CN105506048B - A kind of fermentation process preparing beta carotene using Blakeslea trispora - Google Patents
A kind of fermentation process preparing beta carotene using Blakeslea trispora Download PDFInfo
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Abstract
The invention discloses a kind of fermentation process that beta carotene is prepared using Blakeslea trispora comprising following steps: (1) actication of culture；(2) first order seed culture；(3) secondary seed culture；(4) fermented and cultured.The present invention is not high to equipment requirement, cost input is smaller, and raw material sources are extensive, at low cost, and technique significantly improves the yield of beta carotene compared with before-improvement, therefore, it can be achieved that large-scale production.
Description
Technical field
The present invention relates to a kind of fermentation process for preparing beta carotene, utilize Blakeslea trispora more particularly to a kind of
Prepare the fermentation process of beta carotene.
Background technique
Beta carotene is a kind of fat-soluble unsaturated aliphatic hydrocarbon, is the typical generation of carotenoid race
Most generally existing most stable of natural pigment in table and nature is widely present in green and yellow vegetable and fruit.
Its molecular formula is C40H56, molecular weight 536.88, appearance is in peony to kermesinus, Yi Fasheng unstable to oxygen, light and heat
Redox reaction is not readily dissolved in water, bronsted lowry acids and bases bronsted lowry, is soluble in chloroform, benzene and vegetable oil, is slightly soluble in ethyl alcohol and ether.In addition to normal
Other than colorant, beta carotene also has multiple biological activities, is a kind of closely related with human health medicinal to have
Imitate ingredient.The study found that since the molecular formula of beta carotene contains 2 β-ionone rings and 4 isoprene side chains, it is intermediate
Two vitamin A molecules are generated after fracture, can be used as the precursor substance of vitamin A after being absorbed by the body；In addition, its point
The unsaturated structure contained in son makes it have stronger antioxidant activity and removes the ability of free radical, can be effectively pre-
Prevent, delay and treat certain diseases, especially cancer, while can also improve the immune function of body.Therefore, food, drug,
Cosmetics and health products trade are widely used.
Natural beta carotene is compared with the beta carotene of synthesis, since its is from a wealth of sources, is free of carcinogenic substance
Matter, cost is relatively low and physiological activity is good, occupies leading position on the market.In recent years, the life of domestic and international natural beta-carotin
The method for mostly using microbial fermentation is produced, carrying out fermenting and producing using microorganism, there is with short production cycle, process to be easy to control, no
It influenced by region and weather, be easy to carry out the advantages such as scale operation.During the experiment, researcher has been obtained for one
Have productive strain excellent, such as Blakeslea trispora, mycobacterium, red pseudomonas and red phaffia rhodozyma, wherein
The performance of Blakeslea trispora is preferable, and wide use is also obtained in production.
Blakeslea trispora (Blakeslea trispora) belongs to Mucoales, Ji Mei section, is unicellular lower fungus.One
It is grown rapidly under fixed condition of culture, biggish biomass, the production suitable for scale can be reached in a relatively short period of time.This
Outside, there are one being characterized in that Blakeslea trispora belongs to mycelium anastomosis bacterium, it is divided into positive and negative 2 kinds, both unisexuality bacterial strains are in shape
Without apparent difference in state, individually culture is able to carry out vegetative propagation, the mycelium of positive and negative bacterial strain when their mixed fermentations
It contacts with each other, heterothallism occurs and carries out sexual propagation, can be obtained the beta carotene of high yield.Currently, being permitted both at home and abroad
More scholars are setting about exploring the process conditions for improving Blakeslea trispora producing beta-carotene by fermentation, so that it is more suitable for industry
Change mass production.Due to the growth characteristics of filamentous fungi Blakeslea trispora itself, fermentation medium mostly uses the biggish object of viscosity
Matter (such as starch, soy meal) serves as carbon nitrogen source, to be conducive to the extension of filamentous fungi mycelia, ultimately forms mycelium pellet, but
The biggish fermentation liquid most time of viscosity belongs to non-newtonian fluid, has large effect to the transmitting of oxygen；And before fermentation
Phase, the fast-growth of thallus keep its demand to dissolved oxygen and nutriment relatively high, as the increase of cell concentration further adds
Big fermentation broth viscosity, becomes the key factor of limitation thalli growth, Product formation so as to cause dissolved oxygen, or even cause to be metabolized different
Often, the yield of target product is influenced.
Therefore, in the fermentation process for producing beta carotene as bacterial strain using Blakeslea trispora, researcher multi-pass mistake
Mutagenesis screening strain excellent changes the whipped form of fermentation, dissolved oxygen supply mode, adds the carrier of oxygen during the fermentation or adds
Add the surfactant that can increase somatic cells permeability to improve the utilization rate of oxygen concentration and oxygen in fermentation liquid, these are studied
There is certain effect to the improvement of fermentation process, but there is also such or such deficiency or final low outputs, or
Person is to cause since the device is complicated at high cost, is also had plenty of comprehensive without carrying out to fermentation process only for one aspect
Consider.The technique that the form of a kind of pair of stirrer paddle is changed is disclosed in China patent CN1331343A, using upper middle layer
For the Combined stirring paddle leaf that wide leaf is pusher, lower layer is turbine type, the mass-transfer performance of fermentation liquid is improved, but changes stirring
It can only be suitble to the industry characteristics of the bacterial strain after form, be unfavorable for comprehensive utilization of fermentor；Pass through in patent CN102787158A
Colloid mill high speed shearing emulsification is carried out to improve the oxygen concentration of fermentation liquid entrainment to the culture medium before inoculation, which wants equipment
Ask relatively high, cost input is larger and does not carry out excessive exploration to the technique of fermentation, mainly to subsequent extraction process
It conducts in-depth research；Patent CN1193048A carries out Blakeslea trispora fermentation preparation β-carrot using airlift fermentor
Element, this method can preferably avoid shearing force, be conducive to the growth of mycelia to obtain biggish biomass, but final production
Measure it is lower, be also not easy carry out scale mass production.
Summary of the invention
The purpose of the present invention is to provide a kind of fermentation process that beta carotene is prepared using Blakeslea trispora, i.e., logical
Starch is carried out pretreatment and obtains the fermentation medium of modest viscosity, and combined by the formula and preparation process for crossing adjustment culture medium
The growth change rule of thallus itself carries out feed supplement, thus improve dissolved oxygen concentration and it is made to obtain reasonable, efficient utilization, it should
Method is significant to the output results for improving beta carotene.
The purpose of the present invention can be achieved through the following technical solutions: a kind of to prepare β-carrot using Blakeslea trispora
The fermentation process of element comprising following steps: (1) actication of culture；(2) first order seed culture；(3) secondary seed culture；(4) it sends out
Ferment culture；Wherein,
(1) actication of culture: aseptically, the positive and negative bacterial strain of Blakeslea trispora is inoculated into inclined-plane PDA culture medium respectively
On, it is placed in incubator, 26~30 DEG C of 4~5d of culture, after the positive and negative bacterial strain of Blakeslea trispora grows spore respectively, sterile
Under the conditions of, slant medium miospore is scraped with sterile saline, uniform spore suspension is configured to, makes positive and negative
The concentration of bacterium spore suspension is respectively as follows: positive bacterium 104A spore/mL, negative bacterium 105A spore/mL；Wherein, Blakeslea trispora
(Blakeslea trispora) positive and negative bacterial strain is purchased from ATCC, and number is respectively ATCC 14271 (+), ATCC 14272 (-)；
(2) positive and negative bacterial strain first order seed culture: is respectively connected to seeding tank, the training of positive bacterial strain in a manner of spore suspension
Supporting temperature is 28 DEG C, and revolving speed is 400~600r/min, and ventilatory capacity is 0.6~0.8vvm, and tank pressure is 0.03~0.06MPa, culture
Time is 20~36h；The cultivation temperature of negative bacterial strain is 28 DEG C, and revolving speed is 500~700r/min, and ventilatory capacity is 0.6~0.8vvm,
Tank pressure is 0.03~0.06MPa, and incubation time is 20~36h, obtains the positive and negative bacterial strain primary seed solution of Blakeslea trispora；
(3) step (2) secondary seed culture: are cultivated to the obtained positive and negative bacterial strain level-one kind of the Blakeslea trispora
Sub- liquid combined inoculation is into secondary seed tank, the positive bacterial strain primary seed solution of Blakeslea trispora and the negative bacterium of the Blakeslea trispora
The inoculation volume ratio of strain primary seed solution is 1:5~1:10, and inoculum concentration is 10~15%, and incubation time is 10~20h, cultivates item
Part are as follows: 28 DEG C, revolving speed is 600~900r/min, and ventilatory capacity is 0.6~0.8vvm, and tank pressure is 0.03~0.06MPa, obtains two
Grade seed liquor；
(4) fermented and cultured: being accessed the secondary seed solution in fermentor using pressure differential method, and inoculum concentration is 10~15%,
96~120h, condition of culture are cultivated under conditions of 5.5~7.0 pH are as follows: 28 DEG C, revolving speed and ventilatory capacity with thallus growth feelings
Condition is controlled；Fermentation process needs to fill into vegetable oil, fills into starch hydrolyzate according to sampling inspection results, according to the life of thallus
Long situation carries out benefit solvent or diluent；Wherein, fermentation medium form are as follows: the starch hydrolyzate, soyabean protein powder, fish meal, vegetable oil,
Copper sulphate, magnesium sulfate, potassium dihydrogen phosphate, VE, emulsifier.
Specifically, including following parts by weight of component in every liter of primary-seed medium of the step (2): glucose 22~
26g, the 26~30g of soyabean protein powder, the 0.8~1.0g of magnesium sulfate, the 3~5g of potassium dihydrogen phosphate, calcium chloride 0.06
~0.1g, the 0.06~0.1g of copper sulphate, the VE0.025~0.05g, the 0.5~2g of emulsifier；The step (3)
It include following parts by weight of component: the 22~26g of glucose in every liter of secondary seed medium, the soyabean protein powder 26~
30g, the 0.8~1.0g of magnesium sulfate, the 3~5g of potassium dihydrogen phosphate, the 0.06~0.1g of calcium chloride, the copper sulphate
0.06~0.1g, the VE0.025~0.05g, the 0.5~2g of emulsifier.
Specifically, specifically including following component in described every liter of fermentation medium of step (4): the starch hydrolyzate
0.2~0.4L, the 12~18g of soyabean protein powder, the 26~32g of fish meal, the 20~40g of vegetable oil, the copper sulphate
0.06~0.08g, the 0.8~1.0g of magnesium sulfate, the 3~5g of potassium dihydrogen phosphate, the VEIt is 0.025~0.05g, described
0.5~2g of emulsifier；And adjusting the fermentation medium pH with sodium hydroxide or potassium hydroxide is 5.5~7.0.
Specifically, the starch hydrolyzate comprises the following steps: starch and water being configured to starch milk, salt is then added
Acid react after mixing evenly, after reaction, in the sodium hydroxide or the potassium hydroxide and obtained pH be 6.5-7.5's
The starch hydrolyzate.
Further, the mass percent concentration of the starch milk is 15~20%, and the hydrochloric acid additive amount is the shallow lake
The 0.4~0.6% of powder cream volume total amount, the hydrochloric acid are commercially available concentrated hydrochloric acid, the reaction temperature of the starch milk and the hydrochloric acid
At 40~60 DEG C, the reaction time is 1~3h for control.
Specifically, the starch is any one in cornstarch, sweet potato starch or potato starch.
Specifically, in step (4) fermentation process revolving speed and ventilatory capacity adjusting strategy are as follows: earlier fermentation, revolving speed are
120~150r/min, ventilatory capacity are 0.6~0.8vvm, and tank pressure is 0.03~0.06MPa；As fermentation carries out, observed to microscopy
A large amount of mycelium are sturdy and mycelium in color when largely having accumulated yellow, i.e., when fermentation time reaches 50~60h, by revolving speed tune
Section is 170~250r/min, and ventilatory capacity is adjusted to 0.8~1.2vvm, and tank pressure regulation is 0.03~0.06MPa, then fills into institute
Solvent or diluent is stated, the solvent or diluent includes alpha, beta-lonone and malic acid, and the amount for once filling into the alpha, beta-lonone is fermentating liquid volume
The 0.1~0.4% of total amount, the amount for once filling into the malic acid is the 0.1~0.3% of the fermentating liquid volume total amount.
Specifically, once filling into the vegetable oil, every liter of fermentation liquid when fermenting 30~40h in the step (4)
In fill into vegetable oil described in 1~3g；During the fermentation, when sample detection residual sugar content is lower than 5.0g/L, one or more times
The starch hydrolyzate is filled into, controlling residual sugar content in the fermentation liquid is 6~10g/L.
Specifically, the vegetable oil is any one or a few in corn oil, soybean oil, sunflower oil or palm oil；
The emulsifier is Span series product or TWEEN Series product.
Compared with prior art, the present invention has as follows using the fermentation process that Blakeslea trispora prepares beta carotene
Advantage and significant progress:
Firstly, the present invention is handled starch acidification hydrolization before fermentation medium is prepared, promote starch molecule anti-in acidolysis
Glycosidic bond is broken during answering, and is drastically reduced so as to cause the degree of polymerization of starch molecule, starch viscosity value sharply declines, Ke Yiyou
It is quick at fermentation initial stage to can satisfy mycelium to improve the concentration of dissolved oxygen for the physical property that effect ground improves fermentation liquid
To the consumption of oxygen when accumulation；Meanwhile the main component of starch hydrolyzate is glucose, followed by a small amount of maltose and other one
The compound carbohydrates such as a little disaccharides, oligosaccharide, these substances can meet thallus fast-growth in earlier fermentation, and the incomplete water in part
The starch of solution becomes at a slow speed using carbon source after then accumulating certain biomass in thallus, is conducive to Product formation.Therefore, starch
Pre-treatment operation has taken into account the nutritive peculiarity and Product formation of production bacterium, to substantially increase the yield of beta carotene.
Secondly, the present invention ventilates on the basis of broth viscosity reduces according to the growing state of thallus stage by stage
The change of amount, not only improve thalli growth and Product formation reduces energy consumption again, has saved the energy；And starch is directly used in feed supplement
Hydrolysate is other than it can increase to the fermentation unit of beta carotene, and compared with feed supplement glucose, feed supplement amount is
It reduces, to also reduce cost of material.
Third, the present invention is not high to equipment requirement, cost input is smaller, and raw material sources are extensive, at low cost, β-carrot
Technique significantly improves the yield of element compared with before-improvement, therefore, it can be achieved that large-scale production.
Specific embodiment
The present invention is described in detail combined with specific embodiments below, but following embodiment is not constituted to of the invention
Limitation.
Embodiment 1: a kind of fermentation process preparing beta carotene using Blakeslea trispora comprising following steps:
(1) actication of culture；(2) first order seed culture；(3) secondary seed culture；(4) fermented and cultured；Wherein,
(1) actication of culture: aseptically, the positive and negative bacterial strain of Blakeslea trispora is inoculated into inclined-plane PDA culture medium respectively
On (potato dextrose agar is suitable for fungi and grows), it is placed in incubator, 26 DEG C of culture 4d, to Blakeslea trispora
After positive and negative bacterial strain grows spore respectively, aseptically, slant medium miospore is scraped down with sterile saline
Come, is configured to uniform spore suspension, the concentration of positive and negative bacterium spore suspension is made to be respectively as follows: positive bacterium 104A spore/mL, negative bacterium 105
A spore/mL；Wherein, Blakeslea trispora (Blakeslea trispora) positive and negative bacterial strain is purchased from ATCC, and number is respectively ATCC
14271 (+), ATCC14272 (-)；
(2) positive and negative bacterial strain first order seed culture: is respectively connected to seeding tank, the training of positive bacterial strain in a manner of spore suspension
Supporting temperature is 28 DEG C, revolving speed 400r/min, ventilatory capacity 0.6vvm, and tank pressure is 0.03MPa, incubation time 36h；Negative bacterial strain
Cultivation temperature be 28 DEG C, revolving speed 500r/min, ventilatory capacity 0.8vvm, tank pressure is 0.0~0.06MPa, and incubation time is
20h obtains the positive and negative bacterial strain primary seed solution of Blakeslea trispora；Include following parts by weight of component in every liter of primary-seed medium:
Glucose 22g, soyabean protein powder 26g, magnesium sulfate 0.8g, potassium dihydrogen phosphate 3g, calcium chloride 0.06g, copper sulphate 0.06g, VE
0.025g, Tween-60 0.5g；
(3) secondary seed culture: the positive and negative bacterial strain primary seed solution of Blakeslea trispora that step (2) culture obtains is mixed
It is inoculated into secondary seed tank, the positive bacterial strain primary seed solution of Blakeslea trispora connects with the negative bacterial strain primary seed solution of Blakeslea trispora
Kind volume ratio is 1:10, inoculum concentration 10%, incubation time 20h, condition of culture are as follows: 28 DEG C, revolving speed 900r/min, ventilation
Amount is 0.8vvm, and tank pressure is 0.03MPa, obtains secondary seed solution；It include following parts by weight group in every liter of secondary seed medium
Point: glucose 22g, soyabean protein powder 26g, magnesium sulfate 0.8g, potassium dihydrogen phosphate 3g, calcium chloride 0.06g, copper sulphate 0.06g, VE
0.025g, Tween-60 0.5g.
(4) fermented and cultured: the secondary seed solution is accessed in fermentor using pressure differential method, inoculum concentration 10%, in pH
96h, condition of culture are cultivated under conditions of 5.5 are as follows: 28 DEG C, revolving speed and ventilatory capacity are controlled with the growing state of thallus, revolving speed
With the adjusting strategy of ventilatory capacity are as follows: earlier fermentation, revolving speed 120r/min, ventilatory capacity 0.6vvm, tank pressure are 0.03MPa；With
Fermentation carry out, when microscopy observes that a large amount of mycelium are sturdy and color largely accumulates yellow in mycelium, i.e. fermentation time
It is 170r/min by rotational speed regulation when reaching 56h, ventilatory capacity is adjusted to 0.8vvm, then tank pressure regulation 0.06MPa is filled into
Solvent or diluent, solvent or diluent include alpha, beta-lonone and malic acid, and the amount for once filling into alpha, beta-lonone is fermentating liquid volume total amount
0.1%, the amount for once filling into malic acid is the 0.1% of fermentating liquid volume total amount.Corn oil is once filled into when fermenting 30h, often
It rises in fermentation liquid and fills into 1g vegetable oil；It is sampled during the fermentation every 4h and surveys residual sugar, sample detection residual sugar content is lower than 5.0g/
When L, starch hydrolyzate is filled into one or more times, and controlling residual sugar content in fermentation liquid is 6~10g/L.
Following component: starch hydrolyzate 0.2L, soyabean protein powder 12g, fish meal is specifically included in every liter of fermentation medium
26g, corn oil 20g, copper sulphate 0.06g, magnesium sulfate 0.8g, potassium dihydrogen phosphate 3g, VE0.025g, Tween-60 0.5g；It is used in combination
It is 5.5 that sodium hydroxide, which adjusts fermentation medium pH,.
Wherein, starch hydrolyzate comprises the following steps: cornstarch and water are configured to starch milk, the quality of starch milk
Percent concentration is 20%, and hydrochloric acid is then added, and hydrochloric acid additive amount is the 0.6% of starch milk volume total amount, and hydrochloric acid is commercially available dense
Hydrochloric acid, starch milk react after mixing evenly with hydrochloric acid, and reaction temperature control is at 40 DEG C, reaction time 1h.After reaction, it uses
The starch hydrolyzate for being 6.5 with obtained pH in sodium hydroxide.
It collects fermentation liquid and obtains thallus, measure dry weight and reach 59.5g/L, high performance liquid chromatography detects β-Hu in fermentation liquid
For radish primitive unit cell up to 3.55g/L, percentage of the beta carotene in dry mycelium is 5.97%.
Embodiment 2: a kind of fermentation process preparing beta carotene using Blakeslea trispora comprising following steps:
(1) actication of culture；(2) first order seed culture；(3) secondary seed culture；(4) fermented and cultured；Wherein,
(1) actication of culture: aseptically, the positive and negative bacterial strain of Blakeslea trispora is inoculated into inclined-plane PDA culture medium respectively
On (potato dextrose agar is suitable for fungi and grows), it is placed in incubator, 30 DEG C of culture 5d, to Blakeslea trispora
After positive and negative bacterial strain grows spore respectively, aseptically, slant medium miospore is scraped down with sterile saline
Come, is configured to uniform spore suspension, the concentration of positive and negative bacterium spore suspension is made to be respectively as follows: positive bacterium 104A spore/mL, negative bacterium 105
A spore/mL；Wherein, Blakeslea trispora (Blakeslea trispora) positive and negative bacterial strain is purchased from ATCC, and number is respectively ATCC
14271 (+), ATCC14272 (-)；
(2) positive and negative bacterial strain first order seed culture: is respectively connected to seeding tank, the training of positive bacterial strain in a manner of spore suspension
Supporting temperature is 28 DEG C, revolving speed 600r/min, ventilatory capacity 0.6vvm, and tank pressure is 0.06MPa, incubation time 20h；Negative bacterial strain
Cultivation temperature be 28 DEG C, revolving speed 700r/min, ventilatory capacity 0.8vvm, tank pressure is 0.06MPa, and incubation time 20h obtains
To the positive and negative bacterial strain primary seed solution of Blakeslea trispora；It include following parts by weight of component: glucose in every liter of primary-seed medium
26g, soyabean protein powder 30g, magnesium sulfate 1.0g, potassium dihydrogen phosphate 5g, calcium chloride 0.1g, copper sulphate 0.1g, VE0.05g, Span-
402g；
(3) secondary seed culture: the positive and negative bacterial strain primary seed solution of Blakeslea trispora that step (2) culture obtains is mixed
It is inoculated into secondary seed tank, the positive bacterial strain primary seed solution of Blakeslea trispora connects with the negative bacterial strain primary seed solution of Blakeslea trispora
Kind volume ratio is 1:5, inoculum concentration 15%, incubation time 10h, condition of culture are as follows: 28 DEG C, revolving speed 900r/min, ventilation
Amount is 0.8vvm, and tank pressure is 0.06MPa, obtains secondary seed solution；It include following parts by weight group in every liter of secondary seed medium
Point: glucose 26g, soyabean protein powder 30g, magnesium sulfate 1.0g, potassium dihydrogen phosphate 5g, calcium chloride 0.1g, copper sulphate 0.1g,
VE0.05g, Span -402g.
(4) fermented and cultured: the secondary seed solution is accessed in fermentor using pressure differential method, inoculum concentration 15%, in pH
120h, condition of culture are cultivated under conditions of 7.0 are as follows: 28 DEG C, revolving speed and ventilatory capacity are controlled with the growing state of thallus, revolving speed
With the adjusting strategy of ventilatory capacity are as follows: earlier fermentation, revolving speed 150r/min, ventilatory capacity 0.8vvm, tank pressure are 0.06MPa；With
Fermentation carry out, when microscopy observes that a large amount of mycelium are sturdy and color largely accumulates yellow in mycelium, i.e. fermentation time
It is 250r/min by rotational speed regulation when reaching 60h, ventilatory capacity is adjusted to 1.2vvm, then tank pressure regulation 0.06MPa is filled into
Solvent or diluent, solvent or diluent include alpha, beta-lonone and malic acid, and the amount for once filling into alpha, beta-lonone is fermentating liquid volume total amount
0.4%, the amount for once filling into malic acid is the 0.3% of fermentating liquid volume total amount.Soybean oil is once filled into when fermenting 30h, often
It rises in fermentation liquid and fills into 1g soybean oil；It is sampled during the fermentation every 4h and surveys residual sugar, sample detection residual sugar content is lower than 5.0g/
When L, starch hydrolyzate is filled into one or more times, and controlling residual sugar content in fermentation liquid is 6~10g/L.
Following component: starch hydrolyzate 0.4L, soyabean protein powder 18g, fish meal is specifically included in every liter of fermentation medium
32g, soybean oil 40g, copper sulphate 0.08g, magnesium sulfate 1.0g, potassium dihydrogen phosphate 5g, VE0.05g, Span -402g；And use hydrogen-oxygen
Changing potassium to adjust fermentation medium pH is 7.0.
Wherein, starch hydrolyzate comprises the following steps: sweet potato starch and water are configured to starch milk, the quality of starch milk
Percent concentration is 15%, and hydrochloric acid is then added, and hydrochloric acid additive amount is the 0.4% of starch milk volume total amount, and hydrochloric acid is commercially available dense
Hydrochloric acid, starch milk react after mixing evenly with hydrochloric acid, and reaction temperature control is at 40 DEG C, reaction time 3h.After reaction, it uses
The starch hydrolyzate for being 7.5 with obtained pH in potassium hydroxide.
It collects fermentation liquid and obtains thallus, measure dry weight and reach 65.2g/L, high performance liquid chromatography detects β-Hu in fermentation liquid
For radish primitive unit cell up to 4.68g/L, percentage of the beta carotene in dry mycelium is 7.18%.
Embodiment 3: a kind of fermentation process preparing beta carotene using Blakeslea trispora comprising following steps:
(1) actication of culture；(2) first order seed culture；(3) secondary seed culture；(4) fermented and cultured；Wherein,
(1) actication of culture: aseptically, the positive and negative bacterial strain of Blakeslea trispora is inoculated into inclined-plane PDA culture medium respectively
On (potato dextrose agar is suitable for fungi and grows), it is placed in incubator, 28 DEG C of culture 4d, to Blakeslea trispora
After positive and negative bacterial strain grows spore respectively, aseptically, slant medium miospore is scraped down with sterile saline
Come, is configured to uniform spore suspension, the concentration of positive and negative bacterium spore suspension is made to be respectively as follows: positive bacterium 104A spore/mL, negative bacterium 105
A spore/mL；Wherein, Blakeslea trispora (Blakeslea trispora) positive and negative bacterial strain is purchased from ATCC, and number is respectively ATCC
14271 (+), ATCC14272 (-)；
(2) positive and negative bacterial strain first order seed culture: is respectively connected to seeding tank, the training of positive bacterial strain in a manner of spore suspension
Supporting temperature is 28 DEG C, revolving speed 500r/min, ventilatory capacity 0.7vvm, and tank pressure is 0.04MPa, incubation time 28h；Negative bacterial strain
Cultivation temperature be 28 DEG C, revolving speed 600r/min, ventilatory capacity 0.7vvm, tank pressure is 5MPa, and incubation time 28h obtains
The positive and negative bacterial strain primary seed solution of Blakeslea trispora；It include following parts by weight of component: glucose in every liter of primary-seed medium
24g, soyabean protein powder 28g, magnesium sulfate 0.9g, potassium dihydrogen phosphate 4g, calcium chloride 0.08g, copper sulphate 0.08g, VE0.035g,
Span -401.5g；
(3) secondary seed culture: the positive and negative bacterial strain primary seed solution of Blakeslea trispora that step (2) culture obtains is mixed
It is inoculated into secondary seed tank, the positive bacterial strain primary seed solution of Blakeslea trispora connects with the negative bacterial strain primary seed solution of Blakeslea trispora
Kind volume ratio is 1:8, inoculum concentration 12%, incubation time 15h, condition of culture are as follows: 28 DEG C, revolving speed 750r/min, ventilation
Amount is 0.7vvm, and tank pressure is 0.05MPa, obtains secondary seed solution；It include following parts by weight group in every liter of secondary seed medium
Point: glucose 24g, soyabean protein powder 28g, magnesium sulfate 0.9g, potassium dihydrogen phosphate 4g, calcium chloride 0.08g, copper sulphate 0.08g, VE
0.035g, Span -401.5g.
(4) fermented and cultured: the secondary seed solution is accessed in fermentor using pressure differential method, inoculum concentration 12%, in pH
105h, condition of culture are cultivated under conditions of 6 are as follows: 28 DEG C, revolving speed and ventilatory capacity are controlled with the growing state of thallus, revolving speed and
The adjusting strategy of ventilatory capacity are as follows: earlier fermentation, revolving speed 130r/min, ventilatory capacity 0.7vvm, tank pressure are 0.04MPa；With
Fermentation carries out, and when color largely accumulates yellow in and mycelium sturdy when a large amount of mycelium of microscopy observation, i.e., fermentation time reaches
It is 210r/min by rotational speed regulation when to 55h, ventilatory capacity is adjusted to 1.0vvm, then tank pressure regulation 0.04MPa is filled into dilute
Material, solvent or diluent include alpha, beta-lonone and malic acid, and the amount for once filling into alpha, beta-lonone is the 0.2% of fermentating liquid volume total amount,
The amount for once filling into malic acid is the 0.2% of fermentating liquid volume total amount.Sunflower oil, every liter of hair are once filled into when fermenting 35h
2g sunflower oil is filled into zymotic fluid；It is sampled during the fermentation every 4h and surveys residual sugar, sample detection residual sugar content is lower than 5.0g/L
When, starch hydrolyzate is filled into one or more times, and controlling residual sugar content in fermentation liquid is 6~10g/L.
Following component: starch hydrolyzate 0.3L, soyabean protein powder 15g, fish meal is specifically included in every liter of fermentation medium
29g, sunflower oil 30g, copper sulphate 0.07g, magnesium sulfate 0.9g, potassium dihydrogen phosphate 4g, VE0.035g, Span -401g；It is used in combination
It is 6 that sodium hydroxide, which adjusts fermentation medium pH,.
Wherein, starch hydrolyzate comprises the following steps: potato starch and water are configured to starch milk, the matter of starch milk
Measuring percent concentration is 18%, and hydrochloric acid is then added, and hydrochloric acid additive amount is the 0.5% of starch milk volume total amount, and hydrochloric acid is commercially available
Concentrated hydrochloric acid, starch milk react after mixing evenly with hydrochloric acid, and reaction temperature control is at 50 DEG C, reaction time 2h.After reaction,
With the starch hydrolyzate for being 7 with obtained pH in sodium hydroxide.
It collects fermentation liquid and obtains thallus, measure dry weight and reach 62.3g/L, high performance liquid chromatography detects β-Hu in fermentation liquid
For radish primitive unit cell up to 4.01g/L, percentage of the beta carotene in dry mycelium is 6.44%.
Comparative example: this embodiment differs from embodiment 1 in that:
1) fermentation medium uses the starch of equivalent to substitute starch hydrolyzate in step (4), and intermediate sample detection residual sugar is low
Glucose is filled into when 5g/L, and makes the control range of residual sugar in 6~10g/L；2) in step (4) fermentation process technique control
Strategy are as follows: revolving speed 200r/min, ventilatory capacity 1.0vvm, tank press 0.06MPa, and 28 DEG C of temperature, fermentation 120h knot under the conditions of pH 6.5
Beam fills into 0.1% alpha, beta-lonone and 0.1% malic acid in 50h in the process；Remaining step and parameter are identical.
Fermentation liquid is collected after fermentation and obtains thallus, and measuring dry weight is 80.3g/L, high performance liquid chromatography detection fermentation
Beta carotene unit only has 1.72g/L in liquid, and percentage of the beta carotene in dry mycelium is 2.14%, hence it is evident that lower than real
Apply percentage of the beta carotene of the acquisition of example 1 in dry mycelium.
Claims (7)
1. a kind of fermentation process for preparing beta carotene using Blakeslea trispora, which is characterized in that it includes the following steps:
(1) actication of culture；(2) first order seed culture；(3) secondary seed culture；(4) fermented and cultured；Wherein,
(1) actication of culture: aseptically, the positive and negative bacterial strain of Blakeslea trispora is inoculated into respectively in inclined-plane PDA culture medium,
It is placed in incubator, 26~30 DEG C of 4~5d of culture, after the positive and negative bacterial strain of Blakeslea trispora grows spore respectively, in aseptic condition
Under, slant medium miospore is scraped with sterile saline, is configured to uniform spore suspension, makes positive and negative bacterium spore
The concentration of sub- suspension is respectively as follows: positive bacterium 104A spore/mL, negative bacterium 105A spore/mL；
(2) positive and negative bacterial strain first order seed culture: is respectively connected to seeding tank, the culture temperature of positive bacterial strain in a manner of spore suspension
Degree is 28 DEG C, and revolving speed is 400~600r/min, and ventilatory capacity is 0.6~0.8vvm, and tank pressure is 0.03~0.06MPa, incubation time
For 20~36h；The cultivation temperature of negative bacterial strain is 28 DEG C, and revolving speed is 500~700r/min, and ventilatory capacity is 0.6~0.8vvm, tank pressure
For 0.03~0.06MPa, incubation time is 20~36h, obtains the positive and negative bacterial strain primary seed solution of Blakeslea trispora, every liter of level-one
Including following parts by weight of component in seed culture medium: 22~26g of glucose, 26~30g of soyabean protein powder, magnesium sulfate 0.8~
1.0g, 3~5g of potassium dihydrogen phosphate, 0.06~0.1g of calcium chloride, copper sulphate 0.025~0.05g of 0.06~0.1g, VE, emulsifier
0.5~2g；
(3) step (2) secondary seed culture: are cultivated to the obtained positive and negative bacterial strain primary seed solution of the Blakeslea trispora
Combined inoculation is into secondary seed tank, the positive bacterial strain primary seed solution of Blakeslea trispora and the negative bacterial strain one of the Blakeslea trispora
The inoculation volume ratio of grade seed liquor is 1:5~1:10, and inoculum concentration is 10~15%, and incubation time is 10~20h, condition of culture
Are as follows: 28 DEG C, revolving speed is 600~900r/min, and ventilatory capacity is 0.6~0.8vvm, and tank pressure is 0.03~0.06MPa, obtains second level
Seed liquor, includes following parts by weight of component: 22~26g of glucose in every liter of secondary seed medium, and 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, copper sulphate 0.06~0.1g, VE
0.025~0.05g, 0.5~2g of emulsifier；
(4) fermented and cultured: the secondary seed solution is accessed in fermentor using pressure differential method, inoculum concentration is 10~15%, in pH
96~120h, condition of culture are cultivated under conditions of 5.5~7.0 are as follows: 28 DEG C, revolving speed and ventilatory capacity are carried out with the growing state of thallus
Control；Fermentation process needs to fill into vegetable oil, fills into starch hydrolyzate according to sampling inspection results, according to the growing state of thallus
Benefit solvent or diluent is carried out, the solvent or diluent includes alpha, beta-lonone and malic acid；Wherein, fermentation medium forms are as follows: the Starch Hydrolysis
Liquid, soyabean protein powder, fish meal, vegetable oil, copper sulphate, magnesium sulfate, potassium dihydrogen phosphate, VE, emulsifier, every liter of fermented and cultured
Following component: 0.2~0.4L of starch hydrolyzate, 12~18g of soyabean protein powder, 26~32g of fish meal, vegetable oil is specifically included in base
20~40g, 0.06~0.08g of copper sulphate, 0.8~1.0g of magnesium sulfate, 3~5g of potassium dihydrogen phosphate, VE0.025~0.05g, emulsification
0.5~2g of agent；And adjusting the fermentation medium pH with sodium hydroxide or potassium hydroxide is 5.5~7.0.
2. a kind of fermentation process for preparing beta carotene using Blakeslea trispora according to claim 1, feature
It is, the starch hydrolyzate comprises the following steps: starch and water is configured to starch milk, hydrochloric acid is then added and stirs evenly
After react, after reaction, in the sodium hydroxide or the potassium hydroxide and obtained pH be 6.5-7.5 the starch water
Solve liquid.
3. a kind of fermentation process for preparing beta carotene using Blakeslea trispora according to claim 2, feature
It is, the mass percent concentration of the starch milk is 15~20%, and the hydrochloric acid additive amount is the starch milk volume total amount
0.4~0.6%, at 40~60 DEG C, the reaction time is 1~3h for the reaction temperature control of the starch milk and the hydrochloric acid.
4. a kind of fermentation process for preparing beta carotene using Blakeslea trispora according to claim 3, feature
It is, the starch is any one in cornstarch, sweet potato starch or potato starch.
5. a kind of fermentation process for preparing beta carotene using Blakeslea trispora according to claim 1, feature
It is, the adjusting strategy of revolving speed and ventilatory capacity in step (4) fermentation process are as follows: earlier fermentation, revolving speed are 120~150r/
Min, ventilatory capacity are 0.6~0.8vvm, and tank pressure is 0.03~0.06MPa；When fermentation time reaches 50~60h, by rotational speed regulation
For 170~250r/min, ventilatory capacity is adjusted to 0.8~1.2vvm, and tank pressure regulation is 0.03~0.06MPa, then fills into described
Solvent or diluent, the solvent or diluent include alpha, beta-lonone and malic acid, and the amount for once filling into the alpha, beta-lonone is that fermentating liquid volume is total
The 0.1~0.4% of amount, the amount for once filling into the malic acid is the 0.1~0.3% of the fermentating liquid volume total amount.
6. a kind of fermentation process for preparing beta carotene using Blakeslea trispora according to claim 1, feature
It is, in the step (4), the vegetable oil is once filled into when fermenting 30~40h, fill into 1 in every liter of fermentation liquid~
Vegetable oil described in 3g；During the fermentation, when sample detection residual sugar content is lower than 5.0g/L, the shallow lake is filled into one or more times
Powder hydrolyzate, controlling residual sugar content in the fermentation liquid is 6~10g/L.
7. a kind of fermentation process for preparing beta carotene using Blakeslea trispora according to claim 1 or 6, special
Sign is that the vegetable oil is any one or a few in corn oil, soybean oil, sunflower oil or palm oil；The emulsification
Agent is Span series product or TWEEN Series product.
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| CN109536559A (en) * | 2017-09-22 | 2019-03-29 | 嘉必优生物技术（武汉）股份有限公司 | Method for preparing β -carotene |
| CN108342445B (en) * | 2018-04-09 | 2021-09-21 | 中国科学院合肥物质科学研究院 | Method for producing beta-carotene by Blakeslea trispora fermentation and beta-carotene |
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