CN109805184A - A kind of attapulgite/phytic acid multienzyme complex additive and its preparation method and application - Google Patents
A kind of attapulgite/phytic acid multienzyme complex additive and its preparation method and application Download PDFInfo
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- CN109805184A CN109805184A CN201910195978.0A CN201910195978A CN109805184A CN 109805184 A CN109805184 A CN 109805184A CN 201910195978 A CN201910195978 A CN 201910195978A CN 109805184 A CN109805184 A CN 109805184A
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- attapulgite
- phytase
- phytic acid
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Abstract
The invention discloses a kind of attapulgite/phytic acid multienzyme complex additives and its preparation method and application, the preparation of the compound additive includes the following steps: for phytase to be dissolved in buffer, it is centrifuged after stirring, taking supernatant is phytase solution, adds attapulgite and NaCl or CaCl2Mixed liquor is stirred oscillating reactions at room temperature by solution, is centrifuged, and freeze-drying pulverizes and sieves, obtains compound additive.Attapulgite prepared by the present invention/phytic acid multienzyme complex additive can protect phytase, can effectively reduce the loss of phytase in Feed Manufacturing process, enhance stability, improve the utilization rate of phytase, can be used as the novel form production application of feed addictive.
Description
Technical field
The invention belongs to feed addictive applied technical fields, and in particular to a kind of attapulgite/phytic acid multienzyme complex adds
Add agent and its preparation method and application.
Background technique
Feed addictive refers to a small amount of or micro substance added in Feed Manufacturing processing, use process, in feed
Dosage is seldom but effect is significant.Feed addictive is the raw material that modern feed industry necessarily uses, to reinforcing basal feed nutrition
Value improves breeding performonce fo animals, guarantees animal health, saves feed cost, and improvement livestock products quality etc. has apparent
Effect.Feed addictive mainly include mineral element class, enzyme preparation, vitamins, amino acids, antibiotics, antioxidant,
Microorganism etc..
Attapulgite is common natural mineral feed, is most used for animal husbandry initially as the adhesive of granulation, due to
Its specific surface area and cation exchange capacity (CEC) and to organic matter there are stronger suction-operated, the growth as promotion animal later
With the feed addictive and selective absorbent of health.It can be promoted as the additive of mixed fodder with its distinctive physical property
It into the metabolism of animal body, improves food conversion ratio, keeps animal appetite vigorous, fur is plump, and weight gain is fast, delivers for sale morning, drops
Low feeding cost.Also there is excellent adsorptivity simultaneously, can effectively absorb colibacillus, the enterotoxin of animal, play anti-
The effect that epidemic disease is cured the disease, deinsectization is sterilized, and livestock is provided and grows necessary microelement；As the binder of pellet, have
Good cohesive force had not only reduced Feed Manufacturing cost, but also the utilization rate of feed can be improved, it can adsorb the ammonium ion in water,
Anti- water pollution, antirancidity.In the production process of premixed feed, recessed soil can save a large amount of grain as premixing feedstuff carrier
Food, can also keep vitamin etc. not fail, microelement does not scatter and disappear；As compound fertilizer's binder, granulating and forming rate is high, the time
Short, made granule strength is high, and surface smoothness is good, and the fertilizer conservation time is long, therefore this binder, can improve production capacity, reduces
Power consumption can also save the dosage of the high prices raw material such as talcum powder, ammonium chloride.
Phytase is novel feed addictive, can effectively improve the utilization efficiency of phosphorus in feed grain raw material, is reduced outer
The additive amount of source phosphorus can also reduce the discharge of phosphorus in feces of livestock and poultry, preserve the ecological environment.Nature phytase includes two kinds,
3- phytase and 6- phytase, the two are entirely different phytic acid enzyme products, 3- phytase be mainly derived from most of bacteriums or
The biology of person fungi, 6- phytase, which is mainly derived from extraction generation or Escherichia coli, the two in plant, essential distinction.It is feeding
Phytase preparation is usually directly to dry the tunning of phytase to be made, mainly 3- phytase, in addition to containing more
Substance in addition to phytase, such as aspergillus niger, Pichia pastoris, glucose.Phytase is more sensitive to variations such as humidity, storage
Shi Jun is placed under shady and cool, drying condition, and phytase activity present in plant feed is often because of feed processing (high temperature), storage etc.
The influence of factor and destroyed, cause the utilization rate of phosphorus in feed to substantially reduce.Meanwhile the phytase in feed often with
Divalent mineral ions such as Ca2+, Mg2+, Cu2+The stable complex compound of equal formation, this just affects the absorption of feed Mineral Elements.
On the one hand phytate phosphorus can be converted to Phos by phytase, promote the absorption of phosphorus, on the other hand also relieve phytate to Fe2+Deng
The inhibition that mineral element absorbs.
Have more research, attapulgite and phytic acid respectively as feed addictive now for attapulgite and phytase
Enzyme is common feed addictive, and attapulgite/feeding phytic acid multienzyme complex does not find temporarily to report as feed addictive.
Directly attapulgite and phytase are mixed in the prior art and are added in feed, phytase cannot in Feed Manufacturing process
It is protected well, the influence in Feed Manufacturing process due to temperature humidity and pH etc. can cause in varying degrees
Phytase activity loss, influence the performance of phytase effect.In addition, adding other feed addictive types, it is easy
The problem of now new secure context.
Summary of the invention
Goal of the invention: being more easily damaged in feed processing production and storage for phytase, lead to active reduction,
For above-mentioned drawback, the present invention provides a kind of attapulgite/phytic acid multienzyme complex additive and preparation method thereof, the compound by
Attapulgite and phytase are prepared through magnetic agitation with absorption is vibrated, and phytase passes through with attapulgite vibrates suction-operated, and two
Person comes into full contact with, and attapulgite has stronger adsorptivity, is acted synergistically by ion exchange and physical absorption by feeding phytic acid
Enzyme is adsorbed to attapulgite's surface, and obtained attapulgite/phytic acid multienzyme complex can effectively reduce phytase in Feed Manufacturing
Loss in the process enhances stability, improves its utilization rate.
The present invention also provides the applications of prepared attapulgite/phytic acid multienzyme complex additive.
Technical solution: to achieve the goals above, a kind of attapulgite/phytic acid multienzyme complex additive as described herein
Preparation method, include the following steps: for phytase to be dissolved in buffer, be centrifuged after stirring, take supernatant be phytase solution,
Add attapulgite and CaCl2Mixed liquor is stirred oscillating reactions at room temperature by solution, is centrifuged, and freeze-drying pulverizes and sieves,
Obtain compound additive.
Preferably, the attapulgite for being 2.1~2.2 to density of the attapulgite.
Preferably, the attapulgite is common concave-convex stick soil or acidification attapulgite.
The quality that attapulgite is improved in the present invention, will do it is certain acidified modified, it is recessed after acid activation
Convex stick soil is not only purified, and certain change can also occur for physico-chemical property, and attapulgite is reunited existing under the action of an acid
As being very significantly improved, start to become fluffy, stick crystalline substance obviously appears, and the brilliant hole between stick crystalline substance of stick is deepened and expanded
Greatly, so that its purity and specific surface area be made to be greatly improved.It is good to be acidified attapulgite load-carrying properties, the moderate grain of pH value
Uniformly, alternative corn flour uses degree as feed addictive, because the elements such as lead, mercury are lower than national standard in attapulgite,
It can also save food, reduce feed cost, provide animal necessary microelement, prevent animal from having loose bowels, it can also be largely
Ground is maintained at the ingredient or potency of vitamin in Feed Manufacturing process, microelement.
Preferably, the phytase is feeding phytase or sterling phytase.Feeding plant can be used in the present invention
Sour enzyme or sterling phytase, since price is higher, usually select feeding phytase although sterling phytic acid enzyme effect is more preferable
Using production.
Wherein, the concentration of phytase is 4~6mg/mL in the supernatant；The buffer generally selects the second of pH5.0
Generally 100mL acetate buffer (pH5.0) is added using feeding 4~6g of phytase in acid buffer, the phytase.
Wherein, the dosage of the attapulgite is 0.30~0.80g.
Wherein, wherein NaCl or CaCl2Solution, which is added in phytase solution, guarantees that concentration NaCl or CaCl2 are dense
Degree is 0.05~0.1mol/L.Sodium chloride or calcium chloride, which is added, can effectively enhance the activity after phytase is adsorbed.
Preferably, the hunting speed of mixed liquor stirring oscillating reactions is in 150~200r/min, most preferably oscillation speed
Rate 150r/min.
Further, the attapulgite for being 2.20 to density of the attapulgite, specific preparation step are as follows:
(1) attapulgite purifies: taking the attapulgite raw ore that crushed, soaks in water, mass fraction 20~25% is made
Suspension, high speed dispersion mashing, slurry take out, and standing sedimentation removes supernatant liquor, and centrifugation is dried for standby；
(2) heavy-fluid is centrifugated: taking Du's column heavy-fluid to pour into separatory funnel, the attapulgite for then obtaining step (1)
It is slowly poured into after being infiltrated with methanol, stirs, be placed in centrifuge separation, taking relative density is 2.1~2.20 attapulgite, is dried standby
With.
Attapulgite/plant prepared by attapulgite of the present invention/phytic acid multienzyme complex additive preparation method
Sour multienzyme complex additive.
Attapulgite/plant prepared by attapulgite of the present invention/phytic acid multienzyme complex additive preparation method
Sour multienzyme complex additive is as the application in feedstuff additive product.
The attapulgite and phytase being used in the present invention are common feed addictives, and attapulgite has relatively strong
Suction-operated, the suction-operated generated using method attapulgite of the invention to phytase can be protected to a certain extent
The activity of phytase enables phytase effect normally to play, and avoids in Feed Manufacturing process since temperature is wet
The influence of degree and pH etc. can cause the loss of phytase activity in varying degrees.
The present invention selects attapulgite and phytase to prepare composite feed additive for raw material, because being not desired to additionally introduce it
His new feed addictive type, the problem of avoiding the occurrence of new secure context.
In addition, safety is low directly using will lead to zoogenetic infection pathogen by the phytase that microbial fermentation produces.
In the other factors in feed processing pelletization as the moisture in Granulation time and feed can influence feeding phytase
Activity.Feeding phytase and attapulgite, which are prepared composite feed additive, can increase the stability of phytase, make it preferably
Play catalytic activity.Feeding phytase and attapulgite come into full contact with the two, attapulgite has by oscillation suction-operated
Stronger adsorptivity is acted synergistically by ion exchange and physical absorption feeding phytase being adsorbed to attapulgite's surface, obtained
The attapulgite arrived/feeding phytic acid multienzyme complex.
Raw material of the invention is by commercially available, and wherein attapulgite raw ore is purchased from Jiangsu Hua Zheng mineral products Co., Ltd；
Feeding phytase is purchased from GuangDong YiDuoLi Biology Science Co., Ltd, other are commercially available to commonly use feeding phytase；Phytic acid
Enzyme is purchased in Hefei BASF Biotechnology Co., Ltd；
The utility model has the advantages that compared with prior art, the present invention has the advantage that
(1) primary raw material of the present invention is attapulgite and phytase, can replace the plant directly added in feed mixed process
Sour enzyme reduces additive amount.
(2) preparation method of the present invention is magnetic agitation and constant temperature oscillation, and preparation condition is mild, and technical process is simple, is easy to
It accomplishes scale production.
(3) minerals needed for attapulgite prepared by the present invention/phytic acid multienzyme complex additive can also provide body.
(4) attapulgite prepared by the present invention/phytic acid multienzyme complex additive temperature tolerance, proteolytic relatively from
By phytase apparent increase, the drop of the phytase activity due to caused by the factors such as high temperature in Feed Manufacturing process is effectively avoided
It is low, it is more advantageous to Feed Manufacturing and storage.
(5) the optimum pH media range of attapulgite prepared by the present invention/phytic acid multienzyme complex additive is on 5.0 left sides
The right side, it is consistent with the optimum activity medium range of pH5.0~5.5 of phytase.
(6) present invention does not introduce other additional additives and cheap using attapulgite as compound absorption carrier；Together
When into cross processing select take relative density be 2.1-2.2 attapulgite, eliminate under the impurity of bulky grain and this relative density
Attapulgite good dispersion, it is more preferable to the adsorption effect of phytase, in addition, pH of buffer selected by the present invention be 5.0, in this ring
Phytase can keep preferable activity under border.
(7) attapulgite prepared by the present invention/feeding phytic acid multienzyme complex additive be added to enzyme activity in feed with it is recessed
Convex stick soil with feeding phytase directly through simple physical it is mixed be added in feed compared with temperature tolerance, protease hydrolytic
Tolerance is significantly raised.
(8) attapulgite prepared by the present invention/phytic acid multienzyme complex additive as feed addictive novel form,
Safely and effectively feedstuff additive product can be prepared, meanwhile, the present invention is also that the development of composite feed additive provides
New thinking.
Detailed description of the invention
Fig. 1 attapulgite prepared by the present invention/feeding phytic acid multienzyme complex additive pH adaptability schematic diagram；(note: real
Testing group is attapulgite/feeding phytic acid multienzyme complex prepared by embodiment 2, and feeding phytase group is simple feeding phytase, right
According to group for without the attapulgite/feeding phytic acid multienzyme complex for adding CaCl2 solution；- ■-experimental group-●-feeding phytic acid
Enzyme group-▲-control group).
Specific embodiment
Explanation is further explained to the present invention below with reference to embodiment.
Embodiment 1
Attapulgite purifying: the attapulgite raw ore that crushed 150 meshes is taken, is soaked in water, mass fraction 20% is made
Suspension, high speed dispersion mashing (primary every 10min mashing) 30min, slurry takes out, and it is clear to remove upper layer by standing sedimentation 3h
Liquid, 10000r/min are centrifuged 15min, are dried for standby；
Heavy-fluid centrifuge separation: taking 10mL relative density is that 2.20 Du's column heavy-fluid (mercuric iodixde-liquor kalii iodide) is poured into point
In liquid funnel, slowly pours into separatory funnel after above-mentioned attapulgite is then infiltrated 10min with methanol, is stirred with glass bar,
It is placed under 2000r/min and is centrifugated 12h, the attapulgite for taking relative density to be about 2.20 is dried for standby；
The preparation of compound: it takes feeding phytase 4g to be placed in the 100mL acetate buffer of pH5.0, is placed in magnetic agitation
Stir 30min on device, 4000r/min is centrifuged 30min, and it is about 5.3mg/mL that take supernatant, which be feeding phytase degree) Solutions Solution；
By 0.3g attapulgite, phytase solution (the wherein CaCl of 20mLpH5.0 is added2Solution concentration be 0.05mol/L), then at
Oscillating reactions 150r/min is carried out in constant temperature oscillation instrument, 4000r/min is centrifuged 30min, is freeze-dried 12h, crushed 200 mesh
Sieve, obtaining compound is attapulgite/feeding phytic acid multienzyme complex additive.
Embodiment 2
Attapulgite purifying: the attapulgite raw ore that crushed 150 meshes is taken, is soaked in water, mass fraction 25% is made
Suspension, high speed dispersion mashing (primary every 10min mashing) 30min, slurry takes out, and it is clear to remove upper layer by standing sedimentation 3h
Liquid, 10000r/min are centrifuged 15min, are dried for standby；
Heavy-fluid centrifuge separation: taking 10mL relative density is that 2.20 Du's column heavy-fluid (mercuric iodixde-liquor kalii iodide) is poured into point
In liquid funnel, slowly pours into separatory funnel after above-mentioned attapulgite is then infiltrated 10min with methanol, is stirred with glass bar,
It is placed under 2000r/min and is centrifugated 12h, the attapulgite for taking relative density to be about 2.20 is dried for standby；
The preparation of compound: it takes feeding phytase 5g to be placed in the acetate buffer of pH5.0, is placed on magnetic stirring apparatus and stirs
30min is mixed, 4000r/min is centrifuged 30min, and taking supernatant is feeding phytase solution；By 0.5g attapulgite, it is added
20mLpH5.0 phytase solution (wherein CaCl2Solution concentration is 0.08mol/L), it is vibrated in constant temperature oscillation instrument
150r/min is reacted, 4000r/min is centrifuged 30min, is freeze-dried 12h, crushed 200 meshes, and obtaining compound is bumps
Stick soil/feeding phytic acid multienzyme complex additive.
Embodiment 3
Attapulgite purifying: the attapulgite raw ore that crushed 150 meshes is taken, is soaked in water, mass fraction is made about
25% suspension, high speed dispersion mashing (primary every 10min mashing) 30min, slurry take out, standing sedimentation 3h, remove on
Layer clear liquid, 10000r/min are centrifuged 15min, are dried for standby；
Attapulgite acidification: taking purifying attapulgite, is that 3:1 is added according to solid-to-liquid ratio (sulfuric acid solution and attapulgite ratio)
2h is activated at the sulfuric acid solution that mass fraction is 25%, with 80 DEG C, between pH about 4~5, centrifugation is ground after dry for rinsing
200 meshes are spare；
Heavy-fluid centrifuge separation: taking 10mL relative density is that 2.10 Du's column heavy-fluid (mercuric iodixde-liquor kalii iodide) is poured into point
In liquid funnel, slowly pours into separatory funnel after above-mentioned attapulgite is then infiltrated 10min with methanol, is stirred with glass bar,
It is placed under 2000r/min and is centrifugated 12h, the attapulgite for taking relative density to be about 2.10 is dried for standby；
The preparation of compound: it takes feeding phytase 6g to be placed in the acetate buffer of pH5.0, is placed on magnetic stirring apparatus and stirs
30min is mixed, 4000r/min is centrifuged 30min, and taking supernatant is feeding phytase (concentration is about 5.6mg/mL) solution；By 0.8g
Phytase solution (the wherein CaCl of 20mLpH5.0 is added in attapulgite2Solution concentration is 0.1mol/L), then at constant temperature oscillation
Oscillating reactions 150r/min is carried out in instrument, 4000r/min is centrifuged 30min, is freeze-dried 12h, crushed 200 meshes, answered
Closing object is to be acidified attapulgite/feeding phytic acid multienzyme complex additive.
Embodiment 4
Embodiment 4 is same as Example 2, the difference is that it is molten that feeding phytase solution is substituted for sterling phytase
Liquid finally obtains attapulgite/phytic acid multienzyme complex additive.
Embodiment 5
Embodiment 4 is same as Example 3, the difference is that it is molten that feeding phytase solution is substituted for sterling phytase
Liquid finally obtains acidification attapulgite/phytic acid multienzyme complex additive.
Embodiment 6
Embodiment 5 is same as Example 2, the difference is that CaCl2Solution is substituted for NaCl solution.
Test example 1
The determination of feeding phytase content
It takes 20.55g anhydrous sodium acetate to be dissolved in 900mL deionized water, with salt acid for adjusting pH to 5.0, is settled to
1000mL.The feeding phytase preparation of 1~6g is weighed, 5.0 acetate buffer of 100mL pH is added, is placed on magnetic stirring apparatus and stirs
30min is mixed, 4000r/min is centrifuged 30min, takes supernatant 1mL, by Coomassie brilliant blue G250 decoration method, surveys feeding in supernatant
Phytase protein content.
The feeding phytic acid enzyme concentration of table 1
As shown in Table 1, when feeding phytase additive amount is 4.00~6.00g in 100mL acetate buffer, phytase egg
White content tends towards stability, and selects the amount of feeding phytase for 4~6g/100mL acetate buffer (pH5.0).
Test example 2
The feeding phytase of 5g is weighed into the buffer of different pH, is placed in magnetic stirrer 30min, 4000r/min
It is centrifuged 30min, taking supernatant is feeding phytase solution, takes the feeding phytase solution of 25mL difference pH value, and it is recessed that 0.5g is added
Convex stick soil and 2mL CaCl2Solution 25 DEG C of constant temperature, is placed on magnetic stirring apparatus and stirs 30min, with 150r/ in constant temperature oscillation instrument
Min vibrates 120min, and 4000r/min is centrifuged 15min, takes supernatant 1mL, according to coomassie brilliant blue G250 decoration method, in survey
Feeding phytase protein content in clear liquid.The results are shown in Table 2.Adsorbance and adsorption rate are calculated according to the following formula.
Adsorbance q (mg)=(C0-C)×V
C0The initial concentration of phytase, mg/mL in-sample solution；
C-sample solution actual measurement light absorption value corresponding phytase in standard curve concentration, mg/mL；
V-sample solution volume, mL
Adsorption rate (%)=m/q
M-phytase quality, mg；
The amount of q-attapulgite absorption phytase, mg
Influence of 2 pH of table to absorption
As shown in Table 2, attapulgite reduces the adsorbance of feeding phytase with the increase of pH, concave convex rod when pH3
Soil is 41.97% to the adsorption rate of feeding phytase to attapulgite when the adsorption rate of feeding phytase is 84.25%, pH7, is examined
The optimal pH of phytase activity is considered between 5~5.5, so selecting pH for 5.0 buffer is that test buffer is optimal.
Test example 3
The determination of hunting speed
It weighs in the feeding phytase of 5g and 100mL acetate buffer (pH5.0), is placed in magnetic stirrer 30min,
4000r/min is centrifuged 30min, and taking supernatant is feeding phytase solution, and the feeding phytase solution of 25mL, pH 5.0 is taken to be added
0.5g attapulgite and 2mL CaCl2Solution, is placed on magnetic stirring apparatus and stirs 30min by 25 DEG C of constant temperature, then hunting speed difference
To vibrate 120min under 50,100,150,200r/min in constant temperature oscillation instrument, 4000r/min is centrifuged 15min, takes supernatant
1mL surveys the feeding phytase protein content in supernatant according to coomassie brilliant blue G250 decoration method.The results are shown in Table 3.
Influence of 3 hunting speed of table to absorption
As shown in Table 3, for hunting speed in 150~200r/min, attapulgite is close to the adsorption rate of feeding phytase,
Adsorption rate no longer changes with the variation of hunting speed, and attapulgite substantially remains in the adsorption rate of feeding phytase
80.00% or more, so selecting duration of oscillation optimal for 150r/min.
Test example 4
Composite temperature tolerance test
Microbe-derived phytase, property have differences, most microorganism because of the difference in zymogenic bacteria
The optimum temperature of phytase all concentrates between 40~70 DEG C, this test mainly measures attapulgite/feeding phytic acid multienzyme complex
The enzymatic activity at 70 DEG C is exposed to freely feeding phytase.Take out attapulgite/feeding phytase prepared by 1.0g embodiment 2
Compound is exposed at 70 DEG C, and the enzyme activity of feeding phytase is surveyed after 0.50,2.00,4.00h.Feeding phytase activity
Measuring method refers to GB/T18634-2009 " measurement-spectrophotometry of feeding phytase activity ".It is surveyed certainly under the conditions of
By feeding phytase group, control group 1, control group 2, control group 3, control group 4, control group 5 temperature tolerance.As a result such as table 4
It is shown.
4 temperature tolerance of table
Note: experimental group is attapulgite/phytic acid multienzyme complex prepared by embodiment 2, and feeding phytase group is simple feeding
Phytase, control group 1 are attapulgite and mixture of the phytase directly after physical mixed, and control group 2 is not add
CaCl2Attapulgite/phytic acid multienzyme complex of solution preparation (other are same as Example 2)；It is prepared by control group 3 and embodiment 2
Attapulgite/phytic acid multienzyme complex it is identical, difference select relative density 1.5 or so attapulgite be raw material；It is right
Identical as attapulgite/feeding phytic acid multienzyme complex prepared by embodiment 2 according to group 4, difference is selecting relative density 2.5 left
Right attapulgite is raw material.5 bibliography of control group (natural smectite/phytase complex carrier preparation and its phytate phosphorus water
Solve activity research, Zhang Wenzhong, nonmetallic ore, in January, 2013) scheme, montmorillonite and phytase are substituted for the recessed of embodiment 1
Convex stick soil and feeding phytase and keep usage amount consistent.
As shown in Table 4, under 70 DEG C of hot conditions, the enzyme activity retention rate at three time points herein of control group 1 is respectively less than
Attapulgite/feeding phytic acid multienzyme complex, because since attapulgite and the direct physical mixed of phytase do not have phytase absorption
The environment of attapulgite cannot be adsorbed effectively, while directly physical mixed is more easily damaged the activity of phytase, leads
Activation reduces, if directly attapulgite and phytase are added in feed, phytase cannot in Feed Manufacturing process
It is protected well, since temperature influences to cause phytase activity in varying degrees in Feed Manufacturing process
Loss.
Experimental group is high compared with control group 2 with respect to enzyme activity, the reason is that calcium ion can promote phytic acid enzyme-to-substrate to combine, from
And improve the activity of enzyme.Start in 0.50h, after feeding phytase group is 60.89%, 4.00h with respect to enzyme activity, feeding phytase group
Opposite enzyme activity only has 16.47%, and it is irreversible to show that the microscopic three-dimensional amino acid residue structure of feeding phytase protein occurs
Change, enzyme activity is caused to be lost.Attapulgite/feeding phytic acid multienzyme complex after exposure 4.00h, still there is 31.37% at 70 DEG C
Opposite enzyme activity, the more feeding phytase of attapulgite/feeding phytase temperature tolerance significantly improve.
Control group 3 and 4 enzyme activity of control group are lower, and the attapulgite for being primarily due to the opposite relative density contains much greatly
The impurity of particle, and the attapulgite bad dispersibility under this relative density are opposite to the adsorbance of phytase with this condition to add
It is small.
The method of control group 5 is ineffective due to causing similar to direct physical mixed, and enzyme activity loss is serious.
In addition, adopting acidification attapulgite prepared with embodiment 3/feeding phytic acid multienzyme complex carries out temperature tolerance test
Opposite enzyme activity: 0.50h 88.18%；2.00h being 55.87%；4.00h being 35.48%.
The attapulgite prepared using embodiment 4/phytic acid multienzyme complex is carried out temperature tolerance and tests opposite enzyme activity:
0.50h is 90.05%；2.00h being 55.47%；4.00h being 36.74%.
Test example 5
The white enzyme hydrolysis tolerance test of compound stomach
The different livestock and poultry gastric emptying times are generally in 2.00~6.00h or so, i.e., feeding phytase playing a role in stomach
Time be 2.00~6.00h, but be not precluded in small intestine and large intestine there are still, and feeding phytase meet water i.e. be catalyzed
Reaction, so the feeding phytase activity in this test measurement protease hydrolytic 24.00h.Concave convex rod prepared by 1.0g embodiment 2
Soil/phytic acid multienzyme complex, is dissolved in the buffer of pH2.0, and enough pepsins are added, and (pepsin additive amount is phytase
500 times of enzyme activity), hydrolysis temperature is 37 DEG C, measurement 2.00,6.00, the enzyme activity after 24.00h.Freely feeding plant is surveyed under the conditions of
The pepsin hydrolysis tolerance of sour enzyme, control group 1, control group 2, control group 3, control group 4, control group 5.Feeding phytase enzyme
Measuring method living is the same as embodiment 4.The results are shown in Table 5.
5 pepsin hydrolysis tolerance of table
Note: experimental group is attapulgite/phytic acid multienzyme complex prepared by embodiment 2, and free phytase group is simple feeding
Phytase, control group 1 are attapulgite and mixture of the feeding phytase directly after physical mixed, and control group 2 is not add
Add CaCl2Attapulgite/phytic acid multienzyme complex of solution preparation (other are same as Example 2)；Control group 3 and embodiment 2 are made
Standby attapulgite/phytic acid multienzyme complex is identical, and difference is raw material in the attapulgite for selecting relative density 1.5 or so；
Control group 4 is identical as attapulgite/phytic acid multienzyme complex prepared by embodiment 2, and difference is selecting relative density 2.5 or so
Attapulgite be raw material.5 bibliography of control group (natural smectite/phytase complex carrier preparation and its phytate phosphorus hydrolysis
Activity research, Zhang Wenzhong, nonmetallic ore, in January, 2013) scheme, montmorillonite and phytase are substituted for the bumps of embodiment 2
Stick soil and feeding phytase and keep usage amount consistent.
The opposite enzyme activity of the above results show control group 1 three periods herein is respectively less than experimental group and feeding phytase
Group, because since attapulgite and the direct physical mixed of phytase do not have the environment of phytase absorption attapulgite, it cannot be effective
It is adsorbed, while directly physical mixed is more easily damaged the activity of phytase, leads to active reduction, if directly will be concave-convex
Stick soil and phytase are added in feed, and phytase cannot be protected well in Feed Manufacturing process, raw in feed
It produces in process since pH influences the loss that can cause phytase activity in varying degrees.
Experimental group is high compared with control group 2 with respect to enzyme activity, the reason is that calcium ion can promote phytic acid enzyme-to-substrate to combine, from
And improve the activity of enzyme.Under the same terms, after hydrolyzing 2.00h, attapulgite/feeding phytic acid multienzyme complex and feeding phytase
The opposite enzyme activity of group is respectively 97.63% and 70.19%, after hydrolyzing 24.00h, attapulgite/feeding phytic acid multienzyme complex phase
To enzyme activity, still more feeding phytase group is high with respect to enzyme activity, it is seen then that the protease hydrolytic tolerance of compound is more preferable.
Control group 3 and 4 enzyme activity of control group are lower, and the attapulgite for being primarily due to the opposite relative density contains much greatly
The impurity of particle, and the attapulgite bad dispersibility under this relative density are opposite to the adsorbance of phytase with this condition to add
It is small.
The method of control group 5 is ineffective due to causing similar to direct physical mixed, and enzyme activity loss is serious.
In addition, adopting acidification attapulgite/feeding phytic acid multienzyme complex prepared with embodiment 3 and being prepared using embodiment 4
Attapulgite/phytic acid multienzyme complex carry out pepsin hydrolysis tolerance test it is suitable with the result of embodiment 2 with respect to enzyme activity.
Test example 6
Complex media pH compatibility test
Livestock and poultry digestive organs body fluid pH generally between 3.5~7.0, take 1.0g embodiment 2 prepare attapulgite/it is feeding
Phytic acid multienzyme complex is respectively placed in the glycine-HCI buffer of pH3, the acetate buffer of pH4,5 and 6, the Tris- salt of pH7
In acid buffer, mixed liquor is placed in constant temperature oscillation instrument, 200r/min oscillation stirring 2.00h takes supernatant, surveys compound
Enzyme activity.Feeding phytase activity measuring method is the same as test example 4.As a result as shown in Figure 1.
Fig. 1 the result shows that, under the conditions of same pH, the most suitable medium pH of attapulgite/feeding phytic acid multienzyme complex 5.0 a left side
It is right.Gastrointestinal tract of livestock and fowls pH is generally 3.5~7.0, it was demonstrated that attapulgite/phytic acid multienzyme complex can play enzyme under gastrointestinal tract environment
Activity, and the feeding phytase in compound is consistent with the pH range of the feeding phytase optimal activity of sterling.Control group experiment
As a result low compared with attapulgite/phytic acid multienzyme complex with respect to enzyme activity, the reason is that calcium ion can promote phytic acid enzyme-to-substrate mutually to tie
It closes, to improve the activity of enzyme.Most suitable medium pH be mainly to the evaluation index of each compound, so this test example be not related to
The control group of attapulgite and mixture of the feeding phytase sterling after physical mixed is tested.
In addition, selecting the attapulgite of relative density 1.5 or so and 2.5 or so is that raw material is made with respect to enzyme activity compared with embodiment 2
Standby attapulgite/feeding phytase is relatively low.
Claims (10)
1. a kind of attapulgite/phytic acid multienzyme complex additive preparation method, which comprises the steps of: will plant
Sour enzyme is dissolved in buffer, is centrifuged after stirring, and taking supernatant is phytase solution, add attapulgite and NaCl or
CaCl2Mixed liquor is stirred oscillating reactions at room temperature by solution, is centrifuged, and freeze-drying pulverizes and sieves, and obtains compound addition
Agent.
2. attapulgite according to claim 1/phytic acid multienzyme complex additive preparation method, which is characterized in that institute
The relative density that takes for stating attapulgite is 2.10~2.20 attapulgite.
3. attapulgite according to claim 1/phytic acid multienzyme complex additive preparation method, which is characterized in that institute
Stating attapulgite is attapulgite or acidification attapulgite.
4. attapulgite according to claim 1/phytic acid multienzyme complex additive preparation method, which is characterized in that institute
Stating phytase is feeding phytase or sterling phytase.
5. attapulgite according to claim 1/phytic acid multienzyme complex additive preparation method, which is characterized in that institute
Stating buffer is preferably acetate buffer, 4~6mg/mL of concentration of phytase in supernatant.
6. attapulgite according to claim 1/phytic acid multienzyme complex additive preparation method, which is characterized in that institute
The dosage for stating attapulgite is 0.30~0.80g.
7. attapulgite according to claim 1/phytic acid multienzyme complex additive preparation method, which is characterized in that institute
State NaCl or CaCl2Solution is added to guarantee concentration NaCl or CaCl in phytase solution2Concentration is 0.05~0.1mol/
L。
8. attapulgite according to claim 2/phytic acid multienzyme complex additive preparation method, which is characterized in that institute
State the attapulgite for being 2.10~2.20 to density of attapulgite, specific preparation step are as follows:
(1) attapulgite purifies: taking the attapulgite raw ore that crushed, soaks in water, it is suspended that mass fraction 20~25% is made
Liquid, high speed dispersion mashing, slurry take out, and standing sedimentation removes supernatant liquor, and centrifugation is dried for standby；
(2) heavy-fluid is centrifugated: taking Du's column heavy-fluid to pour into separatory funnel, the attapulgite for then obtaining step (1) is with first
It is slowly poured into after alcohol infiltration, stirs, be placed in centrifuge separation, taking relative density is 2.10~2.20 attapulgite, is dried for standby.
9. concave convex rod prepared by a kind of attapulgite described in claim 1/phytic acid multienzyme complex additive preparation method
Soil/phytic acid multienzyme complex additive.
10. concave convex rod prepared by a kind of attapulgite described in claim 1/phytic acid multienzyme complex additive preparation method
Soil/phytic acid multienzyme complex additive is as the application in feedstuff additive product.
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