Characterization, Antioxidant Capacity and Protective Effect of Peptides from Cordyceps militaris Cultivated with Tussah Pupa on Oxidative Injured HepG2 Cells.

The antioxidant capacity and protective effect of peptides from protein hydrolysate of Cordyceps militaris cultivated with tussah pupa (ECPs) on H2O2-injured HepG2 cells were studied. Results indicated ECP1 (<3 kDa) presented the strongest antioxidant activity compared with other molecular weight peptides. Pretreated with ECPs observably enhanced survival rates and reduced apoptosis rates of HepG2 cells. ECPs treatment decreased the ROS level, MDA content and increased CAT and GSH-Px activities of HepG2 cells. Besides, the morphologies of natural peptides from C. militaris cultivated with tussah pupa (NCP1) and ECP1 were observed by scanning electron microscopy (SEM). Characterization results suggested the structure of NCP1 was changed by enzymatic hydrolysis treatment. Most of hydrophobic and acidic amino acids contents (ACC) in ECP1 were also observably improved by enzymatic hydrolysis. In conclusion, low molecular weight peptides had potential value in the development of cosmetics and health food.

Characteristic analysis and fermentation optimization of a novel Aureobasidium pullulans RM1603 with high pullulan yield.

A high-yielding microbial polysaccharide-producing strain, named RM1603, was isolated from rhizosphere soil and identified by morphological and phylogenetic analysis. The extracellular polysaccharides (EPS) were identified by thin-layer chromatography and infrared spectroscopy. The fermentation conditions were optimized by single factor experiments in shake flasks and a 5-L fermentor. The results of morphological and phylogenetic tree analysis showed that RM1603 was a strain of Aureobasidium pullulans. Its microbial polysaccharide was identified as pullulan, and the EPS production capacity reached 33.07 ± 1.03 g L-1 in shake flasks. The fermentation conditions were optimized in a 5-L fermentor, and were found to encompass an initial pH of 6.5, aeration rate of 2 vvm, rotor speed of 600 rpm, and inoculum size of 2 %. Under these conditions, the pullulan yield of RM1603 reached 62.52 ± 0.24 g L-1. Thus, this study contributes RM1603 as a new isolation with high-yielding pullulan and potential application value in biotechnology.

Systemic metabolic engineering of Enterobacter aerogenes for efficient 2,3-butanediol production.

2,3-Butanediol (2,3-BDO) is an important gateway molecule for many chemical derivatives. Currently, microbial production is gradually being recognized as a green and sustainable alternative to petrochemical synthesis, but the titer, yield, and productivity of microbial 2,3-BDO remain suboptimal. Here, we used systemic metabolic engineering strategies to debottleneck the 2,3-BDO production in Enterobacter aerogenes. Firstly, the pyruvate metabolic network was reconstructed by deleting genes for by-product synthesis to improve the flux toward 2,3-BDO synthesis, which resulted in a 90% increase of the product titer. Secondly, the 2,3-BDO productivity of the IAM1183-LPCT/D was increased by 55% due to the heterologous expression of DR1558 which boosted cell resistance to abiotic stress. Thirdly, carbon sources were optimized to further improve the yield of target products. The IAM1183-LPCT/D showed the highest titer of 2,3-BDO from sucrose, 20% higher than that from glucose, and the yield of 2,3-BDO reached 0.49 g/g. Finally, the titer of 2,3-BDO of IAM1183-LPCT/D in a 5-L fermenter reached 22.93 g/L, 85% higher than the wild-type strain, and the titer of by-products except ethanol was very low. KEY POINTS: Deletion of five key genes in E. aerogenes improved 2,3-BDO production The titer of 2,3-BDO was increased by 90% by regulating metabolic flux Response regulator DR1558 was expressed to increase 2,3-BDO productivity.

Optimization of hydrogen production in Enterobacter aerogenes by Complex I peripheral fragments destruction and maeA overexpression.

As a concentrated energy source with high added value, hydrogen has great development prospects, with special emphasis on sustainable microbial production as a replacement for traditional fossil fuels. In this study, λ-Red recombination was used to alter the activity of Complex I by single and combined knockout of nuoE, nuoF and nuoG. In addition, the conversion of malic to pyruvic acid was promoted by overexpressing the maeA gene, which could increase the content of NADH and formic acid in the bacterial cells. Compared to the original strain, hydrogen production was 65% higher in the optimized strain IAM1183-EFG/M, in which the flux of the formic acid pathway was increased by 257%, the flux of the NADH pathway was increased by 13%, and the content of metabolites also changed significantly. In further bioreactor, the total hydrogen production of the scale-up IAM1183-EFG/M after 44 h of fermentation was 4.76 L, which increased by 18% compared with the starting strain. This study provides a new direction for future exploration of microbial hydrogen production by combinatorial modification of multiple genes.

Metabolomics reveals the effect of hypobaric treatment on energy metabolism in vibration-injured 'Huangguan' pears.

Mechanical damage caused by vibration during transportation can destroy organization structure and reduce the fruit quality. The objective was to reveal the mechanism of hypobaric treatment on energy metabolism in vibration-injured 'Huangguan' pears based on metabolomics. Results showed that hypobaric treatment delayed the decline of adenosine triphosphate (ATP) content, energy charge (EC), H+-ATPase and Ca2+-ATPase activities comparing to untreated samples. Metabolomics data indicated there were 83 significant differential metabolites between untreated samples and hypobaric treated ones. KEGG analysis results showed significant differential metabolites were associated with 14 pathways. Key metabolites and pathways analysis revealed these up-regulated amino acids were related to amino acid metabolism, biosynthesis of secondary metabolites and membrane transport. These pathways were activated observably by hypobaric treatment. The results indicated hypobaric treatment slowed energy consumption in vibration-injured samples, which was in relation to the accumulation of amino acids. The findings provide a feasible preservation technology for vibration-injured fruit.

Regulation of carbon flux and NADH/NAD+ supply to enhance 2,3-butanediol production in Enterobacter aerogenes.

As a valuable platform chemical, 2,3-Butanediol (2,3-BDO) has a variety of industrial applications, and its microbial production is particularly attractive as an alternative to petroleum-based production. In this study, the regulation of intracellular carbon flux and NADH/NAD+ was used to increase the 2,3-BDO production of Enterobacter aerogenes. The genes encoding lactate dehydrogenase (ldh) and pyruvate formate lyase (pfl) were disrupted using the λ-Red recombination method and CRISPR-Cas9 to reduce the production of several byproducts and the consumption of NADH. Knockout of ldh or pfl increased intracellular NADH/NAD+ by 111 % and 113 %, respectively. Moreover, two important genes in the 2,3-BDO biosynthesis pathway, acetolactate synthase (budB) and acetoin reductase (budC), were overexpressed in E. aerogenes to further amply the metabolic flux toward 2,3-BDO production. And the overexpression of budB or budC increased intracellular NADH/NAD+ by 46 % and 57 %, respectively. In shake-flask cultivation with sucrose as carbon source, the 2,3-BDO titer of the IAM1183-LPBC was 3.55 times that of the wild type. In the 5-L fermenter, the maximal 2,3-BDO production produced by the IAM1183-LPBC was 2.88 times that of the original strain. This work offers new ideas for promoting the biosynthesis of 2,3-BDO for industrial applications.

Infection Process and Genome Assembly Provide Insights into the Pathogenic Mechanism of Destructive Mycoparasite Calcarisporium cordycipiticola with Host Specificity.

Calcarisporium cordycipiticola is the pathogen in the white mildew disease of Cordyceps militaris, one of the popular mushrooms. This disease frequently occurs and there is no effective method for disease prevention and control. In the present study, C. militaris is found to be the only host of C. cordycipiticola, indicating strict host specificity. The infection process was monitored by fluorescent labeling and scanning and transmission electron microscopes. C. cordycipiticola can invade into the gaps among hyphae of the fruiting bodies of the host and fill them gradually. It can degrade the hyphae of the host by both direct contact and noncontact. The parasitism is initially biotrophic, and then necrotrophic as mycoparasitic interaction progresses. The approximate chromosome-level genome assembly of C. cordycipiticola yielded an N50 length of 5.45 Mbp and a total size of 34.51 Mbp, encoding 10,443 proteins. Phylogenomic analysis revealed that C. cordycipiticola is phylogenetically close to its specific host, C. militaris. A comparative genomic analysis showed that the number of CAZymes of C. cordycipiticola was much less than in other mycoparasites, which might be attributed to its host specificity. Secondary metabolite cluster analysis disclosed the great biosynthetic capabilities and potential mycotoxin production capability. This study provides insights into the potential pathogenesis and interaction between mycoparasite and its host.

Metabolic Engineering of Enterobacter aerogenes for Improved 2,3-Butanediol Production by Manipulating NADH Levels and Overexpressing the Small RNA RyhB.

Biotechnological production of 2,3-butanediol (2,3-BD), a versatile platform bio-chemical and a potential biofuel, is limited due to by-product toxicity. In this study, we aimed to redirect the metabolic flux toward 2,3-BD in Enterobacter aerogenes (E. aerogenes) by increasing the intracellular NADH pool. Increasing the NADH/NAD+ ratio by knocking out the NADH dehydrogenase genes (nuoC/nuoD) enhanced 2,3-BD production by up to 67% compared with wild-type E. aerogenes. When lactate dehydrogenase (ldh) was knocked out, the yield of 2,3-BD was increased by 71.2% compared to the wild type. Metabolic flux analysis revealed that upregulated expression of the sRNA RyhB led to a noteworthy shift in metabolism. The 2,3-BD titer of the best mutant Ea-2 was almost seven times higher than that of the parent strain in a 5-L fermenter. In this study, an effective metabolic engineering strategy for improved 2,3-BD production was implemented by increasing the NADH/NAD+ ratio and blocking competing pathways.

Comparison of metabolism substances in Cordyceps sinensis and Cordyceps militaris cultivated with tussah pupa based on LC-MS.

The objective of our study was to compare the chemical composition of Cordyceps sinensis (C. sinensis) and Cordyceps militaris (C. militaris) cultivated with tussah pupa by using metabonomics technology in order to clarify the similarity and difference of the two medicinal materials from the whole metabolite level. The results showed that there were 25 different metabolites among the 69 metabolites that were highly expressed in C. militaris cultivated with tussah pupa compared with C. sinensis in both positive and negative ion modes. Analysis results of partial differential metabolites pathways indicated that 16 differential metabolites were involved in multiple pathways, such as histidine metabolism, arginine biosynthesis, tyrosine metabolism, glyoxylate and dicarboxylate metabolism, phenylpropanoid biosynthesis, pyruvate metabolism, etc. Therefore, the composition of C. militaris cultivated with tussah pupa had significant advantage comparing with C. sinensis, which demonstrated that high-priced C. sinensis could be substituted with C. militaris cultivated with tussah pupa to some extent. PRACTICAL APPLICATIONS: This study comprehensively compared the chemical composition of Cordyceps sinensis (C. sinensis) and Cordyceps militaris (C. militaris) cultivated with tussah pupa by using metabonomics technology in order to clarify the similarity and difference of the two medicinal materials from the whole metabolite level. The experimental results provide a theoretical basis and scientific support for whether C. sinensis can be substituted with C. militaris cultivated with tussah pupa in clinical practice.

Effect of mechanical vibration on postharvest quality and volatile compounds of blueberry fruit.

The objective of the study was to investigate the effect of mechanical vibration on postharvest quality and volatile compounds in blueberries (Vaccinium spp. Berkeley). Ethylene production, respiratory rate, firmness, decay incidence, soluble solids content (SSC), titratable acid content (TAC), flavonoid content, total phenols content (TPC), enzyme activity and volatile compounds of blueberry fruit were determined. Results showed that mechanical vibration resulted in the increase of ethylene production, respiratory rate, decay incidence and the decrease of firmness, enzyme activity, SSC, TAC, flavonoid content, TPC in blueberries comparing with the control. Moreover, mechanical vibration improved the relative content of alcohols and decreased the relative content of esters in comparison with the control group. In conclusion, the effect of transport vibration on blueberry quality was conspicuous. The quality of blueberry fruit gradually decreased with the extension of vibration time during the transportation, which seriously reduced storage life and commodity value of blueberries.

Influence of drop shock on physiological responses and genes expression of apple fruit.

The aim of our study was to investigate the effect of drop shock on physiological responses and genes expression in harvested apple fruit stored at 20 ±â€¯2 °C. Ethylene production, respiratory rate, firmness, soluble solid content, relative electrical conductivity, LOX (lipoxygenase) activity, MDA (malondialdehyde) content, variation of volatile compounds, ethylene biosynthetic genes, and ethylene receptor genes of apple fruit were examined. The results indicated that drop shock observably resulted in the increase of ethylene production, respiratory rate, soluble solid content, relative electrical conductivity, LOX activity, MDA content and gene expression levels in apples. Furthermore, drop shock significantly decreased firmness and high-intensitive drop shock stimulated the accumulation of aldehydes and esters in harvested apples. Overall, the greater impact on apple quality is the effect of larger amplitude of shock during truck transportation, which seriously reduced storage life and quality of postharvest apples.

Effect of ultrasonic treatment combined with 1-methylcyclopropene (1-MCP) on storage quality and ethylene receptors gene expression in harvested apple fruit.

The objective of our study was to investigate the effectiveness of ultrasonic treatment combined with 1-methylcyclopropene (1-MCP) on storage quality and gene expression of postharvest apples compared with ultrasonic or 1-MCP treatment alone. Ethylene production, respiratory rate, firmness, soluble solid content (SSC), peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) activity, and ethylene receptors gene expression of apples were tested. The results indicated 1-MCP or ultrasonic treatment-restrained ethylene production and respiratory rate, improved firmness, suppressed the escalation of SSC, enhanced the activities of POD, SOD, and CAT, suppressed the gene expression level of MdETR1 and MdERS1 compared with the untreated samples. Furthermore, effect of the combination was more observable than that of ultrasonic or 1-MCP alone. In a word, the results proved the technology of ultrasonic plus 1-MCP was effective to enhance the storage quality and lengthen the life span of apples. PRACTICAL APPLICATIONS: Jonah king apple has the characteristics of crisp texture, long shelf life, high sugar content, and solidity-acid ratio, which is suitable for the taste of east and southeast Asian. However, as for a kind of respiration climacteric fruit, the commercial quality of Jonah king apple dramatically drops during the later stage of storage due to its softening, superficial scald, and perishability. One of the primary problems in apple preservation is the reduction of microbial growth, which directly affects the internal quality of apple fruit. The aim of present work is to estimate the effect of ultrasonic plus 1-MCP treatment on storage quality and life span of apple fruit, as well as to provide theoretical basis to enhance the storage quality and lengthen the life span of apples.

Effectiveness of lysozyme coatings and 1-MCP treatments on storage and preservation of kiwifruit.

The objective of the present study was to investigate the effectiveness of lysozyme coatings and 1-MCP on storage and preservation of kiwifruit stored at 4 ±â€¯1 °C and 90-95% RH for 20 d. Ethylene production, respiratory rate, decay incidence, weight loss, firmness, chlorophyll, soluble solid, titratable acid, ascorbic acid, total bacterial count, ascorbate peroxidase (APX), superoxide dismutase (SOD) and catalase (CAT) activity of treated kiwifruit were examined. The results showed that lysozyme coatings or 1-MCP treatment inhibited ethylene production and respiratory rate, delayed the increase of decay incidence, weight loss, soluble solid and total bacterial count, improved firmness, chlorophyll, titratable acid, ascorbic acid content, APX, SOD and CAT activity during the storage compared with the untreated kiwifruit in different degree. Moreover, the combined effect of lysozyme coatings and 1-MCP was more excellent than that of lysozyme coatings or 1-MCP alone. In conclusion, our present results indicated that the combined treatment of lysozyme coatings and 1-MCP may be an efficient way to improve the postharvest quality and prolong the shelf life of kiwifruit.

Evaluation of 1-methylcyclopropene (1-MCP) treatment combined with nano-packaging on quality of pleurotus eryngii.

The aim of the present study was to investigate the effect of 1-methylcyclopropene (1-MCP) combined with nano-packaging on quality of pleurotus eryngii stored at 4 ± 1 °C for 12 days. Texture, respiration rate, soluble protein, soluble sugar, weight loss, soluble solid, malondialdehyde (MDA) content, polyphenol oxidase (PPO), superoxide dismutase (SOD) and catalase (CAT) activity of treated pleurotus eryngii were determined. The results suggested that 1-MCP treatment combined with nano-packaging reduce respiration rate, weight loss and MDA content, delayed the decrease of soluble protein content, and maintained soluble sugar and soluble solid content of treated pleurotus eryngii during the storage at 4 ± 1 °C compared with the untreated samples. The efficiency of the combined treatment (1-MCP + nano-packaging) was better than that of 1-MCP or nano-packaging alone. Moreover, 1-MCP plus nano-packaging treatment effectively improved SOD and CAT activities, and suppressed the increase of PPO activity in pleurotus eryngii. Therefore, present results indicated that 1-MCP plus nano-packaging treatment may be an effective technology on maintaining commercial quality and lengthening shelf life of pleurotus eryngii.

Modulation of Actinidia arguta fruit ripening by three ethylene biosynthesis inhibitors.

The purpose of this study was to investigate the effect of postharvest application of 1-methylecyclopropene (1-MCP), 1-pentylcyclopropene (1-PentCP) and 1-octylcyclopropene (1-OCP), two structural analogues of 1-MCP, on Actinidia arguta (Siebold et Zucc.) Planch. ex. Miq. Fruit post-harvest ripening and antioxidant activity. The results showed that these two structural analogues, just as 1-MCP, exerted their effect in a concentration-dependent manner. The most effective concentration of 1-MCP, 1-PentCP or 1-OCP treatment was 1.2 µl L(-1), 1.2 µl L(-1) and 0.8 µl L(-1), respectively. But, 0.8 µl L(-1) 1-OCP was found to be more potent in postponing the appearances of respiration rate peak and ethylene production peak, delaying the softening and weight loss, suppressing the activities of ACC synthase (ACS) and ACC oxidase (ACO), maintaining higher glutathione (GSH) content, activities of glutathione reductase (GR) and ascorbate peroxidase (APX), though slightly inferior to 1.2 µl L(-1) 1-MCP.