A method for extracting Blakeslea trispora product and Blakeslea trispora product extracted thereby

Abstract

The present invention provides a method for extracting a Blakeslea trispora product and a Blakeslea trispora product extracted therefrom. The method for extracting a Blakeslea trispora product comprises the following steps: drying, sterilizing and enzymolyzing a fermentation broth obtained by fermenting Blakeslea trispora to obtain an extract. The present invention adopts a pretreatment method of drying and sterilizing, and then combines it with enzymolysis to break the cell wall of Blakeslea trispora, thereby realizing the effective extraction of carotenoids from Blakeslea trispora, and the carotenoid extraction rate can reach more than 90%.

Description

Technical Field

[0001] The invention belongs to the technical field of purification, and particularly relates to a method for extracting a Blakeslea trispora product. Background Art

[0002] Carotenoids are a class of compounds with 040 tetraterpenoid structures and their derivatives, which have been widely studied as antioxidants. They are composed of 8 isoprenoid units and are yellow, orange-red or red pigments. Lycopene and β-carotene are very representative carotenoids, which can effectively quench singlet oxygen and scavenge free radicals in the human body, thereby playing an anti-cancer and anti-tumor role and activating immune cells. They are called "plant gold".

[0003] At present, one way to prepare carotenoids is to extract them from plants, such as carrots, corn, sea buckthorn, tomatoes, etc. Taking lycopene as an example, even the tomato with the highest lycopene content has a content of only about 100 mg / kg. Large-scale plant extraction will cause a large waste of plant resources, and the plant components are complex, resulting in low purity of the extracted products. Therefore, considering factors such as quality, technology, production, and resource costs, the prospect of extracting and producing carotenoids from natural plants is not optimistic. Lycopene can be artificially synthesized through the Wittig olefination reaction, but the chemical synthesis process route is long, the reaction conditions are relatively harsh, the yield is low, and the product carries a large amount of toxic residues, which has a great impact on food safety.

[0004] Microorganisms have the characteristics of fast growth and reproduction, short growth cycle, high yield, few impurities, and can be produced on a large scale. They can overcome the shortcomings of tomato extraction and chemical synthesis of lycopene and are very suitable for industrial production. Their cultivation method is simple, the raw material source is abundant, the price is low, and the economic benefit is high.

[0005] Blakeslea trispora can produce β-carotene and lycopene, and is currently widely used as a common bacterial species for the industrialization of both. Blakeslea trispora belongs to Mucor fungi and can grow into thick and long hyphae. Conventional cell wall breaking methods used for ordinary microorganisms are difficult to achieve good results, and carotenoids exist in the cell as tiny crystals, so Blakeslea trispora needs to be thoroughly broken.

[0006] According to literature reports, the prior art mainly uses enzyme method, physical homogenization or grinding, and acid method. Among them, the acid method requires long-term lysis of the bacteria in a strong acid solution, and carotenoids are polyene structures and are easily destroyed under long-term strong acid conditions; the enzymatic wall breaking in the prior art, such as the document "Compound Enzyme Wall Breaking Treatment of Blakeslea trispora", claims that its extraction rate can reach 90%; physical homogenization or grinding means are currently the most commonly used wall breaking means for Blakeslea trispora. The applicant has also tried to use high-pressure homogenization and the use of enzymatic hydrolysis means, as described in CN110407659A, to extract lycopene from Blakeslea trispora and obtain a good yield. Although the combination of enzymatic hydrolysis means can reduce the pressure of the homogenization process to a certain extent, the energy consumption generated by high-pressure homogenization, the processing level, and the loss of equipment caused by temperature cannot be ignored for large-scale production.

[0007] Therefore, the applicant wants to develop a method for breaking the cell wall of Blakeslea trispora mainly by enzymatic means, which can completely break the cell wall and enable carotenoids to be smoothly extracted under mild conditions. Summary of the invention

[0008] During the research process of the applicant, it was found that the composite enzyme cell wall breaking method given in the prior art could not achieve the good effect it claimed. The reason may be that the strain treated by the applicant is a mutated Blakeslea trispora strain, and after domestication, its carotenoid production is higher than that of most conventional Blakeslea trispora strains. Its carotenoid content can reach more than 7% through fermentation regulation. Carotenoids exist in the form of crystals in the mold body. Correspondingly, the mutated Blakeslea trispora strain has evolved a thicker cell wall to bear the pressure brought by the high-content product, so it is difficult to achieve the effect reported in the literature by only using the composite enzyme method. In order to solve the problem that the prior art cannot solve, the applicant proposes a new extraction method for this Blakeslea trispora, which combines pre-treatment methods such as spray drying and enzymolysis to break the cell wall of Blakeslea trispora, and has a good effect on cell wall breaking.

[0009] The strain targeted by the present invention was deposited in China Center for Type Culture Collection (CCTCC) on August 8, 2014, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being: Blakeslea trispora BT7251 (+) CCTCC M 2014378; Blakeslea trispora BT7603 (-) CCTCC M 2014379.

[0010] To achieve this object, the present invention adopts the following technical solutions:

[0011] In a first aspect, the present invention provides a method for extracting a Blakeslea trispora product, comprising the following steps: drying, sterilizing and enzymolyzing a fermentation broth obtained by fermenting Blakeslea trispora to obtain an extract.

[0012] In the present invention, carotenoid fermentation broth is obtained by fermentation of Blakeslea trispora. For the specific fermentation method, see (patent CN104531538A). The technology of obtaining product lycopene by adding a blocking agent to prevent lycopene from being converted into β-carotene during the fermentation of Blakeslea trispora is well known to those skilled in the art. The content of β-carotene and lycopene in the product of Blakeslea trispora can be determined by the regulation of the fermentation process. Based on this, the applicant has conducted a series of fermentation studies, such as patents CN108034686B, CN107937469B, etc., and fermentation broths of Blakeslea trispora with different contents can be obtained according to the purpose.

[0013] In the present invention, the cell water content is reduced to less than 5% by the drying process, so that the cell wall structure of the bacterial cell is denatured, and the cell wall structure is well destroyed, which is beneficial to the subsequent enzymatic hydrolysis and is easier to cooperate with solvent extraction, thereby further improving the carotenoid yield. Sterilization can also be called cell inactivation, and inactivated bacteria are easier to enzymatic hydrolysis. Usually, cell inactivation in the field of microorganisms or microbial oils and fats requires 20-30 minutes whether it is high pressure sterilization or pasteurization, but if only long-term inactivation is performed, carotenoids will be destroyed in large quantities. However, the present application is spray-dried and then sterilized to fully avoid carotenoids from being destroyed, thereby greatly improving the carotenoid extraction rate.

[0014] Preferably, the water content of the dry bacteria obtained by drying is less than 5wt%, for example, it can be 5wt%, 4.5wt%, 4wt%, 3.5wt%, 3wt%, 2.5wt%, 2wt%, 1.5wt%, 1wt%, 0.5wt%, 0.2wt%, 0.1wt%, 0.05wt%, 0wt%, etc.

[0015] In the present invention, the drying may be direct spray drying, or vacuum drying after filtering the bacterial cells, and the water content of the dried bacterial cells after drying needs to be ensured to be 5wt%.

[0016] Preferably, the drying is spray drying.

[0017] In the present invention, spray drying is preferably used because the advantages of spray drying are large processing capacity and short time required. The processing capacity of an ordinary pilot tower can be 1 kg / h, and the processing capacity of a production-level spray tower can reach 70 kg / h.

[0018] Preferably, the sterilization is high pressure steam sterilization.

[0019] Preferably, the temperature of the high-pressure steam sterilization treatment is 100-150°C (for example, it can be 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, etc.), the pressure is 0.1-0.2MPa (for example, it can be 0.1MPa, 0.12MPa, 0.14MPa, 0.16MPa, 0.18MPa, 0.2MPa, etc.), and the time is 5-10min (for example, it can be 5min, 6min, 7min, 8min, 9min, 10min, etc.).

[0020] Preferably, the enzymes used in the enzymolysis include composite protease and glucanase.

[0021] Preferably, the total enzyme activity of the composite protease is 400-800U / mL, for example, it can be 400U / mL, 420U / mL, 440U / mL, 460U / mL, 480U / mL, 500U / mL, 550U / mL, 600U / mL, 650U / mL, 700U / mL, 750U / mL, 800U / mL and the like.

[0022] Preferably, the enzymatic activity of the glucanase is 0.5-10U / mL, for example, it can be 0.5U / mL, 1U / mL, 2U / mL, 3U / mL, 4U / mL, 5U / mL, 6U / mL, 7U / mL, 8U / mL, 9U / mL, 10U / mL and the like.

[0023] Preferably, the composite protease comprises alkaline protease, neutral protease and papain.

[0024] Preferably, the activity ratio of the alkaline protease, neutral protease and papain is 1:1:(0.5-0.8), for example, it can be 1:1:0.5, 1:1:0.55, 1:1:0.6, 1:1:0.65, 1:1:0.7, 1:1:0.75, 1:1:0.8, etc.

[0025] Preferably, the enzymatic hydrolysis temperature is 35-65°C, for example, it can be 35°C, 40°C, 45°C, 50°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, etc., and the enzymatic hydrolysis time is 12-24h, for example, it can be 12h, 14h, 16h, 18h, 20h, 22h, 24h, etc.

[0026] Preferably, the dry bacteria obtained by drying need to be diluted before sterilization, and the dilution solvent is water.

[0027] Preferably, the water content of the diluted liquid obtained by the dilution is 70-90wt%, for example, it can be 70wt%, 72wt%, 74wt%, 76wt%, 78wt%, 80wt%, 82wt%, 84wt%, 86wt%, 88wt%, 90wt% and the like.

[0028] Preferably, the pH value needs to be adjusted to 5-8 before enzymatic hydrolysis, for example, it can be 5, 5.5, 6, 6.5, 7, 7.5, 8, etc.

[0029] In the present invention, the pH adjustment here means that the dry bacteria obtained by drying need to be diluted before sterilization, and the pH of the dilution obtained by sterilization needs to be adjusted before enzymatic hydrolysis.

[0030] Preferably, the pH is adjusted using a base.

[0031] Preferably, the base comprises any one of sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate or potassium bicarbonate, or a combination of at least two thereof.

[0032] Preferably, solid-liquid separation is required after the enzymatic hydrolysis.

[0033] Preferably, the solid-liquid separation method is centrifugation.

[0034] In the present invention, the enzymatic hydrolysis is further followed by centrifugation: the reaction solution after the enzymatic hydrolysis is centrifuged.

[0035] Preferably, the centrifugal rotation speed is 8000-10000 r / min, for example, it can be 8000 r / min, 8500 r / min, 9000 r / min, 9500 r / min, 10000 r / min, etc., and the centrifugal time is 5-20 min, for example, it can be 5 min, 6 min, 8 min, 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, etc.

[0036] Preferably, extraction and purification are further performed after the solid-liquid separation: the upper crude crystals obtained by layering are extracted with an organic solvent, desolventized, and filtered to obtain crystals containing carotenoids.

[0037] Preferably, the organic solvent for extraction is ethyl acetate.

[0038] Preferably, the extraction temperature is 60-70°C (for example, it can be 60°C, 62°C, 64°C, 66°C, 68°C, 70°C, etc.), the number of extractions is more than 2 times (for example, it can be 2 times, 4 times, 6 times, 8 times, 10 times, etc.), and the extraction time is more than 10 minutes (for example, it can be 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, etc.).

[0039] In consideration of purity, the crude crystals may be washed before extraction, or the crystals after solvent removal and filtration may be washed a second time. The washing solvent is ethanol. Since carotenoids are poorly soluble in ethanol, the extraction rate is basically not affected.

[0040] Preferably, the solvents used in the first washing and the second washing are both ethanol.

[0041] Preferably, the mass ratio of the upper crude crystals to the solvent of the first washing is 1:(2-5), for example, it can be 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc.

[0042] Preferably, the mass ratio of the crystals obtained by filtration to the solvent of the first washing is 1:(10-30), for example, it can be 1:10, 1:12, 1:15, 1:18, 1:20, 1:23, 1:25, 1:27, 1:30, etc.

[0043] Preferably, the method for extracting the Blakeslea trispora product comprises the following steps:

[0044] (1) Drying: Drying the fermentation liquid obtained by fermenting Blakeslea trispora to obtain dry bacterial cells with a water content of less than 5 wt %;

[0045] (2) Sterilization: diluting the dried product to obtain a dilution having a water content of 80-90 wt%, and then sterilizing the dilution;

[0046] (3) Enzymatic hydrolysis: The pH of the sterilized fermentation broth is adjusted to 5-8, and then mixed with alkaline protease, neutral protease, papain and glucanase, and the enzymatic hydrolysis reaction is carried out at 55-60°C for 12-24 hours;

[0047] (4) Centrifugal stratification: centrifuge the enzymatic hydrolyzate at a speed of 8000-10000 r / min for 5-20 min to obtain the upper layer of coarse crystals;

[0048] (5) The upper crude crystals are extracted with ethyl acetate at 60-70° C. for more than two times, desolventized, and filtered to obtain crystals containing carotenoids.

[0049] In a second aspect, the present invention provides a Blakeslea trispora product, wherein the Blakeslea trispora product is obtained by the extraction method of the Blakeslea trispora product as described in the first aspect.

[0050] Preferably, the Blakeslea trispora product is carotenoid crystals.

[0051] In the present invention, the final product contains lycopene and β-carotene at the same time. Since the properties of the two are very similar, the extraction method of the Blakeslea trispora product can extract the two at the same time. Therefore, only lycopene is used as an example in all the following indicators.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The extraction method of the Blakeslea trispora product of the present invention adopts spray drying to make the bacterial cell wall easier to destroy, and the combined use of composite protease and glucanase has a good effect on cell wall breaking;

[0054] (2) The method for extracting Blakeslea trispora products of the present invention effectively breaks the cell walls of Blakeslea trispora, making it easier to extract with solvents, and the lycopene yield is as high as more than 90%;

[0055] (2) The method for extracting the Blakeslea trispora product of the present invention has a fast raw material production speed, high product quality, few subsequent treatment processes required, is easy to mass produce, has a high return on investment, and can be produced continuously. DETAILED DESCRIPTION

[0056] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0057] The products of the following examples contain both lycopene and β-carotene. Since the properties of the two are very similar, the present method can extract both of them at the same time without any special selective characteristics. Therefore, only lycopene is used as an example in the indicators.

[0058] Example 1

[0059] The present embodiment provides a method for extracting a product of Blakeslea trispora, and the method for extracting a product of Blakeslea trispora specifically comprises the following steps:

[0060] (1) spray drying 500 g of Blakeslea trispora fermentation broth to a water content of 3 wt %;

[0061] The lycopene content in the fermentation broth of Blakeslea trispora is 0.588 wt %; the spray drying is carried out using a spray drying tower with an inlet air temperature of 175° C., an outlet air temperature of 65° C., and a processing capacity of 1 kg / h;

[0062] (2) diluting the spray-dried product to a water content of 85 wt % and a lycopene content of 0.588%, and then sterilizing at 121° C. and 0.12 MPa for 5 min;

[0063] (3) first adjusting the pH of the fermentation broth after high-pressure sterilization to 6 with sodium hydroxide, then adding composite protease and glucanase for enzymatic hydrolysis at 55°C for 18 hours to obtain an enzymatic hydrolyzate;

[0064] In the enzymatic hydrolysis reaction solution: the composite protease is a combination of 210U / mL alkaline protease, 210U / mL neutral protease and 180U / mL papain, and the glucanase is 3U / mL glucanase;

[0065] (4) Centrifuge the enzymatic hydrolysate at 8000 r / min for 10 min and allow to stand to separate into three layers: upper, middle and lower;

[0066] (5) Take the upper crude crystals, add 400 mL of ethanol to the upper crude crystals, centrifuge to separate the ethanol, obtain 110 g of dehydrated crude crystals, add 2 L of ethyl acetate to extract four times, the extraction temperature is 68 ° C, stir for 15 min, collect the extract, desolventize, collect the desolventized mother liquor, filter to obtain crystals, wash the crystals with 20 times the mass of ethanol for three times to obtain purified crystals, the purified crystals contain 2.7 g of lycopene, and the extraction rate is 91.8%.

[0067] Example 2

[0068] The present embodiment provides a method for extracting a product of Blakeslea trispora, and the method for extracting a product of Blakeslea trispora specifically comprises the following steps:

[0069] (1) 500 g of Blakeslea trispora fermentation broth was spray dried to a water content of 5 wt %.

[0070] The lycopene content in the fermentation broth of Blakeslea trispora is 1.17 wt %; the spray drying is carried out using a spray drying tower with an inlet air temperature of 175° C., an outlet air temperature of 65° C., and a processing capacity of 1 kg / h;

[0071] (2) diluting the spray-dried product to a water content of 70 wt % and a lycopene content of 1.17%, and then sterilizing at 121° C. and 0.12 MPa for 10 min;

[0072] (3) first adjusting the pH of the fermentation broth after high-pressure sterilization to 6.5 with sodium hydroxide, then adding composite protease and glucanase for enzymatic hydrolysis at 60°C for 18 hours to obtain an enzymatic hydrolyzate;

[0073] In the enzymatic hydrolysis reaction solution: the composite protease is a combination of 300U / mL alkaline protease, 300U / mL neutral protease and 180U / mL papain, and the glucanase is 5U / mL glucanase;

[0074] (4) Centrifuge the enzymatic hydrolysate at 8000 r / min for 10 min and allow to stand to separate into three layers: upper, middle and lower;

[0075] (5) Take the upper crude crystals, add 400 mL of ethanol to the upper crude crystals, centrifuge to separate the ethanol, obtain 110 g of dehydrated crude crystals, add 2 L of ethyl acetate to extract four times, the extraction temperature is 68 ° C, stir for 15 min, collect the extract, desolventize, collect the desolventized mother liquor, filter to obtain crystals, wash the crystals with 20 times the mass of ethanol for three times to obtain purified crystals, the purified crystals contain 2.5 g of lycopene, and the extraction rate is 90.2%.

[0076] Example 3

[0077] This embodiment provides a method for extracting a product of Blakeslea trispora, which is different from Embodiment 1 only in that, in step (1), the dried bacteria are dried to a water content of 10 wt%.

[0078] Example 4

[0079] This embodiment provides a method for extracting a Blakeslea trispora product, which is different from Embodiment 1 only in that, in step (2), the product is diluted to a water content of 55 wt % and sterilized directly.

[0080] Example 5

[0081] This embodiment provides a method for extracting a product of Blakeslea trispora, which is different from Embodiment 1 only in that the composite protease is a combination of 210 U / mL alkaline protease and 180 U / mL papain.

[0082] Example 6

[0083] This embodiment provides a method for extracting a product of Blakeslea trispora, which is different from Embodiment 1 only in that the composite protease is a combination of 210 U / mL neutral protease and 180 U / mL papain.

[0084] Example 7

[0085] This embodiment provides a method for extracting a Blakeslea trispora product, which is different from Embodiment 1 only in that the composite protease is a combination of 210 U / mL alkaline protease and 180 U / mL neutral protease.

[0086] Example 8

[0087] This embodiment provides a method for extracting a product of Blakeslea trispora, which is different from Example 1 only in that 3 U / mL of glucanase is not added.

[0088] Example 9

[0089] This example provides a method for extracting a product of Blakeslea trispora, which is different from Example 1 only in that 3 U / mL of glucanase is replaced by 70 U / mL of phospholipase.

[0090] Example 10

[0091] The present embodiment provides a method for extracting a product of Blakeslea trispora, which is different from Example 1 only in that the enzymes involved in the enzymatic hydrolysis are neutral protease, cellulase and phospholipase, wherein the enzyme activity of the neutral protease is 210 U / mL, the enzyme activity of the cellulase is 350 U / mL, and the enzyme activity of the phospholipase is 70 U / mL.

[0092] Comparative Example 1

[0093] This comparative example provides a method for extracting a product of Blakeslea trispora, and the method for extracting a product of Blakeslea trispora specifically comprises the following steps:

[0094] (1) Concentrating 500 g of fermentation broth to a water content of 85 wt% and a lycopene content of 0.588%;

[0095] (2) Sterilize at 121°C and 0.12 MPa for 5 min;

[0096] (3) first adjusting the pH of the fermentation broth after high-pressure sterilization to 6 with sodium hydroxide, then adding composite protease and glucanase for enzymatic hydrolysis at 55°C for 18 hours to obtain an enzymatic hydrolyzate;

[0097] In the enzymatic hydrolysis reaction solution: the composite protease is a combination of 210U / mL alkaline protease, 210U / mL neutral protease and 180U / mL papain, and the glucanase is 3U / mL glucanase;

[0098] (4) Centrifuge the enzymatic hydrolysate at 8000 r / min for 10 min and allow to stand to separate into three layers: upper, middle and lower;

[0099] (5) Take the upper crude crystals, add 400 mL of ethanol to the upper crude crystals, centrifuge to separate the ethanol, obtain 110 g of dehydrated crude crystals, add 2 L of ethyl acetate to extract four times, the extraction temperature is 68 ° C, stir for 15 min, collect the extract, desolventize, collect the desolventized mother liquor, filter to obtain crystals, wash the crystals with 20 times the mass of ethanol for three times to obtain purified crystals, the purified crystals contain 2.23 g of lycopene, and the extraction rate is 76%.

[0100] Comparative Example 2

[0101] This comparative example provides a method for extracting a product of Blakeslea trispora, and the method for extracting a product of Blakeslea trispora specifically comprises the following steps:

[0102] (1) spray drying 500 g of fermentation broth until the water content is 3%, and the lycopene content in the fermentation broth is 0.588%;

[0103] The spray drying is carried out using a spray dryer with a power of 15 kW, an air inlet temperature of 175°C, an air outlet temperature of 65°C, and a processing capacity of 1 kg / h;

[0104] (2) diluting the spray-dried product to a water content of 85 wt % and a lycopene content of 0.588%, and then sterilizing at 121° C. and 0.12 MPa for 5 min;

[0105] (3) The pH of the fermentation liquid after high-pressure sterilization was adjusted to 6 with sodium hydroxide, and 2 L of ethyl acetate was added for extraction four times at an extraction temperature of 68°C. The mixture was stirred for 15 min, and the extract was collected. The extract was desolvated, and the desolvated mother liquor was collected and filtered to obtain crystals. The crystals were washed three times with 20 times the mass of ethanol to obtain purified crystals. The purified crystals contained 0.17 g of lycopene, and the extraction rate was 5.8%.

[0106] Comparative Example 3

[0107] This comparative example provides a method for extracting a product of Blakeslea trispora, and the method for extracting a product of Blakeslea trispora specifically comprises the following steps:

[0108] (1) spray drying 500 g of fermentation liquid until the water content is 3%; the lycopene content in the fermentation liquid is 0.588%;

[0109] The spray drying is carried out using a spray dryer with a power of 15 kW, an air inlet temperature of 175°C, an air outlet temperature of 65°C, and a processing capacity of 1 kg / h;

[0110] (2) diluting the spray-dried product to a water content of 85 wt % and a lycopene content of 0.588%;

[0111] (3) first adjusting the pH of the fermentation broth after high-pressure sterilization to 6 with sodium hydroxide, then adding composite protease and glucanase for enzymatic hydrolysis at 55°C for 18 hours to obtain an enzymatic hydrolyzate;

[0112] In the enzymatic hydrolysis reaction solution: the composite protease is a combination of 210U / mL alkaline protease, 210U / mL neutral protease and 180U / mL papain, and the glucanase is 3U / mL glucanase;

[0113] (4) Centrifuging the enzymatic hydrolysate at 8000 r / min for 10 min, the enzymatic hydrolysate is separated into three layers: upper, middle and lower layers;

[0114] (5) Take the upper crude crystals, add 400 mL of ethanol to the upper crude crystals, centrifuge to separate the ethanol, obtain 110 g of dehydrated crude crystals, add 2 L of ethyl acetate to extract four times, the extraction temperature is 68 ° C, stir for 15 min, collect the extract, desolventize, collect the desolventized mother liquor, filter to obtain crystals, wash the crystals with 20 times the mass of ethanol for three times to obtain purified crystals, the purified crystals contain 2.3 g of lycopene, and the extraction rate is 78%.

[0115] Performance Testing

[0116] The purity and extraction rate of lycopene in the purified crystals obtained in the above Examples 1-10 and Comparative Examples 1-3 were tested by HPLC. The specific test results are shown in Table 1 below:

[0117] Table 1

[0118]

[0119]

[0120] From the test data in Table 1, it can be seen that the extraction method of the present invention uses spray drying to make the bacterial cell wall easier to destroy; the combined use of composite protease and glucanase has a good effect on cell wall breaking. The method effectively breaks the cell wall of Blakeslea trispora, making it easier to cooperate with solvent extraction, and the lycopene yield is as high as more than 90%.

[0121] From the comparison between Example 1 and Examples 3-4, it can be seen that if the temperature during spraying is too low, it may lead to incomplete drying, which in turn leads to incomplete cell wall breaking; if the temperature during spraying is too high, although the drying is complete, the following situations may occur.

[0122] From the comparison between Example 1 and Examples 5-7, it can be seen that if the lycopene is not redissolved after drying, or the redissolved concentration is not within the preferred range of the present application, the enzymatic hydrolysis will not be able to fully break the cell wall, thereby resulting in a decrease in the yield of lycopene.

[0123] From the comparison between Example 1 and Examples 8-12, it can be seen that the composite enzymatic hydrolysis using alkaline protease, neutral protease, papain and glucanase can further improve the cell wall breaking effect.

[0124] From the comparison between Example 1 and Comparative Examples 1-3, it can be seen that the present invention adopts a treatment mode of drying, sterilization and enzymatic hydrolysis to fully destroy the cell wall. Without any of the operations, the lycopene yield is very low, with a maximum of only 78%.

[0125] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the extraction method of the Blakeslea trispora product and the Blakeslea trispora product extracted therefrom, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for extracting a product of Blakeslea trispora, characterized in that: The method for extracting the Blakeslea trispora product comprises the following steps: drying, sterilizing and enzymolyzing the fermentation liquid obtained by fermenting Blakeslea trispora to obtain an extract; The dried bacteria obtained by drying need to be diluted before sterilization, and the dilution solvent is water; the water content of the diluted solution obtained by dilution is 70-90wt%; the water content of the dried bacteria obtained by drying is less than 5wt%; the sterilization is high-pressure steam sterilization, the sterilization temperature is 100-150°C, and the sterilization time is 5-10min The enzymes used in the enzymatic hydrolysis include composite protease and glucanase; the composite protease includes alkaline protease, neutral protease and papain; the temperature of the enzymatic hydrolysis is 35-65°C, and the time of the enzymatic hydrolysis is 12-24h; and the pH needs to be adjusted to 5-8 before the enzymatic hydrolysis.

2. The method for extracting the Blakeslea trispora product according to claim 1, wherein The total enzyme activity of the composite protease is 400-800 U / mL.

3. The method for extracting the Blakeslea trispora product according to claim 1, characterized in that The enzymatic activity of the dextranase is 0.5-10 U / mL.

4. The method for extracting the Blakeslea trispora product according to claim 1, wherein The activity ratio of the alkaline protease, the neutral protease and the papain is 1:1:(0.5-0.8).

5. The method for extracting the Blakeslea trispora product according to claim 1, characterized in that After the enzymolysis, solid-liquid separation is required.

6. The method for extracting the Blakeslea trispora product according to claim 5, characterized in that: The solid-liquid separation method is centrifugation.

7. The method for extracting the Blakeslea trispora product according to claim 6, characterized in that: The centrifugal speed is 8000-10000 r / min, and the centrifugal time is 5-20 min.

8. The method for extracting the Blakeslea trispora product according to claim 5, characterized in that: After the solid-liquid separation, extraction and purification are required: the upper layer of crude crystals obtained by layering are extracted with an organic solvent, desolventized, and filtered to obtain crystals containing carotenoids.

9. The method for extracting the Blakeslea trispora product according to claim 8, characterized in that: The organic solvent for extraction is ethyl acetate.

10. The method for extracting the Blakeslea trispora product according to claim 8, characterized in that: The extraction temperature is 60-70° C., and the extraction times are more than 2 times.

11. The method for extracting the Blakeslea trispora product according to claim 1, characterized in that: The extraction method of the Blakeslea trispora product comprises the following steps: (1) Drying: Drying the fermentation broth obtained by fermenting Blakeslea trispora to obtain dry bacterial cells with a water content of less than 5 wt %; (2) Sterilization: diluting the dried product to obtain a dilution having a water content of 80-90 wt%, and then subjecting the dilution to high temperature steam sterilization; (3) Enzymatic hydrolysis: The pH of the sterilized fermentation broth is adjusted to 5-8, and then mixed with alkaline protease, neutral protease, papain and glucanase, and the enzymatic hydrolysis reaction is carried out at 55-60°C for 12-24 hours; (4) Centrifugal stratification: centrifuge the enzymatic hydrolyzate at a speed of 8000-10000 r / min for 5-20 min to obtain the upper layer of coarse crystals; (5) The upper crude crystals are extracted with ethyl acetate at 60-70° C. for more than two times, desolventized, and filtered to obtain crystals containing carotenoids.

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