CN103074314A - Method for producing feruloyl esterase through Aspergillus niger fermentation - Google Patents
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- CN103074314A CN103074314A CN2012105946858A CN201210594685A CN103074314A CN 103074314 A CN103074314 A CN 103074314A CN 2012105946858 A CN2012105946858 A CN 2012105946858A CN 201210594685 A CN201210594685 A CN 201210594685A CN 103074314 A CN103074314 A CN 103074314A
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Abstract
The invention discloses a method for producing a feruloyl esterase through Aspergillus niger fermentation comprises the following step of inoculating an Aspergillus niger ZJUQH2012147 spore suspension into a fermentation medium for fermentation, wherein the collection number of the Aspergillus niger ZJUQH2012147 is CGMCC No.6152, the fermentation medium is added with microparticles, and the microparticles can be at least one of talcum powder, kaoline, bauxite, titanate, quartz sand, carbon powder and silicon carbide. Liquid added with the microparticles is taken as the fermentation medium for producing the feruloyl esterase through the Aspergillus niger fermentation; and in a fermentation process, mycelia are relatively small, oxygen and nutrition are smoothly supplied and a strain grows and metabolizes vigorously, so that the effect of enzyme production through the fermentation is effectively improved, the level of producing the feruloyl esterase by the strain is approximately twice of that under original conditions, the industrial production of the feruloyl esterase can be achieved, and a reference is provided for fermentation production of other Aspergillus.
Description
Technical field
The present invention relates to biological technical field, relate in particular to the method that a kind of fermentation of Aspergillus niger is produced feruloyl esterase.
Background technology
Feruloyl esterase claims again Ferulic acid esterase, and it is a kind of Procaine esterase, refers to be hydrolyzed the ester bond in Ferulic acid methylester, oligosaccharide ferulic acid ester and the polysaccharide ferulic acid ester, with the free enzyme out of forulic acid.With feruloyl esterase process vegetal raw material bright prospect is arranged in the industry such as food, feed and papermaking.
Mould is the general designation of the filamentous fungus that can cause that species go mouldy, is the part of fungi.Every being grown in is fine hair shape, spider reticulation or cotton-shaped mycelial bacterium colony on the substratum, all be referred to as mould.The mould distribution in the nature is very extensive, also closely bound up with people's life, as being used for traditional wine brewing, sauce processed and making nonstaple food and other leavened food etc., they also have very widely in industry uses, as produce microbiotic, biological pesticide, natural pigment and various enzymes etc. with mold fermentation.But the liquid fermenting of mould exists many shortcomings, and, mycelia high such as broth viscosity formed agglomerate, oxygen supply is not enough, the nutrition transmission is not smooth etc., and this has just limited its application, how to address this problem the focus that has become current research.
The application for a patent for invention of publication number CN 102796673A discloses a strain feruloyl esterase superior strain and has utilized this bacterial strain to carry out the method that liquid fermenting prepares feruloyl esterase, this bacterial strain is viride (Trichodermaviride) JBSH-001, preserving number is CGMCCNo.5612, utilizes this bacterial strain can produce by liquid fermenting and obtains feruloyl esterase.Feruloyl esterase produces and operation is induced in accumulation but use in the method, needs during the fermentation to add inducible factor, and need constantly add carbon source, nitrogenous source, and operation is more loaded down with trivial details.
The patent of invention of notification number CN 102286442B discloses a kind of method of producing feruloyl esterase by fermentation of aspergillus fumigatus, comprise: activated Aspergillus fumigatus (Aspergillus fumigatus) CGMCCNo.3.5835 is inoculated in the nutrient solution, and cultivated 2～6 days at 25～30 ℃ of bottom fermentations; Wherein, described nutrient solution is comprised of Semen Maydis powder or wheat bran and basic liquid nutrient medium.The application for a patent for invention of publication number CN102796670A discloses a kind of Aspergillus niger strain, called after aspergillus niger (Aspergillus niger) ZJUQH2012147, and deposit number is CGMCC No.6152; This application also discloses the method that adopts this bacterial strain to produce feruloyl esterase, comprising: aspergillus niger ZJUQH201217 access seed culture medium is carried out multiplication culture, obtain seed liquor; Seed liquor is accessed fermention medium carry out fermentation culture.These two kinds of methods can both fermentative production obtain feruloyl esterase, but exist the common shortcoming of mould liquid fermenting in its fermenting process, i.e. the easy conglomeration of mycelia, and oxygen, nutrition supply are not enough, and the activity that causes producing feruloyl esterase is still not high.
For this reason, explore suitable technique fermentation process is improved, can not only improve the fermentative production of feruloyl esterase, and the liquid fermenting of other mould is also had certain reference value.
Summary of the invention
The invention provides a kind of fermentation of Aspergillus niger and produce the method for feruloyl esterase, solve existing technique broth viscosity height, the easy conglomeration of mycelia, oxygen and nutrition supply deficiency, produce the low problem of enzyme level.
A kind of fermentation of Aspergillus niger is produced the method for feruloyl esterase, comprising: will carry out fermentation culture in aspergillus niger (Aspergillus niger) the ZJUQH2012147 spore suspension access fermention medium;
Wherein, the preserving number of described aspergillus niger ZJUQH2012147 is CGMCC No.6152;
Be added with microparticle in the described fermention medium; Microparticle is at least a in talcum powder, kaolin, alumina, titanate, quartz sand, carbon dust and the silicon carbide.
Microparticle (Microparticles) refers to the particle of size about 10000 to 100 orders.In fermention medium, add an amount of microparticle, can improve the fungal hyphae clustering phenomena, thereby for mould provides sufficient oxygen and nutritive substance, improve and produce enzyme efficient.
Described aspergillus niger ZJUQH2012147 has been deposited in China Committee for Culture Collection of Microorganisms 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.This bacterial strain belongs to Deuteromycotina, hyphomycetes, and hyphomycetales, Aspergillus, very fast in wort agar substratum (MEA) growth, cultivated 7 days under 25 ℃ of dark conditions, colony diameter 58-61mm, quality is velvet-like; The conidial head chocolate, the initial stage is spherical, the 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 the top capsule is spherical, and most surfaces can be educated, and conidial fructification is double-deck, and conidium is subsphaeroidal, and is shallow to brown, tool spinule, 3.0-5.5 μ m; There are no spermatium.Disclose this bacterial strain in the application for a patent for invention of publication number CN 102796670A, and disclose in detail its screening, cultural method, form and biological property etc., this bacterial strain can ferment and produce feruloyl esterase.
Described aspergillus niger ZJUQH2012147 spore suspension can prepare by the following method: aspergillus niger ZJUQH2012147 is inoculated on the PDA slant medium, activated in 25-30 ℃ of constant temperature culture 6-8 days; The bacterial strain of activation scraped to make concentration in the sterilized water be 10
6-10
7The spore suspension of individual/mL.
The inoculum size of described aspergillus niger ZJUQH2012147 spore suspension is 1-10%, is beneficial to the growth and breeding of spore.
In volume 1L, described fermention medium comprises: the vat liquor 200-300mL of dregs of beans, wheat bran, corn ear or corn stalk, nitrogenous source 5-20g, inorganic salt 5-15g.The raw material sources such as dregs of beans, wheat bran, corn ear or corn stalk are extensive, with low cost, and its vat liquor can not only provide carbon source for strain growth, and contains a large amount of esterification forulic acid, can induce aspergillus niger expression and secretion feruloyl esterase.
Described nitrogenous source can be organic nitrogen source, inorganic nitrogen-sourced or their mixture, and organic nitrogen source can be selected peptone, yeast powder or their mixture, inorganic nitrogen-sourcedly can select ammonium salt, such as ammonium sulfate, ammonium chloride, SODIUMNITRATE etc.
Described inorganic salt mainly comprise the various elements that bacterial cell growth is required, and such as P, Na, K, Mg, Ca etc., its consumption can be with reference to the prescription of conventional mould medium.
Preferably, in volume 1L, described fermention medium comprises: the vat liquor 200-300mL of dregs of beans, wheat bran, corn ear or corn stalk, peptone 8-12g, KH
2PO
41-4g, Na
2HPO
412H
2O 5-10g, NaCl 0-0.4g, MgSO
47H
2O 0-0.4g, CaCl
20-0.2g.
More preferably, in volume 1L, described fermention medium comprises: the vat liquor 226mL of dregs of beans, peptone 10.35g, KH
2PO
43g, Na
2HPO
412H
2O 8g, NaCl 0.1g, MgSO
47H
2O 0.2g, CaCl
20.05g.
Described vat liquor can prepare by the following method: dregs of beans, wheat bran, corn ear or corn stalk are pulverized, then added water boil 0.5 hour, cross leaching filtrate, obtain vat liquor.
These microparticle particle diameters of described talcum powder, kaolin, alumina, titanate, quartz sand, carbon dust and silicon carbide are little, can make mycelia form framboid, and are larger with extraneous contact area, are convenient to oxygen supply and nutritive substance transmission; And these microparticle inertia are better, can not affect the carrying out of fermentation reaction.Preferably, described microparticle is at least a in talcum powder, kaolin and the alumina, and it is better that these three kinds of microparticles are used for producing the feruloyl esterase effect, and be convenient to industrial application.
The particle diameter of described microparticle can be the 100-10000 order; Be preferably the 100-1000 order; 200-800 order more preferably.Particle diameter is too small, and is not obvious to fermentation improvement effect; Particle diameter is excessive, and mycelia is difficult for forming framboid.
The concentration of described microparticle in fermention medium can be 0.1-100g/L; Be preferably 0.1-20g/L; 0.1-1.0g/L more preferably; Most preferably be 0.5-1.0g/L.The microparticle addition is too little, and action effect is not obvious; Add too much, then suppress the growth of thalline.
Described microparticle can add in the fermention medium before fermentation culture.With the separately sterilization of fermention medium and microparticle, during spore suspension to be inoculated microparticle is added in the fermention medium, operate more conveniently, be convenient to suitability for industrialized production.
Described microparticle also can add in the fermention medium in fermentation culture in 1-2 days.Spore suspension accessed cultivated first in the fermention medium 1-2 days, be convenient to the spore Fast-propagation and reach certain cell concentration, this moment, spore was in logarithmic phase.Add microparticle at logarithmic phase, can not affect the spore breeding in early stage, and the microparticle that adds can make spore form preferably framboid, be convenient to follow-up metabolism and generate feruloyl esterase.
Fermentation culture conditions affects fecundity and the enzymatic productivity of bacterial strain.The culture temperature of described fermentation culture can be 25-30 ℃, and incubation time is 3-7 days; Preferably, culture temperature is 28 ℃, and incubation time is 5 days.
Described fermentation culture can adopt the shaking table shaking culture, also can utilize the fermentor tank large-scale industrialization to cultivate.When adopting the shaking table shaking culture, shaking speed is preferably 100-200r/min, is beneficial to thalline and disperses.
After fermentation culture was finished, feruloyl esterase was present in the fermented liquid, can realize separation and purification by conventional enzyme separation method.
The present invention as fermention medium, adopts aspergillus niger ZJUQH2012147 to produce feruloyl esterase by the liquid fermenting mode with the liquid that is added with an amount of concentration, an amount of big or small microparticle under suitable culture condition.
Adopt the inventive method, hypha body is less, oxygen and nutritive substance supply are smooth and easy, strain growth, metabolism are vigorous, effectively improved the enzymatic production effect, the level of bacterial strain product feruloyl esterase is about the twice under former the having ready conditions, and can realize the suitability for industrialized production of feruloyl esterase, and also the fermentative production for other aspergillus provides reference.
Embodiment
In following examples, used aspergillus niger ZJUQH2012147 has been deposited in China Committee for Culture Collection of Microorganisms 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 CGMCCNo.6152.
The screening of this bacterial strain, cultural method, form and biological property etc. are open in the application for a patent for invention of publication number CN102796670A.Particularly, the form of this bacterial strain and biological property are: belong to Deuteromycotina, and hyphomycetes, hyphomycetales, Aspergillus, very fast in wort agar substratum (MEA) growth, cultivated 7 days under 25 ℃ of dark conditions, colony diameter 58-61mm, quality is velvet-like; The conidial head chocolate, the initial stage is spherical, the 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 the top capsule is spherical, and most surfaces can be educated, and conidial fructification is double-deck, and conidium is subsphaeroidal, and is shallow to brown, tool spinule, 3.0-5.5 μ m; There are no spermatium.
Embodiment 1
In the present embodiment, microparticle is selected 800 purpose talcum powder.
(1) preparation of spore suspension: aspergillus niger ZJUQH2012147 is inoculated on the PDA slant medium, activated in 7 days in 28 ℃ of constant temperature culture; The bacterial strain of activation scraped to make concentration in the sterilized water be 10
6The spore suspension of individual/mL.
(2) preparation of vat liquor: dregs of beans is pulverized, then added water, in electric furnace, boiled 0.5 hour, with eight layers of filtered through gauze, get filtrate, be the vat liquor of dregs of beans.
(3) preparation of fermention medium: in volume 1L, fermention medium comprises: the vat liquor 226mL of dregs of beans, peptone 10.35g, KH
2PO
43g, Na
2HPO
412H
2O 8g, NaCl0.1g, MgSO
47H
2O 0.2g, CaCl
20.05g, the pH nature.
(4) sterilization: fermention medium and talcum powder are separated sterilization (121 ℃, 20min).
(5) talcum powder is added in the fermention medium, make talcous concentration reach respectively 0.1g/L, 0.5g/L, 1g/L, 5g/L, 10g/L, 20g/L.
(6) according to 10% inoculum size above-mentioned spore suspension is accessed in the cooled fermention medium, in 28 ℃, 180rpm fermentation culture 7 days.
In sampling in the 3rd, 5,7 day of fermentation culture, measure the feruloyl esterase enzyme and live successively, the results are shown in Table 1.
By the result as can be known, add that enzyme that 800 order talcum powder make feruloyl esterase is lived and live than enzyme all improving, especially at the 7th day, under the talcum powder concentration of 0.5g/L, the work of feruloyl esterase enzyme reaches the highest, has improved compared with the control 46%; At the 5th day, under the 0.5g/L concentration, reach the highest than enzyme work, improved compared with the control 52%.
Table 1
Embodiment 2
In the present embodiment, microparticle is selected 200-300 purpose kaolin.
(1) preparation of spore suspension: aspergillus niger ZJUQH2012147 is inoculated on the PDA slant medium, activated in 7 days in 28 ℃ of constant temperature culture; The bacterial strain of activation scraped to make concentration in the sterilized water be 10
6The spore suspension of individual/mL.
(2) preparation of vat liquor: dregs of beans is pulverized, then added water, in electric furnace, boiled 0.5 hour, with eight layers of filtered through gauze, get filtrate, be the vat liquor of dregs of beans.
(3) preparation of fermention medium: in volume 1L, fermention medium comprises: the vat liquor 226mL of dregs of beans, peptone 10.35g, KH
2PO
43g, Na
2HPO
412H
2O 8g, NaCl0.1g, MgSO
47H
2O 0.2g, CaCl
20.05g, the pH nature.
(4) sterilization: fermention medium and kaolin are separated sterilization (121 ℃, 20min).
(5) kaolin is added in the fermention medium, make kaolinic concentration reach respectively 0.1g/L, 0.5g/L, 1g/L, 5g/L, 10g/L, 20g/L.
(6) according to 10% inoculum size above-mentioned spore suspension is accessed in the cooled fermention medium, in 28 ℃, 180rpm fermentation culture 7 days.
In sampling in the 3rd, 5,7 day of fermentation culture, measure the feruloyl esterase enzyme and live successively, the results are shown in Table 2.
By the result as can be known, add 200-300 purpose kaolin and also make the feruloyl esterase enzyme live and improve than enzyme work, especially at the 7th day, under the kaolin concentration of 1g/L, the enzyme work of feruloyl esterase reaches the highest, has improved compared with the control 87%; At the 5th day, under the kaolin concentration of 0.5g/L, reach the highest than enzyme work, improved compared with the control 79%.
Table 2
Embodiment 3
In the present embodiment, microparticle is selected 300 purpose alumina.
(1) preparation of spore suspension: aspergillus niger ZJUQH2012147 is inoculated on the PDA slant medium, activated in 7 days in 28 ℃ of constant temperature culture; The bacterial strain of activation scraped to make concentration in the sterilized water be 10
6The spore suspension of individual/mL.
(2) preparation of vat liquor: dregs of beans is pulverized, then added water, in electric furnace, boiled 0.5 hour, with eight layers of filtered through gauze, get filtrate, be the vat liquor of dregs of beans.
(3) preparation of fermention medium: in volume 1L, fermention medium comprises: the vat liquor 226mL of dregs of beans, peptone 10.35g, KH
2PO
43g, Na
2HPO
412H
2O 8g, NaCl0.1g, MgSO
47H
2O 0.2g, CaCl
20.05g, the pH nature.
(4) sterilization: fermention medium and alumina are separated sterilization (121 ℃, 20min).
(5) alumina is added in the fermention medium, make the concentration of alumina reach respectively 0.1g/L, 0.5g/L, 1g/L, 5g/L, 10g/L, 20g/L.
(6) according to 10% inoculum size above-mentioned spore suspension is accessed in the cooled fermention medium, in 28 ℃, 180rpm fermentation culture 7 days.
In sampling in the 3rd, 5,7 day of fermentation culture, measure the feruloyl esterase enzyme and live successively, the results are shown in Table 3.
By the result as can be known, add the enzyme that 300 purpose alumina also make feruloyl esterase and live and improve than enzyme work, especially at the 7th day, under the concentration of 0.5g/L, enzyme work reaches the highest, has improved compared with the control 52%; At the 5th day, under the concentration of 0.5g/L, improved 56% than enzyme work.
Table 3
Claims (10)
1. the method that fermentation of Aspergillus niger is produced feruloyl esterase comprises: will carry out fermentation culture in aspergillus niger (Aspergillus niger) the ZJUQH2012147 spore suspension access fermention medium;
Wherein, the preserving number of described aspergillus niger ZJUQH2012147 is CGMCC No.6152;
Be added with microparticle in the described fermention medium; Microparticle is at least a in talcum powder, kaolin, alumina, titanate, quartz sand, carbon dust and the silicon carbide.
2. method according to claim 1 is characterized in that, the inoculum size of described aspergillus niger ZJUQH2012147 spore suspension is 1-10%.
3. method according to claim 1 is characterized in that, in volume 1L, described fermention medium comprises: the vat liquor 200-300mL of dregs of beans, wheat bran, corn ear or corn stalk, nitrogenous source 5-20g, inorganic salt 5-15g.
4. method according to claim 1 is characterized in that, described microparticle is at least a in talcum powder, kaolin and the alumina.
5. method according to claim 1 is characterized in that, the particle diameter of described microparticle is the 100-10000 order.
6. method according to claim 1 is characterized in that, the concentration of described microparticle in fermention medium is 0.1-100g/L.
7. method according to claim 1 is characterized in that, described microparticle adds in the fermention medium before fermentation culture.
8. method according to claim 1 is characterized in that, described microparticle added in the fermention medium in fermentation culture in 1-2 days.
9. method according to claim 1 is characterized in that, the culture temperature of described fermentation culture is 25-30 ℃, and incubation time is 3-7 days.
10. method according to claim 1 is characterized in that, described fermentation culture is the shaking table shaking culture, and shaking speed is 100-200r/min.
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| CN113186236A (en) * | 2021-05-28 | 2021-07-30 | 汕头大学 | Method for increasing yield of monascus yellow pigment by adding particles into monascus fermentation broth |
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| CN102796670A (en) * | 2012-07-25 | 2012-11-28 | 浙江大学 | Aspergillus niger strain and application thereof |
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Application publication date: 20130501 |