Extraction method and application of black garlic extract

Abstract

The invention discloses an extraction method and application of a black garlic extract. The extraction method comprises the following steps: fermenting fresh garlic into black garlic in a high-temperature and high-humidity liquid state fermentation manner, then inactivating an activating enzyme allinase in the black garlic by adopting a microwave enzyme inactivation method, selecting ethanol with the volume fraction of 26% as an extraction solvent, centrifuging a solution containing the black garlic extract to obtain supernate, combining, and drying until the weight is constant, so as to obtain the black garlic extract. According to the invention, alliin in the black garlic extract is taken as an object, a microwave enzyme deactivation process preparation method is optimized, the action mechanism of the black garlic extract is explored through combination of network pharmacology target function prediction and in-vivo and in-vitro antioxidant experiments, and meanwhile, the black garlic extract is applied to staple food for pets for the first time; the effects in the large health fields such as aging resistance are explored, and the actual effects and industrial production are verified through feeding tests of pet cats.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an extraction method of black garlic extract and application thereof. Background Art

[0002] Garlic may be one of the earliest known medicinal plants and has been used to treat a variety of human ailments since ancient times. Its primary medicinal benefits include lowering blood pressure and cholesterol, fighting infection, and preventing cancer. Garlic contains approximately 33 active sulfur-containing compounds, the primary of which is alliin (S-allyl-L-cysteine ​​Sulfoxid, SACS), which is rapidly absorbed and metabolized. Alliin, a precursor to allicin, is readily soluble in water, odorless, and non-volatile. Its stereochemical structure is centered around carbon and sulfur atoms, and it is the primary source of medicinal and flavor compounds produced by plants in the Allicaceae family in response to tissue damage. Studies have shown that garlic can lower total cholesterol levels by approximately 10% and improve the ratio of high-density lipoprotein (HDL) to low-density lipoprotein (LDL). Garlic can be used as a mild antihypertensive agent, lowering blood pressure by 5-7%. Garlic also inhibits platelet aggregation and enhances fibrinolytic activity, thereby reducing thrombosis in damaged endothelial tissue. Another important use of garlic is its antidiabetic properties. Garlic regulates blood sugar levels through multiple mechanisms. In vitro studies and animal data also suggest that garlic may help prevent certain solid tumors. Therefore, garlic has also shown effectiveness in cancer prevention. Other proposed uses for garlic include liver protection, anthelmintic, anti-inflammatory, antioxidant, antifungal, and wound healing.

[0003] Compared to fresh garlic, black garlic is a food product made by processing fresh garlic under high temperature and humidity for a certain period of time. This processing results in a softer and sweeter taste, reducing the original pungent odor. While retaining its ingredients, it also enhances its nutritional value. Black garlic exhibits higher antioxidant activity than fresh garlic, but its efficacy and mechanism of action warrant further investigation. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the present invention aims to provide a method for extracting black garlic extract and application thereof.

[0005] The technical solution for achieving the purpose of the present invention is as follows: A method for extracting black garlic extract comprises the following steps: firstly fermenting fresh garlic into black garlic by a high-temperature and high-humidity liquid fermentation method, then inactivating the activating enzyme allinase in the black garlic by a microwave inactivation method, selecting 26% by volume ethanol as an extraction solvent, centrifuging a solution containing the black garlic extract to obtain a supernatant, combining the supernatants, and drying to a constant weight to obtain the black garlic extract.

[0006] The extraction method has a material-liquid ratio of 5:3, a fermentation temperature of 80° C., and a fermentation time of 7 days.

[0007] A functional cat food comprises 0.1% by weight of black garlic extract and is used for preventing and improving liver function of pet cats.

[0008] The cat food is a complete cat food for basic cats containing 0.1% black garlic extract by weight, and 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.

[0009] 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.

[0010] A functional cat food, wherein the basic cat food contains 0.1% by mass of black garlic extract for anti-aging.

[0011] The application of black garlic extract in establishing a Caenorhabditis elegans model is used to explore the mechanism by which allicin prolongs the lifespan of Caenorhabditis elegans and improves stress response.

[0012] The application of black garlic extract in establishing a Caenorhabditis elegans model was used to explore the mechanism by which allicin improves and alleviates liver lesions.

[0013] Beneficial effects of the present invention: This paper takes alliin in black garlic extract as the target, optimizes the preparation method of microwave enzyme inactivation process, explores the mechanism of action 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 staple food for the first time to explore its effects in anti-aging and other health fields. The actual effect and industrial production are verified through feeding experiments on pet cats. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Effect of different material-liquid ratios on total acid in liquid fermented black garlic.

[0015] Figure 2 Effects of different temperatures on total acid in liquid fermented black garlic.

[0016] Figure 3(A) shows the antioxidant activity of black garlic extract (reducing power of alliin).

[0017] Figure 3 (B) is the antioxidant property test of black garlic extract (reducing power of VC standard solution).

[0018] Figure 3 (C) shows the antioxidant activity of black garlic extract (the relationship between alliin dilution and DPPH scavenging rate).

[0019] Figure 3 (D) is the antioxidant activity test of black garlic extract (relationship between VC concentration and DPPH scavenging rate).

[0020] Figure 3 (E) is the antioxidant property test of black garlic extract (ABTS free radical scavenging ability of black garlic extract).

[0021] Figure 3 (F) is the antioxidant activity test of black garlic extract (ABTS free radical scavenging ability of VC solution).

[0022] Figure 4 (The antioxidant protective effect of garlic extract on Caenorhabditis elegans (survival curves of Caenorhabditis elegans in different treatment groups).

[0023] Figure 5(A) is a network pharmacology analysis of the mechanism of alliin in treating alcoholic liver disease (venny diagram of the intersection of alliin and ALD targets).

[0024] Figure 5(B) is a network pharmacology analysis of the mechanism of alliin in treating alcoholic liver disease (PPI network diagram of alliin targets and ALD intersection targets).

[0025] Figure 5(C) is a network pharmacology analysis of the mechanism of alliin in treating alcoholic liver disease (biological process (BP) enrichment analysis bubble diagram).

[0026] Figure 5(D) shows the network pharmacology analysis of the mechanism of alliin in treating alcoholic liver disease (cellular component (CC) enrichment analysis bubble diagram).

[0027] Figure 5(E) is a network pharmacology analysis of the mechanism of alliin in treating alcoholic liver disease (molecular function (MF) enrichment analysis bubble diagram).

[0028] Figure 5(F) is the network pharmacology analysis of the mechanism of alliin in treating alcoholic liver disease (KEGG enrichment analysis bubble diagram). DETAILED DESCRIPTION

[0029] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0030] Example 1: Optimization of black garlic production process and alliin extraction Step 1: Black garlic fermentation. High-quality garlic is screened and one to two layers of skin and stalks are removed. After cleaning, the garlic is fermented in a high-temperature, high-humidity liquid state. Finally, quality inspection is performed to obtain the finished black garlic. The processing humidity is 80%, the processing temperature is 70°C, and the processing time is 168 hours (i.e., 7 days). The fermentation parameters are as follows: material-liquid ratios of 5:0, 5:2, 5:3, and 5:4; the fermentation temperatures are 70°C, 80°C, and 90°C, and the fermentation cycle is 7 days. The goal of this example is to optimize the extraction method to improve the fermentation efficiency of the active substances in black garlic. Therefore, fermentation parameters were assessed by measuring the total acid content (g / kg).

[0031] Step 2: In this experiment, the microwave inactivation method was selected. The microwave power was set to 300W, and the inactivation time was set to 30s, 60s, and 90s.

[0032] Step 3: Drying the black garlic extract: Use 26% ethanol by volume as the extraction solvent and extract for 60 minutes. Centrifuge the solution containing the black garlic extract to obtain the supernatant, then combine the supernatants and dry them to a constant weight to obtain a solid powder of the black garlic extract.

[0033] Step 4: Prepare the black garlic extract solution. Accurately weigh 10.0 g of black garlic solid powder sample and add 100 mL of 10% methanol (volume fraction). Grind the sample using a juicer until it becomes a thin paste. Ultrasonicate (70% intensity) at room temperature for 15 minutes, then filter through filter paper. Transfer the clarified filtrate to a centrifuge tube and centrifuge at 8000 rpm for 15 minutes. Add the supernatant to a 250 mL volumetric flask and adjust to the mark with the extract. This step is used to determine the antioxidant capacity of the black garlic extract.

[0034] The test 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 keys affecting the extraction efficiency. The purpose of fermentation is to increase the content of allicin, and the total acid is used to evaluate the results. Figure 1 and Figure 2 As shown in the figure, under different fermentation parameters, the total acid content of most groups reached its peak around day 6 of fermentation. When the acidity of black garlic reached 50g / kg, the time required for the material-liquid ratios of 5:0, 5:1, 5:2, 5:3, and 5:4 was 5 days, 5 days, 7 days, 7 days, and more than 7 days, respectively. It can be seen from the trend (e.g. Figure 1 As shown in the figure), the higher the water content, the longer it takes to reach the same acidity. When the material-liquid ratio is 5:0, the fermentation time to peak is the shortest, and when it is 5:4, it is the longest. Considering the economic value, when the material-liquid ratio is 5:3, the time to peak is moderate, and the amount of black garlic added is also low. In addition, the fermentation efficiency at different temperatures was also compared (such as Figure 2As shown in the figure, the higher the temperature, the shorter the time it takes for the total acid to reach its peak. Considering the economic benefits, the fermentation temperature was selected to be 80℃. Too high a temperature (90℃) has no significant effect on improving the fermentation efficiency and is costly. Too low a temperature (70℃) will reduce the fermentation efficiency and take a longer time.

[0035] Example 2, Antioxidant Capacity Detection of Black Garlic Extract Reducing power determination The black garlic extract was diluted to different concentrations. 36 mL of the gradient-diluted black garlic extract was added to 247 mL of phosphate buffer (0.2 mol / L, pH 6.6) and 250 mL of 1% potassium ferricyanide solution, and mixed thoroughly. The mixture was reacted at 50°C for 30 minutes. 250 μL of 10% trichloroacetic acid was added, and the mixture was allowed to stand for 10 minutes. 100 μL of the supernatant was added to 100 μL of deionized water, followed by 20 μL of 10% ferric chloride solution. The resulting solution was then added to an ELISA plate in three separate batches, and the absorbance was measured at 700 nm. A series of VC solutions were used in place of the black garlic extract, with all other conditions remaining the same. The resulting solutions served as controls. This is shown in Figures 3(A) and 3(B).

[0036] 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid, ABTS) activity assay Take 1mL of black garlic extract and 1mg / L of VC solution and dilute them to different concentration gradients respectively. Take 8μL of gradient diluted black garlic extract, add 200μL of ABTS free radical PBS solution and 12μL of PBS solution and mix well. At the same time, deionized water was used instead of black garlic extract as a blank group. Take 8μL of black garlic extract and add 212μL of PBS solution and mix well as a model control. After adding the sample, react at 30℃ for 1h, and measure the absorbance value at 734nm. The black garlic extract was replaced by gradient diluted VC solution, and other conditions were the same. The resulting solution was used as a positive control. Two parallels were made for each mass concentration. The results are as follows Figure 3(E) 、 3(F) shown.

[0037] 2,2-Diphenyl-1-picrylhydrazyl (DPPH) activity assay 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.

[0038] 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.

[0039] Example 3: Detection of the protective effect of black garlic extract on oxidative damage in Caenorhabditis elegans 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.

[0040] ① Nematode amplification and synchronization 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.

[0041] ② Regulation of antioxidant levels in nematodes by black garlic extract 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.

[0042] 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.

[0043] 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)

[0044] Example 4: Target screening and network construction using alcoholic liver disease as a model Alliin targets were identified using a "fishing" approach using the CHEMBL database. Alliin targets were collected using databases such as SwissTargetPrediction, resulting in 146 targets. A search for "alcoholic liver disease" in the OMIM and GeneCards databases yielded 1,211 targets. Eighty-eight common alliin-alcoholic liver disease targets were identified and imported into the String platform. Analysis was performed using the "Organism" option, selecting "Homo Sapiens." Network topology analysis was performed using Cytoscape 3.9.1 software.

[0045] To elucidate the biological functions of key targets of alliin and ALD and their relationships with various signaling pathways in vivo, we entered the intersection of alliin and ALD targets into the DAVID online database (https: / / david.ncifcrf.gov / home.jsp), entered the target gene names, and restricted the species to "homo sapiens." Genetic enrichment (G0) data were generated and exported. Representative entries were selected for G0 (gene ort o1oa) biological process enrichment analysis to identify the biological processes involved in alliin and ALD at the targets, their molecular functions, and cellular locations. G0 functional enrichment analysis bubbles were mapped using the online mapping website BioLadder (https: / / www.bioladder.cn / web / # / pro / cloud). Furthermore, "KEGG_PATHWAY" was selected within the Pathway category in the DAVID online database (https: / / david.ncifcrf.gov / home.jsp). By limiting the species to "homoszpiens", the KEGG (Kyoto Encsclopedia of Genes and Genomes) signaling pathway enrichment results can be obtained. The results are then exported and the KEGG pathway is mapped and analyzed in detail to explore the biological processes and in vivo signaling pathways involved in the potential targets of alliin for the treatment of alcoholic liver disease.

[0046] To further explore the medical applications of alliin, this study used ALD as a model and analyzed the potential targets and pathways through which alliin may affect the disease through network pharmacology. Protein-protein interaction (PPI) relationships, gene function, and pathway enrichment were further analyzed for the 88 common alliin-ALD targets identified (Figures 5(A)-5(F)).

[0047] The results suggest that, within the gene ontology functional domain, alliin may improve symptoms in patients with alcoholic liver disease and prevent further liver cell damage by improving synaptic chemical transmission, locomotory behavior, and responsiveness to xenobiotic stimuli. Furthermore, within the KEGG pathway domain, alliin may enhance the liver cell's ability to resist oxidative damage and alleviate the symptoms of alcoholic liver disease by regulating neuroactive ligand-receptor interaction, glutamate synapses, and other metabolic pathways including glutamate metabolism.

[0048] According to the Body Condition Score (BCS) system for cats, overweight domestic cats score 6 or above, meaning their ribs are not obvious, they have obvious fat accumulation, a slightly flat waist, a slightly protruding abdomen, and most cats have mild fatty liver. Fifteen overweight cats (BCS>6) of similar age and weight were selected and randomly divided into three groups of five each. Group A served as the control group and was fed a basic complete cat food; Group B served as a low-dose experimental group and was fed a basic complete cat food containing 0.1% black garlic extract by weight; and Group C served as a high-dose experimental group and was fed a basic complete cat food containing 0.3% black garlic extract. These groups were fed continuously for six weeks.

[0049] The formula of basic complete cat food for cats is as follows: Raw material composition: frozen chicken breast 80%, frozen chicken heart 5.0%, frozen chicken liver 5%, sweet potato flour 3%, egg yolk powder 1%, chicken fat 1%, seaweed polysaccharide 0.2%, cellulose 0.2%, yeast extract 0.2%.

[0050] Additive ingredients: 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-tocopheryl acetate, vitamin K3, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, D-calcium pantothenate, biotin, folic acid.

[0051] The test indicators are shown in Tables 2 and 3: body weight, food intake, feces, and liver function indicators (alanine aminotransferase ALT, aspartate aminotransferase AST, alkaline phosphatase ALP, glutamyl transferase GGT, and total bilirubin TBIL).

[0052]

[0053]

[0054] The feeding results are as follows. Before feeding, the average weights of the three groups were close, ranging from 6.31 to 6.43 kg. After feeding, the weights of the low-dose and high-dose groups decreased slightly, while the control group remained almost the same, and the stool was normal. In terms of liver function indicators, the average total bilirubin (TBIL) of the 15 cats before feeding was close to the critical value, indicating that some experimental cats may have certain inflammation or damage to their liver function, suggesting that it may be related to overweight. After 6 weeks of feeding, the TBIL of the low-dose and high-dose groups decreased 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 damage. The average value of the experimental cats is within the normal range. 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, which was basically unchanged. In addition, the levels of aspartate aminotransferase AST, alkaline phosphatase ALP and glutamyl transferase GGT in the low-dose and high-dose groups were reduced to varying degrees. The feeding results show that adding different doses of black garlic extract can improve some liver function indicators of overweight cats, but the difference in the improvement effect between adding 0.1% and 0.3% is not obvious, suggesting that black garlic extract has a certain preventive and therapeutic effect on cat liver function.

[0055] The above examples are only some specific embodiments of the present invention. It should be noted that the present invention is not limited to the above examples. There are many other variations, including changes in the parameters of the black garlic extract process (without affecting the core process), the application of black garlic extract on other pet models (normal-sized dogs and cats, obese-sized dogs and cats, etc.), and changes in the addition ratio of black garlic extract. The above content is only an example and explanation of the present invention. Those skilled in the art may make various modifications or supplements to the described specific embodiments or replace them in a similar manner. As long as they do not deviate from the invention or exceed the scope defined by the claims, they should all fall within the scope of protection of the present invention.

Claims

1. An application of black garlic extract in establishing a Caenorhabditis elegans model, characterized in that: The invention is used to explore the mechanism by which alliin prolongs the lifespan of Caenorhabditis elegans and improves stress response; the extraction method of black garlic extract comprises the following steps: 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% volume fraction 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 are as follows: a material-liquid ratio of 5:3, a fermentation temperature of 80°C, and a fermentation time of 7 days.

2. An application of black garlic extract in establishing a Caenorhabditis elegans model, characterized in that: Used to explore the mechanism by which alliin improves and alleviates liver lesions; the extraction method of black garlic extract has the following steps: 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% volume fraction ethanol is selected as the extraction solvent, and 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 are as follows: a material-liquid ratio of 5:3, a fermentation temperature of 80°C, and a fermentation time of 7 days.

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