Extraction method of black garlic extract and application thereof

Abstract

The application discloses an extraction method of black garlic extract and application thereof. The extraction method comprises the following steps: fermenting fresh garlic into black garlic by using a high-temperature and high-humidity liquid fermentation mode, inactivating activated enzyme allinase in the black garlic by using a microwave enzyme inactivation method, selecting 26% ethanol as an extraction solvent, centrifuging a solution containing the black garlic extract to obtain supernatant, and combining and drying the supernatant to constant weight to obtain the black garlic extract. The application takes alliin as an object in the black garlic extract, optimizes a method of preparing the black garlic extract by using the microwave enzyme inactivation method, explores an action mechanism of the black garlic extract by combining in-vivo and in-vitro antioxidant experiments and network pharmacology target function prediction, applies the black garlic extract to pet main food for the first time, explores effects in the anti-aging and other health fields, and verifies actual effects and industrial production by feeding experiments of pet cats.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to an extraction method of black garlic extract and application thereof. BACKGROUND

[0002] Garlic is probably one of the earliest medicinal plants known, and has been used since ancient times to treat a variety of diseases in humans. The main medicinal effects of garlic include lowering blood pressure and cholesterol, resisting infection, and preventing cancer. Garlic contains about 33 active sulfur-containing compounds, of which the main component S-allyl-L-cysteine sulfoxid (SACS, Allicin) can be rapidly absorbed and metabolized. S-allyl-L-cysteine sulfoxid is a precursor of allicin, easily soluble in water, odorless, non-volatile, and has a stereochemical structure centered on carbon and sulfur atoms, and is the main source of medicinal and flavor compounds produced by plants in the Alliaceae family when the tissue is damaged. Studies have shown that garlic can reduce total cholesterol concentration by about 10%, and is beneficial to improving the ratio of high-density lipoprotein (HDL) and low-density lipoprotein (LDL). Garlic can be used as a mild antihypertensive drug, which can reduce blood pressure by 5-7%. Garlic also inhibits platelet aggregation and enhances fibrinolytic activity, thereby reducing thrombosis on the damaged endothelium. Another important use of garlic is anti-diabetes. Garlic controls blood sugar levels through multiple mechanisms. In vitro studies and animal data also suggest that garlic may help prevent certain solid tumors. Therefore, garlic also shows effectiveness in cancer prevention. Other proposed uses of garlic include liver protection, deworming, anti-inflammatory, antioxidant, antifungal, and wound healing, etc.

[0003] Compared with fresh garlic, black garlic is a food made from fresh garlic under high temperature and high humidity conditions for a certain period of time. After processing, the taste of garlic becomes soft and sweet, reducing the irritating odor of garlic, while retaining the ingredients, further improving the nutritional value of garlic. The antioxidant activity of black garlic is higher than that of fresh garlic, but its efficacy and mechanism of action need further study. SUMMARY

[0004] In order to overcome the deficiencies of the prior art, the purpose of the present application is to provide an extraction method of black garlic extract and application thereof.

[0005] The technical solution of the present application to achieve its purpose is as follows:

[0006] An extraction method of black garlic extract, the steps are as follows: fresh garlic is first fermented into black garlic by using high-temperature and high-humidity liquid fermentation method, then microwave enzyme inactivation method is used to inactivate the activated enzyme allinase in black garlic, and 26% ethanol by volume is selected as the extraction solvent. The supernatant obtained by centrifuging the solution containing black garlic extract is combined and dried to constant weight, which is the black garlic extract.

[0007] The extraction method, the ratio of material to liquid is 5:3, the fermentation temperature is 80 DEG C, and the fermentation time is 7 days.

[0008] A functional cat food, containing 0.1% black garlic extract by mass percentage, is used for preventing and improving the liver function of pet cats.

[0009] The cat food is a special full-value cat food for base cats, containing 0.1% black garlic extract by mass percentage, and the formula is as follows:

[0010] Frozen chicken breast 80%, frozen chicken heart 5.0%, frozen chicken liver 5%, sweet potato powder 3%, egg yolk powder 1%, chicken oil 1%, fucoidan 0.2%, cellulose 0.2%, yeast extract 0.2%, 0.1% black garlic extract, and the rest is additives.

[0011] The additives include taurine, vitamin A acetate, vitamin D3, DL-a-tocopherol acetate, vitamin B1, vitamin B2, vitamin B6, vitamin B12, nicotinic acid, D-calcium pantothenate, folic acid, D-biotin, glycine iron chelate, glycine copper chelate, manganese amino acid complex, zinc amino acid complex, calcium iodate, selenium yeast, choline chloride, calcium carbonate, rosemary extract.

[0012] A functional cat food, the base cat food contains 0.1% black garlic extract by mass percentage, and is used for anti-aging.

[0013] Application of black garlic extract in establishing a Caenorhabditis elegans model is used to explore the mechanism of allicin in prolonging the life of Caenorhabditis elegans and improving stress response.

[0014] Application of black garlic extract in establishing a Caenorhabditis elegans model is used to explore the mechanism of allicin in improving and relieving liver lesions.

[0015] The beneficial effects of the present application are:

[0016] The present application takes allin as the object in black garlic extract, optimizes the microwave enzyme inactivation process preparation method, explores the action mechanism of black garlic extract through network pharmacology target function prediction combined with in vivo and in vitro antioxidant experiments, and applies black garlic extract to pet main food for the first time, explores the effect in the field of anti-aging and other health, and verifies the actual effect and industrial production through pet cat feeding test. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Effect of different material-liquid ratios on total acid of liquid-state fermented black garlic.

[0018] Figure 2 Effect of different temperatures on total acid of liquid-state fermented black garlic.

[0019] Figure 3(A) is the detection of the antioxidant performance of black garlic extract (reducing power of allin).

[0020] Figure 3(B) is the detection of the antioxidant performance of black garlic extract (reducing power of VC standard solution).

[0021] Figure 3(C) is the detection of the antioxidant performance of black garlic extract (relationship between dilution multiple of allin and DPPH clearance rate).

[0022] Figure 3(D) is the detection of the antioxidant performance of black garlic extract (relationship between concentration of VC and DPPH clearance rate).

[0023] Figure 3(E) is the detection of the antioxidant performance of black garlic extract (ABTS free radical scavenging ability of black garlic extract).

[0024] Figure 3(F) is the detection of the antioxidant performance of black garlic extract (ABTS free radical scavenging ability of VC solution).

[0025] Figure 4 Figure 4 is the antioxidant protection effect of garlic extract on C. elegans (survival curve of C. elegans in different treatment groups).

[0026] Figure 5(A) is the mechanism of allin for treating alcoholic liver disease analyzed by network pharmacology (venn diagram of intersection of allin and ALD target points).

[0027] Figure 5(B) is the mechanism of allin for treating alcoholic liver disease analyzed by network pharmacology (PPI network diagram of intersection of allin target points and ALD intersection target points).

[0028] Figure 5(C) is the mechanism of allin for treating alcoholic liver disease analyzed by network pharmacology (bubble chart of biological process (BP) enrichment analysis).

[0029] Figure 5(D) is the mechanism of allin for treating alcoholic liver disease analyzed by network pharmacology (bubble chart of cellular component (CC) enrichment analysis).

[0030] Figure 5(E) is the mechanism of allin for treating alcoholic liver disease analyzed by network pharmacology (bubble chart of molecular function (MF) enrichment analysis).

[0031] Figure 5(F) is the mechanism of allin for treating alcoholic liver disease analyzed by network pharmacology (bubble chart of KEGG enrichment analysis). DETAILED DESCRIPTION

[0032] The specific embodiments of the present application will be further described in detail below with reference to the accompanying drawings.

[0033] Example 1, Black Garlic Processing Optimization and Alliin Extraction

[0034] Step 1: Black garlic fermentation. High-quality garlic was selected and the 1-2 layers of skin and stem were removed, then washed and subjected to high-temperature and high-humidity liquid fermentation, and finally quality inspection to obtain finished black garlic. The processing humidity was 80%, the processing temperature was 70°C, and the processing time was 168 h (i.e. 7 days). The fermentation parameters were as follows: the solid-liquid ratio was 5:0, 5:2, 5:3 and 5:4; the fermentation temperature was 70°C, 80°C and 90°C, and the fermentation period was 7 days. The purpose of this example was to optimize the extraction method to improve the fermentation efficiency of active substances in black garlic, so the total acid (g / kg) content was determined to evaluate the fermentation parameters.

[0035] Step 2: Microwave enzyme inactivation method was selected in this experiment. The microwave power was 300W, and the enzyme inactivation time was 30s, 60s and 90s, respectively.

[0036] Step 3: Black garlic extract drying. Volume fraction 26% ethanol was selected as the extraction solvent, and the extraction was carried out for 60 minutes. The black garlic extract solution was centrifuged to obtain the supernatant, which was then combined and dried to constant weight, resulting in black garlic extract solid powder.

[0037] Step 4: Preparation of black garlic extract solution. Accurately weigh 10.0 g of black garlic solid powder sample, add 100 mL of 10% pure methanol (volume fraction), and crush it to a thin paste with a juicer. After ultrasonic treatment (intensity 70%) at room temperature for 15 min, filter with filter paper, and centrifuge the clear filtrate at 8000 r·min-1 for 15 min. The supernatant was diluted to 250 mL in a volumetric flask, and the extract was diluted to the mark. This step is used for antioxidant capacity determination of black garlic extract.

[0038] The detection results are as follows: the purpose of this extraction method is to improve the extraction efficiency of allicin, so the fermentation efficiency is one of the key factors affecting the extraction efficiency. The purpose of fermentation is to increase the content of allicin, and the total acid is used as the evaluation index. The results are shown in Figure 1 and Figure 2 Under different fermentation parameters, the total acid content of most groups reached the peak value after 6 days of fermentation. When the acidity of black garlic reached 50 g / kg, the time required for 5:0, 5:1, 5:2, 5:3 and 5:4 solid-liquid ratio was 5 days, 5 days, 7 days, 7 days and more than 7 days, respectively. As shown in Figure 1 , the higher the moisture content, the longer the time required to reach the same acidity. The fermentation reached the peak time shortest at a solid-liquid ratio of 5:0, and longest at a solid-liquid ratio of 5:4. Considering the economic value, the peak time was moderate at a solid-liquid ratio of 5:3, and the black garlic addition was also lower. In addition, the fermentation efficiency under different temperatures was also compared (as shown in Figure 2The higher the temperature, the shorter the time to reach the peak of total acid, considering the economic benefits, the fermentation temperature is selected as 80℃, the temperature is too high (90℃), the fermentation efficiency is not significantly improved and the cost is high, the fermentation temperature is too low (70℃), the fermentation efficiency is reduced and the fermentation time is longer.

[0039] Example 2, antioxidant capacity detection of black garlic extract

[0040] Reducing power determination

[0041] The black garlic extract solution was diluted to different concentration gradients. 36 mL of the gradient-diluted black garlic extract solution was taken, 247 mL of phosphate buffer (0.2 mol / L, pH = 6.6) and 250 mL of 1% potassium ferricyanide solution were added, and the mixture was mixed. Under the condition of 50℃, react for 30 min, add 250 μL of 10% trichloroacetic acid, stand for 10 min. Take 100 μL of supernatant, add 100 μL of deionized water, add 20 μL of 10% ferric trichloride solution, and divide the prepared solution into three times into the enzyme-labeled plate, and measure the absorbance at 700 nm. The same conditions as above, except that the black garlic extract solution is replaced by a series of concentration VC solution, and the obtained solution is used as a control. As shown in Figure 3(A) and Figure 3(B).

[0042] 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid, ABTS) activity determination

[0043] 1 mL of black garlic extract solution and 1 mg / L of VC solution were diluted to different concentration gradients, 8 μL of gradient-diluted black garlic extract solution was added to 200 μL of ABTS free radical PBS solution and 12 μL of PBS solution and mixed, at the same time, deionized water was used instead of black garlic extract solution as a blank group, 8 μL of black garlic extract solution was added to 212 μL of PBS solution and mixed, as a model control. After adding the sample, react for 1 h at 30℃, and measure the absorbance at 734 nm. The same conditions as above, except that the black garlic extract solution is replaced by a gradient-diluted VC solution, and the obtained solution is used as a positive control. Each mass concentration was done in duplicate. The results are shown in Figure 3(E) , 3(F) .

[0044] 2,2-Diphenyl-1-picrylhydrazyl (DPPH) activity determination

[0045] The black garlic extract was diluted to different concentration gradients. Take 400 μL of gradient diluted black garlic extract, add 800 μL, 0.1 mmol / L DPPH-methanol solution and 90 μL, 50 mmol / L Tris-HCl buffer and mix well. Place in a 25°C incubator away from light for 30 minutes, and measure the absorbance at 517 nm. Three parallel experiments were performed in each group. The VC stock solution was diluted with different mass concentrations of VC solution instead of black garlic extract as a positive control, and two parallel experiments were performed for each mass concentration. The results are as follows. Figure 3(C) 、 3(D) shown.

[0046] The test results show that black garlic extract contains high concentrations of alliin, as well as phenolic and flavonoid compounds, and exhibits strong superoxide dismutase (SOD) activity. These compounds enhance the antioxidant capacity of black garlic extract. Therefore, its antioxidant activity was evaluated using multiple in vitro antioxidant indices, including reducing power, DPPH free radical scavenging rate, and ABTS free radical scavenging rate. The results show that the DPPH free radical scavenging rate decreases with increasing dilution of the black garlic extract. At a 50x dilution, the DPPH free radical scavenging rate reaches 95%, comparable to that of a 0.1 mg / mL vitamin C solution. Furthermore, the reducing power and ABTS free radical scavenging rate of the black garlic extract are also close to those of a standard vitamin C solution. These results demonstrate that black garlic extract exhibits excellent in vitro antioxidant activity, with the 50x dilution being comparable to that of vitamin C, suggesting significant potential for application.

[0047] Example 3: Detection of the protective effect of black garlic extract on oxidative damage in Caenorhabditis elegans

[0048] Model organism Caenorhabditis elegans (C. elegans): As a health food, black garlic has gradually become more popular in recent years. The unique sulfur-containing active substance alliin has antioxidant, anti-inflammatory and immunomodulatory effects. The unique advantages of C. elegans make it an ideal biological model for anti-aging research. A large number of anti-aging drugs have been screened through C. elegans. Through lifespan experiments, the effect of alliin and related sulfides on the lifespan of nematodes can be explored. The gene regulatory network of C. elegans is very sensitive to changes in environmental factors and produces stress responses to some environmental factors such as drugs, toxins, temperature, and stress. By studying the stress response mechanism of C. elegans, we can gain a deeper understanding of the molecular basis of biological adaptation to the environment and the impact of the environment on health and disease.

[0049] ① Nematode amplification and synchronization

[0050] Take a piece of prepared NGM medium for nematodes, add 800 μl of E. coli OP50 solution, shake well, and air dry. Prepare a clean, nematode-rich medium in advance. Using a sterilized scalpel, cut a small piece from an area with a high concentration of nematodes and place it upside down in the center of a new medium. Incubate in a 20°C incubator for 2 days. Select a medium containing a large number of egg-laying hermaphroditic nematodes. Wash the medium several times with M9 buffer, then add 2 mL of nematode lysis buffer. After lysis, transfer the washed eggs to NGM medium for nematodes and incubate in a 20°C incubator for 48 hours.

[0051] ② Regulation of antioxidant levels in nematodes by black garlic extract

[0052] A blank control group, a model group, and five groups of NGM culture media containing 40uL / mL, 80uL / mL, and 160uL / mL black garlic extract were prepared respectively. About 20 synchronized Caenorhabditis elegans were selected from each group. Then, 10μL of 15% hydrogen peroxide was added to the culture medium of each group. The number of surviving Caenorhabditis elegans was recorded every half hour until all the nematodes died. Survival curves were drawn based on the survival and death time of the nematodes.

[0053] The experimental results are as follows: The oxidative damage model of Caenorhabditis elegans was established by stimulating it with hydrogen peroxide, and the nematodes were protected by three concentrations of black garlic extract: low (40μl / mol), medium (80μl / mol), and high (160μl / mol). The results showed that ( Figure 4 (Table 1) Oxidative damage caused by hydrogen peroxide significantly reduced the average lifespan of nematodes from 178 minutes to 96 minutes. However, treatment with black garlic extract, particularly in the medium and high-dose groups, demonstrated significant antioxidant protection. The average lifespan of nematodes in the medium-dose group was significantly higher than that of the model group, while the high-dose group increased the average lifespan to a level insignificantly different from that of the blank group. This demonstrates that the in vivo antioxidant activity of black garlic extract has been strongly validated, suggesting its potential role in anti-aging and lifespan extension.

[0054] Table 1 shows the antioxidant protective effect of garlic extract on Caenorhabditis elegans (average lifespan and maximum lifespan of Caenorhabditis elegans in different treatment groups)

[0055]

[0056] Example 4: Target screening and network construction using alcoholic liver disease as a model

[0057] The target points of allitride were obtained by using the CHEMBL database in a "fishing" way, and the target points of allitride were collected through the SwissTargetPrediction database, obtaining 146 target points. Through the OMIM database and GeneCards data database, the disease "alcoholic liver disease" was obtained, and the target point 1211 was obtained. After sorting, 88 common target points of allitride and alcoholic liver disease were obtained, which were imported into the String platform, and the "Organism" option was selected as "Homo Sapiens" for analysis. The network topology analysis was performed by using Cytoscape 3.9.1 software.

[0058] In order to clarify the biological function of the key target points of allitride and alcoholic liver disease and its relationship with the signal pathways in vivo, the intersection target points of allitride and alcoholic liver disease were input into the DAVID online database (https: / / david.ncifcrf.gov / home.jsp), the name list of target genes was input and the species was limited to "homo sapiens", the G0 enrichment data was obtained, which was exported, and the representative items were selected for G0 (gene or tola) biological process enrichment analysis. The biological reaction processes, molecular functions and cell locations of allitride and alcoholic liver disease in target points were obtained. In addition, the G0 function enrichment analysis bubble was drawn by using the online drawing website-BioLadder (https: / / www.bioladder.cn / web / # / pro / cloud). At the same time, in the Pathwany of the DAVID online database (https: / / david.ncifcrf.gov / home.jsp), "KEGG_PATHWAY" was selected, and the species was limited to "homoszpiens", so that the KEGG (Kyoto Encyclopedia of Genes and Genecmes) signal pathway enrichment result was obtained. After exporting the result, the KEGG pathway was mapped and analyzed in detail, and the biological processes and involved signal pathways of the potential target points of allitride in treating alcoholic liver disease were explored.

[0059] In order to further explore the medical application of allitride, the alcoholic liver disease was taken as a model, and the possible target points and pathways of allitride in treating the disease were analyzed by network pharmacology. The 88 common target points of allitride and alcoholic liver disease were further analyzed by protein-protein interaction (PPI) relationship and gene function and pathway enrichment analysis (Figures 5(A)-5(F)).

[0060] The results show that in the field of gene ontology function, alliin may improve the symptoms of alcoholic liver disease patients by improving chemical synaptic transmission, locomotory behavior and the ability to respond to xenobiotic stimuli, and preventing further damage to liver cells. In addition, in terms of KEGG pathways, alliin may improve the antioxidant damage resistance of liver cells by regulating neuroactive ligand-receptor interaction, glutamatergic synapse and other metabolic pathways including glutamate metabolism, and alleviate the symptoms of alcoholic liver disease.

[0061] According to the cat body condition score system (Body Condition Score, BCS), the score of overweight domestic cats is more than 6, that is, the ribs are not obvious, the cat has obvious fat accumulation, the waist is slightly flat, the abdomen is slightly protruding, and most of the cats are accompanied by mild fatty liver. Select 15 overweight (BCS>6) cats with similar age and weight, and randomly divide them into 3 groups, 5 cats in each group. Group A is the control group, fed with basic cat special complete cat food, group B is the low-dose experimental group, fed with basic cat special complete cat food containing 0.1% black garlic extract, and group C is the high-dose experimental group, fed with basic cat special complete cat food containing 0.3% black garlic extract, for 6 weeks.

[0062] The basic cat special complete cat food formula is as follows:

[0063] Raw material composition: frozen chicken breast 80%, frozen chicken heart 5.0%, frozen chicken liver 5%, sweet potato powder 3%, egg yolk powder 1%, chicken oil 1%, algin polysaccharide 0.2%, cellulose 0.2%, yeast extract 0.2%.

[0064] Additive composition: glycerol, taurine, salt, L-calcium lactate, ferrous sulfate, zinc sulfate, copper sulfate, magnesium sulfate, manganese sulfate, potassium iodide, choline chloride, vitamin A acetate, vitamin D3, dl-α-tocopherol acetate, vitamin K3, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, D-calcium pantothenate, biotin, folic acid.

[0065] The detection indexes are as shown in Tables 2 and 3: body weight, feed intake, feces condition, liver function indexes (alanine aminotransferase ALT, aspartate aminotransferase AST, alkaline phosphatase ALP, glutamyl transferase GGT, total bilirubin TBIL).

[0066]

[0067]

[0068] The feeding results are as follows: before feeding, the average weight of the three groups is close, between 6.31-6.43 kg, after feeding, the weight of the low-dose group and the high-dose group decreases slightly, and the weight of the control group is almost unchanged, and the feces is normal. In terms of liver function indicators, the average total bilirubin TBIL of 15 cats before feeding is close to the critical value, indicating that the liver function of some experimental cats may have certain inflammation or damage, suggesting that it may be related to overweight, after feeding for 6 weeks, the TBIL of the low-dose group and the high-dose group decreases to a certain extent, by 0.82 and 0.66 respectively. In terms of other liver function indicators, alanine aminotransferase ALT is one of the most sensitive indicators of liver function damage, the average value of the experimental cats is within the normal range, among which the low-dose group decreased by 11.27 after 8 weeks of feeding, the high-dose group decreased by 12.31, and the control group decreased by 0.82, basically unchanged. In addition, the aspartate aminotransferase AST, alkaline phosphatase ALP and glutamyl transferase GGT of the low-dose group and the high-dose group decreased to varying degrees, this feeding result shows that the addition of different doses of black garlic extract can improve some liver function indicators of overweight cats, but the improvement effect of 0.1% and 0.3% is not obvious, suggesting that black garlic extract has a certain preventive and therapeutic effect on the liver function of cats.

[0069] The above only lists some specific embodiments of the present application, it should be noted that the present application is not limited to the above embodiments, there can be many other variations, including changes in the parameters of the black garlic extract process (without affecting the core process), application of black garlic extract on other pet models (normal size dogs and cats, obese dogs and cats, etc.), changing the addition ratio of black garlic extract. The above content is only an example and description of the present application, those skilled in the art can make various modifications or supplements to the described specific embodiments or use similar ways to replace, as long as it does not deviate from the invention or exceed the scope defined by the present claims, it should belong to the protection scope of the present application.

Claims

1. A method for extracting black garlic extract, characterized in that, The steps are as follows: fresh garlic is first fermented into black garlic using a high-temperature and high-humidity liquid fermentation method, and then the activating enzyme allinase in the black garlic is inactivated by a microwave inactivation method. 26% by volume ethanol is selected as the extraction solvent. The solution containing the black garlic extract is centrifuged to obtain the supernatant, which is then combined and dried to a constant weight to obtain the black garlic extract. The fermentation parameters were as follows: material-liquid ratio of 5:3, fermentation temperature of 80°C, and fermentation time of 7 days.

2. A functional cat food, characterized in that: The invention comprises 0.1% by mass of black garlic extract, and is used for preventing and improving the liver function of pet cats, wherein the black garlic extract is prepared according to the extraction method according to claim 1.

3. The cat food according to claim 2, wherein This is a complete cat food for cats containing 0.1% black garlic extract by mass. The formula is as follows: Frozen chicken breast 80%, frozen chicken heart 5.0%, frozen chicken liver 5%, sweet potato powder 3%, egg yolk powder 1%, chicken fat 1%, seaweed polysaccharide 0.2%, cellulose 0.2%, yeast extract 0.2%, black garlic extract 0.1%, the remainder is additives.

4. The cat food according to claim 3, wherein The additives include taurine, vitamin A acetate, vitamin D3, DL-a-tocopheryl acetate, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, D-calcium pantothenate, folic acid, D-biotin, glycine iron chelate, glycine copper chelate, amino acid manganese complex, amino acid zinc complex, calcium iodate, yeast selenium, choline chloride, calcium carbonate, and rosemary extract.

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