A method for fermenting hemp seeds

Abstract

The present invention relates to a method for fermenting hemp seeds. The method for fermenting hemp seeds comprises the following steps: sterilizing the hemp seeds, wherein the sterilization condition is sterilizing at 121°C for 30 minutes; fermenting the hemp seeds using Rhizopus oryzae, wherein the fermentation conditions include: an inoculation amount of 2%-10% of Rhizopus oryzae, a water content of 8%-43%; a fermentation temperature of 20-35°C; and a fermentation time of 4-20 hours. The mixture obtained by fermenting the hemp seeds can be used to prepare superfine powder of hemp seeds fermented by Rhizopus oryzae. The fermented hemp seeds and the superfine powder of hemp seeds fermented by Rhizopus oryzae prepared by the above method can be used in the fields of medicine, food, health care products, and the like, and can be used to prepare products such as lipid-lowering products, blood sugar-lowering products, and antioxidant products. The fermented hemp seeds and the superfine powder thereof prepared by the method provided by the present invention have the advantages of stronger efficacy, better free radical scavenging effect, and more conducive to production and application.

Description

[0001] This invention is a divisional application. The original Chinese invention patent application number is: 202311206608.5, the application date is: September 19, 2023, and the patent name at the time of application is: A method for fermenting hemp seeds and its application. Technical Field

[0002] The invention belongs to the field of processing technology, and in particular relates to a method for fermenting hemp seeds. Background Art

[0003] Hemp seeds (Cannabis sativa L.) are mature seeds of the mulberry plant hemp. They are rich in high-quality unsaturated fatty acids, proteins, amino acids, sugars, insoluble fibers, vitamins and nutritional minerals. According to the Compendium of Materia Medica, hemp seeds have the effect of strengthening the body. Long-term use can make people healthy and youthful. Hemp seeds can not only moisturize dryness, but also replenish deficiency. In clinical practice, they have therapeutic effects on symptoms such as thirst, constipation caused by dry intestines, dysentery, scabies, rheumatism, stranguria, epilepsy, and irregular menstruation. After the textual research of herbal literature, medical and pharmaceutical literature of all dynasties believed that hemp seeds are a dual-purpose medicine for tonic, fitness, and beauty. Hemp seeds have great application potential and are rich in nutrients such as fatty acids, proteins, carbohydrates, natural antioxidants (phytosterols, trienols, carotene, phospholipids, polyphenols), etc. Among all oilseed crops, only hemp seeds contain this long-chain polyunsaturated fatty acid, with a fatty acid content of 25%-45%, and a ratio of linoleic acid to linolenic acid of about 3:1, which is recognized by WHO as the best dynamic balance of ω-3 and ω-6 essential fatty acids, and is the best ratio required for body metabolism. Hemp protein is rich in various essential amino acids, especially arginine, which meets WHO's requirements for amino acids required by infants.

[0004] Rhizopus oryzae Went et Pr.Geer l. is a fungus widely found in soil, air and other environments. It belongs to the genus Rhizopus of the family Mucorales. Rhizopus oryzae can generally grow normally at 30-35°C, and the most suitable temperature for its growth is 37°C.

[0005] However, there is no report on the use of Rhizopus oryzae in the fermentation of hemp seeds. In addition, due to unreasonable process settings, the efficacy needs to be further improved, and its physical and chemical properties are not suitable for further production, which limits the further application of hemp seeds. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a method for fermenting hemp seeds and application thereof, so as to further improve the efficacy and physical and chemical properties of fermented hemp seeds and superfine powder thereof.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] The invention provides a method for fermenting hemp seeds, which comprises the following steps: fermenting the hemp seeds by using Rhizopus oryzae.

[0009] The beneficial effects of adopting the above technical scheme include: the Rhizopus oryzae fermentation agent is a safe bacterial preparation that does not affect health. During the fermentation process, the Rhizopus oryzae hydrolyzes starch, protein, lipids, etc. in the hemp seeds, and the Rhizopus oryzae with a well-developed enzyme system converts macromolecules into small molecules. The present invention achieves the effect of enhancing the efficacy and promoting the conversion of components through the method of fermenting hemp seeds with Rhizopus oryzae.

[0010] Further, the fermentation conditions include any one or more of the following: Rhizopus oryzae inoculation amount 2%-10%, water content 8%-43%, fermentation temperature 20-35°C, fermentation time 4-20h;

[0011] Preferably, the inoculation amount of Rhizopus oryzae is 6-8%, the water content is 15-36%, the fermentation temperature is 30-35° C., and the fermentation time is 8-16 h;

[0012] Optimally, the inoculation amount of Rhizopus oryzae is 6.9-7%, the water content is 17%, the fermentation temperature is 34.66-35°C, and the fermentation time is 12h.

[0013] Furthermore, the method further includes sterilizing the hemp seeds before fermentation, wherein the sterilization condition is 121° C. for 30 minutes.

[0014] The beneficial effects of adopting the above technical solution include:

[0015] The present invention uses hemp seeds as raw materials, and uses Rhizopus oryzae to ferment the seeds. By optimizing the process, it is found that the fermentation product prepared by the above process can significantly improve the combination rate of sodium glycocholate and sodium taurocholate. After fermentation by Rhizopus oryzae, the contents of hemp seed flavonoids, hemp seed polysaccharides, hemp seed total fatty acids, hemp seed total amino acids, hemp seed oligopeptides, etc. are significantly improved. The fermented product has higher blood lipid-lowering, blood sugar-lowering, and antioxidant effects than unfermented hemp seeds. It is beneficial to prepare medicines, foods, health products, etc., and improves the added value of hemp seeds.

[0016] The invention provides a method for preparing ultrafine powder of hemp seeds fermented by Rhizopus oryzae, which comprises the following steps: ultrafinely grinding a mixture obtained by fermenting the hemp seeds by Rhizopus oryzae.

[0017] Furthermore, the ultrafine grinding comprises the following steps: mixing the mixture after Rhizopus oryzae fermentation of hemp seeds with water-soluble starch, grinding, then adding water to mix, homogenizing and filtering, freeze-drying the filtrate, ultrafine grinding, and obtaining ultrafine powder of hemp seeds fermented by Rhizopus oryzae.

[0018] The beneficial effects of adopting the above technical solution include: adding water-soluble starch can increase the dispersibility and water solubility of the ultrafine powder of the fermentation product.

[0019] Specifically, the method may include the following steps: mixing a mixture of hemp seeds fermented by Rhizopus oryzae with water-soluble starch at a mass ratio of 4:6, crushing for 5 minutes, taking out and adding water to mix at a mass ratio of 1:20, homogenizing with a high-pressure homogenizer for 20 minutes, filtering with a 450-mesh filter, freeze-drying the filtrate for 48 hours to remove moisture, and then crushing with a vibrating drug superfine powder machine for 15 minutes at a temperature of 10°C to obtain Rhizopus oryzae fermented hemp seeds superfine powder.

[0020] The beneficial effects of adopting the above technical scheme include: the physicochemical properties of the ultrafine powder of hemp seeds fermented by Rhizopus oryzae prepared by the above method are improved and improved. Compared with ordinary powder, the degree of organizational structure fragmentation of ultrafine powder particles is relatively large, and the obtained particle size is significantly smaller; at the same time, compared with ordinary powder, the wettability, water holding capacity, water solubility, and swelling power of ultrafine powder are significantly improved. By measuring the angle of repose and the angle of sliding friction, it is found that the fluidity of ultrafine powder is also improved, which can meet the basic needs of production. In addition, the loose density and tap density of ultrafine powder are also good, higher than ordinary powder, and more conducive to human absorption. Ultrafine grinding treatment greatly improves the production adaptability of ultrafine powder. At the same time, ultrafine grinding treatment also significantly improves the effects of lowering blood lipids, lowering blood sugar and anti-oxidation. It is conducive to the deep processing of hemp seeds and the development of functional products.

[0021] The invention provides a fermented hemp seed, and the fermented hemp seed can be prepared by adopting the method.

[0022] The present invention provides one or more applications of the fermented hemp seeds in preparing products for lowering blood lipids, lowering blood sugar, anti-oxidation, preventing obesity, and preventing intestinal cancer.

[0023] The invention provides an ultrafine hemp seed powder fermented by Rhizopus oryzae. The ultrafine hemp seed powder fermented by Rhizopus oryzae can be prepared by adopting the method.

[0024] The present invention provides one or more applications of the Rhizopus oryzae fermented hemp seed superfine powder in preparing blood lipid lowering products, blood sugar lowering products, anti-oxidation, obesity prevention, and intestinal cancer prevention products.

[0025] In the above, the product can be a pharmaceutical preparation, food, health product, cosmetics, etc. Fermented hemp seeds or superfine powder thereof can be used alone or in combination with other components.

[0026] The beneficial effects of adopting the above technical solution include: compared with unfermented hemp seeds, fermented hemp seeds and their ultrafine powders have significantly improved effects in lowering blood lipids, lowering blood sugar, and anti-oxidation. They can be used to prepare products with related functions and are widely used in the fields of medicine, food, health products, cosmetics, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 These are the experimental results of the effect of Rhizopus oryzae inoculation amount on bile salt binding rate. In each group, from left to right are the experimental results of sodium glycocholate and sodium taurocholate.

[0028] Figure 2 These are the experimental results of the effect of fermentation time on bile salt binding rate. In each group, from left to right are the experimental results of sodium glycocholate and sodium taurocholate.

[0029] Figure 3 These are the experimental results of the effect of water content on bile salt binding rate. In each group, from left to right are the experimental results of sodium glycocholate and sodium taurocholate.

[0030] Figure 4 These are the experimental results of the effect of fermentation temperature on bile salt binding rate. In each group, from left to right are the experimental results of sodium glycocholate and sodium taurocholate.

[0031] Figure 5 This is a diagram of significant factors affecting the binding rate of sodium glycocholate.

[0032] Figure 6 This is a diagram of significant factors affecting sodium taurocholate binding rate.

[0033] Figure 7 The response surface and contour plot are the interaction between factor A and factor B.

[0034] Figure 8 This is a comparison chart of bile salt binding rates of unfermented hemp seeds and fermented hemp seeds.

[0035] Fig. 9 This is the finished product of hemp seed powder fermented by Rhizopus oryzae.

[0036] Fig.10 This is the finished product of hemp seed ultrafine powder fermented by Rhizopus oryzae.

[0037] Fig.11 This is the ultrafine powder particle size detection diagram.

[0038] Fig.12 Potential diagram of ultrafine powder stability test.

[0039] Fig.13 These are the experimental results of the binding rates of unfermented hemp seeds, hemp seeds fermented by Rhizopus oryzae and ultrafine powder to sodium cholate.

[0040] Fig.14 The inhibitory effects of unfermented hemp seeds, hemp seeds fermented by Rhizopus oryzae and ultrafine powder on pancreatic lipase activity.

[0041] Fig.15 The inhibitory effects of unfermented hemp seeds, hemp seeds fermented by Rhizopus oryzae and ultrafine powder on micelle solubility.

[0042] Fig.16 IC for unfermented hemp seeds, hemp seeds fermented by Rhizopus oryzae, and ultrafine powder 50 value.

[0043] Fig.17 The results show the inhibitory effects of acarbose, unfermented hemp seeds, hemp seeds fermented by Rhizopus oryzae and superfine powder on α-glucosidase.

[0044] Fig.18 The results show the inhibitory effects of acarbose, unfermented hemp seeds, hemp seeds fermented by Rhizopus oryzae and superfine powder on α-amylase.

[0045] Fig.19 The scavenging effects of Vc control solution, unfermented hemp seeds, hemp seeds fermented by Rhizopus oryzae and ultrafine powder on DPPH free radicals.

[0046] Fig. 20 ABTS was tested for Vc control solution, unfermented hemp seeds, hemp seeds fermented by Rhizopus oryzae and superfine powder + Free radical scavenging effect. DETAILED DESCRIPTION

[0047] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0048] The present invention uses Rhizopus oryzae to ferment hemp seeds, optimizes the fermentation process with bile salt binding rate as an indicator, compares the main components of the obtained product with those of unfermented hemp seeds, then prepares ultrafine powder and detects its properties, and finally conducts in vitro lipid-lowering, blood sugar-lowering and antioxidant efficacy research, and the main contents include:

[0049] (1) The process of fermenting hemp seeds with Rhizopus oryzae was optimized, and the lipid-lowering effect of the fermented object in vitro (binding rate of sodium glycocholate and sodium taurocholate) was used as the index. The four influencing factors of Rhizopus oryzae inoculation amount, fermentation time, fermentation temperature and water content were studied through single factor experiments. Then, the Rhizopus oryzae inoculation amount, water content and fermentation temperature were selected as the three factors for the next response surface experimental design through the Plackett-Burman experiment. On this basis, a response surface optimization experiment was carried out, and a verification experiment was carried out to obtain the optimal extraction process conditions: Rhizopus oryzae inoculation amount 6.9%, water content 17%, fermentation temperature 34.66℃, and fermentation time 12h. Under this optimal condition, the actual value of the average sodium glycocholate binding rate was 75.64%±2.33%, and the actual value of the sodium taurocholate binding rate was 65.77%±3.45%. Compared with unfermented hemp seeds, the binding rate of sodium glycocholate in fermented hemp seeds increased by about 37.60%, and the binding rate of sodium taurocholate increased by about 37.25%.

[0050] (2) The content of components of hemp seeds before and after fermentation was measured, and the results showed that the content of flavonoids, polysaccharides, amino acids, fatty acids, and oligopeptides in fermented hemp seeds increased, while the protein content decreased. The largest changes were in polysaccharides and flavonoids, which increased by about 62.69% and 45.56%, respectively, the amino acid content increased by about 7.14%, the fatty acid content increased by about 11.94%, the oligopeptide content increased by about 7.84%, and the protein content decreased by about 4.54%.

[0051] (3) The fermented hemp seed product was prepared into fermented hemp seed ultrafine powder by using ultrafine grinding technology, and the particle size of the ultrafine powder was measured to be 837.1nm; the ultrafine powder wetting time was 18.76s±1.82s; the moisture content of the ultrafine powder was 1.55%±0.05%; the water holding capacity was 4.25±0.18 (g / g); the swelling power was 5.61±0.27 (mL / g) and the water solubility was 77.02%±0.25%; the repose angle of the ultrafine powder was 38.71° and the sliding friction angle was 50.24°; the bulk density of the ultrafine powder was 0.51±0.23 (g / cm 3 ) and true density 0.60±0.12(g / cm 3 ). Potential detection proves that it has good dispersibility in water; it means that the particle size of ultrafine powder is smaller, the hydration properties such as wettability, water holding capacity, water solubility, and swelling power are better, and the fluidity of the powder is also better. The above indicators are better than ordinary powders, proving the advantages of ultrafine powder in dosage form.

[0052] (4) In vitro lipid-lowering indicators included sodium bileate binding rate, pancreatic lipase inhibition rate, and cholesterol micelle solubility inhibition rate. The results of the pancreatic lipase activity inhibition experiment showed that the IC 50 The value is 8.054mg / mL, the IC value of hemp seeds fermented by Rhizopus oryzae 50The value is 1.255mg / mL, the IC value of ultrafine powder 50 The value is 0.605 mg / mL. The results of the cholesterol micelle solubility inhibition activity experiment showed that the IC 50 The value is 11.758mg / mL, the IC value of hemp seeds fermented by Rhizopus oryzae 50 The value is 7.436mg / mL, and the IC value of ultrafine powder is 50 The value is 3.171 mg / mL. It can be seen that the strength of blood lipid inhibition activity is: ultrafine powder > Rhizopus oryzae fermented hemp seeds > unfermented hemp seeds.

[0053] (5) The study found that the fermented hemp seed ultrafine powder has good anti-oxidation ability and can reduce blood sugar in vitro. The ultrafine powder has an inhibition rate of 87.53% ± 2.20% on the activity of α-glucosidase and 67.70% ± 2.12% on the activity of α-amylase. The ultrafine powder has a scavenging rate of 90.05% ± 1.85% on DPPH and 90.05% ± 1.85% on ABTS. + The clearance rate can reach 97.13%±1.99%.

[0054] In summary, the present invention studies and determines the optimal fermentation process of hemp seeds, studies the changes in its nutritional components, and prepares it into ultrafine powder. Physical and chemical indicators prove that the ultrafine powder has the advantages of good water solubility, lowering blood lipids, lowering blood sugar, and anti-oxidation, which is conducive to the further application of hemp seeds.

[0055] The following is an introduction through specific examples. The experimental methods used in each example are conventional experimental methods in the art unless otherwise specified. The materials, reagents, and instruments used are conventional materials, reagents, and instruments in the art unless otherwise specified, and can be obtained through commercial channels or prepared by conventional methods.

[0056] The UV spectrophotometer (model CARY-100) was purchased from VARIAN, USA; the Kjeldahl nitrogen analyzer (model Kjeltec8400) was purchased from FOSS, Denmark; the amino acid automatic analyzer (model S433D) was purchased from Sykam, Germany; the vibration drug ultrafine pulverizer (model GYB40-10S) was purchased from Jinan Longwei Pharmaceutical Equipment Co., Ltd.; the moisture meter (model HB43-S) was purchased from Shanghai Yidian Analytical Instrument Co., Ltd.; the quick freezer (model SSF-12) was purchased from S&M, Germany. IGMA Company; multifunctional pulverizer (model LX-07A) was purchased from Shanghai Jiangxin Technology Co., Ltd.; electric constant temperature blast drying oven (model DHG-9240A) was purchased from Suzhou Devos Oven Manufacturing Co., Ltd.; intelligent powder characteristics tester (model BT-1001) and laser particle size distribution analyzer (model BT-2001) were purchased from Dandong Better Instrument Co., Ltd.; high-pressure homogenizer (model GYB40-10S) was purchased from Guangzhou Dongzheng Chemical Co., Ltd.

[0057] Hemp seeds were purchased from Yunnan Hanmeng Co., Ltd.; Rhizopus oryzae was purchased from Yantai Dejian Biotechnology Co., Ltd.; 4-nitrophenyl laurate, trypsin, sodium glycocholate, and sodium taurocholate were all purchased from Shanghai Chemical Reagent Company; DPPH, Vc, and ABTS + , oleic acid, sodium acetate, pepsin, and phosphate buffer were purchased from Sinopharm Chemical Reagent Co., Ltd.; rutin standard, glucose reference, aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine, cysteine, and tryptophan were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; petroleum ether, ninhydrin, sodium citrate, and water-soluble starch were purchased from Tianjin Komiou Chemical Reagent Co., Ltd.; α-amylase, α-glucosidase, acarbose, cholesterol, Triton X-100, sodium taurocholate, and pancreatic lipase were purchased from Shanghai McLean Biotechnology Co., Ltd.; deionized water for the experiment was purchased from Wahaha Group. All the above reagents were analytically pure.

[0058] Example 1 Optimization of the process of fermenting hemp seeds with Rhizopus oryzae

[0059] 1.1 Experimental methods

[0060] (1) Sterilization of hemp seeds: Weigh 10 g of hemp seeds respectively, put them in a conical bottle, seal them with a sealing film, and then sterilize them at high temperature, 121°C for 30 minutes. After sterilization, put them on a clean workbench to cool and wait for inoculation.

[0061] (2) Preparation of standard curve:

[0062] Take 2 mL of sodium glycocholate standard solution of different concentrations (0.03, 0.06, 0.12, 0.18, 0.24, 0.30, 0.36, 0.42, 0.48 mmol / L) in a volumetric flask, add 6 mL of 60% sulfuric acid, place in a water bath at 70°C for 20 minutes, then place in an ice bath for 5 minutes, and measure the absorbance at a wavelength of 387 nm using an ultraviolet spectrophotometer. Draw a sodium glycocholate standard curve with the bile salt content as the horizontal axis and the absorbance as the vertical axis.

[0063] Take 2 mL of sodium taurocholate standard solution of different concentrations (0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 mmol / L) in a volumetric flask, add 6 mL of 60% sulfuric acid, and place in a water bath at 70°C for 20 minutes and then in an ice bath for 5 minutes. Measure the absorbance at a wavelength of 387 nm using an ultraviolet spectrophotometer. Draw a sodium taurocholate standard curve with the bile salt content as the horizontal axis and the absorbance as the vertical axis.

[0064] (3) Determination of bile salt content: 0.5 g of the fermented crude powder was placed in a volumetric flask, 3 mL of 10 mg / mL pepsin (dissolved in 0.1 mol / L phosphate buffer at pH 6.5) and 1 mL of 0.01 mol / L hydrochloric acid were added, and the sample was placed in a constant temperature water bath at 37°C and subjected to shaking digestion for one hour to simulate the effect of gastric digestion. The pH of the solution was then adjusted to 6.3 with 0.1 mol / L NaOH solution, and 4 mL of 10 mg / mL trypsin (dissolved in 0.1 mol / L phosphate buffer at pH 6.5) was added, and the sample was placed in a constant temperature water bath at 37°C and subjected to shaking digestion for one hour to simulate the effect of intestinal digestion. Finally, add 4 mL of 0.4 mmol / L sodium glycocholate and 4 mL of 0.5 mmol / L sodium taurocholate to the sample, place the sample in a constant temperature water bath at 37°C for one hour of shaking digestion, transfer the mixed solution to a centrifuge tube, centrifuge at 10,000 r / min for 20 min, take the supernatant and filter it with a 0.45 um aqueous needle filter, measure the absorbance at a wavelength of 387 nm with an ultraviolet spectrophotometer, and calculate the content of sodium glycocholate and sodium taurocholate. The calculation method of bile salt binding rate is as follows:

[0065]

[0066] (4) Single-factor experimental design of Rhizopus oryzae fermentation of hemp seeds: Taking the binding rate of sodium glycocholate and sodium taurocholate as indicators, the effects of Rhizopus oryzae inoculation amount (the fermentation system included Rhizopus oryzae, hemp seeds and water, and the inoculation amount was 2%, 4%, 6%, 8% and 10% of the fermentation system, respectively, all in mass percentage), fermentation time (4h, 8h, 12h, 16h, 20h), fermentation temperature (20℃, 25℃, 30℃, 35℃, 40℃), and water content of the fermentation system (8%, 15%, 22%, 36%, 43%) on the experimental indicators were studied in turn.

[0067] (5) Plackett-Burman experimental design: Based on the single-factor experiment, the binding rate of sodium glycocholate and sodium taurocholate was used as indicators, and the factors of Rhizopus oryzae inoculation amount, fermentation temperature, fermentation time and water content were selected as independent variables to design the Plackett-Burman experiment. By analyzing the results, the most significant factors were screened out.

[0068] (6) Response surface experimental design: Based on the Plackett-Burman test, the binding rate of sodium glycocholate and sodium taurocholate was used as the response value, and the independent variables A, B, and C were used as the influencing factors. A response surface experiment with three factors and three levels was designed. The Box-Behnken experimental factors and levels are shown in Table 1, and the experimental design scheme is shown in Table 2.

[0069] Table 1. Box-Behnken test factors and levels for fermentation process optimization

[0070]

[0071] Table 2 Response surface experimental design scheme

[0072]

[0073] 1.2 Experimental Results and Analysis

[0074] (1) Sodium glycocholate standard curve: The standard curve equation of sodium glycocholate is y=2.6642x+0.0561, R 2 =0.9968, where x is the sodium glycocholate content (mmol / L) and y is the absorbance.

[0075] (2) Taurocholate sodium standard curve: The standard curve equation of taurocholate sodium is y=2.4524x+0.0367, R 2 =0.9985, where x is the sodium taurocholate content (mmol / L) and y is the absorbance.

[0076] (3) Single factor test results and analysis

[0077] ① Inoculation amount of Rhizopus oryzae: Figure 1 It can be seen that when other conditions are the same (i.e. fermentation time 10h, fermentation temperature 30°C, water content 15%), when the Rhizopus oryzae inoculation amount is 2%-8%, the binding rates of sodium glycocholate and sodium taurocholate are constantly improving. When the Rhizopus oryzae inoculation amount is 8%, the sodium glycocholate binding rate is 65.91%±1.22%, and the sodium taurocholate binding rate is 64.40%±1.41%. At this time, the binding rates of the two bile salts are the best. When the Rhizopus oryzae inoculation amount is 10%, the binding rates of sodium glycocholate and sodium taurocholate begin to decline. Therefore, the Rhizopus oryzae inoculation amount of 8% is selected to carry out the next fermentation test.

[0078] ② Fermentation time: Figure 2 It can be seen that when other conditions are the same (i.e., inoculation volume 8%, fermentation temperature 30°C, water content 15%), when the fermentation time is 4-12h, the binding rates of sodium glycocholate and sodium taurocholate are continuously increasing. When the fermentation time is 12h, the binding rate of sodium glycocholate is 70.13%±2.01%, and the binding rate of sodium taurocholate is 65.86%±1.74%. At this time, the binding rates of the two bile salts are the best. When the fermentation time is 12-20h, the binding rates of sodium glycocholate and sodium taurocholate begin to decrease, so the fermentation time of 12h is selected for the experiment.

[0079] ③Water content: Figure 3 It can be seen that under the same other conditions (fermentation time 12h, fermentation temperature 30°C, inoculation amount 8%), when the water content is 8%-22%, the binding rates of sodium glycocholate and sodium taurocholate are constantly increasing. When the water content is 22%, the binding rate of sodium glycocholate is 71.54%±1.91%, and the binding rate of sodium taurocholate is 77.4%±2.11%. At this time, the binding rates of the two bile salts are the best. When the water content is 22%-43%, the binding rates of sodium glycocholate and sodium taurocholate continue to decrease, so a water content of 22% is selected for fermentation.

[0080] ④ Fermentation temperature: Figure 4 It can be seen that when other conditions are the same (i.e., fermentation time 12h, inoculation size 8%, water content 22%), when the fermentation temperature is 20°C-35°C, the binding rates of sodium glycocholate and sodium taurocholate are continuously increasing. When the fermentation temperature is 35°C, the binding rate of sodium glycocholate is 71.92%±1.42%, and the binding rate of sodium taurocholate is 72.65%±2.06%. At this time, the binding rates of the two bile salts are the best. When the fermentation temperature is 35°C-45°C, the binding rates of sodium glycocholate and sodium taurocholate continue to decrease, so the fermentation temperature is selected to be 35°C for fermentation.

[0081] (4) Fermentation process optimization Plackett-Burman test results and analysis

[0082] According to the results of the single factor test, the Plackett-Burman test was designed with the sodium glycocholate binding rate and sodium taurocholate binding rate as the experimental response values. The experimental design method and results are shown in Table 3. The results of the screened significant factors are shown in Figure 5 and Figure 6 .

[0083] Table 3 Factor levels and results of Plackett-Burman test design

[0084]

[0085] Depend on Figure 5 It can be seen that the top three factors that have the most obvious impact on the binding rate of sodium glycocholate are water content (C), Rhizopus oryzae inoculation amount (A) and fermentation temperature (D). Figure 6 It can be seen that the top three factors that have the most obvious impact on the binding rate of sodium taurocholate are fermentation temperature (D), water content (C), and Rhizopus oryzae inoculation amount (A). It can be seen that these three factors have a more significant impact on the binding rate of sodium taurocholate, so Rhizopus oryzae inoculation amount (A), water content (C) and fermentation temperature (D) are selected as the three factors for the next response surface experimental design.

[0086] (5) Results and analysis of response surface experiments for fermentation process optimization

[0087] ① Response surface Box-Behnken experimental design

[0088] Based on the results of the Plackett-Burman test, the binding rate of sodium glycocholate (Y1) and the binding rate of sodium taurocholate (Y2) were used as the response values. The factors of Rhizopus oryzae inoculation amount (A), water content (B) and fermentation temperature (C) were selected as independent variables. Design-Expert 11 was used to carry out a 3-factor 3-level response surface experimental design. The results of the Box-Behnken test are shown in Table 4.

[0089] Table 4 Response surface experimental design and results

[0090]

[0091] ②Response surface interaction analysis

[0092] Taking the sodium glycocholate binding rate (Y1) as the response value, the equation obtained by quadratic polynomial regression fitting is: Y1 = 63.69-9.48A-11.86B-2.84C+0.8950AB-0.8600AC+2.05BC-2.95A 2 -8.56B 2 -17.37C 2 .

[0093] As shown in Table 5, the model is significant (P<0.05), and the lack of fit term is not significant (P=0.3892>0.05), which shows that the model has a good fit and can better reflect the change rules of sodium glycocholate binding rate and various factors. In addition, the determination coefficient R 2 =0.9305, indicating that the regression equation simulates the test well, the test error is relatively small, and the correction determination coefficient R 2 adj = 0.8410. It can be seen that the predicted value of the software is correlated with the actual value of the test, and the correlation is very high. In addition, the coefficient of variation CV is 12.48%, indicating that the variability of the test is 12.48%, which can be used in the model to analyze and predict the process of fermenting hemp seeds with Rhizopus oryzae. From the P value, it can be obtained that the linear terms A and B of the equation, and the quadratic term B 2 The effect on the binding rate of sodium glycocholate was significant (P<0.05), while other factors had no significant effect (P>0.05). Therefore, the regression equation can be used to predict the binding rate of sodium glycocholate under different fermentation conditions.

[0094] Table 5 Variance analysis table of regression model

[0095]

[0096]

[0097] Note: * indicates significant difference (P < 0.05); ** indicates extremely significant difference (P < 0.01); coefficient of variation CV = 12.48%, R 2 =0.9305, R 2 adj=0.8410.

[0098] Taking the sodium taurocholate binding rate (Y2) as the response value, the equation obtained by quadratic polynomial regression fitting is: Y2 = 64.67 + 1.99A-11.89B-1.57C + 4.71AB-2.08AC + 0.1475BC-4.53A 2 -15.66B2-9.20C 2 .

[0099] As shown in Table 6, the model is significant (P<0.0001<0.05), and the lack of fit term is not significant (P=0.7558>0.05), which shows that the model has a good fit and can better reflect the change law of sodium taurocholate binding rate and various factors. It can be seen that the determination coefficient R 2 =0.9895, which shows that the linear regression equation simulates the test well, the error of the test results is small, and the correction determination coefficient R 2 adj = 0.9759, from which it can be seen that the predicted value of the software is correlated with the actual value of the experiment, and the correlation is very high. In addition, the coefficient of variation CV is 4.13%, indicating that the variability of the experiment is 4.13%, which can be used for the model to conduct the subsequent analysis and prediction of the hemp seed fermentation process of Rhizopus oryzae. From the P value, it can be seen that the first-order term A of the equation has a significant effect on the binding rate of sodium taurocholate (P < 0.05), the first-order term B of the equation, the interaction term AB, and the quadratic term A of the equation have a significant effect on the binding rate of sodium taurocholate (P < 0.05). 2 , B 2 , C 2 The effect on the binding rate of sodium taurocholate was extremely significant (P<0.01), while other factors had no significant effect (P>0.05). From the F value, it can be seen that the order of influence of each factor on the binding rate of sodium taurocholate is water content> Rhizopus oryzae inoculation amount> fermentation temperature. Therefore, the regression equation can be used to predict the binding rate of sodium taurocholate under different fermentation conditions.

[0100] Table 6 Analysis of variance of regression model

[0101]

[0102] Note: * indicates significant difference (P < 0.05); ** indicates extremely significant difference (P < 0.01); coefficient of variation CV = 4.13%, R 2 =0.9895, R 2 adj=0.9759.

[0103] Depend on Figure 7 It can be seen that the contour map of the interaction between Rhizopus oryzae inoculation amount and water content is elliptical, which proves that the interaction is significant and consistent with the variance results of the regression model. In the 3D response surface diagram, it can be seen that the sodium taurocholate binding rate first increases and then decreases with the increase of Rhizopus oryzae inoculation amount and water content.

[0104] ③Optimal process conditions and verification experiments

[0105] Using Design-Expert 11 software, the optimal fermentation conditions were predicted by optimization: Rhizopus oryzae inoculation amount 6.9%, water content 17%, fermentation temperature 34.66℃, and fermentation time 12h. Under this optimal fermentation condition, the software predicted the binding rate of sodium glycocholate to be 72.44%, and the predicted binding rate of sodium taurocholate to be 65.39%. Based on actual conditions, the optimal fermentation conditions are: Rhizopus oryzae inoculation amount 6.9%, water content 17%, fermentation temperature 35℃, and fermentation time 12h. Three parallel validation tests were carried out under this fermentation process condition, and the actual value of the average sodium glycocholate binding rate was 75.64%±2.33%, and the actual value of the sodium taurocholate binding rate was 65.77%±3.45%. The sodium glycocholate binding rate was within 5% of the model predicted value, and the sodium taurocholate binding rate was within 1% of the model predicted value, which was basically consistent with the model prediction, indicating that the equation has a good fit with the actual experimental results, and the optimal parameters obtained through the experiment have good stability and reliability, indicating that the model is effective and feasible.

[0106] (6) Results of bile salt binding rate of unfermented hemp seeds: Figure 8 As shown, the experimental results show that the binding rate of unfermented hemp seeds to sodium glycocholate is 34.84% ± 3.59%, while the binding rate of hemp seeds fermented by Rhizopus oryzae to sodium glycocholate is 75.64% ± 2.33%; the binding rate of unfermented hemp seeds to sodium taurocholate is 28.52% ± 2.38%, while the binding rate of hemp seeds fermented by Rhizopus oryzae to sodium glycocholate is 65.77% ± 3.45%. It can be seen that after fermentation by Rhizopus oryzae, the binding rate of sodium glycocholate and sodium taurocholate is significantly improved.

[0107] In summary, the present embodiment uses sodium glycocholate and sodium taurocholate binding rates as indicators, and takes Rhizopus oryzae inoculum, fermentation time, water content, and fermentation temperature as single factors to optimize the fermentation process. Rhizopus oryzae inoculum: 2%, 4%, 6%, 8%, 10%, the highest cholate binding rate of 8% inoculum, sodium glycocholate binding rate is 65.91% ± 1.22%, and sodium taurocholate binding rate is 64.4% ± 1.41%. Fermentation time: 4h, 8h, 12h, 16h, 20h, the highest cholate binding rate of 12h, sodium glycocholate binding rate is 70.13% ± 2.01%, and sodium taurocholate binding rate is 65.86% ± 1.74%. Water content: 8%, 15%, 22%, 29%, 36%, 43%, the highest bile salt binding rate was obtained at 22% water content, the binding rate of sodium glycocholate was 71.54%±1.91%, and the binding rate of sodium taurocholate was 77.4%±2.11%. Fermentation temperature: 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, the highest bile salt binding rate was obtained at 35℃ water content, the binding rate of sodium glycocholate was 71.92%±1.42%, and the binding rate of sodium taurocholate was 72.65%±2.06%.

[0108] Through the Plackett-Burman test, the inoculation amount of Rhizopus oryzae, water content, and fermentation temperature were obtained as three significant factors, and a 3-factor 3-level response surface experiment was designed based on this. After the response surface experiment, the optimal conditions were as follows: the optimal fermentation conditions obtained by optimization were 6.9% Rhizopus oryzae inoculation amount, 17% water content, 34.66°C fermentation temperature, and 12h fermentation time. Under these optimal conditions, the actual value of the average sodium glycocholate binding rate was 75.64% ± 2.33%, and the actual value of the sodium taurocholate binding rate was 65.77% ± 3.45%.

[0109] In the response surface design, both sodium glycocholate binding rate and sodium taurocholate binding rate were selected as response values ​​for process optimization, and it was impossible to simultaneously meet the two indicators to obtain the maximum value. Because the sodium glycocholate binding rate is more important in cholesterol binding, the parameter with the highest sodium glycocholate binding rate was selected in the response surface experimental results. At this time, the sodium taurocholate binding rate was lower than the maximum value of the single factor experiment.

[0110] Through comparative experiments, it was found that the binding rate of unfermented hemp seeds to sodium glycocholate was 34.84%±3.59%, and the binding rate of hemp seeds fermented by Rhizopus oryzae to sodium glycocholate was 72.44%±2.33%; the binding rate of unfermented hemp seeds to sodium taurocholate was 28.52%±2.38%, and the binding rate of hemp seeds fermented by Rhizopus oryzae to sodium glycocholate was 65.77%±3.45%. It was concluded that hemp seeds fermented by Rhizopus oryzae significantly increased the binding rate of sodium glycocholate and sodium taurocholate.

[0111] Example 2 Determination of the content of each component before and after Rhizopus oryzae fermentation of hemp seeds

[0112] This embodiment is aimed at the rich nutrients in hemp seeds, and some components that may have the effect of lowering blood lipids are determined before and after Rhizopus oryzae fermentation. The Rhizopus oryzae fermentation conditions are: inoculation amount 7%, water content 17%, fermentation temperature 35°C, and fermentation time 12h; comparative analysis is performed to study the components that affect the blood lipid-lowering effect of hemp seeds, so as to facilitate the further application of hemp seeds.

[0113] 2.1 Experimental methods

[0114] (1) Determination of flavonoid content: The total flavonoid content in hemp seeds was determined by sodium nitrite-aluminum chloride complex spectrophotometry. When sodium nitrite is present in the solution, when the pH of the solution is neutral or weakly alkaline, flavonoid compounds will form chelates with the aluminum salts contained therein. After adding NaOH, the solution will appear red-orange. Within a certain concentration range, the flavonoid content is proportional to the absorbance value.

[0115] ①Prepare rutin standard stock solution: accurately weigh 5 mg of rutin standard, dissolve it in 60% ethanol solution to make up to 100 mL. Prepare a standard stock solution with a concentration of 50 mg / L and store it in a refrigerator at 4°C away from light.

[0116] ② Make a standard curve: Accurately pipette a certain amount of rutin standard solution into 10mL volumetric flasks to prepare a standard series of 2mg / mL, 4mg / mL, 6mg / mL, 8mg / mL, 10mg / mL, and 12mg / mL, add ethanol solution to make the total volume of the solution 5mL, then add 0.3mL of sodium nitrite solution, shake well, let stand for 8min, add 0.3mL of aluminum chloride solution, shake well, let stand for 10min, add 4mL of sodium hydroxide solution, dilute to the scale with ethanol solution, shake well, let stand for 10min. Measure its absorbance at 510nm, and draw a standard curve with concentration as the horizontal axis and absorbance as the vertical axis.

[0117] ③Prepare the sample: weigh 5 g of the hemp seed sample before and after fermentation and place them in a 50 mL volumetric flask, add 30 mL of 60% ethanol solution, perform ultrasound at 280 V and 60° C. for 90 min, make up the volume with 60% ethanol, centrifuge until clear, filter and set aside.

[0118] ④ Determination of sample solution: Pipette 1 mL of the test solution before and after fermentation into a volumetric flask, add ethanol solution to a total volume of 5 mL, add 0.3 mL of sodium nitrite solution, shake well and let stand for 8 minutes, then add 0.3 mL of aluminum chloride solution, shake well and let stand for 10 minutes, add 4 mL of sodium hydroxide solution, dilute to the scale with ethanol solution, shake well and let stand for 10 minutes. Measure the absorbance at 510 nm and calculate the concentration of total flavonoids in the sample according to the standard curve.

[0119] ⑤ Calculation of total flavonoid content

[0120]

[0121] Where: X is the content of total flavonoids in hemp seeds, in milligrams per kilogram (mg / kg);

[0122] c—the total flavonoid concentration of the sample solution obtained by calculation (mg / L);

[0123] 10—dilution multiple of the test solution;

[0124] m—mass of hemp seed sample, in grams (g);

[0125] 0.05—total volume of the extract of the hemp seed sample, in (L);

[0126] H—mass fraction of water in hemp seeds (%).

[0127] (2) Determination of polysaccharide content

[0128] ①Prepare standard glucose solution: Accurately weigh 10 mg of anhydrous glucose and place it in a beaker. Dissolve it with distilled water and transfer it to a 100 mL volumetric flask to make a glucose standard solution with a concentration of 0.1 mg / mL.

[0129] ②Prepare standard curve: Accurately pipette a certain amount of glucose standard solution into 10mL volumetric flasks, make up to 1.0mL with water, then add 1mL of 5% phenol solution and 3mL of sulfuric acid to prepare a standard series with concentrations of 0.01mg / mL, 0.02mg / mL, 0.03mg / mL, 0.04mg / mL, 0.05mg / mL, and 0.06mg / mL. After oscillation and mixing, heat in a 100℃ water bath for 10min, and draw the glucose standard curve with the concentration of glucose solution as the horizontal axis and the absorbance value as the vertical axis.

[0130] ③Sample determination:

[0131] Unfermented hemp seed powder and Rhizopus oryzae fermented hemp seed powder were prepared respectively. The preparation method was freeze drying, pre-freeze drying at -55°C for 12h and post-freeze drying at 35°C for 8h.

[0132] Take 1.0g of unfermented hemp seed powder and 1.0g of hemp seed powder fermented by Rhizopus oryzae, place them in volumetric flasks, add anhydrous ethanol at a material ratio of 1g:30mL, extract for 40min at 280V and 46°C with an ultrasonic extractor, centrifuge for 15min at 8000r / min, retain the supernatant, add 50mL of water to the remaining residue, ultrasonically extract 3 times, 30min each time, centrifuge for 10min at 4000r / min in a centrifuge, combine the supernatants of the three times, mix and draw 1mL into a 25mL volumetric flask, add water to the scale, shake well, and obtain the sample determination liquid. Then accurately draw 1mL of the sample determination liquid into a volumetric flask, add 1mL of 5% phenol solution and 3mL of 80% sulfuric acid solution respectively, shake and mix, heat in a 100°C water bath for 10min, and measure the absorbance after cooling to room temperature.

[0133] ④ Calculation of polysaccharide content

[0134]

[0135] Where: W is the polysaccharide content in hemp seeds, in grams per 100 grams (g / 100g);

[0136] P—the calculated concentration of the sample solution in milligrams per milliliter (mg / mL);

[0137] V1—volume of sample test solution, in milliliters (mL);

[0138] V2—volume of sample extract, in milliliters (mL);

[0139] V3—The volume of sample extract used to prepare the sample test solution, in milliliters (mL);

[0140] m—sample mass, in grams (g);

[0141] 0.9—Correction factor for converting glucose to dextran.

[0142] (3) Determination of protein content: The protein content was determined by the Kjeldahl method. 0.149 g of hemp seed samples before and after fermentation were accurately weighed and placed in a digestion tube. 8 mL of sulfuric acid, 3 g of potassium sulfate, and 0.3 g of copper sulfate were added to the tubes, respectively. The tubes were placed in a 420°C digestion furnace for digestion for 1 h. After digestion, the samples were taken out and cooled, and then mounted on a Kjeldahl nitrogen analyzer for titration. After the titration, the protein content was determined.

[0143] (4) Determination of fatty acid content: Weigh 2 g of hemp seed samples before fermentation and 2 g of hemp seed samples fermented with Rhizopus oryzae, respectively, and place the two samples in defatted filter paper bags, respectively, and extract them for 4 hours using a Soxhlet extraction apparatus with petroleum ether as the extractant. Then, place the filter paper bags in a 105°C oven and dry them to constant weight.

[0144] (5) Determination of amino acid content:

[0145] ① Preparation of mixed amino acid standard stock solution: Preparation of mixed amino acid standard stock solution (1umol / mL): Accurately weigh individual amino acid standards (accurate to 0.00001g) in the same 50mL beaker, pipette 8.3mL of 6mol / L hydrochloric acid solution to dissolve, then transfer to a 250mL volumetric flask, dilute to the mark with water, and mix well.

[0146] ②Prepare mixed amino acid standard working solution: Preparation of mixed amino acid standard working solution (100nmol / mL): Accurately pipette 1.0mL of mixed amino acid standard stock solution into a 10mL volumetric flask, then add pH 2.2 sodium citrate buffer solution to the scale, mix well, and obtain the standard liquid for loading the machine.

[0147] ③ Determination of samples: Accurately weigh the hemp seed samples before and after fermentation in a hydrolysis tube, control the protein content in the sample between 10-20 mg, and then add 12 mL of 6 mol / L hydrochloric acid solution. Freeze the hydrolysis tube with a refrigerant for 5 minutes, and evacuate it with a vacuum pump (close to 0 Pa) and inject nitrogen. After three times of evacuation and nitrogen injection, the nitrogen is full. After sealing, place the hydrolysis tube in an electric blast thermostat, hydrolyze it at a temperature of 110℃±1℃ for 22 hours, then take it out and cool it to room temperature. Open the hydrolysis tube and filter it into a 50mL volumetric flask, then add water to the scale and shake it well. Accurately extract 1.0 mL of the filtrate into a 25mL test tube, dry it under reduced pressure with a test tube concentrator at a high temperature of 50℃, then dissolve it with 2 mL of water, then dehydrate it under reduced pressure, and finally evaporate it to dryness. Dissolve with 1.0mL-2.0mL pH 2.2 sodium citrate buffer solution, shake and mix, filter with a 0.22μm filter membrane and put into the sample injection bottle of the instrument, which is the sample test solution. At the detection wavelength of 570nm and 440nm, inject the mixed amino acid standard working solution and the sample test solution into the amino acid analyzer in the same volume, respectively, and use the external standard method to calculate the concentration of amino acids in the sample test solution using the peak area. The content of each amino acid in the sample test solution is calculated according to the following formula:

[0148]

[0149] Where: C i—The content of amino acid i in the sample assay solution, in nanomoles per milliliter (nmol / mL);

[0150] A i —The peak area of ​​amino acid i in the sample determination solution;

[0151] A s —The peak area of ​​amino acid s of amino acid standard working solution;

[0152] c s —Amino acid content of amino acid standard working solution, in nanomoles per milliliter (nmol / mL).

[0153] (6) Determination of oligopeptide content

[0154] ① Determination of acid-soluble protein content: Accurately weigh 1.0 g of unfermented hemp seeds and 1.0 g of hemp seeds fermented by Rhizopus oryzae into a volumetric flask, add 15% trichloroacetic acid solution, dissolve and dilute to 50 mL, mix well and let stand for 10 min, centrifuge the two sample solutions at 9000 r / min for 25 min, take 10 mL of the two sample centrifuges respectively, and determine the protein content therein by Kjeldahl method, calculate the content of acid-soluble protein in the two samples, and the protein conversion factor is 6.25.

[0155] ② Determination of oil-free amino acids: Accurately weigh 1.0 g of unfermented and fermented hemp seeds in a volumetric flask, dissolve them evenly with 20 mL of 5% trichloroacetic acid solution, transfer the two sample solutions to 100 mL volumetric flasks, and then make up to volume with 0.002 mol / L hydrochloric acid solution. Then, centrifuge the two sample solutions at 9000 r / min for 15 min and take the supernatant.

[0156] ③Result calculation

[0157] The oligopeptide content X1 is calculated according to the following formula:

[0158]

[0159] Where: X1—the content of oligopeptide in the sample, in grams per hundred grams (g / 100g);

[0160] C2—the content of acid-soluble protein in the sample, in grams per hundred grams (g / 100g);

[0161] C3—the content of amino acids in the sample, g / 100g;

[0162] w—water content in the sample, grams per hundred grams (g / 100g).

[0163] 2.2 Experimental Results and Analysis

[0164] (1) Results and analysis of flavonoids, polysaccharides, and protein contents

[0165] ① Glucose content standard curve: The standard curve equation of glucose is y=8.8534x+0.0397, R 2 =0.9962, where x is the glucose concentration (mg / mL) and y is the absorbance.

[0166] ② Flavonoid content standard curve: The standard curve equation of flavonoids is y=0.0067x-0.0056, R 2 =0.9994, where x is mg / mL and y is AU.

[0167] As shown in Table 7, the polysaccharide content of hemp seeds increased significantly after fermentation by Rhizopus oryzae, which may be due to the fact that Rhizopus oryzae is an enzyme with a well-developed enzyme system, which may convert starch into polysaccharides during the fermentation process, so the polysaccharide content of hemp seeds increased after fermentation; the flavonoid content of hemp seeds also increased significantly after fermentation by Rhizopus oryzae, which may be due to the destruction of the combination of antioxidant active substances and the matrix during the fermentation process, resulting in an increase in the content of flavonoids; the protein content of hemp seeds decreased after fermentation by Rhizopus oryzae, which may be due to the conversion of some large molecular proteins into small molecular amino acids through the catalytic action of enzymes during the fermentation process. The nutritional value of protein is related to the quality of its total protein as well as the content and composition of the amino acids it contains.

[0168] Table 7 Contents of flavonoids, polysaccharides and proteins

[0169]

[0170] (2) Results and analysis of fatty acid content

[0171] Through experimental determination, the content of fatty acids in hemp seeds without fermentation and hemp seeds fermented by Rhizopus oryzae is shown in Table 8. Hemp seeds contain a large amount of fatty acids. It can be seen from Table 8 that the total fatty acid content accounts for about 40%. Among these fatty acids, linoleic acid has the highest content, and its proportion in the whole fatty acid exceeds 50%, which has anti-thrombotic and anti-arrhythmic effects; linolenic acid is the fatty acid in hemp seeds second only to linoleic acid, accounting for less than 20%, followed by oleic acid, accounting for less than 15%, and palmitic acid and stearic acid account for the smallest proportion, about 5% and 3%. It can be found from Table 8 that the content of oleic acid and linoleic acid in hemp seeds fermented by Rhizopus oryzae has increased, while the content of linolenic acid, stearic acid and palmitic acid has decreased. The ratio of linoleic acid to linolenic acid is about 3:1, which is the best ratio required by the human body, which fully explains that the composition of various fatty acids in hemp seeds fermented by Rhizopus oryzae has changed, indicating that Rhizopus oryzae can affect the content of fatty acids after fermentation.

[0172] Table 8 Fatty acid content

[0173]

[0174] (3) Results and analysis of amino acid content

[0175] Through experimental determination, the content of amino acids in hemp seeds that have not been fermented and hemp seeds fermented by Rhizopus oryzae is shown in Table 9. As can be seen from Table 9, the total amount of amino acids accounts for about 30% of hemp seeds. These amino acids are divided into many categories, so the nutritional value of protein depends on the composition of these amino acids in addition to the total content. Among the 16 amino acids, the content of glutamic acid is the highest (6.43-6.81g / 100g), and it also contains a large amount of arginine (3.74-4.18g / 100g) and aspartic acid (3.27-3.65g / 100g). For the nerve center and cerebral cortex, glutamic acid is a good supplementary substance that can delay the consumption of energy substances, thereby promoting the recovery of physical fitness after strenuous exercise. Arginine can promote the growth and development of children. Hemp seeds are a good food for supplementing arginine, and aspartic acid can be converted into glutamic acid in the human body, which can further enhance the anti-fatigue effect of hemp seeds. The content of leucine, isoleucine and valine is also slightly higher than that of soybeans. These three substances work together to repair muscles, lower blood sugar, enhance the body's energy, and promote the growth and development of children. Hemp seeds are rich in threonine and isoleucine, which have a good effect on the repair and growth of thin physiques. The amino acid content of hemp seeds is similar to that of soybeans, but it also has other advantages that soybeans do not have, such as: because hemp protein does not contain inhibitory factors of tryptophan, it will not have any effect on protein absorption, and hemp seeds do not have many oligosaccharides in soybeans, which will not cause bloating and nausea. It can be seen from Table 9 that the total amount of amino acids in hemp seeds after fermentation by Rhizopus oryzae is increased, and the content of most amino acids is significantly increased, which fully explains the increase in the amino acid content of hemp seeds after fermentation by Rhizopus oryzae. The possible reason is that after fermentation, macromolecules such as protein in hemp seeds are transformed into small molecular amino acids.

[0176] Table 9 Content of 16 kinds of amino acids

[0177]

[0178] (4) Results and analysis of oligopeptides

[0179] Through various experimental measurements, the content of acid-soluble protein and the content of oil-free amino acids were obtained, and the content of oligopeptides was calculated by formula. It can be seen from Table 10 that the content of oligopeptides in hemp seeds fermented by Rhizopus oryzae increased by about 7.8% compared with that before fermentation. Recent studies have shown that in the digestive tract, proteins are absorbed by the human body in the form of oligopeptides during enzymatic hydrolysis, and their role in the intestine is much greater than that of complete free amino acids. Therefore, their role in the intestine has received more and more attention. Therefore, the increase in the content of oligopeptides in hemp seeds fermented by Rhizopus oryzae may be due to the fact that Rhizopus oryzae contains a large amount of enzymes, and the protein in the hemp seeds is converted into oligopeptides through enzymatic hydrolysis during the fermentation process, so the hemp seeds fermented by Rhizopus oryzae may be more easily absorbed by our body.

[0180] Table 10 Contents of acid-soluble proteins, free amino acids and oligopeptides

[0181]

[0182] In this embodiment, flavonoids, polysaccharides, proteins, five fatty acids, sixteen amino acids, and oligopeptide components in hemp seeds before fermentation and hemp seeds fermented by Rhizopus oryzae are measured, and the content changes before and after fermentation are analyzed and compared, and the following conclusions are obtained: the flavonoid content of hemp seeds after Rhizopus oryzae fermentation is increased by 350.4 mg / kg, an increase of about 45.6%; the polysaccharide content of hemp seeds after Rhizopus oryzae fermentation is increased by 0.42 g / 100 g, an increase of about 62.7%; the protein content of hemp seeds after Rhizopus oryzae fermentation is reduced by 1.45%; the total fatty acid content of hemp seeds after Rhizopus oryzae fermentation is increased by 4.37 g / 100 g, an increase of about 11.94%; among different types of fatty acids, the contents of linolenic acid, stearic acid and palmitic acid are reduced, and the contents of oleic acid and linoleic acid are increased; the total amino acid content of hemp seeds after Rhizopus oryzae fermentation is increased by 2.1 g / 100 g, an increase of 7.14%. Except for methionine and proline, the contents of other types of amino acids have increased to varying degrees; the oligopeptide content of hemp seeds after fermentation by Rhizopus oryzae increased by 0.08g / 100g, an increase of about 7.84%. According to the above embodiment, the lipid-lowering ability of the fermented product of hemp seeds fermented by Rhizopus oryzae is higher than that of unfermented hemp seeds. Combined with the substantial increase in flavonoids and polysaccharides in hemp seeds after fermentation by Rhizopus oryzae, it can be explained that the increase of flavonoids and polysaccharides in the fermented product has a promoting effect on the enhancement of its lipid-lowering ability.

[0183] Example 3 Preparation and physicochemical properties of superfine hemp seed powder fermented by Rhizopus oryzae

[0184] In this embodiment, hemp seeds fermented by Rhizopus oryzae are processed to be prepared into ultrafine powder, and then the prepared ultrafine powder is subjected to physical and chemical indexes such as particle size, wettability, powder moisture, water holding capacity, water solubility, swelling power, angle of repose and sliding friction angle, loose density and tap density to explore whether the properties of the prepared ultrafine powder are good, so as to conduct further research later.

[0185] The product of hemp seeds fermented with Rhizopus oryzae in this embodiment is prepared under the following fermentation conditions: inoculation amount 7%, water content 17%, fermentation temperature 35° C., and fermentation time 12 h.

[0186] The common powder involved in this embodiment is prepared by the following method: pulverizing the powder three times with a traditional Chinese medicine pulverizer, each time for 15 seconds and passing through a 100-mesh sieve.

[0187] 3.1 Experimental methods

[0188] (1) A preparation process of superfine hemp seed powder fermented by Rhizopus oryzae comprises the following steps: mixing a mixture of hemp seeds fermented by Rhizopus oryzae with water-soluble starch in a mass ratio of 4:6, placing the mixture in a grinder and grinding it for 5 min, taking it out and adding water, wherein the mass ratio of the ground powder to water is 1:20, mixing, homogenizing it with a high-pressure homogenizer at 60 MPa for 20 min, filtering it with a 450-mesh filter screen, freeze-drying the filtrate for 48 h to remove moisture, and then grinding it with a vibrating drug superfine powder machine (temperature 10° C., grinding time 15 min) to obtain superfine hemp seed powder fermented by Rhizopus oryzae, taking the powder not ground by the vibrating drug superfine powder machine as ordinary powder, and measuring the physical and chemical indicators of the two.

[0189] (2) Determination of physical and chemical indicators of ultrafine powder

[0190] ① Determination of particle size: The particle sizes of ordinary powder and ultrafine powder were measured by wet method using a laser particle size distribution analyzer, and the particle size was evaluated by specific surface area and span value.

[0191] ② Determination of wettability: First, add 50 mL of distilled water to the culture dish, and then add 1.0 g of ordinary powder and ultrafine powder respectively, and measure the time (s) required for the two powders to be completely wetted by water. Each sample is measured in parallel 3 times.

[0192] ③ Determination of powder moisture content: Accurately weigh 1.0g of ordinary powder and ultrafine powder, and spread them flat in weighing bottles with constant weight, put them into a drying oven at 105℃ for constant weight drying, so as to calculate their moisture content.

[0193] ④ Determination of water holding capacity: Accurately weigh 0.50g of ordinary powder and ultrafine powder respectively and place them in a 50mL centrifuge tube. The weighed mass is m1. Add 50mL of distilled water to the centrifuge tube and shake well. Centrifuge at 5000r / min for 15min in a centrifuge. Discard the supernatant and weigh the weight of the remaining sediment as m2. Each sample is measured three times in parallel and calculated according to the following formula:

[0194]

[0195] ⑤ Determination of water solubility: Accurately weigh 1.0g of ordinary powder and ultrafine powder respectively, evenly disperse each in 50mL of distilled water, shake in a 80℃ water bath for 30min to fully dissolve, centrifuge at 6000r / min for 10min, take the supernatant and place it in a 105℃ drying oven to dry to constant weight, and weigh its mass. The water solubility calculation formula is as follows:

[0196]

[0197] ⑥ Determination of expansion force: accurately weigh 1.0g of ordinary powder and ultrafine powder respectively, put them into a 50mL measuring cylinder, record the volume as V1, add 50mL of distilled water, stir evenly and let stand at room temperature for 24h, record the volume of ordinary powder and ultrafine powder as V2, measure each sample in parallel 3 times, and calculate according to the following formula:

[0198]

[0199] ⑦ Determination of the angle of repose: Use the BT1001 intelligent powder property tester, fix a funnel at a certain distance from the horizontal plane, take appropriate amounts of ordinary powder and ultrafine powder respectively, the powder first passes through the filter, flows to the discharge port, falls on the sample table and slowly turns into a cone, until the powder accumulates on the sample table into a symmetrical cone, and the highest point reaches the bottom of the funnel, then stop feeding. After the feeding is completed, it is automatically measured. Each sample is measured 3 times in parallel to calculate the angle of repose.

[0200] ⑧ Determination of sliding friction angle: Use BT1001 intelligent powder property tester, weigh 3g of ordinary powder and ultrafine powder respectively and put them into the center of the glass plate, then gently lift one end of the glass plate until 90% of the powder falls off, so that the angle between the lifted glass plate and the horizontal table top will be automatically measured, that is, the sliding friction angle of the sample, each sample is measured 3 times in parallel, and finally the sliding friction angle is calculated.

[0201] ⑨ Determination of bulk density: Let the common powder and ultrafine powder to be tested fall freely into 10mL (V) measuring cylinders respectively until they are full, accurately weigh the mass (m) of the two powders, and measure each sample 3 times in parallel. Calculate according to the following formula:

[0202]

[0203] ⑩ Determination of tap density: Use the BT1001 intelligent powder performance tester and the fixed volume method. Place a 100mL empty beaker on an electronic balance and weigh it, and read the mass of the empty cup. Connect the 100mL empty cup to a 100mL extension tube to form a vibration density assembly. Then add appropriate amounts of ordinary powder and ultrafine powder to the assembly, respectively, until it reaches more than half, and start tapping. Stop tapping until the surface of the powder in the extension tube no longer drops. Use a scraper to scrape the mouth of the 100mL container flat, and read the mass of the full cup. The bulk density of powder (ρ) refers to the ratio of the mass of powder (m) to the volume of the volume occupied by the powder (V), that is, ρ = m / V. When the mass of the powder is constant, the bulk density of the powder decreases with the decrease of the particle size.

[0204] 3.2 Experimental Results and Analysis

[0205] (1) Pictures of hemp seed regular powder and superfine powder fermented by Rhizopus oryzae

[0206] Ordinary powder: Fig. 9 It can be seen that the crushing strength of ordinary powder is small, the powder is coarse, and it is easy to form lumps. Fig.10 It can be seen that the ultrafine powder has strong crushing strength, fine powder and high dispersion.

[0207] (2) Analysis of powder particle size results

[0208] Depend on Fig.11 It can be seen that the particle size of ultrafine powder is 837.1nm, which is relatively small. Compared with general particles, it has a larger specific surface area, good solubility and high bioavailability. Fig.12 It can be seen that the scattered points and the point curve fit well, indicating that the dispersibility and stability of the ultrafine powder in the aqueous solution are relatively good.

[0209] (3) Results and analysis of powder wettability

[0210] Wettability is an important indicator to measure the properties of powder. It generally refers to the time required for a certain amount of powder to be completely immersed in water. Generally speaking, the shorter the time required for wetting, the stronger the wettability of the powder. Due to its high wettability, the components in the powder are easier to digest and absorb, so it has been widely used in various medicines. After testing, the wetting time of ordinary powder is 27.41s±1.64s, and the wetting time of ultrafine powder is 18.76s±1.82s. The wetting time of ultrafine powder is shorter than that of ordinary powder, indicating that its wettability is stronger than that of ordinary powder, and it can be dissolved at a faster speed, which has more dosage form advantages in the preparation of medicines. In addition, the smaller the particle size of the powder, the stronger the wettability, which shows that the wettability changes inversely with the particle size. This may be because the ultrafine grinding technology makes the powder have a high wall breaking rate, thereby exposing more water-soluble substances, which can quickly merge with water and dissolve in water, thereby reducing the dissolution time and improving the wettability of the powder.

[0211] (4) Results and analysis of hydration properties of powders

[0212] The hydration properties of powders include moisture content, water solubility (WS I), water holding capacity (WHC) and swelling capacity (SC). As shown in Table 11, the moisture content of powders is related to the moisture content rate of powders. The moisture content of ordinary powders and ultrafine powders is very low; the water holding capacity of powders is an important parameter of their hydrophilicity, reflecting their binding performance with water, and plays a very critical role in the storage and granulation of powder raw materials. As can be seen from Table 11, the water holding capacity of ordinary powders is less than that of ultrafine powders, which shows that ultrafine grinding can significantly improve the water holding capacity of powders. The reason for this phenomenon is probably because the strong mechanical force of ultrafine grinding effectively improves the surface energy of powder particles. At the same time, there will be more activation points on the surface of powder particles, which will greatly accelerate the interaction between hydrophilic groups on the surface of particles, such as hydroxyl groups, and water molecules. In this process, because of the strong mechanical force, the ultrafine powder has a high wall breaking rate. The macromolecular substances such as protein, polysaccharide, cellulose, etc. in the powder will interact with water to form a tight network structure, which locks the water molecules firmly in it and reduces the loss of water. Ordinary powder uses the traditional crushing method, so the degree of crushing is relatively low, and the particles of the powder are larger and the binding force with water is weaker, resulting in lower water retention.

[0213] The water solubility of the powder also reflects its properties. It can be seen from Table 11 that as the powder is broken more and more, the particle diameter becomes smaller and smaller, and the water solubility of the powder becomes larger and larger, which shows that the water solubility of the powder is negatively correlated with the particle size. Among the two powders, the water solubility of the ultrafine powder reaches 77.02% ± 0.25%, while the water solubility of the ordinary powder reaches 68.49% ± 0.18%. Compared with the ordinary powder, the water solubility of the ultrafine powder is increased by about 1.73 times, indicating that the water solubility of the ultrafine powder is significantly better than that of the ordinary powder. This is because the surface area contact of ultrafine powder becomes larger, which increases the exposure of some hydrophilic groups on the surface of the powder and the contact with water, thereby increasing the water solubility of the powder; in addition, strong pressure and shear force are generated during ultrafine grinding, which makes the hydrophilic groups of the powder more exposed and increases the contact area with water, thereby improving the water solubility of the powder; in addition, during the ultrafine grinding process, due to the huge pressure and shear force generated by the ultrafine grinding, some water-insoluble components in the powder, such as cellulose, have local chain breaks and melt, and are then transformed into soluble components. Therefore, the water solubility of ultrafine powder becomes greater.

[0214] The expansion force represents the ability of the powder to absorb water and expand, which is an important parameter of the powder. As can be seen from Table 11, the expansion force of ultrafine powder is significantly higher than that of ordinary powder. This may be because after ultrafine grinding, some hydrophilic groups and some macromolecules of the powder are exposed more, resulting in an increase in the contact site and contact area between the powder and water. After dissolving in water, the spatial structure of the molecules expands and extends, so the expansion force also increases. Generally speaking, powders with greater expansion force have better suspension properties and better stability after dissolving in water; moreover, due to the greater expansion force, a feeling of fullness will be produced when digested in the stomach, which will have an adverse effect on the digestion and absorption of other substances. Therefore, it has a certain preventive effect on obesity, intestinal cancer and other diseases.

[0215] Table 11 Hydration properties of ordinary powder and ultrafine powder: moisture content, water holding capacity, water solubility, and swelling power

[0216]

[0217] (5) Analysis of the results of the angle of repose and sliding friction angle of the powder

[0218] The angle of repose and the sliding friction angle are indicators that reflect the flow properties of ultrafine powders. The size depends on the particle size and surface properties of the powder. When the angle of repose increases, the friction of the powder also increases, and the flowability of the powder becomes poor. Under normal circumstances, when the angle of repose is less than 30°, its flowability is very good, and when the angle of repose is greater than 45°, its flowability is very poor. However, in actual production operations, when the angle of repose is less than 40°, the demand for flowability can be met. It can be seen from Table 12 that as the particle size decreases, the angle of repose and the sliding friction angle both increase significantly. Among them, the angle of repose decreases from 47.44° of ordinary powder to 38.71° of ultrafine powder, and the sliding friction angle increases from 45.82° of ordinary powder to 50.24° of ultrafine powder, indicating that the flowability of ultrafine powder is significantly better than that of ordinary powder. Ultrafine powders with an angle of repose of less than 40° can meet the requirements of daily production. After being crushed by strong mechanical force, the specific surface area of ​​the powder increases, and the electrostatic adsorption between particles increases, thereby increasing the surface aggregation force and the friction coefficient, so the sliding friction of the powder is relatively large. However, by increasing the angle of repose and sliding friction angle of the powder, the adsorption of the powder is improved, and the quality of the product after being made will be more stable. More importantly, there is usually no stratification after mixing evenly.

[0219] Table 12 Angle of repose and sliding friction angle of ordinary powder and ultrafine powder

[0220]

[0221] (6) Analysis of loose density and tap density of powders

[0222] The bulk density and tap density are very important for powder filling capsules and tablets. They are two important indicators for evaluating the filling effect of powders and are closely related to the particle size and microstructure of the powder. The smaller the bulk density and tap density, the looser the powder; the larger the bulk density and tap density, the more beneficial to the filling effect. Under the same volume, the powder has a smaller particle size and a smaller spacing, the higher the density and the larger the mass; the higher the bulk density and the higher the tap density, the more conducive to digestion and absorption by the human body. As can be seen from Table 13, the bulk density and tap density of ultrafine powder are much larger than those of ordinary powder, which shows that ultrafine powder can effectively reduce the particle size and increase the gap between particles, thereby improving its filling performance and being more conducive to absorption in the human body.

[0223] Table 13 Bulk density and tap density of ordinary powder and ultrafine powder

[0224]

[0225] The present embodiment has carried out the preparation of superfine powder to the fire hemp seeds after Rhizopus oryzae fermentation, and then the physical and chemical indicators of superfine powder have been detected. It can be seen that the superfine grinding technology has a certain improvement effect on the physical and chemical characteristics of the fire hemp seeds after Rhizopus oryzae fermentation. Compared with ordinary powder, the organizational structure fragmentation degree of superfine powder particles is relatively large, and the obtained particle size is obviously smaller; Compared with ordinary powder, the wettability, water holding capacity, water solubility and swelling power of superfine powder have all been significantly improved. By measuring the angle of repose and the sliding friction angle, it is found that the fluidity of superfine powder also becomes better, which can meet the basic needs of production. In addition, the loose density and tap density of superfine powder are also better, higher than ordinary powder, and more conducive to human body absorption. Therefore, the superfine grinding process improves the production adaptability of superfine powder to a great extent, and has a very important effect on the deep processing of fire hemp seeds and functional product development.

[0226] Example 4: Hypolipidemic Effect

[0227] This example mainly discusses the in vitro lipid-lowering effect, including the sodium bileate binding rate, the pancreatic lipase inhibition rate and the cholesterol micelle solubility inhibition rate. By comparing the effects of unfermented hemp seeds, hemp seeds fermented by Rhizopus oryzae and ultrafine powder on these three indicators, the most effective in vitro lipid-lowering effect is obtained.

[0228] The product of hemp seeds fermented by Rhizopus oryzae in this embodiment is prepared under the following fermentation conditions: inoculation amount 7%, water content 17%, fermentation temperature 35°C, and fermentation time 12h. The ultrafine powder is prepared by referring to the method of Example 3 using the product of hemp seeds fermented by Rhizopus oryzae as raw material.

[0229] 4.1 Experimental methods

[0230] (1) Determination of sodium cholate binding rate

[0231] Preparation of standard curve: Take 2 mL of sodium cholate standard solution of different concentrations (0.03, 0.06, 0.12, 0.18, 0.24, 0.30 mmol / L) in a volumetric flask, add 6 mL of 60% sulfuric acid, place in a 70°C water bath for 20 minutes, then place in an ice bath for 5 minutes, and measure the absorbance value at a wavelength of 387 nm using an ultraviolet spectrophotometer. Draw a standard curve of sodium cholate with the bile salt content as the abscissa and the absorbance value as the ordinate. Calculate the binding rate of sodium cholate using the same method as in Example 1.

[0232] (2) Determination of pancreatic lipase activity inhibition rate

[0233] Preparation of the main solution: 0.08% (w / v) 4-nitrophenyl laurate (p-PNL) solution: Weigh 0.4 g p-PNL and add it to a sodium acetate solution containing 1% Triton X-100 (the concentration of sodium acetate is 5 mmol / L), boil it in a boiling water bath for 1 min to help it dissolve, cool to room temperature, make up to 50 mL with 5 mmol / L sodium acetate solution, and store it in a refrigerator at 4°C. 3 mL of phosphate buffer solution with pH 7.4, 3 mL of 0.08% lauric acid 4-nitrophenyl ester solution, and 3 mL of 1.0 mg / mL pancreatic lipase solution were taken respectively, and mixed evenly with a certain amount of hemp seeds before and after fermentation. The sample masses were 4.5 mg, 9.0 mg, 18 mg, 27 mg, 54 mg, 72 mg, and 90 mg, respectively, and the sample solutions with concentrations of 0.5, 1, 2, 4, 6, 8, and 10 mg / mL were prepared. The samples were placed in a constant temperature water bath at 37°C for 30 min, and then the absorbance value was measured at 410 nm and recorded as A. At the same time, the absorbance at 410 nm without adding the sample was A1, and the absorbance at 410 nm with adding the sample but not adding the pancreatic lipase solution (replaced by phosphate buffer) was A0, and the pancreatic lipase inhibition rate was calculated according to the formula:

[0234]

[0235] Where, C—pancreatic lipase inhibition rate, %;

[0236] A—absorbance value of sample group;

[0237] A1—absorbance value of blank group;

[0238] A0—absorbance value of control group.

[0239] (3) Determination of the inhibition rate of cholesterol micelle solubility

[0240] To prepare 10 mL of cholesterol micelle solution, take 7.7 mg of cholesterol, 53.8 mg of sodium taurocholate, 77.1 mg of sodium chloride, 14.1 mg of oleic acid, and 7.5 mL of 0.02 mol pH 7.4 phosphate buffer, so that 1 mL of the solution contains 2 mmol / L cholesterol, 10 mmol / L sodium taurocholate, 132 mmol / L sodium chloride, 5 mmol / L oleic acid and 15 mmol / L phosphate buffer. Then, perform ultrasonic homogenization at 160 W for 50 min and place in a 37 ° C environment overnight. A certain amount of unfermented hemp seed powder and fermented hemp seed powder were prepared into sample solutions with concentrations of 1, 2, 4, 6, 8, 10, and 12 mg / mL, respectively. A certain amount was added to the micelle solution, and the mixed solution was shaken and cultured at 37°C for 2 hours, centrifuged at 14000 r / min for 20 minutes, and the supernatant was collected. The cholesterol content in the micelle was determined using a cholesterol determination kit. The cholesterol solubility of the sample solution was A, and the cholesterol solubility without adding the sample solution was A0. As the control group, the inhibition rate was calculated as follows:

[0241]

[0242] Where: A—absorbance value of sample solution;

[0243] A0—absorbance value of control group.

[0244] (4)IC 50 Worth calculating: IC 50 The value refers to the concentration at which the sample inhibition effect reaches half. The experimental results obtained by measuring the inhibition rate of pancreatic lipase and cholesterol micelle solubility are compared with the concentrations of unfermented hemp seed solution, Rhizopus oryzae fermented hemp seed solution and ultrafine powder solution, respectively, and input them into IC 50 The IC values ​​of the unfermented hemp seed solution, the hemp seed solution fermented by Rhizopus oryzae and the superfine powder solution were calculated in the calculator software. 50 value.

[0245] 4.2 Experimental Results and Analysis

[0246] (1) Evaluation of the effect of sodium cholate binding in vitro

[0247] ① Sodium cholate standard curve

[0248] The standard curve equation of sodium cholate is y=2.9861+0.0193, R 2 =0.9994, where x is the sodium cholate concentration mmol / L and y is the absorbance.

[0249] ② Analysis of sodium bile acid binding rate results: Fig.13As shown, the sodium cholate binding rate of unfermented hemp seeds is 22.45%±2.21%, the sodium cholate binding rate of hemp seeds fermented by Rhizopus oryzae is 65.83%±1.97%, and the binding rate of ultrafine sodium cholate is 70.15%±2.32%. It proves that the optimized process of hemp seeds fermented by Rhizopus oryzae can increase the binding rate of cholate and improve its lipid-lowering effect.

[0250] (2) Analysis of the effect of pancreatic lipase activity inhibition rate

[0251] Depend on Fig.14 It can be seen that the inhibitory effect of unfermented hemp seeds, hemp seeds fermented by Rhizopus oryzae and superfine powder on pancreatic lipase is concentration-dependent and increases with increasing concentration. When the concentration of unfermented hemp seed solution is less than 8 mg / mL, its inhibitory effect on pancreatic lipase activity increases rapidly with increasing concentration. After 8 mg / mL, the dependence of the inhibitory effect on concentration weakens. At 10 mg / mL, the inhibition rate reaches 65.75% ± 1.94%. The inhibitory effect of hemp seeds fermented by Rhizopus oryzae on pancreatic lipase is significantly stronger, and also shows a dose relationship. After 4 mg / mL, a plateau phase gradually appears. At a concentration of 10 mg / mL, the inhibition rate is 93.16% ± 2.17%. Superfine powder has the best inhibitory effect on pancreatic lipase activity. When the concentration reaches 8 mg / mL, the inhibition rate reaches 99.46% ± 1.39%, proving that hemp seeds fermented by Rhizopus oryzae do improve the inhibitory effect of pancreatic lipase activity, and superfine powder makes the drug more effective. The strength is: ultrafine powder > hemp seeds fermented by Rhizopus oryzae > unfermented hemp seeds. This may be because after fermentation by Rhizopus oryzae, some macromolecular substances in hemp seeds are converted into small molecular substances, which have a stronger effect.

[0252] (3) Analysis of the effect of cholesterol micelle solubility inhibition rate

[0253] This experiment simulated the conditions of the human small intestine in vitro and compared the cholesterol binding ability of unfermented hemp seeds, hemp seeds fermented by Rhizopus oryzae and ultrafine powder. Fig.15 It can be seen that the three samples have different binding abilities to cholesterol, and the solubility of cholesterol decreases with increasing concentration. This shows that all three samples can reduce the solubility of cholesterol in micellar solution. After comparing their binding abilities to cholesterol, it was found that the ultrafine powder has the strongest binding ability to cholesterol, with an inhibition rate of 85.95%±2.03% at a concentration of 12 mg / mL. The binding ability of hemp seeds fermented by Rhizopus oryzae to cholesterol is stronger than that of unfermented hemp seeds, reaching 69.51%±1.48% at a concentration of 12 mg / mL. Fig.15 It can be seen that the binding force of hemp seeds before fermentation is the weakest, and the solubility inhibition rate of cholesterol micelles is 56.31%±2.12% at a concentration of 12 mg / mL. The strength is: ultrafine powder > Rhizopus oryzae fermented hemp seeds > unfermented hemp seeds.

[0254] (4)IC 50 Calculation results and analysis

[0255] like Fig.16 As shown, after IC 50 IC value calculator for unfermented hemp seeds in the pancreatic lipase activity inhibition test 50 The value is 8.054mg / mL, the IC value of hemp seeds fermented by Rhizopus oryzae 50 The value is 1.255mg / mL, the IC value of ultrafine powder 50 The value is 0.605 mg / mL. In the cholesterol micelle solubility inhibition activity experiment, the IC 50 The value is 11.758mg / mL, the IC value of hemp seeds fermented by Rhizopus oryzae 50 The value is 7.436mg / mL, and the IC value of ultrafine powder is 50 The value is 3.171 mg / mL. It can be seen that the strength of the inhibitory activity is: ultrafine powder > Rhizopus oryzae fermented hemp seeds > unfermented hemp seeds.

[0256] The present embodiment mainly performs an in vitro hypolipidemic experiment, because the binding rate experiments of sodium glycocholate and sodium taurocholate were done before, so the present embodiment section only studies the remaining cholate, and the binding rate of sodium cholate obtained by unfermented hemp seeds is 22.45% ± 2.21%, the binding rate of sodium cholate obtained by fermenting hemp seeds with Rhizopus oryzae is 65.83% ± 1.97%, and the binding rate of sodium cholate obtained by superfine powder is 70.15% ± 2.32%, indicating that the binding rate of sodium cholate obtained by superfine powder is the best. When the inhibition rate concentration of superfine powder to pancreatic lipase is 8mg / mL, the inhibition rate reaches 99.46% ± 1.39%, when the concentration of hemp seeds fermented with Rhizopus oryzae is 10mg / mL, the inhibition rate is 93.16% ± 2.17%, and when the concentration of unfermented hemp seeds is 10mg / mL, the inhibition rate is 65.75% ± 1.94%. The inhibition of cholesterol micelle solubility can reach up to 85.95% ± 2.03% when the concentration of ultrafine powder solution is 10mg / mL, while the inhibition rate of unfermented hemp seeds is 56.32% ± 2.12%, and the inhibition rate of hemp seeds fermented by Rhizopus oryzae is 69.51% ± 1.48%. The experimental results show that the inhibition of hemp seeds fermented by Rhizopus oryzae on pancreatic lipase and cholesterol micelles is improved, thereby improving the ability to lower blood lipids. Since ultrafine powder makes the particles of the powder smaller and easier to be digested and absorbed by the human body, the effect of lowering blood lipids is better.

[0257] Example 5 Hypoglycemic and Antioxidant Effects

[0258] This example focuses on the ability of unfermented hemp seeds, Rhizopus oryzae fermented hemp seeds and superfine powder to reduce blood sugar in vitro and have antioxidant activity in vitro. In vitro blood sugar reduction mainly includes the inhibition of α-glucosidase and α-amylase activity. By contrasting with the hypoglycemic drug acarbose, the strength of the three substances in reducing blood sugar was studied. In vitro antioxidant capacity was compared with the traditional antioxidant Vc to explore the three substances' effects on DPPH and ABTS. + The strength of the free radical scavenging ability provides a reference for the further development and utilization of hemp seeds.

[0259] The hemp seeds fermented by Rhizopus oryzae in this embodiment are prepared under the following fermentation conditions: inoculation amount 7%, water content 17%, fermentation temperature 35°C, and fermentation time 12h. The superfine powder is prepared by referring to the method of Example 3 using the product of hemp seeds fermented by Rhizopus oryzae as raw material.

[0260] 5.1 Experimental Methods

[0261] 5.1.1 In vitro hypoglycemic activity studies

[0262] (1) Determination of α-glucosidase inhibition activity

[0263] 0.5 mL of different mass concentrations (1, 5, 10, 15, 20 mg / mL) of unfermented hemp seeds, Rhizopus oryzae fermented hemp seeds, and ultrafine powder solution were uniformly mixed with 0.5 mL of 0.1 U / mL α-glucosidase solution (dissolved in 25 mmol / L pH 6.8 phosphate buffer), mixed and reacted in a 37 ° C water bath for 10 minutes, and then 0.5 mL of 5 mmol / L p-NPG solution (dissolved in 0.1 mol / L pH 6.8 phosphate buffer) was added, reacted in a 37 ° C water bath for 15 minutes, and then 1 mL of 0.1 mol / L sodium carbonate solution was added to terminate the reaction. The absorbance value was measured at 405 nm by ultraviolet spectrophotometer, acarbose was used as a positive control, and an equal volume of 0.1 mol / L pH 6.8 phosphate buffer was used instead of α-glucosidase as a control group. The inhibition rate formula is as follows:

[0264]

[0265] Where: A—absorbance of sample group;

[0266] A0—absorbance of control group;

[0267] A1—absorbance of the mixed reaction of PBS buffer and p-NPG solution with enzyme solution;

[0268] A2—Absorbance of the mixed reaction of PBS buffer and p-NPG solution.

[0269] (2) Determination of α-amylase inhibition activity

[0270] 1mL of unfermented hemp seeds, hemp seeds fermented by Rhizopus oryzae, and ultrafine powder solution of different mass concentrations (1, 5, 10, 15, 20mg / mL) were mixed with 2mL of 1U / mL α-amylase solution (dissolved in pH 6.8 phosphate buffer) in a 37℃ water bath for 10min, and then 1mL of 1% soluble starch solution was added, mixed and reacted in a 37℃ water bath for 10min, and then 1mL of DNS reagent was added, and the reaction was terminated by heating in a boiling water bath for 5min, and the volume was fixed to 10mL. After cooling to room temperature, the absorbance value was measured at 540nm, and acarbose was used as a positive control, and an equal volume of 0.1mol / L pH 6.8 phosphate buffer was used instead of α-amylase as a control group. The inhibition rate formula is as follows:

[0271]

[0272] Where: A—absorbance of sample group;

[0273] A0—absorbance of control group.

[0274] 5.1.2 In vitro antioxidant activity studies

[0275] (1) DPPH in vitro antioxidant test

[0276] ① Preparation of DPPH test solution: Accurately weigh 3.94 mg DPPH into a 100 mL volumetric flask, and then dilute to volume with anhydrous ethanol to obtain a 0.1 mmol / L DPPH ethanol solution.

[0277] ② Vc control experiment: Accurately weigh 25 mg of Vc into a 10 mL volumetric flask, dilute to volume with deionized water to prepare a 2.5 mg / mL Vc reference solution; then dilute with deionized water to make 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1.0 mg / mL reference solutions.

[0278] ③ Preparation of sample solution: Prepare the sample into solutions of 0.0625 mg / mL, 1.0 mg / mL, 10 mg / mL, 20 mg / mL, 40 mg / mL, 60 mg / mL, and 80 mg / mL, add 70% ethanol solution at a solid-liquid ratio of 1:25 to dissolve, ultrasonically extract at 280 W, 45 ° C for 1 h, then centrifuge at 8000 r / min for 20 min, and take the supernatant for use.

[0279] ④Determination of DPPH scavenging ability: Take 2mL of each concentration of Vc control solution, unfermented hemp seed solution, Rhizopus oryzae fermented hemp seed solution and superfine powder solution, add 2mL of DPPH solution, mix thoroughly, seal and place in dark for 30min, measure the absorbance value of each sample at 517nm, and bring the result into the DPPH scavenging rate formula to calculate the scavenging rate of each concentration of Vc control solution, unfermented hemp seed solution, Rhizopus oryzae fermented hemp seed solution and superfine powder solution. The scavenging rate calculation formula is as follows:

[0280]

[0281] Where: A0—absorbance of 2mL DPPH solution and 2mL anhydrous ethanol;

[0282] A1—Absorbance of 2 mL sample solution and 2 mL anhydrous ethanol;

[0283] A2—Absorbance of 2 mL DPPH solution and 2 mL sample solution.

[0284] (2)ABTS + In vitro antioxidant assay

[0285] ①ABTS + Preparation of test solution: Accurately weigh 6.62 mg of potassium persulfate and ABTS + 38.4 mg was dissolved in a 10 mL volumetric flask and fixed to volume with distilled water to prepare ABTS + After reacting at room temperature in the dark for 16 h, dilute ABTS with 1 mmol / L PBS buffer at pH 7.4. + The absorbance of the mother solution is 0.70 at 734 nm. + About 0.02, that is, ABTS + Test solution.

[0286] ② Vc control experiment: Accurately weigh 25 mg of Vc into a 10 mL volumetric flask, dilute with deionized water to make a 2.5 mg / mL Vc reference solution; then dilute with deionized water to make 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1.0 mg / mL reference solutions.

[0287] ③ Preparation of sample solution: Prepare the sample into solutions of 0.0625 mg / mL, 1.0 mg / mL, 10 mg / mL, 20 mg / mL, 40 mg / mL, 60 mg / mL, and 80 mg / mL, add 70% ethanol solution at a solid-liquid ratio of 1:25 to dissolve, ultrasonically extract at 280 W, 45 ° C for 1 h, then centrifuge at 8000 r / min for 20 min, and take the supernatant for use.

[0288] ④ABTS + Determination of scavenging ability: Pipette 4.8 mL ABTS + The test solution was added with 0.2 mL of each concentration of Vc control solution, unfermented hemp seed solution, Rhizopus oryzae fermented hemp seed solution and ultrafine powder sample solution, and then all the liquids were ultrasonically oscillated for 30 seconds, sealed and protected from light, and reacted for 6 minutes. The absorbance of each sample was detected at a wavelength of 734 nm using an ultraviolet spectrophotometer, and the absorbance value was recorded as A. The blank was zeroed with 1 mmol / L phosphate buffer with a pH value of 7.4, and the results were brought into ABTS + The clearance rate formula is used to calculate the clearance rates of Vc control solution, unfermented hemp seed solution, Rhizopus oryzae fermented hemp seed solution and superfine powder solution at each concentration. The clearance rate calculation formula is as follows:

[0289]

[0290] (3)IC 50 Calculation of values

[0291] IC 50 The value refers to the concentration at which the sample can scavenge half of the free radicals, and the inhibitory capacity of α-glucosidase and α-amylase, DPPH and ABTS + The experimental results obtained from the scavenging capacity determination and the different concentrations of each sample were input into the IC 50 The value calculator software was used to calculate the inhibitory capacity of acarbose control solution, Vc control solution, unfermented hemp seed solution, Rhizopus oryzae fermented hemp seed solution and superfine powder solution on α-glucosidase and α-amylase, DPPH and ABTS + IC scavenging capability 50 value.

[0292] 5.2 Experimental Results and Analysis

[0293] (1) Evaluation of α-glucosidase activity inhibition effect

[0294] Depend on Fig.17It can be seen that the four solutions have different inhibitory effects on α-glucosidase, and are dose-dependent. As the mass concentration of the solution increases, the inhibition rates of acarbose, unfermented hemp seeds, hemp seeds fermented by Rhizopus oryzae, and superfine powder on α-glucosidase continue to increase. At different concentrations of 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, and 20 mg / mL, the inhibition rates of unfermented hemp seeds on α-glucosidase were 20.06% ± 1.96%, 46.38% ± 2.21%, 50.35% ± 2.15%, 56.25% ± 2.18%, and 66.08% ± 1.76%, respectively. The inhibition rates of hemp seeds fermented by Rhizopus oryzae on α-glucosidase were 31.91% ± 1.69%, 54.48% ± 2.09%, 61.58% ± 1.57%, 71.41% ± 2.27%, and 75.46% ± 1.59%, respectively. The inhibition rates of superfine powder on α-glucosidase were 27.94%±2.36%, 44.71%±2.35%, 47.85%±2.39%, 71.05%±2.21%, 87.53%±2.20%, respectively. The inhibition rates of acarbose on α-glucosidase were 38.75%±2.16%, 62.84%±2.53%, 78.81%±2.12%, 86.52%±1.67%, 95.08%±3.14% at the concentrations of 0.1mg / mL, 0.25mg / mL, 0.5mg / mL, 0.75mg / mL, and 1.0mg / mL, respectively. Therefore, in the α-glucosidase activity inhibition effect experiment, the strength of the inhibition rate was: acarbose>superfine powder>rhizopus oryzae fermented hemp seeds>unfermented hemp seeds.

[0295] Acarbose is a hypoglycemic drug. As a drug, it has clear contraindications and multiple precautions, and there are adverse reactions. However, superfine powder and Rhizopus oryzae fermented hemp seeds are made from hemp seeds, which are both medicinal and edible. It is safer and has no adverse reactions. In addition to the effect of lowering blood sugar, it also has the effects of lowering blood lipids, anti-oxidation, preventing obesity, and preventing intestinal cancer. It has more advantages in the fields of food and health products.

[0296] (2) Evaluation of α-amylase activity inhibition effect

[0297] Depend on Fig.18It can be seen that the four solutions have different inhibitory effects on α-amylase and are dose-dependent. As the mass concentration of the solution increases, the inhibition rates of unfermented hemp seeds, hemp seeds fermented by Rhizopus oryzae, superfine powder and acarbose on α-amylase continue to increase. At different concentrations of 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL and 20 mg / mL, the inhibition rates of unfermented hemp seeds on α-amylase were 3.23% ± 0.14%, 10.13% ± 1.02%, 20.04% ± 1.27%, 31.94% ± 2.03% and 40.76% ± 2.12%, respectively, and the inhibition rates of hemp seeds fermented by Rhizopus oryzae on α-amylase were 10.80% ± 1.04%, 14.93% ± 1.26%, 31.60% ± 1.52%, 44.60% ± 2.20%, 53.13% ± 2 .07%; the inhibition rates of superfine powder on α-amylase were 14.61%±0.64%, 24.25%±1.08%, 40.88%±1.16%, 55.13%±2.13%, 67.70%±2.12% respectively; the inhibition rates of acarbose on α-amylase at concentrations of 0.1mg / mL, 0.25mg / mL, 0.5mg / mL, and 0.75mg / mL were 34.88%±2.35%, 48.99%±2.28%, 62.61%±2.45%, 74.18%±2.31%, and 80.42%±1.88% respectively. Therefore, in the experiment of α-amylase activity inhibition, the strength of the inhibition rate was: acarbose>superfine powder>rhizopus oryzae fermented hemp seeds>unfermented hemp seeds.

[0298] (3) Results and analysis of DPPH in vitro antioxidant experiment

[0299] The experimental results are as follows Fig.19As shown in the figure, the scavenging rates of 5 concentrations of Vc solution were 35.78% ± 2.15%, 42.31% ± 2.14%, 58.60% ± 1.88%, 74.36% ± 1.86%, and 90.68% ± 1.63%, respectively. It can be seen that with the increase of the concentration of Vc solution, the scavenging ability of DPPH free radicals continued to increase; the scavenging rates of 7 concentrations of unfermented hemp seeds were 0.21% ± 0.06%, 8.24% ± 0.21%, 37.06% ± 1.18%, 41.32% ± 2.30%, 52.24% ± 2.32%, 62.35% ± 2.40%, and 63.92% ± 1.69%, respectively. It can be seen that the scavenging ability of DPPH free radicals was relatively weak; the scavenging rates of 7 concentrations of hemp seeds fermented by Rhizopus oryzae were 0.23% ± 0.06%, 8.31% ± 0.44%, 3 2.96%±1.15%, 41.41%±1.85%, 54.46%±2.08%, 62.94%±2.43%, 70.70%±1.96%. When the concentration reached 80 mg / mL, the clearance rate reached more than 70%, and the clearance rate of DPPH free radicals was significantly higher than that of unfermented hemp seeds. The clearance rates of 7 concentrations of superfine powder were 9.67±0.69%, 26.84±1.01%, 47.75±1.40%, 55.53±1.98%, 73.53%±2.11%, 79.76%±1.77%, 90.05%±1.85%. When the concentration was low, the clearance ability of superfine powder was lower than that of Vc solution, but with the continuous increase of concentration, the superfine powder had a clearance ability similar to that of Vc solution, indicating that the superfine powder had a better clearance ability for DPPH. Therefore, in the DPPH in vitro antioxidant experiment, the strength of the free radical scavenging ability is: Vc>superfine powder>Rhizopus oryzae fermented hemp seeds>unfermented hemp seeds. When evaluating antioxidant activity, using Vc as a control is the most commonly used experimental method. The present invention uses Vc as a control to prove that superfine powder and Rhizopus oryzae fermented hemp seeds have antioxidant properties. In addition to antioxidant activity, Rhizopus oryzae fermented hemp seeds and superfine powder have other nutritional components and functions that Vc does not have. For example: superfine powder and Rhizopus oryzae fermented hemp seeds also contain active ingredients such as flavonoids, polysaccharides and small molecule peptides. In addition to antioxidant, they also have the effects of lowering blood sugar, lowering blood lipids, preventing obesity, and preventing intestinal cancer.

[0300] (4)ABTS + In vitro antioxidant test results and analysis

[0301] The experimental results are as follows Fig. 20 As shown in the figure, the clearance rates of the five concentrations of Vc solution were 31.40±1.19%, 59.21%±2.23%, 95.74%±1.87%, 99.78%±2.11%, and 99.91%±1.85%, respectively. It can be seen that as the concentration of Vc solution increases, its clearance rate of ABTS+ The free radical scavenging ability of Vc solution is continuously enhanced. + It has a strong scavenging ability for free radicals; the scavenging rates of 7 concentrations of unfermented hemp seeds are 0.14% ± 0.01%, 9.06% ± 0.61%, 44.84% ± 1.91%, 65.08% ± 2.45%, 79.68% ± 2.11%, 84.71% ± 2.26%, 84.94% ± 2.03%, which shows that low concentrations are the most effective for ABTS + The free radical scavenging ability is weak, and the scavenging rate gradually increases with the increase of concentration, indicating that it has a strong effect on ABTS + Free radicals have a certain scavenging ability; the scavenging rates of 7 concentrations of hemp seeds fermented by Rhizopus oryzae were 0.47% ± 0.13%, 13.51% ± 1.76%, 48.28% ± 2.07%, 65.36% ± 2.14%, 81.38% ± 1.88%, 92.48% ± 2.06%, 97.03% ± 1.72%; the scavenging rates of 7 concentrations of superfine powder were 9.61% ± 0.62%, 31.75% ± 1.71%, 47.13% ± 1.95%, 65.63% ± 1.91%, 82.25% ± 2.07%, 92.92% ± 2.35%, 97.13% ± 1.99%. It can be seen that hemp seeds fermented by Rhizopus oryzae and superfine powder have a certain scavenging ability against ABTS + The scavenging ability of hemp seeds and ultrafine powder fermented with Rhizopus oryzae was significantly improved, and it had a similar scavenging ability to that of Vc solution, indicating that the scavenging ability of hemp seeds and ultrafine powder fermented with Rhizopus oryzae was + Therefore, in ABTS + In the in vitro antioxidant experiment, the ability to scavenge free radicals is as follows: Vc>ultrafine powder>hemp seeds fermented with Rhizopus oryzae>unfermented hemp seeds.

[0302] (5)IC 50 Calculation results and analysis

[0303] As shown in Table 14, through the α-glucosidase activity inhibition experiment, after IC 50 IC value calculator to calculate the IC value of acarbose 50 The value is 0.158mg / mL, and the IC of unfermented hemp seeds 50 The value is 8.255mg / mL, the IC value of hemp seeds fermented by Rhizopus oryzae 50 The value is 23.646mg / mL, and the IC value of ultrafine powder is 50 The value is 4.620mg / mL. In the α-amylase activity inhibition experiment, the IC 50 The value is 0.231mg / mL, and the IC of unfermented hemp seeds 50 The value is 34.594mg / mL, the IC value of hemp seeds fermented by Rhizopus oryzae 50The value is 24.080mg / mL, the IC value of ultrafine powder 50 The value is 12.241 mg / mL. It can be seen that the strength of the hypoglycemic activity inhibition ability is: acarbose>superfine powder>rhizopus oryzae fermented hemp seeds>unfermented hemp seeds.

[0304] Table 14 IC of acarbose, unfermented hemp seeds, Rhizopus oryzae fermented hemp seeds and superfine powder on the inhibition of α-glucosidase and α-amylase activities 50 value

[0305]

[0306] As shown in Table 15, after IC 50 IC value calculator for calculating the IC value of Vc in the DPPH in vitro antioxidant test 50 The value is 0.145mg / mL, the IC of unfermented hemp seeds 50 The value is 29.882mg / mL, the IC value of hemp seeds fermented by Rhizopus oryzae 50 The value is 27.926mg / mL, and the IC value of ultrafine powder is 50 The value is 5.554 mg / mL. + In the in vitro antioxidant test, the IC 50 The value is 0.089mg / mL, and the IC of unfermented hemp seeds 50 The value is 12.827mg / mL, the IC value of hemp seeds fermented by Rhizopus oryzae 50 The value is 7.346mg / mL, and the IC value of ultrafine powder is 50 The value is 2.870 mg / mL. It can be seen that the ability to remove free radicals is as follows: Vc>ultrafine powder>rhizopus oryzae fermented hemp seeds>unfermented hemp seeds.

[0307] Table 15 IC of Vc control solution, unfermented hemp seeds, Rhizopus oryzae fermented hemp seeds and superfine powder 50 value

[0308]

[0309] The results of the in vitro hypoglycemic experiment showed that the inhibitory strength of the activity of α-glucosidase and α-amylase was as follows: acarbose>superfine powder>rhizopus oryzae fermented hemp seeds>unfermented hemp seeds. The inhibition rate of superfine powder on the activity of α-glucosidase reached 87.53%±2.20%, and the inhibition rate on the activity of α-amylase could reach 67.70%±2.12%, indicating that the superfine powder of rhizopus oryzae fermented hemp seeds has a good hypoglycemic effect; the results of the in vitro antioxidant experiment showed that the superfine powder had a good inhibitory effect on DPPH and ABTS. +The scavenging ability of the samples is as follows: Vc>ultrafine powder>rhizopus oryzae fermented hemp seeds>unfermented hemp seeds. The scavenging rate of ultrafine powder on DPPH is 90.05%±1.25%, and the scavenging rate on ABTS is + The clearance rate can reach 97.13%±1.99%, indicating that the ultrafine hemp seed powder fermented by Rhizopus oryzae has good antioxidant activity.

[0310] In summary, the present invention uses hemp seeds as raw materials, adopts Rhizopus oryzae for fermentation, finds the fermentation conditions with the highest bile salt binding rate, and then makes the fermented product into ultrafine powder, and explores the advantages of the ultrafine powder dosage form through the determination of some powder properties; and through the changes in the content of components before and after fermentation, determines which components are affected by fermentation, so as to provide a reference for further research on hemp seeds; and then conducts preliminary research on lowering blood lipids, lowering blood sugar and anti-oxidation.

[0311] (1) The fermented product was subjected to a study on the bile salt binding rate, with the binding rate of sodium glycocholate and sodium taurocholate as indicators. First, a single factor test was conducted, with the inoculation amount of Rhizopus oryzae, fermentation time, water content, and fermentation temperature as influencing factors, and the optimal fermentation conditions were found to be 8% Rhizopus oryzae inoculation amount, 12h fermentation time, 22% water content, and 35°C fermentation temperature; the Plackett-Burman test was then conducted, and the three significant influencing factors with the highest binding rate of sodium glycocholate and sodium taurocholate were obtained: Rhizopus oryzae inoculation amount, water content, and fermentation temperature; then a 3-factor 3-level response surface test was conducted, and finally the binding rate of fermented hemp seeds to sodium glycocholate was 75.64%±2.77%, and the binding rate of sodium taurocholate was 65.77%±3.45%. Compared with unfermented hemp seeds, the binding rate of sodium glycocholate and sodium taurocholate of fermented hemp seeds increased by about 37.60%, and the binding rate of sodium taurocholate increased by about 37.25%. This shows that hemp seeds themselves have the effect of lowering blood lipids, but after fermentation with Rhizopus oryzae, their effect of lowering blood lipids is significantly improved.

[0312] (2) The components before and after fermentation of Rhizopus oryzae were measured and compared. It was found that flavonoids, polysaccharides, amino acids, fatty acids, and oligopeptides increased to a certain extent after fermentation of Rhizopus oryzae. The largest changes were polysaccharides and flavonoids, which increased by about 62.69% and 45.56%, respectively. The amino acid content increased by about 7.14%, the fatty acid content increased by about 11.94%, and the oligopeptide content increased by about 7.84%. In addition, the protein content decreased by about 4.54%. Rhizopus oryzae is a mold with a well-developed enzyme system, which can catalyze the components in hemp seeds and thus transform the components. Combined with the fact that the lipid-lowering ability of the fermented product of hemp seeds fermented by Rhizopus oryzae is higher than that of unfermented hemp seeds, it shows that the flavonoids and polysaccharides of hemp seeds fermented by Rhizopus oryzae are greatly increased, which can explain that the increase of flavonoids and polysaccharides in the fermented product has a promoting effect on the enhancement of its lipid-lowering ability.

[0313] (3) The hemp seeds fermented by Rhizopus oryzae were ultrafinely ground, and the particle size of the ultrafine powder was measured to be 837.1 nm; the wetting time of the ultrafine powder was 18.76 s ± 1.82 s; the moisture content of the ultrafine powder was 1.55% ± 0.05%; the water holding capacity was 4.25 ± 0.18 (g / g); the swelling power was 5.61 ± 0.27 (mL / g) and the water solubility was 77.02% ± 0.25%; the repose angle of the ultrafine powder was 38.71° and the sliding friction angle was 50.24°;. The bulk density of the ultrafine powder was 0.51 ± 0.23 (g / cm 3 ) and true density 0.60±0.12(g / cm 3 ). The particle size of the powder is smaller, and the hydration properties such as wettability, water retention, water solubility, and swelling power are better. The fluidity of the powder is also better, which is more conducive to absorption in the human body and can enhance the efficacy. In the subsequent production, it can be used in the food and pharmaceutical industries. The above indicators are better than ordinary powders, proving the advantages of ultrafine powder in dosage form.

[0314] (4) A preliminary study on in vitro lipid-lowering was conducted. The present invention mainly studied the binding rate of sodium cholate, the inhibition rate of pancreatic lipase and the inhibition rate of cholesterol micelle solubility. It was found that the order of in vitro lipid-lowering effect was: ultrafine powder > hemp seeds fermented by Rhizopus oryzae > unfermented hemp seeds. This is consistent with the original intention of preparing ultrafine powder to improve drug efficacy. It proves that the ultrafine grinding technology increases the surface area of ​​the powder, which is more conducive to improving drug efficacy.

[0315] (5) Preliminary studies on hypoglycemic and antioxidant effects in vitro have been conducted, and it has been found that the hypoglycemic effect is good. The ultrafine powder has an inhibition rate of 87.53% ± 2.20% on the activity of α-glucosidase and 67.70% ± 2.12% on the activity of α-amylase. The free radical scavenging ability is relatively better. The scavenging rate of ultrafine powder on DPPH is 90.05% ± 1.85%, and the scavenging rate of ultrafine powder on ABTS is 90. + The scavenging rate can reach 97.13% ± 1.99%. The flavonoid content is increased after fermentation by Rhizopus oryzae, and flavonoids have a better free radical scavenging effect, so the free radical scavenging ability of hemp seeds is improved after fermentation.

[0316] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for fermenting hemp seeds, characterized in that: The following steps are involved: The hemp seeds were sterilized at 121°C for 30 minutes. Hemp seeds were fermented with Rhizopus oryzae, and the fermentation conditions included: Rhizopus oryzae inoculation amount of 2%-10%, water content of 8%-43%, fermentation temperature of 20-35°C, and fermentation time of 4-20 h.

2. A method for fermenting hemp seeds according to claim 1, characterized in that: The fermentation conditions include: Rhizopus oryzae inoculation amount of 6%-8%, water content of 15%-36%, fermentation temperature of 30-35°C, and fermentation time of 8-16h.

3. A method for fermenting hemp seeds according to claim 2, characterized in that: The fermentation conditions include: Rhizopus oryzae inoculation amount of 6.9%-7%, water content of 17%, fermentation temperature of 34.66-35°C, and fermentation time of 12 h.

4. A method for preparing superfine hemp seed powder fermented by Rhizopus oryzae, characterized in that: The following steps are involved: The mixture of hemp seeds fermented by Rhizopus oryzae is ultrafinely ground; the hemp seeds fermented by Rhizopus oryzae are prepared by the method according to any one of claims 1 to 3.

5. The method for preparing superfine hemp seed powder fermented by Rhizopus oryzae according to claim 4, characterized in that: The ultrafine grinding comprises the following method: mixing a mixture of hemp seeds fermented by Rhizopus oryzae with water-soluble starch, grinding the mixture, adding water to mix the mixture, homogenizing the mixture and filtering the mixture, freeze-drying the mixture, and ultrafine grinding the mixture to obtain ultrafine hemp seeds fermented by Rhizopus oryzae powder.

6. The method for preparing superfine hemp seed powder fermented by Rhizopus oryzae according to claim 5, characterized in that: The mixture of hemp seeds fermented by Rhizopus oryzae was mixed with water-soluble starch in a mass ratio of 4:6, and the crushing time was 5 min. After being taken out, water was added in a mass ratio of 1:20 and mixed, and the homogenization time was 20 min. The mixture was filtered through a 450-mesh filter, and the filtrate was freeze-dried for 48 h to remove moisture. The mixture was crushed by a vibrating drug ultrafine powder machine at a temperature of 10°C and a crushing time of 15 min to obtain ultrafine hemp seed powder fermented by Rhizopus oryzae.

7. A fermented hemp seed, characterized in that: Prepared by the method according to any one of claims 1 to 3.

8. Use of the fermented hemp seeds according to claim 7 in the preparation of one or more of products that help maintain healthy blood lipid levels, products that help maintain healthy blood sugar levels, and products that help anti-oxidation.

9. A superfine hemp seed powder fermented by Rhizopus oryzae, characterized in that: Prepared by the method according to any one of claims 4 to 6.

10. Use of the ultrafine hemp seed powder fermented by Rhizopus oryzae according to claim 9 in the preparation of one or more of products that help maintain healthy blood lipid levels, products that help maintain healthy blood sugar levels, and products that help anti-oxidation.

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