CN110283854A - A kind of fermentation medium and its application and the method for preparing lycopene using Blakeslea trispora fermentation - Google Patents

Abstract

The invention belongs to lycopene production field, a kind of fermentation medium and its application and the method for preparing lycopene using Blakeslea trispora fermentation are disclosed.Contain starch ingredients, bean powder component, fish meal, vegetable oil, copper sulphate, magnesium sulfate, potassium dihydrogen phosphate, VE and emulsifier in the fermentation medium, the starch ingredients are starch and/or starch hydrolyzate, the bean powder group is divided into bean powder and/or bean powder hydrolyzate, and at least one of the starch ingredients and bean powder component are its hydrolyzate.Dissolved oxygen concentration can be improved using fermentation medium provided by the invention and it is made to obtain reasonable, efficient utilization, to improve the yield of lycopene.

Description

A kind of fermentation medium and its application and utilize Blakeslea trispora fermentation preparation tomato The method of red pigment

Technical field

The invention belongs to lycopene production fields, and in particular to a kind of fermentation medium and its application and utilize three spore cloth The method for drawing mold fermentation to prepare lycopene.

Background technique

Carotenoid is the important pigment being widely present in nature, is one kind using isoprene as molecular center, leads to Cross the compound with similar structure that the modes such as end cyclisation, the rotation and isomerization of oxygenation or key derive.And tomato red Element is one kind important in carotenoid.Lycopene is a kind of fat-soluble antioxidant, can be by many plants and micro- Biosynthesis, but cannot be synthesized by animals and humans, because being found in tomato the lycopene that is otherwise known as at first, in human body each group Knitting also has more distribution in organ, the unsaturated structure contained in molecule makes it have stronger antioxidant activity and removes certainly By the ability of base, it can effectively prevent, delay and treat certain diseases, especially cancer, while can also improve exempting from for body Epidemic disease function.Therefore, lycopene is widely used in food, drug, cosmetics and health products trade.

At present there are mainly three types of the synthetic methods of lycopene: chemical synthesis, natural extraction method and microbial fermentation Synthetic method.Wherein, lycopene is prepared since by-product is more using chemical synthesis, food safety risk is big, before market Scape limitation is larger；And tomato resources shortage and the unstable of Lycopene in Tomatoes content can all seriously affect natural extraction The industrialized production of method；Compared with first two method, lycopene is produced using microbial fermentation synthetic method, is had not by season It limits, is with short production cycle, the advantages that polluting in production process can be reduced, being a desirable route for realizing industrialized production.

In recent years, the production of domestic and international lycopene mostly uses microbial fermentation synthetic method, is fermented using microorganism There is with short production cycle, process to be easy to control, do not influenced by region and weather production, it is excellent to be easy to carry out scale operation etc. Gesture.During the experiment, researcher has been obtained for some productive strain excellents of tool, such as Blakeslea trispora, greatly Enterobacteria, red pseudomonas and saccharomyces cerevisiae etc., wherein the performance of Blakeslea trispora is preferable, also obtains in production wide Use.

Blakeslea trispora (Blakesleatrispora) belongs to Mucoales, Ji Mei section, is unicellular lower fungus.Three spores Bradley is mould to be grown rapidly under certain condition of culture, can be reached biggish biomass in a relatively short period of time, is suitable for rule The production of modelling.In addition, being divided into positive and negative two kinds, this two there are one being characterized in that Blakeslea trispora belongs to mycelium anastomosis bacterium Kind unisexuality bacterial strain is morphologically without apparent difference, and individually culture is able to carry out vegetative propagation, just when their mixed fermentations The mycelium of negative bacterial strain contacts with each other, and heterothallism occurs and carries out sexual propagation, in the process cooperation addition cyclisation inhibitor It can be obtained the lycopene of certain yield.Currently, many scholars at home and abroad are setting about exploring raising Blakeslea trispora fermentation The process conditions for producing lycopene, so that it is more suitable for industrialized production.

The fermentation medium of Blakeslea trispora mostly uses starch, soy meal etc. to serve as carbon nitrogen source, but this fermented and cultured Base has large effect to the transmitting of oxygen, and in earlier fermentation, the fast-growth of thallus makes its need to dissolved oxygen and nutriment Ask relatively high, dissolved oxygen becomes the key factor of limitation thalli growth, Product formation, or even causes metabolic disorder, influences target The yield of product.

Therefore, in the fermentation process for producing lycopene as bacterial strain using Blakeslea trispora, researcher multi-pass, which is crossed, to be lured Become screening strain excellent or change the whipped form of fermentation, dissolved oxygen supply mode, adds the carrier of oxygen or addition during the fermentation The surfactant of somatic cells permeability can be increased to improve the utilization rate of oxygen concentration and oxygen in fermentation liquid, these researchs pair The improvement of fermentation process has the effect of certain, but there is also such or such deficiency or final low outputs, or The substance of person's addition has certain injury to thallus itself, or causes since the device is complicated at high cost, also has plenty of only needle To one aspect without comprehensively considering to fermentation process.The form of a kind of pair of stirrer paddle is disclosed in CN1331343A The technique being changed uses the Combined stirring paddle leaf that middle layer is pusher for wide leaf, lower layer is turbine type, although energy in this way The mass-transfer performance of fermentation liquid is enough improved, but the industry characteristics of the bacterial strain can only be suitble to after changing whipped form, is unfavorable for fermenting Comprehensive utilization of tank.CN101555490A adds active carbon in fermentation liquid to increase dissolved oxygen content, but this method has meeting Increase equipment loss and maintenance cost, the problems such as extraction process is complicated.

Summary of the invention

The purpose of the invention is to overcome the prior art that can increase using the carrier of oxygen or addition is added during the fermentation Add the surfactant of somatic cells permeability to improve disadvantages described above existing for the utilization rate of oxygen concentration and oxygen in fermentation liquid, and The method for providing one kind new fermentation medium and its application and preparing lycopene using Blakeslea trispora fermentation, passes through tune The formula of whole culture medium, by fermentation medium starch and/or bean powder carry out pretreatment and obtain starch hydrolyzate and/or bean powder Hydrolyzate, to improve dissolved oxygen concentration to improve the yield of lycopene.

Specifically, the present invention provides a kind of fermentation mediums, wherein in the fermentation medium containing starch ingredients, Bean powder component, fish meal, vegetable oil, copper sulphate, magnesium sulfate, potassium dihydrogen phosphate, VE and emulsifier, the starch ingredients are starch And/or starch hydrolyzate, the bean powder group are divided into bean powder and/or bean powder hydrolyzate, and in the starch ingredients and bean powder component At least one be its hydrolyzate.

Further, starch hydrolyzate, bean powder hydrolyzate, fish meal, vegetable oil, sulfuric acid are contained in the fermentation medium Copper, magnesium sulfate, potassium dihydrogen phosphate, VE and emulsifier.

Further, in every liter of fermentation medium containing 0.2~0.4L of starch hydrolyzate, bean powder hydrolyzate 0.1~ 0.3L, 26~32g of fish meal, 20~40g of vegetable oil, 0.06~0.08g of copper sulphate, 0.8~1.0g of magnesium sulfate, potassium dihydrogen phosphate 3 0.5~2g of 0.025~0.05g of~5g, VE and emulsifier, and the pH value of the fermentation medium is 5.5~7.0.

Further, the starch hydrolyzate is made in accordance with the following methods: starch and water being configured to starch milk, later will Acidification hydrolization reaction is carried out after the starch milk and acid solution mixing, after reaction to acidification hydrolization, by the pH of acidification hydrolization liquid Value is adjusted to 6.5~7.5 to get the starch hydrolyzate.

Further, the starch is selected from least one of cornstarch, sweet potato starch and potato starch.

Further, the concentration of the starch milk is 15~20wt%.

Further, the acid solution additive amount is the 0.4~0.6% of the starch milk volume total amount, and the acid solution is dense Spend the concentrated hydrochloric acid of 36~38wt%.

Further, it is 40~60 DEG C that the condition of acidification hydrolization reaction, which includes reaction temperature, the reaction time is 1~ 3h。

Further, the bean powder hydrolyzate is made in accordance with the following methods: bean powder and water are configured to bean powder suspension, it Afterwards will the bean powder suspension and acid solution mix after carry out hydrolysis, to hydrolysis after, by the pH value tune of hydrolyzate Section is to 6.5~7.5 to get the bean powder hydrolyzate.

Further, the bean powder is selected from least one of beancake powder, soyabean protein powder and bean cake powder.

Further, the concentration of the bean powder suspension is 20~30wt%.

Further, the acid solution additive amount is the 1.0~1.5% of the bean powder suspension volume total amount, the acid solution For the concentrated hydrochloric acid of 36~38wt% of concentration.

Further, it is 70~100 DEG C that the condition of the hydrolysis, which includes reaction temperature, and the reaction time is 3~5h, instead Supplement water maintains the constancy of volume of bean powder suspension during answering.

The present invention also provides the fermentation mediums to prepare answering in lycopene in culture Blakeslea trispora fermentation With.

In addition, the present invention also provides a kind of method for preparing lycopene using Blakeslea trispora fermentation, this method Including the positive bacterial strain of Blakeslea trispora and negative bacterial strain are successively carried out actication of culture and seed expansion culture, wherein this method is also Resulting seed liquor is cultivated using above-mentioned fermentation medium progress fermented and cultured including expanding the seed.

Further, the mode of the fermented and cultured be by the seed liquor access fermentor in, inoculum concentration be 10~ 15%, 96~120h of fermented and cultured under conditions of temperature is 26~30 DEG C, pH value is 5.5~7.0, in fermented and cultured process In, revolving speed and ventilatory capacity are controlled according to the growing state of thallus and fill into solvent or diluent, carbon source is filled into according to sampling inspection results, obtains Fermentation liquid containing lycopene.

Further, revolving speed and ventilatory capacity and the mode for filling into solvent or diluent are controlled according to the growing state of thallus are as follows: before fermentation Phase, revolving speed are 110~140r/min, and ventilatory capacity is 0.6~0.8vvm, and tank pressure is 0.03~0.06MPa；Work as fermented incubation time It is 150~250r/min by rotational speed regulation when reaching 30~50h, ventilatory capacity is adjusted to 0.8~1.2vvm, and tank pressure regulation is Then 0.03~0.06MPa fills into solvent or diluent.

Further, in the solvent or diluent containing solvent or diluent A and solvent or diluent B and optional solvent or diluent C, the solvent or diluent A be selected from imidazoles, At least one of piperidines, N-methylmorpholine and nicotine, the solvent or diluent B are selected from α-ionone, alpha, beta-lonone, isoniazid and take off At least one of acid is fallen, the solvent or diluent C is fumaric acid.

Further, the amount for once filling into the solvent or diluent A is 0.02~0.4% (g/100mL) of fermentating liquid volume total amount.

Further, the amount for once filling into the solvent or diluent B is 0.05~0.4% (g/100mL) of fermentating liquid volume total amount.

Further, the amount for once filling into the solvent or diluent C is 0.08~0.2% (g/100mL) of fermentating liquid volume total amount.

Further, the carbon source is the starch hydrolyzate.

Further, the mode of carbon source is filled into according to sampling inspection results are as follows: sample detection residual sugar content, when the residual sugar When content is lower than 4~6g/L, the starch hydrolyzate is filled into one or more times, and the residual sugar content control in fermentation liquid is existed 6~10g/L.

Further, the method provided by the invention for preparing lycopene using Blakeslea trispora fermentation further includes sending out Vegetable oil is disposably filled into when ferment 30~40h of culture, the amount of the vegetable oil filled into every liter of fermentation medium is 1~3g.

Further, the vegetable oil is selected from least one of corn oil, soybean oil, sunflower oil and palm oil.

Further, the mode of the actication of culture are as follows: aseptically, by the positive bacterial strain of Blakeslea trispora and negative bacterium Strain is inoculated on slant medium respectively, be placed in incubator and cultivated at 26~30 DEG C, just to Blakeslea trispora After bacterial strain and negative bacterial strain grow spore respectively, aseptically, the spore in slant medium is scraped with sterile saline It washes, being configured to concentration respectively is 10³~10⁵The positive bacterium spore suspension and concentration of a spore/mL is 10⁴~10⁶A spore The negative bacterium spore suspension of son/mL.

Further, it includes first order seed culture and secondary seed culture that the seed, which expands culture,；

Further, the mode of the first order seed culture are as follows: by the positive bacterium spore suspension and negative bacterium spore suspension point It Jie Ru not be cultivated in first class seed pot；The condition of culture of the positive bacterium spore suspension includes that cultivation temperature is 26~30 DEG C, Revolving speed be 400~600r/min, ventilatory capacity be 0.6~0.8vvm, tank pressure be 0.03~0.06MPa, incubation time be 20~ 36h；The condition of culture of the negative bacterium spore suspension includes that cultivation temperature is 26~30 DEG C, and revolving speed is 500~700r/min, ventilation Amount is 0.6~0.8vvm, and tank pressure is 0.03~0.06MPa, and incubation time is 20~36h, is respectively obtaining Blakeslea trispora just Bacterial strain primary seed solution and negative bacterial strain primary seed solution.

Further, the mode of the secondary seed culture are as follows: by the positive bacterial strain primary seed solution of the Blakeslea trispora It is cultivated with negative bacterial strain primary seed solution combined inoculation into secondary seed tank, the positive bacterial strain level-one kind of the Blakeslea trispora The inoculation volume ratio of sub- liquid and negative bacterial strain primary seed solution is 1:5~1:10, and inoculum concentration is 10~15%, cultivation temperature is 26~ 30 DEG C, revolving speed is 600~900r/min, and ventilatory capacity is 0.6~0.8vvm, and tank pressure is 0.03~0.06MPa, incubation time 10 ~20h, obtains secondary seed solution.

Compared with prior art, beneficial effects of the present invention are as follows:

Starch in fermentation medium is substituted using starch hydrolyzate and/or bean powder is used bean powder hydrolyzate by the present invention Substitution, can be improved the concentration of dissolved oxygen in this way, meets consumption of the mycelium when fermenting initial stage Rapid Accumulation to oxygen.Together When, the main component of starch hydrolyzate is glucose, the glycoconjugates such as followed by a small amount of maltose and some other disaccharides, oligosaccharide Class contains free amino acid in bean powder hydrolyzate, these substances can meet the needs of thallus fast-growth in earlier fermentation, and The starch and bean powder of the non-complete hydrolysis in part become at a slow speed using carbon nitrogen source after then running up to certain biomass in thallus, have Conducive to the synthesis of lycopene.Therefore, the pre-treatment operation of starch and bean powder has taken into account the nutritive peculiarity of production bacterium and product closes At to substantially increase the yield of lycopene.In addition, provided by the invention utilize Blakeslea trispora fermentation preparation tomato The method of red pigment is not high to equipment requirement, input cost is smaller, and raw material sources are extensive, at low cost, the yield of lycopene compared with It is significantly improved before process modification, and can realize large-scale production.

A preferred embodiment of the invention, starch and/or bean powder in the fermentation medium use starch water On the basis of solving liquid and/or bean powder hydrolyzate substitution, revolving speed, ventilatory capacity and tank pressure are carried out stage by stage according to the growing state of thallus The adjustment (i.e. earlier fermentation and fermentation later period, using different revolving speed and ventilatory capacity and comparable tank pressure) of amount, is more advantageous to Thalli growth and Product formation can reduce energy consumption to improve the fermentation unit of lycopene, energy saving.

A preferred embodiment of the invention, it is dilute by adding when fermented incubation time reaches 30~50h Material is conducive to the fermentation unit for improving lycopene.

According to another preferred method of implementation of the present invention, when directlyed adopt during fermented and cultured starch hydrolyzate make When carrying out feed supplement for carbon source, other than being more advantageous to the fermentation unit for improving lycopene, compared with feed supplement glucose, mend Doses decreases, to also reduce cost of material.

Specific embodiment

Contain starch ingredients, bean powder component, fish meal, vegetable oil, copper sulphate, sulfuric acid in fermentation medium provided by the invention Magnesium, potassium dihydrogen phosphate, VE and emulsifier, the starch ingredients are starch and/or starch hydrolyzate, and the bean powder group is divided into bean powder And/or bean powder hydrolyzate, and at least one of the starch ingredients and bean powder component are its hydrolyzate (that is, the starch group It is divided into starch hydrolyzate and bean powder group is divided into bean powder or the starch ingredients are starch and bean powder group is divided into bean powder hydrolyzate, Or the starch ingredients are starch hydrolyzate and bean powder group is divided into bean powder hydrolyzate).It is particularly preferred that the starch ingredients are Starch hydrolyzate and bean powder group is divided into bean powder hydrolyzate, that is, hydrolyzed in the fermentation medium containing starch hydrolyzate, bean powder Liquid, fish meal, vegetable oil, copper sulphate, magnesium sulfate, potassium dihydrogen phosphate, VE and emulsifier, are more advantageous to yield of lycopene at this time It improves.Further, 0.2~0.4L of starch hydrolyzate, 0.1~0.3L of bean powder hydrolyzate, fish are contained in every liter of fermentation medium 26~32g of powder, 20~40g of vegetable oil, 0.06~0.08g of copper sulphate, 0.8~1.0g of magnesium sulfate, 3~5g of potassium dihydrogen phosphate, VE 0.5~2g of 0.025~0.05g and emulsifier, and the pH value of the fermentation medium is 5.5~7.0.

The starch hydrolyzate refers to that starch, which is carried out acidification hydrolization, handles resulting incomplete hydrolysate.The starch Hydrolyzate can be commercially available, and can also be prepared according to existing various methods.It is according to the present invention a kind of specific Embodiment, the starch hydrolyzate are made in accordance with the following methods: starch and water being configured to starch milk, later by the starch Acidification hydrolization reaction is carried out after cream and acid solution mixing after reaction to acidification hydrolization to be adjusted to the pH value of acidification hydrolization liquid 6.5~7.5.Wherein, the specific example of the starch includes but is not limited to: in cornstarch, sweet potato starch and potato starch At least one.The concentration of the starch milk can be 15~20wt%.The acid solution additive amount is preferably the starch milk body The 0.4~0.6% of product total amount.The acid solution is specifically as follows at least one of hydrochloric acid solution, sulfuric acid solution and nitric acid solution, The particularly preferably concentrated hydrochloric acid of 36~38wt% of concentration.In addition, the condition of the acidification hydrolization reaction generally includes reaction temperature Preferably 40~60 DEG C, the reaction time is preferably 1~3h.

The bean powder hydrolyzate refers to that bean powder, which is carried out heating acidification hydrolization, handles resulting incomplete hydrolysate.It is described Bean powder hydrolyzate can be commercially available, and can also be prepared according to existing various methods.One kind according to the present invention Specific embodiment, the bean powder hydrolyzate are made in accordance with the following methods: bean powder and water being configured to bean powder suspension, later will The bean powder suspension and acid solution mixing after carry out hydrolysis, to hydrolysis after, the pH value of hydrolyzate is adjusted to 6.5~7.5 to get the bean powder hydrolyzate.Wherein, the specific example of the bean powder includes but is not limited to: beancake powder, soybean egg At least one of white powder and bean cake powder.The concentration of the bean powder suspension can be 20~30wt%.The acid solution additive amount The 1.0~1.5% of the preferably described bean powder suspension volume total amount.The acid solution is specifically as follows hydrochloric acid solution, sulfuric acid solution At least one of with nitric acid solution, the particularly preferably concentrated hydrochloric acid of 36~38wt% of concentration.In addition, the item of the hydrolysis It is preferably 70~100 DEG C that part, which generally includes reaction temperature, and the reaction time is preferably 3~5h, and water is supplemented during reaction and maintains bean powder Suspension constancy of volume.

The present invention also provides the fermentation mediums to prepare answering in lycopene in culture Blakeslea trispora fermentation With.

In addition, the present invention also provides a kind of method for preparing lycopene using Blakeslea trispora fermentation, this method Including the positive bacterial strain of Blakeslea trispora and negative bacterial strain are successively carried out actication of culture, seed expands culture and fermented and cultured.

In the present invention, the positive and negative bacterial strain of the Blakeslea trispora (Blakesleatrispora) is commercially available available bacterium Strain, for example, can be respectively the positive and negative bacterial strain of ATCC14271 (+) and the Blakeslea trispora of ATCC14272 (-) for number, it can Think that number is respectively the positive and negative bacterial strain of the Blakeslea trispora of CCTCC AF 97006 (+) and CCTCC AF 96002 (-), also It can be respectively the positive and negative bacterial strain of the Blakeslea trispora of NRRL 2895 (+) and NRRL 2896 (-) for number.

The detailed process of the actication of culture includes: aseptically, by the positive bacterial strain and negative bacterial strain of Blakeslea trispora Be inoculated on slant medium respectively, be placed in incubator and cultivated at 26~30 DEG C, the positive bacterium to Blakeslea trispora Strain and negative bacterial strain are grown after spore respectively (it is generally necessary to 4~5d), aseptically, with sterile saline by inclined-plane culture Spore in base scrapes, and being configured to concentration respectively is 10³~10⁵The positive bacterium spore suspension and concentration of a spore/mL be 10⁴~10⁶The negative bacterium spore suspension of a spore/mL.Wherein, the slant medium is specifically as follows inclined-plane PDA culture medium.

It includes first order seed culture and secondary seed culture that the seed, which expands culture,.

The detailed process of the first order seed culture includes: to connect the positive bacterium spore suspension and negative bacterium spore suspension respectively Enter in first class seed pot and is cultivated；The condition of culture of the positive bacterium spore suspension includes that cultivation temperature is 26~30 DEG C, revolving speed For 400~600r/min, ventilatory capacity is 0.6~0.8vvm, and tank pressure is 0.03~0.06MPa, and incubation time is 20~36h；Institute It is 26~30 DEG C that the condition of culture for stating negative bacterium spore suspension, which includes cultivation temperature, and revolving speed is 500~700r/min, and ventilatory capacity is 0.6~0.8vvm, tank pressure are 0.03~0.06MPa, and incubation time is 20~36h, respectively obtain the positive bacterial strain of Blakeslea trispora Primary seed solution and negative bacterial strain primary seed solution.The inoculum concentration of the positive bacterium spore suspension and negative bacterium spore suspension usually can be each From independently being 10~15%.Preferably comprised in every liter of primary-seed medium 22~26g of glucose, soyabean protein powder 26~ 30g, 0.8~1.0g of magnesium sulfate, 3~5g of potassium dihydrogen phosphate, 0.06~0.1g of calcium chloride, 0.06~0.1g of copper sulphate, VE 0.5~2g of 0.025~0.05g and emulsifier.

The detailed process of the secondary seed culture includes: by the positive bacterial strain primary seed solution of the Blakeslea trispora and to bear The combined inoculation of bacterial strain primary seed solution is cultivated into secondary seed tank, the positive bacterial strain primary seed solution of the Blakeslea trispora Inoculation volume ratio with negative bacterial strain primary seed solution is 1:5~1:10, and inoculum concentration is 10~15%, and cultivation temperature is 26~30 DEG C, revolving speed be 600~900r/min, ventilatory capacity be 0.6~0.8vvm, tank pressure be 0.03~0.06MPa, incubation time be 10~ 20h obtains secondary seed solution.Wherein, 22~26g of glucose, soyabean protein powder are preferably comprised in every liter of secondary seed medium 26~30g, 0.8~1.0g of magnesium sulfate, 3~5g of potassium dihydrogen phosphate, 0.06~0.1g of calcium chloride, 0.06~0.1g of copper sulphate, VE 0.5~2g of 0.025~0.05g and emulsifier.

The detailed process of the fermented and cultured includes: to be accessed the secondary seed solution in fermentor using pressure differential method, is connect Kind amount is 10~15%, and 96~120h of fermented and cultured under conditions of temperature is 26~30 DEG C, pH value is 5.5~7.0 is fermenting In incubation, revolving speed and ventilatory capacity are controlled according to the growing state of thallus and fill into solvent or diluent, is filled into according to sampling inspection results Carbon source obtains the fermentation liquid containing lycopene.

A preferred embodiment of the invention controls revolving speed and ventilatory capacity according to the growing state of thallus and fills into The mode of solvent or diluent are as follows: earlier fermentation, revolving speed are 110~140r/min, and ventilatory capacity is 0.6~0.8vvm, tank pressure for 0.03~ 0.06MPa；It is 150~250r/min by rotational speed regulation when fermented incubation time reaches 30~50h, ventilatory capacity is adjusted to 0.8 ~1.2vvm, tank pressure regulation are 0.03~0.06MPa, then fill into solvent or diluent.Wherein, reach 30~50h in fermented incubation time When fill into solvent or diluent purpose be yield in order to improve lycopene.Containing solvent or diluent A and solvent or diluent B and optionally in the solvent or diluent Solvent or diluent C is preferably made of solvent or diluent A, solvent or diluent B and solvent or diluent C.The solvent or diluent A is in imidazoles, piperidines, N-methylmorpholine and nicotine At least one, preferably imidazoles.The solvent or diluent B in α-ionone, alpha, beta-lonone, isoniazid and abscisic acid at least One kind, preferably alpha, beta-lonone.The solvent or diluent C is fumaric acid.It wherein, can be into one when containing solvent or diluent C in the solvent or diluent Step promotes the raising of yield of lycopene.In addition, the amount for once filling into the solvent or diluent A is preferably the 0.02 of fermentating liquid volume total amount ~0.4% (g/100mL), that is, contain 0.02~0.4g solvent or diluent A in 100mL fermentation liquid.The amount for once filling into the solvent or diluent B is excellent It is selected as 0.05~0.4% (g/100mL) of fermentating liquid volume total amount, that is, contain 0.05~0.4g solvent or diluent B in 100mL fermentation liquid. The amount for once filling into the solvent or diluent C is preferably 0.08~0.2% (g/100mL) of fermentating liquid volume total amount, that is, 100mL fermentation Contain 0.08~0.2g solvent or diluent C in liquid.When earlier fermentation and fermented incubation time reach 30~50h using as above preferred side Formula is adjusted revolving speed, ventilatory capacity and tank pressure, not only improves thalli growth and Product formation to improve the fermentation of lycopene Unit, and can reduce energy consumption, it is energy saving.

In the present invention, the fermented and cultured process needs to fill into carbon source according to sampling inspection results.Wherein, the carbon source It can be starch hydrolyzate, or glucose, preferably starch hydrolyzate are more advantageous to mentioning for yield of lycopene in this way It is high.Generally, the mode of supplementary carbon source is sample detection residual sugar content, when the residual sugar content is lower than 4~6g/L, is divided primary Or the starch hydrolyzate is repeatedly filled into, the residual sugar content in fermentation liquid is controlled in 6~10g/L.Wherein, detection residual sugar contains The time interval of amount can be 2~8h.

According to a kind of preferred embodiment, the side provided by the invention that lycopene is prepared using Blakeslea trispora fermentation Method further includes that vegetable oil is disposably filled into 30~40h of fermented and cultured, the vegetable oil filled into every liter of fermentation medium Amount be 1~3g.The purpose for filling into vegetable oil is to improve yield.The specific example of the vegetable oil includes but is not limited to: At least one of corn oil, soybean oil, sunflower oil and palm oil.

In the present invention, it is emulsified contained in the primary-seed medium, secondary seed medium and fermentation medium The type of agent may be the same or different, and can be each independently Span series emulsifier and/or TWEEN Series emulsification Agent.

The embodiment of the present invention is described below in detail, the examples of the embodiments are intended to be used to explain the present invention, and cannot It is interpreted as limitation of the present invention.In the examples where no specific technique or condition is specified, described according to the literature in the art Technology or conditions or carried out according to product description.Reagents or instruments used without specified manufacturer is that can lead to Cross the conventional products of commercially available acquisition.

Embodiment 1: a method of lycopene being prepared using Blakeslea trispora fermentation, includes the following steps: (1) bacterium Kind activation；(2) first order seed culture；(3) secondary seed culture；(4) fermented and cultured.

(1) actication of culture: aseptically, the positive bacterial strain of Blakeslea trispora and negative bacterial strain are inoculated into inclined-plane respectively In PDA culture medium (potato dextrose agar, similarly hereinafter), be placed in incubator, 4d is cultivated at 26 DEG C, to three After the mould positive bacterial strain of spore Bradley and negative bacterial strain grow spore respectively, aseptically, with sterile saline by inclined-plane culture Base miospore scrapes, and is configured to uniform spore suspension respectively, makes the concentration of positive bacterium spore suspension and negative bacterium spore suspension Be positive bacterium 10 respectively⁴A spore/mL and negative bacterium 10⁵A spore/mL.Wherein, Blakeslea trispora (Blakeslea trispora) Positive bacterial strain and negative bacterial strain are purchased from ATCC, and number is respectively ATCC 14271 (+) and ATCC 14272 (-).

(2) first order seed culture: positive bacterium spore suspension and negative bacterium spore suspension are respectively connected to carry out in first class seed pot The cultivation temperature of culture, positive bacterium spore suspension is 28 DEG C, revolving speed 400r/min, ventilatory capacity 0.6vvm, and tank pressure is 0.03MPa, incubation time 36h；The cultivation temperature of negative bacterium spore suspension is 28 DEG C, revolving speed 500r/min, and ventilatory capacity is 0.8vvm, tank pressure be 0.04MPa, incubation time 36h, respectively obtain Blakeslea trispora positive bacterial strain primary seed solution and negative bacterium Strain primary seed solution；Contain following component: glucose 22g, soyabean protein powder 26g, magnesium sulfate in every liter of primary-seed medium 0.8g, potassium dihydrogen phosphate 3g, calcium chloride 0.06g, copper sulphate 0.06g, VE 0.025g and Tween-60 0.5g.

(3) secondary seed culture: the positive bacterial strain primary seed solution for the Blakeslea trispora that step (2) culture is obtained and negative bacterium Strain primary seed solution combined inoculation is cultivated into secondary seed tank, the positive bacterial strain primary seed solution and three spores of Blakeslea trispora The inoculation volume ratio of the mould negative bacterial strain primary seed solution of Bradley is 1:10, and inoculum concentration 10%, cultivation temperature is 28 DEG C, and revolving speed is 600r/min, ventilatory capacity 0.6vvm, tank pressure is 0.03MPa, and incubation time 20h obtains secondary seed solution；Every liter of second level kind Contain following component: glucose 22g, soyabean protein powder 26g, magnesium sulfate 0.8g, potassium dihydrogen phosphate 3g, calcium chloride in sub- culture medium 0.06g, copper sulphate 0.06g, VE 0.025g and Tween-60 0.5g.

(4) fermented and cultured: the secondary seed solution is accessed in fermentor using pressure differential method, inoculum concentration 10%, in temperature Fermented and cultured 96h under conditions of degree is 28 DEG C, pH value is 5.5, revolving speed, ventilatory capacity and tank pressure are adjusted with the growing state of thallus Section adjusts strategy are as follows: earlier fermentation, revolving speed 110r/min, ventilatory capacity 0.6vvm, tank pressure are 0.03MPa；With ferment into It goes, when color has started to accumulate yellow in and mycelium sturdy when a large amount of mycelium of microscopy observation, i.e., fermentation time reaches 40h When, it is 150r/min by rotational speed regulation, ventilatory capacity is adjusted to 0.8vvm, and then tank pressure regulation 0.03MPa fills into solvent or diluent, institute It states solvent or diluent to be made of alpha, beta-lonone, imidazoles and fumaric acid, the amount for once filling into alpha, beta-lonone is fermentating liquid volume total amount 0.1% (g/100mL), the amount for once filling into imidazoles is 0.06% (g/100mL) of fermentating liquid volume total amount, once fills into rich horse The amount of acid is 0.08% (g/100mL) of fermentating liquid volume total amount.Corn oil, every liter of fermentation liquid are once filled into when fermenting 30h In the amount of corn oil that fills into be 1g；It is sampled during the fermentation every 4h and surveys residual sugar content, when sample detection residual sugar content is low When 5.0g/L, starch hydrolyzate is filled into one or more times, by residual sugar content control in fermentation liquid in 6~10g/L.

Contain following component: the starch hydrolyzate 0.2L, bean powder hydrolyzate 0.1L, fish meal in every liter of fermentation medium 26g, corn oil 20g, copper sulphate 0.06g, magnesium sulfate 0.8g, potassium dihydrogen phosphate 3g, VE 0.025g and Tween-60 0.5g, and The pH value of fermentation medium is adjusted to 5.5 with sodium hydroxide.

The starch hydrolyzate comprises the following steps: it is 20% that cornstarch and water, which are configured to mass percent concentration, Starch milk, then be added hydrochloric acid (commercial concentration be 36~38wt% concentrated hydrochloric acid, similarly hereinafter), hydrochloric acid additive amount be starch milk body The 0.6% of product total amount, starch milk reacts 1h at 60 DEG C after mixing evenly with hydrochloric acid, to after reaction, in sodium hydroxide The starch hydrolyzate for being 6.5 with obtained pH value.

The bean powder hydrolyzate comprises the following steps: soyabean protein powder, which is configured to mass percent concentration with water, is 30% soyabean protein powder suspension, is then added hydrochloric acid, and hydrochloric acid additive amount is soyabean protein powder suspension volume total amount 1.5%, soyabean protein powder suspension reacts 3h with hydrochloric acid at 100 DEG C after mixing evenly, during which supplements water and maintains suspended liquid Product is constant.After reaction, with the soyabean protein powder hydrolyzate for being 6.5 with obtained pH value in sodium hydroxide.

It collects fermentation liquid and obtains thallus, measure dry weight and reach 61.3g/L, high performance liquid chromatography detects tomato in fermentation liquid For red pigment unit up to 4.37g/L, mass percent of the lycopene in dry mycelium is 7.13%.

Embodiment 2: a method of lycopene being prepared using Blakeslea trispora fermentation, includes the following steps: (1) bacterium Kind activation；(2) first order seed culture；(3) secondary seed culture；(4) fermented and cultured.

(1) actication of culture: aseptically, the positive bacterial strain of Blakeslea trispora and negative bacterial strain are inoculated into inclined-plane respectively In PDA culture medium, be placed in incubator, cultivate 5d at 27 DEG C, distinguish to the positive bacterial strain of Blakeslea trispora and negative bacterial strain After growing spore, aseptically, slant medium miospore is scraped with sterile saline, be configured to respectively Even spore suspension makes the concentration of positive bacterium spore suspension and negative bacterium spore suspension be positive respectively bacterium 10⁴A spore/mL and negative bacterium 10⁵ A spore/mL.Wherein, the positive bacterial strain of Blakeslea trispora (Blakeslea trispora) and negative bacterial strain are purchased from ATCC, number point It Wei not ATCC 14271 (+) and ATCC 14272 (-).

(2) first order seed culture: positive bacterium spore suspension and negative bacterium spore suspension are respectively connected to carry out in first class seed pot The cultivation temperature of culture, positive bacterium spore suspension is 28 DEG C, revolving speed 450r/min, ventilatory capacity 0.8vvm, and tank pressure is 0.05MPa, incubation time 32h；The cultivation temperature of negative bacterium spore suspension is 28 DEG C, revolving speed 550r/min, and ventilatory capacity is 0.7vvm, tank pressure be 0.06MPa, incubation time 32h, respectively obtain Blakeslea trispora positive bacterial strain primary seed solution and negative bacterium Strain primary seed solution；Contain following component: glucose 23g, soyabean protein powder 27g, magnesium sulfate in every liter of primary-seed medium 0.8g, potassium dihydrogen phosphate 3g, calcium chloride 0.06g, copper sulphate 0.06g, VE 0.025g and Tween-60 0.5g.

(3) secondary seed culture: the positive bacterial strain primary seed solution for the Blakeslea trispora that step (2) culture is obtained and negative bacterium Strain primary seed solution combined inoculation is cultivated into secondary seed tank, the positive bacterial strain primary seed solution and three spores of Blakeslea trispora The inoculation volume ratio of the mould negative bacterial strain primary seed solution of Bradley is 1:6, and inoculum concentration 12%, cultivation temperature is 28 DEG C, and revolving speed is 650r/min, ventilatory capacity 0.7vvm, tank pressure is 0.04MPa, and incubation time 17h obtains secondary seed solution；Every liter of second level kind In sub- culture medium contain following parts by weight of component: glucose 23g, soyabean protein powder 26g, magnesium sulfate 0.8g, potassium dihydrogen phosphate 3g, Calcium chloride 0.06g, copper sulphate 0.06g, VE 0.025g and Tween-60 0.5g.

(4) fermented and cultured: the secondary seed solution is accessed in fermentor using pressure differential method, inoculum concentration 13%, in temperature Fermented and cultured 100h under conditions of degree is 28 DEG C, pH value is 6.0, revolving speed, ventilatory capacity and tank pressure are carried out with the growing state of thallus It adjusts, adjusts strategy are as follows: earlier fermentation, revolving speed 130r/min, ventilatory capacity 0.7vvm, tank pressure are 0.05MPa；With fermentation It carries out, when color has started to accumulate yellow in and mycelium sturdy when a large amount of mycelium of microscopy observation, i.e., fermentation time reaches 45h When, it is 170r/min by rotational speed regulation, ventilatory capacity is adjusted to 0.8vvm, and then tank pressure regulation 0.05MPa fills into solvent or diluent, institute It states solvent or diluent to be made of alpha, beta-lonone, imidazoles and fumaric acid, the amount for once filling into alpha, beta-lonone is fermentating liquid volume total amount 0.2% (g/100mL), the amount for once filling into imidazoles is 0.15% (g/100mL) of fermentating liquid volume total amount, once fills into rich horse The amount of acid is 0.15% (g/100mL) of fermentating liquid volume total amount.Sunflower oil, every liter of fermentation are once filled into when fermenting 35h The amount of the sunflower oil filled into liquid is 1g；It is sampled during the fermentation every 4h and surveys residual sugar content, when sample detection residual sugar contains When amount is lower than 5.0g/L, starch hydrolyzate is filled into one or more times, by residual sugar content control in fermentation liquid in 6~10g/L.

Contain following component: the starch hydrolyzate 0.2L, soyabean protein powder 40g, fish meal in every liter of fermentation medium 26g, sunflower oil 20g, copper sulphate 0.06g, magnesium sulfate 0.8g, potassium dihydrogen phosphate 3g, VE 0.025g and Tween-60 0.5g, And the pH value of fermentation medium is adjusted to 6.0 with sodium hydroxide.

The starch hydrolyzate comprises the following steps: it is 17% that sweet potato starch and water, which are configured to mass percent concentration, Starch milk, be then added hydrochloric acid, hydrochloric acid additive amount is the 0.5% of starch milk volume total amount, and starch milk is stirred evenly with hydrochloric acid 2h is reacted at 50 DEG C afterwards, to after reaction, in sodium hydroxide and starch hydrolyzate that obtained pH value is 7.0.

It collects fermentation liquid and obtains thallus, measure dry weight and reach 64.7g/L, high performance liquid chromatography detects tomato in fermentation liquid For red pigment unit up to 3.62g/L, mass percent of the lycopene in dry mycelium is 5.60%.

Embodiment 3: a method of lycopene being prepared using Blakeslea trispora fermentation, includes the following steps: (1) bacterium Kind activation；(2) first order seed culture；(3) secondary seed culture；(4) fermented and cultured.

(1) actication of culture: aseptically, the positive bacterial strain of Blakeslea trispora and negative bacterial strain are inoculated into inclined-plane respectively In PDA culture medium, be placed in incubator, cultivate 5d at 30 DEG C, distinguish to the positive bacterial strain of Blakeslea trispora and negative bacterial strain After growing spore, aseptically, slant medium miospore is scraped with sterile saline, be configured to respectively Even spore suspension makes the concentration of positive bacterium spore suspension and negative bacterium spore suspension be positive respectively bacterium 10⁴A spore/mL and negative bacterium 10⁵ A spore/mL.Wherein, the positive bacterial strain of Blakeslea trispora (Blakeslea trispora) and negative bacterial strain are purchased from ATCC, number point It Wei not ATCC 14271 (+) and ATCC 14272 (-).

(2) first order seed culture: positive bacterium spore suspension and negative bacterium spore suspension are respectively connected to cultivate in seeding tank, The cultivation temperature of positive bacterium spore suspension is 28 DEG C, revolving speed 600r/min, ventilatory capacity 0.6vvm, and tank pressure is 0.06MPa, culture Time is 20h；The cultivation temperature of negative bacterium spore suspension is 28 DEG C, revolving speed 700r/min, ventilatory capacity 0.8vvm, and tank pressure is 0.06MPa, incubation time 20h respectively obtain the positive bacterial strain primary seed solution and negative bacterial strain primary seed solution of Blakeslea trispora； Contain following component: glucose 26g, soyabean protein powder 30g, magnesium sulfate 1.0g, biphosphate in every liter of primary-seed medium Potassium 5g, calcium chloride 0.1g, -40 2.0g of copper sulphate 0.1g, VE 0.05g and Span；

(3) secondary seed culture: the positive bacterial strain primary seed solution for the Blakeslea trispora that step (2) culture is obtained and negative bacterium Strain primary seed solution combined inoculation is cultivated into secondary seed tank, the positive bacterial strain primary seed solution and three spores of Blakeslea trispora The inoculation volume ratio of the mould negative bacterial strain primary seed solution of Bradley is 1:5, and inoculum concentration 15%, cultivation temperature is 28 DEG C, and revolving speed is 900r/min, ventilatory capacity 0.8vvm, tank pressure is 0.06MPa, and incubation time 10h obtains secondary seed solution；Every liter of second level kind Contain following component: glucose 26g, soyabean protein powder 30g, magnesium sulfate 1.0g, potassium dihydrogen phosphate 5g, calcium chloride in sub- culture medium 0.1g, -40 2.0g of copper sulphate 0.1g, VE 0.05g and Span.

(4) fermented and cultured: the secondary seed solution is accessed in fermentor using pressure differential method, inoculum concentration 15%, in temperature Fermented and cultured 120h under conditions of degree is 28 DEG C, pH value is 7.0, revolving speed, ventilatory capacity and tank pressure are carried out with the growing state of thallus It adjusts, adjusts strategy are as follows: earlier fermentation, revolving speed 140r/min, ventilatory capacity 0.8vvm, tank pressure are 0.06MPa；With fermentation It carries out, when color has started to accumulate yellow in and mycelium sturdy when a large amount of mycelium of microscopy observation, i.e., fermentation time reaches 35h When, it is 250r/min by rotational speed regulation, ventilatory capacity is adjusted to 1.2vvm, and then tank pressure regulation 0.06MPa fills into solvent or diluent, institute It states solvent or diluent to be made of alpha, beta-lonone, imidazoles and fumaric acid, the amount for once filling into alpha, beta-lonone is fermentating liquid volume total amount 0.4% (g/100mL), the amount for once filling into imidazoles is 0.25% (g/100mL) of fermentating liquid volume total amount, once fills into rich horse The amount of acid is 0.2% (g/100mL) of fermentating liquid volume total amount.Soybean oil is once filled into when fermenting 35h, in every liter of fermentation liquid The amount of the soybean oil filled into is 1g；It is sampled during the fermentation every 4h and surveys residual sugar content, when sample detection residual sugar content is lower than When 5.0g/L, starch hydrolyzate is filled into one or more times, by residual sugar content control in fermentation liquid in 6~10g/L.

Contain following component: the starch hydrolyzate 0.4L, bean powder hydrolyzate 0.3L, fish meal in every liter of fermentation medium 32g, soybean oil 40g, copper sulphate 0.08g, magnesium sulfate 1.0g, -40 2g of potassium dihydrogen phosphate 5g, VE 0.05g and Span, and use hydrogen The pH value of fermentation medium is adjusted to 7.0 by sodium oxide molybdena.

The starch hydrolyzate comprises the following steps: it is 15% that sweet potato starch and water, which are configured to mass percent concentration, Starch milk, be then added hydrochloric acid, hydrochloric acid additive amount is the 0.4% of starch milk volume total amount, and starch milk is stirred evenly with hydrochloric acid 3h is reacted at 40 DEG C afterwards, to after reaction, in sodium hydroxide and starch hydrolyzate that obtained pH value is 7.5.

The bean powder hydrolyzate comprises the following steps: soyabean protein powder, which is configured to mass percent concentration with water, is 20% soyabean protein powder suspension, is then added hydrochloric acid, and hydrochloric acid additive amount is soyabean protein powder suspension volume total amount 1.0%, soyabean protein powder suspension reacts 5h with hydrochloric acid at 80 DEG C after mixing evenly, during which supplements water and maintains suspension volume It is constant.After reaction, with the soyabean protein powder hydrolyzate for being 7.5 with obtained pH value in sodium hydroxide.

It collects fermentation liquid and obtains thallus, measure dry weight and reach 58.4g/L, high performance liquid chromatography detects tomato in fermentation liquid For red pigment unit up to 5.06g/L, mass percent of the lycopene in dry mycelium is 8.66%.

Embodiment 4: a method of lycopene being prepared using Blakeslea trispora fermentation, includes the following steps: (1) bacterium Kind activation；(2) first order seed culture；(3) secondary seed culture；(4) fermented and cultured.

(1) actication of culture: aseptically, the positive bacterial strain of Blakeslea trispora and negative bacterial strain are inoculated into inclined-plane respectively In PDA culture medium, be placed in incubator, cultivate 5d at 28 DEG C, distinguish to the positive bacterial strain of Blakeslea trispora and negative bacterial strain After growing spore, aseptically, slant medium miospore is scraped with sterile saline, be configured to respectively Even spore suspension makes the concentration of positive bacterium spore suspension and negative bacterium spore suspension be positive respectively bacterium 10⁴A spore/mL and negative bacterium 10⁵ A spore/mL.Wherein, the positive bacterial strain of Blakeslea trispora (Blakeslea trispora) and negative bacterial strain are purchased from ATCC, number point It Wei not ATCC 14271 (+) and ATCC 14272 (-).

(2) first order seed culture: positive bacterium spore suspension and negative bacterium spore suspension are respectively connected to carry out in first class seed pot The cultivation temperature of culture, positive bacterium spore suspension is 28 DEG C, revolving speed 550r/min, ventilatory capacity 0.7vvm, and tank pressure is 0.06MPa, incubation time are for 24 hours；The cultivation temperature of negative bacterium spore suspension is 28 DEG C, revolving speed 650r/min, and ventilatory capacity is 0.6vvm, tank pressure are 0.05MPa, and incubation time is the positive bacterial strain primary seed solution for respectively obtaining Blakeslea trispora for 24 hours and negative bacterium Strain primary seed solution；Contain following component: glucose 25g, soyabean protein powder 29g, magnesium sulfate in every liter of primary-seed medium 1.0g, potassium dihydrogen phosphate 5g, calcium chloride 0.1g, -40 2.0g of copper sulphate 0.1g, VE 0.05g and Span.

(3) secondary seed culture: the positive bacterial strain primary seed solution for the Blakeslea trispora that step (2) culture is obtained and negative bacterium Strain primary seed solution combined inoculation is cultivated into secondary seed tank, the positive bacterial strain primary seed solution and three spores of Blakeslea trispora The inoculation volume ratio of the mould negative bacterial strain primary seed solution of Bradley is 1:7, and inoculum concentration 14%, cultivation temperature is 28 DEG C, and revolving speed is 800r/min, ventilatory capacity 0.8vvm, tank pressure is 0.06MPa, and incubation time 14h obtains secondary seed solution；Every liter of second level kind Contain following component: glucose 25g, soyabean protein powder 28g, magnesium sulfate 1.0g, potassium dihydrogen phosphate 5g, calcium chloride in sub- culture medium 0.1g, -40 2.0g of copper sulphate 0.1g, VE 0.05g and Span.

(4) fermented and cultured: the secondary seed solution is accessed in fermentor using pressure differential method, inoculum concentration 14%, in temperature Fermented and cultured 110h under conditions of degree is 28 DEG C, pH value is 6.5, revolving speed, ventilatory capacity and tank pressure are carried out with the growing state of thallus It adjusts, adjusts strategy are as follows: earlier fermentation, revolving speed 140r/min, ventilatory capacity 0.8vvm, tank pressure are 0.06MPa；With fermentation It carries out, when color has started to accumulate yellow in and mycelium sturdy when a large amount of mycelium of microscopy observation, i.e., fermentation time reaches 45h When, it is 230r/min by rotational speed regulation, ventilatory capacity is adjusted to 1.0vvm, and then tank pressure regulation 0.06MPa fills into solvent or diluent, institute It states solvent or diluent to be made of alpha, beta-lonone, imidazoles and fumaric acid, the amount for once filling into alpha, beta-lonone is fermentating liquid volume total amount 0.4% (g/100mL), the amount for once filling into imidazoles is 0.25% (g/100mL) of fermentating liquid volume total amount, once fills into rich horse The amount of acid is 0.2% (g/100mL) of fermentating liquid volume total amount.Soybean oil is once filled into when fermenting 30h, in every liter of fermentation liquid The amount of the soybean oil filled into is 2g；It is sampled during the fermentation every 4h and surveys residual sugar, when sample detection residual sugar content is lower than 5.0g/ When L, glucose is filled into one or more times, by residual sugar content control in fermentation liquid in 6~10g/L.

Contain following component: starch 45g, bean powder hydrolyzate 0.2L, fish meal 29g, soybean oil in every liter of fermentation medium 30g, copper sulphate 0.07g, magnesium sulfate 0.9g, -40 1.0g of potassium dihydrogen phosphate 4g, VE 0.035g and Span, and use sodium hydroxide The pH value of fermentation medium is adjusted to 6.5.

The bean powder hydrolyzate comprises the following steps: soyabean protein powder, which is configured to mass percent concentration with water, is 28% soyabean protein powder suspension, is then added hydrochloric acid, and hydrochloric acid additive amount is soyabean protein powder suspension volume total amount 1.3%, soyabean protein powder suspension reacts 4h with hydrochloric acid at 90 DEG C after mixing evenly, during which supplements water and maintains suspension volume It is constant.After reaction, with the soyabean protein powder hydrolyzate for being 7.0 with obtained pH value in sodium hydroxide.

It collects fermentation liquid and obtains thallus, measure dry weight and reach 59.5g/L, high performance liquid chromatography detects tomato in fermentation liquid For red pigment unit up to 2.64g/L, mass percent of the lycopene in dry mycelium is 4.44%.

Embodiment 5: a method of lycopene being prepared using Blakeslea trispora fermentation, includes the following steps: (1) bacterium Kind activation；(2) first order seed culture；(3) secondary seed culture；(4) fermented and cultured.

(1) actication of culture: aseptically, the positive bacterial strain of Blakeslea trispora and negative bacterial strain are inoculated into inclined-plane respectively In PDA culture medium, be placed in incubator, cultivate 4d at 29 DEG C, distinguish to the positive bacterial strain of Blakeslea trispora and negative bacterial strain After growing spore, aseptically, slant medium miospore is scraped with sterile saline, be configured to respectively Even spore suspension makes the concentration of positive bacterium spore suspension and negative bacterium spore suspension be positive respectively bacterium 10⁴A spore/mL and negative bacterium 10⁵ A spore/mL.Wherein, the positive bacterial strain of Blakeslea trispora (Blakeslea trispora) and negative bacterial strain are purchased from ATCC, number point It Wei not ATCC 14271 (+) and ATCC 14272 (-).

(2) first order seed culture: positive bacterium spore suspension and negative bacterium spore suspension are respectively connected to carry out in first class seed pot The cultivation temperature of culture, positive bacterium spore suspension is 28 DEG C, revolving speed 500r/min, ventilatory capacity 0.7vvm, and tank pressure is 0.04MPa, incubation time 36h；The cultivation temperature of negative bacterium spore suspension is 28 DEG C, revolving speed 600r/min, and ventilatory capacity is 0.7vvm, tank pressure be 0.05MPa, incubation time 36h, respectively obtain Blakeslea trispora positive bacterial strain primary seed solution and negative bacterium Strain primary seed solution；Contain following component: glucose 24g, soyabean protein powder 28g, magnesium sulfate in every liter of primary-seed medium 0.9g, potassium dihydrogen phosphate 4g, calcium chloride 0.08g, -40 1.5g of copper sulphate 0.08g, VE 0.035g and Span.

(3) secondary seed culture: the positive bacterial strain primary seed solution for the Blakeslea trispora that step (2) culture is obtained and negative bacterium Strain primary seed solution combined inoculation is cultivated into secondary seed tank, the positive bacterial strain primary seed solution and three spores of Blakeslea trispora The inoculation volume ratio of the mould negative bacterial strain primary seed solution of Bradley is 1:8, and inoculum concentration 12%, cultivation temperature is 28 DEG C, and revolving speed is 750r/min, ventilatory capacity 0.7vvm, tank pressure is 0.05MPa, and incubation time 15h obtains secondary seed solution；Every liter of second level kind Contain following component: glucose 24g, soyabean protein powder 28g, magnesium sulfate 0.9g, potassium dihydrogen phosphate 4g, calcium chloride in sub- culture medium 0.08g, -40 1.5g of copper sulphate 0.08g, VE 0.035g and Span.

(4) fermented and cultured: the secondary seed solution is accessed in fermentor using pressure differential method, inoculum concentration 12%, in temperature Fermented and cultured 105h under conditions of degree is 28 DEG C, pH value is 6.0, revolving speed, ventilatory capacity and tank pressure are carried out with the growing state of thallus It adjusts, adjusts strategy are as follows: earlier fermentation, revolving speed 120r/min, ventilatory capacity 0.7vvm, tank pressure are 0.04MPa；With fermentation It carries out, when color has started to accumulate yellow in and mycelium sturdy when a large amount of mycelium of microscopy observation, i.e., fermentation time reaches 50h When, it is 200r/min by rotational speed regulation, ventilatory capacity is adjusted to 1.0vvm, and then tank pressure regulation 0.04MPa fills into solvent or diluent, institute It states solvent or diluent to be made of alpha, beta-lonone, imidazoles and fumaric acid, the amount for once filling into alpha, beta-lonone is fermentating liquid volume total amount 0.2% (g/100mL), the amount for once filling into imidazoles is 0.15% (g/100mL) of fermentating liquid volume total amount, once fills into rich horse The amount of acid is 0.15% (g/100mL) of fermentating liquid volume total amount.Sunflower oil, every liter of fermentation are once filled into when fermenting 40h The amount of the sunflower oil filled into liquid is 2g；It is sampled during the fermentation every 4h and surveys residual sugar content, when sample detection residual sugar contains When amount is lower than 5.0g/L, starch hydrolyzate is filled into one or more times, by residual sugar content control in fermentation liquid in 6~10g/L.

Contain following component: the starch hydrolyzate 0.3L, bean powder hydrolyzate 0.2L, fish meal in every liter of fermentation medium 29g, sunflower oil 30g, copper sulphate 0.07g, magnesium sulfate 0.9g, -40 1.0g of potassium dihydrogen phosphate 4g, VE 0.035g and Span, And the pH value of fermentation medium is adjusted to 6.0 with sodium hydroxide.

The starch hydrolyzate comprises the following steps: potato starch, which is configured to mass percent concentration with water, is Then hydrochloric acid is added in 18% starch milk, hydrochloric acid additive amount is the 0.5% of starch milk volume total amount, and starch milk and hydrochloric acid stir 2h is reacted at 50 DEG C after uniformly, to after reaction, in sodium hydroxide and starch hydrolyzate that obtained pH value is 7.0.

The bean powder hydrolyzate comprises the following steps: it is 24% that beancake powder and water, which are configured to mass percent concentration, Then hydrochloric acid is added in beancake powder suspension, hydrochloric acid additive amount is the 1.2% of beancake powder suspension volume total amount, and beancake powder is suspended Liquid reacts 5h with hydrochloric acid at 70 DEG C after mixing evenly, during which supplements water and maintains suspension constancy of volume.After reaction, hydrogen is used The beancake powder hydrolyzate for being 7.0 with obtained pH value in sodium oxide molybdena.

It collects fermentation liquid and obtains thallus, measure dry weight and reach 60.6g/L, high performance liquid chromatography detects tomato in fermentation liquid For red pigment unit up to 4.56g/L, mass percent of the lycopene in dry mycelium is 7.52%.

Embodiment 6~13 and comparative example 1~2

In embodiment 6~13 and comparative example 1~2, only part test condition is different from embodiment 1, remaining same embodiment 1, biomass, yield and content such as 1 institute of table in dry mycelium of experimental condition difference and gained fermentation lycopene Show.

Table 1

Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example Property, it is not considered as limiting the invention, those skilled in the art are not departing from the principle of the present invention and objective In the case where can make changes, modifications, alterations, and variations to the above described embodiments within the scope of the invention.

Claims (11)

1. a kind of fermentation medium, which is characterized in that contain starch ingredients, bean powder component, fish meal, plant in the fermentation medium Object oil, copper sulphate, magnesium sulfate, potassium dihydrogen phosphate, VE and emulsifier, the starch ingredients are starch and/or starch hydrolyzate, institute It states bean powder group and is divided into bean powder and/or bean powder hydrolyzate, and at least one of the starch ingredients and bean powder component hydrolyze for it Liquid.

2. fermentation medium according to claim 1, which is characterized in that contain Starch Hydrolysis in the fermentation medium Liquid, bean powder hydrolyzate, fish meal, vegetable oil, copper sulphate, magnesium sulfate, potassium dihydrogen phosphate, VE and emulsifier；Preferably, described in every liter Contain 0.2~0.4L of starch hydrolyzate, 0.1~0.3L of bean powder hydrolyzate, 26~32g of fish meal, vegetable oil 20 in fermentation medium ~40g, 0.06~0.08g of copper sulphate, 0.8~1.0g of magnesium sulfate, 3~5g of potassium dihydrogen phosphate, 0.025~0.05g of VE and emulsification 0.5~2g of agent, and the pH value of the fermentation medium is 5.5~7.0.

3. fermentation medium according to claim 1 or 2, which is characterized in that the starch hydrolyzate is in accordance with the following methods It is made: starch and water is configured to starch milk, acidification hydrolization reaction is carried out after later mixing the starch milk and acid solution, to acid After changing hydrolysis, the pH value of acidification hydrolization liquid is adjusted to 6.5~7.5 to get the starch hydrolyzate；

Preferably, the starch is selected from least one of cornstarch, sweet potato starch and potato starch；

Preferably, the concentration of the starch milk is 15~20wt%；

Preferably, the acid solution additive amount be the starch milk volume total amount 0.4~0.6%, the acid solution be concentration 36~ The concentrated hydrochloric acid of 38wt%；

Preferably, it is 40~60 DEG C that the condition of the acidification hydrolization reaction, which includes reaction temperature, and the reaction time is 1~3h.

4. fermentation medium according to claim 1 or 2, which is characterized in that the bean powder hydrolyzate is in accordance with the following methods It is made: bean powder and water is configured to bean powder suspension, carry out hydrolysis after later mixing the bean powder suspension and acid solution, After to hydrolysis, the pH value of hydrolyzate is adjusted to 6.5~7.5 to get the bean powder hydrolyzate；

Preferably, the bean powder is selected from least one of beancake powder, soyabean protein powder and bean cake powder；

Preferably, the concentration of the bean powder suspension is 20~30wt%；

Preferably, the acid solution additive amount is the 1.0~1.5% of the bean powder suspension volume total amount, and the acid solution is concentration The concentrated hydrochloric acid of 36~38wt%；

Preferably, it is 70~100 DEG C that the condition of the hydrolysis, which includes reaction temperature, and the reaction time is 3~5h, during reaction It supplements water and maintains the constancy of volume of bean powder suspension.

5. fermentation medium described in any one of Claims 1 to 4 prepares tomato red in culture Blakeslea trispora fermentation Application in element.

6. a kind of method for preparing lycopene using Blakeslea trispora fermentation, this method includes by the positive bacterium of Blakeslea trispora Strain and negative bacterial strain successively carry out actication of culture and seed expands culture, which is characterized in that this method further includes expanding the seed Resulting seed liquor is cultivated greatly, and fermented and cultured is carried out using fermentation medium described in any one of Claims 1 to 4.

7. the method according to claim 6 for preparing lycopene using Blakeslea trispora fermentation, which is characterized in that institute State fermented and cultured mode be by the seed liquor access fermentor in, inoculum concentration be 10~15%, temperature be 26~30 DEG C, PH value is 96~120h of fermented and cultured under conditions of 5.5~7.0, during fermented and cultured, according to the growing state control of thallus Revolving speed and ventilatory capacity processed simultaneously fill into solvent or diluent, fill into carbon source according to sampling inspection results, obtain the fermentation liquid containing lycopene.

8. the method according to claim 7 for preparing lycopene using Blakeslea trispora fermentation, which is characterized in that root Revolving speed and ventilatory capacity and the mode for filling into solvent or diluent are controlled according to the growing state of thallus are as follows: earlier fermentation, revolving speed are 110~140r/ Min, ventilatory capacity are 0.6~0.8vvm, and tank pressure is 0.03~0.06MPa；When fermented incubation time reaches 30~50h, will turn Speed is adjusted to 150~250r/min, and ventilatory capacity is adjusted to 0.8~1.2vvm, and tank pressure regulation is 0.03~0.06MPa, then mends Enter solvent or diluent；

Preferably, in the solvent or diluent containing solvent or diluent A and solvent or diluent B and optional solvent or diluent C, the solvent or diluent A be selected from imidazoles, piperidines, At least one of N-methylmorpholine and nicotine, the solvent or diluent B are selected from α-ionone, alpha, beta-lonone, isoniazid and abscisic acid At least one of, the solvent or diluent C is fumaric acid；

Preferably, the amount for once filling into the solvent or diluent A is 0.02~0.4% (g/100mL) of fermentating liquid volume total amount；

Preferably, the amount for once filling into the solvent or diluent B is 0.05~0.4% (g/100mL) of fermentating liquid volume total amount；

Preferably, the amount for once filling into the solvent or diluent C is 0.08~0.2% (g/100mL) of fermentating liquid volume total amount.

9. the method according to claim 7 for preparing lycopene using Blakeslea trispora fermentation, which is characterized in that institute Stating carbon source is the starch hydrolyzate；The mode of carbon source is filled into according to sampling inspection results are as follows: sample detection residual sugar content works as institute When stating residual sugar content lower than 4~6g/L, the starch hydrolyzate is filled into one or more times, by the residual sugar content in fermentation liquid Control is in 6~10g/L.

10. the method according to claim 7 for preparing lycopene using Blakeslea trispora fermentation, which is characterized in that This method further includes that vegetable oil is disposably filled into the 30~40h of fermented and cultured, is filled into every liter of fermentation medium Vegetable oil amount be 1~3g；Preferably, the vegetable oil in corn oil, soybean oil, sunflower oil and palm oil extremely Few one kind.

11. the side of lycopene is prepared using Blakeslea trispora fermentation according to any one of claim 6~10 Method, which is characterized in that the mode of the actication of culture are as follows: aseptically, by the positive bacterial strain and negative bacterial strain of Blakeslea trispora Be inoculated on slant medium respectively, be placed in incubator and cultivated at 26~30 DEG C, the positive bacterium to Blakeslea trispora After strain and negative bacterial strain grow spore respectively, aseptically, the spore in slant medium is scraped with sterile saline Get off, being configured to concentration respectively is 10³~10⁵The positive bacterium spore suspension and concentration of a spore/mL is 10⁴~10⁶A spore/ The negative bacterium spore suspension of mL；

It includes first order seed culture and secondary seed culture that the seed, which expands culture,；

The mode of the first order seed culture are as follows: the positive bacterium spore suspension and negative bacterium spore suspension are respectively connected to first order seed It is cultivated in tank；The condition of culture of the positive bacterium spore suspension include cultivation temperature be 26~30 DEG C, revolving speed be 400~ 600r/min, ventilatory capacity are 0.6~0.8vvm, and tank pressure is 0.03~0.06MPa, and incubation time is 20~36h；The negative bacterium spore The condition of culture of sub- suspension include cultivation temperature be 26~30 DEG C, revolving speed be 500~700r/min, ventilatory capacity be 0.6~ 0.8vvm, tank pressure are 0.03~0.06MPa, and incubation time is 20~36h, respectively obtain the positive bacterial strain level-one kind of Blakeslea trispora Sub- liquid and negative bacterial strain primary seed solution；

The mode of the secondary seed culture are as follows: by the positive bacterial strain primary seed solution and negative bacterial strain level-one kind of the Blakeslea trispora Sub- liquid combined inoculation is cultivated into secondary seed tank, the positive bacterial strain primary seed solution of the Blakeslea trispora and negative bacterial strain one The inoculation volume ratio of grade seed liquor is 1:5~1:10, and inoculum concentration is 10~15%, and cultivation temperature is 26~30 DEG C, revolving speed 600 ~900r/min, ventilatory capacity are 0.6~0.8vvm, and tank pressure is 0.03~0.06MPa, and incubation time is 10~20h, obtain second level Seed liquor.