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
Method for optimizing inulin fermentation medium to increase yield of dry bacterial cellulose films and improve quality of dry bacterial cellulose films Download PDFInfo
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Abstract
本发明公开了一种优化菊粉发酵培养基提高细菌纤维素干膜产率、质量的方法,通过采用Plackett-Burman试验设计方法,对发酵细菌纤维素的菊粉发酵培养基中主要组分进行筛选,将效应显著的无水乙醇作为关键因素进一步优化,获得优化的菊粉发酵培养基配方配比。用优化了的菊粉发酵培养基发酵细菌纤维素的产率为5.54g/L,是菊粉培养基基料发酵细菌纤维素干膜产率0.26g/L的21.31倍,比基础培养基发酵细菌纤维素干膜产率4.80g/L提高15.42%,主要质量指标优于基础培养基培养的细菌纤维素,用优化的菊粉培养基制备细菌纤维素在持水性、结晶度、热稳定性等性质方面优于用基础培养基制备的细菌纤维素。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
技术领域 technical field
本发明涉及细菌纤维素生产领域,具体地说,是一种优化菊粉发酵培养基提高细菌纤维素干膜产率、质量的方法。 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.
背景技术 Background technique
椰子水是最早用于生产细菌纤维素的原料,也是目前普遍使用的原料。近年来,细菌纤维素凭借其优良的特性被广泛运用于医疗、纺织、环保、造纸等诸多领域,导致细菌纤维素需要量巨大,使得有限的椰子水资源早已供不应求,且以椰子水为主要原料具有地域性限制,无法满足市场扩增的需要。寻找替代椰子水的细菌纤维素原料成为研究的一大热点。本发明人所在课题组前期研究表明,菊粉可用于培养细菌纤维素,但用纯菊粉(菊粉培养基基料)发酵细菌纤维素时,产量仅为0.26g/L,产量远低于基础培养基(化学合成培养基)的4.80g/L。本发明针对菊粉培养细菌纤维素干膜产率低的问题,以常用的木醋杆菌为试验菌株,对菊粉培养基基料进行优化,将所培养的细菌纤维素与基础培养基发酵的细菌纤维素在产量和产品特性方面进行比较,以期找到一种提高细菌纤维素干膜产率、质量的方法。 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.
发明内容 Contents of the invention
本发明的目的在于针对目前椰子水生产细菌纤维素存在生产周期长、产量低、劳动强度大、生产成本高、受原料季节性和地域性限制的问题,研究用其他原料代替椰子水,生产出产量高、质量好的细菌纤维素。具体地说是利用菊粉代替椰子水,通过优化菊粉发酵培养基提高细菌纤维素干膜产率和质量的方法。 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.
本发明一种优化菊粉发酵培养基提高细菌纤维素干膜产率、质量的方法,是通过采用Plackett-Burman试验设计方法,对发酵细菌纤维素的菊粉发酵培养基中主要组分进行筛选,将效应显著因子作为关键因素进一步优化,探讨菊粉发酵培养基各组分的最优配比,获得优化的菊粉发酵培养基配方配比为:酵母膏10g,蛋白胨8.0g,磷酸二氢钾4.3g,硫酸镁2.5g,用菊粉培养基基料配成1000ml,121℃灭菌20-30min,灭菌冷却后无菌地加入无水乙醇30ml,用优化了的菊粉发酵培养基发酵细菌纤维素的产率为5.54g/L,是菊粉培养基基料发酵细菌纤维素干膜产率0.26g/L的21.31倍,比基础培养基发酵细菌纤维素干膜产率4.80g/L提高了15.42%,主要质量指标优于基础培养基培养的细菌纤维素。 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.
所述的Plackett-Burman试验设计方法是以蛋白胨、酵母膏、KH2PO4、MgSO4、FeSO4、柠檬酸、无水乙醇7种成分作为Plackett-Burman试验的7个因素,每个因素取高、低2个水平,以细菌纤维素干膜产率g/L为响应值,按照Plackett-Burman试验设计进行12组试验,得到其中显著影响的因素为无水乙醇。 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.
将效应显著因子作为关键因素进一步优化,是对显著影响细菌纤维素干膜产率的无水乙醇进行优化,通过考察2.2%-4.0%v/v无水乙醇添加量对细菌纤维素干膜产率的影响,发现细菌纤维素干膜产率随无水乙醇添加量的增加呈现先增加后减小的过程。当无水乙醇添加量为3.0%v/v时,细菌纤维素干膜产率达到最高值。 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.
上述所指菊粉培养基基料的制备方法为:经DNS法测菊粉的总糖含量为78.82g/100g菊粉,将此菊粉63.5g溶于1000ml蒸馏水中,所得菊粉溶液总糖含量为50g/L。 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.
上述所指基础培养基配方为:蔗糖50g,酵母膏10g,蛋白胨8.0g,磷酸二氢钾4.3g,硫酸镁2.5g,用蒸馏水配成1000ml,灭菌冷却后加入无水乙醇30ml,利用基础培养基发酵细菌纤维素的产量为4.80g/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.
上述所指的细菌纤维素主要质量指标为持水性、结晶度、热稳定性。 The main quality indicators of bacterial cellulose referred to above are water holding capacity, crystallinity and thermal stability.
用优化的菊粉培养基制备细菌纤维素的方法为: The method for preparing bacterial cellulose with optimized inulin medium is:
(1)配制优化的菊粉发酵培养基(简称发酵培养基)1000ml; (1) Prepare 1000ml of optimized inulin fermentation medium (referred to as fermentation medium);
(2)加入80ml经过培养24小时的木醋杆菌种子液; (2) Add 80ml of Acetobacter xylinum seed solution cultivated for 24 hours;
(3)于30℃恒温培养箱静置培养8天; (3) Culture in a constant temperature incubator at 30°C for 8 days;
(4)收集细菌纤维素膜,用蒸馏水反复冲洗除去膜表面的培养基及杂质后,置于80℃,0.1mol/L的NaOH溶液中,维持2h,以除去菌体蛋白和残余培养基,至膜呈乳白色半透明状,冷却至室温; (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)将步骤(4)的细菌纤维素膜用0.1mol/LHCl中和30min,蒸馏水充分洗涤; (5) Neutralize the bacterial cellulose membrane in step (4) with 0.1mol/L HCl for 30 minutes, and wash fully with distilled water;
(6)将步骤(5)在80℃下干燥至恒重即得成品。 (6) Dry the step (5) at 80°C to constant weight to obtain the finished product.
有益效果Beneficial effect
本发明提供了以菊粉为基础原料经过优化,以优化菊粉培养基发酵细菌纤维素的方法,能够达到提高细菌纤维素干膜产率和质量的目的,解决了细菌纤维素生产中地域性限制的难题,增加了细菌纤维素的产量,降低了生产成本,提高了经济效益。 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.
附图说明 Description of drawings
图1无水乙醇添加量对细菌纤维素干膜产率的影响 Figure 1 The effect of the amount of absolute ethanol added on the dry film yield of bacterial cellulose
图2基础培养基细菌纤维素的TG-DTA图谱 Figure 2 TG-DTA pattern of basal medium bacterial cellulose
图3菊粉培养基细菌纤维素的TG-DTA图谱。 Figure 3 TG-DTA pattern of bacterial cellulose in inulin medium.
具体实施方式 detailed description
实施例1菊粉培养基基料的制备: The preparation of embodiment 1 inulin culture medium base material:
经DNS法测菊粉的总糖含量为78.82g/100g菊粉,将此菊粉63.5g溶于1000ml蒸馏水中,所得菊粉溶液总糖含量为50g/L。 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.
实施例2基础培养基的制备: The preparation of embodiment 2 basal medium:
取蔗糖50g,酵母膏10g,蛋白胨8.0g,磷酸二氢钾4.3g,硫酸镁2.5g,用蒸馏水配成1000mL,灭菌冷却后加入无水乙醇3%v/v。 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.
实施例3用菊粉培养基基料发酵细菌纤维素: Embodiment 3 ferments bacterial cellulose with inulin medium base material:
(1)制备菊粉发酵培养基基料1000ml; (1) Prepare 1000ml of inulin fermentation medium base material;
(2)加入80ml木醋杆菌种子液; (2) Add 80ml of Acetobacter xylinum seed solution;
(3)于30℃恒温培养箱静置培养8天; (3) Culture in a constant temperature incubator at 30°C for 8 days;
(4)收集细菌纤维素膜,用蒸馏水反复冲洗除去膜表面的培养基及杂质后,置于80℃,0.1mol/L的NaOH溶液中,维持2h,以除去菌体蛋白和残余培养基,至膜呈乳白色半透明状,冷却至室温; (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)将步骤(4)的细菌纤维素膜用0.1mol/LHCl中和30min,蒸馏水充分洗涤; (5) Neutralize the bacterial cellulose membrane in step (4) with 0.1mol/L HCl for 30 minutes, and wash fully with distilled water;
(6)将步骤(5)在80℃下干燥至恒重即得成品。 (6) Dry the step (5) at 80°C to constant weight to obtain the finished product.
利用菊粉培养基基料发酵细菌纤维素干膜产率为0.26g/L,远远小于基础培养基4.80g/L的产量。由于菊粉培养基基料的总糖含量与基础培养基的相同,都为50g/L,说明导致菊粉培养基基料发酵细菌纤维素干膜产率不高的原因是除糖类外其他营养成分。 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.
实施例4Plackett-Burman试验设计 Example 4 Plackett-Burman experimental design
在单因素试验的基础上,以蛋白胨、酵母膏、KH2PO4、MgSO4、FeSO4、柠檬酸、无水乙醇7种成分作为Plackett-Burman试验的7个因素,每个因素取高、低2个水平,因素水平见表1。以细菌纤维素干膜产率R(g/L干膜重)为响应值,按照Plackett-Burman试验设计进行12组试验,Plackett-Burman试验设计及响应值见表2。利用DesignExpert7.0对Plackett-Burman试验数据进行分析,经整理后如表3。 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.
经软件分析主效应,无水乙醇(P=0.0002)的效应显著(见表3),P值小于0.10,可以作为进一步优化的关键因素。其他因素对结果影响不大(P>0.10),在进一步研究中作为条件因素考虑。 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.
注:*表示0.10水平上的差异 Note: * indicates the difference at the 0.10 level
实施例5关键因素无水乙醇添加量的确定: The determination of embodiment 5 key factors dehydrated alcohol addition:
由Plackett-Burman试验可知,在7个检测因素中,无水乙醇为最显著的影响因素,其他因素对结果影响不大,确定其他因素的添加量为高水平和低水平添加量的平均值,即酵母膏为12.5g/L、蛋白胨为10.0g/L、KH2PO4为6.5g/L、MgSO4为3.1g/L、FeSO4为0.3g/L、柠檬酸为0.3g/L。考察2.2%-4.0%v/v无水乙醇添加量对细菌纤维素干膜产率的影响,结果见图1。如图1所示,细菌纤维素干膜产率随无水乙醇添加量的增加呈现先增加后减小的过程。当无水乙醇添加量为3.0%v/v时,细菌纤维素干膜产率达到最高值5.54g/L,是未优化前的(0.26g/L)的21.31倍。 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).
实施例6用优化菊粉发酵培养基生产细菌纤维素: Embodiment 6 produces bacterial cellulose with optimization inulin fermentation medium:
(1)配制优化的菊粉发酵培养基(简称发酵培养基)1000ml; (1) Prepare 1000ml of optimized inulin fermentation medium (referred to as fermentation medium);
(2)加入80ml经过培养24小时的木醋杆菌种子液; (2) Add 80ml of Acetobacter xylinum seed solution cultivated for 24 hours;
(3)于30℃恒温培养箱静置培养8天; (3) Culture in a constant temperature incubator at 30°C for 8 days;
(4)收集细菌纤维素膜,用蒸馏水反复冲洗除去膜表面的培养基及杂质后,置于80℃,0.1mol/L的NaOH溶液中,维持2h,以除去菌体蛋白和残余培养基,至膜呈乳白色半透明状,冷却至室温; (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)将步骤(4)的细菌纤维素膜用0.1mol/LHCl中和30min,蒸馏水充分洗涤; (5) Neutralize the bacterial cellulose membrane in step (4) with 0.1mol/L HCl for 30 minutes, and wash fully with distilled water;
(6)将步骤(5)在80℃下干燥至恒重即得成品。 (6) Dry the step (5) at 80°C to constant weight to obtain the finished product.
实施例7优化菊粉发酵培养基生产细菌纤维素性能检测 Example 7 Optimizing the Inulin Fermentation Medium to Produce Bacterial Cellulose Performance Detection
(1)细菌纤维素持水性和复水性分析 (1) Analysis of water holding capacity and rehydration property of bacterial cellulose
细菌纤维素持水性和复水性分析结果见表4。由表4可知,与基础培养基相比,菊粉培养基培养的细菌纤维素在持水性和复水性方面略高于基础培养基培养的细菌纤维素。 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)细菌纤维素结晶度分析 (2) Bacterial cellulose crystallinity analysis
根据X-射线衍射实验数据,分别根据公式计算各培养基细菌纤维素的结晶度,结果见表5。与基础培养基相比,菊粉培养基发酵的细菌纤维素结晶度83.99%,比基础培养基的76.27%提高了10.12%。 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)细菌纤维素热稳定性分析 (3) Thermal stability analysis of bacterial cellulose
图2、图3为基础培养基和菊粉培养基发酵的细菌纤维素的TG-DTA图谱,由图2、图3的细菌纤维素TG曲线可知,所测样品的质量随温度变化可以分为三个阶段:0~170℃内的质量轻度损耗,170~550℃内的质量快速损耗阶段,550~800℃之间的质量轻微损耗阶段。在0~170℃阶段菊粉培养基、基础培养基所得的细菌纤维素的最大失重速率温度分别为65.1℃和46.9℃,表明该阶段的质量损失主要是细菌纤维素脱水产生的,故称失水阶段,由于所测样品没有达到恒重干燥的程度或者是因为吸收了空气中的水分,所以造成了最大失重速率处的温度不同;170~550℃阶段,最大失重速率处温度分别为347.5℃、333.5℃,此阶段质量降低速度快,持续时间长,为细菌纤维素的降解阶段,对于材料热稳定性采用最大失重速率法来衡量,最大失重速率处温度越高,细菌纤维素的热稳定性越好,说明热稳定性方面菊粉培养基优于基础培养基。 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.
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