CN102888388B - Method for producing feruloyl esterase by solid fermentation - Google Patents
Method for producing feruloyl esterase by solid fermentation Download PDFInfo
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- CN102888388B CN102888388B CN201210316415.0A CN201210316415A CN102888388B CN 102888388 B CN102888388 B CN 102888388B CN 201210316415 A CN201210316415 A CN 201210316415A CN 102888388 B CN102888388 B CN 102888388B
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- fermentation
- feruloyl esterase
- aspergillus niger
- seed liquor
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Abstract
The invention discloses a method for producing feruloyl esterase by solid fermentation. The method comprises the following steps of: preparing seed liquor, inoculating and fermenting; the fermentation strain is Aspergllus niger CGMCC No.6152; and a culture medium of fermentation consists of a carbon source, a nitrogen source, inorganic salt and sterile water, wherein the carbon source is at least one of bran, corn cob powder, bean pulp and straw powder. The feruloyl esterase produced by the method is high in enzyme activity; the raw materials used by fermentation are not needed to be subjected to special pretreatment; agricultural byproducts are directly used for fermentation; a process is simple; and the production cost and pollution are reduced.
Description
Technical field
The present invention relates to a kind of method of producing feruloyl esterase, relate in particular to a kind of method of solid fermentation production feruloyl esterase.
Background technology
Feruloyl esterase claims again Ferulic acid esterase, is a kind of Procaine esterase, can be hydrolyzed the ester bond in Ferulic acid methylester, oligosaccharide ferulic acid ester and polysaccharide ferulic acid ester, thereby forulic acid is dissociated out.Utilize feruloyl esterase to carry out process vegetal raw material has bright prospect in the industrial production such as food, feed and papermaking, and feruloyl esterase has become study hotspot in recent years.
Plant cell wall is a tomograph being comprised of polysaccharide network, and these polysaccharide comprise Mierocrystalline cellulose, hemicellulose, and xylogen etc., are connected by hydroxycinnamic acid class ester bond between them, so there is a large amount of compound of phenolic acid in plant cell wall.In foodstuffs industry, by using feruloyl esterase to carry out degrading plant cell walls, can obtain a large amount of compound of phenolic acid.Because compound of phenolic acid has anti-oxidant, to remove human body free radical, uvioresistant, the effect such as antibacterial and antiviral, the concern that is subject to people more, be widely used in the aspects such as medicine, makeup and foodstuff additive, especially forulic acid, it not only can be used as antioxidant, also have as antithrombotic, some physiological actions such as anticancer, it can also serve as the precursor of synthesis of vanillin simultaneously.
In fodder industry, feruloyl esterase can be broken mutual being cross-linked between hemicellulose and xylogen, thereby makes the mechanism of cell walls become loose, and therefore feruloyl esterase can be used as additive use.Feruloyl esterase is accelerated the degradation speed of animal to food, can also improve the digestible degree of animal to feed simultaneously, and reduces the loss of the nutritive substance of discharging with ight soil, thereby improves the utilization ratio of animal to feed.In paper industry, use the removal xylogen that feruloyl esterase can part, reduce pulping process pharmaceutical chemicals as the usage quantity of chemical bleaching agent, increased the whiteness of paper pulp, the quality of paper is further improved.
According to incompletely statistics, produce every year 7.0 hundred million tons of agricultural crop straws (resource such as wheat straw, corn ear, grass stalk), the waste of farm crop source mill also reaches 1.5 hundred million tons, how further to utilize them, and they are turned waste into wealth, and has become the focus of current research.
The source of feruloyl esterase very extensively, since people are for the first time in the culture at streptomyces olivaceus, found that feruloyl esterase can discharge after forulic acid from wheat bran, separated in the extracellular enzyme of the multiple-microorganism such as and streptomycete mould at aspergillus niger, side spore and obtained Ferulic acid methylester up to now.The production of feruloyl esterase at present mainly adopts liquid fermenting, but the enzyme of feruloyl esterase is alive generally lower, and compared with solid fermentation, the fermentation period of liquid fermenting is longer simultaneously, and cost is higher, and fermentation condition requires also high.Publication number is that the Chinese patent literature of CN 101200712A has reported that employing process for solid state fermentation produces feruloyl esterase, obtained good effect, but it is still lower that enzyme is lived, and the agricultural byproducts raw material that adopts of fermentation needs first ferment again, virtually brought energy consumption after the quick-fried processing of vapour.
Summary of the invention
The invention provides a kind of solid fermentation and produce the method for feruloyl esterase, solved existing technique product enzyme enzyme alive low, fermentation raw material need be through the problem of special pre-treatment.
Solid fermentation is produced a method for feruloyl esterase, comprises preparation seed liquor, inoculation and fermentation, and the bacterial classification of described fermentation is aspergillus niger (Aspergillus niger) CGMCC No.6152; The substratum of described fermentation is comprised of carbon source, nitrogenous source, inorganic salt and sterilized water, and wherein carbon source is at least one in wheat bran, corn ear powder, dregs of beans and rice straw powder.
The bacterial classification of fermentation use affects enzymatic productivity, the present invention bacterial classification aspergillus niger (Aspergillus niger) CGMCC No.6152 used is a strain live high-enzyme strain, this bacterial strain is that the product enzyme higher bacterial strain alive by a strain being separated in soil carries out gamma-radiation mutagenesis, and screening obtains.
This strain classification called after aspergillus niger (Aspergillus niger), bacterial strain number is ZJUQH2012147, this bacterial strain was deposited in the China Committee for Culture Collection of Microorganisms's common micro-organisms center (referred to as CGMCC) that is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 05 23rd, 2012, deposit number is CGMCC No.6152.
Substratum of the present invention adopts wheat bran, corn ear powder, dregs of beans and rice straw powder etc. as carbon source, except providing for thalli growth essential energy, also contains a large amount of esterification forulic acid, is suitable for producing feruloyl esterase.Can select following several array configuration: 1, corn ear powder and wheat bran; 2, corn ear powder and dregs of beans; 3, wheat bran, corn ear powder and rice straw powder.Preferably select the mixing of corn ear powder and wheat bran, weight ratio is 1:2～2:1.
For bacterial classification mixes with matrix, before fermentation, need first prepare seed liquor, the described method of preparing seed liquor is: aspergillus niger (Aspergillus niger) CGMCC No.6152 is cultivated and produced after spore, wash lower spore with water and make spore suspension, concentration is generally 10
6～10
7individual/mL.
The inoculum size that is 1～5% according to volume ratio by spore suspension is inoculated in liquid seed culture medium, shaking culture on the shaking table that is 150～200r/min at rotating speed, and temperature is generally 28～30 ℃, and the time is generally 15～20h.Wherein, seed liquor substratum can be selected PDA substratum.
After seed prepares, can ferment, the seed liquor making is inoculated in the substratum of solid fermentation, mix secondary fermentation, inoculum size is generally 0.1～1mL seed liquor/5g substratum, and the temperature of fermentation is 25～30 ℃, 4～7 days time.
The key that completes the object of the invention is to select suitable bacterial strain and carbon source, and all the other compositions of the substratum of fermentation can be conventional selections, wherein nitrogenous source can be selected peptone, yeast extract paste etc., also can select inorganic nitrogen-sourced, in inorganic salt, should comprise necessary mineral element, as sodium, potassium, magnesium, calcium etc.
Preferably, in weight part, the substratum of described fermentation consists of: 2～5 parts of carbon sources, 0.1～0.4 part, nitrogenous source, 0.1～0.2 part, inorganic salt, 15～20 parts, water.
Inorganic salt are wherein by KH
2pO
4, Na
2hPO
47H
2o, NaC 1, MgSO
47H
2o and CaC1
2composition; Preferred, in weight part, described inorganic salt consist of: KH
2pO
40.08～0.12 part, Na
2hPO
47H
20.06 part of O, NaC10.001 part, MgSO
47H
20.002 part of O, CaC1
20.0005 part.
For the ease of storage with use, after ferment, can fermenting culture is dry, generally under 35～45 ℃ of air-flows, to dry to water content lower than 10%, pulverizing is made Powdered.
Compared with prior art, beneficial effect of the present invention is:
(1) enzyme of the feruloyl esterase that the present invention produces is lived high, be 10～30 times that under existing solid fermentation condition, report, and more existing feruloyl esterase is heat-resisting, and temperature reaches 40 ℃, and optimal pH is 5.5 in addition.
(2) raw material of the present invention does not need through special pre-treatment, directly utilizes agricultural by-products fermentation, and technique is simple, has reduced pollution when having reduced production cost.
(3) the present invention has carried out system optimization to solid fermentation process, has obtained culture condition and the technical parameter of applicable suitability for industrialized production.
Embodiment
One, substratum
Slant medium: fresh potato 200g, glucose 20g, agar 20g, distilled water 1000ml, pH nature.
Liquid seed culture medium: fresh potato 200g, glucose 20g, distilled water 1000ml, pH nature.
Primary dcreening operation substratum: KH
2pO
43g, Na
2hPO
47H
2o 6g, NaC1 0.1g, peptone 8g, MgSO
47H
2o 0.2g, CaC1
20.05g, agar 20g, distilled water 1000ml, aseptic dimethyl formamide solution (mass volume ratio the is 10%) 0.3ml containing Ferulic acid methylester, pH nature.
Sieve again substratum: dregs of beans 20g, KH
2pO
43g, Na
2hPO
47H
2o 6g, NaC1 0.1g, peptone 8g, MgSO
47H
2o 0.2g, CaC1
20.05g, distilled water 1000ml, pH nature.
Two, the separation and purification of bacterial strain
From the pedotheque of paper mill, dregs of beans source mill, wheatland and brewery, collect 50 duplicate samples and first through proliferated culture medium, cultivate 120h, dilution spread is in the single bacterium colony of the dull and stereotyped formation of czapek's solution, select again 50 bacterium colony dibblings on primary dcreening operation substratum, the bacterium colony of selection display white transparent circle carries out shaking flask and sieves again, obtaining a strain product enzyme the highest bacterial strain alive is W1, measures fermented liquid ferulic acid ester enzyme activity (28 ℃ of cultivations of this bacterial classification enzyme work in 120 hours reaches 162.84U/L).Through gamma-radiation mutagenesis (dosage 700Gy), dilute respectively 10 times of bacterium liquid, 10
2doubly, 10
3doubly, 10
4doubly, 10
5doubly, 10
6doubly, then be coated with bacterium liquid in primary dcreening operation substratum, cultivate appropriate time, the bacterium colony of selecting the white transparent circle of generation sieves (adopt and sieve again culture medium culturing) again in shaking flask, cultivate and after 168 hours, measure feruloyl esterase enzyme and live, obtain a strain enzyme the highest bacterial strain alive, called after ZJUQH2012147, its enzyme work reaches 213.07U/L.
Three, the evaluation of bacterial strain
The bacterial strain of above-mentioned separation is Aspergillus, very fast from the upper growth of wort agar substratum (MEA), under 25 ℃ of dark conditions, cultivate 7 days, and colony diameter 58～61mm, quality is velvet-like; Conidial head chocolate, the initial stage is spherical, later stage cracking, the bacterium colony back side is without any pigment; Bacterial strain has podocyte, and conidiophore is tall and big, wide 7.5～12.8 μ m, and wall is smooth, and top capsule is spherical, and most surfaces can be educated, conidial fructification bilayer, conidium is subsphaeroidal, shallow to brown, tool spinule, 3.0～5.5 μ m.There are no spermatium.The 28SrRNA D1D2 region sequence of measuring is as shown in sequence table SEQ ID NO.1.
The experimental datas such as comprehensive its colonial morphology, microscopic features and rRNA gene order are comprehensively analyzed, and can be accredited as aspergillus niger, and called after aspergillus niger (Aspergillus niger) ZJUQH2012147.This bacterial strain has been deposited in the China Committee for Culture Collection of Microorganisms's common micro-organisms center (referred to as CGMCC) that is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 05 23rd, 2012, deposit number is CGMCC No.6152.
Four, solid fermentation is produced the method for feruloyl esterase
(1) preparation of spore suspension:
By preserving number be CGMCC No.6152 aspergillus niger to the layout line method be inoculated on slant medium, in 28 ℃ of constant temperature culture 7 days, now bacterial strain was ripe, with spore under aseptic washing, vibrates and breaks up spore, making concentration is 10
6～10
7the spore suspension of individual/ml, stand-by.
(2) seed preparation:
First by liquid seed culture medium through 121 ℃, 20min sterilizing, more cooling; Then the inoculum size that is 5% according to volume ratio (V/V) accesses spore suspension in above-mentioned cooled liquid seed culture medium, in 28 ℃, cultivates 18 hours on the shaking table that rotating speed is 200rpm, obtains liquid seeds.
(3) fermentation of feruloyl esterase
The substratum of preparation fermentation use;
The bottleneck of triangular flask of the substratum that fermentation use is housed is covered with 8 layers of gauze, by fermention medium after 121 ℃ of sterilizing 20min, cooling, then according to 0.1～1mL seed liquor/5g culture medium inoculated amount, aforesaid liquid seed is accessed in cooling fermention medium, fully stirring and evenly mixing, cultivates 6 days in 28 ℃.
(4) aftertreatment:
It is 10% that the product of fermentation gained is dried to moisture weight content under the low-temperature airflow of 35～45 ℃, obtains ferulic acid ester zymin after pulverizing.
The solids of gained after fermentation step is stirred with stirrer, take 1g left and right, add the citrate buffer of pH6.0, the 1h that vibrates in the shaking bath of 28 ℃, filter paper filtering obtains crude enzyme liquid.Alive to measure enzyme by damping fluid dilution certain multiple.
The enzyme activity determination of feruloyl esterase is as follows: because of substrate Ferulic acid methylester water insoluble, therefore dissolve and be mixed with 50mM with one-level Chromatographic Pure Methanol.Used the pH6.0 citrate buffer dilution of 90ml, 0.1M.The Ferulic acid methylester solution of getting after 2mL dilution is heated to 40 ℃ again, adds the enzyme liquid of 10 times of 1mL dilutions.Mixture reacts 30min at 40 ℃, reheats to 100 ℃ termination reaction after 10min.Reaction mixture is cooling in room temperature.Take distilled water, replace substrate solution system as blank.
Forulic acid product after enzyme digestion reaction adopts HPLC quantitative analysis, and correct in this test.The high-efficient liquid phase chromatogram condition adopting in experiment is: adopt acetonitrile and 0.5% formic acid as moving phase, filter, by degassed half an hour ultrasonication under the solvent normal temperature having filtered before using with 0.45 μ m mocromembrane.Premenstruum test gropes to determine that chromatographic condition is: moving phase is acetonitrile and 0.5% formic acid (volume ratio 30:70), C18 reversed-phase column (250 × 4.6mm, 4 μ m), 30 ℃ of detected temperatures, sample size 10 μ L, detect wavelength 317nm.
The enzyme activity of feruloyl esterase is defined as under 40 ℃, pH6.0 condition, and it is an enzyme activity unit that per minute hydrolysis substrate Ferulic acid methylester produces the needed enzyme amount of 1 μ mol forulic acid.
Embodiment 1
The aspergillus niger CGMCC No.6152 obtaining take separation and purification is bacterial classification, collect spore suspension, prepare after seed, seed is inoculated into and in fermention medium, carries out fermentation culture, after having fermented, make ferulic acid ester zymin, the solids survey enzyme of getting after fermentation step is lived.
The preparation process of fermention medium is: in the triangular flask of 250ml, pack wheat bran that 5g carbon source is respectively 2.8g and the corn ear powder of 2.2g into, 0.4g peptone, 0.12g KH
2pO
4, 0.06gNa
2hPO
47H
2o, 0.001g NaC1,0.002g MgSO
47H2O, 0.0005g CaC1
2then to the water that adds 15ml in bottle, pH nature.
After measured, the enzyme of feruloyl esterase is lived as 22.04U/g dry medium.
Embodiment 2
The aspergillus niger CGMCC No.6152 obtaining take separation and purification is bacterial classification, collect spore suspension, prepare after seed, seed is inoculated into and in fermention medium, carries out fermentation culture, after having fermented, make ferulic acid ester zymin, the solids survey enzyme of getting after fermentation step is lived.
The preparation process of fermention medium is: in the triangular flask of 250ml, pack dregs of beans that 5g carbon source is respectively 2.5g and the corn ear powder of 2.5g into, 0.2g peptone, 0.08g KH
2pO
4, 0.06gNa
2hPO
47H
2o, 0.001g NaC1,0.002g MgSO
47H
2o, 0.0005g CaC1
2then to the water pH nature that adds 20ml in bottle.
After measured, the enzyme of feruloyl esterase is lived as 2.99U/g dry medium.
Embodiment 3
The aspergillus niger CGMCC No.6152 obtaining take separation and purification is bacterial classification, collect spore suspension, prepare after seed, seed is inoculated into and in fermention medium, carries out fermentation culture, after having fermented, make ferulic acid ester zymin, the solids survey enzyme of getting after fermentation step is lived.
The preparation process of fermention medium is: in the triangular flask of 250ml, pack the wheat bran that 5g carbon source is respectively 1.67g, the corn ear powder of 1.67g and the rice straw powder of 1.66g into, 0.2g peptone, 0.08gKH
2pO
4, 0.06g Na
2hPO
47H
2o, 0.001g NaC1,0.002g MgSO
47H
2o, 0.0005gCaC1
2, then to the water that adds 15ml in bottle, pH nature.
After measured, the enzyme of feruloyl esterase is lived as 9.28U/g dry medium.
Finally, it is also to be noted that, what more than enumerate is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (9)
1. solid fermentation is produced a method for feruloyl esterase, comprises preparation seed liquor, inoculation and fermentation, it is characterized in that, the bacterial classification of described fermentation is aspergillus niger (Aspergillus niger) CGMCC No.6152; In weight part, the substratum of described fermentation consists of: 15～20 parts of 2～5 parts of carbon sources, 0.1～0.4 part, nitrogenous source, 0.1～0.2 part, inorganic salt and sterilized waters, wherein carbon source is at least one in wheat bran, corn ear powder, dregs of beans and rice straw powder.
2. the method for claim 1, is characterized in that, described nitrogenous source is peptone.
3. the method for claim 1, is characterized in that, described inorganic salt are by KH
2pO
4, Na
2hPO
47H
2o, NaC1, MgSO
47H
2o and CaC1
2composition.
4. method as claimed in claim 3, is characterized in that, in weight part, described inorganic salt consist of: KH
2pO
40.08～0.12 part, Na
2hPO
47H
2o0.06 part, NaC10.001 part, MgSO
47H
2o0.002 part and CaC1
20.0005 part.
5. the method for claim 1, it is characterized in that, the described method of preparing seed liquor is: the spore suspension of aspergillus niger (Aspergillus niger) CGMCC No.6152 is provided, spore suspension is inoculated in liquid nutrient medium, shaking culture 15～20h, makes seed liquor.
6. the method for claim 1, is characterized in that, inoculum size is 0.1～1mL seed liquor/5g substratum.
7. the method for claim 1, is characterized in that, the temperature of described fermentation is 25～30 ℃, and the time is 4～7 days.
8. the method for claim 1, is characterized in that, after having fermented, fermenting culture is dried to water content lower than 10% under 35～45 ℃ of air-flows.
9. the method for claim 1, is characterized in that, described carbon source is the mixture of wheat bran and corn ear powder.
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| CN105543293A (en) * | 2016-03-10 | 2016-05-04 | 南京林业大学 | Method for preparing ferulic acid |
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