CN115777919A - Preparation method and quality detection method of angelica keiskei enzyme - Google Patents
Preparation method and quality detection method of angelica keiskei enzyme Download PDFInfo
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- CN115777919A CN115777919A CN202211594877.9A CN202211594877A CN115777919A CN 115777919 A CN115777919 A CN 115777919A CN 202211594877 A CN202211594877 A CN 202211594877A CN 115777919 A CN115777919 A CN 115777919A
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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Abstract
The invention belongs to the technical field of functional foods, and particularly relates to a preparation method and a quality detection method of angelica keiskei ferment, which only take angelica keiskei as a raw material, generate the angelica keiskei ferment with high content of total flavonoids and superoxide dismutase (SOD) through an ultrahigh pressure and fermentation process, greatly improve the antioxidation and free radical elimination effects of the ferment, enable the ferment to have the effect of repairing alcoholic liver injury, and have the advantages of high safety, no toxic or side effect, low production cost and high quality controllability; meanwhile, the liver protection effect of the angelica keiskei ferment is comprehensively evaluated through mouse weight, liver function indexes, serum four indexes and liver function indexes obtained through mouse pre-experiments so as to ensure the quality and safety of the ferment.
Description
Technical Field
The invention belongs to the technical field of functional foods, and particularly relates to a preparation method and a quality detection method of angelica keiskei ferment.
Background
With the rising incidence of alcoholic liver injury, the intervention prevention and repair of liver diseases caused by the alcoholic liver injury have important practical significance for guaranteeing the physical health of people and developing the healthy development of wine culture of Chinese nationality.
In medicine, the method for preventing and treating the alcoholic liver comprises the steps of giving up the wine and carrying out corresponding drug treatment according to the morbidity degree of the alcoholic liver. Glucocorticoid is needed to be taken if the disease is serious, and anti-inflammatory drugs for protecting liver are needed to be taken if the disease is mild or moderate, such as MEIDOTAI, polyene phosphatidyl choline, glycyrrhizic acid preparation, silymarin, N-acetylcysteine, glutathione, S-adenosyl methionine, etc., and simultaneously, the nutritional ingredients such as vitamin B, vitamin C, vitamin K, folic acid, trace elements, etc. are supplemented. However, the drugs generally have side effects, for example, the common side effects of glucocorticoids include hypertension, elevated blood sugar, increased potassium excretion, myasthenia, gastric ulcer, mental excitation, dysphoria, insomnia, decreased immunity, susceptibility to infection, osteoporosis, cataract, glaucoma and the like. Adverse reactions of skin allergy are observed in clinical experiments, and symptoms such as alanine aminotransferase increase and aspartate aminotransferase increase may occur. The Chinese herbal medicines are compounded and decocted to obtain juice, and the juice is used for relieving alcoholism and protecting liver. For example, kudzu root, astragalus root, ganoderma lucidum, gardenia, schisandra fruit, motherwort, notoginseng, licorice, pinellia tuber, coptis, poria, dried orange peel, bamboo shavings, peach kernel, bupleurum root, red peony root, salvia root, hawthorn, chicken's gizzard-membrane, medicated leaven, white peony root, amomum fruit, curcuma aromatica and the like can be selected as the prescription of the herbal medicine. Patent application CN201710168541.9 discloses a health food with an auxiliary protection effect on alcoholic liver injury, which is characterized in that hovenia dulcis thunb, tremella, kudzuvine root, liquorice, schisandra chinensis, hericium erinaceus and chitosan oligosaccharide are used as main components; patent application CN201610870110.2 discloses a traditional Chinese medicine for treating alcoholic liver disease, which comprises 10 parts of rhizoma rhei, 30 parts of coltsfoot herb, 10 parts of rhizoma corydalis, 10 parts of rhizoma zedoariae, 12 parts of herba artemisiae scopariae, 12 parts of semen vaccariae, 12 parts of inversely hung candle, and 12 parts of radix curcumae. Patent application CN1410456288.3 relates to a traditional Chinese medicine for treating alcoholic liver, which is prepared from the following raw material medicines in parts by weight: 20-40 parts of feather cockscomb seed, 7-13 parts of semen coicis, 5-7 parts of anoectochilus roxburghii, 10-20 parts of semen cuscutae, 12-18 parts of sculellaria barbata, 10-20 parts of semen oroxyli, 12-18 parts of citron, 6-14 parts of semen hoveniae, 15-25 parts of an nanchan, 10-20 parts of morinda officinalis, 15-25 parts of pokeberry root, 10-20 parts of winged euonymus twig, 12-18 parts of radix puerariae, 15-25 parts of ringing dog, 5-13 parts of galium aparine, 8-16 parts of cherokee rose fruit, 15-25 parts of zedoary, 15-25 parts of allium macrostemon and 15-25 parts of chinarum herb. Soaking the 19 raw materials in water, decocting, and removing residues to obtain filtrate. Patent application CN201410543094.7 discloses a traditional Chinese medicine formula for treating alcoholic liver, which comprises the following components in parts by weight: 8 g of Chinese yam, 6 g of astragalus, 7 g of schisandra chinensis, 5g of white peony root, 7 g of hawthorn, 9 g of tuckahoe, 6 g of red peony root, 6 g of sea buckthorn, 7 g of liquorice, 7 g of salvia miltiorrhiza, 6 g of bighead atractylodes rhizome and 6 g of chrysanthemum. The preparation and taking method comprises the following steps: putting all materials into a marmite, and adding 3 cups of water; boiling with strong fire, decocting with slow fire for 10min, and collecting decoction; then the traditional Chinese medicine is decocted again by the same method, and the two medicines are neutralized together for oral administration. However, the herbs or prescriptions are empirical medicine, have no definite functional components, have low curative effect, slow effect, lack evaluation basis and take time for decoction, and are not easy to be accepted by drinkers due to bad smell and taste.
In recent years, enzymes are a functional food. The definition of the edible plant enzyme in the ' enzyme product classification guide "(QB/T5324) standard implemented in 2016 (9 months) of China is ' an enzyme product which is prepared by taking plants which can be used for food processing as main raw materials, adding or not adding auxiliary materials and carrying out microbial fermentation on the main raw materials and contains specific bioactive components and can be eaten by human beings '. The patent application CN202010165035 discloses a functional fermented beverage for relieving alcoholism and protecting liver and a preparation method thereof, wherein the fermented beverage comprises raw materials of 510g of red dates, 1015g of kudzuvine root powder and the balance of water; the probiotics used for fermentation of the functional fermented beverage for dispelling the effects of alcohol and protecting the liver is one of lactobacillus plantarum or pediococcus acidilactici. Although the application belongs to the enzyme category, the traditional Chinese medicine components of kudzu root and red date are used as raw materials, so that the curative effect is limited, the functional components are not clear enough, and the key evaluation basis for the effect is lacked.
Angelica keiskei Koidzumi (Angelica keiskei Koidzumi) is a perennial herbaceous plant originating in the same place of Japan, and approved as a new food material resource by the examination of the national Committee for health and wellness in 2019. The angelica keiskei koidzumi is rich in nutrition, is rich in flavonoid active substances, and has good health-care effects of resisting oxidation, resisting tumors and the like. However, the existing angelica keiskei koidzumi tea and angelica keiskei koidzumi powder are low in total flavone content and insufficient in oxidation resistance, and the effect of preventing and repairing liver diseases caused by alcoholic liver injury is still achieved, so that the effect of the angelica keiskei koidzumi product on protecting the liver is still greatly challenged.
Disclosure of Invention
The invention provides a preparation method and a quality detection method of angelica keiskei ferment aiming at the defects of the prior art.
The method is realized by the following technical scheme:
a first object of the present invention is to provide: a preparation method of angelica keiskei ferment comprises the following steps:
(1) Preparing strains:
s1 activating lactobacillus plantarum: culturing lactobacillus plantarum glycerol strain in 50mL MRS liquid culture medium for 22h according to 3% inoculation amount to obtain activated lactobacillus plantarum for later use;
s2, yeast activation: mixing and dissolving high-activity dry yeast and sterile water according to a mass-volume ratio (g/mL) of 1;
(2) Preparing raw materials:
s1, pretreatment of raw materials: cleaning collected fresh folium Artemisiae Argyi and stems, cutting into 2-5cm segments, and breaking cell wall with ultra-high pressure equipment to obtain cell wall-broken material
S2, pulping the wall-broken material, finely grinding, concentrating in vacuum, quickly freezing at-40 ℃, subliming by ice, and crushing to reach the fineness of about 300 meshes to obtain the angelica keiskei freeze-dried powder for later use;
(3) Fermentation:
s1, adding sterile water into the angelica keiskei freeze-dried powder, adding activated yeast liquid and activated plant lactobacillus liquid, uniformly stirring the mixed materials, and standing and fermenting for 70-100 hours at 37 ℃ to obtain fermentation liquor;
s2, filtering, blending, sterilizing and packaging the fermentation liquor to obtain the angelica keiskei enzyme product.
Further, the working conditions of the wall breaking treatment are as follows: the pressure is 350-650Mpa, and the time is 5-20min.
Further, the mass-volume ratio of the angelica keiskei freeze-dried powder to sterile water is 10-15 (kg/L).
The fermentation capacity of the high-activity dry yeast is more than or equal to 600mL/h.
Further, the total inoculation amount of the activated yeast and lactobacillus plantarum is 7-10%, and the strain ratio of the yeast liquid to the lactobacillus plantarum liquid is 1.
Further, the filtration is carried out by adopting diatomite or 400-800 meshes of filter cloth; the blending is to blend the taste by adding 0 to 2 percent of honey; the sterilization is homogenizing sterilization treatment by 600MPaHPP sterilization for 15-30 min.
A second object of the present invention is to provide: a quality detection method of angelica keiskei ferment comprises the following steps:
(1) Evaluation and test of quality index of angelica keiskei enzyme product
S1 adopts NaNO 2 -Al(NO 3 ) 3 Measuring the content of total flavonoids in the angelica keiskei ferment by using a NaOH chromogenic method;
s2, measuring the SOD enzyme activity by using a hydroxylamine color development method;
(2) Testing the liver protection function by adopting a mouse pre-experiment method:
s1 animal grouping and intervention
After 6-week-old SPF male Kunming mice are subjected to adaptive feeding for 7 days, randomly dividing the mice into 5 groups of 10 mice, continuously performing experiment feeding for 6 weeks for 42 days, and weighing the mice after fasting for 24 hours without water inhibition after the experiment feeding is finished; the 5 groups were fed as follows:
tomorrow leaf enzyme group (FAK): enzyme stock solution is 10mL/kg/d and 50% ethanol is 7mL/kg/d;
normal group (N): normal saline of equal volume to the FAK group;
model group 1 (ML): the volume of the normal saline and 40% ethanol which are equal to that of the FAK group is 7mL/kg/d;
model group 2 (MM): the volume of the normal saline and 50% ethanol which are equal to that of the FAK group is 7mL/kg/d;
model group 3 (MH): the volume of the normal saline and 50% ethanol which are equal to that of the FAK group is 10mL/kg/d;
s2 sample Collection
Picking eyeball and collecting blood, centrifuging at 3000r/min for 15min, separating serum, packaging, and storing in refrigerator at-80 deg.C; immediately after sacrifice, the weight of the liver, kidney and spleen was measured; rapidly placing the liver into liquid nitrogen and keeping the liver at-80 ℃;
s3, calculating a liver index according to a formula of mouse liver mass/mouse body mass multiplied by 100%;
s4 serum (TG, TC, ALT, AST) test, and the serum index experiment is designed as follows:
centrifuging blood at 4 deg.C at 3000rpm for 15min, collecting serum from the supernatant, and detecting Triglyceride (TG), total Cholesterol (TC), aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) levels in the serum;
s5 liver function (TG, ALT and AST) test, the liver function index experiment design is as follows:
liver homogenate was prepared with a high-speed homogenizer on ice using physiological saline, and then centrifuged at 1000 × g and 4 ℃ for 10min to obtain a supernatant, and Triglyceride (TG), total Cholesterol (TC), aspartate Aminotransferase (AST), and alanine Aminotransferase (ALT) levels of the liver were measured, respectively.
The adaptive feeding is free to eat and drink water under the condition of 12h light and dark circulation, the temperature is controlled to be 22-25 ℃, the humidity is controlled to be 50% -60%.
Has the advantages that:
the angelica keiskei enzyme disclosed by the invention has the effect of repairing alcoholic liver injury, and is high in safety, free of toxic and side effects, low in production cost and high in quality controllability.
1. In the aspect of raw materials, the enzyme is prepared from the angelica keiskei, has the advantages of definite types of active ingredients and controllable content of the active ingredients, is high in enzyme safety and free of toxic and side effects, and saves a preparation process for a traditional Chinese medicine compound requiring synergistic interaction of multiple traditional Chinese medicines, so that the raw material cost is reduced, the additional value of the angelica keiskei product is improved, and the enzyme is suitable for industrial production.
2. In the aspect of the method, the angelica keiskei ferment with high content of total flavonoids and superoxide dismutase (SOD) is generated by the process of combining ultrahigh pressure and fermentation, so that the effects of oxidation resistance and free radical elimination of the ferment are greatly improved, and the liver protection capability of the ferment is greatly improved.
Specifically, the method comprises the following steps: a large amount of superoxide dismutase (SOD) is generated by a fermentation process, and the defect that the traditional product only depends on flavonoid molecules to avoid the deficiency of damaged liver by antioxidation is overcome. Because SOD can effectively remove free radicals, flavonoids have strong antioxidation, and SOD combines with flavones to play a synergistic effect, the angelica keiskei ferment greatly improves the antioxidation value, and has more obvious effects of improving and repairing alcoholic liver injury.
3. On the aspect of quality control, the liver protection effect of the angelica keiskei ferment is comprehensively evaluated according to the mouse weight, liver function index, serum four index and liver function three indexes obtained by a mouse pre-experiment, and the prevention and repair effect mechanism of the angelica keiskei ferment product on alcoholic liver function injury is explained through the synergistic effect of the total flavone content and the SOD value for the first time.
Detailed Description
The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.
Example 1
A preparation method of angelica keiskei ferment comprises the following steps:
(1) Preparing strains:
s1 activating lactobacillus plantarum: culturing lactobacillus plantarum glycerol strain in 50mL MRS liquid culture medium for 22h according to 3% inoculation amount to obtain activated lactobacillus plantarum for later use;
s2, yeast activation: mixing and dissolving high-activity dry yeast (fermentation capacity is more than or equal to 600 mL/h) and sterile water according to the proportion of 1;
(2) Preparing raw materials:
s1, raw material pretreatment: cleaning collected fresh angelica keiskei leaves and stems, and cutting into 3cm;
s2, performing wall breaking treatment for 15min by using ultrahigh pressure equipment under the constant pressure of 450 MPa;
s3, pulping the wall-broken material obtained in the step, finely grinding, performing vacuum concentration, performing quick-freezing treatment at the temperature of minus 40 ℃, then performing ice sublimation, and crushing to obtain the angelica keiskei freeze-dried powder for later use, wherein the fineness of the angelica keiskei freeze-dried powder is about 300 meshes;
(3) The fermentation process comprises the following steps:
s1, adding sterile water into the angelica keiskei freeze-dried powder, and stirring to form a feed liquid, wherein the powder-water ratio is 13; adding activated yeast liquid and plant lactobacillus liquid (strain ratio is 1;
s2, filtering the fermentation liquor, adding 1% of honey, and blending the taste; sterilizing under 600MPa for 15min, and aseptic packaging to obtain Angelica keiskei ferment;
(4) Quality index evaluation and test of angelica keiskei enzyme product
The total flavone content in Angelica keiskei enzyme adopts NaNO 2 -Al(NO 3 ) 3 -NaOH chromogenic assay;
measuring the SOD enzyme activity by using a hydroxylamine color development method;
see table 1 for specific data.
Example 2
A preparation method of angelica keiskei ferment comprises the following steps:
(1) Preparing strains:
s1 activating lactobacillus plantarum: culturing lactobacillus plantarum glycerol strain in 50mL MRS liquid culture medium for 22h according to 3% inoculation amount to obtain activated lactobacillus plantarum for later use;
s2, yeast activation: mixing and dissolving high-activity dry yeast (fermentation capacity is more than or equal to 600 mL/h) and sterile water according to the proportion of 1;
(2) Preparing raw materials:
s1, raw material pretreatment: cleaning collected fresh angelica keiskei leaves and stems, and cutting into 2cm;
s2, breaking the cell wall by an ultrahigh pressure device under the constant pressure of 350MPa, and sterilizing for 20min;
s3, pulping the wall-broken material obtained in the step, finely grinding, concentrating in vacuum, quickly freezing at-40 ℃, subliming in ice, and crushing to reach the fineness of about 300 meshes to obtain the angelica keiskei freeze-dried powder for later use;
(3) The fermentation process comprises the following steps:
s1, adding sterile water into the angelica keiskei freeze-dried powder, and stirring to obtain a feed liquid, wherein the powder-water ratio is 10; adding activated yeast liquid and plant lactobacillus liquid (strain ratio is 1;
s2, filtering the fermentation liquor, adding honey to 0%, and keeping the original taste; then sterilizing for 15min under 600MPa and carrying out aseptic packaging to obtain angelica keiskei ferment;
(4) Evaluation and test of quality index of angelica keiskei enzyme product
The total flavone content in the angelica keiskei enzyme adopts NaNO 2 -Al(NO 3 ) 3 -NaOH chromogenic assay;
the SSOD enzyme activity is measured by a hydroxylamine color development method;
see table 1 for specific data.
Example 3
A preparation method of angelica keiskei ferment comprises the following steps:
(1) Preparing strains:
s1 activating lactobacillus plantarum: culturing lactobacillus plantarum glycerol strain in 50mL MRS liquid culture medium for 22h according to 3% inoculation amount to obtain activated lactobacillus plantarum for later use;
s2, yeast activation: mixing and dissolving high-activity dry yeast (fermentation capacity is more than or equal to 600 mL/h) and sterile water according to the proportion of 1;
(2) Preparing raw materials:
s1, pretreatment of raw materials: cleaning collected fresh angelica keiskei leaves and stems, and cutting into 4cm;
s2, breaking the wall under the constant pressure of 550MPa by using ultrahigh pressure equipment, and performing sterilization treatment for 10min;
s3, pulping the wall-broken material obtained in the step, finely grinding, performing vacuum concentration, performing quick-freezing treatment at the temperature of minus 40 ℃, then performing ice sublimation, and crushing to obtain the angelica keiskei freeze-dried powder for later use, wherein the fineness of the angelica keiskei freeze-dried powder is about 300 meshes;
(3) The fermentation process comprises the following steps:
s1, adding sterile water into the angelica keiskei freeze-dried powder, and stirring to obtain a feed liquid, wherein the powder-water ratio is 15; adding activated yeast liquid and plant lactobacillus liquid (strain ratio is 1;
s2, filtering the fermentation liquor, adding 0.5% of honey, and blending the taste; then sterilizing for 15min under 600MPa and carrying out aseptic packaging to obtain angelica keiskei ferment;
(4) Evaluation and test of quality index of angelica keiskei enzyme product
The total flavone content in Angelica keiskei enzyme adopts NaNO 2 -Al(NO 3 ) 3 -NaOH chromogenic assay;
measuring the SOD enzyme activity by using a hydroxylamine color development method;
see table 1 for specific data.
Example 4
A preparation method of angelica keiskei ferment comprises the following steps:
(1) Preparing strains:
s1 activating lactobacillus plantarum: culturing lactobacillus plantarum glycerol strain in 50 mM MRS liquid culture medium for 22h according to 3% inoculum concentration to obtain activated lactobacillus plantarum for later use;
s2, yeast activation: mixing and dissolving high-activity dry yeast (fermentation capacity is more than or equal to 600 mL/h) and sterile water according to the proportion of 1;
(2) Preparing raw materials:
s1, raw material pretreatment: cleaning collected fresh angelica keiskei leaves and stems, and cutting into 5cm;
s2, breaking the cell wall and sterilizing for 5min by using ultrahigh pressure equipment under the constant pressure of 650 MPa;
s3, pulping the wall-broken material obtained in the step, finely grinding, concentrating in vacuum, quickly freezing at-40 ℃, subliming in ice, and crushing to reach the fineness of about 300 meshes to obtain the angelica keiskei freeze-dried powder for later use;
(3) The fermentation process comprises the following steps:
s1, adding sterile water into the angelica keiskei freeze-dried powder, and stirring to form a feed liquid, wherein the powder-water ratio is 13; adding activated yeast liquid and plant lactobacillus liquid (strain ratio is 1;
s2, filtering the fermentation liquor, adding 2% of honey, and blending the taste; sterilizing under 600MPa for 15min, and aseptic packaging to obtain Angelica keiskei enzyme product;
(4) Quality index evaluation and test of angelica keiskei enzyme product
The total flavone content in the angelica keiskei enzyme adopts NaNO 2 -Al(NO 3 ) 3 -NaOH chromogenic assay;
measuring the SOD enzyme activity by using a hydroxylamine color development method;
see table 1 for specific data.
Example 5
A preparation method of angelica keiskei ferment comprises the following steps:
this example is a comparison of the non-ultra high pressure wall breaking of reference example 1, conducted as follows:
(1) Preparing strains:
s1 activating lactobacillus plantarum: culturing lactobacillus plantarum glycerol strain in 50mL MRS liquid culture medium for 22h according to 3% inoculation amount to obtain activated lactobacillus plantarum for later use;
s2, yeast activation: mixing and dissolving high-activity dry yeast (fermentation capacity is more than or equal to 600 mL/h) and sterile water according to the proportion of 1;
(2) Preparing raw materials:
s1, pretreatment of raw materials: cleaning collected fresh angelica keiskei leaves and stems, and cutting into 3cm;
s2, directly pulping the cut sections obtained in the step, finely grinding, carrying out vacuum concentration, carrying out quick-freezing treatment at the temperature of minus 40 ℃, then carrying out ice sublimation, and crushing to obtain the angelica keiskei freeze-dried powder for later use, wherein the fineness of the angelica keiskei freeze-dried powder is about 300 meshes;
(3) The fermentation process comprises the following steps:
s1, adding sterile water into the angelica keiskei freeze-dried powder, and stirring to obtain a feed liquid, wherein the powder-water ratio is 13; adding activated yeast liquid and plant lactobacillus liquid (strain ratio is 1;
s2, filtering the fermentation liquor, adding 1% of honey, and blending the taste; sterilizing at 600MPa for 15min under ultrahigh pressure, and aseptically packaging to obtain non-wall-broken Angelica keiskei ferment;
(4) Quality index evaluation and test of enzyme product of non-wall-broken angelica keiskei koidzumi
The total flavone content in the enzyme of non-wall-broken Angelica keiskei adopts NaNO 2 -Al(NO 3 ) 3 -NaOH chromogenic assay;
measuring the SOD enzyme activity by a hydroxylamine chromogenic method;
see table 1 for specific data.
Example 6
A preparation method of angelica keiskei primary pulp comprises the following steps:
this example is a non-fermented control according to example 1, operating as follows:
(1) Preparing raw materials:
pretreatment of raw materials: cleaning collected fresh angelica keiskei leaves and stems, and cutting into 3cm;
(2) Ultrahigh pressure wall breaking:
breaking cell wall under 450MPa and constant pressure by ultra-high pressure equipment, and sterilizing for 15min;
(3) Pulping and juice extraction:
pulping the wall-broken materials obtained in the above steps, finely grinding, and rolling to obtain juice, namely angelica keiskei products, namely raw juice;
(4) Quality index evaluation and test of primary pulp product
The total flavone content in the raw pulp adopts NaNO 2 -Al(NO 3 ) 3 -NaOH chromogenic assay;
measuring the SOD enzyme activity by using a hydroxylamine color development method;
see table 1 for specific data.
Example 7
In order to examine the effect of enzymes on preventing and repairing liver function, a mouse pre-experiment was performed using the enzyme sample obtained in example 1, and the following operations were performed:
1. animal grouping and intervention
SPF male Kunming mice (6 weeks old), controlling temperature (22-25 ℃) and humidity (50% -60%), circulating light and shade for 12h, freely eating and drinking water, after adaptive feeding for 7d, randomly dividing the mice into 5 groups of 10 mice each;
experimental feeding of group 5 was as follows:
normal group (N): and normal saline with the same volume as the ferment group.
Model group (M):
(ML) physiological saline +40% ethanol at the same volume as the ferment group 7mL/kg/d.
(MM) saline +50% ethanol in equal volume to the enzyme group and 7mL/kg/d.
(MH) saline +50% ethanol 10mL/kg/d equal volume to that of the ferment group.
(FAK) angelica keiskei enzyme group: the ferment stock solution is 10mL/kg/d +50% ethanol is 7mL/kg/d.
Continuously feeding for 6 weeks in a free diet for 42 days in an experiment, weighing, and recording the weight;
2. sample collection
After the experiment is finished, the mice are fasted for 24 hours without water supply; weighing; picking eyeball and collecting blood, centrifuging at 3000r/min for 15min, separating serum, packaging, and storing in refrigerator at-80 deg.C; immediately after sacrifice, liver, kidney and spleen weights were measured; the liver was quickly placed in liquid nitrogen and kept at-80 ℃ until further analysis;
3. mouse body weight, liver index test, experimental design and operation were as follows:
mouse weight: record initial and 42d body weights;
liver index = mouse liver mass/mouse body mass × 100%.
4. Serum (TG, TC, ALT, AST) test, serum index experimental design as follows:
the blood was centrifuged at 3000rpm for 15min at 4 ℃ and then serum was collected from the supernatant. Detecting the level of Triglyceride (TG), total Cholesterol (TC), glutamic-oxalacetic transaminase (AST) and alanine Aminotransferase (ALT) in serum;
5. liver function (TG, ALT, AST) test, liver function index experimental design and operation are as follows:
liver homogenate was prepared with physiological saline on ice with a high speed homogenizer and then centrifuged at 1000 Xg and 4 ℃ for 10min to obtain supernatant. Triglyceride (TG), total Cholesterol (TC), aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) levels of the liver were measured separately.
See tables 2-4 for the above experimental data;
TABLE 1 comparison of technical indexes of total flavone and SOD enzyme activity in each example
The cellular senescence process can be truncated or delayed by antioxidant and anti-free radical pathways, depending on the cellular current senescence mechanism. Therefore, the higher the total flavone value and the higher the SOD value in the angelica keiskei ferment, the stronger the anti-cell aging effect of the product. As can be seen from Table 1: by the ultra-high pressure wall breaking process (example 6), not only the total flavonoids are not at the maximum value, but also the SOD value is zero. On the contrary, the total flavone and SOD values are lower only by fermentation without wall breaking treatment. Only by the double process of wall breaking and fermentation (examples 1-4) can the total flavonoids and SOD values be kept in the high value range. The higher the ultrahigh pressure is, the higher the total flavone value is, but considering that the higher the ultrahigh pressure is, the higher the requirement on equipment is, the higher the energy consumption is, and the higher the cost is, the cost performance is considered in the actual process, so that the cost performance is considered in the preferred process. In the four embodiments of the patent application, the embodiment 1 basically accords with the principle, the total flavone value and the SOD value are large, the process conditions are mild, and the process belongs to an energy-saving and efficient green process.
TABLE 2 comparison of body weight and liver index in mouse Pre-experiment in example 7
* Data in parentheses are initial body weight.
If a large amount of alcohol is drunk for a long time, the incoordination between the spleen and the stomach can be caused, the symptoms such as nausea, vomiting, inappetence and the like are caused, the ingested alcohol is absorbed by the stomach and the small intestine and enters the liver through mesentery and portal vein, and the liver is the main target organ of the alcohol. Excessive drinking can result in increased permeability of hepatocytes, damage to hepatocytes and even necrosis. The metabolism of alcohol in the liver leads to enlargement of the liver, resulting in an increase in the liver index. The weight gain of the N group is 17.3%, which is a normal value, compared with the weight gain of the MH group, which is 8.0%, with the increase of the alcohol content, the weight is reduced and the increasing rule is presented. The weight of FAK is slightly changed and slightly increased compared with that of MM group, which indicates that alcohol causes the spleen and stomach to be suitable for reducing the weight, and the enzyme of Angelica keiskei Koidz has the effect of weakening or counteracting the side effect.
The liver indexes of the N groups are the lowest and belong to the normal condition, compared with the N groups, the liver indexes of the ML, MM and MH groups are obviously increased and are increased along with the increase of the alcohol content, and the dosage correlation is shown, which indicates that the alcohol has obvious damage to the liver function; compared with the group M, the liver index of the FAK group is reduced, and the Angelica keiskei Koidz enzyme FAK is proved to have a repairing effect on alcoholic liver injury.
Table 3 example 7 serum index technical index comparison
Because the liver is a metabolic organ of alcohol and fat, excessive intake of alcohol can cause the reduction of the metabolic function of liver fat, lead to the accumulation of lipid substances in the liver, further cause fatty lesion of the liver, and directly cause the increase of Triglyceride (TG) and Total Cholesterol (TC) indexes in blood. ALT and AST are two important transaminases present in the cytoplasm and mitochondria of the liver, and are normally present in low serum levels, and when hepatocytes are damaged or necrotic, ALT and AST infiltrate the blood. Therefore, the level of ALT and AST in serum is a specific indicator of the degree of damage to liver cells.
The N groups of indices were regarded as normal values, and the higher the four indices compared with the N group, the more serious the hepatic function damage. As can be seen from the data in Table 3, the TG indexes of ML, MM and MH groups increase with the increase of the alcohol content, and the alcoholic injury tends to increase. Meanwhile, the Angelica keiskei ferment FAK group can reduce four indexes compared with ML, MM and MH groups. Wherein, after FAK is dried, the TG content is obviously reduced, and the TC, AST and ALT contents also show the trend of reducing to different degrees. Therefore, the angelica keiskei enzyme FAK intervention has obvious improvement and repair effects on the alcoholic liver injury of mice.
Table 4 example 7 liver function index comparison
In clinical medicine, when ALT and AST are increased, liver damage is indicated; AST/ALT > 1, indicating liver necrosis. The N groups of indices were considered as normal values, and the higher the three indices compared to the N group, the more severe the liver function impairment was. As can be seen from the data in Table 4, the three indexes in the livers of ML, MM and MH groups show an increasing trend along with the increase of the alcohol content, but the indexes show an obvious decreasing trend after FAK intervention. Therefore, the angelica keiskei ferment FAK has a remarkable improvement or repair effect on the alcoholic liver injury of the mice.
It should be noted that the embodiments described in this patent only serve to put into effect one-to-three actions, and the processes implemented by changing the process conditions or adding other food materials and food additives thereof still belong to the protection scope of this patent application.
Claims (7)
1. The preparation method of the angelica keiskei ferment is characterized by comprising the following steps of:
(1) Preparing strains:
s1 activating lactobacillus plantarum: culturing lactobacillus plantarum glycerol strain in 50 mM MRS liquid culture medium for 22h according to 3% inoculum size to obtain activated lactobacillus plantarum for later use;
s2, yeast activation: mixing and dissolving high-activity dry yeast (fermentation capacity is more than or equal to 600 mL/h) and sterile water according to a mass-volume ratio (g/mL) of 1;
(2) Preparing raw materials:
s1, pretreatment of raw materials: cleaning collected fresh Angelica keiskei leaves and stems, cutting into 2-5cm small segments, and breaking cell wall with ultrahigh pressure equipment to obtain cell wall-broken material
S2, pulping the wall-broken material, finely grinding, concentrating in vacuum, quickly freezing at-40 ℃, subliming by ice, and crushing to reach the fineness of about 300 meshes to obtain the angelica keiskei freeze-dried powder for later use;
(3) Fermentation:
s1, adding sterile water into the angelica keiskei freeze-dried powder, adding activated yeast liquid and activated plant lactobacillus liquid, uniformly stirring the mixed materials, and standing and fermenting for 70-100 hours at 37 ℃ to obtain fermentation liquor;
s2, filtering, blending, sterilizing and packaging the fermentation liquor to obtain the angelica keiskei enzyme product.
2. The method for preparing angelica keiskei ferment as claimed in claim 1, wherein the working conditions of the wall-breaking treatment are as follows: the pressure is 350-650Mpa, and the time is 5-20min.
3. The method for preparing angelica keiskei ferment as claimed in claim 1, wherein the mass-to-volume ratio of the angelica keiskei freeze-dried powder to the sterile water is 10-15 (kg/L).
4. The method according to claim 1, wherein the total inoculum size of the activated yeast and lactobacillus plantarum is 7-10%, and the ratio of the yeast liquid to the lactobacillus plantarum strain is 1.
5. The method for preparing the angelica keiskei ferment as claimed in claim 1, wherein the filtration is performed by using diatomite or 400-800 mesh filter cloth; the blending is to blend the taste by adding 0 to 2 percent of honey; the sterilization is homogenizing sterilization treatment by 600MPaHPP sterilization for 15-30 min.
6. The method for detecting the quality of angelica keiskei ferment as claimed in any one of claims 1 to 5, comprising the steps of:
(1) Evaluation and test of quality index of angelica keiskei enzyme product
S1 adopts NaNO 2 -Al(NO 3 ) 3 Measuring the content of total flavonoids in the angelica keiskei ferment by using a NaOH chromogenic method;
s2, measuring the SOD enzyme activity by using a hydroxylamine color development method;
(2) Testing the liver protection function by adopting a mouse pre-experiment method:
s1 animal grouping and intervention
After 6-week-old SPF male Kunming mice are subjected to adaptive feeding for 7 days, randomly dividing the mice into 5 groups of 10 mice, continuously performing experiment feeding for 6 weeks for 42 days, and weighing the mice after fasting for 24 hours without water inhibition after the experiment feeding is finished; the 5 groups were fed as follows:
tomorrow leaf enzyme group (FAK): enzyme stock solution 10mL/kg/d +50% ethanol 7mL/kg/d;
normal group (N): normal saline of equal volume to the FAK group;
model group 1 (ML): the volume of the normal saline and 40% ethanol which are equal to that of the FAK group is 7mL/kg/d;
model set 2 (MM): 7mL/kg/d of physiological saline and 50% ethanol with the same volume as the FAK group;
model group 3 (MH): the volume of the normal saline and 50% ethanol which are equal to that of the FAK group is 10mL/kg/d;
s2 sample Collection
Picking eyeball and collecting blood, centrifuging at 3000r/min for 15min, separating serum, packaging, and storing in refrigerator at-80 deg.C; immediately after sacrifice, liver, kidney and spleen weights were measured; rapidly placing the liver into liquid nitrogen and keeping the liver at-80 ℃;
s3, calculating a liver index according to a formula of mouse liver mass/mouse body mass multiplied by 100%;
s4 serum (TG, TC, ALT, AST) test, and the serum index experiment is designed as follows:
centrifuging blood at 4 deg.C at 3000rpm for 15min, collecting serum from the supernatant, and detecting Triglyceride (TG), total Cholesterol (TC), aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) levels in the serum;
s5 liver function (TG, ALT and AST) test, the liver function index experiment design is as follows:
liver homogenate was prepared on ice using a high speed homogenizer using physiological saline, and then centrifuged at 1000 xg and 4 ℃ for 10min to obtain a supernatant, and Triglyceride (TG), total Cholesterol (TC), aspartate Aminotransferase (AST), and alanine Aminotransferase (ALT) levels of the liver were measured, respectively.
7. The method for detecting the quality of angelica keiskei ferment as claimed in claim 1, wherein the adaptive feeding is free eating and drinking water under 12h light and dark cycle conditions with the temperature controlled at 22-25 ℃ and the humidity controlled at 50% -60%.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105124571A (en) * | 2015-07-24 | 2015-12-09 | 唐山师范学院 | Preparation method of sport nutrient supplement agent by fermenting angelica keiskei through lactic acid bacteria |
| CN106333355A (en) * | 2015-07-06 | 2017-01-18 | 莫沉鹏 | Preparation method of pumpkin angelica keiskei enzyme |
| CN112006274A (en) * | 2020-08-24 | 2020-12-01 | 上海交通大学 | Preparation method of plant enzyme |
| CN113197272A (en) * | 2021-05-18 | 2021-08-03 | 青岛三丰明日叶农业科技有限公司 | Small peptide and angelica keiskei freeze-dried powder beverage capable of activating blood vessels and resisting aging and preparation method thereof |
| CN114522218A (en) * | 2022-03-14 | 2022-05-24 | 唐建 | Composition for quickly dispelling effects of alcohol, protecting liver and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106333355A (en) * | 2015-07-06 | 2017-01-18 | 莫沉鹏 | Preparation method of pumpkin angelica keiskei enzyme |
| CN105124571A (en) * | 2015-07-24 | 2015-12-09 | 唐山师范学院 | Preparation method of sport nutrient supplement agent by fermenting angelica keiskei through lactic acid bacteria |
| CN112006274A (en) * | 2020-08-24 | 2020-12-01 | 上海交通大学 | Preparation method of plant enzyme |
| CN113197272A (en) * | 2021-05-18 | 2021-08-03 | 青岛三丰明日叶农业科技有限公司 | Small peptide and angelica keiskei freeze-dried powder beverage capable of activating blood vessels and resisting aging and preparation method thereof |
| CN114522218A (en) * | 2022-03-14 | 2022-05-24 | 唐建 | Composition for quickly dispelling effects of alcohol, protecting liver and preparation method thereof |
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