阅读说明：本技术 一种布氏乳杆菌S-层蛋白修饰的香芹酚/β-环糊精脂质体及其制备方法和抗菌用途 (Lactobacillus buchneri S-layer protein modified carvacrol/β -cyclodextrin liposome and preparation method and antibacterial application thereof ) 是由 胡源 饶胜其 杨振泉 焦新安 孙美玲 李华祥 高亚军 高璐 伍能建 于 2020-05-09 设计创作，主要内容包括：本发明公开了一种布氏乳杆菌S-层蛋白修饰的香芹酚/β-环糊精脂质体及其制备方法和抗菌用途,所述香芹酚/β-环糊精脂质体主要由以下重量份比例的原料,并且利用食品上可接受的溶剂制成：布氏乳杆菌S-层蛋白1～5份,香芹酚2～5份,β-环糊精10～30份,尤特奇RL100,大豆卵磷脂20～50份,胆固醇4～10份。本发明通过利用β环糊精将香芹酚包埋后,再将香芹酚/β-环糊精的包合物包封于脂质体中,进一步提高香芹酚的可溶性,从而提高脂质体中香芹酚的包封率,提高其利用率,再由布氏乳杆菌S-层蛋白修饰至脂质体表面,可以提高其稳定性和储存性能,布氏乳杆菌S-层蛋白与香芹酚协同抑菌,将显著提高脂质体的抑菌效果,并可对预防致病菌的产生起到可持续释放的效果。(The invention discloses a carvacrol/β -cyclodextrin liposome modified by lactobacillus buchneri S-layer protein, a preparation method and an antibacterial application thereof, wherein the carvacrol/β -cyclodextrin liposome is mainly prepared from the following raw materials in parts by weight and by using a solvent acceptable to food, namely 1-5 parts of lactobacillus buchneri S-layer protein, 2-5 parts of carvacrol, 10-30 parts of β -cyclodextrin, Ewing R L, 20-50 parts of soybean lecithin and 4-10 parts of cholesterol.)

1. A lactobacillus buchneri S-layer protein modified carvacrol/β -cyclodextrin liposome is characterized by being mainly prepared from the following raw materials in parts by weight by utilizing a solvent acceptable for food:

1-5 parts of lactobacillus buchneri S-layer protein, 2-5 parts of carvacrol, 10-30 parts of β -cyclodextrin, 20-50 parts of soybean lecithin, 20-50 parts of Utex R L10020-50 parts, and 4-10 parts of cholesterol.

2. The lactobacillus buchneri S-layer protein modified carvacrol/β -cyclodextrin liposome of claim 1, wherein the food acceptable solvent is selected from absolute ethanol and ultra pure water.

3. The lactobacillus buchneri S-layer protein modified carvacrol/β -cyclodextrin liposome of claim 1, wherein carvacrol and β -cyclodextrin are prepared into a carvacrol/β -cyclodextrin inclusion compound, then soybean lecithin and cholesterol are utilized to load the carvacrol/β -cyclodextrin inclusion compound into the liposome, and finally lactobacillus buchneri S-layer protein is modified on the surface of the liposome to obtain the lactobacillus buchneri S-layer protein modified carvacrol/β -cyclodextrin liposome.

4. The method for preparing lactobacillus buchneri S-layer protein modified carvacrol/β -cyclodextrin liposome of claim 1, comprising the following steps:

a. preparing β -cyclodextrin inclusion compound of carvacrol, namely carvacrol/β -cyclodextrin inclusion compound, by taking β -cyclodextrin, carvacrol and ultrapure water as solvents;

b. c, re-dissolving the carvacrol/β -cyclodextrin inclusion compound obtained in the step a in ultrapure water to prepare a carvacrol/β -cyclodextrin inclusion compound aqueous solution, then preparing a soybean lecithin, Eudragit R L100 and cholesterol absolute ethyl alcohol mixed solution, injecting the mixed solution into the carvacrol/β -cyclodextrin inclusion compound aqueous solution, and stirring;

c. and (c) evaporating the solution prepared in the step (b) under reduced pressure to remove ethanol, and filtering by using a centrifugal membrane and a microporous filter membrane to obtain the carvacrol/β -cyclodextrin liposome solution.

d. Dissolving the lactobacillus buchneri S-layer protein freeze-dried powder in carvacrol/β -cyclodextrin liposome solution, and stirring to obtain the S-layer protein modified carvacrol/β -cyclodextrin liposome.

5. The preparation method of the lactobacillus buchneri S-layer protein modified carvacrol/β -cyclodextrin liposome of claim 4, wherein in the step a, β -cyclodextrin is firstly added into ultrapure water to swell, so as to prepare β -cyclodextrin solution with the mass volume ratio of 0.01% -99%, then carvacrol is added into the β -cyclodextrin solution, and after uniform mixing and freeze drying, the carvacrol/β -cyclodextrin inclusion compound is obtained.

6. The method for preparing L.buchneri S-layer protein modified carvacrol/β -cyclodextrin liposome of claim 4, wherein in step b, the soybean lecithin, Ewing R L100 and cholesterol are added into 50-100 parts of absolute ethyl alcohol to prepare a mixed solution, and then the mixed solution is injected into the carvacrol/β -cyclodextrin inclusion compound aqueous solution at a speed of 0.1-1 m L/min, and the volume ratio of the absolute ethyl alcohol mixed solution to the carvacrol/β -cyclodextrin inclusion compound aqueous solution is 1: 0.1-2, and stirring is carried out.

7. The method for preparing the lactobacillus buchneri S-layer protein modified carvacrol/β -cyclodextrin liposome according to claim 4, wherein the reduced pressure evaporation temperature in step c is 35-70 ℃, and the reduced pressure evaporation time is 20-40 min.

8. The preparation method of the lactobacillus buchneri S-layer protein modified carvacrol/β -cyclodextrin liposome of claim 4, wherein in the step d, the lactobacillus buchneri S-layer protein is soluble protein extracted from lactobacillus buchneri, and the protein is freeze-dried into powder, and the stirring is performed for 1-4 h at 20-25 ℃ by magnetic stirring.

9. Use of a lactobacillus buchneri S-layer protein modified carvacrol/β -cyclodextrin liposome according to any of claims 1 to 3 as an antibacterial agent.

Technical Field

The invention belongs to the technical field of carvacrol liposome, and particularly relates to an S-layer protein modified carvacrol/β -cyclodextrin liposome, a preparation method and an antibacterial application thereof.

Background

Carvacrol (car) is a colorless to pale yellow liquid with a thymol flavor, and is commonly found in essential oils of origanum vulgaris, thyme, camomile, and the like. At present, carvacrol is reported to have the effects of resisting oxidation, resisting bacteria, expelling parasites, relieving ischemia reperfusion injury of brain and spinal cord of rats and the like. Because the perfume has no toxic or side effect, no residue and no pollution to the environment, the perfume is often used as perfume to be added into daily necessities such as cosmetics and the like; the seasoning is often used as a natural seasoning spice, so that fishy smell in seafood, meat, fish and other foods can be removed, and the flavor of dishes is increased; is often used as a bacteriostatic agent and a preservative in food preservation. However, the carvacrol is insoluble in water, is easy to volatilize and oxidize in the air, has poor stability and the like, and greatly influences the bioavailability and the application range of the carvacrol.

Disclosure of Invention

The invention aims to solve the technical problems and provides a carvacrol/β -cyclodextrin liposome modified by lactobacillus buchneri S-layer protein, a preparation method and an antibacterial application thereof.

The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

a lactobacillus buchneri S-layer protein modified carvacrol/β -cyclodextrin liposome is mainly prepared from the following raw materials in parts by weight by utilizing a food-acceptable solvent:

1-5 parts of lactobacillus buchneri S-layer protein, 2-5 parts of carvacrol, 10-30 parts of β -cyclodextrin, 20-50 parts of soybean lecithin, 20-50 parts of Utex R L10020-50 parts, and 4-10 parts of cholesterol.

Preferably, the food acceptable solvent is selected from the group consisting of absolute ethanol and ultra pure water.

Preferably, carvacrol and β -cyclodextrin are firstly prepared into a carvacrol/β -cyclodextrin inclusion compound, then soybean lecithin, Eudragit R L100 and cholesterol are utilized to load the carvacrol/β -cyclodextrin inclusion compound into liposome, and finally S-layer protein is modified into the carvacrol/β -cyclodextrin liposome to obtain the S-layer protein modified carvacrol/β -cyclodextrin liposome.

The preparation method of the lactobacillus buchneri S-layer protein modified carvacrol/β -cyclodextrin liposome comprises the following steps:

a. preparing β -cyclodextrin inclusion compound of carvacrol, namely carvacrol/β -cyclodextrin inclusion compound, by taking β -cyclodextrin, carvacrol and ultrapure water as solvents;

b. c, re-dissolving the carvacrol/β -cyclodextrin inclusion compound obtained in the step a in ultrapure water to prepare a carvacrol/β -cyclodextrin inclusion compound aqueous solution, then preparing a soybean lecithin, Eudragit R L100 and cholesterol absolute ethyl alcohol mixed solution, injecting the mixed solution into the carvacrol/β -cyclodextrin inclusion compound aqueous solution, and stirring;

c. and (c) evaporating the solution prepared in the step (b) under reduced pressure to remove ethanol, and filtering by using a centrifugal membrane and a microporous filter membrane to obtain the carvacrol/β -cyclodextrin liposome solution.

d. Dissolving the lactobacillus buchneri S-layer protein freeze-dried powder in carvacrol/β -cyclodextrin liposome solution, and stirring to obtain the S-layer protein modified carvacrol/β -cyclodextrin liposome.

Preferably, in the step a, β -cyclodextrin is added into ultrapure water to swell, so that β -cyclodextrin solution with the mass volume ratio of 0.01-99% is prepared, carvacrol is added into the β -cyclodextrin solution, and the carvacrol/β -cyclodextrin inclusion compound is obtained after uniform mixing and freeze drying.

Preferably, in the step b, the soybean lecithin, the Eudragit R L100 and the cholesterol are added into 50-100 parts of absolute ethyl alcohol to prepare a mixed solution, then the mixed solution is injected into the carvacrol/β -cyclodextrin inclusion compound aqueous solution at the speed of 0.1-1 m L/min, and the volume ratio of the absolute ethyl alcohol mixed solution to the carvacrol/β -cyclodextrin inclusion compound aqueous solution is 1: 0.1-2, and the mixture is stirred.

Preferably, in the step c, the reduced pressure evaporation temperature is 35-70 ℃, and the reduced pressure evaporation time is 20-40 min.

Preferably, in step d, the lactobacillus buchneri S-layer protein is a soluble protein extracted from lactobacillus buchneri, and is freeze-dried into powder, and further preferably, the molecular weight of the S-layer protein is 40-200 kDa. The stirring is magnetic stirring for 1-4 h at 20-25 ℃.

Further, the extraction method of the lactobacillus buchneri S-layer protein comprises the following steps:

1) culturing lactobacillus buchneri: the MRS solid culture medium and the MRS liquid culture medium are sequentially utilized to culture the lactobacillus buchneri, until the last logarithmic phase of the growth of the lactobacillus buchneri, three generations of strains are continuously inoculated to improve the activity of the strains, and the third generation of lactobacillus culture solution is reserved.

2) And (3) S-layer protein extraction, namely treating the cultured lactobacillus buchneri with L iCl solution and CuHCl solution in sequence, collecting supernatant, dialyzing, centrifuging, collecting solution, and freeze-drying to obtain the lactobacillus buchneri S-layer protein.

The resulting S-layer protein was analyzed by SDS-PAGE, and the protein concentration of the solution was determined by BCA protein concentration determination kit three times each to obtain precision.

Preferably, in step 1), the lactobacillus buchneri strain is cultured in the MRS liquid medium for 18 to 23 hours.

Preferably, in the step 2), the concentration of the L iCl solution is 5-10 mol/m L and the concentration of the solution is 1-6mol/m L.

Wherein, the swelling time in the step a is preferably 1min to 48 h.

Wherein, in the step a, carvacrol can be uniformly mixed (i.e. completely dissolved) in the solvent by adopting a conventional method, for example, the carvacrol is completely dissolved in the solvent by adopting methods such as standing, heating, stirring, ultrasound or grinding.

Wherein, the decompression evaporation temperature in the step c is too high, which can cause the oxidation of the phospholipid, and the temperature is too low, which can not reach the phase transition temperature to form the liposome.

The blank liposome is prepared by adding soybean lecithin, cholesterol and Eudragit R L100 into 50-100 parts of absolute ethyl alcohol to prepare a mixed solution, then injecting the mixed solution into ultrapure water at the speed of 0.1-1 m L/min, stirring and evaporating under reduced pressure until the ethyl alcohol is completely volatilized.

The invention finally provides the application of the S-layer protein modified carvacrol/β -cyclodextrin liposome as an antibacterial agent.

The invention firstly prepares carvacrol and β -cyclodextrin into carvacrol/β -cyclodextrin inclusion compound, then carries out loading on β -cyclodextrin/carvacrol inclusion compound through nano liposome and self-assembles S-layer protein on the surface of liposome, and finally prepares S-layer protein modified carvacrol/β -cyclodextrin liposome with nano particle size.

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. aiming at the problems that carvacrol is not easy to dissolve in water, is easy to volatilize and has low utilization rate, cyclodextrin liposome double-layer embedding is adopted, so that the dissolving problem of carvacrol is greatly increased, the utilization rate of carvacrol is improved, waste is reduced, and the cost is reduced to play an effective role.

2. The novel carvacrol/β -cyclodextrin liposome antibacterial agent is prepared by a cyclodextrin liposome delivery system, so that the biological activity of the carvacrol is improved, the slow release characteristic is realized, and the storage life is prolonged.

3. The invention makes full use of the characteristics of the cyclodextrin liposome, not only improves the edible value of carvacrol, but also provides a new way for improving the added value of the antibacterial property of carvacrol.

4. The invention utilizes the S-layer protein of the lactobacillus buchneri to modify the carvacrol/β -cyclodextrin liposome, can improve the stability and the adhesion property to bacteria, and leads the prepared liposome to have stronger stability.

Drawings

FIG. 1: and (3) extracting the lactobacillus buchneri S-layer protein.

FIG. 2 is the entrapment efficiency of S-layer protein modified carvacrol/β cyclodextrin liposome.

Figure 3 stability of carvacrol liposomes and S-layer protein modified carvacrol/β cyclodextrin liposomes.

FIG. 4: carvacrol release curves in different liposomes.

Detailed Description

The technical solution of the present invention is further described in detail by the following specific examples.

The culture method of lactobacillus buchneri in the following examples is as follows:

diluting Lactobacillus buchneri suspension to a certain gradient, coating the gradient on MRS solid culture medium, culturing in 37 deg.C constant temperature incubator for 48h, selecting single colony, inoculating in MRS liquid culture medium, standing in 37 deg.C constant temperature incubator for a certain time to late logarithmic phase of Lactobacillus buchneri growth, continuously inoculating third generation lactobacillus to improve strain activity, and storing the third generation lactobacillus culture solution in 4 deg.C refrigerator for use