CN104694607B - A kind of method that solid state fermentation prepares beta carotene - Google Patents

Abstract

The invention relates to the field of bioengineering, in particular to a method for solid-state fermentation of β-carotene by using B. trispora. It is characterized in that the liquid fermentation medium in the traditional process is replaced by a solid fermentation medium, and after cultivation and fermentation, the obtained solid fermentation product containing mycelia can be directly dried under reduced pressure for extracting β-carotene therein. Compared with liquid fermentation, the process of obtaining mycelium by filtration or centrifugation is reduced, and the amount of water used in the process and the discharge of waste water are greatly reduced, which is conducive to the protection of my country's limited fresh water resources, especially suitable for water-scarce areas in northern my country promotion and application.

Description

A method for preparing β-carotene by solid-state fermentation

technical field

The invention relates to the field of bioengineering, in particular to a method for solid-state fermentation of beta-carotene by using B. trispora.

Background technique

The molecular formula of β-carotene is C ₄₀ H ₅₅ . It is an effective physiological antioxidant. It has the functions of anti-aging, preventing and treating cardiovascular diseases, anti-tumor, enhancing the body's specific and non-specific immune functions, regulating inflammatory reactions, and preventing radiation. It can prevent night blindness caused by vitamin A deficiency and promote the normal maturation of epithelial cells. It is also an ideal food coloring agent and nutritional supplement.

At present, there are three main methods for producing β-carotene, namely chemical synthesis, natural product extraction and microbial fermentation. Among them, the microbial fermentation method has the advantages of stable production, little influence by season, climate, time and geographical factors, short production cycle, relatively easy quality control, etc., and has increasingly attracted the attention of researchers and manufacturers. At present, the isolated β-carotene-producing microorganisms have been systematically compared and studied at home and abroad, and through mutagenesis screening and fermentation research, some microbial strains with application value have been found. Among them, B. trispora Due to the advantages of safe production and application of the obtained β-carotene, large biomass, and high β-carotene yield, it has become the main industrial application strain for preparing natural β-carotene by biological fermentation (CN 103484520A).

Through literature search, it is found that the current domestic and foreign studies on the fermentation of B. trispora producing β-carotene focus on liquid fermentation, also known as submerged fermentation or aeration and stirring fermentation. Shake culture on the bed, or put it in a fermenter, and pass in sterile air under stirring, so that the mycelium can grow in the liquid medium and produce β-carotene. Since β-carotene is almost insoluble in water, β-carotene is deposited in the mycelium during the fermentation process, and it is necessary to collect the mycelium and then extract β-carotene from it. In the process of traditional β-carotene liquid fermentation, to increase the yield needs to proceed from two aspects: 1) increase the content (biomass) of mycelium in the fermentation broth; 2) increase the content of β-carotene in the mycelium However, no matter which method is used, the fermentation product needs to be filtered or centrifuged, the bacteria are collected, dried and then extracted, and a large amount of aqueous liquid obtained after filtration or centrifugation is discarded as waste liquid.

CN 103484520A discloses a carotene fermentation process using a fermentation accelerator. The fermentation medium is placed on a 230rpm shaker for fermentation and cultivation. The fermentation level is improved by adding accelerators such as salicylic acid, and the yield can reach more than 2.25 g/L.

CN102787158A discloses a method for producing β-carotene by fermentation. The spore suspension of B. trispora is inoculated in a fermenter to ferment to prepare mycelium. The fermented liquid is adjusted to alkaline with an organic base, and wet Mycelium is treated with a hydrophobic organic solvent, and then the β-carotene in the mycelium is extracted with an ester organic solvent.

CN102757995A discloses a method for preparing β-carotene by fermenting Branella trispora trispora, using liquid culture medium containing bran extract, maltose, magnesium sulfate, etc. to obtain β-carotene with higher content Mycelium, the dry weight of mycelium can reach 21.5g/L～32.5g/L, and the content of β-carotene in dry mycelium can reach 2.6%～4.8% (w/w)

Publication No. CN1331343A discloses a method: inoculate the positive and negative strains of filamentous fungi in a stirred fermenter, use the upper and middle layers as a wide-leaf propulsion type, and the lower layer as a turbine-type combined stirring blade, which can increase the fermentation liquid. Excellent mass transfer performance and gas-liquid contact efficiency, more suitable for mycelium growth.

CN1193048A discloses a method: by cultivating positive and negative strains of B. trispora in an air-lift fermenter to ferment and produce β-carotene, and the yield can reach 1.5 g/L.

The methods used in the above-mentioned patents all use liquid fermentation medium, need to consume more water resources, and will produce a large amount of fermentation waste liquid.

Contents of the invention

The invention discloses a method for fermenting β-carotene by using B. trispora B. trispora, which is characterized in that a solid fermentation medium is used instead of a liquid fermentation medium in a traditional process, and the obtained solid containing mycelia The fermented product can be directly dried under reduced pressure for the extraction of β-carotene. Compared with liquid fermentation, the process of obtaining mycelium by filtration or centrifugation is reduced, and the amount of water used in the process and the discharge of waste water are greatly reduced, which is conducive to the protection of my country's limited fresh water resources, especially suitable for water-scarce areas in northern my country promotion and application.

The method of the present invention comprises: comprising: inoculating with B. trispora through seed culture first, then inoculating in the culture medium, collecting the thalline after fermentation, extracting, and obtaining, wherein the culture medium is a loose and porous solid culture medium, The strains were B. trispora (+) bacteria (ATCC 14059, purchased from China Common Microorganism Culture Collection Management Center) and B. trispora (-) bacteria (ATCC 14060, purchased from China Common Microbiology Culture Collection Center). Collection Management Center).

Wherein the fermentation and cultivation time of the strains on the solid medium is preferably 5-8 days, and the fermentation temperature is preferably 24-28°C.

The composition of the solid fermentation medium is preferably: the content of millet is 30-50%, the content of bran is 2-10%, the content of soybean oil is 5-20%, the content of Tween 80 is 5-20%, and the balance is who, in percentage is a percentage by weight.

The most preferred solid medium for fermentation: per kilogram of solid fermentation medium is made of 400g of millet, 50g of bran, 100g of soybean oil, 100g of Tween 80, 10g of soybean cake powder, and mixed with water to 1kg.

In the present invention, the weight ratio ratio of B. trispora (+) bacteria and B. trispora (-) bacteria inoculated on the solid medium is preferably 1:5 to 1:8. More preferably 1:7.

The described seed culture can use conventional methods, preferably: B. trispora (+) bacterium and (-) bacterium are respectively inoculated in the PDA (potato agar medium) plate, 26 ℃ static culture 40～50h, The mycelia covered the plate.

The culture is collected after fermentation, and the extraction method of the product β-carotene can be extracted by reference to the literature. The preferred method is as follows: the solid culture is dried under reduced pressure at 60 ° C, and the β-carotene is extracted with 2 times the volume of ethyl acetate after crushing. Carotene was extracted three times respectively, and the extracts were combined to obtain an extract containing β-carotene. Further, the crude product of β-carotene can be obtained by concentrating under reduced pressure and drying in vacuum

The present invention directly uses solid medium to ferment to obtain mycelium, avoids the process of liquid fermentation centrifugation or filtration to obtain mycelium, reduces the waste of water resources, and there is almost no waste water discharge in the whole process, which has little environmental pollution, especially It is suitable for popularization and application in water-deficient areas in northern my country. The present invention also optimizes the solid medium and fermentation conditions that are suitable for fermentation, and the yield can reach 8.3g/kg, which is 14.8 times that of conventional solid medium such as PDA fermentation yield (0.56g/kg), and is the highest yield of bran solid medium. (0.34g/kg) 24.4 times, also higher than the 3.0g/kg output of liquid fermentation, detailed contrast result sees table 1.

Comparative Test:

Taking liquid culture (control 1), solid PDA medium fermentation (control 2), and bran solid medium fermentation (control 3) as references, the effects of different fermentation methods and culture conditions on yield and process were compared.

Control 1: The medium is a liquid fermentation medium, the composition of which is millet flour 8%, glucose 2%, vegetable oil 8%, KH ₂ PO ₄ 0.05%, KCl 0.05%, Tween-80 10%, pH 7.0.

Control 2: solid PDA medium, prepared by peeling 200g of potatoes, chopping them, adding 1L of water to boil for 20min, filtering through 8 layers of gauze, removing the filtrate, adding 20g of glucose, 20g of agar powder, and pouring into a plate after sterilization.

Control 3: Bran solid culture medium, composed of 360g of bran, 140g of corn flour, and 500g of water, sterilized after being heated and stirred evenly for later use.

Technical Example 1: Solid medium A, composed of 400g millet, 50g bran, 100g soybean oil, 100g Tween 80, 10g soybean cake powder, 0.5g KH ₂ PO ₄ , 0.5g KCl, add water to 1kg.

Technical Example 2: Solid medium B, consisting of 350g millet, 60g bran, 80g soybean oil, 120g Tween 80, 20g soybean cake powder, 0.5g KH ₂ PO ₄ , 0.5g KCl, and add water to 1kg.

Inoculate the (+) bacterium and (-) bacterium seed of B. trispora into the fermentation medium according to the ratio of 1:7 or 1:6 (only used in technical example 2) and mix well, except for control 1 Except for shaking culture at 220rpm in a shaker, the rest were cultured statically at 26°C for 5-7 days. After liquid fermentation, the product is collected by centrifugation and the mycelium is collected by centrifugation. After solid fermentation, the fermentation product is directly collected and dried in a vacuum oven at 60°C. Dried thalline was ground and leached with ethyl acetate to extract the β-carotene therein and determined by HPLC. The results are shown in Table 1:

Table 1 Comparison of different fermentation conditions

*The density of fermentation broth is calculated based on the density of water 1g/cm ³

As can be seen from the results in Table 1, using the medium of the present technology invention, the solid fermentation yield is much higher than that of commonly used solid fermentation medium, such as PDA medium and bran medium. Compared with the liquid fermentation method, the solid fermentation method used in this technology not only has higher output and lower water consumption, but also reduces the filtration (or centrifugation) steps in the process and reduces the discharge of waste liquid. In addition, it can be seen that the formulations of millet, bran and other ingredients in the medium have a greater impact on the production of β-carotene. The preferred formulation is the formulation of medium A, that is, 400g of millet, 50g of bran, 100g of soybean oil, Tween 80: 100g, soybean cake powder 10g, KH ₂ PO ₄ 0.5g, KCl 0.5g, add water to 1kg.

Detailed ways

Example 1

Incline medium: PDA medium, the preparation method is 200g of peeled potatoes, chopped, add 1L of water and boil for 20min, filter with 8 layers of gauze, remove the filtrate, add 20g of glucose, 20g of agar powder, heat and melt, mix well and fill Put it into a test tube and prepare a PDA slope after sterilization.

Seed medium: Same as the slant medium, sterilized and poured into a plate to prepare a PDA plate.

Fermentation medium: 400g millet, 50g bran, 100g soybean oil, 100g Tween 80, 10g soybean cake powder, 0.5g KH ₂ PO ₄ , 0.5g KCl, add water to 1kg. Stir the prepared medium evenly and spread it on a stainless steel tray, cover with eight layers of gauze, steam in a steamer for 10 minutes, take it out and break it into loose granules with a spatula, cover with eight layers of gauze, and sterilize at 121°C for 25 minutes .

(1) Activation of bacteria

Take out the freeze-dried tube of the preserved B. trispora (+) bacteria (ATCC 14059), pick a small amount of bacteria with a sterile inoculation needle on the ultra-clean bench, apply it on the PDA slope, and place it at 26-28°C Cultivate in an incubator for 3-5 days. After the mycelia cover the slope, prepare an activated slope of positive bacteria and place it in a refrigerator at 4°C for use. Prepare the activated slant of B. trispora (—) bacteria (ATCC 14060) in the same way, and store it in a refrigerator at 4°C for later use.

(2) Seed cultivation

Use a sterile inoculation needle to pick a small amount of activated (+) fungal mycelium on the slope, spread it on a PDA plate, and culture it statically at 26°C for 40-50 hours. After the mycelia cover the plate, make (+) fungal seeds . Prepare (—) bacteria seeds in the same way.

(3) Cultivation and fermentation

Scrape out the (+) bacteria and (-) bacteria seeds grown on the PDA plate together with the culture medium, mix them according to the weight ratio of 1:7, mash and mix them with a sterile spatula to make mixed seeds. Shovel the mixed seeds with a spatula, add them to the fermentation medium, add about 50 g of the mixed seeds per kg of fermentation medium, stir well, cover with eight layers of gauze, and put them in a constant temperature incubator at 26°C for 5 days.

(4) Product extraction

Break up the solid culture obtained after fermentation on the tray with a shovel, place it in a vacuum oven at 60°C and dry it under reduced pressure, take it out and grind it to obtain a red powder, then add 2 times the volume of ethyl acetate for extraction for 1 hour, extract 3 times, combined extracts. The β-carotene in the extract was determined by HPLC.

According to the above method, the yield of β-carotene obtained by fermentation per kg of fermentation medium is 8.3g. The fermentation product is a dry mycelium mass, which can be dried under reduced pressure and ground to obtain a red powder, which can be directly extracted with organic solvents such as ethyl acetate to extract β-carotene, and there is no waste water discharge in the whole process.

Claims (6)

1. A method for preparing beta-carotene, comprising: culturing by seeds with B. trispora, then inoculating in the culture medium, collecting the thalline after fermentation, extracting to obtain final product, characterized in that culturing The base is a loose and porous solid medium, and the bacterial classification is B. trispora (+) bacterium and B. trispora (—) bacterium; wherein the solid medium consists of: millet 400g, bran 50g, soybean oil 100g, Tween 80 is 100g, soybean meal powder 10g, KH ₂ PO ₄ 0.5g, KCl 0.5g add water to 1kg; or the composition of solid medium is: millet 350g, bran 60g, soybean oil 80g, Tween 80 is 120g 1. Soybean cake powder 20g, KH ₂ PO ₄ 0.5g, KCl 0.5g, add water to 1kg. 2. The method according to claim 1, wherein the bacterial classification is fermented and cultivated on a solid medium for 5-8 days, and the fermentation temperature is 24-28° C. 3. The method according to claim 1, wherein the weight ratio ratio of the three-spora Borealis (+) bacterium and the three-spora Borealis (-) bacterium inoculated on the solid medium is 1:5～1:8. 4. the method for claim 3, wherein three spores Bradley's mold (+) bacterium and three spores Bradley's mold (-) bacterium inoculum weight ratio are 1:7. 5. The method of claim 1, wherein seed cultivation comprises: inoculating B. trispora (+) bacterium and (-) bacterium into PDA plates respectively, and statically culturing at 26° C. for 40 to 50 hours to obtain final product. 6. The method of claim 1, wherein the extraction method of the product β-carotene after collecting the thalline is as follows: the solid culture is dried under reduced pressure at 60° C., and the β-carotene is leached with ethyl acetate after crushing.