CN113373197B - Application of Spodoptera frugiperda in production of carotenoid and exopolysaccharide - Google Patents
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Abstract
The invention discloses an application of Spodoptera latus in production of carotenoid and extracellular polysaccharide, belonging to the field of microorganisms and microbial 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 latus in production of carotenoid and extracellular polysaccharide, belonging to the field of microorganisms and microbial fermentation.
Background
Microbial carotenoids are carotenoids which are stored in cells by oleaginous microorganisms such as yeast, algae, molds, and bacteria. The characteristics and advantages of the microbial carotenoid are mainly reflected in that: plant-derived fiddleCompared with the carotene product, 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, plant growing season conditions and the like; 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 mainly focuses on the production of high value-added functional carotenoids by microorganisms, such as cryptodino cohnii (cryptococcus cohnii), schizochytrium sp, and klucothecium wukenii (Ulkenia sp.) strains 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 -1 And 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. The current research has shown that the structure of exopolysaccharides of microorganisms has remarkable diversity, but only a small number of them have been studied for potential of industrial development, such as xanthan gum and dextran, etc [3,4] . The microbial exopolysaccharide has been industrialized with shorter production cycle and more convenience than other polysaccharides of natural originThe extraction method and the production process of the microbial exopolysaccharide are carried out in a fermentation tank, the production land of the microbial exopolysaccharide does not compete with other grains and crops, and the microbial exopolysaccharide and the production method can also fully utilize other agricultural waste resources as the raw materials for the production. In addition, due to the unique structure and physicochemical properties of the exopolysaccharide, the exopolysaccharide is widely applied to the aspects of foods, biomaterials, rheology 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-mild irradiation on the mechanism and bioactives of the polysaccharide from blackcurrant fractions [ J]Ultrasonics Sonochemistry,2018, 49. 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 carotenoids derived from microorganisms have not been largely limited to high production costs by large-scale application, more and more studies have been focused on optimization of fermentation conditions and reduction of production costs by using inexpensive raw materials. However, the production of carotenoids is always reduced by using cheap raw materials, and the production cost is further increased by producing a plurality of unusable byproducts while producing carotenoids by using 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 pararosaeus (Sporidiobolus pararosaeus) as a fermentation strain to carry out fermentation production in a reaction system containing corn steep liquor and glucose; the Zuojing yeast is preserved in China Center for Type Culture Collection (CCTCC) at 2021, 6 months and 17 days, and the preservation number is CCTCC NO: m2021729.
In one embodiment, the throw-locked yeast cultured to the logarithmic phase is inoculated into the fermentation system at 5-15% of the fermentation system and cultured at 25-30 ℃ for 60-70 h.
In one embodiment, the OD is 600 The 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 64h.
In one embodiment, the initial concentrations of glucose and corn steep liquor in the fermentation system are 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 within 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 within 18-56h of fermentation, and the residual sugar content in the fermentation system is controlled to be 20g/L.
In one embodiment, dissolved oxygen is controlled between 15% and 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 the 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 64h.
The invention has the beneficial effects that: the invention selects a Spodophyllum yeast screened from vinegar as a fermentation strain for producing carotenoid and extracellular polysaccharide, and the Spodophyllum yeast strain is subjected to high-density fermentation culture, so that the cell length of 64h can reach more than 80g/L, 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 carotenoid and exopolysaccharide by culturing Spodoptera glauca
The culture medium is selected as follows:
(1) Slant activation medium (g/L): glucose 20, peptone 1, yeast extract 1, pH6.0.
(2) Liquid seed medium (g/L): 40 portions of glucose, 20 portions of corn steep liquor and KH 2 PO 4 1，MgSO 4 ·7H 2 O0.5, 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) And (3) a feed culture medium:
(1) glucose: the concentration was 800g/L.
(2) Corn steep liquor: the concentration was 100g/L.
(5) The culture method comprises the following steps:
(1) slant activation culture:
the strain is inoculated to an activated slant culture medium from a preservation slant and cultured for 48 hours at the temperature of 28 ℃.
Liquid seed culture: inoculating to seed culture medium, shaking at 28 deg.C (100 r/min, reciprocating shaking table, amplitude of 8 cm) for culturing 169h 600 Is 0.4-0.5.
(2) 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 2; 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
(1) 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 incarnatum, and detecting carotenoid composition in Spodophyllum incarnatum oil with Hitachi L-2000 high performance liquid chromatography equipped with DAD detector and C18 column (25 mm × 4.6 mm), wherein the carotenoid content is about 900 μ g/g dry thallus weight.
(2) Extracellular polysaccharide
Determination of extracellular polysaccharide production: taking 10mL of fermentation supernatant, adding 20mL of absolute ethanol, 6000 r.min -1 Centrifuging 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 -1 (ii) a 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 -1 Feeding the corn steep liquor from the beginning of fermentation to 24 hours, and stopping feeding the corn steep liquor in the rest time.
Determination of extracellular polysaccharide production: 10mL of fermentation supernatant is added with 20mL of absolute ethyl alcohol, 6000 r.min -1 Centrifuging 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 -1 Is 44.3 percent higher than the optimized premise. 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 one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (8)
1. A method for increasing carotenoid yield is characterized by that the sporophore is thrown by near pink lock (Sporobolomyces) (A. Clarkii)Sporidioboluspararoseus) ZQHL is a fermentation strain, and is produced in a reaction system containing corn steep liquor and glucose through fermentation; the near pink locked sporotrichum tosporum ZQHL was preserved in the China center for type culture Collection in 2021, 6 and 17, and the preservation number is CCTCC NO: m2021729, wherein the initial concentrations of the glucose and the corn steep liquor are 50 to 70g/L and 5 to 15g/L respectively.
2. The method according to claim 1, characterized in that the near-pink Sporobolomyces cultured to logarithmic phase is inoculated into the fermentation system according to 5 to 15 percent of the fermentation system, and cultured for 60 to 70h at 25 to 30 ℃.
3. The method as claimed in claim 2, wherein glucose is supplemented within 15 to 60 hours of fermentation, and the residual sugar content in the fermentation system is controlled to be 15 to 20g/L.
4. The method according to any one of claims 1 to 3, wherein the dissolved oxygen is controlled to be 15 to 25% in the reaction process.
5. A method for improving the yield of exopolysaccharides is characterized in that the sporotrichum roseum ZQHL is used as a fermentation strain and is fermented and produced in a reaction system containing corn steep liquor and glucose; the near pink locked sporotrichum tosporum ZQHL was preserved in the China center for type culture Collection in 2021, 6 and 17, and the preservation number is CCTCC NO: m2021729, wherein the initial concentrations of the glucose and the corn steep liquor are 50 to 70g/L and 15 to 25g/L respectively.
6. The method as claimed in claim 5, wherein the corn steep liquor is supplemented within 20 to 25 hours after the start of fermentation, the concentration of the corn steep liquor is 80 to 120g/L, and the feeding speed is 0.1 to 0.2L-h -1 。
7. The method as claimed in claim 6, wherein the dissolved oxygen is controlled to be 15 to 25% during the reaction.
8. The method according to any one of claims 5 to 7, wherein the reaction time is not less than 60 hours.
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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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| 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 |
Non-Patent Citations (4)
| Title |
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| Sporidiobolus pararoseus WZ012产类胡萝卜素发酵条件的优化;魏华等;《食品科技》;20120820;第37卷(第08期);第22-26、31页 * |
| Sporidiobolus pararoseus发酵产物中类胡萝卜素的分离与鉴定;李晓芹等;《食品与发酵工业》;20111231;第37卷;第25-30页 * |
| 氮源种类及溶氧水平对锁掷酵母产类胡萝卜素组分的影响;王小波等;《工业微生物》;20131222;第43卷(第06期);第43-48页 * |
| 锁掷孢酵母产胞外多糖发酵条件优化;裴芳艺等;《中国酿造》;20200625;第39卷(第06期);第99-105页 * |
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