CN108795819B - Compound microorganism culture and application thereof in production of carotenoid - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and discloses a compound microorganism culture for preparing carotenoid, which comprises bacillus subtilis, rhodopseudomonas capsulata and rhodotorula glutinis. The composite microbial culture of the invention improves the yield of carotenoid and reduces the culture cost.

Description

Compound microorganism culture and application thereof in production of carotenoid

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a compound microorganism culture and application thereof in production of carotenoids.

Background

Carotenoids are fat-soluble pigments, most of which are insoluble in water, soluble in organic solvents and yellow, orange or red polyene compounds, and become important natural pigments due to wide distribution, various structures and more functions. It is present in both plants, animals and microorganisms. The carotenoid has various biological effects due to the structural characteristics, and researches show that the carotenoid can have the effects of preventing and treating heart diseases, cancers, cataract and several human chronic diseases and other biological activity effects. In addition, carotenoid as an excellent food additive and a nutritional supplement for improving human nutrition, its excellent quality and effect have long been recognized by various countries in the world and are known as the most promising antioxidant. Carotenoids have long been favored by people because of their good application prospects and excellent functions.

The chemically synthesized carotene is difficult to be absorbed by human bodies, has low physiological activity, can be synthesized into intermediate products harmful to the human bodies, is not easy to separate, has certain toxic or side effect, and is not popularized and used on a large scale. Naturally occurring carotene has no toxic or side effect and is more and more valued by people. At present, the sources of natural carotenoids are mainly extracted from plants and microbial fermentation. The plant extract is obtained by extracting carotenoid from carotenoid-rich plant such as carrot. The microbial fermentation extraction refers to the production of carotenoid by the fermentation of microorganisms such as Blakeslea trispora, seaweed of Dunaliella, yeast and the like. Chinese invention patent CN102702777A discloses a method for extracting carotenoid from plant material by enzymatic hydrolysis, firstly homogenizing fresh plant material and water by a homogenizer, and crushing by an ultrasonic crusher; adding cellulase, pectinase and surfactant for enzymolysis, adding diatomite into the enzymolysis liquid, filtering, and vacuum drying the filtrate to obtain carotenoid; the method has the defects of more raw materials consumption, high cost, larger influence of the raw materials and the like. Since the process for extracting carotenoids from natural plants is complicated and has low yield and is greatly influenced by raw materials, the production of carotenoids by microbial fermentation has become a research hotspot. For example, chinese patent CN104805168A discloses a method for producing carotenoids by fermentation using photosynthetic bacteria, wherein the yield of carotenoids is significantly increased by adjusting the culture conditions to be micro-aerobic. Some researches have also been conducted on mixed strain fermentation, such as "research on producing carotenoids by mixed solid fermentation of yam skins, science and technology in food industry in 2014", which adopts mixed solid fermentation of neurospora crassa and lactobacillus plantarum to produce carotenoids, wherein the solid fermentation time is 60 hours, the yield is less than 200ug/g of culture, and the yield is relatively low. The method is limited by the content of carotenoid from plant sources, the price of the current natural carotene is higher, which is twice of that of chemicals, the cost is relatively higher, and the microbial fermentation method has the defects of low yield and high culture cost, so how to improve the yield of the carotenoid in the microorganism and reduce the fermentation cost is a technical problem to be solved.

Disclosure of Invention

The invention aims to overcome the defects of carotenoid production in the prior art and provides a compound microorganism culture and application thereof in producing carotenoid.

The invention is realized by the following technical scheme:

a composite microbial culture comprising Bacillus subtilis, Rhodopseudomonas capsulata, and Rhodotorula glutinis.

Further, the complex microbial culture is used for preparing carotenoids.

Specifically, the composite microbial culture is prepared according to the following steps:

step 1) firstly, crushing rice straws to 1-5cm, and then carrying out steam explosion pretreatment;

step 2), sieving the rice straws subjected to steam explosion treatment by a 50-mesh sieve, collecting undersize products, and mixing the undersize products with the vinasse according to the ratio of 3: 1 to obtain a mixture, adding the mixture into water which accounts for 2-3 times of the weight of the mixture, heating to 60-70 ℃, stirring for 60-90min at 100rpm under the condition of heat preservation, then heating to 121 ℃, preserving heat for 3-5min, and naturally cooling to room temperature to obtain a culture solution;

step 3) inoculating the bacillus subtilis seed solution into a photosynthetic culture fermentation tank containing the culture solution according to the inoculation amount of 5-7% for culture for 24-36h, and then inoculating the rhodopseudomonas capsulata seed solution and the rhodotorula glutinis seed solution, wherein the inoculation amounts are 8-10%; continuously culturing for 24-36h to obtain the composite microorganism culture.

Preferably, the first and second electrodes are formed of a metal,

the steam explosion pretreatment conditions are as follows: the pressure is 1.5-1.8MPa, and the retention time is 5-10 min.

Preferably, the first and second electrodes are formed of a metal,

the culture conditions are that the temperature is 30 ℃, the dissolved oxygen concentration is 1-3 mg/L, and the illumination intensity is 2000-3000 lux.

Preferably, the first and second electrodes are formed of a metal,

the preparation method of the bacillus subtilis seed solution comprises the steps of inoculating bacillus subtilis to L B solid culture medium for culture to obtain single colonies, and selecting the single colonies to inoculate the single colonies to L B liquid culture medium for culture to obtain the bacillus subtilis seed solution.

Preferably, the first and second electrodes are formed of a metal,

the preparation method of the rhodopseudomonas capsulata seed solution comprises the following steps: and (3) streaking and inoculating the rhodopseudomonas capsulata to a beef extract peptone agar culture medium for culture, then selecting colonies, and inoculating the colonies to a liquid seed culture medium for seed culture to obtain a rhodopseudomonas capsulata seed solution.

Preferably, the first and second electrodes are formed of a metal,

the liquid seed culture medium comprises 3 g/L of glucose, 2 g/L of yeast extract, 0.5 g/L of ammonium chloride, 0.1 g/L of monopotassium phosphate, 0.1 g/L of magnesium sulfate heptahydrate and 0.01 g/L of ferrous sulfate heptahydrate.

Preferably, the first and second electrodes are formed of a metal,

the preparation method of the Rhodotorula glutinis strain seed liquid comprises the following steps: inoculating rhodotorula glutinis to a YPDA solid culture medium for culture to obtain a single colony; and (3) selecting a single colony, inoculating the single colony to a YPD liquid culture medium for culture, and culturing at 30 ℃ for 24 hours to obtain Rhodotorula glutinis strain seed liquid.

The strains specifically adopted in the embodiment of the invention are bacillus subtilis ATCC6633, rhodopseudomonas capsulata ATCC33762 and rhodotorula glutinis ATCC 32759. The preparation method of the seed liquid of the strain is not limited to the method described in the examples, and can be performed according to the conventional preparation method in the field.

Compared with the prior art, the invention has the advantages that the invention mainly comprises but is not limited to the following aspects:

the steam explosion pretreatment is used for partially degrading the hemicellulose and the lignin, destroying the wrapping effect of the lignin and the hemicellulose on the cellulose, destroying the crystalline region of the cellulose, and increasing the porosity and the internal surface area of the raw material, thereby being more beneficial to the subsequent enzyme hydrolysis of the cellulose;

the invention carries out crushing and blasting treatment on agricultural waste straws, combines with vinasse, can provide normal growth conditions for bacillus subtilis, has relatively low raw materials and can reduce the enterprise cost;

under the stress condition of proper nutrient deficiency or hypoxia condition, more carotenoid and other substances can be produced in the bacterial cells, and then the slow proliferation of the bacterial strains is easily caused, so that the yield of the carotenoid, the proliferation of the bacterial cells and the culture cost need to be balanced in industrial production;

rhodopseudomonas capsulata and rhodotorula glutinis are strains for producing carotenoid, and the rhodotorula capsulata and the rhodotorula glutinis can be symbiotic and can utilize xylose as a fermentation substrate, but reducing sugar nutrients such as xylose in the straw treatment product are insufficient and are not beneficial to the growth of the strains; the method comprises the following steps of firstly, fermenting straws and vinasse by using bacillus subtilis, wherein the bacillus subtilis can produce cellulase and protease, a carbon source in a culture solution is mainly cellulose and lignin components, the bacillus subtilis can produce enzyme with high efficiency under the stress condition of relatively insufficient carbohydrate components, the cellulase can produce reducing sugar by using the cellulose as a substrate for rhodopseudomonas capsulata and rhodotorula glutinis, the rhodopseudomonas capsulata and the rhodotorula glutinis have a nutrition competition relationship, and a large amount of carotene substances can be produced intracellularly under the stress condition; the protease produced by the bacillus subtilis can also carry out enzymolysis on vinasse, and an enzymolysis product can be used as a nitrogen source; the three strains can be symbiotically coordinated in a culture system, and the proliferation of the strains and the generation of the carotenoids are promoted.

Drawings

FIG. 1: the influence of mixed culture time on the biomass yield of thalli;

FIG. 2: effect of mixed culture time on carotenoid production.

Detailed Description

Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.

Example 1

A composite microbial culture comprising bacillus subtilis, rhodopseudomonas capsulata, and rhodotorula glutinis;

firstly, crushing rice straws to 2cm, and then placing under the conditions of 1.5MPa of pressure and 10min of residence time for steam explosion pretreatment;

sieving the rice straws subjected to steam explosion treatment by a 50-mesh sieve, collecting undersize products, and mixing the undersize products with vinasse according to the weight ratio of 3: 1 to obtain a mixture, adding the mixture into water which accounts for 2 times of the weight of the mixture, heating to 60 ℃, stirring for 90min at 100rpm under the condition of heat preservation, then heating to 121 ℃, preserving heat for 5min, and naturally cooling to room temperature to obtain a culture solution;

inoculating the Bacillus subtilis seed solution into a photosynthetic culture fermentation tank containing the culture solution according to the inoculation amount of 7 percent (volume ratio) for culture at the temperature of 30 ℃, the dissolved oxygen concentration of 1 mg/L, the illumination intensity of 2000lux and the culture time of 36 hours, then inoculating the rhodopseudomonas capsulata seed solution and the rhodotorula glutinis seed solution, wherein the inoculation amounts are 10 percent (volume ratio), and continuously culturing for 36 hours to obtain the composite microbial culture.

The preparation method of the Bacillus subtilis seed solution comprises inoculating Bacillus subtilis to L B solid culture medium for culture to obtain single colony, selecting the single colony, inoculating to L B liquid culture medium for culture to obtain thallus concentration of 1 × 10⁸cfu/ml of bacillus subtilis seed solution;

the preparation method of the rhodopseudomonas capsulata seed solution comprises the steps of streaking rhodopseudomonas capsulata, inoculating the streaked rhodopseudomonas capsulata to a beef extract peptone agar culture medium for culture, then selecting colonies, inoculating the colonies to a liquid seed culture medium for seed culture, and obtaining the thallus concentration of 3 × 10⁸The liquid seed culture medium comprises 3 g/L of glucose, 2 g/L of yeast extract, 0.5 g/L of ammonium chloride, 0.1 g/L of monopotassium phosphate, 0.1 g/L of magnesium sulfate heptahydrate and 0.01 g/L of ferrous sulfate heptahydrate;

the preparation method of the Rhodotorula glutinis strain seed liquid comprises the following steps: inoculating rhodotorula glutinis to a YPDA solid culture medium for culture to obtain a single colony; picking single colony to inoculate YPD liquidCulturing on culture medium at 30 deg.C for 24 hr to obtain thallus concentration of 5 × 10⁷cfu/ml Rhodotorula glutinis strain seed liquid.

Example 2

A composite microbial culture comprising bacillus subtilis, rhodopseudomonas capsulata, and rhodotorula glutinis;

firstly, crushing rice straws to 5cm, and then placing under the conditions of 1.8MPa of pressure and 5min of residence time for steam explosion pretreatment;

sieving the rice straws subjected to steam explosion treatment by a 50-mesh sieve, collecting undersize products, and mixing the undersize products with vinasse according to the weight ratio of 3: 1 to obtain a mixture, adding the mixture into water which accounts for 3 times of the weight of the mixture, heating to 70 ℃, stirring for 80min at 100rpm under the condition of heat preservation, then heating to 121 ℃, preserving heat for 4min, and naturally cooling to room temperature to obtain a culture solution;

inoculating the bacillus subtilis seed solution into a photosynthetic culture fermentation tank containing a culture solution according to the inoculation amount of 6 percent (volume ratio) for culture at the temperature of 30 ℃, the dissolved oxygen concentration of 2 mg/L, the illumination intensity of 3000lux and the culture time of 24 hours, then inoculating the rhodopseudomonas capsulata seed solution and the rhodotorula glutinis seed solution, wherein the inoculation amounts are 8 percent (volume ratio), and continuously culturing for 30 hours to obtain the composite microbial culture.

The preparation method of the Bacillus subtilis seed solution comprises inoculating Bacillus subtilis to L B solid culture medium for culture to obtain single colony, selecting the single colony, inoculating to L B liquid culture medium for culture to obtain thallus concentration of 2 × 10⁸cfu/ml of bacillus subtilis seed solution;

the preparation method of the rhodopseudomonas capsulata seed solution comprises the steps of streaking rhodopseudomonas capsulata, inoculating the streaked rhodopseudomonas capsulata to a beef extract peptone agar culture medium for culture, then selecting colonies, inoculating the colonies to a liquid seed culture medium for seed culture, and obtaining the thallus concentration of 3 × 10⁸The liquid seed culture medium comprises 3 g/L of glucose, 2 g/L of yeast extract, 0.5 g/L of ammonium chloride, 0.1 g/L of monopotassium phosphate, 0.1 g/L of magnesium sulfate heptahydrate and 0.01 g/L of ferrous sulfate heptahydrate;

the above-mentionedThe Rhodotorula glutinis strain seed solution is prepared by inoculating Rhodotorula glutinis to YPDA culture medium for culturing to obtain single colony, selecting single colony, inoculating to YPD liquid culture medium for culturing at 30 deg.C for 24 hr to obtain strain with a concentration of 7 × 10⁷cfu/ml Rhodotorula glutinis strain seed liquid.

Example 3

The changes of the main components of the rice straw by the blasting are shown in the table 1:

TABLE 1

Index (I)	Before blasting	After blasting
			Hemicellulose and cellulose%	53.7	40.5
Lignin%	27.9	22.1
			Oligo-xylose%	0	3.4

And (4) conclusion: the blasting causes the cell wall of the straw to be damaged, and part of hemicellulose and cellulose are degraded and dissolved out, so that the subsequent enzymolysis of cellulose by cellulase is facilitated; the crystallinity and polymerization degree of cellulose are reduced in the process of blasting pretreatment, and the hemicellulose is degraded into monosaccharide and oligosaccharide through self-hydrolysis and can be used as a carbon source of a strain.

Example 4

Determination of biomass of bacteria and yield of carotenoid:

centrifuging the compound microorganism culture at 8000rpm and 4 deg.C for 20min, discarding supernatant, drying at 45 deg.C for 36h to reach constant weight, accurately weighing thallus 0.5g, adding 2 mol/L hydrochloric acid 2m L, mixing, vacuum filtering, washing with water to neutrality, collecting residue, adding 10m L acetone into residue, leaching at room temperature for 30min, centrifuging twice, mixing the two centrifugated supernatants, adding acetone to desired volume 25m L, mixing, using acetone as blank control, measuring absorbance at 461nm, and measuring carotenoid content (mg/g thallus biomass) = (A)_λmax×D×V）/（1000×0.16×W）。

In the formula A_λmaxAbsorbance at the maximum absorption wavelength of carotenoid, V-amount of solvent used for extraction (m L), -D-dilution factor in measurement of sample, W-cell weight (g), and 0.16-molar extinction coefficient of carotenoid.

Setting a control group to verify the synergistic effect of each strain, wherein the control group 1: the preparation method is the same as that of the example 1 only by adopting the bacillus subtilis and the rhodopseudomonas capsulata and not adding the rhodotorula glutinis; control group 2: the preparation method is the same as that of example 1 only by using bacillus subtilis and rhodotorula glutinis without adding rhodopseudomonas capsulata. Specific detection results are shown in table 2:

TABLE 2

Group of	Bacterial biomass dry weight g/L	Yield mg/g bacterial biomass of carotenoid
			Example 1	12.7	2.08
Example 2	12.1	1.96
			Control group 1	10.9	1.65
Control group 2	9.8	1.50

As shown in Table 2, in examples 1-2, the biomass and carotenoid yields were higher than those of controls 1-2, and the carotenoid yields were significantly higher than those of controls 1-2, wherein the dry weight of biomass was 12.7 g/L in example 1, which was 16.5% and 29.6% higher than those of controls 1 and 2, respectively, and the carotene yields were 2.08mg/g biomass, which was 26.1% and 38.7% higher than those of controls 1 and 2, respectively.

Example 5

The effect of the mixed culture time of the three strains on the biomass of the bacteria and the yield of the carotenoid.

Taking the method of example 1 as an example, different mixed culture time points, specifically 6, 12,18,24,30,36 and 42h, were set. As shown in the figure 1-2, the increase of the biomass of the thallus is obvious within 12 hours, but the yield of the carotenoid is maintained at a lower level, the biomass of the thallus and the yield of the carotenoid are obviously increased along with the increase of the culture time, and the increase of the biomass of the thallus is slowed after the culture time is 24 hours, probably because the culture components in the culture solution are reduced, the proliferation rate of the strain is reduced under the condition of nutrient deficiency and stress, but the yield of the carotenoid is continuously increased, the biomass of the thallus reaches the peak value when the strain is cultured for 36 hours, and then the biomass of the thallus is reduced, probably because the strain is dead, and correspondingly, the yield of the carotenoid is also reduced.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (1)

1. A complex microbial culture for the production of carotenoids, which is produced according to the following steps:

step 1) firstly, crushing rice straws to 1-5cm, and then carrying out steam explosion pretreatment, wherein the pressure is 1.5-1.8MPa, and the retention time is 5-10 min;

step 2), sieving the rice straws subjected to steam explosion treatment by a 50-mesh sieve, collecting undersize products, and mixing the undersize products with the vinasse according to the ratio of 3: 1 to obtain a mixture, adding the mixture into water which accounts for 2-3 times of the weight of the mixture, heating to 60-70 ℃, stirring for 60-90min at 100rpm under the condition of heat preservation, then heating to 121 ℃, preserving heat for 3-5min, and naturally cooling to room temperature to obtain a culture solution;

step 3) inoculating the bacillus subtilis seed solution into a photosynthetic culture fermentation tank containing the culture solution according to the inoculation amount of 5-7% for culture for 24-36h, and then inoculating the rhodopseudomonas capsulata seed solution and the rhodotorula glutinis seed solution, wherein the inoculation amounts are 8-10%; continuously culturing for 24-36h to obtain a composite microorganism culture;

the preparation method of the bacillus subtilis seed solution comprises the steps of inoculating bacillus subtilis to L B solid culture medium for culture to obtain single colony, selecting the single colony to be inoculated to L B liquid culture medium for culture to obtain the bacillus subtilis seed solution;

the preparation method of the rhodopseudomonas capsulata seed solution comprises the following steps: inoculating rhodopseudomonas capsulata to a beef extract peptone agar culture medium for culture by streaking, then selecting colonies, inoculating the colonies to a liquid seed culture medium for seed culture to obtain rhodopseudomonas capsulata seed solution;

the liquid seed culture medium comprises 3 g/L of glucose, 2 g/L of yeast extract, 0.5 g/L of ammonium chloride, 0.1 g/L of monopotassium phosphate, 0.1 g/L of magnesium sulfate heptahydrate and 0.01 g/L of ferrous sulfate heptahydrate;

the preparation method of the Rhodotorula glutinis strain seed liquid comprises the following steps: inoculating rhodotorula glutinis to a YPDA solid culture medium for culture to obtain a single colony; and (3) selecting a single colony, inoculating the single colony to a YPD liquid culture medium for culture, and culturing at 30 ℃ for 24 hours to obtain Rhodotorula glutinis strain seed liquid.

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