CN103468589B - Strain for producing hemicellulase and micro fermentation method of strain - Google Patents
Strain for producing hemicellulase and micro fermentation method of strain Download PDFInfo
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- CN103468589B CN103468589B CN201310425377.7A CN201310425377A CN103468589B CN 103468589 B CN103468589 B CN 103468589B CN 201310425377 A CN201310425377 A CN 201310425377A CN 103468589 B CN103468589 B CN 103468589B
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Abstract
The invention discloses a wild aspergillus aculeatus strain for producing a hemicellulase based on degradation of citrus sac garments and a micro fermentation method of the wild aspergillus aculeatus strain. The aspergillus aculeatus strain ZC-1005 is obtained from citrus wastes (sac garments, citrus peels and the like) embedded in the soil of the Yuelu mountain in Hunan province in a screening manner. The preservation number of the aspergillus aculeatus strain ZC-1005 is CCTCCM2013324. A crude enzyme liquid for obtaining the hemicellulase with a high yield is prepared in a micro liquid fermentation manner by utilizing bran as a sole carbon source. The obtained hemicellulase preparation capable of degrading the citrus sac garments has a certain application value for the sac garment degradation during citrus processing, and also provides a way for the comprehensive utilization of abandoned biomass. In addition, the research on the application feasibility of the method for producing the hemicellulase in the micro fermentation manner is verified.
Description
Technical field
The invention belongs to fermentable and produce zymotechnic field, be specifically related to a strain based on the product hemicellulase bacterial strain of degraded orange skin and adopt this bacterial strain to carry out miniature fermentation to produce the method for hemicellulase.
Background technology
China's orange yield has reached more than 2,000 ten thousand tons, occupies first place in the world, and China is also the main product ground of oranges and tangerines can simultaneously.Hunan Province is the oranges and tangerines main producing region of China, also occupy national prostatitis to the deep processing of oranges and tangerines and correlation technique exploitation.In the production process of oranges and tangerines can, excystation clothing technology is the key in whole production, and it directly determines the quality of finished product can.
Orange skin is the semitransparent thin film that one deck of tangerine lobe outside surface parcel is made up of Mierocrystalline cellulose, hemicellulose and pectin, and the enzyme that therefore can be used for excystation clothing has cellulase, hemicellulase, polygalacturonase etc.Main employing is polygalacturonase, cellulase at present, and the research both at home and abroad about other orange skin microorganism of degrading is fewer, lacks specificity and systematicness on the whole to the research of the degrading enzyme of capsule clothing.Adopt common polygalacturonase and cellulase to process capsule clothing, the treatment time is long often, and capsule clothing degradation effect is undesirable, and specificity is poor, cost is higher, brings certain burden to production; But along with the development of enzyme engineering and International Food Safety Control more and more come into one's own, enzymic degradation orange skin is applied gradually at food production field such as oranges and tangerines cans, this just needs the zymin of efficient, the single-minded degraded orange skin of energy, wherein just comprises hemicellulase (zytase).
Hemicellulase refers to that energy degradation of hemicellulose generates the general name of one group of enzyme of the material such as wood sugar and seminose, produces primarily of bacterium and mold fermentation.Hemicellulose is that occurring in nature is only second to cellulosic renewable organic resource, with Mierocrystalline cellulose (β ~ 1,4 dextran backbone) compare, its structure is very complicated with composition, comprise xylan, mannosans, arabinan and wood and gather the various ingredients such as glucose, and wherein maximum with the industrial relation such as xylan and food.
Strain improvement is the element task that hemicellulase is produced.The microbial profile of producing hemicellulase is very wide, has tens genus, more than 100 kinds, comprises bacterium, actinomycetes, fungi and some yeast.Current research more clearly have genus bacillus, streptomycete, aspergillus, sickle-like bacteria, Split-gill, the mould and pore fungus of wood etc.
It is adopt 250mL triangular flask (liquid amount 50 ~ 100mL) to carry out shaker fermentation that traditional laboratory fermentable produces enzyme research method, and required raw material and pharmaceutical chemicals consumption are comparatively large, and product enzyme enzyme stability alive is not high.Adopt miniature system to ferment, not only saved fermentation costs, the different fermentations batch stability that product enzyme enzyme is lived is higher simultaneously.
Summary of the invention
A plant height is the object of the present invention is to provide to produce the microorganism Aspergillus aculeatus strains A spergillus aculeatusZC-1005 of hemicellulase.
The microorganism Aspergillus aculeatus bacterial strain that the present invention relates to submits preservation on July 9th, 2013.Depositary institution's title: China typical culture collection center; Preservation address: China, Wuhan, Wuhan University; Deposit number is CCTCC NO:M2013324, and Classification And Nomenclature is: microorganism Aspergillus aculeatus Aspergillus aculeatus ZC-1005.
Another object of the present invention is to the deficiency existed for existing enzymic degradation orange skin technology, the method that a species specificity is strong, the bacterial strain of high yield hemicellulase carries out miniature fermentation product hemicellulase is provided.The method comprises the following steps:
The dry wheat bran powder getting water content≤5% 3 ~ 5% joins in fermentation culture by mass percentage, and fermentation culture adds NaNO according in every 1L deionized water
37g, K
2hPO
41g, KCl0.5g, MgSO
47H
2o0.5g, FeSO
47H
2the proportions of O0.01g, inoculation microorganism Aspergillus aculeatus spore suspension after autoclaving cooling, inoculum density is 1.0 × 10
4~ 1.0 × 10
6individual spore/mL nutrient solution; 48 ~ 72h is cultivated, temperature 34 ~ 36 DEG C, rotating speed 150 ~ 190r/min in constant-temperature table.
The preparation process of described microorganism Aspergillus aculeatus spore suspension: microorganism Aspergillus aculeatus spore inoculating to slant medium is cultivated 3 ~ 4 days for 34 ~ 36 DEG C; With microorganism Aspergillus aculeatus spore under aseptic washing, make spore suspension, refrigerate in 4 DEG C for subsequent use.
The initial pH of described fermentation culture is 6.0 ~ 7.0,121 DEG C of autoclaving 15 ~ 25min.
Fermenting container adopts 50mL Erlenmeyer flask, liquid amount 8 ~ 12mL, 6 layers of gauze sealing.
Bacterial strain screening process of the present invention is as follows:
(1) primary dcreening operation: utilize the oranges and tangerines waste (capsule clothing, orange peel etc.) be embedded in the soil of Yue Lu mountain, Hunan, carry out enrichment culture (34 ~ 36 DEG C, 2 ~ 3 days), after the nutrient solution gradient dilution after enrichment culture, get 10
-6~ 10
-8gradient dilution liquid 0.2mL coats primary dcreening operation plate culture medium: cultivate after 3 ~ 5 days, get single bacterium colony and carry out flat-plate spotting for 34 ~ 36 DEG C, cultivates after 3 days, adds 0.1% congo red staining 15min for 34 ~ 36 DEG C, and then reject dye liquor adds 1M NaCl washing 15min.Finally select the primary dcreening operation bacterial strain producing transparent circle, carry out continuous print plate streaking purifying bacterial strain.Primary dcreening operation plate culture medium adds NaNO according in every 1L deionized water
33g, K
2hPO
41g, KCl0.5g, MgSO
47H
2o0.5g, FeSO
47H
2the proportions of O0.01g, xylan 10g, agar 20g.
(2) multiple sieve: with the orange skin of drying (40 ~ 60 order) for substrate, primary dcreening operation substratum (not adding xylan and agar) is nutrient solution, the bacterial strain that primary dcreening operation is obtained carry out 50mL triangular flask (liquid amount 8 ~ 10mL), 34 ~ 36 DEG C, 150 ~ 190r/min shaking flask sieves again, surveys xylanase activity after 72h.Enzyme live the highest namely for the purpose of bacterial strain.
(3) identification of strains: the rDNA sequence of being carried out the bacterial strain selected by pcr amplification by a pair ITS universal primer ITS1 and ITS4, then the sequence that obtains of sequencing analysis and BLSAT are analyzed, and showing this bacterium is microorganism Aspergillus aculeatus.
(4) crude enzyme liquid extracting method: by the tunning 8000r/min of the object bacterial strain that multiple sieve obtains, centrifugal 10 ~ 15min removes solid substance, and supernatant liquor is crude enzyme liquid.
(5) enzyme activity analytical procedure: get suitable damping fluid dilution enzyme liquid 2mL add 2mL1.0%(W/V) xylan solution (this solution is by pH=5.5, concentration 0.1M acetic acid-sodium acetate buffer solution configures) in 25mL color-comparison tube, 5mL DNS termination reaction is added after 50 DEG C of reaction 20min, boiling water bath 5min, flowing water cools, keep the skin wet to scale with distilled water, 540nm place measures light absorption value.Blank is for before adding enzyme liquid, and first add the DNS termination reaction of 5mL, other steps are identical.Enzyme is lived and is defined: 1mL crude enzyme liquid is a Ge Meihuo unit (U) at the raw 1 μm of ol wood sugar of 1min bottom exploded produce.
The present invention is based on xylan is that sole carbon source carries out primary dcreening operation, with capsule clothing powder for sole carbon source carries out multiple sieve.The bacterial strain screened is compared with the bacterium of some similar functions in existing disclosed technology, and produce hemicellulase enzyme ability comparatively by force, fermentation time is 55-65h, slightly short compared with this field laboratory ferment, is generally 72h.
Accompanying drawing explanation
Fig. 1 is the crude enzyme liquid degraded capsule clothing experimental conditions figure that strain fermentation of the present invention produces;
Fig. 2 is bacterial strain PDA slat chain conveyor of the present invention 2 days bacterium colony figure.
Embodiment
Following examples are intended to the present invention instead of limitation of the invention further are described, the present invention can implement by the either type described in summary of the invention.
Embodiment 1:
, in 35 DEG C of growths, with microorganism Aspergillus aculeatus spore under aseptic washing, make on microorganism Aspergillus aculeatus spore inoculating to slant medium of the present invention pityrosporion ovale suspension and be diluted to 4.24 × 10 after 4 days
7individual/mL, refrigerates in 4 DEG C for subsequent use.
Get water content≤5% dry wheat bran 0.4g(40 ~ 60 order) to put into and be equipped with in the 50mL triangular flask of 10mL fermentation culture, fermentation culture adds NaNO according in every 1L deionized water
33g, K
2hPO
41g, KCl0.5g, MgSO
47H
2o0.5g, FeSO
47H
2the proportions of O0.01g, pH6.0 ~ 7.0,121 DEG C of autoclaving 20min; Aseptically add microorganism Aspergillus aculeatus spore suspension 24 μ L after cooling, nutrient solution inoculum density is 1.0 × 10
5individual/mL; 50mL Erlenmeyer flask, liquid amount 8 ~ 12mL, 6 layers of gauze sealing.Postvaccinal shaking flask is cultivated 60h in constant-temperature table, and set temperature is 35 DEG C, rotating speed 170r/min; Filtered or centrifugal (getting 1mL for enzyme activity determination) by nutrient solution, adopt DNS method to measure xylanase content, recording xylanase activity is 120.9U/mL.
Embodiment 2:
, in 35 DEG C of growths, with microorganism Aspergillus aculeatus spore under aseptic washing, make on microorganism Aspergillus aculeatus spore inoculating to slant medium of the present invention pityrosporion ovale suspension and be diluted to 4.24 × 10 after 4 days
7individual/mL, refrigerates in 4 DEG C for subsequent use.
Get water content≤5% dry wheat bran 0.4g(40 ~ 60 order) to put into and be equipped with in the 50mL triangular flask of 10mL fermentation culture, fermentation culture adds NaNO according in every 1L deionized water
37g, K
2hPO
41g, KCl0.5g, MgSO
47H
2o0.5g, FeSO
47H
2the proportions of O0.01g, pH6.0 ~ 7.0,121 DEG C of autoclaving 20min; Aseptically add microorganism Aspergillus aculeatus spore suspension 24 μ L after cooling, nutrient solution inoculum density is 1.0 × 10
5individual/mL; 50mL Erlenmeyer flask, liquid amount 8 ~ 12mL, 6 layers of gauze sealing.Postvaccinal shaking flask is cultivated 60h in constant-temperature table, and set temperature is 35 DEG C, rotating speed 170r/min; Filtered or centrifugal (getting 1mL for enzyme activity determination) by nutrient solution, adopt DNS method to measure xylanase content, recording xylanase activity is 139.6U/mL.
Embodiment 3:
, in 35 DEG C of growths, with microorganism Aspergillus aculeatus spore under aseptic washing, make on microorganism Aspergillus aculeatus spore inoculating to slant medium of the present invention pityrosporion ovale suspension and be diluted to 4.24 × 10 after 4 days
7individual/mL, refrigerates in 4 DEG C for subsequent use.
Get water content≤5% dry wheat bran 0.4g(40 ~ 60 order) to put into and be equipped with in the 50mL triangular flask of 10mL fermentation culture, fermentation culture adds NaNO according in every 1L deionized water
39g, K
2hPO
41g, KCl0.5g, MgSO
47H
2o0.5g, FeSO
47H
2the proportions of O0.01g, pH6.0 ~ 7.0, autoclaving 20min; Aseptically add microorganism Aspergillus aculeatus spore suspension 24 μ L after cooling, nutrient solution inoculum density is 1.0 × 10
5individual/mL; 50mL Erlenmeyer flask, liquid amount 8 ~ 12mL, 6 layers of gauze sealing.Postvaccinal shaking flask is cultivated 60h in constant-temperature table, and set temperature is 35 DEG C, rotating speed 170r/min; Filtered or centrifugal (getting 1mL for enzyme activity determination) by nutrient solution, adopt DNS method to measure xylanase content, recording xylanase activity is 116.3U/mL.
Embodiment 4:
The thick enzyme that bacterial strain of the present invention produces can effectively be degraded orange skin:
(1) capsule clothing degradation experiment is raw materials used is satsuma mandarin, is purchased from supermarket, Changsha.Tangerine lobe is manually peeled off, go network for subsequent use.
(2) crude enzyme liquid is prepared according to embodiment 2.
(3) hydrolyzed solution preparation: crude enzyme liquid forms with distilled water proportioning by a certain percentage.It is 10%, 20%, 30%, 40%, 50% that crude enzyme liquid concentration gradient sets gradually.(accounting for the ratio of sample hydrolyzed solution).
(4) DeR solid-liquid ratio is tangerine lobe: hydrolyzed solution (w/w)=1:2; The DeR time is 3h; DeR temperature is 50 DEG C of water-baths.
Capsule clothing degraded situation is as shown in Figure 1: wherein A sample is that peel manually is left away the tangerine lobe after network, without water bath processing.B sample is that peel manually is left away network, and the tangerine lobe after 50 DEG C do not add crude enzyme liquid water-bath, A, B are sample blank.No. 1-5 represents crude enzyme liquid concentration is successively 10%-50% sample, and each sample is two parallel.
Result: as can be seen from picture, 1-4 sample all has capsule clothing in various degree to remain, and only have No. 5 samples (enzymolysis 3h, crude enzyme liquid addition 50%) hydrolysis result best, tangerine lobe capsule clothing enzymolysis is complete, and pulp is complete, not destroyed.Above situation illustrates, the crude enzyme liquid adopting the inventive method to prepare is more remarkable to orange skin degradation effect.
Claims (5)
1. hemicellulase bacterial strain microorganism Aspergillus aculeatus Aspergillus aculeatus ZC-1005 is produced in a strain, and deposit number is CCTCC NO:M 2013324.
2. adopt the miniature fermentation of microorganism Aspergillus aculeatus bacterial strain according to claim 1 to produce a method for hemicellulase, it is characterized in that, comprise the following steps:
The dry wheat bran powder getting water content≤5% 3 ~ 5% joins in fermentation culture by mass percentage, and fermentation culture adds NaNO according in every 1L deionized water
37g, K
2hPO
41g, KCl 0.5g, MgSO
47H
2o0.5g, FeSO
47H
2the proportions of O 0.01g, inoculation microorganism Aspergillus aculeatus spore suspension after autoclaving cooling, inoculum density is 1.0 × 10
4~ 1.0 × 10
6individual spore/mL nutrient solution; 48 ~ 72h is cultivated, temperature 34 ~ 36 DEG C, rotating speed 150 ~ 190r/min in constant-temperature table.
3. method according to claim 2, is characterized in that: the preparation process of described microorganism Aspergillus aculeatus spore suspension: microorganism Aspergillus aculeatus spore inoculating to slant medium is cultivated 3 ~ 4 days for 34 ~ 36 DEG C; With microorganism Aspergillus aculeatus spore under aseptic washing, make spore suspension, refrigerate in 4 DEG C for subsequent use.
4. method according to claim 2, is characterized in that: the initial pH of described fermentation culture is 6.0 ~ 7.0,121 DEG C of autoclaving 15 ~ 25min.
5. method according to claim 2, is characterized in that: fermenting container adopts 50mL Erlenmeyer flask, liquid amount 8 ~ 12mL, 6 layers of gauze sealing.
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