CN105420310A - Method for improving yield and quality of bacterial cellulose dry film by optimizing rice saccharification liquid fermentation culture medium - Google Patents
Method for improving yield and quality of bacterial cellulose dry film by optimizing rice saccharification liquid fermentation culture medium Download PDFInfo
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Abstract
The invention discloses a method for improving the yield and quality of a bacterial cellulose dry film by optimizing a rice saccharification liquid fermentation culture medium. Through adoption of the Plackett-Burman experiment design and the Box-Behnken experiment design, the main ingredients of the rice saccharification liquid fermentation culture medium for fermentation of bacterial cellulose are screened, and the proportions of all the components are optimized, so that a formula composition for the rice saccharification liquid fermentation culture medium is obtained. When the optimized rice saccharification liquid fermentation culture medium is utilized for fermentation of the bacterial cellulose, the bacterial cellulose yield reaches 7.08 g/L which is 18.5 times of the bacterial cellulose yield (0.382 g/L) reached through fermentation by a rice saccharification liquid fermentation culture medium base material, and is 47.5% higher than the bacterial cellulose yield (4.80 g/L) reached through fermentation by a basic culture medium; the main quality indicators achieved through fermentation by the optimized rice saccharification liquid fermentation culture medium is superior to those achieved through fermentation by the basic culture medium; compared with that prepared by utilizing the basic culture medium, the bacterial cellulose prepared by utilizing the optimized rice saccharification liquid fermentation culture medium has higher properties such as water binding capacity, crystallinity and thermal stability.
Description
Technical field
The present invention relates to bacteria cellulose production field, specifically, is a kind of method optimizing rice saccharified liquid fermention medium raising bacteria cellulose dry film productive rate, quality.
Background technology
Sucus Cocois is the earliest for the production of the raw material of bacteria cellulose, is also the raw material generally used at present.In recent years, bacteria cellulose relies on its excellent characteristic to be widely used in numerous areas such as medical treatment, weaving, environmental protection, papermaking, cause bacteria cellulose requirement huge, supply falls short of demand already to make limited Sucus Cocois resource, and be that main raw material has region restriction with Sucus Cocois, the needs of market amplification cannot be met.Find the large focus that the bacteria cellulose raw material substituting Sucus Cocois becomes research.The present inventor place seminar early-stage Study shows, rice saccharified liquid can be used for culturing bacterium Mierocrystalline cellulose, but low conversion rate when there is concentration height, the problem yielded poorly when concentration is low.The present invention is directed to the cellulosic problem of rice saccharified liquid culturing bacterium, with conventional acetobacter xylinum for test strain, optimize rice saccharified liquid fermention medium, the bacteria cellulose that the bacteria cellulose cultivate the rice saccharified liquid fermention medium of optimization and basic medium ferment compares in output and product performance, finds a kind of method improving bacteria cellulose dry film productive rate, quality.
Summary of the invention
The object of the invention is to produce bacteria cellulose for current Sucus Cocois and have that the production cycle grow, yields poorly, labour intensity is large, production cost is high, seasonal and region limits by raw material problem, study and replace Sucus Cocois with other raw materials, produce that output is high, the measured bacteria cellulose of matter.Specifically utilizing rice saccharified liquid to replace Sucus Cocois, improving the method for bacteria cellulose dry film productive rate and quality by optimizing rice saccharified liquid fermention medium.
The present invention's one is optimized rice saccharified liquid fermention medium and is improved bacteria cellulose dry film productive rate, the method of quality, by adopting Plackett-Burman test design and Box-Behnken test design method, the rice saccharified liquid fermention medium main ingredient of fermented bacterial cellulose is screened, optimize the proportioning of each component, obtain the rice saccharified liquid fermentative medium formula proportioning optimized, its formulation ratio is: yeast extract paste 13.1g, peptone 10.0g, potassium primary phosphate 5.7g, magnesium sulfate 3.1g, ferrous sulfate 0.3g, citric acid 0.3g, dehydrated alcohol 40ml, 1000ml is mixed with rice saccharification liquid culture medium base-material, be 7.08g/L with the productive rate of the rice saccharified liquid fermention medium fermented bacterial cellulose dry film optimized, it is 18.5 times of rice saccharification liquid culture medium base-material fermented bacterial cellulose dry film productive rate 0.382g/L, 47.5% is improved than basic medium fermented bacterial cellulose dry film productive rate 4.80g/L, primary quality measure is better than the bacteria cellulose of base culture base.
Described Plackett-Burman test design method is with peptone, yeast extract paste, KH
2pO
4, MgSO
4, FeSO
4, 7 factors testing as Plackett-Burman of citric acid, dehydrated alcohol 7 kinds of compositions, each factor gets high and low 2 levels, with bacteria cellulose dry film productive rate g/L for response value, carry out 12 groups of tests according to Plackett-Burman test design, four factors obtaining wherein remarkably influenced are yeast extract paste, KH
2pO
4, FeSO
4and dehydrated alcohol.
Described Box-Behnken test design is on Plackett-Burman design experiment basis, on the principal element yeast extract paste, the KH that affect bacteria cellulose dry film productive rate
2pO
4, FeSO
4with dehydrated alcohol by Box-Behnken test design method do four factors, three levels Box-Behnken test, carry out 29 groups of tests altogether, utilize Design-Exper7.0 software to carry out multiple regression matching to testing data, obtain bacteria cellulose dry film productive rate
yto independent variable(s) yeast extract paste
x 1 , KH
2pO
4 x 2 , FeSO
4 x 3 , dehydrated alcohol
x 4 multiple regression equation: y=6.63+0.31x
1-0.13x
2-0.035x
3+ 0.11x
4-0.01x
1x
2-0.008x
1x
3-0.007x
1x
4+ 0.11x
2x
3+ 0.14x
2x
4+ 0.036x
3x
4-0.67x
1 2-0.37x
2 2-0.33x
3 2-0.82x
4 2, independent variable(s) and response value have remarkable linear relationship, have the dependency of height between predictor and trial value
r 2=0.9306, the theoretical prediction of bacteria cellulose fermentative production can be applied to, after acquisition returns nonlinear model and response surface, for obtaining the mass concentration of substratum the best, to the regression fit equation of gained respectively to each independent variable(s) x
1, x
2, x
3, x
4ask extreme value, show that the optimum addition affecting bacteria cellulose dry film productive rate principal element is yeast extract paste 13.1g/L, KH by extreme point
2pO
45.7g/L, FeSO
40.3g/L, dehydrated alcohol 40.4ml/L.
The preparation method of described rice saccharification liquid culture medium base-material is as follows:
(1) rice is pulverized, and adds water, be heated to 80-90 DEG C in the ratio of 1:12, and gelatinization 30min obtains rice and sticks with paste;
(2) rice paste is cooled to 60 DEG C, adjusts pH to 4.0-4.5;
(3) ratio adding 300u saccharifying enzyme in every g rice adds saccharifying enzyme, and 60 DEG C of insulation saccharification to iodine liquid detect not aobvious blue look, survey saccharified liquid total sugar content by DNS method;
(4) adjusting saccharified liquid total sugar content with water is that namely 50g/L obtains rice saccharification liquid culture medium base-material.
The basal medium formulation of indication is: sucrose 50g, yeast extract paste 10g, peptone 8.0g, potassium primary phosphate 4.3g, magnesium sulfate 2.5g, is made into 1000mL with distilled water, add dehydrated alcohol 3%v/v after sterilizing cooling, utilize the output of basic medium fermented bacterial cellulose for 4.80g/L.
Above-mentioned indication bacteria cellulose primary quality measure is retentiveness, degree of crystallinity, thermostability.
The method preparing bacteria cellulose with the rice saccharified liquid fermention medium optimized is:
(1) by the rice saccharified liquid fermention medium that the rice saccharified liquid fermentative medium formula proportioning preparation optimized is optimized, fermention medium 1000ml is called for short;
(2) fermentation culture of getting step (1) gained, based on 121 DEG C of sterilizing 20-30min, is chilled to room temperature;
(3) the acetobacter xylinum seed liquor of 80ml through cultivating 24 hours is added;
(4) in 30 DEG C of constant incubator quiescent culture 8 days;
(5) collect bacteria cellulose film, repeatedly rinse except after the substratum on striping surface and impurity with distilled water, be placed in 80 DEG C, in the NaOH solution of 0.1mol/L, maintain 2h, to remove tropina and residual media, be creamy white to film translucent, be cooled to room temperature;
(6) by the bacteria cellulose film 0.1mol/LHCl of step (5) and 30min, distilled water fully washs;
(7) step (6) is dried to constant weight at 80 DEG C to get product.
beneficial effect
The invention provides and optimize the method that rice saccharification liquid culture medium is fermenting raw materials bacteria cellulose, the object improving bacteria cellulose dry film productive rate and quality can be reached, solve a difficult problem for region restriction in bacteria cellulose production, add the output of bacteria cellulose, reduce production cost, improve economic benefit.
Accompanying drawing explanation
The TG-DTA collection of illustrative plates of Fig. 1 basic medium bacteria cellulose
The TG-DTA collection of illustrative plates of Fig. 2 rice saccharification liquid culture medium bacteria cellulose.
Embodiment
embodiment 1the preparation method of rice saccharification liquid culture medium base-material is as follows:
(1) rice is pulverized, and adds water, be heated to 80-90 DEG C in the ratio of 1:12, and gelatinization 30min obtains rice and sticks with paste;
(2) rice paste is cooled to 60 DEG C, adjusts pH to 4.0-4.5;
(3) ratio adding 300u saccharifying enzyme in every g rice adds saccharifying enzyme, and 60 DEG C of insulation saccharification to iodine liquid detect not aobvious blue look, survey saccharified liquid total sugar content by DNS method;
(4) adjusting saccharified liquid total sugar content with water is that namely 50g/L obtains rice saccharification liquid culture medium base-material.
embodiment 2basic medium is prepared:
Get sucrose 50g, yeast extract paste 10g, peptone 8.0g, potassium primary phosphate 4.3g, magnesium sulfate 2.5g, is made into 1000mL with distilled water, adds dehydrated alcohol 3%v/v after sterilizing cooling.If preparation basic medium 2000ml, each component increases in proportion.
embodiment 3:with rice saccharification liquid culture medium base-material fermented bacterial cellulose
(1) rice saccharified liquid fermention medium base-material 1000ml is prepared by the preparation method of rice saccharification liquid culture medium base-material;
(2) fermentation culture of getting step (1) gained, based on 121 DEG C of sterilizing 20-30min, is chilled to room temperature;
(3) the acetobacter xylinum seed liquor of 80ml through cultivating 24 hours is added;
(4) in 30 DEG C of constant incubator quiescent culture 8 days;
(5) collect bacteria cellulose film, repeatedly rinse except after the substratum on striping surface and impurity with distilled water, be placed in 80 DEG C, in the NaOH solution of 0.1mol/L, maintain 2h, to remove tropina and residual media, be creamy white to film translucent, be cooled to room temperature;
(6) by the bacteria cellulose film 0.1mol/LHCl of step (5) and 30min, distilled water fully washs;
(7) step (6) is dried to constant weight at 80 DEG C to get product.
Utilize rice saccharification liquid culture medium base-material fermented bacterial cellulose dry film productive rate to be 0.382g/L, be far smaller than the output of basic medium 4.80g/L.Due to the total sugar content of rice saccharification liquid culture medium base-material and the identical of basic medium, be all 50g/L, illustrate and cause the not high reason of rice saccharification liquid culture medium base-material fermented bacterial cellulose dry film productive rate to be other nutritive ingredients in addition to sugars.
embodiment 4plackett-Burman test design:
On the basis of single factor experiment, with peptone, yeast extract paste, KH
2pO
4, MgSO
4, FeSO
4, 7 factors testing as Plackett-Burman of citric acid, dehydrated alcohol 7 kinds of compositions, each factor gets high and low 2 levels, and level of factor is in table 1.With bacteria cellulose dry film productive rate R(g/L dry film weight) for response value, carry out 12 groups of tests according to Plackett-Burman test design, Plackett-Burman test design and response value are in table 2.DesignExpert7.0 is utilized to analyze Plackett-Burman testing data, as table 3 after arranging.
Through software analysis, in main effect, yeast extract paste (
p=0.0683), KH
2pO
4(
p=0.0765), FeSO
4(
p=0.0511), dehydrated alcohol (
p=0.0018) effect is remarkable,
pvalue is all less than 0.10, can as the key factor optimized further.Other factors to Influence on test result little (
p>0.10), then in studying further consider as condition element.
Note: * represents the difference in 0.10 level
embodiment 5box-Behnken test design:
On the basis of Plackett-Burman test, by Box-Behnken test design, 29 groups of tests are carried out to the principal element affecting acetobacter xylinum metabolism synthesis bacteria cellulose in rice saccharification liquid culture medium, experimental factor level and coding are in table 4, test-results utilizes Design-Exper7.0 software to carry out multiple regression matching to testing data in table 5., obtain bacteria cellulose dry film productive rate (
y) to independent variable(s) yeast extract paste (
x 1 ), KH
2pO
4(
x 2 ), FeSO
4(
x 3 ), dehydrated alcohol (
x 4 ) multiple regression equation: y=6.63+0.31x
1-0.13x
2-0.035x
3+ 0.11x
4-0.01x
1x
2-0.008x
1x
3-0.007x
1x
4+ 0.11x
2x
3+ 0.14x
2x
4+ 0.036x
3x
4-0.67x
1 2-0.37x
2 2-0.33x
3 2-0.82x
4 2.
By variance analysis known (table 6), this model extremely significantly (
p< 0.0001), lose intend item not significantly (
p=0.2721), have between predictor and trial value high correlation (
r 2=0.9306), well, independent variable(s) and response value have remarkable linear relationship for model and actual matching, have the dependency of height, can be applied to the theoretical prediction of bacteria cellulose fermentative production between predictor and trial value.
Regression coefficient test of significance result (table 7) shows, in model parameter, and x
1, x
2, x
1 2, x
2 2, x
3 2, x
4 2extremely remarkable to bacteria cellulose dry film yield impact.After acquisition returns nonlinear model and response surface, for obtaining the mass concentration of substratum the best, to the regression fit equation of gained respectively to each independent variable(s) x
1, x
2, x
3, x
4ask extreme value, obtain extreme point and be respectively: x
1=0.24, x
2=-0.32, x
3=1, x
4=0.04, namely yeast extract paste addition is 13.1g/L, KH
2pO
4addition is 5.7g/L, FeSO
4addition is 0.3g/L, and dehydrated alcohol addition is 40.4ml/L.Under optimum substratum fermentation condition, the maximum production of theoretical bacteria cellulose is 6.66g/L.Substratum after optimization is yeast extract paste 13.1g/L, peptone 10g/L, KH
2pO
45.7g/L, MgSO
43.1g/L, FeSO
40.3g/L, citric acid 0.3g/L, dehydrated alcohol 40.0ml/L, be mixed with 1000mL with rice saccharification liquid culture medium base-material.Through actual measurement, the bacteria cellulose mean yield of the fermention medium after optimization is 7.08g/L, relatively predictor, is 18.5 times (0.382g/L) before optimizing.
Note: * * represents the difference in 0.05 level
embodiment 6 is usedthe rice saccharified liquid fermention medium optimized prepares the method for bacteria cellulose:
(1) by the rice saccharified liquid fermention medium that the rice saccharified liquid fermentative medium formula proportioning preparation optimized is optimized, fermention medium 1000ml is called for short;
(2) fermentation culture of getting step (1) gained, based on 121 DEG C of sterilizing 20-30min, is chilled to room temperature;
(3) the acetobacter xylinum seed liquor of 80ml through cultivating 24 hours is added;
(4) in 30 DEG C of constant incubator quiescent culture 8 days;
(5) collect bacteria cellulose film, repeatedly rinse except after the substratum on striping surface and impurity with distilled water, be placed in 80 DEG C, in the NaOH solution of 0.1mol/L, maintain 2h, to remove tropina and residual media, be creamy white to film translucent, be cooled to room temperature;
(6) by the bacteria cellulose film 0.1mol/LHCl of step (5) and 30min, distilled water fully washs;
(7) step (6) is dried to constant weight at 80 DEG C to get product.
embodiment 7optimize rice saccharified liquid fermention medium and produce bacteria cellulose Performance Detection:
Step (1) bacteria cellulose retentiveness and rehydration analysis
Bacteria cellulose retentiveness and rehydration analytical results are in table 8.As shown in Table 8, compared with basic medium, rice saccharification liquid culture medium cultivate bacteria cellulose in retentiveness and rehydration a little more than the bacteria cellulose of base culture base.
(2) bacteria cellulose crystallinity analysis
According to X-ray diffraction experimental data, respectively according to the degree of crystallinity of each substratum bacteria cellulose of formulae discovery, the results are shown in Table 9.Compared with basic medium, the bacteria cellulose degree of crystallinity 86.24% of rice saccharification liquid culture medium fermentation, improves 13.07% than 76.27% of basic medium.
(3) bacteria cellulose thermal stability analysis
The TG-DTA collection of illustrative plates of the bacteria cellulose of substratum and the fermentation of rice saccharification liquid culture medium based on Fig. 1, Fig. 2, from the bacteria cellulose TG curve of Fig. 1, Fig. 2, the quality of institute's test sample product varies with temperature and can be divided into three phases: loss that the quality in 0 ~ 170 DEG C is slight, the quality rapid deterioration stage in 170 ~ 550 DEG C, the slight loss stage of the quality between 550 ~ 800 DEG C.60.0 DEG C and 46.9 DEG C are respectively in the maximum weight loss rate temperature of the bacteria cellulose of 0 ~ 170 DEG C of stage rice saccharification liquid culture medium, basic medium gained, the mass loss showing this stage mainly bacteria cellulose dehydration produces, therefore claim the dehydration stage, due to institute's test sample product do not reach constant weight drying degree or because absorb moisture in air, so the temperature causing maximum weight loss rate place is different; 170 ~ 550 DEG C of stages, maximum weight loss rate place temperature is respectively 346.8 DEG C, 333.5 DEG C, this stage quality underspeeds soon, time length is long, for the degradation period of bacteria cellulose, adopt maximum weight loss rate method to weigh for material thermostability, maximum weight loss rate place temperature is higher, the thermostability of bacteria cellulose is better, illustrates that thermostability aspect rice saccharification liquid culture medium is better than basic medium.
Claims (7)
1. optimize rice saccharified liquid fermention medium raising bacteria cellulose dry film productive rate for one kind, the method of quality, it is characterized in that by adopting Plackett-Burman test design and Box-Behnken test design method, the rice saccharified liquid fermention medium main ingredient of fermented bacterial cellulose is screened, optimize the proportioning of each component, obtain the rice saccharified liquid fermentative medium formula proportioning optimized, its formulation ratio is: yeast extract paste 13.1g, peptone 10.0g, potassium primary phosphate 5.7g, magnesium sulfate 3.1g, ferrous sulfate 0.3g, citric acid 0.3g, dehydrated alcohol 40ml, 1000ml is mixed with rice saccharification liquid culture medium base-material, be 7.08g/L by the output of the rice saccharified liquid fermention medium fermented bacterial cellulose dry film optimized, it is 18.5 times of rice saccharification liquid culture medium base-material fermented bacterial cellulose dry film productive rate 0.382g/L, 47.5% is improved than basic medium fermented bacterial cellulose dry film productive rate 4.80g/L, primary quality measure is better than the bacteria cellulose of base culture base.
2. a kind of method optimizing rice saccharified liquid fermention medium raising bacteria cellulose dry film productive rate, quality according to claims 1, is characterized in that described Plackett-Burman test design method is with peptone, yeast extract paste, KH
2pO
4, MgSO
4, FeSO
4, 7 factors testing as Plackett-Burman of citric acid, dehydrated alcohol 7 kinds of compositions, each factor gets high and low 2 levels, with bacteria cellulose dry film productive rate g/L for response value, carry out 12 groups of tests according to Plackett-Burman test design, four factors obtaining wherein remarkably influenced are yeast extract paste, KH
2pO
4, FeSO
4and dehydrated alcohol.
3. a kind of method optimizing rice saccharified liquid fermention medium raising bacteria cellulose dry film productive rate, quality according to claims 1, it is characterized in that described Box-Behnken test design is on Plackett-Burman design experiment basis, on the principal element yeast extract paste, the KH that affect bacteria cellulose dry film productive rate
2pO
4, FeSO
4with dehydrated alcohol by Box-Behnken test design method do four factors, three levels Box-Behnken test, carry out 29 groups of tests altogether, utilize Design-Exper7.0 software to carry out multiple regression matching to testing data, obtain bacteria cellulose dry film productive rate
yto independent variable(s) yeast extract paste
x 1 , KH
2pO
4 x 2 , FeSO
4 x 3 , dehydrated alcohol
x 4 multiple regression equation: y=6.63+0.31x
1-0.13x
2-0.035x
3+ 0.11x
4-0.01x
1x
2-0.008x
1x
3-0.007x
1x
4+ 0.11x
2x
3+ 0.14x
2x
4+ 0.036x
3x
4-0.67x
1 2-0.37x
2 2-0.33x
3 2-0.82x
4 2, independent variable(s) and response value have remarkable linear relationship, have the dependency of height between predictor and trial value
r 2=0.9306, the theoretical prediction of bacteria cellulose fermentative production can be applied to, after acquisition returns nonlinear model and response surface, for obtaining the mass concentration of substratum the best, to the regression fit equation of gained respectively to each independent variable(s) x
1, x
2, x
3, x
4ask extreme value, show that the optimum addition affecting bacteria cellulose dry film productive rate principal element is yeast extract paste 13.1g/L, KH by extreme point
2pO
45.7g/L, FeSO
40.3g/L, dehydrated alcohol 40.4ml/L.
4. a kind of method optimizing rice saccharified liquid fermention medium raising bacteria cellulose dry film productive rate, quality according to claims 1, is characterized in that the preparation method of indication rice saccharification liquid culture medium base-material is as follows:
(1) rice is pulverized, and adds water, be heated to 80-90 DEG C in the ratio of 1:12, and gelatinization 30min obtains rice and sticks with paste;
(2) rice paste is cooled to 60 DEG C, adjusts pH to 4.0-4.5;
(3) ratio adding 300u saccharifying enzyme in every g rice adds saccharifying enzyme, and 60 DEG C of insulation saccharification to iodine liquid detect not aobvious blue look, survey saccharified liquid total sugar content by DNS method;
(4) adjusting saccharified liquid total sugar content with water is that namely 50g/L obtains rice saccharification liquid culture medium base-material.
5. a kind of method optimizing rice saccharified liquid fermention medium raising bacteria cellulose dry film productive rate, quality according to claims 1, it is characterized in that indication basal medium formulation is: sucrose 50g, yeast extract paste 10g, peptone 8.0g, potassium primary phosphate 4.3g, magnesium sulfate 2.5g, is made into 1000mL with distilled water, add dehydrated alcohol 3%v/v after sterilizing cooling, utilize the productive rate of basic medium fermented bacterial cellulose dry film to be 4.80g/L.
6. a kind of method optimizing rice saccharified liquid fermention medium raising bacteria cellulose dry film productive rate, quality according to claims 1, is characterized in that indication bacteria cellulose primary quality measure is retentiveness, degree of crystallinity, thermostability.
7. a kind of method optimizing rice saccharified liquid fermention medium raising bacteria cellulose dry film productive rate, quality according to claims 1, is characterized in that the method preparing bacteria cellulose with the rice saccharification liquid culture medium optimized is:
(1) by the rice saccharified liquid fermention medium that the rice saccharified liquid fermentative medium formula proportioning preparation optimized is optimized, fermention medium 1000ml is called for short;
(2) fermentation culture of getting step (1) gained, based on 121 DEG C of sterilizing 20-30min, is chilled to room temperature;
(3) the acetobacter xylinum seed liquor of 80ml through cultivating 24 hours is added;
(4) in 30 DEG C of constant incubator quiescent culture 8 days;
(5) collect bacteria cellulose film, repeatedly rinse except after the substratum on striping surface and impurity with distilled water, be placed in 80 DEG C, in the NaOH solution of 0.1mol/L, maintain 2h, to remove tropina and residual media, be creamy white to film translucent, be cooled to room temperature;
(6) by the bacteria cellulose film 0.1mol/LHCl of step (5) and 30min, distilled water fully washs;
(7) step (6) is dried to constant weight at 80 DEG C to get product.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110205349A (en) * | 2019-06-26 | 2019-09-06 | 浙江科技学院 | A method of bacteria cellulose is prepared using rice bran hydrolyzate fermentation |
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