Preparation method of cordyceps militaris extract and application of cordyceps militaris extract in nitrite adsorption products

Abstract

The invention discloses a preparation method of a cordyceps militaris extract and application of the cordyceps militaris extract in a nitrite adsorption product. Cordyceps militaris sporocarp powder is added into water, heating and mixing are conducted, alpha-amylase, protease and amyloglucosidase are added for enzymolysis, suction filtration is conducted to collect filtrate, precipitate is washed, then the filtrate is collected and concentrated, and a concentrated solution is obtained; and adding ethanol, standing and precipitating, centrifugally collecting the precipitate, adding water for redissolving, and performing vacuum freeze drying to obtain the cordyceps militaris soluble dietary fiber extract. According to the method, the soluble dietary fiber in the cordyceps militaris is extracted by adopting an enzyme method, the extraction rate is high, the concentration of an extracting solution influences the activity of the prepared soluble dietary fiber, and the soluble dietary fiber obtained by the method has more excellent nitrite adsorption activity and has a good market application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of dietary fiber, and specifically relates to a preparation method of cordyceps militaris extract and its application in products for adsorbing nitrite. Background Art

[0002] Dietary fiber is a special nutrient, specifically referring to polysaccharide substances in carbohydrates that cannot be decomposed by human digestive enzymes. Cellulose, hemicellulose, lignin, etc. all belong to dietary fiber, and there are currently hundreds of them.

[0003] Dietary Fiber (DF) also belongs to dietary fiber. Since it cannot directly provide nutrition for the human body and cannot produce energy, it was once regarded as a "non-nutrient substance" and has not been given enough attention for a long time. With the in-depth research of the nutrition field, dietary fiber has been recognized as the seventh nutrient, ranking alongside the traditional six nutrients of protein, fat, carbohydrates, vitamins, minerals, and water. DF is divided into soluble dietary fiber (SDF) and insoluble dietary fiber (IDF). SDF can dissolve in water to form a colloid or viscous solution, which helps to reduce cholesterol and blood sugar levels in the blood and is beneficial to cardiovascular health. Summary of the Invention

[0004] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments.

[0005] As one aspect of the present invention, the present invention provides a preparation method of cordyceps militaris extract, which is characterized in that: adding cordyceps militaris fruiting body powder into water, heating and mixing, adding α-amylase, protease and amyloglucosidase for enzymatic hydrolysis, filtering to collect the filtrate, washing the precipitate and then collecting the filtrate, concentrating to obtain a concentrated solution, adding ethanol and standing for precipitation, centrifuging to collect the precipitate, redissolving with water, and vacuum freeze-drying to obtain cordyceps militaris soluble dietary fiber extract.

[0006] As a preferred scheme of the preparation method of cordyceps militaris extract described in the present invention: when adding cordyceps militaris fruiting body powder into water, the material-liquid ratio is 1:30 - 70.

[0007] As a preferred scheme of the preparation method of cordyceps militaris extract described in the present invention: when adding cordyceps militaris fruiting body powder into water, the material-liquid ratio is 1:40 - 50.

[0008] As a preferred scheme of the preparation method of cordyceps militaris extract described in the present invention: the heating and mixing is carried out at 50 - 60 °C for 1 - 2 h.

[0009] As a preferred embodiment of the preparation method of the Cordyceps militaris extract of the present invention: adding α-amylase, protease and amyloglucosidase for enzymatic hydrolysis includes adding α-amylase for enzymatic hydrolysis at pH = 6.5 and 95 - 100 °C for 30 - 40 min, cooling to 50 - 60 °C, adding protease for enzymatic hydrolysis for 30 - 40 min, adjusting the pH = 4.5, and adding amyloglucosidase for enzymatic hydrolysis at 55 - 60 °C for 30 - 40 min.

[0010] As a preferred embodiment of the preparation method of the Cordyceps militaris extract of the present invention: the concentration to obtain a concentrated solution, and the mass concentration of Cordyceps militaris fruiting body powder in the concentrated solution is 5.5 - 7%.

[0011] As a preferred embodiment of the preparation method of the Cordyceps militaris extract of the present invention: adding ethanol and allowing it to stand for precipitation, and the ethanol concentration in the concentrated solution after adding ethanol is 76%.

[0012] As a preferred embodiment of the preparation method of the Cordyceps militaris extract of the present invention: adding ethanol and allowing it to stand for precipitation is carried out at 2 - 8 °C for 8 - 14 h.

[0013] The beneficial effects of the present invention: The present invention uses an enzymatic method to extract soluble dietary fiber from Cordyceps militaris, with a high extraction rate, and it is found that the concentration of the extraction solution affects the activity of the prepared soluble dietary fiber. The soluble dietary fiber obtained by the method of the present invention has more excellent nitrite adsorption activity and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Among them:

[0015] Figure 1 is the influence of different extraction methods on the yield of soluble dietary fiber from Cordyceps militaris.

[0016] Figure 2 is the influence of different extraction methods on the ability of soluble dietary fiber from Cordyceps militaris to adsorb nitrite in a pH 7 environment.

[0017] Figure 3 is the influence of different concentration ratios on the extraction rate of dietary fiber from Cordyceps militaris by enzymatic extraction.

[0018] Figure 4 is the influence of different concentration ratios on the ability of dietary fiber from Cordyceps militaris extracted by enzymatic extraction to adsorb nitrite in a pH 7 environment. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention will be given in conjunction with specific embodiments.

[0020] 1. Experimental method:

[0021] 1.1 Pretreatment of Cordyceps militaris samples:

[0022] Wash the surface of Cordyceps militaris fruiting bodies with clear water, place them in an oven and dry to a constant weight, crush them with a pulverizer and pass through a 60-mesh sieve to obtain Cordyceps militaris fruiting body powder.

[0023] 1.2 Extraction method of soluble dietary fiber:

[0024] 1.2.1 Alkaline extraction method of soluble dietary fiber:

[0025] Weigh 3 g of the sieved Cordyceps militaris fruiting body powder, add 5wt% NaOH or 5wt% Na 2 CO 3 aqueous solution according to a solid-liquid ratio of 1:40, stir magnetically at 40 °C for 2 h, centrifuge to collect the supernatant, add 1 mol / L hydrochloric acid to neutralize the pH to 7.0, concentrate to one-fourth of the volume, add 4 times the volume of 95% ethanol for alcohol precipitation to make the final ethanol concentration reach 76%, stand at 4 °C for 12 h, centrifuge at 300 rpm for 10 min to collect the precipitate, redissolve with 100 mL of water (stir magnetically at 60 °C for 1 h), rotary evaporate at 55 °C until it dissolves into a block, and obtain the crude extract of soluble dietary fiber after vacuum freeze-drying.

[0026] 1.2.2 Enzymatic extraction method of soluble dietary fiber:

[0027] Weigh 3 g of the sieved Cordyceps militaris fruiting body powder, add distilled water according to a solid-liquid ratio (mass ratio) of 1:40, stir magnetically at 50 °C for 1 h, adjust the pH to 6.5, add 250 μL of 4×10 4 u / g α-amylase (CAS: 9000-85-5), oscillate at 95 °C for 40 min, cool down to 60 °C, add 500 μL of 1.5×10 5 u / g neutral protease, oscillate for 30 min, adjust the pH to 4.5, add 500 μL of 1×10 5Glucoamylase (CAS: 9032-08-0), oscillate at 60 °C for 35 min. After enzymatic hydrolysis, filter by suction to collect the filtrate, wash the precipitate twice with 50 mL of hot water at 70 °C and collect the filtrate. Concentrate the collected filtrate to one-fourth of its volume by water bath heating, add 4 volumes of 95% ethanol for alcohol precipitation to make the final concentration of ethanol in the alcohol precipitation system reach 76%, let stand at 4 °C for 12 h, centrifuge at 300 rpm for 10 min to collect the precipitate, redissolve with 100 mL of water (magnetic stirring at 60 °C for 1 h), rotary evaporate at 55 °C until it dissolves into lumps, and obtain the crude extract of soluble dietary fiber after vacuum freeze-drying.

[0028] 1.3 Concentration of dietary fiber extract:

[0029] Weigh 3 g of the powdered fruiting bodies of Cordyceps militaris after sieving, add distilled water according to a solid-liquid ratio of 1:40, stir magnetically at 50 °C for 1 h, adjust the pH to 6.5, add 250 μL of 4×10 4 U / g α-amylase (CAS: 9000-85-5), oscillate at 95 °C for 40 min, cool to 60 °C, add 500 μL of 1.5×10 5 U / g neutral protease, oscillate for 30 min, adjust the pH to 4.5, add 500 μL of 1×10 5 U / g glucoamylase (CAS: 9032-08-0), oscillate at 60 °C for 35 min. After enzymatic hydrolysis, filter by suction to collect the filtrate, wash the precipitate twice with 50 mL of hot water at 70 °C and collect the filtrate. Concentrate the filtrate to 1 / 3 volume, 2 / 9 volume or 1 / 6 volume of the original filtrate respectively to obtain the concentrated solution. Then add 4 volumes of 95% ethanol for alcohol precipitation to make the final concentration of ethanol in the alcohol precipitation system reach 76%, let stand at 4 °C for 12 h, centrifuge at 300 rpm for 10 min to collect the precipitate, redissolve with 100 mL of water (magnetic stirring at 60 °C for 1 h), rotary evaporate at 55 °C until it dissolves into lumps, and obtain the crude extract of soluble dietary fiber after vacuum freeze-drying.

[0030] 1.4 Nitrite adsorption experiment:

[0031] Prepare sodium nitrite standard solutions with different concentration gradients according to Table 1, add 2 mL of 4 mg / mL sulfanilic acid solution, mix well and let stand in the dark for 5 min, then add 1 mL of 2 mg / mL N-(1-Naphthyl)ethylenediamine dihydrochloride solution, add distilled water to make up to 10 mL with 6 mL and mix well. After standing in the dark for 15 min, measure the absorbance at 538 nm and draw the standard curve.

[0032] Table 1 Preparation table of sodium nitrite standard solution

[0033]

[0034] Adjust the 20 μg / mL sodium nitrite solution to pH = 7 with 1 mol / L sodium hydroxide. Take 100 mg of each soluble dietary fiber crude extract sample and mix it with 25 mL of sodium nitrite at pH = 7. The working concentration of the sample is 4 mg / mL. Shake it at 37 °C and 120 r / min for 2 h. After centrifuging at 4000 r / min for 20 min at room temperature, take 1 mL of the supernatant, add 2 mL of 4 mg / mL sulfanilic acid solution, mix well and let it stand in the dark for 5 min. Then add 1 mL of 2 mg / mL N-(1-Naphthyl)ethylenediamine dihydrochloride solution, and make up the volume to 10 mL with 6 mL of distilled water and mix well. After standing in the dark for 15 min, measure the absorbance at 538 nm. The adsorption capacity of nitrite was measured according to the following formula:

[0035]

[0036] 1.5 Determination of the yield of soluble dietary fiber:

[0037] Weigh the mass of the soluble dietary fiber of Cordyceps militaris after freeze-drying, and calculate the yield of soluble dietary fiber according to the following formula:

[0038]

[0039] 2. Experimental results:

[0040] 2.1 Effects of four extraction methods on the yield of dietary fiber from Cordyceps militaris:

[0041] As Figure 1 shown, Figure 1 are the effects of different extraction methods on the yield of soluble dietary fiber from Cordyceps militaris (*P < 0.05 vs enzyme extraction group). The yields of soluble dietary fiber from Cordyceps militaris by enzyme extraction, NaOH extraction and Na 2 CO 3 extraction were 13.22%, 5.87% and 3.160% respectively. Among them, the yield of enzyme extraction was the highest, which was 125.20% higher than that of NaOH extraction and 318.48% higher than that of Na 2 CO 3 extraction respectively.

[0042] 2.2 Effects of four extraction methods on the ability of dietary fiber from Cordyceps militaris to adsorb nitrite:

[0043] The reaction system at pH = 7 simulates the pH environment in the intestine. Therefore, it explores the adsorption ability of Cordyceps militaris samples to nitrite in the intestinal environment. Figure 2 are the effects of different extraction methods on the ability of soluble dietary fiber from Cordyceps militaris to adsorb nitrite in the pH 7 environment (*P < 0.05 vs enzyme extraction group). The enzyme extraction, NaOH extraction and Na 2 CO 3The adsorption values of Cordyceps militaris soluble dietary fiber obtained by extraction method for nitrite under pH 7 condition were 326.78 μg / g, 46.54 μg / g and 152.93 μg / g respectively. Among them, the adsorption rate of the enzymatic extraction method was the highest, which was 602.15% higher than that of the NaOH extraction method and 113.68% higher than that of the extraction method 1. 2 CO 3 extraction method 1.

[0044] 2.3 Effect of different concentration ratios on the extraction rate of Cordyceps militaris dietary fiber by enzymatic method:

[0045] As Figure 3 shown, the extraction solutions were concentrated at ratios of 3∶1, 4.5∶1 or 6∶1 (i.e., concentrated to 1 / 3 volume, 2 / 9 volume or 1 / 6 volume of the filtrate respectively), and the yields of soluble dietary fiber were 10.05%, 15.42% and 12.12% respectively. Concentrating the extraction solution at 4.5∶1, the yield of soluble dietary fiber was 53.43% and 27.23% higher than that at 3∶1 and 6∶1 respectively. Figure 3 Effect of different concentration ratios on the extraction rate of Cordyceps militaris dietary fiber by enzymatic method (*P < 0.05 vs 4.5∶1 volume concentration group).

[0046] 2.4 Effect of different concentration ratios on the ability of Cordyceps militaris dietary fiber extracted by enzymatic method to adsorb nitrite in pH 7 environment:

[0047] Figure 4 Effect of different concentration ratios on the ability of Cordyceps militaris dietary fiber extracted by enzymatic method to adsorb nitrite in pH 7 environment (*P < 0.05 vs 4.5∶1 volume concentration group). As Figure 4As shown, when the extraction solution is concentrated at a ratio of 3:1, 4.5:1 or 6:1 (i.e., concentrated to 1 / 3 volume, 2 / 9 volume or 1 / 6 volume of the filtrate respectively), the adsorption values of soluble dietary fiber for nitrite under the condition of pH = 7 are 250.11, 342.63 and 315.77 μg / g respectively. Among them, the nitrite adsorption effect of the 4.5:1 concentration ratio is the best, which is 36.99% higher than that of the 3:1 concentration method and 8.51% higher than that of the 6:1 concentration method respectively, and the results have significant differences. This may be because the intermolecular voids are significantly related to the concentration of the extraction solution. Therefore, at a lower extraction solution concentration, the molecules in the solution cannot form a network structure, while at a higher extraction solution concentration, the intermolecular voids are too small, and the formed network structure is relatively tight, resulting in certain differences in the molecular structure of the SDF samples obtained from extraction solutions with different concentrations using ethanol. There is a strong correlation between the adsorption capacity of the sample for nitrite and the molecular structure of the SDF sample. The present invention discovers that the extraction solution concentrated at a ratio of 4.5:1 can obtain an SDF sample through ethanol precipitation with 76% ethanol, which can form a better network-like molecular structure and exhibit better nitrite adsorption capacity.

[0048] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A method for preparing a Cordyceps militaris extract, characterized in that: The Cordyceps militaris fruiting body powder is added into water, heated and mixed, α-amylase, protease and amyloglucosidase are added for enzymolysis, the filtrate is collected by suction filtration, the precipitate is washed and the filtrate is collected, concentrated to obtain a concentrated solution, ethanol is added to stand and precipitate, the precipitate is collected by centrifugation, water is added for redissolution, and vacuum freeze-drying is performed to obtain a Cordyceps militaris soluble dietary fiber extract.

2. The method for preparing the Cordyceps militaris extract according to claim 1, characterized in that: The Cordyceps militaris fruiting body powder is added into water, and the solid-liquid ratio is 1:30-70.

3. The method for preparing the Cordyceps militaris extract according to claim 1, characterized in that: The Cordyceps militaris fruiting body powder is added into water, and the solid-liquid ratio is 1:40-50.

4. The method for preparing the Cordyceps militaris extract according to any one of claims 1 to 3, characterized in that: The heating and mixing is heating and mixing at 50-60° C. for 1-2 hours.

5. The method for preparing the Cordyceps militaris extract according to any one of claims 1 to 3, characterized in that: The method of adding α-amylase, protease and amyloglucosidase for enzymolysis comprises adding α-amylase for enzymolysis for 30 to 40 minutes at pH=6.5 and 95 to 100° C., cooling to 50 to 60° C., adding protease for enzymolysis for 30 to 40 minutes, adjusting pH=4.5, and adding amyloglucosidase for enzymolysis for 30 to 40 minutes at 55 to 60° C.

6. The method for preparing the Cordyceps militaris extract according to any one of claims 1 to 3, characterized in that: The concentration obtains a concentrated solution, in which the mass concentration of the Cordyceps militaris fruiting body powder is 5.5-7%.

7. The method for preparing the Cordyceps militaris extract according to any one of claims 1 to 3, characterized in that: The ethanol was added and allowed to stand for precipitation, and the ethanol concentration in the concentrated solution after the addition of ethanol was 76%.

8. The method for preparing the Cordyceps militaris extract according to any one of claims 1 to 3, characterized in that: The step of adding ethanol and allowing to precipitate is allowing to stand at 2-8° C. for 8-14 hours.

9. The Cordyceps militaris extract prepared by the method for preparing the Cordyceps militaris extract according to claim 1.

10. Use of the Cordyceps militaris extract prepared by the method for preparing the Cordyceps militaris extract according to claim 1 in adsorbing nitrite.

Images