CN114522195A - Chinese chestnut peel pigment nano intestinal probiotic accelerant with Chinese chestnut almond glycoprotein coating and preparation and application thereof - Google Patents
Chinese chestnut peel pigment nano intestinal probiotic accelerant with Chinese chestnut almond glycoprotein coating and preparation and application thereof Download PDFInfo
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- CN114522195A CN114522195A CN202210101388.9A CN202210101388A CN114522195A CN 114522195 A CN114522195 A CN 114522195A CN 202210101388 A CN202210101388 A CN 202210101388A CN 114522195 A CN114522195 A CN 114522195A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
- A61K36/736—Prunus, e.g. plum, cherry, peach, apricot or almond
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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
- A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
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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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention belongs to the technical field of probiotic promoters, and discloses a chestnut almond glycoprotein coated chestnut peel pigment nano intestinal probiotic promoter as well as preparation and application thereof. The method comprises the following steps: 1) extracting pigment from the chestnut peel to obtain the chestnut peel pigment; 2) extracting semen Castaneae and semen Armeniacae amarum in buffer solution with pH of 7-10 to obtain mixture of polysaccharide and protein; 3) reacting polysaccharide and protein in the mixture to obtain chestnut almond glycoprotein conjugate; 4) respectively preparing the chestnut peel pigment and the chestnut almond glycoprotein conjugate into solutions, mixing the chestnut peel pigment solution and the chestnut almond glycoprotein conjugate solution under the condition of stirring, and filtering by using a microporous filter membrane to obtain the intestinal probiotic accelerant. The accelerant of the invention can inhibit Escherichia coli after being orally taken, and promote the obvious proliferation of intestinal lactobacillus and bifidobacteria. The accelerant is used for preparing the intestinal flora regulating medicine.
Description
Technical Field
The invention belongs to the technical field of probiotic promoters, and particularly relates to a chestnut peel pigment nano intestinal probiotic promoter coated with chestnut almond glycoprotein, and a preparation method and application thereof.
Background
The intestinal flora is a normal microorganism in the intestinal tract of a host and is of various types. Generally, healthy adults have about 10 microorganisms in their intestines14About 10 times of the number of human cells, can form a complex and unique system in vivo to regulate the normal physiological functions of the host, and the composition, metabolism and the like of the flora are also influenced by the surrounding environment of the host. The intestinal probiotics comprise lactobacillus, bifidobacterium and the like, are indispensable elements for human health, can synthesize various vitamins, participate in the digestion of food, promote the intestinal peristalsis, inhibit the growth of pathogenic flora, decompose harmful and toxic substances and the like. By regulating intestinal flora and increasing probiotics, intestinal diseases caused by various pathogenic bacteria can be inhibited, and metabolism and immunity of human body can be regulated.
The Chinese chestnut and the almond have the functions of regulating the gastrointestinal function and relaxing the bowels, and CN107156408A and CN112273567A both disclose a Chinese chestnut beverage which has the function of regulating the intestinal flora; CN104905352A and CN108208504A disclose a method for preparing a health beverage, respectively, containing raw materials of almonds, having the function of regulating intestinal flora, however, after these products are taken orally, because the chestnuts and almonds contain some functional components and are unstable, and are easily destroyed in the environment of gastrointestinal tract, the purpose of inhibiting harmful bacteria in intestinal tract and promoting the proliferation of probiotics can not be achieved by oral administration.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide a preparation method of a chestnut peel pigment nano intestinal probiotic accelerant coated with chestnut almond glycoprotein.
The invention also aims to provide the application of the chestnut almond glycoprotein coated with chestnut peel pigment nano intestinal probiotic accelerant in preparing intestinal flora regulating medicines.
The purpose of the invention is realized by the following technical scheme.
A nanometer intestinal canal probiotic promoter containing chestnut peel pigment coated with chestnut almond glycoprotein is a nanometer preparation comprising chestnut polysaccharide and almond protein conjugate as shell and chestnut peel pigment as core.
The preparation method of the chestnut peel pigment nano intestinal probiotic accelerant with the chestnut almond glycoprotein coated plate comprises the following steps:
(1) extracting pigment in the chestnut peel by adopting an ethanol water solution to obtain the chestnut peel pigment; the ethanol water solution in the step (1) is 60-80% ethanol water solution in volume fraction; the mass ratio of the chestnut peels to the ethanol water solution is 1: (8-20) times, wherein the extraction condition is leaching for 2-5h at 60-80 ℃; filtering after extraction, and vacuum drying the filtrate (at 50-60 deg.C and 0.01-0.1MPa for 3-5 hr) to obtain chestnut peel pigment;
(2) extracting semen Castaneae and semen Armeniacae amarum in buffer solution with pH of 7-10 to obtain mixture of polysaccharide and protein; the mass ratio of the Chinese chestnuts to the almonds in the step (2) is 1: (0.5-3), preferably 1: (0.8-3); the mass ratio of the total mass of the Chinese chestnuts and the almonds to the buffer solution is 1: (10-30); after extraction, filtering, dialyzing, freezing and drying to obtain a polysaccharide and protein mixture; the dialysis refers to dialysis with membrane with molecular weight cut-off of 50-200kDa for 24-48h, and the freeze drying refers to freeze drying of dialyzed cut-off solution for 24-48 h; the temperature of freeze drying is-10 to-50 ℃.
The Chinese chestnut and the almond are crushed and sieved before extraction, and the mesh number of the sieving is 10-30 meshes; the extraction condition is that the extraction is carried out for 1 to 3 hours at the temperature of between 50 and 60 ℃;
(3) reacting the mixture of polysaccharide and protein at 50-70 deg.C and relative humidity of 65-85% for 24-72 hr to obtain semen Castaneae and semen Armeniacae amarum glycoprotein conjugate;
(4) preparing a solution of chestnut peel pigment by adopting an ethanol water solution with the volume fraction of 60-80% to obtain a chestnut peel pigment solution; preparing the Chinese chestnut almond glycoprotein conjugate into a solution by adopting a pH7-10 buffer solution to obtain a Chinese chestnut almond glycoprotein conjugate solution; mixing the chestnut peel pigment solution and the chestnut almond glycoprotein conjugate solution under the condition of stirring, and filtering by using a microporous filter membrane to obtain the chestnut peel pigment nano intestinal probiotic accelerant coated with the chestnut almond glycoprotein.
The mass ratio of the chestnut peel pigment to the chestnut almond glycoprotein conjugate in the step (4) is 1: (3-20), preferably 1: (4-20).
The mass concentration of the chestnut peel pigment solution in the step (4) is 5-10%, and the mass concentration of the chestnut almond glycoprotein conjugate solution is 5-10%.
The rotating speed of the stirring in the step (4) is 5000-; the mixing is stirring mixing, and the mixing time is 10-60 min; the mixing is to add the chestnut peel pigment solution into the chestnut almond glycoprotein conjugate solution and stir and mix.
The microporous filter membrane in the step (4) is a microporous filter membrane with the diameter of 0.1-0.5 mu m;
the buffer solution in the steps (2) and (4) is one of phosphate buffer solution and Tris-HCl buffer solution.
The application of the chestnut peel pigment nano intestinal probiotic accelerant with the chestnut almond glycoprotein coated with chestnut peel pigment in the preparation of the intestinal flora regulating medicine can inhibit Escherichia coli after oral administration so as to promote the obvious proliferation of intestinal lactobacillus and bifidobacteria, thereby preventing diseases caused by harmful bacteria in the intestinal tract.
The principle of the invention is as follows: extracting chestnut peel with ethanol to obtain pigment product, extracting chestnut and almond with buffer solution, dialyzing and separating to obtain mixture of chestnut polysaccharide (the chestnut polysaccharide is glucose-fructose-mannose-xylose-arabinose, and has terminal reducibility) and almond protein, and performing Maillard reaction to connect the chestnut polysaccharide and the almond protein to form chestnut and almond glycoprotein conjugate. After the chestnut peel pigment and the chestnut almond glycoprotein conjugate are subjected to nano emulsification, the chestnut almond glycoprotein conjugate has amphipathy, hydrophilic glycoprotein is outside, and hydrophobic groups are connected with pigment molecules inside, so that a nano structure with the chestnut almond glycoprotein as a shell and the chestnut peel pigment as a core is formed. The chestnut almond glycoprotein is acidic glycoprotein which is stable in gastric juice and is hydrolyzed in intestinal tracts to release pigment, the glycoprotein can strengthen the adhesion with bacteria, and the chestnut peel pigment is a polyphenol compound and can inhibit harmful bacteria, thereby promoting the proliferation of intestinal probiotics and keeping the health of human bodies.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention prepares a chestnut peel pigment nano preparation coated with chestnut almond glycoprotein, which keeps the stability of chestnut peel pigment in gastric juice.
(2) The invention overcomes the defects that the Chinese chestnut polysaccharide or the almond protein only can provide single nutrition and can not inhibit harmful bacteria, and can simultaneously realize the proliferation of beneficial bacteria in intestinal tracts and the inhibition of harmful bacteria.
(3) The method has mild reaction conditions and is easy for industrial production.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1
(1) Mixing the chestnut peel with 80% ethanol water solution (the mass ratio of the chestnut peel to the ethanol water solution is 1: 8), extracting at 60 deg.C for 5h, filtering, and drying the filtrate at 50 deg.C under 0.01MPa for 3h to obtain chestnut peel pigment;
(2) chinese chestnut (pulp) and almond are mixed according to the mass ratio of 1: 1, crushing, sieving with a 10-mesh sieve, adding 50-time pH7 phosphate buffer solution with the mass 10 times of that of the mixture, extracting for 3h, filtering, dialyzing the filtrate with a membrane with the molecular weight cutoff of 50kDa for 48h, and freeze-drying the cutoff solution for 24h (the freeze-drying temperature is-50 ℃), thus obtaining a mixture of chestnut almond polysaccharide and protein;
(3) reacting the mixture of chestnut almond polysaccharide and protein at 50 ℃ and 65% of relative humidity for 72h to obtain a chestnut almond glycoprotein conjugate;
(4) preparing 10g of chestnut peel pigment into a 5% solution by using an ethanol water solution with the volume fraction of 70%; preparing 50g of Chinese chestnut almond glycoprotein conjugate into a 5% solution by using a pH7 phosphoric acid buffer solution, adding a Chinese chestnut peel pigment solution into the Chinese chestnut almond glycoprotein conjugate solution under stirring at 5000 r/min, stirring for 10min, and then filtering through a 0.1 mu m microporous filter membrane to obtain the Chinese chestnut peel pigment nano intestinal probiotic promoter with the Chinese chestnut glycoprotein coated plate.
Example 2
(1) Mixing the chestnut peels with an ethanol aqueous solution with the volume fraction of 60% being 20 times of the mass of the chestnut peels, leaching for 2 hours at 80 ℃, filtering, and drying the filtrate for 5 hours at 60 ℃ under 0.1MPa to obtain chestnut peel pigment;
(2) chinese chestnut and almond are mixed according to the mass ratio of 1: 3, crushing and sieving the mixture by a 30-mesh sieve, adding 60-DEG C pH10 phosphate buffer solution with the mass 30 times of that of the mixture, extracting the mixture for 1h, filtering the mixture, dialyzing the filtrate for 24h by using a membrane with the molecular weight cutoff of 200kDa, and freeze-drying the cutoff solution for 48h to obtain a mixture of chestnut almond polysaccharide and protein;
(3) reacting the mixture of chestnut almond polysaccharide and protein at 70 ℃ and 85% of relative humidity for 24h to obtain a chestnut almond glycoprotein conjugate;
(4) preparing 10g of Chinese chestnut peel pigment into 10 wt% of solution by using 80% ethanol aqueous solution in volume fraction; preparing 200g of chestnut almond glycoprotein conjugate into 10 wt% solution by using a pH10 phosphate buffer solution; adding the chestnut peel pigment solution into the chestnut almond glycoprotein conjugate solution under the stirring of 20000 revolutions per minute, stirring for 60min, and filtering with 0.5 μm microporous membrane to obtain the jujube glycoprotein coated chestnut peel pigment nano intestinal probiotic promoter.
Example 3
(1) Adding 70% ethanol water solution with volume fraction of 10 times of the chestnut peel, extracting at 70 deg.C for 3 hr, filtering, and drying the filtrate at 55 deg.C under 0.05MPa for 4 hr to obtain chestnut peel pigment;
(2) chinese chestnut and almond are mixed according to the mass ratio of 1: 2, crushing and sieving the mixture by a 20-mesh sieve, adding a Tris-HCl buffer solution with the mass of 20 times of the mixture and the pH value of 8 at 55 ℃ for extracting for 2 hours, filtering, dialyzing the filtrate by a membrane with the cut-off molecular weight of 100kDa for 36 hours, and freeze-drying the cut-off solution for 36 hours to obtain a chestnut almond polysaccharide and protein mixture;
(3) reacting the mixture of chestnut almond polysaccharide and protein at 60 ℃ and 75% of relative humidity for 48h to obtain a chestnut almond glycoprotein conjugate;
(4) preparing 10g of Chinese chestnut peel pigment into 8 wt% solution by using 75% ethanol water solution in volume fraction; preparing 100g of chestnut almond glycoprotein conjugate into 8 wt% solution by using a pH8Tris-HCl buffer solution; adding the chestnut peel pigment solution into the chestnut almond glycoprotein conjugate solution under stirring at 10000 r/min, stirring for 40min, and filtering with 0.3 μm microporous membrane to obtain the jujube glycoprotein coated chestnut peel pigment nano intestinal probiotic promoter.
Example 4
(1) Adding 65% ethanol water solution with volume fraction 12 times of the chestnut peel, extracting at 75 deg.C for 3 hr, filtering, and drying the filtrate at 50 deg.C under 0.03MPa for 3.5 hr to obtain chestnut peel pigment;
(2) chinese chestnut and almond are mixed according to the mass ratio of 1: 1.5 pulverizing, sieving with 15 mesh sieve, extracting with 50 deg.C pH9 phosphate buffer solution 15 times the mass of the mixture for 1.5h, filtering, dialyzing the filtrate with 80kDa membrane for 40h, and freeze drying the retentate for 30h to obtain a mixture of polysaccharide and protein of semen Castaneae;
(3) reacting the mixture of chestnut almond polysaccharide and protein at 55 ℃ and 70% of relative humidity for 40h to obtain a chestnut almond glycoprotein conjugate;
(4) preparing 10g of chestnut peel pigment into 6 wt% solution by using 60% ethanol aqueous solution in volume fraction; preparing 80g of chestnut almond glycoprotein conjugate into 6 wt% solution by using a pH9 phosphate buffer solution; adding the chestnut peel pigment solution into the chestnut almond glycoprotein conjugate solution under stirring at 6000 rpm, stirring for 30min, and filtering with 0.2 μm microporous membrane to obtain the jujube glycoprotein coated chestnut peel pigment nano intestinal probiotic promoter.
Example 5
(1) Adding a 75% ethanol aqueous solution with volume fraction 16 times of the chestnut peel, leaching at 65 ℃ for 4h, filtering, and drying the filtrate at 60 ℃ under 0.06MPa for 4.5h to obtain chestnut peel pigment;
(2) chinese chestnut and almond are mixed according to the mass ratio of 1: 2.5 crushing and sieving with a 25-mesh sieve, adding 60-DEG C pH7.5Tris-HCl buffer solution with the mass of the mixture being 25 times of the mass of the mixture, extracting for 2.5h, filtering, dialyzing the filtrate for 30h by using a membrane with the molecular weight cutoff of 150kDa, and freeze-drying the cutoff solution for 40h to obtain a mixture of chestnut almond polysaccharide and protein;
(3) reacting the mixture of chestnut almond polysaccharide and protein at 65 ℃ and with the relative humidity of 80% for 30h to obtain a chestnut almond glycoprotein conjugate;
(4) preparing 10g of chestnut peel pigment into 8 wt% solution by using 65% ethanol water solution in volume fraction; preparing 160g of chestnut almond glycoprotein conjugate into 8 wt% solution by using pH7.5Tris-HCl buffer solution; adding the chestnut peel pigment solution into the chestnut almond glycoprotein conjugate solution under the stirring of 12000 r/min, stirring for 25min, and filtering with 0.4 μm microporous membrane to obtain the jujube glycoprotein coated chestnut peel pigment nano intestinal probiotic promoter.
Comparative example 1
The preparation method of example 1 was followed, except that step (3) was omitted, and the chestnut almond glycoprotein conjugate in step (4) was replaced with the chestnut almond polysaccharide and protein mixture in step (2), to obtain a product of control 1.
Comparative example 2
The procedure of example 1 was followed except that the chestnut almond glycoprotein conjugate was replaced with lecithin in step (4), and the product obtained was control 2.
Test 1 compositional analysis of the chestnut almond glycoprotein conjugates prepared in examples 1-5
The method comprises the following steps: taking a proper amount of the Chinese chestnut almond glycoprotein conjugate prepared in the embodiment 1-5, measuring the sugar content in the sample according to the agricultural industry standard of the people's republic of China-the measurement (NY/T1676-2008) of the content of crude polysaccharide in edible fungi, and measuring the protein content in the sample according to the export and import inspection industry standard of the people's republic of China-the measurement of the protein content in milk, eggs and bean food by the Coomassie brilliant blue method (SN/T3926-2006).
As a result: the contents of sugar and protein in the chestnut almond glycoprotein conjugate prepared in examples 1-5 are shown in Table 1, and each 100g of the product contains 22.8-33.8g of sugar and 62.4-75.6g of protein, which indicates that the conjugate mainly comprises sugar and protein.
TABLE 1 Chinese chestnut almond glycoprotein conjugate content composition
| Sample source | Sugar content% | Protein content% |
| Example 1 | 33.8 | 62.4 |
| Example 2 | 22.8 | 75.6 |
| Example 3 | 26.4 | 67.5 |
| Example 4 | 29.2 | 64.3 |
| Example 5 | 24.5 | 71.2 |
Test 2 determination of nanoparticle size and amount of chestnut peel pigment-loaded in chestnut almond glycoprotein coated chestnut peel pigment nano intestinal probiotic accelerator prepared in examples 1 to 5
The method comprises the following steps: the Chinese chestnut almond glycoprotein coated carrier Chinese chestnut peel pigment nano preparation prepared in the embodiment 1-5, the comparison 1 and the comparison 2 are measured for particle size by a Malvern nano particle sizer, because the Chinese chestnut peel pigment belongs to anthocyanin compounds, a proper amount of Chinese chestnut peel pigment is removed by a micro-column centrifugation method, the content of the Chinese chestnut peel pigment is measured according to the ultraviolet/visible spectrophotometry method for measuring proanthocyanidins in DB 12/T885-2019 plant extracts, and the drug loading amount is calculated.
As a result: the grain diameter and the chestnut peel pigment nano intestinal canal probiotic promoter of the chestnut almond glycoprotein coated carrier prepared in the embodiments 1-5 are shown in table 2, the grain diameter is 216-284nm, the drug-loading rate is 4.8-10.4%, and the product is the nano preparation loaded with the chestnut peel pigment. The comparative example 1 has no drug loading, which indicates that the Chinese chestnut polysaccharide and the almond protein have no entrapment capability; control 2 has a small drug loading, indicating that the liposomes can entrap the pigment from the chestnut peel, but the drug loading is low.
TABLE 2 particle size and chestnut peel pigment loading of the accelerators of examples 1-5 and comparative examples 1-2
Test 3 determination of stability of chestnut peel pigment nano intestinal probiotic accelerator coated with chestnut almond glycoprotein prepared in examples 1 to 5 in simulated gastrointestinal fluids
The method comprises the following steps: the stability of the chestnut peel pigment nano preparation coated with chestnut almond glycoprotein is evaluated by adopting simulated artificial gastric juice (containing 1% pepsin and pH being 1.2) and simulated artificial intestinal juice (containing 1% trypsin and pH being 6.8). 1mL of the chestnut almond glycoprotein coated carrier chestnut peel pigment nano preparation prepared in the examples 1 to 5 and the comparative example 2 were added to 4mL of simulated artificial gastric fluid or intestinal fluid, incubated at 37 ℃ under oscillation at 100rpm, 200. mu.L of the sample was taken at intervals of 2 hours, the exuded free pigment was removed by a microcolumn centrifugation method, the obtained filtrate was transferred to a 5mL volumetric flask, and the volume was determined by methanol and demulsification by ultrasound. The content of the pigment in the chestnut peel is determined by adopting the ultraviolet/visible spectrophotometry method for determining the procyanidine in the DB 12/T885-2019 plant extract, and the residual pigment percentage of each time point of the preparation is calculated by taking the drug concentration at the time 0 as 100%.
As a result: the chestnut almond glycoprotein coated with chestnut pericarp pigment nano intestinal probiotic accelerant prepared in the examples 1-5 still has more than 80% of pigment retained in simulated gastric fluid within 4 hours, but is degraded in intestinal fluid faster, and the result is shown in table 3. The product has the effect of improving gastric juice stability, and can quickly release pigment in intestinal tract. Comparative example 2 released the drug rapidly in gastrointestinal fluids, indicating that the liposomes were unstable in gastrointestinal fluids.
Table 3 retention of pigment in simulated gastrointestinal fluids (%)
Test 4 the effect of the chestnut peel pigment nano intestinal probiotic promoter coated with chestnut almond glycoprotein prepared in example 1
The method comprises the following steps: 35 male SD rats (250 + -25 g) were randomly assigned to 7 groups of 5 rats. The group 1 was given normal rat diet, the group 2 was given high fat diet (87.6% standard diet, 2% cholesterol, 0.2% sodium cholate, 10% lard), the group 3-7 was given high fat diet, and then the chestnut almond polysaccharide and protein mixture, chestnut peel pigment, chestnut almond glycoprotein conjugate, chestnut peel pigment-coated chestnut peel pigment nano intestinal probiotic accelerator prepared in example 1, and the control 2 were separately gavaged, at a dose of 1g/100g body weight twice a day. The rat feces were taken at 15 days and 30 days, respectively, subjected to 16sRNA metagenomic sequencing of the intestinal flora and probiotics (such as bifidobacterium and lactobacillus), and analyzed for abundance (relative percentage) of various probiotics and escherichia coli.
As a result: the results are shown in table 4, which shows that the group 2 model rats have significantly reduced probiotics but increased escherichia coli compared to the normal group, and the groups 3-4 and 7 have improved probiotics but have little difference from the normal group; the group 5 is remarkably increased, the group 6 is optimal, which shows that the Chinese chestnut almond glycoprotein conjugate can provide more comprehensive nutrition for intestinal probiotics and promote the proliferation of the intestinal probiotics, and the Chinese chestnut almond glycoprotein coated with the Chinese chestnut peel pigment nano intestinal probiotics accelerant not only can provide various nutrition, but also can better promote the proliferation of the intestinal probiotics by inhibiting harmful flora Escherichia coli through the Chinese chestnut peel pigment.
Table 4 relative abundance of probiotics in rat feces (%)
The above examples of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A preparation method of a chestnut peel pigment nano intestinal probiotic accelerant coated with chestnut almond glycoprotein is characterized by comprising the following steps: the method comprises the following steps:
(1) extracting pigment in the chestnut peel by adopting an ethanol water solution to obtain the chestnut peel pigment;
(2) extracting semen Castaneae and semen Armeniacae amarum in buffer solution with pH of 7-10 to obtain mixture of polysaccharide and protein;
(3) reacting the mixture of polysaccharide and protein at 50-70 deg.C and relative humidity of 65-85% for 24-72 hr to obtain semen Castaneae glycoprotein conjugate;
(4) preparing a solution of chestnut peel pigment by adopting an ethanol water solution with the volume fraction of 60-80% to obtain a chestnut peel pigment solution; preparing the Chinese chestnut almond glycoprotein conjugate into a solution by adopting a pH7-10 buffer solution to obtain a Chinese chestnut almond glycoprotein conjugate solution; mixing the chestnut peel pigment solution and the chestnut almond glycoprotein conjugate solution under the condition of stirring, and filtering by using a microporous filter membrane to obtain the chestnut peel pigment nano intestinal probiotic accelerant coated with the chestnut almond glycoprotein.
2. The preparation method of the chestnut almond glycoprotein coated chestnut peel pigment nano intestinal probiotic accelerant as claimed in claim 1, is characterized in that: the mass ratio of the Chinese chestnuts to the almonds in the step (2) is 1: (0.5-3);
the mass ratio of the chestnut peel pigment to the chestnut almond glycoprotein conjugate in the step (4) is 1: (3-20).
3. The preparation method of the chestnut almond glycoprotein coated chestnut peel pigment nano intestinal probiotic accelerant as claimed in claim 1, is characterized in that:
the rotating speed of the stirring in the step (4) is 5000-;
the buffer solution in the steps (2) and (4) is one of phosphate buffer solution and Tris-HCl buffer solution.
4. The preparation method of the chestnut almond glycoprotein coated chestnut peel pigment nano intestinal probiotic accelerant as claimed in claim 1, is characterized in that: in the step (4), the mass concentration of the chestnut peel pigment solution is 5-10%, and the mass concentration of the chestnut almond glycoprotein conjugate solution is 5-10%;
the mixing in the step (4) is stirring and mixing, and the mixing time is 10-60 min;
the microporous filter membrane in the step (4) is a 0.1-0.5 μm microporous filter membrane.
5. The preparation method of the chestnut almond glycoprotein coated chestnut peel pigment nano intestinal probiotic accelerant as claimed in claim 1, is characterized in that:
the ethanol water solution in the step (1) is 60-80% ethanol water solution in volume fraction;
and (3) after the extraction in the step (2), filtering, dialyzing, and freeze-drying to obtain a polysaccharide and protein mixture.
6. The preparation method of the chestnut almond glycoprotein coated chestnut peel pigment nano intestinal probiotic accelerant as claimed in claim 5, is characterized in that: the dialysis refers to dialysis with membrane with molecular weight cut-off of 50-200kDa for 24-48h, and the freeze drying refers to freeze drying of dialyzed cut-off solution for 24-48 h; the temperature of freeze drying is-10 to-50 ℃.
7. The preparation method of the chestnut almond glycoprotein coated chestnut peel pigment nano intestinal probiotic accelerant as claimed in claim 1, is characterized in that: the mass ratio of the total mass of the Chinese chestnuts and the almonds to the buffer solution in the step (2) is 1: (10-30);
the mass ratio of the chestnut peels to the ethanol water solution in the step (1) is 1: (8-20) times, wherein the extraction condition in the step (1) is leaching for 2-5h at 60-80 ℃;
the extraction condition in the step (2) is extraction at 50-60 ℃ for 1-3 h.
8. The preparation method of the chestnut almond glycoprotein coated chestnut peel pigment nano intestinal probiotic accelerant as claimed in claim 1, is characterized in that: filtering after the extraction in the step (1), and drying the filtrate for 3-5h at 50-60 ℃ under 0.01-0.1MPa to obtain chestnut peel pigment;
and (3) crushing and sieving the Chinese chestnuts and the almonds before extraction in the step (2), wherein the sieved meshes are 10-30 meshes.
9. A chestnut almond glycoprotein coated carrier chestnut peel pigment nano intestinal probiotic accelerant obtained by the preparation method of any one of claims 1 to 8 is characterized in that: the nanometer preparation is prepared with the combination of Chinese chestnut polysaccharide and almond protein as shell and Chinese chestnut peel pigment as kernel.
10. The application of the chestnut almond glycoprotein coated chestnut peel pigment nano intestinal probiotic accelerant as claimed in claim 9, is characterized in that: the chestnut almond glycoprotein coated with chestnut peel pigment nano intestinal probiotic accelerant is used for preparing intestinal flora regulating medicines and inhibiting escherichia coli so as to promote the proliferation of intestinal lactobacilli and bifidobacteria.
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