WO2018028402A1

WO2018028402A1 - Method for preparing beta carotene crystals

Info

Publication number: WO2018028402A1
Application number: PCT/CN2017/093522
Authority: WO
Inventors: 李翔宇; 汪志明; 陆姝欢; 田勇; 周强; 易德伟
Original assignee: 嘉必优生物技术（武汉）股份有限公司
Priority date: 2016-08-09
Filing date: 2017-07-19
Publication date: 2018-02-15
Also published as: CN106278975A

Abstract

A method for preparing beta carotene crystals, comprising the following steps: (1) inoculating and fermenting a microbial strain, and (2) performing solid-liquid separation of the fermentation broth to obtain wet bacteria; (3) drying the wet bacteria to obtain dry bacteria; (4) using an organic solvent to perform extraction on the dry bacteria; (5) performing solid-liquid separation to obtain an extract solution; (6) evaporating and crystallising the extract solution to obtain crystal mother liquor; (7) performing solid-liquid separation of the crystal mother liquor to obtain a crude wet crystal; (8) washing the crude wet crystal in organic solvent, and vacuum drying to obtain the beta carotene crystals. An antioxidant is added in steps (3), (4), and (6). Antioxidant is added in some or all of the steps of drying, solid-liquid separation, and evaporating and crystallising, reducing the loss of beta carotene in the extraction process and improving the beta carotene yield; the purity and quality of the obtained beta carotene crystals is high.

Description

Method for preparing β-carotene crystals

Cross-reference to related applications

The present application claims priority to Chinese Patent Application No. Serial No.

Technical field

The invention relates to a method for preparing β-carotene crystals, in particular to a method for preparing microbial-derived β-carotene crystals.

Background technique

Carotenoids are nutrients that are widely found in plants, animals, and microorganisms and are closely related to human health. They mainly include β-carotene, lycopene, and lutein. Carotenoids not only have high medicinal value such as anti-cancer and anti-oxidation, but also an important source of vitamin A in the human body. As a food additive and a nutrient enhancer, carotenoids have been recognized by international organizations such as the FDA, the European Community, and the WHO. In recent years, carotenoids have been widely used in medicine, food, health care products, and cosmetics.

Natural carotenoids are mainly from plants. However, plant raw materials have shortcomings such as insufficient supply and low content, and cannot be industrially utilized on a large scale. Microorganisms have the advantages of fast growth, high content, and no seasonal influence. Therefore, it is an ideal method to produce carotenoids by microbial fermentation.

At present, the main processes for extracting carotenoids from B. trispora include: high-quality homogenization of wet cells mixed with water, wet and dry cells and organic solvent extraction. After the crude extract is obtained, the steps of separation and purification are carried out to obtain a high content of crystals. Chinese Patent No. 01804173.6 discloses the separation of a carotenoid crystal, which is mixed with water and then homogenized by a high pressure homogenizer to extract a mixture of carotenoids, and then the mixture is washed with ethanol and brine. The method has high temperature when extracting carotenoids, and is easy to cause oxidative degradation of carotenoids, and is difficult to separate and purify, and the yield is extremely low. Chinese Patent Application No. 201210180281.4 No. discloses a method for extracting β-carotene by using high pressure steam to break the wall. However, this method uses higher temperatures and pressures, which tends to oxidize β-carotene, resulting in low product yield and affecting product quality.

The existing preparation of β-carotene crystals by microbial fermentation has the following disadvantages: high temperature treatment is required in the drying or extraction process, and it is easy to cause light and oxidative degradation of carotenoids, resulting in low yield and poor product quality. . Therefore, there is a need to consider an improved way to avoid loss of carotenoids during the preparation process.

Summary of the invention

It is an object of the present invention to provide a process for preparing β-carotene crystals which can reduce the oxidation of β-carotene and improve the yield and quality.

In order to achieve the above object, a method for producing a β-carotene crystal of the present invention comprises the following steps:

(1) Inoculating the microbial strains,

(2) performing solid-liquid separation of the fermentation liquid to obtain a wet bacterial cell;

(3) drying the wet cells to obtain dried cells;

(4) extracting the dried cells using an organic solvent;

(5) obtaining an extract after solid-liquid separation;

(6) The extract is crystallized by evaporation to obtain a crystallization mother liquor;

(7) The crystallization mother liquid is subjected to solid-liquid separation to obtain coarse wet crystals;

(8) The crude wet crystals are washed with an organic solvent and dried under vacuum to obtain β-carotene crystals;

In at least one of the steps (3), (4), and (6), an antioxidant is added.

The method of preparing β-carotene crystals as described above, and the steps (3), (4) and (6) are all added with an antioxidant.

The method for preparing β-carotene crystals as described above, and in the step (1), the microbial species is B. trispora, Dunaliella salina or yeast.

a method for preparing β-carotene crystals as described above, and an antioxidant in the step (3) The amount added is from 0.1% to 5% by weight of the wet cells.

The method of preparing β-carotene crystals as described above, the antioxidant is dibutylhydroxytoluene, vitamin E, vitamin C palmitate, ethyl p-hydroxybenzoate or rosemary.

For the trispora, the antioxidant is preferably one of ethyl p-hydroxybenzoate, rosemary, vitamin E, dibutylhydroxytoluene, used in the above steps (3), (4), In at least one of the steps 6), it is further preferred to use dibutylhydroxytoluene, vitamin E and ethyl p-hydroxybenzoate for use in steps (3), (4) and (6), respectively, more preferably two. Butylated hydroxytoluene is used in step (3), vitamin E is used in step (4), and ethyl p-hydroxybenzoate is used in step (6).

For Dunaliella salina, the antioxidant is preferably dibutylhydroxytoluene or vitamin E, which is used in at least one of the above steps (3), (4), and (6).

For yeast, the antioxidant is preferably vitamin E, vitamin C palmitate, dibutylhydroxytoluene or rosemary, which is used in at least one of the above steps (3), (4), (6) in.

The method for producing β-carotene crystals as described above, in the step (4) or (6), the antioxidant is added in an amount of 0.1% to 5% by weight of the dry cells.

The method for preparing β-carotene crystals as described above, in the step (4), the mass ratio of the dry cells to the organic solvent is 1:3-1:30.

The method for preparing β-carotene crystals as described above, and in the step (4), the extraction is simultaneously performed by a mechanical crushing process, and the average particle diameter of the cells after mechanical crushing is not higher than 300 μm.

The method for preparing β-carotene crystals as described above, in the step (4), the organic solvent is hexane, dichloromethane, petroleum ether, ethyl acetate or acetone.

The method for preparing β-carotene of the invention has the following beneficial effects: due to the oxidative degradation of β-carotene, a series of volatile aroma substances are mainly produced, such as β-ionone, dihydro kiwi lactone, isophorone, oxidized Vorketone and so on. The large amount of these oxidative degradation products has a great influence on the purification of β-carotene, which affects the purity of β-carotene. Therefore, some or all of the antioxidants are added during the drying, solid-liquid separation and evaporative crystallization. The agent not only reduces the loss of β-carotene during the extraction process, but also increases the yield of β-carotene. The extraction rate of β-carotene crystals is increased to 64.1%, preferably to 66.1%, and further preferably to 69.1%. Further, it is further preferably increased to 73.3%, and more preferably to 80.1%, and a β-carotene crystal having higher purity and better quality can be obtained, wherein the purity of the obtained β-carotene is increased to 97.3%, preferably elevated. Up to 97.7%, further preferably increased to 98.1%, more preferably increased to 99.1%.

detailed description

The present invention will be further described in detail below with reference to specific examples.

Yield calculation and standard or method for detecting purity

1. Detection method of β-carotene content of bacterial body weight: accurately weigh 0.01~0.03g sample, accurate 0.0001g, after being broken by glass homogenizer, extract with ethyl acetate, and repeatedly extract until the sample is completely colorless. , set to 25ml. Then use a pipette to take 1ml and dilute to a certain multiple and then dilute to volume. Using ethyl acetate as a reference, the solution after constant volume was poured into a cuvette, the absorbance was measured at the maximum absorption wavelength in the range of 455 nm ± 2 nm, and then β in the cells was calculated according to the β-carotene standard curve. - Carotene content.

2. Calculation method of β-carotene extraction rate:

Extraction rate (%) = m1/(m2*w)

Wherein: m1: obtaining the crystal weight of β-carotene;

M2: the weight of β-carotene dry cells;

w: content of β-carotene in β-carotene dry cells.

3, β-carotene crystal purity detection method: refer to GB 28310-2012 national food safety standard food additive β-carotene (fermentation method).

Example 1

Using Dunaliella salina as a fermentation strain, follow the steps below:

1) Fermentation of Dunaliella salina was carried out, and the fermentation broth was centrifuged to obtain a wet biomass of 75.7%.

2) Take 1 kg of wet cells, add 1 g of dibutylhydroxytoluene (antioxidant) to the wet cells, mix well, and dry in vacuo to obtain 269.3 g of dry cells. The conditions under vacuum drying are: temperature 80 ° C, negative pressure -0.085 MPa.

3) 200 g of the above dried cells were taken, added to a 3 L four-necked flask, and 600 mL of dichloromethane was added thereto, and the mixture was crushed by a high-speed shear to an average particle diameter of 181 μm, and suction-filtered to obtain a extract and a wet extract. The slag and the wet slag were subjected to secondary extraction with 2 L of dichloromethane, and suction-filtered to obtain a second extract.

4) Combine the two extracts and evaporate the crystals at 50 ° C, -0.08 MPa to obtain a crystallization mother liquor.

5) The crystallization mother liquid was suction filtered to obtain a wet crystal, and the wet crystal was washed with 20 ml of ethyl acetate, and suction-filtered again to obtain a wet crystal. The wet crystal was dried under vacuum at 80 ° C, -0.085 MPa for 2 hours to obtain a finished carotenoid crystal. After detection, the extraction rate of β-carotene crystals was 66.1%, and the purity of β-carotene was 97.3%.

6) According to the same process as above, no control agent was added for the control test, and the extraction rate of β-carotene crystal was calculated to be 60.9%, and the purity of β-carotene was 95.9%.

Example 2

Take yeast as the fermentation strain, and then follow the steps below:

1) The yeast was inoculated and fermented, and the fermentation broth was centrifuged to obtain a wet cell having a water content of 80.3%.

2) Take the above 10kg wet cells, add 75g of vitamin E (antioxidant) to the wet bacteria body, mix well and stir evenly, and freeze-dry to obtain 2.17kg dry cells. The freeze-drying conditions are: vacuum 20Pa, trap temperature is - Dry at 40 ° C for 18 h.

3) Take 1kg of dried bacteria, put it into a 50L reactor, add 30L of n-hexane, circulate it to the average particle size of 217um with a sand mill, and centrifuge to obtain a extract and wet residue. The slag was continuously added with 30 L of n-hexane, and extracted at 55 ° C for 1 h, and centrifuged to obtain a second extract and wet slag.

4) Combining the extracts and the second extracts, evaporating and crystallizing at 43 ° C, -0.085 MPa, to obtain a crystallization mother liquor.

5) Centrifuging the above crystallization mother liquid to obtain wet crystals, and using 500 ml of n-hexane for wet crystals The mixture was stirred and washed again to obtain wet crystals, and the wet crystals were vacuum dried at 80 ° C, -0.085 MPa for 2 hours to obtain a finished carotenoid crystal. The yield of carotenoid crystals was calculated to be 69.1%, and the purity of β-carotene was 97.7%.

6) According to the same process as above, a control test was carried out without adding an antioxidant, and the yield of β-carotene crystal was calculated to be 62.1%, and the purity of β-carotene was determined to be 96.1%.

Example 3

Take yeast as the fermentation strain, and then follow the steps below:

1) The yeast was inoculated and fermented, and the fermentation broth was centrifuged to obtain a wet cell having a water content of 80.1%.

2) Take the above 50 kg of wet cells, add 150 g of vitamin C palmitate (antioxidant) to the wet fungus, mix well and stir well, and spray dry to obtain 20.78 kg of dry cells. The spray drying conditions are: hot air temperature 130 ° C, material The temperature was 65 ° C, the fan frequency was 45 Hz, and the feed rate was 5 kg / h.

3) Take 5kg of dry bacteria, put it into a 100L reaction kettle, add 50L of n-hexane, circulate it to the average particle size of 193um by a sand mill, and centrifuge to obtain a extract and wet residue. The slag was continuously added with 50 L of n-hexane, and extracted at 55 ° C for 1 h, and centrifuged to obtain a second extract and wet slag.

5) The above crystallization mother liquid was centrifuged to obtain wet crystals, the wet crystals were washed with 250 ml of ethanol, and centrifuged again to obtain wet crystals. The wet crystals were vacuum dried at 80 ° C, -0.085 MPa for 2 hours to obtain finished carotenoid crystals. The yield of carotenoid crystals was calculated to be 69.1%, and the purity of β-carotene was 97.7%.

6) According to the same process as above, a control test was carried out without adding an antioxidant, and the yield of β-carotene crystal was calculated to be 61.7%, and the purity of β-carotene was determined to be 96.1%.

Example 4

Use the species of B. trispora as a fermentation strain, and then follow the steps below:

1) Inoculating B. trispora, fermenting the fermentation broth through a plate frame to obtain a water content of 55.1% wet cells.

2) 500 kg of wet cells were crushed with a pulverizer, 25 kg of ethyl p-hydroxybenzoate (antioxidant) was added and uniformly mixed, and then dried by boiling to obtain 241.1 kg of dry cells. The boiling drying conditions were: hot air temperature 120 ° C, material temperature 55 ° C, fan frequency 50Hz.

3) Take 5 kg of the above dried cells, put them into a 200 L reactor, add 100 L of dichloromethane, and crush them with a high shear colloid mill to an average particle size of 300 μm, and raise the temperature to 55 ° C for 1 hour. After sedimentation and separation, the upper extract was filtered with a 5um bag filter and a 0.5um filter plug to obtain a extract. The lower wet slag was continuously added with 100L petroleum ether, extracted at 55 ° C for 1.5 h, and the extract was mixed with 5 um bag. The filter and the 0.5 um filter plug filter were filtered to obtain a second extract and a wet residue.

4) The two extracts were combined and evaporated to crystallize at 48 ° C, -0.08 MPa to obtain a crystallization mother liquor.

5) The crystallization mother liquid is suction filtered to obtain coarse and wet crystals, and the coarse wet crystals are washed with 500 ml of petroleum ether, and then suction-filtered to obtain wet crystals. The wet crystals are dried under vacuum at 80 ° C, -0.085 MPa for 2 hours to obtain finished carotenoid crystals. . The extraction rate of the carotenoid crystal was calculated to be 73.3%, and the purity of the beta-carotene was determined to be 98.1%.

6) According to the same process as above, no control agent was added for the control test, and the extraction rate of β-carotene crystal was calculated to be 62.9%, and the purity of β-carotene was determined to be 96.9%.

Example 5

Using Dunaliella salina as a fermentation strain, follow the steps below:

2) 1 kg of wet cells were taken and dried under vacuum to obtain 269.3 g of dried cells, and the conditions of vacuum drying were as follows: temperature 80 ° C, negative pressure -0.085 MPa.

3) 200 g of the above dried cells were added, 0.2 g of vitamin E (antioxidant) was added thereto, and added to a 3 L four-necked flask, and 600 mL of ethyl acetate was added thereto, and the mixture was crushed to an average particle diameter of the cells by a high speed shear. At 151 um, a extraction extract and wet slag were obtained by suction filtration. The wet slag was subjected to secondary extraction with 2 L of ethyl acetate, and suction-filtered to obtain a second extract.

5) The crystallization mother liquid was suction filtered to obtain wet crystals, and the wet crystals were washed with 20 ml of ethanol, and suction-filtered again to obtain wet crystals. The wet crystals were dried under vacuum at 80 ° C, -0.085 MPa for 2 hours to obtain a finished carotenoid crystal. After testing, the extraction rate of β-carotene crystals was 64.1%, and the purity of β-carotene was 97.3%.

Example 6

Take yeast as the fermentation strain, and then follow the steps below:

1) The yeast is inoculated and fermented, and the fermentation liquid is centrifuged to obtain a wet cell having a water content of 80.5%.

2) Take the above 10 kg of wet cells, mix well and stir well, and freeze-dry to obtain 2.07 kg of dry cells. The freeze-drying conditions are: vacuum 20 Pa, trap temperature is -40 ° C, and dried for 20 h.

3) Take 1kg of dried bacteria, put it into a 30L reactor, add 50g of dibutylhydroxytoluene (antioxidant), add 30L of n-hexane, and crush it with a sand mill to the average particle size of 133um, centrifuge. A extract and wet slag were separated, and the wet slag was continuously added with 30 L of n-hexane, and extracted at 55 ° C for 1 h, and centrifuged to obtain a second extract and wet slag.

5) The above crystallization mother liquid was centrifuged to obtain wet crystals, and the wet crystals were washed with 250 ml of n-hexane, and centrifuged again to obtain wet crystals. The wet crystals were vacuum dried at 80 ° C, -0.085 MPa for 2 hours to obtain finished carotenoid crystals. The yield of carotenoid crystals was calculated to be 69.1%, and the purity of β-carotene was 97.7%.

6) According to the same process as above, a control test was carried out without adding an antioxidant, and the yield of β-carotene crystal was calculated to be 63.3%, and the purity of β-carotene was determined to be 96.1%.

Example 7

Take yeast as the fermentation strain, and then follow the steps below:

2) Take the above 50kg wet cells, mix thoroughly and stir well, and spray dry to obtain 20.78kg dry cells. The spray drying conditions are: hot air temperature 130°C, material temperature 65°C, fan frequency 45Hz, feed rate 5kg/ h.

3) Take 5kg of dried cells, put them into a 100L reactor, add 100g of vitamin C palmitate (antioxidant), add 50L of n-hexane, and pulverize with a sand mill to the average particle size of the cells is 153um, centrifuge A extract and wet slag were separated, and the wet slag was continuously added with 50 L of n-hexane, and extracted at 55 ° C for 1 h, and centrifuged to obtain a second extract and wet slag.

5) The above crystallization mother liquid was centrifuged to obtain wet crystals, and the wet crystals were washed with 250 ml of ethyl acetate, centrifuged again to obtain wet crystals, and the wet crystals were vacuum dried at 80 ° C, -0.085 MPa for 2 hours to obtain a finished carotenoid crystal. The yield of carotenoid crystals was calculated to be 69.1%, and the purity of β-carotene was 97.7%.

Example 8

1) Fermentation of B. trispora was carried out, and the fermentation broth was subjected to pressure filtration through a plate frame to obtain a wet cell having a water content of 55.1%.

2) 500 kg of wet cells were crushed with a pulverizer and dried by boiling to obtain 233.1 kg of dry cells. The boiling drying conditions were: hot air temperature 120 ° C, material temperature 55 ° C, fan frequency 50 Hz.

3) Take 50 kg of the above dried cells, put them into a 1000 L reactor, add 1 kg of rosemary (antioxidant), add 500 L of petroleum ether, and crush with a high shear colloid mill. The average particle size of the cells was 231 um, and the temperature was raised to 55 ° C for 1 h. After sedimentation and separation, the upper extract was filtered with a 5 um bag filter and a 0.5 um filter to obtain a extract, and the lower layer of wet slag continued. 500 L of petroleum ether was added and extracted at 55 ° C for 1.5 h. The extract mixture was filtered through a 5 um bag filter and a 0.5 um filter plug to obtain a second extract and wet slag.

5) The crystallization mother liquid was suction filtered to obtain coarse and wet crystals, and the crude wet crystals were further filtered with 5 L of ethanol to obtain wet crystals. The wet crystals were dried under vacuum at 80 ° C, -0.085 MPa for 2 hours to obtain a finished carotenoid crystal. The extraction rate of the carotenoid crystal was calculated to be 73.3%, and the purity of the beta-carotene was determined to be 98.1%.

Example 9

Using Dunaliella salina as a fermentation strain, follow the steps below:

1) Fermentation of Dunaliella salina was carried out, and the fermentation broth was centrifuged to obtain a wet biomass of 75.9%.

2) 1 kg of wet cells were taken and dried under vacuum to obtain 267.3 g of dried cells, and the conditions of vacuum drying were as follows: temperature 80 ° C, negative pressure -0.085 MPa.

4) The extracts were combined twice, 0.2 g of vitamin E (antioxidant) was added thereto, and the crystals were evaporated under the conditions of 50 ° C and -0.08 MPa to obtain a crystal mother liquid.

5) The crystallization mother liquid was suction filtered to obtain wet crystals, the wet crystals were washed with 50 ml of ethanol, and the wet crystals were again filtered by suction, and the wet crystals were dried under vacuum at 80 ° C, -0.085 MPa for 2 h. The finished carotenoid crystals are obtained. After testing, the extraction rate of β-carotene crystals was 64.1%, and the purity of β-carotene was 97.3%.

Example 10

Take yeast as the fermentation strain, and then follow the steps below:

2) Take the above 10 kg of wet cells, mix thoroughly and stir well, and freeze-dry to obtain 2.1 kg of dried cells. The freeze-drying conditions are: vacuum 20 Pa, trap temperature is -40 ° C, and dried for 20 h.

3) Take 1kg of dried bacteria, put it into a 30L reactor, add 30L of n-hexane, circulate it with a sand mill to an average particle size of 300um, and centrifuge to obtain a extract and wet residue. The slag was continuously added with 30 L of n-hexane, and extracted at 55 ° C for 1 h, and centrifuged to obtain a second extract and wet slag.

4) Combining the extracts and the second extracts, adding 50 g of dibutylhydroxytoluene (antioxidant), evaporating and crystallizing at 43 ° C, -0.085 MPa, to obtain a crystallization mother liquor.

Example 11

Take yeast as the fermentation strain, and then follow the steps below:

1) The yeast was inoculated and fermented, and the fermentation broth was centrifuged to obtain a wet cell having a water content of 80.9%.

3) Take 5kg of dried bacteria, put it into a 100L reactor, add 50L of n-hexane, circulate and crush it with a sand mill to an average particle size of 300um, and centrifuge to obtain a extract and wet residue. The slag was continuously added with 50 L of n-hexane, and extracted at 55 ° C for 1 h, and centrifuged to obtain a second extract and wet slag.

4) Combining the extracts and the second extracts, adding 100 g of vitamin C palmitate (antioxidant), evaporating and crystallizing at 43 ° C, -0.085 MPa, to obtain a crystallization mother liquor.

Example 12

3) Take 50kg of the above dried bacteria, put it into a 2000L reaction kettle, add 1000L petroleum ether, crush it with high shear colloid mill to the average particle size of the bacteria is 231um, then heat up to 55 °C, extract for 1h, After sedimentation and separation, the upper extract mixture was filtered with a 5um bag filter and a 0.5um filter plug to obtain a extract, and 600L petroleum ether was continuously added to the lower wet slag, and extracted at 55 ° C for 1.5 h to extract the mixture. The second extract and the wet residue were obtained by filtration using a 5 um bag filter and a 0.5 um filter plug filter.

4) Combine the two extracts, add 2 kg of rosemary (antioxidant), and evaporate and crystallize at 48 ° C, -0.08 MPa to obtain a crystallization mother liquor.

5) The crystallization mother liquid was suction filtered to obtain coarse and wet crystals, and the crude wet crystals were washed with 10 L of ethyl acetate, and again filtered to obtain wet crystals. The wet crystals were dried under vacuum at 80 ° C, -0.085 MPa for 2 h to obtain a finished carotenoid. Crystal. The extraction rate of the carotenoid crystal was calculated to be 73.1%, and the purity of the β-carotene was determined to be 98.1%.

6) According to the same process as above, the control experiment was carried out without adding an antioxidant, and the extraction rate of β-carotene crystal was calculated to be 61.9%, and the purity of β-carotene was determined to be 96.9%.

Example 13

3) Take 200kg of the above dried bacteria, put it into 5000L reaction kettle, add 2000L petroleum ether, crush it with high shear colloid mill to the average particle size of the bacteria is 231um, then heat up to 55 °C, extract for 1h, After sedimentation and separation, the upper extract mixture was filtered with a 5um bag filter and a 0.5um filter plug to obtain a extract, and 2000L petroleum ether was continuously added to the lower wet slag, and extracted at 55 ° C for 1.5 h to extract the mixture. The second extract and the wet residue were obtained by filtration using a 5 um bag filter and a 0.5 um filter plug filter.

4) Combine the two extracts, add 5 kg of ethyl p-hydroxybenzoate (antioxidant), and evaporate and crystallize at 48 ° C, -0.08 MPa to obtain a crystallization mother liquor.

5) The crystallization mother liquid is suction filtered to obtain coarse and wet crystals, and the coarse wet crystals are washed with 500 ml of dichloromethane, and then suction-filtered to obtain wet crystals. The wet crystals are dried under vacuum at 80 ° C, -0.085 MPa for 2 hours to obtain a finished carotenoid. Crystal. The extraction rate of the carotenoid crystal was calculated to be 73.3%, and the purity of the beta-carotene was determined to be 98.1%.

6) According to the same process as above, no antioxidant is added for the control test, and β- is calculated. The extraction rate of carotene crystals was 62.9%, and the purity of β-carotene was determined to be 96.9%.

Example 14

Using Dunaliella salina as a fermentation strain, follow the steps below:

2) 1 kg of wet cells were added, 0.5 g of dibutylhydroxytoluene (antioxidant) was added, mixed uniformly, and dried under vacuum to obtain 261.3 g of dry cells, and the conditions of vacuum drying were as follows: temperature 80 ° C, negative pressure -0.085 MPa.

3) Take 200 g of the above dried cells, add to a 3 L four-necked flask, add 0.5 g of vitamin E (antioxidant), add 600 mL of dichloromethane, and crush with a high-speed shear to an average particle size of 181 μm. The extract and the wet residue were obtained by suction filtration, and the wet residue was subjected to secondary extraction with 2 L of dichloromethane, and filtered to obtain a second extract.

5) The crystallization mother liquid was suction filtered to obtain wet crystals, and the wet crystals were washed with 20 ml of dichloromethane, and suction-filtered again to obtain wet crystals. The wet crystals were dried under vacuum at 80 ° C, -0.085 MPa for 2 hours to obtain a finished carotenoid crystal. After testing, the extraction rate of β-carotene crystals was 64.1%, and the purity of β-carotene was 97.3%.

Example 15

Take yeast as the fermentation strain, and then follow the steps below:

2) Take the above 10kg wet cells, add 50g of Vc palmitate (antioxidant), mix well and stir evenly, and spray dry to obtain 2.18kg dry cells. The spray drying conditions are: hot air temperature 130°C, material temperature 65°C, fan The frequency is 45 Hz and the feed rate is 5 kg/h.

3) Take 2kg of dried bacteria, put into a 50L reaction kettle, add 20L of n-hexane, circulate with a sand mill to the average particle size of the bacteria is 300um, then extract and stir for 1h at 550, centrifuge to obtain a extraction The liquid and wet slag, the wet slag were continuously added with 20 L of n-hexane, and extracted at 55 ° C for 1 h, and centrifuged to obtain a second extract and a wet slag.

4) Combine the extracts of the two extracts and the second extract, add 50 g of rosemary (antioxidant), and evaporate the crystals at 43 ° C, -0.085 MPa to obtain a crystallization mother liquor.

Example 16

2) 50 kg of wet cells were crushed by a pulverizer, and dried by boiling to obtain 23.1 kg of dry cells. The boiling drying conditions were: hot air temperature 120 ° C, material temperature 55 ° C, fan frequency 50 Hz.

3) Take 10 kg of the above dried cells, put them into a 200 L reactor, add 250 g of ethyl p-hydroxybenzoate (antioxidant), add 100 L of petroleum ether, and crush to the average cell size with a high shear colloid mill cycle. The diameter is 231 um, then the temperature is raised to 55 ° C, and extracted for 1 h. After sedimentation and separation, the upper extract mixture is filtered with a 5 um bag filter and a 0.5 um filter plug to obtain a extract, which is continuously added to the lower layer of wet slag. 100 L of petroleum ether was extracted at 55 ° C for 1.5 h, and the extract mixture was filtered through a 5 um bag filter and a 0.5 um filter plug to obtain a second extract and wet slag.

4) Combine the two extracts, add 250 g of vitamin E (antioxidant), and evaporate and crystallize at 48 ° C, -0.08 MPa to obtain a crystallization mother liquor.

5) The mother liquor is filtered by suction to obtain coarse and wet crystals. The coarse and wet crystals are washed with 2 L of dichloromethane, and then filtered again to obtain wet crystals. The wet crystals are dried under vacuum at 80 ° C, -0.085 MPa for 2 h to obtain the finished carotenoids. Crystal. The extraction rate of the carotenoid crystal was calculated to be 73.3%, and the purity of the beta-carotene was determined to be 98.1%.

Example 17

2) 500 kg of wet cells were crushed with a pulverizer, 2.5 kg of dibutylhydroxytoluene (antioxidant) was added, mixed uniformly, and dried by boiling to obtain 233.1 kg of dry cells. The boiling drying conditions were: hot air temperature 120 ° C, material temperature 55 ° C, fan frequency 50Hz.

3) Take 200kg of the above dried bacteria, put it into a 5000L reaction kettle, add 1kg of vitamin E (antioxidant), add 2000L of petroleum ether, and crush it with high shear colloid mill to the average particle size of the bacteria is 231um, then The temperature was raised to 55 ° C, extracted for 1 h, and after separation and sedimentation, the upper extract mixture was filtered with a 5 um bag filter and a 0.5 um filter plug to obtain a extract, and 2000 L of petroleum ether was continuously added to the lower layer of wet slag. After extraction at 55 ° C for 1.5 h, the extract mixture was filtered through a 5 um bag filter and a 0.5 um filter plug to obtain a second extract and wet slag.

4) Combine the two extracts, add 1 kg of ethyl p-hydroxybenzoate (antioxidant), and evaporate and crystallize at 48 ° C, -0.08 MPa to obtain a crystallization mother liquor.

5) The crystallization mother liquid is suction filtered to obtain coarse and wet crystals, and the coarse wet crystals are washed with 500 ml of dichloromethane, and then suction-filtered to obtain wet crystals. The wet crystals are dried under vacuum at 80 ° C, -0.085 MPa for 2 hours to obtain a finished carotenoid. Crystal. The extraction rate of the carotenoid crystal was calculated to be 80.1%, and the purity of the β-carotene was determined to be 99.1%.

6) According to the same process as above, no antioxidant is added for the control test, and β- is calculated. The extraction rate of carotene crystals was 61.9%, and the purity of β-carotene was 96.9%.

The method for preparing β-carotene of the invention has the following beneficial effects: in the steps of drying, solid-liquid separation and evaporative crystallization, some or all of the antioxidants are added, the loss of β-carotene during the extraction process is reduced, and β- is improved. The yield of carotene and the β-carotene crystal obtained at the same time are higher in purity and better in quality.

The antioxidants mentioned in the present invention are not limited to the types exemplified in the examples, and other products having the same effects in use are within the scope of the present invention.

The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., which are included in the spirit and scope of the present invention, should be included in the present invention. Within the scope of protection.

Industrial applicability

The invention provides a method for preparing β-carotene crystals, comprising the steps of: (1) inoculating a microbial strain to ferment, (2) performing solid-liquid separation of the fermentation liquid to obtain a wet bacterial body; and (3) wet bacteria After drying, the dried cells are obtained; (4) extracting the dried cells with an organic solvent; (5) obtaining an extract after solid-liquid separation; (6) evaporating and crystallizing the extract to obtain a crystallization mother liquid; (7) crystallization mother liquid is solidified The liquid is separated to obtain coarse wet crystals; (8) the crude wet crystals are washed with an organic solvent, and dried under vacuum to obtain β-carotene crystals; and in steps (3), (4) and (6), antioxidants are added. The invention reduces the loss of β-carotene in the extraction process, improves the yield of β-carotene, and at the same time, the obtained β-carotene crystal has higher purity and better quality. The method of the invention can be applied to the field of carotenoid extraction on a large scale, and has broad market prospects.

Claims

A method of preparing beta-carotene crystals, comprising the steps of:

(1) inoculating the microbial strains;

(2) performing solid-liquid separation of the fermentation liquid to obtain a wet bacterial cell;

(3) dried cells are obtained after drying the wet cells;

(4) extracting the dried cells using an organic solvent;

(5) obtaining an extract after solid-liquid separation;

(6) evaporating and crystallizing the extract to obtain a crystallization mother liquor;

(7) The crystallization mother liquid is subjected to solid-liquid separation to obtain coarse wet crystals;

(8) The crude wet crystals are washed with an organic solvent and dried under vacuum to obtain β-carotene crystals;

It is characterized in that an antioxidant is added to each of the steps (3), (4) and (6).
The method for preparing β-carotene crystal according to claim 1, wherein in the step (1), the microbial strain is B. trispora, Dunaliella salina or yeast.
The method for producing a β-carotene crystal according to claim 1, wherein the antioxidant is added in an amount of from 0.1% to 5% by weight based on the weight of the wet cells in the step (3).
The method for producing a β-carotene crystal according to claim 1, wherein in the steps (4) and (6), the antioxidant is added in an amount of from 0.1% to 5% by weight based on the dry cells.
The method for producing a β-carotene crystal according to claim 1, wherein the antioxidant is dibutylhydroxytoluene, ethyl p-hydroxybenzoate, vitamin C palmitate, vitamin E or rosemary. .
The method for preparing β-carotene crystal according to claim 1, wherein in step (4), the mass ratio of the dry cells to the organic solvent is 1:3-1:30.
The method for preparing a β-carotene crystal according to claim 1, wherein in the step (4), the extraction is simultaneously performed by mechanical disruption, and the average particle diameter of the cells after mechanical disruption is not higher than 300 μm.
The method for preparing β-carotene crystal according to claim 1, wherein in the step (4), the organic solvent is hexane, dichloromethane, petroleum ether or ethyl acetate. Or acetone.
The method for producing a β-carotene crystal according to claim 1, wherein in the step (5), the wet slag obtained by the solid-liquid separation is repeatedly subjected to extraction to obtain an extract, and all the extracts are combined.