CN104805026A - Bacterial strain producing beta-galactosidase and method for preparing high-purity galactooligosaccharide - Google Patents
Bacterial strain producing beta-galactosidase and method for preparing high-purity galactooligosaccharide Download PDFInfo
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Abstract
The invention discloses a bacterial strain producing beta-galactosidase and a method for preparing high-purity galactooligosaccharide. The bacterial strain producing beta-galactosidase is pichia pastoris SMD1168H-pGAZaA-lac, and the preservation number is CGMCC No. 8349. The invention also discloses the method for preparing high-purity galactooligosaccharide. The method includes the following steps: using pichia pastoris strain SMD1168H-pGAZaA-lac to prepare beta-galactosidase; using beta-galactosidase to catalyze lactose, so as to synthesize galactooligosaccharide; purifying galactooligosaccharide mixed liquid through Kluyveromyces marxianus; refining to obtain galactooligosaccharide with high purity. The purity of galactooligosaccharide obtained through the method is equal to or greater than 90%, and the yield is equal to or greater than 30%.
Description
Technical field
The present invention relates to a kind of preparation method of oligomeric galactose, specifically utilize the Pichi strain of restructuring Aspergillus candidus beta-galactosidase gene to produce the production method that beta-galactosidase enzymes catalyzes and synthesizes oligomeric galactose, belong to microbial technology field.
Background technology
Oligomeric galactose (Galactooligosaccharides, GOS) there is low sugariness, low in calories, low cariogenic tooth, hot acid are stablized and food safety etc. is special character, be the nutrition source and effective multiplicaiton factor that in human intestinal, the probiotics such as bifidus bacillus, Lactobacillus acidophilus is fabulous.It can improve the digestion and absorption function of human intestinal, and a kind of novel functional food additives of Chang Zuowei, is called as " bifidus factor " that food therapy effect is best.In September, 2008, according to the regulation of " Food Hygiene Law of the People's Republic of China " and " new resource food management method ", approval GOS is new resource food.Nowadays be widely used in the industry-by-industries such as food, healthcare products, milk-product, medicine, beverage, feed, its production application has huge market outlook.
Industrial general employing aspergillus oryzae, Kluyveromyces fragilis etc. produce oligomeric galactose.The concentration that GOS initially generates mostly is about 30%, in order to obtain highly purified GOS, needs to carry out separation and purification to GOS.Its method comprises chromatogram column technique, Nanofiltering membrane, enzyme process and microbe fermentation method.Wherein microbe fermentation method is simple to operate, and cost is lower, and its principle utilizes microorganism to need metabolism monose or lactose to obtain the energy grown, thus assorted sugared composition is removed the object reaching purifying.In prior art, the beta-galactosidase enzymes that patent literature different strain produces catalyzes and synthesizes GOS, and the beta-galactosidase enzymes that the people such as Jiang Bo utilize expense formula shigella to produce prepares oligomeric galactose, productive rate reaches 44.5%; The people such as Hispanic Rodriguez-Colinas B utilize the beta-galactosidase enzymes from Kluyveromyces lactis to catalyze and synthesize oligomeric galactose, freeze-drying after yeast cell Ethanol Treatment, and the maximum yield obtaining oligomeric galactose is 44%.For the monosaccharide component in GOS mixed solution, there are the fermentation consumption glucose wherein such as patent utilization yeast, but semi-lactosi is not well separated with lactose.Meanwhile, China carries out industrialization research to GOS from the nineties in 20th century, but highly purified GOS does not also realize suitability for industrialized production.
Summary of the invention
For overcoming prior art Problems existing, the object of the present invention is to provide a kind of Pichi strain producing beta-galactosidase gene.Invention also provides a kind of apply above-mentioned bacterial strains produce beta-galactosidase enzymes catalysis high density lactose hydrolysis and turn glycosides, synthesis GOS production method.
The bacterial strain of product beta-galactosidase enzymes of the present invention is pichia spp (pichia pastoris) SMD1168H-pGAZaA-lac, this bacterial strain is transformed in pichia spp SMD1168H by genetic engineering means by the beta-galactosidase gene be cloned in Aspergillus candidus thalline, the Pichi strain of the high yield beta-galactosidase enzymes obtained.This bacterial strain is deposited in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 17th, 2013, Institute of Microorganism, Academia Sinica), culture presevation is numbered CGMCC No.8349.
The present invention utilizes above-mentioned bacterial strains to prepare the method for high-purity oligomate, it is characterized in that, (one) utilizes Pichi strain SMD1168H-pGAZaA-lac to prepare beta-galactosidase enzymes; (2) beta-galactosidase enzymes catalysing lactose synthesis of oligonucleotides semi-lactosi; (3) by kluyveromyces marxianus purifying oligomeric galactose mixed solution; (4) refiningly highly purified oligomeric galactose is obtained.
Further, described (one) utilizes Pichi strain SMD1168H-pGAZaA-lac to prepare beta-galactosidase enzymes, specifically comprises the following steps:
(1) Pichi strain SMD1168H-pGAZaA-lac is inoculated in seed culture medium, under 28-30 DEG C of condition, cultivates 12-24h, obtain seed liquor;
(2) inoculum size of the seed liquor of step (1) 5-10% is in mass ratio transferred in fermention medium, at 28-30 DEG C, under pressure 0.1-0.2MPa, dissolved oxygen 0.1%-20% condition, cultivates 48-96h, obtain fermented liquid;
(3) by centrifugal for the fermented liquid of step (2), supernatant liquor is beta-galactosidase enzymes crude enzyme liquid;
(4) the crude enzyme liquid ultrafiltration and concentration of step (3), lyophilize are obtained the powder of beta-galactosidase enzymes.
Wherein, the seed culture based component in described step (1) is as follows: yeast powder 1-10g/L, peptone 1-20g/L, glucose 5-20g/L; Fermentation medium components in described step (2) is as follows: KH
2pO
45-20g/L, CaCl
20.5-0.93g/L, MgSO
41-15g/L, glycerine 5-20g/L, glucose 5-20g/L.
Further, described (two) beta-galactosidase enzymes catalysing lactose synthesis of oligonucleotides semi-lactosi, specifically comprises the following steps:
(1) the beta-galactosidase enzymes powder after freeze-drying being joined concentration is in the lactose solution of 200-450g/L, obtains mixed solution; The add-on of described beta-galactosidase enzymes is every gram of lactose 10-80U;
(2) mixed solution that step (1) is obtained is reacted 20-48h under the condition of 30-45 DEG C, obtain the thick liquid of oligomeric galactose.
Further, described (three) are specially by kluyveromyces marxianus purifying oligomeric galactose mixed solution: refer to cultured kluyveromyces marxianus wet thallus to be linked in the enzyme that the goes out thick liquid of oligomeric galactose alive with the inoculum size of mass ratio 5-10%, cultivate 12-24h for 28-30 DEG C, after removing thalline, supernatant liquor is highly purified oligomeric galactose solution.
Wherein, described kluyveromyces marxianus yeast culture method is specially: be inoculated in by kluyveromyces marxianus in seed culture medium, after 28-30 DEG C of cultivation 12-20h, then transfer in lactose medium with the inoculum size of 10-15%, cultivate 48-96h, under aseptic condition, collect thalline for 28-30 DEG C.Described seed culture medium adopts the substratum that yeast is conventional, as YPD liquid nutrient medium; Described lactose medium composition is, yeast powder 1-10g/L, peptone 1-20g/L, lactose 5-20g/L.
Further, obtain highly purified oligomeric galactose after described (four) refining oligomeric galactose mixed solution, be specially: by high-purity oligomate solution concentrated by rotary evaporation, decolouring, alcohol precipitation, dry, after pulverizing, obtain high-purity oligomate powder.
Wherein, described decolouring refers to decolours to oligomeric galactose solution with under 0.5-2% gac room temperature; Described alcohol precipitation refers to the oligomeric galactose solution after by decolouring, precipitates under high velocity agitation, and carry out second dehydration to the precipitation 1-2 times of industrial spirit obtained with the industrial spirit of 3-5 times of volume; Described oven dry refer to after by alcohol precipitation be deposited in 45-65 DEG C at dry.
Beneficial effect of the present invention:
1, utilize Pichi strain SMD1168H-pGAZaA-lac to produce beta-galactosidase enzymes to catalyze and synthesize GOS, yield reaches more than 30%.By K.marxianus purifying GOS mixed solution, GOS solution after industrial spirit precipitation concentration, obtains the GOS powder that purity is more than 90%, and the production having filled up China high purity GOS is blank, facilitate the application of GOS in food, for society brings huge economic benefit.
2, because GOS itself has high viscosity, common spray-drying process needs to add auxiliary material such as maltodextrin etc. could obtain solid-state GOS.Comparatively speaking, alcohol deposition method technique disclosed by the invention is simple, and GOS purity is higher with yield, provides the production technique of an optimization.
Embodiment
In order to better set forth the proposed by the invention method preparing high-purity oligomate, below by case study on implementation, it is described further.
The structure of embodiment 1 Pichi strain SMD1168H-pGAZaA-lac and preservation
From Aspergillus candidus thalline, extract genome, and then obtain beta-galactosidase gene, electrophoresis is verified; By pcr amplification goal gene fragment, be connected with plasmid vector pGAPZaA after carrying out double digestion, and transformation of E. coli, transform picking mono-clonal successfully, verify after extracting plasmid.Cultivate and transform successful intestinal bacteria, extract plasmid, to go forward side by side line linearity, then the linearization plasmid electricity containing goal gene is transformed in pichia spp SMD1168H, by adding X-gal in the medium, utilize colour developing principle to screen there being the bacterial strain of betagalactosidase activity, the dark bacterium that gets colors carries out postsearch screening; Express saturate recombinant yeast pichia pastoris bacterium, beta-galactosidase enzymes enzyme is measured to its fermented supernatant fluid and lives, choose active high bacterial strain, finally obtain the Pichi strain SMD1168H-pGAZaA-lac of high yield beta-galactosidase enzymes.
This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 17th, 2013 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), culture presevation is numbered CGMCC No.8349.
Embodiment 2
(1) 10L fermentor tank enlarged culturing Pichi strain SMD1168H-pGAZaA-lac prepares beta-galactosidase enzymes: will be stored in the Pichi strain SMD1168H-pGAZaA-lac glycerine pipe access seed culture medium of-80 DEG C, after cultivating 16h under 30 DEG C of conditions, obtain seed liquor; Then be linked in 10L fermentor tank with the inoculum size of mass ratio 10%, regulate pH to 7.0,30 DEG C, cultivate 72h under pressure 0.15Mpa, dissolved oxygen 5-10% condition, obtain fermented liquid; The centrifugal 10min of fermented liquid 10000r/min, collects supernatant and is beta-galactosidase enzymes crude enzyme liquid.Beta-galactosidase enzymes crude enzyme liquid by concentrated 10 times of the ultra-filtration membrane of 10kDa, by concentrated solution lyophilize, obtains the beta-galactosidase enzymes powder of 75000U/g under 4 DEG C of conditions.
Wherein, above-mentioned seed culture based component is as follows: yeast powder 5g/L, peptone 10g/L, glucose 15g/L; Fermentation medium components is as follows: KH
2pO
46g/L, CaCl
20.7g/L, MgSO
42g/L, glycerine 10g/L, glucose 15g/L.
(2) 1L concentration is the beta-galactosidase enzymes powder adding 10U/g lactose in 200g/L lactose solution, and under the condition of 30 DEG C, reaction 20h, obtains the thick liquid of GOS.Detect through TLC and HPLC, its composition is the GOS of a small amount of glucose, semi-lactosi, a large amount of unreacted lactose and generation, and its GOS content is 30%.
(3) K.marxianus glycerine pipe is inoculated in seed culture medium, transfers in lactose medium, cultivate 72h, collect thalline under aseptic condition for 30 DEG C after 30 DEG C of cultivation 16h with the inoculum size of mass ratio 12%; Described lactose medium composition is, yeast powder 8g/L, peptone 10g/L, lactose 10g/L.
(4), after the GOS mixed solution of preparation being diluted 2 times, sterilizing, pH is adjusted to 7.0.The K.marxianus wet thallus of collection is joined in the GOS mixed solution of sterilizing with the addition of mass ratio 5%, cultivates 12h, centrifugal segregation thalline for 30 DEG C.Be detected as the highly purified GOS solution not comprising monose and lactose by TLC and HPLC, purity is 90%.By after the GOS solution concentrated by rotary evaporation that obtains, decolour under adding the gac room temperature of 0.5% 4h, and suction filtration removes gac, obtains water white GOS solution.By it under high velocity agitation by 3 times of volume dehydrated alcohol precipitations, and after utilizing 1 times of volume dehydrated alcohol second dehydration, dry, pulverize and obtain highly purified GOS powder, purity is 90%, and yield is 30%.
Embodiment 3
(1) 10L fermentor tank enlarged culturing Pichi strain SMD1168H-pGAZaA-lac prepares beta-galactosidase enzymes, and fermented supernatant fluid concentrates postlyophilization and obtains beta-galactosidase enzymes pressed powder, method with step in embodiment 2 (1) described in.
(2) with 4L concentration for 350g/L lactose solution is for substrate, add 40U/g lactose beta-galactosidase enzymes powder catalytic generate GOS, temperature of reaction is 37 DEG C, and the reaction times is 36h, obtains the thick liquid of GOS; Detect through TLC and HPLC, its composition is the GOS of a small amount of glucose, semi-lactosi, a large amount of unreacted lactose and generation, and its GOS content is 32%.
(3) 10L fermentor tank enlarged culturing K.marxianus, cultural method is with in embodiment 2 described in step (3);
(4) the GOS mixed solution handled well is squeezed into purifying in yeast fermentation tank, K.marxianus wet thallus inoculum size is 7%, 30 DEG C and cultivates 18h.Concentrated 10 times of the degerming rear supernatant of ceramic membrane filter, decolour under adding the gac room temperature of 1% 2h, and suction filtration removes gac, obtains water white GOS solution.Utilize 3 times of industrial spirit alcohol precipitations under high-speed stirring, after 2 times of industrial spirit second dehydrations, dry, pulverize and obtain highly purified GOS powder, purity is 93%, and yield is 33%.
Embodiment 4
(1) 10L fermentor tank enlarged culturing Pichi strain SMD1168H-pGAZaA-lac prepares beta-galactosidase enzymes, and fermented supernatant fluid concentrates postlyophilization and obtains beta-galactosidase enzymes pressed powder, method with step in embodiment 2 (1) described in.
(2) with 8L concentration for 450g/L lactose solution is for substrate, add 80U/g lactose beta-galactosidase enzymes powder catalytic generate GOS, temperature of reaction is 45 DEG C, and the reaction times is 48h, obtains the thick liquid of GOS; Detect through TLC and HPLC, its composition is the GOS of a small amount of glucose, semi-lactosi, a large amount of unreacted lactose and generation, and its GOS content is 33%;
(3) 10L fermentor tank enlarged culturing K.marxianus, cultural method is with in embodiment 2 described in step (3);
(4) the GOS mixed solution to be purified handled well is squeezed into purifying in yeast fermentation tank, K.marxianus wet thallus inoculum size is 10%, 30 DEG C and cultivates 24h.Concentrated 10 times of the degerming rear supernatant of ceramic membrane filter, decolour under adding the gac room temperature of 2% 4h, and suction filtration removes gac, obtains water white GOS solution.Utilize 5 times of industrial spirit alcohol precipitations under high-speed stirring, after 2 times of industrial spirit second dehydrations, dry, pulverize and obtain highly purified GOS powder, purity is 95%, and yield is 33%.
Be more than embody rule example of the present invention, protection scope of the present invention is not constituted any limitation.The technical scheme that all employing equivalents or equivalence are replaced and formed, all drops within scope of patent protection of the present invention.
Claims (10)
1. produce a beta-galactosidase strain, it is pichia spp (pichia pastoris) SMD1168H-pGAZaA-lac, and the deposit number of described bacterial strain is CGMCC No.8349.
2. utilize the bacterial strain described in claim 1 to prepare a method for high-purity oligomate, it is characterized in that, (one) utilizes Pichi strain SMD1168H-pGAZaA-lac to prepare beta-galactosidase enzymes; (2) beta-galactosidase enzymes catalysing lactose synthesis of oligonucleotides semi-lactosi; (3) by kluyveromyces marxianus purifying oligomeric galactose mixed solution; (4) refiningly highly purified oligomeric galactose is obtained.
3. prepare the method for high-purity oligomate as claimed in claim 2, it is characterized in that, described step (one) specifically comprise the following steps:
(1) Pichi strain SMD1168H-pGAZaA-lac is inoculated in seed culture medium, under 28-30 DEG C of condition, cultivates 12-24h, obtain seed liquor;
(2) inoculum size of the seed liquor of step (1) 5-10% is in mass ratio transferred in fermention medium, at 28-30 DEG C, under pressure 0.1-0.2MPa, dissolved oxygen 0.1-20% condition, cultivates 48-96h, obtain fermented liquid;
(3) by centrifugal for the fermented liquid of step (2), supernatant liquor is beta-galactosidase enzymes crude enzyme liquid;
(4) the crude enzyme liquid ultrafiltration and concentration of step (3), lyophilize are obtained the powder of beta-galactosidase enzymes.
4. prepare the method for high-purity oligomate as claimed in claim 3, it is characterized in that, the seed culture based component in described step (1) is as follows: yeast powder 1-10g/L, peptone 1-20g/L, glucose 5-20g/L; Fermentation medium components in described step (2) is as follows: KH
2pO
45-20g/L, CaCl
20.5-0.93g/L, MgSO
41-15g/L, glycerine 5-20g/L, glucose 5-20g/L.
5. prepare the method for high-purity oligomate as claimed in claim 2, it is characterized in that, described step (two) specifically comprise the following steps:
(1) beta-galactosidase enzymes being joined concentration is in the lactose solution of 200-450g/L, obtains mixed solution; The add-on of described beta-galactosidase enzymes is every gram of lactose 10-80U;
(2) mixed solution that step (1) is obtained is reacted 20-48h under the condition of 30-45 DEG C, obtain the thick liquid of oligomeric galactose.
6. prepare the method for high-purity oligomate as claimed in claim 5, it is characterized in that, described step (three) be specially: be linked in the enzyme that the goes out thick liquid of oligomeric galactose alive by cultured kluyveromyces marxianus wet thallus with the inoculum size of mass ratio 5-10%, cultivate 12-24h for 28-30 DEG C, after removing thalline, supernatant liquor is highly purified oligomeric galactose solution.
7. the described method preparing high-purity oligomate as claimed in claim 6, it is characterized in that, described kluyveromyces marxianus wet thallus cultural method is specially: be inoculated in by kluyveromyces marxianus in seed culture medium, after 28-30 DEG C of cultivation 12-20h, then transfer in lactose medium with the inoculum size of 10-15%, cultivate 48-96h, under aseptic condition, collect thalline for 28-30 DEG C; Described lactose medium composition is, yeast powder 1-10g/L, peptone 1-20g/L, lactose 5-20g/L.
8. as the method preparing high-purity oligomate in claim 5-7 as described in any one, it is characterized in that, described step (four) be specially: by high-purity oligomate solution concentrated by rotary evaporation, decolouring, alcohol precipitation, dries, obtains high-purity oligomate powder after pulverizing.
9. prepare the method for high-purity oligomate as claimed in claim 8, it is characterized in that, described alcohol precipitation refers to the oligomeric galactose solution after by decolouring, precipitates under high velocity agitation, and carry out second dehydration to the precipitation 1-2 times of industrial spirit obtained with the industrial spirit of 3-5 times of volume.
10. prepare the method for high-purity oligomate as claimed in claim 8, it is characterized in that, described decolouring refers to decolours to oligomeric galactose solution with under 0.5-2% gac room temperature; Described oven dry refer to after by alcohol precipitation be deposited in 45-65 DEG C at dry.
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Cited By (7)
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| CN107523595A (en) * | 2017-08-21 | 2017-12-29 | 天津大学 | A kind of environment-friendly preparation method thereof of high-purity oligomate |
| CN107828834A (en) * | 2016-12-23 | 2018-03-23 | 南通励成生物工程有限公司 | A kind of preparation method of galactooligosaccharide |
| CN108138208A (en) * | 2016-01-12 | 2018-06-08 | 维塔鲁斯营养有限公司 | By the method for lactose production galactooligosacchari(es |
| CN108524664A (en) * | 2018-05-31 | 2018-09-14 | 南京中生生物科技有限公司 | It is a kind of that there is the composition and its preparation method and application for adjusting enteron aisle and cardiovascular function |
| CN111334541A (en) * | 2020-02-24 | 2020-06-26 | 天津大学 | Method for preparing high-purity galactooligosaccharide by using β -galactosidase |
| CN112251481A (en) * | 2020-11-13 | 2021-01-22 | 广东省农业科学院蚕业与农产品加工研究所 | Preparation method of galacto-oligosaccharide |
| CN112493489A (en) * | 2020-12-21 | 2021-03-16 | 广东省农业科学院蚕业与农产品加工研究所 | Preparation method of synbiotics |
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| CN108138208A (en) * | 2016-01-12 | 2018-06-08 | 维塔鲁斯营养有限公司 | By the method for lactose production galactooligosacchari(es |
| CN107828834A (en) * | 2016-12-23 | 2018-03-23 | 南通励成生物工程有限公司 | A kind of preparation method of galactooligosaccharide |
| CN107523595A (en) * | 2017-08-21 | 2017-12-29 | 天津大学 | A kind of environment-friendly preparation method thereof of high-purity oligomate |
| CN108524664A (en) * | 2018-05-31 | 2018-09-14 | 南京中生生物科技有限公司 | It is a kind of that there is the composition and its preparation method and application for adjusting enteron aisle and cardiovascular function |
| CN111334541A (en) * | 2020-02-24 | 2020-06-26 | 天津大学 | Method for preparing high-purity galactooligosaccharide by using β -galactosidase |
| CN112251481A (en) * | 2020-11-13 | 2021-01-22 | 广东省农业科学院蚕业与农产品加工研究所 | Preparation method of galacto-oligosaccharide |
| CN112493489A (en) * | 2020-12-21 | 2021-03-16 | 广东省农业科学院蚕业与农产品加工研究所 | Preparation method of synbiotics |
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