Preparation method of garlic polysaccharide and application thereof

Abstract

The present application belongs to the technical field of natural product extraction and preparation, and particularly relates to a preparation method of garlic polysaccharide and application thereof. The present application finds through experimental research that the garlic polysaccharide product prepared by taking garlic (such as black garlic and fresh garlic) as raw material not only has good antioxidant activity, but also has prebiotic effect and can effectively promote the growth of lactic acid bacteria such as lactobacillus plantarum and lactobacillus casei. In particular, the antioxidant and prebiotic effect of the black garlic polysaccharide product prepared by taking black garlic as raw material is better, so that the garlic polysaccharide product can be widely applied in the fields of food, medicine, daily chemical product and feed, and the comprehensive utilization value of garlic, especially black garlic, is effectively improved, and therefore the present application has wide and good practical application value and prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural product extraction and preparation, and particularly relates to a preparation method of garlic polysaccharide and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art already known to a person of ordinary skill in the art.

[0003] Garlic is the underground bulb of Allium sativum L., a perennial herb of the Liliaceae Allium genus. Allium Sativum Garlic is a pungent, warm-acting medicinal herb that enters the spleen, stomach and intestines. It has the functions of removing stagnation, warming the spleen and stomach, eliminating accumulation, detoxifying and killing insects. Garlic is a popular spice vegetable that is widely cultivated around the world. Garlic is not only a safe, non-toxic, non-polluting and non-residual seasoning, but also a healthy food with low cost, no toxicity, no side effects and unique pharmacological and nutritional health functions. Black garlic is a deep-processed product of fresh garlic, which is made by ripening in a high-temperature and high-humidity environment. It has a sweet and sour taste, and the irritating garlic odor is removed. The content of nutrients such as sugar, protein and polyphenol is also increased to some extent, which is welcomed by consumers.

[0004] Plant polysaccharides, also known as plant polysaccharides, are polysaccharides with a degree of polymerization of more than 10 produced by plant cell metabolism. Currently, polysaccharides have been extracted from hundreds of plants. Studies have shown that plant polysaccharides have immunomodulatory, antitumor, anti-aging, hypoglycemic and anti-inflammatory activities. However, the inventors found that there is still less research on the biological activity and efficacy of garlic polysaccharides. Therefore, it is of great value and significance to study garlic functional products with garlic polysaccharides as the main functional ingredient to further improve the utilization rate of garlic, increase the added value of garlic products and extend the garlic industry chain. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides a preparation method of garlic polysaccharide and application thereof. The inventors have found through experimental research that the garlic polysaccharide product prepared from garlic (such as black garlic and fresh garlic) not only has good antioxidant activity, but also has prebiotic effect, which can effectively promote the growth of lactic acid bacteria such as Lactobacillus plantarum and Lactobacillus casei. Therefore, it can be widely used in actual production and life. Based on the above research results, the present application is completed.

[0006] To achieve the above technical purposes, the technical solutions adopted by the present application are as follows:

[0007] The first aspect of the present application provides an application of garlic polysaccharide in an antioxidant and / or a product for promoting growth and reproduction of probiotics.

[0008] The garlic can be fresh garlic or black garlic. Specifically, the present application finds that the garlic polysaccharide has good reducing capacity, total antioxidant capacity and free radical (hydroxyl radical, DPPH radical, superoxide anion radical and ABTS radical) scavenging activity, that is, good antioxidant performance. In addition, the garlic polysaccharide has a prebiotic effect and can effectively promote the growth and reproduction of probiotics, such as lactic acid bacteria (Lactobacillus plantarum and Lactobacillus casei), and increase the lactic acid content of lactic acid bacteria fermentation liquor. In particular, the antioxidant performance and prebiotic effect of black garlic polysaccharide prepared from black garlic are better than those of fresh garlic polysaccharide prepared from fresh garlic.

[0009] The second aspect of the present application provides a preparation method of garlic polysaccharide, which comprises obtaining the garlic polysaccharide from garlic through water extraction, alcohol precipitation and a purification step.

[0010] The garlic can be fresh garlic or black garlic, and preferably black garlic.

[0011] The purification step comprises removing protein by Sevage method.

[0012] Specifically, the preparation method comprises:

[0013] S1, crushing garlic and adding water, then heating and extracting, and centrifuging to obtain supernatant;

[0014] S2, concentrating the supernatant obtained in step S1, adding ethanol, precipitating, and centrifuging;

[0015] S3, drying and crushing the precipitate obtained after centrifugation in step S2, re-dissolving, centrifuging to obtain supernatant, and freeze-drying to obtain crude garlic polysaccharide;

[0016] S4, dissolving the crude garlic polysaccharide obtained in step S3 in water and adding Sevage reagent to remove protein, and freeze-drying to obtain.

[0017] The third aspect of the present application provides a product with antioxidant performance and / or prebiotic effect, which comprises the above-mentioned garlic polysaccharide.

[0018] The prebiotic effect specifically means promoting the growth and reproduction of lactic acid bacteria and increasing the lactic acid production of lactic acid bacteria.

[0019] The lactic acid bacteria include, but are not limited to, Lactobacillus plantarum and Lactobacillus casei.

[0020] The product includes health food and medicine.

[0021] The above one or more technical solutions have the following beneficial technical effects:

[0022] The technical scheme has good antioxidant effect and prebiotic effect, and the black garlic polysaccharide prepared from black garlic has better antioxidant effect and prebiotic effect, so that the garlic polysaccharide can be widely applied to the fields of health-care food and medicine, and the comprehensive utilization value of garlic, especially black garlic, is effectively improved, and therefore, the garlic polysaccharide has wide and good practical application value and prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] The drawings constituting a part of the specification of the present application are used to provide further understanding of the present application, and the illustrative embodiments of the present application and the description thereof are used to explain the present application, and do not constitute improper limitation on the present application.

[0024] Figure 1 The table is the reducing power determination results of BGPS and FGPS in the embodiment 2 of the present application; wherein (a) is BGPS, and (b) is FGPS.

[0025] Figure 2 The table is the total antioxidant capacity determination results of BGPS and FGPS in the embodiment 3 of the present application; wherein (a) is BGPS, and (b) is FGPS.

[0026] Figure 3 The table is the hydroxyl radical scavenging rate determination results of BGPS and FGPS in the embodiment 4 of the present application; wherein (a) is BGPS, and (b) is FGPS.

[0027] Figure 4 The table is the DPPH radical scavenging rate determination results of BGPS and FGPS in the embodiment 5 of the present application; wherein (a) is BGPS, and (b) is FGPS.

[0028] Figure 5 The table is the superoxide anion radical scavenging rate determination results of BGPS and FGPS in the embodiment 6 of the present application; wherein (a) is BGPS, and (b) is FGPS.

[0029] Figure 6 The table is the ABTS radical scavenging rate determination results of BGPS and FGPS in the embodiment 7 of the present application; wherein (a) is BGPS, and (b) is FGPS.

[0030] Figure 7 The table is the growth promotion effect determination of BGPS and FGPS on Lactobacillus acidophilus in the embodiment 8 of the present application; wherein A is the growth promotion effect of BGPS / FGPS on Lactobacillus acidophilus; B is the influence of BGPS / FGPS on pH value in the fermentation process; C is the influence of BGPS / FGPS on OD value in the fermentation process; and D is the influence of BGPS / FGPS on the growth of Lactobacillus acidophilus. L. plantarum L. plantarum L. plantarum The table is the growth promotion effect determination of BGPS and FGPS on Lactobacillus acidophilus in the embodiment 8 of the present application; wherein A is the growth promotion effect of BGPS / FGPS on Lactobacillus acidophilus; B is the influence of BGPS / FGPS on pH value in the fermentation process; C is the influence of BGPS / FGPS on OD value in the fermentation process; and D is the influence of BGPS / FGPS on the growth of Lactobacillus acidophilus.​​L. plantarum Effect of lactic acid content during fermentation. NC: negative control group (carbon-free medium); PC: positive control group (inulin prebiotic medium); BGPS sample group (BGPS prebiotic medium); FGPS sample group (FGPS prebiotic medium). P <0.05, P <0.01 compared with NC; a P : BGPS compared with FGPS P <0.01. Data represent mean ± SD (n = 3).

[0031] Figure 8 are BGPS and FGPS, respectively, for the growth promotion effect of BGPS and FGPS on Lactobacillus bulgaricus in Example 8 of the present application; wherein, A is the effect of BGPS / FGPS on the pH value during fermentation; B is the effect of BGPS / FGPS on the OD value during fermentation; C is the effect of BGPS / FGPS on the lactic acid content during fermentation. L. casei L. casei L. casei L. casei Effect of lactic acid content during fermentation. NC: negative control group (carbon-free medium); PC: positive control group (inulin prebiotic medium); BGPS sample group (BGPS prebiotic medium); FGPS sample group (FGPS prebiotic medium). P <0.05, P <0.01 compared with NC; a P : BGPS compared with FGPS P< 0.01. Data represent mean ± SD (n = 3).

[0032] Figure 9 are the IC spectra of BGPS and FGPS in Example 9 of the present application; wherein, (a) is BGPS, and in the figure, 3: Ara; 5: Glc; 6: Xyl; 7: Man; 8: Fru; (b) is FGPS, and in the figure, 2: Rha; 3: Ara; 4: Gal; 5: Glc; 6: Xyl; 8: Fru.

[0033] Figure 10 are the HPSEC-MALLS chromatograms of BGPS and FGPS in Example 10 of the present application; wherein, (a) is BGPS, and (b) is FGPS.

[0034] Figure 11 are the results of the maximum absorption wavelength of the complex of BGPS and FGPS with Congo red in different alkaline environments in Example 11 of the present application; wherein, (a) is BGPS, and (b) is FGPS.

[0035] Figure 12 ​​​The images show the UV-Vis spectra of the BGPS and FGPS reactants with I2-KI in Example 12 of this invention; where (a) is BGPS and (b) is FGPS. Detailed Implementation

[0036] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for the purpose of describing specific embodiments and not for limiting the scope of protection of the present invention.

[0038] In a typical embodiment of the present invention, the application of garlic polysaccharide in antioxidant and / or probiotic growth and reproduction promotion products is provided.

[0039] The garlic can be fresh garlic or black garlic.

[0040] Specifically, this invention has discovered that garlic polysaccharides not only possess excellent reducing power, total antioxidant capacity, and free radical scavenging activity (hydroxyl radicals, DPPH radicals, superoxide anion radicals, and ABTS radicals), demonstrating good antioxidant properties, but also exhibit prebiotic activity. This effectively promotes the growth and reproduction of probiotics, such as lactic acid bacteria (Lactobacillus plantarum and Lactobacillus casei), increasing the lactic acid content of lactic acid bacteria fermentation broth. In particular, black garlic polysaccharides prepared from black garlic exhibit superior antioxidant and prebiotic effects compared to fresh garlic polysaccharides prepared from fresh garlic.

[0041] The products include health foods and pharmaceuticals.

[0042] It should be noted that the garlic polysaccharide in this invention is a polysaccharide component extracted from garlic (including fresh garlic and black garlic), and its extraction and preparation process is not specifically limited.

[0043] In another specific embodiment of the present invention, a method for preparing garlic polysaccharide is provided, the method comprising obtaining the garlic polysaccharide from garlic by water extraction, alcohol precipitation and purification steps.

[0044] The garlic can be fresh garlic and black garlic; preferably black garlic.

[0045] The purification step includes removing protein by using Sevage method.

[0046] In another embodiment of the present application, the preparation method comprises:

[0047] S1, crushing garlic with water and heating to extract, centrifuging to obtain supernatant;

[0048] S2, concentrating the supernatant obtained in step S1, adding ethanol, precipitating, and centrifuging;

[0049] S3, drying and crushing the precipitate obtained after centrifuging in step S2, re-dissolving, centrifuging to obtain supernatant, and freeze-drying to obtain crude garlic polysaccharide;

[0050] S4, dissolving the crude garlic polysaccharide obtained in step S3 in water and adding Sevage reagent to remove protein, and freeze-drying to obtain.

[0051] In step S1, the mass-volume ratio of garlic to water is 1:10-30, g / mL; preferably 1:20. The crushing can be performed by grinding and ultrasonic crushing, so that the active ingredients such as garlic polysaccharide are fully dissolved. The ultrasonic crushing conditions can be 100-500 W, ultrasonic treatment for 1-30 min, preferably 300 W ultrasonic treatment for 15 min.

[0052] The heating extraction method is to extract at 80-100℃ for 1-4 h, preferably 90℃ for 2 h. The above heating extraction can be performed by water bath method, and the supernatant is obtained by centrifugation. The centrifugation method can be 8000-12000 r / min centrifugation at low temperature (such as 4℃) for 5-15 min; preferably 10000 r / min centrifugation for 10 min. It should be noted that in order to fully extract the garlic polysaccharide, the above extraction method can be repeated 2-4 times. The supernatant obtained after each extraction and centrifugation is combined to obtain the total supernatant, which then enters step S2.

[0053] In the step S2, the supernatant can be concentrated by reduced pressure concentration to 1 / 2-2 / 3 of the original volume, and then 2-4 times (preferably 3 times) volume of ethanol solution is added. To further improve the extraction efficiency, the ethanol is preferably high-concentration ethanol, such as 60%-99.5% ethanol solution, and 95% ethanol (i.e. edible alcohol) is further preferred. The ethanol precipitation time can be 1-24 h, preferably 12 h. The centrifugation conditions are 8000-12000 r / min centrifugation at low temperature (e.g. 4°C) for 5-15 min, preferably 10000 r / min centrifugation for 10 min.

[0054] In the step S3, drying can be performed by drying, such as drying the precipitate at 50-60°C (preferably 55°C). The centrifugation conditions are 8000-12000 r / min centrifugation at low temperature (e.g. 4°C) for 5-15 min, preferably 10000 r / min centrifugation for 10 min.

[0055] In the step S4, the mass-volume ratio of garlic crude polysaccharide to water is 1:10-30, g / mL, preferably 1:20. To ensure rapid and sufficient dissolution of the crude polysaccharide, water bath heating can be used to obtain a garlic crude polysaccharide solution. The volume ratio of the garlic crude polysaccharide solution to Sevage reagent is 2-8:1, preferably 4:1. To remove as much protein as possible and obtain garlic polysaccharide with higher purity, the above purification step can be repeated 1-15 times.

[0056] Experiments have shown that the black garlic polysaccharide BGPS obtained by the above preparation method is mainly composed of Ara, Glc, Xyl, Man and Fru, and its Mn, Mp, Mw and Mz are 7.78×10 4 Da, 4.92×10 4 Da, 9.59×10 4 Da, 1.38×10 5 Da; the fresh garlic polysaccharide FGPS is mainly composed of Rha, Ara, Gal, Glc, Xyl and Fru, and its Mn, Mp, Mw and Mz are 7.55×10 3 Da, 9.66×10 3 Da, 1.64×10 4 Da, 3.20×10 5 Da; and both the black garlic polysaccharide and the fresh garlic polysaccharide contain long side chains and more branches, but do not have a triple helix structure.

[0057] Meanwhile, the inventors have proved by experiments that the garlic polysaccharide components prepared by the above method all have good antioxidant and prebiotic effects. In particular, the antioxidant performance and prebiotic effect of the black garlic polysaccharide prepared by taking black garlic as the raw material are superior to those of the fresh garlic polysaccharide prepared by taking fresh garlic as the raw material.

[0058] In another specific embodiment of the present application, a product having antioxidant performance and / or prebiotic effect is provided, and the product comprises the above garlic polysaccharide.

[0059] The prebiotic effect specifically manifests in promoting the growth and reproduction of lactic acid bacteria and improving the lactic acid yield of lactic acid bacteria.

[0060] The lactic acid bacteria include but are not limited to Lactobacillus plantarum and Lactobacillus casei.

[0061] The product includes health foods and medicines.

[0062] It should be noted that in the present application, the term "food" should be understood in a broad sense, and can be understood as any edible form, so the food includes ordinary food and special food, the special food includes health food and special medical purpose formula food; and the ordinary food is food suitable for all people relative to the special food.

[0063] The medicine can be administered in unit dose forms, liquid dosage forms, solid dosage forms. The liquid dosage form can be true solution, colloid, microparticle, emulsion, suspension, etc. Other dosage forms such as tablets, capsules, dripping pills, pills, powders, emulsions, granules, suppositories, clathrates, landfill agents, etc.

[0064] The daily chemical product can be laundry detergent, personal hygiene cleaner and cosmetic, etc., specifically toothpaste, mouthwash, disinfectant, shampoo, hair cream, hair gel, shower gel, soap, facial cleanser, facial mask, facial cream, sunscreen, etc.

[0065] The feed is the food of animals raised in agriculture or animal husbandry. The garlic polysaccharide of the present application can be added to any kind of feed as a feed additive, and the feed includes but is not limited to complete compound feed, concentrated feed and premixed feed.

[0066] The present application is further explained and described by the following examples, but does not constitute a limitation on the present application. It should be understood that the examples are only used to illustrate the present application and not to limit the scope of the present application.

[0067] Example 1

[0068] Extraction and purification of black garlic and fresh garlic polysaccharide

[0069] An appropriate amount of black garlic and fresh garlic was weighed, and deionized water was added at a ratio of 1:20 (black garlic / fresh garlic:deionized water, m:v, g / mL). The mixture was ground and ultrasonically broken (ultrasonic power 300 W, ultrasonic time 15 min), and then placed in a 90°C water bath for 2 h. Subsequently, centrifugation (10000 r / min, 10 min, 4°C) was performed to obtain the supernatant. The above steps were repeated three times, and the supernatants were combined and concentrated to 1 / 2-2 / 3 of the original volume under reduced pressure. Three volumes of anhydrous ethanol were added to precipitate the polysaccharides for 12 h. Subsequently, centrifugation (10000 r / min, 10 min, 4°C) was performed, and the precipitate was dried at 55°C. After being ground, the precipitate was redissolved in deionized water, and centrifugation (10000 r / min, 10 min, 4°C) was performed. Finally, the supernatant was freeze-dried to obtain black garlic crude polysaccharides and fresh garlic crude polysaccharides.

[0070] The extracted black garlic and fresh garlic crude polysaccharides were added to deionized water at a ratio of 1:20 (crude polysaccharides:deionized water, m:v, g / mL), and a crude polysaccharide solution was obtained by water bath at 55°C for 2 h. The crude polysaccharide solution was added to Sevage reagent (dichloromethane:n-butanol=5:1, v:v) at a ratio of 4:1 (v:v), and after being shaken for 15 min, centrifugation (10000 r / min, 10 min, 4°C) was performed. The organic phase and the denatured protein emulsion layer at the interface between the organic phase and the aqueous phase were removed, and the supernatant was retained. The above steps were repeated 4-6 times until the emulsion layer at the interface between the organic phase and the aqueous phase disappeared. The obtained polysaccharide solution was vacuum freeze-dried to obtain black garlic polysaccharides (BGPS) and fresh garlic polysaccharides (FGPS). The extraction rate of BGPS and FGPS was calculated by formula 1.

[0071] (1)

[0072] The polysaccharide content of BGPS and FGPS was determined by the phenol-sulfuric acid method, and the polysaccharide content of BGPS and FGPS was calculated by formula 2.

[0073] (2)

[0074] The extraction rate of BGPS was 14.39±0.83%, and the polysaccharide content was 60.33±3.16%. The extraction rate of FGPS was 8.02±0.40%, and the polysaccharide content was 64.57±3.23%.

[0075] Example 2

[0076] Reducing power experiment of BGPS and FGPS

[0077] BGPS and FGPS were configured into a series (500, 1000, 1500, 2000, 2500, 3000 mg / L) of polysaccharide sample solution with different concentrations, and Vc solution with the same concentration was prepared.

[0078] The Prussian blue method was used for determination. 1 mL of polysaccharide sample solution with different concentrations (500-3000 mg / L) was mixed with 2.5 mL of phosphate buffer (0.2 mol / L, pH 6.6), 1 mL of potassium ferricyanide solution (10 g / L) to form a mixture, which was placed at 50°C for 20 min. 2 mL of trichloroacetic acid solution (100 g / L) and 1.2 mL of ferric trichloride solution (1 g / L) were added to the mixture, and Vc was used as a positive control. The absorbance of the resulting solution was measured at 700 nm.

[0079] The results of the reducing power determination of BGPS and FGPS are shown in Figure 1 .

[0080] The antioxidant activity is closely related to the reducing power, and the antioxidant activity of antioxidants increases with the increase of their reducing power. Therefore, the antioxidant activity of antioxidants can be indirectly reflected by determining their reducing power. Potassium ferricyanide is usually added to the solution of antioxidants, and under the action of antioxidants, Fe 3+ in the solution will generate Fe 2+ , and Fe 2+ has an absorption peak at 700 nm, which can be measured by an enzyme label instrument. With the increase of absorbance, the content of Fe 2+ in the solution increases, and the reducing power of the solution also increases. As can be seen from Figure 1 , the reducing power of BGPS and FGPS increases with the increase of polysaccharide concentration, therefore, BGPS and FGPS both have reducing power.

[0081] As can be seen from Figure 1 (a), the reducing power of BGPS is 1.53 at a concentration of 3000 mg / L, which is 66.21% of the reducing power of Vc. As can be seen from Figure 1 (b), the reducing power of FGPS is 1.08, which is 46.78% of the reducing power of Vc.

[0082] Example 3

[0083] Experiment of total antioxidant capacity of BGPS and FGPS

[0084] 0.4 mL of polysaccharide sample solutions of different concentrations (500–3000 mg / L) were mixed thoroughly with 4 mL of a mixed solution (containing 0.6 mol / L sulfuric acid, 28 mmol / L trisodium phosphate, and 4 mmol / L ammonium molybdate) and reacted at 95 °C for 90 min. Using vitamin C as a positive control, the absorbance of the solution was recorded at 695 nm after cooling.

[0085] The results of the total antioxidant capacity determination of BGPS and FGPS are as follows: Figure 2 As shown.

[0086] from Figure 2 As can be seen, the total antioxidant capacity of BGPS and FGPS increases with the increase of polysaccharide concentration within the experimental concentration range. Therefore, both BGPS and FGPS have total antioxidant capacity.

[0087] Depend on Figure 2 (a) shows that at a concentration of 3000 mg / L, the antioxidant capacity of BGPS is 2.51, which is 77.38 ± 3.10% of the total antioxidant capacity of Vc. From... Figure 2 (b) It can be seen that the antioxidant capacity of FGPS is 1.98, which is 61.12±3.06% of the total antioxidant capacity of Vc.

[0088] Example 4

[0089] Hydroxyl radical scavenging experiments of BGPS and FGPS

[0090] The Fenton method was used for determination. 1 mL of polysaccharide sample solutions of different concentrations (500–3000 mg / L) were mixed with 1 mL of FeSO4 solution (9 mmol / L), 1 mL of salicylic acid-ethanol solution (9 mmol / L), and 1 mL of H2O2 solution (8.8 mmol / L). After reacting at 37 °C for 30 min, the mixture was centrifuged at 10000 r / min for 10 min, and the absorbance (A) was recorded at 510 nm. Vitamin C was used as a positive control, and the sample was zeroed with deionized water. The hydroxyl radical scavenging rate (SR) was calculated using Equation 3.

[0091] (3)

[0092] In the formula: A0 is the absorbance of the blank control (deionized water instead of the sample), and A is the absorbance of the mixture of the sample solution and the reaction solution.

[0093] The results of the hydroxyl radical scavenging rate determination of BGPS and FGPS are as follows: Figure 3 As shown.

[0094] from Figure 3It can be seen that the hydroxyl radical scavenging rate of BGPS and FGPS is proportional to the polysaccharide concentration in the experimental concentration range, and the hydroxyl radical scavenging rate of polysaccharide increases with the increase of polysaccharide concentration. The half maximal effective concentration (EC 50 ) represents the polysaccharide sample concentration when the radical scavenging rate reaches 50%.

[0095] From Figure 3 (a), it can be seen that the calculation of EC 50 value can obtain the EC 50 value of BGPS is 376.11±18.81 mg / L. And the hydroxyl radical scavenging rate of BGPS can reach 75.48% at the highest concentration (3000 mg / L). From Figure 3 (b), it can be seen that the calculation of EC 50 value can obtain the EC 50 value of FGPS is 555.37±27.77 mg / L. And the hydroxyl radical scavenging rate of FGPS can reach 72.77% at the highest concentration (3000 mg / L).

[0096] Example 5

[0097] DPPH radical scavenging experiment of BGPS and FGPS

[0098] 2 mL of polysaccharide sample solution with different concentrations (500-3000 mg / L) and 2 mL of DPPH ethanol solution (0.2 mmol / L) were reacted in the dark for 30 min, and the absorbance (A) was measured at 517 nm. Vc was used as a positive control, and deionized water was used instead of samples as a blank group, and anhydrous ethanol was used instead of DPPH ethanol solution as a control group. The DPPH radical scavenging rate (SR) was calculated by formula 4.

[0099] (4)

[0100] In the formula: A is the absorbance of the mixture of sample solution and DPPH ethanol solution, A 对 is the absorbance of the mixture of sample solution and ethanol, and A 空 is the absorbance of the mixture of deionized water and DPPH ethanol solution. The DPPH radical scavenging rate determination results of BGPS and FGPS are shown in Figure 4 .

[0101] DPPH is a kind of stable active free radical, its ethanol solution color is purple, it needs to be stored at low temperature in the dark, its electronic structure is single. When free radical scavenger exists, the single electron of DPPH is captured, the color becomes light, the absorbance at the maximum light absorption wavelength decreases, and the decrease degree is linear. The decrease of absorbance level indicates the increase of antioxidant capacity, so as to evaluate the antioxidant capacity of the test sample. This antioxidant capacity is expressed in inhibition rate, and the greater the inhibition rate is, the stronger the antioxidant capacity is. From Figure 4 It can be seen that the DPPH free radical scavenging rate of BGPS and FGPS increases with the increase of polysaccharide concentration, therefore, BGPS and FGPS both have DPPH free radical scavenging capacity.

[0102] From Figure 4 (a), it can be seen that the calculation of EC 50 The EC 50 value of BGPS is 467.83±23.39 mg / L. And the DPPH free radical scavenging capacity of BGPS can reach 72.67% at the highest concentration (3000 mg / L). From Figure 4 (b), it can be seen that the calculation of EC 50 The EC 50 value of FGPS is 1369.66±54.79 mg / L. The DPPH free radical scavenging capacity of FGPS can reach 60.53% at the highest concentration (3000 mg / L).

[0103] Example 6

[0104] SOD experiment of BGPS and FGPS

[0105] 1 mL of polysaccharide sample solution with different concentrations (500-3000 mg / L) and 2 mL of Tris-HCl buffer (50 mmol / L, pH 8.2) were reacted at 25℃ for 20 min, and then 0.4 mL of pyrogallol solution (5 mmol / L) was rapidly mixed, the absorbance was measured at 325 nm every 20 s for 9 times. Vc was used as a positive control, and deionized water instead of sample was used as a blank control. The superoxide anion free radical scavenging rate (SR) was calculated by formula 5.

[0106] (5)

[0107] In the formula: S0 is the slope of the absorbance of the blank control (deionized water instead of sample), and S is the slope of the absorbance after mixing the sample solution with the reaction solution.

[0108] O 2-The results of radical scavenging rate determination are shown in Table 1. Figure 5

[0109] As can be seen from Figure 5 , the BGPS and FGPS have obvious dose-effect relationship in the superoxide anion radical scavenging ability, which indicates that the BGPS and FGPS have superoxide anion radical scavenging ability. - -

[0110] As can be seen from Figure 5 (a), the maximum superoxide anion radical scavenging rate of BGPS is 48.76% at the concentration of 3000 mg / L. As can be seen from Figure 5 (b), the maximum superoxide anion radical scavenging rate of FGPS is 39.92%.

[0111] Example 7

[0112] ABTS radical scavenging experiment of BGPS and FGPS

[0113] To prepare ABTS working solution, equal volume of potassium persulfate solution (4.9 mmol / L) and ABTS solution (7 mmol / L) are mixed, and after reaction in the dark for 20 h, diluted with phosphate buffer (0.1 mol / L, pH 7.4) to the absorbance at 734 nm of 0.70±0.02. 1 mL of polysaccharide sample solution with different concentrations (500-3000 mg / L) is mixed with 3 mL of ABTS working solution, and then reacted in the dark for 6 min, and the absorbance (A) is measured at 734 nm. Deionized water is used instead of the sample as a blank group, and the phosphate buffer is used instead of the ABTS working solution as a control group, and Vc is used as a positive control. The ABTS radical scavenging rate (SR) is calculated by formula 6.

[0114] (6)

[0115] In the formula: A is the absorbance of the sample solution mixed with the ABTS working solution, A 对 is the absorbance of the sample solution mixed with the phosphate buffer, and A 空 is the absorbance of deionized water mixed with the ABTS working solution.

[0116] The results of ABTS radical scavenging rate determination of BGPS and FGPS are shown in Table 2. Figure 6

[0117] ​​​​It has been shown that K2S2O8 forms stable ABTS free radicals with ABTS, and the reaction of the antioxidant substance with ABTS can reduce the color of the reaction system. ABTS free radical itself has a maximum absorption peak at 734 nm. If the absorbance of the reaction system at 734 nm decreases, it means that the substance has reacted with ABTS, thereby fading the reaction system, so the substance has antioxidant capacity. From Figure 6 It can be seen that both BGPS and FGPS have ABTS free radical scavenging capacity.

[0118] From Figure 6 (a), it can be seen that the EC 50 value of BGPS is 1022.21±40.89 mg / L. The ABTS free radical scavenging rate of BGPS can reach 65.38% at the highest concentration (3000 mg / L). From Figure 6 (b), it can be seen that the EC 50 value of FGPS is 1765.52±70.62 mg / L. In addition, the ABTS free radical scavenging rate of FGPS can reach 57.60% at the highest concentration (3000 mg / L).

[0119] Example 8

[0120] Prebiotic effect of BGPS and FGPS

[0121] Growth promoting effect of Lactobacillus plantarum L. plantarum and Lactobacillus casei L. casei

[0122] Single colonies on the plate were inoculated into MRS broth medium, and cultured at 37℃ in a shaking incubator at a speed of 150 r / min for 48 h. The cultured bacterial liquid was inoculated into different groups at a concentration of 1%. NC: negative control group (carbon-free culture medium); PC: positive control group (inulin prebiotic culture medium); BGPS sample group (BGPS prebiotic culture medium); FGPS sample group (FGPS prebiotic culture medium). The samples were cultured at 37℃ in a shaking incubator at a speed of 150 r / min for 120 h. Every 24 h, the pH of the fermented bacterial liquid was measured with a pH meter, and the change in lactic acid content was measured with a biological sensor analyzer. The fermented liquid was centrifuged (10000 r / min, 10 min, 4℃), and the supernatant was discarded. After washing with PBS buffer until colorless, the bacterial density of the fermented liquid was measured at a wavelength of 600 nm with a microplate reader.

[0123] The results of the prebiotic effect of BGPS and FGPS are shown in Figure 7 and Figure 8 .

[0124] ​The experimental results of BGPS and FGPS prebiotics are as follows: Figure 7 and Figure 8 As shown in the figure, both BGPS and FGPS have certain prebiotic effects. It can be seen that the cell density of the BGPS fermentation broth is significantly higher than that of the FGPS fermentation broth. P <0.01), the lactic acid content of BGPS fermentation broth was significantly higher than that of FGPS fermentation broth ( P <0.01). Therefore, BGPS promotes L. plantarum and L. casei Its growth effect is better than that of FGPS.

[0125] Example 9

[0126] Determination of monosaccharide composition of BGPS and its components, and FGPS and its components.

[0127] Weigh 5 mg of each polysaccharide sample (BGPS and FGPS) into a chromatographic vial, add 1 mL of 2.5 mol / L trifluoroacetic acid solution, and heat at 60 °C for 1 h. After purging with nitrogen to dry, add methanol, and then purge again. Repeat the methanol washing step 2-3 times. Dissolve in sterile water and transfer to a chromatographic vial for analysis. Ion chromatography (IC) system was used to analyze the monosaccharide components using a conductivity detector. A Dionex™ CarboPac™ PA20 (150 × 3.0 mm, 10 μm) liquid chromatography column was used to detect the monosaccharide components at a flow rate of 0.5 mL / min. The mobile phase consisted of mobile phase A (0.1 mol / L NaOH) and mobile phase B (0.1 mol / L NaOH, 0.2 mol / L NaAc), with an injection volume of 5 μL and a column temperature of 30 °C. Elution was performed using a linear gradient program: 0 min, 95% A; 30 min, 80% A; 30.1–45 min, 60% A; 45.1–60 min, 95% A. The monosaccharide composition of the polysaccharide samples was calculated by comparison with standards.

[0128] BGPS is mainly composed of Ara, Glc, Xyl, Man and Fru, with a molar ratio of 11.60:260.16:1:39.67:1410.30.

[0129] FGPS is mainly composed of Rha, Ara, Gal, Glc, Xyl and Fru, with a molar ratio of 4.02:1:7.83:143.50:2.00:1509.13.

[0130] Example 10

[0131] Molecular weight determination of BGPS and components thereof, FGPS and components thereof

[0132] The molecular weight of BGPS and FGPS was determined by 18-angle laser light scattering high gel permeation chromatography system with 18-angle laser light scattering detector and refractive index detector. The mobile phase was double distilled water, the flow rate was 1 mL / min, the sample was passed through Shodex SBOHPAK-806-803 column (8 mm x 300 mm), the column temperature was 40°C, and dextran was used as standard. Astra software was used to calculate the molecular weight distribution.

[0133] The Mn, Mp, Mw, Mz of BGPS were 7.78 x 10 4 Da, 4.92 x 10 4 Da, 9.59 x 10 4 Da, 1.38 x 10 5 Da

[0134] The Mn, Mp, Mw, Mz of FGPS were 7.55 x 10 3 Da, 9.66 x 10 3 Da, 1.64 x 10 4 Da, 3.20 x 10 5 Da.

[0135] Example 11

[0136] Triple helix structure analysis of BGPS and FGPS

[0137] Congo red test was used to detect the helical conformation of polysaccharide samples (BGPS, FGPS) in aqueous solution. The Congo red solution (80 μmol / L, 1 mL) was mixed evenly with the polysaccharide sample solution (1 mg / mL, 1 mL), and 1 mL of NaOH solution of different concentrations was added to it, to adjust the concentration of NaOH in the mixed solution to 0-1.0 mol / L. Deionized water instead of sample was used as a blank control, and after equilibration at room temperature for 5 min, the maximum absorption wavelength (λmax) of the mixed solution at each NaOH concentration was determined by an enzyme marker (Dynex Spectra MR) in the range of 485-520 nm.

[0138] Congo red test can be used to detect whether there is a triple helix conformation in polysaccharide. Polysaccharide with triple helix conformation can form a complex with Congo red under weak alkaline conditions, and the maximum absorption wavelength is red-shifted compared with that under non-alkaline conditions. From the red shift of the maximum absorption wavelength, the helical conformation of the polysaccharide can be determined. Figure 11It can be seen that in the range of 0~3 mol / L NaOH solution concentration, the maximum absorption wavelength of BGPS and FGPS mixed with Congo red does not appear red shift phenomenon with the increase of NaOH solution concentration, indicating that BGPS and FGPS do not have triple helix structure.

[0139] Example 12

[0140] Branching structure analysis of BGPS and FGPS

[0141] Iodine-potassium iodide reaction was used to detect the branching structure of polysaccharide samples (BGPS, FGPS). 1.2 mL of I2 (0.02%) containing KI (0.2%) solution was added to the polysaccharide sample solution (2 mg / mL, 2 mL), and after mixing, ultraviolet scanning was performed at 230 nm~600 nm.

[0142] Iodine-potassium iodide experiment was used to detect whether the sugar had longer side chains and more branches. If the polysaccharide sample had a maximum absorption peak between 230 nm~400 nm after reaction with I2-KI, and no maximum absorption at 565 nm, it indicated that the polysaccharide sample had more branches and longer side chains; if the polysaccharide sample had a maximum absorption peak at 565 nm after reaction with I2-KI, the result was the opposite.

[0143] As shown in Figure 12 , the maximum absorption peaks of BGPS and I2-KI reactants were all near 288 nm and 351 nm, and there was no maximum absorption at 565 nm, indicating the presence of longer side chains and more branches. The maximum absorption peaks of FGPS and I2-KI reactants were all near 288 nm and 352 nm, and there was no maximum absorption at 565 nm, also indicating the presence of longer side chains and more branches.

[0144] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not to limit it. Although the present application has been described in detail with reference to the examples given, ordinary skilled in the art can modify or equivalently replace the technical solutions of the present application according to the needs without departing from the spirit and scope of the present application.

Claims

1. Use of black garlic polysaccharide in the preparation of an antioxidant and a product for promoting the growth and reproduction of probiotics, wherein, The probiotics are Lactobacillus plantarum and Lactobacillus casei. The preparation method of the black garlic polysaccharide comprises the following steps: adding deionized water into black garlic according to a ratio of 1:20, g / mL, grinding uniformly, performing ultrasonic crushing at an ultrasonic power of 300 W for 15 min, then placing in a 90 DEG C water bath for 2 h, then centrifuging at 10000 r / min for 10 min at 4 DEG C, and taking supernatant; extracting 3 times according to the above steps, combining the supernatants, and concentrating to 1 / 2-2 / 3 volume under reduced pressure, then adding 3 times volume of anhydrous ethanol to precipitate for 12 h, then centrifuging at 10000 r / min for 10 min at 4 DEG C, and drying the precipitate at 55 DEG C, then crushing, redissolving with deionized water, centrifuging at 10000 r / min for 10 min at 4 DEG C, and finally freeze-drying the supernatant to obtain black garlic crude polysaccharide. The black garlic polysaccharide is obtained by the following steps: adding deionized water into the extracted black garlic crude polysaccharide according to a ratio of 1:20, g / mL, water-bathing in a 55 DEG C water bath for 2 h to obtain a crude polysaccharide solution; adding Sevage reagent into the crude polysaccharide solution according to a ratio of 4:1, v:v, wherein the Sevage reagent is composed of dichloromethane and n-butanol according to a ratio of 5:1, v:v, fully shaking for 15 min, then centrifuging at 10000 r / min for 10 min at 4 DEG C, removing the organic phase and the denatured protein emulsion layer at the interface of the organic phase and the aqueous phase, and retaining the supernatant; repeating the above steps 4-6 times until the emulsion layer at the interface disappears; and vacuum freeze-drying the obtained polysaccharide solution to obtain the black garlic polysaccharide. The product comprises health-care food and medicine.

2. Use according to claim 1, wherein ​

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