CN102936587A - Method for producing low-temperature pectinase and low-temperature clear apple juice from shaddock peel powder - Google Patents
Method for producing low-temperature pectinase and low-temperature clear apple juice from shaddock peel powder Download PDFInfo
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- CN102936587A CN102936587A CN2012104813789A CN201210481378A CN102936587A CN 102936587 A CN102936587 A CN 102936587A CN 2012104813789 A CN2012104813789 A CN 2012104813789A CN 201210481378 A CN201210481378 A CN 201210481378A CN 102936587 A CN102936587 A CN 102936587A
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Abstract
The invention relates to a method for producing low-temperature pectinase and low-temperature clear apple juice from shaddock peel powder. Shaddock peel powder is used as a carbon source to induce Penicillium aculeatum to produce low-temperature pectinase, and the low-temperature pectinase is utilized to clarify fresh apple juice at low temperature. The fermentation strain is Penicillium aculeatum; the fermentation medium is prepared from 0.5-2% of shaddock peel powder (w/v), 0.5-2% of peptone, 0.2% of magnesium sulfate heptahydrate, 0.2% of potassium dihydrogen phosphate and 0.01% of anhydrous calcium chloride the pH value of which is 5.5; and the fermentation conditions are as follows: the inoculum size is 2% (v/v), the fermentation time is 75-147 hours, the fermentation temperature is 30 DEG C, and the rotation speed is 150rpm. The low-temperature pectinase reacts with fresh apple juice at 10 DEG C, 20 DEG C or 30 DEG C for 2 hours to obtain the clear apple juice with the light transmittance of 43.3%, 53.3% or 62.4%; and thus, the clarifying effect is obvious.
Description
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Technical field
The present invention relates to microbial fermentation field and food processing field, specifically a kind of method of utilizing the pomelo peel powder to produce low temperature pectic enzyme and low temperature clarification Sucus Mali pumilae.
Background technology
Pectin (Pectin) be in the plant tissue with the water-fast class material of the combinations such as Mierocrystalline cellulose, hemicellulose, xylogen and protein.Pectin extensively is present in higher plant, in the cereal such as fruit, soybean and corn.Polygalacturonase (Pectinase) refers to the one group of multienzyme complex that can work in coordination with decompose pectin, generally includes pectin lyase (PL), polygalacturonase (PG) and Rohapect MPE (PE) etc.Polygalacturonase just sees nineteen thirty, is used for preparing drinks and fruit juice.General fruit juice nature pH value is 3.0～4.0, so foodstuffs industry mostly is acid pectase with polygalacturonase.In recent years, along with the adjustment of China's national policy and the industrial structure, fruit juice and fruit wine industry obtain fast development.Since the planning of farming industry Eleventh Five-Year Development Plan, concentrated source mill is set up in the raw material main producing region, the work in-process such as the concentrated garden spgarden stuff of development and fruit, vegetable juice.Polygalacturonase is as the important enzyme preparation of fruit and vegetable juice processing, and market demand is huge.
Shaddock is more common in south, and all there are a large amount of cultivations on the ground such as the Guangdong of China, Guangxi, Fujian, Jiangxi, Hunan, Zhejiang, Sichuan now.Pomelo peel pectin content is higher, but shaddock is after edible, and shaddock ped often goes out of use and is not utilized effectively.Thereby utilize pomelo peel to produce polygalacturonase, and with low cost, have potential exploitation and be worth.
Apple all has cultivation all over the world, be main in the world one of the kind that cultivates apple trees.Apple is the fruit of China's output maximum, and apple juice also begins to enter the flourish stage.Apple juice clarifying is the critical process that determines quality product, the defecation method that uses at present mainly contains centrifugal separation, filtration method and adds the method for scavenging agent, unless but before adopting these processing steps, fruit and vegetable juice has been carried out enzymatic treatment or purifying treatment, otherwise use separately these processing steps infeasible or uneconomic often.Under cold condition, fresh apple juice is carried out enzyme process clarification, can reduce not only that microorganism grows therein and the probability that pollutes, local flavor and the quality that can also save the energy and keep Sucus Mali pumilae.
Summary of the invention
The purpose of this invention is to provide a kind of method of utilizing the pomelo peel powder to produce low temperature pectic enzyme and low temperature clarification Sucus Mali pumilae.
The present invention utilizes the pomelo peel powder to be carbon source, and utilizing sour jujube spore Penicillium notatum (Penicillium aculeatum) is bacterial classification, and fermentative production low temperature pectic enzyme, fermentation unit reach 16.5U mL
1, concrete grammar is as follows:
(1) be that 28～32 ℃, rotating speed are under 120～180rpm condition in temperature, sour jujube spore Penicillium notatum is cultivated 36～60h in the seed activation substratum;
(2) seed liquor after will activating is inoculated in the fermention medium with 1.0～3.0%(v/v) inoculum sizes, is that 28～32 ℃, rotating speed are under 120～180rpm condition in temperature, cultivates 75～147h;
(3) the centrifugal supernatant liquor that obtains behind the thalline of removing is low temperature pectic enzyme enzyme liquid;
Described fermention medium is (w/v): pomelo peel powder 0.5～2%, peptone 0.5～2%, magnesium sulfate heptahydrate 0.2%, potassium primary phosphate 0.2%, Calcium Chloride Powder Anhydrous 0.01%, pH4.0～6.0.
Described seed activation substratum is (w/v): glucose 0.8%, pectin 0.2%, ammonium sulfate 0.5%, magnesium sulfate heptahydrate 0.2%, potassium primary phosphate 0.2%, Calcium Chloride Powder Anhydrous 0.01%, pH nature.
The method concrete steps of low temperature clarification Sucus Mali pumilae are as follows:
(1) with apple peel, is whittled into 1.5cm
3Fritter, weigh;
(2) add water (w/v=1/1), add again 0.5%(w/v) xitix;
(3) with juice extractor the apple piece is pressed into juice;
(4) with eight layers of gauze apple residue is filtered, obtain fresh apple juice;
(5) every milliliter of fresh apple juice adds 3～7U polygalacturonase enzyme liquid, at 10～30 ℃ of lower insulation reaction 1～3h;
(6) reaction namely obtains clarified apple juices after finishing.
Behind 10 ℃, 20 ℃ and 30 ℃ of lower insulation reaction 2h, the Sucus Mali pumilae transmittance of clarification is respectively 43.3%, 53.3% and 62.4%.
The present invention uses pomelo peel to induce sour jujube spore Penicillium to have the polygalacturonase of low temperature active as carbon source, and this enzyme clarifying effect to fresh apple juice under low temperature environment is remarkable.To the clarification of fresh apple juice, can reduce not only that microorganism grows therein and the probability that pollutes, local flavor and the quality that can also save the energy and keep Sucus Mali pumilae under the cold condition.
Description of drawings
Fig. 1: the pH of low temperature pectic enzyme of the present invention is active.
Fig. 2: the pH stability of low temperature pectic enzyme of the present invention.
Fig. 3: the temperature of low temperature pectic enzyme of the present invention is active.
Fig. 4: low temperature pectic enzyme is to the clarification rate of fresh apple juice under the cold condition of the present invention.
Embodiment
Embodiment 1:
1, the preparation of low temperature pectic enzyme enzyme liquid:
(1) with 10 of-20 ℃ of preservation
6Individual sour jujube spore Penicillium notatum spore suspension is inoculated in 50mL seed activation substratum, and under 30 ℃ of temperature, rotating speed 150rpm condition, sour jujube spore Penicillium notatum is cultivated 48h in the seed activation substratum;
(2) the seed liquor 4mL after will activating is inoculated in the 200mL fermention medium, 30 ℃ of temperature, under the rotating speed 150rpm condition, cultivates 76h;
(3) the centrifugal supernatant liquor that obtains behind the thalline of removing is low temperature pectic enzyme enzyme liquid.
Described seed activation substratum (w/v): glucose 0.8%, pectin 0.2%, ammonium sulfate 0.5%, magnesium sulfate heptahydrate 0.2%, potassium primary phosphate 0.2%, Calcium Chloride Powder Anhydrous 0.01%, pH nature.
Described fermention medium (w/v): pomelo peel powder 1.0%, peptone 0.5%, magnesium sulfate heptahydrate 0.2%, potassium primary phosphate 0.2%, Calcium Chloride Powder Anhydrous 0.01%, pH5.5.
2, enzyme activity determination method
(1) adds 0.2mM CaCl Pectin(Sigma company 0.1M pH5.0 glycine-NaOH damping fluid preparation 0.5%(w/v)),
2, then draw 900 μ L preheating 5min under 60 ℃ of conditions;
(2) add an amount of low temperature pectic enzyme enzyme of 100 μ L liquid, reaction 10min;
(3) add 1.5mL 3,5-dinitrosalicylic acid (DNS) termination reaction is boiled 5min;
(4) reaction system is cooled under 540nm, measures the OD value after the room temperature;
(5) 1 enzyme units alive (U) are defined as the required enzyme amount of per minute bottom exploded deposits yields 1 μ mol galacturonic acid under given condition, the enzyme activity calculation formula:
Enzyme activity (U/mL)=2.9936X * N/(T * V)
In the formula: X---absorbancy;
N---enzyme liquid extension rate;
V---enzyme concentration in the reaction (mL);
T---the enzyme-substrate reactions time (min).
The low temperature pectic enzyme enzyme activity for preparing is 16.5U/mL.
3, the optimal pH of low temperature pectic enzyme and pH Stability Determination
The optimal pH of enzyme is measured: low temperature pectic enzyme is carried out enzymatic reaction under 37 ℃ and in the damping fluid of 0.1M pH1.0 – 8.0.The pH Stability Determination of enzyme: enzyme liquid is placed the damping fluid of 0.1M pH1.0～10.0, at 37 ℃ of lower 1h that process, then under pH5.0 and 60 ℃, carry out enzymatic reaction, with untreated enzyme liquid in contrast.Damping fluid is 0.1M glycine-NaOH and glycine-HCl.To contain 0.2mM CaCl
20.5%(w/v) Pectin be substrate, reaction 10min measures the zymologic property of low temperature pectic enzyme.The result shows: the optimal pH of this enzyme is 5.0, at pH3.0～5.5 scope endoenzyme activity keepings more than 83% (Fig. 1); Damping fluid through pH3.0～8.0 is processed 1h, and enzyme work remains in (Fig. 2) more than 77%.
4, the optimum temperuture of low temperature pectic enzyme is measured
The optimum temperuture of enzyme is measured: in the damping fluid of pH5.0, carry out enzymatic reaction under 10～70 ℃.To contain 0.2mM CaCl
20.5%(w/v) Pectin be substrate, reaction 10min measures the zymologic property of low temperature pectic enzyme.The result shows: the optimum temperuture of this enzyme is 60 ℃, all has activity in 10-70 ℃ of scope, wherein relative enzyme residue 7.4%(Fig. 3 that lives 10 ℃ the time).
Embodiment 2
1, the preparation of low temperature pectic enzyme enzyme liquid:
(1) with 10 of-20 ℃ of preservation
6Individual sour jujube spore Penicillium notatum spore suspension is inoculated in 50mL seed activation substratum, and under 28 ℃ of temperature, rotating speed 120rpm condition, sour jujube spore Penicillium notatum is cultivated 36h in the seed activation substratum;
(2) the seed liquor 2mL after will activating is inoculated in the 200mL fermention medium, 28 ℃ of temperature, under the rotating speed 130rpm condition, cultivates 88h;
(3) the centrifugal supernatant liquor that obtains behind the thalline of removing is low temperature pectic enzyme enzyme liquid.
Described seed activation substratum (w/v): glucose 0.8%, pectin 0.2%, ammonium sulfate 0.5%, magnesium sulfate heptahydrate 0.2%, potassium primary phosphate 0.2%, Calcium Chloride Powder Anhydrous 0.01%, pH nature.
Described fermention medium (w/v): pomelo peel powder 0.5%, peptone 1%, magnesium sulfate heptahydrate 0.2%, potassium primary phosphate 0.2%, Calcium Chloride Powder Anhydrous 0.01%, pH4.0.
2, enzyme activity determination method
(1) adds 0.2mM CaCl Pectin(Sigma company 0.1M pH5.0 glycine-NaOH damping fluid preparation 0.5%(w/v)),
2, then draw 900 μ L preheating 5min under 60 ℃ of conditions;
(2) add an amount of low temperature pectic enzyme enzyme of 100 μ L liquid, reaction 10min;
(3) add 1.5mL 3,5-dinitrosalicylic acid (DNS) termination reaction is boiled 5min;
(4) reaction system is cooled under 540nm, measures the OD value after the room temperature;
(5) 1 enzyme units alive (U) are defined as the required enzyme amount of per minute bottom exploded deposits yields 1 μ mol galacturonic acid under given condition, the enzyme activity calculation formula:
Enzyme activity (U/mL)=2.9936X * N/(T * V)
In the formula: X---absorbancy;
N---enzyme liquid extension rate;
V---enzyme concentration in the reaction (mL);
T---the enzyme-substrate reactions time (min).
The low temperature pectic enzyme enzyme activity for preparing is 7.7U/mL.
Embodiment 3
1, the preparation of low temperature pectic enzyme enzyme liquid:
(1) with 10 of-20 ℃ of preservation
6Individual sour jujube spore Penicillium notatum spore suspension is inoculated in 50mL seed activation substratum, and under 32 ℃ of temperature, rotating speed 180rpm condition, sour jujube spore Penicillium notatum is cultivated 60h in the seed activation substratum;
(2) the seed liquor 6mL after will activating is inoculated in the 200mL fermention medium, 32 ℃ of temperature, under the rotating speed 180rpm condition, cultivates 147h;
(3) the centrifugal supernatant liquor that obtains behind the thalline of removing is low temperature pectic enzyme enzyme liquid.
Described seed activation substratum (w/v): glucose 0.8%, pectin 0.2%, ammonium sulfate 0.5%, magnesium sulfate heptahydrate 0.2%, potassium primary phosphate 0.2%, Calcium Chloride Powder Anhydrous 0.01%, pH nature.
Described fermention medium (w/v): pomelo peel powder 2%, peptone 2%, magnesium sulfate heptahydrate 0.2%, potassium primary phosphate 0.2%, Calcium Chloride Powder Anhydrous 0.01%, pH6.0.
2, enzyme activity determination method
(1) adds 0.2mM CaCl Pectin(Sigma company 0.1M pH5.0 glycine-NaOH damping fluid preparation 0.5%(w/v)),
2, then draw 900 μ L preheating 5min under 60 ℃ of conditions;
(2) add an amount of low temperature pectic enzyme enzyme of 100 μ L liquid, reaction 10min;
(3) add 1.5mL 3,5-dinitrosalicylic acid (DNS) termination reaction is boiled 5min;
(4) reaction system is cooled under 540nm, measures the OD value after the room temperature;
(5) 1 enzyme units alive (U) are defined as the required enzyme amount of per minute bottom exploded deposits yields 1 μ mol galacturonic acid under given condition, the enzyme activity calculation formula:
Enzyme activity (U/mL)=2.9936X * N/(T * V)
In the formula: X---absorbancy;
N---enzyme liquid extension rate;
V---enzyme concentration in the reaction (mL);
T---the enzyme-substrate reactions time (min).
The low temperature pectic enzyme enzyme activity for preparing is 14.0U/mL.
Embodiment 4
Low temperature pectic enzyme is to the clarification of fresh apple juice under the cold condition
(1) with apple peel, is whittled into 1.5cm
3Fritter, take by weighing 30g apple piece;
(2) add 30mL water, add again the xitix of 0.15g;
(3) with juice extractor the apple piece is pressed into juice;
(4) with eight layers of gauze apple residue is filtered, obtain fresh apple juice;
(5) get the 5mL fresh apple juice, add 5U low temperature pectic enzyme enzyme liquid, respectively at 10 ℃, 20 ℃ and 30 ℃ of insulation reaction 2h; Get simultaneously the 5mL fresh apple juice, add 5U and boil the enzyme liquid of 10min deactivation, respectively at 10 ℃, 20 ℃ and 30 ℃ of insulation reaction 2h in contrast;
(6) reaction namely obtains clarified apple juices after finishing.Take water as contrast, under 660nm, measure absorbancy, calculate transmittance.Transmittance (%)=10
2-OD, under 10 ℃, 20 ℃ and 30 ℃, the fresh apple juice transmittance that the low temperature pectic enzyme enzyme liquid of deactivation is processed is all 4.4%, and the fresh apple juice transmittance that low temperature pectic enzyme enzyme liquid is processed be respectively 43.3%, 53.3% and 62.4%(Fig. 4).
The addition of low temperature pectic enzyme enzyme liquid can also be 3U or 7U, or other numerical value among 3U～7U, can obtain the 4 akin fresh apple juice transmittances with embodiment.
Claims (3)
1. the production method of a low temperature pectic enzyme is characterized in that carrying out according to the following steps:
(1) is that 28～32 ℃, rotating speed are under 120～180rpm condition in temperature, sour jujube spore Penicillium notatum (Penicillium aculeatum) is cultivated 36～60h in the seed activation substratum;
(2) seed liquor after will activating is inoculated in the fermention medium with 1.0～3.0%(v/v) inoculum sizes, is that 28～32 ℃, rotating speed are under 120～180rpm condition in temperature, cultivates 75～147h;
(3) the centrifugal supernatant liquor that obtains behind the thalline of removing is low temperature pectic enzyme enzyme liquid;
Described fermention medium is (w/v): pomelo peel powder 0.5～2%, peptone 0.5～2%, magnesium sulfate heptahydrate 0.2%, potassium primary phosphate 0.2%, Calcium Chloride Powder Anhydrous 0.01%, pH value 4.0～6.0.
2. the production method of low temperature pectic enzyme according to claim 1 is characterized in that described activation medium is (w/v): glucose 0.8%, pectin 0.2%, ammonium sulfate 0.5%, magnesium sulfate heptahydrate 0.2%, potassium primary phosphate 0.2%, Calcium Chloride Powder Anhydrous 0.01%, the pH nature.
3. the method with low temperature pectic enzyme claimed in claim 1 clarification fresh apple juice is characterized in that adding 3～7U polygalacturonase enzyme liquid in every milliliter of fresh apple juice, namely obtains clarified apple juices behind 10 ℃ or 20 ℃ or 30 ℃ of lower insulation reaction 1～3h.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104256801A (en) * | 2014-10-13 | 2015-01-07 | 宁波海通食品科技有限公司 | Method for increasing juice yield and anthocyanidin content of blueberry pulp |
| CN106119215A (en) * | 2016-06-30 | 2016-11-16 | 安徽工程大学 | A kind of fungus fermentation lacquer producing enzyme liquid culture medium and its preparation method and application |
| CN107668466A (en) * | 2017-10-20 | 2018-02-09 | 江苏连恒生物科技有限公司 | A kind of blackberry fruit juice defecation method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1613381A (en) * | 2004-12-08 | 2005-05-11 | 河北科技师范学院 | Fresh apple juice production |
| WO2009141156A2 (en) * | 2008-05-23 | 2009-11-26 | Ab Enzymes Gmbh | Use of pectinolytic enzymes for the treatment of fruit and vegetable mash and enzyme sequences therefor |
| CN102154124A (en) * | 2011-03-28 | 2011-08-17 | 浙江师范大学 | Penicillium oxallcum Po-5 strain and application thereof |
-
2012
- 2012-11-23 CN CN2012104813789A patent/CN102936587A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1613381A (en) * | 2004-12-08 | 2005-05-11 | 河北科技师范学院 | Fresh apple juice production |
| WO2009141156A2 (en) * | 2008-05-23 | 2009-11-26 | Ab Enzymes Gmbh | Use of pectinolytic enzymes for the treatment of fruit and vegetable mash and enzyme sequences therefor |
| CN102037122A (en) * | 2008-05-23 | 2011-04-27 | Ab酶有限公司 | Use of pectinolytic enzymes for the treatment of fruit and vegetable mash and enzyme sequences therefor |
| CN102154124A (en) * | 2011-03-28 | 2011-08-17 | 浙江师范大学 | Penicillium oxallcum Po-5 strain and application thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104256801A (en) * | 2014-10-13 | 2015-01-07 | 宁波海通食品科技有限公司 | Method for increasing juice yield and anthocyanidin content of blueberry pulp |
| CN106119215A (en) * | 2016-06-30 | 2016-11-16 | 安徽工程大学 | A kind of fungus fermentation lacquer producing enzyme liquid culture medium and its preparation method and application |
| CN107668466A (en) * | 2017-10-20 | 2018-02-09 | 江苏连恒生物科技有限公司 | A kind of blackberry fruit juice defecation method |
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Application publication date: 20130220 |