The efficacy of prevention for colon cancer based on the microbiota therapy and the antitumor mechanisms with intervention of dietary Lactobacillus.

Gut microbiota and their secreted metabolites have an influence on the initiation and progression of colon cancer. Probiotics are extensively perceived as a potential microbiota-modulation strategy to promote the health of the host, while the effectiveness of preventing colon cancer based on microbiota therapy has not been confirmed, and antitumor mechanisms influenced by microbiota and their metabolites with the intervention of probiotics remain to be further investigated. In vitro, Lactobacillus (JY300-8 and JMR-01) significantly inhibited the proliferation of CT26, HT29, and HCT116 cells. Moreover, we studied the prevention and therapy efficiency of Lactobacillus and its underlying antitumor mechanism through the alteration of gut microbiota and their metabolites regulated by Lactobacillus in colon cancer models in mice. We demonstrated that the pre-administration of Lactobacillus (JY300-8 and JMR-01) for 20 days before establishing tumor models resulted in an 86.21% reduction in tumor formation rate compared to tumor control group. Subsequently, continuous oral administration of living Lactobacillus significantly suppresses tumor growth, and tumor volumes decrease by 65.2%. Microbiome and metabolome analyses reveal that Lactobacillus suppresses colonic tumorigenesis and progression through the modulation of gut microbiota homeostasis and metabolites, including the down-regulation of secondary bile acids, sphingosine 1-phosphate (S1P), and pyrimidine metabolism, as well as the production of anticarcinogenic compounds in tumor-bearing mice. Additionally, metabolome analyses of Lactobacillus (JY300-8 and JMR-01) indicate that living Lactobacillus could reduce the relative abundance of alanine and L-serine to suppress tumor progression by regulating the tumor microenvironment, including down-regulation of pyrimidine metabolism and S1P signaling in cancer. These findings provide a potential prevention strategy and therapeutic target for colon cancer through the intervention of dietary Lactobacillus. IMPORTANCE The modulation of gut microbiota and metabolites has a significant influence on the progression of colon cancer. Our research indicated that the intervention of probiotics is a potentially feasible strategy for preventing colon cancer. We have also revealed the underlying antitumor mechanism through the alteration of gut microbiota and their metabolites, which could lead to broader biomedical impacts on the prevention and therapy of colon cancer with microbiota-based therapy regulated by probiotics.

Biosynthesis and Pharmacological Activities of Flavonoids, Triterpene Saponins and Polysaccharides Derived from Astragalus membranaceus.

Astragalus membranaceus (A. membranaceus), a well-known traditional herbal medicine, has been widely used in ailments for more than 2000 years. The main bioactive compounds including flavonoids, triterpene saponins and polysaccharides obtained from A. membranaceus have shown a wide range of biological activities and pharmacological effects. These bioactive compounds have a significant role in protecting the liver, immunomodulation, anticancer, antidiabetic, antiviral, antiinflammatory, antioxidant and anti-cardiovascular activities. The flavonoids are initially synthesized through the phenylpropanoid pathway, followed by catalysis with corresponding enzymes, while the triterpenoid saponins, especially astragalosides, are synthesized through the universal upstream pathways of mevalonate (MVA) and methylerythritol phosphate (MEP), and the downstream pathway of triterpenoid skeleton formation and modification. Moreover, the Astragalus polysaccharide (APS) possesses multiple pharmacological activities. In this review, we comprehensively discussed the biosynthesis pathway of flavonoids and triterpenoid saponins, and the structural features of polysaccharides in A. membranaceus. We further systematically summarized the pharmacological effects of bioactive ingredients in A. membranaceus, which laid the foundation for the development of clinical candidate agents. Finally, we proposed potential strategies of heterologous biosynthesis to improve the industrialized production and sustainable supply of natural products with pharmacological activities from A. membranaceus, thereby providing an important guide for their future development trend.

Light-Induced Flavonoid Biosynthesis in Sinopodophyllum hexandrum with High-Altitude Adaptation.

Sinopodophyllum hexandrum is a perennial alpine herb producing the anti-cancer metabolite podophyllotoxin (PPT). Although the adaptation of S. hexandrum to high altitudes has been demonstrated and the effects of temperature, precipitation, and UV-B light on plant growth and metabolite accumulation have been studied, knowledge on the role of flavonoid biosynthesis in adapting to high altitudes is limited. In this study, light intensity, amount and type of flavonoids, and differentially expressed proteins (DEPs) and genes (DEGs) at 2300 and 3300 m were analyzed by HPLC, proteomic, transcriptomic, and qRT-PCR analysis. We found that higher light intensity correlated with greater flavonoid, flavonol, and anthocyanin content as well as higher anthocyanin to total flavonoid and flavonol ratios observed at the higher altitude. Based on proteomic and transcriptomic analyses, nine DEPs and 41 DEGs were identified to be involved in flavonoid biosynthesis and light response at 3300 m. The relative expression of nine genes (PAL, CHS1, IFRL, ANS, MYB4, BHLH137, CYP6, PPO1, and ABCB19) involved in flavonoid biosynthesis and seven genes (HSP18.1, HSP70, UBC4, ERF5, ERF9, APX3, and EX2) involved in light stress were observed to be up-regulated at 3300 m compared with 2300 m. These findings indicate that light intensity may play a regulatory role in enhancing flavonoid accumulation that allows S. hexandrum to adapt to elevated-altitude coupled with high light intensity.

L. reuteri JMR-01 adjuvant 12C6+ irradiation exerts anti-colon carcinoma effects by modulating the gut microbiota in mice.

BACKGROUND: Probiotics such as Lactobacillus could modulate the intestinal microbiota and have been considered as an effective strategy for ameliorating colon carcinoma. Nevertheless, its efficiency remains the biggest challenge. METHODS: We investigated the therapeutic efficacy of Lactobacillus reuteri JMR-01 adjuvant 12C6+ irradiation on CT-26 syngeneic mouse models. Meanwhile, intestinal flora and innate immunity were examined to outline mechanisms. RESULTS: Anti-proliferation effect of live probiotic combined with inactivated probiotic JMR-01 (LP + IP) on CT-26 reached a maximum of 39.55% among other experiment groups at 24 h when the ratio of cell to CFU was 1:1 in vitro. These activities have been fully validated in vivo, tumor-bearing mice treated by 12C6+ irradiation combining with living and inactivated probiotics JMR-01 (IR + LP + IP) for 50-day held the highest survival rate (71.4%) and complete remission rate (14.3%). We also demonstrated significant fluctuation in gut microbiota, including the decreased abundance of Bacteroides fragilis and Clostridium perfringens related to tumorigenesis and development, and the increased abundance of Lactobacillus and Bifidobacterium closely associated with health restoration in fecal of mice treated with JMR-01 LP + IP adjuvant 12C6+ irradiation (IR + LP + IP). Similarly, the decreasing nitroreductase activities and increasing short chain fatty acids (SCFAs) concentrations were observed in IR + LP + IP group compared with tumor control group, which further confirmed the changes of gut microbiota. Additionally, we found that the strongest stimulation index of splenocyte (2.47) and the phagocytosis index peritoneal macrophage (3.68) were achieved by LP + IP compared with single live JMR-01 (LP) and inactivated JMR-01 (IP). CONCLUSIONS: JMR-01 LP + IP adjuvant 12C6+ irradiation could mitigate cancer progression by modulating innate immunity as well as intestinal flora.

Integrative transcriptome and proteome analyses of Trichoderma longibrachiatum LC and its cellulase hyper-producing mutants generated by heavy ion mutagenesis reveal the key genes involved in cellulolytic enzymes regulation.

BACKGROUND: The major challenge of facing the efficient utilization of biomass is the high cost of cellulolytic enzyme, while the Trichoderma longibrachiatum plays an essential role in the production of industrial enzymes and biomass recycling. RESULTS: The cellulase hyper­producing mutants of LC-M4 and LC-M16 derived from the wild type T. longibrachiatum LC strain through heavy ion mutagenesis exhibited the high-efficiency secretion ability of cellulase and hemicellulose. The FPase activities of LC-M4 (4.51 IU/mL) and LC-M16 (4.16 IU/mL) mutants increased by 46.91% and 35.5% when compared to the LC strain, respectively. Moreover, these two cellulase hyper-producing mutants showed faster growth rate on the cellulosic substrates (Avicel and CMC-Na) plate than that of LC strain. Therefore, an integrative transcriptome and proteome profiling analysis of T. longibrachiatum LC and its cellulase hyper­producing mutant LC-M4 and LC-M16 were employed to reveal the key genes involved in cellulolytic enzymes regulation. It was showed that the transcriptome and proteome profiles changed dramatically between the wild strain and mutant strains. Notably, the overlapped genes obtained from integrative analysis identified that the protein processing in ER involved in protein secretory pathway, starch and sucrose metabolism pathway and N-glycan biosynthesis pathway were significantly changed both in cellulase hyper-producing mutants and thereby improving the enzyme secretion efficiency, which maybe the main reason of cellulase hyper-production in LC-M4 and LC-M16 mutants. In addition, the three DEGs/DEPs (PDI, Sec61, VIP36) related with protein secretion in ER and two DEGs/DEPs (OST, MOGS) related with N-glycan biosynthesis were identified as key candidate genes participating in enzyme protein biosynthesis and secretion. CONCLUSIONS: In this study, a hypothetical secretory model of cellulase protein in filamentous fungi was established on the basis of DEGs/DEPs and key genes identified from the omics analysis, which were of great guidance on the rational genetic engineering and/or breeding of filamentous fungi for improving cellulase production.

Effects of microbial inoculants on the fermentation characteristics and microbial communities of sweet sorghum bagasse silage.

Sweet sorghum bagasse (SSB) is a promising raw material for silage fermentation due to its high residual nutritive, but the efficient fermentation strategy of SSB has not been reported yet. This study evaluated the effects of microbial inoculant on the fermentation quality, chemical composition and microbial community of SSB silage. The silage inoculated with isolated lactic acid bacteria (LpE) achieved better fermentation than that of commercial inoculant A, B (CIA, CIB) and untreatment, including low pH value, high levels of lactic acid and water soluble carbohydrates (WSC) content, which demonstrated that the LpE inoculant could contribute to the preservation of nutrition and the manipulation of fermentation process of SSB. In addition, the results of microbial community analysis indicated that the LpE inoculant significantly changed the composition and diversity of bacteria in SSB silage. After ensiling, the LpE inoculated silage were dominated by Lactobacillus(95.71%), Weissella(0.19%). These results were of great guiding significance aiming for high-quality silage production using SSB materials on the basis of target-based regulation methods.

Pretreatment of sweet sorghum straw and its enzymatic digestion: insight into the structural changes and visualization of hydrolysis process.

BACKGROUND: The efficient utilization of lignocellulosic biomass for biofuel production has received increasing attention. Previous studies have investigated the pretreatment process of biomass, but the detailed enzymatic hydrolysis process of pretreated biomass remains largely unclear. Thus, this study investigated the pretreatment efficiency of dilute alkali, acid, hydrogen peroxide and its ultimate effects on enzymatic hydrolysis. Furthermore, to better understand the enzymatic digestion process of alkali-pretreated sweet sorghum straw (SSS), multimodal microscopy techniques were used to visualize the enzymatic hydrolysis process. RESULT: After pretreatment with alkali, an enzymatic hydrolysis efficiency of 86.44% was obtained, which increased by 99.54% compared to the untreated straw (43.23%). The FTIR, XRD and SEM characterization revealed a sequence of microstructural changes occurring in plant cell walls after pretreatment, including the destruction of lignin-polysaccharide interactions, the increase of porosity and crystallinity, and reduction of recalcitrance. During the course of hydrolysis, the cellulase dissolved the cell walls in the same manner and the digestion firstly occurred from the middle of cell walls and then toward the cell wall corners. The CLSM coupled with fluorescent labeling demonstrated that the sclerenchyma cells and vascular bundles in natural SSS were highly lignified, which caused the nonproductive bindings of cellulase on lignin. However, the efficient delignification significantly increased the accessibility and digestibility of cellulase to biomass, thereby improving the saccharification efficiency. CONCLUSION: This work will be helpful in investigating the biomass pretreatment and its structural characterization. In addition, the visualization results of the enzymatic hydrolysis process of pretreated lignocellulose could be used for guidance to explore the lignocellulosic biomass processing and large-scale biofuel production.

Enhancing enzymatic hydrolysis yield of sweet sorghum straw polysaccharides by heavy ion beams irradiation pretreatment.

A deeper understanding of the pretreatment process of lignocellulosic biomass could enhance the production efficiency of biofuels. Sweet sorghum straws (SSS) were subjected to heavy ion beams irradiation (HIBI) pretreatment and then hydrolyzed with 2.4 FPU of cellulase. Notably, the pretreatment has been proved to increase enzymatic digestibility of SSS. The reducing sugar yield and hydrolysis yield of SSS pretreated by 600 Gray (Gy) of HIBI reached to 7.23 mg/mL and 34.43% for 36 h, respectively, which was significantly higher than that of the untreated SSS (4.93 mg/mL of reducing sugar and 23.47% of hydrolysis yield). Additionally, the analysis of pretreated SSS showed that the destruction of amorphous region and surface ultrastructure as well as the transformation of polymorphs (Iα →Iß) of cellulose I were major effects on SSS by HIBI pretreatment. The results demonstrated that HIBI could be chosen as an effective physical pretreatment process for enhancing enzymatic hydrolysis yield of lignocellulosic biomass.

Effects of heavy-ion beam irradiation on avermectin B1a and its analogues production by Streptomyces avermitilis.

The biggest challenge in anabolism research is to improve the stability and safety of microbial metabolite production on an industrial scale. One class of metabolites, avermectins, are produced by Streptomyces avermitilis. In this study, an avermectin B1a-high-producing mutant was produced using heavy ion mutagenesis and selected based on LTQ-MS and HPLC-UV method. The mutants ZJAV-Y-147 and ZJAV-Y-HS, obtained after subjecting the spores of S. avermitilis to 70 Gy of 12C6+ heavy ion irradiation, were found to best improve the avermectin B1a production (4822.23 µg/mL and 4632.17 µg/mL, respectively). These two mutants' yielded of avermectin B1a were 2-fold high than the original strains. The DNA of the original and mutant strains were analyzed by RAPD technique with four random primers after irradiated with ion beam irradiation. The results show that different high-titer S. avermitilis strains contain different genetic modifications. In addition, the mutation position, mutation type and sequence context of all mutations of aveC, aveD, aveI, aveR gene in two mutants S.avermitilis were researched, and the production of avermectin B1a and its analogues of wild-type and mutants were analyzed by fermenting 240 h, which was suggested that the partial base deletion of aveI gene may be the key sites for increasing avermectin B1a production after the 12C6+-ion irradiation. All these modifications promote increased avermectin biosynthesis, leading to multiple high-titer S. avermitilis strains. The results demonstrate that this is an effective approach to engineer S. avermitilis as a host for the biological production of commercial analogs.

Addition of aluminum oxide microparticles to Trichoderma viride My preculture enhances cellulase production and influences fungal morphology.

Morphological engineering techniques have recently become popular, since they are used to increase the production of a variety of metabolites and enzymes when fungi are grown in submerged cultures. This study aimed to facilitate cellulase production by adding aluminum oxide to Trichoderma viride My precultures.The results showed that the highest cellulase activity was achieved when aluminum oxide at 10 g/L was used, and the activities of cellulase for filter paper and endoglucanase activity assays increased from 519.11 to 607.35 U/mL by 17.1%, and from 810.08 U/mL to 917.59 U/mL by 13.3%, compared with the control, respectively. Addition of aluminum oxide decreased the size of T. viride My pellets and increased the final pH. The changes in pellet diameter after the addition of different concentrations of aluminum oxide were fitted using a modified exponential decay model, which could precisely predict the pellet size by controlling aluminum oxide concentration.The optimum concentration of microparticles, and therefore pellet size, could significantly improve cellulase production, which is an encouraging step towards commercial cellulase production.

Study of a High-Yield Cellulase System Created by Heavy-Ion Irradiation-Induced Mutagenesis of Aspergillus niger and Mixed Fermentation with Trichoderma reesei.

The aim of this study was to evaluate and validate the efficiency of 12C6+ irradiation of Aspergillus niger (A. niger) or mutagenesis via mixed Trichoderma viride (T. viride) culturing as well as a liquid cultivation method for cellulase production via mixed Trichoderma reesei (T. reesei) and A. niger culture fermentation. The first mutagenesis approach was employed to optimize yield from a cellulase-producing strain via heavy-ion mutagenesis and high-throughput screening, and the second was to effectively achieve enzymatic hydrolysis of cellulase from a mixed culture of mutant T. viride and A. niger. We found that 12C6+-ion irradiation induced changes in cellulase biosynthesis in A. niger but had no effect on the time course of the synthesis. It is notable that the exoglucanases (CBH) activities of A. niger strains H11-1 and H differed (6.71 U/mL vs. 6.01 U/mL) and were significantly higher than that of A. niger mutant H3-1. Compared with strain H, the filter paper assay (FPA), endoglucanase (EG) and ß-glucosidase (BGL) activities of mutant strain H11-1 were increased by 250.26%, 30.26% and 34.91%, respectively. A mixed culture system was successfully optimized, and the best ratio of T. reesei to A. niger was 5:1 for 96 h with simultaneous inoculation. The BGL activity of the mixed culture increased after 72 h. At 96 h, the FPA and BGL activities of the mixed culture were 689.00 and 797.15 U/mL, respectively, significantly higher than those of monocultures, which were 408.70 and 646.98 U/mL for T. reesei and 447.29 and 658.89 U/mL for A. niger, respectively. The EG activity of the mixed culture was 2342.81 U/mL, a value that was significantly higher than that of monocultures at 2206.57 U/mL for T. reesei and 1727.62 U/mL for A. niger. In summary, cellulose production and hydrolysis yields were significantly enhanced by the proposed combination scheme.