CN117679352A - Biological enzyme-based composition, hypoglycemic toothpaste containing same and preparation method thereof - Google Patents
Biological enzyme-based composition, hypoglycemic toothpaste containing same and preparation method thereof Download PDFInfo
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- CN117679352A CN117679352A CN202311708365.5A CN202311708365A CN117679352A CN 117679352 A CN117679352 A CN 117679352A CN 202311708365 A CN202311708365 A CN 202311708365A CN 117679352 A CN117679352 A CN 117679352A
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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
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a biological enzyme-based composition, hypoglycemic toothpaste containing the composition and a preparation method thereof, belonging to the technical field of toothpaste. The composite material is prepared from the following raw materials in parts by weight: 5-15 parts of biological enzyme microspheres, 2-10 parts of probiotics, 3-7 parts of active ferment and 1-3 parts of antibacterial peptide. The biological enzyme-based composition has good sterilization, bacteriostasis and antibacterial effects, can decompose food residues, reduce metabolic acid production, avoid gum irritation, tooth allergy and tooth enamel damage, can prepare the blood glucose-reducing toothpaste, has a certain blood glucose-reducing, lipid-reducing and pressure-regulating effect, and can absorb health-care active ingredients in the modes of early and late gargling, tooth brushing and the like by people, thereby having the advantages of small side effect, easy acceptance and the like.
Description
Technical Field
The invention relates to the technical field of toothpaste, in particular to a biological enzyme-based composition, a hypoglycemic toothpaste containing the composition and a preparation method thereof.
Background
Most diabetics are accompanied with oral diseases, and the probability of the diabetics suffering from periodontitis is more than twice that of normal people. Because blood sugar is not effectively controlled, red swelling is easy to appear at the edge of the gum, and a granulomatous proliferation state is presented at the same time, so that the gum is stripped and bleeding is caused, abscess can appear at the periodontal, dental calculus can be deposited in a short time, periodontal membranes are damaged to a certain extent, periodontal strips can be formed, teeth are loose and weak, and even the teeth of the whole mouth of an early patient fall off.
The canker sore can also induce diabetes, saliva in the oral cavity of a diabetic patient is obviously reduced, the oral mucosa is in a dry state and is sticky, the stimulation effect is reduced, and the defending ability of the mucosa to external stimulation is also reduced. In addition, the body is in a water shortage state and can cause canker sore due to excessive internal heat. Diabetes also causes disturbances in gastrointestinal function and digestive system diseases, decreases the ability to digest and absorb nutrients, and decreases resistance and immunity, thereby allowing oral pathogenic bacteria to enter while they are deficient, resulting in oral ulcers.
Diabetes patients have bad breath, which is a bad breath, due to disturbed metabolism and increased ketone body in the body, and the oral cavity emits a taste similar to that of rotten apples, thus affecting social contact. Halitosis of diabetics is not caused by fermentation of food residues, and is mainly caused by reduction of insulin secretion amount, and carbohydrates in the body are not easily decomposed, so that fat metabolism is very active, and acetone ingredient is generated. Acetone will be expelled from the mouth through the lungs. In addition, hyperglycemia can cause blockage of capillaries, the oral cavity can not be supplied with sufficient oxygen, so that a large amount of anaerobic bacteria can be propagated, and halitosis is induced.
Diabetes and oral disease are causal to each other. Oral care such as daily tooth brushing, tooth washing and oral sterilization can only improve the dental and periodontal diseases locally, and long-term oral antibacterial drugs are easy to generate bacterial drug resistance and interfere with the normal flora ecological balance of the oral cavity to induce oral fungal infection. Therefore, the new method for preventing and treating oral diseases and preventing or relieving diabetes mellitus is discussed, and has important scientific value and profound social significance.
Toothpaste is the most commonly used oral cleaning product in daily life of people, and is a paste product which is applied to the surface of teeth in a friction manner and is used for cleaning the main purpose; generally in gel form, the cleaning agent is generally smeared on a toothbrush, and the surface of teeth is cleaned by the action of mechanical friction of the toothbrush, so that the teeth and the periphery of the teeth are cleaned, and the oral cavity is cleaned and refreshed. The toothpaste has mainly focused on eliminating stomatitis and oral malodor, whitening teeth, strengthening teeth, and preventing caries. In the process of gargling, tooth brushing or buccal tablet, the active ingredients in the toothpaste are permeated and absorbed into blood through sublingual veins, capillaries and oral mucosa, so that the first pass elimination effect of organs such as liver and kidney on the active ingredients is avoided, and meanwhile, the burden of the liver and kidney can be relieved.
Probiotics are well known to a wide range of consumers and have various health promoting functions, including improving intestinal flora structure, treating diarrhea, constipation, improving body immunity, etc. More than 30 species of probiotics have been approved by the country for use in health foods, mainly including lactobacillus (lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus casei, lactobacillus paracasei, lactobacillus plantarum, lactobacillus grignard, lactobacillus reuteri, lactobacillus bulgaricus, lactobacillus johnsonii and the like), bifidobacterium (bifidobacterium bifidum, bifidobacterium longum, bifidobacterium breve, bifidobacterium adolescentis, bifidobacterium infantis and the like), other gram positive bacteria (bacillus coagulans, propionibacterium freudenreichii, enterococcus faecalis, lactococcus lactis, streptococcus thermophilus and the like) and yeast (kluyveromyces marxianus and the like). Some probiotics have been shown to have in vivo hypoglycemic effects, such as lactobacillus reuteri, lactobacillus rhamnosus, and the like.
In conclusion, through scientific formula and reasonable collocation, the oral care product which has better effect, is nontoxic, has the function of reducing blood sugar and preventing and treating type II diabetes is developed, and has urgency.
Disclosure of Invention
The invention aims to provide a biological enzyme-based composition, blood glucose-reducing toothpaste containing the composition and a preparation method thereof, which have good sterilization, bacteriostasis and antibacterial effects, decompose food residues, reduce metabolic acidogenesis, avoid gum irritation, tooth allergy and damage to tooth enamel, reduce blood glucose and saccharification hemoglobin, achieve the effects of regulating blood glucose, regulating oral flora, resisting inflammation and resisting oxidation, achieve the effects of reducing blood glucose, reducing lipid and regulating pressure, absorb health-care active ingredients in the modes of early and late gargling, brushing teeth and the like, and have the advantages of small side effect, easy acceptance and the like.
The technical scheme of the invention is realized as follows:
the invention provides a biological enzyme-based composition which is prepared from the following raw materials in parts by weight: 5-15 parts of biological enzyme microspheres, 2-10 parts of probiotics, 3-7 parts of active ferment and 1-3 parts of antibacterial peptide;
the biological enzyme microsphere is prepared by chemically modifying lysozyme, performing oligomerization to obtain oligomeric modified lysozyme, performing Maillard reaction with xanthan gum, mixing with cellulase, trypsin and dextranase, and embedding in crosslinked starch microsphere;
the active ferment is an active ferment obtained by extracting carob, mulberry leaf and rhodiola rosea.
As a further improvement of the invention, the preparation method of the biological enzyme microsphere comprises the following steps:
s1, preparing modified lysozyme: dissolving lactic acid, ethylenediamine tetraacetic acid and cinnamic acid in alkali liquor, regulating the pH value of the solution, adding 1-ethyl (3-dimethylaminopropyl) -3-carbodiimide, adding lysozyme under the condition of heating and stirring, stirring for reaction, centrifuging, and dialyzing to obtain modified lysozyme;
s2, preparation of oligomeric modified lysozyme: dissolving the modified lysozyme prepared in the step S1 in water, adding hydrogen peroxide, heating, stirring, reacting, and dialyzing to obtain the oligomeric modified lysozyme;
S3, preparing cross-linked oligomeric modified lysozyme: dissolving the oligomeric modified lysozyme and the arabinose prepared in the step S2 in PBS solution, heating and stirring for reaction, adding ammonium sulfate until the system is saturated, precipitating, filtering, washing and drying to prepare the crosslinking oligomeric modified lysozyme;
s4, preparation of biological enzyme: uniformly mixing the cross-linked oligomeric modified lysozyme, cellulase, trypsin and dextranase prepared in the step S3 to prepare biological enzyme;
s5, preparing biological enzyme microspheres: adding the biological enzyme, starch and emulsifier prepared in the step S4 into alkali liquor, heating and dissolving, and uniformly mixing to prepare a water phase; adding the water phase into fish oil, emulsifying, dripping n-butanol and epichlorohydrin, stirring for reaction, centrifuging, washing, and drying to obtain the biological enzyme microsphere.
As a further improvement of the invention, in the step S1, the mass ratio of the lactic acid, the ethylenediamine tetraacetic acid, the cinnamic acid, the 1-ethyl (3-dimethylaminopropyl) -3-carbodiimide and the lysozyme is 3-5:7-10:5-7:35-45:15-22, the alkali liquor is 7-12% NaOH or KOH solution, the pH value of the solution is adjusted to 7.2-7.5, the temperature of heating and stirring is 35-40 ℃, the stirring reaction time is 5-7 hours, and the aperture of a dialysis bag for dialysis is 9-10kDa; and in the step S2, hydrogen peroxide is added to the system, the concentration of the hydrogen peroxide is 1-3wt%, the temperature of the heating and stirring reaction is 55-60 ℃, the time is 2-4h, and the aperture of the dialysis bag for dialysis is 9-10kDa.
As a further improvement of the invention, in the step S3, the mass ratio of the oligomeric modified lysozyme to the arabinose is 10-12:7-10, the PBS buffer solution is PBS buffer solution with pH=8-8.5, the temperature of the heating and stirring reaction is 60-65 ℃ and the time is 10-12h; the mass ratio of the cross-linked oligomeric modified lysozyme to the cellulase to the trypsin to the dextranase in the step S4 is 15-20:3-5:5-7:2-3.
As a further improvement of the invention, in the step S5, the mass ratio of the biological enzyme, the starch, the emulsifying agent, the alkali liquor, the fish oil, the n-butyl alcohol and the epichlorohydrin is 12-15:17-22:1-2:150-200:250-300:3-5:5-7, the alkali liquor is 5-7wt% NaOH or KOH solution, the temperature of the stirring reaction is 50-55 ℃ and the time is 3-5h.
Preferably, the emulsifier is at least one selected from the group consisting of Tween-20, tween-40, tween-60, tween-80.
As a further improvement of the invention, the preparation method of the active ferment comprises the following steps:
respectively cleaning carob, mulberry leaf and rhodiola rosea, drying, crushing to obtain mixed powder, adding the mixed powder into water, adding compound enzyme for enzymolysis, sterilizing, inoculating lactobacillus paracasei and bifidobacterium longum strain seed liquid, carrying out enzyme-assisted fermentation culture, filtering a product, washing solid residues, collecting bacterial liquid, mixing the bacterial liquid with filtrate, and freeze-drying to obtain the active ferment.
As a further improvement of the invention, the mass ratio of the carob to the mulberry leaf to the rhodiola rosea is 7-10:3-5:5-7, the compound enzyme is cellulase and pectase, the mass ratio of the compound enzyme is 7-10:3-5, the addition amount of the compound enzyme is 3-5wt% of the total mass of the system, the inoculation amounts of lactobacillus paracasei and bifidobacterium longum strain seed liquid are respectively 2-4v/v% and 1-3v/v%, and the bacterial content of the strain seed liquid is 10 8 -10 9 cfu/mL, wherein the conditions of enzyme-assisted fermentation culture are 37-39 ℃,100-120r/min and enzyme-assisted fermentation culture for 48-56h.
As a further improvement of the invention, the probiotics comprise lactobacillus reuteri, lactobacillus rhamnosus and lactobacillus acidophilus, and the mass ratio is 5-7:3-5:2-4.
The invention further protects a hypoglycemic toothpaste containing the biological enzyme-based composition, which is prepared from the following raw materials in parts by weight: 20-25 parts of biological enzyme-based composition,1-2 parts of lactobacillus reuteri, 1-2 parts of lactobacillus rhamnosus and lactobacillus acidophilus 1-2 parts of,70-95 parts of matrix and 2-4 parts of sodium hyaluronate; the matrix is prepared from the following raw materials in percentage by mass: 12-60% of friction agent, 5-60% of humectant, 0.5-4% of thickening agent, 1-5.5% of foaming agent, 0-2% of spice, 0-1% of sweetener, 0-1% of food color, 0-0.5% of preservative and the balance of deionized water.
Preferably, the friction agent is at least one selected from calcium carbonate, precipitated silica, water-insoluble sodium metaphosphate and nano-hydroxyapatite; the humectant is one or more selected from glycerol, propylene glycol, sorbitol and xylitol; the thickener is at least one selected from sodium carboxymethyl cellulose, carbopol resin, carboxyethyl cellulose, hydroxypropyl guar, carrageenan and thickening silica; the foaming agent is at least one selected from sodium lauryl sulfate, sodium lauroyl sarcosinate and sodium methyl cocoa butter based taurine; the perfume is at least one selected from fructus Citri Sarcodactylis, orange oil, tangerine oil, perilla oil, spearmint oil, wintergreen oil, oleum Caryophylli, oleum Cinnamomi, oleum Foeniculi, oleum Menthae Dementholatum, mentholum, borneolum Syntheticum, lauraldehyde, citral, vanillin, amyl acetate; the sweetener is at least one selected from sucrose, saccharin, sodium cyclamate, xylitol, stevioside, disodium glycyrrhizinate, tripotassium glycyrrhizinate and trisodium glycyrrhizinate; the preservative is at least one selected from parahydroxybenzoate, sodium benzoate, potassium sorbate and sorbitol. The edible pigment is purple, pink, blue or green.
The invention further provides a preparation method of the hypoglycemic toothpaste, which comprises the following steps:
(1) Mixing and stirring the components in the matrix for 2-3h to obtain the matrix for toothpaste;
(2) Mixing and stirring all the components in the biological enzyme-based composition for 0.5-1h to obtain the biological enzyme-based composition;
(3) A substrate for toothpaste,Lactobacillus reuteri, lactobacillus rhamnosus, lactobacillus acidophilus,Mixing the biological enzyme-based composition and sodium hyaluronate, stirring for 20-30min, placing in a vacuum paste maker, maintaining vacuum degree at-0.09 MPa to-0.096 MPa, homogenizing, stirring for 20-30min, and packaging to obtain blood sugar reducing toothpaste.
The invention has the following beneficial effects:
because the two bacterial cell walls have different structural components, lysozyme mainly acts on gram-positive bacteria and has little effect on gram-negative bacteria, when the lysozyme acts on the gram-positive bacteria, beta-1, 4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine can be destroyed, so that the stability of the cell walls of the gram-positive bacteria is reduced, the imbalance of osmotic pressure inside and outside the cell walls is broken, the content escapes, and the bacteria are dissolved and killed; however, although gram-positive bacteria only have one layer of peptidoglycan chain, the outer layer of the gram-positive bacteria is wrapped and protected by thicker lipopolysaccharide substances, so that lysozyme cannot crack the cell wall peptidoglycan of the gram-positive bacteria, and the sterilization effect of the lysozyme on the gram-positive bacteria is not obvious.
According to the lysozyme disclosed by the invention, firstly, chemical modification reaction is carried out on the lysozyme, and lactic acid, ethylenediamine tetraacetic acid and cinnamic acid, the ethylenediamine tetraacetic acid structure can be used as a cell membrane interfering agent, and salt bridge divalent cations between lipopolysaccharide on the cell surface are chelated, so that the cell membrane of the bacterium is destabilized, and the tolerance of part of bacteria to the lysozyme is regulated; lactic acid structure can form bond with lipopolysaccharide on bacterial wall to increase cell membrane permeability, and the synergistic effect can leak bacterial content to cause bacterial death; the cinnamic acid modification improves the antibacterial effect of the prepared modified lysozyme on gram-negative bacteria by changing the hydrophobicity of the lysozyme.
Furthermore, the prepared modified lysozyme is prepared into an oligomer, namely a dimer or a trimer through the reaction of hydrogen peroxide under the heating condition, so that the structure of the lysozyme is changed, the hydrophobicity is increased, the reaction with the bacterial cell wall is improved, the integrity of a lipid layer is damaged, and the bacterial cell membrane is disturbed to cause death.
And then the prepared oligomeric modified lysozyme and arabinose undergo Maillard reaction, so that the formed cross-linked product can obviously improve the antibacterial performance of the prepared cross-linked oligomeric modified lysozyme, broaden the antibacterial spectrum, and meanwhile, the arabinose can be decomposed and enter a human body, so that sugar in the human body is rapidly decomposed and digested, the absorption of sugar is inhibited, the rapid reduction of blood sugar in the human body is facilitated, and an auxiliary improvement effect is achieved.
Then, the invention mixes the crosslinking oligomerization modified lysozyme, the cellulase, the trypsin and the dextranase to prepare the biological enzyme, in addition, under the synergistic effect of the cellulase, the trypsin and the dextranase, the decomposition of oral residues can be promoted, the inflammation can be relieved, the dextranase can reduce exogenous pigments of teeth, remove tooth stains, improve the phenomenon of tooth yellow and clean teeth. By improving oral flora, the oral cavity self-healing agent has the function of activating oral cavity self-healing, and can prevent and relieve various dental problems.
The prepared biological enzyme is embedded by the crosslinked starch, so that the stability of the biological enzyme can be greatly improved, and the inactivation and invalidation of the enzyme in the storage process are avoided, thereby greatly improving the effectiveness of the hypoglycemic toothpaste.
The blood sugar reducing and health care effects of the edible traditional Chinese medicine extract are increasingly accepted. Scientists found that hyperglycemic patients had low concentrations of D-chiro-inositol in urination, muscle, hemodialysis and that carob contained D-chiro-inositol. The D-chiro-inositol (DCI) has the functions of promoting liver lipid metabolism, obviously enhancing insulin sensitivity, reducing blood sugar, improving ovulation condition of polycystic ovary syndrome (PCOS) patients, regulating hormone balance, improving menstrual disorder, and resisting oxidation, aging and inflammation. The carob extract has hypoglycemic and sensitization effects, which is proved to be due to the DCI containing active ingredients. Similarly, rhodiola rosea extract and mulberry leaf extract have also been shown to have hypoglycemic and type II diabetes preventing functions.
The active ferment disclosed by the invention takes medicinal and edible raw materials as main components, and comprises carob, mulberry leaf and rhodiola rosea, wherein the carob extract contains D-chiro-inositol, so that the carob extract can assist insulin to normally act, reduce insulin resistance, increase insulin activity in cells, reduce insulin content in blood and reduce glucose concentration, and has a good blood glucose reducing effect. The mulberry leaf extract can inhibit sugar metabolism enzyme, stabilize and reduce fasting blood glucose and postprandial blood glucose peak, wherein flavonoids, phytosterol, alkaloid and the like can inhibit fatty liver formation, reduce blood viscosity and inhibit atherosclerosis plaque formation. The rhodiola rosea extract contains components such as the zoysin, the aglycone tyrosol, the salidroside and the like, can enhance the secretion of insulin, improve the sensitivity of the insulin, promote the metabolism, effectively reduce the blood sugar and help to control diabetes.
In the preparation of the active ferment, under the synergistic effect of complex enzymes (including cellulase and pectase), the carob, the mulberry leaf and the rhodiola rosea can promote the rupture of plant cell walls, promote the enzymolysis and inactivation of the carob anti-nutritional factors, promote the dissolution of active components including D-chiral inositol, flavonoid, phytosterol, alkaloid, zoavidine, aglycone tyrosol, salidroside and the like, further promote the proliferation of probiotics under the action of enzyme-assisted fermentation, improve the generation of beneficial hypoglycemic components such as short-chain fatty acid and the like, promote the crushing of plant cell walls and the decomposition of the carob anti-nutritional factors, greatly reduce the toxicity of carob, improve the effect of the prepared active ferment on regulating blood sugar, blood fat and blood pressure, inhibit the growth of oral pathogenic bacteria, eliminate oral inflammation and oral odor, whiten teeth, strengthen teeth and prevent decayed teeth.
By adding the biological enzyme microspheres, under the action of oral amylase, the starch ball enzymolysis releases the biological enzyme, so that the biological enzyme has good sterilization, bacteriostasis and antibacterial effects, decomposes food residues, reduces metabolic acid production, and avoids gum irritation, tooth allergy and tooth enamel damage; the added probiotics have the function of occupying space, reduce the adhesion of pathogenic bacteria in the oral cavity to teeth or oral mucosa, inhibit the formation of biological films by the pathogenic bacteria, reduce gingival inflammation and tissue damage, reduce oxidation potential and inflammation conditions in vivo, improve insulin resistance, further reduce blood sugar and saccharification hemoglobin, and achieve the effects of regulating blood sugar, adjusting oral flora, resisting inflammation and resisting oxidation. The antibacterial peptide is added to have the effect of killing growth and reproduction of oral pathogenic bacteria; the addition of the active ferment can further strengthen the efficacy of probiotics and the effects of reducing blood sugar, blood fat and pressure. The health care active ingredients are absorbed by people in the modes of gargling in the morning and evening, brushing teeth and the like, and the health care active ingredients have the advantages of small side effect, easy acceptance and the like.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The starch is potato starch, purchased from the company of the biological engineering of Lactobacillus paracasei, bifidobacterium longum.
Lactobacillus paracasei, 100 hundred million cfu/g, bifidobacterium longum, 100 hundred million cfu/g, lactobacillus reuteri, 100 hundred million cfu/g, lactobacillus rhamnosus, 100 hundred million cfu/g, lactobacillus acidophilus, 100 hundred million cfu/g, purchased from Jia Yi bioengineering Co., ltd.
The preparation method of lactobacillus paracasei and bifidobacterium longum strain seed liquid comprises the following steps: inoculating the strain into Gao's culture medium, activating and culturing at 37deg.C and 100r/min for 24 hr to obtain strain with a bacterial content of 10 8 -10 9 cfu/mL strain seed solution.
Lysozyme, 2 ten thousand U/g, cellulase, 1 ten thousand U/g, pectase, 1 ten thousand U/g, purchased from Nanning Donghua bioscience, inc. Trypsin, 2U/g, purchased from Shanghai Seiyaka Biotechnology Co. Dextranase, 3 ten thousand U/g, purchased from Shandong Fengtai Biotechnology Co.
Preparation example 1 preparation of biological enzyme microspheres
The method comprises the following steps:
s1, preparing modified lysozyme: dissolving 3 parts by weight of lactic acid, 7 parts by weight of ethylenediamine tetraacetic acid and 5 parts by weight of cinnamic acid in 200 parts by weight of 7% NaOH solution, regulating the pH value of the solution to 7.2, adding 35 parts by weight of 1-ethyl (3-dimethylaminopropyl) -3-carbodiimide, heating to 35 ℃, adding 15 parts by weight of lysozyme under stirring, stirring for reacting for 5 hours, centrifuging, dialyzing by using a dialysis bag with the aperture of 9kDa, and freeze-drying the dialyzate to obtain modified lysozyme;
S2, preparation of oligomeric modified lysozyme: dissolving 15 parts by weight of the modified lysozyme prepared in the step S1 in 200 parts by weight of water, adding hydrogen peroxide until the concentration of the hydrogen peroxide in the system is 1wt%, heating to 55 ℃, stirring for reaction for 2 hours, dialyzing with a dialysis bag with the aperture of 9kDa, and freeze-drying the dialysate to obtain the oligomeric modified lysozyme;
s3, preparing cross-linked oligomeric modified lysozyme: dissolving 10 parts by weight of the oligomeric modified lysozyme prepared in the step S2 and 7 parts by weight of arabinose in 200 parts by weight of PBS buffer solution with pH=8, heating to 60 ℃, stirring and reacting for 10 hours, adding ammonium sulfate until the system is saturated, precipitating, filtering, washing and drying to prepare the crosslinking oligomeric modified lysozyme;
s4, preparation of biological enzyme: 15 parts by weight of the cross-linked oligomeric modified lysozyme prepared in the step S3, 3 parts by weight of cellulase, 5 parts by weight of trypsin and 2 parts by weight of dextranase are stirred and mixed for 20 minutes to prepare biological enzyme;
s5, preparing biological enzyme microspheres: adding 12 parts by weight of the biological enzyme prepared in the step S4, 17 parts by weight of starch and 1 part by weight of tween-20 into 150 parts by weight of 5wt% NaOH solution, heating and dissolving, and uniformly mixing to prepare a water phase; adding the water phase into 250 parts by weight of fish oil, emulsifying for 10min at 10000r/min, dropwise adding 3 parts by weight of n-butanol and 5 parts by weight of epichlorohydrin, stirring at 50 ℃ for reaction for 3h, centrifuging, washing and drying to obtain the biological enzyme microsphere.
Preparation example 2 preparation of biological enzyme microspheres
The method comprises the following steps:
s1, preparing modified lysozyme: dissolving 5 parts by weight of lactic acid, 10 parts by weight of ethylenediamine tetraacetic acid and 7 parts by weight of cinnamic acid in 200 parts by weight of 12% KOH solution, regulating the pH value of the solution to 7.5, adding 45 parts by weight of 1-ethyl (3-dimethylaminopropyl) -3-carbodiimide, heating to 40 ℃, adding 22 parts by weight of lysozyme under stirring, stirring for reaction for 7 hours, centrifuging, dialyzing with a dialysis bag with the aperture of 10kDa, and freeze-drying the dialysate to obtain modified lysozyme;
s2, preparation of oligomeric modified lysozyme: dissolving 15 parts by weight of the modified lysozyme prepared in the step S1 in 200 parts by weight of water, adding hydrogen peroxide until the concentration of the hydrogen peroxide in the system is 3wt%, heating to 60 ℃, stirring for reaction for 4 hours, dialyzing with a dialysis bag with the aperture of 10kDa, and freeze-drying the dialysate to obtain the oligomeric modified lysozyme;
s3, preparing cross-linked oligomeric modified lysozyme: dissolving 12 parts by weight of the oligomeric modified lysozyme prepared in the step S2 and 10 parts by weight of arabinose in 200 parts by weight of PBS buffer solution with pH=8.5, heating to 65 ℃, stirring and reacting for 12 hours, adding ammonium sulfate until the system is saturated, precipitating, filtering, washing and drying to prepare the crosslinking oligomeric modified lysozyme;
S4, preparation of biological enzyme: stirring and mixing 20 parts by weight of the cross-linked oligomeric modified lysozyme prepared in the step S3, 5 parts by weight of cellulase, 7 parts by weight of trypsin and 3 parts by weight of dextranase for 20 minutes to prepare biological enzyme;
s5, preparing biological enzyme microspheres: adding 15 parts by weight of the biological enzyme prepared in the step S4, 22 parts by weight of starch and 2 parts by weight of tween-40 into 200 parts by weight of 7wt% KOH solution, heating and dissolving, and uniformly mixing to prepare a water phase; adding the water phase into 300 parts by weight of fish oil, emulsifying for 10min at 10000r/min, dropwise adding 5 parts by weight of n-butanol and 7 parts by weight of epichlorohydrin, stirring at 55 ℃ for reaction for 5h, centrifuging, washing and drying to obtain the biological enzyme microsphere.
Preparation example 3 preparation of biological enzyme microspheres
The method comprises the following steps:
s1, preparing modified lysozyme: dissolving 4 parts by weight of lactic acid, 8.5 parts by weight of ethylenediamine tetraacetic acid and 6 parts by weight of cinnamic acid in 200 parts by weight of 10% NaOH solution, regulating the pH value of the solution to 7.35, adding 40 parts by weight of 1-ethyl (3-dimethylaminopropyl) -3-carbodiimide, heating to 37 ℃, adding 20 parts by weight of lysozyme under stirring, stirring for reaction for 6 hours, centrifuging, dialyzing with a dialysis bag with the aperture of 10kDa, and freeze-drying the dialysate to obtain modified lysozyme;
S2, preparation of oligomeric modified lysozyme: dissolving 15 parts by weight of the modified lysozyme prepared in the step S1 in 200 parts by weight of water, adding hydrogen peroxide until the concentration of the hydrogen peroxide in the system is 2wt%, heating to 57 ℃, stirring for reaction for 3 hours, dialyzing with a dialysis bag with the aperture of 10kDa, and freeze-drying the dialysate to obtain the oligomeric modified lysozyme;
s3, preparing cross-linked oligomeric modified lysozyme: dissolving 11 parts by weight of the oligomeric modified lysozyme prepared in the step S2 and 8.5 parts by weight of arabinose in 200 parts by weight of PBS buffer solution with pH=8.2, heating to 62 ℃, stirring and reacting for 11 hours, adding ammonium sulfate until the system is saturated, precipitating, filtering, washing and drying to prepare the crosslinking oligomeric modified lysozyme;
s4, preparation of biological enzyme: 17 parts by weight of the cross-linked oligomeric modified lysozyme prepared in the step S3, 4 parts by weight of cellulase, 6 parts by weight of trypsin and 2.5 parts by weight of dextranase are stirred and mixed for 20 minutes to prepare the biological enzyme;
s5, preparing biological enzyme microspheres: 13.5 parts by weight of the biological enzyme prepared in the step S4, 20 parts by weight of starch and 1.5 parts by weight of tween-80 are added into 170 parts by weight of 6wt% NaOH solution, heated and dissolved, and uniformly mixed to prepare a water phase; adding the water phase into 270 parts by weight of fish oil, emulsifying for 10min at 10000r/min, dropwise adding 4 parts by weight of n-butanol and 6 parts by weight of epichlorohydrin, stirring at 52 ℃ for reaction for 4h, centrifuging, washing and drying to obtain the biological enzyme microsphere.
Comparative preparation example 1
The difference from preparation example 3 is that lactic acid was not added in step S1.
The method comprises the following steps:
s1, preparing modified lysozyme: dissolving 8.5 parts by weight of ethylenediamine tetraacetic acid and 6 parts by weight of cinnamic acid in 200 parts by weight of 10% NaOH solution, regulating the pH value of the solution to 7.35, adding 40 parts by weight of 1-ethyl (3-dimethylaminopropyl) -3-carbodiimide, heating to 37 ℃, adding 20 parts by weight of lysozyme under stirring, stirring for reaction for 6 hours, centrifuging, dialyzing by using a dialysis bag with the aperture of 10kDa, and freeze-drying the dialysate to obtain the modified lysozyme.
Comparative preparation example 2
In comparison with preparation example 3, ethylenediamine tetraacetic acid was not added in step S1.
The method comprises the following steps:
s1, preparing modified lysozyme: 4 parts by weight of lactic acid and 6 parts by weight of cinnamic acid are dissolved in 200 parts by weight of 10% NaOH solution, the pH value of the solution is regulated to 7.35, 40 parts by weight of 1-ethyl (3-dimethylaminopropyl) -3-carbodiimide is added, the temperature is heated to 37 ℃, 20 parts by weight of lysozyme is added under stirring, the reaction is stirred for 6 hours, the reaction is carried out centrifugally, dialysis is carried out by a dialysis bag with the aperture of 10kDa, and the dialyzate is freeze-dried, so that the modified lysozyme is obtained.
Comparative preparation example 3
In comparison with preparation example 3, the difference is that cinnamic acid is not added in step S1.
The method comprises the following steps:
s1, preparing modified lysozyme: 4 parts by weight of lactic acid and 8.5 parts by weight of ethylenediamine tetraacetic acid are dissolved in 200 parts by weight of 10% NaOH solution, the pH value of the solution is regulated to 7.35, 40 parts by weight of 1-ethyl (3-dimethylaminopropyl) -3-carbodiimide is added, the temperature is heated to 37 ℃, 20 parts by weight of lysozyme is added under the stirring condition, the reaction is carried out for 6 hours, the centrifugation is carried out, the dialysis is carried out by a dialysis bag with the aperture of 10kDa, and the dialyzate is freeze-dried, thus obtaining the modified lysozyme.
Comparative preparation example 4
In comparison with preparation example 3, the difference is that step S1 is not performed.
The method comprises the following steps:
s1, preparation of oligomeric modified lysozyme: dissolving 15 parts by weight of lysozyme in 200 parts by weight of water, adding hydrogen peroxide until the concentration of the hydrogen peroxide in the system is 2wt%, heating to 57 ℃, stirring for reaction for 3 hours, dialyzing with a dialysis bag with the aperture of 10kDa, and freeze-drying the dialysate to obtain the oligomeric lysozyme;
s2, preparing cross-linked oligomeric modified lysozyme: dissolving 11 parts by weight of the oligolysozyme prepared in the step S1 and 8.5 parts by weight of arabinose in 200 parts by weight of PBS buffer solution with pH=8.2, heating to 62 ℃, stirring and reacting for 11 hours, adding ammonium sulfate until the system is saturated, precipitating, filtering, washing and drying to prepare the cross-linked oligolysozyme;
S3, preparation of biological enzyme: 17 parts by weight of the cross-linked oligomeric lysozyme prepared in the step S2, 4 parts by weight of cellulase, 6 parts by weight of trypsin and 2.5 parts by weight of dextranase are stirred and mixed for 20 minutes to prepare the biological enzyme;
s4, preparing biological enzyme microspheres: 13.5 parts by weight of the biological enzyme prepared in the step S3, 20 parts by weight of starch and 1.5 parts by weight of tween-80 are added into 170 parts by weight of 6wt% NaOH solution, heated and dissolved, and uniformly mixed to prepare a water phase; adding the water phase into 270 parts by weight of fish oil, emulsifying for 10min at 10000r/min, dropwise adding 4 parts by weight of n-butanol and 6 parts by weight of epichlorohydrin, stirring at 52 ℃ for reaction for 4h, centrifuging, washing and drying to obtain the biological enzyme microsphere.
Comparative preparation example 5
In comparison with preparation example 3, the difference is that step S2 is not performed.
The method comprises the following steps:
s1, preparing modified lysozyme: dissolving 4 parts by weight of lactic acid, 8.5 parts by weight of ethylenediamine tetraacetic acid and 6 parts by weight of cinnamic acid in 200 parts by weight of 10% NaOH solution, regulating the pH value of the solution to 7.35, adding 40 parts by weight of 1-ethyl (3-dimethylaminopropyl) -3-carbodiimide, heating to 37 ℃, adding 20 parts by weight of lysozyme under stirring, stirring for reaction for 6 hours, centrifuging, dialyzing with a dialysis bag with the aperture of 10kDa, and freeze-drying the dialysate to obtain modified lysozyme;
S2, preparing cross-linked modified lysozyme: dissolving 11 parts by weight of the oligomeric lysozyme prepared in the step S1 and 8.5 parts by weight of arabinose in 200 parts by weight of PBS buffer solution with pH=8.2, heating to 62 ℃, stirring and reacting for 11 hours, adding ammonium sulfate until the system is saturated, precipitating, filtering, washing and drying to prepare the crosslinking modified lysozyme;
s3, preparation of biological enzyme: 17 parts by weight of the cross-linked modified lysozyme prepared in the step S3, 4 parts by weight of cellulase, 6 parts by weight of trypsin and 2.5 parts by weight of dextranase are stirred and mixed for 20 minutes to prepare the biological enzyme;
s4, preparing biological enzyme microspheres: 13.5 parts by weight of the biological enzyme prepared in the step S3, 20 parts by weight of starch and 1.5 parts by weight of tween-80 are added into 170 parts by weight of 6wt% NaOH solution, heated and dissolved, and uniformly mixed to prepare a water phase; adding the water phase into 270 parts by weight of fish oil, emulsifying for 10min at 10000r/min, dropwise adding 4 parts by weight of n-butanol and 6 parts by weight of epichlorohydrin, stirring at 52 ℃ for reaction for 4h, centrifuging, washing and drying to obtain the biological enzyme microsphere.
Comparative preparation example 6
In comparison with preparation example 3, the difference is that step S3 is not performed.
The method comprises the following steps:
s1, preparing modified lysozyme: dissolving 4 parts by weight of lactic acid, 8.5 parts by weight of ethylenediamine tetraacetic acid and 6 parts by weight of cinnamic acid in 200 parts by weight of 10% NaOH solution, regulating the pH value of the solution to 7.35, adding 40 parts by weight of 1-ethyl (3-dimethylaminopropyl) -3-carbodiimide, heating to 37 ℃, adding 20 parts by weight of lysozyme under stirring, stirring for reaction for 6 hours, centrifuging, dialyzing with a dialysis bag with the aperture of 10kDa, and freeze-drying the dialysate to obtain modified lysozyme;
S2, preparation of oligomeric modified lysozyme: dissolving 15 parts by weight of the modified lysozyme prepared in the step S1 in 200 parts by weight of water, adding hydrogen peroxide until the concentration of the hydrogen peroxide in the system is 2wt%, heating to 57 ℃, stirring for reaction for 3 hours, dialyzing with a dialysis bag with the aperture of 10kDa, and freeze-drying the dialysate to obtain the oligomeric modified lysozyme;
s3, preparation of biological enzyme: 17 parts by weight of the oligomeric modified lysozyme prepared in the step S2, 4 parts by weight of cellulase, 6 parts by weight of trypsin and 2.5 parts by weight of dextranase are stirred and mixed for 20 minutes to prepare the biological enzyme;
s4, preparing biological enzyme microspheres: 13.5 parts by weight of the biological enzyme prepared in the step S3, 20 parts by weight of starch and 1.5 parts by weight of tween-80 are added into 170 parts by weight of 6wt% NaOH solution, heated and dissolved, and uniformly mixed to prepare a water phase; adding the water phase into 270 parts by weight of fish oil, emulsifying for 10min at 10000r/min, dropwise adding 4 parts by weight of n-butanol and 6 parts by weight of epichlorohydrin, stirring at 52 ℃ for reaction for 4h, centrifuging, washing and drying to obtain the biological enzyme microsphere.
Comparative preparation example 7
In comparison with preparation example 3, the difference is that no dextranase was added in step S4.
The method comprises the following steps:
s4, preparation of biological enzyme: 19.5 parts by weight of the cross-linked oligomeric modified lysozyme prepared in the step S3, 4 parts by weight of cellulase and 6 parts by weight of trypsin are stirred and mixed for 20 minutes to prepare the biological enzyme.
Comparative preparation example 8
In comparison with preparation example 3, the difference is that the crosslinking oligomodified lysozyme was not added in step S4.
The method comprises the following steps:
s4, preparation of biological enzyme: 4 parts by weight of cellulase, 6 parts by weight of trypsin and 19.5 parts by weight of dextranase are stirred and mixed for 20 minutes to prepare the biological enzyme.
Comparative preparation example 9
In comparison with preparation example 3, the difference is that no cellulase and trypsin are added in step S4.
The method comprises the following steps:
s4, preparation of biological enzyme: 17 parts by weight of the cross-linked oligomeric modified lysozyme prepared in the step S3 and 2.5 parts by weight of dextranase are stirred and mixed for 20 minutes to prepare the biological enzyme.
Comparative preparation example 10
In comparison with preparation example 3, the difference is that step S5 is not performed.
The method comprises the following steps:
s1, preparing modified lysozyme: dissolving 4 parts by weight of lactic acid, 8.5 parts by weight of ethylenediamine tetraacetic acid and 6 parts by weight of cinnamic acid in 200 parts by weight of 10% NaOH solution, regulating the pH value of the solution to 7.35, adding 40 parts by weight of 1-ethyl (3-dimethylaminopropyl) -3-carbodiimide, heating to 37 ℃, adding 20 parts by weight of lysozyme under stirring, stirring for reaction for 6 hours, centrifuging, dialyzing with a dialysis bag with the aperture of 10kDa, and freeze-drying the dialysate to obtain modified lysozyme;
S2, preparation of oligomeric modified lysozyme: dissolving 15 parts by weight of the modified lysozyme prepared in the step S1 in 200 parts by weight of water, adding hydrogen peroxide until the concentration of the hydrogen peroxide in the system is 2wt%, heating to 57 ℃, stirring for reaction for 3 hours, dialyzing with a dialysis bag with the aperture of 10kDa, and freeze-drying the dialysate to obtain the oligomeric modified lysozyme;
s3, preparing cross-linked oligomeric modified lysozyme: dissolving 11 parts by weight of the oligomeric modified lysozyme prepared in the step S2 and 8.5 parts by weight of arabinose in 200 parts by weight of PBS buffer solution with pH=8.2, heating to 62 ℃, stirring and reacting for 11 hours, adding ammonium sulfate until the system is saturated, precipitating, filtering, washing and drying to prepare the crosslinking oligomeric modified lysozyme;
s4, preparation of biological enzyme: 17 parts by weight of the cross-linked oligomeric modified lysozyme prepared in the step S3, 4 parts by weight of cellulase, 6 parts by weight of trypsin and 2.5 parts by weight of dextranase are stirred and mixed for 20 minutes to prepare the biological enzyme.
Test example 1 bacteriostatic Effect against pathogenic bacteria
Culture of common pathogenic bacteria in the oral cavity of diabetics: culture of common pathogenic bacteria in the oral cavity of diabetics: streptococcus mutans (ATCC 25175), actinomycetes viscosus (ATCC 27044), porphyromonas gingivalis (BNCC 353909), acinetobacter actinomycetes (BNCC 336945) and Clostridium nucleatum (BNCC 336949) are respectively inoculated into BHI liquid culture medium, anaerobic cultured for 48 hours at 37 ℃, and centrifuged to obtain fresh bacterial cultures. The cells were washed twice with ph=7.0 phosphate buffer, and then resuspended with ph=7.0 phosphate buffer to the initial absorbance OD of the bacterial suspension 600 Between 0.5 and 0.6 for standby.
Pretreatment: 10mg of the biological enzyme microspheres prepared in preparation examples 1 to 3 and comparative preparation examples 1 to 10 were added to 100mL of an aqueous solution containing 1wt% amylase, and subjected to enzymolysis at 37℃for 2 minutes to obtain a sample solution.
Bacteriostasis test: preparing a BHI culture medium with the agar content of 0.7%, and sterilizing; the isothermal temperature is reduced to below 45 ℃, 0.2 percent (volume ratio) of bacterial suspension of oral pathogenic bacteria is added, and the mixture is uniformly shaken; and immersing a sterile filter paper sheet with the diameter of 7mm in the sample solution, taking out, drying, taking 7mL, pouring the 7mL onto a bottom agar plate, standing for solidification, placing the prepared paper sheet, carrying out anaerobic culture for 48h at 37 ℃, and observing the diameter (mm) of a bacteriostasis ring.
The results are shown in Table 1.
TABLE 1
Therefore, the biological enzyme microspheres prepared in preparation examples 1-3 release biological enzymes after enzymolysis, and have a certain antibacterial effect on pathogenic bacteria.
Test example 2 agglutination effect against oral pathogenic bacteria
Culture of common pathogenic bacteria in the oral cavity of diabetics: culture of common pathogenic bacteria in the oral cavity of diabetics: streptococcus mutans (ATCC 25175), actinomycetes viscosus (ATCC 27044), porphyromonas gingivalis (BNCC 353909), acinetobacter actinomycetes (BNCC 336945) and Clostridium nucleatum (BNCC 336949), respectively inoculating into BHI liquid culture medium, anaerobic culturing at 37deg.C for 48 hr, centrifuging Fresh cell cultures were obtained. The cells were washed twice with ph=7.0 phosphate buffer, and then resuspended with ph=7.0 phosphate buffer to the initial absorbance OD of the bacterial suspension 600 Between 0.5 and 0.6 for standby;
pretreatment: 10mg of the biological enzyme microspheres prepared in preparation examples 1 to 3 and comparative preparation examples 1 to 10 were added to 100mL of an aqueous solution containing 1wt% amylase, and subjected to enzymolysis at 37℃for 2 minutes to obtain a sample solution.
Agglutination test: 100. Mu.L of the sample solution was added to a 24-well plate, and 300. Mu.L of the pathogenic bacterial suspension was added as a reaction sample. Another sample solution, 100. Mu.L, was added to a 24-well plate and mixed with 300. Mu.L buffer as a control, 2 replicates for each control and sample. The 24-well plate was placed in a microplate thermostatted shaker at 400rpm, room temperature and incubated with shaking. Every 30min of oscillation, stopping for 30min, observing the state of the pore plate, and measuring OD 600 . An equal amount of pathogenic bacterial suspension was added to the wells of the plate as a negative control. Three samples were run in parallel. After 6 hours, 100. Mu.L of the upper suspension was aspirated to determine the absorbance OD 600 The aggregation ratio (R) was calculated.
R＝1-A t /A 0 。
R is the agglutination rate,%; a is that t Absorbance at time t; a is that 0 The absorbance is the initial absorbance of the pathogenic bacteria suspension.
TABLE 2
As is clear from the above table, in preparation examples 1 to 3, floc condensation points were found in the well plates of 5 pathogenic bacteria of Streptococcus mutans, porphyromonas gingivalis, actinomyces viscosus, clostridium nucleatum and Actinobacillus semiactinomyces. Therefore, the biological enzyme microsphere releases biological enzyme after enzymolysis, and can be aggregated with streptococcus mutans, actinomyces viscosus, porphyromonas gingivalis, actinomyces conglomerate and clostridium nucleatum, the aggregation rate reaches 80.6% after 6 hours, and the pathogenic bacteria quantity in the oral cavity can be obviously reduced.
Preparation example 4 preparation of active ferment
The method comprises the following steps:
respectively cleaning 7 parts by weight of carob, 3 parts by weight of mulberry leaf and 5 parts by weight of rhodiola rosea, drying, crushing to obtain mixed powder, adding 200 parts by weight of water, adding compound enzyme, carrying out enzymolysis for 2 hours at 50 ℃, sterilizing, inoculating lactobacillus paracasei and bifidobacterium longum strain seed liquid, wherein the inoculum sizes of the lactobacillus paracasei and the bifidobacterium longum strain seed liquid are respectively 2v/v% and 1v/v%,37 ℃, and carrying out enzyme-assisted fermentation culture for 48 hours at 100 r/min. Filtering the product, washing the solid residue to collect bacterial liquid, mixing the bacterial liquid with the filtrate, and freeze-drying to obtain the active ferment.
The compound enzyme is cellulase and pectase, and the mass ratio is 7:3.
Preparation example 5 preparation of active ferment
The method comprises the following steps:
respectively cleaning 10 parts by weight of carob, 5 parts by weight of mulberry leaf and 7 parts by weight of rhodiola rosea, drying, crushing to obtain mixed powder, adding 200 parts by weight of water, adding compound enzyme, carrying out enzymolysis for 2 hours at 50 ℃, sterilizing, inoculating lactobacillus paracasei and bifidobacterium longum strain seed liquid, wherein the inoculum sizes of the lactobacillus paracasei and the bifidobacterium longum strain seed liquid are respectively 4v/v% and 3v/v%,39 ℃,120r/min, and carrying out enzyme-assisted fermentation culture for 56 hours. Filtering the product, washing the solid residue to collect bacterial liquid, mixing the bacterial liquid with the filtrate, and freeze-drying to obtain the active ferment.
The compound enzyme is cellulase and pectase, and the mass ratio is 10:5.
Preparation example 6 preparation of active ferment
The method comprises the following steps:
8.5 parts by weight of carob, 4 parts by weight of mulberry leaf and 6 parts by weight of rhodiola rosea are respectively washed, dried and crushed to obtain mixed powder, the mixed powder is added into 200 parts by weight of water, compound enzyme is added, enzymolysis is carried out for 2 hours at 50 ℃, the addition amount is 4wt% of the total mass of the system, sterilization is carried out, lactobacillus paracasei and bifidobacterium longum seed liquid are inoculated, the inoculation amounts of the lactobacillus paracasei and bifidobacterium longum seed liquid are respectively 3v/v% and 2v/v%, the inoculation amount of the lactobacillus paracasei and bifidobacterium longum seed liquid is 38 ℃, and the fermentation and cultivation are carried out for 52 hours by enzyme assistance. Filtering the product, washing the solid residue to collect bacterial liquid, mixing the bacterial liquid with the filtrate, and freeze-drying to obtain the active ferment.
The compound enzyme is cellulase and pectase, and the mass ratio is 8.5:4.
Preparation example 7
The difference from preparation example 6 is that the complex enzyme is a single cellulase.
Preparation example 8
The difference compared to preparation example 6 is that the complex enzyme is a single pectase.
Comparative preparation 11
In comparison with preparation example 6, the complex enzyme was not added.
The method comprises the following steps:
8.5 parts by weight of carob, 4 parts by weight of mulberry leaf and 6 parts by weight of rhodiola rosea are respectively washed, dried and crushed to obtain mixed powder, the mixed powder is added into 200 parts by weight of water, and the mixed powder is sterilized and inoculated with lactobacillus paracasei and bifidobacterium longum seed liquid, wherein the inoculation amounts of the lactobacillus paracasei and bifidobacterium longum seed liquid are respectively 3v/v% and 2v/v%, the temperature is 38 ℃, and the enzyme assisted fermentation culture is carried out for 52h. Filtering the product, washing the solid residue to collect bacterial liquid, mixing the bacterial liquid with the filtrate, and freeze-drying to obtain the active ferment.
Comparative preparation example 12
The difference compared with preparation example 6 is that the seed solution of Lactobacillus paracasei strain was not inoculated.
The method comprises the following steps:
8.5 parts by weight of carob, 4 parts by weight of mulberry leaf and 6 parts by weight of rhodiola rosea are respectively washed, dried and crushed to obtain mixed powder, the mixed powder is added into 200 parts by weight of water, compound enzyme is added, enzymolysis is carried out for 2 hours at 50 ℃, the addition amount is 4wt% of the total mass of the system, sterilization and inoculation are carried out on bifidobacterium longum strain seed liquid, the inoculation amount of the bifidobacterium longum strain seed liquid is 5v/v%, the inoculation amount of the bifidobacterium longum strain seed liquid is 38 ℃, and the fermentation and the culture are carried out for 52 hours by enzyme assistance. Filtering the product, washing the solid residue to collect bacterial liquid, mixing the bacterial liquid with the filtrate, and freeze-drying to obtain the active ferment.
Comparative preparation example 13
The difference from preparation example 6 is that the seed solution of Bifidobacterium longum strain was not inoculated.
The method comprises the following steps:
8.5 parts by weight of carob, 4 parts by weight of mulberry leaf and 6 parts by weight of rhodiola rosea are respectively washed, dried and crushed to obtain mixed powder, the mixed powder is added into 200 parts by weight of water, compound enzyme is added, enzymolysis is carried out for 2 hours at 50 ℃, the addition amount is 4wt% of the total mass of the system, sterilization and inoculation are carried out on lactobacillus paracasei strain seed liquid, the inoculation amount of the lactobacillus paracasei strain seed liquid is 5v/v%, the inoculation amount of the lactobacillus paracasei strain seed liquid is 38 ℃, and the enzyme-assisted fermentation culture is carried out for 52 hours. Filtering the product, washing the solid residue to collect bacterial liquid, mixing the bacterial liquid with the filtrate, and freeze-drying to obtain the active ferment.
Comparative preparation example 14
The difference from preparation example 6 is that lactobacillus paracasei and bifidobacterium longum seed solution were not inoculated.
The method comprises the following steps:
8.5 parts by weight of carob, 4 parts by weight of mulberry leaf and 6 parts by weight of rhodiola rosea are respectively washed, dried and crushed to obtain mixed powder, the mixed powder is added into 200 parts by weight of water, compound enzyme is added, enzymolysis is carried out for 2 hours at 50 ℃, the addition amount is 4wt% of the total mass of the system, the product is filtered, the solid residue is washed to collect bacterial liquid, and the bacterial liquid and filtrate are mixed and freeze-dried to obtain the active ferment.
Comparative preparation example 15
The difference compared to preparation example 6 is that no carob is added.
The method comprises the following steps:
cleaning, drying and crushing 4 parts by weight of mulberry leaves and 6 parts by weight of rhodiola rosea respectively to obtain mixed powder, adding 200 parts by weight of water, adding compound enzyme for enzymolysis, sterilizing, inoculating lactobacillus paracasei and bifidobacterium longum strain seed liquid, wherein the inoculation amounts of the lactobacillus paracasei and bifidobacterium longum strain seed liquid are 3v/v percent and 2v/v percent respectively, and culturing for 52 hours at 38 ℃ under the enzyme-assisted fermentation condition of 110 r/min. Filtering the product, washing the solid residue to collect bacterial liquid, mixing the bacterial liquid with the filtrate, and freeze-drying to obtain the active ferment.
Comparative preparation example 16
The difference from preparation example 6 is that no mulberry leaf was added.
The method comprises the following steps:
8.5 parts by weight of carob and 6 parts by weight of rhodiola rosea are respectively washed, dried and crushed to obtain mixed powder, the mixed powder is added into 200 parts by weight of water for enzymolysis, the added amount is 4% by weight of the total mass of the system, sterilization and inoculation of lactobacillus paracasei and bifidobacterium longum strain seed liquid are carried out, the inoculation amounts of the lactobacillus paracasei and bifidobacterium longum strain seed liquid are respectively 3v/v% and 2v/v%, the temperature is 38 ℃, the temperature is 110r/min, and the enzyme-assisted fermentation culture is carried out for 52 hours. Filtering the product, washing the solid residue to collect bacterial liquid, mixing the bacterial liquid with the filtrate, and freeze-drying to obtain the active ferment.
Comparative preparation example 17
Compared with preparation example 6, the difference is that rhodiola root is not added.
The method comprises the following steps:
8.5 parts by weight of carob and 4 parts by weight of mulberry leaf are respectively washed, dried and crushed to obtain mixed powder, the mixed powder is added into 200 parts by weight of water for enzymolysis, the added amount is 4% by weight of the total mass of the system, sterilization and inoculation of lactobacillus paracasei and bifidobacterium longum strain seed liquid are carried out, the inoculation amounts of the lactobacillus paracasei and bifidobacterium longum strain seed liquid are respectively 3v/v% and 2v/v%, the temperature is 38 ℃, the temperature is 110r/min, and the enzyme-assisted fermentation culture is carried out for 52 hours. Filtering the product, washing the solid residue to collect bacterial liquid, mixing the bacterial liquid with the filtrate, and freeze-drying to obtain the active ferment.
Test example 3 mouse test
1. Hypoglycemic test on adrenergic hyperglycemic mice
90 healthy mice were randomly divided into a normal group, an epinephrine group, a metformin hydrochloride group (125 mg/kg. D) and preparation examples 6 to 10, and comparative examples 11 to 17 (100 mg/kg. D of active ferment prepared in preparation examples 6 to 10 and comparative examples 11 to 17 were administered), 6 mice each were administered by intragastric administration 1 time per day, and normal group and epinephrine group were administered with an equivalent amount of physiological saline, followed by continuous administration for 2 weeks. After the administration of 2.0 hours after the administration of 2mg/kg of norepinephrine was injected into the abdominal cavity except normal group normal saline, after 30 minutes, the orbit was bled and the blood glucose level was measured.
The results are shown in Table 3.
TABLE 3 Table 3
| Group of | Fasting blood glucose level (mmol/L) |
| Normal group | 5.58±0.81 |
| Epinephrine group | 11.92±1.02* |
| Metformin hydrochloride group | 7.95±1.15# |
| Preparation example 6 | 5.97±0.94# |
| Preparation example 7 | 6.01±1.07# |
| Preparation example 8 | 5.94±1.01# |
| Preparation example 9 | 6.85±1.16 |
| Preparation example 10 | 6.94±1.14 |
| Comparative preparation 11 | 7.10±1.04 |
| Comparative preparation example 12 | 7.02±1.16 |
| Comparative preparation example 13 | 7.05±1.09 |
| Comparative preparation example 14 | 7.21±0.99 |
| Comparative preparation example 15 | 8.10±1.04 |
| Comparative preparation example 16 | 7.82±1.12 |
| Comparative preparation example 17 | 7.93±1.02 |
Annotation: * P <0.05 compared to the normal group; # is P <0.05 compared to model group.
As is clear from the above table, the active ferment prepared in preparation examples 6 to 8 of the present invention can significantly reduce the fasting blood glucose level of adrenergic hyperglycemia mice.
2. 100 healthy mice are selected, after 1 week of adaptive feeding, 90 mice are selected to be fasted for 12 hours, 200mg/kg of tetraoxypyrimidine is injected into the abdominal cavity, after 3 days, the mice are fasted for 5 hours, the tail tips are sheared to take blood and measure blood sugar, and the mice with the empty abdominal blood sugar above 11.1mmol/L are screened as diabetes model mice, and the total number of the mice is 84. The mice were randomly divided into a model group, a metformin hydrochloride group (125 mg/kg. D) and preparation examples 6-10, and comparative examples 11-17 (100 mg/kg. D) of active ferment obtained in preparation examples 6-10 and comparative examples 11-17 were administered, and 6 mice each were administered by intragastric administration 1 time a day, and 10 healthy mice were taken as normal groups. Normal and model groups were given equal amounts of physiological saline, and 2 weeks after continuous administration. The eyes were bled 12h before the last dose and blood glucose levels were measured 2h after the last dose.
The results are shown in Table 4.
TABLE 4 Table 4
Annotation: * P <0.05 compared to the normal group; # is P <0.05 compared to model group.
As shown in the table above, the active ferment prepared in preparation examples 6-8 of the invention can obviously reduce the fasting blood glucose value of mice with hyperglycemia caused by tetraoxypyrimidine.
Example 1
The present example provides a bio-enzyme-based composition prepared by uniformly mixing 5 parts by weight of the bio-enzyme microsphere prepared in preparation example 1, 2 parts by weight of probiotics, 3 parts by weight of the active ferment prepared in preparation example 4, and 1 part by weight of antibacterial peptide. The probiotics comprise lactobacillus reuteri, lactobacillus rhamnosus and lactobacillus acidophilus, and the mass ratio is 5:3:2.
Example 2
The present example provides a bio-enzyme-based composition prepared by uniformly mixing 15 parts by weight of the bio-enzyme microsphere prepared in preparation example 2, 10 parts by weight of probiotics, 7 parts by weight of the active ferment prepared in preparation example 5, and 3 parts by weight of the antibacterial peptide. The probiotics comprise lactobacillus reuteri, lactobacillus rhamnosus and lactobacillus acidophilus, and the mass ratio is 7:5:4.
Example 3
The present example provides a bio-enzyme-based composition prepared by uniformly mixing 10 parts by weight of the bio-enzyme microsphere prepared in preparation example 3, 7 parts by weight of probiotics, 5 parts by weight of the active ferment prepared in preparation example 6, and 2 parts by weight of the antibacterial peptide. The probiotics comprise lactobacillus reuteri, lactobacillus rhamnosus and lactobacillus acidophilus, and the mass ratio is 6:4:3.
Examples 4 to 5
The difference compared with example 3 is that the active ferment is replaced by the active ferment prepared in preparation examples 7 and 8.
Comparative examples 1 to 10
The difference compared with example 3 is that the bio-enzyme microspheres are replaced with the bio-enzyme microspheres prepared in comparative preparation examples 1 to 10.
Comparative examples 11 to 17
The difference compared to example 3 is that the active ferment is replaced by the active ferment prepared in comparative preparation examples 11 to 17.
Examples 6 to 10
A blood glucose reducing toothpaste containing biological enzyme-based composition is provided.
The composition of the matrix is shown in Table 5 below.
TABLE 5
Examples 6 to 10 the biological enzyme-based compositions prepared in examples 1 to 5 were used in this order, respectively.
The preparation method comprises the following steps:
(1) Mixing and stirring all the components in the matrix for 3 hours to obtain the matrix for toothpaste;
(2) 80 parts by weight of a toothpaste matrix,1.5 parts by weight of Lactobacillus reuteri, 1.5 parts by weight of Lactobacillus rhamnosus, 1.5 parts by weight of Lactobacillus acidophilus,Mixing 18 parts by weight of the biological enzyme-based composition and 2 parts by weight of sodium hyaluronate, stirring for 30min, placing in a vacuum paste making machine, maintaining the vacuum degree at-0.092 MPa, homogenizing, stirring for 30min, and packaging to obtain the hypoglycemic toothpaste.
Comparative examples 18 to 34
The difference compared to example 10 is that the bio-enzyme based composition was replaced with the bio-enzyme based compositions prepared in comparative examples 1 to 17.
Test example 3 health-care and therapeutic effects on diabetes and its complications of cardiovascular and cerebrovascular diseases induced by diabetes
Test object: 230 diabetics were selected for trial and observation.
The toothpaste using method comprises the following steps: patients were randomly divided into 23 groups and brushed with the hypoglycemic toothpastes prepared in examples 6-10 and comparative examples 18-34, 2g each, for 2-5 minutes.
Treatment course: 60 days is a treatment course, and each day of teeth is brushed once in the morning and evening.
Testing fasting blood glucose and postprandial blood glucose levels were tested on the first day after a brushing session using the hypoglycemic toothpastes prepared in examples 6-10 and comparative examples 18-34.
The results are shown in Table 6.
TABLE 6
As can be seen from the above table, after one treatment course, the blood glucose level of the blood glucose-lowering toothpaste prepared by the embodiment of the invention is obviously reduced after the use * p < 0.05), and blood glucose of examples 6-8 was significantly reduced and diabetes was significantly alleviated.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. The biological enzyme-based composition is characterized by being prepared from the following raw materials in parts by weight: 5-15 parts of biological enzyme microspheres, 2-10 parts of probiotics, 3-7 parts of active ferment and 1-3 parts of antibacterial peptide;
the biological enzyme microsphere is prepared by chemically modifying lysozyme, performing oligomerization to obtain oligomeric modified lysozyme, performing Maillard reaction with xanthan gum, mixing with cellulase, trypsin and dextranase, and embedding in crosslinked starch microsphere;
The active ferment is an active ferment obtained by extracting carob, mulberry leaf and rhodiola rosea.
2. The bio-enzyme based composition according to claim 1, wherein the bio-enzyme microsphere is prepared by the following method:
s1, preparing modified lysozyme: dissolving lactic acid, ethylenediamine tetraacetic acid and cinnamic acid in alkali liquor, regulating the pH value of the solution, adding 1-ethyl (3-dimethylaminopropyl) -3-carbodiimide, adding lysozyme under the condition of heating and stirring, stirring for reaction, centrifuging, and dialyzing to obtain modified lysozyme;
s2, preparation of oligomeric modified lysozyme: dissolving the modified lysozyme prepared in the step S1 in water, adding hydrogen peroxide, heating, stirring, reacting, and dialyzing to obtain the oligomeric modified lysozyme;
s3, preparing cross-linked oligomeric modified lysozyme: dissolving the oligomeric modified lysozyme and the arabinose prepared in the step S2 in PBS solution, heating and stirring for reaction, adding ammonium sulfate until the system is saturated, precipitating, filtering, washing and drying to prepare the crosslinking oligomeric modified lysozyme;
s4, preparation of biological enzyme: uniformly mixing the cross-linked oligomeric modified lysozyme, cellulase, trypsin and dextranase prepared in the step S3 to prepare biological enzyme;
s5, preparing biological enzyme microspheres: adding the biological enzyme, starch and emulsifier prepared in the step S4 into alkali liquor, heating and dissolving, and uniformly mixing to prepare a water phase; adding the water phase into fish oil, emulsifying, dripping n-butanol and epichlorohydrin, stirring for reaction, centrifuging, washing, and drying to obtain the biological enzyme microsphere.
3. The biological enzyme-based composition according to claim 2, wherein in step S1, the mass ratio of lactic acid, ethylenediamine tetraacetic acid, cinnamic acid, 1-ethyl (3-dimethylaminopropyl) -3-carbodiimide, lysozyme is 3-5:7-10:5-7:35-45:15-22, the alkaline solution is 7-12% NaOH or KOH solution, the pH of the solution is adjusted to 7.2-7.5, the temperature of the heating and stirring is 35-40 ℃, the time of the stirring reaction is 5-7 hours, and the dialysis bag pore size of the dialysis is 9-10KDa; and in the step S2, hydrogen peroxide is added to the system, the concentration of the hydrogen peroxide is 1-3wt%, the temperature of the heating and stirring reaction is 55-60 ℃, the time is 2-4h, and the aperture of the dialysis bag for dialysis is 9-10kDa.
4. The biological enzyme-based composition according to claim 2, wherein in the step S3, the mass ratio of the oligomeric modified lysozyme to the arabinose is 10-12:7-10, the PBS buffer solution is a PBS buffer solution with ph=8-8.5, the temperature of the heating and stirring reaction is 60-65 ℃ for 10-12 hours; the mass ratio of the cross-linked oligomeric modified lysozyme to the cellulase to the trypsin to the dextranase in the step S4 is 15-20:3-5:5-7:2-3.
5. The bio-enzyme based composition according to claim 2, wherein the mass ratio of the bio-enzyme, starch, emulsifier, alkali solution, fish oil, n-butanol and epichlorohydrin in the step S5 is 12-15:17-22:1-2:150-200:250-300:3-5:5-7, the alkali solution is 5-7wt% NaOH or KOH solution, and the stirring reaction is performed at 50-55 ℃ for 3-5 hours.
6. The biological enzyme-based composition according to claim 1, wherein the active ferment is prepared by the following method:
respectively cleaning carob, mulberry leaf and rhodiola rosea, drying, crushing to obtain mixed powder, adding the mixed powder into water, adding compound enzyme for enzymolysis, sterilizing, inoculating lactobacillus paracasei and bifidobacterium longum strain seed liquid, carrying out enzyme-assisted fermentation culture, filtering a product, washing solid residues, collecting bacterial liquid, mixing the bacterial liquid with filtrate, and freeze-drying to obtain the active ferment.
7. The biological enzyme-based composition according to claim 6, wherein theThe mass ratio of the carob seed to the mulberry leaf to the rhodiola rosea is 7-10:3-5:5-7, the mass ratio of the compound enzyme to the pectase is 7-10:3-5, the addition amount of the compound enzyme is 3-5wt% of the total mass of the system, the inoculation amounts of lactobacillus paracasei and bifidobacterium longum strain seed liquid are respectively 2-4v/v% and 1-3v/v%, and the bacterial content of the strain seed liquid is 10 8 -10 9 cfu/mL, wherein the conditions of enzyme-assisted fermentation culture are 37-39 ℃,100-120r/min and enzyme-assisted fermentation culture for 48-56h.
8. The biological enzyme-based composition according to claim 1, wherein the probiotics comprise lactobacillus reuteri, lactobacillus rhamnosus, lactobacillus acidophilus, in a mass ratio of 5-7:3-5:2-4.
9. A hypoglycemic toothpaste comprising the bioenzyme-based composition according to any one of claims 1 to 8, characterized by being prepared from the following raw materials by weight: 20-25 parts of biological enzyme-based composition,1-2 parts of lactobacillus reuteri, 1-2 parts of lactobacillus rhamnosus, 1-2 parts of lactobacillus acidophilus,70-95 parts of matrix and 2-4 parts of sodium hyaluronate; the matrix is prepared from the following raw materials in percentage by mass: 12-60% of friction agent, 5-60% of humectant, 0.5-4% of thickening agent, 1-5.5% of foaming agent, 0-2% of spice, 0-1% of sweetener, 0-1% of food color, 0-0.5% of preservative and the balance of deionized water.
10. A method for preparing the hypoglycemic toothpaste according to claim 9, comprising the steps of:
(1) Mixing and stirring the components in the matrix for 2-3h to obtain the matrix for toothpaste;
(2) Mixing and stirring all the components in the biological enzyme-based composition for 0.5-1h to obtain the biological enzyme-based composition;
(3)a substrate for toothpaste,Lactobacillus reuteri, lactobacillus rhamnosus, lactobacillus acidophilus,Mixing and stirring the biological enzyme-based composition and sodium hyaluronate for 20-30min, placing into a vacuum paste maker, and maintaining vacuum degree at-0.09 MPa to-0.09 Homogenizing under 6MPa, stirring for 20-30min, and packaging to obtain the final product.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311708365.5A CN117679352A (en) | 2023-12-13 | 2023-12-13 | Biological enzyme-based composition, hypoglycemic toothpaste containing same and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311708365.5A CN117679352A (en) | 2023-12-13 | 2023-12-13 | Biological enzyme-based composition, hypoglycemic toothpaste containing same and preparation method thereof |
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