CN102994400A - 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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- CN102994400A CN102994400A CN2012105590735A CN201210559073A CN102994400A CN 102994400 A CN102994400 A CN 102994400A CN 2012105590735 A CN2012105590735 A CN 2012105590735A CN 201210559073 A CN201210559073 A CN 201210559073A CN 102994400 A CN102994400 A CN 102994400A
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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 the traditional technology productions such as hand peeling and soda acid excystation clothing for a long time.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 are the problems such as labour intensive, production efficiency are low, unstable product quality in artificial peeling and soda acid excystation clothing technique.At present industrialization can production excystation 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.Simultaneously, because of a large amount of alkali that use, 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, meet with easily technology barriers and green barrier, weakened the competitive edge of China's oranges and tangerines canning industry in the world market.Simultaneously, in the oranges and tangerines processing, produce the skin slag that accounts for oranges and tangerines amount of finish about 30%, usually all be dropped, not only caused the wasting of resources, also brought pollution to environment.
Excystation clothing technique is one of its technical difficult points in the navel orange processing.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; Because the difference of peel of Citrus reticulata Blanco and Peel of Navel Orange structure causes 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, takes off to solve in the prior art soda acid that navel orange capsule clothing explained hereafter efficient 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, adopting degraded navel orange capsule clothing microorganism to cultivate through strain fermentation cultivation, liquid submerged fermentation or solid state fermentation makes.
Further, the step that further comprises activation, preparation spore suspension, seed culture before liquid submerged fermentation or solid state fermentation are cultivated.
Further, this zymin comprises: with zymin through ultrafiltration, concentrated make concentrated enzyme preparation, or enzyme preparation is become 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, may further comprise the steps: it is 8 * 10 that zymin is diluted to enzyme work
3～10 * 10
3U/g: the zymin after will diluting is mixed with the ratio of navel orange take mass ratio as 3:1 of peeling, 45 ~ 50 ℃ of insulation 90 ~ 120min
There are the unsafe factors such as heavy-metal residual in tradition soda acid excystation clothing technique, and the zymin that adopts the microorganism preparation of degraded navel orange capsule clothing of the present invention does not relate to a large amount of uses of soda acid, carry out removing of navel orange capsule clothing by the 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, and is 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
Need to prove that in the situation of not conflicting, embodiment and the feature among the embodiment among the application can make up 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 obtains by the following steps screening: screen aimed strain from rotting the samples such as navel orange surface, soil, then after passing through ultraviolet mutagenesis, obtain navel orange capsule clothing is had the bacterial strain of efficient degradation effect by screening repeatedly.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, just be white, rear overstrike is until black heavy fleece shape, the colourless or central slightly tawny in the back side.The conidial head brown-black is radial, and conidiophore is different in size.The top capsule is spherical, double-deck stigma.The conidium brown is spherical.The globe-roof capsule is formed on the top, covers one deck metulae and one deck stigma on it comprehensively, and long on the stigma have bunchiness memnonious spherical, diameter 2.5～4.0 μ m.Conidial head is spherical, diameter 700～800 μ m, brown-black.The conidial head brown-black is radial, and conidiophore is different in size.Conidiophore stretches out in matrix and is about 1～3mm, 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 the 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 the above-mentioned aspergillus niger C-2 of employing as fermentation strain, prepares degraded navel orange capsule clothing zymin through liquid submerged fermentation or solid state fermentation cultivation.What play a major role in the subtractive process of navel orange capsule clothing is this aspergillus niger C-2 and metabolite thereof, because this preparation also can adopt ordinary method of the prior art to carry out, 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, the step that further comprises activation, preparation spore suspension, seed culture before liquid submerged fermentation or solid state fermentation are cultivated, increase the increased activity that these steps can make bacterial strain, the production efficiency that is conducive to zymin improves, and makes zymin keep higher activity in follow-up use procedure.
A kind of typical embodiment according to the present invention, activation comprises: aspergillus niger C-2 is inoculated in the substratum activates, 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 can make the fast quick-recovery of ability of this production by biological enzyme normal by this activation procedure.A kind of typical embodiment according to the present invention, seed culture comprises: the spore suspension of activation is inoculated in the seed liquid nutrient medium, the pH value of control 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: will carry out through the seed liquid culture medium inoculated that contains aspergillus niger C-2 of seed culture submerged fermentation and cultivate in fermentation flask, fermentation culture conditions is: temperature is 30 ℃ in the 12h, behind the 12h 28 ℃; The pH value is 7.0～7.2; Pressure position 0.06MPa～0.08MPa; 180r/m in the stirring velocity 12h, 200r/m behind the 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, have 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 that contains aspergillus niger C-2 through described seed culture is inoculated in the solid-state fermentation culture medium, cultivate 108h for 30 ℃, it is bent to obtain aspergillus niger C-2 solid state fermentation, described solid state fermentation triton makes described zymin after crossing cryodrying and pulverizing, or obtains described zymin by lixiviate in the 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 during fermentation ends, substratum is the wet stock state, and purpose production concentration height and extraction process are simple.
A kind of typical embodiment according to the present invention, the substratum of activation usefulness is the solid slant culture base; The step of preparation spore suspension comprises: adopt sterilized water to wash the spore of aspergillus niger C-2, and be diluted to 2～3 * 10
6Individual/mL makes spore suspension, and with spore suspension be kept at 4 ℃ for subsequent use.
Preferably, in the seed culture step spore suspension is inoculated in the seed liquid nutrient medium by 5% inoculum size; To be inoculated in by 8%～10% inoculum size through the seed liquid nutrient medium that contains aspergillus niger C-2 of seed culture in the liquid submerged fermentation culturing step and carry out submerged fermentation in the fermentation flask and cultivate.This inoculum size neither can cause the waste of bacterial classification, is conducive to again the rapid multiplying of aspergillus niger C-2.
Preferably, activation usefulness comprise the component of following content as the substratum of carbon source take navel orange capsule clothing powder: (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 the substratum 30min that sterilizes under 121 ℃ of temperature of activation usefulness makes.This kind 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 can make the fast quick-recovery of ability of this microorganisms navel orange capsule clothing degrading enzyme normal by this activation procedure.What preferably, the seed liquid culture was used comprises the component of following content take navel orange capsule clothing powder as the substratum of carbon source: (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 the seed liquid nutrient medium 30min that sterilizes under 121 ℃ of temperature makes.This kind 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, further comprise according to actual needs after the step 4): with the ultrafiltration of zymin process, the concentrated concentrated enzyme preparation that makes, or enzyme preparation become pulvis or granule etc., concentrated enzyme preparation is convenient to be used, be more convenient for preservation and the transportation such as pulvis or granule.
Zymin enzymolysis efficiency by the inventive method preparation is higher; And the zymin that makes through the method after enzymic fermentation liquid is through filtration sterilization, ultrafiltration and concentration, rotproofing room temperature preservation after 6 months enzyme live and 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 may further comprise the steps: the 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: the navel orange fruit ball of the residual capsule clothing of will peeling is 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 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 maintenance constant temperature 90～120min, obtain excystation clothing navel orange fruit ball, after the fruit ball warp is crossed dispersion machine and is processed to the orange juice born of the same parents, this zymin can be reused 3～5 times under these conditions.
The present invention's technique to its enzymatic production in to the seed selection of degraded navel orange capsule clothing microorganism strains is optimized, and suitable large-scale industrialization is produced this zymin.
According to a kind of typical embodiment of the present invention, provide a kind of substratum for degraded navel orange capsule clothing microorganism.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 sterilization 30min makes described substratum under 121 ℃ of temperature.
When being used 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 sterilization 30min makes described substratum under 121 ℃ of temperature.
Further specify beneficial effect of the present invention below in conjunction with embodiment.
The substratum of activation usefulness 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 the substratum 30min that sterilizes under 121 ℃ of temperature of activation usefulness makes.This kind 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 the seed liquid nutrient medium 30min that sterilizes under 121 ℃ of temperature makes.This kind 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
Aspergillus niger C-2 bacterial strain CCTCCNO:M 2012160 fermented liquids of the present invention are to the degraded of navel orange capsule clothing in the present embodiment investigation 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 the substratum 30min that sterilizes under 121 ℃ of temperature of activation usefulness makes.This kind activation medium is suitable for the activation of aspergillus niger C-2.Substratum effect in the said components scope is similar.
The 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 the seed liquid nutrient medium 30min that sterilizes under 121 ℃ of temperature makes.This kind 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 the fresh solid slant culture base and activates, in the described reactivation process, the pH value of control solid slant culture base is 7.0～7.2, activation temperature is controlled at 28 ℃～29 ℃, activation culture 2～3 days, wash aspergillus niger C-2 spore with sterilized water, the preparation spore suspension also is diluted to 2～3 * 10
6Individual/mL, and with its be kept at 4 ℃ for subsequent use;
(2) seed culture: the aspergillus niger C-2 spore suspension after the above-mentioned activation is inoculated in the seed liquid nutrient medium by 5% inoculum size, the pH value of control seed culture medium is 7.0～7.2, temperature during 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 the substratum.
(3) liquid submerged fermentation is cultivated: the seed aspergillus niger C-2 bacterial classification after the above-mentioned cultivation is inoculated by the inoculum size of liquid submerged fermentation substratum 8%～10% carried out the submerged fermentation cultivation in the fermentation flask, the prescription of described liquid submerged fermentation substratum is identical with described seed liquid nutrient medium, fermentation culture conditions is: temperature is 30 ℃ in the 12h, behind the 12h 28 ℃; Automatically regulate pH 7.0～7.2; Tank pressure 0.06MPa～0.08MPa; 180r/m in the stirring velocity 12h, 200r/m behind the 12h; Air flow 1:1.0～1.5; Fermentation time 108～120h.
The whole process incubation time is greatly about 10 days, the enzyme liquid of liquid submerged fermentation gained is used immediately, the resulting enzyme liquid of described fermentation is obtained concentrated enzyme liquid through ultrafiltration and concentration, or the different degraded navel orange capsule clothing zymin that obtains by other processing modes, such as pulvis or granule etc., for subsequent use.
Navel orange 50kg through the skiving machine punching two ends and after peeling, obtains the navel orange fruit ball that about 40kg contains the capsule clothing, and is 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, after the fruit ball warp is crossed dispersion machine and is processed to the orange juice born of the same parents, this zymin can be reused 3～5 times under these conditions.
Embodiment 2
Aspergillus niger C-2 bacterial strain CCTCCNO:M 2012160 fermented liquids of the present invention are to the degraded of navel orange capsule clothing in the present embodiment investigation 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 the substratum 30min that sterilizes under 121 ℃ of temperature of activation usefulness makes.This kind activation medium is suitable for the activation of aspergillus niger C-2.Substratum effect in the said components scope is similar.
The 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 the seed liquid nutrient medium 30min that sterilizes under 121 ℃ of temperature makes.This kind 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 the fresh solid slant culture base and activates, in the described reactivation process, the pH value of control solid slant culture base is 7.0～7.2, activation temperature is controlled at 29 ℃～30 ℃, activation culture 2～3 days, wash aspergillus niger C-2 spore with sterilized water, the preparation spore suspension also is diluted to 2～3 * 10
6Individual/mL, and with its be kept at 4 ℃ for subsequent use;
(2) seed culture: the aspergillus niger C-2 spore suspension after the above-mentioned activation is inoculated in the seed liquid nutrient medium by 5% inoculum size, the pH value of control seed culture medium is 7.0～7.2, temperature during 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 the substratum.
(3) liquid submerged fermentation is cultivated: the seed aspergillus niger C-2 bacterial classification after the above-mentioned cultivation is inoculated by the inoculum size of liquid submerged fermentation substratum 8%～10% carried out the submerged fermentation cultivation in the fermentation flask, the prescription of described liquid submerged fermentation substratum is identical with described seed liquid nutrient medium, fermentation culture conditions is: temperature is 30 ℃ in the 12h, behind the 12h 28 ℃; Automatically regulate pH 7.0～7.2; Tank pressure 0.06MPa～0.08MPa; 180r/m in the stirring velocity 12h, 200r/m behind the 12h; Air flow 1:1.0～1.5; Fermentation time 108～120h.
The whole process incubation time is greatly about 10 days, the enzyme liquid of liquid submerged fermentation gained is used immediately, the resulting enzyme liquid of described fermentation is obtained concentrated enzyme liquid through ultrafiltration and concentration, or the different degraded navel orange capsule clothing zymin that obtains by other processing modes, such as pulvis or granule etc., for subsequent use.
Navel orange 50kg through the skiving machine punching two ends and after peeling, obtains the navel orange fruit ball that about 40kg contains the capsule clothing, and is 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, after the fruit ball warp is crossed dispersion machine and is processed to the orange juice born of the same parents, this zymin can be reused 3～5 times under these conditions.
Embodiment 3
Aspergillus niger C-2 bacterial strain CCTCC NO:M 2012160 solid state fermentation songs of the present invention are to the degraded of navel orange capsule clothing in the present embodiment investigation 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 the substratum of the activation usefulness 30min that sterilizes under 121 ℃ of temperature makes.This kind activation medium is suitable for the activation of aspergillus niger C-2.Substratum effect in the said components scope is similar.
The 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 the seed liquid nutrient medium 30min that sterilizes under 121 ℃ of temperature makes.This kind 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 the fresh solid slant culture base and activates, in the described reactivation process, the pH value of control solid slant culture base is 7.0～7.2, activation temperature is controlled at 28 ℃～30 ℃, activation culture 2～3 days, wash aspergillus niger C-2 spore with sterilized water, the preparation spore suspension also is diluted to 2～3 * 10
6Individual/mL, and with its be kept at 4 ℃ for subsequent use;
(2) seed culture: with the aspergillus niger C-2 spore suspension after the above-mentioned activation by 5% inoculum size be inoculated in sterilization by wheat wheat bran 95 weight parts, navel orange pomace or fruit peel powder 180 weight parts, ammonium sulfate 2 weight parts, sal epsom 0.5 weight part, dipotassium hydrogen phosphate 1 weight part, more than each component add entry to mix and regulate moisture humidity thoroughly be 60%.(initial pH 6.5) cultivates 48h for 30 ℃.(3) solid state fermentation is cultivated: the aspergillus niger C-2 seed of above-mentioned activation is bent by in the 10% inoculum size access 50kg solid-state fermentation culture medium, each component of described solid-state fermentation culture medium is identical with above-mentioned solid-state seed culture medium component, after the seed song mixes with solid-state fermentation culture medium, cultivate 108h for 30 ℃, it is bent to obtain aspergillus niger C-2 solid state fermentation, this solid state fermentation song uses after can and pulverizing by cryodrying, also can obtain to use behind the liquid enzymes liquid by lixiviate in the 0.5mol/L pH=6.5 phosphoric acid buffer.
Navel orange 50kg through the skiving machine punching two ends and after peeling, obtains the navel orange fruit ball that about 40kg contains the capsule clothing, and is for subsequent use.The bent 12kg of the aspergillus niger C-2 solid state fermentation of above-mentioned cryodrying, pulverizing 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 maintenance constant temperature 90～120min, obtain excystation clothing navel orange fruit ball, the fruit ball warp is crossed and is obtained the orange juice born of the same parents after dispersion machine is processed, and this aspergillus niger C-2 solid state fermentation song can be reused 3～5 times under these conditions.
Embodiment 4
Aspergillus niger C-2 bacterial strain CCTCC NO:M 2012160 fermented liquids of the present invention are to the degraded of orange skin in the present embodiment investigation 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, through washing, peeling behind the hot soup, distinguish is also removed train of thought on the tangerine lobe, and it is for subsequent use to obtain the about 35kg of tangerine lobe.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 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 Examples
What adopt in this Comparative Examples is traditional fungi culture medium, and aspergillus niger C-2 bacterial strain CCTCC NO:M 2012160 fermented liquids of the present invention are to the degradation effect of navel orange capsule clothing in the investigation liquid submerged fermentation liquid.
Traditional fungus culture based formulas: take by weighing the 200g potato, clean the peeling chopping, add water 1000mL and boil half hour, filtered through gauze, add again 15g glucose (activation 15g agar), abundant filtered through gauze while hot after the dissolving, 121 ℃ of sterilizations are 20 minutes after the packing.
It is identical with embodiment 1 with method to adopt traditional fungus culture based formulas to prepare the step of zymin of aspergillus niger C-2 bacterial strain of the present invention.
Navel orange 100kg through the skiving machine punching two ends and after peeling, obtains really ball of navel orange that about 80kg contains the capsule clothing, be equally divided into two parts for subsequent use.The fermenting enzyme preparation that respectively the different culture media Fermented Condensed is 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 Examples 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, have economical and social double meaning.
To sum up, compared with prior art, the invention has the advantages that:
But 1, degraded navel orange capsule clothing micro-organism enzyme preparation large-scale industrialization of the present invention production, after degerming, anticorrosion and concentration, zymin is preserved 6 months its enzymic activitys at normal temperature and is 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 compares with soda acid technique, and the nutritive ingredient behind the 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 the residual Safety of Food Quality technology barriers of the objectionable impuritiess such as heavy metal that traditional soda acid technique causes and the environmental pollution that the soda acid discharging causes, 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 promotes Safety of Food Quality for preserving the ecological environment, and keeps the original local flavor of navel orange, and the aspects such as promotion navel orange intensive processing development are significant.
The above is the preferred embodiments of the present invention only, is not limited to the present invention, and 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., all should be included within 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 CCTCCNO:M 2012160.
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 cultivate through strain fermentation cultivation, liquid submerged fermentation or solid state fermentation and makes.
5. zymin according to claim 4 is characterized in that, further comprises the step of activation, preparation spore suspension, seed culture before described liquid submerged fermentation or solid state fermentation are cultivated.
6. zymin according to claim 5 is characterized in that, further comprises: with described zymin through ultrafiltration, concentratedly make concentrated enzyme preparation, or described enzyme preparation is become pulvis or granule.
7. one kind such as each described degraded navel orange capsule clothing zymin application on navel orange, oranges and tangerines excystation clothing of claim 3 to 6.
8. the application of zymin according to claim 7 is characterized in that, may further comprise the steps:
It is 8 * 10 that described zymin is diluted to enzyme work
3～10 * 10
3U/g: the described zymin after will diluting is mixed with the ratio of navel orange take mass ratio as 3:1 of peeling, 45 ~ 50 ℃ of insulation 90 ~ 120min.
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| CN104498372A (en) * | 2015-01-06 | 2015-04-08 | 邵阳学院 | Fusarium oxysporum BM201, compound pectinase produced by fusarium oxysporum BM201 and preparation method and application of compound pectinase |
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