CN117018062B - Method for fermenting hemp seeds and application thereof - Google Patents
Method for fermenting hemp seeds and application thereof Download PDFInfo
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- CN117018062B CN117018062B CN202311206608.5A CN202311206608A CN117018062B CN 117018062 B CN117018062 B CN 117018062B CN 202311206608 A CN202311206608 A CN 202311206608A CN 117018062 B CN117018062 B CN 117018062B
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- rhizopus oryzae
- powder
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- seeds
- fermented
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Abstract
The invention relates to a method for fermenting hemp seeds and application thereof. The method for fermenting hemp seeds comprises the following steps: fermenting hemp seeds by rhizopus oryzae. The mixture obtained by fermenting the hemp seeds can be used for preparing rhizopus oryzae fermented hemp seed superfine powder. The fermented cannabis seed and rhizopus oryzae fermented cannabis seed superfine powder prepared by the method can be used in the fields of medicines, foods, health care products and the like, and can be used for preparing products such as blood lipid reducing products, blood sugar reducing products, antioxidation products and the like. The fermented hemp seeds and the superfine powder thereof prepared by the method have the advantages of stronger drug effect, better free radical scavenging effect, more favorable production and application and the like.
Description
Technical Field
The invention belongs to the technical field of processing, and particularly relates to a method for fermenting hemp seeds and application thereof.
Background
Hemp seeds (Cannabis sativa L.) are mature seeds of Cannabis sativa, moraceae, which are rich in high quality unsaturated fatty acids, proteins, amino acids, sugars, insoluble fibers, vitamins and nutritional minerals. According to the description of the compendium of materia medica, the hemp seeds have the effect of strengthening the body, and can make people healthy and youth forever after long-term administration. The hemp seeds can moisten dryness and tonify deficiency, and can be used for treating symptoms such as diabetes, constipation due to intestinal dryness, dysentery, scabies, wind arthralgia, heat stranguria, epilepsy, irregular menstruation and the like clinically. According to the examination of the herbal literature, the traditional Chinese medicine and the medical literature consider that the cannabis sativa seeds are a medicine and food dual-purpose medicine for tonifying, body building, beautifying and the like. Hemp seeds have great application potential and are rich in nutrient components such as fatty acid, protein, carbohydrate, natural antioxidants (phytosterol, trienol, carotene, phospholipid, polyphenol) and the like. Of all oilseed type crops, only cannabis contains this long chain polyunsaturated fatty acid, with a fatty acid content of between 25% and 45%, with a linoleic acid to linolenic acid ratio of about 3:1, the best dynamic balance of omega-3 and omega-6 essential fatty acids identified by WHO being the best ratio for metabolic needs of the body. The hemp protein is rich in various essential amino acids, in particular arginine, which meets the requirement of WHO on amino acids required by infants.
Rhizopus oryzae (Rhizopus oryzae Went et pr. Geer.) is a fungus widely existing in soil, air, etc., belonging to genus rhizopus of family trichoderma of order trichoderma, rhizopus oryzae normally grows at 30-35 deg.c, and the most suitable growth temperature is 37 deg.c.
However, there is no report of rhizopus oryzae for fermentation of hemp seeds. In addition, due to unreasonable process setting and the like, the problems that the efficacy needs to be further improved, the physicochemical properties of the hemp seeds are not suitable for further production and the like exist, and the further application of the hemp seeds is limited.
Disclosure of Invention
In view of the problems existing in the prior art, the invention provides a method for fermenting hemp seeds and application thereof, and further improves the efficacy and physicochemical properties of the fermented hemp seeds and superfine powder thereof.
The technical scheme for solving the technical problems is as follows:
The invention provides a method for fermenting hemp seeds, which comprises the following steps: fermenting hemp seeds by rhizopus oryzae.
The beneficial effects of adopting the technical scheme include: rhizopus oryzae leavening agent is a safe strain preparation which does not affect the health of human body, and in the fermentation process, rhizopus oryzae can hydrolyze starch, protein, lipid and the like in hemp seeds, and rhizopus oryzae with developed enzyme system can convert macromolecular substances into small molecules. The invention achieves the effects of enhancing the drug effect and promoting the component conversion by the method of fermenting the hemp seeds by rhizopus oryzae.
Further, the fermentation conditions include any one or any several of the following: 2-10% of rhizopus oryzae inoculum size, 8-43% of water content, 20-35 ℃ of fermentation temperature and 4-20h of fermentation time;
preferably, rhizopus oryzae inoculum size is 6-8%, water content is 15-36%, fermentation temperature is 30-35 ℃, fermentation time is 8-16h;
optimally, rhizopus oryzae inoculum size is 6.9-7%, water content is 17%, fermentation temperature is 34.66-35 ℃, and fermentation time is 12h.
Further, the method comprises the step of sterilizing the hemp seeds before fermentation.
The beneficial effects of adopting the technical scheme include:
According to the invention, hemp seeds are used as raw materials, rhizopus oryzae is used for fermentation, and the fermentation product prepared by the process can be found to be capable of remarkably improving the combination ratio of sodium glycocholate and sodium taurocholate through optimizing the process. After rhizopus oryzae fermentation, the contents of hemp seed flavone, hemp seed polysaccharide, hemp seed total fatty acid, hemp seed total amino acid, hemp seed oligopeptide and the like are all obviously improved. The fermented product has higher effects of reducing blood lipid, reducing blood sugar and resisting oxidation than unfermented fructus Cannabis. Is beneficial to preparing medicines, foods, health care products and the like, and improves the added value of the hemp seeds.
The invention provides a preparation method of rhizopus oryzae fermented cannabis sativa seed superfine powder, which comprises the following steps: superfine pulverizing the mixture of rhizopus fermented hemp seeds.
Further, the superfine grinding comprises the following steps: mixing the mixture obtained after fermenting the hemp seeds by rhizopus oryzae with water-soluble starch, pulverizing, adding water, mixing, homogenizing, filtering, lyophilizing the filtrate, and micronizing to obtain superfine powder of the hemp seeds by rhizopus oryzae fermentation.
The beneficial effects of adopting the technical scheme include: the addition of water-soluble starch can increase the dispersibility and water solubility of the superfine powder of the fermentation product.
Specifically, the method comprises the following steps: mixing the mixture obtained after the rhizopus oryzae fermentation of the hemp seeds with water-soluble starch in a mass ratio of 4:6, crushing for 5min, taking out, adding water, mixing in a mass ratio of 1:20, homogenizing for 20min by a high-pressure homogenizer, filtering by a 450-mesh filter screen, freeze-drying the filtrate for 48h to remove water, crushing for 15min by a vibration type medicine superfine grinder, and obtaining rhizopus oryzae fermentation of the hemp seeds superfine powder at a temperature of 10 ℃.
The beneficial effects of adopting the technical scheme include: the superfine powder of the rhizopus oryzae fermented hemp seeds prepared by the method has the advantages that the physical and chemical properties are improved, compared with common powder, the superfine powder has larger tissue structure breaking degree, and the obtained particle size is obviously smaller; meanwhile, compared with common powder, the wettability, water holding capacity, water solubility and expansion force of the superfine powder are all obviously improved. By measuring the repose angle and the sliding friction angle, the fluidity of the superfine powder is found to be good, the basic requirement of production can be met, in addition, the apparent density and tap density of the superfine powder are also good, and the superfine powder is higher than that of common powder and is more beneficial to human body absorption. The superfine grinding treatment improves the production adaptability of superfine powder to a great extent. Simultaneously, the superfine grinding treatment also obviously improves the effects of reducing blood fat, reducing blood sugar, resisting oxidation and the like. Is beneficial to deep processing of hemp seeds and functional product development.
The invention provides an application of fermented cannabis sativa seeds in preparing one or more of blood lipid-lowering products, blood sugar-lowering products, antioxidation, obesity prevention and intestinal cancer prevention products.
Further, the fermented cannabis seed may be prepared by the methods described above.
The invention provides application of rhizopus oryzae fermented cannabis sativa seed superfine powder in preparing one or more of blood lipid-lowering products, blood sugar-lowering products, antioxidation, obesity prevention and intestinal cancer prevention products.
Further, rhizopus oryzae fermented cannabis seed superfine powder can be prepared by adopting the method.
Among the above, the product may be a pharmaceutical preparation, food, health product, cosmetic, etc. The fermented hemp seeds or the ultrafine powder thereof can be used alone or in combination with other components.
The beneficial effects of adopting the technical scheme include: compared with unfermented cannabis sativa seeds, the fermented cannabis sativa seeds and the superfine powder thereof have the effects of reducing blood fat, reducing blood sugar, resisting oxidation and the like. Can be used for preparing products with related functions, and can be widely applied to the fields of medicines, foods, health products, cosmetics and the like.
Drawings
FIG. 1 shows experimental results of the effect of rhizopus oryzae inoculum size on cholate binding rate, and the experimental results of sodium glycocholate and sodium taurocholate in each group are shown in sequence from left to right.
Fig. 2 shows experimental results of the effect of fermentation time on cholate binding rate, and the experimental results of sodium glycocholate and sodium taurocholate are sequentially from left to right in each group.
Fig. 3 shows experimental results of the effect of water content on cholate binding rate, and the experimental results of sodium glycocholate and sodium taurocholate are sequentially carried out from left to right in each group.
Fig. 4 shows experimental results of the influence of fermentation temperature on cholate binding rate, and the experimental results of sodium glycocholate and sodium taurocholate are sequentially from left to right in each group.
Fig. 5 is a graph showing the significant factor of sodium glycocholate binding rate.
Fig. 6 is a graph of the significant factor of sodium taurocholate binding rate.
FIG. 7 is a response surface and contour plot for the interaction of factor A and factor B.
FIG. 8 is a graph comparing the binding rates of unfermented and fermented cannabis seed cholate.
FIG. 9 is a diagram of the finished product of rhizopus oryzae fermented cannabis seed common powder.
FIG. 10 is a diagram of the finished product of rhizopus oryzae fermented cannabis seed superfine powder.
FIG. 11 is a graph showing the particle size of ultrafine powder.
Figure 12 is a graph of the stability detection potential of the ultrafine powder.
FIG. 13 is a graph showing the results of experiments on the binding rate of unfermented hemp seeds, rhizopus oryzae fermented hemp seeds, and ultrafine powder to sodium cholate.
FIG. 14 shows the inhibitory effect of unfermented hemp seeds, rhizopus oryzae fermented hemp seeds, and ultrafine powder on pancreatic lipase activity.
FIG. 15 shows the inhibitory effect of unfermented cannabis seed, rhizopus oryzae fermented cannabis seed and ultra-micro powder on micelle solubility.
FIG. 16 is an IC 50 value for unfermented hemp seeds, rhizopus oryzae fermented hemp seeds, and ultrafine powder.
FIG. 17 shows the inhibitory effect of acarbose, unfermented cannabis seed, rhizopus oryzae fermented cannabis seed and ultra-fine powder on alpha-glucosidase.
FIG. 18 shows the inhibitory effect of acarbose, unfermented cannabis seed, rhizopus oryzae fermented cannabis seed and ultra-fine powder on alpha-amylase.
FIG. 19 shows the effect of Vc control, unfermented cannabis seed, rhizopus oryzae fermented cannabis seed and ultra-fine powder on DPPH radical scavenging.
FIG. 20 shows the scavenging effect of Vc control, unfermented cannabis seed, rhizopus oryzae fermented cannabis seed and ultra-fine powder on ABTS + free radicals.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
According to the invention, rhizopus oryzae is utilized to ferment hemp seeds, cholate combination ratio is used as an index to optimize fermentation process, main components of the obtained product are compared with unfermented hemp seeds, then superfine powder preparation and property detection are carried out, and finally in-vitro efficacy researches of reducing blood fat, reducing blood sugar and resisting oxidation are carried out, wherein the main contents include:
(1) The process of fermenting hemp seeds by rhizopus oryzae is optimized, and 4 influencing factors of rhizopus oryzae inoculum size, fermentation time, fermentation temperature and water content are researched by taking the blood lipid reduction outside a fermentation substance as an index (the combination rate of sodium glycocholate and sodium taurocholate) through a single factor test. The rhizopus oryzae inoculum size, water content and fermentation temperature are screened out through a Plackett-Burman experiment and used as three factors for the next response surface test design. On the basis, a response surface optimization experiment is carried out, a verification experiment is carried out, and the optimal extraction process conditions are as follows: rhizopus oryzae inoculum size 6.9%, water content 17%, fermentation temperature 34.66 ℃ and fermentation time 12h. Under the optimal conditions, the actual value of the average sodium glycocholate binding rate is 75.64 +/-2.33%, and the actual value of the sodium taurocholate binding rate is 65.77+/-3.45%. Compared with unfermented cannabis sativa seeds, the sodium glycocholate binding rate of the fermented cannabis sativa seeds is improved by about 37.60%, and the sodium taurocholate binding rate is improved by about 37.25%.
(2) And (3) measuring the component content of the hemp seeds before and after fermentation to obtain the fermented hemp seed flavone, polysaccharide, amino acid, fatty acid and oligopeptide content, and reducing the protein content. The greatest change is polysaccharide and flavone, about 62.69% and 45.56% increase, about 7.14% increase in amino acid content, about 11.94% increase in fatty acid content, about 7.84% increase in oligopeptide content, and about 4.54% decrease in protein content.
(3) Preparing the fermented cannabis seed product into superfine powder of fermented cannabis seeds by utilizing an superfine grinding technology, and measuring the particle size of the superfine powder to 837.1nm; the wetting time of the superfine powder is 18.76s plus or minus 1.82s; moisture of the superfine powder is 1.55% +/-0.05%; a water retention capacity of 4.25.+ -. 0.18 (g/g); expansion force of 5.61+/-0.27 (mL/g) and water solubility 77.02 +/-0.25%; the repose angle of the superfine powder is 38.71 degrees and the sliding friction angle is 50.24 degrees; the apparent density of the superfine powder is 0.51+/-0.23 (g/cm 3) and the true density is 0.60+/-0.12 (g/cm 3). Potential detection proves that the dispersion in water is good; the particle size of the superfine powder is smaller, the wettability, the water-holding capacity, the water solubility, the expansion force and other hydration properties are better, and the powder fluidity is also better. The indexes are all superior to those of common powder, and the advantages of the superfine powder in the aspect of dosage form are proved.
(4) The in-vitro blood lipid lowering index comprises sodium cholate binding rate, pancreatic lipase inhibition rate and cholesterol micelle solubility inhibition rate. The experimental result of pancrelipase activity inhibition shows that the IC 50 value of unfermented cannabis sativa seeds is 8.054mg/mL, the IC 50 value of rhizopus oryzae fermented cannabis sativa seeds is 1.255mg/mL, and the IC 50 value of superfine powder is 0.605mg/mL. The result of cholesterol micelle solubility inhibition activity experiments shows that the IC 50 value of unfermented cannabis sativa seeds is 11.758mg/mL, the IC 50 value of rhizopus oryzae fermented cannabis sativa seeds is 7.436mg/mL, and the IC 50 value of superfine powder is 3.171mg/mL. From this, the intensity of the blood lipid inhibitory activity was found to be: superfine powder is larger than rhizopus oryzae fermented hemp seeds and unfermented hemp seeds.
(5) The research shows that the hemp seed superfine powder after fermentation has better anti-oxidation capability for reducing blood sugar in vitro. The inhibition rate of the superfine powder on the activity of alpha-glucosidase reaches 87.53 +/-2.20%, the inhibition rate of the superfine powder on the activity of alpha-amylase can reach 67.70+/-2.12%, the clearance rate of the superfine powder on DPPH reaches 90.05+/-1.85%, and the clearance rate of the superfine powder on ABTS + can reach 97.13+/-1.99%.
In conclusion, the research of the invention determines the optimal fermentation process of the cannabis sativa seeds, researches the change of the nutritional ingredients of the cannabis sativa seeds, prepares the cannabis sativa seeds into superfine powder, and proves that the superfine powder has the advantages of good water solubility, blood fat reduction, blood sugar reduction and oxidization resistance through physical and chemical indexes, thereby being beneficial to the further application of the cannabis sativa seeds.
The following is presented by way of specific examples. The experimental methods used in each example are conventional in the art unless specifically indicated. The materials, reagents and apparatus used, unless otherwise specified, are those conventionally known in the art and may be commercially available or prepared by conventional methods.
An ultraviolet spectrophotometer (model CARY-100) was purchased from VARIAN corporation, USA; kjeldahl nitrogen instrument (model Kje ltec 8400) was purchased from Denmark FOSS company; an amino acid autoanalyzer (model S433D) was purchased from Sykam, germany; the vibrating type medicine superfine pulverizer (model GYB 40-10S) is purchased from Jinan Dragon micro pharmaceutical equipment Co., ltd; moisture meter (model HB 43-S) was purchased from Shanghai Instrument electric analysis instruments Co., ltd; quick-freeze boxes (model SSF-12) were purchased from S IGMA, germany; multifunctional pulverizer (model LX-07A) was purchased from Shanghai river communication technologies limited; electrothermal constant temperature blast drying oven (model DHG-9240A), manufactured by dow oven, inc; the intelligent powder characteristic tester (model BT-1001) and the laser particle size distribution tester (model BT-2001) are purchased from Dendong Baite instruments Co., ltd; high pressure homogenizer (model GYB 40-10S) was purchased from Guangzhou City east characterization Co.
Hemp seeds were purchased from yunnan han ban corporation; rhizopus oryzae was purchased from Kadsura Biotechnology Co., ltd; lauric acid 4-nitrophenyl ester, trypsin, sodium glycocholate, sodium taurocholate were all purchased from Shanghai chemical reagent company; DPPH, vc, ABTS +, oleic acid, sodium acetate, pepsin, phosphate buffer were all purchased from national pharmaceutical group chemical reagent company, inc; rutin standard, glucose control, aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine, cysteine, tryptophan were all purchased from Shanghai source leaf biotechnology Co., ltd; petroleum ether, ninhydrin, sodium citrate, and water-soluble starch were all purchased from Miou chemical reagent Co., ltd; alpha-amylase, alpha-glucosidase, acarbose, cholesterol, triton X-100, sodium taurocholate, pancreatic lipase were all purchased from Shanghai Michlin Biotechnology Co., ltd; deionized water was purchased from the child haha group for the experiments. All the reagents are analytically pure.
Example 1 Process optimization of rhizopus oryzae fermented hemp seeds
1.1 Experimental methods
(1) Sterilizing hemp seeds: respectively weighing 10g of hemp seeds, respectively placing them in a conical flask, sealing them with a sealing film, sterilizing at 121deg.C for 30 min, cooling in a clean bench, and waiting for inoculation.
(2) And (3) making a standard curve:
2mL of sodium glycocholate standard solutions with different concentrations, namely 0.03, 0.06, 0.12, 0.18, 0.24, 0.30, 0.36, 0.42 and 0.48mmol/L, are respectively placed in a volumetric flask, 6mL of 60% sulfuric acid is added, water bath is carried out at the temperature of 70 ℃ for 20min, ice bath is carried out for 5min, and the absorbance value is measured at the wavelength of 387nm by an ultraviolet spectrophotometer. And drawing a sodium glycocholate standard curve by taking the cholate content as an abscissa and the absorbance value as an ordinate.
2ML of sodium taurocholate standard solutions with different concentrations, namely 0.05, 0.10, 0.15, 0.20, 0.25, 0.30,0.35, 0.40 and 0.45mmol/L, are respectively placed in volumetric flasks, 6mL of 60% sulfuric acid is added, water bath is carried out at the temperature of 70 ℃ for 20min, ice bath is carried out for 5min, and the absorbance value is measured at the wavelength of 387nm by an ultraviolet spectrophotometer. And drawing a sodium taurocholate standard curve by taking the cholate content as an abscissa and the absorbance as an ordinate.
(3) Determining cholate content: taking 0.5g of fermented coarse powder in a volumetric flask, adding 3mL of pepsin (0.1 mol/L phosphate buffer solution dissolved in pH 6.5) and 1mL of hydrochloric acid (0.01 mol/L), placing the sample in a constant-temperature water bath at 37 ℃ and continuing to perform shaking digestion for one hour to simulate the effect of stomach digestion. Then, the pH of the solution was adjusted to 6.3 with a 0.1mol/L NaOH solution, and then 4mL of trypsin (0.1 mol/L phosphate buffer solution dissolved in pH 6.5) was added in an amount of 10mg/mL, and the sample was placed in a constant temperature water bath at 37℃and subjected to shaking digestion for one hour to simulate the effect of intestinal digestion. Finally, adding 4mL of 0.4mmol/L sodium glycocholate and 4mL of 0.5mmol/L sodium taurocholate into the sample, placing the sample into a constant-temperature water bath at 37 ℃ for continuous one-hour shaking digestion, transferring the mixed solution into a centrifuge tube, centrifuging for 20min at 10000r/min, taking the supernatant, filtering by using a 0.45um aqueous needle filter, measuring absorbance value at 387nm wavelength by using an ultraviolet spectrophotometer, and calculating the content of sodium glycocholate and sodium taurocholate. The cholate binding rate was calculated as follows:
(4) Design of a rhizopus oryzae fermented cannabis seed single-factor test: the effects of rhizopus oryzae inoculum size (fermentation system comprises rhizopus oryzae, cannabis sativa seeds and water, inoculum size is 2%, 4%, 6%, 8%, 10% of fermentation system respectively, and mass percent), fermentation time (4 h, 8h, 12h, 16h, 20 h), fermentation temperature (20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃) and water content (8%, 15%, 22%, 36%, 43%) of fermentation system on test indexes are sequentially studied by taking the binding rate of sodium glycocholate and sodium taurocholate as indexes.
(5) Plackett-Burman test design: based on a single-factor test, the sodium glycocholate binding rate and the sodium taurocholate binding rate are used as indexes, rhizopus oryzae inoculum size, fermentation temperature, fermentation time and water content are selected as independent variables, a Plackett-Burman test is designed, and the most obvious influencing factors are screened out through analysis of results.
(6) Response surface test design: on the basis of the Plackett-Burman test, a response surface test with the level of 3 factors and 3 is designed by taking the combination ratio of sodium glycocholate and sodium taurocholate as a response value and the independent variable A, B, C as an influence factor, wherein the factors and the levels of the Box-Behnken test are shown in Table 1 and the test design scheme are shown in Table 2.
TABLE 1 fermentation process optimization Box-Behnken test factors and level Table
Table 2 response surface test design
1.2 Experimental results and analysis
(1) Sodium glycocholate standard curve: the standard curve equation of sodium glycocholate is y=2.6642x+0.0561, and r 2 = 0.9968, where x is sodium glycocholate content (mmol/L), and y is absorbance.
(2) Sodium taurocholate standard curve: the standard curve equation for sodium taurocholate is y=2.4524x+0.0367, and r 2 =0.9985, where x is sodium taurocholate content (mmol/L) and y is absorbance.
(3) Single factor test results and analysis
① Rhizopus oryzae inoculum size: as can be seen from FIG. 1, under the same conditions (i.e., fermentation time of 10h, fermentation temperature of 30 ℃ and water content of 15%), the binding rate of sodium glycocholate and sodium taurocholate was continuously increased when the rhizopus oryzae inoculum size was 2% -8%, the binding rate of sodium glycocholate was 65.91% + -1.22% and the binding rate of sodium taurocholate was 64.40% + -1.41%, and at this time, the binding rates of both cholate were the best, and the binding rates of sodium glycocholate and sodium taurocholate began to decrease when the rhizopus oryzae inoculum size was 10%, so that rhizopus oryzae inoculum size was 8% was selected for the next fermentation test.
② Fermentation time: as is clear from FIG. 2, under the same conditions (i.e., inoculum size 8%, fermentation temperature 30 ℃ C., water content 15%), the binding ratio of sodium glycocholate and sodium taurocholate was continuously increased for 4-12 hours, and the binding ratio of sodium glycocholate was 70.13% + -2.01% and the binding ratio of sodium taurocholate was 65.86% + -1.74% for 12 hours, and the binding ratio of both cholate was the best, and the binding ratio of sodium glycocholate and sodium taurocholate began to decrease for 12-20 hours, so that the test was conducted with a selected fermentation time of 12 hours.
③ Water content: as can be seen from FIG. 3, under the same conditions (fermentation time 12h, fermentation temperature 30 ℃ C., inoculum size 8%), the binding ratio of sodium glycocholate and sodium taurocholate was continuously increased when the water content was 8% -22%, the binding ratio of sodium glycocholate was 71.54% + -1.91% when the water content was 22%, the binding ratio of sodium taurocholate was 77.4% + -2.11%, and the binding ratio of both cholate was the best, and the binding ratio of sodium glycocholate and sodium taurocholate was continuously decreased when the water content was 22% -43%, so that fermentation was performed with a water content of 22% selected.
④ Fermentation temperature: as can be seen from FIG. 4, under the same conditions (i.e., fermentation time of 12h, inoculum size of 8%, water content of 22%), the binding rate of sodium glycocholate and sodium taurocholate was continuously increased at a fermentation temperature of 20℃to 35℃and at a fermentation temperature of 35℃the binding rate of sodium glycocholate was 71.92% + -1.42% and the binding rate of sodium taurocholate was 72.65% + -2.06%, and at this time, the binding rate of both cholate was the best, and at a fermentation temperature of 35℃to 45℃the binding rate of sodium glycocholate and sodium taurocholate was continuously decreased, so that fermentation was carried out at a fermentation temperature of 35℃was selected.
(4) Fermentation process optimization Plackett-Burman test results and analysis
According to the single factor test result, the Plackett-Burman test is designed by taking the sodium glycocholate binding rate and the sodium taurocholate binding rate as the response values of the test, the test design method and the result are shown in Table 3, and the screened significant factor results are shown in FIG. 5 and FIG. 6.
TABLE 3 factor level and results for Plackett-Burman test design
As is clear from FIG. 5, the first three factors that have the most significant effect on sodium glycocholate binding rate are water content (C), rhizopus oryzae inoculum size (A) and fermentation temperature (D), respectively, and as is clear from FIG. 6, the first three factors that have the most significant effect on sodium taurocholate binding rate are fermentation temperature (D), water content (C) and rhizopus oryzae inoculum size (A). It can be seen that these three factors have a more pronounced effect on the sodium taurocholate binding rate, so rhizopus oryzae inoculum size (a), water content (C) and fermentation temperature (D) were chosen as three factors for the next response surface test design.
(5) Fermentation process optimization response surface test results and analysis
① Response surface Box-Behnken test design
Based on Plackett-Burman test results, sodium glycocholate binding rate (Y 1) and sodium taurocholate binding rate (Y 2) are used as response values, rhizopus oryzae inoculum size (A), water content (B) and fermentation temperature (C) are selected as independent variables, des ign-Expert 11 is used for carrying out 3-factor 3 horizontal response surface test design, and Box-Behnken test results are shown in Table 4.
TABLE 4 response surface test design and results Table
② Response surface interaction analysis
The sodium glycocholate binding rate (Y 1) is taken as a response value, and the equation is obtained by quadratic polynomial regression fitting ：Y1＝63.69-9.48A-11.86B-2.84C+0.8950AB-0.8600AC+2.05BC-2.95A2-8.56B2-17.37C2.
As can be seen from table 5, the model is remarkable (P < 0.05), and the mismatch term is not remarkable (p=0.3892 > 0.05), which can indicate that the model has a good fitting degree, and can reflect the change rule of sodium glycocholate binding rate and each factor relatively well. In addition, the determination coefficient R 2 = 0.9305 of the model shows that the regression equation has better simulation condition for the test, the error of the test is smaller, the correction determination coefficient R 2 adj= 0.8410, and it can be seen that the predicted value of the software is related with the actual value of the test, and the correlation is high. In addition, the variation coefficient C.V. is 12.48%, which shows that the variability of the test is 12.48%, and the model can be used for carrying out subsequent analysis and prediction on the rhizopus oryzae fermented hemp seed process. From the P value, the first term a and B of the equation, the second term B 2, has a significant effect on the binding rate of sodium glycocholate (P < 0.05), and other factors have no significant effect (P > 0.05). Therefore, the regression equation can be used for predicting the sodium glycocholate binding rate under different fermentation conditions.
TABLE 5 regression model analysis of variance table
Note that: * Indicating significant differences (P < 0.05); * Represents that the difference is very significant (P < 0.01); coefficient of variation c.v. =12.48%, R 2＝0.9305,R2 adj= 0.8410.
Taking the sodium taurocholate binding rate (Y 2) as a response value, and obtaining an equation by a second polynomial regression fit: y 2＝64.67+1.99A-11.89B-1.57C+4.71AB-2.08AC+0.1475BC-4.53A2-15.66B2-9.20C2.
As shown in table 6, the model is significant (P <0.0001< 0.05), the mismatch term is insignificant (p=0.7558 > 0.05), which can indicate that the fitting degree of the model is better, and the change rule of sodium taurocholate binding rate and each factor can be reflected better. The determination coefficient R 2 = 0.9895 of the model can be seen, which indicates that the linear regression equation has good simulation conditions of the test, the error of the obtained test result is small, the correction determination coefficient R 2 adj= 0.9759, and the correlation between the predicted value of the software and the actual value of the test is high. In addition, the coefficient of variation c.v. is 4.13%, indicating that the variability of the test is 4.13% and can be used for the model for subsequent analysis and prediction of the rhizopus oryzae fermented cannabis seed process. From the P value, the primary term a of the equation has a significant effect on sodium taurocholate binding rate (P < 0.05), the primary term B of the equation has an interactive term AB, and the secondary term a 2、B2、C2 of the equation has an extremely significant effect on sodium taurocholate binding rate (P < 0.01) and other factors have no significant effect (P > 0.05). From the F value, the influence sequence of each factor on the sodium taurocholate binding rate is that the water content is more than the rhizopus oryzae inoculum size and the fermentation temperature. Therefore, the regression equation can be used for predicting the sodium taurocholate binding rate under different fermentation conditions.
TABLE 6 regression model analysis of variance
Note that: * Indicating significant differences (P < 0.05); * Represents that the difference is very significant (P < 0.01); coefficient of variation c.v. =4.13%, R 2＝0.9895,R2 adj= 0.9759.
From fig. 7, it can be seen that the contour plot of rhizopus oryzae inoculum size and water content interaction is oval in shape, demonstrating significant interaction, consistent with regression model variance results. In the 3D response surface graph, the tendency that the binding rate of sodium taurocholate increases and decreases with the increase of rhizopus oryzae inoculum size and water content can be seen visually.
③ Optimal process conditions and verification experiments
The optimum fermentation conditions are predicted by optimizing by using Des ign-Expert 11 software: rhizopus oryzae inoculum size 6.9%, water content 17%, fermentation temperature 34.66 ℃ and fermentation time 12h. Under the optimal fermentation conditions, the predicted value of the software for sodium glycocholate binding rate is 72.44%, and the predicted value for sodium taurocholate binding rate is 65.39%. The optimal fermentation conditions from the practical point of view are as follows: rhizopus oryzae inoculum size 6.9%, water content 17%, fermentation temperature 35 ℃ and fermentation time 12h. And carrying out 3 parallel verification tests according to the fermentation process conditions to obtain an average sodium glycocholate actual value of 75.64 +/-2.33%, and a sodium taurocholate actual value of 65.77+/-3.45%, wherein the sodium glycocholate actual value is within 5% of a model predicted value, and the sodium taurocholate binding rate is within 1% of the model predicted value, so that the model is basically consistent with the model prediction, the equation is well fitted with the actual test result, and the optimal parameters obtained through the tests have good stability and reliability, so that the model is effective and feasible.
(6) Cholate binding rate results for unfermented cannabis seeds: as shown in fig. 8, the binding rate of unfermented cannabis sativa seeds to sodium glycocholate was found to be 34.84% ± 3.59% by experiment, while the binding rate of rhizopus oryzae fermented cannabis sativa seeds to sodium glycocholate was 75.64% ± 2.33%; the binding rate of unfermented cannabis seed to sodium taurocholate was 28.52% + -2.38%, while the binding rate of rhizopus oryzae fermented cannabis seed to sodium glycocholate was 65.77% + -3.45%. It can be seen that the binding rate of sodium glycocholate and sodium taurocholate is obviously improved after rhizopus oryzae fermentation.
In summary, in this embodiment, the combination ratio of sodium glycocholate and sodium taurocholate is used as an index, the rhizopus oryzae inoculation amount, the fermentation time, the water content and the fermentation temperature are used as single factors to optimize the fermentation process. Rhizopus oryzae inoculum size: 2%, 4%, 6%, 8%, 10% and the highest cholate binding rate of 8% of inoculum size was obtained, the sodium glycocholate binding rate was 65.91% + -1.22%, and the sodium taurocholate binding rate was 64.4% + -1.41%. Fermentation time: 4h, 8h, 12h, 16h and 20h, the highest binding rate of 12h cholate, the binding rate of sodium glycocholate of 70.13 percent+/-2.01 percent and the binding rate of sodium taurocholate of 65.86 percent+/-1.74 percent are obtained. Water content: 8%, 15%, 22%, 29%, 36%, 43%, the highest binding rate of cholate with 22% water content, 71.54% + -1.91% sodium glycocholate and 77.4% + -2.11% sodium taurocholate. Fermentation temperature: the binding rate of the cholate with the water content of 35 ℃ is highest at 20 ℃,25 ℃,30 ℃, 35 ℃,40 ℃ and 45 ℃, and the binding rate of sodium glycocholate is 71.92% +/-1.42%, and the binding rate of sodium taurocholate is 72.65% +/-2.06%.
The Plackett-Burman test was used to design a 3-factor 3 horizontal response surface test, with three significant factors of rhizopus oryzae inoculum size, water content, and fermentation temperature. The optimization conditions after the response surface experiment are as follows: the optimal fermentation condition is obtained by optimizing the rhizopus oryzae inoculum size of 6.9%, the water content of 17%, the fermentation temperature of 34.66 ℃ and the fermentation time of 12h. Under the optimal conditions, the actual value of the average sodium glycocholate binding rate is 75.64 +/-2.33%, and the actual value of the sodium taurocholate binding rate is 65.77+/-3.45%.
In the design of the response surface, two indexes of the sodium glycocholate binding rate and the sodium taurocholate binding rate are simultaneously selected as response values for process optimization, and the two indexes cannot be simultaneously met to obtain the maximum value. And because the sodium glycocholate binding rate is more important in cholesterol binding, the parameter with the highest sodium glycocholate binding rate is selected from the response surface experimental result, and at the moment, the sodium taurocholate binding rate is lower than the maximum value of a single factor experiment.
The combination ratio of unfermented cannabis sativa seeds to sodium glycocholate is 34.84% +/-3.59% and the combination ratio of rhizopus oryzae fermented cannabis sativa seeds to sodium glycocholate is 72.44% +/-2.33% measured by a comparison experiment; the binding rate of unfermented cannabis sativa seeds to sodium taurocholate is 28.52 +/-2.38%, and the binding rate of rhizopus oryzae fermented cannabis sativa seeds to sodium glycocholate is 65.77+/-3.45%. Concluding that: the rhizopus oryzae fermented cannabis sativa seeds remarkably improve the combination rate of sodium glycocholate and sodium taurocholate.
Example 2 determination of the content of Components before and after rhizopus oryzae fermentation of hemp seed
The embodiment aims at rich nutrient substances in hemp seeds, and can possibly measure the content of some components with the function of reducing blood fat before and after rhizopus oryzae fermentation, wherein the rhizopus oryzae fermentation conditions are as follows: the inoculation amount is 7%, the water content is 17%, the fermentation temperature is 35 ℃, and the fermentation time is 12 hours; and (3) performing comparative analysis to research the components affecting the blood lipid reducing effect of the cannabis seeds so as to be beneficial to further application of the cannabis seeds.
2.1 Experimental methods
(1) Measuring the flavone content: the total flavone content in the hemp seeds was determined by sodium nitrite-aluminum chloride complex spectrophotometry. Under the condition that the pH value of the solution is neutral or weak alkaline in the presence of sodium nitrite in the solution, the flavonoid compound can form a chelate with the contained aluminum salt, and after NaOH is added, the solution can be reddish orange, and the content of the flavonoid is in direct proportion to the absorbance value within a certain concentration range.
① Preparing rutin standard stock solution: accurately weighing 5mg of rutin standard substance, and dissolving with 60% ethanol solution to volume to 100mL. Preparing a standard stock solution with the concentration of 50mg/L, and storing in a refrigerator at the temperature of 4 ℃ in a dark place.
② And (3) making a standard curve: accurately sucking a certain amount of rutin standard solution, respectively placing the rutin standard solution into a 10mL volumetric flask to prepare standard series of 2mg/mL,4mg/mL,6mg/mL,8mg/mL,10mg/mL and 12mg/mL, adding ethanol solution to ensure that the total volume of the solution is 5mL, adding 0.3mL of sodium nitrite solution, shaking uniformly, standing for 8min, adding 0.3mL of aluminum chloride solution, shaking uniformly, standing for 10min, adding 4mL of sodium hydroxide solution, fixing the volume to a scale by using ethanol solution, shaking uniformly, and standing for 10min. The absorbance was measured at 510nm, and a standard curve was drawn with the concentration as the abscissa and the absorbance as the ordinate.
③ Preparing a sample: weighing 5g of hemp seed samples before and after fermentation, respectively placing in 50mL volumetric flasks, adding 30mL of 60% ethanol solution, performing ultrasonic treatment at 280V and 60 ℃ for 90min, performing constant volume treatment with 60% ethanol, centrifuging to clarify, and filtering for later use.
④ Measurement of sample solution: respectively sucking 1mL of liquid to be detected before and after fermentation in a volumetric flask, adding ethanol solution until the total volume is 5mL, adding 0.3mL of sodium nitrite solution, shaking uniformly, standing for 8min, adding 0.3mL of aluminum chloride solution, shaking uniformly, standing for 10min, adding 4mL of sodium hydroxide solution, fixing the volume to a scale by using the ethanol solution, shaking uniformly, and standing for 10min. Absorbance was measured at 510nm and the concentration of total flavonoids in the samples was calculated from the standard curve.
⑤ Calculation of total flavone content
Wherein: the content of total flavonoids in X-hemp seeds is expressed in milligrams per kilogram (mg/kg);
c-total flavone concentration (mg/L) of the sample solution calculated;
10-dilution multiple of the liquid to be measured;
m-mass of cannabis seed sample in grams (g);
0.05-total volume of extract of hemp seed sample, unit is (L);
h-mass fraction (%) of moisture in hemp seed.
(2) Determination of polysaccharide content
① Glucose standard solution was prepared: accurately weighing 10mg of anhydrous glucose, placing in a beaker, dissolving with distilled water, transferring to a 100mL volumetric flask for constant volume, and preparing into glucose standard solution with concentration of 0.1 mg/mL.
② And (3) making a standard curve: respectively precisely absorbing a certain amount of glucose standard solution, placing the glucose standard solution into a 10mL volumetric flask, respectively adding water to be filled to 1.0mL, respectively adding 1mL of 5% phenol solution and 3mL of sulfuric acid to prepare standard series with the concentration of 0.01mg/mL,0.02mg/mL,0.03mg/mL,0.04mg/mL,0.05mg/mL and 0.06mg/mL, shaking and uniformly mixing, placing the materials into a water bath kettle with the temperature of 100 ℃ and heating for 10min, taking the concentration of the glucose solution as an abscissa and the absorbance value as an ordinate, and drawing a glucose standard curve.
③ Sample measurement:
Respectively preparing unfermented cannabis seed powder and rhizopus oryzae fermented cannabis seed powder. The preparation method comprises freeze drying, pre-lyophilizing at-55deg.C for 12 hr, and post-lyophilizing at 35deg.C for 8 hr.
Taking 1.0g of unfermented cannabis sativa seed powder and 1.0g of rhizopus oryzae fermented cannabis sativa seed powder, respectively placing the materials in volumetric flasks, adding absolute ethyl alcohol according to the material ratio of 1g to 30mL, extracting for 40min at 280V and 46 ℃ by an ultrasonic extractor, centrifuging for 15min at 8000r/min, retaining supernatant, adding 50mL of water into the residual residue, ultrasonically extracting for 3 times for 30min each time, centrifuging for 10min at 4000r/min each time in a centrifuge, combining the three supernatants, absorbing 1mL to 25mL volumetric flasks after uniform mixing, adding water to scale, and shaking uniformly to obtain a sample measuring solution. Then accurately sucking 1mL of the sample measuring solution into a volumetric flask, respectively adding 1mL of 5% phenol solution and 3mL of 80% sulfuric acid solution, shaking and mixing uniformly, heating in a water bath kettle at 100 ℃ for 10min, cooling to room temperature, and measuring absorbance.
④ Calculation of polysaccharide content
Wherein: w-polysaccharide content in hemp seeds in grams per hundred grams (g/100 g);
p-calculated concentration of sample solution in milligrams per milliliter (mg/mL);
v 1 -volume of sample measurement fluid in milliliters (mL);
V 2 -volume of sample extract in milliliters (mL);
V 3 —the volume of sample extract used to prepare the sample assay solution in milliliters (mL);
m-sample mass in grams (g);
0.9-glucose is converted into a correction factor for dextran.
(3) Measuring protein content: measuring protein content by Kjeldahl nitrogen method, precisely weighing 0.149g of hemp seed sample before and after fermentation in a digestion tube, respectively adding 8mL of sulfuric acid, 3g of potassium sulfate and 0.3g of copper sulfate into the tube, placing the tube on a digestion furnace at 420 ℃ for digestion for 1h, taking out after digestion, cooling, mounting the tube on a Kjeldahl nitrogen instrument for titration, and measuring protein content after the completion.
(4) Determination of fatty acid content: 2g of hemp seed samples before fermentation and after rhizopus oryzae fermentation are respectively weighed, the two samples to be detected are respectively placed in a degreasing filter paper bag, a Soxhlet extraction device is utilized, petroleum ether is used as an extractant for extraction for 4 hours, and then the filter paper bag is placed in a 105 ℃ oven for drying until the weight is constant.
(5) Determining the amino acid content:
① Preparing a mixed amino acid standard stock solution: mixed amino acid standard stock preparation (1 umol/mL): accurately weighing single amino acid standard substances (0.00001 g) respectively, putting into a 50mL beaker, sucking 8.3mL of 6mol/L hydrochloric acid solution for dissolution, transferring to a 250mL volumetric flask, diluting with water, fixing volume to scale, and mixing uniformly.
② Preparing a mixed amino acid standard working solution: preparation of mixed amino acid standard working solution (100 nmol/mL): accurately sucking 1.0mL of the mixed amino acid standard stock solution into a10 mL volumetric flask, adding a sodium citrate buffer solution with pH of 2.2 to a fixed volume to scale, and uniformly mixing to obtain the standard machine feeding solution.
③ Measuring a sample: accurately weighing the hemp seed samples before and after fermentation in a hydrolysis tube, controlling the protein content in the samples to be between 10 and 20mg, and adding 12mL of 6mol/L hydrochloric acid solution. Freezing the hydrolysis tube with refrigerant for 5min, pumping vacuum (near 0 Pa) and then injecting nitrogen, sealing, placing the hydrolysis tube in an electrothermal blast incubator, hydrolyzing at 110+ -1deg.C for 22h, taking out, and cooling to room temperature. Opening the hydrolysis tube, filtering out to a 50mL volumetric flask, adding water to a fixed volume to a scale, and shaking uniformly. 1.0mL of the filtrate was accurately drawn into a 25mL test tube, dried under reduced pressure with a tube concentrator at a high temperature of 50℃and then dissolved with 2mL of water, dehydrated under reduced pressure, and finally evaporated to dryness. Dissolving with 1.0mL-2.0mL of sodium citrate buffer solution with pH of 2.2, shaking, mixing, filtering with 0.22 μm filter membrane, and placing into sample bottle of instrument to obtain sample measurement solution. And (3) respectively injecting the mixed amino acid standard working solution and the sample measuring solution into an amino acid analyzer at the detection wavelength of 570nm and 440nm according to the same capacity, and calculating the concentration of the amino acid in the sample measuring solution by using the peak area by adopting an external standard method. The content of each amino acid in the sample measurement solution is calculated according to the following formula:
wherein: c i -the content of amino acid i in nanomole per milliliter (nmol/mL) of a sample measurement solution;
A i -peak area of amino acid i of sample measurement solution;
a s peak area of amino acid s of the amino acid standard working solution;
c s -amino acid standard working solution the content of amino acid s is in nanomoles per milliliter (nmol/mL).
(6) Determination of oligopeptide content
① Determination of acid soluble protein content: accurately weighing 1.0g of each of unfermented and rhizopus oryzae fermented hemp seeds in a volumetric flask, respectively adding 15% trichloroacetic acid solution, dissolving and fixing the volume to 50mL, uniformly mixing, standing for 10min, centrifuging the two sample solutions at 9000r/min for 25min, respectively taking 10mL of the two sample centrifugate, measuring the protein content in the two sample centrifugate by using a Kjeldahl nitrogen method, and calculating the acid soluble protein content in the two samples, wherein the conversion coefficient of the protein is 6.25.
② Determination of oil-free amino acids: and (3) weighing 1.0g of unfermented and rhizopus oryzae fermented cannabis sativa seeds in a volumetric flask, respectively and uniformly dissolving the seeds in 20mL of 5% trichloroacetic acid solution, respectively transferring the two sample solutions into 100mL volumetric flasks, fixing the volume by using 0.002mol/L hydrochloric acid solution, and centrifuging the two sample solutions at 9000r/min for 15min to obtain supernatant.
③ Result calculation
The oligopeptide content X 1 was calculated according to the following formula:
Wherein: x 1 -the content of oligopeptide in grams per hundred grams (g/100 g) in the sample;
C 2 -the content of acid soluble protein in the sample in grams per hundred grams (g/100 g);
C 3 -content of oil-free amino acids in the sample, g per hundred g (g/100 g);
w-moisture in sample, grams per hundred grams (g/100 g).
2.2 Experimental results and analysis
(1) Results and analysis of flavone, polysaccharide, protein content
① Glucose content standard curve: the standard curve equation for glucose is y=8.8534x+0.0397, and r 2 =0.9962, where x is glucose concentration (mg/mL) and y is absorbance.
② Flavone content standard curve: the standard curve equation of the flavone is y=0.0067x-0.0056, and R 2 =0.9994, wherein x is mg/mL, and y is AU.
As shown in table 7, the polysaccharide of cannabis sativa seeds increased significantly after rhizopus oryzae fermentation, probably because rhizopus oryzae is an enzyme with developed enzyme system, and starch may be converted into polysaccharide during fermentation, so the polysaccharide content of cannabis sativa seeds after fermentation increased; the flavone content of hemp seeds after rhizopus oryzae fermentation is also obviously increased, probably because the combination of antioxidant active substances and matrixes is destroyed in the fermentation process, so that the flavone content is increased; the reduced protein content after fermentation of cannabis sativa seeds is probably due to the conversion of some macromolecular proteins into small amino acids by enzymatic catalysis during fermentation. The nutritional value of a protein is related to the content and composition of the amino acids it contains, in addition to the mass of its total protein.
TABLE 7 content of flavone, polysaccharide and protein
(2) Results and analysis of fatty acid content
The content of fatty acids in the fermented hemp seeds and rhizopus oryzae fermented hemp seeds, which were not subjected to the fermentation, was determined by the experiment, is shown in table 8. The hemp seeds contained a large amount of fatty acids, and as can be seen from Table 8, the total fatty acid content was about 40%, among these fatty acids, the linoleic acid content was the greatest, and it was more than 50% of the total fatty acids, having antithrombotic and antiarrhythmic effects; linolenic acid is the fatty acid next to linoleic acid in hemp seeds, with a ratio of less than 20%, then oleic acid, with a ratio of less than 15%, with the minimum ratio of palmitic acid and stearic acid, about 5% and about 3%. Through the findings in Table 8, the contents of oleic acid and linoleic acid in the rhizopus oryzae fermented hemp seeds are increased, the contents of linolenic acid, stearic acid and palmitic acid are reduced, the ratio of the linoleic acid to the linolenic acid is about 3:1, and the ratio is the optimal ratio required by human bodies, which fully shows that the composition of various fatty acids of the hemp seeds after rhizopus oryzae fermentation is changed, and the content of the fatty acids can be influenced after rhizopus oryzae fermentation.
TABLE 8 fatty acid content
(3) Results and analysis of amino acid content
The amino acid content of the fermented cannabis seed and rhizopus oryzae fermented cannabis seed was determined experimentally and is shown in table 9. As can be seen from Table 9, the total amount of amino acids is about 30% of the hemp seeds, and these amino acids are classified into a large variety, so that the nutritional value in proteins depends on the constitution of these amino acids in addition to the total content. The content of glutamic acid in the 16 amino acids is highest (6.43-6.81 g/100 g), a large amount of arginine (3.74-4.18 g/100 g) and aspartic acid (3.27-3.65 g/100 g) are contained, and for nerve centers and cerebral cortex, glutamic acid is a good supplementary substance, can delay the consumption of energy substances, thereby playing a role in promoting physical recovery after strenuous exercise, arginine can promote the growth and development ability of children, hemp seeds are a good food for supplementing arginine, and aspartic acid can be converted into glutamic acid in human bodies, so that the anti-fatigue effect of hemp seeds can be further enhanced. The contents of leucine, isoleucine and valine are slightly higher, and the three substances act together, so that the muscle can be repaired, the blood sugar can be reduced, the body energy can be enhanced, and the growth and development of children can be promoted. The hemp seeds are rich in threonine and isoleucine, and have good effects on restoring and growing lean body mass. Hemp seeds have similar amino acid content as soybean, but they also possess other advantages not possessed by soybean, such as: because the hemp protein does not contain the inhibition factor of tryptophan, the protein absorption is not affected, and the hemp seed does not contain many oligosaccharides in soybean, so that the stomach distention and nausea are not caused. As can be seen from Table 9, the total amount of amino acids in the hemp seeds after fermentation of Rhizopus oryzae is increased, and the content of most of amino acids is significantly increased, which fully shows that the amino acid content of the hemp seeds after fermentation of Rhizopus oryzae is increased, probably because macromolecular substances such as proteins in the hemp seeds are converted into small molecular amino acids after fermentation.
TABLE 9 content of 16 amino acids
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(4) Results and analysis of oligopeptides
The content of acid soluble protein and the content of oil-free amino acid are obtained through each experiment measurement, the content of oligopeptide is obtained through formula calculation, and the content of oligopeptide in rhizopus oryzae fermented hemp seeds is improved by about 7.8% compared with the content before fermentation as can be seen from table 10. Recent studies have shown that in the digestive tract, proteins are absorbed by the human body in the form of oligopeptides during enzymatic hydrolysis, whose effect in the intestine is far greater than that of the complete free amino acids, and therefore their effect in the intestine is of increasing interest. Thus, the increased oligopeptide content in rhizopus oryzae fermented cannabis seeds may be due to the high amount of enzymes contained in rhizopus oryzae, which converts proteins in cannabis seeds to oligopeptides during fermentation by enzymatic hydrolysis, so rhizopus oryzae fermented cannabis seeds may be more easily absorbed by our body.
TABLE 10 content of acid soluble proteins, free amino acids and oligopeptides
In this example, the following conclusion is obtained by measuring the flavone, polysaccharide, protein, five fatty acids, sixteen amino acids and oligopeptide components in hemp seeds before fermentation and rhizopus oryzae fermented hemp seeds, and analyzing and comparing the content changes before and after fermentation: the flavone content of hemp seeds after rhizopus oryzae fermentation is increased by 350.4mg/kg, which is increased by about 45.6%; the polysaccharide content of hemp seeds after rhizopus oryzae fermentation is increased by 0.42g/100g and is increased by about 62.7%; the protein content of hemp seeds after rhizopus oryzae fermentation is reduced by 1.45%; the total fatty acid content of the hemp seeds after rhizopus oryzae fermentation is increased by 4.37g/100g and is increased by about 11.94%; in different kinds of fatty acids, the content of linolenic acid, stearic acid and palmitic acid is reduced, and in addition, the content of oleic acid and linoleic acid is increased; the total amino acid content of hemp seeds after rhizopus oryzae fermentation is increased by 2.1g/100g and 7.14%. The contents of other amino acids except methionine and proline are increased to different degrees; the content of hemp seed oligopeptide after rhizopus oryzae fermentation is increased by 0.08g/100g and is increased by about 7.84%. According to the above examples, the fermented product of rhizopus oryzae fermented cannabis seed has a higher lipid-lowering ability than unfermented cannabis seed. The combination of the great improvement of the flavone and the polysaccharide of the hemp seeds after the rhizopus oryzae fermentation can prove that the improvement of the flavone and the polysaccharide in the fermentation product has a promotion effect on the improvement of the blood fat reducing capability.
EXAMPLE 3 preparation of Rhizopus oryzae fermented cannabis seed ultrafine powder and physical and chemical Property study
In the embodiment, hemp seeds fermented by rhizopus oryzae are prepared into superfine powder through certain treatment, and then the physicochemical indexes of particle size, wettability, powder moisture, water holding capacity, water solubility, swelling power, repose angle, sliding friction angle, apparent density and tap density of the prepared superfine powder are measured to investigate whether the prepared superfine powder is excellent in properties or not so as to carry out deeper study later.
The product of the rhizopus oryzae after fermenting hemp seeds is prepared by adopting the following fermentation conditions: the inoculation amount is 7%, the water content is 17%, the fermentation temperature is 35 ℃, and the fermentation time is 12h.
The preparation method of the common powder comprises the following steps: pulverizing with Chinese medicinal pulverizer for 3 times, and sieving with 100 mesh sieve for 15s each time.
3.1 Experimental methods
(1) The preparation process of rhizopus oryzae fermented cannabis sativa seed superfine powder comprises the following steps: mixing the mixture obtained after rhizopus oryzae fermentation of hemp seeds with water-soluble starch in a mass ratio of 4:6, pulverizing in a pulverizer for 5min, taking out, adding water, homogenizing under 60Mpa for 20min, filtering with 450 mesh filter screen, lyophilizing the filtrate for 48 hr to remove water, pulverizing with a vibrating type medicine superfine pulverizer (temperature 10 deg.C and pulverizing time 15 min), and measuring physicochemical indexes of the two powder without vibration type medicine superfine pulverizer.
(2) Determination of physicochemical index of superfine powder
① Measurement of particle size: the particle diameters of the common powder and the ultrafine powder were measured by a wet method by using a laser particle size distribution analyzer, and the particle diameters were evaluated by specific surface area and span values.
② Determination of wettability: 50mL of distilled water was first added to the petri dish, and then 1.0g of ordinary powder and ultrafine powder were added, respectively, and the time(s) required for the two powders to be completely soaked with water was measured, 3 times per sample.
③ Powder moisture determination: accurately weighing 1.0g of common powder and superfine powder, respectively laying the common powder and the superfine powder in weighing bottles with constant weight, and putting the weighing bottles into a drying oven with 105 ℃ for constant weight drying, thereby calculating the moisture content of the common powder and the superfine powder.
④ Determination of water retention: accurately weighing 0.50g of common powder and superfine powder respectively, placing the common powder and superfine powder into a 50mL centrifuge tube, adding 50mL of distilled water into the centrifuge tube, shaking uniformly, centrifuging for 15min at 5000r/min in the centrifuge, discarding supernatant, weighing the rest precipitate, measuring m 2 in parallel for 3 times, and calculating according to the following formula:
⑤ Determination of water solubility: respectively precisely weighing 1.0g of common powder and superfine powder, uniformly dispersing in 50mL of distilled water, oscillating in 80 ℃ water bath for 30min to fully dissolve, centrifuging in a centrifuge for 10min at 6000r/min, and drying the supernatant in a 105 ℃ drying oven to constant weight to obtain the mass. The water solubility calculation formula is as follows:
⑥ Measurement of expansion force: 1.0g of common powder and superfine powder are respectively weighed precisely, put into a 50mL measuring cylinder, the volume is recorded as V 1, distilled water is added into the cylinder for 50mL, the mixture is stirred uniformly and then kept stand for 24 hours at room temperature, the volumes of the common powder and the superfine powder are recorded as V 2, each sample is measured in parallel for 3 times, and the calculation is carried out according to the following formula:
⑦ Measurement of angle of repose: the intelligent powder characteristic tester for BT1001 is characterized in that a funnel is fixed at a certain distance from a horizontal plane, proper amounts of common powder and superfine powder are respectively taken, the powder firstly passes through a filter screen and flows to a discharge port, and then falls onto a sample table to be gradually changed into a cone shape until the powder is piled up on the sample table to form a symmetrical cone shape, the lowest end of the funnel is reached to stop feeding, after feeding is completed, automatic measurement is carried out, each sample is measured for 3 times in parallel, and the repose angle is calculated.
⑧ Sliding friction angle measurement: and 3g of common powder and superfine powder are respectively weighed by adopting the BT1001 intelligent powder characteristic tester and are put into the center of the glass plate, then one end of the glass plate is gently lifted until 90% of powder falls off, thus the angle between the lifted glass plate and a horizontal tabletop, namely the sliding friction angle of a sample, is automatically measured, each sample is parallelly measured for 3 times, and finally the sliding friction angle is calculated.
⑨ Determination of bulk Density: and (3) freely scattering the common powder to be detected and the superfine powder, respectively filling the common powder and the superfine powder into a 10mL (V) measuring cylinder until the common powder and the superfine powder are fully filled, accurately weighing the mass (m) of the two kinds of powder, and parallelly measuring each sample for 3 times. The calculation is performed according to the following formula:
⑩ Determination of tap Density: using a BT1001 type intelligent powder performance tester, selecting a fixed-volume method, placing a 100mL empty beaker on an electronic balance for weighing, and reading the mass of the empty beaker; and connecting a 100mL empty cup with a 100mL extension cylinder to form a vibration density assembly, then adding a proper amount of common powder and superfine powder into the assembly to reach more than half, starting compaction, stopping compaction until the surface of the powder in the extension cylinder does not drop any more, scraping the opening of a 100mL container with a scraper, and reading the mass of the full cup. The bulk density (ρ) of powder is the ratio of the mass (m) of powder to the volume (V) of the volume occupied by the powder, i.e., ρ=m/V. When the powder is of a certain quality, the bulk density of the powder decreases with decreasing particle size.
3.2 Experimental results and analysis
(1) Rhizopus oryzae fermented cannabis sativa seed common powder and superfine powder picture
Ordinary powder: as can be seen from fig. 9, the ordinary powder has a small crushing strength, and the powder is thick and easily agglomerated. Superfine powder: as can be seen from FIG. 10, the superfine powder has high pulverizing strength, fine powder and high dispersity.
(2) Analysis of results of particle size of powder
As is clear from FIG. 11, the ultrafine powder has a particle size of 837.1nm, a smaller particle size, a larger specific surface area than that of the conventional particles, and has the advantages of good solubility and high bioavailability. As can be seen from fig. 12, the scattered points and the point location curves are well fitted, which indicates that the dispersibility and the stability of the superfine powder in the aqueous solution are good.
(3) Results and analysis of powder wettability
Wettability is an important measure of the properties of a powder and generally refers to the time required for a certain amount of powder to be fully immersed in water. In general, the shorter the wetting time, the more wetting the powder. The components in the powder are more easily digested and absorbed due to the higher wettability, so that the powder is widely used in various medicines. The detection shows that the wetting time of the common powder is 27.41s plus or minus 1.64s, the wetting time of the superfine powder is 18.76s plus or minus 1.82s, and the wetting time of the superfine powder is shorter than that of the common powder, so that the superfine powder has higher wettability than that of the common powder, can be dissolved more quickly, and has the advantage of dosage form in the aspect of preparing medicines. In addition, the smaller the particle size of the powder, the stronger the wettability, which indicates that the wettability varies inversely with the particle size, probably because the superfine pulverizing technology makes the wall breaking rate of the powder high, so that more water-soluble substances are exposed, which can be quickly fused with water and dissolved in water, thereby reducing the dissolution time and improving the wettability of the powder.
(4) Results and analysis of hydration Properties of powders
The hydration properties of the powder include the moisture, water solubility (WS I), water retention (WHC) and expansion force (SC) of the powder. As shown in table 11, the moisture content of the powder is related to the moisture content of the powder, and the moisture content of the common powder and the superfine powder is very low; the water holding capacity of the powder is an important parameter of the hydrophilicity of the powder, reflects the combination property of the powder and moisture, and plays a very critical role in the processes of storage, granulation and the like of powder raw materials. As can be seen from table 11, the water holding capacity of the common powder is smaller than that of the superfine powder, which indicates that the superfine powder can obviously improve the water holding capacity of the powder, and the reason for this phenomenon is probably that the surface energy of the powder particles is effectively improved due to the strong mechanical force of the superfine powder, and meanwhile, more activation points exist on the surfaces of the powder particles, which can greatly accelerate the interaction of hydrophilic groups, such as hydroxyl groups, on the surfaces of the particles with water molecules. In the process, because of strong mechanical force, the wall breaking rate of the superfine powder is high, and macromolecular substances such as protein, polysaccharide, cellulose and the like in the powder can interact with water to form a compact network structure, so that water molecules are firmly locked in the network structure, and the loss of water is reduced. The common powder is crushed by the traditional crushing method, so that the crushing degree is relatively low, the particles of the powder are larger, and the binding force with water is weaker, so that the water holding capacity is lower.
The water solubility of the powder also reflects the property of the powder, and as can be seen from table 11, as the crushing degree of the powder is higher and higher, the particle diameter is smaller and the water solubility of the powder is larger, which indicates that the water solubility of the powder is inversely related to the particle size, in the two kinds of powder, the water solubility of the superfine powder reaches 77.02% ± 0.25%, and the water solubility of the common powder reaches 68.49% ± 0.18%, compared with the common powder, the water solubility of the superfine powder is improved by about 1.73 times, and the water solubility of the superfine powder is obviously better than that of the common powder. This is because the surface area of the ultrafine powder is greatly contacted, the exposure of hydrophilic groups on the surface part of the powder is increased, and the contact with water is increased, so that the water solubility of the powder is increased; in addition, strong pressure and shearing force are generated during superfine grinding, so that hydrophilic groups of the powder are exposed more, the contact area with water is increased, and the water solubility of the powder is improved; in addition, in the superfine pulverizing process, some water-insoluble components in the powder, such as cellulose and the like, generate partial chain breakage due to the huge pressure and shearing force generated by superfine pulverizing, and generate a phenomenon of melting, and further, are converted into soluble components, so that the water solubility of the superfine powder becomes greater.
The expansion force represents the ability of the powder to expand by absorbing water, and is an important parameter of the powder. As can be seen from table 11, the swelling power of the ultrafine powder is significantly higher than that of the conventional powder, probably because some hydrophilic groups and some macromolecules of the powder are exposed more after the ultrafine powder is subjected to the ultrafine powder, resulting in an increase in contact area and contact area of the powder with water, and expansion and extension of the spatial structure of the molecules after the dissolution in water, so that the swelling power is also increased. Generally, the powder with larger expansion force has better suspension performance and better stability after being dissolved in water; in addition, the stomach-invigorating and stomach-nourishing health-care food has a large swelling force, and can generate a feeling of satiety when being digested in the stomach, so that the stomach-invigorating and stomach-nourishing health-care food has adverse effects on digestion and absorption of other substances, and has a certain prevention effect on diseases such as obesity, intestinal cancer and the like.
TABLE 11 hydration Properties of ordinary and superfine powders moisture, water-holding Properties, water-solubility and swelling Properties
(5) Analysis of results of repose angle and sliding friction angle of powder
The repose angle and the sliding friction angle are indexes reflecting the flowing property of the superfine powder, and the size is that the particle size and the surface property of the powder, when the repose angle is increased, the friction force of the powder is increased, and the fluidity of the powder is poorer. In general, the fluidity is very good when the angle of repose is < 30 DEG, and very poor when the angle of repose is > 45 deg. However, in actual production operation, when the repose angle is less than 40 degrees, the requirement of mobility can be met. As can be seen from table 12, as the particle size becomes smaller, both the angle of repose and the sliding friction angle increase significantly, wherein the angle of repose decreased from 47.44 ° for the normal powder to 38.71 ° for the ultra-fine powder and the sliding friction angle increased from 45.82 ° for the normal powder to 50.24 ° for the ultra-fine powder, indicating that the flowability of the ultra-fine powder is significantly better than that of the normal powder. The superfine powder has an repose angle smaller than 40 degrees, so that the requirement of daily production can be met, the specific surface area of the powder is increased after the powder is crushed by strong mechanical force, the electrostatic adsorption effect among particles is increased, the surface cohesion is increased, the friction coefficient is increased, and the sliding friction of the powder is larger. However, by increasing the repose angle and the sliding friction angle of the powder, the adsorptivity of the powder is improved, the quality of the manufactured product is more stable, and more importantly, layering phenomenon usually does not occur after uniform mixing.
TABLE 12 repose angle and sliding Friction angle of ordinary powders and ultrafine powders
(6) Analysis of results of bulk and tap Density of powder
The apparent density and tap density are important for powder filling capsules and tablets, are two important indexes for evaluating the powder filling effect, and are closely related to the particle size and microstructure of the powder. The smaller the loose density and the tap density is, the loose the powder is; the high bulk and tap densities are more advantageous for the filling effect. Under the same volume, the powder has smaller particle size and smaller interval, so that the density is larger and the quality is larger; the loose packing density is higher, and the tap density is higher, so that the digestion and absorption are more favorable for human bodies. It can be seen from table 13 that the apparent bulk density and tap density of the ultrafine powder are much higher than those of the conventional powder, which means that the ultrafine powder can effectively reduce the particle size and increase the gaps between particles, thereby improving the filling property thereof and being more advantageous for absorption in the human body.
TABLE 13 apparent bulk and tap Density of Normal powders and ultra micropowder
According to the embodiment, the superfine powder is prepared from the hemp seeds fermented by the rhizopus oryzae, and then the physicochemical indexes of the superfine powder are detected, so that the superfine grinding technology has a certain effect of improving the physicochemical properties of the hemp seeds fermented by the rhizopus oryzae, and compared with the common powder, the superfine powder has the advantages that the tissue structure breaking degree of superfine powder particles is larger, and the obtained particle size is obviously smaller; meanwhile, compared with common powder, the wettability, water holding capacity, water solubility and expansion force of the superfine powder are all obviously improved. By measuring the repose angle and the sliding friction angle, the fluidity of the superfine powder is found to be good, the basic requirement of production can be met, in addition, the apparent density and tap density of the superfine powder are also good, and the superfine powder is higher than that of common powder and is more beneficial to human body absorption. Therefore, the superfine grinding treatment improves the production adaptability of superfine powder to a great extent, and plays an important role in deep processing of hemp seeds and development of functional products.
EXAMPLE 4 hypolipidemic action
The embodiment mainly discusses the conditions of reducing blood fat in vitro, including sodium cholate binding rate, pancrelipase inhibition rate and cholesterol micelle solubility inhibition rate, and the most effective in reducing blood fat in vitro is obtained by comparing the effects of unfermented cannabis sativa seeds, rhizopus oryzae fermented cannabis sativa seeds and superfine powder on the three indexes.
The product of the rhizopus oryzae after fermenting hemp seeds is prepared by adopting the following fermentation conditions: the inoculation amount is 7%, the water content is 17%, the fermentation temperature is 35 ℃, and the fermentation time is 12h. The superfine powder is prepared by taking a product obtained by fermenting hemp seeds by rhizopus oryzae as a raw material according to the method of the example 3.
4.1 Experimental methods
(1) Determination method of sodium cholate binding rate
And (3) manufacturing a standard curve: 2mL of sodium cholate standard solutions with different concentrations of 0.03, 0.06, 0.12, 0.18, 0.24 and 0.30mmol/L are respectively put into volumetric flasks, 6mL of 60% sulfuric acid is added, the solution is put into a water bath at 70 ℃ for 20min and then is subjected to ice bath for 5min, and an absorbance value is measured at 387nm wavelength by an ultraviolet spectrophotometer. And drawing a standard curve of sodium cholate by taking the cholate content as an abscissa and the absorbance value as an ordinate. The binding rate of sodium cholate was calculated by the same method as in example 1.
(2) Method for measuring pancreatic lipase activity inhibition rate
Preparing a main solution: 0.08% (w/v) lauric acid 4-nitrophenyl ester (p-PNL) solution: weighing 0.4g p-PNL, adding into 1% sodium acetate solution containing triton X-100 (sodium acetate concentration is 5 mmol/L), boiling water bath for 1min to aid dissolution, cooling to room temperature, metering volume to 50mL with 5mmol/L sodium acetate solution, and storing in refrigerator at 4deg.C. 3mL of phosphate buffer solution with pH of 7.4 and 3mL of 0.08% lauric acid 4-nitrophenyl ester solution, 3mL of pancreatic lipase solution with 1.0mg/mL are respectively sucked, and uniformly mixed with a certain amount of hemp seeds before and after fermentation, wherein the mass of the sample is 4.5mg,9.0mg,18mg,27mg,54mg,72mg and 90mg respectively, so as to prepare sample solutions with the concentrations of 0.5, 1,2, 4, 6, 8 and 10mg/mL, the sample solutions are subjected to constant temperature water bath at 37 ℃ for 30min, and then the absorbance value is measured at 410nm and recorded as A. Meanwhile, the absorbance of the sample without being added at 410nm is A 1, the absorbance of the sample without being added with pancreatic lipase solution (replaced by phosphate buffer solution) at 410nm is A 0, and the pancreatic lipase inhibition rate is calculated according to the formula:
Wherein, the inhibition rate of C-pancreatic lipase,%;
A-absorbance values of the sample set;
A 1 -blank absorbance values;
a 0 -absorbance values of control group.
(3) Inhibition rate measurement of cholesterol micelle solubility
10ML of a cholesterol micelle solution was prepared, 7.7mg of cholesterol, 53.8mg of sodium taurocholate, 77.1mg of sodium chloride, 14.1mg of oleic acid and 7.5mL of a phosphate buffer solution of pH 7.4 of 0.02mol were taken so that 1mL of the solution contained 2mmol/L of cholesterol, 10mmol/L of sodium taurocholate, 132mmol/L of sodium chloride, 5mmol/L of oleic acid and 15mmol/L of phosphate buffer solution, followed by ultrasonic homogenization at 160W for 50 minutes and standing overnight at 37 ℃. Preparing a certain amount of unfermented cannabis seed powder and fermented cannabis seed powder into sample solutions with the concentration of 1,2, 4, 6, 8, 10 and 12mg/mL respectively, adding a certain amount into a micelle solution, carrying out shaking culture on the solution after uniform mixing at 37 ℃ for 2 hours, centrifuging at 14000r/min for 20min, collecting supernatant, measuring the cholesterol content in the micelle by using a cholesterol measuring kit, wherein the cholesterol solubility of the sample solution is A, the cholesterol solubility of the sample solution is A 0, and the inhibition rate is calculated by using the following formula as a control group:
wherein: a-absorbance value of the sample solution;
a 0 -absorbance values of control group.
(4) IC 50 deserves calculation: the IC 50 value refers to the concentration at which the inhibitory effect of the sample reaches half, and the experimental results obtained by measuring the solubility inhibitory rate of pancrelipase and cholesterol micelle and the unfermented cannabis sativa seed solution, the rhizopus oryzae fermented cannabis sativa seed solution and the superfine powder solution of each concentration are respectively input into IC 50 calculator software to calculate, so that the IC 50 value of the unfermented cannabis sativa seed solution, the rhizopus oryzae fermented cannabis sativa seed solution and the superfine powder solution is obtained.
4.2 Experimental results and analysis
(1) Evaluation of in vitro sodium cholate-binding Effect
① Sodium cholate standard curve
The sodium cholate standard curve equation is y=2.9861+0.0193, and r 2 =0.9994, where x is sodium cholate concentration mmol/L and y is absorbance.
② Sodium cholate binding rate results analysis: as shown in fig. 13, the sodium cholate binding rate of unfermented cannabis sativa seeds was 22.45% ± 2.21%, the sodium cholate binding rate of rhizopus oryzae fermented cannabis sativa seeds was 65.83% ± 1.97%, and the sodium cholate binding rate of ultrafine powder was 70.15% ± 2.32%. Proved by the optimized process of fermenting hemp seeds by rhizopus oryzae, the combination rate of cholate can be improved, and the efficacy of reducing blood fat is improved.
(2) Effect analysis of pancreatic lipase Activity inhibition Rate
As can be seen from fig. 14, the inhibition effect of unfermented cannabis sativa seeds, rhizopus oryzae fermented cannabis sativa seeds, and ultra fine powder all showed a concentration-dependent relationship to pancreatic lipase, and increased with increasing concentration. The inhibition effect of unfermented cannabis sativa seed solution increases with the concentration when the concentration is less than 8mg/mL, the inhibition effect increases rapidly, the dependence of the inhibition effect on the concentration is weakened after 8mg/mL, the inhibition rate reaches 65.75% +/-1.94% when 10mg/mL, the inhibition effect of rhizopus oryzae fermented cannabis sativa seeds on the pancreatic lipase is obviously stronger, the dosage relationship is also presented, the platform period gradually appears after 4mg/mL, the inhibition rate is 93.16% +/-2.17% when the concentration is 10mg/mL, the inhibition effect of superfine powder on the pancreatic lipase activity is best, and the inhibition rate reaches 99.46% +/-1.39% when the concentration reaches 8mg/mL, so that the inhibition effect of rhizopus oryzae fermented cannabis sativa seeds on the pancreatic lipase is truly improved, and the efficacy of the superfine powder is stronger. The strength is as follows: superfine powder > rhizopus oryzae fermented hemp seeds > unfermented hemp seeds. It is possible that some macromolecular substances of the hemp seeds are converted into small molecular substances after the rhizopus oryzae is fermented, so that the effect is stronger.
(3) Effect analysis of cholesterol micelle solubility inhibition ratio
The experiment simulates human intestinal conditions in vitro and compares the binding capacity of unfermented cannabis sativa seeds, rhizopus oryzae fermented cannabis sativa seeds, superfine powder and cholesterol. As can be seen in fig. 15, the binding capacity of the three samples to cholesterol was different and the solubility to cholesterol was continuously decreased with increasing concentration. It is shown that all three samples can reduce the solubility of cholesterol in micelle solution, and the binding capacity of the three samples and cholesterol is compared, so that the binding capacity of superfine powder and cholesterol is the strongest, the inhibition rate reaches 85.95 +/-2.03% at the concentration of 12mg/mL, the binding rate of rhizopus oryzae fermented hemp seeds and cholesterol is stronger than that of unfermented hemp seeds, the binding rate reaches 69.51 +/-1.48% at the concentration of 12mg/mL, the binding force of the hemp seeds before fermentation is the weakest, and the inhibition rate of the solubility of the superfine powder and cholesterol micelle is 56.31 +/-2.12% at the concentration of 12mg/mL is shown in FIG. 15. The strength is as follows: superfine powder > rhizopus oryzae fermented hemp seeds > unfermented hemp seeds.
(4) IC 50 value calculation and analysis
As shown in FIG. 16, in the pancreatic lipase activity inhibition test, the IC 50 value of unfermented cannabis sativa seeds was 8.054mg/mL, the IC 50 value of Rhizopus oryzae fermented cannabis sativa seeds was 1.255mg/mL, and the IC 50 value of ultrafine powder was 0.605mg/mL, as calculated by the IC 50 value calculator. In cholesterol micelle solubility inhibition activity experiments, the IC 50 value of unfermented cannabis sativa seeds is 11.758mg/mL, the IC 50 value of rhizopus oryzae fermented cannabis sativa seeds is 7.436mg/mL, and the IC 50 value of superfine powder is 3.171mg/mL. From this, the inhibition activity was found to be as follows: superfine powder is larger than rhizopus oryzae fermented hemp seeds and unfermented hemp seeds.
In this example, an in vitro blood lipid lowering experiment was mainly performed, and since the experiment of the binding rate of sodium glycocholate and sodium taurocholate was performed before, the section of this example was studied only for the remaining one cholate, and the obtained unfermented cannabis sativa seed had a binding rate of 22.45% ± 2.21%, a binding rate of rhizopus oryzae fermented cannabis sativa seed sodium cholate of 65.83% ± 1.97%, and a binding rate of ultrafine sodium cholate of 70.15% ± 2.32%, indicating that the binding rate of ultrafine sodium cholate was the best. The inhibition rate of the superfine powder to pancreatic lipase reaches 99.46% +/-1.39% when the concentration of the superfine powder is 8mg/mL, the inhibition rate of the rhizopus oryzae fermented hemp seeds is 93.16% +/-2.17% when the concentration of the rhizopus oryzae fermented hemp seeds is 10mg/mL, and the inhibition rate of the superfine powder to the pancreatic lipase is 65.75% +/-1.94% when the concentration of the unfermented hemp seeds is 10 mg/mL. The highest concentration of the superfine powder solution can reach 85.95 +/-2.03 percent when the concentration of the superfine powder solution is 10mg/mL, the inhibition rate of unfermented hemp seeds is 56.32+/-2.12 percent, and the inhibition rate of rhizopus oryzae fermented hemp seeds is 69.51 +/-1.48 percent. Experimental results prove that the inhibition effect of rhizopus oryzae fermented cannabis sativa seeds on pancreatic lipase and cholesterol micelle is improved, so that the blood fat reducing capability is improved. The superfine powder makes the particles of the powder smaller and is easier to be digested and absorbed by human bodies, so the blood fat reducing effect is better.
EXAMPLE 5 hypoglycemic and antioxidant effects
This example focused on the ability of three of unfermented cannabis sativa seeds, rhizopus oryzae fermented cannabis sativa seeds, and superfine powder to reduce blood glucose and antioxidant activity in vitro. In vitro blood sugar reduction mainly comprises inhibition of alpha-glucosidase and alpha-amylase activities, and the strength of three substances on blood sugar reduction capacity is studied by contrast with an hypoglycemic drug acarbose. The in vitro antioxidant capacity is compared with the traditional antioxidant Vc, the strength of three substances on DPPH and ABTS + free radical scavenging capacity is explored, and a reference is provided for further development and utilization of cannabis sativa seeds.
The rhizopus oryzae fermented hemp seeds related in this example were prepared using the following fermentation conditions: the inoculation amount is 7%, the water content is 17%, the fermentation temperature is 35 ℃, and the fermentation time is 12h. The superfine powder is prepared by taking a product obtained by fermenting hemp seeds by rhizopus oryzae as a raw material according to the method of the example 3.
5.1 Experimental methods
5.1.1 In vitro hypoglycemic Activity Studies
(1) Activity assay for inhibiting alpha-glucosidase
0.5ML of unfermented hemp seeds, rhizopus oryzae fermented hemp seeds and superfine powder solution with 0.5mL of 0.1U/mL of alpha-glucosidase solution (dissolved in 25mmol/L phosphate buffer solution with pH of 6.8) are uniformly mixed, mixed and reacted for 10min in a 37 ℃ water bath, then 0.5mL of p-NPG solution (dissolved in 0.1mol/L phosphate buffer solution with pH of 6.8) is added, reacted for 15min in a 37 ℃ water bath, and then 1mL of sodium carbonate solution with concentration of 0.1mol/L is added to terminate the reaction. The absorbance value is measured at 405nm by an ultraviolet spectrophotometer, acarbose is used as a positive control, and an equal volume of 0.1mol/L phosphate buffer solution with pH of 6.8 is used as a control group instead of alpha-glucosidase. The inhibition rate formula is as follows:
Wherein: a—absorbance of the sample set;
A 0 -absorbance of control;
Absorbance of mixed reaction of A 1 -PBS buffer solution and p-NPG solution with enzyme solution;
Absorbance of a 2 -PBS buffer and p-NPG solution mixed reaction.
(2) Activity assay for inhibiting alpha-amylase
1ML of unfermented hemp seeds, rhizopus oryzae fermented hemp seeds and superfine powder solution with different mass concentrations (1, 5, 10, 15 and 20 mg/mL) are respectively mixed with 2mL of 1U/mL alpha-amylase solution (dissolved in phosphate buffer with pH of 6.8) to react for 10min in a 37 ℃ water bath, 1mL of 1% soluble starch solution is added to react for 10min in the 37 ℃ water bath, 1mL of DNS reagent is added, and the boiling water bath is heated for 5min to terminate the reaction, and the volume is fixed to 10mL. After cooling to room temperature, the absorbance was measured at 540nm, acarbose was used as a positive control, and an equal volume of 0.1mol/L phosphate buffer pH 6.8 was used as a control instead of alpha-amylase. The inhibition rate formula is as follows:
Wherein: a—absorbance of the sample set;
a 0 -absorbance of control group.
5.1.2 In vitro antioxidant Activity Studies
(1) DPPH in vitro antioxidation experiment
① Preparation of DPPH test solution: 3.94mg of DPPH is precisely weighed and put into a volumetric flask of 100mL, and then the volumetric flask is fixed with absolute ethyl alcohol to obtain a solution of DPPH in ethanol of 0.1 mmol/L.
② Vc control experiment: precisely weighing 25mg of Vc in a 10mL volumetric flask, and fixing the volume by deionized water to prepare 2.5mg/mL Vc reference substance solution; then diluted with deionized water into reference substance solutions of 0.0625mg/mL,0.125mg/mL,0.25mg/mL,0.5mg/mL and 1.0mg/mL in sequence.
③ Preparation of sample solution: the sample is prepared into 0.0625mg/mL,1.0mg/mL,10mg/mL,20mg/mL,40mg/mL,60mg/mL and 80mg/mL solution, 70% ethanol solution is added for dissolution according to the feed liquid ratio of 1:25, ultrasonic extraction is carried out for 1h at the temperature of 280W and 45 ℃, and centrifugation is carried out for 20min at 8000r/min, and supernatant fluid is taken for standby.
④ Measurement of DPPH removal ability: respectively sucking 2mL of Vc control solution, unfermented cannabis sativa seed solution, rhizopus oryzae fermented cannabis sativa seed solution and superfine powder solution, adding 2mL of DPPH solution, fully and uniformly mixing, sealing and placing for 30min in a dark place, measuring the absorbance value of each sample at 517nm, taking the obtained result into a DPPH clearance formula, and calculating the clearance of Vc control solution, unfermented cannabis sativa seed solution, rhizopus oryzae fermented cannabis sativa seed solution and superfine powder solution with each concentration. The clearance rate calculation formula is as follows:
Wherein: absorbance of A 0 -2 mL DPPH solution and 2mL absolute ethanol;
Absorbance of A 1 -2 mL sample solution and 2mL absolute ethanol;
a 2 -2 mL of DPPH solution and 2mL of sample solution.
(2) ABTS + in vitro antioxidant experiment
①ABTS+ Preparation of the test solution: accurately weighing 6.62mg of potassium persulfate and 3835.4 mg of ABTS + in a 10mL volumetric flask, dissolving with distilled water, and fixing the volume to prepare an ABTS + mother solution. After 16h of reaction at room temperature in a dark place, the absorbance value of the ABTS + mother solution is about 0.70+/-0.02 at the wavelength of 734nm by diluting with 1mmol/L PBS buffer solution with pH of 7.4, thus obtaining the ABTS + test solution.
② Vc control experiment: precisely weighing 25mg of Vc in a 10mL volumetric flask, and carrying out volume fixing by using deionized water to prepare 2.5mg/mL Vc reference substance solution; then diluted with deionized water into reference substance solutions of 0.0625mg/mL,0.125mg/mL,0.25mg/mL,0.5mg/mL and 1.0mg/mL in sequence.
③ Preparation of sample solution: the sample is prepared into 0.0625mg/mL,1.0mg/mL,10mg/mL,20mg/mL,40mg/mL,60mg/mL and 80mg/mL solution, 70% ethanol solution is added for dissolution according to the feed liquid ratio of 1:25, ultrasonic extraction is carried out for 1h at the temperature of 280W and 45 ℃, and centrifugation is carried out for 20min at 8000r/min, and supernatant fluid is taken for standby.
④ABTS+ Determination of clearance ability: absorbing 4.8mL of ABTS + test solution by a pipette, respectively adding 0.2mL of Vc control solution with various concentrations, unfermented cannabis sativa seed solution, rhizopus oryzae fermented cannabis sativa seed solution and sample solution of superfine powder, carrying out ultrasonic oscillation on all the liquids for 30s, sealing and light-shielding treatment, detecting the absorbance of each sample at 734nm wavelength by an ultraviolet spectrophotometer after reaction for 6min, recording the absorbance value as A, zeroing the blank by using phosphate buffer solution with the pH value of 7.4, and taking the result into an ABTS + clearance formula, and calculating the clearance rate of Vc control solution with various concentrations, unfermented cannabis sativa seed solution, rhizopus oryzae fermented cannabis sativa seed solution and superfine powder solution, wherein the clearance rate is calculated as follows:
(3) Calculation of IC 50 values
The IC 50 value refers to the concentration when the free radical removal of the sample reaches half, the experimental results obtained by measuring the alpha-glucosidase and alpha-amylase inhibition capability, DPPH and ABTS + removal capability and the different concentrations of each sample are respectively input into IC 50 value calculator software to calculate so as to obtain the IC 50 value of the acarbose control solution, vc control solution, unfermented hemp seed solution, rhizopus oryzae fermented hemp seed solution and superfine powder solution on the alpha-glucosidase and alpha-amylase inhibition capability, DPPH and ABTS + removal capability.
5.2 Experimental results and analysis
(1) Evaluation of alpha-glucosidase Activity inhibitory Effect
As can be seen from fig. 17, the four solutions all had different inhibitory effects on α -glucosidase and had a dose-dependent relationship. Along with the increase of the mass concentration of the solution, the inhibition rate of the acarbose, the unfermented cannabis sativa seeds, the rhizopus oryzae fermented cannabis sativa seeds and the superfine powder on the alpha-glucosidase is continuously increased. The inhibition rate of unfermented cannabis sativa seeds to alpha-glucosidase is 20.06% +/-1.96%, 46.38% +/-2.21%, 50.35% +/-2.15%, 56.25% +/-2.18%, 66.08% +/-1.76% respectively, 31.91% +/-1.69%, 54.48% +/-2.09%, 61.58% +/-1.57%, 71.41% +/-2.27% and 75.46% +/-1.59% respectively at different concentrations of 1mg/mL,5mg/mL,10mg/mL,15mg/mL and 20 mg/mL; the inhibition rate of the superfine powder to the alpha-glucosidase is 27.94% +/-2.36%, 44.71% +/-2.35%, 47.85% +/-2.39%, 71.05% +/-2.21%, 87.53% +/-2.20%, and the inhibition rate of the acarbose to the alpha-glucosidase is 38.75% +/-2.16%, 62.84% +/-2.53%, 78.81% +/-2.12%, 86.52% +/-1.67% and 95.08% +/-3.14% under the conditions that the concentration of acarbose is 0.1mg/mL,0.25mg/mL,0.5mg/mL and 1.0 mg/mL. Therefore, in the experiment of the inhibition effect of the alpha-glucosidase activity, the inhibition rate is as follows: acarbose > superfine powder > rhizopus oryzae fermented hemp seeds > unfermented hemp seeds.
Acarbose is a hypoglycemic agent, and has definite contraindications and various precautions as a medicine, and has adverse reactions. However, the superfine powder and the rhizopus oryzae fermented cannabis seeds are prepared from the cannabis seeds with homology of medicine and food as raw materials, so that the safety is better, adverse reactions are avoided, the effects of reducing blood fat, resisting oxidization, preventing obesity, preventing intestinal cancer and the like are achieved, and the superfine powder and the rhizopus oryzae fermented cannabis seeds have more advantages in the fields of foods, health care products and the like.
(2) Evaluation of alpha-Amylase Activity inhibitory Effect
As can be seen from fig. 18, the 4 solutions all had different inhibitory effects on α -amylase and had a dose-dependent relationship. With the increase of the mass concentration of the solution, the inhibition rate of the unfermented cannabis seeds, rhizopus oryzae fermented cannabis seeds, superfine powder and acarbose on the alpha-amylase is continuously increased. The inhibition rate of unfermented hemp seeds to alpha-amylase is 3.23 percent+/-0.14 percent, 10.13 percent+/-1.02 percent, 20.04 percent+/-1.27 percent, 31.94 percent+/-2.03 percent, 40.76 percent+/-2.12 percent, and the inhibition rate of rhizopus oryzae fermented hemp seeds to alpha-amylase is 10.80 percent+/-1.04 percent, 14.93 percent+/-1.26 percent, 31.60 percent+/-1.52 percent, 44.60 percent+/-2.20 percent and 53.13 percent+/-2.07 percent under the different concentrations of 1mg/mL,5mg/mL,10mg/mL,15mg/mL and 20mg/mL respectively; the inhibition rate of the superfine powder to the alpha-amylase is 14.61% +/-0.64%, 24.25% +/-1.08%, 40.88% +/-1.16%, 55.13% +/-2.13%, 67.70% +/-2.12%, and the inhibition rate of the acarbose to the alpha-amylase is 34.88% +/-2.35%, 48.99% +/-2.28%, 62.61% +/-2.45%, 74.18% +/-2.31% and 80.42% +/-1.88% under the conditions that the concentration of acarbose is 0.1mg/mL,0.25mg/mL,0.5mg/mL and 0.75 mg/mL. Therefore, in the experiment of the inhibition effect of the alpha-amylase, the inhibition ratio is as follows: acarbose > superfine powder > rhizopus oryzae fermented hemp seeds > unfermented hemp seeds.
(3) DPPH in vitro antioxidation experimental result and analysis
As shown in fig. 19, the scavenging rate of the Vc solution with 5 concentrations is 35.78% ± 2.15%,42.31% ± 2.14%,58.60% ± 1.88%,74.36% ± 1.86%,90.68% ± 1.63%, respectively, and it can be seen that the scavenging force of DPPH free radicals is continuously enhanced as the Vc solution concentration is increased; the clearance rate of 7 concentrations of unfermented hemp seeds is 0.21% ± 0.06%,8.24% ± 0.21%,37.06% ± 1.18%,41.32% ± 2.30%,52.24% ± 2.32%,62.35% ± 2.40%,63.92% ± 1.69%, respectively, and it can be seen that the clearance rate of DPPH free radicals is weak; the clearance rate of 7 concentrations of rhizopus oryzae fermented hemp seeds is 0.23% +/-0.06%, 8.31% +/-0.44%, 32.96% +/-1.15%, 41.41% +/-1.85%, 54.46% +/-2.08%, 62.94% +/-2.43% and 70.70% +/-1.96%, and when the concentration reaches 80mg/mL, the clearance rate of the rhizopus oryzae fermented hemp seeds reaches more than 70%, and the clearance rate of DPPH free radicals is obviously higher than that of unfermented hemp seeds; the clearance rate of the superfine powder with 7 concentrations is 9.67+/-0.69%, 26.84+/-1.01%, 47.75+/-1.40%, 55.53+/-1.98%, 73.53+/-2.11%, 79.76+/-1.77% and 90.05+/-1.85%, respectively, and when the concentration is lower, the clearance capacity of the superfine powder is lower than that of Vc solution, but along with the continuous increase of the concentration, the superfine powder has similar clearance capacity to that of Vc solution, which indicates that the clearance capacity of the superfine powder to DPPH is better. Therefore, in DPPH in vitro antioxidation experiments, the ability to remove free radicals is as follows: vc is more than superfine powder is more than rhizopus oryzae fermented hemp seeds is more than unfermented hemp seeds. When evaluating the antioxidant activity, vc is used as a reference as the most common experimental method, and the superfine powder and the rhizopus oryzae fermented hemp seeds can be proved to have the antioxidant property by using Vc as the reference. Besides the antioxidant activity, the rhizopus oryzae fermented hemp seeds and the superfine powder also have other nutritional components and effects which are not possessed by Vc. For example: the superfine powder and rhizopus oryzae fermented hemp seeds also contain active ingredients such as flavone, polysaccharide, small molecular peptide and the like, and have the effects of reducing blood sugar, reducing blood fat, preventing obesity, preventing intestinal cancer and the like besides antioxidation.
(4) ABTS + in vitro antioxidation experimental results and analysis
The experimental results are shown in fig. 20, the scavenging rates of the Vc solutions with 5 concentrations are 31.40 +/-1.19%, 59.21 +/-2.23%, 95.74+/-1.87%, 99.78+/-2.11% and 99.91+/-1.85%, respectively, and the scavenging capability of the Vc solution on the ABTS + free radical is enhanced continuously along with the increasing of the Vc solution concentration, and the Vc solution has very strong scavenging capability on the ABTS + free radical; the clearance rate of 7 concentrations of unfermented hemp seeds is 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%, respectively, and the clearance rate is weaker than that of ABTS + free radical at low concentration, and gradually increases along with the increase of concentration, so that the clearance rate has a certain clearance capacity to ABTS + free radical; the clearance rate of 7 concentrations of rhizopus oryzae fermented hemp seeds is 0.47% +/-0.13%, 13.51% +/-1.76%, 48.28% +/-2.07%, 65.36% +/-2.14%, 81.38% +/-1.88%, 92.48% +/-2.06% and 97.03% +/-1.72% respectively; the clearance rate of 7 concentrations of the superfine powder is 9.61% +/-0.62%, 31.75% +/-1.71%, 47.13% +/-1.95%, 65.63% +/-1.91%, 82.25% +/-2.07%, 92.92% +/-2.35% and 97.13% +/-1.99%, respectively, and the clearance capability of the rhizopus oryzae fermented hemp seeds and the superfine powder on ABTS + is obviously improved, and the clearance capability of the rhizopus oryzae fermented hemp seeds and the superfine powder is similar to that of Vc solution, so that the clearance capability of the rhizopus oryzae fermented hemp seeds and the superfine powder on ABTS + is better. Therefore, in ABTS + in vitro antioxidant experiments, the strength of the ability to scavenge free radicals is: vc is more than superfine powder is more than rhizopus oryzae fermented hemp seeds is more than unfermented hemp seeds.
(5) IC 50 value calculation and analysis
As shown in Table 14, the alpha-glucosidase activity inhibition test shows that the IC 50 value calculator calculates that the acarbose IC 50 value is 0.158mg/mL, the unfermented hemp seed IC 50 value is 8.255mg/mL, the Rhizopus oryzae fermented hemp seed IC 50 value is 23.646mg/mL, and the superfine powder IC 50 value is 4.620mg/mL. In the alpha-amylase activity inhibition experiment, the IC 50 value of acarbose is 0.231mg/mL, the IC 50 value of unfermented cannabis sativa seeds is 34.594mg/mL, the IC 50 value of rhizopus oryzae fermented cannabis sativa seeds is 24.080mg/mL, and the IC 50 value of superfine powder is 12.241mg/mL. From this, the level of the blood glucose lowering activity inhibitory ability was found to be: acarbose > superfine powder > rhizopus oryzae fermented hemp seeds > unfermented hemp seeds.
Table 14 IC 50 values for inhibition of alpha-glucosidase and alpha-amylase activity by acarbose, unfermented cannabis seed, rhizopus oryzae fermented cannabis seed and superfine powder
As shown in Table 15, in DPPH in vitro antioxidation test, the IC 50 value calculator calculates that the IC 50 value of Vc is 0.145mg/mL, the IC 50 value of unfermented cannabis sativa seeds is 29.882mg/mL, the IC 50 value of rhizopus oryzae fermented cannabis sativa seeds is 27.926mg/mL, and the IC 50 value of superfine powder is 5.554mg/mL. In an ABTS + in-vitro antioxidation experiment, the IC 50 value of Vc is 0.089mg/mL, the IC 50 value of unfermented cannabis sativa seeds is 12.827mg/mL, the IC 50 value of rhizopus oryzae fermented cannabis sativa seeds is 7.346mg/mL, and the IC 50 value of superfine powder is 2.870mg/mL. From this, the ability to remove free radicals is as follows: vc is more than superfine powder is more than rhizopus oryzae fermented hemp seeds is more than unfermented hemp seeds.
Table 15 Vc IC 50 values for control solution, unfermented Cannabis sativa seed, rhizopus oryzae fermented Cannabis sativa seed, and ultrafine powder
The results of in vitro hypoglycemic experiments show that the activity inhibition of alpha-glucosidase and alpha-amylase is as follows: acarbose > superfine powder > rhizopus oryzae fermented hemp seeds > unfermented hemp seeds. The activity inhibition rate of the superfine powder on alpha-glucosidase reaches 87.53 +/-2.20 percent, and the activity inhibition rate on alpha-amylase can reach 67.70+/-2.12 percent, which shows that the rhizopus oryzae fermented cannabis sativa seed superfine powder has better blood sugar reducing effect; the results of in vitro antioxidant experiments show that the scavenging ability to DPPH and ABTS + is as follows: vc is more than superfine powder is more than rhizopus oryzae fermented hemp seeds is more than unfermented hemp seeds. The clearance rate of the superfine powder to DPPH reaches 90.05+/-1.25 percent, and the clearance rate to ABTS + can reach 97.13+/-1.99 percent, which shows that the rhizopus oryzae fermented hemp seed superfine powder has better antioxidation activity.
In summary, the invention takes hemp seeds as raw materials, adopts rhizopus oryzae seeds for fermentation, finds the fermentation condition with the highest binding rate with cholate, then prepares the fermented product into superfine powder, and discovers the advantages of the superfine powder in the dosage form through the measurement of some powder properties; and determining which components are affected by fermentation through the component content change before and after fermentation, so as to provide reference for further research on the hemp seeds; and then, carrying out preliminary researches on reducing blood fat, reducing blood sugar and resisting oxidization.
(1) The method comprises the steps of (1) researching the binding rate of cholate of a fermented product, taking the binding rate of sodium glycocholate and the binding rate of sodium taurocholate as indexes, firstly, carrying out a single factor test, and respectively taking rhizopus oryzae inoculation amount, fermentation time, water content and fermentation temperature as influencing factors, wherein the optimal fermentation conditions are found that the rhizopus oryzae inoculation amount is 8%, the fermentation time is 12 hours, the water content is 22%, and the fermentation temperature is 35 ℃; the Plackett-Burman test performed later gave three significant influencing factors with the highest binding rate to sodium glycocholate sodium taurocholate: rhizopus oryzae inoculum size, water content, fermentation temperature; and then carrying out a 3-factor 3 horizontal response surface test to finally obtain the fermentation hemp seed with the sodium glycocholate binding rate of 75.64 +/-2.77 percent and the sodium taurocholate binding rate of 65.77+/-3.45 percent. Compared with unfermented cannabis sativa seeds, the sodium glycocholate binding rate of the fermented cannabis sativa seeds is improved by about 37.60%, and the sodium taurocholate binding rate is improved by about 37.25%. The hemp seeds have the specific blood lipid reducing effect, but the effect of reducing the blood lipid of the hemp seeds is remarkably improved after the rhizopus oryzae is fermented.
(2) The content of the components before and after the fermentation of the rhizopus oryzae is measured and compared, and the flavone, the polysaccharide, the amino acid, the fatty acid and the oligopeptide are increased to a certain extent after the fermentation of the rhizopus oryzae, wherein the maximum change is about 62.69 percent and 45.56 percent of the polysaccharide and the flavone, the amino acid content is increased by about 7.14 percent, the fatty acid content is increased by about 11.94 percent, the oligopeptide content is increased by about 7.84 percent, and the protein content is reduced by about 4.54 percent. Rhizopus oryzae is a mold with developed enzyme system, and can catalyze components in hemp seeds, so that the components are converted. The blood lipid reducing capability of the fermentation product of the rhizopus oryzae fermented cannabis seed is higher than that of the unfermented cannabis seed, so that the rhizopus oryzae fermented cannabis seed flavone and polysaccharide are greatly improved, and the improvement of the flavone and polysaccharide in the fermentation product can be proved to have a promotion effect on the enhancement of the blood lipid reducing capability.
(3) Micronizing rhizopus oryzae fermented hemp seeds to obtain superfine powder with particle diameter of 837.1nm; the wetting time of the superfine powder is 18.76s plus or minus 1.82s; moisture of the superfine powder is 1.55% +/-0.05%; a water retention capacity of 4.25.+ -. 0.18 (g/g); expansion force of 5.61+/-0.27 (mL/g) and water solubility 77.02 +/-0.25%; the repose angle of the superfine powder is 38.71 degrees and the sliding friction angle is 50.24 degrees; . The apparent density of the superfine powder is 0.51+/-0.23 (g/cm 3) and the true density is 0.60+/-0.12 (g/cm 3). The particle size of the powder is smaller, the hydration properties such as wettability, water retention capacity, water solubility, expansion force and the like are better, the fluidity of the powder is also better, the powder is more beneficial to absorption in a human body, the drug effect can be enhanced, and the powder can be applied to the food and medicine industries in the subsequent production. The indexes are all superior to those of common powder, and the advantages of the superfine powder in the aspect of dosage form are proved.
(4) Preliminary researches on in-vitro blood fat reduction are carried out, and the invention mainly researches on the binding rate of sodium cholate, the inhibition rate of pancreatic lipase and the inhibition rate of cholesterol micelle solubility, and discovers that the in-vitro blood fat reduction sequence is as follows: the superfine powder is more than rhizopus oryzae fermented cannabis sativa seeds more than unfermented cannabis sativa seeds, which is consistent with the original purpose of improving the efficacy of the prior superfine powder preparation, and proves that the surface area of the powder is increased by the superfine powder technology, thereby being more beneficial to improving the efficacy of the drug.
(5) Preliminary researches on in-vitro blood sugar reduction and in-vitro oxidation resistance show that the effect of reducing blood sugar is better, the activity inhibition rate of the superfine powder on alpha-glucosidase reaches 87.53 +/-2.20%, the activity inhibition rate on alpha-amylase reaches 67.70+/-2.12%, the scavenging capacity on free radicals is relatively better, the scavenging rate of the superfine powder on DPPH reaches 90.05+/-1.85%, and the scavenging rate on ABTS + reaches 97.13+/-1.99%. The rhizopus oryzae has improved flavone content after fermentation, and has good free radical scavenging effect, so that the free radical scavenging capability of the fermented hemp seeds is improved.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (7)
1. A method of fermenting cannabis sativa seeds, comprising the steps of: fermenting hemp seeds by rhizopus oryzae; fermentation conditions include;
(1) 2% -10% of rhizopus oryzae inoculum size;
(2) The water content is 8% -43%;
(3) The fermentation temperature is 20-35 ℃;
(4) The fermentation time is 4-20 h.
2. A method of fermenting cannabis seed according to claim 1, wherein the fermentation conditions comprise;
(1) 6% -8% of rhizopus oryzae inoculum size;
(2) The water content is 15-36%;
(3) The fermentation temperature is 30-35 ℃;
(4) The fermentation time is 8-16h.
3. A method of fermenting cannabis seed as claimed in claim 2, wherein the fermentation conditions comprise;
(1) Rhizopus oryzae inoculum size 6.9-7%;
(2) The water content is 17%;
(3) Fermenting at 34.66-35deg.C;
(4) Fermentation time 12 h.
4. A method of fermenting cannabis seed according to any of claims 1-3, further comprising the step of sterilizing the cannabis seed prior to fermentation.
5. The preparation method of rhizopus oryzae fermented cannabis sativa seed superfine powder is characterized by comprising the following steps of: superfine pulverizing the mixture of rhizopus oryzae fermented hemp seeds; the rhizopus oryzae fermented cannabis seed is prepared by the method of any one of claims 1-4.
6. The method for preparing rhizopus oryzae fermented cannabis seed superfine powder as claimed in claim 5, wherein the superfine grinding comprises the following steps: mixing the mixture obtained after fermenting the hemp seeds by rhizopus oryzae with water-soluble starch, pulverizing, adding water, mixing, homogenizing, filtering, lyophilizing the filtrate, and micronizing to obtain superfine powder of the hemp seeds by rhizopus oryzae fermentation.
7. The method for preparing the rhizopus oryzae fermented cannabis seed superfine powder according to claim 6, wherein the mixture after rhizopus oryzae fermentation cannabis seed is mixed with water-soluble starch in a mass ratio of 4:6, the crushing time is 5min, water is added after the mixture is taken out and mixed in a mass ratio of 1:20, the homogenization time is 20: 20 min, a 450-mesh filter screen is adopted for filtration, the filtrate is freeze-dried 48h for removing water, and the obtained rhizopus oryzae fermented cannabis seed superfine powder is obtained by crushing the filtrate by a vibrating type drug superfine grinder at a temperature of 10 ℃ for 15 min.
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