50 results about "Bile salt hydrolase" patented technology

The invention relates to a method for preparing cholesterol reducing eggnog fermenting drink by utilizing lactobacillus and microzyme which produce bile salt hydrolase, belonging to a functional drinkwhich is prepared from the following ingredients: 15% of whole egg pulp, 35% of milk, 7% of saccharose, 0.3% of CMC-Na. and 0.2% of carrageenin, wherein the ratio of the raw materials (the whole eggplug and the milk) to water is 1:1 (W/V). The primary fermentation condition is described as following: streptococcus thermophilus Tx, Lactococcus lactis subsp.lactis KS4 and Lactobacillus casei KL1 are mixed in proportion of 1:1:1, the inoculum size is 3%, and then the mixture is fermented for 8h at the temperature of 43 DEG C. The secondary fermentation condition is described as following: Kluyveromyces marxianus M3 and K1 are compounded in proportion of 1:1 or the inoculum size of the single strain is 1%, and then the composite is fermented for 24-26 h at the temperature of 30 DEG C (mixture strain) or 32-34 DEG C; after ripening is carried out for 1 day at the temperature of 4 DEG C, the reducing rate of raw material cholesterol reaches as high as 89.3%. By utilizing the characteristics of strain high-yielding BSH, exopolysaccharide and the like, the invention has the efficiencies of immunological regulation, cholesterol reducing, tumor prevention and the like, and the problem of egg smell is not only solved, but also the content of raw material cholesterol is efficiently reduced through egg pulp proper sterilization and eggnog secondary fermentation. The eggnog drink preparedby the invention has simple fermentation process, short period and lower cost and is suitable for industrialized production.

the invention discloses a lactic acid bacteria bile salt hydrolase and manufacturing method and specific manufacturing strain, which comprises the following steps: 1) fermenting Lactobacillus casei (KTx CGMCCNo..1739); gathering bacteria; 2) suspending bacterial through phosphoric buffer; grinding in the icy bath through supersonic wave; collecting the rough enzyme liquid; sedimenting protein through ammonia sulfate; centrifuging to obtain the product.

The invention discloses a high-bile salt resistance strain and bile salt hydrolase genes. The preservation number of the strain is CGMCCNo.8198. The strain has high acid resistance, high bile salt resistance, high nitrate reducing capability and the like. Three types of bile salt hydrolase genes of the strain are amplified, and are cloned, sequenced and heterogeneously expressed, so that generated bile salt hydrolases have high activity. The strain and the bile salt hydrolases can be used in the fields of food, medicine, agriculture and the like as food and feed leavening agents, microecological preparations, medicament expression carriers, feed additives and the like; the bile salt hydrolases can be developed into recombinant protein products, and can also be used for the molecular modification of lactic acid bacteria strains as selection markers of lactic acid bacteria gene engineering expression systems.

The invention provides an extraction method of bile salt hydrolase (BSH) and extracellular polysaccharide (EPS) in lactococcus lactis and streptococcus thermophilus, and the BSH and the EPS acting as functional factors and the EPS as a natural stabilizer in a functional dairy product are applicable for developing and producing novel functional food. The method utilizes lactococcus lactis subsp. lactis KS4 (CGMCC No.1807) and streptococcus thermophilus Tx (CGMCC No.1810) of the high-yield BSH and EPS, and obtains lactobacillus bile salt hydrolase and extracellular polysaccharide crude products through optimizing fermentation conditions and the extraction method. The adoption of the optimum ultrasonic cell disruption extraction method can solve the problem of difficult extraction of intracellular bile salt hydrolase; and the extraction rate of EPS is increased by optimizing the extraction conditions of EPS. The fermentation technique and the extraction method for preparing the BSH and EPS are simple, the fermentation period is rather short, and the method has low requirements on equipment and cost, and is applied to the industrialized production.

The invention discloses lactic acid bacteria for producing bile salt hydrolase as well as a screening method and application thereof, belonging to the technical field of food biology. In the invention, an improved qualitative analysis and quantitative analysis combined method can be used for accurately screening a bile salt hydrolase bacterial strain in high efficiency; the method disclosed by the invention is used for screening a plant lactic acid bacterium strain with higher activity of bile salt hydrolase; and the bacterial strain and the bile salt hydrolase produced by the bacterial strain can be applied to the field of foods and can be used for reducing the content of cholesterol of human serum.

The invention discloses a bile salt hydrolase (BSH) mutant and a use thereof, and belongs to the technical field of gene engineering. Through comparison analysis of a BSH amino acid sequence and homology modeling and overlay analysis of a 3-D structure, a BSH substrate specific amino acid mutation site is determined, through a site-specific mutagenesis technology, the substrate specific amino acid mutates into a nonpolar amino acid, and the mutant is transferred into a host bacterium so that a mutant strain is obtained. The BSH produced by the mutant is active enzyme. Compared with a wild-type bacterium, the mutant has a reduced glycine-combined bile salt hydrolysis capability and an improved taurine-combined bile salt hydrolysis capability. The BSH mutant lays a foundation for improving a taurine-combined bile salt hydrolysis capability of BSH and uncovering a substrate combination mechanism of BSH.

The invention discloses a method for producing bile salt hydrolase by fermentation of twin-arginine signal peptides and application thereof, and belongs to the technical field of gene engineering. According to the method provided by the invention, a twin-arginine signal peptide gene and a bile salt hydrolase gene are fused, the fusion gene is transformed into the E.coli host to obtain recombinant bacteria, and recombinant bacteria is used for fermentation for producing bile salt hydrolase. The bile salt hydrolase is successfully secreted to the outside of the cells of the recombinant bacteria, and the extracellular BSH activity of the recombinant E.coli BL21 (DE3) pLysS (pET-20b (+)-dmsA-bsh) reaches the highest value of 0.72 +/- 0.05 U/mL. The invention lays foundation for the study on isolation and purification and enzymatic characteristics of BSH, and provides effective methods for secretion expression of other heterologous protein.

The invention relates to a method for preparing a bile salt hydrolase lactobacillus powder starter, which is suitable for producing powder starters for fermented dairy products such as functional yoghourt and probiotics preparations. The method comprises the following steps of: performing activation and amplification culture on Lactobacillus casei KL1, Lactococcus lactis lactis KS4 and Streptococcus thermophiles Tx, which are screened from Tibetan kefir and are strains of bile salt hydrolase; performing high density culture by using an automatic fermentation tank; performing centrifugal concentration and adding a freeze-drying protective agent for prefreezing and freeze drying to prepare the bile salt hydrolase lactobacillus powder starter. After the powder starter prepared by the method is used for fermenting milk, the obtained yoghourt has sticky, fine and smooth mouthfeel, moderate sweet and sour taste, and thick ester aroma.

The invention discloses lactic acid bacteria strains capable of inhibiting multiple drug-resistant food-borne pathogenic bacteria in a broad-spectrum manner and an application of the lactic acid bacteria strains. The strains have functions of inhibiting the activity of multiple drug-resistant bacteria and food-borne pathogenic bacteria in a broad spectrum manner, and are named as lactobacillus plantarum GS083, lactobacillus plantarum GS086, lactobacillus plantarum GS090 and lactobacillus fermentum GF110. The lactic acid bacteria strains provided by the invention has a very good bacteriostaticeffect on multiple drug-resistant bacteria and food-borne pathogenic bacteria, and can inhibit gram-negative bacteria and gram-positive bacteria; the strains also have the characteristics of bile salthydrolase activity, acid resistance, bile salt resistance, low drug resistance, no hemolysis, no toxicity gene and the like, enrich the lactobacillus strain resource library, and have wide application prospects in the industries of food, cosmetics, medicines and the like.

The invention provides Lactobacillus casei J1 capable of reducing cholesterol. According to a technical scheme in the invention, the Lactobacillus casei J1 is separated and screened from Tibetan kefir (also called as kefir grain) cultured in a common household and is preserved in China General Microbiological Culture Collection Center on September 11, 2006, with an accession number of CGMCC No. 1808. The invention also discloses a method for producing probiotic yogurt by using the Lactobacillus casei J1. On the basis of no change of common yoghourt fermentation process, the yoghourt capable of reducing serum cholesterol can be prepared by utilizing the characteristic that the J1 strain produces bile salt hydrolase, and the problem of precipitation of whey of the yoghourt is overcome. The method for producing the probiotic yogurt has the advantages of simple process, a short fermentation period, low cost and applicability to large-scale industrial production.

The invention discloses a gene engineering bacteria capable of producing bile salt hydrolase and a construction method and application thereof, and belongs to the technical field of gene engineering. According to the gene engineering bacteria capable of producing the bile salt hydrolase and the construction method and application thereof, by adopting a recombinant DNA technology, the bile salt hydrolase (bsh) of Bifidobacterium infantis KL412 is connected to a prokaryotic expression vector pET-22b(+) in a gene-cloning mode and converted into Escherichia coli BL21(DE3), and recombinant Escherichia coli BL21(DE3)-pET-22b(+)-bsh capable of producing the high-activity bile salt hydrolase is obtained through screening and identifying. 2% inoculation amount of seed liquid is inoculated to a fermentation medium, the recombinant bacteria is induced for 32 h to reach the highest enzyme activity on the condition that the temperature is 20 DEG C, OD<600> is 2.0, and IPTG is 1 mmol/L, and the enzyme activities of the recombinant bacteria for glycodeoxycholic acid sodium (GDCA) and taurodeoxycholic acid sodium (TDCA) are 135.2 U/mL and 121.3 U/mL respectively. Therefore, a good foundation is laid for large-scale production of the bile salt hydrolase and using the bile salt hydrolase as functional food to decrease serum cholesterol.

The invention relates to a preparation method of a bile salt hydrolase-yielding lactobacillus paracasei dry powder living bacterium preparation, wherein the preparation method is suitable for the production of functional probiotic lactic acid bacterium leavening agents and microecologics for eating or feeding. The method comprises the following steps: carrying out activation, cultivation enlargement, high-density fermentation and centrifugal concentration on bile salt hydrolase-yielding lactobacillus paracasei KL1-Liu CGMCC No.7029 screened from Tibetan kefir, adding a freeze-drying protective additive, pre-freezing, freeze-drying and the like. According to the method, a nitrogen source in an improved MRS culture medium is completely replaced with rapeseed peptone, so that the production cost is lowered; and production and fermentation conditions and a formula of the freeze-drying protective additive are optimized, so that the number of living bacteria of the KL1-Liu strain can reach above 10<10>CFU/g, and the survival rate can reach above 85%. The prepared living bacterium preparation is low in cost and high in living bacterium content, and is a high-quality probiotic living bacterium preparation/leavening agent with a serum cholesterol lowering function.

The invention relates to a method for extracting bile salt hydrolase (BSH) of two kinds of yeasts and a production technology of functional kefir, applicable to the production of functional kefir and sour kefir in fermented dairy products. The invention adopts high-throughput screening technique and phthalic aldehyde method to acquire two kinds of yeasts which can lower cholesterol efficiently and have higher capacity of fermenting lactose, and Oxford cup method to acquire strains yielding the BSH highly through secondary screening. The invention optimizes the fermentation conditions and extraction method for the two kinds of yeasts to produce the BSH, and adopts proper cracking method by ultrasonic wave, which solves the problem that the cell walls of the yeasts are difficult to be cracked, and the cracking effect can be compared with that of the high pressure cell cracker. Kluyveromyces Marxianus K1 (CGMCC No 1812) and M3 (CGMCC No 1811) which can lower the cholesterol efficiently can yield the BSH highly and can ferment lactose to yield ethanol highly, are adopted to prepare the kefir of high quality that has the function of lowering the cholesterol. The fermentation process of preparing the functional kefir is simple with shorter fermentation period and lower cost. Thus, the fermentation process is applicable to mass industrial production.

The invention relates to a preparation method of a Chinese-date probiotics tablet of lactobacillus paracasei capable of producing bile salt hydrolase. The preparation method is suitable for producing microecological preparations such as a functional probiotic lactic bacteria living bacteria agent. A formula of the Chinese-date probiotics tablet consists of lactobacillus paracasei KL1-Liu probiotics bacterial powder and auxiliary materials such as Chinese-date powder, fructo-oligosaccharide, xylitol, sucrose, citric acid, microcrystalline cellulose and maltodextrin. The preparation method of the Chinese-date probiotics tablet comprises the following steps: preparing the probiotics bacterial powder from lactobacillus paracasei KL1-Liu CGMCC No.7029 capable of producing the bile salt hydrolase by virtue of processes such as activating, performing enlarged culture, performing high-density fermentation, performing centrifugal concentration, adding a freeze-drying protective additive, pre-freezing, and freeze-drying; mixing the crushed and screened auxiliary materials with the probiotics bacterial powder, thereby obtaining a mixture, and then pressing the mixture into the Chinese-date probiotics tablet. The Chinese-date probiotics tablet prepared by the preparation method disclosed by the invention has good storage stability and a refrigeration living bacteria quality guarantee period of over one year at 4 DEG C, not only can bring the health effects of lowering the serum cholesterol of the products into play, but also can bring the medicinal and edible nutritional and health effects of Chinese-dates into play. Moreover, the preparation method of the Chinese-date probiotics tablet disclosed by the invention has a convenient raw material source, a simple process, a short preparation period and simple operation, and is suitable for industrial production.

The invention relates to an extraction method of BSH (Bile Salt Hydrolase) contained in lactobacillus salivarius. The extraction method is suitable for developing and producing a novel functional food by taking BSH as a functional factor in functional milk products. The extraction method comprises the following step of: acquiring lactobacillus bile salt hydrolase through optimized fermentation condition and extraction method by utilizing the Lactobacillus salivarius LMG14476 capable of producing the BSH. By adopting suitable ultrasonic cell disruption, the extraction method disclosed by the invention solves the problem that the intracellular bile salt hydrolase is difficult to extract and enhances the extraction rate of the BSH. The BSH prepared through the extraction method has the advantages of simple fermentation process and extraction method, shorter fermentation period, low requirement on equipment, lower cost and suitability for industrialized production.

The invention discloses a lactobacillus casei and a culture medium thereof. The bacterial strain is stored in China General Microbiological Culture Collection Center with the registration number of CGMCC NO. 3993; the Latin name of the strain is Lactobacillus casei L7-13; and the bacterial strain has the morphological characteristics of gram positive bacteria, no spore grown, no flagella, no motion, rough bacterial colony, offwhite or light yellow. The invention also discloses the complete sequence of 16s ribosome DNA (16S rDNA) of the bacteria; the lactobacillus casei is high in lactic acid generation speed and high in acid productivity, and further high in reproduction speed, high in activity and good in stress resistance; besides, the lactobacillus casei has the function of improving the gastrointestinal tract and thereby is capable of inhibiting colonization of harmful bacteria; the lactobacillus casei is capable of generating bile salt hydrolase and decomposing bound cholic acid into free cholic acid; and, the free cholic acid and cholesterol can form a composite so as to settle down together; therefore, the lactobacillus casei is capable of reducing cholesterol.

The invention discloses a method for directionally and rapidly screening lactobacillus plantarum capable of producing bile salt hydrolase, and belongs to the technical field of microorganisms. The invention comprises the following steps: (1) taking metagenome DNA of a sample as a template, performing PCR amplification on BSHL/BSHR by utilizing a primer, and screening to obtain the sample with a BSH gene; (2) taking the sample as a separating object, and purifying to obtain a suspected plant lactobacillus strain; (3) taking the strain genome DNA as a template, performing PCR amplification on the BSHL/BSHR by utilizing the primer, and screening to obtain a suspected strain with the BSH gene; (4) performing molecular identification on the strain and screening to obtain the lactobacillus plantarum capable of producing bile salt hydrolase. According to the method, most samples which do not contain the lactobacillus plantarum capable of producing the BSH can be filtered out through the firststep of amplification of metagenome-specific fragments, so that blind screening is avoided, the screening range is greatly reduced, and the method has the advantages of directionality, rapidness, easiness, convenience, low workload and the like.

The invention discloses a method for extracting bile acid by using ox bile efficient enzyme, and belongs to the technical field of animal bile effective extraction. The method comprises the followingsteps of mixing raw materials and a compound enzyme preparation, adjusting the pH value, carrying out enzymolysis and stirring adjustment, and carrying out solid-liquid separation to obtain an extracting solution; carrying out enzyme deactivation, adding bile salt hydrolase for enzymolysis, adjusting the pH value, performing heating, performing stirring saponification, performing centrifugation, and adjusting the pH value to obtain a supernatant; adding hydrogen peroxide, performing uniform stirring and mixing, reacting at room temperature, and performing filtration to obtain a treatment solution; slowly adding butyl acetate, performing stirring, then adjusting the pH value of the solution, and carrying out solid-liquid separation to obtain solid matter; and performing centrifugation and water washing, and performing drying until the water content is not higher than 1%, thereby obtaining finished bile acid extract. According to the invention, the enzyme preparation is adopted for extraction, so that the product purity is improved, impurities are reduced, the product yield is increased, pollution caused by participation of toxic substances is fundamentally avoided, and the problem of environmental pollution caused by strong acid and strong alkali is reduced; and the method is simple in process, low in investment and low in cost.

The invention relates to an extraction method of bile salt hydrolase (BSH) and extracellular polysaccharide (EPS) in Lactococcus lactis and Streptococcus thermophilus, which is suitable for developing and producing novel functional foods by using BSH and EPS as functional factors and using EPS as a natural stabilizer in functional dairy products. In the invention, Lactococcus lactis subsp. lactis KS4 (CGMCC No.1807) and Streptococcus thermophilus Tx (CGMCC No.1810) of high-yield BSH and EPS are utilized, and the fermentation conditions and the extraction method are optimized to obtain crude products of BSH and EPS of lactic acid bacteria. A proper ultrasonic cell breakage extraction method is adopted to solve the problem that intracellular BSH can not be extracted easily; and the extraction conditions of the EPS are optimized to improve the extraction ratio of the EPS. The invention has simple fermentation process and extraction method for preparing BSH and EPS, shorter fermentation period, low requirements for equipment and lower cost, and is suitable for industrialized production.

The invention provides a lactic acid bacterium double-gene expression box which is used for conducting expression on an exogenous gene in the lactic acid bacterium after being inserted in the exogenous gene. The lactic acid bacterium double-gene expression box comprises a first expression box and a second expression box. The first expression box comprises a pldh promoter and a first terminator; and the second expression box comprises a p23 promoter and a second terminator. The invention further provides an expression carrier comprising the lactic acid bacterium double-gene expression box and a construction method of the expression carrier. By means of the expression carrier comprising the lactic acid bacterium double-gene expression box, coexpression of two objective exogenous genes in the lactic acid bacterium can be achieved, lactobacillus casei can express two kinds of bile salt hydrolase and show bile salt degradation activity.

The invention discloses a bile salt hydrolase mutant with improved enzyme activity, and belongs to the technical field of enzyme engineering. The bile salt hydrolase mutant has the advantages that in a site-directed mutation mode, asparagine near active sites inside bile salt hydrolase molecules mutates into phenylalanine, tyrosine and tryptophan with high hydrophobicity; valine mutates into isoleucine; the molecule inside hydrophobicity is changed; the enzyme activity of the bacterial strain expression bile salt hydrolase is obviously improved; the enzyme producing capability of the modified bacterial strain is obviously improved; the enzyme activity is at least improved to 91.32U/mL; the enzyme activity improvement of a compound mutant strain pET-22b(+)-bsh-V59I/N19Y is most obvious and the enzyme activity reaches 141.87 U/mL; the enzyme activity is improved by 71.6 percent through being compared with that of an original strain; the production efficiency can be improved; and the bile salt hydrolase mutant is more suitable for industrial application.