CN102994400B - Microorganism capable of degrading navel orange segment membrane and enzymic preparation containing microorganism as well as application - Google Patents
Microorganism capable of degrading navel orange segment membrane and enzymic preparation containing microorganism as well as application Download PDFInfo
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- CN102994400B CN102994400B CN201210559073.5A CN201210559073A CN102994400B CN 102994400 B CN102994400 B CN 102994400B CN 201210559073 A CN201210559073 A CN 201210559073A CN 102994400 B CN102994400 B CN 102994400B
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Abstract
The invention discloses a microorganism capable of degrading a navel orange segment membrane and an enzymic preparation containing the microorganism as well as an application. The preservation name of the microorganism is Aspergillus sp.C-2 and the preservation unit is China Center for Type Culture Collection; and the preservation number is CCTCC NO: M 2012160. The enzymic preparation prepared from the microorganism capable of degrading the navel orange segment membrane does not use a lot of acid and alkali and can remove the navel orange segment membrane through a biological enzymolysis effect, so that the production efficiency can be greatly improved, the crushing rate of navel oranges is reduced and the product quality is stable; and the production cost can be reduced, no residues exist, the microorganism is environment-friendly, the safety is high and the environment is not polluted. The utilization of the enzymic preparation is simple and fast; the effect on the navel orange segment membrane is efficient and safe; and the degradation is complete and inner orange juice cells are not damaged.
Description
Technical field
The present invention relates to technical field of bioengineering, in particular to a kind of degrade navel orange capsule clothing microorganism, the zymin that contains it and application.
Background technology
China's oranges and tangerines processing is following for a long time the traditional technology such as hand peeling and soda acid excystation clothing and is producing.The acid-alkali treatment method of existing acid-base method excystation clothing mainly contains: classical soda acid two step method, the heavy sour two step method of improvement, phosphoric acid salt NaOH single stage method, and EDTA or Na
2-EDTA(ethylenediamine tetraacetic acid (EDTA) or disodium ethylene diamine tetraacetate) remove skin method for the low alkali of auxiliary agent.There is the problems such as labour intensive, production efficiency are low, unstable product quality in artificial peeling and soda acid excystation clothing technique.The excystation of industrialization can production at present clothing water loss is large, and product water loss per ton reaches 60 ~ 80 tons, not only increases product cost, and has aggravated the shortage of tap water resource.Meanwhile, because of a large amount of alkali that uses, produce a large amount of industrial alkali liquid waste water, increased the production cost of product, also contaminate environment, has affected the security of product, easily meet with technology barriers and green barrier, weakened the competitive edge of China's oranges and tangerines canning industry in world market.Meanwhile, in oranges and tangerines processing, produce the skin slag that accounts for oranges and tangerines amount of finish 30% left and right, be conventionally all dropped, not only caused the wasting of resources, also brought pollution to environment.
In navel orange processing, excystation clothing technique is one of its technical difficult points.Orange juice born of the same parents' production technology mainly adopts the technique of traditional oranges and tangerines can acid-alkali treatment at present, has hand peeling, distinguish, the problem that production efficiency is low; Soda acid capsule clothing water consumption is large, and environmental pollution is serious, and Product Safety is low; Due to the difference of peel of Citrus reticulata Blanco and Peel of Navel Orange structure, cause existing oranges and tangerines enzyme process to go capsule clothing technology to be not suitable for navel orange orange juice born of the same parents' processing.
Summary of the invention
The present invention aims to provide a kind of degrade navel orange capsule clothing microorganism, the zymin that contains it and application, and to solve in prior art, the de-navel orange capsule clothing explained hereafter efficiency of soda acid is low, unstable product quality and have environment and the technical problem of food safety.
According to an aspect of the present invention, provide a kind of degraded navel orange capsule clothing microorganism.The preservation name of this microorganism is called aspergillus niger C-2(Aspergillus sp.C-2), depositary institution is Chinese Typical Representative culture collection center, preserving number is CCTCC NO:M2012160.
According to another aspect of the present invention, provide a kind of application of navel orange capsule clothing microorganism in preparation degraded navel orange capsule clothing zymin of degrading.
According to a further aspect of the invention, provide a kind of degraded navel orange capsule clothing zymin.This degraded navel orange capsule clothing zymin adopts above-mentioned degraded navel orange capsule clothing microorganism to make.
Further, adopt degraded navel orange capsule clothing microorganism to cultivate and make through strain fermentation cultivation, liquid submerged fermentation or solid state fermentation.
Further, liquid submerged fermentation or solid state fermentation before cultivating, further comprise activation, prepare spore suspension, the step of seed culture.
Further, this zymin comprises: by zymin through ultrafiltration, concentrated make concentrated enzyme preparation, or enzyme preparation is become to pulvis or granule.
According to a further aspect of the invention, provide the application of a kind of above-mentioned degraded navel orange capsule clothing zymin on navel orange, oranges and tangerines excystation clothing.
Further, comprise the following steps: it is 8 × 10 that zymin is diluted to enzyme work
3～10 × 10
3u/g: the zymin after dilution is mixed to 45 ~ 50 ℃ of insulation 90 ~ 120min with the ratio of navel orange take mass ratio as 3:1 of peeling
There is the unsafe factors such as heavy-metal residual in tradition soda acid excystation clothing technique, and the zymin that adopts the microorganism of degraded navel orange capsule clothing of the present invention to prepare does not relate to a large amount of uses of soda acid, carry out removing of navel orange capsule clothing by biological enzymolysis effect, therefore greatly improving production efficiency, reduce navel orange fruit percentage of damage, make constant product quality, and can reduce production costs, noresidue, environmental protection, safe, free from environmental pollution.The use of this zymin is simple and convenient, efficient, complete to the effect of navel orange capsule clothing, complete decomposition, and do not damage inner orange juice born of the same parents.It is simple that degraded navel orange capsule clothing fermentation using enzyme produces enzymatic process, and can reach the target of energy-saving and cost-reducing, water saving, protection of the environment, cleaner production.
Embodiment
It should be noted that, in the situation that not conflicting, the feature in embodiment and embodiment in the application can combine mutually.Describe the present invention in detail below in conjunction with embodiment.
A kind of typical embodiment according to the present invention, provides a kind of degraded navel orange capsule clothing microorganism.The preservation name of this microorganism is called aspergillus niger C-2(Aspergillus sp.C-2), depositary institution is Chinese Typical Representative culture collection center, and preserving number is CCTCC NO:M 2012160, and preservation day is on May 7th, 2012.
Degraded navel orange capsule clothing microorganism of the present invention is screened acquisition by following steps: the samples such as navel orange surface, soil, screen aimed strain from rotting, then after ultraviolet mutagenesis, obtain by screening repeatedly the bacterial strain that navel orange capsule clothing is had to efficient degradation effect.Mutatis mutandis bacterial strain of the present invention is through being accredited as aspergillus niger C-2(Aspergillus sp.C-2), its main biological property is: on solid medium, cultivate 3d～5d, bacterium colony spreads rapidly, be just white, rear overstrike is until black heavy fleece shape, the colourless or central slightly tawny in the back side.Conidial head brown-black is radial, and conidiophore is different in size.Top capsule is spherical, double-deck stigma.Conidium brown is spherical.Globe-roof capsule is formed on top, covers one deck metulae and one deck stigma on it comprehensively, and long on stigma have bunchiness memnonious spherical, diameter 2.5～4.0 μ m.Conidial head is spherical, diameter 700～800 μ m, brown-black.Conidial head brown-black is radial, and conidiophore is different in size.Conidiophore stretches out and is about 1～3mm in matrix, wall thickness and smooth.
A kind of typical embodiment according to the present invention, provides a kind of degraded navel orange capsule clothing zymin.This zymin adopts above-mentioned degraded navel orange capsule clothing microorganism to make.Because this degraded navel orange capsule clothing zymin does not relate to a large amount of uses of soda acid, carry out removing of navel orange capsule clothing by biological enzymolysis effect, therefore greatly improving production efficiency, reduce navel orange fruit percentage of damage, make constant product quality, and can reduce production costs, noresidue, environmental protection, safe, free from environmental pollution.Preferably, this zymin adopts above-mentioned degraded navel orange capsule clothing microorganism to make through liquid submerged fermentation or solid state fermentation cultivation.
A kind of typical embodiment according to the present invention, provides a kind of preparation method of the navel orange capsule clothing zymin of degrading.This preparation method comprises that the above-mentioned aspergillus niger C-2 of employing is as fermentation strain, prepares degraded navel orange capsule clothing zymin through liquid submerged fermentation or solid state fermentation cultivation.What in the subtractive process of navel orange capsule clothing, play a major role is this aspergillus niger C-2 and metabolite thereof, because prepare and also can adopt ordinary method of the prior art to carry out for this reason, preferably, aspergillus niger C-2 prepares degraded navel orange capsule clothing zymin through liquid submerged fermentation or solid state fermentation cultivation, because the preparation method of these zymins is simple, be convenient to suitability for industrialized production, and can not cause environmental pollution.Preferably, liquid submerged fermentation or solid state fermentation further comprise activation before cultivating, prepare spore suspension, the step of seed culture, increase the increased activity that these steps can make bacterial strain, the production efficiency that is conducive to zymin improves, and in follow-up use procedure, makes zymin keep higher activity.
A kind of typical embodiment according to the present invention, activation comprises: aspergillus niger C-2 is inoculated in substratum and is activated, and the pH value of substratum is 7.0～7.2, and activation temperature is controlled at 28 ℃～30 ℃, and soak time is 1～2 day.The product enzyme aspergillus niger C-2 bacterium cell of freezing, by this activation procedure, can make the fast quick-recovery of ability of this production by biological enzyme normal.A kind of typical embodiment according to the present invention, seed culture comprises: the spore suspension of activation is inoculated in seed liquid nutrient medium, the pH value of controlling seed liquid nutrient medium is 7.0～7.2, culture temperature is controlled at 28 ℃～30 ℃, 200r/m cultivates 12～16h, finally obtain the purebred son of some amount and quality, can be used as the bacterial classification that large scale fermentation is produced.A kind of typical embodiment according to the present invention, liquid submerged fermentation is cultivated and is comprised: the seed liquid culture medium inoculated containing aspergillus niger C-2 through seed culture is carried out to submerged fermentation cultivation in fermentation flask, fermentation culture conditions is: temperature is 30 ℃ in 12h, after 12h 28 ℃; PH value is 7.0～7.2; Pressure position 0.06MPa～0.08MPa; 180r/m in stirring velocity 12h, 200r/m after 12h; Air flow 1:1.0～1.5; Fermentation time 108～120h, makes zymin.Can guarantee nutrient uniformly distributing and oxygen abundance by liquid submerged fermentation, there is the advantages such as enzymatic production is with short production cycle, output is high, benefit is large.A kind of typical embodiment according to the present invention, the step that solid state fermentation is cultivated comprises: the described seed solid medium containing aspergillus niger C-2 through described seed culture is inoculated in solid-state fermentation culture medium, cultivate 108h for 30 ℃, obtain aspergillus niger C-2 solid state fermentation song, described solid state fermentation triton makes described zymin after crossing cryodrying and pulverizing, or obtains described zymin by lixiviate in 0.5mol/L pH=6.5 phosphoric acid buffer.Solid state fermentation simulation bacterial classification self-sow environment, makes microorganism keep the growth conditions similar to nature, and when fermentation ends, substratum is wet stock state, and object production concentration is high and extraction process is simple.
A kind of typical embodiment according to the present invention, the substratum of activation use is solid slant culture base; The step of preparing spore suspension comprises: adopt the spore of aspergillus niger C-2 under aseptic washing, and be diluted to 2～3 × 10
6individual/mL makes spore suspension, and by spore suspension be kept at 4 ℃ for subsequent use.
Preferably, in seed culture step, spore suspension is inoculated in seed liquid nutrient medium by 5% inoculum size; In liquid submerged fermentation culturing step by being inoculated in and carrying out submerged fermentation cultivation in fermentation flask by 8%～10% inoculum size containing the seed liquid nutrient medium of aspergillus niger C-2 through seed culture.This inoculum size neither can cause the waste of bacterial classification, is conducive to again the rapid multiplying of aspergillus niger C-2.
Preferably, the component that comprises following content take navel orange capsule clothing powder as the substratum of carbon source of activation use: (NH
4)
2sO
42g/L～2.5g/L, MgSO
40.6g/L～1g/L, K
2hPO
41g/L～2g/L, NaCl 1g/L～2g/L, navel orange capsule clothing powder 6g/L～10g/L and agar 15g/L～20g/L, and substratum sterilizing 30min at 121 ℃ of temperature of activation use makes.This kind of activation medium is suitable for the activation of aspergillus niger C-2, because take navel orange capsule clothing powder as carbon source, the product enzyme aspergillus niger C-2 bacterium cell of freezing, by this activation procedure, can make the fast quick-recovery of ability of this microorganisms navel orange capsule clothing degrading enzyme normal.Preferably, the component that comprises following content take navel orange capsule clothing powder as the substratum of carbon source that seed liquid culture is used: (NH
4)
2sO
42g/L～2.5g/L, MgSO
40.6g/L～1g/L, K
2hPO
41g/L～2g/L, NaCl 1g/L～2g/L, navel orange capsule clothing powder or Peel of Navel Orange pomace 15g/L～20g/L; And seed liquid nutrient medium sterilizing 30min at 121 ℃ of temperature makes.This kind of seed liquid nutrient medium is suitable for the cultivation of aspergillus niger C-2, finally obtains the purebred son of some amount and quality, can be used as the bacterial classification that large scale fermentation is produced.And also can adopt this substratum to carry out in the process that liquid submerged fermentation is cultivated.Because take navel orange capsule clothing powder as carbon source, can make the navel orange capsule clothing degrading enzyme of this microorganisms keep high vigor.A kind of typical embodiment according to the present invention, after step 4), further comprise according to actual needs: by the ultrafiltration of zymin process, the concentrated concentrated enzyme preparation that makes, or enzyme preparation is become to pulvis or granule etc., concentrated enzyme preparation is easy to use, be more convenient for preservation and the transportation such as pulvis or granule.
The zymin enzymolysis efficiency of preparing by the inventive method is higher; And the zymin making through the method enzymic fermentation liquid after filtration sterilization, ultrafiltration and concentration, rotproofing room temperature preservation after 6 months enzyme live still keep more than 95%.
A kind of typical embodiment according to the present invention, the application of above-mentioned degraded navel orange capsule clothing zymin on navel orange, oranges and tangerines excystation clothing.Preferably, the application of zymin comprises the following steps: zymin dilution is rear and peel navel orange in 45 ~ 50 ℃ of water bath heat preservation 90 ~ 120min.
A kind of typical embodiment according to the present invention, the concrete grammar of degraded navel orange capsule clothing is: by for subsequent use the navel orange fruit ball of the residual capsule clothing of peeling, concentrated fermenting enzyme preparation is stirred after with 5%～8% concentration dilution, jacketed kettle vapour is bathed and is warmed up to 45 ℃, then add the navel orange (solid-liquid ratio 1:3) of peeling, with citric acid adjust pH to 4.5, under 45～50 ℃ of conditions of temperature, constantly stir and keep constant temperature 90～120min, obtain excystation clothing navel orange fruit ball, it is after treatment to orange juice born of the same parents that fruit ball warp is crossed dispersion machine, this zymin can be reused 3～5 times under these conditions.
The present invention is optimized the technique of its enzymatic production in to the seed selection of degraded navel orange capsule clothing microorganism strains, and applicable large-scale industrialization is produced this zymin.
The typical embodiment of one according to the present invention, provides a kind of substratum for the navel orange capsule clothing microorganism of degrading.This substratum is for take navel orange capsule clothing powder or Peel of Navel Orange pomace as carbon source.
During as activation of microorganism, preferably include the component of following content: (NH
4)
2sO
42g/L～2.5g/L, MgSO
40.6g/L～1g/L, K
2hPO
41g/L～2g/L, NaCl 1g/L～2g/L, navel orange capsule clothing powder 6g/L～10g/L and agar 15g/mL～20g/mL, and at 121 ℃ of temperature, sterilizing 30min makes described substratum.
During for the liquid cultivation of seed and deep fermentation cultivation, preferably include the component of following content.(NH
4)
2sO
42g/L～2.5g/L, MgSO
40.6g/L～1g/L, K
2hPO
41g/L～2g/L, NaCl 1g/L～2g/L, navel orange capsule clothing powder or Peel of Navel Orange pomace 15g/L～20g/L; And sterilizing 30min makes described substratum at 121 ℃ of temperature.
Further illustrate beneficial effect of the present invention below in conjunction with embodiment.
The substratum of activation use comprises the component of following content: (NH
4)
2sO
42g/L～2.5g/L, MgSO
40.6g/L～1g/L, K
2hPO
41g/L～2g/L, NaCl 1g/L～2g/L, navel orange capsule clothing powder 6g/L～10g/L and agar 15g/L～20g/L, and substratum sterilizing 30min at 121 ℃ of temperature of activation use makes.This kind of activation medium is suitable for the activation of aspergillus niger C-2.
Seed liquid nutrient medium and deep fermentation are cultivated the component that comprises following content: (NH
4)
2sO
42g/L～2.5g/L, MgSO
40.6g/L～1g/L, K
2hPO
41g/L～2g/L, NaCl 1g/L～2g/L, navel orange capsule clothing powder or Peel of Navel Orange pomace 15g/L～20g/L; And seed liquid nutrient medium sterilizing 30min at 121 ℃ of temperature makes.This kind of seed liquid nutrient medium is suitable for the cultivation of aspergillus niger C-2, and also can adopt this substratum to carry out in the process of liquid submerged fermentation cultivation.
Embodiment 1
The present embodiment is investigated the degraded of aspergillus niger C-2 bacterial strain CCTCCNO:M of the present invention 2012160 fermented liquids to navel orange capsule clothing in liquid submerged fermentation liquid.Adopt following methods to prepare a kind of zymin of aspergillus niger C-2 bacterial strain of the present invention:
Activation medium comprises the component of following content: (NH
4)
2sO
42g/L, MgSO
40.6g/L, K
2hPO
41g/L, NaCl1g/L, navel orange capsule clothing powder 6g/L and agar 15g/L, and substratum sterilizing 30min at 121 ℃ of temperature of activation use makes.This kind of activation medium is suitable for the activation of aspergillus niger C-2.Substratum effect within the scope of said components is similar.
Seed liquid nutrient medium comprises the component of following content: (NH
4)
2sO
42g/L, MgSO
40.6g/L, K
2hPO
41g/L, NaCl 1g/L, navel orange capsule clothing powder or Peel of Navel Orange pomace 15g/L; And seed liquid nutrient medium sterilizing 30min at 121 ℃ of temperature makes.This kind of seed liquid nutrient medium is suitable for the cultivation of aspergillus niger C-2, and also can adopt this substratum to carry out in the process of liquid submerged fermentation cultivation.
Step is as follows:
(1) activation: the aspergillus niger C-2 seed that described seed selection is obtained is inoculated in fresh solid slant culture base and activates, in described reactivation process, the pH value of controlling solid slant culture base is 7.0～7.2, activation temperature is controlled at 28 ℃～29 ℃, activation culture 2～3 days, with aspergillus niger C-2 spore under aseptic washing, prepare spore suspension and be diluted to 2～3 × 10
6individual/mL, and be kept at 4 ℃ for subsequent use;
(2) seed culture: the aspergillus niger C-2 spore suspension after above-mentioned activation is inoculated in seed liquid nutrient medium by 5% inoculum size, the pH value of controlling seed culture medium is 7.0～7.2, temperature when seed culture is controlled at 28 ℃～30 ℃, and 200r/m cultivates 12～16h; Navel orange capsule clothing powder or Peel of Navel Orange pomace 15g/L in substratum.
(3) liquid submerged fermentation is cultivated: the seed aspergillus niger C-2 bacterial classification after above-mentioned cultivation is inoculated and in fermentation flask, carried out submerged fermentation cultivation by the inoculum size of liquid submerged fermentation substratum 8%～10%, the formula of described liquid submerged fermentation substratum is identical with described seed liquid nutrient medium, fermentation culture conditions is: temperature is 30 ℃ in 12h, after 12h 28 ℃; Automatically regulate pH 7.0～7.2; Tank pressure 0.06MPa～0.08MPa; 180r/m in stirring velocity 12h, 200r/m after 12h; Air flow 1:1.0～1.5; Fermentation time 108～120h.
Whole process incubation time is greatly about 10 days, the enzyme liquid of liquid submerged fermentation gained is used immediately, and the enzyme liquid that described fermentation is obtained obtains concentrated enzyme liquid through ultrafiltration and concentration, or the different degraded navel orange capsule clothing zymin obtaining by other processing modes, as pulvis or granule etc., for subsequent use.
Navel orange 50kg, through skiving machine punching two ends and after peeling, obtains the navel orange fruit ball of about 40kg containing capsule clothing, for subsequent use.Concentrated fermenting enzyme preparation is stirred after with 5%～8% concentration dilution, jacketed kettle vapour is bathed and is warmed up to 40 ℃, then add the navel orange (solid-liquid ratio 1:3) of peeling, with citric acid adjust pH to 4.5, under 45～50 ℃ of conditions of temperature, constantly stir and keep constant temperature 90～120min, obtain excystation clothing navel orange fruit ball, it is after treatment to orange juice born of the same parents that fruit ball warp is crossed dispersion machine, and this zymin can be reused 3～5 times under these conditions.
Embodiment 2
The present embodiment is investigated the degraded of aspergillus niger C-2 bacterial strain CCTCCNO:M of the present invention 2012160 fermented liquids to navel orange capsule clothing in liquid submerged fermentation liquid.Adopt following methods to prepare a kind of zymin of aspergillus niger C-2 bacterial strain of the present invention:
Activation medium comprises the component of following content: (NH
4)
2sO
42.5g/L, MgSO
41g/L, K
2hPO
42g/L, NaCl2g/L, navel orange capsule clothing powder 10g/L and agar 20g/L, and substratum sterilizing 30min at 121 ℃ of temperature of activation use makes.This kind of activation medium is suitable for the activation of aspergillus niger C-2.Substratum effect within the scope of said components is similar.
Seed liquid nutrient medium comprises the component of following content: (NH
4)
2sO
42.5g/L, MgSO
41g/L, K
2hPO
42g/L, 2g/L, navel orange capsule clothing powder or Peel of Navel Orange pomace 20g/L; And seed liquid nutrient medium sterilizing 30min at 121 ℃ of temperature makes.This kind of seed liquid nutrient medium is suitable for the cultivation of aspergillus niger C-2, and also can adopt this substratum to carry out in the process of liquid submerged fermentation cultivation.
Step is as follows:
(1) activation: the aspergillus niger C-2 seed that described seed selection is obtained is inoculated in fresh solid slant culture base and activates, in described reactivation process, the pH value of controlling solid slant culture base is 7.0～7.2, activation temperature is controlled at 29 ℃～30 ℃, activation culture 2～3 days, with aspergillus niger C-2 spore under aseptic washing, prepare spore suspension and be diluted to 2～3 × 10
6individual/mL, and be kept at 4 ℃ for subsequent use;
(2) seed culture: the aspergillus niger C-2 spore suspension after above-mentioned activation is inoculated in seed liquid nutrient medium by 5% inoculum size, the pH value of controlling seed culture medium is 7.0～7.2, temperature when seed culture is controlled at 28 ℃～30 ℃, and 200r/m cultivates 12～16h; Navel orange capsule clothing powder or Peel of Navel Orange pomace 20g/L in substratum.
(3) liquid submerged fermentation is cultivated: the seed aspergillus niger C-2 bacterial classification after above-mentioned cultivation is inoculated and in fermentation flask, carried out submerged fermentation cultivation by the inoculum size of liquid submerged fermentation substratum 8%～10%, the formula of described liquid submerged fermentation substratum is identical with described seed liquid nutrient medium, fermentation culture conditions is: temperature is 30 ℃ in 12h, after 12h 28 ℃; Automatically regulate pH 7.0～7.2; Tank pressure 0.06MPa～0.08MPa; 180r/m in stirring velocity 12h, 200r/m after 12h; Air flow 1:1.0～1.5; Fermentation time 108～120h.
Whole process incubation time is greatly about 10 days, the enzyme liquid of liquid submerged fermentation gained is used immediately, and the enzyme liquid that described fermentation is obtained obtains concentrated enzyme liquid through ultrafiltration and concentration, or the different degraded navel orange capsule clothing zymin obtaining by other processing modes, as pulvis or granule etc., for subsequent use.
Navel orange 50kg, through skiving machine punching two ends and after peeling, obtains the navel orange fruit ball of about 40kg containing capsule clothing, for subsequent use.Concentrated fermenting enzyme preparation is stirred after with 5%～8% concentration dilution, jacketed kettle vapour is bathed and is warmed up to 40 ℃, then add the navel orange (solid-liquid ratio 1:3) of peeling, with citric acid adjust pH to 4.5, under 45～50 ℃ of conditions of temperature, constantly stir and keep constant temperature 90～120min, obtain excystation clothing navel orange fruit ball, it is after treatment to orange juice born of the same parents that fruit ball warp is crossed dispersion machine, and this zymin can be reused 3～5 times under these conditions.
Embodiment 3
The present embodiment is investigated aspergillus niger C-2 bacterial strain CCTCC NO:M of the present invention 2012160 degradeds of solid state fermentation song to navel orange capsule clothing in solid state fermentation.Adopt following methods to prepare a kind of aspergillus niger C-2 solid koji of the present invention:
Activation medium comprises the component of following content: (NH
4)
2sO
42.3g/L, MgSO
40.8g/L, K
2hPO
41.5g/L, NaCl1.5g/L, navel orange capsule clothing powder 8g/L and agar 18g/L, and substratum sterilizing 30min at 121 ℃ of temperature of activation use makes.This kind of activation medium is suitable for the activation of aspergillus niger C-2.Substratum effect within the scope of said components is similar.
Seed liquid nutrient medium comprises the component of following content: (NH
4)
2sO
42.3g/L, MgSO
40.8g/L, K
2hPO
41.5g/L, NaCl 1.5g/L, navel orange capsule clothing powder or Peel of Navel Orange pomace 18g/L; And seed liquid nutrient medium sterilizing 30min at 121 ℃ of temperature makes.This kind of seed liquid nutrient medium is suitable for the cultivation of aspergillus niger C-2, and also can adopt this substratum to carry out in the process of liquid submerged fermentation cultivation.
Step is as follows:
(1) activation: the aspergillus niger C-2 seed that described seed selection is obtained is inoculated in fresh solid slant culture base and activates, in described reactivation process, the pH value of controlling solid slant culture base is 7.0～7.2, activation temperature is controlled at 28 ℃～30 ℃, activation culture 2～3 days, with aspergillus niger C-2 spore under aseptic washing, prepare spore suspension and be diluted to 2～3 × 10
6individual/mL, and be kept at 4 ℃ for subsequent use;
(2) seed culture: by the aspergillus niger C-2 spore suspension after above-mentioned activation by 5% inoculum size be inoculated in sterilizing by wheat wheat bran 95 weight parts, navel orange pomace or fruit peel powder 180 weight parts, ammonium sulfate 2 weight parts, magnesium sulfate 0.5 weight part, dipotassium hydrogen phosphate 1 weight part, it is 60% that above each component adds water to mix and regulate moisture humidity thoroughly.(initial pH 6.5), cultivates 48h for 30 ℃.(3) solid state fermentation is cultivated: access in 50kg solid-state fermentation culture medium by 10% inoculum size bent the aspergillus niger C-2 seed of above-mentioned activation, the each component of described solid-state fermentation culture medium is identical with above-mentioned solid-state seed culture medium component, after seed song mixes with solid-state fermentation culture medium, cultivate 108h for 30 ℃, obtain aspergillus niger C-2 solid state fermentation song, this solid state fermentation song uses after can and pulverizing by cryodrying, also can be by using after lixiviate acquisition liquid enzymes liquid in 0.5mol/L pH=6.5 phosphoric acid buffer.
Navel orange 50kg, through skiving machine punching two ends and after peeling, obtains the navel orange fruit ball of about 40kg containing capsule clothing, for subsequent use.Bent the aspergillus niger C-2 solid state fermentation of above-mentioned cryodrying, pulverizing 12kg is added in the physiological saline of 120kg, stir, jacketed kettle vapour is bathed and is warmed up to 40 ℃, then add the navel orange (solid-liquid ratio 1:3) of peeling, with citric acid adjust pH to 4.5, under 45～50 ℃ of conditions of temperature, constantly stir and keep constant temperature 90～120min, obtain excystation clothing navel orange fruit ball, fruit ball warp is crossed after dispersion machine is processed and is obtained orange juice born of the same parents, and this aspergillus niger C-2 solid state fermentation song can be reused 3～5 times under these conditions.
Embodiment 4
The present embodiment is investigated the degraded of aspergillus niger C-2 bacterial strain CCTCC NO:M of the present invention 2012160 fermented liquids to orange skin in liquid submerged fermentation liquid.Adopt following methods to prepare a kind of zymin of aspergillus niger C-2 bacterial strain of the present invention: preparation process is with embodiment 1.
Snowy peak tangerine orange 50kg, peeling after washing, hot soup, distinguish is also removed the train of thought on tangerine lobe, obtains the about 35kg of tangerine lobe for subsequent use.Concentrated fermenting enzyme preparation is stirred after with 5%～8% concentration dilution, jacketed kettle vapour is bathed and is warmed up to 40 ℃, then add 35kg peeling except train of thought tangerine lobe (solid-liquid ratio 1:3), with citric acid adjust pH to 4.5, under 40～45 ℃ of conditions of temperature, constantly stir and keep constant temperature 90～100min, obtain full excystation clothing tangerine lobe, interior born of the same parents' percentage of damage is low, and has kept the original local flavor of oranges and tangerines and color and luster.This zymin can be reused 3～5 times under these conditions.
Comparative example
What in this comparative example, adopt is traditional fungi culture medium, investigates the degradation effect of aspergillus niger C-2 bacterial strain CCTCC NO:M of the present invention 2012160 fermented liquids to navel orange capsule clothing in liquid submerged fermentation liquid.
Traditional fungus culture based formulas: take 200g potato, clean peeling chopping, the 1000mL that adds water boils half hour, filtered through gauze, add again 15g glucose (activation 15g agar), filtered through gauze while hot after fully dissolving, 121 ℃ of sterilizings 20 minutes after packing.
Adopt traditional fungus culture based formulas to prepare the step of zymin of aspergillus niger C-2 bacterial strain of the present invention identical with embodiment 1 with method.
Navel orange 100kg, through skiving machine punching two ends and after peeling, obtains the navel orange fruit ball of about 80kg containing capsule clothing, be equally divided into two parts for subsequent use.The fermenting enzyme preparation respectively different culture media Fermented Condensed being obtained stirs after with 5%～8% concentration dilution, jacketed kettle vapour is bathed and is warmed up to 40 ℃, then add the navel orange (solid-liquid ratio 1:3) of peeling, with citric acid adjust pH to 4.5, under 45～50 ℃ of conditions of temperature, constantly stir and keep constant temperature 90～120min.
Experimental result by embodiment 1 to 4 and comparative example can be found out, by traditional fungi culture medium formula, fermenting liquid to navel orange capsule clothing do not observe degradation effect, and obtain the navel orange fruit ball of full excystation clothing by culture medium prescription fermentation liquor treatment provided by the present invention.Find the culture medium prescription of the present invention navel orange capsule clothing successful of not only degrading by simultaneous test, also utilize navel orange pomace enzymatic production, turn waste into wealth, both improved the added value of navel orange industry, protected environment, there is economical and social double meaning.
To sum up, compared with prior art, the invention has the advantages that:
1, degraded navel orange capsule clothing micro-organism enzyme preparation of the present invention can large-scale industrialization be produced, and after degerming, anticorrosion and concentration, zymin is preserved 6 months its enzymic activitys at normal temperature and kept more than 95%, and easy to use, production cost is low.
2, this zymin is efficient, complete to navel orange capsule clothing Degradation, thoroughly decomposes, and does not damage inner orange juice born of the same parents, enhances productivity, and reduces labour intensity, reduces production costs.
3, the present invention is compared with soda acid technique, and nutritive ingredient after biological enzyme formulation degraded capsule clothing is destroyed and run off and be better than traditional acid-base method, and it has kept the original local flavor of navel orange and color and luster.
4, avoid residual Safety of Food Quality technology barriers and the soda acid of the objectionable impuritiess such as heavy metal that traditional soda acid technique causes to discharge the environmental pollution causing, saved a large amount of water resourcess, realized the unification of economic benefit, ecological benefits and social benefit.
5, the culture medium prescription of the present invention navel orange capsule clothing successful of not only degrading, also utilizes navel orange pomace enzymatic production, turns waste into wealth, and has both improved the added value of navel orange industry, has protected environment, has economical and social double meaning.
On the whole, aspergillus niger C-2 bacterial strain of the present invention and enzymatic production thereof have the advantages such as production cost is little, easy to use, navel orange capsule clothing good degrading effect, are adapted at national Orange Producing enterprise and promote the use of on a large scale.The present invention, for preserving the ecological environment, promotes Safety of Food Quality, keeps the original local flavor of navel orange, promotes that the aspects such as navel orange intensive processing development are significant.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (8)
1. a degraded navel orange capsule clothing microorganism, is characterized in that, the preservation name of described microorganism is called aspergillus niger C-2, and depositary institution is Chinese Typical Representative culture collection center, and preserving number is CCTCC NO:M2012160.
2. the application of degraded navel orange capsule clothing microorganism as claimed in claim 1 in preparation degraded navel orange capsule clothing zymin.
3. a degraded navel orange capsule clothing zymin, is characterized in that, adopts degraded navel orange capsule clothing microorganism as claimed in claim 1 to make.
4. zymin according to claim 3, is characterized in that, adopts described degraded navel orange capsule clothing microorganism to make through liquid submerged fermentation or solid state fermentation cultivation.
5. zymin according to claim 4, is characterized in that, described liquid submerged fermentation or solid state fermentation further comprise activation before cultivating, prepare spore suspension, the step of seed culture.
6. zymin according to claim 5, is characterized in that, further comprises: by described zymin through ultrafiltration, concentratedly make concentrated enzyme preparation, or described enzyme preparation is become to pulvis or granule.
7. the degraded navel orange capsule clothing zymin as described in claim 3 to the 6 any one application on navel orange, oranges and tangerines excystation clothing.
8. the application of zymin according to claim 7, is characterized in that, comprises the following steps:
It is 8 × 10 that described zymin is diluted to enzyme work
3～10 × 10
3u/g, mixes the described zymin after dilution with the ratio of navel orange take mass ratio as 3:1 of peeling, 45～50 ℃ of insulation 90～120min.
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| CN104498372B (en) * | 2015-01-06 | 2017-08-04 | 邵阳学院 | Fusarium oxysporum BM201, its compound pectinase produced and compound pectinase preparation method and application |
| CN105462866B (en) * | 2015-12-24 | 2018-11-20 | 邵阳学院 | The activation culture original washing powder of yeast strain and activation, excystation clothing medium powder and application |
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| CN1271774A (en) * | 2000-06-01 | 2000-11-01 | 中国科学院生态环境研究中心 | Process for preparing pectinase by solid-state fermentation of fruit or vegetable dregs |
| CN101011181A (en) * | 2007-02-13 | 2007-08-08 | 湖南省农产品加工研究所 | Process for bacteria degradation of orange capsule clothes |
| CN101596013A (en) * | 2009-06-18 | 2009-12-09 | 浙江省农业科学院 | A kind of fermentation-enzymolysis coupling connection removes the method for oranges and tangerines tangerine sheet capsule clothing |
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| CN1271774A (en) * | 2000-06-01 | 2000-11-01 | 中国科学院生态环境研究中心 | Process for preparing pectinase by solid-state fermentation of fruit or vegetable dregs |
| CN101011181A (en) * | 2007-02-13 | 2007-08-08 | 湖南省农产品加工研究所 | Process for bacteria degradation of orange capsule clothes |
| CN101596013A (en) * | 2009-06-18 | 2009-12-09 | 浙江省农业科学院 | A kind of fermentation-enzymolysis coupling connection removes the method for oranges and tangerines tangerine sheet capsule clothing |
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Application publication date: 20130327 Assignee: Hunan nine Sheng food Co., Ltd. Assignor: Hunan Liwen Food Co., Ltd. Contract record no.: 2015430000071 Denomination of invention: Microorganism capable of degrading navel orange segment membrane and enzymic preparation containing microorganism as well as application Granted publication date: 20140618 License type: Exclusive License Record date: 20150611 |
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