[Objective]In order to improve expression vector of L.lactis at food grade and widen application range of original system.[Method] Taking pNZ8149 as basis,the promoter PnisA of pNZ8149 was inserted L.lactis MG1363 and...[Objective]In order to improve expression vector of L.lactis at food grade and widen application range of original system.[Method] Taking pNZ8149 as basis,the promoter PnisA of pNZ8149 was inserted L.lactis MG1363 and SPusp45 of unknown secretory protein in downstream.Through PCR technology,specific primers were used to delete restriction sites between promoter sequence and signal peptide gene sequence and ensure better distance between SD sequence and start codon to construct secreting expression vector pNZS.The reporter gene gus was recombined into multiple cloning site of pNZS to construct pNZS-gus and L.lactis was transformed by electroporation.10 ng/ml nisin was used for induction culture,then culture solution was conducted GUS staining test.[Result]The new constructed L.lactis N3900/pNZS-gus system could express active GUS protein and GUS protein could be secreted out of cell.[Conclusion]The successful construction of this system lays foundation for secretion expression study of protein and oral vaccine research.展开更多
The flotation separation of chalcopyrite from monoclinic pyrrhotite using food-grade guar gum(FGG) as a depressant was studied through flotation tests, kinetic studies, dynamic potential measurements, adsorption exper...The flotation separation of chalcopyrite from monoclinic pyrrhotite using food-grade guar gum(FGG) as a depressant was studied through flotation tests, kinetic studies, dynamic potential measurements, adsorption experiments, and infrared spectral analyses. The microflotation results showed that the flotation separation of chalcopyrite from monoclinic pyrrhotite could not be realized by adding mixed aerofloat(CSU11) alone. The depressant FGG exhibited a selective depression effect on monoclinic pyrrhotite by controlling the pulp pH range from 5.0 to 6.0, with a maximum floatability variation of 79.36% in the presence of CSU11. The flotation kinetics, zeta-potential, adsorption, and infrared spectroscopy studies revealed that the FGG could absorb more strongly on the surface of monoclinic pyrrhotite than on the surface of chalcopyrite. In addition, the results revealed that the interaction of FGG with the monoclinic pyrrhotite surface was governed primarily by strong chemisorption, whereas FGG mainly bonded to chalcopyrite through hydrogen bonding. This difference was responsible for the excellent depression selectivity of FGG toward monoclinic pyrrhotite flotation and weak depression effect toward chalcopyrite flotation.展开更多
The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi...The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase （GALl） and phosphoglycerate kinase 1 （PGK1） promoters in different orientations was constructed. The β-1,3-1,4-glucanase gene from Bacillus subtilis was fused to α-agglutinin and ex- pressed under the control of the GALl promoter, α-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the α-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of β-1,3-1,4-glucanase. The analysis of β-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that β-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed β-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of β-1,3-1,4-glucanase displayed in the recombinant yeast cells was en- hanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.展开更多
Construction of a food-grade expression vector for application to lactic acid bacteria（LAB） is of importance for dairy fermentation system. An α-galactosidase（aga） gene encoding an enzyme degrading melibiose was ...Construction of a food-grade expression vector for application to lactic acid bacteria（LAB） is of importance for dairy fermentation system. An α-galactosidase（aga） gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid p RAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36 e to substitute the p rimary antibiotic selectable marker of pMG36 e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long α-amylase（amy） gene from Ba cillus li cheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. Th e selection efficiency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU ？g-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putati ve molecular weight of α-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of α-amylase by expressing of amy gene of pMG36-aga-amy.展开更多
L-arabinose isomerase (L-AI) is the key enzyme for D-galactose isomerization of D-tagatose by biological method. In this research, Lactobacillus plantarum WU14 with high D-tagatose yield was identified as Lactobacillu...L-arabinose isomerase (L-AI) is the key enzyme for D-galactose isomerization of D-tagatose by biological method. In this research, Lactobacillus plantarum WU14 with high D-tagatose yield was identified as Lactobacillus plantarum was isolated from the number of lactic acid bacteria from pickled vegetables. The crude L-arabinose isomerase activity of Lactobacillus plantarum WU14 with high D-tagatose yield was 13.95 U/mL under the optimal temperature 60°C, pH 7.17 and substrate concentration 0.8 mol/L, and the conversion rate of 56.12% could be gained after 28 hours. Protein structure and specific of L-Arabinose Isomerase of Lactobacillus plantarum WU14 were researched. The results showed that L-arabinose isomerase is mainly composed of alpha helix and random coil. Then the recombinant L-AI gene was inserted into the food-grade expression vector pRNA48 and expressed in L. lactis NZ9000 successfully. The target protein expression reached the maximum amount when the induced concentration of nisin reaches 30 ng/mL after 12 h. And the crude enzyme activity of recombinant bacteria reached 6.21 U/mL under 60°C. Otherwise the optimal conversion rate recombinant of L. lactis NZ9000/pRNA48-L-AI can reach 39.21% under the temperature of 50°C, pH 7.17 and D-galactose concentration was 0.6 mol/L.展开更多
In this paper, processes for producing a food-grade glucose solution through enzymatic hydrolysis of celluloserich solids obtained from rice straws are presented. The rice straws were pretreated by acid-catalyzed stea...In this paper, processes for producing a food-grade glucose solution through enzymatic hydrolysis of celluloserich solids obtained from rice straws are presented. The rice straws were pretreated by acid-catalyzed steam explosion, and the reaction efficiency, toxicity control, and process economic feasibility were studied. Mass transfer resistance to the hydrolysis reaction was reduced by grinding with glass beads. A higher glucose concentration could be obtained by feeding more cellulose in the hydrolysis reaction; however, this also resulted in the production of undesired byproducts. Thus, a soaking process for the cellulose solids in water was developed to effectively reduce the generation of byproducts in the hydrolysis reaction. The resulting food-grade glucose solution can provide 414 kilocalories per liter, and could be used during a food-shortage crisis in the future.The current production cost is estimated to be 0.82 USD·L^(-1).展开更多
As the utilization of maltodextrins in food,pharmaceuticals,and agriculture continues to expand,substantial research has been conducted to enhance their functionality.Among the methods presented,the one-pot approach f...As the utilization of maltodextrins in food,pharmaceuticals,and agriculture continues to expand,substantial research has been conducted to enhance their functionality.Among the methods presented,the one-pot approach for the synthesis of nonreducing maltoheptaose offers a novel solution to the challenge of maltodextrins with varying degrees of polymerization and reducing ends.Nevertheless,the key enzyme in this method was currently only expressed in Escherichia coli,which restricts the applicability of this method in the food and pharmaceutical industries.In this study,the food-grade expression of cyclomaltodextrinase(CDase,EC 3.2.1.54),one of the key enzymes,was achieved using Bacillus subtilis as a host.The enzymatic properties of the recombinant CDase were then investigated,and the extracellular secretion of the CDase was enhanced in order to make it more widely available for use in the food industry.The enzyme exhibited optimal activity at a pH of 8.0 and a temperature range of 35-45℃.After incubation at 25-35℃for 10 h,90%of the enzyme activity was retained.Additionally,the enzyme retained 80%of its initial activity after 24 h at pH 5.5-9.5.Finally,Cu2+completely inhibited the enzyme activity.The extracellular secretion efficiency of recombinant CDase was significantly increased by the addition of Mn^(2+)to the fermentation medium.The percentage of extracellular enzyme activity increased to 63.75%when the final concentration of Mn^(2+)in the fermentation medium was 5 mM,which was 5.3-fold higher than that of the unadded one.展开更多
Food-grade tracers have been developed as an identification technology for grain traceability from original harvest to final destination for transportation.The characteristics of food-grade tracers must be able to sat...Food-grade tracers have been developed as an identification technology for grain traceability from original harvest to final destination for transportation.The characteristics of food-grade tracers must be able to satisfy the environmental demands for grain traceability.To optimize the food-grade tracer production process,the effects of direct compression formulation and load on the mechanical characteristics were studied using response surface methodology(RSM)with central composite design(CCD).Among the four tested formulations,Formulations#2(consisting of 35.00%lactose 100 mesh,64.50%microcrystalline cellulose 102 and 0.50%magnesium stearate)and#4(consisting of 38.00%lactose 100 mesh,50.00%microcrystalline cellulose 102,11.00%pregelatinized starch and 1.00%magnesium stearate)were selected for tracer production based on their physical properties as powders.The value of Carr’s flowability index was 68 for both Formulations#2 and#4,which was the highest among all the formulations.Therefore,Formulations#2 and#4 also had the best powder flowability.The magnesium stearate ratio(1.00%-3.00%)and pressure(6.00-16.00 kgf)were used as independent variables to detect changes in the breaking rate,peak shear force and friction coefficient of tracers compressed by the selected formulations.The optimal production parameters could be achieved at a magnesium stearate ratio of 2.25%and pressure of 16.00 kgf for Formulation#2 and at a magnesium stearate ratio of 1.02%and pressure of 16.00 kgf for Formulation#4.Under these optimal conditions,the tracers had good impact characteristics(breaking rate),compression characteristics(peak shear force)and frictional characteristics(friction coefficient).Moreover,Formulation#2 was more suitable for production because compared to Formulation#4,its breaking rate and friction coefficient values were lower,and its peak shear force value was higher.展开更多
The purpose of this study was to optimize the coating process of food-grade tracers to manufacture tracers with good physical,mechanical and practical properties and an excellent appearance.The effects of the coating ...The purpose of this study was to optimize the coating process of food-grade tracers to manufacture tracers with good physical,mechanical and practical properties and an excellent appearance.The effects of the coating weight gain(1.00%-5.00%),coating solution spray rate(1.50-7.50 g/min)and tablet bed temperature(30℃-40℃)on the coating appearance quality,moisture absorption rate,friction coefficient,peak shear force,breaking rate,barcode recognition rate,transport wear rate and transport recognition rate were analysed using a Box-Behnken design(BBD)of response surface methodology(RSM).The experimental data were fitted to quadratic polynomial models by multiple regression analysis.The mathematical models of the barcode recognition rate,transport wear rate and transport recognition rate exhibited no statistically significant difference in these data.The optimum coating parameters were as follows:a 5.00%coating weight gain,spray rate of 5.47 g/min and tablet bed temperature of 35.42℃.Under the optimized conditions,the tracers had a good appearance(coating appearance quality),moisture resistance(moisture absorption rate),and frictional(friction coefficient),compression(peak shear force),and impact characteristics(breaking rate).展开更多
Mycotoxins in grains pose severe threat to human and animal health.Previous studies showed that manganese peroxidases had degradation capability for various mycotoxins.In this work,to effectively detoxify aflatoxin B1...Mycotoxins in grains pose severe threat to human and animal health.Previous studies showed that manganese peroxidases had degradation capability for various mycotoxins.In this work,to effectively detoxify aflatoxin B1(AFB1),zearalenone(ZEN)and deoxynivalenol(DON),the manganese peroxidases PhcMnp and IrlMnp from Phanerochaete chrysosporium and Irpex lacteus were successfully expressed in Pichia pastoris in a food-grade manner,and were used for degradation tests with the three mycotoxins.The fermentation supernatants containing PhcMnp achieved degradation ratios of 76.56%,70.78%and 48.93%for AFB1,ZEN and DON respectively,while that containing IrlMnp achieved degradation ratios of 46.72%,45.13%and 41.64%correspondingly.The inducible expression conditions for the enzymes and the reaction parameters for the mycotoxin degradations were both optimized.Furthermore,the fermentation supernatants of strain Pichia pastoris GS115(pPIC9K-Phcmnp)were used for mycotoxin degradations in contaminated peanut samples(pH 4.5)at 40℃ for 10 h,resulting in apparent detoxification effects.The residual concentrations of AFB1,ZEN and DON in peanut samples after degradations were 9.42,127.68 and 636.71μg/kg respectively,all of which remained below the legal limits.Results in this study demonstrated that the enzymes PhcMnp and IrlMnp have potentials in mycotoxins detoxification in feed industry.展开更多
Protein-glutaminase(PG)is an enzyme used to enhance the functional properties of food proteins,such as emulsification,solubility and foaming.However,the use of PG in the food industry is limited because of its low cat...Protein-glutaminase(PG)is an enzyme used to enhance the functional properties of food proteins,such as emulsification,solubility and foaming.However,the use of PG in the food industry is limited because of its low catalytic activity and expression levels.In this study,a mutant library of PG was systematically constructed and screened through a structure-guided semi-rational design.This resulted in the identification of a mutant strain,A291S,which exhibited a 45.71%increase in activity.Moreover,a food-grade expression system for PG was established in Bacillus subtilis,demonstrating PG expression levels comparable to those obtained using plasmid-based expression systems that utilized antibiotics.The activity of PG remained above 99%even after the plates were incubated at ambient temperature for 5 days.However,the antibiotic-based expression system retained only 36%of PG activity.The efficacy of fed-batch fermentation for PG production was also evaluated and a maximum activity of 8.15 U/mL was obtained.These findings highlight the significance of PG modification and potential applications of the food-grade expression system in the food industry.展开更多
γ-Aminobutyric acid(GABA)is a bioactive compound with diverse physiological functions.It has a wide range of applica-tions in food and medicine and mainly biosynthesized through glutamate decarboxylase(GAD)catalysis....γ-Aminobutyric acid(GABA)is a bioactive compound with diverse physiological functions.It has a wide range of applica-tions in food and medicine and mainly biosynthesized through glutamate decarboxylase(GAD)catalysis.Bacillus subtilis is recognized for its robust secretion capabilities and high food safety standards,making it a prevalent choice for recombinant protein expression.In traditional industrial enzyme production in B.subtilis,antibiotics are required to sustain plasmid stability.However,the incorporation of antibiotics fails to align with the criteria applicable to enzymes for use in the food industry.To eliminate the need for antibiotics in the production of GAD preparations,we constructed a marker-free recom-binant strain B.subtilis WS9D-GAD.This strain was developed based on antibiotic-free multi-copy gene expression vector pUBDAL-amyL and d-alanine racemase(dal)-deficient B.subtilis WS9D,ultimately enabling the food-grade expression of GAD.To enhance GAD expression levels,we integrated the gadA expression cassette into the genome of B.subtilis using the Cre/lox system method.Additionally,strain WS9C6D-GAD was generated through the co-expression of free plasmid and genome integration,which carried the free plasmid pUBDAL-gadA and featured six copies of the gadA expression cassette within its genome.The enzyme activity during shake flask fermentation reached 28.15 U mL^(−1),while the enzyme activity in high-density 3-L fermenter culture reached 199.49 U mL^(−1),marking the highest level of food-grade GAD expression with a multitude of potential applications.This study presents an effective strategy for the expression of food-grade industrial enzymes in B.subtilis.展开更多
Phase behavior of scCO2 microemul- sion formed with food grade surfactant sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) was studied Critical microemulsion concentration (cμc) was de- duced from the dependence of pre...Phase behavior of scCO2 microemul- sion formed with food grade surfactant sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) was studied Critical microemulsion concentration (cμc) was de- duced from the dependence of pressure of cloud points on the concentration of surfactant AOT at con- stant temperature and water concentration. The re- sults show that there are transition points on the cloud point curve in a very narrow range of concen- tration of surfactant AOT. The transition points were changed with the temperature and water concentra- tion. These phenomena show that lower temperature is suitable to forming microemulsion droplet and the microemulsion with high water concentration is likely to absorb more surfactants to structure the interface.展开更多
基金Supported by Doctoral Fund of Jilin Agricultural University(20070193005)~~
文摘[Objective]In order to improve expression vector of L.lactis at food grade and widen application range of original system.[Method] Taking pNZ8149 as basis,the promoter PnisA of pNZ8149 was inserted L.lactis MG1363 and SPusp45 of unknown secretory protein in downstream.Through PCR technology,specific primers were used to delete restriction sites between promoter sequence and signal peptide gene sequence and ensure better distance between SD sequence and start codon to construct secreting expression vector pNZS.The reporter gene gus was recombined into multiple cloning site of pNZS to construct pNZS-gus and L.lactis was transformed by electroporation.10 ng/ml nisin was used for induction culture,then culture solution was conducted GUS staining test.[Result]The new constructed L.lactis N3900/pNZS-gus system could express active GUS protein and GUS protein could be secreted out of cell.[Conclusion]The successful construction of this system lays foundation for secretion expression study of protein and oral vaccine research.
基金support of the National Key Technology R&D Program of China(No.2015BAB12B02)the Science and Technology Planning Project Guangdong Province,China(No.2013B090800016)
文摘The flotation separation of chalcopyrite from monoclinic pyrrhotite using food-grade guar gum(FGG) as a depressant was studied through flotation tests, kinetic studies, dynamic potential measurements, adsorption experiments, and infrared spectral analyses. The microflotation results showed that the flotation separation of chalcopyrite from monoclinic pyrrhotite could not be realized by adding mixed aerofloat(CSU11) alone. The depressant FGG exhibited a selective depression effect on monoclinic pyrrhotite by controlling the pulp pH range from 5.0 to 6.0, with a maximum floatability variation of 79.36% in the presence of CSU11. The flotation kinetics, zeta-potential, adsorption, and infrared spectroscopy studies revealed that the FGG could absorb more strongly on the surface of monoclinic pyrrhotite than on the surface of chalcopyrite. In addition, the results revealed that the interaction of FGG with the monoclinic pyrrhotite surface was governed primarily by strong chemisorption, whereas FGG mainly bonded to chalcopyrite through hydrogen bonding. This difference was responsible for the excellent depression selectivity of FGG toward monoclinic pyrrhotite flotation and weak depression effect toward chalcopyrite flotation.
基金Project (No.2006AA10Z316) supported by the Hi-Tech Research and Development Program (863) of China
文摘The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase （GALl） and phosphoglycerate kinase 1 （PGK1） promoters in different orientations was constructed. The β-1,3-1,4-glucanase gene from Bacillus subtilis was fused to α-agglutinin and ex- pressed under the control of the GALl promoter, α-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the α-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of β-1,3-1,4-glucanase. The analysis of β-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that β-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed β-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of β-1,3-1,4-glucanase displayed in the recombinant yeast cells was en- hanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.
基金supported by the National High-Tech R&D Program of China (863 Program, 2006AA10Z317)
文摘Construction of a food-grade expression vector for application to lactic acid bacteria（LAB） is of importance for dairy fermentation system. An α-galactosidase（aga） gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid p RAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36 e to substitute the p rimary antibiotic selectable marker of pMG36 e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long α-amylase（amy） gene from Ba cillus li cheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. Th e selection efficiency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU ？g-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putati ve molecular weight of α-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of α-amylase by expressing of amy gene of pMG36-aga-amy.
文摘L-arabinose isomerase (L-AI) is the key enzyme for D-galactose isomerization of D-tagatose by biological method. In this research, Lactobacillus plantarum WU14 with high D-tagatose yield was identified as Lactobacillus plantarum was isolated from the number of lactic acid bacteria from pickled vegetables. The crude L-arabinose isomerase activity of Lactobacillus plantarum WU14 with high D-tagatose yield was 13.95 U/mL under the optimal temperature 60°C, pH 7.17 and substrate concentration 0.8 mol/L, and the conversion rate of 56.12% could be gained after 28 hours. Protein structure and specific of L-Arabinose Isomerase of Lactobacillus plantarum WU14 were researched. The results showed that L-arabinose isomerase is mainly composed of alpha helix and random coil. Then the recombinant L-AI gene was inserted into the food-grade expression vector pRNA48 and expressed in L. lactis NZ9000 successfully. The target protein expression reached the maximum amount when the induced concentration of nisin reaches 30 ng/mL after 12 h. And the crude enzyme activity of recombinant bacteria reached 6.21 U/mL under 60°C. Otherwise the optimal conversion rate recombinant of L. lactis NZ9000/pRNA48-L-AI can reach 39.21% under the temperature of 50°C, pH 7.17 and D-galactose concentration was 0.6 mol/L.
基金the Ministry of Science and Technology of Taiwan for financially supporting this research under Contract No.NSC-1022623-E-002-012-ET
文摘In this paper, processes for producing a food-grade glucose solution through enzymatic hydrolysis of celluloserich solids obtained from rice straws are presented. The rice straws were pretreated by acid-catalyzed steam explosion, and the reaction efficiency, toxicity control, and process economic feasibility were studied. Mass transfer resistance to the hydrolysis reaction was reduced by grinding with glass beads. A higher glucose concentration could be obtained by feeding more cellulose in the hydrolysis reaction; however, this also resulted in the production of undesired byproducts. Thus, a soaking process for the cellulose solids in water was developed to effectively reduce the generation of byproducts in the hydrolysis reaction. The resulting food-grade glucose solution can provide 414 kilocalories per liter, and could be used during a food-shortage crisis in the future.The current production cost is estimated to be 0.82 USD·L^(-1).
文摘As the utilization of maltodextrins in food,pharmaceuticals,and agriculture continues to expand,substantial research has been conducted to enhance their functionality.Among the methods presented,the one-pot approach for the synthesis of nonreducing maltoheptaose offers a novel solution to the challenge of maltodextrins with varying degrees of polymerization and reducing ends.Nevertheless,the key enzyme in this method was currently only expressed in Escherichia coli,which restricts the applicability of this method in the food and pharmaceutical industries.In this study,the food-grade expression of cyclomaltodextrinase(CDase,EC 3.2.1.54),one of the key enzymes,was achieved using Bacillus subtilis as a host.The enzymatic properties of the recombinant CDase were then investigated,and the extracellular secretion of the CDase was enhanced in order to make it more widely available for use in the food industry.The enzyme exhibited optimal activity at a pH of 8.0 and a temperature range of 35-45℃.After incubation at 25-35℃for 10 h,90%of the enzyme activity was retained.Additionally,the enzyme retained 80%of its initial activity after 24 h at pH 5.5-9.5.Finally,Cu2+completely inhibited the enzyme activity.The extracellular secretion efficiency of recombinant CDase was significantly increased by the addition of Mn^(2+)to the fermentation medium.The percentage of extracellular enzyme activity increased to 63.75%when the final concentration of Mn^(2+)in the fermentation medium was 5 mM,which was 5.3-fold higher than that of the unadded one.
基金This work was supported by the National Natural Science Foundation of China(31401610)the Fundamental Research Funds for the Central Universities of China(KJON201557)+1 种基金the Outstanding Youth Foundation Science and Technology Fund of College of Engineering at Nanjing Agricultural University(YQ201603)the Jiangsu Agriculture Science and Technology Innovation Fund(CX(16)1059).
文摘Food-grade tracers have been developed as an identification technology for grain traceability from original harvest to final destination for transportation.The characteristics of food-grade tracers must be able to satisfy the environmental demands for grain traceability.To optimize the food-grade tracer production process,the effects of direct compression formulation and load on the mechanical characteristics were studied using response surface methodology(RSM)with central composite design(CCD).Among the four tested formulations,Formulations#2(consisting of 35.00%lactose 100 mesh,64.50%microcrystalline cellulose 102 and 0.50%magnesium stearate)and#4(consisting of 38.00%lactose 100 mesh,50.00%microcrystalline cellulose 102,11.00%pregelatinized starch and 1.00%magnesium stearate)were selected for tracer production based on their physical properties as powders.The value of Carr’s flowability index was 68 for both Formulations#2 and#4,which was the highest among all the formulations.Therefore,Formulations#2 and#4 also had the best powder flowability.The magnesium stearate ratio(1.00%-3.00%)and pressure(6.00-16.00 kgf)were used as independent variables to detect changes in the breaking rate,peak shear force and friction coefficient of tracers compressed by the selected formulations.The optimal production parameters could be achieved at a magnesium stearate ratio of 2.25%and pressure of 16.00 kgf for Formulation#2 and at a magnesium stearate ratio of 1.02%and pressure of 16.00 kgf for Formulation#4.Under these optimal conditions,the tracers had good impact characteristics(breaking rate),compression characteristics(peak shear force)and frictional characteristics(friction coefficient).Moreover,Formulation#2 was more suitable for production because compared to Formulation#4,its breaking rate and friction coefficient values were lower,and its peak shear force value was higher.
基金The authors gratefully acknowledge the support of the National Natural Science Foundation of China(31401610)the Fundamental Research Funds for the Central Universities of China(KJON201557)+1 种基金the Outstanding Youth Foundation Science and Technology Fund of College of Engineering at Nanjing Agricultural University(YQ201603)the Jiangsu Agriculture Science and Technology Innovation Fund(CX(17)1103).
文摘The purpose of this study was to optimize the coating process of food-grade tracers to manufacture tracers with good physical,mechanical and practical properties and an excellent appearance.The effects of the coating weight gain(1.00%-5.00%),coating solution spray rate(1.50-7.50 g/min)and tablet bed temperature(30℃-40℃)on the coating appearance quality,moisture absorption rate,friction coefficient,peak shear force,breaking rate,barcode recognition rate,transport wear rate and transport recognition rate were analysed using a Box-Behnken design(BBD)of response surface methodology(RSM).The experimental data were fitted to quadratic polynomial models by multiple regression analysis.The mathematical models of the barcode recognition rate,transport wear rate and transport recognition rate exhibited no statistically significant difference in these data.The optimum coating parameters were as follows:a 5.00%coating weight gain,spray rate of 5.47 g/min and tablet bed temperature of 35.42℃.Under the optimized conditions,the tracers had a good appearance(coating appearance quality),moisture resistance(moisture absorption rate),and frictional(friction coefficient),compression(peak shear force),and impact characteristics(breaking rate).
基金supported by the National Key Research and Development Program of China(2021YFE0101800)the S&T Support Program of Jiangsu Province(BE2022324).
文摘Mycotoxins in grains pose severe threat to human and animal health.Previous studies showed that manganese peroxidases had degradation capability for various mycotoxins.In this work,to effectively detoxify aflatoxin B1(AFB1),zearalenone(ZEN)and deoxynivalenol(DON),the manganese peroxidases PhcMnp and IrlMnp from Phanerochaete chrysosporium and Irpex lacteus were successfully expressed in Pichia pastoris in a food-grade manner,and were used for degradation tests with the three mycotoxins.The fermentation supernatants containing PhcMnp achieved degradation ratios of 76.56%,70.78%and 48.93%for AFB1,ZEN and DON respectively,while that containing IrlMnp achieved degradation ratios of 46.72%,45.13%and 41.64%correspondingly.The inducible expression conditions for the enzymes and the reaction parameters for the mycotoxin degradations were both optimized.Furthermore,the fermentation supernatants of strain Pichia pastoris GS115(pPIC9K-Phcmnp)were used for mycotoxin degradations in contaminated peanut samples(pH 4.5)at 40℃ for 10 h,resulting in apparent detoxification effects.The residual concentrations of AFB1,ZEN and DON in peanut samples after degradations were 9.42,127.68 and 636.71μg/kg respectively,all of which remained below the legal limits.Results in this study demonstrated that the enzymes PhcMnp and IrlMnp have potentials in mycotoxins detoxification in feed industry.
基金financially supported by the National Natural Science Foundation of China(32172153)the National Key Research and Development Program of China(2021YFC2101400)the Natural Science Foundation of Jiangsu Province(BK20202002).
文摘Protein-glutaminase(PG)is an enzyme used to enhance the functional properties of food proteins,such as emulsification,solubility and foaming.However,the use of PG in the food industry is limited because of its low catalytic activity and expression levels.In this study,a mutant library of PG was systematically constructed and screened through a structure-guided semi-rational design.This resulted in the identification of a mutant strain,A291S,which exhibited a 45.71%increase in activity.Moreover,a food-grade expression system for PG was established in Bacillus subtilis,demonstrating PG expression levels comparable to those obtained using plasmid-based expression systems that utilized antibiotics.The activity of PG remained above 99%even after the plates were incubated at ambient temperature for 5 days.However,the antibiotic-based expression system retained only 36%of PG activity.The efficacy of fed-batch fermentation for PG production was also evaluated and a maximum activity of 8.15 U/mL was obtained.These findings highlight the significance of PG modification and potential applications of the food-grade expression system in the food industry.
基金supported by grants from the National Natural Science Foundation of China(32272264,31730067,31901633)the Natural Science Foundation of Jiangsu Province(BE2023686)+1 种基金the Fundamental Research Funds for the Central Universities(JUSRP122012)Shenzhen Institute of Synthetic Biology Scientific Research Program(DWKF20210004).
文摘γ-Aminobutyric acid(GABA)is a bioactive compound with diverse physiological functions.It has a wide range of applica-tions in food and medicine and mainly biosynthesized through glutamate decarboxylase(GAD)catalysis.Bacillus subtilis is recognized for its robust secretion capabilities and high food safety standards,making it a prevalent choice for recombinant protein expression.In traditional industrial enzyme production in B.subtilis,antibiotics are required to sustain plasmid stability.However,the incorporation of antibiotics fails to align with the criteria applicable to enzymes for use in the food industry.To eliminate the need for antibiotics in the production of GAD preparations,we constructed a marker-free recom-binant strain B.subtilis WS9D-GAD.This strain was developed based on antibiotic-free multi-copy gene expression vector pUBDAL-amyL and d-alanine racemase(dal)-deficient B.subtilis WS9D,ultimately enabling the food-grade expression of GAD.To enhance GAD expression levels,we integrated the gadA expression cassette into the genome of B.subtilis using the Cre/lox system method.Additionally,strain WS9C6D-GAD was generated through the co-expression of free plasmid and genome integration,which carried the free plasmid pUBDAL-gadA and featured six copies of the gadA expression cassette within its genome.The enzyme activity during shake flask fermentation reached 28.15 U mL^(−1),while the enzyme activity in high-density 3-L fermenter culture reached 199.49 U mL^(−1),marking the highest level of food-grade GAD expression with a multitude of potential applications.This study presents an effective strategy for the expression of food-grade industrial enzymes in B.subtilis.
基金This work was supported by the National Natural Science Foundation of China （Grant Nos. 20573056, 20473035 and 20273032）the New Technique Foundation of Jiangsu Province （Grant No. BG-2005041）.
文摘Phase behavior of scCO2 microemul- sion formed with food grade surfactant sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) was studied Critical microemulsion concentration (cμc) was de- duced from the dependence of pressure of cloud points on the concentration of surfactant AOT at con- stant temperature and water concentration. The re- sults show that there are transition points on the cloud point curve in a very narrow range of concen- tration of surfactant AOT. The transition points were changed with the temperature and water concentra- tion. These phenomena show that lower temperature is suitable to forming microemulsion droplet and the microemulsion with high water concentration is likely to absorb more surfactants to structure the interface.
