CN117487868A - Method for co-producing 2' -fucosyllactose by using D-psicose fermentation supernatant - Google Patents
Method for co-producing 2' -fucosyllactose by using D-psicose fermentation supernatant Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 116
- 230000004151 fermentation Effects 0.000 title claims abstract description 116
- 229940062827 2'-fucosyllactose Drugs 0.000 title claims abstract description 54
- HWHQUWQCBPAQQH-UHFFFAOYSA-N 2-O-alpha-L-Fucosyl-lactose Natural products OC1C(O)C(O)C(C)OC1OC1C(O)C(O)C(CO)OC1OC(C(O)CO)C(O)C(O)C=O HWHQUWQCBPAQQH-UHFFFAOYSA-N 0.000 title claims abstract description 54
- HWHQUWQCBPAQQH-BWRPKUOHSA-N 2-fucosyllactose Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O HWHQUWQCBPAQQH-BWRPKUOHSA-N 0.000 title claims abstract description 54
- SNFSYLYCDAVZGP-UHFFFAOYSA-N UNPD26986 Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(OC(O)C(O)C2O)CO)OC(CO)C(O)C1O SNFSYLYCDAVZGP-UHFFFAOYSA-N 0.000 title claims abstract description 54
- 239000006228 supernatant Substances 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 31
- BJHIKXHVCXFQLS-PUFIMZNGSA-N D-psicose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C(=O)CO BJHIKXHVCXFQLS-PUFIMZNGSA-N 0.000 title claims description 34
- 230000000813 microbial effect Effects 0.000 claims abstract description 19
- 239000001888 Peptone Substances 0.000 claims abstract description 18
- 108010080698 Peptones Proteins 0.000 claims abstract description 18
- 235000019319 peptone Nutrition 0.000 claims abstract description 18
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 17
- 239000000843 powder Substances 0.000 claims abstract description 17
- 239000000463 material Substances 0.000 claims abstract description 8
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 50
- 239000008101 lactose Substances 0.000 claims description 50
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 44
- 239000008103 glucose Substances 0.000 claims description 44
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 34
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 33
- 239000007788 liquid Substances 0.000 claims description 31
- 230000001954 sterilising effect Effects 0.000 claims description 14
- 230000008569 process Effects 0.000 claims description 13
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 12
- 230000001276 controlling effect Effects 0.000 claims description 11
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- 239000001301 oxygen Substances 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
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- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 abstract description 26
- 238000006243 chemical reaction Methods 0.000 abstract description 9
- 239000002609 medium Substances 0.000 abstract description 3
- 239000000049 pigment Substances 0.000 abstract 1
- 150000003839 salts Chemical class 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract 1
- 241000588724 Escherichia coli Species 0.000 description 8
- 238000009630 liquid culture Methods 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 4
- 235000020256 human milk Nutrition 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 210000004251 human milk Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000010865 sewage Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000009423 ventilation Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- LKDRXBCSQODPBY-JDJSBBGDSA-N D-allulose Chemical compound OCC1(O)OC[C@@H](O)[C@@H](O)[C@H]1O LKDRXBCSQODPBY-JDJSBBGDSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013350 formula milk Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000029219 regulation of pH Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y501/00—Racemaces and epimerases (5.1)
- C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
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- General Engineering & Computer Science (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the field of microbial fermentation, in particular to a method for co-producing 2' -fucosyllactose by utilizing D-psinase fermentation supernatant, which comprises the steps of firstly centrifugally collecting the D-psinase fermentation supernatant, decoloring to remove pigments, then adding yeast powder and peptone into the decolored material night for fermentation to prepare the 2' -fucosyllactose, and controlling the addition amount of the yeast powder and the peptone and the fermentation time to improve the conversion rate of the 2' -fucosyllactose, wherein all inorganic salts and 50% of materials of a fermentation medium of the yeast powder and the peptone can be saved by using a culture medium compounded with water.
Description
Technical Field
The invention relates to the technical field of microbial fermentation preparation, in particular to a method for co-producing 2' -fucosyllactose by utilizing D-psicose fermentation supernatant.
Background
2' -fucosyllactose is a human milk oligosaccharide with a probiotic function, is the most abundant human milk oligosaccharide secreted by human breast milk, and plays a very important role in the growth and development process of infants. Research shows that the 2' -fucosyllactose can be used as an effective inhibitor for bacteria or viruses to adhere to epithelial cells, can enhance the immunity of organisms, can be utilized by microorganisms, can improve intestinal flora, and can stimulate the development and repair of cerebral nerves and improve learning and memory capacity. In view of the role in the growth and development of infants, efficient and economical production of 2' -fucosyllactose has become a research hotspot.
Currently, the synthesis of 2' -fucosyllactose has the limitations of microbial fermentation, chemical synthesis and natural extraction, and chemical synthesis, including difficulty in precisely controlling glycosidic bonds, cumbersome activation and (de) protection steps, use of toxic reagents, and the like, are difficult to popularize. Natural extraction method is limited by human milk, and cannot form large-scale production, so that market demands are difficult to meet. The industrial synthesis of 2' -fucosyllactose from the head by using microbial cells is a relatively economical and mature method at present. 2' -fucosyllactose produced by microbial fermentation has been approved in tens of countries such as the united states, and is widely used in infant formulas and dietary supplements in more than 160 countries worldwide, and has received great attention. China is formally evaluated by the safety of the compound as a food nutrition enhancer in 2023 and 10 and 7 days. However, although the method uses substrates such as glucose or glycerol with lower cost, the final yield is relatively low and the production cost is obviously higher than other types of prebiotics products due to the metabolic flux and the loss of intermediate products caused by the longer catalytic step of the head synthesis route.
In order to solve the problem, research on the fermentation process shows that, considering that the fermentation of the epimerase for preparing D-psicose by microbial fermentation is finished, only the thalli are collected for converting fructose to prepare psicose, and the supernatant liquid needs to be discharged. The supernatant contains a large amount of ions brought by pH regulation in the process, a small amount of unutilized protein and other nutrient substances, which are needed by the microbial fermentation to synthesize the 2 '-fucosyllactose, and the 2' -fucosyllactose is synthesized by repeatedly utilizing the supernatant through microbial fermentation, so that the environmental protection pressure brought by the excretion can be relieved, and the addition amount of the 2 '-fucosyllactose, inorganic salt and peptone can be reduced, so that the production cost of the 2' -fucosyllactose synthesized by microbial fermentation can be reduced.
Disclosure of Invention
In order to achieve the above object, the present invention provides a method for co-producing 2' -fucosyllactose by fermenting supernatant with D-psicose.
The invention adopts the following scheme:
a method for co-producing 2' -fucosyllactose by using D-psicose fermentation supernatant, comprising the following steps:
(1) Preparing a fermentation supernatant of the D-psicose epimerase;
(2) Adding activated carbon into the collected supernatant, preserving the temperature at 60-75 ℃ for 30-60min, and filtering by a plate filter press;
(3) Taking filtered supernatant, adding yeast powder and peptone, regulating the pH of the feed liquid to 6.5-7.2, and sterilizing at high temperature in a fermentation tank to prepare a bottom material of 2' -fucosyllactose by microbial fermentation;
(4) Sterilizing glucose and lactose solution at high temperature, and then adding the sterilized glucose and lactose solution into the fermentation bed charge according to a proportion;
(5) Inoculating the cultured seed liquid into a fermentation tank according to a certain inoculation amount, and fermenting and culturing, wherein lactose and glucose are continuously added in the process, so that the concentration of lactose and glucose in the fermentation liquid is maintained at 10-20g/L;
(6) Stopping feeding lactose and glucose when the fermentation is carried out for 70-90h and the content of 2' -fucosyllactose in the fermentation liquor is slowly increased, and maintaining the concentration at 3-5g/L;
(7) When the lactose and glucose content in the fermentation liquid is reduced to below 3g/L, the fermentation culture is ended.
The D-psicose epimerase in step (1) is an intracellular enzyme.
The pH of the 2' -fucosyllactose fermentation medium in the step (3) is controlled by adopting phosphoric acid and ammonia water.
The addition amount of the activated carbon is 0.05-0.15% of the volume of the collected fermentation supernatant.
The addition amount of the yeast powder in the step (3) is 1-1.5% W/V, and the addition amount of peptone is 0.5-0.8% W/V.
The high-temperature sterilization temperature in the step (3) is 117-118 ℃ and the time is 20min.
In the step (4), the concentration of glucose and lactose is 65-80% and 25-30% respectively, and the initial fermentation content is controlled to be 20-30g/L and 10-20g/L respectively.
The high-temperature sterilization temperature of glucose and lactose in the step (4) is 115-118 ℃ and the time is 20min.
The OD of the seed liquid cultured in the step (5) 600 The nm is more than or equal to 3.0, the pH is 6.3-8.0, and the inoculation amount is 8-10%.
In the step (5), the pH value in the fermentation culture process is controlled to be 6.5-7.5, the dissolved oxygen is controlled to be 10-30%, and the dissolved oxygen is regulated by the rotating speed, the tank pressure and the air quantity.
The invention has the following beneficial effects:
(1) The environmental protection treatment of the D-psicose epimerase fermentation supernatant is realized, the conductance of the fermentation supernatant of the D-psicose epimerase prepared by microbial fermentation is up to 11-15ms/cm, and if the fermentation supernatant is directly discharged to a sewer for sewage treatment, the sewage treatment cost is too high;
(2) The invention prepares 2' -fucosyllactose by repeatedly using the compound as a part of the ingredients of 2' -fucosyllactose by microbial fermentation, which not only solves the environmental protection pressure caused by the outward discharge of the D-psicose epimerase fermentation supernatant, but also further reduces the raw material cost of 2' -fucosyllactose by microbial synthesis, wherein phosphate can be completely saved, and when yeast powder and peptone are saved by 50% of the original process, the fermentation level of the original process can be reached, and better economic benefit is obtained while the sewage treatment pressure is reduced;
(3) The conversion rate of the 2' -fucosyllactose is increased.
Detailed Description
The present invention will be described in detail with reference to specific examples. The working principle of the invention is as follows: the fermentation supernatant of the D-psicose epimeric intracellular enzyme is prepared by microbial fermentation for dosing, so that the addition amount of inorganic salt, yeast powder and peptone in the fermentation dosing is reduced, and the fermentation cost of preparing 2' -fucosyl lactose by microbial fermentation is reduced.
Example 1: e.coli K-12MG1655BLBYZT6 is inoculated into a seed liquid culture medium, and cultured for 8 hours at 37 ℃ and 200rmp to obtain seed liquid; inoculating 8% seed solution into fermentation culture medium, wherein the fermentation culture medium comprises D-psicose epimerase fermentation supernatant, decolorizing and sterilizing the D-psicose epimerase fermentation supernatant, adding 20g/L sterile glucose, 10g/L sterile lactose, shaking flask at 30+ -1deg.C and 220rpm for fermentation culture for 60h, and ending fermentation culture when lactose and glucose content in fermentation liquid is reduced to below 3g/L, wherein the fermentation end feed liquid has pH of 5.68, lactose content of 8.3g/L, glucose content of 10.5g/L and bacterial density OD 600 18, 2' -fucosyllactose 1.35g/L.
The K-12MG1655BLBYZT6 is stored in China Committee for culture Collection of microorganisms in 2023, 8 and 31 days, and the preservation number is CGMCC NO28317.
Example 2: e.coli K-12MG1655BLBYZT6 is inoculated into a seed liquid culture medium, and cultured for 5-10h at 37 ℃ and 200rmp to obtain seed liquid; inoculating the collected seed solution into a fermentation medium with the composition of D-psicose in an inoculation amount of 8 percentAdding 15g/L yeast powder and 7g/L peptone into the decolorized feed liquid of the D-psicose epimerase fermentation supernatant, sterilizing the feed liquid separately, adding glucose and lactose according to the addition amount of 20g/L and 11g/L respectively, shaking and fermenting at 30+/-1 ℃ and 200rpm for 60 hours, ending the fermentation culture when the lactose and glucose content in the fermentation liquid is reduced to below 3g/L, wherein the fermentation end feed liquid is pH7.27, the lactose content is 0.42g/L, the glucose content is 0g/L and the bacterial density OD 600 35, 11.21g/L of 2 '-fucosyllactose, and 106% of conversion rate of 2' -fucosyllactose to lactose.
Example 3: e.coli K-12MG1655BLBYZT6 is inoculated into a seed liquid culture medium, and cultured for 5-10h at 37 ℃ and 200rmp to obtain seed liquid; inoculating 8% of seed solution into a fermentation culture medium, wherein the fermentation culture medium comprises D-psicose epimerase fermentation supernatant, adding 10g/L of yeast powder and 5g/L of peptone into the decolorized material liquid of the D-psicose epimerase fermentation supernatant, independently sterilizing glucose and lactose, adding the glucose and lactose according to the addition amount of 20g/L and 11g/L, after-seeding pH7.15, the temperature of 30+/-1 ℃ and the pressure of 200rpm and the tank pressure of 0.05, starting fermentation culture at a ventilation ratio of 1:0.5, continuously adding lactose and glucose in the process, controlling the content of the lactose and glucose in the fermentation liquid to be 10-20g/L, controlling the pH value of the 2 '-fucosyllactose fermentation culture medium prepared by microbial fermentation to be 6.5-7.5 through phosphoric acid and ammonia water, stopping feeding lactose after 80h of fermentation culture, controlling the glucose content to be 3-5g/L, placing the lactose in a 115h tank, and controlling the conversion rate of 2' -fucosyllactose to lactose to be 71.79g/L and 110% of lactose.
Example 4: e.coli K-12MG1655BLBYZT6 is inoculated into a seed liquid culture medium, and cultured for 5-10h at 37 ℃ and 200rmp to obtain seed liquid; the seed solution is inoculated into a fermentation culture medium with an inoculum size of 8%, the fermentation culture medium is composed of D-psicose epimerase fermentation supernatant, 15g/L yeast powder and 5g/L peptone are added into the decolorized feed liquid of the D-psicose epimerase fermentation supernatant, glucose and lactose are independently sterilized and then added according to the addition amount of 20g/L and 11g/L, the fermentation culture is started by the aeration ratio of 1:0.5 at the temperature of 30+/-1 ℃ and the pressure of 200rpm, lactose and glucose are continuously added in the process, the content of the 2 '-fucosyllactose fermentation culture medium prepared by microbial fermentation is controlled between 10-20g/L, the pH value of the 2' -fucosyllactose fermentation culture medium is controlled between 6.5-7.5 by phosphoric acid and ammonia water, after 80h of fermentation culture, the glucose content is controlled to 3-5g/L, the lactose is placed in a pot for 113h, and the conversion rate of the 2 '-fucosyllactose is 68.33g/L, and the 2' -fucosyllactose is 102%.
Example 5: e.coli K-12MG1655BLBYZT6 is inoculated into a seed liquid culture medium, and cultured for 5-10h at 37 ℃ and 200rmp to obtain seed liquid; inoculating 8% of seed solution into a fermentation culture medium, inoculating the fermentation culture medium into a D-psicose epimerase fermentation supernatant, decoloring the D-psicose epimerase fermentation supernatant, adding 24g/L of yeast powder and 12g/L of peptone into the material liquid, independently sterilizing glucose and lactose, adding the glucose and lactose according to the addition amounts of 24g/L and 12g/L, fermenting and culturing at the temperature of 30+/-1 ℃ and at the speed of 200rpm and the pressure of 0.05 in a ventilation ratio of 1:0.5, continuously adding lactose and glucose in the process to control the content of the lactose and glucose in the fermentation liquid to be 10-20g/L, stopping adding lactose in the process, controlling the content of glucose to be 3-5g/L, placing the glucose and lactose in a tank for 113h, and controlling the conversion rate of 2' -fucosyllactose to lactose to be 112 percent.
Comparative example 1
E.coli K-12MG1655BLBYZT6 is inoculated into a seed liquid culture medium, and cultured for 5-10h at 37 ℃ and 200rmp to obtain seed liquid; inoculating 8% of seed liquid into a fermentation culture medium, inoculating 25g/L of yeast powder, 15g/L of peptone, 12.5g/L of dipotassium hydrogen phosphate and 2.4g/L of monopotassium hydrogen phosphate to the fermentation culture medium, independently sterilizing, adding 22g/L of sterile glucose and 15g/L of sterile lactose, fermenting and culturing at 30+/-1 ℃ at 200rpm and a pot pressure of 0.05, continuously adding lactose and glucose in the fermentation culture medium, controlling the content of lactose and glucose in the fermentation culture medium to be 10-20g/L, controlling the pH of the fermentation culture medium to be 6.5-7.5 through phosphoric acid and ammonia water, stopping adding lactose after 86h of fermentation culture, controlling the content of glucose to be 3-5g/L, and placing 69.93g/L of 2 '-fucosyllactose in a pot for 116h, wherein the conversion rate of 2' -fucosyllactose to lactose is 107.5%.
Comparative example 2
E.coli K-12MG1655BLBYZT6 is inoculated into a seed liquid culture medium, and cultured for 5-10h at 37 ℃ and 200rmp to obtain seed liquid; inoculating 8% of seed solution into a fermentation culture medium, fermenting and culturing to obtain a substrate material, wherein the substrate material comprises 12.5g/L of dipotassium hydrogen phosphate and 2.4g/L of monopotassium phosphate, independently sterilizing, adding 22g/L of sterile glucose and 15g/L of sterile lactose, fermenting and culturing at the temperature of 30+/-1 ℃ at 200rpm and the tank pressure of 0.05 after the seed solution is inoculated, wherein the ventilation ratio is 1:0.5, the pH of the process is controlled between 6.5-7.5 through phosphoric acid and ammonia water, the glucose content is 3.5g/L, the lactose 5g/L and the conversion rate of 2' -fucosyllactose to lactose is 14.5%, and the embodiment does not contain yeast powder and peptone.
According to the embodiment and the comparative example, yeast powder and peptone with different contents are respectively added as culture media to be connected with a good K-12MG1655BLBYZT6 escherichia coli seed liquid, a certain amount of lactose and glucose are added for fermentation, shake flask control verification and fermentation tank amplification verification are carried out, and the feasibility of co-producing 2 '-fucosyllactose by utilizing alonase supernatant is verified, wherein the yeast powder, the peptone, the addition amount and the fermentation time can influence the conversion rate of the 2' -fucosyllactose.
Table 1 shake flask control results:
。
Claims (10)
1. a method for co-producing 2' -fucosyllactose by using D-psicose fermentation supernatant, comprising the steps of:
(1) Preparing a fermentation supernatant of the D-psicose epimerase;
(2) Adding activated carbon into the collected supernatant, preserving the temperature at 60-75 ℃ for 30-60min, and filtering by a plate filter press;
(3) Taking filtered supernatant, adding yeast powder and peptone, regulating the pH of the feed liquid to 6.5-7.2, and sterilizing at high temperature in a fermentation tank to prepare a bottom material of 2' -fucosyllactose by microbial fermentation;
(4) Sterilizing glucose and lactose solution at high temperature, and then adding the sterilized glucose and lactose solution into the fermentation bed charge according to a proportion;
(5) Inoculating the cultured seed liquid into a fermentation tank according to a certain inoculation amount, and fermenting and culturing, wherein lactose and glucose are continuously added in the process, so that the concentration of lactose and glucose in the fermentation liquid is maintained at 10-20g/L;
(6) Stopping feeding lactose and glucose when the fermentation is carried out for 70-90h and the content of 2' -fucosyllactose in the fermentation liquor is slowly increased, and maintaining the concentration at 3-5g/L;
(7) When the lactose and glucose content in the fermentation liquid is reduced to below 3g/L, the fermentation culture is ended.
2. A method for co-producing 2' -fucosyllactose using D-psicose fermentation supernatant according to claim 1, characterized in that: the D-psicose epimerase in the step (1) is an intracellular enzyme.
3. A method for co-producing 2' -fucosyllactose using D-psicose fermentation supernatant according to claim 1, characterized in that: and (3) controlling the pH of the 2' -fucosyllactose fermentation medium prepared by microbial fermentation in the step (3) by adopting phosphoric acid and ammonia water.
4. A method for co-producing 2' -fucosyllactose using D-psicose fermentation supernatant according to claim 1, characterized in that: the addition amount of the activated carbon is 0.05-0.15% of the volume of the collected fermentation supernatant.
5. A method for co-producing 2' -fucosyllactose using D-psicose fermentation supernatant according to claim 1, characterized in that: the addition amount of the yeast powder in the step (3) is 1-1.5% W/V, and the addition amount of peptone is 0.5-0.8% W/V.
6. A method for co-producing 2' -fucosyllactose using D-psicose fermentation supernatant according to claim 1, characterized in that: and (3) sterilizing at 117-118 ℃ for 20min.
7. A method for co-producing 2' -fucosyllactose using D-psicose fermentation supernatant according to claim 1, characterized in that: the concentration of glucose and lactose in the step (4) is 65-80% and 25-30%, respectively, and the initial fermentation content is controlled to be 20-30g/L and 10-20g/L, respectively.
8. A method for co-producing 2' -fucosyllactose using D-psicose fermentation supernatant according to claim 1, characterized in that: the high-temperature sterilization temperature of glucose and lactose in the step (4) is 115-118 ℃ and the time is 20min.
9. A method for co-producing 2' -fucosyllactose using D-psicose fermentation supernatant according to claim 1, characterized in that: the OD of the seed liquid cultured in the step (5) 600 The nm is more than or equal to 3.0, the pH is 6.3-8.0, and the inoculation amount is 8-10%.
10. A method for co-producing 2' -fucosyllactose using D-psicose fermentation supernatant according to claim 1, characterized in that: in the step (5), the pH value in the fermentation culture process is controlled to be 6.5-7.5, the dissolved oxygen is controlled to be 10-30%, and the dissolved oxygen is regulated by the rotating speed, the tank pressure and the air quantity.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1331344A (en) * | 2000-06-22 | 2002-01-16 | 上海化工研究院 | Process for preparing natural beta-carotin by cyclic utilization of fermented waste liquid |
| CN102660519A (en) * | 2012-05-02 | 2012-09-12 | 东华大学 | Method for preparing biological enzyme by utilizing fermentation waste liquid |
| CN106434494A (en) * | 2016-12-02 | 2017-02-22 | 山东百龙创园生物科技股份有限公司 | Bacillus subtilis, as well as culture method and application thereof |
| CN115725484A (en) * | 2022-08-12 | 2023-03-03 | 武汉茵智生物技术有限公司 | Enzyme mutation expression engineering bacterium for synthesizing D-psicose and application thereof |
| CN117089503A (en) * | 2023-10-17 | 2023-11-21 | 保龄宝生物股份有限公司 | Escherichia coli K-12 MG1655 BLBYZT6 and application thereof |
-
2023
- 2023-12-28 CN CN202311826648.XA patent/CN117487868A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1331344A (en) * | 2000-06-22 | 2002-01-16 | 上海化工研究院 | Process for preparing natural beta-carotin by cyclic utilization of fermented waste liquid |
| CN102660519A (en) * | 2012-05-02 | 2012-09-12 | 东华大学 | Method for preparing biological enzyme by utilizing fermentation waste liquid |
| CN106434494A (en) * | 2016-12-02 | 2017-02-22 | 山东百龙创园生物科技股份有限公司 | Bacillus subtilis, as well as culture method and application thereof |
| CN115725484A (en) * | 2022-08-12 | 2023-03-03 | 武汉茵智生物技术有限公司 | Enzyme mutation expression engineering bacterium for synthesizing D-psicose and application thereof |
| CN117089503A (en) * | 2023-10-17 | 2023-11-21 | 保龄宝生物股份有限公司 | Escherichia coli K-12 MG1655 BLBYZT6 and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| P.S.R. BABU等: "Effect of Recycling of Fermentation Broth for the Production of Penicillin Amidase", PROCESS BIOCHEMISTRY, vol. 26, 31 December 1991 (1991-12-31), pages 7 - 14 * |
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