A fermentation process of cistanche

Abstract

The application provides a fermentation process of Cistanche, which comprises the following steps: (1) crushing Cistanche medicinal materials, preparing a suspension, and then adding a lignin-degrading enzyme to perform enzymolysis to obtain an enzymolysis liquid; (2) performing sterilization treatment on the enzymolysis liquid, then adding activated strains and a culture medium to perform fermentation, and obtaining a Cistanche fermentation liquid after the fermentation is completed. The fermentation liquid obtained by the fermentation process has a high content of total flavones and polysaccharides, and has important application significance.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a fermentation process for Cistanche deserticola. Background Technology

[0002] Cistanche deserticola Ma., a tall herbaceous plant belonging to the genus Cistanche in the family Orobanchaceae, has high medicinal value. According to the "Compendium of Materia Medica" and "Rihuazi Materia Medica," Cistanche deserticola can be used to treat kidney yang deficiency, depletion of essence and blood, cold pain in the lower back and knees, tinnitus, blurred vision, leukorrhea, frequent urination, metrorrhagia, infertility, and constipation. Cistanche deserticola can also be used to make porridge and infuse alcohol. In addition, it is a precious traditional Chinese medicine, known as "desert ginseng," and has considerable economic value. Using Cistanche deserticola as raw material, and employing enzymatic hydrolysis and microbial fermentation techniques for deeper processing, not only is the extraction rate of polysaccharides and flavonoids improved, but large, poorly absorbed molecules are also broken down into smaller, more easily absorbed molecules. This enhances its anti-aging, anti-fatigue, anti-Alzheimer's disease, immune-boosting, liver-protecting, and gastrointestinal motility-promoting effects, providing a theoretical basis for the development of functional foods with huge market potential. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a fermentation process for Cistanche deserticola, wherein the fermentation broth obtained by the fermentation process has a high content of total flavonoids and polysaccharides.

[0004] The technical solution of the present invention is as follows:

[0005] A fermentation process for Cistanche deserticola includes the following steps:

[0006] (1) Cistanche deserticola is pulverized and prepared into a suspension. Then, lignin-degrading enzyme is added for enzymatic hydrolysis to obtain the hydrolysate.

[0007] (2) The enzymatic hydrolysate is first sterilized, and then activated bacteria and culture medium are added for fermentation. After fermentation, the fermentation liquid of Cistanche deserticola is obtained.

[0008] Preferably, in step (1), the lignin-degrading enzyme is cellulase, laccase, or a combination thereof.

[0009] As a further preferred option, in step (1), the lignin-degrading enzyme is a combination of cellulase and laccase, and the mass ratio of cellulase to laccase is 2 to 7:1.

[0010] The amount of lignin-degrading enzyme added is 5% to 8%.

[0011] Preferably, in step (1), the temperature of enzymatic hydrolysis is 55℃~70℃ and the time of enzymatic hydrolysis is 60~120min.

[0012] Preferably, in step (2), the bacterial strain is a mixture of Lactobacillus rhamnosus and Lactobacillus plantarum, with a mass ratio of 1 to 3:1.

[0013] Preferably, in step (2), the inoculation amount of the bacterial strain is 9-11%.

[0014] Preferably, in step (2), the fermentation temperature is 35-37℃ and the fermentation time is 1.5-2.5 days.

[0015] Preferably, in step (2), the initial pH value of fermentation is 5.5 to 6.5.

[0016] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0017] This invention provides a fermentation process for Cistanche deserticola, which involves sequential enzymatic hydrolysis and fermentation steps. By controlling the process parameters of enzymatic hydrolysis and fermentation, the extraction rate of total flavonoids and polysaccharides in the fermentation broth is effectively improved, which has important application significance. Detailed Implementation

[0018] Example 1: Optimization of the enzymatic hydrolysis process of Cistanche deserticola

[0019] The experiment used a single-factor experimental method to investigate the key factors affecting the enzymatic hydrolysis of Astragalus membranaceus, including the type of enzyme, hydrolysis time, hydrolysis temperature, and amount of enzyme added.

[0020] (1) Effect of enzyme type on total flavonoids and polysaccharide content of Cistanche deserticola

[0021] Cistanche deserticola was pulverized, and about 2g was weighed accurately. An aqueous solution was prepared according to a material-to-liquid ratio of 1:12.5. Cellulase and laccase were mixed in different proportions (1:1, 1:2, 1:3, 2:1, 3:1), with a total addition of 6%. The mixture was enzymatically hydrolyzed in a constant temperature water bath at 50℃ for 90 minutes. The optimal enzyme type was determined by using the total flavonoid and polysaccharide content as indicators. The optimal mixing ratio of cellulase and laccase was 3:1.

[0022] (2) Effect of enzyme dosage on the total flavonoid and polysaccharide content of Cistanche deserticola

[0023] Cistanche deserticola was pulverized, and about 2g was weighed accurately. It was then prepared into a suspension at a material-to-liquid ratio of 1:12.5. The ratio of cellulase to laccase was 3:1. The enzyme addition amounts were 5%, 6%, 7%, and 8%, respectively. The mixture was enzymatically hydrolyzed in a constant temperature water bath at 50°C for 90 minutes. The optimal enzyme addition amount of 7% was determined based on the total flavonoid and polysaccharide content.

[0024] (3) Effect of Cistanche deserticola powder particle size on total flavonoid content

[0025] Cistanche deserticola was pulverized to 60 mesh, 80 mesh, and 100 mesh. 2g of the pulverized material was weighed and prepared into a suspension at a material-to-liquid ratio of 1:12.5. Cellulase and laccase were mixed in a 4:1 ratio, and 7% of the mixture was added. The mixture was then enzymatically hydrolyzed in a constant temperature water bath at 50℃ for 90 minutes. The optimal pulverization particle size of 80 mesh was determined based on the content of total flavonoids and polysaccharides.

[0026] (4) Effect of enzymatic hydrolysis time on the content of total flavonoids and polysaccharides in Cistanche deserticola

[0027] Cistanche deserticola was pulverized, and approximately 2g was weighed accurately. A suspension was prepared at a material-to-liquid ratio of 1:12.5. The enzymatic hydrolysis times were 60min, 90min, 120min, and 150min, with a cellulase to laccase ratio of 3:1. 7% cellulase and laccase were added, and the mixture was enzymatically hydrolyzed in a constant temperature water bath at 50℃. The optimal enzymatic hydrolysis time of 90min was determined based on the total flavonoid and polysaccharide content.

[0028] (5) Effect of enzymatic hydrolysis temperature on the content of total flavonoids and polysaccharides in Cistanche deserticola

[0029] Cistanche deserticola was pulverized, and approximately 2g was weighed precisely. A suspension was prepared at a material-to-liquid ratio of 1:12.5. The hydrolysis temperatures were controlled at 55℃, 60℃, 65℃, and 70℃, with a cellulase to laccase ratio of 3:1. 7% cellulase and laccase were added. The hydrolysis was carried out in a constant temperature water bath for 90 minutes. The optimal hydrolysis temperature of 60℃ was determined based on the total flavonoid and polysaccharide content.

[0030] (6) Orthogonal experiment

[0031] An orthogonal experiment was conducted using a 4-factor, 3-level orthogonal experimental table, with the content of total flavonoids and polysaccharides as indicators, to determine the optimal enzymatic hydrolysis process.

[0032]

[0033] The optimal enzyme ratio of 5:1, enzyme dosage of 5%, hydrolysis time of 100 min, and hydrolysis temperature of 65 degrees Celsius were determined. After optimization of the hydrolysis, the extraction rate of isoflavones increased by 67% and the extraction rate of polysaccharides increased by 53%.

[0034] Example 2: Screening of Cistanche deserticola fermentation strains

[0035] (1) Screening of Cistanche deserticola fermentation bacteria

[0036] Cistanche deserticola was pulverized, and approximately 2g of the appropriate particle size was weighed accurately. A suspension was prepared at a material-to-liquid ratio of 1:12.5. Then, 7% cellulase and laccase (in a 3:1 ratio) were added. The mixture was enzymatically hydrolyzed in a constant-temperature water bath at 60℃ for 90 minutes. The solution was then transferred to a sterilized conical flask, sealed, and sterilized in a high-pressure steam sterilizer at 121℃ for 15 minutes. After cooling to room temperature, the solution was sterilized under ultraviolet light in a clean bench for 20 minutes. Activated Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus delbrueckii, and Bifidobacterium breve were inoculated. The mixture was then cultured in a constant-temperature shaker for 48 hours. The optimal fermentation strain was determined to be a mixture of Lactobacillus rhamnosus and Lactobacillus plantarum, based on the total flavonoid and polysaccharide content.

[0037] (2) Effects of different proportions of two optimal strains on the fermentation of Cistanche deserticola

[0038] Cistanche deserticola was pulverized, and approximately 2g of 80-mesh pulverized Cistanche deserticola was accurately weighed. A suspension was prepared at a material-to-liquid ratio of 1:12.5. Then, 7% cellulase and laccase (in a 3:1 ratio) were added. The mixture was enzymatically hydrolyzed in a constant-temperature water bath at 60℃ for 90 minutes. The solution was then transferred to a sterilized conical flask, sealed, and sterilized in a high-pressure steam sterilizer at 121℃ for 15 minutes. After cooling to room temperature, the solution was sterilized under ultraviolet light in a clean bench for 20 minutes. The optimal ratio of activated Rhamnus to plant bacteria was then inoculated at ratios of 1:0, 0:1, 1:1, 1:2, 2; 1, 1:3, 3:1 and cultured in a constant-temperature shaker for 48 hours. The optimal fermentation ratio of Rhamnus to plant bacteria (2:1) was determined using the total flavonoid and polysaccharide content as indicators.

[0039] Example 3: Optimization of Cistanche deserticola fermentation culture medium

[0040] (1) Effect of carbon source type on the content of total flavonoids and polysaccharides

[0041] Cistanche deserticola was pulverized, and approximately 2g of 80-mesh Cistanche deserticola was weighed precisely. A suspension was prepared at a material-to-liquid ratio of 1:12.5. The amount of cellulase and laccase added was 7% (the ratio of cellulase to laccase was 3:1). The mixture was enzymatically hydrolyzed in a constant temperature water bath at 60℃ for 90 minutes with an inoculation volume of 5mL. Four carbon sources were selected: glucose, maltose, sucrose, and lactose. Each carbon source was added to the hydrolysate at a dosage of 2%. Fermentation was carried out at 37℃ for 48 hours. The optimal carbon source, maltose, was determined based on the content of total flavonoids and polysaccharides.

[0042] (2) Effect of nitrogen source type on the content of total flavonoids and polysaccharides

[0043] Cistanche deserticola was pulverized, and approximately 2g of 80-mesh Cistanche deserticola was weighed precisely. A suspension was prepared at a material-to-liquid ratio of 1:12.5. The addition of cellulase and laccase was 7% (the ratio of cellulase to laccase was 3:1). The mixture was enzymatically hydrolyzed in a constant temperature water bath at 60℃ for 90 minutes with an inoculation volume of 5mL. Four nitrogen sources were selected: urea, yeast powder, soybean powder, and soybean peptone. Each of these was added to the hydrolysate at a dosage of 1%. Fermentation was carried out at 37℃ for 48 hours. The optimal nitrogen source, soybean powder, was determined based on the content of total flavonoids and polysaccharides.

[0044] (3) Effect of carbon source addition on the content of total flavonoids and polysaccharides

[0045] Cistanche deserticola was pulverized, and approximately 2g of 80-mesh pulverized Cistanche deserticola was weighed precisely. A suspension was prepared at a material-to-liquid ratio of 1:12.5. The amount of cellulase and laccase added was 7% (the ratio of cellulase to laccase was 3:1). Enzymatic hydrolysis was carried out in a constant temperature water bath at 60℃ for 90 minutes, with an inoculation volume of 5mL. The amounts of maltose added were selected as 1%, 2%, 3%, 4%, and 5%, respectively. Fermentation was carried out at 37℃ for 48 hours. The optimal amount of carbon source added was determined to be 3%, based on the content of total flavonoids and polysaccharides.

[0046] (4) Effect of nitrogen source addition on the content of total flavonoids and polysaccharides

[0047] Cistanche deserticola was pulverized, and approximately 2g of 80-mesh Cistanche deserticola was weighed precisely. A suspension was prepared at a material-to-liquid ratio of 1:12.5. The addition of cellulase and laccase was 7% (the ratio of cellulase to laccase was 3:1). Enzymatic hydrolysis was carried out in a constant temperature water bath at 60℃ for 90 minutes, with an inoculation volume of 5mL. Soybean flour was added at different amounts of 1%, 2%, 3%, 4%, and 5%, respectively. Fermentation was carried out at 37℃ for 48 hours. The optimal nitrogen source addition was determined to be 3%, based on the content of total flavonoids and polysaccharides.

[0048] Example 4: Optimization of Cistanche fermentation conditions

[0049] (1) Effect of inoculum size on the content of total flavonoids and polysaccharides

[0050] Cistanche deserticola was pulverized, and approximately 2g of 80-mesh pulverized Cistanche deserticola was accurately weighed and prepared into a suspension at a material-to-liquid ratio of 1:12.5. The amount of cellulase and laccase added was 7% (the ratio of cellulase to laccase was 3:1). Enzymatic hydrolysis was carried out in a constant temperature water bath at 60℃ for 90 minutes. The initial pH was 6.0. The inoculum amounts were 2%, 4%, 6%, 8%, and 10%, respectively. Fermentation was carried out at 37℃ for 48 hours. The optimal inoculum amount of 10% was determined based on the content of total flavonoids and polysaccharides.

[0051] (2) Effect of fermentation temperature on the content of total flavonoids and polysaccharides

[0052] Cistanche deserticola was pulverized, and approximately 2g of 80-mesh pulverized Cistanche deserticola was accurately weighed and prepared into a suspension at a material-to-liquid ratio of 1:12.5. The amount of cellulase and laccase added was 7% (the ratio of cellulase to laccase was 3:1). Enzymatic hydrolysis was carried out in a constant temperature water bath at 60℃ for 90min. The initial pH was 6.0, and the inoculum volume was 2ml. Fermentation was carried out at 36, 36.5, 37, 37.5, and 38℃ for 48 hours respectively. The optimal fermentation temperature of 36℃ was determined based on the content of total flavonoids and polysaccharides.

[0053] (3) Effect of fermentation time on the content of total flavonoids and polysaccharides

[0054] Cistanche deserticola was pulverized, and approximately 2g of 80-mesh Cistanche deserticola was weighed precisely. A suspension was prepared at a material-to-liquid ratio of 1:12.5. The amount of cellulase and laccase added was 7% (the ratio of cellulase to laccase was 3:1). Enzymatic hydrolysis was carried out in a constant temperature water bath at 60℃ for 90 minutes, with an inoculation amount of 5mL. The fermentation times were 1d, 2d, 3d, 4d, and 5d, respectively, at 36℃. The optimal fermentation time of 2d was determined based on the content of total flavonoids and polysaccharides.

[0055] (4) Effect of initial pH on the content of total flavonoids and polysaccharides

[0056] Cistanche deserticola was pulverized, with 20% Cistanche deserticola added and 7% cellulase and laccase added (cellulase to laccase ratio of 3:1). Enzymatic hydrolysis was carried out in a constant temperature water bath at 60℃ for 90 min with an inoculation volume of 5 mL. The initial pH was 5.5 and 6.0. The optimal fermentation process was optimized based on the content of total flavonoids and polysaccharides to 6.5, 7.0, and 7.5. Fermentation was carried out at 36℃, and the initial pH was determined to be 6 based on the content of total flavonoids and polysaccharides.

[0057] After conducting single-factor experiments, experiments were carried out using the orthogonal experimental method.

[0058]

[0059] After optimization, the optimal conditions are 6%, 37 degrees Celsius, pH 6.5, and 2 days.

Claims

1. A fermentation process for Cistanche deserticola, characterized in that, Includes the following steps: (1) Cistanche deserticola is pulverized and prepared into a suspension. Then, lignin-degrading enzyme is added for enzymatic hydrolysis to obtain the hydrolysate. In step (1), the lignin-degrading enzyme is a combination of cellulase and laccase, with a mass ratio of cellulase to laccase of 5:

1. The amount of lignin-degrading enzyme added is 5%; (2) The enzyme hydrolysate is first sterilized, and then activated bacteria and culture medium are added for fermentation. After fermentation, the fermentation liquid of Cistanche deserticola is obtained. In step (2), the bacterial strain is a mixture of Lactobacillus rhamnosus and Lactobacillus plantarum, with a mass ratio of 1 to 3:1; In step (1), the enzymatic hydrolysis temperature is 65℃ and the enzymatic hydrolysis time is 100 min.

2. The fermentation process according to claim 1, characterized in that, In step (2), the inoculation amount of the strain is 5-10%.

3. The fermentation process according to claim 1, characterized in that, In step (2), the fermentation temperature is 35~37℃ and the fermentation time is 1~3 days.

4. The fermentation process according to claim 1, characterized in that, In step (2), the initial pH value of fermentation is 5.5~6.5.

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