CN105524847A - Aureobasidium pullulans AP172-1B and method for producing fructo-oligosaccharide by virtue of aureobasidium pullulans - Google Patents
Aureobasidium pullulans AP172-1B and method for producing fructo-oligosaccharide by virtue of aureobasidium pullulans Download PDFInfo
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Abstract
The invention discloses aureobasidium pullulans AP172-1B, wherein the aureobasidium pullulans AP172-1B is preserved in China General Microbiological Culture Collection Center on November 25, 2015 with preservation number of CGMCC No.11739. The invention also provides a method for producing fructo-oligosaccharide by virtue of the aureobasidium pullulans, and the fructo-oligosaccharide produced through fermentation culture of the aureobasidium pullulans. The method disclosed by the invention, which prepares the fructo-oligosaccharide by virtue of a microorganism fermentation process, optimizes a high-producing strain fermentation process, determines the technological parameters of fermentation and improves the yield of the fructo-oligosaccharide, and the method that biomass is no less than 72.4g/L and yield is no less than 65.9% achieves domestic advanced level. The yield of the fructo-oligosaccharide is effectively improved, and the purity of the fructo-oligosaccharide is no less than 93%.
Description
Technical field
The invention belongs to industrial biotechnology field, relate to a kind of Aureobasidium pullulans, also relate to a kind of method utilizing Aureobasidium pullulans fermentation to produce oligofructose.
Background technology
Oligofructose (fructooligsacchride, FOS) also known as oligofructose or FOS, be by fructose monomers by β glycosidic link connect into not by fructose polymer that endogenous enzyme is hydrolyzed.Molecular formula is G-F-Fn, n=1 ~ 3 or Fm, m=2 ~ 7, (G, F represent glucosyl group and fructosyl respectively, and n represents glucose unit number, and m represents fructose units number).Oligofructose is the functional oligose that a typical case meets prebiotics.Its nourishing function one is a kind of two qi multiplicaiton factor based on it, can improve the microorganism species in enteron aisle, suppress the growth of harmful bacteria, reduce and suppress the generation of septic matter in intestines; It two is a kind of water miscible food fibres, reduces blood fat, improves lipid metabolism, reduces Blood Cholesterol and triglyceride content.Can reduce hepatotoxin in addition, generate anticancer organic acid in intestines, have significant preventing cancer function, mammary cancer, colorectal carcinoma are had to preventive and therapeutic action and increase the resultant quantity of category-B VITAMIN, and improve the immunoregulation effect of human body, inhibition tumor cell grows.Oligofructose is the food ingredients of the GRAS rank of FDA accreditation, and security is high.Confirm as a kind of functional food ingredient in the world.
Oligofructose can the balance of flora in control agent, thus suppresses the growth of the spoilage organism such as Salmonellas in intestines, improves intestinal environment, reduces the growth of corrupt substance in intestines, promote to prevent constipation by intestines peristalsis; Face's acne, blackspot etc. that these characteristics internal secretion imbalance that is bad for Digestive tract and that cause produces have well to be improved and eliminating effect.Oligofructose is a kind of good water-soluble dietary fibre, there is the effect reducing serum cholesterol and blood fat, its mechanism is that oligofructose is fermented by intestinal bacterium, produce propionic acid, hinder the synthesis of cholesterol, impel cholesterol to change to bile acide, increase bile acide output, thus can reduce blood fat.So oligofructose has good improvement result for a series of diseases, such as hypertension, cardiovascular disorder etc. caused because blood fat is high; Taking oligofructose can help human body to the absorption of mineral substance as elements such as calcium, iron, magnesium, and promote the eliminating of vivotoxin, its mechanism is that oligofructose is produced lactic acid by fermentation using bacteria in large intestine, lactic acid can dissolve the mineral substance such as calcium, iron, magnesium in intestines, promote that human body is to the absorption of mineral substance, the activity of β – glucuroide can also be reduced, be conducive to the eliminating of the carcinogenic Toxic waste such as indoles in intestines, sub-whistle base amine; In addition, within 1997, ministry of Health of China food supervision and inspection proved, oligofructose has the effect significantly improving antibody forming cell's number and NK cytoactive and enhancing immunologic function.Based on more than oligofructose various physiological function, become functional foodstuff popular in the world in recent years.Can be used as the manufacture that functional ingredient and bifidus bacillus multiplicaiton factor are widely used in functional foodstuff, medicine and healthcare products.
At present, oligofructose is most is that raw material extraction and isolation obtains with inulin, inulin be fructose molecule with the chain Polylevulosan of fructoside key connecting, end is glucose molecule.Inulin extensively distributes at occurring in nature, is mainly present in the plants such as onion, banana, Garden Dahlia, jerusalem artichoke.Wherein jerusalem artichoke is the maximum plant of inulin content, accounts for 80% of stem tuber dry weight, abundance.Although take inulin as the oligofructose industrialization of raw material production, production technique is perfect not, and the purity of Inulin oligofructose is not high enough.In current production technique, subject matter is present in the process procedure such as the desalination of inulin extracting solution and the purification refine of Inulin oligofructose.Being containing by products such as a certain amount of glucose, fructose, sucrose in the oligofructose product of raw material production with inulin, is that Inulin oligofructose produces upper problem demanding prompt solution.And the research of the preparation method of industrialization oligofructose there is not yet report.Because Inulin oligofructose product composition is complicated and often character is comparatively close, its separation and purification just becomes more difficult, and conventional separation technique such as crystallization process is difficult to be suitable for.Though the purity of existing several functionalities oligose product reaches more than 80% at present, because production cost is high, volume of production and marketing is low.
Summary of the invention
For this reason, an object of the present invention is to provide a kind of Aureobasidium pullulans AureobasidiumpullulansAP172-1B.
Another object of the present invention is to provide a kind of method utilizing Aureobasidium pullulans to produce oligofructose.
Technical scheme provided by the invention is:
A kind of Aureobasidium pullulans AureobasidiumpullulansAP172-1B, this Aureobasidium pullulans AureobasidiumpullulansAP172-1B is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is: CGMCCNo.11739, the preservation time is: on November 25th, 2015, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Utilize described Aureobasidium pullulans to produce a method for oligofructose, by described Aureobasidium pullulans fermentation culture, produce oligofructose.
Preferably, in described method, described fermentation culture comprises the steps:
1) Aureobasidium pullulans first order seed is inoculated in the seed culture medium of 250mL, cultivates 24-36h in temperature 28-30 DEG C and rotating speed 180-200r/min, obtain Aureobasidium pullulans secondary seed;
2) according to the inoculum size of 1.0%-2.0% by step 1) in the Aureobasidium pullulans secondary seed access fermentor tank that obtains, under temperature 26-30 DEG C of condition, cultivate 60h-72h, and obtain fermented liquid;
3) from described step 2) in the fermented liquid that obtains purifying obtain oligofructose.
Preferably, in described method, described step 2) in, the volume of described fermentor tank is 100L, and the liquid amount of described fermentor tank is 40-60L.
Preferably, in described method, described step 2) in, the air flow in culturing process is 1:0.3 ~ 0.8 (v/v).
Preferably, in described method, described step 2) in, initial speed is 150-200r/min, use the 3-5% glucose accounting for described fermented liquid weight to carry out feed supplement, and control pH is 6.0 after fermentation 24h.
Preferably, in described method, described step 3) in, the method that purifying obtains oligofructose from fermented liquid comprises: first fermented liquid is heated to 80 DEG C, 48-60h is placed under being cooling conditions after being cooled to room temperature, collect upper pellucid syrup afterwards, then pellucid syrup decolorizing with activated carbon is gone up, fructose-transferring enzyme is added again in the liquid after decolouring, the mass volume ratio of the liquid after the consumption of described fructose-transferring enzyme and described decolouring is pellucid syrup on 0.6-1.0g/L, and carries out enzyme reaction 10-15h under temperature 50 C, pH5.5 condition.
Preferably, in described method, described step 3) in, the pH value of described fermented liquid is 6.5-7.5.
Preferably, in described method, described step 3) in, described cooling conditions is 4 DEG C.
Preferably, in described method, described step 3) in, collect described upper pellucid syrup by 10min centrifugal under 5000-6000r/min.
The production cost of the oligofructose prepared according to method of the present invention is low
The present invention at least has the following advantages:
1. the present invention is that microbe fermentation method prepares oligofructose, oligofructose is recognized has raising body immunity, not digested absorption, improve lipid metabolism, prevention decayed tooth waits excellent properties, now by as functional ingredient widespread use, to its research and development, there is good economic worth and application potential.
2. the present invention is optimized superior strain zymotechnique, determines the processing parameter of fermentation, and improve the output of oligofructose, biomass is not less than 72.4g/L, and yield is not less than 65.9%, reaches domestically leading level.
3. the present invention adopts cold settling and enzymolysis process, and can carry out at a lower temperature, normal-temperature operation, device are simple, energy consumption is low, cost is low, and selectivity is high, effectively improves the yield of oligofructose, oligofructose purity is not less than 93%.
Accompanying drawing explanation
Fig. 1 is the schema utilizing Aureobasidium pullulans to produce the method for oligofructose in the present invention.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, can implement according to this with reference to specification sheets word to make those skilled in the art.
The invention provides a kind of Aureobasidium pullulans (Aureobasidiumpullulans) AP172-1B, this Aureobasidium pullulans (Aureobasidiumpullulans) AP172-1B is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is: CGMCCNo.11739, and the preservation time is: on November 25th, 2015.
The present invention also provides a kind of method utilizing described Aureobasidium pullulans to produce oligofructose, by described Aureobasidium pullulans fermentation culture, produces oligofructose.
In one of them embodiment of the present invention, as preferably, described fermentation culture comprises the steps:
1) Aureobasidium pullulans first order seed is inoculated in the seed culture medium of 250mL, cultivates 24-36h in temperature 28-30 DEG C and rotating speed 180-200r/min, obtain Aureobasidium pullulans secondary seed;
2) according to the inoculum size of 1.0%-2.0% by step 1) in the Aureobasidium pullulans secondary seed access fermentor tank that obtains, under temperature 26-30 DEG C of condition, cultivate 60h-72h, and obtain fermented liquid;
3) from described step 2) in the fermented liquid that obtains purifying obtain oligofructose.
In such scheme, as preferably, described step 2) in, the volume of described fermentor tank is 100L, and the liquid amount of described fermentor tank is 40-60L.
In one of them embodiment of the present invention, as preferably, described step 2) in, the air flow in culturing process is 1:0.3 ~ 0.8 (v/v).
In one of them embodiment of the present invention, as preferably, described step 2) in, initial speed is 150-200r/min, use the 3-5% glucose accounting for described fermented liquid weight to carry out feed supplement, and control pH is 6.0 after fermentation 24h.
In one of them embodiment of the present invention, as preferably, described step 3) in, the method that purifying obtains oligofructose from fermented liquid comprises: first fermented liquid is heated to 80 DEG C, 48-60h is placed under being cooling conditions after being cooled to room temperature, collect upper pellucid syrup afterwards, then pellucid syrup decolorizing with activated carbon is gone up, fructose-transferring enzyme is added again in the liquid after decolouring, the mass volume ratio of the liquid after the consumption of described fructose-transferring enzyme and described decolouring is pellucid syrup on 0.6-1.0g/L, and carries out enzyme reaction 10-15h under temperature 50 C, pH5.5 condition.
In one of them embodiment of the present invention, as preferably, described step 3) in, the pH value of described fermented liquid is 6.5-7.5.
In one of them embodiment of the present invention, as preferably, described step 3) in, described cooling conditions is 4 DEG C.
In one of them embodiment of the present invention, as preferably, described step 3) in, collect described upper pellucid syrup by 10min centrifugal under 5000-6000r/min.
As shown in Figure 1, the invention provides a kind of method utilizing this Aureobasidium pullulans to produce oligofructose, comprise the following steps:
1, fermentation strain: adopt the Aureobasidium pullulans AP172-1B built voluntarily
2, substratum:
(1) slant medium: sucrose 20-40g/L, magnesium sulfate 3-5g/L, Repone K 3-5g/L, magnesium sulfate 0.1-0.2g/L, potassium primary phosphate 0.1-0.2g/L, peptone 200-210g/L, agar 18-20g, 1L is settled to, natural pH, 121 DEG C of sterilizing 15-25min with distilled water.
(2) seed culture medium: sucrose 50-70g/L, yeast leaching powder 20-40g/L, magnesium sulfate 3-5g/L, potassium primary phosphate 0.1-0.8g/L, pH5.5 ~ 6.5,121 DEG C of sterilizing 15-25min.
(3) fermention medium: sucrose 45-70g/L, yeast leaching powder 20-40g/L, magnesium sulfate 1-5g/L, ammonium sulfate 3-8g/L, potassium primary phosphate 0.1-0.8g/L, pH5.5 ~ 6.5,115 DEG C of sterilizing 15-25min.
3, bacterial strain screening:
Slant medium is used to be activated by Aureobasidium pullulans, prepare spore suspension by the Aureobasidium pullulans of slant culture and inoculate seed culture medium, be inoculated in 50mL seed culture medium afterwards, 28-30 DEG C, 180-200r/min cultivates 16-20h, detect biomass (biomass (g/L)=dry cell weight (g)/fermentating liquid volume (L)), seed culture medium biomass being not less than 70g/L is inoculated in screening culture medium; (screening culture medium forms: magnesium sulfate 3-5g/L with volume fraction 10% inoculum size, seed culture medium to be inoculated in screening culture medium, SODIUMNITRATE 3-5g/L, L-potassium primary phosphate 0.1-0.2g/L, agar 18-20g, sucrose 450-500g/L, 115 DEG C of sterilizing 15-25min), 28 DEG C, 140-150r/min ferments 24-30h; Fermented liquid is detected and (detects with high-efficient liquid phase technique, moving phase is acetonitrile/water: 70:30, v/v, flow velocity is 0.8mL/min, 30 DEG C of column temperatures), after glucose, sucrose and oligofructose go out peak, calculate the content of each sugar component with areas of peak normalization method, select to obtain the highest Aureobasidium pullulans AP172-1B of oligofructose content.
4, fermentation culture:
Be inoculated in the seed culture medium of 250mL by the Aureobasidium pullulans AP172-1B of slant culture, 28-30 DEG C, 180-200r/min cultivate 24-36h; By the inoculum size of 1.0%-2.0%, the access of aforesaid liquid bacterial classification is equipped with in the 100L automatic fermenter of liquid fermentation medium, at liquid amount 40-60L, 60h-72h is cultivated under culture temperature 26-30 DEG C of condition, air flow 1:0.3 ~ 0.8 (v/v), after fermentation 24h, 3-5% glucose carries out feed supplement, control pH is 6.0, and initial speed is 150-200r/min.
Biomass (g/L)=dry cell weight (g)/fermentating liquid volume (L)
In fermented liquid, mycelial biomass is not less than 72g/L, and yield is not less than 65%.
Purifying: fermented liquid 30L is heated to 80 DEG C under neutrallty condition (pH value is 6.5-7.5), and heating is the dissolving in order to promote oligofructose, so that more containing oligofructose in the supernatant liquor after centrifugal.Refrigerator (4 DEG C) 48-60h is placed after being cooled to room temperature, difference collecting precipitation and supernatant liquor after the centrifugal 10min of 5000-6000r/min, upper pellucid syrup decolorizing with activated carbon, adds 3U/g fructose-transferring enzyme in the liquid after decolouring, its add-on is pellucid syrup on 0.6-1.0g/L.50 DEG C, carry out enzyme reaction 10-15h under pH5.5 condition, HPLC (condition: moving phase is acetonitrile/water (70: 30, v/v), flow velocity 0.8mL/min, 30-35 DEG C of column temperature), measure glucose, sucrose, kestose, GF3, GF4 content, the total content of oligofructose is not less than 85%.
Embodiment 1
The access of aforesaid liquid bacterial classification is equipped with in liquid fermentation medium 100L automatic fermenter by the inoculum size by 2.0%, 8h is cultivated under liquid amount 60L, culture temperature 30 DEG C of conditions, air flow 1:0.4 (v/v), feed supplement is carried out with 5% glucose in fermenting process, control pH is 6.0, and initial speed is 200r/min.Compound concentration is respectively the fermented extracted liquid 30L of 45%, in neutral conditions with being heated to 80 DEG C, places refrigerator (8 DEG C) 48h after being cooled to room temperature.Difference collecting precipitation and supernatant liquor after the centrifugal 10min of 5000r/min, decolour under upper pellucid syrup 1.0kg/10L granular activated charcoal normal temperature shaking table 100-140r/min 8h, 3U/g fructose-transferring enzyme is added in liquid after decolouring, at 50 DEG C, enzyme reaction 10-h is carried out under pH5.5 condition, HPLC (condition: moving phase is acetonitrile/water (75: 25, v/v), flow velocity 1.0mL/min, 35 DEG C of column temperatures), measure glucose, sucrose, kestose, GF3, GF4 content, oligofructose total content is 85.63%, mycelial biomass 73.54g/L in fermented liquid, yield 66.46%.
Oligofructose prepared by the present invention builds Aureobasidium pullulans (Aureobasiumpullulans) biological fermentation voluntarily to obtain, have selected liquid culture based formulas preferably and corresponding ferment control parameter, adopt cold settling and enzymolysis process in leaching process, gained oligofructose purity is not less than 93%.
Although embodiment of the present invention are open as above, but it is not restricted to listed in specification sheets and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the legend described.
Claims (10)
1. an Aureobasidium pullulans AureobasidiumpullulansAP172-1B, this Aureobasidium pullulans AureobasidiumpullulansAP172-1B is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is: CGMCCNo.11739, and the preservation time is: on November 25th, 2015.
2. utilize Aureobasidium pullulans as claimed in claim 1 to produce a method for oligofructose, it is characterized in that, by Aureobasidium pullulans fermentation culture according to claim 1, produce oligofructose.
3. method as claimed in claim 2, it is characterized in that, described fermentation culture comprises the steps:
1) Aureobasidium pullulans first order seed is inoculated in the seed culture medium of 250mL, cultivates 24-36h in temperature 28-30 DEG C and rotating speed 180-200r/min, obtain Aureobasidium pullulans secondary seed;
2) according to the inoculum size of 1.0%-2.0% by step 1) in the Aureobasidium pullulans secondary seed access fermentor tank that obtains, under temperature 26-30 DEG C of condition, cultivate 60h-72h, and obtain fermented liquid;
3) from described step 2) in the fermented liquid that obtains purifying obtain oligofructose.
4. method as claimed in claim 3, is characterized in that, described step 2) in, the volume of described fermentor tank is 100L, and the liquid amount of described fermentor tank is 40-60L.
5. method as claimed in claim 3, is characterized in that, described step 2) in, the air flow in culturing process is 1:0.3 ~ 0.8 (v/v).
6. method as claimed in claim 3, is characterized in that, described step 2) in, initial speed is 150-200r/min, use the 3-5% glucose accounting for described fermented liquid weight to carry out feed supplement, and control pH is 6.0 after fermentation 24h.
7. the method as described in as arbitrary in claim 3 to 6, it is characterized in that, described step 3) in, the method that purifying obtains oligofructose from fermented liquid comprises: first fermented liquid is heated to 80 DEG C, 48-60h is placed under being cooling conditions after being cooled to room temperature, collect upper pellucid syrup afterwards, then pellucid syrup decolorizing with activated carbon is gone up, fructose-transferring enzyme is added again in the liquid after decolouring, the mass volume ratio of the liquid after the consumption of described fructose-transferring enzyme and described decolouring is pellucid syrup on 0.6-1.0g/L, and in temperature 50 C, enzyme reaction 10-15h is carried out under pH5.5 condition.
8. method as claimed in claim 7, is characterized in that, described step 3) in, the pH value of described fermented liquid is 6.5-7.5.
9. method as claimed in claim 7, is characterized in that, described step 3) in, described cooling conditions is 4 DEG C.
10. method as claimed in claim 7, is characterized in that, described step 3) in, collect described upper pellucid syrup by 10min centrifugal under 5000-6000r/min.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1265422A (en) * | 2000-02-01 | 2000-09-06 | 中国农业科学院饲料研究所 | New bacterial strain producing fructose transferase and fructose transferease producing process |
| US20050006927A1 (en) * | 2003-03-19 | 2005-01-13 | Granger Sean Elliot | Clip assembly for adjustably attaching a cladding to a vehicle |
| WO2011051965A2 (en) * | 2009-10-09 | 2011-05-05 | Tata Chemicals Ltd. | A process for production of fructo-oligosaccharides |
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- 2016-01-25 CN CN201610047919.5A patent/CN105524847A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1265422A (en) * | 2000-02-01 | 2000-09-06 | 中国农业科学院饲料研究所 | New bacterial strain producing fructose transferase and fructose transferease producing process |
| US20050006927A1 (en) * | 2003-03-19 | 2005-01-13 | Granger Sean Elliot | Clip assembly for adjustably attaching a cladding to a vehicle |
| WO2011051965A2 (en) * | 2009-10-09 | 2011-05-05 | Tata Chemicals Ltd. | A process for production of fructo-oligosaccharides |
Non-Patent Citations (6)
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Application publication date: 20160427 |