CN1329519C - Gamma-linolenic acid dry microbial powder enriched with organic celenium and its producing process - Google Patents

Abstract

The invention relates to a dry bacterial powder of gamma-linolenic acid rich in organic selenium, a production process thereof, and a mutagenic strain used for biosynthesizing the bacterial powder. Using the mutagenized CGMCC No. 1652, silvery mildew C. californica was used to carry out selenium-enriched fermentation on a medium containing organic carbon sources, organic nitrogen sources, inorganic salts, and sodium selenite to obtain γ- The linolenic acid bacteria are then dried by microwave vacuum freeze-sublimation technology to obtain γ-linolenic acid dried bacteria powder with an oil content of 35-39%, a γ-linolenic acid content of 15-18%, and a selenium content of 139-330μg/g. After multiple production tests, the present invention has mature technology, simple process, environmental protection, is suitable for large-scale industrial production, and has the characteristics of wide source of raw materials, low cost, good quality and the like. Its product, γ-linolenic acid dried bacteria powder rich in organic selenium, can be widely used in medicine, food, feed additives and other fields.

Description

γ-linolenic acid dried bacteria powder rich in organic selenium and its production process

technical field

The present invention relates to a kind of gamma-linolenic acid dried bacteria powder and its production process, in particular to a kind of gamma-linolenic acid dried bacteria powder product rich in organic selenium fermented by the mutagenic strain of C. and its craft.

Background technique

Gamma-Linolenic Acid (Gamma-Linolenic Acid), GLA for short, molecular formula: C ₁₈ H ₃₀ O ₂ , is a kind of octadecatrienoic acid, which belongs to polyene active substances. GLA is an essential unsaturated fatty acid for the human body, and it is a large class of substances that regulate the vitality of human cells. It has the effects of treating hypertension, hyperlipidemia, hyperglycemia, diabetes, correcting lipid metabolism disorders, treating liver cirrhosis and nephrotic syndrome, inhibiting the proliferation of cancer cells, and enhancing human immune function.

In the prior art, gamma-linolenic acid has been extracted from plants such as evening primrose, but due to the lack of raw materials, the output of gamma-linolenic acid is limited, which is far from meeting the needs of domestic and foreign markets. In recent years, a method of biosynthesizing γ-linolenic acid has been developed, using Mortierella, C. chrysalis and other microbial strains to ferment and produce γ-linolenic acid powder. The main component of biosynthesized γ-linolenic acid, in addition to polyene linoleic acid (the main component of Maitong) and linolenic acid, also contains anti-mutation, anti-radiation, anti-fatigue, anti-aging superoxide dismutase (SOD ), high-molecular polysaccharides that enhance the body's immunity, as well as proteins, various amino acids, vitamins and trace elements. The comprehensive utilization of its multiple active ingredients has been proved by a large number of clinical and pathological experiments to have remarkable curative effect without any toxic and side effects, and does not contain any hormones.

Selenium is an indispensable trace element for human and animal life activities. Scientific research shows that more than 40 kinds of diseases are related to selenium. Selenium deficiency can cause anemia, cardiovascular and cerebrovascular diseases, liver disease, diabetes, cancer, poor growth in children, and premature aging of the human body. Selenium is the active center of various antioxidant enzymes, and its antioxidant capacity is 400 times that of Ve. It can effectively inhibit the division and proliferation of cancer cells and enhance human immunity. Selenium supplementation can purify blood and enhance myocardial power. At present, some people use pure linolenic acid and selenium to synthesize selenized linolenic acid by chemical method, and obtain a product with obvious antitumor effect. However, there are chemical lysozyme residues in the product obtained by chemical synthesis, which is harmful to the human body, and this problem has not been solved yet.

Contents of the invention

One of purpose of the present invention is to provide a kind of novel technology that utilizes microbial fermentation method (industrialization) to produce the gamma-linolenic acid dry bacterial powder that is rich in organic selenium; γ-linolenic acid dry bacterial powder product containing organic selenium; the third purpose is to provide a selenium-resistant bacterial strain with high yield of GLA obtained through mutagenesis.

The present invention adopts following technical scheme to solve above-mentioned invention object:

1. strain mutagenesis: Cunninghamella echinulata (Cunninghamella echinulata) CGMCC NO.1652 used in the present invention is based on the AS3.2473 Cunninghamella echinulata provided by the General Microbiology Center of China Microbiological Culture Collection Committee as the original bacteria species obtained by mutagenesis. The specific mutagenesis process is as follows: the young mycelium is processed to obtain protoplasts, and after microwave and nitrosoguanidine composite treatment, the treated strain suspension is coated with sodium selenite 10mg/L-200mg/L Cultivate on PDA plates, pick out the strains with obvious differences in morphology, and then obtain them through shake flask re-selection. Compared with the original strain, the strain has the characteristics of fast growth, large number of spores, strong hyphae, and stable genetic properties after subculture.

The strain was cultured on PDA medium at a temperature of 28°C. On the 2nd day, the diameter of the colony was 2-5cm, the mycelium was off-white, and some conidia formed; on the 3rd-4th day, the diameter of the colony was 4-10cm, a large number of conidia formed, and the colony was dark gray.

The hyphae have no septa at the beginning, and later form septa. The hyphae are thick and branched, about 11-19 microns in diameter. Cyst peduncle erect, branched umbrella-shaped. The sporangia are solitary and spherical. Conidia are spherical or oval, with long spines and can fall off. The spores are 9.5-13.2 microns in diameter. Conidia cymose, quincunx or irregular.

2. Seed solution culture: inoculate the prepared spore suspension in the seed tank for cultivation. The composition of the medium includes: 3-5% of sucrose (or glucose), 0.05-0.1% of peptone, 0.01-0.03% of potassium dihydrogen phosphate, 0.01-0.03% of magnesium sulfate, 0.03-0.05% of calcium carbonate, and 0.0005% of sodium selenite -0.001%, pH 5.5-6, temperature 22-30°C, incubation time 24-36 hours.

3. Fermentation tank culture: put the well-grown seed liquid into the fermentation tank. The composition of the fermentation medium includes: 8-15% of sucrose (or starch or molasses), 1-5% of peanut powder, 1-5% of peptone, 0.01-0.03% of potassium dihydrogen phosphate, 0.01-0.03% of magnesium sulfate, carbonic acid Calcium 0.03-0.05%, sodium selenite 0.0005-0.001%, trace growth factor, PH5.5-6, temperature 22-30℃, air volume 1: 0.05-0.5v/v, stirring speed 150-200rpm, tank pressure 0.02-0.08mpa, culture time 96-100 hours.

4. Thalline collection: the obtained fresh gamma-linolenic acid bacteria are dried by microwave vacuum freeze-sublimation drying method. The reaction parameters are as follows:

Vacuum chamber: diameter 1.6m, length 13m, vacuum degree 30mbar

Input microwave frequency: 2450MHz±50MHz

Input microwave power: 48KW±2KW

Conveyors are carried out using glass reinforced PTFE conveyor belts.

Put the γ-linolenic acid fresh bacteria pre-frozen at -5°C on the conveyor belt, input microwave energy at a low pressure of 80-100T, heat for 40 minutes, and keep the temperature at 15-20% of the hot air convection, from the inside to the outside Evaporate water. The drying time is 4-6 hours in total.

Product technical index of the present invention is as follows: in every 100g dried bacteria powder,

Oil content: 35-39%

Gamma-linolenic acid content: 15-18%

Selenium content: 139-330μg/g

Compound amino acid: 3.5-6.8g

Proteoglycan: 4-7g

Superoxide dismutase (SOD): 543.7μg/g

Adopt above-mentioned technical scheme to implement the present invention, its main beneficial effect is as follows:

1. Use the mutagenized bacterial strain to ferment and produce γ-linolenic acid. While screening high-yield GLA strains, culture the bacterial strain with a medium containing high-concentration selenium, and screen out selenium-containing selenium-resistant bacterial strains (common C. Yinhan mold can not tolerate selenium growth), and adopted corresponding conditions in the fermentation culture, obtained the gamma-linolenic acid dry bacterial powder product that not only contains GLA but also is rich in organic selenium, wherein the content of gamma-linolenic acid is 12-15 %, up to 18%. At the same time, it is rich in selenium, and the selenium content is as high as 139-330μg/g. Experiments have proved that the synergistic effect of various substances with physiological functions can significantly improve the efficacy of the product.

2. During the preparation process, the main raw materials (carbon source and nitrogen source) are all natural organic matter, and no inorganic raw materials are used, which improves the safety of the product and is environmentally friendly.

3. In view of the characteristics of high oil content and easy oxidation of the product, the product is dried by microwave vacuum freeze-sublimation drying method. This method is an organic combination of vacuum microwave drying and vacuum freeze-sublimation drying, where microwaves mainly play the role of heating. In the past, in the vacuum freezing and sublimation technology, devices such as electric heating or heat pump units were used for heating. After using microwaves, the above devices can be eliminated. The basic principle of freeze-drying is based on the three-state change of water, and the three phases can be converted to each other and can coexist. When water is at the triple point (temperature 0.01°C, water vapor pressure 610.5Pa), water, ice, and water vapor can coexist and balance each other. In a high vacuum state, using the principle of sublimation, the water in the pre-frozen γ-linolenic acid bacteria can be directly sublimated into water vapor in the ice state and removed without melting the ice, so as to achieve the purpose of freeze-drying. Freeze-dried γ-linolenic acid bacteria form a spongy shape, no drying shrinkage, excellent rehydration, and very little water content. After corresponding packaging, it can be stored and transported at room temperature for a long time. Compared with other drying methods, the advantages of microwave vacuum freeze sublimation drying are as follows:

Many heat-sensitive substances will not be denatured or inactivated;

When drying at low temperature, some volatile components in the gamma-linolenic acid bacteria lose very little;

At low temperature, the growth of microorganisms and the action of enzymes cannot be carried out, so the product can maintain its original state;

Due to drying in a frozen state, the volume of the product is almost unchanged, and the original structure of the gamma-linolenic acid bacteria is maintained, and no concentration phenomenon occurs;

Since the water in the gamma-linolenic acid cells exists in the form of ice crystals after pre-freezing, the inorganic salts originally dissolved in water are evenly distributed in the gamma-linolenic acid cells. During sublimation, the dissolved substances in water are precipitated, which avoids the phenomenon of surface hardening caused by the precipitation of inorganic salts carried by the internal moisture of γ-linolenic acid cells to the surface in the general drying method;

The dried gamma-linolenic acid bacteria are loose and porous, in the shape of a sponge, and dissolve quickly and completely after adding water, and almost immediately return to the original state;

Since the drying is carried out under vacuum, there is very little oxygen, so some easily oxidizable substances are protected;

Drying can remove more than 95-99% of water, so that the dried gamma-linolenic acid bacteria can be stored for a long time without deterioration.

Due to the existence of the above-mentioned advantages, the method of drying can increase the technical indicators of the gamma-linolenic acid dried bacteria powder by 1-3 percentage points, and effectively increase the added value of the gamma-linolenic acid dried bacteria powder.

4. Compared with chemically synthesized selenized linolenic acid, γ-linolenic acid dried bacteria powder rich in organic selenium obtained by biological fermentation method has no chemical lysozyme residue, no toxic and side effects on the human body, and a variety of physiological The synergistic effect of functional substances can obviously improve the efficacy of the product.

5. In the whole process of production and fermentation, a sterile area is set up and a fully enclosed operation is adopted, and the computer monitors the whole process, which can ensure the quality of the dried γ-linolenic acid powder and reduce the cost of industrial production.

Cunninghamellaechinulata used in the present invention has been preserved in the General Microorganism Center of China Committee for the Collection of Microorganisms on March 16, 2006, and the preservation number is CGMCC NO.1652.

Detailed ways

The following examples further illustrate the invention.

Embodiment 1: 1 ton fermenter produces thalline

1. Inoculate the slant strain CGMCC NO.1652 into a Kirschner flask for expanded cultivation at a temperature of 22-30°C for 3-5 days.

2. Inoculate the bacteria suspension into the seed tank, the volume of the seed tank is 100 liters, medium: 4 kg of sucrose, 0.4 kg of peptone, 20 g of potassium dihydrogen phosphate, 20 g of magnesium sulfate, 35 g of calcium carbonate, selenous acid Sodium 2.1g, pH 5.5-6, temperature 25-30°C, air volume 1:0.5v/v, stirring speed 150-200rpm, incubation time 32 hours.

3. Transfer the seed liquid to a fermenter with a volume of 1000 liters. Medium: 72kg sucrose, 4kg peanut cake powder, 200g potassium dihydrogen phosphate, 200g magnesium sulfate, 350g calcium carbonate, 21g sodium selenite, 2.3g growth-promoting factor, PH5.5-6, temperature 22-30℃, air volume 1: 0.25-0.55v/V, stirring speed 150-200rpm, tank pressure 0.02-0.08mpa, culture for 96 hours.

4. Bacterial cells are collected using microwave vacuum freeze-sublimation technology, input microwave frequency 2400MHz, input microwave power 46KW, heat for 40 minutes, and maintain the temperature of 15-20% hot air convection, and dry for 4-6 hours. 31.5 kg of bacteria were obtained, wherein the oil content per 100 g of dry bacteria powder was 35.5%, the GLA content was 12.5%, and the selenium content was 139.2 μg/g.

Embodiment 2: 5 tons of fermentation tanks produce thalline

1. Inoculate the slant strain CGMCC NO.1652 into a Kirschner flask for expanded cultivation at a temperature of 22-30°C for 3 days.

2. Inoculate the bacteria suspension into the seed tank, the volume of the seed tank is 500 liters, the medium: sucrose 15kg, peptone 3.5kg, potassium dihydrogen phosphate 90g, magnesium sulfate 90g, calcium carbonate 150g, selenous acid Sodium 12g, pH 5.5-6, temperature 22-30°C, air volume 1:0.3v/v, stirring speed 150-200rpm, tank pressure 0.02-0.08mpa, incubation time 28 hours.

3. Transfer the seed solution to a fermenter with a capacity of 5000 liters. Medium: 400kg sucrose, 35kg peanut cake powder, 900g potassium dihydrogen phosphate, 900g magnesium sulfate, 1500g calcium carbonate, 120g sodium selenite, 10.5g growth-promoting factor, PH5.5-6, temperature 22-30℃, fermentation When the sugar content is 4%, add 100kg of sterilized molasses, air volume 1: 0.1-0.3v/v, stirring speed 150-200rpm, tank pressure 0.02-0.08mpa, and cultivate for 100 hours.

4. Bacteria collection, with embodiment 1. 128 kg of bacteria were obtained, wherein in every 100 g of dry bacteria powder, the oil content was 32.3%, the GLA content was 18.1%, and the selenium content was 142.4 μg/g.

Embodiment 3: 5 tons of fermentation tanks produce thalline

1. Inoculate the slant strain CGMCC NO.1652 into a Kirschner flask for expanded cultivation at a temperature of 22-30°C for 3-4 days.

2. Inoculate the bacteria suspension into the seed tank, the volume of the seed tank is 500 liters, the medium: sucrose 15kg, peptone 3.5kg, potassium dihydrogen phosphate 90g, magnesium sulfate 90g, calcium carbonate 150g, selenous acid Sodium 12g, pH 5.5-6, temperature 25-30°C, air volume 1:0.3v/v, stirring speed 150-200rpm, tank pressure 0.02-0.08mpa, incubation time 30 hours.

3. Transfer the seed solution to a fermenter with a volume of 5000 liters. Medium: sucrose 400kg, peanut cake powder 35kg, potassium dihydrogen phosphate 900g, magnesium sulfate 900g, calcium carbonate 1500g, sodium selenite 120g, growth-promoting factor 10.5g, pH5.5-6, temperature 25-30℃, fermentation When the sugar content is 10%, add 80kg of sterilized molasses, air volume 1: 0.1-0.3v/v, stirring speed 150-200rpm, tank pressure 0.02-0.08mpa, and cultivate for 100 hours.

4. Bacteria collection, with embodiment 1. 120 kg of thalline was obtained, wherein the oil content per 100 g of dry bacterial powder was 37.3%, the GLA content was 16.2%, and the selenium content was 168.5 μg/g.

Embodiment 4: 5 tons of fermentation tanks produce thalline

1. Inoculate the slant strain CGMCC NO.1652 into a Kirschner flask for expanded cultivation at a temperature of 22-30°C for 3-4 days.

2. Inoculate the bacteria suspension into the seed tank with the temperature of 22° C. and the pressure of the tank at 0.05 mpa, and the rest are the same as in Example 3.

3. Transfer the seed solution to a fermenter, where the temperature is 30°C, the tank pressure is 0.08mpa, and the stirring speed is 180rpm, and the rest are the same as in Example 3.

4. Bacteria collection, with embodiment 1. 118 kg of thalline was obtained, wherein the oil content per 100 g of dry bacterial powder was 35.2%, the GLA content was 17.0%, and the selenium content was 156.3 μg/g.

Comparative example 1: 1 ton fermenter produces thalline

1. Same as Example 1. 2. Same as Example 1. 3. Same as Example 1.

4. Bacteria were collected by using the fluidized bed drying method. The drying time is 6-8 hours, and the temperature is 50-70°C.

Result comparison

Fluidized bed drying method Microwave Vacuum Freeze Sublimation Drying drying method open, susceptible to bacteria Vacuum, not easy to infect bacteria heat sensitive substances Easy to oxidize Not easily oxidized fat content 33.0% 35.5% GLA content 10.2% 12.5% Selenium content 110.4μg/g 139.2μg/g Compound Amino Acid Content 3.0g 5.8g proteoglycan content 3.2g 6.9g SOD content 540.1μg/g 543.7μg/g storage method Cryopreservation Store at room temperature

Comparative example 2: 5 tons of fermentation tanks produce thalline

1. Same as embodiment 3.

2. Same as embodiment 3.

3. Same as embodiment 3.

4. Bacteria were collected by using the fluidized bed drying method. The drying time is 6-8 hours, and the temperature is 50-70°C.

Result comparison

Fluidized bed drying method Microwave Vacuum Freeze Sublimation Drying fat content 32.7% 37.3% GLA content 9.8% 16.2% Selenium content 140.0μg/g 168.5μg/g Compound Amino Acid Content 4.5g 6.8g proteoglycan content 3.6g. 7.0g SOD content 538.2μg/g 543.7μg/g

It should be noted that the present invention can be summarized in other specific forms without departing from the spirit or main characteristics of the present invention. Therefore, no matter from which point of view, the above-mentioned embodiments of the present invention can only be considered as illustrations of the present invention rather than limiting the present invention.

Claims (6)

1. production method that is rich in the gamma-linolenic acid of organoselenium is characterized in that: adopting cunninghamella echinulata (Cunninghamella echinulata) CGMCC NO.1652 is that bacterial strain carries out rich selenium fermentation, may further comprise the steps:

(1) spawn culture: slant strains inserted carry out enlarged culturing in the Kolle flask;

(2) planting liquid cultivates: spore suspension is inoculated in the seeding tank cultivates, substratum is a carbon source with glucose and/or sucrose, is nitrogenous source with the peptone, and adds Sodium Selenite, PH5.5-6, and temperature 22-30 ℃, incubation time is 24-36 hour;

(3) fermentation culture: will cultivate in well-grown seed liquor access fermentor tank, substratum is so that any one or a few is a carbon source in sucrose, molasses, the starch, with groundnut meal and/or peptone is nitrogenous source, and the interpolation Sodium Selenite, PH5.5-6, temperature 22-30 ℃, tank pressure 0.02-0.08mpa, air quantity 1:0.05-0.5v/v, stirring velocity 150-200rpm, incubation time 96-100 hour;

(4) microorganism collection: adopt microwave vacuum freeze sublimation drying method drying, wherein the condition of microwave vacuum freeze sublimation drying is: input microwave frequency 2450MHz ± 50MHz, input microwave power 48KW ± 2KW, microwave heating 40 minutes, and the hot air convection of maintenance temperature 15-20%, be 4-6 hour time of drying, must be rich in the gamma-linolenic acid dry bacterial powder of organoselenium.

2. production method according to claim 1, the condition of wherein said spawn culture is: the PDA substratum, temperature 22-30 ℃, cultivated 3-5 days.

3. production method according to claim 1, wherein seed culture medium comprises: sucrose 3-5%, peptone 0.05-0.1%, potassium primary phosphate 0.01-0.03%, sal epsom 0.01-0.03%, lime carbonate 0.03-0.05%, Sodium Selenite 0.0005-0.001%.

4. production method according to claim 1, wherein fermention medium comprises: sucrose 8-15%, groundnut meal 1-5%, peptone 1-5%, potassium primary phosphate 0.01-0.03%, sal epsom 0.01-0.03%, lime carbonate 0.03-0.05%, Sodium Selenite 0.0005-0.001%, somatomedin trace, incubation time 96 hours.

5. according to any gamma-linolenic acid dry bacterial powder that described method is produced of claim 1-4.

6. cunninghamella echinulata (Cunninghamella echinulata) CGMCC NO.1652.