CN102796716A - Method for preparing tannase - Google Patents

Abstract

The invention, relating to tannase, provides a method for preparing tannase, comprising the following steps: inoculating aspergillus niger in a medium, conducting fermental cultivation to obtain a medium containing tannase, and separating the medium containing tannase to obtain the tannase. The medium is a mixture of tea stalk powder and a salt solution. The step of separating the medium containing tannase comprises: adding a citric acid-sodium citrate buffer in the medium containing tannase, oscillating and extracting, filtering with filter paper to obtain a crude enzyme liquid; decoloring the crude enzyme liquid, filtering and conducting sterilization; and carrying out fractional precipitation on the crude enzyme liquid with ammonium sulfate or conducting ultrafiltration and concentration to obtain the tannase. According to the invention, the innovative use of solid fermentation of tea stalks for producing tannase opens up a new approach for resource utilization of agricultural waste tea stalks; the conditions of using solid fermentation of tea stalks for producing tannase are optimized, the enzyme activity under optimized conditions reaches 62U/gds, which is 5.17 times the enzyme activity before optimizing.

Description

A kind of method for preparing tannase

Technical field

The present invention relates to tannase, particularly a kind of method for preparing tannase.

Background technology

Tannase (Tannase, EC 3.1.1.20) full name tannin ester acyl lytic enzyme can generate gallic acid and glucose by the gallic acid by hydrolyzing tannin.Tannase has distribution quite widely at occurring in nature, the people is just arranged carrying out finding that mycelia contains tannase when black mold is cultivated as far back as the twenties in 20th century, finds each quasi-microorganism later on again successively, comprises that fungi, yeast, bacterium can both produce tannase; Tannase also is present in some vegetable materials that are rich in tannin, for example can detect tannase in the tea leaf extract.

Raising along with expanding economy and people's living standard; Tannase has been widely used in fields such as beverage, wine brewing, food, medicine, chemical industry, process hides, makeup; Particularly in preparation medicinal intermediate gallic acid and Food Antioxidant Propyl Gallate; And, significant application value is arranged at aspects such as " cream down " of handling tealeaves and beer depositions.But the efficient of producing tannase at present is not high, and market value is expensive, has hindered the application of tannase.

Tea be China special product, YO has reached more than 100 ten thousand tons, in the Tea Processing process tea stalks produce thereupon and output much larger than tealeaves.Present tea stalks also do not obtain reasonable use the pillow except being used on a small quantity making, and generally are used as agricultural wastes and abandon, and so a large amount of tea stalks are dropped the serious waste that has not only caused resource and have also brought environmental pollution.Contain 18%～36% tannin in the tea stalks, tannase is a kind of inducible enzyme, and inductor is a tannin, and therefore, the tea stalks that are rich in tannin can induce black mold to produce tannase as a kind of basic medium well.

Summary of the invention

The object of the present invention is to provide a kind of method for preparing tannase.

The said method for preparing tannase comprises: black mold is inoculated in the substratum, obtains to contain the tannase substratum after the fermentation culture, separate and contain tannase substratum acquisition tannase.

Said black mold available from Chinese industrial microbial strains preservation administrative center (China Center of Industrial Culture Collection, CICC).Said substratum is the mixture of tea stalks powder and salts solution.Comprise NH in the said salts solution ₄NO ₃, MgSO ₄7H ₂O, KH ₂PO ₄, NaCl, the NH in the said salts solution ₄NO ₃, MgSO ₄7H ₂O, KH ₂PO ₄, NaCl massfraction (w/w) be followed successively by 1%, 0.1%, 0.1%, 0.1%.The quality of said tea stalks is 1g ︰ (1-2) mL with the ratio of the volume of salts solution, preferred 1g ︰ 1.8 mL.

The condition of said fermentation culture is salts solution pH 6.06, the ratio of salts solution and substratum (v/w) 1.8,28 ℃ of culture temperature, inoculum size 2 mL (1 * 10 ⁸Individual spore/mL), the NH of 5.69% (w/v) ₄Cl is as nitrogenous source, and the alpha-lactose of 5% (w/v) is as additional carbon, 250 mL triangular flask loading amounts, 5 g, and incubation time is 96 h.

The step that said separation contains the tannase substratum comprises: the adding of citric acid-sodium citrate damping fluid contained in the tannase substratum, and the vibration lixiviate, filter paper filtering promptly gets crude enzyme liquid.With the gained crude enzyme liquid decolour, filtration sterilization.The gained crude enzyme liquid carries out ammonium sulfate precipitation or ultrafiltration and concentration again, gets tannase.

Novelty of the present invention ground adopts the tea stalks solid state fermentation to produce tannase, has opened up the new road of the agricultural wastes tea stalks utilizations of resources.The present invention has optimized the condition that the tea stalks solid state fermentation is produced tannase, and enzyme work reaches 62 U/gds under optimized conditions, is 5.17 times before optimizing.Improve the yield of fermentative prodn tannase, reduced cost.

Description of drawings

Fig. 1 is a gallic acid visible region abosrption spectrogram.In Fig. 1, X-coordinate is wavelength (wavelength), and unit is nm, and ordinate zou is absorbancy (Abs).

Fig. 2 is wavelength 520 nm place gallic acid canonical plottings.In Fig. 2, X-coordinate is a gallic acid concentration, and unit is μ mol/L, and ordinate zou is an absorbancy, and linear regression equation is y=0.0016x-0.0358, coefficient R ²=0.9992.

Fig. 3 is tannin enzyme activity and a living weight change curve under the starting condition.In Fig. 3, X-coordinate is fermentation time (h), and left ordinate zou is tannase vigor (U/dgs), and right ordinate zou is GS content (mg/gds).Curve ▲ be the tannase vigor, curve ■ is a GS content.

Fig. 4 is that the tea stalks content of powder is to producing the figure that influences of enzyme.In Fig. 4, X-coordinate is tea stalks content of powder (%), ordinate zou tannase vigor (U/dgs).

Fig. 5 is that salts solution pH is to producing the figure that influences of enzyme.In Fig. 5, X-coordinate is salts solution pH, ordinate zou tannase vigor (U/gds).

Fig. 6 is that degree of wetting is to producing the figure that influences of enzyme.In Fig. 6, X-coordinate is liquid-solid ratio (being the ratio of salts solution and tea stalks powder), ordinate zou tannase vigor (U/gds).

Fig. 7 is that loading amount is to producing the figure that influences of enzyme.In Fig. 7, X-coordinate is loading amount (g), ordinate zou tannase vigor (U/gds).

Fig. 8 is that temperature is to producing the figure that influences of enzyme.In Fig. 8, X-coordinate be temperature (℃), ordinate zou tannase vigor (U/gds).

Fig. 9 is that inoculum size is to producing the figure that influences of enzyme.In Fig. 9, X-coordinate is inoculum size (mL), ordinate zou tannase vigor (U/gds).

Figure 10 is that different nitrogen sources is to producing the figure that influences of enzyme.In Figure 10, and the different nitrogenous source of X-coordinate (1%, w/v), from left to right be followed successively by analysis for soybean powder, peptone, NaNO ₃, NH ₄NO ₃, (NH ₄) ₂SO ₄, urea, NH ₄Cl, ordinate zou tannase vigor (U/gds).

Figure 11 is NH ₄The Cl addition is to producing the figure that influences of enzyme.In Figure 11, X-coordinate is NH ₄The Cl addition (%, w/v), ordinate zou tannase vigor (U/gds).

Figure 12 is that the different carbon sources of extra interpolation are to producing the figure that influences of enzyme.In Figure 12, X-coordinate is different carbon sources, is followed successively by from left to right, does not add the control group of extra carbon source, glucose, sucrose, alpha-lactose, SANMALT-S, Zulkovsky starch, ordinate zou tannase vigor (U/gds).

Figure 13 is that the alpha-lactose addition is to producing the figure that influences of enzyme.In Figure 13, X-coordinate be the alpha-lactose addition (%, w/v), ordinate zou tannase vigor (U/gds).

Figure 14 is different N H ₄The Cl addition is to producing the figure that influences of enzyme.In Figure 14, X-coordinate is NH ₄The Cl addition (%, w/v), ordinate zou tannase vigor (U/gds).

Figure 15 is that salts solution pH is to producing the figure that influences of enzyme.In Figure 15, X-coordinate is salts solution pH, ordinate zou tannase vigor (U/gds).

Figure 16 is tannin enzyme activity and a living weight change curve under the optimal conditions.In Figure 16, X-coordinate is fermentation time (h), and left ordinate zou is tannase vigor (U/dgs), and right ordinate zou is GS content (mg/gds).Curve ▲ be the tannase vigor, curve ■ is a GS content.

Embodiment

For making those skilled in the art can further understand the present invention, enumerate specific embodiment of the present invention below, and cooperate Figure of description, specify technical scheme of the present invention.Need to stress: embodiment is not the exhaustive of the possible embodiment of technical scheme of the present invention institute, so be not used in restriction protection scope of the present invention.

Embodiment

1 materials and methods

1.1 bacterial classification

Black mold ( Aspergillus niger) (China Center of Industrial Culture Collection, CICC), bacterial classification is kept on the potato dextrose agar slant medium under 4 ℃ of conditions from Chinese industrial microbial strains preservation administrative center in purchase.

1.2 substratum

Slant medium: take by weighing 200 g yams, clean the peeling chopping, add water 1000 mL and boil half hour; Filtered through gauze; Filtrating adds 20 g glucose again and 20 g agar continue to boil, and fully dissolves back packing test tube, about 5 mL of every test tube; Take out test tube pendulum inclined-plane behind 121 ℃ of sterilization 20 min, the cooling back is stored subsequent use.

Basic medium: the tea stalks oven dry is pulverized, and the tea stalks powder that 120 order separation sieves leak down is basic medium.

Fermention medium: take by weighing 5 g tea stalks, the powder that 120 order separation sieves leak down is pulverized in oven dry, as medium base, takes by weighing 1% (w/w) NH in the triangular flask of 250 mL that pack into ₄NO ₃, 0.1% (w/w) MgSO ₄7H ₂O, 0.1% (w/w) KH ₂PO ₄, 0.1% (w/w) NaCl, add 5 mL zero(ppm) water, regulating the initial pH of salts solution is 5.0, salts solution is added in the triangular flask and stirs with the tea stalks powder, 121 ℃ of sterilization 20 min.

1.3 main agents

Tenox PG, rhodanine are purchased in Tokyo HuaCheng Industry Co., Ltd; Tea stalks are purchased in Anxi County tea place, Quanzhou City, Fujian Province; Gallic acid is purchased the traditional Chinese medicines Group Co.,Ltd in Shanghai; Methyl ethyl diketone is purchased in Chemical Reagent Co., Ltd., Sinopharm Group; Para diaminobenzene formaldehyde is purchased the fine chemicals engineering and technological research development centre in Guangdong Province; Analysis for soybean powder is purchased in the Xiamen City, Fujian Province everybody happy supermarket of Jimei District; Other reagent, an ammonium nitrate, SODIUMNITRATE, ammonium sulfate, ammonium chloride, SANMALT-S, alpha-lactose, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5, potassium primary phosphate, sodium-chlor etc. are purchased the traditional Chinese medicines Group Co.,Ltd in Shanghai; Reagent is analytical pure.

1.4 main solution and preparation thereof

The citrate buffer solution of (1) 0.05 mol/L pH 5.0

Accurately take by weighing 2.6274 g Hydrocerol As with zero(ppm) water constant volume in 250 mL volumetric flasks; Take by weighing 7.3550 g Trisodium Citrates again with zero(ppm) water constant volume in 500 mL volumetric flasks.Then two kinds of solution are approximately mixed according to volume ratio 1:2, utilize these two kinds of solution that the pH value of mixing solutions is adjusted to 5.0 respectively;

The Tenox PG of (2) 0.01 mol/L (PG) solution

Accurately take by weighing citrate buffer solution in 100 mL volumetric flasks the constant volume of 0.212 g PG, hydrotropy in 50 oC thermostat water baths with pH 5.0;

The methyl alcohol rhodanine solution of (3) 0.667% (w/v)

Accurately take by weighing 0.667 g rhodanine, with methyl alcohol constant volume in 100 mL volumetric flasks;

(4) 0.7 mol/L KOH solution

Accurately take by weighing 3.9277 g KOH with zero(ppm) water constant volume in 100 mL volumetric flasks;

(5) 1 mol/L NaOH solution

Accurately take by weighing 40 g NaOH and be settled to 2 L with zero(ppm) water;

1.5 TP

1.5.1 the preparation of monospore bacteria suspension

With 4 ℃ down the slant strains of storage be inoculated in and cultivate 4 d down at 30 ℃ on the slant medium and obtain ripe spore, wash spore with sterile distilled water, be put in the Erlenmeyer flask that sterile glass beads is housed, 30 min fully vibrate.After treating that spore is fully broken up, survey bacteria suspension OD ₆₀₀Value, and adjust it with SPSS ODValue to 2.0 promptly gets the monospore bacteria suspension.

1.5.2 tea stalks solid state fermentation

After fermention medium prepares, 121 ℃ of sterilization 20 min, the cooling back is inoculation 1 mL aspergillus niger spore suspension (1 * 10 under aseptic condition ⁸Individual/mL), mixing is placed on to leave standstill in 30 ℃ of incubators cultivates 120 h.

1.5.3 gallic acid absorption peak wavelength determination

Compound concentration is 10 ^-4The gallic acid aqueous solution of mol/L adds in the gallic acid solution and test tube of 0.5 mL, adds 0.3 mL methyl alcohol rhodanine (0.667% then; W/v) solution; Test tube is put into 30 ℃ of water-bath insulation 5 min, then in test tube, adds the 0.7 mol/L KOH solution of 0.2 mL again, and test tube is incubated 5 min again; Add 4 mL distilled water dilutings at last, survey peak value.

1.5.4 gallic acid standard curve making

With the citric acid solution preparation gallic acid standardized solution of pH 5.0, from 12 different concentration of 40-260 μ mol/L preparation.(0.667%, w/v), test tube reacts 5 min to adding 0.3 mL methyl alcohol rhodanine solution under 30 ℃ of conditions after in test tube, adding 0.5 mL gallic acid solution.The 0.7mol/L KOH solution of 0.2 mL is added in all test tubes; Constant temperature 5 min under 30 ℃ of conditions once more; Add 4 mL zero(ppm) water in the last test tube; Is blank with zero(ppm) water, under 520 nm, measures the light absorption value of the correspondence of each concentration, obtain gallic acid solution typical curve with spectrophotometer.

1.5.5 crude enzyme liquid extracts

Take out the substratum of having cultivated the product tannase of 120 h in the constant incubator, in triangular flask, add the citric acid-sodium citrate damping fluid of 100 mL pH 5.0,180 r/min vibration lixiviate, 1 h promptly gets crude enzyme liquid with the qualitative filter paper filtration under 30 ℃ of conditions.

1.5.6 the mensuration of tannase vigor

According to Sharma reported method (SHARMA S; BHAT T; DAWRA R. A spectrophotometric method for assay of tannase using rhodanine [J]. Analytical Biochemistry; 2000,279 (1): 85-89.) measure the tannase vigor.The principle of the method is: gallic acid with form look group material around the pellet effect, measure the tannase vigor based on the variation of look group material.The test tube of getting three cleanings is labeled as blank pipe, testing tube, control tube respectively.Tenox PG solution and the crude enzyme liquid of 0.01 mol/L were put into 30 ℃ of thermostat water bath preheating 10 min earlier before the reaction beginning.In three test tubes that are labeled, all add 0.25 mL Tenox PG solution; Then 0.25 mL citrate buffer solution is added in the blank pipe; 0.25 the mL crude enzyme liquid joins in the testing tube, and three test tubes are all placed 30 ℃ of water-bath insulation 5 min, in all test tubes, all adds 0.3 mL methyl alcohol rhodanine (0.667% w/v)) solution; Add the methyl alcohol rhodanine all keep 5 min in 30 ℃ of water-baths; KOH solution 0.2 mL that adds 0.7 mol/L after insulation is accomplished keeps 5 min in 30 ℃ of water-baths, only in control tube, add 0.25 mL crude enzyme liquid then.All test tubes are all used 9 mL distilled water dilutings at last, behind insulation 10 min under 30 ℃, under 520 nm wavelength, do blank with zero(ppm) water, measure light absorption value.3 parallel laboratory tests are all done in all tests, get arithmetical av.PM under 30 ℃ of conditions is produced the required enzyme amount of 1 μ mol gallic acid be defined as enzyme unit alive.The vigor of tannase is calculated by the variation of light absorption value:

△ A ₅₂₀=(A? _test-A? _blank)-(A? _control?-?A? _blank)

1.5.7 the mensuration of living weight

According to document (Wei Peilian; Cen Peilin; Sheng Chunqi. the comparison [J] of 3 kinds of solid state fermentation living weight mensuration methods. food and biotechnology journal, 2006,25 (1)) measure living weight through the content of measuring GS: precision takes by weighing dried fermented product 0.3 g and adds 2 mL, 60% H ₂SO ₄Solution, 25 ℃ are soaked 24 h, the mixture diluted after the immersion to H ₂SO ₄Concentration be 1 mol/L, place 250 mL triangular flasks, 9.8 * 10 ⁴The Pa high pressure heats 1 h, and the cooling back is neutralized to pH 7.0 with 1 mol/L NaOH solution, and constant volume is to 100 mL.Get 2 mL appearance liquid (blank is 2 mL zero(ppm) water) and add 1 mL methyl ethyl diketone reagent (3.5 mL methyl ethyl diketones+50 mL, 1.2 mol/L Na ₂CO ₃Solution), boiling water bath heats 30 min, and the cooling back adds 2 mL absolute ethyl alcohols, (1.333 g para diaminobenzene formaldehyde are dissolved in the mixed solution of 25 mL absolute ethyl alcohols and 25 mL concentrated hydrochloric acids 1 mL para diaminobenzene formaldehyde reagent; Preserve in the brown bottle; Existing with join at present), vibration adds 4 mL absolute ethyl alcohols again; 60 ℃ of insulation 1 h, 495 nm places measure absorbance.

1.5.8 single factor optimization of tannase zymotechnique

Tea stalks powder 5 g, 1% (w/w) NH ₄NO ₃, 0.1% (w/w) MgSO ₄7H ₂O, 0.1% (w/w) KH ₂PO ₄, the initial pH of 0.1% (w/w), 5 mL zero(ppm) water, solution 5.0,30 ℃ of leavening temperatures, fermentation time 120 h be optimized as initial fermentation condition.Have fermentation time (0 –, 168 h), tea stalks content of powder (0 – 100%), loading amount (3 –, 13 g), the initial pH of salts solution (3 – 8), degree of wetting (the ratio 1-2 of salts solution and substratum), leavening temperature (33 ℃ of 20 –), inoculum size (0.5 –, 3 mL) in optimizing, add nitrogenous source and additional carbon.The influence of independent a certain factor and the result that the optimization in each step all utilizes one-step optimization to go out are only surveyed in each step optimization.

2 results and analysis.

2.1 the making of gallic acid typical curve

The look group material property absorption peak of mentioning the formation of gallic acid and rhodanine in the Sharma method is rolled into a ball the charateristic avsorption band of material at 520 nm in order to detect this look, has carried out the surface sweeping of visible region wavelength in the experiment, and the surface sweeping result is as shown in Figure 1.Can know charateristic avsorption band that look that gallic acid and rhodanine form rolls into a ball material at 520 nm by figure, this explains that this measuring method is a vigor feasible, that can be used to measure tannase under this experiment condition.

Accurately measure the tannase vigor, need to make a gallic acid typical curve.The gallic acid typical curve of making is as shown in Figure 2.Can know the wavelength 520 nm place gallic acid concentration light absorption value linear dependence corresponding by figure with it.The gained data are carried out obtaining behind the linear regression analysis linear regression equation:

y?=?0.0016x?-?0.0358?(R ²?=?0.9992)

Coefficient R ²≈ 1, shows that gallic acid concentration (x) light absorption value (y) linear dependence corresponding with it is remarkable.

The 5 g tea stalks powder of in the triangular flask of 250 mL, packing into after the salts solution that adds 5 mL pH 5.0 stirs and sterilizes, insert the spore liquid of 1 mL, under 30 ℃ of conditions, cultivate 168 h, and per 24 h sampling is once surveyed tannase vigor and living weight.Living weight is to confirm through the content of measuring amino grape.GS is the element of cell walls, and the amount of its content and cell is proportionate, thus can through the GS content of measuring cell walls confirm the black mold living weight how much.Test result alive to enzyme in the fermenting process and living weight is as shown in Figure 3.Fig. 3 shows that along with the prolongation of time, tannase output increases, and the tannase vigor reaches maximum (12 U/gds) when fermentation 120 h.By scheming to see that also producing the enzyme curve is the same with the living weight variation tendency, enzyme is lived and living weight all is at 120 h places peak to be arranged, and produces enzyme and behind 120 h, slightly descends, and living weight also is to tend towards stability and slightly decline at 120 h.This explanation is in the fermenting process of tannase, and yield of enzyme and living weight are proportionate.

2.3 the tea stalks content of powder is to producing the influence of enzyme

The experiment of tea stalks powder and wheat bran co-fermentation has been carried out in this research; Tea stalks powder and wheat bran be totally 5 g; Make the tea stalks powder account for total material proportion (tea stalks powder quality/5 g * 100%) and be respectively 10%, 30%, 50%, 60%, 70%, 80%, 90%, 100%; At 30 ℃ of condition bottom fermentations, 120 h that ferment, measure the tannase vigor, the result is as shown in Figure 4.Can be seen that by Fig. 4 along with the raising of tea stalks powder proportion, the tannase vigor raises gradually, be 100% to be basic medium when all not adding wheat bran with the tea stalks powder when the tea stalks content of powder, and the tannase vigor is maximum.Based on above result, it is 100% that subsequent experimental is selected the tea stalks content of powder.

2.4 salts solution pH is to producing the influence of enzyme

The initial pH that regulates salts solution respectively is 3.0,4.0,5.0,6.0,7.0 and 8.0, adds 5 mL salts solutions in every flask culture, and spore suspension 1 mL is inserted in the sterilization back, cultivates 120 h for 30 ℃, extracts crude enzyme liquid and surveys enzyme activity, and the result sees Fig. 5.Can be known that by Fig. 5 the pH 6.0 of salts solution helps black mold most to produce tannase, under this pH condition, tannase has maximum vigor 16 U/gds.Can find out also that from Fig. 5 pH 5.0-6.0 is the environment that relatively is fit to produce enzyme.Based on above result, it is 6.0 that subsequent experimental is selected the pH of salts solution.

2.5 degree of wetting is to producing the influence of enzyme

The pH of salts solution is adjusted to 6.0; Make the ratio (v/w) of salts solution and solid medium be 1:1,1.3:1,1.5:1,1.8:1 and 2:1 respectively; Be added to salts solution in proportion in the solid medium, spore suspension 1 mL is inserted in the sterilization back that stirs, and cultivates 120 h for 30 ℃; Extract crude enzyme liquid and survey enzyme activity, the result sees Fig. 6.Can know when liquid-solid ratio is 1.8:1 that by Fig. 6 tannase has maximum vigor (17 U/gds),, degree of wetting is crossed the low or too high enzyme that all is unfavorable for producing.Water is the essential composition of cell synthetic, and when carrying out solid state fermentation, the degree of wetting of substratum is a very The key factor, affects microbial growth and produces enzyme.To cross when low in the solid medium water cut low when degree of wetting, thereby do not have competent water to supply the microorganism growth utilization to influence the product enzyme.Water cut is higher in the substratum when degree of wetting is too high, so just cause substratum stir uneven, agglomerating phenomenon is serious, when substratum is agglomerating, is enclosed in inner mikrobe and can not get sufficient oxygen and supply growth utilization, has hindered the product enzyme.Based on above result, it is 1.8:1 that subsequent experimental is selected the ratio of salts solution and solid medium.

2.6 loading amount is to producing the influence of enzyme

In 250 mL triangular flasks, adorn substratum 3 g, 5 g, 7 g, 9 g, 11 g and 13 g respectively, inoculation is placed on 30 ℃ and cultivates down in proportion, cultivates the vigor of measuring tannase in the substratum behind 120 h, and the result is as shown in Figure 7.Result by Fig. 7 can find out that loading amount is produced tannase to the tea stalks solid state fermentation has very remarkable influence, and the synthetic of tannase lacked and more helped to loading amount.For black mold, competent oxygen supply helps the synthetic of tannase; Few loading amount then helps distributing of substratum internal heat, prevents that temperature is too high and causes the bad and influence of thalli growth to produce enzyme.But loading amount very little, increased the area of solid fermentation, is unfavorable for the reduction of production cost.In the large-scale production process of reality, can come the improvement condition through adding forced ventilation and moisturizing.Taking all factors into consideration this experiment still selects loading amount at 5 g.

2.7 temperature is to producing the influence of enzyme

Optimize in former steps under the condition that, make solid state fermentation under the temperature condition of 20 ℃, 22 ℃, 25 ℃, 28 ℃, 30 ℃ and 33 ℃, carry out respectively, cultivate the vigor of measuring tannase in the substratum behind 120 h, temperature is as shown in Figure 8 to the influence of producing enzyme.The result of Fig. 8 shows that tannase has maximum vigor (18 U/gds) when culture temperature is 28 ℃.Observe in the culturing process and find, the too low black mold poor growth of temperature, mycelia is seldom; 25 ℃～30 ℃ these scopes relatively are fit to produce enzyme, and the black mold growth is vigorous; Black mold was grown fast when temperature was too high, but spore is too many, and media surface has been full of spore.The result is that the optimum temperuture of black mold product tannase is 28 ℃ in this research, and based on above result, it is 28 ℃ that subsequent experimental is selected culture temperature.

2.8 inoculum size is to producing the influence of enzyme

Use sterile distilled water to wash the aspergillus niger spore on the inclined-plane that to process concentration be 1 * 10 ⁸The aspergillus niger spore suspension of individual/mL adds 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL and 3 mL spore suspensions respectively in 5 g substratum.Under 28 ℃ of conditions, measure the vigor of tannase in the substratum behind cultivation 120 h, inoculum size is as shown in Figure 9 to the influence of producing enzyme.Can find out that by Fig. 9 enzyme activity is the highest when inoculating 2 mL aspergillus niger spore suspensions, is 18 U/gds.A suitable inoculum size is very important during the fermentation.The spore suspension that inserts more for a long time, mycelial growth is very fast, possibly be the meta-bolites accumulation too much, can suppress the growth of later stage thalline on the contrary, thereby suppress the product enzyme in later stage.Inoculum size is too little, and mycelial growth is slow, has prolonged growth cycle and has caused yield of enzyme to reduce.Based on above result, it is 2 mL that subsequent experimental is selected inoculum size.

2.9 different nitrogen sources is to producing the influence of enzyme

In substratum, add 1% different nitrogen sources respectively: analysis for soybean powder, peptone, NaNO ₃, (NH ₄) ₂SO ₄, urea, NH ₄Cl replaces NH ₄NO ₃, shown in figure 10 to the influence of producing tannase.Can know by figure, add NH ₄It is the most desirable to produce the enzyme situation during Cl.Further studied different NH ₄The Cl addition is to producing the influence of enzyme, and the result is shown in figure 11.When the research different nitrogen sources influences producing enzyme, add NH ₄It is the most desirable to produce the enzyme situation during Cl, and this moment, maximum enzyme work was 25 U/gds.Further studied different NH ₄The Cl addition is worked as NH to producing the influence of enzyme ₄The addition of Cl is to have maximum enzyme live at 5% o'clock, and this moment, enzyme work was 37 U/gds.Work as NH ₄Enzyme was lived and is sharply descended when the addition of Cl increased in 5% continued.Based on above result, subsequent experimental selects to add 5% NH ₄Cl replaces original NH ₄NO ₃

2.10 extra interpolation carbon source is to producing the influence of enzyme

The different carbon sources of extra interpolation under the situation of inductor tea stalks powder as carbon source; Be respectively glucose, sucrose, alpha-lactose, SANMALT-S and Zulkovsky starch; Addition is 1%; Study producing the enzyme situation, the result is shown in figure 12 for the product enzyme, can know that by figure the substratum product enzyme situation of having added alpha-lactose is best.Further studied different alpha-lactose additions to producing the influence of enzyme, the result is shown in figure 13.When the extra interpolation carbon source of research influenced producing enzyme, it is best as producing enzyme under the situation of additional carbon that substratum adds 1% alpha-lactose, and this moment, maximum enzyme work was 45 U/gds.Further studied the alpha-lactose addition to producing the influence of enzyme, when addition is 5%, had maximum enzyme to live, this moment, enzyme work was 53 U/gds.After the addition of alpha-lactose is greater than 5%, lives and reduce on the contrary along with the increase enzyme of addition.Based on above result, subsequent experimental selects extra interpolation 5% alpha-lactose as additional carbon.

2.11 black mold utilizes tea stalks powder solid state fermentation to produce the response surface optimization of tannase

Influence black mold and utilize the principal element of tea stalks powder solid state fermentation product tannase to be fermentation time, culture temperature, salts solution pH, initial wetting degree, inoculum size, loading amount and additional carbon nitrogenous source, table 2.2 has been listed the result that these factors are optimized.

The single factor optimum result of table 1

Parameter	Parameter area	Optimum value
			Fermentation time (h)	0-168	120
Tea stalks content of powder (%)	10-100	100
			Salts solution pH	3-8	6
Liquid-solid ratio	1-2	1.8
			Loading amount (g)	3-13	5
Culture temperature (℃)	20-33	28
			Inoculum size (mL)	0.5-3.5	2
The nitrogenous source kind	Ammonium chloride, an ammonium nitrate, ammonium sulfate, SODIUMNITRATE, analysis for soybean powder, peptone	Ammonium chloride
			Ammonium chloride concentration (%)	0.5-20	5
The additional carbon kind	Glucose, sucrose, SANMALT-S, alpha-lactose, Zulkovsky starch	Alpha-lactose
			Alpha-lactose concentration (%)	0-20	5

Carry out variance analysis with 17.0 pairs of experiment of single factor data of SPSS; With qualitative factor fermentation time 120 h, 250mL triangular flask loading amount 5 g, 28 ℃ of leavening temperatures, nitrogenous source kind ammonium chloride, additional carbon kind alpha-lactose with influence less factor substratum initial wetting degree, inoculum size, alpha-lactose concentration and be taken as constant, select the variables NH of remarkably influenced ₄Cl addition and salts solution pH are used for the response surface design.

For more approaching maximum respective regions, to NH ₄These two factors of Cl addition and salts solution pH have carried out dwindling the experiment of horizontal extent again, and experimental result is shown in Figure 14 and 15.

Optimization Experiment Design adopts Response Surface Method (RSM), and used software is Design-Expert 8.0, implementation model for center combination design (Central Composite Design, CCD).NH is chosen in this test ₄2 factors of Cl addition and salts solution pH adopt the response surface analysis method of 2 factors, 3 levels, are that response value is done the response surface design with the tannase vigor, and fermentation condition is optimized, and need 13 tests altogether.Choose the experiment of factor level design response surface according to phase shipping order factor result, empirical factor and design levels see Table 3.NH ₄Cl addition and salts solution pH convert as follows: X ₁=NH ₄The Cl addition, X ₂=salts solution pH.By EQUATION x _i=(X _i-X ₀)/X encodes to independent variable(s), x _iBe the encoded radio of independent variable(s), X _iBe actual value.X ₀Be the actual value of independent variable(s), experimental center Dian Chu just, X is the change step of independent variable(s) in the test.

Table 2 response surface design factor water-glass

(1) foundation of regression model and test of significance

MV with 3 experiment gained tannase enzyme activities is response value (Y).The center combination design is seen table 3 with the result.

Experimental design of table 3 response surface and result

After adopting Design-Expert 8.0 program his-and-hers watches, 3 response values and each factor to carry out regression fit, obtain the tannase vigor to NH ₄Cl addition (x ₁) and salts solution pH (x ₂) the multinomial regression equation of secondary of encoded radio is: Y=55.10+2.08x ₁+ 8.824 * 10 ^-3x ₂+ 0.89 x ₁x ₂-2.36 x ₁ ²-2.41x ₂ ²

This model is carried out variance analysis, and the result sees table 4, and the coefficient test of significance result of regression equation sees table 5.Can find out by table 4, model P=0.0005 0.01, show that regression model is extremely remarkable, lose and intend a P=0.1671 0.05, it is not remarkable that model loses the plan degree.Coefficient R ²=0.9337,93.37% testing data equation explanation thus is described, the model-fitting degree is good; Experimental error is little; This model is fit to, and regression equation can be described the true relation between each factor and the response value preferably, can utilize this model to confirm optimal conditions of fermentation.The adjustment coefficient of determination R of model _Adj ²Value explains that for=0.8864 the variation of response value has 88.64% to derive from selected independent variable(s).The tolerance range of the variation coefficient (CV) expression test, its value is big more, and the safety of test-results is low more.This test CV=2.00% in tolerance interval, explains that test-results is reliable, can produce the work of tannase enzyme to tea stalks powder solid state fermentation with this model and analyze and predict.

Can know an once x of model by the test of significance of regression equation coefficient ₁(P<0.01) influence extremely remarkable, x ₂(P>0.05) influence not remarkable; Quadratic term x ₁ ², x ₂ ²(P<0.01) influence all extremely remarkable; A mutual x ₁x ₂(P>0.05) influence not remarkable.Estimated value x according to equation coefficient ₁=2.08 and x ₂=0.0088, can know the influence NH that the tannase enzyme is lived ₄The Cl addition is greater than salts solution pH.

Table 4 regression model analysis of variance table

Annotate: * * is extremely significantly (P < 0.01), * be significantly under (P < 0.05) together.

Table 5 regression equation coefficient test of significance table

Coefficient entry	The coefficient estimation value	Degree of freedom	Standard error	The F value	The P value	Significance
							Constant term	55.10	1	0.47	?	?	?
x 1	2.08	1	0.37	31.76	0.0008	**
							x 2	0.0088	1	0.37	0.0006	0.9816	?
x 1x 2	0.89	1	0.52	2.91	0.1318	?
							x 1 2	-2.36	1	0.40	35.41	0.0006	**
x 2 2	-2.41	1	0.40	36.90	0.0005	**

(2) confirmatory experiment

Utilize the numerical characteristic of Design expert 8.0.5.0, the minimum and maximum value of each factor and the span of tannase vigor are seen table 6.

The optimization range of table 6 maximum enzyme vigor

Project	NH 4Cl addition (%)	Salts solution pH	Tannase vigor (U/gds)
				Target	Scope	Scope	Peak
Lower limit	3.5	5.25	46.321
				The upper limit	6.5	6.75	200

Draw at NH through software ₄Under Cl addition 5.69%, salts solution pH 6.06 conditions, the predictor Y that maximum tannase is lived is 55.58 U/gds.Safety for check response surface method gained result; Under the top condition that above-mentioned response surface analysis method is tried to achieve, carry out solid state fermentation (repeated experiments 5 times); The actual average tannase vigor that records is 56.7 U/gds; With the theoretical prediction value mutually ratio error be 2.0%, experimental value meets predictor in 95% fiducial interval, thus the model that shows the optimization of response surface analysis method is suitable for tea stalks powder solid-state fermentation process.Therefore, the tea stalks powder solid-state fermentation process parameter of optimizing based on response surface has practical value accurately and reliably.

2.12 the time curve of tannase under the optimized process conditions

Comprehensive above parameters, 5 g tea stalks powder as basic medium, salts solution pH be 6.06, liquid-solid ratio is the NH of 1.8:1, adding 5.69% ₄The alpha-lactose that Cl does nitrogenous source, adding 5% is 28 ℃ as extra carbon source, inoculum size 2 mL, culture temperature.Adopt the fermentation condition after optimizing to utilize the tea stalks powder to carry out solid state fermentation to black mold, fermentation time is 168 h, and whenever at a distance from 24 h sampling one-time detection tannase activity and living weight, the result is shown in figure 16.The result shows 96 h after the vertex of enzyme activity appears at fermentation; Be 62 U/gds; It is thus clear that the condition after employing is optimized is fermented and not only shortened fermentation period but also improved enzyme activity greatly, enzyme activity 62 U/gds are (12 U/gds) before optimizing 5.17 times.The trend that also can be found out black mold product tannase under optimized conditions by figure is consistent with living weight, and yield of enzyme and living weight are proportionate in the fermenting process of tannase, and this is consistent with optimizing preceding result.

3 brief summaries

The method that adopts experiment of single factor to combine with response surface optimization; Obtaining black mold utilizes the optimum parameter condition of tea stalks powder solid state fermentation product tannase to be: salts solution pH 6.06; The ratio of salts solution and substratum (v/w) 1.8,28 ℃ of culture temperature, inoculum size 2 mL (1 * 10 ⁸Individual spore/ml), the NH of 5.69% (w/v) ₄Cl is as nitrogenous source, and the alpha-lactose of 5% (w/v) is as additional carbon, 250 mL triangular flask loading amounts, 5 g, and incubation time is 96 h, enzyme work reaches 62 U/gds under optimized conditions), be 5.17 times before optimizing.

4 tannases separate

4.1 the extraction of crude enzyme liquid

Take out the solid medium that contains tannase of having cultivated 120 h in the constant incubator, in triangular flask, add the citric acid-sodium citrate damping fluid of 100 mL pH 5.0,180 r/min vibration lixiviate, 1 h promptly gets crude enzyme liquid with the qualitative filter paper filtration under 30 ℃ of conditions;

4.2 the decolouring of enzyme liquid

The gac that in the crude enzyme liquid that extracts, adds 1% (w/v), 1 h is placed at 4 ℃ in the back that stirs, and filters with qualitative filter paper more afterwards;

4.3 the Sterile Filtration of enzyme liquid

Through the water film suction filtration in 0.8,0.45 and 0.22 μ m aperture, in Bechtop accomplish successively by entire operation for enzyme liquid after the decolouring;

4.4 ammonium sulfate precipitation

In order to confirm an ammonium sulfate saturation ratio scope, tannase is precipitated out in this scope, need it is carried out an ammonium sulfate precipitation.Get 8 parts of first enzyme liquid, every part 100 mL adds (NH respectively ₄) ₂SO ₄Saturation ratio to 30%-100% adds during interpolation while stirring, adds continued and stirs 0.5 h; This operates on the ice bath and carries out, and leaves standstill 3 h then, in 4 ℃, 10; Frozen centrifugation 20 min under 000 * g condition with certain volume 0.01 mol/L citrate buffer solution dissolution precipitation, survey enzyme respectively and live;

4.5 dialysis

(1) pre-treatment of dialysis tubing and preservation

Cut the dialysis tubing of suitable length (10-20 cm), boil 10 min with 2 % (w/v) sodium hydrogencarbonate and 1 mmol/L EDTA (pH 8.0), then with the thorough rinsing of deionized water; Boil 10 min in 1 mmol/L EDTA (pH 8.0) lining again, treat dialysis tubing cooling after, soak to remove Deproteinization with saline water; And rinse well with deionized water; Be placed on 4 ℃ of preservations in 50% ethanol then, guarantee that dialysis tubing is immersed in the liquid all the time, in order to avoid dialysis tubing is killed;

(2) dialysis desalting

Adopt the dialysis tubing dialysis desalting; Deposition is with minimum damping fluid dissolving; Sample after the dissolving is packed in the dialysis tubing of handling, and two ends are sealed with clip, place 4 ℃ of 24 h that dialyse with same buffer; Change damping fluid during this time 4 times, the volume of each damping fluid of changing is about 20 times of the interior sample volume of dialysis tubing.Dialysis is to detecting no SO with BaCl2 ₄ ^2-Till;

4.6 concentrating of enzyme liquid

The A ammonium sulfate precipitation method

Enzyme liquid after the degerming slowly adds the ammonium sulfate through pulverizing while stirring; To saturation concentration be 40%; Continue to stir 0.5 h, this operates on the ice bath and carries out, and leaves standstill 3 h then; It is 90% that the supernatant that frozen centrifugation 20 min collect under 4 ℃, 10,000 * g condition continues to add ammonium sulfate to saturation concentration.Repeat above operation, collecting precipitation dissolves the back with the citrate buffer solution of the pH 5.0 of certain volume to deposition and dialyses;

B ultrafiltration and concentration method

Enzyme liquid through Sterile Filtration directly carries out ultrafiltration, and ultrafiltration is carried out in the chromatography cabinet, and operation steps is following:

(1) flushing of ultra-filtration membrane

Need before the sample ultrafiltration wash ultra-filtration membrane, the pH that uses deionized water rinsing to filtered solution got final product in 7.0 o'clock;

(2) ultrafiltration of sample

The ultra-filtration membrane intake pressure is controlled at 25-30 Psi, and return pressure is controlled at 5-10 Psi, will note liquid volume in the ultrafiltration groove in the ultra-filtration process constantly

(3) cleaning of film and preservation

Using concentration is the NaOH cleaning ultra-filtration membrane of 0.5 mol/L, and scavenging period is 0.5 h, and the pH that uses washed with de-ionized water ultra-filtration membrane to filtered solution then is 7.0, takes off 4 ℃ of preservations of ultra-filtration membrane afterwards.

4.7 the drying of enzyme liquid

The A vacuum lyophilization

The enzyme liquid that concentrates 2,5,10 times is put into freeze drier, dry about 2 d, with the citrate buffer solution dissolving of the solid enzyme that obtains with an amount of pH 5.0, the spectrophotometric determination enzyme activity is used in the back;

The B spraying drying

With ultrafiltration and concentration the enzyme liquid of certain multiple, adjust to certain solid content with stopping composition (like maltodextrin, beta-cyclodextrin, skim-milk etc.) and carry out spraying drying.Spraying drying condition starting condition is: 110 ℃ of EATs, cleansing pin 3 seconds/inferior, input speed 170 mL/h, spraying gun pressure 0.4 MPa.Spray-dired condition can be carried out certain adjustment along with the different needs of experiment.

Above embodiment is to specific descriptions of the present invention, only is used to technical scheme of the present invention is described, and be not the exhaustive of technical scheme of the present invention, so be not used in restriction protection scope of the present invention.

Claims (10)

1. a method for preparing tannase is characterized in that comprising: black mold is inoculated in the substratum, obtains to contain the tannase substratum after the fermentation culture, separate and contain tannase substratum acquisition tannase.

2. the method for preparing tannase as claimed in claim 1 is characterized in that said substratum is the mixture of tea stalks powder and salts solution.

3. the method for preparing tannase as claimed in claim 2 is characterized in that comprising NH in the said salts solution ₄NO ₃, MgSO ₄7H ₂O, KH ₂PO ₄, NaCl.

4. the method for preparing tannase as claimed in claim 3 is characterized in that the NH in the said salts solution ₄NO ₃, MgSO ₄7H ₂O, KH ₂PO ₄, NaCl massfraction be followed successively by 1%, 0.1%, 0.1%, 0.1%.

5. the method for preparing tannase as claimed in claim 1, the quality that it is characterized in that said tea stalks is 1g ︰ (1-2) mL with the ratio of the volume of salts solution.

6. the method for preparing tannase as claimed in claim 5, the quality that it is characterized in that said tea stalks is 1g ︰ 1.8 mL with the ratio of the volume of salts solution.

7. the method for preparing tannase as claimed in claim 1, the condition that it is characterized in that said fermentation culture is salts solution pH 6.06, the ratio 1.8 of salts solution and substratum, 28 ℃ of culture temperature, inoculum size 2 mL, 5.69% NH ₄Cl is as nitrogenous source, and 5% alpha-lactose is as additional carbon, 250 mL triangular flask loading amounts, 5 g, and incubation time is 96 h.

8. the method for preparing tannase as claimed in claim 1 is characterized in that the step that said separation contains the tannase substratum comprises: the citric acid-sodium citrate damping fluid added contains in the tannase substratum, and the vibration lixiviate, filter paper filtering promptly gets crude enzyme liquid.

9. the method for preparing tannase as claimed in claim 8, it is characterized in that with the gained crude enzyme liquid decolour, filtration sterilization, carry out ammonium sulfate precipitation or ultrafiltration and concentration again, tannase.

10. the method for preparing tannase as claimed in claim 8 is characterized in that the gained crude enzyme liquid is carried out ultrafiltration and concentration, gets tannase.

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