CN114081924A

CN114081924A - Fermentation product for regulating qi and blood and preparation method and application thereof

Info

Publication number: CN114081924A
Application number: CN202111386850.6A
Authority: CN
Inventors: 严超; 赵星; 李水玲; 王建云; 范会芬; 马若琰; 王岩; 赵林森
Original assignee: Hebei Yiran Biotechnology Co ltd
Current assignee: Hebei Yiran Biotechnology Co ltd
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2022-02-25

Abstract

The invention relates to the technical field of medicinal and edible products, in particular to a fermentation product for regulating qi and blood and a preparation method and application thereof. The fermentation product is prepared by fermenting raw materials after enzymolysis, and the raw materials comprise: rhizoma Polygonati, Ginseng radix Rubri, fructus Jujubae, fructus Lycii and arillus longan. The fermented product has qi and blood regulating effect, and has effects of caring skin by regulating qi and blood. Through the verification of people, the fermented product can obviously improve chloasma, sallow complexion, vigorous grease secretion and other skin problems in a short time, effectively improve the sleep quality and mental state and also improve other various symptoms caused by deficiency of qi and blood.

Description

Fermentation product for regulating qi and blood and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicinal and edible products, in particular to a fermentation product for regulating qi and blood and a preparation method and application thereof.

Background

The theory of traditional Chinese medicine considers that qi and blood are two basic substances in human body and are also the substance basis of normal physiological activities, and the occurrence of diseases is closely related to qi-blood disorder. Deficiency of qi and blood, i.e., deficiency of both qi and blood in traditional Chinese medicine, can cause a series of sub-health reactions, i.e., "unhappy" in traditional Chinese medicine, mild people are listened, poor in resistance, insomnia, rough skin, pale complexion, irregular menstruation, soreness and weakness of waist and knees, etc., and severe people can affect the functions of viscera, thereby causing diseases. The records of the Yuan Dynasty medical book Danxi Xin Fa: "there are many internal and must go outside", and the internal reaction of the human body must be expressed outside. In the theory of traditional Chinese medicine, facial skin is regarded as a mirror reflecting the functions of the five internal organs, and if the functions of the five internal organs are normal, qi, blood and body fluid can be delivered to the facial skin through meridians and collaterals, so that people look bright. However, if the five internal organs are deficient in qi and blood or dysfunctional, the reaction on the skin surface may be dull skin color or rough and dry skin. Therefore, people with healthy body often have good complexion, and traditional Chinese medicine beauty treatment usually focuses on internal regulation and external maintenance, and achieves the aims of beautifying, maintaining beauty and treating root causes by regulating qi and blood and nourishing internal organs.

However, it usually takes a long time to improve the external skin state by regulating qi and blood internally, and especially when safer medicine-food homologous traditional Chinese medicines are adopted, because of mild medicine property, the improvement on sleep state, mental state and poor skin state caused by deficiency of qi and blood needs a longer time, especially the improvement on the skin state needs a longer time, and the time is usually long, and the change can be obvious for several months, so that the user is hard to insist on, the trust degree of the user is reduced along with the lapse of time, and the market popularization of the medicine-food homologous products is extremely unfavorable.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides a fermentation product for regulating qi and blood and a preparation method and application thereof. The fermented product is used for carrying out enzymolysis and fermentation on medicinal and edible raw materials such as rhizoma polygonati, and the like, and the obtained fermented product not only has the effect of regulating qi and blood, but also has quick response, can be used for improving skin problems such as chloasma and dark skin color in a short time, and has the effects of improving sleep quality and improving mental state and energy.

In order to solve the technical problems, in a first aspect, the embodiments of the present invention provide a fermentation product for regulating qi and blood, wherein the fermentation product is prepared by performing enzymolysis on raw materials and then fermenting, and the raw materials include, by weight: 30-60 parts of rhizoma polygonati, 10-20 parts of red ginseng, 10-30 parts of Chinese date, 20-50 parts of medlar and 10-30 parts of longan.

Rhizoma Polygonati is sweet in flavor and neutral in nature, and has effects of invigorating qi, nourishing yin, invigorating spleen, moistening lung, and invigorating kidney. Modern researches show that rhizoma polygonati contains active ingredients such as rhizoma polygonati polysaccharide, saponin, flavone, alkaloid and the like, and has the effects of resisting oxidation, resisting inflammation, regulating blood sugar and blood fat, improving immunity, inhibiting tumors and the like.

Red Ginseng is warm in nature, sweet and slightly bitter in taste. It enters spleen, lung, heart and kidney meridians. Has effects of invigorating qi and nourishing blood.

The Chinese dates are sweet in taste and warm in nature, enter spleen and stomach channels, and have the effects of tonifying middle-jiao and Qi, tonifying spleen and stomach, nourishing blood and soothing nerves, promoting production of body fluid, regulating yingfen and weifen and removing vexation.

The medlar is sweet in taste and neutral in nature, enters liver and kidney meridians, and has the effects of tonifying liver and kidney, replenishing vital essence and improving eyesight. The medlar contains polysaccharide, fatty acid, betaine, carotene, riboflavin, nicotinic acid, amino acid, vitamin C, various trace elements and the like, and has the functions of enhancing the immunologic function, delaying senility, resisting tumors, reducing blood fat, protecting the liver, promoting hematopoiesis and the like.

Longan has sweet taste, mild nature and warm property, and has the effects of invigorating heart and spleen, replenishing qi and blood, invigorating spleen and stomach, and nourishing muscle. Arillus longan contains protein, fat, saccharide, organic acid, crude fiber, vitamins and minerals, and has effects of inhibiting lipid peroxidation and improving antioxidase activity, and has certain antiaging effect.

The raw materials in the fermented product are matched with each other, so that the effects of strengthening body resistance, eliminating pathogenic factors, promoting blood circulation, removing blood stasis, regulating metabolism function and promoting blood circulation can be achieved, the effect of regulating qi and blood can be achieved, and the effects of maintaining beauty and keeping young can be achieved through qi and blood regulation. In vitro experiments and animal experiments prove that the fermentation product has stronger antioxidant capacity and tyrosinase activity inhibiting capacity, can inhibit the secretion of proinflammatory factors IL-6, TNF-alpha and IL-1 beta, promotes the secretion of anti-inflammatory factors IL-10, and can prolong the natural life of fruit flies. Through the verification of people, the fermented product can obviously improve the skin problems of chloasma, sallow complexion, vigorous grease secretion and the like in a short time, effectively improve the sleep quality and the mental state, and can also improve the symptoms of irregular menstruation, dysmenorrheal, red blood silk of eyes and the like caused by deficiency of qi and blood.

According to the first aspect, the rhizoma polygonati, red ginseng, Chinese date, medlar and longan are selected according to the following weight parts: 40-50 parts of rhizoma polygonati, 13-17 parts of red ginseng, 15-25 parts of Chinese date, 30-40 parts of medlar and 15-25 parts of longan.

Preferably, the sealwort, the red ginseng, the Chinese date, the medlar and the longan are prepared according to the following weight parts: 45 parts of rhizoma polygonati, 15 parts of red ginseng, 20 parts of Chinese date, 35 parts of Chinese wolfberry and 20 parts of longan.

In combination with the first aspect, the enzyme for enzymolysis of the raw material may be cellulase and hemicellulase, or cellulase and alpha-amylase, wherein the dosage of the cellulase may be 0.8-1.2% and the dosage of the hemicellulase or alpha-amylase may be 0.3-0.5% on a dry matter basis. The enzymes can fully decompose the cell walls of the raw materials in the enzymolysis process, improve the release amount of effective components and improve the functions of the fermentation product in the aspects of improving sleep, spirit and skin states.

With reference to the first aspect, the fermentation bacteria for fermenting the raw material comprise lactobacillus plantarum Lp45, and may further comprise at least one of lactobacillus rhamnosus LR519 and lactobacillus acidophilus La28, and more preferably lactobacillus plantarum Lp45 and lactobacillus rhamnosus LR519 are combined in a mixing ratio of 1: 1-1: 2. The zymophyte can decompose macromolecular substances in the traditional Chinese medicine raw materials into micromolecular substances which are beneficial to the absorption and utilization of organisms in the fermentation process, the bioavailability and the onset speed of the fermentation product are improved, the effects of improving sleep, spirit and skin states can be achieved under the conditions of low dosage and short taking time, and the effect is better when the lactobacillus plantarum Lp45 and the lactobacillus rhamnosus LR519 are combined for use.

Wherein, the lactobacillus plantarum Lp45 is disclosed in the patent with the application number CN201310587738.8 (a lactobacillus plantarum and application thereof), and the preservation number is CGMCC NO. 8072; lactobacillus rhamnosus LR519 has been disclosed in patent with application number CN201910129081.8 (application of Lactobacillus rhamnosus and its compound bacteria powder in product for preventing and treating vaginitis), with collection number CGMCC No. 15969; lactobacillus acidophilus is disclosed in the patent with the application number of La28CN201610381849.7 (a Lactobacillus acidophilus La28 with immunoregulation and anti-allergic functions and its application), and the preservation number is CGMCC No. 11506.

With reference to the first aspect, the polygonatum sibiricum is polygonatum sibiricum. By using rhizoma polygonati as a raw material, the obtained fermentation liquor has stronger in-vitro oxidation resistance.

In a second aspect, an embodiment of the present invention further provides a preparation method of the qi and blood regulating fermented product, specifically including the following operations:

mixing the raw materials with water, pulping, performing enzymolysis, fermenting, and performing solid-liquid separation to obtain a liquid phase.

With reference to the second aspect, the preparation method may further include steaming the polygonatum sibiricum for 50-80 min before mixing and pulping the raw materials with water. Experiments show that the stimulating taste can be generated after the direct fermentation of the sealwort, and the fermentation after steaming can obviously reduce the stimulating feeling and increase the content of soluble sugar in sealwort fermentation products, thereby improving the sweetness of the product. The steamed polygonatum sibiricum is soaked in water for 6-8 hours according to the ratio of 1:8, so that the polygonatum sibiricum can be softened in hard cores, pulping can be smoothly carried out, cell wall breaking in the enzymolysis process can be promoted, and effective components can be fully released.

With reference to the second aspect, the temperature of the enzymolysis is 40 to 50 ℃, the time of the enzymolysis is 90 to 120min, and the more preferable time of the enzymolysis is 90 to 120 min.

With reference to the second aspect, the fermentation time can be selected from 50 to 60 hours; the fermentation temperature can be 37 +/-1 ℃.

Drawings

FIG. 1 is a comparison of the in vitro antioxidant capacity of example 1 and comparative examples 1 and 2 in effect example 1 of the present invention;

FIG. 2 is a comparison result of in vitro antioxidant capacity of examples 1, 7, 8, 9 and comparative examples 5 to 8 in effect example 1 of the present invention;

FIG. 3 shows the results of comparison of polysaccharide contents of rhizoma Polygonati in examples 1 and 6 and comparative examples 3 and 4 in example 3 of the effect of the present invention;

FIG. 4 is a result of comparing tyrosinase activity inhibition rates of example 1 and comparative example 8 in effect example 4 of the present invention;

FIG. 5 is a comparison result of in vitro anti-inflammatory test of example 1 and comparative example 8 in effect example 5 of the present invention;

FIG. 6 is a graph showing the life span of naturally aging female Drosophila melanogaster in each group in example 6 of the effect of the present invention;

FIG. 7 is a graph showing the life span of naturally aging male Drosophila melanogaster in each group in example 6 of the effect of the present invention;

FIG. 8 is a graph showing the skin condition improving effect of the fermented product obtained in example 1 in example 7 of the present invention;

FIG. 9 shows the effect of improving sleep in the fermented product obtained in example 1 in example 7 of the present invention;

FIG. 10 is a graph showing the effect of improving mental state of the fermented product obtained in example 1 in example 7 of the effect of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Rhizoma Polygonati, also called JITOSHEN, has effects of invigorating spleen, moistening lung, promoting fluid production, quenching thirst, and is sweet in taste, mild in nature, and easily accepted by weak people, and is suitable for people with spleen and stomach deficiency cold, asthenia, listlessness, lung dryness, dry cough, diabetes, etc. Modern researches show that rhizoma polygonati contains active ingredients such as rhizoma polygonati polysaccharide, saponin, flavone, alkaloid and the like, and has the effects of resisting oxidation, resisting inflammation, regulating blood sugar and blood fat, improving immunity, inhibiting tumors and the like. The invention takes the sealwort as the main fermentation substrate, and combines the red ginseng, the Chinese date, the medlar and the longan which have the effects of invigorating qi, activating blood, nourishing yin and tonifying kidney, and can strengthen body resistance to eliminate pathogenic factors, activate blood circulation, dissipate blood stasis, regulate the metabolism function of a human body, improve the blood circulation of the human body, thereby achieving the effects of regulating qi and blood and beautifying.

The active ingredients of the traditional Chinese medicine are complex, and the extract obtained by any extraction method can produce expected effects. According to the invention, while the raw materials are screened, the extraction method of effective components is also investigated by combining different raw material combinations, the raw materials with homology of medicine and food are finally determined to be matched for use, the raw materials containing rhizoma polygonati, red ginseng, Chinese date, medlar and longan are subjected to extraction of the effective components in a mode of enzymolysis and fermentation, and the fermented product is prepared.

The fermented product is proved to have the capacity of inhibiting melanin synthesis key enzyme-tyrosinase and the effects of inflammation inhibition and aging resistance through in vitro cell experiments and fruit of drosophila, and the use effect of the crowd verifies that the fermented product can effectively improve the skin state, the sleep state and the mental state.

The following examples are intended to illustrate the invention in more detail.

In the following examples, Polygonatum sibiricum, Red Ginseng, Zizyphi fructus, Lycii Frutus and Euphoria longana were obtained from Hongmon, Anguo, Chinese medicine science and technology, Inc.

Examples 1 to 5

Embodiments 1 to 5 of the present invention provide qi and blood regulating fermented products, and the raw materials in each embodiment are shown in table 1:

table 1 examples 1 to 5 raw material ratios

	Example 1	Example 2	Example 3	Example 4	Example 5
						Rhizome of Chinese Thalictrifoliate Polygonatum	45	40	50	30	60
Red ginseng	15	17	13	20	10
						Chinese date	20	15	25	10	30
Chinese wolfberry	35	30	40	20	50
						Longan fruit	20	25	15	30	10

The preparation method of the above examples 1 to 5 is:

weighing the raw materials according to the weight part ratio, steaming the polygonatum sibiricum for 30-90 min by using steam, then soaking the polygonatum sibiricum for 8 hours by using 8 times of water, adding other raw materials, and pulping together;

adding enzyme, carrying out enzymolysis at 40-50 ℃ for 90-120 minutes, heating to 115 ℃ for 20min to inactivate enzyme, adding zymophyte, fermenting at 37 +/-1 ℃ for 50-60 h, filtering to remove residues, filling the obtained filtrate, and sterilizing at 90-95 ℃ for 30min to obtain the product.

Specific production parameters of examples 1 to 5 are shown in Table 2.

TABLE 2 examples 1-5 preparation Process parameters

Examples 6 to 9

The raw material ratios of examples 6 to 9 were the same as those of example 1, and the enzyme or fermentation tubes were different from those of example 1. The preparation process parameters of examples 6 to 9 are shown in Table 3.

Table 3 example 6 preparation process parameters

Comparative example

The following comparative examples are fermentation products obtained by carrying out enzymolysis with other enzymes and fermentation with other fermentation strains using other species of polygonatum as a raw material in addition to example 1.

The sealwort types of the comparative examples 1 and 2 are different from the example 1, and the rest raw materials and the preparation process parameters are the same as the example 1. The enzymes used in the enzymatic hydrolysis in comparative examples 3 and 4 are different from those in example 1, and the raw materials and the preparation process parameters are the same as those in example 1, as shown in table 4.

TABLE 4 comparative examples 1 to 4

The fermentation bacteria used in the comparative examples 5 to 7 are different from those in example 1, and the raw materials and the preparation process parameters are the same as those in example 1. Comparative example 8 was not fermented and the starting materials and other preparation process parameters were the same as in example 1, as shown in table 5.

TABLE 5 comparative examples 5 to 8

Effect example 1

The effect examples show the in vitro antioxidant ability of the fermentation products obtained in examples 1, 7, 8 and 9, comparative examples 1 and 2 (different species of polygonatum sibiricum) and comparative examples 5 to 7 (different fermentation tubes).

1. Determination of DPPH radical scavenging Capacity

By adopting the DPPH free radical scavenging capability kit G0128W, DPPH free radicals have single electron, strong absorption is generated at 517nm, and the alcoholic solution is purple. When the free radical scavenger exists, the absorption of the free radical scavenger disappears gradually due to the single electron pairing with the free radical scavenger, and the lighter the color is, namely the lower the A value is, so that the DPPH scavenging capacity in the sample is quantitatively analyzed.

DPPH radical clearance (%) (1- (a)_{Measurement of}-A_{Control of})÷A_{Blank space})×100]％

2. Measurement of OH.scavenging ability

OH. kit G0125W was used to generate OH. with Fenton reaction system, the reaction was as follows:

Fe²⁺+H₂O₂→Fe³⁺+OH·+OH^—

hydroxyl radicals generated by Fenton reaction react with salicylic acid, and products have special absorption at 510 nm. And (3) judging the capability of the test object for removing the hydroxyl radicals by adopting a fixed reaction time method according to the absorbance value of the test object at 510 nm.

Removal rate (%) for OH ═ for control tube a₅₁₀Assay tube A₅₁₀) Control tube A₅₁₀×100％

3. ABTS free radical scavenging ability

The ABTS free radical scavenging kit G0127W is adopted, ABTS is a mediator substance, and K2S2O8 and ABTS are used for directly generating stable cationic free radical ABTS +, and an antioxidant substance reacts with ABTS + to enable a reaction system to fade. Since the maximum absorption wavelength of ABTS +. radical ions is 734nm, the concentration of ABTS +. radical ions can be detected by A734 nm.

Samples were processed according to the instructions and absorbance was measured using a microplate reader. The above kits were purchased from tsuzhou grizzi biotechnology limited.

The test results are shown in fig. 1 and 2.

As can be seen from FIG. 1, in example 1, compared with comparative examples 1 and 2, the Oh & clearing rate, DPPH clearing rate and ABTS clearing rate of Polygonatum cyrtonema Hua are all significantly higher than those of Polygonatum cyrtonema Hua and Curcuma rhizome essence. Meanwhile, according to the taste evaluation results, the fermented products obtained in the examples 1-5 (all adopting polygonatum odoratum) are proper in sweetness and sourness, full in taste, free of bad flavor and optimal in score. By combining the antioxidant capacity and the taste evaluation results, the polygonatum kingianum is the better choice for the fermentation product of the invention.

As can be seen from FIG. 2, compared with comparative examples 5 to 8, the fermented products obtained in examples 1, 7, 8 and 9 have obviously higher antioxidant capacity than that of unfermented comparative example 8 and that of comparative examples 5 to 7 without Lactobacillus plantarum Lp45, and the fermented product fermented by the combination of Lactobacillus plantarum Lp45 and Lactobacillus rhamnosus LR519 has the strongest antioxidant capacity.

Effect example 2

The fermented products obtained in examples 1 to 9 and comparative examples 5 to 7 were evaluated for taste. 10 panelists were healthy and tasted normally, and were not smoking, drinking, or spicy and pungent foods for 12 hours before the evaluation, and the fermented products of examples and comparative examples were scored according to the sensory evaluation criteria of table 6, and the scoring results of each example are shown in table 7.

TABLE 6 sensory evaluation criteria and scores

TABLE 7 scoring results of fermented products obtained in examples and comparative examples

	Taste scoring	Sourness and sweetness scores	Evaluation of mouthfeel	Total score of
					Example 1	29	29	The sour feeling is soft, the medicinal materials have sweet taste and are sour and sweet	58
Example 2	29	27	The sour feeling is soft, the medicinal materials have sweet taste, and the sour and sweet are more harmonious	56
					Example 3	28	28	The sour feeling is soft, the medicinal materials have sweet taste, and the sour and sweet are more harmonious	56
Example 4	25	26	Weak sour taste and obvious sweet taste	51
					Example 5	25	27	Weak sour taste and obvious sweet taste	52
Example 6	22	25	Weak sour taste and obvious sweet taste	47
					Example 7	18	20	The sour taste is weak, but no obvious sweet taste of the medicinal materials exists	38
Example 8	21	24	Weak sour taste and obvious sweet taste	45
					Example 9	23	26	Weak sour taste and obvious sweet taste	49
Comparative example 5	12	11	Strong sour sensation and sour irritation in the mouth	23
					Comparative example 6	9	11	Disharmony of sour feeling in the back	20
Comparative example 7	10	12	The sour feeling is not obvious, and the fermentation flavor is not prominent	22

The results show that the fermented product provided by the embodiment of the invention has good mouthfeel, and is superior to the fermented products of the embodiments, wherein the fermented product fermented by combining lactobacillus plantarum Lp45 and lactobacillus rhamnosus LR519 has optimal mouthfeel, proper sour and sweet taste, and no obvious stimulation and sour taste.

Effect example 3

In the effect example, the fermentation products prepared in examples 1 and 6 and comparative examples 3 and 4 were subjected to crude sugar content measurement, and on the basis of the same raw materials and preparation process parameters, different enzymolysis times were respectively adopted in the enzymolysis process, and the obtained fermentation products were subjected to crude sugar content measurement.

Glucose standard solution preparation: accurately weighing 10mg of anhydrous glucose dried to constant weight, placing in a 100ml volumetric flask, adding distilled water to dissolve and dilute to scale, and shaking uniformly.

Sample preparation: 15ml of each of the fermentation products obtained in examples 1 and 6 and comparative examples 3 and 4 was precisely aspirated, absolute ethanol was added to each of the fermentation products to make the alcohol content reach 80% v/v, the mixture was thoroughly stirred and mixed, and then centrifuged at 4000r/min for 5min, the supernatant was discarded, and the residue was washed with 80% v/v ethanol for 3 times. Dissolving the residue with water, and diluting to 250ml to obtain test solution.

Preparation of a standard curve: respectively and precisely sucking 0.1mg/ml glucose standard solution 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0ml and supplementing water to 2 ml. Then adding 1ml of 5% phenol test solution respectively, shaking up, adding 5.0ml of concentrated sulfuric acid quickly, shaking up, heating in boiling water bath for 1min, taking out, quickly cooling in ice water to room temperature, taking phenol-concentrated sulfuric acid solution as a blank, measuring absorbance at the position of 485nm to obtain a regression equation Y which is 5.5025X +0.1428, and r which is 0.9995.

And (3) sample content determination: preparing a sample solution according to a preparation method of a test sample solution, precisely absorbing 1.0ml of the sample solution into a test tube with a plug, respectively adding 1.0ml of a 5% phenol solution, shaking up, rapidly adding 5.0ml of concentrated sulfuric acid, shaking up, heating in a boiling water bath for 15min, taking out, rapidly cooling in ice water to room temperature, measuring absorbance at a wavelength of 485nm by taking a blank solution of a test machine as a reference, and substituting into a regression equation to calculate the content of crude polysaccharide in the sample.

The results are shown in fig. 3, the content of the polysaccharides from polygonatum sibiricum is used as an evaluation index, the content of the polysaccharides from huangjing in the products obtained in example 1 (cellulase and hemicellulase) and example 6 (cellulase and alpha-amylase) is significantly higher than that in comparative example 3 and comparative example 4, the content of the polysaccharides from huangjing in the products obtained in example 1 is slightly higher than that in example 6, the content of the polysaccharides from huangjing in the products obtained in example 1 is significantly higher than that in the products obtained in example 6 when the products are subjected to enzymolysis at 50 ℃ for 90min, and the content of the polysaccharides from huangjing in the products is not significantly different from that in the products obtained in example 120 min.

Effect example 4

The tyrosinase activity inhibition test was performed on the products obtained in example 1 and comparative example 8.

Tyrosinase is a key enzyme in skin melanin biosynthesis, and oxidizes L-tyrosine into L-dopa, which is then oxidized into quinone, which spontaneously undergoes a series of reactions to form melanin. Therefore, the activity of tyrosinase is inhibited, and the synthesis of melanin can be effectively inhibited.

(1) Reagent: tyrosinase, activity is more than or equal to 1000unit/mg solid; levodopa with purity not less than 98%; tyrosinase solution: prepared by 100 mu g/mL of disodium hydrogen phosphate-citric acid buffer solution with the pH of 6.8 for temporary use; levodopa solution: prepared by a pH6.8 disodium hydrogen phosphate-citric acid buffer solution with the concentration of 1mg/mL, and stored in a dark place; positive control: kojic acid with purity not less than 98.5%.

(2) The experimental method comprises the following steps: the positive control was diluted to 1mg/mL, 0.2mg/mL, 0.04mg/mL, 0.008mg/mL series concentration gradients with pH6.8 disodium phosphate-citric acid buffer to verify the assay system. Treating a test object: the test substance is diluted into a multi-concentration sample by using a disodium hydrogen phosphate-citric acid buffer solution. Referring to Table 8, use 1A 0mL test tube is provided with a sample tube (T) and a sample background (T)₀) Enzyme reaction tube (C) and solvent background (C)₀) For each sample, 3 parallel tubes are required to be set for each sample tube (T) of each concentration to be tested, and 3 parallel tubes are required to be set for the enzyme reaction tube (C). In the sample tube (T) and sample background (T)₀) 1mL of the same concentration of each of the sample solutions, an enzyme reaction tube (C) and a solvent background (C)₀) 1mL of disodium hydrogen phosphate-citric acid buffer was added. Adding 0.5mL of tyrosinase solution into each of the sample tube (T) and the enzyme reaction tube (C), and obtaining a sample background (T)₀) With background of solvent (C)₀) The samples were mixed well with tyrosinase in 0.5mL disodium hydrogen phosphate-citric acid buffer and incubated in a 37 ℃ water bath for 10 min. And sequentially adding 2mL of levodopa solution into each tube, controlling the reaction time of each tube to be 5 minutes, immediately transferring each tube of reaction solution into a cuvette, and measuring the light absorption value at 475 nm.

TABLE 8 sample addition requirements

	T-sample tube	T₀Background of sample	C-enzyme reaction tube	C₀Background of solvent
					Sample solution/ml	1	1	—	—
PBS solution/ml	—	0.5	1	1.5
					Tyrosinase solution/ml	0.5	—	0.5	—
Levodopa solution/ml	2	2	2	2

(3) And (4) calculating a result: tyrosinase inhibition [% 1- (T-T)₀)/(C-C₀)]X 100%, T-sample tube light absorption value, namely the light absorption value of the solution after the sample reacts with tyrosinase; t is a unit of₀-sample background absorbance; c, averaging the light absorption values of the enzyme reaction tube for 3 times, namely the light absorption value of the reaction of tyrosinase and dopa when no sample is added; c₀-background absorbance of solvent.

(4) And (3) test validity verification: each batch of experiment requires the test of adding a positive control, IC of kojic acid₅₀The test system was considered to be effective when the concentration of the sample to be tested (corresponding to the 50% inhibitory effect) was 0.05 mg/mL-0.15 mg/mL.

The tyrosinase activity inhibition rates of the products obtained in the example 1 and the comparative example 8 and the Lp45+ LR519 mixed fermentation liquor are shown in FIG. 4, and it can be seen that the tyrosinase activity inhibition rate of the fermentation liquor obtained by fermenting the raw material of the application with Lactobacillus plantarum Lp45 and LR519 is remarkably higher than that of the fermentation liquor of the comparative example 8 and the strain fermentation liquor which are not fermented, and the tyrosinase inhibition capability of the raw material of the application after fermentation is remarkably improved.

Effect example 5

This effect example the fermentation products obtained in example 1 and comparative example 8 were subjected to in vitro anti-inflammatory tests.

(1) Cells and reagents

RAW264.7 (mouse mononuclear macrophage leukemia cells), wuhan punuosai life science ltd; DMEM high-glucose medium, Invitrogen, usa; fetal bovine serum, diabody (penicillin/streptomycin), trypan blue, shanghai bio-engineering ltd; neutral red kit, bi yun tian; LPS, Sigma, usa; IL-6, IL-10 and TNF-alpha kits, Beijing Sizhenbai Biotech limited.

(2) Cell culture

RAW264.7 cells were cultured in DMEM complete medium containing 10% fetal calf serum and 1% streptomycin double antibody at 37 deg.C and 5% CO₂And culturing in an incubator with saturated humidity, and taking cells in logarithmic growth phase for experiment.

(3) Co-incubation of samples with cells

Cell plating: the concentration of RAW.264.7 cells was adjusted to 2.0X 10⁵cfu/mL, 2mL of cell culture medium per well of a 24-well plate is inoculated, and 2mL of fresh culture medium is replaced after 16-20h of culture for adherence. And randomly setting a control group and an experimental group, adding 100 mu L of sample into the experimental group for intervention, respectively adding DMEM with the same dose into a blank control group and an inflammation model control group, and adding 2 mu L of LPS (lipopolysaccharide) into the model control group for inflammation induction (the final concentration is 1 mu g/mL). After 24h of co-incubation, collecting cell culture solution of each group, centrifuging for 10min at 1000r/min, collecting supernatant, subpackaging in a sterile centrifuge tube, and preserving at-80 ℃ for later use. The secretion of IL-6, IL-10, IL-1. beta. and TNF-. alpha.cytokines was determined according to the ELISA kit instructions.

The results are shown in FIG. 5 and Table 9.

TABLE 9 Effect on macrophage RAW264.7 secretion of inflammatory factors

As can be seen from the results, compared with the blank control, the products obtained in the comparative example 8 and the example 1 can promote the immune cells to secrete the cytokines IL-6, TNF-alpha and IL-10; compared with a model control, the products obtained in the comparative example 8 and the example 1 can promote the cells to produce less proinflammatory factors IL-6 and TNF-alpha and promote the secretion of the inflammation inhibiting factor IL-10; compared with the comparative example 8, the product obtained in the example 1 after fermentation can stimulate macrophages to produce less proinflammatory factors IL-6, TNF-alpha and IL-1 beta, and can promote the secretion of the inflammation inhibiting factor IL-10.

Effect example 6

Effect example the fermented products obtained in examples and comparative examples were subjected to Drosophila anti-aging test

The wild type fruit fly Canton-S is provided by the national academy of sciences SIBCB fruit fly resource and technical platform. Collecting the eclosion copulated fruit flies within 8h, separating male and female, and culturing in an incubator at the culture temperature (25 +/-1) DEG C and the relative humidity of 50% -60%.

(1) Media preparation

Basic culture medium: 81.5g of cane sugar, 9.2g of yeast powder, 1.3g of sodium benzoate, 108.5g of corn flour, 8.2g of agar and 1000ml of pure water;

negative control group (comparative example 8 group): 81.5g of sucrose, 9.2g of yeast powder, 1.3g of sodium benzoate, 108.5g of corn flour, 8.2g of agar and 1000ml of water extract;

example 1 fermentation broth low dose group: 81.5g of cane sugar, 9.2g of yeast powder, 1.3g of sodium benzoate, 108.5g of corn flour, 8.2g of agar, 500ml of fermentation liquor and 500ml of pure water;

example 1 fermentation broth high dose group: 81.5g of sucrose, 9.2g of yeast powder, 1.3g of sodium benzoate, 108.5g of corn flour, 8.2g of agar and 1000ml of fermentation liquor;

(2) grouping fruit flies

Taking 5-day-old male and female fruit flies, and randomly dividing the fruit flies into the 4 groups respectively, wherein each group of male and female flies is provided with 10 pipes and 20 pipes.

(3) Experiment of Drosophila life

The life test of the fruit flies mainly inspects three indexes: and (4) drawing a life-span curve of the fruit flies according to the average life span of the fruit flies, the half death time of the fruit flies and the maximum life span of the fruit flies. The number of fruit fly deaths was recorded every 2d, and the medium was changed 1 time every 4d until all the flies had died.

Average life: average number of total drosophila deaths per group;

half the death time: half the number of days of drosophila death in each group;

maximum life: average number of surviving days of the last dead 20 flies in each group.

Statistical analysis is carried out on the survival rates of the drosophila melanogaster of the blank group, the comparison example 8 group, the fermentation liquor low-dose group in the example 1 and the fermentation liquor high-dose group in the example 1, the results are shown in fig. 6 and fig. 7, the survival curves of the sample group and the blank control group in the male and female drosophila melanogaster can be obviously separated, the curve is integrally higher than that of the comparison example 8 group, namely, the natural life of the drosophila melanogaster can be prolonged by the fermentation liquor with the high and low doses in the example 1 (p is less than 0.05).

The statistical results of the life span of each group of drosophila are shown in table 10.

TABLE 10 Natural aging Life of the groups of Drosophila melanogaster

As can be seen from the results, the high-dose fermentation liquid of example 1 improves the average life span of female drosophila by 12.4%, the maximum life span by 4% and the half death time by 21.3%; the average life of the male drosophila is improved by 12.27 percent, the maximum life is improved by 20.01 percent, and the half death time is improved by 28.46 percent. Compared with the blank group, the comparative example 8 group can not improve the natural life of female drosophila melanogaster, but can improve the maximum life of male drosophila melanogaster by 7.9 percent and the average life by 4.08 percent.

Effect example 7

This effect example was performed by performing population verification on the fermentation product obtained in example 1.

(1) Screening the group of people: screening people who have skin problems such as acne, chloasma and dark skin, poor sleep, poor mental state and the like caused by deficiency of qi and blood;

(2) trial period and feedback: the sample specification is 30 mL/bottle; the product can be drunk after opening the cover, 1 bottle after breakfast and supper, and the administration period is 1 month

Data acquisition: and filling questionnaires before and after trial, performing survey revisit on trial personnel, and counting the improvement conditions of various symptoms and the improvement rate of people. The results are shown in fig. 8 to 10 and table 11.

TABLE 11 trial rate of symptom improvement for the population

Number of people	32 persons
		Rate of improvement of skin problems	75％
Rate of improvement of sleep quality	81.25％
		Improvement of energy state	75％

The result shows that the fermented product obtained by the embodiment of the invention has obvious improvement effect on skin problems caused by deficiency of qi and blood, such as chloasma, sallow complexion, vigorous oil secretion and other symptoms, and the overall effective rate reaches 75%; the effective rate of improving sleep quality reaches 81.25 percent, and the effective rate of improving energy and mental state reaches 75 percent; besides, test-taking personnel feed back the normal menstruation, the relief of dysmenorrheal and the disappearance of red blood filaments of eyes after test-taking, which are symptoms caused by deficiency of qi and blood, so that the fermented oral liquid is proved to have obvious effect on regulating deficiency of qi and blood.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. The fermentation product for regulating qi and blood is characterized by being prepared by fermenting raw materials after enzymolysis, wherein the raw materials comprise the following components in parts by weight: 30-60 parts of rhizoma polygonati, 10-20 parts of red ginseng, 10-30 parts of Chinese date, 20-50 parts of medlar and 10-30 parts of longan.

2. The qi and blood regulating fermented product according to claim 1, wherein the sealwort, red ginseng, Chinese date, medlar and longan are prepared from the following components in parts by weight: 40-50 parts of rhizoma polygonati, 13-17 parts of red ginseng, 15-25 parts of Chinese date, 30-40 parts of medlar and 15-25 parts of longan.

3. The qi and blood regulating fermented product according to claim 2, wherein the sealwort, red ginseng, Chinese date, medlar and longan are prepared in parts by weight as follows: 45 parts of rhizoma polygonati, 15 parts of red ginseng, 20 parts of Chinese date, 35 parts of Chinese wolfberry and 20 parts of longan.

4. The qi and blood regulating fermentation product according to any one of claims 1 to 3, wherein the enzymes for enzymolysis of the raw materials are cellulase and hemicellulase, or cellulase and alpha-amylase.

5. The qi and blood regulating fermented product according to any one of claims 1 to 3, wherein the fermentation bacteria used for fermenting the raw material comprise Lactobacillus plantarum Lp45, and the preservation number of the Lactobacillus plantarum Lp45 is CGMCC NO. 8072.

6. The fermentation product for regulating qi and blood of claim 5, wherein the fermentation tubes for fermenting the raw material further comprise at least one of Lactobacillus rhamnosus LR519 and Lactobacillus acidophilus La28, wherein the Lactobacillus rhamnosus LR519 has a collection number of CGMCC No.15969, and the Lactobacillus acidophilus La28 has a collection number of CGMCC No. 11506.

7. The qi and blood regulating fermented product according to claim 5, wherein the fermenting bacteria used for fermenting the raw material are Lactobacillus plantarum Lp45 and Lactobacillus rhamnosus LR 519.

8. The qi and blood regulating fermented product according to any one of claims 1 to 3, wherein the rhizoma Polygonati is Polygonatum Canaliculatum.

9. The method for producing a qi and blood regulating fermented product according to any one of claims 1 to 8, comprising the steps of:

mixing the raw materials with water, pulping, performing enzymolysis, fermenting, performing solid-liquid separation, and collecting liquid phase.

10. The method for preparing a qi and blood regulating fermented product according to claim 9, further comprising steaming rhizoma Polygonati for 50-80 min before mixing the raw materials with water and pulping.