Dendrobium officinale soluble dietary fiber, and extraction method and application thereof

Abstract

The application belongs to the technical field of traditional Chinese medicine separation and extraction, and particularly relates to a Dendrobium officinale soluble dietary fiber as well as an extraction method and application thereof. The extraction method comprises the following steps: sequentially using alkaline protease, alpha-amylase and cellulase for enzyme hydrolysis on Dendrobium officinale to obtain an enzyme hydrolysate, and after centrifugation, the supernatant is concentrated and alcohol precipitated to obtain a precipitate; and the precipitate contains the Dendrobium officinale soluble dietary fiber. The method can extract the soluble dietary fiber (SDF) from the Dendrobium officinale which is a homology of medicine and food, and the yield of the SDF is as high as 58.253±2.654%, and the utilization value of the Dendrobium officinale processing by-products is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine separation and extraction, and particularly relates to a Dendrobium officinale soluble dietary fiber as well as an extraction method and application thereof. BACKGROUND

[0002] Dendrobium officinale Kimura et Migo Dendrobium officinale ) is a perennial plant belonging to Orchidaceae and Dendrobium, and is located at the top of the nine Chinese magical grasses. The main components of Dendrobium officinale Kimura et Migo are Dendrobium polysaccharide, Dendrobium alkaloid and total amino acid, which can lower blood pressure, lower blood sugar, resist oxidation and improve human immunity. According to traditional Chinese medicine, Dendrobium officinale Kimura et Migo can nourish the stomach, produce saliva, clear heat and nourish yin, and is often used for treating unrelieved heat, heat disease damaging body fluid and dry mouth.

[0003] Dendrobium polysaccharide, as a main bioactive component, is a material basis for exerting important nutritional and healthy effects. Dietary fiber also belongs to polysaccharide, is a carbohydrate polymer composed of 3 or more monomers, cannot be hydrolyzed by digestive enzymes in the small intestine of the human body, and is a substance beneficial to human health extracted or synthesized from plants. Dietary fiber can be divided into soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) according to water solubility. Compared with IDF, SDF has better solubility and viscosity characteristics, can exert greater potential in the fields of health care and food industry, and has more prospects.

[0004] At present, there are many extraction methods of Dendrobium polysaccharide in the prior art, and there are few reports on the extraction method of soluble dietary fiber of Dendrobium officinale Kimura et Migo. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a Dendrobium officinale Kimura et Migo soluble dietary fiber as well as an extraction method and application thereof. The extraction method of the Dendrobium officinale Kimura et Migo soluble dietary fiber provided by the present application has a larger yield of soluble dietary fiber, and improves the utilization value of Dendrobium officinale Kimura et Migo processing by-products.

[0006] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0007] The present application provides an extraction method of a Dendrobium officinale Kimura et Migo soluble dietary fiber, comprising the following steps: sequentially using alkaline protease, alpha-amylase and cellulase for enzymolysis of Dendrobium officinale Kimura et Migo, and after centrifugation, the supernatant is concentrated and alcohol precipitated to obtain a precipitate; the precipitate contains the Dendrobium officinale Kimura et Migo soluble dietary fiber.

[0008] Preferably, the added mass of the alkaline protease is 7% to 9% of the total mass of Dendrobium officinale Kimura et Migo;

[0009] The added mass of the alpha-amylase enzyme is 2-11% of the total mass of the Dendrobium candidum;

[0010] The added mass of the cellulase enzyme is 0.5-2.5% of the total mass of the Dendrobium candidum.

[0011] Preferably, during the enzymolysis of the alkaline protease, the pH of the enzymolysis is 8.0-11.0, the temperature of the enzymolysis is 45-85 DEG C, and the time of the enzymolysis is 55-75 min.

[0012] Preferably, the alpha-amylase comprises a high-temperature-resistant alpha-amylase.

[0013] Preferably, during the enzymolysis of the high-temperature-resistant alpha-amylase, the pH of the enzymolysis is 4.5-6.5, the temperature of the enzymolysis is 60-100 DEG C, and the time of the enzymolysis is 30-90 min.

[0014] Preferably, during the enzymolysis of the cellulase, ultrasonic treatment is assisted.

[0015] Preferably, during the enzymolysis of the cellulase, the pH of the enzymolysis is 4.5-6.5, the temperature of the ultrasonic treatment is 40-80 DEG C, and the time of the ultrasonic treatment is 0.5-2.5 h.

[0016] Preferably, before the enzymolysis by the alkaline protease, the Dendrobium candidum is mixed with water, and the mass-volume ratio of the Dendrobium candidum to water is 0.7-1.3 g:20-50 mL.

[0017] The application provides the Dendrobium candidum soluble dietary fiber prepared by the above extraction method.

[0018] The application also provides application of the above Dendrobium candidum soluble dietary fiber in preparation of medicines with anti-aging activity.

[0019] Beneficial effects: the application provides an extraction method of Dendrobium candidum soluble dietary fiber, the Dendrobium candidum is sequentially subjected to enzymolysis by alkaline protease, alpha-amylase and cellulase, and the soluble dietary fiber (SDF) is extracted from the plant Dendrobium candidum which is homologous in medicine and food, and the yield of the SDF is as high as 58.253±2.654%, and the utilization value of the processing by-product of the Dendrobium candidum is improved.

[0020] The application provides the Dendrobium candidum soluble dietary fiber prepared by the above method, and the dietary fiber has strong anti-aging activity, good physical and chemical properties and functional properties.

[0021] The application also provides application of the above Dendrobium candidum soluble dietary fiber in preparation of medicines with anti-aging activity, and the medicines prepared by using the dietary fiber with anti-aging activity also have anti-aging activity, and the application range is wide. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure is the effect diagram of AB (liquid-solid ratio and cellulase dosage) interaction on the yield of soluble dietary fiber; wherein, the left graph is a surface graph, and the right graph is a contour graph, the same below;

[0023] Figure 2 Figure is the effect diagram of AC (liquid-solid ratio and cellulase hydrolysis ultrasonic temperature) interaction on the yield of soluble dietary fiber;

[0024] Figure 3 Figure is the effect diagram of AD (liquid-solid ratio and cellulase hydrolysis pH) interaction on the yield of soluble dietary fiber;

[0025] Figure 4 Figure is the effect diagram of BC (cellulase dosage and ultrasonic temperature) interaction on the yield of soluble dietary fiber;

[0026] Figure 5 Figure is the effect diagram of BD (cellulase dosage and pH) interaction on the yield of soluble dietary fiber;

[0027] Figure 6 Figure is the effect diagram of CD (cellulase hydrolysis ultrasonic temperature and pH) interaction on the yield of soluble dietary fiber;

[0028] Figure 7 Figure is the effect diagram of Dendrobium candidum soluble dietary fiber on the life of nematodes;

[0029] Figure 8 Figure is the survival curve diagram of nematodes under hydrogen peroxide induction;

[0030] Figure 9 Figure is the determination result diagram of antioxidant enzyme activity in nematode homogenate supernatant; wherein, A is GSH content, B is CAT activity, C is SOD activity, and D is MDA content. DETAILED DESCRIPTION

[0031] The application provides an extraction method of Dendrobium candidum soluble dietary fiber, comprising the following steps: sequentially using alkaline protease, alpha-amylase and cellulase for enzyme hydrolysis of Dendrobium candidum to obtain an enzyme hydrolysis liquid; after centrifugation of the enzyme hydrolysis liquid, the supernatant is concentrated and alcohol precipitated to obtain a precipitate; the precipitate contains the Dendrobium candidum soluble dietary fiber.

[0032] Unless otherwise specified, the application does not have special requirements for the preparation raw materials, and commercially available goods known to those skilled in the art can be used.

[0033] The application preferably further comprises pretreatment of Dendrobium candidum before alkaline protease enzymolysis, and the pretreatment preferably comprises: first drying and crushing Dendrobium candidum stems to obtain Dendrobium candidum powder; and soaking the Dendrobium candidum powder in petroleum ether, centrifuging and second drying to obtain defatted Dendrobium candidum after treatment. The first drying temperature is preferably 40-55 DEG C, more preferably 45 DEG C; and the first drying time is preferably 7-15 days, more preferably 10 days. The crushing preferably further comprises sieving, and the sieve is preferably a No. 3-5 sieve (50-80 mesh), more preferably a No. 4 sieve (60 mesh). When the Dendrobium candidum powder is soaked in petroleum ether, the mass-volume ratio of the Dendrobium candidum powder to petroleum ether is preferably 0.5-1.5 g:10-30 mL, more preferably 1 g:15 mL, and the petroleum ether plays a role in defatting and decolorizing. The centrifugation speed is preferably 5000-10000 r / min, more preferably 8000 r / min; and the centrifugation time is preferably 5-15 min, more preferably 10 min, and the petroleum ether is recovered after centrifugation. The application preferably second dries the precipitate after centrifugation, and the second drying temperature is preferably 40-60 DEG C, more preferably 50 DEG C; and the second drying time is preferably 24-72 h, more preferably 48 h.

[0034] The application preferably mixes the Dendrobium candidum after treatment with water to obtain a mixed solution; and the mass-volume ratio of the Dendrobium candidum to water is 0.7-1.3 g:20-50 mL, more preferably 0.8-1.2 g:30-45 mL, and most preferably 1 g:40 mL.

[0035] The application preferably adjusts the pH of the mixed solution and then adds alkaline protease for enzymolysis. The addition amount of the alkaline protease is preferably 7%-9% of the total mass of the Dendrobium candidum, more preferably 7.5%-8.5%, and most preferably 8%. In the examples of the application, the enzyme activity of the alkaline protease is preferably 200 U / mg. When the alkaline protease is used for enzymolysis, the pH of the enzymolysis is preferably 8.0-11.0, more preferably 8.5-10.0, and most preferably 9.0, and sodium hydroxide is preferably used for adjusting the pH; the temperature of the enzymolysis is preferably 45-85 DEG C, more preferably 45-65 DEG C or 80-85 DEG C, and most preferably 65 DEG C; and the time of the enzymolysis is preferably 55-75 min, more preferably 55-60 min or 60-75 min, and most preferably 55 min, and a water bath is preferably used for maintaining the temperature during enzymolysis. The application uses alkaline protease for enzymolysis to remove the protein of Dendrobium candidum, which is beneficial to the dissolution of soluble dietary fiber.

[0036] The solution after the alkaline protease enzymolysis is preferably adjusted in pH, and then alpha-amylase is added for enzymolysis. The addition amount of the alpha-amylase is preferably 2% to 11% of the total mass of the dendrobium officinale, more preferably 8% to 10.5%, and most preferably 10%. The alpha-amylase preferably includes a high-temperature-resistant alpha-amylase. In the embodiment of the present application, the enzyme activity of the high-temperature-resistant alpha-amylase is preferably greater than or equal to 5000 U / g. In the enzymolysis of the high-temperature-resistant alpha-amylase, the pH is preferably 4.5 to 6.5, more preferably 4.6 to 5.5, and most preferably 4.8. Glacial acetic acid is preferably used for adjusting the pH. The temperature of the enzymolysis is preferably 60 to 100 DEG C, more preferably 80 to 95 DEG C, and most preferably 90 DEG C. The time of the enzymolysis is preferably 30 to 90 min, more preferably 45 to 85 min, and most preferably 75 min. The alpha-amylase enzymolysis is used to remove the starch of the dendrobium officinale, which is beneficial to the dissolution of the soluble dietary fiber.

[0037] The solution after the alpha-amylase enzymolysis is preferably inactivated. The temperature of the inactivation is preferably 90 to 110 DEG C, and more preferably 100 DEG C. The time of the inactivation is preferably 5 to 15 min, and more preferably 10 min. The cellulase is preferably added to the inactivated solution, and then the enzymolysis is performed after adjusting the pH. The addition amount of the cellulase is preferably 0.5% to 2.5% of the total mass of the dendrobium officinale, more preferably 1% to 2%, and most preferably 1.5%. The pH of the enzymolysis is preferably 4.5 to 6.5, more preferably 4.8 to 6.0, and most preferably 5.0. In the embodiment of the present application, the enzyme activity of the cellulase is preferably 100000 U / g. In the cellulase enzymolysis, ultrasonic treatment is preferably assisted. The temperature of the ultrasonic treatment is preferably 40 to 80 DEG C, more preferably 60 to 75 DEG C, and most preferably 70 DEG C. The time of the ultrasonic treatment is preferably 0.5 to 2.5 h, more preferably 1 to 2 h, and most preferably 1 h. In the cellulase enzymolysis, the temperature and the time of the enzymolysis are consistent with the ultrasonic conditions.

[0038] The alkaline protease, the alpha-amylase and the cellulase are sequentially used for the enzymolysis of the dendrobium officinale. If the starch is removed at high temperature first, the protein in the sample will be denatured, and then it will be difficult to remove. The alkaline protease is used to remove the protein in the sample first, and then the alpha-amylase is used to remove the starch in the sample. Finally, the cellulase is used to decompose the cellulose in the sample, so that the insoluble cellulose part is converted into soluble cellulose, thereby the content of the soluble dietary fiber can be significantly improved.

[0039] The cellulase enzyme is decomposed, and the supernatant is obtained after centrifugation of the decomposed solution. Preferably, the supernatant is inactivated before centrifugation, and the inactivation conditions are the same as those of the alpha-amylase enzyme decomposed solution. Preferably, the inactivated solution is centrifuged at a speed of 5000-10000 r / min, more preferably 8000 r / min, and the centrifugation time is preferably 5-10 min, more preferably 6 min. The supernatant after centrifugation is obtained.

[0040] Preferably, the supernatant is concentrated and alcohol precipitated to obtain a precipitate. The precipitate obtained is washed with hot water until neutral, and the temperature of the hot water is preferably 60-80°C, more preferably 70°C. The washing solution after washing is combined with the supernatant. The combined solution is concentrated under reduced pressure to obtain a concentrated solution. The concentration temperature is preferably 40-60°C, more preferably 55°C, the concentration pressure is preferably 0.04-0.1 Pa, more preferably 0.08 Pa, and the residual concentrated solution is 50-100 mL. The concentrated solution is mixed with anhydrous ethanol for alcohol precipitation, and the mass ratio of the concentrated solution to anhydrous ethanol is preferably 0.5-1.5:2-6, more preferably 1:4. The alcohol precipitation temperature is 2-6°C, more preferably 4°C, and the alcohol precipitation time is preferably 10-14 h, more preferably 12 h. The alcohol precipitated solution is centrifuged, and the centrifugation conditions are the same as those of the inactivated solution.

[0041] The precipitate obtained by centrifugation contains the Dendrobium officinale soluble dietary fiber, and the precipitate is dried to obtain the Dendrobium officinale soluble dietary fiber. The drying temperature is preferably 40-60°C, more preferably 50°C, and the drying time is preferably 3-7 d, more preferably 5 d. The Dendrobium officinale soluble dietary fiber is obtained after drying.

[0042] The present application provides a Dendrobium officinale soluble dietary fiber prepared by the above extraction method, which has strong anti-aging activity, good physicochemical properties and functional properties.

[0043] The present application also provides the use of the above Dendrobium officinale soluble dietary fiber in the preparation of a drug with anti-aging activity. The drug prepared by using the dietary fiber with anti-aging activity also has anti-aging activity and a wide range of applications.

[0044] In order to further illustrate the present application, a Dendrobium officinale soluble dietary fiber, its extraction method and application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present application.

[0045] In the embodiments of the present application, each group of conditional screening tests is independently and simultaneously performed.

[0046] Example 1

[0047] Pre-treatment: Dendrobium candidum stems were dried at 45℃ for 10 days, crushed, and passed through a No. 4 sieve (60 mesh) to obtain Dendrobium powder for use. A certain amount of Dendrobium powder was weighed, 1 g: 15 mL of petroleum ether was added for soaking for 10 h, centrifuged at 8000 r / min for 10 min, the petroleum ether was recovered, and the powder was dried at 50℃ for 48 h to obtain defatted Dendrobium powder for use, and the same batch of pre-treated samples was used in the subsequent examples.

[0048] 1 g of the above defatted Dendrobium powder was precisely weighed and placed in a 50 mL centrifuge tube, 1:30 (g / mL) of distilled water was added, 2 mol / L sodium hydroxide was used to adjust the pH to 9.0, 0.08 g of alkaline protease was added, and the mixture was placed in a 55℃ water bath for 55 min, 3 mol / L glacial acetic acid was used to adjust the pH to 4.8, 0.08 g of high-temperature-resistant α-amylase was added, and the mixture was placed in a 90℃ water bath for 45 min, then inactivated at 100℃ for 10 min, 0.01 g of cellulase was added, the pH was adjusted to 5.0, and the mixture was subjected to ultrasonic enzymolysis at 50℃ for 1.5 h, then inactivated at 100℃ for 10 min, centrifuged, and the supernatant was obtained, the precipitate was washed with 70℃ hot water until it was neutral, the washing liquid was combined with the supernatant, and the mixture was concentrated under reduced pressure to 50 mL, 4 times the mass of anhydrous ethanol was added to the concentrated liquid, and the mixture was alcohol-precipitated at 4℃ for 12 h, then centrifuged, and the precipitate was dried at 50℃ to obtain Dendrobium candidum soluble cellulose.

[0049] Example 2

[0050] The procedure of Example 1 was followed, except that the alkaline protease enzymolysis was performed for 65 min.

[0051] Example 3

[0052] The procedure of Example 1 was followed, except that the alkaline protease enzymolysis was performed for 75 min.

[0053] Comparative Example 1

[0054] The procedure of Example 1 was followed, except that the alkaline protease enzymolysis was performed for 30 min.

[0055] Comparative Example 2

[0056] The procedure of Example 1 was followed, except that the alkaline protease enzymolysis was performed for 45 min.

[0057] Test Example 1: Alkaline protease enzymolysis time screening

[0058] The weights of the samples of Examples 1-3 and Comparative Examples 1-2 were measured, and the soluble dietary fiber (SDF) yield was calculated according to the following formula, and the results are shown in Table 1.

[0059] SDF yield (%) = SDF content (g) ÷ defatted dendrobium nobile powder (g) x 100%

[0060] Table 1: Alkaline protease enzymolysis time screening results

[0061]

[0062] As can be seen from Table 1, the SDF yield of Examples 1-3 is significantly higher than that of Comparative Examples 1-2, and it can be seen that the extraction effect is better within the alkaline protease enzymolysis time range of the examples.

[0063] Example 4

[0064] 1 g of defatted dendrobium nobile powder of Example 1 was precisely weighed into a 50 mL centrifuge tube, 1:30 (g / mL) of distilled water was added, 2 mol / L sodium hydroxide was used to adjust the pH to 9.0, 0.08 g of alkaline protease was added, and a 45°C water bath was used for 55 min, 3 mol / L glacial acetic acid was used to adjust the pH to 4.8, 0.08 g of high-temperature-resistant α-amylase was added, a 90°C water bath was used for 45 min, 100°C inactivation was performed for 10 min, 0.01 g of cellulase was added, the pH was adjusted to 5.0, 50°C ultrasonic enzymolysis was performed for 1.5 h, 100°C inactivation was performed for 10 min, 8000 r / min centrifugation was performed for 6 min, the supernatant was obtained, the precipitate was washed with 70°C hot water until it was neutral, the washing liquid and the supernatant were combined, and under the conditions of 55°C and 0.08 Pa, the combined liquid was concentrated under reduced pressure to 50 mL, 4 times the mass of anhydrous ethanol was added to the concentrated liquid, and alcohol precipitation was performed at 4°C for 12 h, centrifugation was performed, the precipitate was taken out and dried at 50°C for 5 d, and dendrobium nobile soluble cellulose was obtained.

[0065] Example 5

[0066] The method of Example 4 was used, except that during alkaline protease enzymolysis, a 55°C water bath was used.

[0067] Example 6

[0068] The method of Example 4 was used, except that during alkaline protease enzymolysis, a 65°C water bath was used.

[0069] Example 7

[0070] The method of Example 4 was used, except that during alkaline protease enzymolysis, a 75°C water bath was used.

[0071] Example 8

[0072] The method of Example 4 was used, except that during alkaline protease enzymolysis, a 85°C water bath was used.

[0073] Test Example 2: Alkaline protease enzymolysis temperature screening

[0074] The sample weight of Examples 5-8 was weighed, and the soluble dietary fiber (SDF) yield was calculated according to the manner of Test Example 1, and the results are shown in Table 2.

[0075] Table 2. Alkaline protease enzymolysis temperature screening results

[0076]

[0077] As can be seen from Table 2, the SDF yields of Examples 5-8 are not significantly different, and it can be seen that the extraction effect is good within the alkaline protease enzymolysis temperature range of the examples.

[0078] Example 9

[0079] 1 g of the defatted dendrobium nobile powder of Example 1 was precisely weighed into a 50 mL centrifuge tube, 1:30 (g / mL) of distilled water was added, 2 mol / L sodium hydroxide was used to adjust the pH to 9.0, 0.08 g of alkaline protease was added, and a 55°C water bath was used for 65 min, 3 mol / L glacial acetic acid was used to adjust the pH to 4.8, 0.02 g of high-temperature-resistant α-amylase was added, a 90°C water bath was used for 45 min, 100°C inactivation was performed for 10 min, 0.01 g of cellulase was added, the pH was adjusted to 5.0, 50°C ultrasonic enzymolysis was performed for 1.5 h, 100°C inactivation was performed for 10 min, 8000 r / min centrifugation was performed for 6 min, the supernatant was obtained, the precipitate was washed with 70°C hot water until it was neutral, the washing liquid and the supernatant were combined, under the conditions of 55°C and 0.08 Pa, the combined liquid was concentrated under reduced pressure to 50 mL, 4 times the mass of anhydrous ethanol was added to the concentrated liquid, and alcohol precipitation was performed at 4°C for 12 h, centrifugation was performed, the precipitate was dried at 50°C for 5 d, and dendrobium nobile soluble cellulose was obtained.

[0080] Example 10

[0081] The procedure of Example 9 was followed, except that 0.04 g of high-temperature-resistant α-amylase was added (i.e., the addition mass of high-temperature-resistant α-amylase is preferably 4% of the total mass of dendrobium nobile).

[0082] Example 11

[0083] The procedure of Example 9 was followed, except that 0.06 g of high-temperature-resistant α-amylase was added (i.e., the addition mass of high-temperature-resistant α-amylase is preferably 6% of the total mass of dendrobium nobile).

[0084] Example 12

[0085] The procedure of Example 9 was followed, except that 0.08 g of high-temperature-resistant α-amylase was added (i.e., the addition mass of high-temperature-resistant α-amylase is preferably 8% of the total mass of dendrobium nobile).

[0086] Example 13

[0087] The procedure of Example 9 was followed, except that 0.1 g of thermostable α-amylase was added (i.e., the added mass of thermostable α-amylase was preferably 10% of the total mass of D. candidum).

[0088] Test Example 3: α-amylase dosage screening

[0089] The sample weights of Examples 9-13 were measured, and the soluble dietary fiber (SDF) yield was calculated according to the procedure of Test Example 1, and the results are shown in Table 3.

[0090] Table 3: α-amylase dosage screening results

[0091]

[0092] As can be seen from Table 3, the SDF yields of Examples 9-13 were all high, and it can be seen that the extraction effect was good within the α-amylase dosage range of the examples.

[0093] Example 14

[0094] 1 g of defatted D. candidum powder of Example 1 was precisely weighed into a 50 mL centrifuge tube, and 1:30 (g / mL) of distilled water was added. The pH was adjusted to 9.0 using 2 mol / L sodium hydroxide, 0.08 g of alkaline protease was added, and the mixture was placed in a 55°C water bath for 65 min. The pH was adjusted to 4.8 using 3 mol / L glacial acetic acid, 0.1 g of thermostable α-amylase was added, and the mixture was placed in a 60°C water bath for 45 min. The mixture was inactivated at 100°C for 10 min, 0.01 g of cellulase was added, the pH was adjusted to 5.0, and the mixture was subjected to ultrasonic enzymatic hydrolysis at 50°C for 1.5 h. The mixture was inactivated at 100°C for 10 min, and was centrifuged at 8000 r / min for 6 min. The supernatant was obtained, and the precipitate was washed with 70°C hot water until it was neutral. The washings and the supernatant were combined, and were concentrated under reduced pressure to 50 mL at 55°C and 0.08 Pa. Anhydrous ethanol was added to the concentrated solution in an amount of 4 times the mass of the concentrated solution, and the mixture was alcohol-precipitated at 4°C for 12 h. The mixture was centrifuged, and the precipitate was dried at 50°C for 5 d to obtain D. candidum soluble cellulose.

[0095] Example 15

[0096] The procedure of Example 14 was followed, except that the α-amylase enzymatic hydrolysis was performed in a 70°C water bath.

[0097] Example 16

[0098] The procedure of Example 14 was followed, except that the α-amylase enzymatic hydrolysis was performed in an 80°C water bath.

[0099] Example 17

[0100] The procedure of Example 14 was followed, except that the α-amylase enzymatic hydrolysis was performed in a 90°C water bath.

[0101] Example 18

[0102] The procedure of Example 14 was followed, except that the α-amylase enzyme hydrolysis was performed in a 100°C water bath.

[0103] Test Example 4 α-amylase enzyme hydrolysis temperature screening

[0104] The sample weights of Examples 14-18 were measured, and the soluble dietary fiber (SDF) yield was calculated according to the procedure of Test Example 1, and the results are shown in Table 4.

[0105] Table 4 α-amylase enzyme hydrolysis temperature screening results

[0106]

[0107] As can be seen from Table 4, the SDF yield of Examples 14-18 is significantly higher, and it can be seen that the extraction effect is better within the α-amylase enzyme hydrolysis temperature range of the examples.

[0108] Example 19

[0109] Example 1 was weighed out, and was precisely measured into a 50 mL centrifuge tube. Distilled water was added at a ratio of 1:30 (g / mL), 2 mol / L sodium hydroxide was used to adjust the pH to 9.0, 0.08 g of alkaline protease was added, and the mixture was placed in a 55°C water bath for 65 min. 3 mol / L glacial acetic acid was used to adjust the pH to 4.8, 0.1 g of high-temperature-resistant α-amylase was added, and the mixture was placed in a 90°C water bath for 30 min. The mixture was inactivated at 100°C for 10 min, 0.01 g of cellulase was added, the pH was adjusted to 5.0, and the mixture was subjected to ultrasonic enzyme hydrolysis at 50°C for 1.5 h. The mixture was inactivated at 100°C for 10 min, was centrifuged at 8000 r / min for 6 min, and the supernatant was obtained. The precipitate was washed with 70°C hot water until it was neutral, and the wash liquid was combined with the supernatant. The combined solution was concentrated under reduced pressure to 50 mL at 55°C and 0.08 Pa, 4 times the mass of anhydrous ethanol was added, and the mixture was alcohol precipitated at 4°C for 12 h. The mixture was centrifuged, and the precipitate was dried at 50°C for 5 d to obtain the D. candidum soluble cellulose.

[0110] Example 20

[0111] The procedure of Example 19 was followed, except that the α-amylase enzyme hydrolysis was performed in a water bath for 45 min.

[0112] Example 21

[0113] The procedure of Example 19 was followed, except that the α-amylase enzyme hydrolysis was performed in a water bath for 60 min.

[0114] Example 22

[0115] The procedure of Example 19 was followed, except that the α-amylase enzyme hydrolysis was performed in a water bath for 75 min.

[0116] Example 23

[0117] The procedure of Example 19 was followed, except that the α-amylase enzyme hydrolysis was performed in a water bath for 90 min.

[0118] Test Example 5 α-amylase enzyme hydrolysis time screening

[0119] The sample weights of Examples 19-23 were measured, and the soluble dietary fiber (SDF) yield was calculated according to the procedure of Test Example 1, and the results are shown in Table 5.

[0120] Table 5 α-amylase enzyme hydrolysis time screening results

[0121]

[0122] As can be seen from Table 5, the SDF yields of Examples 19-23 are not significantly different, and it can be seen that the extraction effect is good within the α-amylase enzyme hydrolysis time range of the examples.

[0123] Example 24

[0124] 1 g of the defatted Dendrobium nobile powder of Example 1 was precisely weighed into a 50 mL centrifuge tube, and distilled water was added at a ratio of 1:20 (g / mL). The pH was adjusted to 9.0 with 2 mol / L sodium hydroxide, 0.08 g of alkaline protease was added, and the mixture was placed in a 55°C water bath for 65 min. The pH was adjusted to 4.8 with 3 mol / L glacial acetic acid, 0.1 g of thermostable α-amylase was added, and the mixture was placed in a 90°C water bath for 75 min. The mixture was inactivated at 100°C for 10 min, 0.01 g of cellulase was added, the pH was adjusted to 5.0, and the mixture was subjected to ultrasonic enzyme hydrolysis at 50°C for 1.5 h. The mixture was inactivated at 100°C for 10 min, centrifuged at 8000 r / min for 6 min, and the supernatant was obtained. The precipitate was washed with 70°C hot water until it was neutral, and the wash liquid was combined with the supernatant. The combined liquid was concentrated under reduced pressure to 50 mL at 55°C and 0.08 Pa, 4 times the mass of anhydrous ethanol was added to the concentrated liquid, and the mixture was alcohol precipitated at 4°C for 12 h. The mixture was centrifuged, and the precipitate was dried at 50°C for 5 d to obtain Dendrobium nobile soluble cellulose.

[0125] Example 25

[0126] The procedure of Example 24 was followed, except that the defatted Dendrobium nobile powder was added to distilled water at a ratio of 1:30 (g / mL).

[0127] Example 26

[0128] The procedure of Example 24 was followed, except that the defatted Dendrobium nobile powder was added to distilled water at a ratio of 1:40 (g / mL).

[0129] Example 27

[0130] The procedure of Example 24 was followed, except that the defatted dendrobium nobile powder was added to distilled water at a ratio of 1:50 (g / mL).

[0131] Comparative Example 3

[0132] The procedure of Example 24 was followed, except that the defatted dendrobium nobile powder was added to distilled water at a ratio of 1:10 (g / mL).

[0133] Test Example 6: Screening of the ratio of material to liquid

[0134] The samples of Examples 24-27 and Comparative Example 3 were weighed, and the yield of soluble dietary fiber (SDF) was calculated in the manner of Test Example 1, and the results are shown in Table 6.

[0135] Table 6: Results of the screening of the ratio of material to liquid

[0136]

[0137] As can be seen from Table 6, the yield of SDF of Examples 24-27 is significantly higher than that of Comparative Example 3, and it can be seen that the extraction effect is better within the range of the ratio of material to liquid of the examples.

[0138] Example 28

[0139] 1 g of the defatted dendrobium nobile powder of Example 1 was weighed accurately into a 50 mL centrifuge tube, distilled water was added at a ratio of 1:40 (g / mL), 2 mol / L sodium hydroxide was used to adjust the pH to 9.0, 0.08 g of alkaline protease was added, and the mixture was placed in a water bath at 55°C for 65 min, 3 mol / L glacial acetic acid was used to adjust the pH to 4.8, 0.1 g of high-temperature-resistant α-amylase was added, and the mixture was placed in a water bath at 90°C for 75 min, and then inactivated at 100°C for 10 min, 0.005 g of cellulase was added, the pH was adjusted to 5.0, and the mixture was subjected to ultrasonic enzymolysis at 50°C for 1.5 h, and then inactivated at 100°C for 10 min, and then centrifuged at 8000 r / min for 6 min, the supernatant was obtained, the precipitate was washed with hot water at 70°C until neutral, the washing liquid was combined with the supernatant, and the mixture was concentrated under reduced pressure at 55°C and 0.08 Pa to 50 mL, 4 times the mass of anhydrous ethanol was added to the concentrated liquid, and the mixture was alcohol-precipitated at 4°C for 12 h, centrifuged, and the precipitate was dried at 50°C for 5 d to obtain soluble cellulose of dendrobium nobile.

[0140] Example 29

[0141] The procedure of Example 28 was followed, except that 0.01 g of cellulase was added (i.e., the amount of cellulase added is preferably 1% of the total mass of the dendrobium nobile).

[0142] Example 30

[0143] The procedure of Example 28 was followed except that 0.015 g of cellulase was added (i.e. the added mass of cellulase is preferably 1.5% of the total mass of Dendrobium candidum).

[0144] Example 31

[0145] The procedure of Example 28 was followed except that 0.02 g of cellulase was added (i.e. the added mass of cellulase is preferably 2% of the total mass of Dendrobium candidum).

[0146] Example 32

[0147] The procedure of Example 28 was followed except that 0.025 g of cellulase was added (i.e. the added mass of cellulase is preferably 2.5% of the total mass of Dendrobium candidum).

[0148] Comparative Example 4

[0149] The procedure of Example 28 was followed except that 0.01 g of complex enzyme was added, the complex enzyme being a 2:1 mixture of cellulase and pectinase (i.e. the added mass of complex enzyme is preferably 1% of the total mass of Dendrobium candidum).

[0150] Comparative Example 5

[0151] The procedure of Example 28 was followed except that 0.015 g of complex enzyme was added, the complex enzyme being a 2:1 mixture of cellulase and pectinase (i.e. the added mass of complex enzyme is preferably 1.5% of the total mass of Dendrobium candidum).

[0152] Comparative Example 6

[0153] The procedure of Example 28 was followed except that 0.02 g of complex enzyme was added, the complex enzyme being a 2:1 mixture of cellulase and pectinase (i.e. the added mass of complex enzyme is preferably 2% of the total mass of Dendrobium candidum).

[0154] Test Example 7: Cellulase dosage screening

[0155] The sample weights of Examples 28~32 and Comparative Examples 4~6 were measured, and the soluble dietary fibre (SDF) yield was calculated in the manner of Test Example 1, and the results are shown in Table 7.

[0156] Table 7: Cellulase or complex enzyme dosage screening results

[0157]

[0158] As can be seen from Table 7, the SDF yields of Examples 28-32 are not significantly different, while the SDF yields of Comparative Examples 4-6 using a composite enzyme, i.e., a combination of cellulase and pectinase, are only 1 / 3-1 / 2 of those of Examples 28-32, which shows that the extraction effects of the cellulase in the dosage range of the examples are all good.

[0159] Example 33

[0160] Take 1 g of the defatted dendrobium candidum powder of Example 1, accurately weigh and put into a 50 mL centrifuge tube, add distilled water at 1:40 (g / mL), adjust the pH to 9.0 with 2 mol / L sodium hydroxide, add 0.08 g of alkaline protease, and then place in a 55°C water bath for 65 min, adjust the pH to 4.8 with 3 mol / L glacial acetic acid, add 0.1 g of high-temperature-resistant α-amylase, and then place in a 90°C water bath for 75 min, inactivate at 100°C for 10 min, add 0.015 g of cellulase, adjust the pH to 5.0, and then perform ultrasonic enzymolysis at 50°C for 0.5 h, inactivate at 100°C for 10 min, centrifuge at 8000 r / min for 6 min, take the supernatant, wash the precipitate with 70°C hot water until neutral, combine the washing liquid with the supernatant, concentrate under reduced pressure to 50 mL at 55°C and 0.08 Pa, add anhydrous ethanol to 4 times the mass of the concentrated liquid, and then perform alcohol precipitation at 4°C for 12 h, centrifuge, take the precipitate, and then dry at 50°C for 5 d to obtain the dendrobium candidum soluble cellulose.

[0161] Example 34

[0162] Perform in the same manner as in Example 33, except that the ultrasonic enzymolysis time of the cellulase is 1 h.

[0163] Example 35

[0164] Perform in the same manner as in Example 33, except that the ultrasonic enzymolysis time of the cellulase is 1.5 h.

[0165] Example 36

[0166] Perform in the same manner as in Example 33, except that the ultrasonic enzymolysis time of the cellulase is 2 h.

[0167] Example 37

[0168] Perform in the same manner as in Example 33, except that the ultrasonic enzymolysis time of the cellulase is 2.5 h.

[0169] Test Example 8: Screening of Ultrasonic Enzymolysis Time of Cellulase

[0170] Weigh the samples of Examples 33-37, and then calculate the soluble dietary fiber (SDF) yield in the same manner as in Test Example 1 to obtain the results shown in Table 8.

[0171] Table 8: Screening Results of Ultrasonic Enzymolysis Time of Cellulase

[0172]

[0173] From Table 8, it can be seen that the SDF yields of Examples 33-37 are not significantly different, and it can be seen that the extraction effects of the cellulase ultrasonic enzymolysis time ranges of the examples are all good.

[0174] Example 38

[0175] 1 g of the defatted dendrobium nobile powder of Example 1 was precisely weighed into a 50 mL centrifuge tube, 1:40 (g / mL) of distilled water was added, 2 mol / L sodium hydroxide was used to adjust the pH to 9.0, 0.08 g of alkaline protease was added, and the mixture was placed in a 55°C water bath for 65 min, 3 mol / L glacial acetic acid was used to adjust the pH to 4.8, 0.1 g of high-temperature-resistant α-amylase was added, and the mixture was placed in a 90°C water bath for 75 min, 100°C inactivation was performed for 10 min, 0.015 g of cellulase was added, the pH was adjusted to 5.0, 40°C ultrasonic enzymolysis was performed for 1.5 h, 100°C inactivation was performed for 10 min, 8000 r / min centrifugation was performed for 6 min, the supernatant was obtained, the precipitate was washed with 70°C hot water until neutral, the washing liquid was combined with the supernatant, the mixture was concentrated to 50 mL under reduced pressure at 55°C and 0.08 Pa, 4 times the mass of anhydrous ethanol was added to the concentrated liquid, and alcohol precipitation was performed at 4°C for 12 h, centrifugation was performed, the precipitate was dried at 50°C for 5 d, and dendrobium nobile soluble cellulose was obtained.

[0176] Example 39

[0177] The procedure of Example 38 was followed, except that the ultrasonic temperature during cellulase enzymolysis was 50°C.

[0178] Example 40

[0179] The procedure of Example 38 was followed, except that the ultrasonic temperature during cellulase enzymolysis was 60°C.

[0180] Example 41

[0181] The procedure of Example 38 was followed, except that the ultrasonic temperature during cellulase enzymolysis was 70°C.

[0182] Example 42

[0183] The procedure of Example 38 was followed, except that the ultrasonic temperature during cellulase enzymolysis was 80°C.

[0184] Test Example 9: Cellulase ultrasonic enzymolysis temperature screening

[0185] The weights of the samples of Examples 38-42 were measured, and the soluble dietary fiber (SDF) yield was calculated according to the procedure of Test Example 1, and the results are shown in Table 9.

[0186] Table 9 Cellulase ultrasonic enzymolysis temperature screening results

[0187]

[0188] As can be seen from Table 9, the SDF yield of Examples 38-42 is significantly higher, and it can be seen that the extraction effect in the cellulase ultrasonic enzymolysis temperature range of the examples is better.

[0189] Test Example 10 Response surface test optimization of Dendrobium dietary fiber extraction process

[0190] 1 Response surface experiment design

[0191] According to the Box-Behnken experiment design principle, in order to optimize the soluble dietary fiber yield under different factor conditions, the liquid-solid ratio, cellulase dosage, ultrasonic temperature, and pH value were selected as the main influencing factors, i.e. A is the mass-volume ratio of Dendrobium officinale powder to water, B is the dosage of cellulase to Dendrobium officinale powder, C is the ultrasonic temperature during cellulase hydrolysis, and D is the pH value during cellulase hydrolysis. The soluble dietary fiber yield was used as the response value, and the process was optimized by the response surface test method. The Box-Behnken experiment design is shown in Table 10.

[0192] Table 10 4-factor 3-level response surface analysis experiment design table

[0193]

[0194] 2 Results analysis

[0195] 2.1 Response surface design and results

[0196] 1g of defatted Dendrobium officinale powder of Example 1 was accurately weighed into a 50mL centrifuge tube, distilled water was added at a ratio of 1:40 (g / mL), 2mol / L sodium hydroxide was used to adjust the pH to 9.0, 0.08g of alkaline protease was added, and it was placed in a 55℃ water bath for 65min, 3mol / L glacial acetic acid was used to adjust the pH to 4.8, 0.1g of high-temperature-resistant α-amylase was added, and it was placed in a 90℃ water bath for 75min, and then inactivated at 100℃ for 10min. The cellulase hydrolysis conditions were optimized according to the Box-Behnken combination design of 4-factor 3-level response surface test, and 29 experiments were carried out according to the process parameters in Table 11. The experimental results are shown in Table 11.

[0197] Table 11 Response surface optimization of soluble dietary fiber yield experiment design and results

[0198]

[0199]

[0200] 2.2 Model establishment and analysis of soluble dietary fiber yield

[0201] The liquid material ratio, cellulase dosage, ultrasonic temperature and pH were respectively set as A, B, C and D, and a quadratic polynomial regression equation was obtained by multiple regression fitting with the soluble dietary fiber yield as the response value:

[0202] Y 可溶性膳食纤维得率 = 56.53 + 3.11 x A - 1.8 x B + 3.08 x C - 2.07 x D + 5.48 x AB - 3.61 x AC + 6.53 x AD + 3.31 x BC + 2.2 x BD - 0.5 x CD - 11.99 x A 2 - 12 x B2- 11.85 x C 2 - 10.88 x D 2

[0203] The regression analysis results of the soluble dietary fiber yield model and regression coefficients are shown in Table 12.

[0204] Table 12 Regression analysis results of the soluble dietary fiber yield model and regression coefficients

[0205]

[0206] Note: P<0.01 is extremely significant, represented by ***, P<0.05 is significant, represented by *, and P>0.05 is not significant, represented by ns

[0207] As shown in Table 12, the model difference is extremely significant (P<0.001), the determination coefficient R2 is 0.9717, indicating that the model fitting degree is excellent, and the test results can be intuitively fitted. The lack of fit is not significant (P>0.05), indicating that the model error is small, the corrected determination coefficient RAdj2 is 0.9434, indicating that the correlation and explanation of the model are very good, and the model can be used for theoretical analysis and prediction.

[0208] The size of F value is an important index for evaluating the influence degree of each variable on the response value. The larger the F value, the higher the contribution of the relevant model component to the response. When the significance test probability P<0.05, it reveals that the variable has a significant influence on the response value, which has mathematical statistical significance. The analysis of the influence of each factor on the soluble dietary fiber yield shows that the liquid material ratio and ultrasonic temperature have extremely significant influence on the soluble dietary fiber yield (P<0.01), the cellulase dosage and pH have significant influence on the soluble dietary fiber yield (P<0.05), and among the four influencing factors, the influence degree on the soluble dietary fiber yield is A>C>D>B, i.e. the liquid material ratio>ultrasonic temperature>pH>cellulase dosage, and the quadratic terms A 2 , B 2 , C​​2 , D 2 The effect of soluble dietary fiber yield was extremely significant (P < 0.01), indicating that the three factors had a non-linear effect on the yield of soluble dietary fiber. The interaction terms AB, AD had a very significant effect on the yield of soluble dietary fiber (P < 0.01), and AC, BC had a significant effect on the yield of soluble dietary fiber (P < 0.05).

[0209] 2.3 Interaction of each factor

[0210] The test results were analyzed by Design-Expert 10.0.3 software to draw response surface graphs. In the 3D graph and contour graph, the color change from blue to red represents the change from less to more of the response value, and the faster the change, the greater the slope, i.e. the more significant the effect on the test results. The interaction between each factor on the yield of soluble dietary fiber can be reflected more intuitively by the 3D response surface graph, so as to find out the best process parameters and the interaction between each parameter. The results of response surface analysis are shown in Figures 1~6 .

[0211] As shown in Figure 1 , in the AB interaction surface, with the increase of liquid-solid ratio, the yield of soluble dietary fiber showed a trend of first increasing and then decreasing. When the liquid-solid ratio was low, with the increase of cellulase dosage, the yield of soluble dietary fiber showed a trend of first slowly increasing and then decreasing, and the decreasing amplitude was larger. When the liquid-solid ratio was high, with the increase of cellulase dosage, the yield of soluble dietary fiber showed a trend of first increasing and then decreasing, and the decreasing amplitude was smaller. Thus, there was a significant interaction between liquid-solid ratio and ultrasonic temperature. Considering only the interaction between the two, when the cellulase dosage was 1.2%~1.8% and the liquid-solid ratio was 1g:35~45mL, the yield of soluble dietary fiber was larger.

[0212] As shown in Figure 2 , in the AC interaction surface, with the increase of ultrasonic temperature, the yield of soluble dietary fiber showed a trend of first increasing and then decreasing. When the ultrasonic temperature was low, with the increase of liquid-solid ratio, the yield of soluble dietary fiber showed a trend of first increasing and then gently decreasing. When the ultrasonic temperature was high, with the increase of liquid-solid ratio, the yield of soluble dietary fiber showed a trend of first increasing and then decreasing. Thus, there was a significant interaction between liquid-solid ratio and ultrasonic temperature. Considering only the interaction between the two, when the ultrasonic temperature was about 65~75℃ and the liquid-solid ratio was 1g:35~45mL, the yield of soluble dietary fiber was larger.

[0213] As shown in Figure 3As shown in the AD interaction surface, the yield of soluble dietary fiber first increases and then decreases with increasing liquid-to-solid ratio. When the liquid-to-solid ratio is low, the yield of soluble dietary fiber first flattens and then decreases with increasing pH. When the liquid-to-solid ratio is high, the yield of soluble dietary fiber first increases and then decreases with increasing pH, and the decrease is relatively small. This indicates a significant interaction between the liquid-to-solid ratio and the ultrasonic temperature. Considering only the interaction between the two, the yield of soluble dietary fiber is higher when the pH is 5.2–5.8 and the liquid-to-solid ratio is 1g:35–45mL.

[0214] like Figure 4 As shown in the BC interaction surface, the yield of soluble dietary fiber initially increases and then decreases with increasing ultrasonic temperature, and similarly, the yield of soluble dietary fiber initially increases and then decreases with increasing cellulase dosage. Furthermore, the change in soluble dietary fiber yield varies with increasing cellulase dosage when the ultrasonic temperature and dosage are different, indicating a significant interaction between ultrasonic temperature and cellulase dosage. Considering only the interaction, the yield of soluble dietary fiber is higher when the cellulase dosage is 1.2%–1.8% and the ultrasonic temperature is 65–75℃.

[0215] like Figure 5 As shown in the BD interaction surface, the yield of soluble dietary fiber first increases and then decreases as pH and cellulase dosage increase. Considering only the interaction between the two, it is known that the yield of soluble dietary fiber is relatively large when the cellulase dosage is 1.2%~1.8% and the pH is 5.2~5.8.

[0216] like Figure 6 As shown in the CD interaction surface, the yield of soluble dietary fiber first increases and then decreases with the increase of ultrasonic temperature and pH. Considering only the interaction between the two, it is known that the yield of soluble dietary fiber is relatively large when the pH is 5.2~5.8 and the ultrasonic temperature is 65~75℃.

[0217] 2.4 Optimal process and verification test results

[0218] The soluble dietary fiber yield maximum value as the optimization goal, by Design-Expert 10.0.3 software to optimize the test, the predicted soluble dietary fiber yield is 80.133%, the predicted value of four factors is liquid material ratio 40.802, cellulase dosage 1.476%, ultrasonic temperature 71.126℃, pH 5.461, in order to determine the accuracy of the model, the optimized parameters are verified, for the convenience of operation, the condition parameters are set as liquid material ratio 40.8, cellulase dosage 1.48%, ultrasonic temperature 71℃, pH 5.5, under this condition, repeated test 3 times, the measured average value of soluble dietary fiber yield is 58.253±2.654%, and the model predicted value 56.953% is within 5% deviation range, which shows that the process parameters optimized by the model are reliable.

[0219] Example 43

[0220] Pre-treatment: Dendrobium candidum stem is dried at 45℃ for 10d, crushed, passed through No. 4 sieve (60 mesh) to obtain Dendrobium powder for use. A certain amount of Dendrobium powder is weighed, soaked in petroleum ether at 1g:15mL for 10h, degreased and degenerated, centrifuged at 8000r / min for 10min, the petroleum ether is recovered, and the powder is dried at 50℃ for 48h to obtain degreased Dendrobium powder for use.

[0221] Single factor screening: 1g of degreased Dendrobium powder is accurately weighed and placed in a 50mL centrifuge tube, 40.8g of distilled water is added, the pH is adjusted to 9.0 with 2mol / L sodium hydroxide, 0.08g of alkaline protease is added, and it is placed in a 65℃ water bath for 55min, then taken out, the pH is adjusted to 4.8 with 3mol / L glacial acetic acid, 0.1g of high-temperature-resistant α-amylase is added, and it is placed in a 90℃ water bath for 75min, then inactivated at 100℃ for 10min, 0.0148g of cellulase is added, the pH is adjusted to 5.5, and it is ultrasonically hydrolyzed at 71℃ for 1h, then inactivated at 100℃ for 10min, centrifuged at 8000r / min for 6min, the supernatant is obtained, the precipitate is washed with 70℃ hot water until neutral, the washing liquid is combined with the supernatant, concentrated to 50mL under reduced pressure at 55℃ and 0.08Pa, 4 times the mass of anhydrous ethanol is added to the concentrated liquid, and alcohol precipitation is carried out at 4℃ for 12h, then centrifuged, and the precipitate is dried at 50℃ for 5d to obtain Dendrobium soluble cellulose.

[0222] Example 11 Study on the Anti-aging Activity of Dendrobium Soluble Dietary Fiber

[0223] Ordinary life experiment: the experiment is divided into blank control group (marked as blank group in the figure), experimental group, that is, using the dendrobium officinale soluble dietary fiber of example 43 (50 μg / mL, 100 μg / mL, 500 μg / mL, marked as SD-1, SD-2, SD-3 in the figure). After the same period of growth, the L4 stage nematodes were randomly selected and placed on the culture dish of each experimental group, 3 parallel samples in each group, about 50-60 nematodes in each culture dish, which was recorded as day 0 at this time. Incubate at 20℃, transfer the nematodes to a new culture dish every day, and observe the survival of the nematodes. When the nematodes are motionless and do not respond after being stimulated by platinum wire for 10s, they are considered dead, the plate is picked out, and the number of deaths is recorded. If the nematodes crawl out of the culture medium, are lost due to crawling out of the culture medium, and are killed due to the hatching of eggs in the body pressing the internal organs, they will not be recorded. The above experiment is repeated 3 times, and the final results are shown in Figure 7 , Tables 13 and 14.

[0224] Table 13 Effect of dendrobium officinale soluble dietary fiber on nematode survival curve

[0225]

[0226] Table 14 Statistical analysis of nematode survival time

[0227]

[0228] From Figure 7 , Tables 13 and 14, it can be seen that the dendrobium officinale soluble dietary fiber of example 43 is 50, 100, 500 μg / mL, which significantly prolongs the maximum lifespan and average lifespan of nematodes compared with the blank control group, indicating that the dendrobium officinale soluble dietary fiber has strong anti-aging activity.

[0229] Test example 12 acute oxidative stress

[0230] The experiment is divided into blank control group (marked as BL in the figure), positive control group (marked as Vc-1 in the figure), and experimental group, that is, using the dendrobium officinale soluble dietary fiber of example 43 (50 μg / mL, 100 μg / mL, 500 μg / mL, marked as SD-1, SD-2, SD-3 in the figure). L4 stage larvae after the same period of growth were picked into each group of NGM, 40-50 in each plate, 3 plates in each group, and after 5d of drug culture, 30 nematodes in each group were transferred to the NGM culture dish containing 0.15% H2O2, the death of nematodes was observed and recorded every 1h, until all nematodes died, and the results are shown in Figure 8 and Table 15.

[0231] Table 15 Nematode survival

[0232]

[0233] By Figure 8 As can be seen from Table 15 and Table 16,

[0234] Test Example 13 Determination of antioxidant enzyme activity

[0235] The test was divided into high, medium and low (50, 100, 500 μg / mL) concentration groups of Dendrobium dietary fiber of Example 43, a VC positive group and a blank control group (labeled as SD-1, SD-2, SD-3, VC-1, BL in the figure).

[0236] L4 larvae after synchronization were picked on different groups of plates, 10 culture dishes per group, about 200 worms per culture dish, and incubated at 20°C, continuously dosed for 5 days, and the plates were rinsed with M9 buffer to collect the nematodes in 1.5 mL centrifuge tubes. After the Caenorhabditis elegans were naturally settled on ice, the supernatant was aspirated, and the Caenorhabditis elegans were washed with sterile distilled water for 3 times, and the upper liquid was aspirated. 1000 μL of distilled water was added to the tube, and the Caenorhabditis elegans were broken by a micro-electric homogenizer for 15 min on ice (the grinding condition was observed, and the grinding time could be increased in time), centrifuged at 4°C, 9700 rpm for 10 min, and the supernatant was collected in a new centrifuge tube. The total protein concentration of the Caenorhabditis elegans was detected by using a BCA quantitative kit. According to the kit instructions, the SOD, CAT, GSH, MDA contents in the homogenate supernatant of the Caenorhabditis elegans were detected, and the results are shown in Table 16. Figure 9 and Table 16.

[0237] Table 16 Antioxidant enzyme activity content in the homogenate supernatant of the nematodes

[0238]

[0239] By Figure 9 As can be seen from Table 15 and Table 16, Figure 9 A shows that the Dendrobium dietary fiber can significantly improve the GSH content in the nematodes compared with the blank control group, and the high concentration group is better than the positive control group, Figure 9 B shows that compared with the blank control group, the low concentration group and the high concentration group of Dendrobium dietary fiber can improve the CAT activity in the nematodes, Figure 9 C shows that the low concentration of Dendrobium dietary fiber can greatly improve the SOD activity in the nematodes compared with the positive control group and the blank control group, Figure 9D indicates that low concentration of dietary fiber can reduce the content of MDA in nematodes, which fully shows that the dendrobium dietary fiber has an anti-aging effect.

[0240] It can be seen that the extraction method of the dendrobium soluble dietary fiber provided by the present application has a large yield of soluble dietary fiber, and the obtained dendrobium soluble dietary fiber has strong anti-aging activity, can be widely applied in the preparation of medicines and / or foods, and improves the utilization value of the processing by-products of dendrobium.

[0241] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiment of the present application, not all the embodiments, and other embodiments can be obtained according to the embodiment without creativity, which belongs to the protection scope of the present application.

Claims

1. A method for extracting Dendrobium candidum soluble dietary fiber, characterized in that, The method comprises the following steps: The Dendrobium candidum stem is first dried and crushed to obtain Dendrobium candidum powder; the Dendrobium candidum powder is soaked in petroleum ether, centrifuged and secondly dried to obtain defatted Dendrobium candidum after treatment; The defatted Dendrobium candidum is sequentially subjected to enzymolysis by using alkaline protease, α-amylase and cellulase to obtain an enzymolysis liquid, and the supernatant after centrifugation is concentrated and alcohol precipitated to obtain a precipitate; the precipitate contains the Dendrobium candidum soluble dietary fiber; The alkaline protease is added in an amount of 7% to 9% of the total mass of the Dendrobium candidum; The α-amylase is added in an amount of 2% to 11% of the total mass of the Dendrobium candidum; the α-amylase comprises a high-temperature-resistant α-amylase; The cellulase is added in an amount of 0.5% to 2.5% of the total mass of the Dendrobium candidum; The cellulase is subjected to ultrasonic treatment during the enzymolysis; Before the enzymolysis by the alkaline protease, the Dendrobium candidum is mixed with water; the mass-volume ratio of the Dendrobium candidum to water is 0.7 to 1.3 g: 20 to 50 mL.

2. The extraction method according to claim 1, characterized in that, During the enzymolysis by the alkaline protease, the pH of the enzymolysis is 8.0 to 11.0, the temperature of the enzymolysis is 45 to 85°C, and the time of the enzymolysis is 55 to 75 min.

3. The extraction method of claim 1, wherein, During the enzymolysis by the high-temperature-resistant α-amylase, the pH of the enzymolysis is 4.5 to 6.5, the temperature of the enzymolysis is 60 to 100°C, and the time of the enzymolysis is 30 to 90 min.

4. The extraction method of claim 1, wherein, During the enzymolysis by the cellulase, the pH of the enzymolysis is 4.5 to 6.5, the temperature of the ultrasonic treatment is 40 to 80°C, and the time of the ultrasonic treatment is 0.5 to 2.5 h.

5. The Dendrobium candidum soluble dietary fiber prepared by the extraction method of any one of claims 1 to 4.

6. The use of the Dendrobium candidum soluble dietary fiber of claim 5 in the preparation of a medicine with anti-aging activity.

7. The use of the Dendrobium candidum soluble dietary fiber of claim 5 in the preparation of an antioxidant food.

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