CN101691538A - Aspergillus oryzae and method for preparing high purity galacto-oligosaccharides by using same - Google Patents
Aspergillus oryzae and method for preparing high purity galacto-oligosaccharides by using same Download PDFInfo
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- CN101691538A CN101691538A CN200910018452A CN200910018452A CN101691538A CN 101691538 A CN101691538 A CN 101691538A CN 200910018452 A CN200910018452 A CN 200910018452A CN 200910018452 A CN200910018452 A CN 200910018452A CN 101691538 A CN101691538 A CN 101691538A
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- oligomeric galactose
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Abstract
The invention relates to a method for preparing high purity galacto-oligosaccharides, comprising the following product separation and purification steps: Aspergillus oryzae fermentation, ceramic membrane ultrafiltration, nanofiltration separation and the like. In the method, the Aspergillus oryzae separated from the soil is adopted as an original strain and the Aspergillus oryzae BLB-21 (with preservation number of CGMCC No.2951), a high efficiency transformed strain obtained through mutation screening in the laboratory, is directly used to ferment high concentration lactose solution, thus avoiding the steps of enzyme purification and the like in the process of preparing the galacto-oligosaccharides by an enzyme method and saving time and labor. The high purity galacto-oligosaccharides are obtained by ultrafiltration and nanofiltration separation, the process is ideal, the conditions are mild and the method has extensive industrial production prospect.
Description
Technical field
The invention belongs to the functional oligose production field, relate generally to the production method that aspergillus oryzae bacterial classification direct fermentation high density lactose solution carries out oligomeric galactose.
Background technology
Oligomeric galactose (Galacto-oligosaccharides, be called for short GOS) belong to non-digestible oligosaccharide, with other non-digestible oligosaccharide compare have specificity promote bifidus bacillus increment in the enteron aisle, produce extensive, safe to use with raw material sources, to advantages such as food-processing thermal treatment are stable.Therefore, produce and use oligomeric galactose and have great potential in field of food industry.The producer of worldwide production oligomeric galactose is mainly in Japan at present, and China is in conceptual phase to the also not mass-producing of production of oligomeric galactose.Oligomeric galactose is to act on lactose by beta-galactosidase enzymes, through hydrolysis and change the glycosides reaction and get.The microorganism that produces beta-galactosidase enzymes has saccharomyces fragilis, lactose yeast, Bacillus circulans and aspergillus oryzae etc.In addition, but also find also synthesis of oligonucleotides semi-lactosi of penicillium funiculosum cellulase.Industrial with production oligomeric galactoses such as aspergillus, Kluyveromyces fragilis (Kluyveromyces fragilis), Kluyveromyces lactis (Kluyveromyces lactis).Resolvase water synthesis of oligonucleotides semi-lactosi commonly used in the industrial production, production technique is ripe relatively, but stability is poor slightly in producing.Current also have the nonaqueous phase of comprising enzyme reaction and enzyme immobilization technology to produce oligomeric galactose.Yet, though the nonaqueous phase enzyme reaction makes the anti-transgalactosidation that is tending towards by control moisture, and increases to some extent than oligomeric galactose in the aqueous solution,, product do not generate because obtaining separating the continuation that makes product can suppress oligomeric galactose, product yield increase rate is little, is difficult to large-scale production; Enzyme immobilization technology has considerable influence to the oligomeric galactose productive rate, but that the raising that has at the manufacturer production result has is opposite, so this technology is also further among the research.The research of fermentative Production oligomeric galactose is less.The research of fermentative Production oligomeric galactose is less, but fermentation method is removed the fermentation of sugar components such as glucose, lactose by the specific performance that utilizes the preferential metabolizable glucose of certain micro-organisms cell, semi-lactosi or lactose, save the steps such as separation and purification of enzyme simultaneously, technology is simple, the equipment input cost is lower.
In the prior art, have to disclose the method that easy quick preparation contains the low-lactose whey powder of oligomeric galactose in the patent documentation.This patent has prepared the beta-galactosidase enzymes crude enzyme liquid and has handled whey powder, has obtained high-load oligomeric galactose in the lactose in reducing whey powder.But the separation for other components such as monose, lactose etc. in the high-content oligomeric galactose solution is also not mentioned, realize industrial production high-purity oligomate product, also needs the high-content oligomeric galactose is carried out further separation and purification treatment.
Utilize fixed yeast to remove the method for monosaccharide component in the oligomeric galactose in the patent documentation, disclose a kind of method of utilizing fixed yeast to remove monosaccharide component in the oligomeric galactose.This patent is that applying immobilized yeast is removed the monose in the oligomeric galactose solution, has removed glucose, thereby improves oligomeric galactose content.This patent has been removed the part monosaccharide component preferably, but semi-lactosi but can't remove, simultaneously disaccharide component lactose is not well separated.Production by Enzymes oligomeric galactose general technology schema is seen Fig. 1.As can be seen, enzyme process reaction is produced in the process of oligomeric galactose, need carry out the separation and purification of enzyme, so the yield of enzyme is the key that influences this technology, and the product separation purification process directly has influence on the height of quality product.
Summary of the invention
The invention provides a kind of aspergillus oryzae bacterial classification (Aspeigillus oryzae) BLB-21, in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, the address is a Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, on March 17 2009 preservation time, protect minus sign CGMCC No.2951.
The present invention provides a kind of application aspergillus oryzae bacterial classification (Aspeigillus oryzae) BLB-21 simultaneously, and the direct fermentation lactose solution prepares the method for oligomeric galactose.The enlarged culturing that comprises aspergillus oryzae bacterial classification (Aspeigillus oryzae) BLB-21, the lactose solution that uses enlarged culturing seed direct fermentation high density with separate purification, processing and preparing high-purity oligomate.
The technical solution used in the present invention comprises the steps:
(1) strain separating and mutagenesis:
1. strain separating: aspergillus oryzae BLB-12 separates from diary farm, Yucheng City soil, and separating used substratum is rose bengal medium.
The separation method of aspergillus oryzae BLB-12 is: take by weighing 10g soil, add in the 90mL sterilized water, concussion shakes up, and moves into Bechtop after the alcohol wipe.Prepare 6 test tubes that the 9mL sterilized water is housed, numbering 1-6.Pipette 1000 μ L suspension liquids with liquid-transfering gun and place test tube No. 1, pipette No. 1 test tube suspension liquid of 1000 μ L again and place test tube No. 2, take turns doing six test tube gradient dilutions.Get 4,5, No. 6 each 200 μ L (each test tube is got 2-4 part) of test tube solution respectively with liquid-transfering gun and place the rose bengal medium flat board, evenly smear with paint daubs.Be cultured to flat board and grow ripe bacterium colony.Identify and separation and purification aspergillus oryzae bacterium colony that with single colony inoculation inclined-plane, and inoculation is shaken bottle, primary dcreening operation mensuration enzyme activity.Select good inclined-plane starting strain-aspergillus oryzae BLB-12 according to shake-flask culture enzyme activity size.
The separation and Culture condition is: rose bengal medium: peptone 5g, glucose 10g, potassium primary phosphate 1g, sal epsom 0.5g, agar 20g, 1/3000 rose-bengal solution 100mL, distilled water 1000mL, paraxin 0.1g, pH nature, 28～30 ℃ of culture temperature.
The slant culture condition is: 5
0Be ' wort agar substratum: malt extract 3g, glucose 10g, yeast extract 3g, peptone 5g, agar 20g, distilled water 1000mL; The pH nature, 28～30 ℃ of culture temperature.
The shake-flask culture condition is: glucose 300-500g/L, yeast extract paste 15g/L, agar powder 15g/L, pH nature (about 5.5), potassium primary phosphate 1g/L, sal epsom 0.5g/L, urea 5g/L.
2. induction mutation of bacterium: aspergillus oryzae BLB-21 is the good superior strain of production through mutagenic treatment starting strain BLB-12 screening gained.Adopt ultraviolet ray+LiCl complex mutation to handle bacterial strain.Concrete steps:
Transfering loop picking aspergillus oryzae BLB-12 slant pore places the 1h that vibrates on the magnetic stirring apparatus in the aseptic triangular flask that contains 2%LiCl solution, thalline is scattered, spore germination.Spore suspension is poured into the aseptic plate that contains aseptic pin.Open the plate lid, the irradiation of 24cm place 0s, 20s, 40s, 60s, 80s, 100s, 120s under the ultraviolet lamp of the good 20W of preheating in advance, different irradiation dose bacterium liquid are diluted to 10
-1, 1h is placed at dark place.Dilute bacterium liquid to 10 respectively with physiological saline
-3, draw the good bacterium liquid 0.1mL coating 5 of dilution
0Be ' wort agar culture medium flat plate, 3 flat boards of each extent of dilution coating.The plate that coats is wrapped with black cloth, put 28 ℃ of biochemical incubators and cultivate about 5d, to growing ripe bacterium colony, the statistics lethality rate.
(2) bacterial screening: with aspergillus oryzae strain BLB-12 is contrast, bacterial strain after the mutagenesis is carried out fermentation culture produce enzyme and change the glycosidase activity detection.
Produce enzyme substratum and culture condition: lactose 10g/L, peptone 5g/L, yeast powder 5g/L, CaCl
20.11g/L, MnSO
40.001g/L, MgSO
47H
2O 0.3g/L, KH
2PO
40.05g/L, FeSO
47H
2O0.03g/L, pH6.0,30 ℃, 180rmp cultivates 50h.The mensuration enzyme is lived.
Changeing glycosidase activity detects: with crude enzyme liquid and 1: 3 (v/v) mixed of lactose solution, change glycosyl reaction 4～10h in 40～60 ℃.Lactose solution concentration is 30% (w/v).Reaction finishes, and the centrifugal 5min of 12000rpm, supernatant liquor carry out thin-layer chromatography (TLC) analysis, to screen active high production bacterial strain.The thin-layer chromatography developping agent is a propyl carbinol: ethanol: water=5: 3: 2, developer is 3 of 20% sulphuric acid soln and 0.5%, 5-orcin mixed solution, filter out the high production bacterial strain of commentaries on classics glycosidase activity, i.e. aspergillus oryzae bacterial classification (Aspeigillus oryzae) BLB-21 by spot colors and size.
(3) enlarged culturing of aspergillus oryzae bacterial classification (Aspeigillus oryzae) BLB-21: first order seed, secondary seed.
Seed culture medium: glucose 300-500g/L, yeast extract paste 15g/L, agar powder 15g/L, pH nature (about 5.5), potassium primary phosphate 1g/L, sal epsom 0.5g/L, urea 5g/L;
Transfering loop picking one ring is cultivated sophisticated slant pore and is carried out the one-level cultivation in the shake-flask seed of 500mL, and behind the cultivation 24h, the amount with 10% (V/V) inserts the 15L fermentor tank again, carries out secondary seed and cultivates.
(4) ferment lactose solution, the preparation oligomeric galactose.
Enlarged culturing gained secondary seed is linked in the lactose solution (20%-40%) with the amount of 10% (V/V), and oligomeric galactose is produced in direct fermentation.Fermentor tank volume 150L, liquid amount are 60%, regulate pH5.5,20～40 ℃ of temperature, mixing speed 250rpm, air flow 200L/h, fermentation 20-40h; Make the fermented liquid that contains oligomeric galactose.
(5) separate purification and processing
1. ceramic membrane ultrafitration is adopted in ultrafiltration, removes macromolecular substance such as enzyme, protein and nucleic acid in thalline and the fermented liquid, tentatively obtains the sugar component mixed solution;
2. the sugar soln of activated carbon decolorizing after to ultrafiltration adds gac by 2%, 80 ℃ of reaction 60min, decolorization filtering;
3. highly acidic cation-weakly-basic anion-storng-acid cation exchange resin is adopted in ion-exchange, and temperature 30-50 ℃, sample size 10L/h, resin height 1m separates desalination, detects through HPLC and can get oligomeric galactose content.This moment, sugar component was glucose, semi-lactosi, lactose, oligomeric galactose;
4. to adopt organic composite nanometer filtering film to separate concentrated for nanofiltration separation, operational condition is: 2.0-3.0MPa pressure, input concentration 20-30%, add water 3-5BV, temperature 20-35 ℃,, oligose such as monose, lactose, gala trisaccharide can be separated by selecting organic composite nanometer filtering film, improve product purity greatly, oligomeric galactose purity can reach more than 80%.
5. evaporation concentration is imitated the falling film type evaporation thickener through the product of nanofiltration separation by six and is got the high-purity oligomate slurry;
6. spraying drying adopts the dry high-purity oligomate solid that gets of press spray drying tower.
Fermentative Production oligomeric galactose technology of the present invention compared with prior art has following advantage:
(1) adopt aspergillus oryzae direct fermentation, saved the process of preparation purifying beta-galactosidase enzymes, technology is simple, is easy to suitability for industrialized production, output height, the cycle is short, efficient is high;
(2) ceramic membrane ultrafitration is removed the more traditional press filtration height of thalline efficient;
(3) adopt organic composite nanometer filter membrane sepn, well realized the separation fully of monose, lactose in the oligomeric galactose solution, product purity can reach 80%.
Description of drawings
Fig. 1 is a Production by Enzymes oligomeric galactose general technology schema;
Fig. 2 is that high-purity oligomate prepares technological process of production figure;
Embodiment
In order better to set forth the preparation method of high-purity oligomate proposed by the invention, the invention will be further described below by embodiment.
Embodiment 1
Bacterial strain BLB-21 access glucose concn is that the seed liquor of 300g/L is carried out enlarged culturing.Secondary seed solution (concentration: mycelia weight in wet base 0.52%) be linked in 20% lactose solution with the amount of 10% (V/V) and ferment, regulate pH5.5,25～30 ℃ of fermentation 40h.Tentatively obtain the sugar component mixed solution through ultrafiltration, adopt highly acidic cation-weakly-basic anion-storng-acid cation exchange resin behind the activated carbon decolorizing, 30～35 ℃ of temperature, sample size 10L/h, resin height 1m separates desalination, detects to such an extent that oligomeric galactose content is 55.80% sugar soln through HPLC.This moment, sugar component was glucose, semi-lactosi, lactose, oligomeric galactose.
Above sugar soln is at 2.0～2.4MPa pressure, and input concentration 20～25% adds water 3BV, adopts cellulose acetate nano filter-membrane to separate under 20～25 ℃ of conditions of temperature.Effectively separated lactose, semi-lactosi and glucose component, product oligomeric galactose purity reaches 81.67%.
Table 1 is used cellulose acetate nano filter-membrane separating oligomeric semi-lactosi
Embodiment 2
Bacterial strain BLB-21 access glucose concn is that the seed liquor of 400g/L is carried out enlarged culturing.Secondary seed solution (concentration: mycelia weight in wet base 0.45%) be linked in 30% lactose solution with the amount of 10% (V/V) and ferment, regulate pH5.5,30～35 ℃ of fermentation 30h.Tentatively obtain the sugar component mixed solution through ultrafiltration, adopt highly acidic cation-weakly-basic anion-storng-acid cation exchange resin behind the activated carbon decolorizing, 35～40 ℃ of temperature, sample size 10L/h, resin height 1m separates desalination, detects to such an extent that oligomeric galactose content is 56.35% sugar soln through HPLC.This moment, sugar component was glucose, semi-lactosi, lactose, oligomeric galactose.
Above sugar soln is at 2.4～2.7MPa pressure, and input concentration 20～25% adds water 4BV, uses cellulose acetate nano filter-membrane to separate under 25～30 ℃ of conditions of temperature.Lactose, semi-lactosi are realized effectively separating with glucose, and product oligomeric galactose purity reaches 82.91%.
Table 2 is used cellulose acetate nano filter-membrane separating oligomeric semi-lactosi
Embodiment 3
Bacterial strain BLB-21 access glucose concn is that the seed liquor of 500g/L is carried out enlarged culturing.Secondary seed solution (concentration: mycelia weight in wet base 0.56%) be linked in 40% lactose solution with the amount of 10% (V/V) and ferment, regulate pH5.5,35～40 ℃ of fermentation 20h.Tentatively obtain the sugar component mixed solution through ultrafiltration, adopt highly acidic cation-weakly-basic anion-storng-acid cation exchange resin behind the activated carbon decolorizing, 45～50 ℃ of temperature, sample size 10L/h, resin height 1m separates desalination, detects to such an extent that oligomeric galactose content is 55.72% sugar soln through HPLC.This moment, sugar component was glucose, semi-lactosi, lactose, oligomeric galactose.
Above sugar soln is at 2.7～3.0MPa pressure, and input concentration 25～30% adds water 5BV, uses cellulose acetate nano filter-membrane to separate under 30～35 ℃ of conditions of temperature.Lactose, semi-lactosi are realized effectively separating with glucose, and product oligomeric galactose purity reaches 80.38%.
Table 3 is used cellulose acetate nano filter-membrane separating oligomeric semi-lactosi
Embodiment 3
Bacterial strain BLB-21 access glucose concn is that the seed liquor of 500g/L is carried out enlarged culturing.Secondary seed solution (concentration: mycelia weight in wet base 0.56%) be linked in 40% lactose solution with the amount of 10% (V/V) and ferment, regulate pH5.5,35～40 ℃ of fermentation 20h.Tentatively obtain the sugar component mixed solution through ultrafiltration, adopt highly acidic cation-weakly-basic anion-storng-acid cation exchange resin behind the activated carbon decolorizing, 45～50 ℃ of temperature, sample size 10L/h, resin height 1m separates desalination, detects to such an extent that oligomeric galactose content is 55.72% sugar soln through HPLC.This moment, sugar component was glucose, semi-lactosi, lactose, oligomeric galactose.
Above sugar soln is at 2.7～3.0MPa pressure, and input concentration 25～30% adds water 5BV, uses cellulose acetate nano filter-membrane to separate under 30～35 ℃ of conditions of temperature.Lactose, semi-lactosi are realized effectively separating with glucose, and product oligomeric galactose purity reaches 80.38%.
Table 3 is used cellulose acetate nano filter-membrane separating oligomeric semi-lactosi
Claims (10)
1. an aspergillus oryzae bacterial classification (Aspeigillus oryzae) BLB-21 was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number CGMCC No.2951 on March 17th, 2009.
2. application aspergillus oryzae bacterial classification (Aspeigillus oryzae) BLB-21 prepares the method for oligomeric galactose, its feature comprises the enlarged culturing of aspergillus oryzae bacterial classification, uses enlarged culturing seed direct fermentation lactose solution and separates purification processing and prepare oligomeric galactose.
3. the method for preparing oligomeric galactose as claimed in claim 2 is characterized in that the enlarged culturing of aspergillus oryzae BLB-21 comprises slant culture, one-level, secondary seed cultivation; Slant medium: 5
0Be ' wort agar substratum: malt extract 3g, glucose 10g, yeast extract 3g, peptone 5g, agar 20g, distilled water 1000mL; The pH nature, 28～30 ℃ of culture temperature; Seed enlarged culturing base: glucose 300-500g/L, yeast extract paste 15g/L, agar powder 15g/L, pH nature (about 5.5), potassium primary phosphate 1g/L, sal epsom 0.5g/L, urea 5g/L.
4. the method for preparing oligomeric galactose as claimed in claim 2 is characterized in that enlarged culturing seed inoculum size: 5-10% (V/V) in the fermenting process; Lactose solution concentration: 20～40%; Fermentation condition is: regulate pH5.5, and temperature 20-40 ℃, fermentation time 20-40h.
5. the method for preparing oligomeric galactose as claimed in claim 2 is characterized in that ceramic membrane filter is adopted in the separation and purification of oligomeric galactose.
6. the method for preparing oligomeric galactose as claimed in claim 2 is characterized in that oligomeric galactose product separation purifying adopts ion-exchange.
7. the method for preparing oligomeric galactose as claimed in claim 2 is characterized in that oligomeric galactose product separation purifying adopts organic composite nanometer filter membrane sepn.
8. the method for preparing oligomeric galactose as claimed in claim 7 is characterized in that organic composite nanometer filtering film is a cellulose acetate nano filter-membrane.
9. the method for preparing oligomeric galactose as claimed in claim 2 is characterized in that adopting six effect falling film type evaporation thickeners to process to such an extent that high-purity oligomate is starched.
10. the method for preparing oligomeric galactose as claimed in claim 2, it is characterized in that adopting press spray drying tower drying process the high-purity oligomate solid.
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