阅读说明：本技术 天然光敏剂竹红菌素纳米杀菌乳液的制备方法 (Preparation method of hypocrellin nano bactericidal emulsion as natural photosensitizer ) 是由 张春玲 方强胜 李睿 袁媛 庄红 于 2021-07-15 设计创作，主要内容包括：本发明的天然光敏剂竹红菌素纳米杀菌乳液的制备方法属于食品杀菌技术领域,先将光敏剂溶于中链甘油三酯(MCT)得到油相,将乳化剂溶于去离子水中得到水相,再将两者混合搅拌均匀制备初乳液,之后采用高速剪切-高压均质技术制备得到天然光敏剂竹红菌素纳米杀菌乳液。本发明所采用的材料均为食品级原料,无毒无害,安全可靠,对人体无副作用,成本低,制备的纳米杀菌乳液易于保存,且对金黄色葡萄糖球菌的杀菌率可以达到90％,能够广泛适用于食品杀菌消毒领域。(The invention discloses a preparation method of a natural photosensitizer hypocrellin nano bactericidal emulsion, belonging to the technical field of food sterilization. The materials adopted by the invention are food-grade raw materials, are nontoxic and harmless, are safe and reliable, have no side effect on human bodies, have low cost, and the prepared nano sterilizing emulsion is easy to store, has the sterilizing rate of staphylococcus aureus reaching 90 percent, and can be widely applied to the field of food sterilization and disinfection.)

1. A method for preparing hypocrellin nano sterilizing emulsion as natural photosensitizer comprises dissolving photosensitizer in medium chain triglyceride to obtain oil phase, dissolving emulsifier in deionized water to obtain water phase, mixing the two to obtain primary emulsion, and preparing into nano sterilizing emulsion by high speed shearing-high pressure homogenizing technology; the photosensitizer is hypocrellin and accounts for 1-3% of the mass of medium-chain triglyceride, and the emulsifier is soybean lecithin and accounts for 2-5% of the mass of deionized water; the oil phase accounts for 10% of the total emulsion mass.

2. The method for preparing hypocrellin nano bactericidal emulsion as natural photosensitizer as claimed in claim 1, wherein the hypocrellin is extracted from tabasheer powder in a long-term leaching way, and the extraction steps are as follows: taking tabasheer as a raw material, beating the tabasheer into powder, placing the powder in a Soxhlet extractor, adding acetone, heating and refluxing at 75-85 ℃, and circulating for 10 hours; placing the product in a rotary steaming bottle, and carrying out rotary steaming at 40 ℃ for 30min to recover acetone; placing the residual product in a beaker, adding petroleum ether, heating while stirring to boil, keeping for 30min, cooling, filtering with Buchner funnel, and repeating for 3 times to obtain initial product; adding toluene and petroleum ether in a volume ratio of 1: 1-1.5 into a beaker of the primary product, recrystallizing at 50 ℃ for 30min, and drying at 70 ℃ for 24h to obtain hypocrellin.

3. The method for preparing hypocrellin nano sterilizing emulsion as natural photosensitizer in claim 1, wherein the mixing and stirring of the two are carried out at 300r/min rotation speed and 55-65 ℃ for 10-15 min.

4. The method for preparing hypocrellin nano sterilizing emulsion as natural photosensitizer in claim 1, wherein the high speed shearing is carried out in a mechanical dispersing machine at 1300r/min for 25 min.

5. The method for preparing a nano bactericidal emulsion of hypocrellin as a natural photosensitizer as defined in claim 1, wherein the high pressure homogenization is performed under a pressure of 15000psi for 10 min.

Technical Field

The invention belongs to the technical field of food sterilization, and particularly relates to a production technology of a hypocrellin nano sterilization emulsion serving as a natural photosensitizer.

Background

With the continuous development of human food production and processing technology, various foods are continuously appeared, but with the continuous discovery of various food-borne bacteria. The food-borne bacteria mainly comprise diarrheagenic escherichia coli, staphylococcus aureus, salmonella, listeria monocytogenes, vibrio parahaemolyticus and other bacteria, and the food-borne bacteria are widely spread in food, particularly on fresh foods, so that various diseases are generated. At present, the existing technology mainly adopts the modes of high temperature, high pressure, ultraviolet irradiation, antibiotics, metal ions and the like for inhibiting food-borne bacteria. These techniques are limited by the severity of the conditions used, and damage the nutrients, ingredients, etc. of the sterilized food. Therefore, people need to find other safer and more reliable sterilization methods.

In recent years, the use of photodynamic inactivation as a non-traditional sterilization means has attracted much attention. The photodynamic inactivation is to irradiate the photosensitizer by using a light source with specific wavelength, so that the photosensitizer generates energy transition after obtaining energy and is converted into an excited state from a ground state, oxygen around the photosensitizer can generate a series of photochemical reactions, and therefore, active oxygen substances such as singlet oxygen, free radicals and the like are generated, and the active oxygen substances can destroy the cell structure of bacteria to cause the death of the bacteria. However, the photosensitizer has poor water solubility and poor stability at high temperature and light, which greatly limits the application of the photosensitizer in photodynamic inactivation. Therefore, it is necessary to use a suitable delivery means for achieving the effect of effectively preserving the photosensitizer.

The nano emulsion is a dispersion solution prepared by mixing an internal phase (oil phase) and an external phase (water phase) and adopting a proper emulsifier and a high-pressure homogenization method, and the particle size is generally 50-500 nm. The nano emulsion has the characteristics of small size, high bioavailability, good light transmittance and the like, and meanwhile, the stability of the nano emulsion is influenced by the internal phase-external phase ratio, the oil phase type, the emulsifier type and the contents.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a preparation method of a natural photosensitizer hypocrellin nano bactericidal emulsion which has good stability and can effectively inhibit staphylococcus aureus.

The technical scheme of the invention is as follows:

a method for preparing hypocrellin nano sterilizing emulsion as natural photosensitizer comprises dissolving photosensitizer in Medium Chain Triglyceride (MCT) to obtain oil phase, dissolving emulsifier in deionized water to obtain water phase, mixing the two to obtain primary emulsion, and preparing into nano sterilizing emulsion by high speed shearing-high pressure homogenizing technology; the photosensitizer is hypocrellin and accounts for 1-3% of the mass of medium-chain triglyceride, and the emulsifier is soybean lecithin and accounts for 2-5% of the mass of deionized water; the oil phase accounts for 10% of the total emulsion mass.

The emulsifier has the function of effectively coating the inner phase according to the existence of the hydrophobic end and the hydrophilic end of the molecular structure of the emulsifier, and the inner phase is isolated from the outer phase, so that the emulsion forms liquid drops. The type of emulsifier is critical to the formation of emulsion droplets and is therefore of concern for the stability of the emulsion. The invention adopts the soybean lecithin as the emulsifier, can achieve the purposes of stable system and small formed particle size, and meanwhile, the soybean lecithin is a nontoxic substance beneficial to human bodies, and the prepared emulsion has small side effect.

Furthermore, the natural photosensitizer hypocrellin used by the invention is extracted from tabasheer powder in a long-term leaching way, and the extraction steps are as follows: taking tabasheer as a raw material, beating the tabasheer into powder, placing the powder in a Soxhlet extractor, adding acetone, heating and refluxing at 75-85 ℃, and circulating for 10 hours; placing the product in a rotary steaming bottle, and carrying out rotary steaming at 40 ℃ for 30min to recover acetone; placing the residual product in a beaker, adding petroleum ether, heating while stirring to boil, keeping for 30min, cooling, filtering with Buchner funnel, and repeating for 3 times to obtain initial product; adding toluene and petroleum ether in a volume ratio of 1: 1-1.5 into a beaker of the primary product, recrystallizing at 50 ℃ for 30min, and drying at 70 ℃ for 24h to obtain hypocrellin. Practice proves that the hypocrellin cannot be completely leached from the tabasheer powder when the heating temperature in the Soxhlet extractor is too low. When the heating temperature is too high, the hypocrellin structure will be destroyed. The optimal heating environment can be provided by heating the tabasheer powder soaked in acetone in the Soxhlet extractor at 75-85 ℃, so that the hypocrellin is extracted from the tabasheer to the maximum extent.

Furthermore, the two-phase mixing and stirring mode of the invention is carried out for 10min to 15min at the rotating speed of 300r/min and the temperature of 55 ℃ to 65 ℃. The choice of the rotation speed of 300r/min is determined by trial and error, at which a complete mixing of the two phases is ensured. The temperature of 55-65 ℃ is selected to be carried out for 10-15 min, so that the two phases can be ensured to have suitable thermodynamic environment and sufficient time combination, and the emulsification effect is improved.

Further, the high-speed shearing technology adopted by the invention needs to be carried out in a mechanical dispersion machine at the rotating speed of 1300r/min for 25 min. Practice proves that when the shearing rotating speed is too low, the liquid drop cannot be reduced to the minimum, the subsequent high-pressure homogenizing technology is not facilitated, and when the shearing rotating speed is too high, the structure of the liquid drop can be damaged, and the dispersion and the formation of the liquid drop are not facilitated. The selection of a shear rate of 1300r/min was determined by trial and error. And 25min is selected to ensure that the emulsion is fully dispersed.

Further, the high pressure homogenization technique employed in the present invention requires 10min at 15000 psi. Can prepare emulsion with high stability and good coating performance.

Compared with the prior sterilization technology, the natural photosensitizer hypocrellin nano sterilization emulsion prepared by the invention has the following advantages:

1. the invention adopts natural photosensitizer hypocrellin as the photosensitizer for photodynamic inactivation, can greatly reduce the production cost and can expand the application range of the traditional Chinese medicine.

2. The invention adopts the nano emulsion as the main carrier for coating the photosensitizer, can effectively preserve the photosensitizer, improves the solubility and the workability of the photosensitizer and reduces the influence of the environment on the photosensitizer.

3. The nano emulsion prepared by the invention can be stably stored at 30-50 ℃, and no obvious photosensitizer degradation phenomenon occurs.

4. The nano emulsion prepared by the invention can stably exist in an acid-base environment without generating a large-scale condensation phenomenon.

5. The nano emulsion prepared by the invention has a sterilization rate of 90% on staphylococcus aureus.

6. The nano emulsion prepared by the invention adopts food-grade raw materials, is non-toxic, harmless, safe and reliable, has no side effect on human body, and can be widely applied to the field of food sterilization and disinfection.

Description of the drawings:

FIG. 1 is a flow chart of the preparation of an embodiment of the present invention.

FIG. 2 is a structural diagram of hypocrellin, a natural photosensitizer, according to the present invention.

FIG. 3 is a structural diagram of an emulsifier according to the present invention.

Fig. 4 is a change in appearance of the natural photosensitizer hypocrellin nano bactericidal emulsion prepared in example 1 of the present invention on the first and seventh days after 30 mL, 40mL, 50mL, 60mL, 70mL, 80mL treatment.

Fig. 5 is a change in appearance of the natural photosensitizer hypocrellin nano bactericidal emulsion prepared in example 1 of the present invention on the first and seventh days after being treated with pH3, pH5, pH7, pH 9.

FIG. 6 shows the colony count of Staphylococcus aureus in culture medium of the natural photosensitizer hypocrellin nano-bactericidal emulsion prepared in example 1 of the present invention under the control of 10min, 20min, 30min, 40min, 50min and 60 min.

Detailed Description

While specific embodiments of the present invention will be described in detail below, it should be apparent that the described embodiments are merely illustrative of some, but not all, embodiments of the present invention. Meanwhile, the specific embodiments described herein are only to explain the present invention and do not limit the present invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

Example 1:

preparing a photosensitizer: the preparation method comprises the following steps of (1) grinding tabasheer serving as a raw material into powder, placing 160g of the powder into a Soxhlet extractor, adding 500mL of acetone, heating by 75mL of acetone, carrying out thermal reflux, and circulating for 10 hours; placing the product in a rotary evaporation bottle, and carrying out rotary evaporation for 30min at 40mL to recover acetone; placing in a beaker, adding 200mL petroleum ether, heating with 80mL petroleum ether, stirring while heating to boil, carrying out 30min, cooling, sucking with a Buchner funnel, filtering, and repeating for 3 times to obtain an initial product; 100mL of toluene and petroleum ether were added to a beaker of the initial product, recrystallized for 30min at 50mL, and dried for 24h at 70mL to obtain hypocrellin.

Preparing a primary emulsion: taking 0.1g hypocrellin powder, adding medium chain triglyceride to 10g, taking 1.8g lecithin, and adding deionized water to 90 g; mixing the oil phase and the water phase, and performing at the rotation speed of 300r/min and the temperature of 55mL for 10 min.

Preparing a nano emulsion: the primary emulsion was sheared at a rotation speed of 1300r/min for 25min in a mechanical disperser and then at a pressure of 15000psi for 10 min.

Example 2:

preparing a photosensitizer: taking tabasheer as a raw material, grinding the tabasheer into powder, putting 160g of the tabasheer into a Soxhlet extractor, adding 500mL of acetone, heating by 85mL of acetone, carrying out thermal reflux, and circulating for 10 hours; placing the product in a rotary evaporation bottle, and carrying out rotary evaporation for 30min at 40mL to recover acetone; placing in a beaker, adding 200mL petroleum ether, heating with 80mL petroleum ether, stirring while heating to boil, carrying out 30min, cooling, sucking with a Buchner funnel, filtering, and repeating for 3 times to obtain an initial product; 100mL of toluene and petroleum ether were added to a beaker of the initial product, recrystallized for 30min at 50mL, and dried for 24h at 70mL to obtain hypocrellin.

Preparing a primary emulsion: taking 0.1g hypocrellin powder, adding medium chain triglyceride to 10g, taking 1.8g lecithin, and adding deionized water to 90 g; mixing the oil phase and the water phase, and performing at the rotation speed of 300r/min and the temperature of 55mL for 10 min.

Preparing a nano emulsion: the primary emulsion was sheared at a rotation speed of 1300r/min for 25min in a mechanical disperser and then at a pressure of 15000psi for 10 min.

Example 3:

preparing a photosensitizer: taking tabasheer as a raw material, grinding the tabasheer into powder, putting 160g of the tabasheer into a Soxhlet extractor, adding 500mL of acetone, heating by 85mL of acetone, carrying out thermal reflux, and circulating for 10 hours; placing the product in a rotary evaporation bottle, and carrying out rotary evaporation for 30min at 40mL to recover acetone; placing in a beaker, adding 200mL petroleum ether, heating with 80mL petroleum ether, stirring while heating to boil, carrying out 30min, cooling, sucking with a Buchner funnel, filtering, and repeating for 3 times to obtain an initial product; 100mL of toluene and petroleum ether were added to a beaker of the initial product, recrystallized for 30min at 50mL, and dried for 24h at 70mL to obtain hypocrellin.

Preparing a primary emulsion: taking 0.2g hypocrellin powder, adding medium chain triglyceride to 10g, taking 1.8g lecithin, and adding deionized water to 90 g; mixing the oil phase and the water phase, and performing at a rotation speed of 300r/min and a temperature of 55 ℃ for 10 min.

Preparing a nano emulsion: the primary emulsion was sheared at a rotation speed of 1300r/min for 25min in a mechanical disperser and then at a pressure of 15000psi for 10 min.

Example 4:

preparing a photosensitizer: taking tabasheer as a raw material, grinding the tabasheer into powder, putting 160g of the tabasheer into a Soxhlet extractor, adding 500mL of acetone, heating by 85mL of acetone, carrying out thermal reflux, and circulating for 10 hours; placing the product in a rotary evaporation bottle, and carrying out rotary evaporation for 30min at 40mL to recover acetone; placing in a beaker, adding 200mL petroleum ether, heating with 80mL petroleum ether, stirring while heating to boil, carrying out 30min, cooling, sucking with a Buchner funnel, filtering, and repeating for 3 times to obtain an initial product; 100mL of toluene and petroleum ether were added to a beaker of the initial product, recrystallized for 30min at 50mL, and dried for 24h at 70mL to obtain hypocrellin.

Preparing a primary emulsion: taking 0.3g hypocrellin powder, adding medium chain triglyceride to 10g, taking 1.8g lecithin, and adding deionized water to 90 g; mixing the oil phase and the water phase, and performing at a rotation speed of 300r/min and a temperature of 55 ℃ for 10 min.

Preparing a nano emulsion: the primary emulsion was sheared at a rotation speed of 1300r/min for 25min in a mechanical disperser and then at a pressure of 15000psi for 10 min.

Example 5:

preparing a photosensitizer: taking tabasheer as a raw material, grinding the tabasheer into powder, putting 160g of the tabasheer into a Soxhlet extractor, adding 500mL of acetone, heating by 85mL of acetone, carrying out thermal reflux, and circulating for 10 hours; placing the product in a rotary evaporation bottle, and carrying out rotary evaporation for 30min at 40mL to recover acetone; placing in a beaker, adding 200mL petroleum ether, heating with 80mL petroleum ether, stirring while heating to boil, carrying out 30min, cooling, sucking with a Buchner funnel, filtering, and repeating for 3 times to obtain an initial product; 100mL of toluene and petroleum ether were added to a beaker of the initial product, recrystallized for 30min at 50mL, and dried for 24h at 70mL to obtain hypocrellin.

Preparing a primary emulsion: taking 0.3g hypocrellin powder, adding medium chain triglyceride to 10g, taking 2.7g lecithin, and adding deionized water to 90 g; mixing the oil phase and the water phase, and performing at a rotation speed of 300r/min and a temperature of 55 ℃ for 10 min.

Preparing a nano emulsion: the primary emulsion was sheared at a rotation speed of 1300r/min for 25min in a mechanical disperser and then at a pressure of 15000psi for 10 min.

Example 6:

preparing a photosensitizer: taking tabasheer as a raw material, grinding the tabasheer into powder, putting 160g of the tabasheer into a Soxhlet extractor, adding 500mL of acetone, heating by 85mL of acetone, carrying out thermal reflux, and circulating for 10 hours; placing the product in a rotary evaporation bottle, and carrying out rotary evaporation for 30min at 40mL to recover acetone; placing in a beaker, adding 200mL petroleum ether, heating with 80mL petroleum ether, stirring while heating to boil, carrying out 30min, cooling, sucking with a Buchner funnel, filtering, and repeating for 3 times to obtain an initial product; 100mL of toluene and petroleum ether were added to a beaker of the initial product, recrystallized for 30min at 50mL, and dried for 24h at 70mL to obtain hypocrellin.

Preparing a primary emulsion: taking 0.3g hypocrellin powder, adding medium chain triglyceride to 10g, taking 3.6g lecithin, and adding deionized water to 90 g; mixing the oil phase and the water phase, and performing at a rotation speed of 300r/min and a temperature of 55 ℃ for 10 min.

Preparing a nano emulsion: the primary emulsion was sheared at a rotation speed of 1300r/min for 25min in a mechanical disperser and then at a pressure of 15000psi for 10 min.

Example 7:

preparing a photosensitizer: taking tabasheer as a raw material, grinding the tabasheer into powder, putting 160g of the tabasheer into a Soxhlet extractor, adding 500mL of acetone, heating by 85mL of acetone, carrying out thermal reflux, and circulating for 10 hours; placing the product in a rotary evaporation bottle, and carrying out rotary evaporation for 30min at 40mL to recover acetone; placing in a beaker, adding 200mL petroleum ether, heating with 80mL petroleum ether, stirring while heating to boil, carrying out 30min, cooling, sucking with a Buchner funnel, filtering, and repeating for 3 times to obtain an initial product; 100mL of toluene and petroleum ether were added to a beaker of the initial product, recrystallized for 30min at 50mL, and dried for 24h at 70mL to obtain hypocrellin.

Preparing a primary emulsion: taking 0.3g hypocrellin powder, adding medium chain triglyceride to 10g, taking 4.5g lecithin, and adding deionized water to 90 g; mixing the oil phase and the water phase, and performing at a rotation speed of 300r/min and a temperature of 55 ℃ for 10 min.

Preparing a nano emulsion: the primary emulsion was sheared at a rotation speed of 1300r/min for 25min in a mechanical disperser and then at a pressure of 15000psi for 10 min.

Example 8:

preparing a photosensitizer: taking tabasheer as a raw material, grinding the tabasheer into powder, putting 160g of the tabasheer into a Soxhlet extractor, adding 500mL of acetone, heating by 85mL of acetone, carrying out thermal reflux, and circulating for 10 hours; placing the product in a rotary evaporation bottle, and carrying out rotary evaporation for 30min at 40mL to recover acetone; placing in a beaker, adding 200mL petroleum ether, heating with 80mL petroleum ether, stirring while heating to boil, carrying out 30min, cooling, sucking with a Buchner funnel, filtering, and repeating for 3 times to obtain an initial product; 100mL of toluene and petroleum ether were added to a beaker of the initial product, recrystallized for 30min at 50mL, and dried for 24h at 70mL to obtain hypocrellin.

Preparing a primary emulsion: taking 0.3g hypocrellin powder, adding medium chain triglyceride to 10g, taking 4.5g lecithin, and adding deionized water to 90 g; mixing the oil phase and the water phase, and performing at 300r/min at 65 deg.C for 10 min.

Preparing a nano emulsion: the primary emulsion was sheared at a rotation speed of 1300r/min for 25min in a mechanical disperser and then at a pressure of 15000psi for 10 min.

Example 9:

preparing a photosensitizer: taking tabasheer as a raw material, grinding the tabasheer into powder, putting 160g of the tabasheer into a Soxhlet extractor, adding 500mL of acetone, heating by 85mL of acetone, carrying out thermal reflux, and circulating for 10 hours; placing the product in a rotary evaporation bottle, and carrying out rotary evaporation for 30min at 40mL to recover acetone; placing in a beaker, adding 200mL petroleum ether, heating with 80mL petroleum ether, stirring while heating to boil, carrying out 30min, cooling, sucking with a Buchner funnel, filtering, and repeating for 3 times to obtain an initial product; 100mL of toluene and petroleum ether were added to a beaker of the initial product, recrystallized for 30min at 50mL, and dried for 24h at 70mL to obtain hypocrellin.

Preparing a primary emulsion: taking 0.3g hypocrellin powder, adding medium chain triglyceride to 10g, taking 4.5g lecithin, and adding deionized water to 90 g; mixing the oil phase and the water phase, and performing at 300r/min at 65 deg.C for 15 min.

Preparing a nano emulsion: the primary emulsion was sheared at a rotation speed of 1300r/min for 25min in a mechanical disperser and then at a pressure of 15000psi for 10 min.

Example 10

The stability of the hypocrellin nano bactericidal emulsion of the natural photosensitizer prepared by the invention is investigated:

the prepared nano emulsion is processed at 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃, and then the emulsion is photographed on the first day and the seventh day respectively, and whether the emulsion is layered or not and whether the photosensitizer is degraded or not are examined. As shown in FIG. 4, it can be seen that the photosensitizer was substantially well preserved without degradation even when treated at a temperature of 30 ℃ to 50 ℃.

And (3) adjusting the prepared nano emulsion to pH3, pH5, pH7 and pH9 by adopting hydrochloric acid and sodium hydroxide solution, photographing each emulsion on the first day and the seventh day respectively, and inspecting whether the emulsions are layered or not and the phenomenon of photosensitizer degradation. As shown in FIG. 5, it can be seen that the emulsion was not delaminated and the photosensitizer was degraded at each pH treatment, and thus the emulsion was stably preserved under acid-base conditions.

Example 11

The bactericidal performance of the hypocrellin nano bactericidal emulsion of the natural photosensitizer is investigated:

the single bacterial colony of staphylococcus aureus is inoculated in 20mL LB liquid culture medium and cultured for 16h at 37 ℃ and 200 rpm. Diluting the bacterial liquid with ultrapure water to make the bacterial liquid content of the target bacterial liquid reach 10^-6。

100 mu L of the bacterial inoculant and 900 mu L of the prepared hypocrellin nano bactericidal emulsion serving as the natural photosensitizer are mixed for 1 h.

The mixed solution is irradiated under LED light with wavelength of 630nm for 0min, 10min, 20min, 30min, 40min, 50min, and 60min, and the irradiation time of 0min is used as a control group. 50. mu.L of the mixture after the light treatment was inoculated into LB solid medium and cultured at 37 ℃ for 24 hours. As shown in FIG. 6, it can be found that the sterilizing effect of the nano emulsion is continuously enhanced with the enhancement of the illumination time, and the sterilizing effect reaches 90% under 60min illumination.