CN101117358A - Method for preparing mannosan - Google Patents
Method for preparing mannosan Download PDFInfo
- Publication number
- CN101117358A CN101117358A CNA2007101218600A CN200710121860A CN101117358A CN 101117358 A CN101117358 A CN 101117358A CN A2007101218600 A CNA2007101218600 A CN A2007101218600A CN 200710121860 A CN200710121860 A CN 200710121860A CN 101117358 A CN101117358 A CN 101117358A
- Authority
- CN
- China
- Prior art keywords
- supernatant liquor
- centrifugal
- enzymolysis
- solution
- obtains
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing mannan. The method has the steps as follow: 1) enzyme hydrolysis and cell wall breaking process: adding lysozyme into yeast cell wall suspending liquid for enzymolysis, centrifugation and collection at a temperature between 35 to 40 DEG C; 2) alkali process: putting the deposition achieved from step 1) into the NaOH solution, and stirring, and then centrifugally collecting the supernatant fluid at a temperature between 75 to 90 DEG C; 3) enzymatic treating or acid treating the supernatant fluid achieved from step 2), and uniformly mixing the treated supernatant fluid with CTAB solution of concentration expressed in percentage by quality and sodium borate-sodium acetate buffer solution of 0.3 to 0.8 percent, and laying aside for 24 to 48 hours, and centrifuging, and drying the supernatant fluid to achieve mannan. The method mentioned in the invention is simple in control, and can determine the preferred data range by considering every influencing factors to ensure the mannan product has high purity and production, and the product has the protein content and fat content less than 1%.
Description
Technical field
The present invention relates to a kind of preparing mannan.
Background technology
Yeast is a kind of unicellular fungi, is the human up to now microorganism the most extensive, that economic worth is the highest of using.International FDA approval cereuisiae fermentum is the microorganism of safety, widespread use in the production of drink and food.At present, China's year consumes about 2000 tons of yeast, produces about 3 tons waste yeast mud.Because yeast cells wall is the main by product of beer manufacturing industry, and now its main methods is sold as the roughage cheapness or directly discharging exactly, caused very big resource waste and pollution to environment.
Had a plurality of results of study to show, purified yeast cells wall has tangible immunologic enhancement to animal, promotes the growth effect but also show.Further evidence shows that its dominant mechanism is the β-1,3/1 that wherein contains, 6-dextran and mannosans have corresponding immunity and growth-promoting effect, and existing a large amount of experimental study results show β-1,3/1,6-dextran and mannosans are obvious results animal body function regulators.And along with the forbidding gradually of feeding antibiotic, safety, alternative biology feed additive become the industry main flow just gradually.
The relative yield of preparation chemical industry method of mannosans is lower, needs more unstable chemicals in the technical process, and safety coefficient is lower.At present, mainly adopt microbial fermentation to prepare mannosans in conjunction with the method for chemical extraction.Main process is a using gene engineering, filter out the higher bacterial strain of mannosans content the cell walls and it is cultivated from multiple barms, after treating its propagation, it is (shatter with yeast cell with ultrasonic technology to collect yeast cells wall, again with shatter yeast cell solution through cleaning and filtering repeatedly, remove bitter substance and impurity, carry out chemical treatment step).Chemically treated step broadly similar is with resuspended after the yeast cells wall cleaning that is obtained, under high-temperature and high-pressure conditions, make its fragmentation, with cold methanol extract white crude extract matter, with the organic substance Deproteinization and with acetate washing, get final product with alcohol extraction again.The step of Chu Liing can reduce the protein content in the products obtained therefrom like this, and content of mannan also guarantees to some extent; Shortcoming is exactly promptly to adopt ultrasonication in initial step, because the toughness of yeast cells wall makes broken wall efficient not high, the finished product yield is also on the low side.In the leaching process of mannosans, widely applied organic substance, thoroughly remove than difficulty and to suitability for industrialized production and challenge.β in traditional yeast cells wall-1,3/1 classifies mannosans as waste in the extraction process of 6-dextran, wastes huge.
Summary of the invention
The purpose of this invention is to provide a kind of preparing mannan.
Preparing mannan provided by the present invention comprises the steps:
1) enzymolysis broken wall treatment: add N,O-Diacetylmuramidase in the suspension with yeast cells wall, under 35～40 ℃, enzymolysis 30 minutes～1 hour, centrifugal collecting precipitation;
2) alkaline purification: the precipitation behind the enzymolysis that step 1) is obtained, in the NaOH of 0.5～1.5mol/L solution, under 75～90 ℃, carried out stir process 2～3 hours, centrifugal then collection supernatant liquor;
3) degreasing, remove albumen: with step 2) after the supernatant liquor that obtains carries out enzymatic treatment or acidic process, with the supernatant liquor that obtains and mass percentage concentration is that 12～20% CTAB solution and mass percentage concentration are that Sodium Tetraborate-sodium acetate buffer of 0.3～0.8% mixes, leave standstill 24～48h, centrifugal, supernatant liquor is carried out drying obtain mannosans;
Described enzymatic treatment is with described step 2) supernatant liquor that obtains adjusts pH value to neutral, and the adding neutral protease is at 35～40 ℃ of enzymolysis processing centrifugal collection supernatant liquor after 2～3 hours; Described acidic process is with described step 2) supernatant liquor that obtains regulates pH value to 5.5 and leaves standstill centrifugal collection supernatant liquor after 30 minutes～1 hour;
In the described method, in the described step 1), described hydrolysis temperature is preferably 37 ℃.
In the described method, in the described step 1), the addition of described N,O-Diacetylmuramidase is that (10000～60000U/g) yeast cells wall dry weights are preferably 1mg/g (20000U/g) yeast cells wall dry weight to 0.5～3mg/g; The concentration of described N,O-Diacetylmuramidase in the suspension of described yeast cells wall is 2000～12000U/ml, is preferably 4000U/ml.
In the described method, in the described step 1), 30 minutes～1 hour.Be preferably 30 minutes.
In the described method, described step 2) in, the concentration of described NaOH solution is preferably 1mol/L; The temperature of described alkaline purification is preferably 85 ℃, and the alkaline purification time is preferably 3 hours.
In the described method, described step 2) in, the product dry weight behind the described enzymolysis is 1g: 5-12ml with the mass/volume of described NaOH solution ratio, is preferably 1g: 5-6ml.
In the described method, in the described step 3), the mass percentage concentration of described CTAB solution is 16%; Described Sodium Tetraborate-sodium acetate buffer is that to contain the quality percentage composition be that 0.5% Sodium Tetraborate and quality percentage composition are the damping fluid of 0.5% sodium acetate; Described supernatant liquor and CTAB solution and Sodium Tetraborate-sodium acetate buffer are 20: 5: 10 mixed according to volume ratio.
In the described method, in the described step 3), the pH value of described acidic process is preferably pH5.5, and described adjusting pH value reagent is acetate or hydrochloric acid soln, is preferably acetate.
In the described method, in the described step 3), the adjusting pH value reagent in the described enzymatic treatment is that the hydrochloric acid soln of 0.1mol/L or quality percentage composition are 36% HAc solution; The addition of the neutral protease in the described enzymatic treatment is 3500～10500U/ml supernatant liquor, is preferably the 3500U/ml supernatant liquor.The hydrolysis temperature of described enzymatic treatment is 37 ℃, and enzymolysis time is 3 hours.
In the described method, comprise that also the precipitation that described step 1) is collected washes with water.
In the aforesaid method, described step 2) the centrifugal precipitation that obtains can be used for preparing water-insoluble beta-1,3/1 in, the 6-dextran, and concrete preparation method can be:
1. the concentration expressed in percentage by volume that is deposited in that acid treatment: with the step 2 in the aforesaid method) obtains is in 2～8% the acetic acid solution, under the room temperature condition, and stir process 2～3 hours, centrifugal collecting precipitation;
2. degreasing, remove albumen: with step 1. after the acid treatment the centrifugal precipitation that obtains with dehydrated alcohol, acetone resuspended successively agitator treating 30 minutes～1 hour respectively, static then 30～60 minutes, use the resuspended agitator treating of ether 30 minutes～1 hour then, left standstill then 12～24 hours, the centrifugal collecting precipitation drying obtains water-insoluble beta-1,3/1, the 6-dextran.
---alkaline purification cell walls---acid treatment (enzyme processing) supernatant liquor---concrete steps such as organic substance degreasing Deproteinization that the present invention adopts the enzymolysis yeast cells wall.The inventive method is simple to operate, considers each influence factor respectively, determines the preference data scope of parameter, makes the mannosans product of acquisition have higher purity (88.6%) and productive rate (18.5%), and protein content, lipid content are all less than 1% in the product.Adopt the enzyme treated cell wall and, can improve the broken ratio of cell walls greatly and reduce further chemically treated difficulty, help improving the finished product yield through the method for alkaline purification supernatant liquor.Method of the present invention-inferior processing industrial by-products---yeast cells wall can obtain mannosans and water-insoluble beta-1,3/1, the 6-dextran simultaneously.Be the immunomodulator of using in the suitable animal production practice through the animal experiment checking.
Description of drawings
Fig. 1 is the preparing mannan schema.
Fig. 2 is a water-insoluble beta-1,3/1, preparation method's schema of 6-dextran.
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
Percentage composition described in the following embodiment if no special instructions, is the quality percentage composition.
The preparation of embodiment 1, mannosans and water-insoluble beta-1,3/1, the preparation of 6-dextran
One, the preparation of mannosans
The preparation manipulation flow process of mannosans as shown in Figure 1, concrete steps are as described below:
1, the enzymolysis of yeast cells wall: with 100g yeast cells wall (product is available from Yichang Angel Yeast stock company), 100mg (20000U/mg) N,O-Diacetylmuramidase (is derived from ovalbumin, Cayman) and 500ml water mix, use at the uniform velocity agitator middling speed (Shanghai Sample Model Factory under 37 ℃ of conditions, galloping horse board adjustable speed agitator, 200～300 rev/mins) stir, churning time is 30 minutes, afterwards with 3000 rev/mins of (Anting Scientific Instrument Factory, Shanghai, DL6000B type refrigerated centrifuge) goes supernatant to obtain 95.3g precipitation in centrifugal 20 minutes, be the product behind the yeast cells wall enzymolysis.
2, alkaline purification: with in the adding of the product 95.3g behind the gained yeast cells wall enzymolysis in the step 1 500ml 1M NaOH solution (promptly being about the ratio interpolation of 1g: 5ml) according to the mass/volume ratio of product dry weight behind the broken wall and 1M NaOH solution, using at the uniform velocity under 85 ℃ of conditions, the agitator middling speed stirs (Shanghai Sample Model Factory, galloping horse board adjustable speed agitator, 200～300 rev/mins), churning time is 3 hours, centrifugal 20 minutes with 3600 rev/mins then, obtain supernatant liquor 510ml and continue on for extracting mannosans, (be used to extract water-insoluble beta-1 with the 29.5g precipitation, 3/1, the 6-dextran).
3, the centrifugal 510ml supernatant liquor that obtains after the described alkaline purification of step 2 is divided into two parts, every part of 255ml adjusts the pH value to neutral (volume increases to about 265ml) with the HCl solution of 0.1mol/L.
4, adjust the pH value to two portions of supernatant liquors of neutral with obtaining in the step 3, carry out following enzymatic treatment or acidic process respectively:
Enzymatic treatment: in supernatant liquor, add 26.5g (927.5KU, the 3500U/ml supernatant liquor) neutral protease (35000U/g), at the uniform velocity the agitator middling speed stirs, 3 hours treatment times, 37 ℃ of treatment temps, enzyme concn 0.1g/mL (3500U/ml) obtains pale brown look suspension liquid, it is pending that centrifugal (centrifugal speed 3000rpm, centrifugation time 10min) gets supernatant liquor.
Acidic process: supernatant liquor is regulated pH value to 5.5 with acetic acid solution (volumn concentration is 36%, commercial ice acetate) left standstill 60 minutes, get pale brown look suspension liquid, it is pending that centrifugal (centrifugal speed 2400rpm, centrifugation time 10min) gets supernatant liquor.
5, the supernatant liquor that step 4 enzymatic treatment or acidic process are obtained is 16% a CTAB solution and to contain the quality percentage composition be that 0.5% Sodium Tetraborate and quality percentage composition are that the Sodium Tetraborate-sodium acetate buffer of 0.5% sodium acetate is that 20: 5: 10 ratio is made into mixing solutions according to volume ratio with the quality percentage composition respectively, the static 48h of room temperature, centrifugal (centrifugal speed 3000rpm, centrifugation time 10min), get supernatant liquor respectively and carry out spraying drying, enzymatic treatment obtains 18.73g mannosans powder as a result, and acidic process obtains 18.36g mannosans powder.
6, adopt total reducing sugar assay method and thin-layer chromatography assay method to determine that the result shows that the purity of the mannosans powder that above-mentioned enzymatic treatment obtains is 88.49% to products therefrom purity; The purity of the mannosans powder that above-mentioned enzymatic treatment obtains is 88.71%; Yield adopts product amount/used cell walls amount to compare, and the result shows that the yield of the mannosans powder that above-mentioned enzymatic treatment obtains is 18.73%; The yield of the mannosans powder that above-mentioned acidic process obtains is 18.36%; Molecular weight is according to fluorescent method (Rinsten, L., T.Stenberg and R.Andersson.2003.Determination of β-glucan molecular weight using SEC with calcofluor detection in cereal extracts.CerealChemistry, 80 (4): 485-490) measure, be about 30KD.Structure detection is as follows through infrared spectra and nuclear magnetic resonance spectroscopy:
-「-α-D-G-(1，6)-α-D-G-(1，6)-α-D-G-(1，6)-α-D-G-
-
|1，2
α-D-G-(1,2)-(perhaps) α-D-G-(1,3)
The protein of product and lipid content adopt national standard method to carry out, and are respectively: protein content is 0.45%, lipid content is 0.33%.
Two, water-insoluble beta-1,3/1, the preparation of 6-dextran
In the above-mentioned steps one after the described alkaline purification of step 2 centrifugal the and precipitation that obtains of washing be used to prepare water-insoluble beta-1,3/1,6-dextran (the concrete operations flow process is asked for an interview Fig. 2).Concrete steps are as described below:
1, acid treatment: the centrifugal precipitated product that obtains after the gained alkaline purification in step-middle step 2 is washed with water three times, obtain the 28.7g precipitation.To precipitate adding 300ml volumn concentration is (promptly to add than the ratio that is about 1g/10ml according to the mass/volume of product dry weight after the alkaline purification and 4% acetic acid solution) in 4% acetic acid solution, use at the uniform velocity agitator (Shanghai Sample Model Factory at ambient temperature, galloping horse board adjustable speed agitator) middling speed (200～300 rev/mins) stirs, churning time is 2 hours, removes supernatant in centrifugal 20 minutes with 3000 rev/mins afterwards.Obtain the 22.2g precipitation, precipitation is washed with water three times.Precipitation is washed with water three times, obtain the 21.5g precipitation.
2, washing with alcohol: the amount of the product after the gained acid treatment in the step 3 (21.5g) according to 0.2-0.25g product (dry weight)/ml joined in the dehydrated alcohol, use at the uniform velocity agitator (Shanghai Sample Model Factory under room temperature (18～25 ℃) condition, galloping horse board adjustable speed agitator) middling speed (200～300 rev/mins) stirs, churning time is 1 hour, removed supernatant, collecting precipitation in centrifugal 20 minutes with 3000 rev/mins of speed afterwards.
3, washing with acetone: the amount of the precipitated product after the gained washing with alcohol in the step 4 according to 0.2-0.25g product (dry weight)/ml joined in the anhydrous propanone, room temperature condition is used at the uniform velocity agitator (Shanghai Sample Model Factory down, galloping horse board adjustable speed agitator) stirs (100～150 rev/mins) at a slow speed, churning time is 1 hour, removed supernatant, collecting precipitation in centrifugal 20 minutes with 2400 rev/mins of speed afterwards.
4, ether washing: in stink cupboard in join in anhydrous diethyl ether according to the amount of 0.2-0.25g product (dry weight)/ml the precipitated product after the gained acetone treatment in the step 5, room temperature condition is used at the uniform velocity agitator (Shanghai Sample Model Factory down, galloping horse board adjustable speed agitator) (100～150 rev/mins) stir at a slow speed, churning time is 1 hour, afterwards container cover lid back room temperature is left standstill 12～24 hours.Then, 2400 rev/mins were removed supernatant in centrifugal 20 minutes, and dry (air-flow dries up or evaporated under reduced pressure or lyophilize) obtains 20.1g water-insoluble beta-1,3/1, the 6-dextran.
5, products therefrom purity adopts total reducing sugar assay method and thin-layer chromatography assay method to determine that the result shows the above-mentioned water-insoluble beta that obtains-1,3/1, and it is 91.5% that the 6-dextran gets purity; Yield adopts product amount/used cell walls amount to compare, and the result shows the above-mentioned water-insoluble beta that obtains-1,3/1, and the yield of 6-dextran is 20.1%; Molecular weight is according to fluorescent method (Rinsten, L., T.Stenberg and R.Andersson.2003.Determination of β-glucanmolecular weight using SEC with calcofluor detection in cereal extracts.CerealChemistry, 80 (4): 485-490) measure, be about 800～850KD.Structure detection is as follows through infrared spectra and nuclear magnetic resonance spectroscopy:
-「-β-D-Glcp-(1，3)-β-D-Glcp-(1，3)-β-D-Glcp-(1，3)-β-D-GlcpNAc-
-
|1，6
β-D-Glcp-(1，6)-β-D-Quip-(1，6)
The protein of product and lipid content adopt national standard method to carry out, and are respectively: protein content is 1.15%, lipid content is 0.43%.
The preparation of embodiment 2, mannosans and water-insoluble beta-1,3/1, the preparation of 6-dextran
One, the preparation of mannosans
The preparation manipulation flow process of mannosans as shown in Figure 1, concrete steps are as described below:
1, the enzymolysis of yeast cells wall: 50g yeast cells wall, 100mg (20000U/mg) N,O-Diacetylmuramidase (are derived from ovalbumin, Cayman) and 250ml water mix, use at the uniform velocity agitator middling speed (Shanghai Sample Model Factory under 35 ℃ of conditions, galloping horse board adjustable speed agitator, 200～300 rev/mins) stir, churning time is 60 minutes, afterwards with 3000 rev/mins of (Anting Scientific Instrument Factory, Shanghai, DL6000B type refrigerated centrifuge) goes supernatant to obtain 47.80g precipitation in centrifugal 25 minutes, be the product behind the yeast cells wall enzymolysis.
2, alkaline purification: the product 47.80g behind the gained yeast cells wall enzymolysis in the step 1 is added (promptly the mass/volume ratio according to product dry weight behind the broken wall and NaOH solution adds for the ratio of 1g: 10ml) in the 500ml 0.5MNaOH solution, using at the uniform velocity under 75 ℃ of conditions, the agitator middling speed stirs (Shanghai Sample Model Factory, galloping horse board adjustable speed agitator, 200～300 rev/mins), churning time is 3 hours, centrifugal 20 minutes with 3600 rev/mins then, obtain supernatant liquor 510ml and continue on for extracting mannosans, (be used to extract water-insoluble beta-1 with the 12.6g precipitation, 3/1, the 6-dextran).
3, the centrifugal 510ml supernatant liquor that obtains after the described alkaline purification of step 2 is divided into two parts, every part of 255ml adjusts the pH value to neutral (volume increases to about 265ml) with the HCl solution of 0.1mol/L.
4, adjust the pH value to two portions of supernatant liquors of neutral with obtaining in the step 3, carry out following enzymatic treatment or acidic process respectively:
Enzymatic treatment: in supernatant liquor, add 26.5g (927.5KU, the 3500U/ml supernatant liquor) neutral protease (35000U/g), at the uniform velocity the agitator middling speed stirs, 2 hours treatment times, 35 ℃ of treatment temps, enzyme concn 0.1g/mL (3500U/ml) obtains pale brown look suspension liquid, it is pending that centrifugal (centrifugal speed 3000rpm, centrifugation time 10min) gets supernatant liquor.
Acidic process: supernatant liquor is regulated pH value to 5.0 with acetic acid solution (volumn concentration is 36%, commercial ice acetate) left standstill 30 minutes, get pale brown look suspension liquid, it is pending that centrifugal (centrifugal speed 2400rpm, centrifugation time 10min) gets supernatant liquor.
5, the supernatant liquor that step 4 enzymatic treatment or acidic process are obtained is 12% a CTAB solution and to contain the quality percentage composition be that 0.3% Sodium Tetraborate and quality percentage composition are that the Sodium Tetraborate-sodium acetate buffer of 0.3% sodium acetate is that 20: 5: 10 ratio is made into mixing solutions according to volume ratio with the quality percentage composition respectively, the static 48h of room temperature, centrifugal (centrifugal speed 3000rpm, centrifugation time 10min), get supernatant liquor respectively and carry out spraying drying, enzymatic treatment obtains 8.82g mannosans powder as a result, and acidic process obtains 8.48g mannosans powder.
6, products therefrom purity adopts total reducing sugar assay method and thin-layer chromatography assay method to determine that the result shows that the purity of the mannosans powder that above-mentioned acidic process obtains is 85.31%; The purity of the mannosans powder that above-mentioned enzymatic treatment obtains is 85.52%; Yield adopts product amount/used cell walls amount to compare, and the result shows that the yield of the mannosans powder that above-mentioned enzymatic treatment obtains is 17.63%, and the yield of the mannosans powder that above-mentioned acidic process obtains is 16.95%; Molecular weight is according to fluorescent method (Rinsten, L., T.Stenberg and R.Andersson.2003.Determination of β-glucan molecular weight using SEC with calcofluor detection in cereal extracts.CerealChemistry, 80 (4): 485-490) measure, be about 30KD.Structure detection is as follows through infrared spectra and nuclear magnetic resonance spectroscopy:
-「-α-D-G-(1，6)-α-D-G-(1，6)-α-D-G-(1，6)-α-D-G-
-
|1，2
α-D-G-(1,2)-(perhaps) α-D-G-(1,3)
The protein of product and lipid content adopt national standard method to carry out, and are respectively: protein content is 0.54%, lipid content is 0.39%.
Two, water-insoluble beta-1,3/1, the preparation of 6-dextran
The method that 12.6g centrifugal and that washing obtains precipitates according to embodiment 1 after the described alkaline purification of step 2 in the above-mentioned steps one prepares water-insoluble beta-1,3/1,6-dextran (the concrete operations flow process is asked for an interview Fig. 2), with the water-insoluble beta-1 that obtains, 3/1, the 6-dextran adopts total reducing sugar assay method and thin-layer chromatography assay method to determine that the result shows the above-mentioned water-insoluble beta that obtains-1,3/1, it is 89.51% that the 6-dextran gets purity; Yield adopts product amount/used cell walls amount to compare, and the result shows the above-mentioned water-insoluble beta that obtains-1,3/1, and the yield of 6-dextran is 17.31%; Molecular weight is to improve fluorescent method (Rinsten, L., T.Stenberg and R.Andersson.2003.Determination of β-glucanmolecular weight using SEC with calcofluor detection in cereal extracts.CerealChemistry, 80 (4): 485-490) measure, be about 800～850KD.Structure detection is as follows through infrared spectra and nuclear magnetic resonance spectroscopy:
-「-β-D-Glcp-(1，3)-β-D-Glcp-(1，3)-β-D-Glcp-(1，3)-β-D-GlcpNAc-
-
|1，6
β-D-Glcp-(1，6)-β-D-Quip-(1，6)
The protein of product and lipid content adopt national standard method to carry out, and are respectively: protein content is 1.14%, lipid content is 0.52%.
The preparation of embodiment 3, mannosans and water-insoluble beta-1,3/1, the preparation of 6-dextran
One, the preparation of mannosans
The preparation manipulation flow process of mannosans as shown in Figure 1, concrete steps are as described below:
1, the enzymolysis of yeast cells wall: 100g yeast cells wall, 300mg (20000U/mg) N,O-Diacetylmuramidase (are derived from ovalbumin, Cayman) and 500ml water mix, use at the uniform velocity agitator middling speed (Shanghai Sample Model Factory under 40 ℃ of conditions, galloping horse board adjustable speed agitator, 200～300 rev/mins) stir, churning time is 30 minutes, afterwards with 3000 rev/mins of (Anting Scientific Instrument Factory, Shanghai, DL6000B type refrigerated centrifuge) goes supernatant to obtain 95.4g precipitation in centrifugal 20 minutes, be the product behind the yeast cells wall enzymolysis.
2, alkaline purification: will (promptly be about the ratio interpolation of 1g: 10ml) in the adding of the product 95.4g behind the gained yeast cells wall enzymolysis in the step 1 1000ml 1.0MNaOH solution according to the mass/volume ratio of product dry weight behind the broken wall and NaOH solution, using at the uniform velocity under 90 ℃ of conditions, the agitator middling speed stirs (Shanghai Sample Model Factory, galloping horse board adjustable speed agitator, 200～300 rev/mins), churning time is 2.5 hours, centrifugal 20 minutes with 3600 rev/mins then, obtain supernatant liquor 510ml and continue on for extracting mannosans, (be used to extract water-insoluble beta-1 with the 28.7g precipitation, 3/1, the 6-dextran).
3, the centrifugal 510ml supernatant liquor that obtains after the described alkaline purification of step 2 is divided into two parts, every part of 255ml adjusts the pH value to neutral (volume increases to about 265ml) with the HCl solution of 0.1mol/L.
4, adjust the pH value to two portions of supernatant liquors of neutral with obtaining in the step 3, carry out following enzymatic treatment or acidic process respectively:
Enzymatic treatment: in supernatant liquor, add 79.5g (2782.5KU, the 10500U/ml supernatant liquor) neutral protease (35000U/g), at the uniform velocity the agitator middling speed stirs, 2 hours treatment times, 40 ℃ of treatment temps, enzyme concn 0.3g/mL (10500U/ml) obtains pale brown look suspension liquid, it is pending that centrifugal (centrifugal speed 3000rpm, centrifugation time 10min) gets supernatant liquor.
Acidic process: supernatant liquor is regulated pH value to 5.5 with acetic acid solution (volumn concentration is 36%) left standstill 40 minutes, get pale brown look suspension liquid, it is pending that centrifugal (centrifugal speed 2400rpm, centrifugation time 10min) gets supernatant liquor.
5, the supernatant liquor that step 4 enzymatic treatment or acidic process are obtained is 20% a CTAB solution and to contain the quality percentage composition be that 0.8% Sodium Tetraborate and quality percentage composition are that the Sodium Tetraborate-sodium acetate buffer of 0.8% sodium acetate is that 20: 5: 10 ratio is made into mixing solutions according to volume ratio with the quality percentage composition respectively, the static 48h of room temperature, centrifugal (centrifugal speed 3000rpm, centrifugation time 10min), get supernatant liquor respectively and carry out spraying drying, enzymatic treatment obtains 17.25g mannosans powder as a result, and acidic process obtains 17.13g mannosans powder.
6, products therefrom purity adopts total reducing sugar assay method and thin-layer chromatography assay method to determine that the result shows that the purity of the mannosans powder that above-mentioned acidic process obtains is 86.63%; The purity of the mannosans powder that above-mentioned enzymatic treatment obtains is 86.47%; Yield adopts product amount/used cell walls amount to compare, and the result is that the yield of the mannosans powder that shows that above-mentioned enzyme process obtains is 17.25%, and the acid system yield is 17.13%; Molecular weight is according to improving fluorescent method (Rinsten, L., T.Stenberg and R.Andersson.2003.Determination of β-glucan molecular weight usingSEC with calcofluor detection in cereal extracts.Cereal Chemistry, 80 (4): 485-490) measure, be about 30KD.Structure detection is as follows through infrared spectra and nuclear magnetic resonance spectroscopy:
-「-α-D-G-(1，6)-α-D-G-(1，6)-α-D-G-(1，6)-α-D-G-
-
|1，2
α-D-G-(1,2)-(perhaps) α-D-G-(1,3)
The protein of product and lipid content adopt national standard method to carry out, and are respectively: protein content is 0.41%, lipid content is 0.39%.
Two, water-insoluble beta-1,3/1, the preparation of 6-dextran
The method that 28.7g centrifugal and that washing obtains precipitates according to embodiment 1 after the described alkaline purification of step 2 in the above-mentioned steps one prepares water-insoluble beta-1,3/1,6-dextran (the concrete operations flow process is asked for an interview Fig. 2), with the water-insoluble beta-1 that obtains, 3/1, the 6-dextran adopts total reducing sugar assay method and thin-layer chromatography assay method to determine that the result shows the above-mentioned water-insoluble beta that obtains-1,3/1, it is 90.37% that the 6-dextran gets purity; Yield adopts product amount/used cell walls amount to compare, and the result shows the above-mentioned water-insoluble beta that obtains-1,3/1, and the yield of 6-dextran is 18.54%; Molecular weight is according to improving fluorescent method (Rinsten, L., T.Stenberg and R.Andersson.2003.Determination of β-glucan molecular weight using SEC with calcofluor detection in cereal extracts.CerealChemistry, 80 (4): 485-490) measure, be about 800～850KD.Structure detection is as follows through infrared spectra and nuclear magnetic resonance spectroscopy:
-「-β-D-Glcp-(1，3)-β-D-Glcp-(1，3)-β-D-Glcp-(1，3)-β-D-GlcpNAc-
-
|1，6
β-D-Glcp-(1，6)-β-D-Quip-(1，6)
The protein of product and lipid content adopt national standard method to carry out, and are respectively: protein content is 1.04%, lipid content is 0.40%.
Claims (10)
1. a preparing mannan comprises the steps:
1) enzymolysis processing: add N,O-Diacetylmuramidase in the suspension with yeast cells wall, under 35～40 ℃, enzymolysis, centrifugal collecting precipitation;
2) alkaline purification: the precipitation behind the enzymolysis that step 1) is obtained, in the NaOH of 0.5～1.5mol/L solution, under 75～90 ℃, carried out stir process 2～3 hours, centrifugal then collection supernatant liquor;
3) with step 2) after the supernatant liquor that obtains carries out enzymatic treatment or acidic process, with the supernatant liquor that obtains and mass percentage concentration is that 12～20% CTAB solution and mass percentage concentration are that Sodium Tetraborate-sodium acetate buffer of 0.3～0.8% mixes, leave standstill 24～48h, centrifugal, supernatant liquor is carried out drying obtain mannosans;
Described enzymatic treatment is with described step 2) supernatant liquor that obtains adjusts pH value to neutral, and the adding neutral protease is at 35～40 ℃ of enzymolysis processing centrifugal collection supernatant liquor after 2～3 hours;
Described acidic process is with described step 2) supernatant liquor that obtains regulates pH value to 5.5 and leaves standstill centrifugal collection supernatant liquor after 30～60 minutes.
2. method according to claim 1 is characterized in that: in the described step 1), described hydrolysis temperature is 37 ℃.
3. method according to claim 2 is characterized in that: in the described step 1), the addition of described N,O-Diacetylmuramidase is 10000～60000U/g yeast cells wall dry weight, is preferably 20000u/g yeast cells wall dry weight; The concentration of described N,O-Diacetylmuramidase in the suspension of described yeast cells wall is 2000～12000U/ml, is preferably 4000U/ml.
4. method according to claim 3 is characterized in that: in the described step 1), described enzymolysis time is 30～60 minutes, is preferably 30 minutes.
5. method according to claim 4 is characterized in that: described step 2), the concentration of described NaOH solution is 1mol/L; The temperature of described alkaline purification is 85 ℃, and the alkaline purification time is 3 hours.
6. method according to claim 5 is characterized in that: described step 2), the product dry weight behind the described enzymolysis is 1g: 5～12ml with the mass/volume of described NaOH solution ratio, is preferably 1g: 5-6ml.
7. method according to claim 6 is characterized in that: in the described step 3), the mass percentage concentration of described CTAB solution is 16%; Described Sodium Tetraborate-sodium acetate buffer is that to contain the quality percentage composition be that 0.5% Sodium Tetraborate and quality percentage composition are the damping fluid of 0.5% sodium acetate; Described supernatant liquor and CTAB solution and Sodium Tetraborate-sodium acetate buffer are 20: 5: 10 mixed according to volume ratio.
8. method according to claim 7 is characterized in that: in the described step 3), the pH value of described acidic process is pH5.5, and described adjusting pH value reagent is acetic acid solution or hydrochloric acid soln, is preferably acetic acid solution.
9. method according to claim 8 is characterized in that: in the described step 3), the adjusting pH value reagent in the described enzymatic treatment is that the hydrochloric acid soln of 0.1mol/L or quality percentage composition are 36% HAc solution; The addition of the neutral protease in the described enzymatic treatment is 3500～10500U/ml supernatant liquor, is preferably the 3500U/ml supernatant liquor; The hydrolysis temperature of described enzymatic treatment is 37 ℃, and enzymolysis time is 3 hours.
10. according to any described method among the claim 1-9, it is characterized in that: in the described method, comprise that also the precipitation that described step 1) is collected washes with water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2007101218600A CN100572398C (en) | 2007-09-17 | 2007-09-17 | A kind of preparing mannan |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2007101218600A CN100572398C (en) | 2007-09-17 | 2007-09-17 | A kind of preparing mannan |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101117358A true CN101117358A (en) | 2008-02-06 |
| CN100572398C CN100572398C (en) | 2009-12-23 |
Family
ID=39053643
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2007101218600A Expired - Fee Related CN100572398C (en) | 2007-09-17 | 2007-09-17 | A kind of preparing mannan |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100572398C (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570769A (en) * | 2008-04-29 | 2009-11-04 | 安琪酵母股份有限公司 | Yeast glucan and mannan and production method thereof |
| CN101407559B (en) * | 2008-11-25 | 2011-01-26 | 天津科技大学 | Method for rapidly preparing yeast beta-1,3-dextran |
| WO2011054255A1 (en) * | 2009-11-06 | 2011-05-12 | 安琪酵母股份有限公司 | Method for preparing yeast mannose protein product |
| CN103757071A (en) * | 2014-01-24 | 2014-04-30 | 侯梦斌 | Method for producing zymosan |
| CN111172219A (en) * | 2020-02-17 | 2020-05-19 | 武汉轻工大学 | Mannan, preparation method and application thereof, and hot dry noodles |
| CN112760346A (en) * | 2020-12-29 | 2021-05-07 | 唐山拓普生物科技有限公司 | Method for extracting yeast cell wall polysaccharide with high immunocompetence |
-
2007
- 2007-09-17 CN CNB2007101218600A patent/CN100572398C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570769A (en) * | 2008-04-29 | 2009-11-04 | 安琪酵母股份有限公司 | Yeast glucan and mannan and production method thereof |
| CN101407559B (en) * | 2008-11-25 | 2011-01-26 | 天津科技大学 | Method for rapidly preparing yeast beta-1,3-dextran |
| WO2011054255A1 (en) * | 2009-11-06 | 2011-05-12 | 安琪酵母股份有限公司 | Method for preparing yeast mannose protein product |
| CN103757071A (en) * | 2014-01-24 | 2014-04-30 | 侯梦斌 | Method for producing zymosan |
| CN103757071B (en) * | 2014-01-24 | 2015-09-30 | 侯梦斌 | A kind of method of producing zymosan |
| CN111172219A (en) * | 2020-02-17 | 2020-05-19 | 武汉轻工大学 | Mannan, preparation method and application thereof, and hot dry noodles |
| CN112760346A (en) * | 2020-12-29 | 2021-05-07 | 唐山拓普生物科技有限公司 | Method for extracting yeast cell wall polysaccharide with high immunocompetence |
| CN112760346B (en) * | 2020-12-29 | 2022-09-09 | 唐山拓普生物科技有限公司 | Method for extracting yeast cell wall polysaccharide with high immunocompetence |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100572398C (en) | 2009-12-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100572398C (en) | A kind of preparing mannan | |
| CN102060934B (en) | Enzymatic extraction method for auricularia auricula polysaccharides | |
| CN101117357B (en) | Method for preparing water-soluble beta-1,3/1,6-dextran | |
| CN100510094C (en) | Production method of konjak mannose using cellulase | |
| Sun et al. | High-efficiency production of Tremella aurantialba polysaccharide through basidiospore fermentation | |
| CN103834713B (en) | A kind of extracting method of tigogenin | |
| CN105431534A (en) | beta-1,3-glucanase, polynucleotide, recombinant vector, transformant, production method for beta-1,3-glucanase, enzyme preparation, and production method for paramylon having reduced molecular weight | |
| CN104846035B (en) | A kind of method that sesbania enzyme process prepares galactomannan oligosaccharide | |
| CN112760311B (en) | Enzyme solution with better enzyme activity ratio of beta-mannase to alpha-galactosidase, and preparation method and application thereof | |
| CN101897431A (en) | Method for simultaneously preparing yeast extract and beta-1,3-glucan | |
| CN108948227A (en) | A kind of method that high-voltage pulse extracts okra pectin | |
| CN101117356B (en) | Method for preparing water-insoluble beta-1,3/1,6-dextran | |
| PT2766471T (en) | Method for the continuous production of cellulases by a filamentous fungus using a carbon substrate obtained from an acid pretreatment | |
| Wang et al. | Saccharogenic refining of Ginkgo biloba leaf residues using a cost-effective enzyme cocktail prepared by the fungal strain A32 isolated from ancient ginkgo biloba tree | |
| CN115073621B (en) | Preparation method of arabinoxylan | |
| CN1322141C (en) | Method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis | |
| CN110093389A (en) | The fermentation method for producing of instant xanthan gum | |
| CN105274075A (en) | Application of aspergillus niger in preparation of beta-fructofuranosidase | |
| Benkő et al. | Thermoascus aurantiacus CBHI/Cel7A production in Trichoderma reesei on alternative carbon sources | |
| US20220186272A1 (en) | Strain of trichoderma reesei and culture method and use thereof | |
| CN110771824A (en) | Gracilaria lemaneiformis dietary fiber and preparation method thereof | |
| Kyslíková et al. | Cell growth and cellulase production in Trichoderma viride on microcrystalline cellulose | |
| CN104172098B (en) | Technique for preparing soy sauce from palm pulp | |
| AU2020102037A4 (en) | A method of efficiently increasing the alpha-glucosidase inhibitor content in fresh mulberry leaves by the solid-state fermentation | |
| CN115851663B (en) | Method for improving sugar and pectin yield of beet root residue preparation by compounding enzyme system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20091223 Termination date: 20160917 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |