CN105368896A - Method for optimizing inulin fermentation medium to increase yield of dry bacterial cellulose films and improve quality of dry bacterial cellulose films - Google Patents

Abstract

The invention discloses a method for optimizing the inulin fermentation medium to improve the yield and quality of bacterial cellulose dry film. By adopting the Plackett-Burman test design method, the main components in the inulin fermentation medium for fermenting bacterial cellulose are tested. After screening, the absolute ethanol with significant effect was further optimized as a key factor to obtain the optimized formula ratio of inulin fermentation medium. The yield of bacterial cellulose fermented with optimized inulin fermentation medium is 5.54g/L, which is 21.31 times of the dry film yield of bacterial cellulose fermented with inulin medium base material 0.26g/L, which is higher than that of basic medium fermentation The dry film yield of bacterial cellulose was 4.80g/L increased by 15.42%, and the main quality index was better than that of bacterial cellulose cultured on basic medium. In terms of properties, it is superior to bacterial cellulose prepared with basal medium.

Description

Method for optimizing inulin fermentation medium to improve bacterial cellulose dry film yield and quality

technical field

The invention relates to the field of bacterial cellulose production, in particular to a method for optimizing the inulin fermentation medium to improve the yield and quality of bacterial cellulose dry film.

Background technique

Coconut water is the earliest raw material used to produce bacterial cellulose, and it is also a widely used raw material at present. In recent years, bacterial cellulose has been widely used in many fields such as medical treatment, textile, environmental protection, papermaking, etc. due to its excellent characteristics, resulting in a huge demand for bacterial cellulose, making the limited coconut water resources already in short supply, and coconut water is used as the main raw material It has geographical restrictions and cannot meet the needs of market expansion. Finding bacterial cellulose raw materials to replace coconut water has become a hot spot of research. The previous research of the inventor's research group shows that inulin can be used to cultivate bacterial cellulose, but when pure inulin (inulin medium base) is used to ferment bacterial cellulose, the yield is only 0.26g/L, which is much lower than that of 4.80g/L of basal medium (chemically synthesized medium). Aiming at the problem of low dry film yield of bacterial cellulose cultured with inulin, the present invention uses the commonly used Acetobacter xylinum as a test strain to optimize the base material of inulin medium, and ferment the cultured bacterial cellulose with the basic medium. Bacterial cellulose is compared in terms of yield and product characteristics, in order to find a method to improve the yield and quality of bacterial cellulose dry film.

Contents of the invention

The purpose of the present invention is to solve the problems of long production cycle, low output, high labor intensity, high production cost, and limited by raw material seasonality and region in the production of bacterial cellulose from coconut water at present, and to study the use of other raw materials instead of coconut water to produce High yield and good quality bacterial cellulose. Specifically, it uses inulin instead of coconut water, and improves the yield and quality of bacterial cellulose dry film by optimizing the inulin fermentation medium.

A method of optimizing the inulin fermentation medium of the present invention to improve the yield and quality of bacterial cellulose dry film is to screen the main components in the inulin fermentation medium for fermenting bacterial cellulose by adopting the Plackett-Burman test design method , take the significant effect factor as the key factor to further optimize, discuss the optimal ratio of each component of the inulin fermentation medium, and obtain the optimized formula ratio of the inulin fermentation medium: yeast extract 10g, peptone 8.0g, dihydrogen phosphate Potassium 4.3g, magnesium sulfate 2.5g, made into 1000ml with inulin medium base material, sterilized at 121°C for 20-30min, after sterilization and cooling, add 30ml of absolute ethanol aseptically, and use optimized inulin fermentation medium The yield of fermented bacterial cellulose is 5.54g/L, which is 21.31 times of the dry film yield of 0.26g/L fermented with inulin medium base material, which is 4.80g higher than the dry film yield of fermented bacterial cellulose with base medium /L increased by 15.42%, and the main quality index was better than that of bacterial cellulose cultured on basic medium.

The Plackett-Burman test design method is to use seven components of peptone, yeast extract, KH ₂ PO ₄ , MgSO ₄ , FeSO ₄ , citric acid, and absolute ethanol as 7 factors of the Plackett-Burman test, and each factor takes There are two levels of high and low, with the bacterial cellulose dry film yield g/L as the response value, 12 groups of experiments were carried out according to the Plackett-Burman test design, and it was found that the most significant factor was absolute ethanol.

Taking the significant effect factor as the key factor to further optimize is to optimize the absolute ethanol that significantly affects the yield of bacterial cellulose dry film. It was found that the dry film yield of bacterial cellulose increased first and then decreased with the increase of absolute ethanol addition. When the addition of absolute ethanol was 3.0% v/v, the dry film yield of bacterial cellulose reached the highest value.

The preparation method of above-mentioned inulin culture medium base material is: the total sugar content of measuring inulin through DNS method is 78.82g/100g inulin, this inulin 63.5g is dissolved in 1000ml distilled water, the total sugar of gained inulin solution The content is 50g/L.

The basic medium formula referred to above is: 50g sucrose, 10g yeast extract, 8.0g peptone, 4.3g potassium dihydrogen phosphate, 2.5g magnesium sulfate, make 1000ml with distilled water, add 30ml of absolute ethanol after sterilization and cooling, use the basic The yield of bacterial cellulose fermented in the culture medium was 4.80g/L.

The main quality indicators of bacterial cellulose referred to above are water holding capacity, crystallinity and thermal stability.

The method for preparing bacterial cellulose with optimized inulin medium is:

(1) Prepare 1000ml of optimized inulin fermentation medium (referred to as fermentation medium);

(2) Add 80ml of Acetobacter xylinum seed solution cultivated for 24 hours;

(3) Culture in a constant temperature incubator at 30°C for 8 days;

(4) Collect the bacterial cellulose membrane, wash it repeatedly with distilled water to remove the medium and impurities on the surface of the membrane, and then place it in 0.1mol/L NaOH solution at 80°C for 2 hours to remove bacterial protein and residual medium. Until the film is milky white and translucent, cool to room temperature;

(5) Neutralize the bacterial cellulose membrane in step (4) with 0.1mol/L HCl for 30 minutes, and wash fully with distilled water;

(6) Dry the step (5) at 80°C to constant weight to obtain the finished product.

Beneficial effect

The invention provides a method for optimizing the inulin medium to ferment bacterial cellulose with inulin as the basic raw material, which can achieve the purpose of improving the dry film yield and quality of bacterial cellulose, and solves the regional problems in the production of bacterial cellulose The difficult problem of limitation increases the output of bacterial cellulose, reduces production cost and improves economic benefit.

Description of drawings

Figure 1 The effect of the amount of absolute ethanol added on the dry film yield of bacterial cellulose

Figure 2 TG-DTA pattern of basal medium bacterial cellulose

Figure 3 TG-DTA pattern of bacterial cellulose in inulin medium.

detailed description

The preparation of embodiment 1 inulin culture medium base material:

The total sugar content of inulin measured by the DNS method is 78.82g/100g inulin, and 63.5g of this inulin is dissolved in 1000ml distilled water, and the total sugar content of the gained inulin solution is 50g/L.

The preparation of embodiment 2 basal medium:

Take 50g of sucrose, 10g of yeast extract, 8.0g of peptone, 4.3g of potassium dihydrogen phosphate, and 2.5g of magnesium sulfate, make 1000mL with distilled water, add 3% v/v of absolute ethanol after sterilization and cooling.

Embodiment 3 ferments bacterial cellulose with inulin medium base material:

(1) Prepare 1000ml of inulin fermentation medium base material;

(2) Add 80ml of Acetobacter xylinum seed solution;

(3) Culture in a constant temperature incubator at 30°C for 8 days;

(4) Collect the bacterial cellulose membrane, wash it repeatedly with distilled water to remove the medium and impurities on the surface of the membrane, and then place it in 0.1mol/L NaOH solution at 80°C for 2 hours to remove bacterial protein and residual medium. Until the film is milky white and translucent, cool to room temperature;

(5) Neutralize the bacterial cellulose membrane in step (4) with 0.1mol/L HCl for 30 minutes, and wash fully with distilled water;

(6) Dry the step (5) at 80°C to constant weight to obtain the finished product.

The dry film yield of bacterial cellulose fermented with inulin medium base material was 0.26g/L, far less than the yield of 4.80g/L of basic medium. Since the total sugar content of the inulin medium base material is the same as that of the basic medium, which is 50g/L, it shows that the reason for the low yield of the bacterial cellulose dry film fermented by the inulin medium base material is that other than sugars nutrient content.

Example 4 Plackett-Burman experimental design

On the basis of the single factor test, peptone, yeast extract, KH ₂ PO ₄ , MgSO ₄ , FeSO ₄ , citric acid, and absolute ethanol were used as seven factors in the Plackett-Burman test. 2 levels lower, and the factor levels are shown in Table 1. Taking the bacterial cellulose dry film yield R (g/L dry film weight) as the response value, 12 groups of experiments were carried out according to the Plackett-Burman test design. The Plackett-Burman test design and response values are shown in Table 2. Using DesignExpert7.0 to analyze the Plackett-Burman test data, after sorting, it is shown in Table 3.

After software analysis of the main effect, the effect of absolute ethanol ( P =0.0002) is significant (see Table 3), and the P value is less than 0.10, which can be used as a key factor for further optimization. Other factors had little effect on the results ( P >0.10) and were considered as conditional factors in further studies.

Note: * indicates the difference at the 0.10 level

The determination of embodiment 5 key factors dehydrated alcohol addition:

From the Plackett-Burman test, it can be seen that among the 7 detection factors, absolute ethanol is the most significant influencing factor, and other factors have little influence on the results. It is determined that the addition of other factors is the average value of high-level and low-level additions. That is, yeast extract is 12.5g/L, peptone is 10.0g/L, _KH2PO4 is 6.5g/L, _MgSO4 is _3.1g /L, _FeSO4 is 0.3g/L, and citric acid is 0.3g/L. The effect of 2.2%-4.0% v/v absolute ethanol addition on the dry film yield of bacterial cellulose was investigated, and the results are shown in Figure 1. As shown in Figure 1, the dry film yield of bacterial cellulose showed a process of first increasing and then decreasing with the increase of absolute ethanol addition. When the addition of absolute ethanol was 3.0% v/v, the bacterial cellulose dry film yield reached the highest value of 5.54g/L, which was 21.31 times that of the unoptimized (0.26g/L).

Embodiment 6 produces bacterial cellulose with optimization inulin fermentation medium:

(1) Prepare 1000ml of optimized inulin fermentation medium (referred to as fermentation medium);

(2) Add 80ml of Acetobacter xylinum seed solution cultivated for 24 hours;

(3) Culture in a constant temperature incubator at 30°C for 8 days;

(4) Collect the bacterial cellulose membrane, wash it repeatedly with distilled water to remove the medium and impurities on the surface of the membrane, and then place it in 0.1mol/L NaOH solution at 80°C for 2 hours to remove bacterial protein and residual medium. Until the film is milky white and translucent, cool to room temperature;

(5) Neutralize the bacterial cellulose membrane in step (4) with 0.1mol/L HCl for 30 minutes, and wash fully with distilled water;

(6) Dry the step (5) at 80°C to constant weight to obtain the finished product.

Example 7 Optimizing the Inulin Fermentation Medium to Produce Bacterial Cellulose Performance Detection

(1) Analysis of water holding capacity and rehydration property of bacterial cellulose

The results of water holding and rehydration analysis of bacterial cellulose are shown in Table 4. It can be seen from Table 4 that compared with the basal medium, the bacterial cellulose cultured in the inulin medium is slightly higher in water holding capacity and rehydration than the BC cultured in the basal medium.

(2) Bacterial cellulose crystallinity analysis

According to the X-ray diffraction experimental data, the crystallinity of bacterial cellulose in each culture medium was calculated according to the formula respectively, and the results are shown in Table 5. Compared with the basal medium, the crystallinity of bacterial cellulose fermented in the inulin medium was 83.99%, which was 10.12% higher than that of the basal medium (76.27%).

(3) Thermal stability analysis of bacterial cellulose

Fig. 2, Fig. 3 are the TG-DTA collection of illustrative plates of the bacterial cellulose fermented by basal medium and inulin medium, by the bacterial cellulose TG curve of Fig. 2, Fig. 3, it can be known that the quality of measured sample changes with temperature and can be divided into Three stages: slight loss of mass within 0-170°C, rapid loss of mass within 170-550°C, slight loss of mass between 550-800°C. The maximum weight loss rate temperature of bacterial cellulose obtained from inulin medium and basal medium at 0-170°C is 65.1°C and 46.9°C respectively, indicating that the mass loss at this stage is mainly caused by the dehydration of bacterial cellulose, so it is called loss. In the water stage, because the measured sample did not reach the degree of constant weight drying or because it absorbed moisture in the air, the temperature at the maximum weight loss rate was different; in the 170-550°C stage, the temperature at the maximum weight loss rate was 347.5°C , 333.5°C, at this stage, the quality decreases rapidly and lasts for a long time, which is the degradation stage of bacterial cellulose. The thermal stability of the material is measured by the maximum weight loss rate method. The higher the temperature at the maximum weight loss rate, the thermal stability of bacterial cellulose The better the performance, the better the thermal stability of the inulin medium than the basal medium.

Claims (7)

1. optimize inulin 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 method, main ingredient in the inulin fermention medium of fermented bacterial cellulose is screened, effect significant factor is optimized further as key factor, inquire into the optimal proportion of each component of inulin fermention medium, obtaining the inulin fermentative medium formula proportioning optimized is: yeast extract paste 10g, peptone 8.0g, potassium primary phosphate 4.3g, magnesium sulfate 2.5g, 1000ml is made into inulin substratum base-material, 121 DEG C of sterilizing 20-30min, dehydrated alcohol 30ml is sterilely added after sterilizing cooling, be 5.54g/L with the productive rate of the inulin fermention medium fermented bacterial cellulose dry film optimized, it is 21.31 times of inulin substratum base-material fermented bacterial cellulose dry film productive rate 0.26g/L, 15.42% is improve 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 inulin 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, the factor obtaining wherein remarkably influenced is dehydrated alcohol.

3. a kind of method optimizing inulin fermention medium raising bacteria cellulose dry film productive rate, quality according to claims 1, it is characterized in that described being optimized further as key factor by effect significant factor, that the dehydrated alcohol of remarkably influenced bacteria cellulose dry film productive rate is optimized, by investigating 2.2%-4.0%v/v dehydrated alcohol addition to the impact of bacteria cellulose dry film productive rate, find that bacteria cellulose dry film productive rate presents with the increase of dehydrated alcohol addition the process first increasing and reduce afterwards;

When dehydrated alcohol addition is 3.0%v/v, bacteria cellulose dry film productive rate reaches maximum.

4. a kind of method optimizing inulin fermention medium raising bacteria cellulose dry film productive rate, quality according to claims 1, it is characterized in that the preparation method of indication inulin substratum base-material is: the total sugar content surveying inulin through DNS method is 78.82g/100g inulin, be dissolved in 1000ml distilled water by this inulin 63.5g, gained inulin solution total sugar content is 50g/L.

5. a kind of method optimizing inulin 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 30ml after sterilizing cooling, utilize the output of basic medium fermented bacterial cellulose for 4.80g/L.

6. a kind of method optimizing inulin fermention medium raising bacteria cellulose dry film productive rate, quality according to claims 1, is characterized in that the bacteria cellulose primary quality measure of indication is retentiveness, degree of crystallinity, thermostability.

7. a kind of method optimizing inulin fermention medium raising bacteria cellulose dry film productive rate, quality according to claims 1, is characterized in that the method for the inulin medium preparing bacteria cellulose with optimization is:

(1) inulin fermention medium (abbreviation fermention medium) 1000ml of preparation optimization;

(2) 80ml is added through the cultivation acetobacter xylinum seed liquor of 24 hours;

(3) in 30 DEG C of constant incubator quiescent culture 8 days;

(4) 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;

(5) by the bacteria cellulose film 0.1mol/LHCl of step (4) and 30min, distilled water fully washs;

(6) step (5) is dried to constant weight at 80 DEG C to get product.