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Background and Aim: Postbiotics are functional bioactive compounds or bioactive molecules with beneficial effects on health and functional activities in humans or livestock, produced by probiotic bacteria or yeast. Several postbiotics, including enzymes, short-chain fatty acids, amino acids, extracellular polysaccharides, microbial cell fragments, and teichoic acids, are currently being widely studied. This study aimed to explore the potential of secondary metabolites of Schleiferilactobacillus harbinensis LH 991 and Pichia kudriavzevii B-5P as lactic acid bacteria (LAB) and yeast isolated from Budu (fermented fish) which can act as postbiotics through in vitro rumen fermentation. Materials and Methods: The method used a completely randomized design 5 × 4, with five treatments and four replications. The substrate diet consisted of 60% forage and 40% concentrate. The culture mixture was 1.3 × 1011 CFU/mL with a 50%:50% ratio of S. harbinensis LH 991 and P. kudriavzevii B-5P. The inoculum concentrations used in this study were 0% (control), 1%, 2%, 3%, and 4%. Treatments are arranged based on differences in inoculum concentration as follows: T0: control (0%); T1: 1%; T2: 2%; T3: 3%; and T4: 4%. Results: The T4 group showed a significant increase (p < 0.01) in short-chain fatty acids (SCFA), including acetate, propionate, butyrate, valerate, isobutyrate, and isovalerate acids, compared with the other treatments. Meanwhile, T4 shows that there is no significant (p > 0.01) effect on in vitro digestibility (in vitro dry matter digestibility, in vitro organic matter digestibility, and in vitro crude fiber digestibility). However, a highly significant (p < 0.01) effect was on volatile fatty acid total, NH3, and microbial crude protein synthesis. Conclusion: It is concluded that the treatment with a 4% inoculum concentration (T4) containing a mixture of S. harbinensis LH 991 and P. kudriavzevii B-5P as LAB and yeast isolated from Budu (fermented fish) in 50%:50% ratio increased SCFA and rumen fermentation significantly, whereas it did not affect in vitro digestibility. Keywords: digestibility, in-vitro, postbiotics, probiotics, short-chain fatty acids.
Background and Aim: Postbiotics are functional bioactive compounds or bioactive molecules with beneficial effects on health and functional activities in humans or livestock, produced by probiotic bacteria or yeast. Several postbiotics, including enzymes, short-chain fatty acids, amino acids, extracellular polysaccharides, microbial cell fragments, and teichoic acids, are currently being widely studied. This study aimed to explore the potential of secondary metabolites of Schleiferilactobacillus harbinensis LH 991 and Pichia kudriavzevii B-5P as lactic acid bacteria (LAB) and yeast isolated from Budu (fermented fish) which can act as postbiotics through in vitro rumen fermentation. Materials and Methods: The method used a completely randomized design 5 × 4, with five treatments and four replications. The substrate diet consisted of 60% forage and 40% concentrate. The culture mixture was 1.3 × 1011 CFU/mL with a 50%:50% ratio of S. harbinensis LH 991 and P. kudriavzevii B-5P. The inoculum concentrations used in this study were 0% (control), 1%, 2%, 3%, and 4%. Treatments are arranged based on differences in inoculum concentration as follows: T0: control (0%); T1: 1%; T2: 2%; T3: 3%; and T4: 4%. Results: The T4 group showed a significant increase (p < 0.01) in short-chain fatty acids (SCFA), including acetate, propionate, butyrate, valerate, isobutyrate, and isovalerate acids, compared with the other treatments. Meanwhile, T4 shows that there is no significant (p > 0.01) effect on in vitro digestibility (in vitro dry matter digestibility, in vitro organic matter digestibility, and in vitro crude fiber digestibility). However, a highly significant (p < 0.01) effect was on volatile fatty acid total, NH3, and microbial crude protein synthesis. Conclusion: It is concluded that the treatment with a 4% inoculum concentration (T4) containing a mixture of S. harbinensis LH 991 and P. kudriavzevii B-5P as LAB and yeast isolated from Budu (fermented fish) in 50%:50% ratio increased SCFA and rumen fermentation significantly, whereas it did not affect in vitro digestibility. Keywords: digestibility, in-vitro, postbiotics, probiotics, short-chain fatty acids.
Background and Aim: Mosaicism, which is characterized by the presence of wild-type and more than one mutant allele, poses a serious problem in zygotic gene modification through the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system. Therefore, we used pig embryos to compare the gene editing efficiencies achieved by combining electroporation and lipofection using different aminopeptidase N (APN)-targeting guide RNA (gRNA) sequences. Materials and Methods: Six gRNAs (gRNA1–6) with different target sequences were designed to target APN. Zona pellucida (ZP)-intact zygotes collected 10 h after the start of in vitro fertilization (IVF) were electroporated with each gRNA to compare their gene editing efficiency. The gRNA sequences that achieved the lowest and highest mutation rates (gRNA4 and gRNA6, respectively) were selected for additional lipofection to assess gene editing efficiency following combined treatment. As ZP removal is essential for lipofection, ZP-free zygotes were electroporated with gRNA4 or gRNA6 10 h after IVF initiation, followed by lipofection with the same gRNAs 24 or 29 h after IVF initiation. The electroporated ZP-intact and ZP-free zygotes were used as controls. Results: gRNA4 and gRNA6 exhibited the lowest and highest mutation rates, respectively. gRNA4-targeted ZP-free embryos subjected to additional lipofection 29 h after IVF initiation exhibited significantly higher total and biallelic mutation rates than ZP-intact embryos that received only electroporation. Additional lipofection of gRNA6-targeted embryos had no obvious effect on mutation rates. Conclusion: Electroporation combined with lipofection using gRNAs with low mutation rates may improve gene editing efficiency in pig embryos. However, the effects may vary based on the timing of gene editing. Keywords: electroporation, guide RNA sequence, lipofection, pig embryo.
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