CN110558320A - Double-histidine/lycium barbarum polysaccharide composite antibacterial agent and preparation method and application thereof - Google Patents
Double-histidine/lycium barbarum polysaccharide composite antibacterial agent and preparation method and application thereof Download PDFInfo
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- CN110558320A CN110558320A CN201910812026.9A CN201910812026A CN110558320A CN 110558320 A CN110558320 A CN 110558320A CN 201910812026 A CN201910812026 A CN 201910812026A CN 110558320 A CN110558320 A CN 110558320A
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- lycium barbarum
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
- A01N43/50—1,3-Diazoles; Hydrogenated 1,3-diazoles
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
- A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
- A23B4/20—Organic compounds; Microorganisms; Enzymes
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Abstract
the invention belongs to the field of food safety, and particularly relates to a preparation method and application of a double-histidine/lycium barbarum polysaccharide composite antibacterial agent. The preparation method of the composite antibacterial agent specifically comprises the following steps: (1) preparing wolfberry polysaccharide: extracting fructus Lycii polysaccharide by ultra-soaking method; (2) preparing a composite antibacterial agent: completely dissolving bi-histidine with diethyl ether, adding sterile distilled water, dissolving lycium barbarum polysaccharide in sterile distilled water, and mixing the two solutions according to a certain proportion to obtain the composite antibacterial agent. Compared with the single substance, the prepared composite antibacterial agent not only saves the cost, but also improves the antibacterial activity. The composite antibacterial agent prepared by the invention has good antibacterial effect in phosphate buffer and pork.
Description
Technical Field
The invention belongs to the field of food safety, and particularly relates to a double-histidine/lycium barbarum polysaccharide composite antibacterial agent, and a preparation method and application thereof.
Background
The elimination and control of the growth and reproduction of harmful bacteria is still an important topic in the scientific field today. Currently, the types of antibacterial agents mainly include inorganic antibacterial agents, organic antibacterial agents and natural antibacterial agents. The composite antibacterial agent can solve the defect of antibacterial performance of a single antibacterial agent and is a new research hotspot by combining the advantages of antibacterial performance of other antibacterial agents.
The medlar is a traditional rare Chinese medicinal material in China, is deciduous shrub, is sweet in taste and mild in nature, and has the effects of nourishing kidney and marrow, nourishing liver and improving eyesight and dispelling wind. Research shows that the medlar contains various nutrient components and trace elements, mainly comprising saccharides, amino acids, trace elements, proteins, vitamins, fat, fatty acids, sterols and the like. Therefore, the medlar has multiple physiological and pharmacological functions. The lycium barbarum polysaccharide is a main effective component in lycium barbarum. A great deal of research is carried out on the extraction and separation, the physicochemical action, the clinical application and the like of the lycium barbarum polysaccharide in China. Wangjianhua (2002) reported that Lycium barbarum polysaccharides can directly scavenge hydroxyl radicals and lipid peroxidation initiated by the same. Gomphrena globosa et al (2006) report that lycium barbarum polysaccharides have a certain inhibition effect on common bacteria and mold.
The structure of the bi-histidine (His-His, HH) comprises two benzene rings, and the two ends of the bi-histidine (His-His, HH) are respectively-NH2and-COOH, so that it exists in a zwitterionic form at the isoelectric point. It is one of novel biological self-assembly materials and also an important element for preparing biological functional nano materials.
According to the invention, the lycium barbarum polysaccharide and the bi-histidine are compounded to obtain the novel antibacterial agent, so that the antibacterial effect can be improved, and the defects of high cost when the bi-histidine is used alone and limited antibacterial types and general antibacterial effect when the lycium barbarum polysaccharide is used alone can be overcome.
Disclosure of Invention
the invention aims to provide a double-histidine/lycium barbarum polysaccharide composite antibacterial agent, a preparation method and application thereof in the field of food safety.
The preparation method of the invention comprises the steps of firstly extracting the lycium barbarum polysaccharide, then mixing the lycium barbarum polysaccharide solution and the double-component amino acid solution according to a proportion, and stirring to obtain the composite antibacterial agent.
The extraction method of the lycium barbarum polysaccharide comprises the steps of pretreating lycium barbarum, namely drying the lycium barbarum at a constant temperature of 70 ℃ for 24 hours, shearing the lycium barbarum, carrying out ethanol reflux degreasing twice for 3 hours each time, and air-drying obtained residues to obtain a lycium barbarum sample; soaking in distilled water for 4 hr, extracting in water bath (80 deg.C) for 1.5 hr, filtering with absorbent cotton, extracting repeatedly for 3 times, discarding residue, mixing filtrates, concentrating by rotary evaporation (70 deg.C), centrifuging (3000r/min, 30min), and discarding residue to obtain supernatant as polysaccharide solution. And (4) carrying out vacuum freeze drying on the polysaccharide solution at (-80 ℃) for 24h to obtain the lycium barbarum polysaccharide.
The wolfberry polysaccharide solution is prepared by dissolving wolfberry polysaccharide in sterile distilled water, so that the concentration of the wolfberry polysaccharide is 35-45 mg/mL.
The preparation method of the double-histidine solution comprises the steps of dissolving the double-histidine in diethyl ether, then placing the solution in a magnetic stirrer, stirring the solution for 1min at the rotating speed of 200rpm, and finally adding sterile distilled water to enable the concentration of the double-histidine solution to be 25-35 mg/mL.
The composite antibacterial agent is prepared by compounding a lycium barbarum polysaccharide solution and a double-component ammonia acid solution, wherein the double-component ammonia acid solution is 0.5-2.0% by mass, the lycium barbarum polysaccharide solution is 1.0-3.0% by mass, and the balance is sterile distilled water, and after mixing, the mixture is magnetically stirred at the rotating speed of 200rpm for 10min to prepare the composite antibacterial agent.
The better antibacterial activity is obtained through the synergistic effect of the two components, and the defects of high cost of singly using the double-component amino acid antibacterial agent and low antibacterial activity of singly using the lycium barbarum polysaccharide are solved.
Drawings
FIG. 1 shows the antibacterial effect of the complex antibacterial agent of the present invention on Escherichia coli and Staphylococcus aureus in PBS.
FIG. 2 shows the antibacterial effect of the complex antibacterial agent of the present invention on Escherichia coli and Staphylococcus aureus in pork.
Detailed Description
The following examples illustrate specific embodiments of the present invention, but the scope of the present invention is not limited thereto.
Example 1 preparation of Lycium barbarum polysaccharides
1 Experimental materials and apparatus
2. Test method
The extraction steps of the cold wolfberry polysaccharide are as follows:
The extraction method of the lycium barbarum polysaccharide comprises the steps of pretreating lycium barbarum, namely drying the lycium barbarum at a constant temperature of 70 ℃ for 24 hours, shearing the lycium barbarum, carrying out ethanol reflux degreasing twice for 3 hours each time, and air-drying the obtained residues to obtain a lycium barbarum sample. Soaking for 4 hr, extracting in water bath (80 deg.C) for 1.5 hr, filtering with absorbent cotton, extracting repeatedly for 3 times, discarding residue, mixing filtrates, concentrating, centrifuging (3000r/min, 30min), and discarding residue to obtain supernatant as polysaccharide solution. And (3) carrying out vacuum freeze drying on the polysaccharide solution to obtain the lycium barbarum polysaccharide.
1) Drying fructus Lycii at 70 deg.C for 24 hr, cutting, defatting with ethanol under reflux twice, each for 3 hr, and air drying the residue to obtain pretreated sample.
2) Weighing a certain amount of pretreated fructus Lycii, soaking for 4 hr, extracting in water bath (80 deg.C) for 1.5 hr, filtering with absorbent cotton, and repeating the extraction for 3 times.
3) Discarding the residue, combining the filtrates, placing the filtrate into a rotary evaporator at 70 deg.C, and concentrating the filtrate to 1/4 of the original volume.
4) Centrifuging the concentrated solution (3000r/min, 30min) to remove residue, and collecting supernatant as polysaccharide solution.
5) Placing the polysaccharide solution in a freeze dryer at-80 deg.C and 20Pa, and freeze drying for 24 hr to obtain fructus Lycii polysaccharide powder.
EXAMPLE 2 preparation of a two-histidine/Lycium barbarum polysaccharide Complex antimicrobial agent
1 Experimental materials and apparatus
2. Test method
The preparation method of the double-histidine/lycium barbarum polysaccharide composite antibacterial agent comprises the following steps:
1) The bishistidine was dissolved in ether and stirred (200rpm) on a magnetic stirrer for 1min at room temperature.
2) Distilled water was added to make the concentration of the bishistidine solution 30 mg/mL.
3) Dissolving Lycium barbarum polysaccharides in sterile distilled water to make Lycium barbarum polysaccharides concentration 40 mg/mL.
4) Mixing in proportion; the two solutions are calculated according to the mass fraction, the two-component amino acid solution is 1.0 percent, the lycium barbarum polysaccharide solution is 2.0 percent, and the balance is sterile distilled water, and after the two solutions are mixed, the magnetic stirring is carried out for 10min (200rpm), so as to obtain the composite antibacterial agent. EXAMPLE 3 evaluation of antibacterial Capacity of Bi-histidine/Lycium Bararum polysaccharide composite antibacterial agent
1 Experimental materials and apparatus
2 method of experiment
1) Evaluation of in vitro anti-Escherichia coli and Staphylococcus aureus effects
Coli and s.aureus were inoculated in nutrient broth liquid medium and cultured at 37 ℃ for 24h and 48h with shaking (150rpm), respectively.
② adding E.coli and S.aureus in logarithmic growth phase into sterile phosphate buffer solution, and diluting to 10 degree of gradient4-105And (3) CFU/mL, and then adding antibacterial agents (a composite antibacterial agent, a double-component amino acid solution and a lycium barbarum polysaccharide solution) to enable the mass fraction of the antibacterial agent in the E.coli bacterial suspension to be 0.5% and the mass fraction of the antibacterial agent in the S.aureus bacterial suspension to be 0.25%.
③ culturing the bacterial suspension added with the antibacterial agent at 37 ℃ for 3d, and then measuring the residual bacteria number by using a plate counting method every 24 h. Suspensions of e.coli and s.aureus bacteria without any treatment served as blank groups.
2) Evaluation of antibacterial Effect in pork
Weighing a certain amount of fresh pork in a homogenizing bag, adding the fresh pork and PBS into the homogenizing bag according to the mass ratio of 1:9, homogenizing and crushing by using a homogenizer, filtering by using gauze, packaging into a test tube in an equal volume manner to obtain pork broth, and sterilizing for 30min by using high-pressure steam at 121 ℃.
② inoculating E.coli and S.aureus to each test tube respectively to make the concentration of E.coli and S.aureus in the test tube be 103CFU/mL。
And thirdly, adding antibacterial agents (a composite antibacterial agent, a double-component amino acid solution and a wolfberry polysaccharide solution) into the test tube to ensure that the mass fraction of the antibacterial agent in the E.coli bacterial suspension is 0.65 percent and the mass fraction of the antibacterial agent in the S.aureus bacterial suspension is 0.5 percent. Coli and s.aureus without any treatment were blank.
And fourthly, placing the test tube into a constant temperature incubator at 37 ℃ for 3 days, and calculating the number of the residual bacteria every 24 hours.
3. Test results
1) Evaluation of in vitro anti-Escherichia coli and Staphylococcus aureus effects
As can be seen from figure 1, the lycium barbarum polysaccharide, the bishistidine and the composite antibacterial agent have an inhibiting effect on escherichia coli and staphylococcus aureus, wherein the antibacterial effect of the composite antibacterial agent is optimal, and the sterilization rates of the lycium barbarum polysaccharide, the bishistidine and the composite antibacterial agent on the escherichia coli and the staphylococcus aureus after 3 days of treatment are respectively 97.84% and 99.49%.
2) Evaluation of antibacterial Effect in pork
The pig broth contains abundant nutrients, and can promote growth and reproduction of Escherichia coli and Staphylococcus aureus. As can be seen from FIG. 2, the numbers of colonies in the blank group stabilized after a rapid rise in 1 d. And compared with a single antibacterial agent, the composite antibacterial agent also has a better antibacterial effect in pork broth. After 3d, compared with the blank group, the sterilization rate of the escherichia coli in the composite antibacterial agent group reaches 98.95%, and the sterilization rate of the staphylococcus aureus in the composite antibacterial agent group reaches 99.96%.
Claims (7)
1. The double-histidine/lycium barbarum polysaccharide composite antibacterial agent is characterized by being prepared by the following steps: firstly, extracting the lycium barbarum polysaccharide, then mixing the lycium barbarum polysaccharide solution and the double-component ammonia acid solution according to a proportion, and stirring to obtain the composite antibacterial agent for treating microbial pollution.
2. The bi-histidine/lycium barbarum polysaccharide composite antibacterial agent of claim 1, wherein: the extraction method of the lycium barbarum polysaccharide comprises the steps of pretreating lycium barbarum, namely drying the lycium barbarum at constant temperature, shearing the lycium barbarum, carrying out ethanol reflux degreasing twice, and air-drying obtained residues to obtain a lycium barbarum sample; soaking the polysaccharide in distilled water, extracting in water bath for h, filtering with absorbent cotton, discarding residue, mixing filtrates, rotary evaporating for concentration, centrifuging, discarding residue, and collecting supernatant as polysaccharide solution; and (3) carrying out vacuum freeze drying on the polysaccharide solution to obtain the lycium barbarum polysaccharide.
3. The bi-histidine/lycium barbarum polysaccharide composite antibacterial agent of claim 2, wherein: the constant-temperature drying refers to drying for 24 hours at a constant temperature of 70 ℃; the time of ethanol reflux degreasing is 3h each time; soaking in distilled water for 4 h; extracting in 80 deg.C water bath for 1.5h, and repeating the extraction for 3 times; the rotary evaporation temperature is 70 ℃; centrifuging at 3000r/min for 30 min; the freeze drying conditions were-80 deg.C, 20Pa, and 24 hr freeze drying time.
4. The bi-histidine/lycium barbarum polysaccharide composite antibacterial agent of claim 1, wherein: the wolfberry polysaccharide solution is prepared by dissolving wolfberry polysaccharide in sterile distilled water, so that the concentration of the wolfberry polysaccharide is 35-45 mg/mL.
5. The bi-histidine/lycium barbarum polysaccharide composite antibacterial agent of claim 1, wherein: the preparation method of the double-histidine solution comprises the steps of dissolving the double-histidine in diethyl ether, then placing the solution in a magnetic stirrer, stirring the solution for 1min at the rotating speed of 200rpm, and finally adding sterile distilled water to enable the concentration of the double-histidine solution to be 25-35 mg/mL.
6. The bi-histidine/lycium barbarum polysaccharide composite antibacterial agent of claim 1, wherein: according to the mass fraction, the double-component amino acid solution is 0.5-2.0%, the lycium barbarum polysaccharide solution is 1.0-3.0%, and the balance is sterile distilled water, and after mixing, the mixture is magnetically stirred for 10min at the rotating speed of 200rpm to prepare the composite antibacterial agent.
7. The use of the bi-histidine/lycium barbarum polysaccharide complex antibacterial agent prepared according to any one of claims 1-6, wherein the bi-histidine/lycium barbarum polysaccharide complex antibacterial agent is used for storage and preservation of pork.
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Citations (4)
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| CN101343313A (en) * | 2008-09-03 | 2009-01-14 | 南京农业大学 | Glucose dipeptide compounds, preparation method and application thereof |
| CN107383236A (en) * | 2017-07-10 | 2017-11-24 | 中国科学院理化技术研究所 | A kind of new type water-solubility natural polysaccharide anti-biotic material and preparation method thereof |
| CN107624767A (en) * | 2017-09-26 | 2018-01-26 | 山东省林业科学研究院 | A kind of application of dipeptide compounds |
| CN108586462A (en) * | 2018-01-11 | 2018-09-28 | 中国农业科学院植物保护研究所 | A kind of Cyclic dipeptides and its application in preparing fungicide |
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- 2019-08-30 CN CN201910812026.9A patent/CN110558320B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101343313A (en) * | 2008-09-03 | 2009-01-14 | 南京农业大学 | Glucose dipeptide compounds, preparation method and application thereof |
| CN107383236A (en) * | 2017-07-10 | 2017-11-24 | 中国科学院理化技术研究所 | A kind of new type water-solubility natural polysaccharide anti-biotic material and preparation method thereof |
| CN107624767A (en) * | 2017-09-26 | 2018-01-26 | 山东省林业科学研究院 | A kind of application of dipeptide compounds |
| CN108586462A (en) * | 2018-01-11 | 2018-09-28 | 中国农业科学院植物保护研究所 | A kind of Cyclic dipeptides and its application in preparing fungicide |
Non-Patent Citations (1)
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