CN104152592A - Xylose for culturing rhizopus oryzae and preparation method thereof - Google Patents
Xylose for culturing rhizopus oryzae and preparation method thereof Download PDFInfo
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- CN104152592A CN104152592A CN201410250130.0A CN201410250130A CN104152592A CN 104152592 A CN104152592 A CN 104152592A CN 201410250130 A CN201410250130 A CN 201410250130A CN 104152592 A CN104152592 A CN 104152592A
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Abstract
The invention discloses a xylose for culturing rhizopus oryzae and a preparation method thereof. The preparation method comprises the following steps of (1) smashing zizania latifolia wastes, then, extracting at the temperature of 80-90 DEG C for 1-2 hours by using 70-90% ethanol, and filtering to obtain a solid matter; (2) extracting the solid matter at the temperature of 60-75 DEG C for 1-2 hours by using methanoic acid, filtering, carrying out solid-liquid separation, carrying out reduced-pressure distillation on the obtained liquid, and recovering the methanoic acid to obtain a solid matter, namely the xylose. According to the invention, the zizania latifolia wastes are used as raw materials, so that not only can the zizania latifolia wastes be recycled, but also the xylose can be efficiently extracted without adding an additional catalyst in a xylose extraction process, the reaction condition is mild, the energy consumption is low, the recovery rate of the xylose is high, no additional catalysis byproducts are generated in the reaction process, and no any pollution and waste are generated in the whole process.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of for cultivating wood sugar of Rhizopus oryzae thalline and preparation method thereof.
Background technology
Along with day by day increasing the weight of of oil crisis, the raw material of Chemical industry worldwide is just progressively changed to biomass resource by petroleum resources.The raw material of biochemical industry is prepared mainly with grain greatly at present starch and sugar are main, cause the situation of " strive grain, strive ground with grain with people (poultry) " day by day serious.Biomass monose, as the main raw material of biochemical industry, has become the bottleneck of restriction Chinese biological chemical industry development.
Lignocellulose comprises waste and residuum (as agricultural crop straw, husk, wheat bran, bagasse etc.), forest (cork and hardwood) and forestry processing waste, the careless class etc. of agriculture production, and annual production is up to more than 1,500 hundred million tons.Through hydrolysis and saccharification, cellulosic material can be converted into monose material, as platform, through chemistry route, can change into fuel oil, combustion gas and chemical; By biocatalysis, can change into large leavened prod and liquid biofuel; Utilize monose crystal that crystallization purifying technology obtains important industrial raw material especially.
In lignocellulose, hemicellulose, content of cellulose are relatively high, can reach the more than 50% of material, but due to content of lignin more (10～20%), its package action to Mierocrystalline cellulose etc., makes lignocellulose in treating processes, difficult.Wherein the existence of xylogen becomes the major obstacles that lignocellulose resource makes full use of, and reach the object of effectively utilizing lignocellulose resource, reduces the cost of processing, and must first solve the degradation problem of xylogen.The method that lignocellulose is processed is sorted out can be divided into Physical, chemical method, physical-chemical method and biological process etc., mainly contains: mechanical disintegration (ball milling, vibration mill etc.), high-temperature liquid state method, high-energy radiation, microwave treatment, pyrolytic decomposition, steam blasting procedure, the fine blasting procedure of ammonia, CO
2explosion, wet oxidation, acid-hydrolysis method, alkaline purification method, superoxide method and organic solvent method etc.Multiplex chemical Treatment in research in early days, environmental pollution is large, immediate development relatively clean production method, comprise the quick-fried method of steaming, organic solvent extraction method etc., energy consumption is larger relatively in preparation process for these class methods, after all, be also that the tough and tensile internal structure of its lignocellulose has increased separated difficulty, simultaneously, the sugar obtaining in order to upper method, if wood sugar, glucose are in carrying out the process of biological utilisation, also needs to carry out detoxification treatment.As the patent of invention of the applications such as Huang He " method of control growthform of filamentous fungi during fermentation process " (grant number: CN102212484B) explicitly point out, the hydrolyzed solution content that lignocellulose hydrolysis obtains is complicated, composition is more, and hydrolysis sugar liquid will just can be used for the cultivation of thalline and the fermentation of fumaric acid after detoxification treatment.
The utilization of resources for wild rice stem waste at present also rarely has report, the present invention proposes and the present invention proposes specially the technique of extracting specially sugared resource for wild rice stem waste.
Summary of the invention
The object of this invention is to provide a kind of use wild rice stem waste is basic raw material, and high efficiency extraction is for cultivating the wood sugar of Rhizopus oryzae thalline.
Another object of the present invention is to provide the preparation method of this wood sugar, and the wood sugar extraction efficiency that the method obtains is high, and cost is low.
A further object of the invention is to provide the cultural method that utilizes this wood sugar to cultivate Rhizopus oryzae thalline.
The present invention can solve sugared Resource Access and subsequent development in lignocellulose resource and utilize technical deficiency.
The object of the invention is to realize in the following manner:
For cultivating a wood sugar for Rhizopus oryzae thalline, this wood sugar prepares by the following method:
1) after wild rice stem waste is pulverized, by concentration, be 70～90% ethanol lixiviate 1～2h under 80～90 ℃ of conditions of temperature, filter, obtain solid substance;
2) by formic acid lixiviate 1～2h under 60～75 ℃ of conditions of temperature for solid substance, filter, solid-liquid separation, carries out underpressure distillation by the liquid obtaining, and reclaims formic acid, obtains solid substance, is wood sugar.Excellent formic acid is lixiviate 2h under temperature 70 C condition.
Described wild rice stem waste is wild rice stem leaf sheath, blade and first sheathing leaf.
The preparation method of above-mentioned wood sugar comprises the following steps:
1) after wild rice stem waste is pulverized, by concentration, be 70～90% ethanol lixiviate 1～2h under 80～90 ℃ of conditions of temperature, filter, obtain solid substance;
2) by formic acid lixiviate 1～2h under 60～75 ℃ of conditions of temperature for the solid substance obtaining, filter, solid-liquid separation, carries out underpressure distillation by the liquid obtaining, and reclaims formic acid, obtains solid substance.Excellent formic acid is lixiviate 2h under temperature 70 C condition.
In the method, described wild rice stem waste can, first 50 ℃ of oven dry of spending the night, be crushed to the particle that particle diameter is 10～20mm.
The solid-to-liquid ratio adopting in alcohol steep process is 1:5～20, preferably 1:10.
The alcohol concn preferably adopting in alcohol steep process is 80～90%.
In aforesaid method, and step " 1) " in temperature, be preferably lixiviate under 90 ℃ of conditions.
The solid-to-liquid ratio that step " 2) " is used formic acid lixiviate to adopt is 1:5～20, is preferably 1:10～20, most preferably 1:10.
Step " 2) " in, preferably by formic acid lixiviate under temperature 70 C condition for solid substance.
A kind of method of utilizing wood sugar that aforesaid method prepares to cultivate Rhizopus oryzae thalline comprises the following steps: wood sugar is carried out to classification cultivation to Rhizopus oryzae, cultivate and divide 2～4 grades, preferably 3 grades, control xylose concentration at 10～15g/L for every grade, total sugar concentration is controlled at 30～50g/L, control peptone 2.0g/L, potassium primary phosphate 0.2g/L, MgSO simultaneously
47H
2o0.2g/L, CaCO
36.0g/L, the every bottled 50ml seed liquor of 250ml shaking flask, 150r/min, 30 ℃ of reactions, cultivate 8～12h for every grade, after cultivation finishes, obtain uniform spherical Rhizopus oryzae thalline.
Above-mentioned solid-to-liquid ratio all refers to mass volume ratio (g/ml).
Formic acid used in the present invention is commercial goods formic acid, and its mass concentration is preferably 86.24%.
The present invention dilutes the wood sugar obtaining, and add therein (a kind of thalline that produces fumaric acid, a kind of thalline for lactic acid producing cultivated), for cultivating Rhizopus oryzae thalline, cultivate 20～40h, can obtain uniformly, diameter is the spherical thalline of 1mm left and right.The fumaric acid that the fermentation of this thalline obtains and lactic acid production all can reach 53.2 and 57.4g/L (80g/L initial glucose concentration), constituent of chitosan in thalline is analyzed to discovery simultaneously, wherein constituent of chitosan can be up to 0.164g/g, than glucose pure culture, increased more than 27%, as can be seen here, utilize this technique can efficiently prepare wood sugar, the wood sugar simultaneously obtaining can carry out the cultivation of Rhizopus oryzae thalline, and the thalline chitosan content of cultivation is also relatively high.Wherein, the quality determining method of the chitosan in thalline is according to grant number: in the patent of invention of CN102212484B " method of control growthform of filamentous fungi during fermentation process ", disclosed method is carried out.
Compared with the prior art, beneficial effect of the present invention: use wild rice stem waste as raw material, not only realized the reproducible utilization of wild rice stem waste, simultaneously in extracting the process of wood sugar, do not need to add extra catalyzer and get final product high efficiency extraction wood sugar, reaction conditions is gentle, energy consumption is lower, the rate of recovery of wood sugar has reached 90～95%, does not also produce extra catalysis by product in reaction process, and formic acid can be through Distillation recovery recycling, therefore, in whole process, do not produce any pollution and waste, reduced production cost, and the wood sugar safety non-toxic obtaining.Meanwhile, the wood sugar obtaining is used for cultivating Rhizopus oryzae thalline, can obtain uniform spherical thalline, and in thalline, chitosan content can be up to 16.4%.
Below example is further described the present invention by experiment:
1, the impact of ethanol on delignification in wild rice stem
(1) impact of alcohol concn on delignification
Get and dry wild rice stem waste (Jiaobai leaf, blade and leaf sheath) 5g, add in 50ml ethanolic soln W:V=1:10; Alcohol concn is respectively 70,80,90,100%, and temperature of reaction is at 90 ℃; Extract 1h, underpressure distillation, dries, and surveys its dry weight, lignin removing rate, and its result is as shown in table 1.A large amount of bibliographical informations utilize alcohol catalysis to prepare the technique of fiber, but xylogen is the complicated indefinite form superpolymer that is difficult to saccharification, it is the component of difficult decomposition in plant materials, and form protective layer around at Mierocrystalline cellulose, affect saccharification of cellulose, xylogen is a kind of aggregate simultaneously, in structure, there are many polar groups, especially more hydroxyl, caused in very strong molecule and intermolecular hydrogen bond, therefore, substantially alcohol catalysis prepare the technique of fiber all will be under comparatively strict condition, as add simultaneously catalytic temperature higher (being advisable for >180 ℃) of acid base catalysator, although these conditions can make xylogen depart from, but also there is degraded in hemicellulose and Mierocrystalline cellulose.
But for wild rice stem waste, contriver but can extract xylogen by choice for use ethanol, and can be chosen in the extraction that realizes xylogen at lower temperature.As can be seen from the table, when alcohol concn is 90%, xylogen removal effect is best, has reached 76%, and the continuation rising along with alcohol concn, is unfavorable for the removal of xylogen on the contrary; As can be seen from the table, the residue content of waste all changes simultaneously, and it may be that in waste, part pectin, protein matter also extract along with solution, and glucide there is no how many losses.
Table 1
(2) impact of temperature of reaction on delignification
Get and dry wild rice stem waste 5g, add in 50ml ethanolic soln, i.e. W:V=1:10 (g/ml); Temperature of reaction is respectively 70,80, and 90,100 ℃, alcohol concn is 90%; Extract 1h, underpressure distillation, dries, and surveys its dry weight, lignin removing rate, and it is as shown in the table for its result:
Table 2
As can be seen from Table 2, temperature not only has material impact to delignification, the removal of other material is also had to vital role simultaneously, when temperature is lower, the more difficult infiltration internal structure of water molecules, the removal effect of other material is not good yet, along with the rising of temperature, extraction effect is originally better, but temperature is elevated to 90 ℃ when above, although lignin removing rate is also increasing, clearance changes little, consider energy consumption problem, select 90 ℃.
2, the research that formic acid extracts wood sugar
Conventionally lignocellulosic material directly takes formic acid to be hydrolyzed, hydrolysis effect is poor, must add certain density extra catalyzer example hydrochloric acid, sulfuric acid etc., and the inventive method is before processing with formic acid, only with ethanol, carry out preliminary treatment, without any catalyzer of extra interpolation.
(1) impact that temperature of reaction is extracted wood sugar
Get the wild rice stem waste 10.0g after alcohol extraction, add formic acid 100ml, i.e. W:V=1:10 (g/ml), differential responses temperature (50,60,70,80 ℃), after reaction 2.0h, sampling detects wood sugar, glucose concn, and calculate recovery rate.Get 5mL vacuum concentration at every turn, finally use 20mL water extraction sugar, adopt HPLC to detect.
Table 3
As can be seen from the table, in formic acid, reaction conditions is comparatively gentle, when temperature is too low, wood sugar hydrolysis efficiency is lower, rising along with temperature, wood sugar hydrolysis effect is originally better, when temperature is 70 ℃, the Xylose Content obtaining is relatively high, but the further rising along with temperature, wood sugar starts to degrade, thereby be embodied in xylose concentration and the rate of recovery is relatively low, now, from glucose content, within extracting 2h in early stage, temperature is higher, glucose concn is higher, visible, rising along with temperature, act on stronger, glucose is more easily degraded.
(2) impact of reaction times on wood sugar extraction
Get the wild rice stem waste 10g after alcohol extraction, add formic acid 100ml, i.e. W:V=1:10 (g/ml), 70 ℃ of temperature of reaction, reaction different time, sampling detects wood sugar, glucose concn, and calculate recovery rate.Get 5mL vacuum concentration at every turn, finally use 20mL water extraction sugar, adopt HPLC to detect.
Table 4
As can be seen from the table, reaction times more in short-term, reacts insufficient, and wood sugar degradation efficiency is now lower, increase along with the reaction times, formic acid acts on starting material comparatively fully, so wood sugar degradation efficiency is more and more higher, but along with the further increase of time, find, the wood sugar rate of recovery is more and more lower, and its reason may be along with hydrolysis time lengthens, and has wood sugar and continues to be degraded to the side reaction generations such as furfural, the de-acetyl generation of hemicellulose carboxylic acid.Meanwhile, along with the increase of hydrolysis time, the Mierocrystalline cellulose in wheat straw can be hydrolyzed into glucose, therefore wood sugar accounts for the per-cent of total reducing sugars, can continue to reduce.
3, wood sugar is cultivated the technique of Rhizopus oryzae thalline
The wood sugar obtaining is for the cultivation of lactic acid producing Rhizopus oryzae thalline, and wherein seed culture medium (g/L) is: it is 10,20,30g/L that wood sugar is diluted to respectively concentration; Peptone 2.0g/L; Potassium primary phosphate 0.2 g/L; MgSO
47H
2o 0.2 g/L; CaCO
36.0g/L; The every bottled 50ml seed liquor of 250ml shaking flask, 150r/min, cultivates 24h for 28 ℃, observes thalli morphology, dry cell weight, after get seed access fermention medium (g/L): glucose 80.0; (NH
4)
2sO
43.0; MgSO
47H
2o 0.25; ZnSO
47H
2o 0.04; Potassium primary phosphate 0.2 and CaCO
350.0, inoculum size is the every bottled 50ml seed liquor of 10%, 250ml shaking flask, 150r/min, and 28 ℃ are cultured to sugar and deplete, and survey lactic acid production, and its result is as shown in table 5:
Table 5
Take pure glucose as contrast, observe all the other glucose cultivations and carry out lactic acid fermented result, find, when xylose concentration is too low, biomass is inadequate, thereby can have influence on the fermentation in later stage, but when xylose concentration is too high, although biomass has reached certain degree, and than the height of contrast, but lactic acid production or on the low side in contrast, be that wood sugar is while carrying out Rhizopus oryzae seed culture, the Rhizopus oryzae cell that wood sugar is cultivated contains more much molecular components, compared to glucose, be more conducive to the accumulation of Rhizopus oryzae biomass, but fermentation stage, when the thalline that Rhizopus oryzae utilizes wood sugar to cultivate ferments, pathways metabolism is easily obstructed, make biomass and fumaric acid accumulation volume lower.Think it may is that higher xylose concentration is while cultivating Rhizopus oryzae, may make the normal pathways metabolism of Rhizopus oryzae change, therefore, on this basis, we adjust seed culture medium, and Rhizopus oryzae seed is cultivated in classification, and total seed concentration is 30g/L, the seed of cultivating ferments, and its result is as shown in table 6:
Table 6
As can be seen from Table 6, if in the culturing process of every one-level, xylose concentration is too high, all easily cause the fermentation in later stage to be obstructed, when controlling three grades of cultivations, the xylose concentration of every grade is all within the scope of 10g/L, this impact that later stage fermentation is obstructed disappears substantially, simultaneously, the thalline obtaining three grades of cultivations is glomeration all, simultaneously, to utilizing endobacillary chitin that wood sugar cultivates and chitosan content all than the height of contrast, therefore, utilize classification to cultivate and not only can guarantee preferably that thalline is spherical, be beneficial to the fermentation of later stage lactic acid, in thalline, chitin and chitosan content are all higher simultaneously, utilize after fermentation ends, thalline can carry out recycling.
Embodiment
By the following examples the present invention is further expalined to explanation.
Embodiment 1
First that wild rice stem waste Jiaobai leaf, blade and leaf sheath is first 50 ℃ of oven dry of spending the night, with pulverizer, be crushed to the particle that particle diameter is 10～20mm, by concentration, be 90% ethanol lixiviate 1h under 90 ℃ of conditions of temperature, solid-to-liquid ratio (g/ml) is 1:10, filter, by formic acid lixiviate 2h under 70 ℃ of conditions for the solid substance obtaining, solid-to-liquid ratio (g/ml), for 1:10 filters, solid-liquid separation, liquid carries out underpressure distillation, after formic acid reclaims, obtains solid substance, this is wood sugar of preparation, and analyzing after testing the rate of recovery obtain wood sugar is 95%.
Embodiment 2
First that wild rice stem waste Jiaobai leaf, blade and leaf sheath is first 50 ℃ of oven dry of spending the night, with pulverizer, be crushed to the particle that particle diameter is 10～20mm, by concentration, be 80% ethanol lixiviate 2h under 80 ℃ of conditions of temperature, solid-to-liquid ratio is 1:10 (g/ml), filter, by formic acid lixiviate 2h under 75 ℃ of conditions for the solid substance obtaining, solid-to-liquid ratio is 1:20 (g/ml), filter, solid-liquid separation, liquid carries out underpressure distillation, after formic acid reclaims, obtains solid substance, this is wood sugar of preparation, and analyzing after testing the rate of recovery obtain wood sugar is 93%.
Embodiment 3
First that wild rice stem waste Jiaobai leaf, blade and leaf sheath is first 50 ℃ of oven dry of spending the night, with pulverizer, be crushed to the particle that particle diameter is 10～20mm, by concentration, be 70% ethanol lixiviate 1h under 90 ℃ of conditions of temperature, solid-to-liquid ratio is 1:5 (g/ml), filter, by formic acid lixiviate 1h under 70 ℃ of conditions for the solid substance obtaining, solid-to-liquid ratio is 1:10 (g/ml), filter, solid-liquid separation, liquid carries out underpressure distillation, after formic acid reclaims, obtains solid substance, this is wood sugar of preparation, and analyzing after testing the rate of recovery obtain wood sugar is 92%.
Embodiment 4
The method of utilizing wood sugar that embodiment 1 method obtains to cultivate Rhizopus oryzae thalline, the method specifically comprises the following steps:
Wood sugar is carried out to classification cultivation to Rhizopus oryzae, cultivate three grades, control xylose concentration at 10～15g/L for every grade, total sugar concentration is controlled at 30～50g/L, controls peptone 2.0g/L simultaneously, potassium primary phosphate 0.2g/L, MgSO
47H
2o0.2g/L, CaCO
36.0g/L, the every bottled 50ml seed liquor of 250ml shaking flask, 150r/min, 30 ℃ of reactions, cultivate 8～12h for every grade, after cultivating and finishing, obtain of uniform sizely, and diameter is the spherical Rhizopus oryzae thalline of 1mm left and right; Constituent of chitosan analysis in thalline is obtained to constituent of chitosan and reached 0.164g/g.
The fumaric acid output that this Rhizopus oryzae thalline fermentation obtains all can reach 53.2g/L, and this Rhizopus oryzae thalline fermentation lactic acid producing output can reach 57.4g/L (80g/L initial glucose concentration).
Claims (10)
1. for cultivating a wood sugar for Rhizopus oryzae thalline, it is characterized in that this wood sugar prepares by the following method:
1) after wild rice stem waste is pulverized, by concentration, be 70～90% ethanol lixiviate 1～2h under 80～90 ℃ of conditions of temperature, filter, obtain solid substance;
2) by formic acid lixiviate 1～2h under 60～75 ℃ of conditions of temperature for solid substance, filter, solid-liquid separation, carries out underpressure distillation by the liquid obtaining, and reclaims formic acid, obtains solid substance, is wood sugar.
2. wood sugar according to claim 1, is characterized in that described wild rice stem waste is wild rice stem leaf sheath, blade and first sheathing leaf.
3. a preparation method for wood sugar claimed in claim 1, is characterized in that the method comprises the following steps:
1) after wild rice stem waste is pulverized, by concentration, be 70～90% ethanol lixiviate 1～2h under 80～90 ℃ of conditions of temperature, filter, obtain solid substance;
2) by formic acid lixiviate 1～2h under 60～75 ℃ of conditions of temperature for the solid substance obtaining, filter, solid-liquid separation, carries out underpressure distillation by the liquid obtaining, and reclaims formic acid, obtains solid substance.
4. the preparation method of wood sugar according to claim 3, is characterized in that described wild rice stem waste is first crushed to the particle that particle diameter is 10～20mm.
5. the preparation method of wood sugar according to claim 3, is characterized in that the solid-to-liquid ratio adopting in alcohol steep process is 1:5～20, preferably 1:10.
6. the preparation method of wood sugar according to claim 3, is characterized in that the alcohol concn adopting in alcohol steep process is 80～90%.
7. the preparation method of wood sugar according to claim 3, is characterized in that step " 1) " is for being lixiviate under 90 ℃ of conditions in temperature.
8. the preparation method of wood sugar according to claim 3, is characterized in that the solid-to-liquid ratio that step " 2) " is used formic acid lixiviate to adopt is 1:5～20, is preferably 1:10～20, most preferably 1:10.
9. the preparation method of wood sugar according to claim 3, is characterized in that in step " 2) ", by formic acid lixiviate under temperature 70 C condition for solid substance.
10. a method of utilizing the wood sugar described in claim 1 to cultivate Rhizopus oryzae thalline, it is characterized in that the method comprises the following steps: wood sugar is carried out to classification cultivation to Rhizopus oryzae, cultivate and divide 2～4 grades, control xylose concentration at 10～15g/L for every grade, total sugar concentration is controlled at 30～50g/L, control peptone 2.0g/L, potassium primary phosphate 0.2g/L, MgSO simultaneously
47H
2o0.2g/L, CaCO
36.0g/L, the every bottled 50ml seed liquor of 250ml shaking flask, 150r/min, 30 ℃ of reactions, cultivate 8～12h for every grade, after cultivation finishes, obtain uniform spherical Rhizopus oryzae thalline.
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