A method for preparing high bioavailability buckwheat flavonoids and phenolic products

Abstract

This invention discloses a method for preparing high-bioavailability buckwheat flavonoids and phenolic products, comprising the following steps: fermentation of buckwheat flour; preparation of fermentation concentrate; preparation of buckwheat extract-β-cyclodextrin complex; addition of buckwheat extract-β-cyclodextrin complex to an external emulsion and stirring; addition of pectin solution; magnetic stirring; and freeze-drying to obtain a buckwheat extract-β-cyclodextrin complex-whey protein-pectin complex system. This invention uses buckwheat as raw material and utilizes Bacillus subtilis fermentation combined with organic solvent extraction to obtain high concentrations of buckwheat flavonoids and polyphenols. A sustained-release system of active ingredients is prepared by encapsulating the active ingredients of buckwheat extract with β-cyclodextrin-whey protein / gum arabic and pectin. In vitro simulated gastrointestinal digestion is used to evaluate the release of nutrients from buckwheat by Bacillus subtilis fermentation combined with organic solvent extraction and the bioavailability of the sustained-release system of active ingredients from buckwheat extract.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of natural active ingredient extraction and preparation, and particularly relates to a preparation method of high-bioavailability bitter buckwheat flavonoids and phenolic products. BACKGROUND

[0002] Bitter buckwheat is known as the "King of Five Cereals" and is a typical representative of Chinese medicine and food. Bitter buckwheat contains polyphenols, flavonoids, rutin, quercetin, chlorogenic acid and other monomer phenolic components, and various important bioactive substances endow bitter buckwheat with functions such as antioxidant, anti-inflammatory and anticancer, and have a positive effect on improving the gastrointestinal function of the human body and preventing cardiovascular and cerebrovascular diseases. It is a difficult problem for the current active substance extraction to improve the extraction rate and leaching effect of active ingredients through pretreatment of raw materials. In addition, the active ingredients in bitter buckwheat are affected by temperature, light, pH and different digestion environments, resulting in a decrease in their bioactivity. Therefore, it is of practical significance and application value to improve the bioavailability of bitter buckwheat active ingredients. SUMMARY

[0003] Therefore, the application provides a preparation method of high-bioavailability bitter buckwheat flavonoids and phenolic products, which improves the content of flavonoids and phenolic active ingredients in bitter buckwheat and the utilization rate.

[0004] The application is implemented by the following technical scheme:

[0005] The preparation method of high-bioavailability bitter buckwheat flavonoids and phenolic products comprises the following steps:

[0006] (1) bitter buckwheat powder fermentation: after the bitter buckwheat powder is dried and crushed, Bacillus subtilis seed liquid is inoculated for fermentation to obtain bitter buckwheat fermentation liquid;

[0007] (2) preparation of fermentation concentrated liquid: the bitter buckwheat fermentation liquid is extracted and concentrated by 60-80% ethanol to obtain bitter buckwheat extract concentrated liquid;

[0008] (3) preparation of bitter buckwheat extract-β-cyclodextrin complex: the bitter buckwheat extract concentrated liquid is dropped into a β-cyclodextrin solution, and magnetic stirring is performed to obtain a bitter buckwheat extract-β-cyclodextrin complex;

[0009] (4) preparation of external emulsion: whey protein and gum arabic are mixed at a mass ratio of 1:3 to prepare an external emulsion;

[0010] (5) slow-release system: the bitter buckwheat extract-β-cyclodextrin complex is added to the external emulsion and stirred, a pectin solution is added, and magnetic stirring is performed, and then freeze-drying is performed to obtain a bitter buckwheat extract-β-cyclodextrin complex-whey protein-pectin complex system.

[0011] Further, the seed liquid is prepared by inoculating Bacillus subtilis into a nutrient broth solid culture medium, activating and culturing at 37 DEG C for 24 hours, inoculating into a nutrient broth culture medium, and culturing at 37 DEG C and 150 r / min for 72 hours.

[0012] Further, the inoculation amount of Bacillus subtilis is 3-5%, and the fermentation condition is 37 DEG C, 150 r / min, and 24 hours.

[0013] Further, the tartary buckwheat fermentation liquid is concentrated by rotary evaporation, and the tartary buckwheat extract concentrate is obtained by extracting tartary buckwheat active substances at a temperature of 90 DEG C, an extraction time of 2 hours, an ethanol volume concentration of 60-80%, and a solid-liquid ratio of 1:3.

[0014] Further, the tartary buckwheat extract concentrate is added into a β-cyclodextrin solution (0.5%, W / V) at a mass ratio of 1:4, and the tartary buckwheat active ingredients are embedded into the hydrophobic interstices of the β-cyclodextrin by magnetic stirring at 40 DEG C for 20-30 minutes, so that the complex protein and arabic gum are obtained, and the tartary buckwheat extract-β-cyclodextrin complex is obtained.

[0015] Further, the whey protein and arabic gum in step (4) are in a mass ratio of 1:3.

[0016] Further, in step (5), the tartary buckwheat extract-β-cyclodextrin complex and the outer emulsion are added in a mass ratio of 1:5-15, and the mass ratio of the outer emulsion and the pectin is 2:1.

[0017] Further, in step (5), the outer emulsion is added, and the pH is 3-5, the temperature is 45 DEG C, the magnetic stirrer is 200 r / min, and the stirring time is 20 minutes; the pectin solution is added, and the pH is 3-5, the temperature is 45 DEG C, the magnetic stirrer is 200 r / min, and the stirring time is 30 minutes, and then the mixture is stored in a refrigerator at 4 DEG C for 24 hours. The pectin is not digested in the front end of the digestive tract, is fermented in the large intestine, is degraded in the colon as an energy source for bacterial activity, but does not generate heat, and the embedding of the pectin is beneficial to the slow release of the active ingredients.

[0018] Beneficial effects

[0019] The present application uses tartary buckwheat as a raw material, and obtains high-concentration tartary buckwheat flavones and polyphenols by Bacillus subtilis fermentation combined with organic solvent extraction. The active ingredient slow-release system is prepared by embedding tartary buckwheat extract active ingredients in β-cyclodextrin-whey protein / arabic gum and pectin, and the release amount of tartary buckwheat nutrients and the bioavailability of the tartary buckwheat extract active ingredient slow-release system are evaluated by in vitro simulation of gastrointestinal digestion. Specific implementation

[0020] The following will make a detailed description of the embodiments of the present application, which are implemented on the premise of the technical solutions of the present application, and give detailed implementation manners and specific operation processes, but the protection scope of the present application is not limited to the following embodiments.

[0021] Example 1 Preparation of tartary buckwheat active substance sustained-release system

[0022] 1. Preparation of tartary buckwheat flour: tartary buckwheat is baked at 50°C until constant weight, and is pulverized by a super-micro pulverizer and passed through a 50-mesh sieve.

[0023] 2. Bacillus subtilis is inoculated into a nutrient broth solid culture medium (CM187, Beijing 1 Bridge) and activated and cultured at 37°C for 24 hours, is inoculated into a nutrient broth culture medium, and is cultured at 37°C and 150 r / min for 72 hours as a seed liquid, which is ready for use.

[0024] 3. 5g of tartary buckwheat flour is accurately weighed, sterilized at 115°C for 30 minutes, cooled to room temperature, 50mL of distilled water is added, and 3%-5% g / mL of Bacillus subtilis seed liquid is inoculated, and then fermented at 37°C and 150 r / min for 24 hours, and the cooled tartary buckwheat fermentation liquid is sterilized and inactivated.

[0025] 4. The tartary buckwheat fermentation liquid is used to extract tartary buckwheat active substances at a temperature of 90°C, an extraction time of 2 hours, an ethanol concentration of 60-80%, and a solid-liquid ratio of 1:3, and the tartary buckwheat extract concentrate is obtained by rotary evaporation and concentration.

[0026] 5. Preparation of tartary buckwheat extract-β-cyclodextrin complex: β-cyclodextrin solution (0.5%, W / V) is added dropwise to the tartary buckwheat extract concentrate at a mass ratio of 1:4, and magnetic stirring is performed at 40°C for 20-30 minutes, so that the tartary buckwheat active ingredients are embedded in the hydrophobic interstices of β-cyclodextrin, and the tartary buckwheat extract-β-cyclodextrin complex is obtained.

[0027] 6. Whey protein and gum arabic are mixed at a mass ratio of 1:3, and an external emulsion is prepared by using a constant-temperature water tank at 45°C.

[0028] 7. The tartary buckwheat extract-β-cyclodextrin complex is added to the external emulsion at a mass ratio of 1:(5-15) under the conditions of pH 3-5 and temperature 45°C, and the mixture is obtained by stirring at 200 r / min for 20 minutes by using a magnetic stirrer.

[0029] 8. Add pectin solution to the above mixture (pectin added at a mass ratio of 2:1 for the external emulsion and pectin in the mixture). Continue stirring for 30 min at pH 3-5, temperature 45 ℃, and magnetic stirrer speed of 200 r / min. Then store in a refrigerator at 4 ℃ for 24 h to ensure the complete assembly of the buckwheat extract-β-cyclodextrin complex-whey protein-pectin complex system. After 24 h, freeze-dry the complex and pass it through a 100-mesh sieve to obtain the sustained-release system of the active ingredients of buckwheat extract.

[0030] Example 2 Evaluation of the sustained-release system of active ingredients from buckwheat extract

[0031] 1. Detection of total flavonoids and total polyphenols

[0032] The total flavonoid and total polyphenol contents in the fermentation broth and buckwheat extract concentrate were determined by standard curve and ultraviolet spectrophotometry, as shown in Tables 1 and 2.

[0033] 2. In vitro simulation of gastric digestion

[0034] Take 1g each of the prepared tartary buckwheat flour, tartary buckwheat fermentation broth, tartary buckwheat extract-β-cyclodextrin complex, and tartary buckwheat extract-β-cyclodextrin complex-whey protein-pectin complex system, add 25 mL of 9 mg / mL NaCl solution, 4 mL of 0.1 mol / L hydrochloric acid solution, and 4 mL of 4 mg / mL pepsin solution (buffer solution: 0.1 mol / L hydrochloric acid), mix well, and determine the pH of the solution to be between 2.0 and 2.5. Incubate at 37 ℃ with shaking at 100 r / min for 1 h. Reserve a portion of the digestive fluid for later use, and use the remainder for simulating intestinal digestion. The encapsulation efficiency is shown in Table 3.

[0035] 2. Simulation of the gastrointestinal transition phase

[0036] The boiled dialysis bag was soaked in ethanol, cleaned, and then 8 mL of 9 mg / mL NaCl and 2 mL of 0.5 mol / L NaHCO3 were added. The two ends were tied tightly and the bag was placed in the digestive gastric juice and incubated in a constant temperature water bath at 37 ℃ and 100 r / min for 45 min.

[0037] 3. In vitro simulation of intestinal fluid digestion

[0038] The pH of the digestive gastric juice was adjusted to the range of 6.5–7.0. 18 mL of a pancreatic-cholesterol enzyme mixture (mixed at a ratio of 0.2 mg / mL pancreatic enzyme and 1.2 mg / mL bile enzyme, with a buffer solution of 0.1 mol / L sodium bicarbonate) was added. The mixture was then incubated at 37 °C with shaking at 100 r / min. Samples were analyzed at 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h. The gastrointestinal release rate is shown in Table 4.

[0039] Table 1 Active ingredient content in fermentation broth

[0040]

[0041] Table 2 Active ingredient content of tartary buckwheat extraction concentrate

[0042]

[0043] Table 3 Tartary buckwheat active ingredient embedding rate

[0044]

[0045] Table 4 Gastrointestinal release rate of tartary buckwheat active ingredient sustained-release composite system

[0046]

[0047] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A method for preparing high bioavailability tartary buckwheat flavonoids and phenolic products, characterized in that, Comprising the following steps: (1) tartary buckwheat powder fermentation: tartary buckwheat powder is dried and crushed, then inoculated with bacillus subtilis seed liquid for fermentation to obtain tartary buckwheat fermentation liquor; the inoculation amount of bacillus subtilis seed liquid is 5%, and the fermentation conditions are 37 ℃, 150 r / min for 24 h; (2) preparation of concentrated solution: tartary buckwheat fermentation liquor is extracted at a temperature of 90 ℃ for 2 h, with an ethanol volume concentration of 80% and a solid-liquid ratio of 1:3, and then concentrated by rotary evaporation to obtain tartary buckwheat extract concentrate; (3) preparation of tartary buckwheat extract-β-cyclodextrin complex: tartary buckwheat extract concentrate is added dropwise into β-cyclodextrin solution at a mass ratio of 1:4, the mass fraction of β-cyclodextrin solution is 0.5%, and magnetic stirring is carried out at 40 ℃ for 20 min, so that the active ingredients of tartary buckwheat are embedded in the hydrophobic gap of β-cyclodextrin, and tartary buckwheat extract-β-cyclodextrin complex is obtained; (4) preparation of external emulsion: whey protein and gum arabic are mixed at a mass ratio of 1:3 to prepare an external emulsion; (5) slow-release system: tartary buckwheat extract-β-cyclodextrin complex is added to the external emulsion, and stirred at 200 r / min under the conditions of pH 4 and temperature 45℃ for 20 min; pectin is added, and stirred at 200 r / min under the conditions of pH 4 and temperature 45℃ for 30 min, and then stored in a refrigerator at 4℃ for 24 h; the mass ratio of tartary buckwheat extract-β-cyclodextrin complex, external emulsion and pectin is 1:10:5, and tartary buckwheat extract active ingredient slow-release system is obtained by freeze-drying.

2. A method of preparing high bioavailability tartary buckwheat flavonoids and phenolic product according to claim 1, characterized in that, The preparation method of the seed liquid is as follows: bacillus subtilis is inoculated into nutrient broth solid culture medium, activated and cultured at 37 ℃ for 24 h, inoculated into nutrient broth culture medium, and cultured at 37 ℃, 150 r / min for 72 h as seed liquid.

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