Optimization of Enzymatic Extraction Process of Polysaccharides from Pseudostellaria heterophylla Fibrous Roots by Response Surface Methodology and Its Pilot Application

[Objectives]To study and optimize the process conditions of enzymatic hydrolysis technology for extracting polysaccharides from Pseudostellaria heterophylla fibrous roots and its application in workshop pilot tests.[Methods]P.heterophylla fibrous roots were taken as the matrix material,and Box Behnken design was used to analyze the extraction time,composite enzyme addition amount,and liquid-solid ratio for response surface optimization experiments,and then applied to the pilot extraction of P.heterophylla fibrous roots.[Results]Response surface analysis showed that all factors had a significant impact on the experimental indicators.The optimal extraction process conditions for polysaccharides from P.heterophylla fibrous roots were extraction time of 2.7 h,compound enzyme addition of 2.5%,and liquid-solid ratio of 32.The yield of polysaccharides from P.heterophylla fibrous roots was 4.83%.The water extraction process of P.heterophylla fibrous roots extraction pilot was used as the control group for response surface optimization of the pilot experiment.The optimization results showed that the extraction time was 3 h,the amount of composite enzyme added was 2.5%,and the liquid-solid ratio was 28.The polysaccharide yield was 4.75%,an increase of 4.63%compared to the control group.[Conclusions]This paper could provide feasibility for the innovation of enzy-matic hydrolysis technology for P.heterophylla fibrous roots and its workshop pilot practice application,as well as a reference for the industrial application of its medicinal resources.

Unique characteristics of gut microbiota in black snub-nosed monkeys(Rhinopithecus strykeri) reveal an enzymatic mechanism of adaptation to dietary vegetation

DEAR EDITOR,The Myanmar or black snub-nosed monkey(Rhinopithecus strykeri) is a recently discovered and critically endangered colobus primate with an unknown gut microbiota. Here, we characterized and compared the gut microbiota of R. strykeri with those of two closely related snub-nosed monkey species.

Structural and antioxidative properties of royal jelly protein by partial enzymatic hydrolysis

The objective of this study was to investigate the structural and antioxidative properties of royal jelly protein(RJP)at different degrees of hydrolysis(DH)by partial enzymatic hydrolysis. RJP was hydrolyzed by alcalase for 0 min, 15 min, 1 h, 5 h and 8 h to obtain hydrolysates at DH of 5.34%, 11.65%, 15.19%, 21.38% and 23.91%, respectively. With the increased DH, the RJP hydrolysates showed elevated antioxidative activities. The molecular weight of RJP hydrolysates was significantly decreased but their primary backbone kept unchanged. Analysis of circular dichroism spectra revealed that the enzymolysis reduced the content of α-helix but increased the contents of β-sheet, β-turn and random coil. Meanwhile, the surface hydrophobicity and fluorescence intensity of RJP hydrolysates were decreased and a red shift occurred. As the enzymolysis continued, the surface morphology of RJP was gradually changed from a sheet-like structure into microparticles. Changes in antioxidative activities and structures generally followed a DH-dependent manner, however these changes became insignificant for samples at DH beyond 20%. Taking into consideration of both effectiveness and productivity, the optimum enzymatic duration was determined at 5 h.

Enzymatic hydrolysis of silkworm pupa and its allergenicity evaluation by animal model with different immunization routes

Silkworm pupa is a nourishing food with high nutritional value,but its consumption has been greatly limited given its allergenicity.Enzyme hydrolytic technique is recognized as an effective method to reduce the allergenicity of protein.In this study,we aimed to investigate the effect of enzymolysis on the allergenicity of silkworm pupa.Crude silkworm pupa protein was extracted through alkali extraction and acid precipitation,which included 5 proteins with the molecular weights ranging from 34 kDa to 76 kDa,and silkworm pupa were then hydrolyzed by alkaline protease.The allergenicity of silkworm pupa protein and its enzymatic hydrolysates was evaluated by establishing BALB/c mice model,and the mice were immunized via intragastric gavage and intraperitoneal injection,respectively.The results indicated that the intraperitoneal inj ection immunization route induced more by detecting with antibodies,histamine and Th2-related cytokines.Moreover,mice treated with silkworm pupa protein peptide displayed no obvious allergic symptoms,indicating that enzyme hydrolytic technique could significantly reduce the allergenicity of silkworm pupa.

Semi-Automated Enzymatic Determination of Ethanol in Beverages: Collaborative Study for RIDA®CUBE Ethanol

Easy and quick methods to quantify ethanol reliably in beverages are always important. In 2022, the EnzytecTM Liquid Ethanol test kit was approved as AOAC Official MethodSM 2017.07 Final Action after a collaborative study was conducted with different beverages such as kombucha, juices, and beer. During set-up of this collaborative test, small sized companies asked to include the RIDA®CUBE Ethanol/RIDA®CUBE SCAN device since it is easy to use, suitable for a few samples only and contains the identical reagents as the EnzytecTM Liquid system. It is applicable to quantify ethanol in diluted kombucha, fruit juices, and alcohol-free beer samples around 0.5% alcohol-by-volume within 12 min. The overall relative reproducibility standard deviation across a wide concentration range for kombucha, was calculated to be 6.29%. Analysis of juices and beer showed an overall higher variation with an estimated overall RSD(R) value by regression of 14.4%. The data obtained by this collaborative study show that the RIDA®CUBE Ethanol in combination with the RIDA®CUBE SCAN device is suitable to quantify ethanol from matrices representing important alcohol-free liquid food categories.

Enzymatic debridement shall not modify the global strategy for mass burn events

We read with interest the letter by Surowiecka et al.[1]about early burn wound excision in mass casualty events.We couldn’t agree more with their statement about the benefit of early burn wound excision.Still,we doubt whether applying this strategy to every patient during a mass burn event could be realistic.Of note,while there is an undisputed consensus that early burn wound excision is the gold standard of burn care,what‘early’actually means is still debated.Depending on the authors,the corresponding time limit typically varies from 24 h to a few days[2,3].

Enzymatic preparation of mono-and diacylglycerols:A review

Monoacylglycerols(MAGs) and diacylglycerols(DAGs) are partial glycerides widely used in food industry. They are safe and non-toxic food emulsifiers, especially for MAGs. MAGs account for approximately 75% of the total emulsifiers in food industry worldwide. DAGs are recognized as functional cooking oils, they can suppress body fat accumulation and postprandial serum triacylglycerols(TAGs) level. The traditional production of MAGs and DAGs is based on the chemical method, which requires high reaction temperature usually up to 200–260 ℃. Such high temperature is not suitable for oil containing heat sensitive polyunsaturated fatty acids. Enzymatic approach has been received increasing attentions. Enzymatic production of partial glycerides to replace chemical processes has been in industry, particularly for DAGs production as the products have been claimed as a functional and nutritional oil. Enzyme technology for the processing of oils and fats has been moved to industry step by step and case by case during the last 20 years. More and more applications are particularly moving into bulky oils and fats processing. At the same time, the cost of enzymes as a commercial product is reducing steadily. This review summarized the recent 15 years advances on the the enzymatic preparation of MAGs and DAGs. The critical process parameters under different reaction routes were presented and emphasized. The reaction media not only increased the homogeneity of the reaction system, but also shifted the reaction equilibrium towards the target product generation, and this part was stated in detail. In addition, the patent evaluation was included, and the application of MAGs and DAGs was covered.

Polyphenols extracted from Aronia melanocarpa by combined enzymatic and ultrasonication and their antioxidant potential and antimicrobial activity

The extraction of polyphenols from Aronia melanocarpa was carried out using a combination of enzymatic and ultrasound.After single-factor and orthogonal design and experiment,the optimized polyphenol extraction conditions were 1%enzyme,1:40 material-to-liquid ratio,55℃,60 min ultrasonication,70%ethanol,and the final extraction amount was 88.634 mg/g,which displayed a 25.15%and 34.08%improvement compared with the single ultrasonication and enzymatic extraction methods,respectively.Significant antibacterial effects of polyphenols were shown against Staphylococcus aureus,Escherichia coli and Bacillus subtilis.Further antioxidation effects were evaluated,and the superoxide anion radical scavenging rate,hydroxyl radical scavenging rate and DPPH free radical scavenging rate reached 45.2%,83.5%and 85.4%,respectively.This combined enzymatic and ultrasonic extraction method exhibited the advantages of high extraction rate,saving solvent consumption and extraction time,but also provided a new method for the development and utilization of natural antimicrobial and antioxidant health products.

Enzymatic Transformation of Notoginsenoside Fe by β-Glucanase

Aim An industrial enzyme β-glucanase was used to transfortn notoginsenoside Fe for the first time. Methods Notoginsenoside Fe was isolated from the leave saponin of Panax notoginseng （Burk.） Chen FH. The enzymatically transformed compounds were detected by HPLC and two transformed compounds were identified as 20 （S） -protopanaxadiol-20- O- α-L-arabinofuranosyl （ 1→6 ） - β-gluco- pyranoside, ginsenoside-Mc） and 20（S）-protopanaxadiol-20-O-β-D-glucopyranoside compound-K （C-K） respectively on the basis of their ^1H NMR and ^13 C NMR spectral data. Results Based on the enzymolytic kinetic curve, the transformation rate of notoginsenoside Fe reached 95% after 24 h. Conclusion The enzymatic transformation pathway of notoginsenoside Fe by β-glucanase has been proposed as notoginsenoside Fe→ginsenoside Mc→C-K.

Enzymatic Degradation of Parvifloside

Parvifloside (1), a new furostanol pentaglycoside, was isolated from the fresh rhizomes of Dioscorea parviflora C. T. Ting. On the basis of spectroscopic and chemical methods, its structure was elucidated as (25R)-26-O-β-glucopyranosyl-furost-5-en-3β,22ξ,26-triol 3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosyl (1→4)-[α-L-rhamnopyranosyl (1→2)]-β-D-glucopyranoside. Six prosapogenins (2-7)were obtained from the enzymatic degradation of 1by cellulase, but only 3 and 4 were obtained by β-glucosidase. The structures of all compounds were determined by spectroscopic data. The activity of the isolated compounds on deformation of mycelia germinated from Pyricularia oaryzae P-2b conidia was evaluated.

Isolating and Screening of Effective Decomposing Strain of Silkworm Chrysalis Protein and Study on Parts of Its Enzymatic Properties

[Objective] The research aimed to obtain an effectively-decomposing strain of silkworm chrysalis protein and discuss its enzymatic properties.[Method] The effectively-decomposing bacteria of protein was isolated from the decayed silkworm chrysalis by using dilution plate and its enzymatic properties were tested after primary screening and second screening.The enzyme activity was determined and the intermediate and small molecule protein content in silkworm chrysalis was measured after solid-state fermentati...

Studies on the Isolation and Screening of α-amylase Producing Strain and Their Enzymatic Properties

[Objective] The aim was to obtain α-amylase producing strains with some excellent properties like high temperature resistance,strong acid resistance,strong alkali resistance,etc..[Method] α-amylase producing strains were isolated and screened; furthermore their enzymatic properties were studied.[Result] 10 strains with an obvious starch hydrolysis cycle were screened out from starch screening plate coated by diluted sample,from which 3 strains with higher α-amylase activity were screened out,that was X6,X8 and X10.As for X6,X8 and X10,their optimum pH values all belonged to neutral,and their optimum temperatures were all 60 ℃.Meanwhile,Ca^2＋ could increase their enzyme thermal stability.When the concentration of Ca^2＋ was 0.02-0.04 mol/L,the enzyme thermal stability of X6 and X8 reached the highest; When the concentration of Ca^2＋ was 0.03-0.04 mol/L,that of X10 reached the highest; When the concentration of Ca^2＋ was increased continuously,those of the 3 strains all decreased.[Conclusion] The research provides theoretical basis for satisfying the demands of different industries for α-amylase with different characteristics.

Advances in Application of Non-aqueous Phase Enzymatic Catalysis in Food Additive Production

Non-aqueous phase enzymatic catalysis technology has been widely ap- plied in the area of food additives production. This paper reviewed the types of re- action medium of non-aqueous phase enzymatic catalysis reaction, introduced the application of non-aqueous phase enzymatic catalysis technology in catalysis of L-ascorbic （isoascorbic） acid esters, short-chain acid esters, sugar esters, vitamin A esters, vi- tamin E esters, and other food additives, and finally predicted the prospects of non- aqueous phase enzymatic catalysis technology.

Effects of Chemical Pretreatment on Enzymatic Saccharification and Fermentation of Forages

[ Objective] To study the effect of pretreatment with chemical substances on enzymatic saccharification of affalfa （ Medic, ago sativa L. ）, sorghum hybrid sudan grass [ Sorghum bicolor （ L. ） Moench x Sorghum sudanese （Piper） Stapf], erect milkvetch （Astraga/us adsurgens Pall. ） and pearl millet （ Pennisetum americanum （ L. ） Leeke）. [ Method ] The forages were pretreated with sulfuric acid at different concentration, and then the content of cellulose, hemicellulose, and lignin were detected and compared with that before pretreatment. The concentration of glucose and ethanol after different fermentation time was also determined. [ Result] After pretreatment, the content of cellulose increased, while that of hemicel- lulose and lignin decreased. After treatment with 1.0% （W/V） sulfuric acid, the four kinds of forages all had the highest concentration of ethanol in the citric acid-sodium citrate buffer system （pH 4.8）. Dudng fermentation process, the concentration of glucose and ethanol first increased and then declined, peaking respectively at 24 h and 48 h post fermentation. [Condusion] Pretreatment promotes the enzymatic saccharification and fermen- tation of alfalfa, sorehum hvbrid sudan orass. Dead millet, and erect milkvetch, and their enerov performance decreases in order.

Effect of Chitosan-Cysteine Conjugate on Enzymatic Degradation and Hypoglycemic effect of Insulin

Aim To evaluate the inhibitory effect of chitosan-cysteine conjugate onenzymatic degradation and hypogly-cemic enhancement effect of insulin. Methods Chitosan-cysteineconjugate was synthesized. The protective effect of the conjugate against degradation of insulin byα-chymotrypsin and trypsin was evaluated in vitro. Insulin enteric- microspheres were prepared byusing O_1 /Q_2 emulsion solvent evaporation method. The hypoglycemic enhancement effect of theconjugate was studied by oral administration of insulin solution or enteric-microspheres to rats.Results The thiol group content of the synthesized conjugate was about 200 μmol·g^(-1) polymer,which showed a strong protective effect on insulin from enzymatic degradation in vitro. Almost allthe insulin incubated in a-chymotrypsin solution or trypsin solution without chitosan-cysteineconjugate was degraded entirely within 1 h and 5 h respectively, whereas above 75% of insulinremained in the same content of the enzymatic solution containing 4 mg·mL^(-1) conjugate. The drugloading of insulin enteric-microspheres was about 7% . In vivo experiment, chitosan-cysteineconjugate (85 μg·kg^(-1)) prolonged the hypoglycemic time of insulin solution orenteric-microspheres when administered simultaneously with the absorption enhancer SNAC. ConclusionChitosan-cysteine conjugate has a marked inhibitory effect on the enzymatic degradation of insulinin vitro, and it displays a significant hypoglycemic enhancement effect on insulin oral formulationin vivo.

Enhanced Enzymatic Hydrolysis of Miscanthus sinensis Following Synergic Pretreatment with ^(60)Co γ-ray Irradiation and Alkaline Hydrogen Peroxide

[Objective] This study aimed to investigate the effects of different pretreat- ments on enzymatic saccharification of Miscanthus sinensis and improve reducing sugar yield in the enzymolysis process. [Method] M. sinensis was pretreated with 60Co y-ray irradiation and alkaline hydrogen peroxide, to analyze their effects on re- ducing sugar yield of enzymatic hydrolysis. [Result] After pretreatment with 400 kGy 60Co y-ray irradiation, reducing sugar yield in the enzymolysis process of M sinensis was 76.24 mg/g; after synergic pretreatment with 400 kGy 60Co y-ray irradiation and alkaline hydrogen peroxide, reducing sugar yield in the enzymolysis process of M. sinensis was 505.08 mg/g, which was improved by 5.6 times compared to that in pretreatment with 400 kGy 60Co y-ray irradiation. Based on process optimization, the optimal hydrolysis conditions were obtained： pretreatment temperature 30 ℃, NaOH concentration 1.2%, hydrogen peroxide concentration 2%, pretreatment time 6 h. [Conclusion] Synergic pretreatment with 60Co y-ray irradiation and alkaline hydrogen peroxide could significantly improve reducing sugar yield in the enzymolysis process of M. sinensis, which provided a new theoretical basis for preparing fuel ethanol with M. sinensis.

Soil enzymatic activities and microbial community structure with different application rates of Cd and Pb

This study focused on the changes of soil microbial diversity and potential inhibitory effects of heavy metals on soil enzymatic activities at different application rates of Cd and/or Pb. The soil used for experiments was collected from Beijing and classified as endoaquepts. Pots containing 500 g of the soil with different Cd and/or Pb application rates were incubated for a period of 0, 2, 9, 12 weeks in a glasshouse and the soil samples were analyzed for individual enzymes, including catalase, alkaline phosphatase and dehydrogenase, and the changes of microbial community structure. Results showed that heavy metals slightly inhibited the enzymatic activities in all the samples spiked with heavy metals. The extent of inhibition increased significantly with increasing level of heavy metals, and varied with the incubation periods. The soil bacterial community structure, as determined by polymerase chain reaction- denaturing gradient gel electrophoresis techniques, was different in the contaminated samples as compared to the control. The highest community change was observed in the samples amended with high level of Cd. Positive correlations were observed among the three enzymatic activities, but negative correlations were found between the amounts of the heavy metals and the enzymatic activities.

Enzymatic vitrectomy for diabetic retinopathy and diabetic macular edema

The aim of this paper is to determine the role of enzymatic vitrectomy performed by intravitreal injection of autologous plasmin enzyme(APE)in the management of diabetic retinopathy and diabetic macular edema(DME).Diabetic patients with proliferative diabetic retinopathy or DME and evident posterior hyaloid adherence to the retinal surface were included.All cases were treated with an initial intravitreal injection of APE and reevaluated one month later,measuring changes in best-corrected visual acuity(BCVA),macular thickness and the status of the posterior hyaloid.A second APE injection was performed in cases with no evident posterior vitreous detachment(PVD)after the initial treatment.Sixty-three eyes were included in the present review.A complete PVD appeared in 38%of cases(24 eyes)after one injection of plasmin and the total increased to 51%(32 eyes)after the second injection,separated at least by one month.The central macular thickness improved in all cases(100%)and BCVA in89%.Finally,in 50%of eyes with proliferative diabetic retinopathy,a high reduction of new vessels regression was observed.Enzymatic vitrectomy could be considered a good therapeutic alternative in diabetic retinopathy and macular edema.

Cloning,expression in Escherichia coli,and enzymatic properties of laccase from Aeromonas hydrophila WL-11

A strain WL-11 with high laccase activity was isolated from activated sludge collected from the effluent treatment plant of a textile and dyeing industry. It was identified as Aeromonas hydrophila by physiological test and 16S rDNA sequence analysis. A gene encoding of laccase from a newly isolated Aeromonas hydrophila WL-11 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 1605 bp encoding a polypeptide comprised of 534 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. The predicted amino acid sequence showed a high homology （more than 60%） with bacterial laccases in the genome and protein databases and the highest degree of similarity （61% identity） was observed with the multicopper oxidase of KlebsieUa sp. 601. When expressed in Escherichia coli, the recombinant enzyme was overproduced in the cytoplasm as soluble and active form. The purified enzyme had an optimum pH of 2.6 and 8.0 for ABTS （2,2＇-azino-bis（3-ethylbenzthiazolinesulfonic acid） and DMP （2,6-dimethoxyphenol）, respectively. The kinetic study on ABTS revealed a higher affinity of this enzyme to this substrate than DMP.

Thinning intensity affects microbial functional diversity and enzymatic activities associated with litter decomposition in a Chinese fir plantation

Microbial functional diversity and enzymatic activities are critical to maintaining material circulation during litter decomposition in forests.Thinning,an important and widely used silvicultural treatment,changes the microclimate and promotes forest renewal.However,how thinning affects microbial functional diversity and enzymatic activities during litter decomposition remains poorly understood.We conducted thinning treatments in a Chinese fir plantation in a subtropical region of China with four levels of tree stem removal（0,30,50,and 70%）,each with three replicates,and the effects of thinning on microbial functional diversity and enzymatic activities were studied 7 years after treatment by collecting litter samples four times over a 1-year period.Microbial functional diversity and enzymatic activities were analyzed using Biolog Ecoplates（Biolog Inc.,Hayward,CA,USA）based on the utilization of 31 carbon substrates.Total microbial abundance during litter decomposition was lower after the thinning treatments than without thinning.Microbial functional diversity did not differ significantly during litter decomposition,but the types of microbial carbon-source utilization did differ significantly with the thinning treatments.Microbial cellulase and invertase activities during litter decomposition were significantly higher under the thinning treatments due to changes in the litter carbon concentration and the ratios of carbon and lignin to nitrogen.The present study demonstrated the important influence of thinning on microbial activities during litter decomposition.Moderate-intensity thinning may maximize vegetation diversity and,in turn,increase the available substrate sources for microbial organisms in litter and promote nutrient cycling in forest ecosystems.