CN115606811A - Preparation method of stichopus japonicus visceral oligopeptide microcapsules and microcapsules - Google Patents
Preparation method of stichopus japonicus visceral oligopeptide microcapsules and microcapsules Download PDFInfo
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- CN115606811A CN115606811A CN202210410395.7A CN202210410395A CN115606811A CN 115606811 A CN115606811 A CN 115606811A CN 202210410395 A CN202210410395 A CN 202210410395A CN 115606811 A CN115606811 A CN 115606811A
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Images
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/001—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste
- A23J1/002—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste from animal waste materials
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/04—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from fish or other sea animals
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/04—Animal proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
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Abstract
The invention discloses a preparation method of stichopus japonicus viscera oligopeptide microcapsules, which is characterized in that lactobacillus plantarum is screened from cowberry fruit residues, bacillus subtilis is obtained from intestinal tracts of stichopus japonicus, yeast is matched in proportion to serve as probiotics for fermentation, the cowberry fruit residues are treated by the probiotics to obtain cowberry fruit residues SDF, the intestinal stichopus japonicus viscera oligopeptides of the stichopus japonicus are treated by the probiotics, the intestinal stichopus japonicus viscera oligopeptides of the stichopus japonicus are taken as core materials, the cowberry fruit residues SDF and sodium alginate are taken as wall materials, and the microcapsules are prepared by an orifice piercing method. The decrease speed of the antioxidant capacity of the microcapsule is far lower than that of the non-embedded stichopus japonicus visceral oligopeptide.
Description
Technical Field
The invention relates to the technical field of microcapsule preparation, in particular to a preparation method of stichopus japonicus visceral oligopeptide microcapsules and microcapsules.
Background
The internal organs of the stichopus japonicus cannot be efficiently utilized in the processing process, the effective yield is low due to the large amount of silt contained in the intestinal tract, and the treatment and recovery procedures are complicated and are often regarded as waste treatment. But in fact, in the same quality of stichopus japonicus organs, the stichopus japonicus intestinal protein content is the highest, the contents of fat and holothurin are higher, and even the stichopus japonicus organs contain taurine which is not contained in body walls. The sea cucumber oligopeptide is obtained in the current market in an enzymolysis mode, the molecular weight is large, but the product is deep in color, heavy in fishy smell and unobvious in antioxidant function, so that the application of the sea cucumber oligopeptide is greatly limited, and the market acceptance is not high.
Disclosure of Invention
The invention provides a preparation method of stichopus japonicus visceral oligopeptide microcapsules and microcapsules, which are used for making up the defects of the prior art.
The invention is realized by the following technical scheme:
a preparation method of stichopus japonicus visceral oligopeptide microcapsules comprises the following steps:
s100, preparing fermentation bacteria, screening lactobacillus plantarum from cowberry fruit residues, obtaining bacillus subtilis from stichopus japonicus intestinal tracts, and proportionally matching commercial yeasts with the lactobacillus plantarum in the commercial yeasts to serve as probiotics for fermentation;
s200, preparing blueberry pomace SDF, treating blueberry pomace by adopting a bacterial-enzyme interaction technology, performing preliminary enzymolysis on raw material pomace, inoculating probiotics for fermentation prepared in S100 for fermentation, and concentrating, extracting ethanol and drying fermentation liquor to obtain SDF;
s300, preparing core material stichopus japonicus viscera oligopeptides, pretreating stichopus japonicus intestines, performing enzymolysis and decoloration by using compound protease, inoculating probiotics for fermentation prepared in S100 for fermentation, taking out fermentation liquor, centrifuging, and freeze-drying supernatant to obtain stichopus japonicus viscera oligopeptide powder;
s400, preparing stichopus japonicus visceral oligopeptide microgel, preparing a solution by taking cowberry fruit residue SDF and sodium alginate as wall materials, inoculating core materials, namely the stichopus japonicus visceral oligopeptides, adding sucrose fatty acid ester to promote dissolution, heating, carrying out ultrasonic treatment, vibrating, fully dissolving, and then preparing the microcapsule by using an orifice method.
The step S100 specifically includes the steps of,
s110, cutting fresh stichopus japonicus intestines, respectively placing contents in the intestines in an EP (EP) tube, diluting the intestines with seawater subjected to suction filtration and sterilization, centrifuging the intestines to obtain supernate, and properly diluting the supernate to obtain a content diluent; diluting the cowberry fruit residue powder with normal saline, and centrifuging to obtain cowberry fruit residue diluent; marking MRS and LB solid culture medium, respectively coating the content diluent and the cowberry fruit residue diluent on the corresponding culture medium, and culturing in an incubator at 37 ℃;
s120, selecting different types of single colonies from the coating plate, streaking and purifying the single colonies in corresponding culture media, and screening to obtain lactobacillus plantarum and bacillus subtilis after sequencing;
s130, preparing the lactobacillus plantarum and the bacillus subtilis obtained in the S120 and the yeast into zymocyte liquid according to a ratio of 1.
The step S200 specifically includes the steps of,
s210, processing cowberry fruit residues by adopting a bacterial enzyme interaction technology, firstly adopting a double-enzyme method, firstly carrying out enzymolysis by using alpha-amylase, then regulating the pH value to be neutral by using NaOH, then carrying out enzymolysis by using alkaline protease, and drying to obtain cowberry fruit residue zymolyte;
s220, designing a single-factor experiment, and analyzing the influence of each factor on the SDF yield of the cowberry fruit residues; carrying out a single-factor test by taking the fermentation time as 12h,24h,36h,48h,60h and 72h, the liquid-material ratio as 2;
s230, designing a 4-factor 3 horizontal orthogonal test to determine an optimal fermentation condition; positioning the optimal fermentation time obtained by the single-factor test according to the fermentation time, selecting the fermentation temperature of 30 ℃,33.5 ℃,37 ℃, the fermentation pH of 6,7 and 8, the bacterial liquid inoculation amount of 8;
s240, preparing the cowberry fruit residue SDF by using the optimal fermentation condition.
The step S300 specifically includes the steps of,
s310, preparing an apostichopus japonicus intestine enzymolysis liquid, preliminarily dissecting the apostichopus japonicus intestine, removing sand, shearing, adding water according to the mass of the materials, and mixing to obtain a homogenate with the concentration of 20-40%; adding 0.2-0.3% of papain, flavourzyme and compound protease, mixing, stirring intermittently, carrying out enzymolysis for 2-4h in a water bath kettle at 54-56 ℃, heating water to above 80 ℃, inactivating enzyme for at least 15min, cooling to room temperature, and filtering to obtain filtrate 1; adding activated carbon powder into the filtrate 1, intermittently stirring for more than 30min, settling for 20min, filtering, and decolorizing to obtain filtrate 2, which is Stichopus japonicus intestine enzymolysis liquid;
s320, designing a single-factor experiment, and analyzing the influence of each factor on the yield of the stichopus japonicus visceral oligopeptide; respectively analyzing the influence of each factor on the yield of the stichopus japonicus visceral oligopeptides by taking the fermentation time as 1D, 2D, 3D, 4D, 5D, 6D, 7D and 8D, the inoculation amount of probiotics for fermentation as 1.5%,3%,4.5%,6%,7.5% and 9%, the fermentation temperature as 26.5 ℃,30 ℃,33.5 ℃,37 ℃,40.5 ℃ and the fermentation pH as 4,5,6,7,8 and 9 as a single-factor test;
s330, according to the single-factor test result, the fermentation time is the optimal fermentation time of the single-factor experiment, the 4-factor 3 horizontal orthogonal test is designed according to the fermentation temperature of 33.5 ℃, the fermentation temperature of 37 ℃, the fermentation temperature of 40.5 ℃, the fermentation PH of 6,7 and 8 and the bacterial liquid inoculation amount of 6%,7.5% and 9%, so as to determine the optimal fermentation condition;
s340, preparing the stichopus japonicus visceral oligopeptide by using the optimal fermentation condition.
The step 4100 specifically includes the steps of,
s410, designing a single-factor experiment to analyze the influence of each factor on the embedding rate; the ratio of the cowberry pomace SDF to the sodium alginate is (1.5, 1, 2, 1, 2); the core material to wall material ratios are 0.1,0.2,0.3,0.4 and 0.5, the mass percentages of the sucrose fatty acid ester, the solidification liquid and the solidification liquid are respectively 0.15%,0.2%,0.25%,0.3% and 0.35%, 1%,2%,3%,4%,5% and 6%, and single-factor tests are carried out to analyze the influence of the factors on the embedding rate;
s420, obtaining an optimal preparation process condition by using a response surface optimization method, selecting a solidification liquid mass fraction A, a sucrose fatty acid ester mass fraction B and a wall-core ratio C on the basis of a single-factor optimal experiment condition, and performing a three-factor three-level response surface experiment; the experimental data are designed according to a Design-Expert8.0.6 program, and the obtained regression equation is as follows: the embedding rate/% =79.29 +0.13A +0.28B +0.41C-0.45A +0.32A C +0.49B +C-2.09 A2-2.09 B2-2.49C 2; drawing a response surface of the regression equation and a contour map thereof to obtain an optimal embedding condition;
s430, preparing microcapsules under the optimal embedding conditions obtained in S410 and S420, controlling the height between the needle tube and the liquid level to be more than or equal to 20 cm, dripping a solidification liquid at a constant speed, stirring at a constant speed to prevent adhesion, and vibrating and solidifying to obtain gel balls; and repeatedly washing the gel balls with normal saline to wash out the surface saline solution, filtering, and drying in a freeze dryer to finally obtain the microcapsule.
The optimal fermentation conditions in the step S200 are PH =7 and the temperature is 33.5 ℃,14% of inoculation is performed according to a feed-liquid ratio of 10, and the fermentation time is 24h.
The optimal fermentation conditions in step S300 were PH =7, and when the temperature was 37 ℃,9% inoculation and 7D fermentation were performed.
The optimal embedding conditions in the step S400 are that the mass fraction of the solidification liquid is 3.51%, the mass fraction of the sucrose fatty acid ester is 0.3%, the core-wall ratio is 0.21, and the ratio of the cowberry fruit residue SDF to the sodium alginate is 1.
In the S420, the mass fraction of the solidified liquid is 3%, 3.5% and 4%, and the mass fraction of the sucrose fatty acid ester is 0.25%,0.3% and 0.35%.
A stichopus japonicus visceral oligopeptide microcapsule is prepared by any one of the methods.
The invention has the following beneficial technical effects:
the preparation method comprises the steps of preparing stichopus japonicus viscera oligopeptide microcapsules by performing a series of processing treatments such as bacterial enzyme interaction on cowberry fruit residues and stichopus japonicus intestines; the embedding rate of the preparation method of the stichopus japonicus visceral oligopeptide microcapsules is 79.25 percent; through intestinal simulation experiments, compared with the non-embedded stichopus japonicus visceral oligopeptide, the prepared cowberry fruit residue dietary fiber stichopus japonicus visceral oligopeptide microcapsule has a good slow release effect in simulated gastric juice and a good release capacity in simulated intestinal juice, and according to a human absorption mode, the in-vitro accumulated release amount of the microcapsule can reach 63.96%; the removal of DPPH and ABTS during storage at room temperature for 60 days was found to be a much slower rate of decrease in the antioxidant capacity of the microcapsules than non-embedded visceral oligopeptides of Stichopus japonicus.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 shows the fermentation time-SDF yield of cowberry fruit residues.
FIG. 2 is a liquid-to-feed ratio-yield chart of cowberry fruit residue SDF.
FIG. 3 is a graph of pH-SDF yield of fermented blueberry pomace.
FIG. 4 is a graph of fermentation temperature-yield of cowberry fruit residue SDF.
Fig. 5 is a graph of the effect of orthogonal data factors.
FIG. 6 is a graph of fermentation time-yield of stichopus japonicus visceral oligopeptide.
FIG. 7 is a graph of inoculum size-yield of visceral oligopeptide from Stichopus japonicus.
FIG. 8 is a graph of fermentation temperature-yield of visceral oligopeptide from Stichopus japonicus.
FIG. 9 is a graph showing the effect of visceral oligopeptide factors on Stichopus japonicus.
FIG. 10 is a diagram of wall material ratio-embedding rate.
FIG. 11 is a graph of core wall ratio versus embedding rate.
FIG. 12 is a diagram of sucrose fatty acid ester mass fraction-embedding rate.
FIG. 13 is a graph of mass fraction of a coagulating liquid versus embedding rate.
FIG. 14 is a graph showing the interaction between the mass fraction of the solidification liquid and the mass fraction of sucrose fatty acid ester with respect to the embedding rate.
FIG. 15 is a contour plot of the interaction of mass fraction of the solidification liquid and mass fraction of sucrose fatty acid ester on the embedding rate.
FIG. 16 is a graph showing the response of the interaction of mass fraction of coagulating liquid and wall-core ratio to embedding rate.
FIG. 17 is a contour plot of the interaction of mass fraction of solidifying liquid with wall-to-core ratio versus embedding rate.
FIG. 18 is a graph showing the response of the interaction of the wall-core ratio and the mass fraction of sucrose fatty acid ester to the embedding rate.
FIG. 19 is a contour plot of the interaction of the wall-core ratio and the mass fraction of sucrose fatty acid ester versus the embedding rate.
FIG. 20 is a graph showing the release efficiency of microcapsules and stichopus japonicus visceral oligopeptides in simulated gastric fluid.
FIG. 21 is a graph of the release efficiency of microcapsules and stichopus japonicus visceral oligopeptides in simulated intestinal fluid.
FIG. 22 is a graph of the cumulative in vitro sustained release of microcapsules.
FIG. 23 is a graph showing the effect of microcapsules on DPPH scavenging effect for different storage times.
FIG. 24 is a graph showing the effect of visceral oligopeptide of Stichopus japonicus on DPPH removal at different storage times.
FIG. 25 is a graph showing the effect of microcapsules on ABTS clearance for different storage times.
FIG. 26 is a graph showing the effect of visceral oligopeptide of Stichopus japonicus on ABTS clearance at different storage times.
Detailed Description
The invention is described in further detail below with reference to specific examples, which are not intended to be limiting of the invention, and any limited number of modifications that can be made by one within the scope of the claims of the invention are within the scope of the claims.
The invention provides a preparation method of stichopus japonicus visceral oligopeptide microcapsules and microcapsules, and the preparation method specifically comprises the following steps:
s100, preparing fermentation bacteria, screening lactobacillus plantarum from cowberry fruit residues, obtaining bacillus subtilis from stichopus japonicus intestinal tracts, and matching commercial yeasts with the lactobacillus plantarum to serve as fermentation probiotics. Wherein the commercial yeast is Angel high-activity dry yeast.
S110, preparing an MRS culture medium and an LB culture medium; cutting fresh Oplopanax elatus nakai intestines into pieces, respectively placing the intestinal wall and the content in the intestines in a 1.5mLEP tube, marking the intestinal wall as No. (1), and marking the content in the intestines as No. (2). Dissolving with 1mL of seawater subjected to suction filtration sterilization to obtain stock solution, centrifuging to obtain supernatant, and diluting according to 10 times gradient to 10-7 of the stock solution concentration to obtain 7 content dilutions. Accurately weighing 0.1g of cowberry fruit residue powder, diluting with 1mL of normal saline, and placing in a refrigerator for cold storage, and recording as number (3) to obtain the cowberry fruit residue diluent. Marking on MRS, LB solid culture medium, coating 20-50 μ L with glass beads or coating rod, culturing in 37 deg.C incubator. After the tubes (1), (2) and (3) were applied to LB and MRS media, respectively, it was found that the tubes (1) and (2) grew well in LB medium and the tube (3) grew well in MRS medium. The final marking pomace corresponds to 1, and the stichopus japonicus intestine corresponds to 2, wherein the stichopus japonicus intestine wall is 2 (1), and the stichopus japonicus intestine content is 2 (2).
S120, selecting different types of single bacterial colonies from the coating flat plate to the coating flat plate, separately culturing, respectively scribing and purifying in corresponding culture media, marking, counting at three positions, wherein the first position corresponds to the type of raw materials, the second position corresponds to the type of bacterial colonies, and the third position is marked as the purification times, if the coating of fruit residues and the coating of No. two fungus rings are marked, the purification is carried out at one time to be 1-2-1; the stichopus japonicus intestine coating bacteria and the first bacteria colony are purified for three times to be 2-1-3, and the purification is needed for 3 times. After detection and sequencing, lactobacillus plantarum and bacillus subtilis are finally selected from various strains. The lactobacillus plantarum is screened from cowberry fruit residues, numbered 1-5-3, cultured by adopting an MRS solid culture medium, and has a white or light yellow colony, a clear and non-diffused edge and a circular shape with the diameter of 1mm to 2 mm. The bacillus subtilis is screened from the intestinal wall of the stichopus japonicus and cultured by adopting an LB solid culture medium, and the bacillus subtilis is numbered 2 (1) -7-3, wherein the bacterial colony is light white, moist and turbid in appearance and is circular in shape of 2 mm-3 mm.
S130, culturing the lactobacillus plantarum, the bacillus subtilis and the yeast obtained in the S130 to logarithmic phase, and preparing a zymocyte liquid according to the volume of 1.
S200, preparing the blueberry pomace SDF (soluble dietary fiber), treating the blueberry pomace by adopting a bacterial enzyme interaction technology, fermenting the raw material pomace after primary enzymolysis, and concentrating, extracting with alcohol and drying fermentation liquor to obtain the SDF.
S210, processing the cowberry fruit residues by adopting a bacterial enzyme interaction technology, firstly performing enzymolysis for 3h at 75 ℃ by adopting a double-enzyme method through 0.3% alpha-amylase, cooling to 50 ℃, adjusting the pH value to be neutral by using NaOH, performing enzymolysis for 3h by using 0.3% alkaline protease, and drying to obtain the cowberry fruit residue zymolyte.
S220, designing a single-factor experiment, and analyzing the influence of each factor on the SDF yield of the cowberry fruit residues.
Firstly analyzing the influence of different fermentation times on the SDF yield of the cowberry pomace, accurately weighing 5g of cowberry pomace zymolyte, putting the cowberry pomace zymolyte into a cleaned 100ml conical flask, adding 40ml of deionized water, adjusting the pH to be PH =6 by using concentrated hydrochloric acid, sterilizing at high temperature and high pressure, inoculating 10% of mixed bacterial liquid, putting the mixture into a constant temperature incubator at 37 ℃, respectively culturing for 12h,24h,36h,48h,60h and 72h, and performing three groups of parallel tests. And taking out the fermentation liquor, centrifuging, evaporating and concentrating, precipitating with ethanol, centrifuging again, finally drying, weighing, and drawing a fermentation time-cowberry fruit residue SDF yield graph, wherein the highest content is the optimal fermentation time. The selected fermentation time was 24h.
Then analyzing the influence of different liquid-material ratios on the yield of the cowberry pomace SDF, accurately weighing 5g of cowberry pomace zymolyte, placing the cowberry pomace zymolyte in a cleaned 100mL conical flask, adding deionized water according to the liquid-material ratio of 2. And taking out the fermentation liquor, centrifuging, evaporating and concentrating, precipitating with ethanol, centrifuging again, drying, weighing, and drawing a liquid-material ratio-cowberry fruit residue SDF yield diagram, wherein the highest content is the optimal liquid-material ratio. The yield of blueberry pomace SDF gradually increased with increasing liquid-to-feed ratio, i.e. with increasing liquid volume in the reaction environment, and peaked at 10 (mL: g) at 9.28%, followed by a slight decrease. It is inferred that the complex bacteria can grow well in a liquid-rich environment and can also dilute the acidic components, but when the bacteria grow beyond a certain limit, the product is too concentrated [69], a small amount of pectin is hydrolyzed, and thus the pectin tends to slightly decrease after reaching a peak value. Therefore, the optimum liquid-to-material ratio was selected to be 10.
Then analyzing the influence of different fermentation pH values on the yield of the SDF of the cowberry fruit residues, accurately weighing 5g of cowberry fruit residue zymolyte, putting the cowberry fruit residue zymolyte into a cleaned 100ml conical flask, adding 50ml of deionized water, adjusting the pH value to pH =4, 5,6,7,8 and 9 by using concentrated hydrochloric acid or 20% sodium hydroxide, sterilizing at high temperature and high pressure, inoculating 10% of mixed bacteria liquid, culturing for 24h in a constant temperature incubator at 37 ℃, and performing three groups of parallel tests. And taking out the fermentation liquor, centrifuging, evaporating and concentrating, precipitating with ethanol, centrifuging again, finally drying, weighing, and drawing a fermentation PH-cowberry fruit residue SDF yield graph, wherein the highest content is the optimal fermentation PH. As PH increased, the cranberry pomace SDF yield exhibited a trend of increasing first and then decreasing, with the highest yield occurring at PH =7, being 10.9%. It is presumed that when the acidity is too high, the growth of fungi is inhibited, the activity of the product such as enzyme is lowered, and the active ingredient of the obtained dietary fiber is partially hydrolyzed, thereby finally determining the fermentation pH to be 7.
Then analyzing the influence of different fermentation temperatures on the SDF yield of the cowberry fruit residues, accurately weighing 5g of cowberry fruit residue zymolyte, putting the cowberry fruit residue zymolyte into a cleaned 100ml conical flask, adding 50ml of deionized water, adjusting the pH to be PH =7 by using concentrated hydrochloric acid, sterilizing at high temperature and high pressure, inoculating 10% of mixed bacteria liquid, putting the mixture into constant-temperature incubators at 26.5 ℃,30 ℃,33.5 ℃,37 ℃,40.5 ℃ and 44 ℃ respectively, culturing for 24h, and performing three groups of parallel tests. And taking out the fermentation liquor, centrifuging, evaporating and concentrating, precipitating with ethanol, centrifuging again, finally drying, weighing, and drawing a fermentation temperature-cowberry fruit residue SDF yield graph, wherein the highest content is the optimal fermentation temperature. The higher the fermentation temperature, the higher the yield of the blueberry pomace SDF; the yield is improved firstly and then slightly reduced and then tends to be stable, the highest value is 9.69% at the temperature of 33.5 ℃, and the growth rate is fastest at the temperature of 30-33.5 ℃; it is known that the activity of lactic acid bacteria is inhibited at higher fermentation temperature [69] and shows a downward trend, while the composite bacterial liquid can still ensure a more stable yield rate at higher fermentation temperature, which compensates for the yield reduction caused by decomposition of products caused by high temperature, so that the composite bacterial liquid has a larger control range and fault tolerance rate in the operation process, and meets the requirements of pilot plant test.
And finally, analyzing the influence of different inoculation amounts on the SDF yield of the cowberry fruit residues, accurately weighing 5g of cowberry fruit residue zymolyte, placing the cowberry fruit residue zymolyte into a cleaned 100ml conical flask, adding 50ml of deionized water, adjusting the pH to be PH =7 by using concentrated hydrochloric acid, sterilizing at high temperature and high pressure, inoculating the cowberry fruit residue zymolyte into the mixed bacterial liquid according to 6%,8%,10%,12%,14% and 16%, placing the cowberry fruit residue zymolyte into a constant-temperature incubator at 33.5 ℃, culturing for 24h, and performing three groups of parallel tests. And taking out the fermentation liquor, centrifuging, evaporating and concentrating, precipitating with ethanol, centrifuging again, finally drying, weighing, and drawing an inoculation amount-cowberry fruit residue SDF yield diagram, wherein the highest content is the optimal fermentation inoculation amount. With the increase of the inoculation amount, the SDF yield of the cowberry fruit residues shows a trend of increasing firstly and then decreasing, reaches a highest value at 14 percent, is 9.8 percent, and increases the speed fastest at 10 to 12 percent. When the inoculation amount is low, the whole concentration of the bacterial liquid is low, and the fermentation process is slow; with the increase of the concentration, the overall concentration of substances such as produced enzyme, acid and the like is increased, and the products are effectively increased; when the concentration of the zymophyte is too high, or unnecessary competition is generated, nutrient substances are rapidly consumed, so that part of zymophyte can not obtain enough nutrition in the later period of fermentation, and the optimal zymophyte liquid inoculation amount is determined to be 14%.
S230, designing 4-factor 3 horizontal orthogonal test to determine the optimal fermentation condition.
Accurately weighing 5g of cowberry fruit residue zymolyte, placing the cowberry fruit residue zymolyte in a cleaned 100ml conical flask, and then selecting fermentation temperature, fermentation pH, bacterial liquid inoculation amount and material-liquid ratio to perform 4-factor 3 horizontal orthogonal test design, wherein an orthogonal table is shown in the following table 1, and three groups of parallel tests are performed to determine the optimal fermentation condition. The fermentation time is 24h.
TABLE 1L 9 (3 4 ) Experiment factor level meter
The data obtained by the orthogonal experiment were analyzed as shown in the following section 2, and an orthogonal data factor effect graph was prepared.
TABLE 2L 9 (3 4 ) Orthogonal data analysis table
The mean value shows that the optimal process for optimizing, fermenting and extracting the blueberry pomace SDF by the orthogonal method is that the pH =7, when the temperature is 33.5 ℃,14% is inoculated according to the feed-liquid ratio of 10 (mL/g).
S240, preparing the cowberry fruit residue SDF by using the optimal fermentation condition.
S300, preparing core material stichopus japonicus visceral oligopeptide.
S310, preparing the stichopus japonicus intestine enzymolysis liquid.
Preliminarily dissecting the intestines of the oplopanax elatus nakai, removing sand, cutting into pieces, adding water according to the mass of the materials, and mixing to obtain homogenate with the concentration of 20-40%; respectively adding 0.2-0.3% of papain, flavourzyme and compound protease into the homogenate, mixing and intermittently stirring, carrying out enzymolysis for 2-4h in a water bath kettle at 54-56 ℃, heating water to above 80 ℃, inactivating enzyme for at least 15min, cooling to room temperature, and filtering to obtain a filtrate 1; adding 5% active carbon powder into the filtrate 1 by mass, intermittently stirring for more than 30min, settling for 20min, filtering, and decolorizing to obtain filtrate 2, i.e. Stichopus japonicus intestine enzymolysis solution.
S320, designing a single-factor experiment, and analyzing the influence of each factor on the yield of the stichopus japonicus visceral oligopeptide.
Firstly, determining the influence of different fermentation times on the yield of stichopus japonicus visceral oligopeptide, accurately measuring 30mL of enzymatic hydrolysate (corresponding to 5g of stichopus japonicus intestine) in a conical flask, and inoculating bacteria according to the following ratio of bacillus subtilis: lactobacillus plantarum: yeast =1, 6% was inoculated in the culture medium 1, PH was adjusted to 7, fermentation temperature was set to 33.5 ℃, fermentation time was 1D, 2D, 3D, 4D, 5D, 6D, 7D, 8D, respectively, and three groups were performed in parallel. Taking out the fermentation liquor, centrifuging, directly using the supernatant for detecting the peptide yield, and freeze-drying the rest part to obtain the stichopus japonicus viscera oligopeptide powder. Calculating the yield of stichopus japonicus visceral oligopeptide, and drawing a fermentation time-stichopus japonicus visceral oligopeptide yield graph. The yield of the stichopus japonicus visceral oligopeptides is increased along with the prolonging of the fermentation time, the yield of the stichopus japonicus visceral oligopeptides reaches the highest at 7D and is 14.2%, and the yield of the stichopus japonicus visceral oligopeptides tends to be gentle and slightly reduced at 8D; therefore, to obtain the highest extraction yield, the optimal fermentation time is finally selected to be 7D.
Then, determining the influence of different inoculation amounts on the yield of stichopus japonicus visceral oligopeptide, accurately measuring 30mL of enzymatic hydrolysate (corresponding to 5g of stichopus japonicus intestine) in a conical flask, and inoculating the bacillus subtilis: lactobacillus plantarum: yeast =1, inoculated with 1.5%,3%,4.5%,6%,7.5%,9%, respectively, adjusted PH to 7, set fermentation temperature at 33.5 ℃, fermentation time 8D, and run in parallel for three groups. Taking out the fermentation liquor, centrifuging, directly using the supernatant for detecting the peptide yield, and freeze-drying the rest part to obtain the stichopus japonicus viscera oligopeptide powder. And calculating the yield of stichopus japonicus visceral oligopeptide, and drawing an inoculation amount-stichopus japonicus visceral oligopeptide yield graph. With the increase of the inoculation amount, the yield of the stichopus japonicus visceral oligopeptide tends to increase firstly and then decrease, the highest value is reached at 7.5 percent, 16.15 percent, and the speed is increased fastest at 4.5 to 6 percent. When the inoculation amount is low, the whole concentration of the bacteria liquid is low, and the stichopus japonicus viscera oligopeptides are mainly the concentration of the peptides in the enzymolysis liquid; with the increase of the concentration, the yield of the stichopus japonicus visceral oligopeptide gradually increases; when the concentration of the zymophyte is too high, the stichopus japonicus intestinal peptide part is consumed as a nutrient substance, so that the stichopus japonicus intestinal peptide part has a descending trend after 7.5 percent; and (4) determining the optimal inoculum size of the zymocyte liquid to be 7.5 percent by combining the above steps.
Then determining the influence of different fermentation temperatures on the yield of the stichopus japonicus visceral oligopeptides, accurately measuring 30mL of enzymatic hydrolysate (corresponding to 5g of stichopus japonicus intestine) in a conical flask, and inoculating the bacillus according to the ratio of bacillus subtilis: lactobacillus plantarum: yeast =1, 7.5% was inoculated, PH was adjusted to 7, fermentation temperatures were set at 26.5 ℃,30 ℃,33.5 ℃,37 ℃,40.5 ℃, and fermentation times were set at 8D, respectively, and three groups were performed in parallel. Taking out the fermentation liquor, centrifuging, directly using the supernatant for detecting the peptide yield, and freeze-drying the rest part to obtain the stichopus japonicus viscera oligopeptide powder. Calculating the yield of the stichopus japonicus visceral oligopeptide, and drawing a fermentation temperature-yield graph of the stichopus japonicus visceral oligopeptide. With the rise of the fermentation temperature, the yield of the stichopus japonicus visceral oligopeptide is shown to rise firstly and then fall; the intestinal peptide yield increases fastest at 33.5-37 ℃, reaches a maximum value of 14% at 37 ℃, and then decreases; it is presumed that at a high fermentation temperature, the growth of fungi is inhibited and the produced enzyme is not optimally reactive, and therefore, the fermentation temperature is selected to be 37 ℃.
And finally, determining the influence of different fermentation pH values on the yield of the stichopus japonicus visceral oligopeptide, accurately measuring 30mL of enzymatic hydrolysate (corresponding to 5g of stichopus japonicus intestine) in a conical flask, and inoculating the bacillus according to the ratio of bacillus subtilis: lactobacillus plantarum: yeast =1, 7.5% was inoculated, PH was adjusted to 4,5,6,7,8,9, respectively, fermentation temperature was set at 37 ℃ and fermentation time was 8D, and three sets of experiments were performed in parallel. Taking out the fermentation liquor, centrifuging, directly using the supernatant for detecting the peptide yield, and freeze-drying the rest part to obtain the stichopus japonicus viscera oligopeptide powder. And calculating the yield of the stichopus japonicus visceral oligopeptides and drawing a fermentation PH-yield graph of the stichopus japonicus visceral oligopeptides. With the increase of the pH value, the yield of the stichopus japonicus visceral oligopeptides tends to increase and then decrease, wherein the intestinal peptide yield is increased at the highest speed when the pH value is 6 to 7, and the highest yield appears at the pH =7 and is 16.2%. It is presumed that when the acidity or basicity is too high, the growth of fungi is inhibited and the activity of the produced enzyme is lowered, and therefore, the fermentation pH is finally determined to be 7.
S330, designing a 4-factor 3 horizontal orthogonal test according to the single-factor test result, and determining the optimal fermentation condition.
Accurately measuring 30mL of enzymolysis liquid in a conical flask, selecting fermentation temperature, fermentation pH and bacterial liquid inoculation amount, carrying out 4-factor 3 horizontal orthogonal test design, and carrying out three groups of parallel tests so as to determine the optimal fermentation condition.
TABLE 3L 9 (3 4 ) Experiment factor level meter
The data analysis of the orthogonal test is shown in the following table 3, and a graph of the effect of visceral oligopeptide factors of stichopus japonicus was prepared.
TABLE 4L 9 (3 4 ) Orthogonal data analysis table
The average value shows that the optimal process for preparing the stichopus japonicus visceral oligopeptide by optimizing fermentation by the orthogonal method is that the pH =7, 9% of inoculation is carried out at the temperature of 37 ℃, and the fermentation is carried out for 7D; centrifuging the fermentation liquid for 15min at 5000r/min, and freeze-drying the final obtained liquid in a vacuum freeze dryer at constant temperature for 48h to obtain the stichopus japonicus viscera oligopeptide.
After 3 times of fermentation tests are repeated according to the formula, the yield of stichopus japonicus visceral oligopeptide reaches the highest value of 16.82%, and the fishy smell of the final product is obviously reduced.
S340, preparing the stichopus japonicus visceral oligopeptide by using the optimal fermentation condition.
S400, preparing stichopus japonicus visceral oligopeptide microgel.
S410, adding 1.5 percent of wall material, accounting for 1.5g, and designing a single-factor experiment to analyze the influence of each factor on the embedding rate;
firstly, analyzing the influence of different wall material ratios on the dietary fiber stichopus japonicus visceral oligopeptide microcapsule embedding rate, and preparing a ratio solution by selecting a ratio of cowberry fruit residue SDF to sodium alginate as 0. Adding stichopus japonicus viscera oligopeptide according to the mass ratio of the core material to the wall material of 0.3, adding 0.25 mass percent of sucrose fatty acid ester to promote dissolution, heating at the constant temperature of 60 ℃, performing ultrasonic treatment and oscillating for 30min, injecting the mixed solution into a needle tube, dropping 2 percent of CaCl2 solidification solution at constant speed, stirring, oscillating and solidifying for 1h, cleaning, filtering, and finally freeze-drying to obtain the microcapsule. And calculating the embedding rate, drawing a wall material ratio-embedding rate graph, and obtaining the optimal ratio of the areas. Through analysis, when the blueberry pomace SDF is added, the ratio of the blueberry pomace SDF to the sodium alginate is 1.
Then analyzing the influence of different core-wall ratios on the dietary fiber stichopus japonicus visceral oligopeptide microcapsule embedding rate, preparing five groups of wall material solutions according to the selected wall material ratio, and controlling the mass ratio of the core material to the wall material to be 0.1,0.2,0.3,0.4 and 0.5; adding viscera oligopeptide of Stichopus japonicus, adding 0.25 wt% sucrose fatty acid ester to promote dissolution, heating at constant temperature of 60 deg.C, performing ultrasonic treatment, shaking for 30min, injecting the mixed solution into a needle tube, dropping 2% CaCl2 coagulating solution at constant speed, stirring, shaking for 1 hr for solidification, cleaning, filtering, and lyophilizing. And calculating the embedding rate, and drawing a core-wall ratio-embedding rate graph. Through analysis, the ratio of the core material to the wall material is selected to be in the range of 0.1 to 0.3, so that a better embedding effect is achieved, and the ratio of the core material to the wall material is preferably selected to be 0.2.
And then analyzing the influence of the mass fraction of the sucrose fatty acid ester on the embedding rate of dietary fiber stichopus japonicus visceral oligopeptide microcapsules, wherein the mass fraction of the sucrose fatty acid ester is calculated according to the following formula: preparing a wall material solution by sodium alginate =1, controlling the mass ratio of a core material to the wall material to be 0.2, adding stichopus japonicus visceral oligopeptide, adding sucrose fatty acid ester with the mass fraction of 0.15%,0.2%,0.25%,0.3% and 0.35% to promote dissolution, heating at a constant temperature of 60 ℃, performing ultrasonic treatment and shaking for 30min, injecting the mixed solution into a needle tube, and dripping 2% CaCl at a constant speed into the needle tube 2 Stirring the coagulating liquid, shaking for solidifying for 1h, cleaning, filtering, and freeze-drying. And calculating the embedding rate, and drawing a sucrose fatty acid ester mass fraction-embedding rate graph. Through analysis, the sucrose fatty acid ester is in the range of 0.25-0.3%, the microcapsule embedding rate is better, and the highest embedding rate is 82.24% when the addition amount of 0.3% is present. The addition of sucrose fatty acid ester can promote the dissolution of wall materials and promote the microcapsule shape; because the wall material has certain pores, the sucrose fatty acid ester can contact with the mixed wall material of sodium alginate and dietary fiber to form a compound under the combined action of multiple intermolecular forces after the sucrose fatty acid ester is added, the pores are filled, and embedding is promoted [44,47 ]]Therefore, the embedding rate always shows an ascending trend before the addition amount is 0.3 percent; when the addition amount of the sucrose fatty acid ester is too high, the molecular chain gaps formed by the wall materials are enlarged, the intermolecular force is reduced, and the embedding effect is greatly reduced, so that the embedding rate is obviously reduced after the excessive amount is addedTrend.
And then analyzing the influence of different coagulating liquid mass fractions on the dietary fiber stichopus japonicus visceral oligopeptide microcapsule embedding rate, wherein the mass fractions are as follows: preparing a wall material solution by sodium alginate =1 and 2, wherein the mass ratio of the core material to the wall material is controlled to be 0.2; adding viscera oligopeptide of Stichopus japonicus, adding 0.3 wt% sucrose fatty acid ester to promote dissolution, heating at constant temperature of 60 deg.C, performing ultrasonic treatment, shaking for 30min, injecting the mixed solution into needle tube, and respectively dropping 1%,2%,3%,4%,5%, and 6% CaCl at constant speed 2 Stirring in coagulating liquid, shaking for solidifying for 1 hr, cleaning, filtering, and lyophilizing. And calculating the embedding rate, and drawing a solidification liquid mass fraction-embedding rate graph. According to analysis, when the mass fraction of the solidification liquid is between 3 and 5 percent, the embedding rate is better than 4 percent, the highest value is achieved, and the embedding rate is 79.28 percent. When the concentration of the solidification liquid is lower, the solidification degree of the microcapsule is low in the same time, and the phenomenon of adhesion and agglomeration is supposed to be generated due to incomplete ion crosslinking; when the concentration of the solidification solution is too high, the outer layer of the gel ball is quickly solidified to form a compact layer, so that the solution is prevented from further permeating; when the microspheres obtained in the two cases are washed by normal saline for multiple times, part of the microspheres are easy to crack and dissolve out.
And S420, obtaining the optimal preparation process condition by using a response surface optimization method.
In order to obtain the optimal embedding process of the microcapsules and explore the interaction effect of various factors, response surface optimization is selected in the experiment, and on the basis of single-factor optimal experiment conditions, the mass fraction A of the solidification liquid, the mass fraction B of the sucrose fatty acid ester and the wall-core ratio C are selected to perform three-factor three-level response surface experiments.
TABLE 5 response surface experiment factor level coding table
Table 6 response surface experimental design and results
TABLE 7 analysis of variance results of response surface fitting regression equation
| Sources of variance | Sum of squares | Degree of freedom | Variance (variance) | F value | P | Significance of |
| Regression model | 74.51 | 9 | 8.28 | 149.69 | ＜0.0001 | significant |
| Mass fraction of A-solidification liquid | 0.12 | 1 | 0.12 | 2.26 | 0.1764 | |
| B-sucrose fatty acid ester mass fraction | 0.62 | 1 | 0.62 | 11.14 | 0.0125 | |
| C-core wall ratio | 1.38 | 1 | 1.38 | 24.91 | 0.0016 | |
| AB | 0.81 | 1 | 0.81 | 14.65 | 0.0065 | |
| AC | 0.41 | 1 | 0.41 | 7.41 | 0.0297 | |
| BC | 0.96 | 1 | 0.96 | 17.37 | 0.0042 | |
| A 2 | 18.33 | 1 | 18.33 | 331.45 | ＜0.0001 | |
| B 2 | 18.42 | 1 | 18.42 | 333.04 | ＜0.0001 | |
| C 2 | 26.14 | 1 | 26.14 | 472.61 | ＜0.0001 | |
| Residual error | 0.39 | 7 | 0.055 | |||
| Item of mistyption | 0.15 | 3 | 0.051 | 0.86 | 0.5296 | notsignificant |
| Pure error | 0.24 | 4 | 0.059 | |||
| Total up to | 74.89 | 16 |
Note: r 2 ＝0.9948，Adj R 2 ＝0.9882，Pred R 2 ＝0.9626
The data table shows that the regression model has good significance, the simulation losing items are not significant, the interactive items are all less than 0.05, and the experimental design is judged to be good; from data relation analysis, R2 is a response value of 99.48%, the two groups of data with the Adj R2 are close to 1, the difference value between the Adj R2 and the Pred R2 is less than 0.2, the C.V.% is 0.31 and less than 10, the adeq predictor is 28.888 and more than 4, and the model is judged to meet the requirement; according to the change of the value P, the influence factors C > B > A on the embedding rate can be obtained, namely the influence order on the embedding rate is as follows: the wall-core ratio is more than the mass fraction of sucrose fatty acid ester and more than the mass fraction of solidification liquid; in conclusion, the model has good fitting and is matched with real data, and the microcapsule embedding rate can be predicted according to the trend.
The experimental data are designed according to a Design-Expert8.0.6 program, and the obtained regression equation is as follows: the embedding rate/% =79.29 +0.13A +0.28B +0.41C-0.45A +0.32A C +0.49B +C-2.09 A2-2.09 B2-2.49C 2. And obtaining a response surface of the regression equation and a contour map thereof.
It can be seen from the response surface and the contour map thereof that the influence of different factors on the embedding rate is as shown in the figure, when one of the core-wall ratio, the mass fraction of the solidification liquid and the mass fraction of the sucrose fatty acid ester is taken as a set condition, the other two values are changed interactively, in the response surface space map, the whole three groups of experiments show paraboloids which rise first and then fall, the trend is obvious, the highest peak exists, namely, the maximum value in the test range can be taken as a stable point.
The shape of the contour line is known to be approximate to an ellipse, the stronger the influence of the interaction of the factors on the embedding rate of the microcapsule is, the larger the influence of the factors is, the smaller the influence is, the BC (mass fraction of sucrose fatty acid ester-wall-core ratio) is greater than AB (mass fraction of solidification liquid-mass fraction of sucrose fatty acid ester) is greater than AC (mass fraction of solidification liquid-wall-core ratio) by combining with the analysis of variance numerical value.
According to the response surface optimization design, the theoretical optimal embedding conditions are finally obtained, wherein the mass fraction of the coagulating liquid is 3.51%, the mass fraction of the sucrose fatty acid ester is 0.3%, the core-wall ratio is 0.21, the theoretical embedding rate can reach 79.3193%, the embedding rate obtained by adopting the same experimental conditions and the final experiment is 79.25%, and is close to the theoretical value, so that the model can be used for optimizing the embedding rate of dietary fiber stichopus japonicus visceral oligopeptide microcapsules.
S430 microcapsules are prepared according to the optimal embedding conditions obtained in S410 and S420. Controlling the height between the needle tube and the liquid level to be more than or equal to 20 cm, dripping a solidification liquid at a constant speed, stirring at a constant speed to prevent adhesion, and vibrating and solidifying for 1h to obtain gel balls; and repeatedly washing the gel balls with normal saline to wash out the surface saline solution, filtering, and drying in a freeze dryer to obtain the microcapsule.
The effect of the microcapsules prepared by the method is verified.
And performing a microcapsule gastrointestinal tract simulation in vitro sustained release experiment.
Firstly, preparing artificial gastric juice and intestinal juice, dissolving 20g of pepsin in 0.164mol of hydrochloric acid, adding water to prepare 1L of solution, and then sterilizing by using a filter membrane to obtain the artificial gastric juice; preparing 50mL of 3.25% potassium dihydrogen phosphate solution, dissolving 10g of trypsin, adjusting the pH value to 7.4, adding water to prepare 1L of solution, and then sterilizing by using a filter membrane to obtain the artificial intestinal juice.
And (3) measuring the release amount of the microcapsules in gastric juice and intestinal juice, respectively taking 1g of microcapsules, dissolving the microcapsules in 100mL of simulated intestinal juice or gastric juice, controlling the temperature at 37 ℃, shaking at a low speed, sampling every 1h to measure the absorbance, recording the change of the peptide content in the solution, and judging the slow release and release effects of the microcapsules. And drawing a corresponding release rate line graph.
With the time being prolonged, the middle part of the bilberry pomace SDF is decomposed under the influence of acidic conditions to generate gaps and promote the dissolution of stichopus japonicus visceral oligopeptides in the microcapsules, so that the concentration of the stichopus japonicus visceral oligopeptides in simulated gastric juice is gradually increased, namely the release amount is gradually increased; and after the non-embedded stichopus japonicus visceral oligopeptide is added with the simulated gastric juice and gradually stirred and uniformly mixed, the concentration of the stichopus japonicus visceral oligopeptide is gradually increased until the stichopus japonicus visceral oligopeptide is completely dissolved in 5 hours, namely the release amount reaches 100 percent. The release amount of the product microcapsule in the artificial gastric juice is lower than that of the non-embedded stichopus japonicus visceral oligopeptide in the whole process, so that the aim of effectively realizing the slow release of the microcapsule in simulated gastric juice can be inferred.
After the microcapsule is added with the artificial intestinal juice, the concentration of the oligopeptide in the internal organs of the stichopus japonicus in the artificial intestinal juice is positively correlated with the release time, namely the longer the release time is, the larger the release amount of the content of the microcapsule is; the non-embedded stichopus japonicus visceral oligopeptides are not uniformly mixed initially, the release amount is small, in the subsequent process of dissolving the stichopus japonicus visceral oligopeptides in the simulated intestinal juice, the concentration of the stichopus japonicus visceral oligopeptides in the simulated intestinal juice is gradually increased until the stichopus japonicus visceral oligopeptides are completely dissolved in 6 hours, and the release amount reaches 100%. In the experiment, the difference between the release amount of the microcapsule and the release amount of the viscera oligopeptide of the non-embedded stichopus japonicus in the artificial intestinal juice is small because the SDF of the blueberry pomace used in the experiment has good swelling capacity and can be broken in the intestinal tract with higher PH, and in addition, a plurality of probiotics are also arranged in the actual intestinal tract of a human body, part of the probiotics can release substances such as pectinase and the like to promote the microcapsule to break, so that the viscera oligopeptide of the stichopus japonicus is further released; the combination of the above results indicates that the microcapsules have good release ability in simulated intestinal fluid.
Claims (10)
1. The preparation method of the stichopus japonicus visceral oligopeptide microcapsules is characterized by comprising the following steps:
s100, preparing fermentation bacteria, screening lactobacillus plantarum from cowberry fruit residues, obtaining bacillus subtilis from stichopus japonicus intestinal tracts, and proportionally matching commercial yeasts with the lactobacillus plantarum in the commercial yeasts to serve as probiotics for fermentation;
s200, preparing cowberry fruit residue SDF, treating cowberry fruit residue by adopting a bacterial enzyme interaction technology, performing primary enzymolysis on raw material fruit residue, inoculating the raw material fruit residue to fermentation probiotics prepared in S100 for fermentation, and concentrating, extracting with alcohol and drying fermentation liquor to obtain SDF;
s300, preparing core material stichopus japonicus viscera oligopeptide, pretreating stichopus japonicus intestines, performing enzymolysis and decoloration by using compound protease, then inoculating probiotics for fermentation prepared in S100 for fermentation, taking out fermentation liquor, centrifuging, and freeze-drying supernatant to obtain stichopus japonicus viscera oligopeptide powder;
s400, preparing stichopus japonicus visceral oligopeptide microgel, preparing a solution by taking cowberry fruit residue SDF and sodium alginate as wall materials, inoculating a core material stichopus japonicus visceral oligopeptide, adding sucrose fatty acid ester to promote dissolution, heating, performing ultrasonic treatment, vibrating and fully dissolving, and preparing the microcapsule by using an orifice method.
2. The method for preparing the stichopus japonicus visceral oligopeptide microcapsule according to claim 1, wherein the step of: the step S100 specifically includes the steps of,
s110, cutting fresh stichopus japonicus intestines, respectively placing contents in the intestines in an EP (EP) tube, diluting the intestines with seawater subjected to suction filtration and sterilization, centrifuging the intestines to obtain supernate, and properly diluting the supernate to obtain a content diluent; diluting and centrifuging cowberry fruit residue powder with normal saline to obtain cowberry fruit residue diluent; marking MRS and LB solid culture medium, respectively coating the content diluent and the cowberry fruit residue diluent on the corresponding culture medium, and culturing in an incubator at 37 ℃;
s120, selecting different types of single colonies from the coating plate, streaking and purifying the single colonies in corresponding culture media, and screening to obtain lactobacillus plantarum and bacillus subtilis after sequencing;
s130, preparing the lactobacillus plantarum, the bacillus subtilis and the yeast obtained in S120 into zymocyte liquid according to a ratio of 1.
3. The method for preparing the stichopus japonicus visceral oligopeptide microcapsule according to claim 1, wherein the step of: the step S200 specifically includes the steps of,
s210, processing cowberry fruit residues by adopting a bacterial enzyme interaction technology, firstly adopting a double-enzyme method, firstly carrying out enzymolysis by using alpha-amylase, then regulating the pH value to be neutral by using NaOH, then carrying out enzymolysis by using alkaline protease, and drying to obtain cowberry fruit residue zymolyte;
s220, designing a single-factor experiment, and analyzing the influence of each factor on the SDF yield of the cowberry fruit residues; carrying out a single-factor test by taking the fermentation time as 12h,24h,36h,48h,60h and 72h, the liquid-material ratio as 2;
s230, designing a 4-factor 3 horizontal orthogonal test to determine an optimal fermentation condition; positioning the optimal fermentation time obtained by the single-factor test according to the fermentation time, and then selecting a fermentation temperature of 30 ℃,33.5 ℃,37 ℃, a fermentation pH of 6,7 and 8, a bacterial liquid inoculation amount of 8;
s240, preparing the blueberry pomace SDF by using the optimal fermentation conditions.
4. The method for preparing the sea cucumber visceral oligopeptide microcapsule according to claim 1, wherein the microcapsule comprises the following components: the step S300 specifically includes the steps of,
s310, preparing an apostichopus japonicus intestine enzymolysis liquid, preliminarily dissecting the apostichopus japonicus intestine, removing sand, shearing into pieces, adding water according to the mass of the materials, and mixing to obtain homogenate with the concentration of 20-40%; adding 0.2-0.3% of papain, flavourzyme and compound protease, mixing and stirring intermittently, carrying out enzymolysis for 2-4h in a water bath kettle at 54-56 ℃, heating water to above 80 ℃, inactivating enzyme for at least 15min, cooling to room temperature, and filtering to obtain filtrate 1; adding activated carbon powder into the filtrate 1, intermittently stirring for more than 30min, settling for 20min, filtering, and decolorizing to obtain filtrate 2, which is Stichopus japonicus intestine enzymolysis liquid;
s320, designing a single-factor experiment, and analyzing the influence of each factor on the yield of the stichopus japonicus visceral oligopeptide; respectively analyzing the influence of each factor on the yield of the stichopus japonicus visceral oligopeptides by taking the fermentation time as 1D, 2D, 3D, 4D, 5D, 6D, 7D and 8D, the inoculation amount of probiotics for fermentation as 1.5%,3%,4.5%,6%,7.5% and 9%, the fermentation temperature as 26.5 ℃,30 ℃,33.5 ℃,37 ℃,40.5 ℃ and the fermentation pH as 4,5,6,7,8 and 9 as a single-factor test;
s330, according to the single-factor test result, the fermentation time is the optimal fermentation time of the single-factor experiment, the 4-factor 3 horizontal orthogonal test is designed according to the fermentation temperature of 33.5 ℃, the fermentation temperature of 37 ℃, the fermentation temperature of 40.5 ℃, the fermentation PH of 6,7 and 8 and the bacterial liquid inoculation amount of 6%,7.5% and 9%, so as to determine the optimal fermentation condition;
s340, preparing the stichopus japonicus visceral oligopeptide by using the optimal fermentation condition.
5. The method for preparing the stichopus japonicus visceral oligopeptide microcapsule according to claim 1, wherein the step of: the step 4100 specifically includes the steps of,
s410, designing a single-factor experiment to analyze the influence of each factor on the embedding rate; the ratio of the cowberry pomace SDF to the sodium alginate is (1.5, 1, 2, 1, 2); the ratio of the core material to the wall material is 0.1,0.2,0.3,0.4 and 0.5, the mass of the sucrose fatty acid ester is 0.15 percent, 0.2 percent, 0.25 percent, 0.3 percent and 0.35 percent, the mass fraction of the solidification liquid is 1 percent, 2 percent, 3 percent, 4 percent, 5 percent and 6 percent are single-factor tests, and the influence of the factors on the embedding rate is analyzed;
s420, obtaining an optimal preparation process condition by using a response surface optimization method, selecting a solidification liquid mass fraction A, a sucrose fatty acid ester mass fraction B and a wall-core ratio C on the basis of a single-factor optimal experiment condition, and performing a three-factor three-level response surface experiment; the experimental data are designed according to a Design-Expert8.0.6 program to obtain a regression equation as follows: the embedding rate/% =79.29 +0.13A +0.28B +0.41C-0.45A +0.32A C +0.49B +C-2.09 A2-2.09 B2-2.49C 2; drawing a response surface of the regression equation and a contour map thereof to obtain an optimal embedding condition;
s430, preparing microcapsules under the optimal embedding conditions obtained in S410 and S420, controlling the height between the needle tube and the liquid level to be more than or equal to 20 cm, dripping a solidification liquid at a constant speed, stirring at a constant speed to prevent adhesion, and vibrating and solidifying to obtain gel balls; and repeatedly washing the gel balls with normal saline to wash out the surface saline solution, filtering, and drying in a freeze dryer to finally obtain the microcapsule.
6. The method for preparing the stichopus japonicus visceral oligopeptide microcapsule according to claim 3, wherein the step of: the optimal fermentation conditions in the step S200 are PH =7 and the temperature is 33.5 ℃,14% of inoculation is performed according to a feed-liquid ratio of 10, and the fermentation time is 24h.
7. The method for preparing the sea cucumber visceral oligopeptide microcapsule according to claim 4, wherein the microcapsule comprises the following components in parts by weight: the optimal fermentation conditions in step S300 were PH =7, and when the temperature was 37 ℃,9% inoculation and 7D fermentation were performed.
8. The method for preparing the stichopus japonicus visceral oligopeptide microcapsule according to claim 4, wherein the step of: the optimal embedding conditions in the step S400 are that the mass fraction of the solidification liquid is 3.51%, the mass fraction of the sucrose fatty acid ester is 0.3%, the core-wall ratio is 0.21, and the ratio of the cowberry fruit residue SDF to the sodium alginate is 1.
9. The method for preparing the sea cucumber visceral oligopeptide microcapsule according to claim 4, wherein the microcapsule comprises the following components in parts by weight: in the S420, the mass fraction of the solidified liquid is 3%, 3.5% and 4%, and the mass fraction of the sucrose fatty acid ester is 0.25%,0.3% and 0.35%.
10. A stichopus japonicus visceral oligopeptide microcapsule is characterized in that: prepared by the process of any one of claims 1 to 9.
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| CN110419716A (en) * | 2019-08-07 | 2019-11-08 | 河北科技大学 | A kind of Brevibacillus laterosporus antibacterial peptide microcapsule preparation method |
| CN110973342A (en) * | 2020-03-05 | 2020-04-10 | 中国科学院烟台海岸带研究所 | Apostichopus japonicus oligopeptide and preparation method and application thereof |
| CN113693243A (en) * | 2020-09-14 | 2021-11-26 | 贵州省中国科学院天然产物化学重点实验室(贵州医科大学天然产物化学重点实验室) | Method for preparing dietary fiber by biotransformation of rosa roxburghii tratt pomace by edible fungi |
| CN113397175A (en) * | 2021-06-30 | 2021-09-17 | 中国科学院烟台海岸带研究所 | Stichopus japonicus oligopeptide cross-linked microcapsule and preparation method and application thereof |
| CN113558246A (en) * | 2021-07-21 | 2021-10-29 | 石河子大学 | Symbiotic bifidobacterium composite microcapsule and preparation method thereof |
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