CN103087922B - Penicillium, cellulose produced by fermenting penicillium in solid state and method thereof for preparing pomelo peel high-ester pectin - Google Patents

Penicillium, cellulose produced by fermenting penicillium in solid state and method thereof for preparing pomelo peel high-ester pectin Download PDF

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CN103087922B
CN103087922B CN201210420183.3A CN201210420183A CN103087922B CN 103087922 B CN103087922 B CN 103087922B CN 201210420183 A CN201210420183 A CN 201210420183A CN 103087922 B CN103087922 B CN 103087922B
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penicillium
pectin
cellulase
powder
straw
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CN103087922A (en
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周茂洪
赵肖为
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Wenzhou University
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Abstract

The invention discloses penicillium, cellulose produced by fermenting penicillium in a solid state and a method thereof for preparing pomelo peel high-ester pectin. A penicillium strain is penicillium (Penicillium sp.) WZUX01 and is preserved in the General Micro-Organism Centre of the China Micro-Organism Strain Preservation and Management Committees; and the preservation number is CGMCC No. 6370. Cellulose is produced by fermenting the penicillium (Penicillium sp.) WZUX01 in the solid state; the cellulose can be used for preparing pomelo peel high-ester pectin; and the content of the methoxy group in the pomelo peel pectin prepared by the method can be up to 30.48%.

Description

The method of Penicillium notatum, solid state fermentation cellulase-producing and shaddock ped high ester pectin processed thereof
Technical field
The invention belongs to zymetology (C12P) technology, be specifically related to a penicillium, its solid state fermentation cellulase-producing, and this cellulase is for the method for shaddock ped high ester pectin processed.
Background technology
Pectin (Pectin) is a kind of wetting ability vegetable jelly, is extensively present in the cell walls of root, stem, leaf, fruit of higher plant.Pectin main component is D-galacturonic acid (D-galactuonic acid), and wherein part galacturonic acid is by esterification, and molecular weight is between 3～180,000.Commercialization pectin has liquid pectin and pectin powder, and the color and luster of pectin is from oyster white to khaki, because of raw material, production technique difference.According to gamma value (DM) difference, pectin is divided into high methoxyl pectin, and (gamma value is greater than 50%, be equivalent to methoxy content and be greater than 7%) and low-methoxy pectin (gamma value is less than 50%, is equivalent to methoxy content and is less than 7%), the latter comprises acid amides pectin.
Pectin has good emulsifying, thickening, stable and pectisation, is widely used in fields such as food, weaving, printing and dyeing, tobacco, metallurgy.Because pectin has the effects such as antibacterial, hemostasis, detumescence, removing toxic substances, antidiarrheal, reducing blood-fat, radioprotective, or a kind of good pharmaceutical preparation matrix, can be used to manufacture laxative, hemostatic agent, toxic metal toxinicide, plasma substitute etc., is medicine and the indispensable auxiliary material of cosmetic industry.Pectin is the main component of water-soluble dietary fibre in human body seven major nutrient, along with the development research of functional polysaccharide, pectin is as water-soluble dietary fibre, is more and more subject to the attention of research and processing industry, has the multiple effects such as good diarrhea, anticancer, treatment diabetes and fat-reducing.
Shaddock is the mature fruit of rue family citrus plant evergreen fruit trees, plants in a large number in the many areas of south China.China's shaddock ped aboundresources, but major part is dropped, and caused environmental pollution.In shaddock ped, pectin content is higher, and contained pectin degree of gelation is high, rank is high.Therefore, from shaddock ped, extract pectin and there is higher economic benefit and social benefit.
From shaddock ped, extracting pectin existing commercialization at present produces, also there are many bibliographical informations, as having reported the acid system obtaining by orthogonal experiment, Zhou Jinhua extracts the processing condition of pomelo-pectin, pectin yield is 14.9%(research and development of natural products, 2006,18:483-486), and for example Zhao Mei etc. has reported and has adopted acid hydrolyzation to extract the processing condition of shaddock ped low-methoxy pectin, pectin yield is 5.51%(food and pharmaceutical, 2008,10(7): 29-31).
Although acid extraction method is still the most frequently used pectin extraction method at present, acid extraction easily causes environmental pollution.Therefore people actively inquire into other extracting method that reduce environmental pollution.As ion exchange method (Sun Run, foodstuffs industry science and technology, 1989, (2): 40-41), microwave extraction method (Dredycfuss M S, Appl Eneerio Nicobio, 1980,38 (1): 13; M Kratchanova, Carbohydrate Polymers, 2004,56:181～185; Kong Zhen, Journal of Zhengzhou Grain College, 2000,21(2): 11-15), these methods are all difficult to realize industrial applications.
The separation method of the most frequently used pectin is ethanol precipitation.Other separation methods also have report, as salting-out process (Deng Hong, Food science, 2002,23 (3): 57-60; Zhang Linghua, pesticide herd product exploitation, 2000, (11): 12～14).Ethanol precipitation produce pectin lighter color, ash oontent few, degree of gelation is high, quality better.Although ethanol usage quantity is larger, if waste ethanol is reclaimed and recycle, can reduce production costs.
Summary of the invention
The technical problem to be solved in the present invention is to adopt Enzymatic Extraction pomelo-pectin, extracts to alleviate acid system the environmental pollution being caused, and extraction yield and acid system approach and even exceed acid system simultaneously.
In order to solve the problems of the technologies described above, the invention provides a penicillium solid state fermentation cellulase-producing and the novel method for the preparation of shaddock ped high ester pectin.Its principal feature, for carrying out solid state fermentation with a penicillium, extracts the cellulase in solid-state culture, obtained by freeze drying cellulase powder.Extract pomelo-pectin with the cellulase powder that makes, with the pectin of ethanol precipitation precipitation zyme extract, precipitation is dried and makes pectin finished product after centrifugal, its methoxy content belongs to high ester pectin after measured.The shaddock ped high ester pectin preparation method that this invention provides, the pectin extraction rate of its pectin powder is higher than the current acid extraction method of report, and alleviates the pollution of acid to environment.
The invention provides a penicillium, this bacterial strain be Penicillium notatum ( penicillium sp.) WZUX01, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO. 6370.
The present invention also provides the method for above-mentioned Penicillium notatum solid state fermentation cellulase-producing, comprises the steps:
1) described bacterial strain Penicillium notatum WZUX01 line is transferred on PDA slant medium, cultivates 3 ~ 6 days at 25 ~ 35 ℃;
2) Penicillium notatum in the culture obtaining with water elution step 1), obtain spore suspension, the straw bran mass of access sterilizing, cultivates 5 ~ 7 days at 25 ~ 35 ℃, and wherein straw bran mass is made up of the composition of following weight ratio: rice straw powder: wheat bran: (NH 4) 2sO 4: water=4 ~ 36:8 ~ 48: 0 ~ 16: 24 ~ 80;
3) step 2) the solid-state culture that obtains adds distilled water to smash to pieces, and 35 ~ 45 ℃ of extracting at constant temperature 2 ~ 10h, filter, and obtain enzyme liquid, obtain cellulase through lyophilize.Preferably, after above-mentioned extracting at constant temperature 2 ~ 10h, be placed in and at 4 ℃, soak 24 h.
Preferably, above-mentioned steps 2) described in the spore amount of spore suspension be 1 × 10 12~ 9 × 10 12individual/mL, the inoculum size of described spore suspension: the envelope-bulk to weight ratio of spore suspension and straw bran mass is 1 mL: 1 ~ 3 g, is preferably 1:2.
Preferably, step 2) described straw bran mass is made up of the composition of following weight ratio: rice straw powder: wheat bran: (NH 4) 2sO 4: water=8:32:8:52.
The cellulase that the method for above-mentioned Penicillium notatum solid state fermentation cellulase-producing obtains.
The present invention also provides the above-mentioned cellulase method for the preparation of shaddock ped high ester pectin, comprises the steps:
1) shaddock ped is dried, pulverized, obtain shaddock ped powder;
2) shaddock ped powder is added in enzyme reactor, add damping fluid, then add described cellulase to extract, then the centrifugal liquid pectin that obtains;
3) regulating step 2) pH of gained liquid pectin is 5 ~ 9, adds ethanol, precipitation, centrifugal throw out, dry, obtain pectin.Sedimentation time is preferably 2 ~ 32h.
Preferably, step 2) described in the pH value of damping fluid be 5 ~ 7, the weightmeasurement ratio of shaddock ped powder and described damping fluid is 5 ~ 20 g:100 mL, the described damping fluid HAC/NaAC damping fluid that more preferably pH value is 7.
Preferably, step 2) described in the weight that adds of cellulase be 1% ~ 3% of described shaddock ped grain weight amount.
Preferably, step 2) in extract condition be: at 35 ~ 45 ℃, extract 24 ~ 48 h.
Preferably, regulating step 2 in step 3)) pH of gained liquid pectin is 5 ~ 9, the concentration of volume percent in liquid pectin is 60 ~ 80% to it to add ethanol.
The present invention can reach following effect: Penicillium notatum ( penicillium sp.) ability of WZUX01 solid state fermentation cellulase-producing is that every gram of solid-state cultivation produce CMC enzyme activity is 8210.667U, the CMC enzyme activity of prepared cellulase powder is 300U/mg; The shaddock ped Steamed dumpling with pork，mushrooms and bamboo shoots glue extraction yield that cellulase powder extracts pomelo-pectin is 19.30%, the pectin deposition rate of ethanol precipitated pectin is 80.23%, the pectin finished product extraction yield of shaddock ped powder is 15.48%, and the methoxy content of gained pectin finished product can reach 30.48%, belongs to high ester pectin.
Accompanying drawing explanation
Fig. 1 be Penicillium notatum ( penicillium sp.) WZUX01 Produced by Solid-state Fermentation cellulase curve.
Fig. 2 is that temperature is to cellulase powder CMC enzyme activity influence curve.
Fig. 3 is that pH is to cellulase powder CMC enzyme activity influence curve.
Bacterial strain preservation
Mould of the present invention ( penicillium sp.) WZUX01, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is CGMCC NO. 6370, preservation date is on July 20th, 2012.
Embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described so that those skilled in the art the present invention may be better understood and can be implemented, but illustrated embodiment is not as a limitation of the invention.
Embodiment mono-: Penicillium notatum ( penicillium sp.) WZUX01 solid state fermentation cellulase-producing
Bacterial strain
Penicillium notatum ( penicillium sp.) WZUX01, in the rice straw compost soil of tea hill, Wenzhou, separate and obtain with environmental science institute by Wenzhou University's life, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO. 6370.
(2) substratum
Potato substratum (PDA substratum): murphy juice 1000mL, agar 20g, pH nature.The preparation of murphy juice: get the fresh peeled potatoes of 200g, be cut into small pieces, add after 1000mL distillation poach 1h, with filtered through gauze, mend to 1000mL with distilled water.
The weight ratio of the each component of straw bran mass is: rice straw powder: wheat bran: (NH 4) 2sO 4: water=4 ~ 36:8 ~ 48:0 ~ 16:24 ~ 80, pH nature.
(3) method
Preservation strain Penicillium notatum WZUX01 line is transferred on PDA slant medium, cultivate 4 days for 30 ℃, the sterilized water of 10mL is added several times in the test tube that has covered with mould, vibration makes spore suspension, the spore quantity of measuring spore suspension, accesses sterilized straw bran mass by spore suspension, stirs, at 25-35 ℃, cultivate 4 days, every day stir culture base 1 to 2 time.Take the solid-state culture 10g obtaining, add the distilled water of 90mL to smash to pieces, lixiviate 4h vibrates in 40 ℃, 150r/min water bath with thermostatic control, be placed in refrigerator (4 ℃) and soak 24 h, filtered through gauze obtains crude enzyme liquid, centrifugal 15 points of kinds under 4000rpm, obtain supernatant liquor (enzyme liquid), measure CMC enzyme activity.
CMC enzyme activity determination method: the enzyme liquid 0.5mL after absorption is suitably diluted, in test tube, adds the citric acid-sodium citrate damping fluid (0.05molL containing 0.5%CMC-Na -1, pH4.4) and 1.5 mL then react after 30 min in 50 ℃ of water-baths, often in vitro add 1.5mLDNS reagent immediately, then in boiling water, boiling after 5 min, using immediately cold water cooling, be settled to 25mL, measure OD value at 520nm place, try to achieve its reducing sugar content from typical curve.CMC enzyme activity unit is defined as hydrocellulose in per minute and generates the enzyme amount of 1 μ g reducing sugar amount.(Wu Hailong etc., science and technology circular, 2008,24 (6): 769～776) take the enzyme liquid of deactivation 10min in 100 ℃ of water-baths as blank.
The spore quantity of spore suspension adopts blood counting chamber method to measure.
The setting of straw bran mass formula and culture condition and the results are shown in following table 1.
Each experimental group straw bran mass total amount is 20g, and the quantity of spore suspension is 1 × 10 12～9 × 10 12/ mL.
Setting and the result of table 1 straw bran mass formula and culture condition
Experimental group Straw: wheat bran: (NH 4) 2SO 4: water (W:W:W:W) Temperature (℃) CMC enzyme activity (U/g)
1 36:24:0:40 25 3960.6665
2 16:24:16:44 25 2390.8890
3 4:16:8:72 25 3140.5000
4 24:36:0:40 30 5270.9445
5 8:32:8:52 30 7220.500
6 12:8:16:64 30 3840.3890
7 12:48:16:24 35 2120.5000
8 24:16:8:52 35 2000.2220
9 8:12:0:80 35 4590.000
In table 1, temperature refers to that spore suspension accesses the culture temperature of sterilized straw bran mass, and CMC enzyme activity refers to the CMC enzyme activity of the solid-state culture of every g.
From table 1, the CMC enzyme activity data of each experimental group can be found out, the best proportioning of straw bran mass is experimental group 5, i.e. rice straw powder: wheat bran: (NH 4) 2sO 4: water=8: 32: 8: 52, pH nature.As take rice straw powder 1.60g, wheat bran 6.40g, (NH 4) 2sO 41.6g, in 250 mL beakers, adds 10.4 mL distilled water, stirs with glass stick.
Under optimal conditions, produce enzyme curve determination, result as shown in Figure 1.Produce enzyme peak value in 144 h left and right by known its of accompanying drawing 1, cultivate 6 days, CMC enzyme activity can reach 8210.667U/g.
Embodiment bis-: the characteristic measurement of cellulase powder
(1) preparation of cellulase powder
By the Penicillium notatum of preservation ( penicillium sp.) WZUX01 transfers in PDA slant medium, cultivates 4 days at 30 ℃, obtains the straw bran mass of filling a prescription by embodiment mono-experimental group 5 of spore suspension access sterilizing with sterilized water wash-out, the spore amount of spore suspension is 1 × 10 12~ 9 × 10 12/ mL, the inoculum size of spore suspension is spore suspension: straw bran mass=1:2 (V:W, mL/g), after stirring, cultivate 6 days at 30 ℃, add distilled water to smash to pieces, lixiviate 4h vibrates in 40 ℃, 150r/min water bath with thermostatic control, be placed under 4 ℃ of low temperature and soak 24 h, filter to obtain crude enzyme liquid, obtain cellulase powder through lyophilize.
(2) cellulase powder optimum temperuture is measured
Take appropriate cellulase powder 0.05molL -1, pH4.4 citric acid-sodium citrate damping fluid be mixed with enzyme liquid, at 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃, measure respectively CMC enzyme activity by embodiment mono-method, the results are shown in Figure 2.As shown in Figure 2, the optimum temperuture of cellulase powder is 35 ~ 45 ℃.
In Fig. 2, the highest CMC enzyme activity × 100% of the CMC enzyme activity that records at enzyme activity=a certain temperature/record.
(3) cellulase powder optimal pH is measured
Respectively take appropriate cellulase powder pH and be respectively 3.0,4.0,5.0,6.0,7.0,8.0,9.0 damping fluid and be mixed with enzyme solution, wherein pH is 3.0,4.0,5.0 and 6.0 use 0.05molL -1citric acid-sodium citrate damping fluid, pH is 7.0,8.0 and 9.0 the 0.05molL that uses -1hAC/NaAc damping fluid, use from prepare cellulase powder solution phase with damping fluid prepare respectively the 0.5% CMC-Na solution of different pH, measure the CMC enzyme activity under different pH by embodiment mono-method (mensuration temperature is 40 ℃), the results are shown in Figure 3.As shown in Figure 3, the optimal pH of cellulase powder is 5 ~ 7.In Fig. 3, the highest CMC enzyme activity × 100% of the CMC enzyme activity that records under enzyme activity=a certain pH/record.
(4) mensuration of cellulase powder CMC enzyme activity
Take appropriate cellulase powder 0.05molL -1, pH 6.0 citric acid-sodium citrate damping fluid be mixed with enzyme liquid, measure CMC enzyme activity by embodiment mono-method (measure temperature be 40 ℃), the CMC enzyme activity of cellulase powder is 300U/mg after measured.
Embodiment tri-: cellulase powder extracts pomelo-pectin
(1) shaddock ped processing
The fresh pomelo peel of Yongjia County, Zhejiang Province is originated from purchase, is cut into fine grained chippings is placed in baking oven and at 60 ℃, dries and (24-72 h), pulverize, obtain shaddock ped powder and preserve with for subsequent use with scissors.
(2) method
Take a certain amount of shaddock ped powder in enzyme reactor, add the HAC/NaAC damping fluid of pH 7.0, shaddock ped powder: damping fluid is 5 ~ 20:100(W:V, g/mL), add again 1% ~ 3% cellulase powder (accounting for the weight percentage of shaddock ped powder) making by embodiment bis-(1) methods, at 35 ~ 45 ℃, extract 24 ~ 48 h, then under 4000rpm, after centrifugal 10min, measure supernatant liquor (liquid pectin) concentration of pectin, calculate the pectin extraction rate of pectin powder.
The mensuration of concentration of pectin: get 0.4mL pectin extracting soln and be settled to 50mL, in the beaker that is added with ice cube and water, put 4 test tubes, each 6 mL vitriol oils that slowly add, cooling limit, limit adds the liquid pectin 1mL of constant volume, wherein 1 adds distilled water to compare, after shaking up, in boiling water, boil 10min, after being cooled to room temperature, respectively add 1mL carbazole ethanol, under room temperature, place 30min, under 530nm, survey OD value, try to achieve concentration of pectin (Gu Fuchang, Food science from typical curve, 1984, (8): 45～46).
Pectin extraction rate (%)=supernatant liquor concentration of pectin (g/mL) × supernatant liquor volume (mL)/pectin powder amount (g) × 100%.
Following table 2 is set and be the results are shown in to the experiment condition of cellulase powder extracting pomelo-pectin.
The experiment condition of table 2 cellulase powder extracting pomelo-pectin is set and result
Experimental group Shaddock ped powder: damping fluid (W:V, as g:mL) Cellulase powder (accounting for shaddock ped powder percentage ratio, %) Extraction time (h) Extraction temperature (℃) Pectin extraction rate (%)
1 5:100 1 24 35 6.30
2 5:100 2 36 40 10.78
3 5:100 3 48 45 7.68
4 10:100 1 48 45 16.74
5 10:100 2 36 40 19.30
6 10:100 3 24 35 14.32
7 20:100 1 48 40 6.86
8 20:100 2 24 45 9.66
9 20:100 3 36 35 5.52
The pectin extraction rate of experimental group 5 pectin powders is the highest as shown in Table 2, reaches 19.30%.
Embodiment tetra-: ethanol precipitation precipitated pectin
Prepare pomelo-pectin liquid by embodiment tri-experimental group 5 methods, respectively getting liquid pectin 100mL adjusting pH is 5 ~ 9, then the concentration of volume percent in liquid pectin reaches 60 ~ 80% to it to add ethanol, precipitation 2 ~ 32 h, precipitation is got after centrifugal 10min after finishing to be deposited in and at 90 ~ 100 ℃, is dried to constant weight and must be dried pectin under 4000rpm, weighs pectin weight and calculates pectin precipitation yield.
Precipitation yield (%)=dry rear pectin amount (g)/liquid pectin concentration of pectin (g/mL) × liquid pectin volume (mL) × 100.
The experiment condition of ethanol precipitation precipitated pectin and the results are shown in following table 3.
Experiment condition and the result of table 3 ethanol precipitation precipitated pectin
Experimental group Precipitation pH Alcohol concn (%) Sedimentation time (h) Pectin deposition rate (%)
1 5 60 2 51.80
2 5 70 8 58.85
3 5 80 32 44.01
4 7 60 32 73.29
5 7 70 8 80.23
6 7 80 2 62.84
7 9 60 32 15.20
8 9 70 2 17.51
9 9 80 8 14.63
As shown in Table 3, the precipitation yield of experimental group 5 pectin is the highest, reaches 80.23%.
Embodiment five: the mensuration of pectin product methoxyl group
The measuring method of pectin product methoxyl group: accurately take the pectin finished product of 0.6-1.0g with analytical balance, after adding 100mL distilled water and dissolving completely, add 2 of 1% phenolphthalein indicators, use 0.1molL -1naOH is titrated in pink 0.5min colour-fast for terminal, then adds 20mL 0.5 molL -1naOH standardized solution, saponification 2.5 h, use 0.5molL -1h 2sO 4standardized solution is titrated to red disappearance, calculates pectin methoxy content (Gu Fuchang, Food science, 1984, (8): 45～46).
Pectin methoxy content (%)=[(M 1v 1-MV) × 0.031]/W × 100
V 1: the volume (mL) of the NaOH standardized solution that saponification adds
M 1: the volumetric molar concentration (molL of the NaOH standardized solution that saponification adds -1)
V: the H that the alkali of titration saponification amount consumes 2sO 4the volume (mL) of standardized solution
M:H 2sO 4volumetric molar concentration (the molL of standardized solution -1)
0.031: the mmole number of methoxyl group
W: the quality (g) of pectin finished product
After measured, the pectin being obtained by method of the present invention, methoxy content is 30% left and right, belongs to high ester pectin.
And according to top condition: take 3 parts of liquid pectins that extract by embodiment tri-experimental group 5 use cellulase powder and through precipitating dried pectin finished product by example four experimental group 5 ethanol, measure its methoxy content, the methoxyl group average content that records pectin is 30.48%.
The above embodiment is only the preferred embodiment for absolutely proving that the present invention lifts, and protection scope of the present invention is not limited to this.What those skilled in the art did on basis of the present invention is equal to alternative or conversion, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.

Claims (4)

1. a penicillium, is characterized in that, this bacterial strain be Penicillium notatum ( penicillium sp.) WZUX01, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO. 6370.
2. the method for Penicillium notatum solid state fermentation cellulase-producing claimed in claim 1, is characterized in that, comprises the steps:
1) described bacterial strain Penicillium notatum WZUX01 line is transferred on PDA slant medium, cultivates 3 ~ 6 days at 25 ~ 35 ℃;
2) Penicillium notatum in the culture obtaining with water elution step 1), obtain spore suspension, the straw bran mass of access sterilizing, cultivates 5 ~ 7 days at 25 ~ 35 ℃, and wherein straw bran mass is made up of the composition of following weight ratio: rice straw powder: wheat bran: (NH 4) 2sO 4: water=4 ~ 36:8 ~ 48: 0 ~ 16: 24 ~ 80;
3) step 2) the solid-state culture that obtains adds distilled water to smash to pieces, and 35 ~ 45 ℃ of extracting at constant temperature 2 ~ 10h, filter to obtain enzyme liquid, obtain cellulase through lyophilize.
3. method according to claim 2, is characterized in that step 2) described in the spore amount of spore suspension be 1 × 10 12~ 9 × 10 12individual/mL, the inoculum size of described spore suspension: the envelope-bulk to weight ratio of spore suspension and straw bran mass is 1mL:1～3g.
4. method according to claim 2, is characterized in that step 2) described straw bran mass is made up of the composition of following weight ratio: rice straw powder: wheat bran: (NH 4) 2sO 4: water=8:32:8:52.
CN201210420183.3A 2012-10-29 2012-10-29 Penicillium, cellulose produced by fermenting penicillium in solid state and method thereof for preparing pomelo peel high-ester pectin Expired - Fee Related CN103087922B (en)

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