CN113373197A - Application of Spodoptera lata in production of carotenoid and exopolysaccharide - Google Patents
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- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
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Abstract
The invention discloses an application of Spodoptera lata in production of carotenoid and exopolysaccharide, belonging to the field of microorganism and microorganism fermentation. The invention selects a Spodophyllum latum screened from vinegar as a fermentation strain for producing carotenoid and extracellular polysaccharide, and the Spodophyllum latum is subjected to high-density fermentation culture, so that the cell length of the cells can reach more than 80g/L for 72-86h, and the carotenoid content can reach 900ug/g of the dry cell weight. Meanwhile, by adjusting the components of the culture medium, extracellular polysaccharide of 7.9-11.4g/L can be obtained, the extracellular polysaccharide is phosphodiester polysaccharide, the polysaccharide can remarkably enhance the effect of immunity, and the additional value of the fermentation process is remarkably improved. The strain has extensive culture conditions, low nutrient requirement and high carotenoid yield, and the byproduct extracellular polysaccharide also has production and application values and good industrial application prospects.
Description
Technical Field
The invention relates to an application of Spodoptera lata in production of carotenoid and exopolysaccharide, belonging to the field of microorganism and microorganism fermentation.
Background
Microbial carotenoids are carotenoids stored intracellularly by oleaginous microorganisms such as yeast, algae, molds, and bacteria. The characteristics and advantages of the microbial carotenoid are mainly reflected in that: compared with carotenoid products from plant sources, the microbial carotenoid does not compete with grain production and does not occupy a large amount of cultivated land; the method is basically not influenced by natural condition changes such as global climate and plant growth season conditions; the cycle for carotenoid production using microorganisms is shorter; microorganisms utilize industrial waste or other inexpensive raw materials as a carbon source; the microorganism can be modified by metabolic engineering method to enrich specific fatty acid and reduce chemical synthesis steps[1]. The current development and application of microbial carotenoids in the field of edible carotenoids has mainly focused on the production of high value-added functional carotenoids by microorganisms, such as cryptodino cohnii (cryptococcus comani), Schizochytrium sp and Ulkenia sp (Ulkenia sp.) for the production of DHA and mortierella alpina (mortierella alpina) for the production of ARA. Less production of functional carotenoid by using yeast and oil-producing yeast. Yeasts are widely regarded as the most suitable species for producing microbial carotenoids, and the most studied carotenoids are yarrowia lipolytica (yarrowia lipolytica), rhodotorula glutinis (rhodotorula glutinis), Cryptococcus curvatus (Cryptococcus curvatus), and streptomyces carotenoids (lipomycstarkeyi). The carotenoid yeast is influenced by strains and culture conditions, the oil production capacity of different oil-producing yeasts is different when fed-batch fermentation is carried out by using glucose as a carbon source, and the yield of the carotenoid is 5-800 ug.L-1And the content of the carotenoid in the cells is also not equal, so that the conversion rate of the carotenoid to the glucose is not high.
In addition, microbial exopolysaccharides are the research target of many disciplines, and the optimization of the production process, the clarification of the biological function, the determination of the biosynthesis pathway and the application development based on the product characteristics are mainly concerned at present. Current research has shown thatThe structure of the biological exopolysaccharide has remarkable diversity, but only a small amount of products such as xanthan gum, glucan and the like are researched for industrial development potential[3,4]. Compared with other polysaccharides from natural sources, the microbial exopolysaccharide which is already industrialized has a shorter production period and a more convenient extraction method, the production process of the microbial exopolysaccharide is carried out in a fermentation tank, the production land of the microbial exopolysaccharide is not competitive with other grains and crops, and the microbial exopolysaccharide can fully utilize other agricultural waste resources as the raw material for producing the microbial exopolysaccharide. In addition, due to the unique structure and physicochemical properties of the exopolysaccharide, the exopolysaccharide is widely applied to the aspects of food, biological materials, rheological modifiers of aqueous systems, medical treatment and the like. The fungi capable of producing exopolysaccharides include Staphylococcus aureus (Aureobasidium) Candida and Cryptococcus. Exopolysaccharides produced by fungi are mostly homopolysaccharides, but heteropolysaccharides are also common. Such polymers alone or in combination with other saccharides (e.g., galactose, xylose) are combined into biopolysaccharides containing D-mannose, uronic acid and non-saccharide substituents (Xu Y, Guo Y, Duan S, et al. effects of ultra-coarse irradiation on the chromatography and bioactives of the polysaccharide from blackcurrants [ J]Ultrasonics Sonochhemistry, 2018,49: 206-. Although the chemical structure of extracellular polysaccharides of fungi has been studied and the biological activity thereof has been reported, it has not been studied in depth.
In the past decades of development, the modern development of machinery has been accompanied by a great consumption of energy, and non-renewable resources such as oil, coal, natural gas are largely developed and applied, and the energy crisis is getting more and more serious. Since the major limitations of the large-scale application of carotenoids derived from microorganisms are high production costs, more and more research is focused on the optimization of fermentation conditions and the reduction of production costs by using inexpensive raw materials. However, the production of carotenoids is always reduced by cheap raw materials, and the production cost is further increased by producing a plurality of unusable byproducts when the carotenoid is produced by the current production strains.
Disclosure of Invention
Aiming at the problems of low production efficiency, high production cost and the like of the existing carotenoid strain produced by microbial fermentation, the invention provides the red aerobic yeast, the strain can produce an extracellular polysaccharide which is phosphodiester polysaccharide while producing carotenoid by fermentation, the polysaccharide can obviously enhance the effect of immunity, and the additional value of the fermentation process is obviously improved.
The invention provides a method for improving the yield of carotenoid, which takes Sporidiobolus pararosaus as a fermentation strain to carry out fermentation production in a reaction system containing corn steep liquor and glucose; the Zygosaccharomyces has been preserved in China Center for Type Culture Collection (CCTCC) at 6 months and 17 days in 2021, with the preservation number of CCTCC NO: m2021729.
In one embodiment, the throw-locked yeast cultured to logarithmic phase is inoculated into the fermentation system according to 5-15% of the fermentation system, and cultured at 25-30 ℃ for 60-70 h.
In one embodiment, the OD is600The throw-locked yeast of 0.4-0.5 is inoculated into the fermentation system according to 10% of the volume of the fermentation system, and cultured at 28 ℃ for 64 h.
In one embodiment, the initial concentration of glucose and corn steep liquor in the fermentation system is 50-70 g/L and 5-15 g/L, respectively.
In one embodiment, the initial concentration of glucose and corn steep liquor in the fermentation system is 60g/L and 10g/L, respectively.
In one embodiment, glucose is supplemented for 15-60 h of fermentation, and the residual sugar content in the fermentation system is controlled to be 15-20 g/L.
In one embodiment, glucose is supplemented for 18-56h of fermentation, and the residual sugar content in the fermentation system is controlled to be 20 g/L.
In one embodiment, the dissolved oxygen is controlled to be 15-25% during the reaction.
In one embodiment, dissolved oxygen is controlled at 20% during the reaction.
The invention provides a method for improving the yield of exopolysaccharide, which takes Spodoptera latum as a fermentation strain and produces the exopolysaccharide by fermentation in a reaction system containing corn steep liquor and glucose; the Zygosaccharomyces has been preserved in China Center for Type Culture Collection (CCTCC) at 6 months and 17 days in 2021, with the preservation number of CCTCC NO: m2021729.
In one embodiment, the initial concentration of glucose and corn steep liquor in the fermentation system is 50-70 g/L and 15-25 g/L, respectively.
In one embodiment, the initial concentration of glucose and corn steep liquor in the fermentation system is 60g/L and 20g/L, respectively.
In one embodiment, the corn steep liquor is supplemented during the period from the beginning of fermentation to 20-25 h, the concentration of the corn steep liquor is 80-120 g/L, and the feeding speed is 0.1-0.2 L.h-1。
In one embodiment, the corn steep liquor is supplemented during the period from the beginning of the fermentation to 24h, the concentration of the corn steep liquor is 100g/L, and the feeding speed is 0.125 L.h-1。
In one embodiment, dissolved oxygen is controlled at 20% during the reaction.
In one embodiment, the reaction time is not less than 60 hours.
In one embodiment, the reaction time is 64 h.
The invention has the beneficial effects that: the invention selects a Spodophyllum latum screened from vinegar as a fermentation strain for producing carotenoid and exopolysaccharide, and the Spodophyllum latum is cultured by high-density fermentation, the cell length of the cell can reach more than 80g/L for 64h, and the carotenoid content can reach 900ug/g of the dry cell weight. Meanwhile, by adjusting the components of the culture medium, extracellular polysaccharide of 7.9-11.4g/L can be obtained, the extracellular polysaccharide is phosphodiester polysaccharide, the polysaccharide can remarkably enhance the effect of immunity, and the additional value of the fermentation process is remarkably improved.
Biological material preservation
The Sporobolomyces pinosylvis classified and named as near-pink Sporobolomyces pinosylvqHL, which is preserved in China center for type culture Collection in 6.17.2021 with the preservation number of CCTCC NO: m2021729, the preservation address is Wuhan university in China.
Detailed Description
Example 1: production of carotenoids and exopolysaccharides by culturing Spodoptera glauca
The culture medium is selected as follows:
(1) slant activation medium (g/L): glucose 20, peptone 1, yeast extract 1, ph 6.0.
(2) Liquid seed medium (g/L): 40 parts of glucose, 20 parts of corn steep liquor and KH2PO41，MgSO4·7H2O0.5, pH6.0, 100mL/500mL triangular flask.
(3) Fermentation medium (25L system) (g/L):
TABLE 1 fermentation Medium composition
Note: when preparing, the reagent is prepared according to the dosage of 30L. Adjusting the pH to 6.0-6.5, adding 0.1-0.3% (v/v) of a defoamer (25 mL);
(4) a supplemented medium:
glucose: the concentration was 800 g/L.
Corn steep liquor: the concentration was 100 g/L.
(5) The culture method comprises the following steps:
slant activating culture:
the strain is inoculated to an activated slant culture medium from a preservation slant and cultured for 48h at 28 ℃.
Liquid seed culture: inoculating to seed culture medium, shaking (100r/min, reciprocating shaking table, amplitude of 8cm) at 28 deg.C, and culturing for 16 hr with OD600Is 0.4-0.5.
Fermentation culture in a fermentation tank: adding 35L fermentation medium into 50L fermentation tank, adding seed solution into the fermentation medium according to inoculum size of 10% (V/V), and culturing at 28 deg.C for 64h, wherein each parameter in the fermentation process is shown in Table 1; and (4) beginning to supplement 800g/L of glucose after fermenting for 18h, and controlling the residual sugar in the fermentation system at 20g/L according to the residual sugar supplement during 18-56 h.
TABLE 2 fermentation conditions parameters
Note: the dissolved oxygen is controlled to be 20 percent in the fermentation process.
(6) Identification of the product
(ii) Carotenoid
Centrifuging the fermentation liquid to obtain thallus, adding one time of alcohol, micronizing and grinding, extracting with n-hexane, collecting organic solvent layer, removing n-hexane under vacuum to obtain carotenoid produced by Spodophyllum inophyllum, and detecting carotenoid composition in Spodophyllum inophyllum oil with L-2000 high performance liquid chromatography equipped with DAD detector and C18 column (25mm × 4.6mm), wherein the carotenoid content is about 900 μ g/g dry thallus weight.
② extracellular polysaccharide
Determination of extracellular polysaccharide production: 10mL of fermentation supernatant is added with 20mL of absolute ethyl alcohol, 6000 r.min-1Centrifuging for 10min, drying the precipitate in an oven at 60 ℃ to constant weight, and detecting the yield of crude extracellular polysaccharide by a phenol-sulfuric acid method, wherein the yield is 7.9 +/-0.4 g/L fermentation liquor.
Example 2
See example 1 for a difference that the initial concentration of corn steep liquor in the fermentation medium is 20 g.L-1B, carrying out the following steps of; meanwhile, corn steep liquor with the concentration of 100g/L is fed in the fermentation process, and the feeding speed of the corn steep liquor is 0.125 L.h-1Feeding is carried out from the beginning of fermentation to 24 hours, and the feeding of the corn steep liquor is stopped in the rest time.
Determination of extracellular polysaccharide production: 10mL of fermentation supernatant is added with 20mL of absolute ethyl alcohol, 6000 r.min-1Centrifuging for 10min, drying the precipitate in an oven at 60 ℃ to constant weight, and detecting the yield of crude extracellular polysaccharide by a phenol-sulfuric acid method. After substrate optimization, the concentration of corn steep liquor is increased, the yield of exopolysaccharides can be improved, and the yield of exopolysaccharides of the Spodoptera glauca is 11.4 +/-0.4 g.L-1Specific bestThe chemical precursor is 44.3 percent higher. The carotenoid yield was about 820. mu.g/g dry cell weight.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for increasing the yield of carotenoid is characterized in that a Sporidiobolus pararosas is taken as a fermentation strain and is fermented and produced in a reaction system containing corn steep liquor and glucose; the Zygosaccharomyces has been preserved in China Center for Type Culture Collection (CCTCC) at 6 months and 17 days in 2021, with the preservation number of CCTCC NO: m2021729.
2. The method according to claim 1, wherein the throw-locked yeast cultured to the logarithmic phase is inoculated into the fermentation system at 5 to 15% of the fermentation system, and cultured at 25 to 30 ℃ for 60 to 70 hours.
3. The method of claim 2, wherein the initial concentrations of glucose and corn steep liquor in the fermentation system are 50-70 g/L and 5-15 g/L, respectively.
4. The method according to claim 3, wherein glucose is supplemented for 15-60 hours of fermentation, and the amount of residual sugar in the fermentation system is controlled to be 15-20 g/L.
5. The method according to any one of claims 1 to 4, wherein dissolved oxygen is controlled to 15 to 25% during the reaction.
6. A method for improving the yield of exopolysaccharide is characterized in that the exopolysaccharide is produced by fermenting in a reaction system containing corn steep liquor and glucose by taking Spodophyllum latum as a fermentation strain; the Zygosaccharomyces has been preserved in China Center for Type Culture Collection (CCTCC) at 6 months and 17 days in 2021, with the preservation number of CCTCC NO: m2021729.
7. The method of claim 6, wherein the initial concentrations of glucose and corn steep liquor in the fermentation system are 50-70 g/L and 15-25 g/L, respectively.
8. The method of claim 7, wherein the corn steep liquor is supplemented during 20-25 h from the beginning of the fermentation, the concentration of the corn steep liquor is 80-120 g/L, and the feeding speed is 0.1-0.2L-h-1。
9. The method according to claim 8, wherein dissolved oxygen is controlled to be 15-25% during the reaction.
10. The process according to any one of claims 6 to 9, wherein the reaction time is not less than 60 hours.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116286413A (en) * | 2023-02-24 | 2023-06-23 | 天津科技大学 | Brevibacterium roseum strain capable of coloring and producing fragrance of soy sauce and application thereof |
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| CN102115716A (en) * | 2010-12-07 | 2011-07-06 | 江南大学 | Sporidiobolus pararoseus bacterial strain and application thereof |
| CN111793569A (en) * | 2020-08-05 | 2020-10-20 | 盐城工学院 | High-yield fermentation method and application of carotenoid high-yield spore-casting yeast strain |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102115716A (en) * | 2010-12-07 | 2011-07-06 | 江南大学 | Sporidiobolus pararoseus bacterial strain and application thereof |
| CN111793569A (en) * | 2020-08-05 | 2020-10-20 | 盐城工学院 | High-yield fermentation method and application of carotenoid high-yield spore-casting yeast strain |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116286413A (en) * | 2023-02-24 | 2023-06-23 | 天津科技大学 | Brevibacterium roseum strain capable of coloring and producing fragrance of soy sauce and application thereof |
| CN116286413B (en) * | 2023-02-24 | 2024-05-28 | 天津科技大学 | Brevibacterium roseum strain capable of coloring and producing fragrance of soy sauce and application thereof |
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