CN1670203A - Food grade xylanase and its production method - Google Patents
Food grade xylanase and its production method Download PDFInfo
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- CN1670203A CN1670203A CN 200510042557 CN200510042557A CN1670203A CN 1670203 A CN1670203 A CN 1670203A CN 200510042557 CN200510042557 CN 200510042557 CN 200510042557 A CN200510042557 A CN 200510042557A CN 1670203 A CN1670203 A CN 1670203A
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Abstract
This invention relates to a food-type xylanase and its preparing process, which adopts Bacilluspumilus as bacterial, accomplishes it by culturing, fermenting, purifying and filtering procedures, with liquid fermenting enzyme active being 2500-3500IU/ml, solid fermenting enzyme active being 50000-100000IU/g, optimum temperature being 45-55Deg.C, optimum PH being 6.5-7.5, loss of enzyme active conserved in normal temperature for 180 days less than 5%. The invention selects Bacilluspumilus as bacterial, which is easy to culture, and needs short fermentation period of 14-18 hours; the strain's property is stable, the active of liquid fermenting enzyme is high, up to 2500-35IU/ml, which all lower the cost.
Description
Technical field:
The invention belongs to bioengineering field, relate to the technology that strain fermentation prepares enzyme.
Background technology:
(1,4-β-D-xylan xylanohydrolase is a class xylan degrading enzyme system EC.3.2.18) to zytase, belongs to hydrolase, comprises inscribe beta-xylanase, circumscribed beta-xylanase and β-xylo-bioses glycosides enzyme.The inscribe beta-xylanase acts on the xylan glycosidic bond of xylan backbone, and hydrolyzed xylan cuts off main intrachain glycosidic link at random, and generates the oligosaccharides that molecular weight varies in size.It mainly is that β-xylo-bioses enzyme is that xylo-bioses is hydrolyzed into two monose from non reducing end cutting-out monose one by one that circumscribed beta-xylanase acts on xylan.Zytase is the latest scientific research of modern biotechnology, the known bacterial classification that can produce zytase comprises multiple fungi and bacterium now, used bacterial classification difference, method difference, zytase physico-chemical property, the molecular weight produced have nothing in common with each other, enzyme activity and catalysis characteristics have sizable difference, optimal pH, optimum temperature and metal ion have nothing in common with each other to the influence of enzymic activity, and different ranges of application also has nothing in common with each other to the requirement of zytase.In recent years, xylan has very tempting application prospect in industries such as foodstuffs industry, fodder industry, paper industry, alcohol industry, zytase is applied to foodstuffs industry as food improver abroad, be used for producing xylo-oligosaccharide and make function sex-health products batching, be used as fodder additives to increase its utilization ratio, be applied to be used in the paper industry pre-bleaching to improve pulp property, be used for hydrolysis agricultural organic waste materials and produce alcohol, people also are applied to it in textile industry with other zymin.
Along with the expansion of market to the zytase range of application, both at home and abroad the bacterial screening of zytase and the research work of fermentation condition are also increased successively, comprehensive relevant bibliographical information content as can be seen, the research of current zytase also exists two aspect deficiencies, the one, enzyme activity is on the low side, the highest 450-750IU/ml that is no more than of bibliographical information data zytase liquid fermenting enzyme activity, and the solid fermentation enzyme activity is 4000-5000IU/g, production cost is higher, does not reach the suitability for industrialized production level; The 2nd, the enzyme extraction state of the art is low, and the product major part is yellow or faint yellow, and wherein contains a large amount of substratum residues and other impurity, does not reach the food-grade enzyme preparation standard-required, influences its application on food.
External Ceng Youyong aspergillus niger A3 bacterial strain is produced the report that zytase is used for foodstuffs industry, but it is because low with the xylanase activity power of its fermentative production, 300-400IU/ml is only arranged, production cycle is more than 48 hours, the cost height, the enzyme optimal reaction PH that produces is 4.6, slant acidity, and foodstuffs industry to produce overwhelming majority be neutrality, be used for foodstuffs industry and be not suitable for.
The 02131439.x patent of invention of the 2002.10.14 of Institute of Microorganism, Academia Sinica application discloses a kind of method of utilizing bacillus pumilus (Bacillus pumilus) fermentative production zytase, xylanase activity Li Keda 650～750IU/ml that this method makes, its range of application is papermaking and fodder industry, owing to purify without strictness in the gained zytase, foreign matter content is higher, still can not satisfy the requirement of foodstuffs industry.
Summary of the invention:
The purpose of this invention is to provide a kind of strong enzyme activity that has, safety non-toxic can be in the zytase and the production method thereof of Applications in Food Industry.
Zytase provided by the present invention is a kind of zytase of food grade, and it adopts bacillus pumilus (Bacillus pumilus) is bacterial classification, makes through operations such as cultivation, fermentation, purification, dissolveds; Its liquid fermenting enzyme activity is 2500～3500IU/ml, and the solid fermentation enzyme activity is 50000～100000IU/g; Optimum temperature is 45～55 ℃, and the suitableeest action pH is 6.5～7.5, and normal temperature is preserved enzyme activity loss in 180 days and is lower than 5%.
In the above-mentioned zytase, heavy metal (in Pb)≤40mg/kg, total number of bacterial colony≤1000cfu/g, coliform≤10MPN/100g, Salmonellas (25g sample) must not detect, and meets foodstuffs industry health, safety standards.
Zytase production method provided by the present invention comprises master operations such as spawn culture, fermentation, purification, dissolved: spawn culture operation wherein: is to produce bacterial classification with the bacillus pumilus, adopts test tube slant bacterial classification → eggplant-shape bottle slant strains cultural method; Fermentation procedure: adopt the bacterium deep layer fermenting process, by seeding tank and fermentor tank second order fermentation; Purification process: increased flocculation technique-in fermented liquid, add flocculation agent, membrane filtration technique-employing ceramic microfiltration membrane and polysulphones hyperfiltration membrane and carried out secondary filtration, enzyme stabilizers technology-in concentrated solution, add stablizer; Dissolved technology-employing edible ethanol dissolved is extracted zytase.
(1) spawn culture:
Adopt bacillus pumilus (Bacilluspumilus) for producing bacterial classification in the zytase production method of the present invention.This bacterial strain belongs to bacillus, bacillus pumilus.By Institute of Microorganism, Academia Sinica this bacterial classification is identified that qualification result is as follows:
This bacterial strain belongs to bacillus, bacillus pumilus (Bacillus pumilus).Be aerobic bacteria, 5～15 ℃ of minimum growth temperatures, 45～49 ℃ of maximum growth temperatures, long 2～3 microns of thalline, wide 0.6～0.7 micron.Give birth in the spore column, do not have obviously and expand Gram-positive.Peritrichous can move, and the liquefy gelatin ability is strong, and hydrolyzed starch not, glucose fermentation produce not aerogenesis of acid, can utilize pectinose, wood sugar, seminose to replace glucose to produce acid, the V-P reacting positive, and the hydrogen peroxide enzyme positive, egg yolk reaction is negative.Can grow in the substratum that contains 7% sodium-chlor, produce alkali on Citrate trianion agar, the nitrate that can not reduce is nitrite, adapts to the pH wide ranges of growth, all can well grow from pH5.0-pH8.0.
Spawn culture adopts test tube slant bacterial classification → eggplant-shape bottle slant strains cultural method in the zytase production method of the present invention, and its nutrient media components and culture condition are as follows:
Nutrient media components: yeast extract paste 0.2%, peptone 1.0%, sodium-chlor 0.2%, agar 1.8-2.0% (weight %), all the other are water.PH7.0～7.2。
Culture condition: 30～32 ℃ of constant temperature culture three days.
(2) fermentation procedure:
Zymotechnique in the zytase production method of the present invention adopts the bacterium deep layer fermenting process, by seeding tank and fermentor tank second order fermentation.Its nutrient media components and fermentation condition are as follows:
Nutrient media components is: washing wheat bran (1.5～3) %, and soybean cake powder (1.5～3) %, corn cob (1～2) %, dipotassium hydrogen phosphate (0.5～1) %, sal epsom (0.1～0.3) %, ammonium sulfate (0.5～1) % (weight %), all the other are water.PH is 6.5～7.5.Sterilising treatment: pressure 1kg/cm
2, the sterilization 30 minutes down of 120～125 ℃ of temperature.
In the above-mentioned fermention medium, corn cob is that the inductor of bacterial classification product zytase can stimulate bacterial classification to produce enzyme; The washing wheat bran is that water is washed the starch in the wheat bran off, provides bacterial classification single carbon source, helps producing enzyme; Sal epsom is that bacterial classification produces zymoexciter, helps improving bacterial classification and produces enzyme power.
Fermenting process and condition are: the operational path that adopts eggplant-shape bottle seed → seeding tank → fermentor tank.About seeding tank fermentation culture 10-11h, begin to carry out microscopy, observe the form of bacterium, measure the bacterium number, the changing conditions of indexs such as pH value and enzyme work.About 13-14hr, bacterial classification reaches logarithmic phase, the switching fermentor tank.Leavening temperature 31-32 ℃, tank pressure 0.5-0.8kg/cm
2, air flow 0-7h is 1: 0.3-0.5 (vvm), 7-15h are 1: 0.7-0.8 (vvm), 15h are later on 1: 0.3-0.5 (vvm).(vvm represents the umber of the logical people's gas comprises liquid volume of per minute, and liquid volume is 1).Fermentation begins to detect enzyme behind the 12h lives, and enzyme work reaches fermentation ends when the highest.
(3) purification process:
Can reach food grade hygienic safety standard in order to ensure zytase product of the present invention, improve in the zytase production method of the present invention and the perfect purification process of zytase in the fermented liquid, it comprises flocculation, filter and remove remaining substratum, thalline and impurity, concentrate absorption, dissolved, filtration, dry, packaging and other steps, its outstanding feature are the innovation improvement of technical solution of the present invention to flocculation, membrane filtration, enzyme stabilization technique.
Its technical process is as follows:
Flocculation agent diatomite is removed thalline and impurity
↓???????????????↓????????????????????????????↑
Fermented liquid → filter press → filtrate → filter press → clear clear enzyme solution → ceramic membrane secondary filtration → ultra-filtration membrane
↓
Remove the substratum residue
Concentrate → concentrated solution → adsorbents adsorb → alcohol dissolved precipitation → filter press → filter cake →
↑
Stablizer
Lyophilize → pulverizing → sterilization → packing → chemical examination detection → finished product xylanase preparation
Specific operation process is: after the fermentation ends, add flocculation agent in fermented liquid, make that remaining substratum flocculates in the fermented liquid, carry out filter press then, remove the fermentation culture based draff; Add the diatomite adsorption foreign protein, filter press obtains clear clear enzyme solution; Carry out the ceramic membrane secondary filtration again, remove thalline and impurity in the fermented liquid; The filtration stillness of night enters ultrafiltration apparatus, and by the ultra-filtration membrane ultrafiltration and concentration, the work of concentrated solution enzyme is controlled between the 10000-20000u/ml; Concentrated solution adds stablizer and sorbent material W-Gum, and starts and stirred 10～20 minutes, adds edible ethanol, and the zytase in the enzyme liquid is carried out dissolved, and the enzyme cake is collected in the press filtration of 60 minutes laggard andante frames, and the enzyme cake is carried out lyophilize, obtains dry enzyme powder.
The feature of flocculation technique is as follows in the above-mentioned purification process:
The composition of flocculation agent and consumption: Sodium phosphate dibasic 1～2%, calcium chloride 0.5～1%, polyacrylamide 0.01～0.03% (accounting for the fermented liquid weight percentage).
Flocculation operating process and condition: at first in fermented liquid, add Sodium phosphate dibasic, start and stirred 20～30 minutes, it is dissolved fully, add calcium chloride then, open and stirred 10～20 minutes, change fermented liquid over to the flocculation jar, regulate ph to 6.5～7.5, slowly add polyacrylamide, open at last and stirred 5～10 minutes, flocculate.
Effect: Sodium phosphate dibasic and calcium chloride can form powerful gel after adding fermented liquid, add polyacrylamide again and further form bigger gel, and substratum residue and most of thalline in the fermented liquid are flocculated, and are convenient to filtration and remove impurity.
In the above-mentioned purification process membrane filtration technique be characterized as follows::
Employing filtration diameter is that the ceramic microfiltration membrane of 0.2 μ m filters for the first time to enzyme liquid, remove impurity and thalline, the polysulphones hyperfiltration membrane that adopts molecular weight to hold back to 10000D again carries out secondary filtration, remove small molecular weight impurity, and enzyme liquid concentrated, 5～35 ℃ of service temperatures, working pressure 2～5kg/cm
2
Enzyme stabilization technique in the above-mentioned purification process-feature of interpolation stablizer is as follows in concentrated solution:
Stabilizer element and consumption: chitosan 0.1-0.5%, cycloheptaamylose 0.5-1% (accounting for the fermented liquid weight percentage).
The function of stabilizer functional characteristics: chitosan has powerful adsorption to enzyme, enzyme is adsorbed, cycloheptaamylose has ring texture enzyme is carried out embedding, enzyme is in the metastable environment, reduce because alcohol is to the destruction of enzyme in the alcoholic extraction process, the enzyme extraction rate can improve 10-15%.
The feature of dissolved technology is as follows in the above-mentioned purification process:
5～8% the W-Gum that adds weight and be enzyme liquid is adsorbents adsorb and adopts edible ethanol that the zytase in the enzyme liquid is carried out dissolved, edible ethanol content is 90～95%, addition makes that the alcohol ultimate density reaches 65～75% in the enzyme liquid, extracts zytase.
Other operating process can be with reference to similar known technology in the above-mentioned purification process.
Zytase of the present invention, be food grade xylanase, its every index all reaches the foodstuffs industry standard-required, and it can be applicable to the multiple field of foodstuffs industry, as is used for bread, steamed bun suitability for industrialized production, can improve and improve the weave construction of bread, steamed bun, increase the internal porosity uniformity coefficient, improve the high-stiffness and the elasticity of bread, steamed bun, make the volume of bread, steamed bun increase 15～20%, improve the whiteness of steamed bun, can replace chemical additive to use.
Zytase target level of product quality of the present invention is as follows:
Outward appearance: pale powder, the amphidromic caking phenomenon of separating.
Physical and chemical index:
The physical and chemical index of table 1 zytase product
| Project | Index |
| Enzyme activity IU/g 〉= | ????50000 |
| Fineness (by 100 mesh standard sieves) % | ????100 |
Enzyme activity unit IU/g: under 50 ℃, pH value 7.2 conditions, the enzyme amount that per minute produces 1 micromole (μ mol) wood sugar is an enzyme activity unit (testing method is attached).
Sanitary index:
The sanitary index of table 2 zytase product
| Heavy metal (in Pb), mg/kg≤ | ????40 |
| Plumbous (in Pb), mg/kg≤ | ????10 |
| Arsenic (in As), mg/kg≤ | ????3 |
| Total number of bacterial colony, cfu/g≤ | ????1000 |
| Coliform, MPN/100g≤ | ????10 |
| Salmonellas (25g sample) | Must not detect |
Quality guaranteed period: preserve under normal temperature condition, the xylanase preparation quality guaranteed period is no less than 180 days (be not less than and indicate enzyme activity) in the quality guaranteed period.
Toxicity, security detect:
(1) bacterium producing multi enzyme preparation virulence test: bacterium producing multi enzyme preparation carries out the bacterial classification virulence test through Chinese disease health and epidemic prevention control center and detects, detected result shows: the BCP of bacillus pumilus cultivates stoste and 5 times of concentrated solution groups and compares with control group separately, the weight of mice there was no significant difference, do not observe simultaneously and tried toxic reaction of mouse or death, promptly this bacterial strain does not produce poison.
(2) Product Safety toxicology test: the zytase fermented liquid of this bacterial strain production and solid phase prod carry out the toxicity proof test through Shandong Center for Disease Control ﹠ Prevention and confirm that this enzyme is safe.Acute toxicity test in mice shows the true border of this sample non-toxic substance; Salmonella reversion test: the result is negative; Mouse polychromatic erythrocytes is little and test-results is negative; The mouse sperm deformity test: the result is negative; Rat 30 days is fed experiment and shows that this sample was fed in 30 days the every observation index of rat is not seen toxic action.
Zytase the best use of condition of the present invention:
(1) temperature is to the influence of enzyme activity
Measured the enzyme activity that enzyme shows under differing temps, the result shows that the optimum temperature of enzyme at 45～55 ℃, sees accompanying drawing 1.
(2) action pH value is to the influence of enzyme activity
Measured the enzyme activity that enzyme shows under different pH environment, the result shows that the suitableeest action pH of enzyme is 6.5～7.5, sees accompanying drawing 2.
Wherein the pH numerical value of X-coordinate Arabic numerals representative is: 1.pH3.82.pH4.8 3.pH5.8 4.pH6.5 5.pH7.0 6.pH7.2 7.pH7.88.pH8.5 9.pH9.0
The stability of zytase of the present invention:
(1) thermostability of liquid xylanase
Respectively under the condition of 4 ℃ and 30-65 ℃, insulation 0.5hr, 1hr and 2hr measure the residual enzyme vigor by standard conditions then with liquid xylanase.The result shows that enzyme is lived more stable at enzyme below 40 ℃, and along with increasing of temperature, the enzyme activity loss is very fast.The results are shown in Table 3.
Table 3: the thermostability of liquid xylanase
| Temperature/℃ | Relative enzyme (0.5hr)/% alive | Relative enzyme (1hr)/% alive | Relative enzyme (2hr)/% alive |
| ????4 | ????100 | ????100 | ????100 |
| ????30 | ????100 | ????100 | ????100 |
| ????40 | ????100 | ????99.4 | ????91.6 |
| ????50 | ????100 | ????90.2 | ????62.5 |
| ????55 | ????92.1 | ????73.7 | ????36.0 |
| ????60 | ????41.3 | ????16.8 | ????9.5 |
| ????65 | ????28.5 | ????8.9 | ????0 |
(2) thermostability of solid zytase
The solid zytase under 85 ℃ of dry heat conditions, is incubated 0,15,30,45 respectively, 60min, measures the residual enzyme vigor by standard conditions then.
The result shows that zytase thermotolerance under solid conditions is better, still has 90% relative enzyme activity behind 85 ℃ of processing 60min, sees accompanying drawing 3.
(3) storage stability of solid zytase
With the dry powder zymin, under dry environment, normal temperature, to deposit, half a year, endoenzyme was lived loss less than 5%, and the changing conditions that enzyme is lived sees Table 4:
Table 4 solid zytase is preserved the experiment enzyme activity and is changed
| | 0 | 60 days | 121 days | 181 days | Inactivation rate |
| ????1 | ??52683 | ??52001 | ??51258 | ??50155 | ??4.8％ |
| ????2 | ??53217 | ??52594 | ??51836 | ??51088 | ??4.0％ |
| ????3 | ??52875 | ??52065 | ??51328 | ??50602 | ??4.3％ |
| Mean value | ??52925 | ??52534 | ??51374 | ??50615 | ??4.37％ |
Zytase of the present invention and production method thereof have following superiority:
1. the selected bacterial strain of the present invention is a bacillus pumilus, stable performance, and enzyme is pure, and product is the inscribe beta-xylanase, and the hydrolyzed xylan product is a functional low polyxylose, and single wood sugar content is low, and oligosaccharide contg reaches 70% of total reducing sugar; The zytase optimum response PH that this bacterial strain produces is neutral, is fit to the processing environment of foodstuff production; This bacterial strain does not produce poison, and enzyme product is nontoxic product, meets the foodstuffs industry standard.
2. this bacterial strain is easily cultivated, and fermentation period is short, and only 14～18 hours, the liquid fermenting enzyme was lived high, reaches 2500～3500IU/ml, and production cost is low.
3. adopt new culture medium prescription in the fermenting process, had the effect of inducing, activating, promoting to produce enzyme, helped improving the product enzyme activity.
4. adopt advanced flocculation technique, membrane filtration technique in the abstraction process of enzyme, improved the purity of enzyme, guaranteed the high enzymatic activity of product, the rate of recovery of enzyme has been reached more than 85～90%, the work of solid zymin enzyme reaches more than 5～100,000 IU/g.
5. adopt novel enzyme stability protective agent, enzyme activity is stable, is convenient to standing storage and uses.
6. added value height, it is the raw material production food grade xylanase that present method adopts cheap agricultural byproducts, can make raw materials cost reduce by 50%.
Description of drawings:
Fig. 1 is the influence of temperature to enzyme activity
Fig. 2 is the influence of pH to enzyme activity
Fig. 3 is the thermostability of solid zytase
Embodiment:
1. strain inclined plane is cultivated embodiment
Preparation strain inclined plane substratum: yeast extract paste 0.2%, peptone 1.0%, sodium-chlor 0.2%, agar 1.8-2.0%, all the other are water, PH7.0.
Substratum is fully dissolved, pour into then in the eggplant-shape bottle, carry out 1kg/cm after wrapping
2 Pressure sterilization 30 minutes is made slant medium, inoculates test tube strains under aseptic condition, 30-32 ℃ of constant temperature culture three days.
2. fermenting process embodiment
Adopt eggplant-shape bottle seed (1) → 2m
3Seeding tank → 20m
3The operational path of fermentor tank, seed tank culture base and fermentor cultivation based formulas: washing wheat bran 2%, soybean cake powder 2%, liquid hemicellulose 1.5%, dipotassium hydrogen phosphate 0.5%, sal epsom 0.1%, ammonium sulfate 0.5%; PH7.2.About seeding tank fermentation culture 10-11h, begin to carry out microscopy, observe the form of bacterium, measure the bacterium number, the changing conditions of indexs such as pH value and enzyme work.About 13-14hr, bacterial classification reaches logarithmic phase, the switching fermentor tank.Leavening temperature 31-32 ℃, tank pressure 0.6kg/cm
2, air flow 0-7h is 1: 0.5vvm, 7-15h are 1: 0.7vvm, 15h are later on 1: 0.5vvm.
(1) variation of the parameter in the fermenting process sees Table 5:
Table 5:20m
3Parameter changes in the ferment tank process
| Time (hr) | PH value | The bacterium number | Enzyme (IU/ml) alive |
| ????12 | ????7.55 | ????50 | ????1094 |
| ????14 | ????7.88 | ????54 | ????2098 |
| ????16 | ????8.06 | ????55 | ????2652 |
| ????18 | ????8.09 | ????53 | ????2932 |
| ????19 | ????8.11 | ????53 | ????2920 |
(2) 20m
3The fermentor tank production data is given an example: (seeing Table 6)
Table 6:5 criticizes 20m
3The fermentor tank production data for example
| Project | Fermented liquid | Pressing filtering liquid | Ultrafiltrated | Dried enzyme powder | Extraction yield % | ||||
| Enzyme IU/ml alive | Quantity m 3 | Enzyme IU/ml alive | Quantity m 3 | Enzyme IU/ml alive | Quantity m 3 | Enzyme IUg alive | Quantity kg | ||
| 03106 batch | 2512.8 | ?13.5 | ?2000.6 | ?16.8 | ?6385.9 | ?5.0 | ?53586.9 | ?550 | ?86.88 |
| 031008 batch | 2748.3 | ?13.5 | ?2208.5 | ?16.8 | ?7402.6 | ?5.0 | ?54397.5 | ?571 | ?87.60 |
| 031010 batch | 2931.2 | ?13.5 | ?2355.4 | ?16.8 | ?7908.2 | ?5.0 | ?99500.8 | ?384 | ?87.39 |
| 031012 batch | 3122.5 | ?13.5 | ?2509.2 | ?16.8 | ?8340.9 | ?5.0 | ?98825.2 | ?372 | ?87.21 |
| 031014 batch | 3420.1 | ?13.5 | ?2748.3 | ?16.8 | ?9230.2 | ?5.0 | ?99082.5 | ?403 | ?86.44 |
3. extraction and cleaning embodiment
After the fermentation ends, at first in fermented liquid, add Sodium phosphate dibasic, start and stirred 20 minutes, it is dissolved fully, add calcium chloride then, open and stirred 10 minutes, change fermented liquid over to the flocculation jar, regulate ph to 7.0-7.2, slowly add polyacrylamide, open at last and stirred 5 minutes, flocculate.The fermentation culture based draff is removed in the press filtration of 30 minutes laggard andante frames; Add the diatomite adsorption foreign protein, filter press obtains clear clear enzyme solution; Carry out the ceramic micro filter membrane filtration again, remove thalline and impurity in the fermented liquid; The filtration stillness of night enters ultrafiltration apparatus, and by the ultra-filtration membrane ultrafiltration and concentration, the concentrated solution enzyme activity is controlled between the 10000-20000IU/ml; In concentrated solution, add 0.3% chitosan, 0.5% cycloheptaamylose and 8% starch, and start and stirred 10 minutes, add 90-95% (v/v) edible ethanol, make the alcohol ultimate density reach 70% (v/v), the enzyme cake is collected in the press filtration of 60 minutes laggard andante frames, and the enzyme cake carried out lyophilize, obtain dry enzyme powder.Its every index is:
| Numbering | Interventions Requested | Unit | Assay |
| ????1 | Enzyme activity | ??IU/g | ????52122 |
| ????2 | Weight loss on drying | ??％ | ????10.0 |
| ????3 | Fineness (by 100 order copper screen clothes) | ??％ | ????100 |
| ????4 | Heavy metal (in Pb) | ??％ | ????＜0.002 |
| ????5 | Arsenic (in As) | ??％ | ????＜0.0003 |
| ????6 | Total plate count | Individual/gram | ????100 |
| ????7 | Intestinal bacteria | Individual/100 grams | Do not detect |
| ????8 | Salmonellas | ??/ | Do not detect |
Attached: xylanase activity power measuring method
Present method is that the oat xylan of producing with Sigma company is a substrate, is standard with D-wood sugar (analytical pure), surveys the reducing sugar amount with the DNS method, reflects the effect vigor of zytase with this.
Concrete grammar: take by weighing an amount of zytase sample, pH7.2 phosphate buffered saline buffer with 0.2mol/ml suitably dilutes back (the about 0.2-0.3IU/ml of general dilution back enzyme liquid), getting 0.5ml adds in the scale test tube, add the 0.5% xylan solution mixing of 2ml with the pH7.2 phosphate buffered saline buffer preparation of 0.2mol/ml, put 50 ℃ of waters bath with thermostatic control 30 minutes, add 2mlDNS reagent after the taking-up immediately and shake up, put into boiling water bath 10 minutes, cooling is settled to 15ml with distilled water then immediately.Not enzyme-added liquid in the blank test tube, treat 50 ℃ transformed add DNS reagent after, add 0.5ml enzyme liquid again, boil together with other sample, be settled to 15ml, under 550nm, survey the OD value.
Under these conditions, each minute the reducing sugar amount that produces to be equivalent to the required enzyme amount of 1 micromole (μ mol) wood sugar be an enzyme activity unit (IU/ml).
Claims (10)
1. a food grade xylanase is characterized in that it adopts bacillus pumilus (Bacilluspumilus) is bacterial classification, makes through cultivation, fermentation, purification, dissolved operation; Its liquid fermenting enzyme activity is 2500～3500IU/g, and the solid phase prod enzyme activity is 50000～100000IU/g; Optimum temperature is 45～55 ℃, and the suitableeest action pH is 6.5～7.5, and normal temperature is preserved enzyme activity loss in 180 days and is lower than 5%.
2. a kind of food grade xylanase as claimed in claim 1 is characterized in that in the zytase, heavy metal (in Pb)≤40mg/kg, total number of bacterial colony≤1000cfu/g, coliform≤10MPN/100g, Salmonellas (25g sample) must not detect, and meets foodstuffs industry health, safety standards.
3. the production method of a kind of food grade xylanase as claimed in claim 1 is characterized in that comprising spawn culture, fermentation, purification, dissolved operation; Spawn culture operation wherein: with the bacillus pumilus is to produce bacterial classification, adopts test tube slant bacterial classification → eggplant-shape bottle slant strains cultural method; Fermentation procedure: adopt the bacterium deep layer fermenting process, by seeding tank and fermentor tank second order fermentation; Purification process: increased flocculation technique-in fermented liquid, add flocculation agent, membrane filtration technique-employing ceramic microfiltration membrane and polysulphones hyperfiltration membrane and carried out secondary filtration, enzyme stabilizers technology-in concentrated solution, add stablizer; Dissolved operation: adopt the edible ethanol dissolved to extract zytase.
4. the production method of a kind of food grade xylanase as claimed in claim 3, it is characterized in that the culture medium prescription that said spawn culture operation is used is: yeast extract paste 0.2%, peptone 1.0%, sodium-chlor 0.2%, agar 1.8-2.0% (weight %), all the other are water; PH7.0～7.2; Culture condition is: 30～32 ℃ of constant temperature culture three days.
5. the production method of a kind of food grade xylanase as claimed in claim 3, it is characterized in that the nutrient media components that said fermentation procedure uses is: washing wheat bran (1.5～3) %, soybean cake powder (1.5～3) %, corn cob (1～2) %, dipotassium hydrogen phosphate (0.5～1) %, sal epsom (0.1～0.3) %, ammonium sulfate (0.5～1) % (weight %), all the other are water; PH is 6.5～7.5; Fermenting process and condition are: adopt eggplant-shape bottle seed (1) → 2m
3Seeding tank → 20m
3The operational path of fermentor tank about seeding tank fermentation culture 10-11h, begins to carry out microscopy, observes the form of bacterium, measures the bacterium number, the changing conditions of indexs such as pH value and enzyme activity, and about 13-14h, bacterial classification reaches logarithmic phase, the switching fermentor tank; Leavening temperature 31-32 ℃, tank pressure 0.5-0.8kg/cm
2, air flow 0-7h is 1: 0.3-0.5 (vvm), 7-15h are 1: 0.7-0.8 (vvm), 15h are later on 1: 0.3-0.5 (vvm), and fermentation begins to detect enzyme activity behind the 12h, and enzyme activity reaches fermentation ends when the highest.
6. the production method of a kind of food grade xylanase as claimed in claim 3, the composition and the consumption that it is characterized in that adding in the said purification process flocculation agent in fermented liquid are Sodium phosphate dibasic 1-2%, calcium chloride 0.5-1%, polyacrylamide 0.01-0.03% (accounting for the fermented liquid weight percentage).
7. the production method of a kind of food grade xylanase as claimed in claim 3, it is characterized in that membrane filtration technique in the said purification process adopts that to filter diameter be that the ceramic microfiltration membrane of 0.2 μ m filters for the first time to enzyme liquid, the polysulphones hyperfiltration membrane that adopts molecular weight to hold back to 10000D again carries out secondary filtration, remove small molecular weight impurity, and enzyme liquid is concentrated.
8. the production method of a kind of food grade xylanase as claimed in claim 3, it is characterized in that in the said purification process in concentrated solution, adding stabilizer element and consumption is chitosan 0.1-0.5%, cycloheptaamylose 0.5-1% (accounting for the fermented liquid weight percentage).
9. the production method of a kind of food grade xylanase as claimed in claim 3, the dissolved technology in the said purification process of it is characterized in that is that to add weight be that 5～8% W-Gum of enzyme liquid is adsorbents adsorb and adopts edible ethanol that the zytase in the enzyme liquid is carried out dissolved, edible ethanol content is 90～95%, addition makes that the alcohol ultimate density reaches 65～75% in the enzyme liquid, extracts zytase.
10. the application of a kind of food grade xylanase as claimed in claim 1 is characterized in that being applied to doing in the foodstuffs industry food improver and foodstuff additive.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2011003784A1 (en) * | 2009-07-09 | 2011-01-13 | Novozymes A/S | Flocculation with divalent salt and phosphate |
| CN101701205B (en) * | 2009-10-30 | 2011-08-17 | 中国农业科学院饲料研究所 | Alkali-resistant xylanase XynE2, genes thereof and application thereof |
| CN103211035A (en) * | 2013-04-12 | 2013-07-24 | 江南大学 | Method for preparing liquid-state chymosin preparation |
| CN103725615A (en) * | 2013-12-30 | 2014-04-16 | 广东海纳川药业股份有限公司 | Bacteria recovery method and flocculation composition for bacteria recovery |
| CN104807764A (en) * | 2015-04-21 | 2015-07-29 | 华南理工大学 | Alkaline xylanase activity determination method |
| CN110612856A (en) * | 2019-10-08 | 2019-12-27 | 河南省农业科学院植物营养与资源环境研究所 | Mother culture medium for promoting growth of edible fungus hyphae |
| CN115851670A (en) * | 2022-11-30 | 2023-03-28 | 山东龙昌动物保健品有限公司 | Xylanase mutant, compound preparation of xylanase mutant and eucommia ulmoides leaf extract and application of xylanase mutant and eucommia ulmoides leaf extract in feed additive |
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- 2005-03-10 CN CN 200510042557 patent/CN1670203A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011003784A1 (en) * | 2009-07-09 | 2011-01-13 | Novozymes A/S | Flocculation with divalent salt and phosphate |
| CN102471755A (en) * | 2009-07-09 | 2012-05-23 | 诺维信公司 | Flocculation with divalent salt and phosphate |
| CN101701205B (en) * | 2009-10-30 | 2011-08-17 | 中国农业科学院饲料研究所 | Alkali-resistant xylanase XynE2, genes thereof and application thereof |
| CN103211035A (en) * | 2013-04-12 | 2013-07-24 | 江南大学 | Method for preparing liquid-state chymosin preparation |
| CN103211035B (en) * | 2013-04-12 | 2014-11-05 | 江南大学 | Method for preparing liquid-state chymosin preparation |
| CN103725615A (en) * | 2013-12-30 | 2014-04-16 | 广东海纳川药业股份有限公司 | Bacteria recovery method and flocculation composition for bacteria recovery |
| CN103725615B (en) * | 2013-12-30 | 2016-06-15 | 广东海纳川生物科技股份有限公司 | A kind of thalline recovery method and the flocculating composition reclaimed for thalline |
| CN104807764A (en) * | 2015-04-21 | 2015-07-29 | 华南理工大学 | Alkaline xylanase activity determination method |
| CN110612856A (en) * | 2019-10-08 | 2019-12-27 | 河南省农业科学院植物营养与资源环境研究所 | Mother culture medium for promoting growth of edible fungus hyphae |
| CN115851670A (en) * | 2022-11-30 | 2023-03-28 | 山东龙昌动物保健品有限公司 | Xylanase mutant, compound preparation of xylanase mutant and eucommia ulmoides leaf extract and application of xylanase mutant and eucommia ulmoides leaf extract in feed additive |
| CN115851670B (en) * | 2022-11-30 | 2024-04-12 | 山东龙昌动物保健品股份有限公司 | Xylanase mutant, compound preparation of xylanase mutant and eucommia ulmoides leaf extract and application of xylanase mutant and eucommia ulmoides leaf extract in feed additive |
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