulation of n-3 fatty acid in transformants was observed under the conditions of optimal omega3-desaturase gene expression. In particular, eicosapentaenoic acid (20:5n-3), an end product of n-3 fatty acids synthesized in M. alpina 1S-4, reached a maximum of 40% of total fatty acids. In conclusion, the ATMT system was found to be effective and suitable for the industrial strain Mortierella alpina 1S-4 and will be a useful tool for basic mutagenesis research and for industrial breeding of this strain.