CN102994461B - For the substratum of navel orange peel microorganism and the preparation method of zymin of degrading - Google Patents
For the substratum of navel orange peel microorganism and the preparation method of zymin of degrading Download PDFInfo
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Abstract
The invention discloses a kind of for the substratum of navel orange peel microorganism and the preparation method of zymin of degrading.Wherein, this preparation method comprises: employing depositary institution is China typical culture collection center, does is preserving number CCTCC? NO:M? the aspergillus niger C-2(Aspergillus of 2012160? sp.C-2) as fermentation strain, cultivate through liquid submerged fermentation or solid state fermentation and prepare degraded navel orange peel zymin.Adopt technical scheme of the present invention; the substratum being carbon source with navel orange peel powder or Peel of Navel Orange pomace carries out cultivation preparation zymin to the microorganism in the present invention; obtained zymin is not only degraded navel orange peel successful; also utilize navel orange pomace enzymatic production; turn waste into wealth; both improve the added value of navel orange industry, protected environment, there is economical and social double meaning.
Description
Technical field
The present invention relates to technical field of bioengineering, in particular to a kind of for the substratum of navel orange peel microorganism and the preparation method of zymin of degrading.
Background technology
China's Citrus procession follows the traditional technology such as hand peeling and soda acid excystation clothing for a long time and produces.The acid-alkali treatment method of existing acid-base method excystation clothing mainly contains: classical soda acid two step method, improvement heavy sour two step method, 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.Current industrialization can produces excystation clothing water loss greatly, and product water loss per ton reaches 60 ~ 80 tons, not only increases product cost, and exacerbates the shortage of drinking water sources.Meanwhile, because using alkali in a large number, produce a large amount of industrial alkali liquid waste water, add the production cost of product, also contaminate environment, have impact on the security of product, easy experience technology barriers and green barrier, weaken the competitive edge of China's oranges and tangerines canning industry in world market.Meanwhile, in Citrus procession is produced, create the skin slag accounting for Citrus procession amount about 30%, be usually all dropped, not only cause the wasting of resources, also bring pollution to environment.
In navel orange processing, excystation clothing technique is one of its technical difficult points.The production technology of current orange juice born of the same parents mainly adopts the technique of traditional oranges and tangerines can acid-alkali treatment, and there is hand peeling, distinguish, 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, existing oranges and tangerines enzyme process is caused to go capsule clothing technology to be not suitable for the processing of navel orange orange juice born of the same parents.
Summary of the invention
The present invention aims to provide a kind of for the substratum of navel orange peel microorganism and the preparation method of zymin of degrading, to prepare the zymin of degraded navel orange peel microorganism efficiently.
According to an invention of the present invention, provide a kind of preparation method of navel orange peel zymin of degrading.This preparation method comprises: employing depositary institution is China typical culture collection center, preserving number is the aspergillus niger C-2(Aspergillussp.C-2 of CCTCCNO:M2012160) as fermentation strain, cultivate through liquid submerged fermentation or solid state fermentation and prepare degraded navel orange peel zymin.
Further, taking a step forward that liquid submerged fermentation or solid state fermentation are cultivated comprises activation, prepares spore suspension, the step of seed culture.
Further, activation comprises: be inoculated in activation medium by aspergillus niger C-2 and activate, and the pH value of activation medium is 7.0 ~ 7.2, and activation temperature controls at 28 DEG C ~ 30 DEG C, and soak time is 1 ~ 2 day; Wherein, the solid slant culture base that activation medium is is carbon source with navel orange peel powder, 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, NaCl1g/L ~ 2g/L, navel orange peel powder 6g/L ~ 10g/L and agar 15g/L ~ 20g/L, and at 121 DEG C of temperature, sterilizing 30min obtains activation medium.
Further, the step preparing spore suspension comprises: the spore adopting aspergillus niger C-2 under aseptic washing, and is diluted to 2 ~ 3 × 10
6individual/mL obtains spore suspension, and spore suspension is kept at 4 DEG C for subsequent use.
Further, seed culture comprises: be inoculated in by spore suspension in seed liquid nutrient medium, and the pH value controlling seed liquid nutrient medium is 7.0 ~ 7.2, and culture temperature controls at 28 DEG C ~ 30 DEG C, and 200r/m cultivates 12 ~ 16h; Or spore suspension is inoculated in seed solid medium, the initial pH value controlling seed solid medium is 6.5, and culture temperature controls at 28 DEG C ~ 30 DEG C, cultivates 48h; Wherein, the substratum that seed liquid nutrient medium is is carbon source with navel orange peel powder, 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, NaCl1g/L ~ 2g/L, navel orange peel powder or Peel of Navel Orange pomace 15g/L ~ 20g/L; And sterilizing 30min obtains seed liquid nutrient medium at 121 DEG C of temperature.
Further, the step that liquid submerged fermentation is cultivated comprises: will carry out Submerged fermentation containing the seed liquid culture medium inoculated of aspergillus niger C-2 through seed culture in fermentation flask, fermentation culture conditions is: in 12h, temperature is 30 DEG C, after 12h 28 DEG C; PH value is 7.0 ~ 7.2; Pressure position 0.06MPa ~ 0.08MPa; 200r/m after 180r/m, 12h in stirring velocity 12h; Air flow 1:1.0 ~ 1.5; Fermentation time 108 ~ 120h, obtained zymin.
Further, the step that solid state fermentation is cultivated comprises: the seed solid medium containing aspergillus niger C-2 through seed culture is inoculated in solid-state fermentation culture medium, cultivate 108h for 30 DEG C, obtain aspergillus niger C-2 solid state fermentation bent, solid state fermentation triton crosses cryodrying and obtained zymin after pulverizing, or obtains zymin by lixiviate in 0.5mol/LpH=6.5 phosphoric acid buffer; Wherein, solid-state fermentation culture medium is prepared by the following method: wheat bran 95 weight part, navel orange pomace or navel orange fruit peel powder 180 weight part, ammonium sulfate 2 weight part, magnesium sulfate 0.5 weight part, dipotassium hydrogen phosphate 1 weight part are added water and mix thoroughly and regulate moisture humidity to be 60%, obtains solid-state fermentation culture medium.
Further, comprising: by zymin through ultrafiltration, concentrated obtained concentrated enzyme preparation, or enzyme preparation is become pulvis or granule.
According to another aspect of the present invention, provide a kind of substratum for navel orange peel microorganism of degrading, substratum is for carbon source with navel orange peel powder or Peel of Navel Orange pomace.
Further, the component of following content is comprised: (NH
4)
2sO
42g/L ~ 2.5g/L, MgSO
40.6g/L ~ 1g/L, K
2hPO
41g/L ~ 2g/L, NaCl1g/L ~ 2g/L, navel orange peel powder 6g/L ~ 10g/L and agar 15g/L ~ 20g/L, and at 121 DEG C of temperature, sterilizing 30min obtains substratum.
Further, the component of following content is comprised: (NH
4)
2sO
42g/L ~ 2.5g/L, MgSO
40.6g/L ~ 1g/L, K
2hPO
41g/L ~ 2g/L, NaCl1g/L ~ 2g/L, navel orange peel powder or Peel of Navel Orange pomace 15g/L ~ 20g/L; And sterilizing 30min obtains substratum at 121 DEG C of temperature.
Adopt technical scheme of the present invention; (preserving number is for CCTCCNO:M2012160 to the microorganism in the present invention for the substratum being carbon source with navel orange peel powder or Peel of Navel Orange pomace; preservation day is on May 7th, 2012) carry out cultivation preparation zymin; obtained zymin is not only degraded navel orange peel successful; also utilize navel orange pomace enzymatic production, turn waste into wealth, both improve the added value of navel orange industry; protect environment, there is economical and social double meaning.
Embodiment
It should be noted that, when not conflicting, the embodiment in the application and the feature in embodiment can combine mutually.The present invention is described in detail below in conjunction with embodiment.
According to a kind of typical embodiment of the present invention, provide a kind of degraded navel orange peel microorganism.The preservation name of this microorganism is called aspergillus niger C-2(Aspergillussp.C-2), depositary institution is China typical culture collection center, and preserving number is CCTCCNO:M2012160, and preservation day is on May 7th, 2012.
Degraded navel orange peel microorganism of the present invention is obtained by following steps screening: from the samples such as rotten navel orange surface, soil, screen aimed strain, then, after ultraviolet mutagenesis, bacterial strain navel orange peel to efficient degradation effect is obtained by screening repeatedly.Mutatis mutandis bacterial strain of the present invention is through being accredited as aspergillus niger C-2(Aspergillussp.C-2), its main biological property is: on solid medium, cultivates 3d ~ 5d, bacterium colony spreads rapidly, be just white, rear overstrike until black heavy fleece shape, the colourless or central authorities' 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, and it covers one deck metulae and one deck stigma comprehensively, and long on stigma have bunchiness memnonious spherical, diameter 2.5 ~ 4.0 μm.Conidial head is spherical, diameter 700 ~ 800 μm, 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.
According to a kind of typical embodiment of the present invention, provide a kind of degraded navel orange peel zymin.This zymin adopts above-mentioned degraded navel orange peel microorganism to obtain.Because this degraded navel orange peel zymin does not relate to a large amount of uses of soda acid, removing of navel orange peel is carried out by biological enzymolysis effect, therefore can greatly improving production efficiency, reduce navel orange fruit percentage of damage, make constant product quality, and can reduce production cost, noresidue, environmental protection, security are high, free from environmental pollution.Preferably, this zymin adopts above-mentioned degraded navel orange peel microorganism to cultivate obtained through liquid submerged fermentation or solid state fermentation.
According to a kind of typical embodiment of the present invention, provide a kind of preparation method of navel orange peel zymin of degrading.This preparation method comprises the above-mentioned aspergillus niger C-2 of employing as fermentation strain, cultivates prepare degraded navel orange peel zymin through liquid submerged fermentation or solid state fermentation.What play a major role in the subtractive process of navel orange peel is this aspergillus niger C-2 and metabolite thereof, also ordinary method of the prior art can be adopted to carry out because preparing for this reason, preferably, aspergillus niger C-2 cultivates through liquid submerged fermentation or solid state fermentation and prepares degraded navel orange peel zymin, because the preparation method of these zymins is simple, be convenient to suitability for industrialized production, and can not environmental pollution be caused.Preferably, taking a step forward that liquid submerged fermentation or solid state fermentation are cultivated comprises activation, prepares spore suspension, the step of seed culture, increase the increased activity that these steps can make bacterial strain, the production efficiency being conducive to zymin improves, and the activity making zymin keep higher in follow-up use procedure.
According to a kind of typical embodiment of the present invention, activation comprises: be inoculated in substratum by aspergillus niger C-2 and activate, and the pH value of substratum is 7.0 ~ 7.2, and activation temperature controls at 28 DEG C ~ 30 DEG C, 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 the ability of this production by biological enzyme normal.
According to a kind of typical embodiment of the present invention, seed culture comprises: be inoculated in by the spore suspension of activation in seed liquid nutrient medium, the pH value controlling seed liquid nutrient medium is 7.0 ~ 7.2, culture temperature controls at 28 DEG C ~ 30 DEG C, 200r/m cultivates 12 ~ 16h, the purebred son of final acquisition some amount and quality, can be used as the bacterial classification that large scale fermentation is produced.According to a kind of typical embodiment of 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 Submerged fermentation in fermentation flask, fermentation culture conditions is: in 12h, temperature is 30 DEG C, after 12h 28 DEG C; PH value is 7.0 ~ 7.2; Pressure position 0.06MPa ~ 0.08MPa; 200r/m after 180r/m, 12h in stirring velocity 12h; Air flow 1:1.0 ~ 1.5; Fermentation time 108 ~ 120h, obtained zymin.Nutrient uniformly distributing and oxygen abundance can be ensured by liquid submerged fermentation, there is the advantages such as enzymatic production is with short production cycle, output is high, benefit is large.
According to a kind of typical embodiment of 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 DEG C, obtain aspergillus niger C-2 solid state fermentation bent, described solid state fermentation triton crosses cryodrying and obtained described zymin after pulverizing, or obtains described zymin by lixiviate in 0.5mol/LpH=6.5 phosphoric acid buffer.Solid state fermentation simulation bacterial classification self-sow environment, make the growth conditions that microorganism keeps similar to nature, during fermentation ends, substratum is wet stock state, and object production concentration is high and extraction process is simple.
According to a kind of typical embodiment of the present invention, the substratum of activation is solid slant culture base; The step preparing spore suspension comprises: the spore adopting aspergillus niger C-2 under aseptic washing, and is diluted to 2 ~ 3 × 10
6individual/mL obtains spore suspension, and spore suspension is kept at 4 DEG C for subsequent use.
Preferably, in seed culture step by spore suspension by 5% inoculum size be inoculated in seed liquid nutrient medium; In liquid submerged fermentation culturing step by through seed culture containing aspergillus niger C-2 seed liquid nutrient medium by 8% ~ 10% inoculum size be inoculated in fermentation flask and carry out Submerged fermentation.This inoculum size both can not cause the waste of bacterial classification, was conducive to again the rapid multiplying of aspergillus niger C-2.
Preferably, the component comprising following content with the navel orange peel powder substratum that is carbon source of activation: (NH
4)
2sO
42g/L ~ 2.5g/L, MgSO
40.6g/L ~ 1g/L, K
2hPO
41g/L ~ 2g/L, NaCl1g/L ~ 2g/L, navel orange peel powder 6g/L ~ 10g/L and agar 15g/L ~ 20g/L, and the substratum activated sterilizing 30min at 121 DEG C of temperature obtains.This kind of activation medium is suitable for the activation of aspergillus niger C-2, because with navel orange peel powder for 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 microorganism generation navel orange peel degrading enzyme normal.Preferably, the component comprising following content with the navel orange peel powder substratum that is carbon source of seed liquid culture: (NH
4)
2sO
42g/L ~ 2.5g/L, MgSO
40.6g/L ~ 1g/L, K
2hPO
41g/L ~ 2g/L, NaCl1g/L ~ 2g/L, navel orange peel powder or Peel of Navel Orange pomace 15g/L ~ 20g/L; And seed liquid nutrient medium sterilizing 30min at 121 DEG C of temperature obtains.This kind of seed liquid nutrient medium is suitable for the cultivation of aspergillus niger C-2, and the final purebred son obtaining some amount and quality, can be used as the bacterial classification that large scale fermentation is produced.And this substratum also can be adopted to carry out in the process that liquid submerged fermentation is cultivated.Because with navel orange peel powder for carbon source, the navel orange peel degrading enzyme that this microorganism can be made to produce keeps high vigor.According to a kind of typical embodiment of the present invention, comprise further according to actual needs after step 4): by zymin through ultrafiltration, concentrated obtained concentrated enzyme preparation, or enzyme preparation is become pulvis or granule etc., concentrated enzyme preparation is easy to use, and pulvis or granule etc. are more convenient for preservation and transport.
The zymin enzymolysis efficiency prepared by the inventive method is higher; And through the obtained zymin of the method enzymic fermentation liquid after filtration sterilization, ultrafiltration and concentration, rotproofing room temperature preservation after 6 months enzyme live and still keep more than 95%.
According to a kind of typical embodiment of the present invention, the application of above-mentioned degraded navel orange peel 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 DEG C of water bath heat preservation 90 ~ 120min.
According to a kind of typical embodiment of the present invention, the concrete grammar of degraded navel orange peel is: by for subsequent use for the navel orange of residual capsule clothing of peeling fruit ball, by concentrated fermenting enzyme preparation to stir after the concentration dilution of 5% ~ 8%, the bath of jacketed kettle vapour is warmed up to 45 DEG C, then the navel orange (solid-liquid ratio 1:3) of peeling is added, with citric acid adjust pH to 4.5, under temperature 45 ~ 50 DEG C of conditions, continuous stirring also keeps constant temperature 90 ~ 120min, obtain excystation clothing navel orange fruit ball, fruit ball warp cross after dispersion machine process to orange juice born of the same parents, this zymin can reuse 3 ~ 5 times under these conditions.
The present invention is optimized the technique of its enzymatic production while to the seed selection of degraded navel orange peel microorganism strains, is applicable to this zymin of industrialization scale operation.
The typical embodiment of one according to the present invention, provides a kind of substratum for navel orange peel microorganism of degrading.This substratum is for carbon source with navel orange peel powder or Peel of Navel Orange pomace.
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, NaCl1g/L ~ 2g/L, navel orange peel powder 6g/L ~ 10g/L and agar 15g/L ~ 20g/L, and at 121 DEG C of temperature, sterilizing 30min obtains described substratum.
When cultivating for seed liquid state, 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, NaCl1g/L ~ 2g/L, navel orange peel powder or Peel of Navel Orange pomace 15g/L ~ 20g/L; And sterilizing 30min obtains described substratum at 121 DEG C of temperature.
Beneficial effect of the present invention is further illustrated below in conjunction with embodiment.
Activation medium 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, NaCl1g/L ~ 2g/L, navel orange peel powder 6g/L ~ 10g/L and agar 15g/L ~ 20g/L, and the substratum activated sterilizing 30min at 121 DEG C of temperature obtains.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 ~ 2.5g/L, MgSO
40.6g/L ~ 1g/L, K
2hPO
41g/L ~ 2g/L, NaCl1g/L ~ 2g/L, navel orange peel powder or Peel of Navel Orange pomace 15g/L ~ 20g/L; And seed liquid nutrient medium sterilizing 30min at 121 DEG C of temperature obtains.This kind of seed liquid nutrient medium is suitable for the cultivation of aspergillus niger C-2, and this substratum also can be adopted to carry out in the process of liquid submerged fermentation cultivation.
Embodiment 1
In the present embodiment investigation liquid submerged fermentation liquid, aspergillus niger C-2 bacterial strain CCTCCNO:M2012160 fermented liquid of the present invention is to the degraded of navel orange peel.Following methods is adopted 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 peel powder 6g/L and agar 15g/L, and the substratum activated sterilizing 30min at 121 DEG C of temperature obtains.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, NaCl1g/L, navel orange peel powder or Peel of Navel Orange pomace 15g/L; And seed liquid nutrient medium sterilizing 30min at 121 DEG C of temperature obtains.This kind of seed liquid nutrient medium is suitable for the cultivation of aspergillus niger C-2, and this substratum also can be adopted to carry out in the process of liquid submerged fermentation cultivation.Step is as follows:
(1) activate: the aspergillus niger C-2 seed described seed selection obtained is inoculated in fresh solid slant culture base and activates, in described reactivation process, the pH value controlling solid slant culture base is 7.0 ~ 7.2, activation temperature controls at 28 DEG C ~ 29 DEG C, 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 DEG C 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 controlling seed culture medium is 7.0 ~ 7.2, temperature during seed culture controls at 28 DEG C ~ 30 DEG C, and 200r/m cultivates 12 ~ 16h; Navel orange peel powder or Peel of Navel Orange pomace 15g/L in substratum.
(3) liquid submerged fermentation is cultivated: inoculated in fermentation flask by the inoculum size of liquid submerged fermentation substratum 8% ~ 10% by the seed aspergillus niger C-2 bacterial classification after above-mentioned cultivation and carry out Submerged fermentation, the formula of described liquid submerged fermentation substratum is identical with described seed liquid nutrient medium, fermentation culture conditions is: in 12h, temperature is 30 DEG C, after 12h 28 DEG C; Automatic adjustment pH7.0 ~ 7.2; Tank pressure 0.06MPa ~ 0.08MPa; 200r/m after 180r/m, 12h in stirring velocity 12h; Air flow 1:1.0 ~ 1.5; Fermentation time 108 ~ 120h.
Whole process incubation time is greatly about 10 days, used immediately by the enzyme liquid of liquid submerged fermentation gained, the enzyme liquid described fermentation obtained obtains concentrated enzyme liquid through ultrafiltration and concentration, or by different degraded navel orange peel zymin that other processing modes obtain, 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.By concentrated fermenting enzyme preparation to stir after the concentration dilution of 5% ~ 8%, the bath of jacketed kettle vapour is warmed up to 40 DEG C, then the navel orange (solid-liquid ratio 1:3) of peeling is added, with citric acid adjust pH to 4.5, under temperature 45 ~ 50 DEG C of conditions, constantly stir and keep constant temperature 90 ~ 120min, obtaining excystation clothing navel orange fruit ball, fruit ball warp cross after dispersion machine process to orange juice born of the same parents, this zymin can reuse 3 ~ 5 times under these conditions.
Embodiment 2
In the present embodiment investigation liquid submerged fermentation liquid, aspergillus niger C-2 bacterial strain CCTCCNO:M2012160 fermented liquid of the present invention is to the degraded of navel orange peel.Following methods is adopted 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 peel powder 10g/L and agar 20g/L, and the substratum activated sterilizing 30min at 121 DEG C of temperature obtains.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 peel powder or Peel of Navel Orange pomace 20g/L; And seed liquid nutrient medium sterilizing 30min at 121 DEG C of temperature obtains.This kind of seed liquid nutrient medium is suitable for the cultivation of aspergillus niger C-2, and this substratum also can be adopted to carry out in the process of liquid submerged fermentation cultivation.Step is as follows:
(1) activate: the aspergillus niger C-2 seed described seed selection obtained is inoculated in fresh solid slant culture base and activates, in described reactivation process, the pH value controlling solid slant culture base is 7.0 ~ 7.2, activation temperature controls at 29 DEG C ~ 30 DEG C, 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 DEG C 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 controlling seed culture medium is 7.0 ~ 7.2, temperature during seed culture controls at 28 DEG C ~ 30 DEG C, and 200r/m cultivates 12 ~ 16h; Navel orange peel powder or Peel of Navel Orange pomace 20g/L in substratum.
(3) liquid submerged fermentation is cultivated: inoculated in fermentation flask by the inoculum size of liquid submerged fermentation substratum 8% ~ 10% by the seed aspergillus niger C-2 bacterial classification after above-mentioned cultivation and carry out Submerged fermentation, the formula of described liquid submerged fermentation substratum is identical with described seed liquid nutrient medium, fermentation culture conditions is: in 12h, temperature is 30 DEG C, after 12h 28 DEG C; Automatic adjustment pH7.0 ~ 7.2; Tank pressure 0.06MPa ~ 0.08MPa; 200r/m after 180r/m, 12h in stirring velocity 12h; Air flow 1:1.0 ~ 1.5; Fermentation time 108 ~ 120h.
Whole process incubation time is greatly about 10 days, used immediately by the enzyme liquid of liquid submerged fermentation gained, the enzyme liquid described fermentation obtained obtains concentrated enzyme liquid through ultrafiltration and concentration, or by different degraded navel orange peel zymin that other processing modes obtain, 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.By concentrated fermenting enzyme preparation to stir after the concentration dilution of 5% ~ 8%, the bath of jacketed kettle vapour is warmed up to 40 DEG C, then the navel orange (solid-liquid ratio 1:3) of peeling is added, with citric acid adjust pH to 4.5, under temperature 45 ~ 50 DEG C of conditions, constantly stir and keep constant temperature 90 ~ 120min, obtaining excystation clothing navel orange fruit ball, fruit ball warp cross after dispersion machine process to orange juice born of the same parents, this zymin can reuse 3 ~ 5 times under these conditions.
Embodiment 3
The present embodiment investigates the bent degraded to navel orange peel of aspergillus niger C-2 bacterial strain CCTCCNO:M2012160 solid state fermentation of the present invention in solid state fermentation.Following methods is adopted 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 peel powder 8g/L and agar 18g/L, and the substratum activated sterilizing 30min at 121 DEG C of temperature obtains.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, NaCl1.5g/L, navel orange peel powder or Peel of Navel Orange pomace 18g/L; And seed liquid nutrient medium sterilizing 30min at 121 DEG C of temperature obtains.This kind of seed liquid nutrient medium is suitable for the cultivation of aspergillus niger C-2, and this substratum also can be adopted to carry out in the process of liquid submerged fermentation cultivation.
Step is as follows:
(1) activate: the aspergillus niger C-2 seed described seed selection obtained is inoculated in fresh solid slant culture base and activates, in described reactivation process, the pH value controlling solid slant culture base is 7.0 ~ 7.2, activation temperature controls at 28 DEG C ~ 30 DEG C, 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 DEG C 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 part, navel orange pomace or fruit peel powder 180 weight part, ammonium sulfate 2 weight part, magnesium sulfate 0.5 weight part, dipotassium hydrogen phosphate 1 weight part, above each component adds water and mixes thoroughly and regulate moisture humidity to be 60%.(initial pH6.5), cultivates 48h for 30 DEG C.
(3) solid state fermentation is cultivated: by the aspergillus niger C-2 seed of above-mentioned activation song by 10% inoculum size access 50kg solid-state fermentation culture medium, 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 DEG C, obtain aspergillus niger C-2 solid state fermentation bent, this solid state fermentation song can use by cryodrying and after pulverizing, and uses after also obtaining liquid enzymes liquid by lixiviate in 0.5mol/LpH=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 for the aspergillus niger C-2 solid state fermentation of above-mentioned cryodrying, pulverizing 12kg is added in the physiological saline of 120kg, stir, the bath of jacketed kettle vapour is warmed up to 40 DEG C, then the navel orange (solid-liquid ratio 1:3) of peeling is added, with citric acid adjust pH to 4.5, under temperature 45 ~ 50 DEG C of conditions, continuous stirring also keeps constant temperature 90 ~ 120min, obtain excystation clothing navel orange fruit ball, fruit ball warp cross after dispersion machine process to orange juice born of the same parents, this aspergillus niger C-2 solid state fermentation song can reuse 3 ~ 5 times under these conditions.
Embodiment 4
In the present embodiment investigation liquid submerged fermentation liquid, aspergillus niger C-2 bacterial strain CCTCCNO:M2012160 fermented liquid of the present invention is to the degraded of orange skin.Following methods is adopted 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 after hot soup, distinguish the train of thought removed on tangerine lobe, obtaining tangerine lobe, to be about 35kg for subsequent use.By concentrated fermenting enzyme preparation to stir after the concentration dilution of 5% ~ 8%, the bath of jacketed kettle vapour is warmed up to 40 DEG C, then 35kg peeling is added except train of thought tangerine lobe (solid-liquid ratio 1:3), with citric acid adjust pH to 4.5, under temperature 40 ~ 45 DEG C of conditions, constantly stir and keep constant temperature 90 ~ 100min, obtaining full excystation clothing tangerine lobe, interior born of the same parents' percentage of damage is low, and maintains the original local flavor of oranges and tangerines and color and luster.This zymin can reuse 3 ~ 5 times under these conditions.
Comparative example
What adopt in this comparative example is traditional fungi culture medium, and in investigation liquid submerged fermentation liquid, aspergillus niger C-2 bacterial strain CCTCCNO:M2012160 fermented liquid of the present invention is to the degradation effect of navel orange peel.
Traditional fungus culture based formulas: take 200g potato, clean peeling chopping, the 1000mL that adds water boils half hour, filtered through gauze, add 15g glucose (activation 15g agar) again, filtered through gauze while hot after fully dissolving, 121 DEG C of sterilizings 20 minutes after packing.
Traditional fungus culture based formulas is adopted to prepare the step of the 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, obtain about 80kg containing capsule clothing navel orange fruit ball, be equally divided into two parts for subsequent use.The fermenting enzyme preparation obtained by different culture media Fermented Condensed is respectively to stir after the concentration dilution of 5% ~ 8%, the bath of jacketed kettle vapour is warmed up to 40 DEG C, then the navel orange (solid-liquid ratio 1:3) of peeling is added, with citric acid adjust pH to 4.5, under temperature 45 ~ 50 DEG C of conditions, constantly stir and keep constant temperature 90 ~ 120min.
Can be found out by the experimental result of embodiment 1 to 4 and comparative example, by traditional fungi culture medium formula, fermenting liquid degradation effect is not observed to navel orange peel, and obtain the navel orange fruit ball of full excystation clothing by culture medium prescription fermentation liquor treatment provided by the present invention.Find that culture medium prescription of the present invention is not only degraded navel orange peel successful by simultaneous test, also utilize navel orange pomace enzymatic production, turn waste into wealth, both improve 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 peel micro-organism enzyme preparation of the present invention can industrialization scale operation, 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 efficient to navel orange peel Degradation, completely, thoroughly decompose, and not damaging internal orange juice born of the same parents, enhance productivity, reduce labour intensity, reduce production cost.
3, the present invention is compared with soda acid technique, and the nutritive ingredient after biological enzyme formulation degraded capsule clothing is destroyed and runs off and is better than traditional acid-base method, and it maintains the original local flavor of navel orange and color and luster.
4, the residual Safety of Food Quality technology barriers and the soda acid that avoid the objectionable impuritiess such as the heavy metal that traditional soda acid technique causes discharge the environmental pollution caused, and save a large amount of water resourcess, achieve the unification of economic benefit, ecological benefits and social benefit.
5, culture medium prescription of the present invention is not only degraded navel orange peel successful, also utilizes navel orange pomace enzymatic production, turns waste into wealth, both improve the added value of navel orange industry, 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 peel 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 amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (3)
1. the preparation method of navel orange peel zymin that degrades, it is characterized in that, employing depositary institution is China typical culture collection center, preserving number is that the aspergillus niger C-2 of CCTCCNO:M2012160 is as fermentation strain, cultivate through liquid submerged fermentation or solid state fermentation and prepare described degraded navel orange peel zymin
Taking a step forward that described liquid submerged fermentation or solid state fermentation are cultivated comprises activation, prepares spore suspension, the step of seed culture;
Described activation comprises:
Be inoculated in activation medium by described aspergillus niger C-2 and activate, the pH value of described activation medium is 7.0 ~ 7.2, and activation temperature controls at 28 DEG C ~ 30 DEG C, and soak time is 1 ~ 2 day;
Wherein, the solid slant culture base that described activation medium is is carbon source with navel orange peel powder, 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, NaCl1g/L ~ 2g/L, navel orange peel powder 6g/L ~ 10g/L and agar 15g/L ~ 20g/L, and sterilizing obtains described activation medium;
The described step preparing spore suspension comprises:
The spore of described aspergillus niger C-2 under adopting aseptic washing, and be diluted to 2 ~ 3 × 10
6individual/mL obtains spore suspension, and is saved backup by described spore suspension;
Described seed culture comprises:
Be inoculated in seed liquid nutrient medium by described spore suspension, the pH value controlling described seed liquid nutrient medium is 7.0 ~ 7.2, and culture temperature controls at 28 DEG C ~ 30 DEG C, and 200r/m cultivates 12 ~ 16h; Or
Be inoculated in seed solid medium by described spore suspension, the initial pH value controlling described seed solid medium is 6.5, and culture temperature controls at 28 DEG C ~ 30 DEG C, cultivates 24 ~ 48h;
Wherein, the substratum that described seed liquid nutrient medium is is carbon source with navel orange peel powder, 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, NaCl1g/L ~ 2g/L, navel orange peel powder or Peel of Navel Orange pomace 15g/L ~ 20g/L; And sterilizing 30min obtains described seed liquid nutrient medium at 121 DEG C of temperature;
The step that described liquid submerged fermentation is cultivated comprises:
The described seed liquid culture medium inoculated containing aspergillus niger C-2 through described seed culture is carried out liquid submerged fermentation cultivation in fermentation flask, liquid submerged fermentation substratum is with described seed liquid nutrient medium, fermentation culture conditions is: in 12h, temperature is 30 DEG C, after 12h 28 DEG C; PH value is 7.0 ~ 7.2; Pressure position 0.06MPa ~ 0.08MPa; 200r/m after 180r/m, 12h in stirring velocity 12h; Air flow 1:1.0 ~ 1.5; Fermentation time 108 ~ 120h, obtained described zymin;
The step that described 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 DEG C, obtain aspergillus niger C-2 solid state fermentation bent, described solid state fermentation triton crosses cryodrying and obtained described zymin after pulverizing, or obtains described zymin by lixiviate in 0.5mol/LpH=6.5 phosphoric acid buffer;
Wherein, described solid-state fermentation culture medium is prepared by the following method: wheat bran 95 weight part, rice straw powder 90 weight part, navel orange pomace or navel orange fruit peel powder 90 weight part, ammonium sulfate 2 weight part, magnesium sulfate 0.5 weight part, dipotassium hydrogen phosphate 1 weight part are added water and mix thoroughly and regulate moisture humidity to be 60%, obtains described solid-state fermentation culture medium.
2. preparation method according to claim 1, is characterized in that, comprises further: by described zymin through ultrafiltration, concentrated obtained concentrated enzyme preparation, or described enzyme preparation is become pulvis or granule.
3. for a substratum for navel orange peel microorganism of degrading, it is characterized in that, described substratum is with navel orange peel powder or Peel of Navel Orange pomace for carbon source,
When described substratum is used as activation of microorganism, comprise 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, NaCl1g/L ~ 2g/L, navel orange peel powder 6g/L ~ 10g/L and agar 15g/L ~ 20g/L, and at 121 DEG C of temperature, sterilizing 30min obtains described substratum;
When described substratum is used for the cultivation of seed liquid state, comprise 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, NaCl1g/L ~ 2g/L, navel orange peel powder or Peel of Navel Orange pomace 15g/L ~ 20g/L; And sterilizing 30min obtains described substratum at 121 DEG C of temperature.
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| Antioxidant potentials of buntan pummelo(Citrus grandis Osbeck) and its ethanolic and acetified fermentation product;JANG H D ET AL;《Food Chem》;20101231;第118卷(第3期);554-558 * |
| 发酵-酶解偶联脱囊衣工艺研究;张志平;《中国优秀硕士学位论文全文数据库（工程科技I辑）》;20120630(第06期);B024-392 * |
| 黑曲霉产果胶酯酶发酵条件的研究;焦云鹏;《中国酿造》;20071231(第12期);28-31 * |
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