Application of starch degrading bacteria from tobacco leaves in improving the flavor of flue-cured tobacco

Starch is an essential factor affecting the quality of flue-cured tobacco, and high starch content can affect the sensory quality and safety. Recently, the degradation of macromolecules in tobacco raw materials by using additional microorganisms to improve their intrinsic quality and safety has become a new research hotspot in the tobacco industry. However, the technical maturity and application scale are limited. Our study analyzed the correlation between microbial community composition and volatile components on the surface of tobacco leaves from 14 different grades in Fujian tobacco-producing areas. The PICRUSt software was utilized to predict the function of the microbial community present in tobacco leaves. Furthermore, dominant strains that produced amylase were screened out, and an enzyme solution was prepared to enhance the flue-cured tobacco flavor. Changes in the content of macromolecules and volatile components were determined, and sensory evaluations were conducted to assess the overall quality of the tobacco leaves. The results showed that the dominant bacterial genera on the surface of Fujian tobacco leaves were Variovorax, Sphingomonas, Bacillus, etc. Bacillus was positively correlated with various volatile components, which contributed to the sweet and aromatic flavors of Fujian flue-cured tobacco. The main genetic functions of Fujian flue-cured tobacco surface bacteria were carbohydrate metabolism and amino acid metabolism. After treating flue-cured tobacco with an enzyme preparation prepared by the fermentation of Paenibacillus amylolyticus A17 #, the content of starch, pectin, and cellulose in flue-cured tobacco decreased significantly compared with the control group. Meanwhile, the content of total soluble sugar and reducing sugar was significantly increased, and the volatile aroma components, such as 3-hydroxy--damascone, 2,3-dihydro-3,5-dihydroxy-6-methyl-4 H-Pyran-4-one, ethyl palmitate, ethyl linolenic acid, etc., were significantly increased. The aroma quality and quantity of flue-cured tobacco were enhanced, while impurities were reduced. The smoke characteristics were improved, with increased fineness, concentration, and moderate strength. The taste characteristics were also improved, with reduced irritation and a better aftertaste. In conclusion, Bacillus, as the dominant genus in the abundance of bacterial communities on tobacco surfaces in Fujian, had an essential impact on the flavor of tobacco leaves by participating in carbohydrate metabolism and finally forming the unique flavor style of flue-cured tobacco in Fujian tobacco-producing areas. Paenibacillus amylolyticus A17 #, a target strain with amylase-producing ability, was screened from the surface of Fujian flue-cured tobacco. The enzyme preparation, produced by the fermentation of Paenibacillus amylolyticus A17 #, was utilized to reduce the content of macromolecules, increase the content of water-soluble total sugar and reducing sugar, and produce a variety of crucial volatile aroma components, which had a significant improvement on the quality of tobacco leaves

Exosome‐derived uterine miR‐218 isolated from cows with endometritis regulates the release of cytokines and chemokines

Summary As an inflammation of the endometrium, endometritis can affect fertility and lead to serious economic losses in the dairy industry. Widely found in various tissues and body fluids, exosomes and exosome micro (mi)RNAs have been shown to play an important regulatory role in the immune responses. As one of differentially expressed exosome miRNAs, miR‐218 is involved in the pathogenesis of bovine endometritis. The mechanisms of miR‐218 in regulating the release of cytokines and chemokines in endometritis, however, are poorly understood. Exosomes were isolated from bovine uterine cavity fluid and verified by transmission electron microscopy. An in vitro lipopolysaccharide‐treated cell model for bovine endometritis was then established to evaluate the correlation between exosome‐derived miR‐218 and the immune responses. We demonstrated that exosomes could be used to deliver miR‐218 from endometrial epithelial cells (EECs) into the uterine microenvironment and adjacent recipient cells to modulate local immune responses. miR‐218 packaged in the exosomes secreted from EECs acts as an inhibitor by blocking immune factors such as interleukin (IL)‐6, IL‐1β, tumour necrosis factor‐α, the chemokines macrophage inflammatory genes (MIP)‐1α and MIP‐1β to maintain the immune balance in the uterus. However, uterine inflammation altered the immunoregulatory mechanism of exosome miR‐218. MiR‐218 is a potential biomarker for the detection of endometritis. Our findings also revealed a new mechanism for the development of endometritis in cows

Epidermal growth factor-mediated mitogen-activated protein kinase3/1 pathway is conducive to in vitro maturation of sheep oocytes.

Epidermal growth factor (EGF) has been shown to facilitate the in vitro maturation of sheep oocytes, and enhance embryo's capability for further development. However, such kind of molecular mechanism has not yet been elucidated. In the present study, we investigated the effect of EGF-mediated mitogen-activated protein kinases 3 and 1 (MAPK3/1) pathway on in vitro maturation of sheep oocytes. U0126, a specific inhibitor of MEK (MAPK kinase), was added into the maturation culture medium to block the EGF-mediated MAPK3/1 pathway with different doses. Then, the nuclear maturation of sheep oocytes was examined. Additionally, the effect of EGF-mediated MAPK3/1 on cytoplasmic maturation was examined though in vitro fertilization and embryonic development. The rate of germinal vesicle breakdown (GVBD) after 6 h of culture with 10⁻⁴ mol/l of U0126 (50.4%) was significantly decreased compared with control (67.2%, p < 0.05), and the first polation body (PB1) extrusion rate after 22 h of culture in drug treatment was also significantly inhibited compared with control (28.6% vs. 48.4%, p < 0.05). However, 10-6 mol/l U0126 had slight effect on oocyte nuclear maturation. The normal distribution rate of α-tubulin in the oocytes after 22 h of in vitro maturation was significantly decreased in the 10⁻⁴ mol/l U0126 group (54%) compared with control (68%, p < 0.05). After in vitro fertilization, the cleavage rate in drug treatments (56.8% in 10⁻⁶ mol/l U0126 group and 42.6% in 10⁻⁴ mol/l U0126 group) was significantly decreased compared with control (72.3%, p < 0.01). The blastocyst rate in 10⁻⁴ mol/l U0126 group (17.6%) was also significantly decreased compared with control (29.9%, p < 0.05). Collectively, these results suggest that EGF-mediated MAPK3/1 pathway is conducive to in vitro maturation of sheep oocytes

Comparative Analyses between Skeletal Muscle miRNAomes from Large White and Min Pigs Revealed MicroRNAs Associated with Postnatal Muscle Hypertrophy

<div><p>The molecular mechanism regulated by microRNAs (miRNAs) that underlies postnatal hypertrophy of skeletal muscle is complex and remains unclear. Here, the miRNAomes of <i>longissimus dorsi</i> muscle collected at five postnatal stages (60, 120, 150, 180, and 210 days after birth) from Large White (commercial breed) and Min pigs (indigenous breed of China) were analyzed by Illumina sequencing. We identified 734 miRNAs comprising 308 annotated miRNAs and 426 novel miRNAs, of which 307 could be considered pig-specific. Comparative analysis between two breeds suggested that 60 and 120 days after birth were important stages for skeletal muscle hypertrophy and intramuscular fat accumulation. A total of 263 miRNAs were significantly differentially expressed between two breeds at one or more developmental stages. In addition, the differentially expressed miRNAs between every two adjacent developmental stages in each breed were determined. Notably, ssc-miR-204 was significantly more highly expressed in Min pig skeletal muscle at all postnatal stages compared with its expression in Large White pig skeletal muscle. Based on gene ontology and KEGG pathway analyses of its predicted target genes, we concluded that ssc-miR-204 may exert an impact on postnatal hypertrophy of skeletal muscle by regulating myoblast proliferation. The results of this study will help in elucidating the mechanism underlying postnatal hypertrophy of skeletal muscle modulated by miRNAs, which could provide valuable information for improvement of pork quality and human myopathy.</p></div

Number of known miRNAs, homologous novel miRNAs, and nonhomologous miRNAs.

<p>Number of known miRNAs, homologous novel miRNAs, and nonhomologous miRNAs.</p

Pairwise comparisons of differentially expressed (DE) miRNAs in skeletal muscle at different developmental stages.

<p>The comparisons were made for Large White and Min pigs. The numbers marked in the overlapping areas indicate the common DE miRNAs.</p

Cluster analysis of miRNA libraries from skeletal muscle of Large White and Min pigs.

<p>The skeletal muscle was collected at different developmental stages. LW, Large White pig; M, Min pig.</p

Overview of the small RNA sequences in the 10 libraries.

<p>Each number in the pie chart represents the percentage of reads of that category in the total small RNA sequences.</p