CN107502631A - A kind of production method of candida utili β D glucans - Google Patents

Abstract

The present invention discloses a kind of production method of candida utili β D glucans.This method is removed the preparation of the murphy juice of the deproteinized after the murphy juice of potato starch, the murphy juice aqueous solution of deproteinized, sterilizing first, is then inoculated with candida utili bacterium（Candida utilis）CICC 1769 is fermented, the intracellular that the filtering fermentation liquor of gained obtains contains the candida utili bacterium thalline of candida utili β D glucans, then extracted using high-temp extracting and ultrasonication synergy broken wall, then through centrifuging, washing, being dried in vacuo, finally give purity and reach as high as 97%, yield reaches as high as 19%, and yield reaches as high as 87% candida utili β D glucans.Workload greatly reduces in the production method, while reduces production cost, turns waste into wealth simultaneously, meets green production demand.

Description

A kind of production method of Candida utilis β-D-glucan

technical field

The invention belongs to the field of biology and relates to a production method of Candida utilis β-D-glucan.

Background technique

Candida utilis, together with Saccharomyces cerevisiae and Kluyveromyces, has been certified as a microorganism that can be used as a food additive by many countries, and it is also a microorganism that can be used in health food permitted by the Chinese health department. can grow without adding any growth factors.

Yeast beta-glucan is a structural polysaccharide of the yeast cell wall. They have different functional properties, such as medical, pharmaceutical, food, cosmetic and other industries. They can also act as biological modifiers of the immune response. In addition, they are also beneficial to antibacterial and anti-inflammatory reactions; promote the absorption of mycotoxins; reduce cholesterol particles in low-density lipoproteins; they also have anti-cancer, anti-mutagenic, anti-oxidative and wound-healing properties.

At present, the yeast culture method for producing yeast β-D-glucan in China mainly adopts the preparation of carbon and nitrogen sources or yeast extract powder peptone glucose medium, which has disadvantages such as high cost of the medium, and if the carbon-nitrogen ratio is prepared during the production process, Since the nutritional conditions such as the carbon-to-nitrogen ratio required by yeast strains are relatively complicated, there are problems such as a large research workload, and the yield, yield and purity of yeast β-D-glucan in most of the existing literature are all low. Relatively low, the highest is only 80%, 17% and 86%.

Contents of the invention

The purpose of the present invention is to provide a kind of production method of Candida utilis β-D-glucan in order to solve technical problems such as high production cost and large research workload in the above-mentioned yeast β-D-glucan production method. The production method, which can use waste potato juice as a medium after deproteinization, thereby solving the problem that in the prior art, a large amount of carbon and nitrogen sources are used for cultivating yeast and the exploration process is relatively cumbersome, thereby greatly reducing the workload. , at the same time reduce the production cost, and turn waste into treasure, meet the needs of green production, and finally get a high yield of Candida utilis, the highest yield of Candida utilis β-D-glucan can reach 87% , the highest yield can reach 19%, and the highest purity can reach 97%.

Technical scheme of the present invention

A production method of Candida utilis β-D-glucan, specifically comprising the following steps:

(1) Preparation of potato juice with potato starch removed

Crush the potatoes with a pulverizer to obtain mashed potatoes, then add distilled water 10 times the mass of potatoes to the mashed potatoes, stir well, and centrifuge at 10,000 r/min for 20 minutes to remove the precipitate, which is potato starch, and the obtained filtrate Stand for 5 days to get the potato juice that removes the potato starch;

(2), preparation of protein-removed potato juice aqueous solution

Filter the potato juice obtained in step (1) without potato starch through 8 layers of gauze, add distilled water to the obtained filtrate, wherein the amount of distilled water added is 5 times the volume of the obtained filtrate, and then control the temperature at 121°C for extraction 1h, to obtain the extract;

The above-mentioned potato juice that removes potato starch can be replaced with the discarded potato juice of potato starch factory;

The above-mentioned extract is filtered with a SHZ-Ш type circulating water vacuum pump at a vacuum of -0.1MPa to remove the protein. Distilled water that is 10 times the volume of the obtained filtrate is added to the obtained filtrate after the suction filtration. After stirring and mixing, the protein-free potato juice aqueous solution is obtained;

(3) Preparation of sterilized protein-free potato juice

Adjust the pH of the protein-free potato juice aqueous solution obtained in step (2) to 4-8, then add glycerin, mix well, control the temperature at 121° C., and sterilize for 15 minutes to obtain sterilized protein-free potato juice;

The alkali used to adjust the pH of the protein-free potato juice aqueous solution is a NaOH aqueous solution with a mass percentage concentration of 1%, and the acid is a 0.1% HCl aqueous solution with a mass percentage concentration;

The added amount of the above-mentioned glycerin is 5-25% of the volume of the deproteinized potato juice aqueous solution whose pH is 4-8;

(4), inoculation fermentation

Inoculate Candida utilis CICC 1769 preserved in a glycerol tube into the sterilized deproteinized potato juice obtained in step (3), and then control the temperature to 24-32°C and the rotation speed to 100-200r/ Min carries out fermentation culture 24h, obtains fermented liquid;

The amount of inoculation is to add 1-8mL of Candida utilis CICC 1769 preserved in glycerol tubes per 100mL of sterilized deproteinized potato juice;

Candida utilis ( Candida utilis ) CICC 1769 stored in each 1mL glycerol tube contains 1.6×10 ⁹ CFU of Candida utilis ( Candida utilis ) CICC 1769;

(5), filtering the fermented liquid obtained in step (4), removing the filtrate, and the obtained filter cake is the cell of Candida utilis containing Candida utilis β-D-glucan;

(6) Under the condition of 121°C, extract the Candida utilis cells containing Candida utilis β-D-glucan in the cells obtained in step (5) with distilled water for 30 minutes, so that The somatic cells of Candida utilis were subjected to cell wall breaking, and then the obtained extract was subjected to ultrasonic treatment at an ultrasonic frequency of 20KHz and 800W for 60min through an ultrasonic cell disruptor, and then centrifuged at 10000r/min for 15min, and the obtained precipitate Wash with distilled water until the effluent is clear, stop the washing, and then control the temperature to -20°C and vacuum to 10 Pa to carry out vacuum freeze-drying to obtain Candida utilis β-D-glucan.

The Candida utilis β-D-glucan obtained by the above production method can improve the texture of food because it has the characteristics of general yeast β-D-glucan, and can be used as a source of dietary fiber in food , as a very good food additive has great development prospects. Further, the obtained Candida utilis β-D-glucan can significantly enhance the non-specific immune system of aquatic animals, and can greatly enhance the resistance of aquatic animals when applied in aquaculture. It is a pollution-free, natural green Approach. Furthermore, the resulting Candida utilis β-D-glucan can use its immunomodulatory activity to promote skin regeneration, regulate cell growth, and then achieve anti-aging effects, that is, the above-mentioned Candida utilis Yeast β-D-glucan can be used in food, feed, cosmetics and aquaculture.

Further, the Candida utilis β-D-glucan obtained by the above production method has the function of natural nutritional fiber because there is no enzyme to degrade β-glucan in the human body. Studies have shown that a high-fiber diet can lower cholesterol and reduce the incidence of chronic diseases such as arthritis and heart disease. When β-glucan enters the mouth, it will start to absorb water and swell. The dietary fiber of β-glucan can increase the transit time of the intestinal tract and slow down the absorption of the intestinal tract, so it can significantly reduce blood fat, blood sugar and improve immunity. It is suitable for people with diabetes, obesity, constipation and high cholesterol Patients with other diseases have a good role in adjuvant therapy.

Furthermore, the Candida utilis β-D-glucan obtained by the above production method can greatly improve the ability to resist bacteria, fungi and viruses because it can stimulate the innate immune system. Furthermore, Candida utilis β-D-glucan can activate T cells, macrophages and natural killer cells, and can also stimulate the differentiation and activation of T lymphocytes, affect and activate the changes in complement, and finally achieve Enhance the effect of the body's cellular and humoral immunity, so the Candida utilis β-D-glucan has health functions such as protecting the liver, protecting the gastric mucosa, improving microcirculation, and enhancing immunity.

Beneficial effects of the present invention

A kind of production method of Candida utilis β-D-glucan of the present invention, because the potato juice of removing starch utilized in the production process is tested, pH value is 4.82, and COD value is 29.8g O ₂ /L and The value of BOD ₅ is 15.6g O ₂ /L, close to pH, COD, BOD ₅ index of the discarded potato juice of potato starch factory, so the production method of Candida utilis β-D-glucan of the present invention can be Use the waste potato juice from the potato starch factory as the raw material of the medium for production, thereby alleviating the environmental pollution problem caused by the waste potato juice, realizing turning waste into wealth, thereby reducing the production cost of yeast β-D-glucan, and The production process is simple to operate and is convenient for large-scale production.

Further, in a production method of Candida utilis β-D-glucan of the present invention, since the Candida utilis cells containing Candida utilis β-D-glucan obtained during the production process By adopting the extraction method of high-temperature extraction and ultrasonic wall breaking synergistically, high-yield and high-purity Candida utilis β-D-glucan can be obtained with a purity of up to 97%. Up to 19%, yield up to 87%.

Description of drawings

Figure 1 Infrared spectrum of Candida utilis β-D-glucan;

Figure 2 UV full scan spectrum of Candida utilis β-D-glucan.

detailed description

The present invention will be further described below through specific embodiments in conjunction with the accompanying drawings, but the present invention is not limited.

The strain used in each embodiment of the present invention is Candida utilis ( Candida utilis ), the strain number CICC1769, which was purchased from China Industrial Microorganism Culture Collection Management Center, and it was preserved in China Industrial Microbiology Culture Collection Management Center. Serial number: ATCC 9950; address of depository institution: Building 6, No. 24 Jiuxianqiao Middle Road, Chaoyang District, Beijing, postcode: 100015, date of supply: July 13, 2015.

Used instrument type and manufacturer's information in each embodiment of the present invention are as follows:

Ultrasonic cell disruptor, model JY99-IIDN, produced by Ningbo Xinzhi Biotechnology Co., Ltd.;

Circulating water vacuum pump, model SHZ-Ш, produced by Shanghai Yarong Biochemical Instruments.

Fourier transform infrared spectrometer, TENSOR series, German Bruker company;

Ultraviolet spectrophotometer, UV8000 type, Shanghai Yuanxi Instrument Co., Ltd.

The mensuration of Candida utilis β-D-glucan content in each embodiment of the present invention, take glucose as standard, adopt phenol-sulfuric acid method to measure the Candida utilis β-D-glucan obtained in each embodiment Sugar content (references: Song Bo, Zhao Xia, Li Guoqiang. Extraction, separation and activity evaluation of a polysaccharide from soft corals in the South China Sea[J]. China Marine Medicine, 2016, 35(5):1-6.DOI:10.13400/ j.cnki.cjmd.2016.05.001.);

Purity (%) of Candida utilis β-D-glucan in each embodiment of the present invention = mass of yeast β-D-glucan ÷ mass of crude polysaccharide obtained in the cell wall of Candida utilis × 100%;

Yield of Candida utilis β-D-glucan (%) = actually obtained mass of Candida utilis β-D-glucan ÷ mass of Candida utilis cell × 100%;

The yield of Candida utilis β-D-glucan (%) = the actual mass of Candida utilis β-D-glucan ÷ the theoretically contained β-glucan in Candida utilis cells The mass of D-glucan × 100%.

Example 1

A production method of Candida utilis β-D-glucan, specifically comprising the following steps:

(1) Preparation of potato juice with potato starch removed

Take 40.0g of potatoes and smash them into mashed potatoes to obtain mashed potatoes, then add 400mL of distilled water to the mashed potatoes, stir and mix well, and centrifuge at 10000r/min for 20min to remove the precipitate, that is, remove the potato starch, and place the obtained filtrate from the removal of potato starch Potato juice with potato starch removed in 5 days;

The pH value of the potato juice obtained above was tested to be 4.82, the COD value was 29.8g O ₂ /L and the BOD ₅ value was 15.6g O ₂ /L;

(2), preparation of protein-removed potato juice aqueous solution

Filter 280 mL of the potato juice obtained in step (1) without potato starch through 8 layers of gauze, take 100 mL of the obtained filtrate, add 500 mL of distilled water, and extract at a temperature of 121 °C for 1 hour to obtain an extract;

The extract obtained above is suction-filtered with a circulating water vacuum pump at a vacuum of -0.1MPa to remove protein. Take 200mL of the filtrate obtained after suction-filtration, add 2000mL of distilled water to it, and mix well to obtain protein-free potatoes. juice solution;

(3) Preparation of sterilized protein-free potato juice

Use 1% NaOH aqueous solution and 0.1% HCl aqueous solution to adjust the pH of the deproteinized potato juice aqueous solution obtained in step (2) to 4, then add glycerin, mix well and control the temperature to 121°C Sterilize for 15 minutes to obtain deproteinized potato juice after sterilization;

The addition of above-mentioned glycerin is 25% of the volume of the potato juice aqueous solution that the pH is 4;

(4), inoculation fermentation

Inoculate Candida utilis CICC 1769 preserved in a glycerol tube into the sterilized deproteinized potato juice obtained in step (3), and then control the temperature at 24°C and the rotation speed at 100r/min for fermentation culture 24h, obtain fermented liquid;

The amount of inoculum is to insert 1 mL of Candida utilis CICC 1769 preserved in a glycerol tube into every 100 mL of sterilized protein-free potato juice;

The number of Candida utilis ( Candida utilis ) CICC 1769 stored in each 1mL glycerol tube is 1.6×10 ⁹ CFU (CFU means the activity per unit volume) number of bacteria);

(5) Filter the fermentation broth obtained in step (4) with a Buchner funnel, remove the filtrate, and obtain a filter cake that contains Candida utilis β-D-glucan in the cells Bacteria;

After determination, calculate by every 1mL fermented liquid, obtain the Candida utilis bacterium that contains Candida utilis β-D-glucan in the cell of 8.20mg;

Candida utilis containing Candida utilis β-D-glucan per mg cell contains 0.0986mg of β-D-glucan;

Each mL of fermentation broth contains 0.5394 mg of Candida utilis β-D-glucan;

(6) Under the condition of 121°C, the ratio of Candida utilis bacteria containing Candida utilis β-D-glucan in the cell: distilled water is 15:100 calculated by mass ratio. Extracting the Candida utilis cells containing Candida utilis β-D-glucan in the cells obtained in step (5) for 30 minutes, so that the cells of the Candida utilis cells are broken down, Then, the frequency of the ultrasonic cell disruptor is 20KHz, the power is 800W, and the ultrasonic treatment is carried out for 60min, and then centrifuged at 10000r/min for 15min, and the precipitate obtained is washed with distilled water until the effluent is clear, and the washing is terminated, and then the temperature is controlled to be -20°C, vacuum freeze-drying at 10 Pa to obtain Candida utilis β-D-glucan.

It was determined that the yield of the Candida utilis β-D-glucan obtained above was 82%, the yield was 13%, and the purity was 96%.

The Candida utilis β-D-glucan obtained above is measured by a Fourier transform infrared spectrometer, and the infrared spectrogram of the obtained Candida utilis β-D-glucan is shown in Figure 1, from It can be seen from Fig. 1 that the extracted dextran has a peak of β-type near 890cm ^-1 , and a peak near 3200cm ^-1 is the characteristic peak absorption peak of glycosidic bonds, which shows that the sample obtained by this method is glycoside Yeast glucan whose bond type is β-configuration;

Adopt ultraviolet spectrophotometer to measure above-mentioned obtained Candida utilis β-D-glucan, the ultraviolet spectrogram of the obtained Candida utilis β-D-glucan is as shown in Figure 2, from the figure It can be seen in 2 that within the wavelength range of 190-400nm, Candida utilis β-D-glucan does not contain impurities such as protein and nucleic acid, which shows that the obtained sample has a high purity.

Example 2

A production method of Candida utilis β-D-glucan, specifically comprising the following steps:

(1) Preparation of potato juice with potato starch removed

With the step (1) of embodiment 1;

(2), preparation of protein-removed potato juice aqueous solution

With the step (2) of embodiment 1;

(3) Preparation of sterilized protein-free potato juice

Use 1% NaOH aqueous solution and 0.1% HCl aqueous solution to adjust the pH of the deproteinized potato juice aqueous solution obtained in step (2) to 8, then add glycerin, mix well and control the temperature to 121°C Sterilize for 15 minutes to obtain deproteinized potato juice after sterilization;

The addition amount of above-mentioned glycerin is 15% of the volume of the potato juice aqueous solution that the pH is 8;

(4), inoculation fermentation

Inoculate Candida utilis CICC 1769 preserved in a glycerol tube into the sterilized deproteinized potato juice obtained in step (3), and then control the temperature at 28°C and the rotation speed at 150r/min for fermentation culture 24h, obtain fermented liquid;

The amount of inoculum is to add Candida utilis CICC 1769 preserved in a 4 mL glycerol tube per 100 mL of sterilized deproteinized potato juice;

Candida utilis ( Candida utilis ) CICC 1769 stored in each 1mL glycerol tube contains 1.6×10 ⁹ CFU of Candida utilis ( Candida utilis ) CICC 1769;

(5) Filter the fermentation broth obtained in step (4) with a Buchner funnel, remove the filtrate, and obtain a filter cake that contains Candida utilis β-D-glucan in the cells Bacteria;

After determination, 10.16mg (dry weight) of Candida utilis containing Candida utilis β-D-glucan in the cell was obtained on the basis of per 1mL fermentation broth;

Candida utilis containing Candida utilis β-D-glucan in each mg cell contains 0.1039mg of β-D-glucan;

Each mL of fermentation broth contains 0.7988 mg of Candida utilis β-D-glucan;

(6) Under the condition of 121°C, the ratio of Candida utilis bacteria containing Candida utilis β-D-glucan in the cell: distilled water is 15:100 calculated by mass ratio. Extracting the Candida utilis cells containing Candida utilis β-D-glucan in the cells obtained in step (5) for 30 minutes, so that the cells of the Candida utilis cells are broken down, Then, the frequency of the ultrasonic cell disruptor is 20KHz, the power is 800W, and the ultrasonic treatment is carried out for 60min, and then centrifuged at 10000r/min for 15min, and the precipitate obtained is washed with distilled water until the effluent is clear, and the washing is terminated, and then the temperature is controlled to be -20°C, vacuum freeze-drying at 10 Pa to obtain Candida utilis β-D-glucan.

It was determined that the yield of the Candida utilis β-D-glucan obtained above was 87%, the yield was 19%, and the purity was 97%.

Example 3

A production method of Candida utilis β-D-glucan, specifically comprising the following steps:

(1) Preparation of potato juice with potato starch removed

With the step (1) of embodiment 1;

(2), preparation of protein-removed potato juice aqueous solution

With the step (2) of embodiment 1;

(3) Preparation of sterilized protein-free potato juice

Use 1% NaOH aqueous solution and 0.1% HCl aqueous solution to adjust the pH of the deproteinized potato juice aqueous solution obtained in step (2) to 7, then add glycerin, mix well and control the temperature to 121°C Sterilize for 15 minutes to obtain deproteinized potato juice after sterilization;

The addition amount of above-mentioned glycerin is 10% of the volume of the potato juice aqueous solution that the pH is 7;

(4), inoculation fermentation

Inoculate Candida utilis CICC 1769 preserved in a glycerol tube into the sterilized deproteinized potato juice obtained in step (3), and then control the temperature at 30°C and the rotation speed at 200r/min for fermentation culture 24h, obtain fermented liquid;

The amount of inoculum is to add Candida utilis ( Candida utilis ) CICC 1769 preserved in a 2mL glycerol tube per 100mL of sterilized deproteinized potato juice;

Candida utilis ( Candida utilis ) CICC 1769 stored in each 1mL glycerol tube contains 1.6×10 ⁹ CFU of Candida utilis ( Candida utilis ) CICC 1769;

(5) Filter the fermentation broth obtained in step (4) with a Buchner funnel, remove the filtrate, and obtain a filter cake that contains Candida utilis β-D-glucan in the cells Bacteria;

After determination, calculate by every 1mL fermented liquid, obtain the Candida utilis bacterium that contains Candida utilis β-D-glucan in the cell of 9.23mg;

0.0998 mg of β-D-glucan in Candida utilis containing Candida utilis β-D-glucan in each mg cell;

That is, each mL of fermented liquid obtained by the production method of the present invention contains 0.6729 mg of Candida utilis β-D-glucan;

(6) Under the condition of 121°C, the ratio of Candida utilis bacteria containing Candida utilis β-D-glucan in the cell: distilled water is 15:100 calculated by mass ratio. Extracting the Candida utilis cells containing Candida utilis β-D-glucan in the cells obtained in step (5) for 30 minutes, so that the cells of the Candida utilis cells are broken down, Then, the frequency of the ultrasonic cell disruptor is 20KHz, the power is 800W, and the ultrasonic treatment is carried out for 60min, and then centrifuged at 10000r/min for 15min, and the precipitate obtained is washed with distilled water until the effluent is clear, and the washing is terminated, and then the temperature is controlled to be -20°C, vacuum freeze-drying at 10 Pa to obtain Candida utilis β-D-glucan.

It was determined that the yield of the Candida utilis β-D-glucan obtained above was 83%, the yield was 17%, and the purity was 97%.

Example 4

A production method of Candida utilis β-D-glucan, specifically comprising the following steps:

(1) Preparation of potato juice with potato starch removed

With the step (1) of embodiment 1;

(2), preparation of protein-removed potato juice aqueous solution

With the step (2) of embodiment 1;

(3) Preparation of sterilized protein-free potato juice

Use 1% NaOH aqueous solution and 0.1% HCl aqueous solution to adjust the pH of the deproteinized potato juice aqueous solution obtained in step (2) to 6, then add glycerin, mix well and control the temperature to 121°C Sterilize for 15 minutes to obtain deproteinized potato juice after sterilization;

The addition amount of above-mentioned glycerol is 20% of the volume of the potato juice aqueous solution that the pH is 7;

(4), inoculation fermentation

Inoculate Candida utilis CICC 1769 preserved in glycerol tubes into the sterilized deproteinized potato juice obtained in step (3), and then control the temperature at 32°C and the rotation speed at 200r/min for fermentation culture 24h, obtain fermented liquid;

The amount of inoculum is to insert Candida utilis CICC 1769 preserved in 8 mL glycerol tubes into every 100 mL of sterilized protein-free potato juice;

Candida utilis ( Candida utilis ) CICC 1769 stored in each 1mL glycerol tube contains 1.6×10 ⁹ CFU of Candida utilis ( Candida utilis ) CICC 1769;

(5) Filter the fermentation broth obtained in step (4) with a Buchner funnel, remove the filtrate, and obtain a filter cake that contains Candida utilis β-D-glucan in the cells Bacteria;

After determination, calculate by every 1mL fermented liquid, obtain the Candida utilis bacterium that contains Candida utilis β-D-glucan in the cell of 7.98mg;

Candida utilis containing Candida utilis β-D-glucan per mg cell, containing 0.0993mg of β-D-glucan;

That is, each mL of fermented liquid obtained by the production method of the present invention contains 0.6236 mg of Candida utilis β-D-glucan;

(6) Under the condition of 121°C, the ratio of Candida utilis bacteria containing Candida utilis β-D-glucan in the cell: distilled water is 15:100 calculated by mass ratio. Extracting the Candida utilis cells containing Candida utilis β-D-glucan in the cells obtained in step (5) for 30 minutes, so that the cells of the Candida utilis cells are broken down, Then, the frequency of the ultrasonic cell disruptor is 20KHz, the power is 800W, and the ultrasonic treatment is carried out for 60min, and then centrifuged at 10000r/min for 15min, and the precipitate obtained is washed with distilled water until the effluent is clear, and the washing is terminated, and then the temperature is controlled to be -20°C, vacuum freeze-drying at 10 Pa to obtain Candida utilis β-D-glucan.

It was determined that the yield of the Candida utilis β-D-glucan obtained above was 83%, the yield was 15%, and the purity was 96%.

Example 5

A production method of Candida utilis β-D-glucan, specifically comprising the following steps:

(1) Preparation of potato juice with potato starch removed

With the step (1) of embodiment 1;

(2), preparation of protein-removed potato juice aqueous solution

With the step (2) of embodiment 1;

(3) Preparation of sterilized protein-free potato juice

Use 1% NaOH aqueous solution and 0.1% HCl aqueous solution to adjust the pH of the deproteinized potato juice aqueous solution obtained in step (2) to 5, then add glycerin, mix well and control the temperature to 121°C Sterilize for 15 minutes to obtain deproteinized potato juice after sterilization;

The added amount of above-mentioned glycerin is 5% of the volume of the potato juice aqueous solution that the pH is 7;

(4), inoculation fermentation

Inoculate Candida utilis CICC 1769 preserved in glycerol tubes into the sterilized protein-free potato juice obtained in step (3), and then control the temperature at 26°C and the rotation speed at 100r/min for fermentation culture 24h, obtain fermented liquid;

The amount of inoculum is to insert Candida utilis ( Candida utilis ) CICC 1769 preserved in a 6mL glycerol tube into every 100mL of sterilized deproteinized potato juice;

Candida utilis ( Candida utilis ) CICC 1769 stored in each 1mL glycerol tube contains 1.6×10 ⁹ CFU of Candida utilis ( Candida utilis ) CICC 1769;

(5) Filter the fermentation broth obtained in step (4) with a Buchner funnel, remove the filtrate, and obtain a filter cake that contains Candida utilis β-D-glucan in the cells Bacteria;

After determination, calculate by every 1mL fermented liquid, obtain the Candida utilis bacterium that contains Candida utilis β-D-glucan in the cell of 8.91mg;

Candida utilis containing Candida utilis β-D-glucan per mg cell, containing 0.0991mg of β-D-glucan;

That is, each mL of fermented liquid obtained by the production method of the present invention contains 0.6021 mg of Candida utilis β-D-glucan;

(6) Under the condition of 121°C, the ratio of Candida utilis bacteria containing Candida utilis β-D-glucan in the cell: distilled water is 15:100 calculated by mass ratio. Extracting the Candida utilis cells containing Candida utilis β-D-glucan in the cells obtained in step (5) for 30 minutes, so that the cells of the Candida utilis cells are broken down, Then, the frequency of the ultrasonic cell disruptor is 20KHz, the power is 800W, and the ultrasonic treatment is carried out for 60min, and then centrifuged at 10000r/min for 15min, and the precipitate obtained is washed with distilled water until the effluent is clear, and the washing is terminated, and then the temperature is controlled to be -20°C, vacuum freeze-drying at 10 Pa to obtain Candida utilis β-D-glucan.

It was determined that the yield of the Candida utilis β-D-glucan obtained above was 83%, the yield was 14%, and the purity was 96%.

In summary, a kind of production method of Candida utilis β-D-glucan of the present invention, because the potato juice raw material of removing starch utilized in the production process is tested, pH value is 4.82, is 29.8g. ₂ /L and the value of BOD ₅ is 15.6g O ₂ /L, which is close to the pH, COD, BOD ₅ index of the potato juice discarded by the potato starch factory, so the Candida utilis β-D-glucan of the present invention The production method of sugar can use the waste potato juice from the potato starch factory as the raw material of the medium for production. By adopting the extraction method of high-temperature extraction and ultrasonic wall breaking synergistically, the high-yield, high-purity, and high-yield Candida utilis yeast β-D-glucan is finally obtained, and the highest yield can be up to 19%, its purity can reach up to 97%, and its yield can reach up to 87%.

The above embodiments are only used to illustrate the content of the present invention. In addition, the present invention also has other implementation modes, as long as those skilled in the art adopt equivalent replacement or equivalent deformation methods due to the technical inspiration involved in the present invention. The technical solutions all fall within the protection scope of the present invention.

Claims (8)

1. a production method of Candida utilis β-D-glucan, is characterized in that specifically comprising the following steps: (1) Preparation of starch-removed potato juice Take a potato grinder and crush it to obtain mashed potatoes, then add distilled water with a mass ratio of 10 times to the mashed potatoes, stir and mix well, remove the potato starch by centrifuging at 10000r/min for 20min, and place the obtained filtrate for 5 days to obtain the removed potato starch. starchy potato juice; (2), preparation of protein-removed potato juice aqueous solution Filter the potato juice obtained in step (1) without potato starch through 8 layers of gauze, add distilled water to the obtained filtrate, wherein the amount of distilled water added is 5 times the volume of the obtained filtrate, and then control the temperature at 121°C for extraction 1h, to obtain the extract; The extract obtained above is suction-filtered with a circulating water vacuum pump at a vacuum of -0.1MPa, and distilled water 10 times the volume of the obtained filtrate is added to the filtrate obtained after suction filtration, and the protein-free potato juice is obtained after stirring and mixing. aqueous solution; (3) Preparation of sterilized protein-free potato juice Adjust the pH of the protein-free potato juice aqueous solution obtained in step (2) to 4-8, then add glycerin, mix well, control the temperature at 121° C., and sterilize for 15 minutes to obtain sterilized protein-free potato juice; The alkali used to adjust the pH of the protein-free potato juice aqueous solution is a NaOH aqueous solution with a mass percentage concentration of 1%, and the acid is a 0.1% HCl aqueous solution with a mass percentage concentration; The added amount of the above-mentioned glycerin is 5-25% of the volume of the deproteinized potato juice aqueous solution whose pH is 4-8; (4), inoculation fermentation Inoculate Candida utilis CICC 1769 preserved in a glycerol tube into the sterilized deproteinized potato juice obtained in step (3), and then control the temperature to 24-32°C and the rotation speed to 100-200r/ Min carries out fermentation culture 24h, obtains fermented liquid; The amount of inoculation is to add 1-8mL of Candida utilis CICC 1769 preserved in glycerol tubes per 100mL of sterilized deproteinized potato juice; Candida utilis ( Candida utilis ) CICC 1769 stored in each 1mL glycerol tube contains 1.6×10 ⁹ CFU of Candida utilis ( Candida utilis ) CICC 1769; (5), filtering the fermented liquid obtained in step (4), removing the filtrate, and the obtained filter cake is the cell of Candida utilis containing Candida utilis β-D-glucan; (6) Under the condition of 121°C, extract the Candida utilis cells containing Candida utilis β-D-glucan in the cells obtained in step (5) with distilled water for 30 minutes, so that The somatic cells of Candida utilis were subjected to cell wall breaking, and then the obtained extract was subjected to ultrasonic treatment at an ultrasonic frequency of 20KHz and 800W for 60min through an ultrasonic cell disruptor, and then centrifuged at 10000r/min for 15min, and the obtained precipitate Wash with distilled water until the effluent is clear, stop the washing, and then control the temperature to -20°C and vacuum to 10 Pa to carry out vacuum freeze-drying to obtain Candida utilis β-D-glucan. 2. A method for cultivating high-yield Candida utilis β-D-glucan as claimed in claim 1, characterized in that the potato juice removed from the starch in step (1) uses the discarded potatoes of the potato starch factory juice instead. 3. a kind of culture method of high yield Candida utilis β-D-glucan as claimed in claim 1, is characterized in that: In step (3): adjust the pH of the deproteinized potato juice aqueous solution obtained in step (2) to 7-8, and the amount of glycerin added is 10-15% of the volume of the deproteinized potato juice aqueous solution with a pH of 7-8; In step (4): control the temperature at 28-30°C, and carry out fermentation at a rotational speed of 150-200r/min; the inoculation amount is 2-4mL of eugenase preserved in glycerin tubes for every 100mL of sterilized deproteinized potato juice. Candida utilis CICC1769. 4. a kind of culture method of high yield Candida utilis β-D-glucan as claimed in claim 3, is characterized in that: In step (3): adjusting the pH of the deproteinized potato juice aqueous solution obtained in step (2) to be 7, and the amount of glycerin added is 10% of the volume of the deproteinized potato juice aqueous solution with a pH of 7; In step (4): control the temperature at 30°C, and carry out fermentation at a rotational speed of 200r/min; the inoculation amount is based on the amount of Candida utilis preserved in 2mL glycerol tubes for every 100mL of sterilized deproteinized potato juice. ) CICC 1769. 5. a kind of culture method of high yield Candida utilis β-D-glucan as claimed in claim 3, is characterized in that: In step (3): the pH of the deproteinized potato juice aqueous solution obtained in step (2) is adjusted to be 8, and the amount of glycerin added is 15% of the volume of the deproteinized potato juice aqueous solution with a pH of 8; In step (4): control the temperature at 28°C, and carry out fermentation at a rotational speed of 150r/min; the inoculation amount is based on the amount of Candida utilis preserved in a 4mL glycerol tube for every 100mL of sterilized deproteinized potato juice. ) CICC 1769. 6. a kind of culture method of high yield Candida utilis β-D-glucan as claimed in claim 1, is characterized in that: In step (3): adjust the pH of the deproteinized potato juice aqueous solution obtained in step (2) to be 7, and the amount of glycerin added is 20% of the volume of the deproteinized potato juice aqueous solution with a pH of 7; In step (4): control the temperature at 32°C, and carry out fermentation at a rotational speed of 200r/min; the inoculation amount is based on the amount of Candida utilis preserved in 8mL glycerol tubes for every 100mL of sterilized deproteinized potato juice. ) CICC 1769. 7. a kind of culture method of high yield Candida utilis β-D-glucan as claimed in claim 1, is characterized in that: In step (3): adjust the pH of the deproteinized potato juice aqueous solution obtained in step (2) to 7, and the amount of glycerin added is 5% of the volume of the deproteinized potato juice aqueous solution with a pH of 7; In step (4): control the temperature at 26°C, and carry out fermentation at a rotational speed of 100r/min; the inoculation amount is based on the amount of Candida utilis preserved in a 6mL glycerol tube for every 100mL of sterilized deproteinized potato juice. ) CICC 1769. 8. a kind of culture method of high yield Candida utilis β-D-glucan as claimed in claim 1, is characterized in that: In step (3): adjust the pH of the deproteinized potato juice aqueous solution obtained in step (2) to 4, and the amount of glycerin added is 25% of the volume of the deproteinized potato juice aqueous solution with a pH of 4; In step (4): control the temperature at 24°C, and carry out fermentation at a rotational speed of 100r/min; the inoculation amount is based on the amount of Candida utilis preserved in a 1mL glycerol tube for every 100mL of sterilized deproteinized potato juice. ) CICC 1769.