Preparation method of medicinal and edible nutrient freeze-dried powder for enhancing macrophage activity

Abstract

The invention relates to a preparation method of medicinal and edible nutrient freeze-dried powder for enhancing macrophage activity. The preparation method comprises the following steps: S1, inoculating lactobacillus plantarum on a weakly selective solid culture medium for culturing; s2, selecting monoclonal lactobacillus plantarum, inoculating the monoclonal lactobacillus plantarum to a lactobacillus plantarum fermentation culture medium, and culturing; s3, when the concentration of the lactobacillus plantarum reaches the optical density value of 0.5, adding rhizoma polygonati powder, lucid ganoderma powder and mulberry leaf powder into the bacterial liquid for conversion; s4, filtering residues of the rhizoma polygonati powder, the lucid ganoderma powder and the mulberry leaf powder by using filter cloth to obtain a compound nutrient clear liquid; and S5, adding a freeze-drying protective agent into the compound nutrient clear liquid, uniformly stirring, and freeze-drying to constant weight to obtain the compound nutrient freeze-dried powder. Through the design, the utilization rate of active ingredients of the medicinal and edible raw materials can be improved by combining microbial conversion with a freeze-drying technology, and the efficient regulation of the activity of macrophages is realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicinal and edible nutrients, and particularly relates to a preparation method of medicinal and edible nutrient freeze-dried powder for enhancing macrophage activity. BACKGROUND

[0002] As the core cells of the body's immune system, the activity of macrophages directly affects the body's immune defense, inflammation regulation and tissue repair function. When the activity of macrophages decreases, the body is prone to have problems such as decreased immune function and increased susceptibility to infection. Therefore, it is of great health significance to develop a nutritional supplement that can enhance the activity of macrophages.

[0003] At present, the common immune-regulating nutrients on the market mostly take medicinal and edible raw materials such as polygonatum, ganoderma lucidum and mulberry leaves as core components. These raw materials are rich in active substances such as polysaccharides, saponins and flavonoids, which have been proven to have the potential to regulate the activity of macrophages. However, the existing preparation technology has the following shortcomings: (1) Low absorption efficiency of active ingredients: The active substances in polygonatum, ganoderma lucidum and other raw materials are mostly macromolecular structures, such as crude polysaccharides and total saponins, which are difficult for the human digestive system to absorb directly, resulting in low bioavailability and inability to fully exert the effect of regulating the activity of macrophages; (2) Limited preparation process: Traditional processes mostly use direct pulverization, water extraction or high-temperature drying, which can easily cause the destruction of active ingredients such as polysaccharide degradation and saponin oxidation, and the product has a short shelf life and is prone to moisture absorption and caking; (3) Low utilization efficiency of strains: In some microbial-assisted extraction technologies, the strain screening is not accurate and the culture conditions are not optimized, resulting in insufficient conversion capacity of microorganisms to raw materials and inability to effectively convert macromolecular components into small molecular active substances. SUMMARY

[0004] To solve the above technical problems, the present application designs a preparation method of medicinal and edible nutrient freeze-dried powder for enhancing macrophage activity, which uses lactobacillus plantarum as a conversion strain and combines precise culture and freeze-drying technology to improve the utilization rate of active ingredients of medicinal and edible raw materials through the combination of microbial conversion and freeze-drying technology, and to achieve efficient regulation of the activity of macrophages.

[0005] The preparation method of medicinal and edible nutrient freeze-dried powder for enhancing macrophage activity comprises: Step S1, inoculate lactobacillus plantarum on a weak selective solid culture medium for culture; Step S2, pick single colony lactobacillus plantarum and inoculate it on a lactobacillus plantarum fermentation medium for culture; Step S3, when the concentration of lactobacillus plantarum reaches an optical density value of 0.5, add polygonatum powder, ganoderma lucidum powder and mulberry leaf powder to the bacterial solution for conversion; Step S4: filter out the residues of the polygonatum sibiricum, ganoderma lucidum and mulberry leaf powder with filter cloth to obtain the complex nutrient liquid; Step S5: add freeze-drying protective agent into the complex nutrient liquid, stir uniformly, and freeze-dry to constant weight to obtain the freeze-dried powder of the complex nutrient.

[0006] Preferably, in the step S1, the temperature for the culture on the weak selective solid medium is 37 degrees Celsius.

[0007] Preferably, in the step S2, the culture conditions of the monoclonal lactobacillus plantarum are as follows: 37 degrees Celsius culture, no aeration and no shaking.

[0008] Preferably, in the step S3, the mass percentage of the polygonatum sibiricum powder, ganoderma lucidum powder and mulberry leaf powder is 1.5% w / v.

[0009] Preferably, in the step S3, the temperature for the transformation is 24 hours.

[0010] Preferably, the weak selective solid medium comprises: peptone, beef extract powder, yeast extract powder, glucose, Tween-80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, manganese sulfate, agar; The ratio is: 10.0 g: 5.0 g: 4.0 g: 20.0 g: 1.0 mL: 2.0 g: 5.0 g: 2.0 g: 0.2 g: 0.05 g: 15.0 g.

[0011] Preferably, the pH value of the weak selective solid medium is 6.0-6.4.

[0012] Preferably, in the step S2, the culture medium of the monoclonal lactobacillus plantarum comprises: whey permeate powder, yeast powder, glucose, corn syrup powder, magnesium sulfate heptahydrate, manganese sulfate monohydrate, sodium acetate trihydrate, dipotassium hydrogen phosphate trihydrate, anhydrous ammonium citrate, L-aspartic acid, L-cysteine, vitamin C; The ratio is: 30 g / L: 20 g / L: 20 g / L: 10 g / L: 0.6 g / L: 0.5 g / L: 3 g / L: 2 g / L: 1 g / L: 0.03 g / L: 0.03 g / L: 0.5 g / L.

[0013] Preferably, in the step S2, the pH value of the monoclonal lactobacillus plantarum is 6.5-6.7; sodium hydroxide solution is used to adjust the acid-base of the culture medium.

[0014] The advantages and effects of the present application are as follows: The preparation method of the macrophage activity enhancing nutraceutical freeze-dried powder designed by the application comprises the following steps: S1, inoculating Lactobacillus plantarum on a weak selective solid culture medium for culture; S2, picking single colony Lactobacillus plantarum and inoculating it on a Lactobacillus plantarum fermentation medium for culture; S3, when the concentration of Lactobacillus plantarum reaches an optical density value of 0.5, adding Huangjing powder, Lingzhi powder and mulberry leaf powder to the bacterial solution for transformation; S4, filtering out the Huangjing powder, Lingzhi powder and mulberry leaf powder residues with filter cloth to obtain a composite nutrient liquid; and S5, adding a freeze-drying protective agent to the composite nutrient liquid, stirring uniformly, and freeze-drying to constant weight to obtain a composite nutrient freeze-dried powder. Through the above design, the application can combine microbial transformation with freeze-drying technology to improve the utilization rate of active ingredients of nutraceutical raw materials and realize efficient regulation of macrophage activity.

[0015] The above description is only a summary of the technical solutions of the application. In order to more clearly understand the technical means of the application, the content of the description can be implemented, and in order to make the above and other purposes, features and advantages of the application more obvious and easy to understand, the following will be described in detail with the preferred embodiments of the application and with the help of the accompanying drawings.

[0016] According to the detailed description of the specific embodiments of the application below in combination with the accompanying drawings, those skilled in the art will more clearly understand the above and other purposes, advantages and features of the application. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without any creative labor. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual proportions.

[0018] Figure 1 The flowchart of the preparation method of the macrophage activity enhancing nutraceutical freeze-dried powder designed by the application; Figure 2 The effect diagram of AMCN on the level of zebrafish larva macrophages. DETAILED DESCRIPTION

[0019] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the following will be combined with the accompanying drawings for the embodiments of the present application to make a clear and complete description of the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. In the following description, specific details such as specific configurations and components are provided only to help a comprehensive understanding of the embodiments of the present application. Therefore, those skilled in the art should understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. In addition, in order to be clear and concise, the description of known functions and structures is omitted in the embodiments.

[0020] It should be understood that the "one embodiment" or "the embodiment" mentioned throughout the specification means that the specific features, structures or characteristics related to the embodiment are included in at least one embodiment of the present application. Therefore, "one embodiment" or "the embodiment" appearing throughout the specification does not necessarily refer to the same embodiment. In addition, these specific features, structures or characteristics can be combined in one or more embodiments in any suitable manner.

[0021] In addition, reference numerals and / or letters can be repeated in different examples in the present application. Such repetition is for the purpose of simplification and clarity, and does not in itself indicate a relationship between the various embodiments and / or arrangements discussed.

[0022] The term "and / or" herein is only a description of the association relationship of the associated objects, which means that there can be three relationships, for example, A and / or B can mean that A exists alone, B exists alone, and A and B exist together. The term " / and" herein is a description of another association relationship of the associated objects, which means that there can be two relationships, for example, A / and B can mean that A exists alone and A and B exist together. In addition, the character " / " herein generally represents an "or" relationship between the associated objects before and after it.

[0023] The term "at least one" herein is only a description of the association relationship of the associated objects, which means that there can be three relationships, for example, at least one of A and B can mean that A exists alone, A and B exist together, and B exists alone.

[0024] It should also be noted that, in this document, relationship terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the terms "include", "contain" or any other variants thereof are intended to cover non-exclusive inclusion.

[0025] Embodiment 1: Please refer to Figure 1The embodiment mainly introduces the specific method of the preparation method of the freeze-dried powder of the food-medicine homologous nutrient for enhancing the activity of macrophages, including: Step S1, inoculate Lactobacillus plantarum on a weak selective solid culture medium for culture; Step S2, pick single colony Lactobacillus plantarum and inoculate it on a Lactobacillus plantarum fermentation medium for culture; Step S3, when the concentration of Lactobacillus plantarum reaches an optical density value of 0.5, add Huangjing powder, Ganoderma lucidum powder and mulberry leaf powder to the bacterial solution for transformation; the core principle of the transformation process is that Lactobacillus plantarum secretes cellulase, β-glucosidase, protease and other enzyme systems in the growth and metabolism process, which can degrade macromolecular active substances such as Huangjing polysaccharide in Huangjing, Ganoderma triterpenes in Ganoderma lucidum and mulberry flavones in mulberry leaves into small molecular fragments (such as oligosaccharides, monoterpenes and small molecular flavones), significantly improving the absorption efficiency in the human body and further enhancing the regulation effect on the activity of macrophages; Step S4: filter out the residues of Huangjing powder, Ganoderma lucidum powder and mulberry leaf powder with filter cloth to obtain a composite nutrient clear liquid; Step S5: add a freeze-drying protective agent to the composite nutrient clear liquid, stir uniformly, and freeze-dry to constant weight to obtain a composite nutrient freeze-dried powder.

[0026] Further, in step S1, the temperature for culture on the weak selective solid culture medium is 37 degrees Celsius; the role of the weak selective solid culture medium is to meet the growth needs of Lactobacillus plantarum through specific carbon and nitrogen source matching (such as glucose providing carbon source and peptone and beef extract powder providing nitrogen source), and to exclude miscellaneous bacteria (such as Escherichia coli and Staphylococcus) through the weak inhibition effect of sodium acetate and triammonium citrate, so as to realize the purification and screening of the target strain.

[0027] Further, in step S2, the culture conditions of the single colony Lactobacillus plantarum are 37 degrees without aeration and without shaking.

[0028] Further, in step S3, the mass percentage of Huangjing powder, Ganoderma lucidum powder and mulberry leaf powder is all 1.5% w / v.

[0029] Further, in step S3, the temperature of the transformation is 24 hours.

[0030] Further, the weak selective solid culture medium includes peptone, beef extract powder, yeast extract powder, glucose, Tween-80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, manganese sulfate and agar; The matching ratio is: 10.0 g: 5.0 g: 4.0 g: 20.0 g: 1.0 mL: 2.0 g: 5.0 g: 2.0 g: 0.2 g: 0.05 g: 15.0 g.

[0031] Further, the pH value of the weak selective solid medium is 6.0-6.4.

[0032] Further, in the step S2, the culture medium of the monoclonal Lactobacillus plantarum comprises whey permeate powder, yeast powder, glucose, corn syrup powder, magnesium sulfate heptahydrate, manganese sulfate monohydrate, sodium acetate trihydrate, dipotassium hydrogen phosphate trihydrate, ammonium citrate anhydrous, L-aspartic acid, L-cysteine, and vitamin C; the whey permeate powder and the corn syrup powder provide high-quality protein and small-molecule peptides to meet the nitrogen demand of the strain for large-scale proliferation; L-cysteine serves as a reducing agent to maintain a low oxidation-reduction potential in the culture medium, which is suitable for the facultative anaerobic characteristics of Lactobacillus plantarum; vitamin C can protect the integrity of the strain cell membrane and reduce the loss of activity during the culture process; and the pH value is controlled at 6.5-6.7, which is more suitable for high-density proliferation of the strain in liquid culture than the acidic environment of the step S1.

[0033] The ratio is: 30 g / L:20 g / L:20 g / L:10 g / L:0.6 g / L:0.5 g / L:3 g / L:2 g / L:1 g / L:0.03 g / L:0.03 g / L:0.5 g / L.

[0034] Further, in the step S2, the pH value of the monoclonal Lactobacillus plantarum is 6.5-6.7; and sodium hydroxide solution is used to adjust the acidity and alkalinity of the culture medium.

[0035] Example 2: Based on example 1, this embodiment mainly introduces the optimization design of the above method.

[0036] Preparation of the weak selective solid medium: weigh protein peptone 10.0 g, beef extract powder 5.0 g, yeast powder 4.0 g, glucose 20.0 g, Tween-80 1.0 mL, potassium phosphate dibasic 2.0 g, sodium acetate 5.0 g, triammonium citrate 2.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, and agar 15.0 g according to the ratio; add 1 L of deionized water, heat and dissolve, and then adjust the pH to 6.2 with 0.1 mol / L sodium hydroxide solution; sterilize at 100°C for 20 min, cool to 50°C, and then pour the plate to prepare the weak selective solid medium. Purification and culture of Lactobacillus plantarum: take Lactobacillus plantarum Vege-start 60 liquid, streak inoculate on the above-mentioned solid medium, and incubate at 37°C for 20 h to form single colonies with a diameter of 1.5 mm. Liquid expansion culture: prepare liquid medium (whey permeate powder 30 g, yeast powder 20 g, glucose 20 g, corn syrup powder 10 g, magnesium sulfate heptahydrate 0.6 g, manganese sulfate monohydrate 0.5 g, sodium acetate trihydrate 3 g, dipotassium hydrogen phosphate trihydrate 2 g, ammonium citrate 1 g, L-aspartic acid 0.03 g, L-cysteine 0.03 g, vitamin C 0.5 g; add 1 L deionized water to dissolve) according to the proportion, adjust pH to 6.6 with 1 mol / L sodium hydroxide solution, sterilize at 100°C for 20 min; pick single colony and inoculate into the liquid medium, incubate at 37°C without aeration for 14 h, measure the OD value of the bacterial solution 600 0.5. Microbial transformation: add 80 mesh Rhizoma Polygonati, Ganoderma lucidum powder, mulberry leaf powder (1.5 g / 100 mL bacterial solution) to the above bacterial solution, and incubate at 37°C for 24 h. Separation and purification: filter the transformed bacterial solution with 100 mesh filter cloth, collect the supernatant, centrifuge at 3000 r / min for 5 min, and remove the small impurities. Freeze-drying preparation: add 10% (w / v) maltodextrin, 10% (w / v) mannitol, and 2% (w / v) D-(+)-trehalose dihydrate to the supernatant, stir at 120 r / min for 12 min, and place in a freeze dryer: pre-freeze at -40°C for 2.5 h, sublimate at 15 Pa vacuum degree and -10°C for 9 h, and desorb at 25°C for 5 h, then crush through a 60 mesh sieve to obtain freeze-dried powder product.

[0037] The macrophage activity enhancing nutraceutical freeze-dried powder increases the number of macrophages in the zebrafish model; further, the effect of the macrophage activity enhancing nutraceutical freeze-dried powder on macrophages is evaluated using Tg (mpeg1:EGFP) transgenic zebrafish larvae. Macrophages are another key cell type in zebrafish that is responsible for phagocytosis of cellular debris. Macrophage-specific gene 1 (mpeg1) is a gene unique to zebrafish. The transgenic strain (mpeg1:EGFP) zebrafish expresses EGFP driven by the mpeg1 promoter, enabling specific visualization of macrophages in vivo. 30 two-day-old transgenic (mpeg1:EGFP) zebrafish larvae are selected and randomly divided into groups. According to the established treatment plan, the larvae are exposed to AMCN solution (macrophage activity enhancing nutraceutical freeze-dried powder) at concentrations of 12.5, 25, and 50 μg / mL with or without the addition of cyclophosphamide, and then the larvae are incubated in a 28°C environment for 48 hours. 10 larvae from each group are taken for microscopic imaging, and after observation using a MZX81 fluorescence microscope, the fluorescence intensity of the tail vein macrophages is quantitatively analyzed using NIS-Elements software.

[0038] In the normal control group, the number of macrophages was the highest, which reflected the basal level of zebrafish larvae Figure 2 ). Compared with the normal control group, the number of macrophages in the model group decreased by about 54%. Notably, the treatment of macrophage activity-enhancing nutraceutical freeze-dried powder significantly restored the number of macrophages, which increased by about 57% compared with the model group.

[0039] Please refer to Figure 2 , Figure 2 Figure for the effect of AMCN on the level of macrophages in zebrafish larvae, (A) Representative images of Tg (mpeg1:EGFP) zebrafish larvae (4 days after) treated with AMCN for 48 hours. The white dashed line area represents the area for quantitative analysis. Scale bar = 100 µm. (B) Quantitative analysis of the level of macrophages by fluorescence intensity through the tail vein. Data are expressed as mean ± standard deviation (n=10 in each group), ***p<0.001, ****p<0.0001 compared with the model group (one-way ANOVA combined with Dunnett's post-hoc test). Wherein AMCN is the abbreviation of macrophage activity-enhancing nutraceutical freeze-dried powder.

[0040] The above only describes the preferred embodiments of the present application and does not limit the protection scope of the present application. For those skilled in the art, the present application can have various changes and variations. Any changes, modifications, replacements, integrations and parameter changes of these embodiments within the spirit and principles of the present application, which can realize the same functions without departing from the principles and spirit of the present application, fall within the protection scope of the present application.​

Claims

1. A process for the preparation of a freeze-dried powder of a nutraceutical nutrient for enhancing macrophage activity, characterized by, The application relates to a method for preparing a compound nutrient powder, comprising the following steps: S1, inoculating Lactobacillus plantarum on a weak selective solid culture medium for culture; S2, picking single colony Lactobacillus plantarum and inoculating on a Lactobacillus plantarum fermentation culture medium for culture; S3, when the concentration of Lactobacillus plantarum reaches an optical density value of 0.5, adding Huangjing powder, Lingzhi powder and mulberry leaf powder into the bacterial liquid for transformation; S4, filtering off the Huangjing powder, Lingzhi powder and mulberry leaf powder residues by using filter cloth to obtain a compound nutrient clear liquid; S5, adding a freeze-drying protective agent into the compound nutrient clear liquid, uniformly stirring, freeze-drying to constant weight, and obtaining a compound nutrient freeze-dried powder.

2. The process for preparing the macrophage activity enhancing pharmacognosy nutrient freeze-dried powder according to claim 1, characterized by, In the step S1, the culture temperature on the weak selective solid culture medium is 37 DEG C.

3. The process for preparing the macrophage activity enhancing medicinal and food isomer nutrient freeze-dried powder according to claim 1, characterized by, In the step S2, the culture condition of the single colony Lactobacillus plantarum is 37 DEG C culture, no aeration and no oscillation.

4. The process for preparing the macrophage activity enhancing medicinal and food isomer nutrient freeze-dried powder according to claim 1, characterized by, In the step S3, the mass percentage of the Huangjing powder, Lingzhi powder and mulberry leaf powder is all 1.5% w / v.

5. The process for preparing the macrophage activity enhancing medicinal and food isomorphic nutrient freeze-dried powder according to claim 1, characterized by, In the step S3, the transformation temperature is 24 hours.

6. The process for preparing the macrophage activity enhancing medicinal and food isomorphic nutrient freeze-dried powder according to claim 1, characterized by, The weak selective solid culture medium comprises peptone, beef extract powder, yeast extract powder, glucose, Tween-80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, manganese sulfate and agar; The proportion is 10.0 g:5.0 g:4.0 g:20.0 g:1.0 mL:2.0 g:5.0 g:2.0 g:0.2 g:0.05 g:15.0 g.

7. The process for preparing the macrophage activity enhancing medicinal and food isomorphic nutrient freeze-dried powder according to claim 1, characterized by, The pH value of the weak selective solid culture medium is 6.0-6.

4.

8. The process for preparing the macrophage activity enhancing medicinal and food isomorphic nutrient freeze-dried powder according to claim 1, characterized by, In the step S2, the culture medium of the single colony Lactobacillus plantarum comprises whey permeate powder, yeast powder, glucose, corn syrup powder, magnesium sulfate heptahydrate, manganese sulfate monohydrate, sodium acetate trihydrate, dipotassium hydrogen phosphate trihydrate, anhydrous ammonium citrate, L-aspartic acid, L-cysteine and vitamin C; The proportion is 30 g / L:20 g / L:20 g / L:10 g / L:0.6 g / L:0.5 g / L:3 g / L:2 g / L:1 g / L:0.03 g / L:0.03 g / L:0.5 g / L.

9. The process for preparing the macrophage activity enhancing pharmacognosy nutrient freeze-dried powder according to claim 8, characterized by, In the step S2, the pH value of the single colony Lactobacillus plantarum is 6.5-6.7; sodium hydroxide solution is used to adjust the acidity and alkalinity of the culture medium.

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