Fermentation method of astaxanthin
By optimizing the fermentation conditions and adding corn slurry dry powder, adjusting the pH value, the content of astaxanthin in the microbial fermentation method is improved, the problem of low astaxanthin content in the existing technology is solved, and more efficient astaxanthin production is achieved .
Patent Information
- Application Number
- CN202411547979.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-01
- Publication Date
- 2025-01-03
AI Technical Summary
The low content of astaxanthin in the existing microbial fermentation methods leads to problems of high production efficiency and cost.
By optimizing the fermentation conditions, including adjusting the stirring speed, adding corn slurry dry powder and adjusting the pH value in the later stage of fermentation, the acid production and accumulation of astaxanthin are increased. The specific steps include 0.5-2.0 ventilation at 28-30℃ under conditions of 28-30℃ vvm, pH 4~6, the rotation speed is gradually adjusted, and the pH value is reduced in the later stage of fermentation. Fermentation bacteria such as red fermentation species are used for 113~168 hours, the charge coefficient is 60~80%, and the fermentation liquid is used. The content of astaxanthin in the group reaches 1050mg/L.
The acid production and accumulation of astaxanthin were significantly improved, the stirring speed and the addition of corn slurry dry powder increased the acid production rate by 27-75% and 3-12% respectively, and the pH reduction in the later stage of fermentation increased by 11-18%. The acid production increases the content of astaxanthin in the fermentation broth.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of astaxanthin production, and in particular to an astaxanthin fermentation method. Background Art
[0002] Astaxanthin is a natural antioxidant, with the chemical name of 3,3'-dihydroxy-β,β'-carotene-4,4'dione. It has many important biological functions such as strong antioxidant, anti-aging, anti-virus, anti-body coloring, etc., and has broad application prospects in the pharmaceutical, chemical, food, aquaculture and other industries.
[0003] Astaxanthin is mainly found in certain marine organisms, such as shellfish, shrimps and algae. In particular, deep-sea organisms such as Arctic shrimp and krill, as well as specific species such as Kinmen shrimp, are good sources of astaxanthin.
[0004] The production process of astaxanthin includes chemical synthesis, natural extraction and microbial fermentation. Among them, although the chemical synthesis method has the advantages of low production cost and high yield, the absorption and utilization rate of the synthesized astaxanthin in the organism is low, and the stability, safety and antioxidant activity are lower than those of the naturally extracted astaxanthin. In addition, the hazardous waste and chemical solvents generated in the chemical synthesis process cause serious pollution to the environment. Although the natural extraction method can extract high-purity astaxanthin, the ash and chitin content in the shells of discarded shrimps and crabs is high, while the astaxanthin content is low, which will cause the extraction process to be complicated and there is a problem of high extraction cost. In addition, traditional extraction methods such as alkali extraction, oil dissolution, and organic solvent method also have problems such as environmental pollution or low product concentration. Compared with traditional chemical synthesis and natural extraction methods, the fermentation method of astaxanthin production can avoid environmental pollution and reduce the use of hazardous waste and chemical solvents; the fermentation method does not rely on the source of raw materials, and will not affect the output and quality due to raw material restrictions, so the cost is lower. At the same time, its operation difficulty is lower, and it has higher feasibility and competitiveness for enterprises; astaxanthin prepared by fermentation production has long been praised for its effect and efficiency and is widely used in the market. Through precise fermentation control and extraction and separation technology, high-purity astaxanthin products can be produced. Therefore, microbial fermentation has become a hot spot in the industry.
[0005] The existing microbial fermentation method has the following shortcomings: the astaxanthin content in the fermentation liquid is low.
[0006] The above background technology is for facilitating the understanding of the present invention and is not a known technology disclosed to the general public before the application of the present invention. Summary of the invention
[0007] In view of the above defects, the present invention provides a method for fermenting astaxanthin, wherein the astaxanthin content in the fermentation liquid is 1050 mg / L.
[0008] The technical solution is: a fermentation method of astaxanthin, comprising the following steps:
[0009] S1, loading the fermentation medium into the fermentation tank with a loading factor of 60-80%, and after sterilization, inoculating the astaxanthin-producing fermentation bacteria seed culture solution with an inoculation amount of 1-10 v / v%;
[0010] S2, fermentation to obtain fermentation liquid, the fermentation conditions are: 28-30°C, ventilation volume of 0.5-2.0vvm, pH 4-6, rotation speed as shown in Table 1, fermentation time of 113-168h, dissolved oxygen controlled at 20-30%;
[0011] In S2, the speed during fermentation is:
[0012] From the beginning of fermentation to 8h of fermentation, 180-220r / min, excluding 8h of fermentation
[0013] Fermentation 8h ~ 24h, 230 ~ 270r / min, excluding fermentation 24h
[0014] Fermentation 24h ~ 30h, 280 ~ 320r / min, excluding fermentation 30h
[0015] Fermentation 30 to 38 hours, 330 to 370 r / min, excluding fermentation 38 hours
[0016] Fermentation 38 to 48 hours, 380 to 420 r / min, excluding fermentation 48 hours
[0017] Fermentation 48h ~ 56h, 480 ~ 520r / min, excluding fermentation 56h
[0018] From 56h of fermentation to the end of fermentation, the speed is 530-570r / min.
[0019] Furthermore, the fermentation bacteria is one of Phaffia rhodozyma, marine algae and bacteria.
[0020] Furthermore, the fermentation medium comprises the following components:
[0021] Glucose 5~30g / L
[0022] Peptone 5~30g / L
[0023] Yeast powder 2~15g / L.
[0024] Furthermore, the fermentation medium also includes:
[0025] Corn steep liquor powder 1~8g / L.
[0026] Furthermore, in S2, the pH in the early and middle stages of fermentation is greater than the pH in the late stage of fermentation.
[0027] Furthermore, the pH in the early and middle fermentation stages minus the pH in the late fermentation stages=0.5-1.5.
[0028] Furthermore, the pH in the early and middle fermentation stages minus the pH in the late fermentation stages=1.
[0029] Furthermore, the start time of the late fermentation period is 90 hours of fermentation.
[0030] Compared with the prior art, the present invention has the following beneficial effects:
[0031] The present invention optimizes astaxanthin and surprisingly finds that a specific stirring speed can increase the acid production of astaxanthin by 27-75%, and adding only a small amount of corn slurry dry powder can increase the acid production of astaxanthin by 3%-12%. Lowering the pH in the late fermentation period is beneficial to the accumulation of astaxanthin and increases the acid production by 11%-18%. DETAILED DESCRIPTION
[0032] As used herein:
[0033] "Prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.
[0034] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.
[0035] When an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values and all integers and fractions within the range.
[0036] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0037] Those skilled in the art will appreciate that the following examples are only used to illustrate the present application and should not be considered to limit the scope of the present application. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents, instruments or microorganisms used is not specified, they are all conventional products that can be purchased commercially.
[0038] In these examples, parts and percentages are by mass unless otherwise indicated.
[0039] "Parts by mass" refers to the basic unit of measurement for expressing the mass ratio of multiple components. 1 part can represent any unit mass, such as 1g or 2.689g. If we say that the mass of component A is a parts and the mass of component B is b parts, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it means that the mass of component A is aK and the mass of component B is bK (K is an arbitrary number, indicating a multiple factor). It should not be misunderstood that, unlike the mass parts, the sum of the mass of all components is not limited to 100 parts.
[0040] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).
[0041] Example 1 Preparation of seed solution
[0042] The Yarrowia lipolytica is inoculated into a YPD medium, and cultured at 28-30° C. and 180-220 rpm / min for 16-24 hours to obtain a seed culture solution.
[0043] In the following Examples 2 to 5 and Comparative Examples 1 to 6, the volume of the fermenter is 50 L. In the fermenter, the loading factor is 70%, which means the loading volume is 35 L. When the fermenter is loaded, after the fermentation medium is loaded, water is added to replenish the liquid level to 35 L (basic common sense in this field).
[0044] Example 2
[0045] A fermentation method for astaxanthin comprises the following steps:
[0046] S1, the fermentation medium is loaded into the fermenter (the loading factor is 70%), and after sterilization, the seed culture solution of Example 1 is inoculated at an inoculation amount of 5 v / v%.
[0047] S2, fermentation to obtain fermentation liquid, the fermentation conditions are: 30°C, ventilation volume at 1.0vvm, pH controlled at about 5, rotation speed as shown in Table 1, fermentation time 144h, dissolved oxygen controlled at 20-30%.
[0048] Table 1 Stirring speed during fermentation
[0049]
[0050]
[0051] In Table 1, the rotation speed is 200 r / min from the beginning of fermentation to 8 h of fermentation, and the rotation speed is 250 r / min from 8 h to 24 h of fermentation, and so on.
[0052] In this embodiment, the fermentation medium is as shown in Table 2:
[0053] Table 2
[0054] Culture medium g / L glucose 20 Peptone 20 Yeast powder 10
[0055] The OD570 and astaxanthin in the fermentation broth were detected. The results are shown in Table 3.
[0056] Table 3
[0057] Time (h) OD570 Potency (mg / L) 0 22 8 45 24 65 160 30 75 38 110 48 131 340 56 132 72 143 605 80 155 96 163 695 104 157 120 149 730 128 159 144 157 790
[0058] Comparative Example 1
[0059] The only difference between this comparative example and Example 2 is that in S2, the rotation speed is constant at 200 r / min.
[0060] The OD570 and astaxanthin in the fermentation broth were detected. The results are shown in Table 4.
[0061] Table 4
[0062] Time (h) OD570 Potency (mg / L) 0 4 0 8 5 24 4 30 4 15 38 4 48 4 56 4 20 72 4 80 4 96 4 25 104 4 120 4 128 4 30 144 4 30
[0063] Comparative Example 2
[0064] The only difference between this comparative example and Example 2 is that in S2, the rotation speed is constant at 300 r / min.
[0065] The OD570 and astaxanthin in the fermentation broth were detected. The results are shown in Table 5.
[0066] Table 5
[0067]
[0068]
[0069] Comparative Example 3
[0070] The only difference between this comparative example and Example 2 is that in S2, the rotation speed is constant at 500 r / min.
[0071] The OD570 and astaxanthin in the fermentation broth were detected. The results are shown in Table 6.
[0072] Table 6
[0073] Time (h) OD570 Potency (mg / L) 0 33 8 57 24 74 150 30 77 38 78 48 82 320 56 88 72 84 485 80 90 96 92 505 104 91 120 95 550 128 101 144 102 620
[0074] Comparative Example 4
[0075] The only difference between this comparative example and Example 2 is that in S2, the rotation speed is constant at 550 r / min.
[0076] The OD570 and astaxanthin in the fermentation broth were detected. The results are shown in Table 7.
[0077] Table 7
[0078]
[0079]
[0080] Example 3
[0081] The difference between this embodiment and embodiment 2 is that, in this embodiment, the fermentation medium is as shown in Table 8:
[0082] Table 8
[0083] Culture medium g / L glucose 20 Peptone 20 Yeast powder 10 Corn Stew Powder 5
[0084] OD570 and astaxanthin in the fermentation broth were detected. The results are shown in Table 9.
[0085] Table 9
[0086] Time (h) OD570 Potency (mg / L) 0 28 8 56 24 68 180 30 85 38 110 48 131 360 56 135 72 147 705 80 155 96 164 805 104 157 120 159 850 128 169 144 187 890
[0087] Comparative Example 5
[0088] The only difference between this comparative example and Example 3 is that in S2, the rotation speed is constant at 300 r / min. OD570 and astaxanthin in the fermentation broth were detected, and the results are shown in Table 10.
[0089] Table 10
[0090]
[0091]
[0092] Comparative Example 6
[0093] The only difference between this comparative example and Example 3 is that in S2, the rotation speed is constant at 500 r / min.
[0094] The OD570 and astaxanthin in the fermentation broth were detected. The results are shown in Table 11.
[0095] Table 11
[0096] Time (h) OD570 Potency (mg / L) 0 53 8 77 24 84 155 30 97 38 98 48 92 340 56 107 72 110 475 80 124 96 135 525 104 144 120 143 570 128 144 144 142 640
[0097] Example 4
[0098] The present embodiment is different from the embodiment 3 in that: in S2, the pH is controlled at about 5 from the start of fermentation to 90h (excluding 90h) of fermentation, and the pH is lowered and controlled at about 4 at 90h or thereafter.
[0099] The OD570 and astaxanthin in the fermentation broth were detected. The results are shown in Table 12.
[0100] Table 12
[0101] Time (h) OD570 Potency (mg / L) 0 28 8 56 24 68 180 30 85 38 110 48 131 360 56 135 72 147 705 80 155 96 164 845 104 167 120 179 970 128 179 144 177 1050
[0102] Example 5
[0103] The present embodiment is different from the embodiment 2 in that: in S2, the pH is controlled at about 5 from the start of fermentation to 90h (excluding 90h) of fermentation, and the pH is lowered and controlled at about 4 at 90h or thereafter.
[0104] OD570 and astaxanthin in the fermentation broth were detected. The results are shown in Table 13.
[0105] Table 13
[0106] Time (h) OD570 Potency (mg / L) 0 22 8 45 24 65 160 30 75 38 110 48 131 340 56 132 72 143 605 80 155 96 163 625 104 157 120 148 720 128 156 144 154 880
[0107] From the comparison of Example 2, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, it can be seen that the astaxanthin content in the fermentation liquid varies with the change of the stirring speed. The stirring conditions of Example 2 are more conducive to the formation of astaxanthin. The amount of astaxanthin in Example 2 is increased by 27-75% compared with Comparative Example 2 and Comparative Example 3. The yield of astaxanthin in Comparative Example 1 and Comparative Example 4 is too low and can be basically ignored. From the comparison of Example 3, Comparative Example 5, and Comparative Example 6 with Example 2, Comparative Example 2, and Comparative Example 3, it can be seen that in the microbial fermentation of astaxanthin, the addition of only 5g / L corn syrup powder increases the amount of astaxanthin by 3%-12%. From the comparison of Example 4 with Example 3, and Example 5 with Example 2, it can be seen that in the microbial fermentation of astaxanthin, lowering the pH value in the late fermentation period is more conducive to the formation of astaxanthin, and lowering the pH value in the late fermentation period increases the amount of astaxanthin by 11%-18% compared with not lowering the pH value.
[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A fermentation method for astaxanthin, characterized in that: The following steps are involved: S1, loading the fermentation medium into the fermentation tank with a loading factor of 60-80%, and after sterilization, inoculating the astaxanthin-producing fermentation bacteria seed culture solution with an inoculation amount of 1-10 v / v%; S2, fermentation to obtain fermentation liquid, the fermentation conditions are: 28-30°C, ventilation volume of 0.5-2.0vvm, pH 4-6, rotation speed as shown in Table 1, fermentation time of 113-168h, dissolved oxygen controlled at 20-30%; In S2, the speed during fermentation is: From the beginning of fermentation to 8h of fermentation, 180-220r / min, excluding 8h of fermentation Fermentation 8h ~ 24h, 230 ~ 270r / min, excluding fermentation 24h Fermentation 24h ~ 30h, 280 ~ 320r / min, excluding fermentation 30h Fermentation 30 to 38 hours, 330 to 370 r / min, excluding fermentation 38 hours Fermentation 38 to 48 hours, 380 to 420 r / min, excluding fermentation 48 hours Fermentation 48h ~ 56h, 480 ~ 520r / min, excluding fermentation 56h From 56h of fermentation to the end of fermentation, the speed is 530-570r / min.
2. The fermentation method of astaxanthin according to claim 1, characterized in that: The fermentation bacteria is one of red Phaffia saccharomyces, marine algae and bacteria.
3. The fermentation method of astaxanthin according to claim 1, characterized in that: The fermentation medium comprises the following components: Glucose 5~30g / L Peptone 5~30g / L Yeast powder 2~15g / L.
4. The fermentation method of astaxanthin according to claim 3, characterized in that: The fermentation medium also includes: 1-8 g / L corn steep liquor dry powder.
5. The astaxanthin fermentation method according to any one of claims 1 to 4, characterized in that: In S2, the pH in the early and middle stages of fermentation is greater than the pH in the late stage of fermentation.
6. The astaxanthin fermentation method according to claim 5, characterized in that: The pH in the initial and middle stages of fermentation minus the pH in the late stage of fermentation = 0.5 to 1.
5.
7. The astaxanthin fermentation method according to claim 6, characterized in that: The pH in the early and middle fermentation stages minus the pH in the late fermentation stages=1.
8. The astaxanthin fermentation method according to claim 5, characterized in that: The start time of the late fermentation period is when the fermentation is carried out for 90 hours.