CN109735593B - Method for simply and rapidly producing PHB/bacterial cellulose composite material - Google Patents
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Description
Translated fromChinese技术领域technical field
本发明属于生物材料领域，具体涉及一种简单快速生产PHB/细菌纤维素复合材料的方法。The invention belongs to the field of biological materials, in particular to a method for simply and rapidly producing PHB/bacterial cellulose composite materials.
背景技术Background technique
新型材料的开发，是21世纪优先发展的科学领域。细菌纤维素(BC)是当今新型生物材料的研究热点之一。利用微生物发酵生产细菌纤维素.并将其应用于医药，在医药、造纸、生物医学工程和食品工业中具有广泛的应用前景。其中，由醋酸菌属产生的细菌纤维素不同于自然界广泛存在的纤维素，其具有如下独特的性质：(1)纤维素纯度高、结晶度高和重合度高，并且以单一纤维存在，这样在制备一些微小纤维产品时非常有利。传统微小纤维产品要从天然纤维出发制备，需要一系列特殊的加工过程。(2)细菌纤维素的纤维直径在0.01～0.1m之间，弹性模数为一般纤维的数倍至十倍以上，并且抗拉强度高。对纤维素的机械性能研究时发现细菌纤维素的扬氏模量高达15×10Pa。(3)具有较高的生物适应性，并且在自然界可直接降解，不污染环境。The development of new materials is a priority scientific field in the 21st century. Bacterial cellulose (BC) is one of the research hotspots of new biomaterials. Using microbial fermentation to produce bacterial cellulose and applying it to medicine has broad application prospects in medicine, papermaking, biomedical engineering and food industry. Among them, the bacterial cellulose produced by Acetobacter is different from the cellulose widely existing in nature, and it has the following unique properties: (1) cellulose has high purity, high crystallinity and high degree of overlap, and exists as a single fiber, so Very advantageous in the preparation of some microfiber products. The preparation of traditional microfiber products from natural fibers requires a series of special processing procedures. (2) The fiber diameter of bacterial cellulose is between 0.01 and 0.1 m, the elastic modulus is several times to ten times that of ordinary fibers, and the tensile strength is high. When studying the mechanical properties of cellulose, it was found that the Young's modulus of bacterial cellulose is as high as 15×10Pa. (3) It has high biological adaptability and can be directly degraded in nature without polluting the environment.
聚β-羟基丁酸酯(PHB)是一种具有生物降解性和可再生性的新型生物塑料，可被多种生物体合成，贮存于细胞内，当碳源贫乏时可以被重新分解作为细胞生长的碳源和能源。尽管PHB被认为是医疗、农业和食品包装等传统塑料的有效替代品，但这种生物聚合物的全面商业化受到其高生产成本的阻碍。为了克服这些局限性，细菌培养生成PHB引起了研究者的广泛兴趣。真养产碱杆菌(Ralstonia eutropha)H16是一种可以积累PHB的优良菌株，能利用果糖大量合成PHB，可达到细胞干重(CDW)的80％。Ralstonia eutropha是一种在土壤和淡水中普遍存在的生物，可以在短暂的缺氧环境中生活。其关键的生活条件之一是它能同时使用有机化合物和H2分子作为能量来源。利用微生物产生代谢产物以其原料易得、环境友好、可再生而表现出非常好的应用前景，如何提高这些代谢产物的产量也引起了众多研究者与企业家的广泛关注。Poly-β-hydroxybutyrate (PHB) is a new bioplastic with biodegradability and renewability. It can be synthesized by a variety of organisms, stored in cells, and can be re-decomposed as cells when carbon sources are poor. Carbon and energy sources for growth. Although PHB is considered an effective alternative to traditional plastics such as medical, agricultural and food packaging, the full commercialization of this biopolymer is hindered by its high production cost. To overcome these limitations, the production of PHB by bacterial culture has attracted widespread interest among researchers. Ralstonia eutropha H16 is an excellent strain that can accumulate PHB. It can synthesize a large amount of PHB using fructose, which can reach 80% of the dry cell weight (CDW). Ralstonia eutropha is a ubiquitous organism in soil and freshwater that can live in a transient anoxic environment. One of its key living conditions is its ability to use both organic compounds andH2 molecules as an energy source. The use of microorganisms to produce metabolites shows a very good application prospect because of their easy availability, environmental friendliness, and reproducibility. How to improve the yield of these metabolites has also attracted extensive attention of many researchers and entrepreneurs.
为了改善这些性能，可以将细菌纤维素和聚β-羟基丁酸酯(PHB)复合。由于两者固有的生物降解性和生物相容性，开发以可持续方式制备的完全可生物降解的“绿色”新型复合材料具有广泛的应用前景。另外，基于聚合物增强的细菌纤维素新型复合材料对于结构应用同样具有非常强的吸引力。对于细菌纤维素与PHB的结合很少有报道，目前，将两者结合所用的方法比较单一，即将购买的细菌纤维素浸泡在用氯仿溶解PHB的溶液中，制得PHB/细菌纤维素的复合材料。To improve these properties, bacterial cellulose can be compounded with poly-β-hydroxybutyrate (PHB). Due to the inherent biodegradability and biocompatibility of both, the development of fully biodegradable "green" novel composites prepared in a sustainable manner has broad application prospects. In addition, novel composites based on polymer-reinforced bacterial cellulose are also very attractive for structural applications. There are few reports on the combination of bacterial cellulose and PHB. At present, the method used to combine the two is relatively simple, that is, soak the purchased bacterial cellulose in the solution of dissolving PHB with chloroform to prepare the composite of PHB/bacterial cellulose. Material.
发明内容SUMMARY OF THE INVENTION
本发明针对现有技术的不足，目的在于提供一种简单快速生产PHB/细菌纤维素复合材料的方法。The present invention aims at providing a simple and fast method for producing PHB/bacterial cellulose composite material in view of the deficiencies of the prior art.
为实现上述发明目的，本发明采用的技术方案为：In order to realize the above-mentioned purpose of the invention, the technical scheme adopted in the present invention is:
一种简单快速生产PHB/细菌纤维素复合材料的方法，包括如下步骤：A simple and rapid method for producing PHB/bacterial cellulose composite material, comprising the following steps:
(1)Ralstonia eutropha菌液的培养：将Ralstonia eutropha菌株在种子培养基中活化，待菌株生长24h后，将所得菌液接种至基础培养基Ⅰ中进行扩大培养至OD600值为1，再将所得菌液接种至基础培养基Ⅰ中重复扩大培养至OD600值为1；(1) Cultivation of Ralstonia eutropha bacterial liquid: the Ralstonia eutropha strain is activated in the seed medium, and after the bacterial strain has grown for 24 hours, the obtained bacterial liquid is inoculated into the basal medium I for expansion to an OD600 value of 1, and then the The obtained bacterial liquid was inoculated into the basal medium I and repeatedly expanded and cultivated to an OD600 value of 1;
(2)Gluconacetobacter xylinus菌液的培养：将葡糖醋酸杆菌Gluconacetobacter xylinus在基础培养基II中活化，待Gluconacetobacter xylinus菌体动态培养生长48h后，将所得菌液接种至基础培养基II中进行动态扩大培养24h，再将所得菌液接种至基础培养基II中重复动态扩大培养24h，再一次将所得菌液接种至基础培养基II中进行动态扩大培养24h；(2) Cultivation of Gluconacetobacter xylinus bacterial liquid: Gluconacetobacter Gluconacetobacter xylinus was activated in basal medium II, and after Gluconacetobacter xylinus bacterial growth was dynamically cultivated for 48 hours, the obtained bacterial liquid was inoculated into basal medium II for dynamic expansion Culture for 24h, then inoculate the obtained bacterial liquid into basal medium II to repeat dynamic expansion culture for 24h, and inoculate the obtained bacterial liquid into basal medium II again for dynamic expansion culture for 24h;
(3)取步骤(1)扩大培养所得Ralstonia eutropha菌液加入到步骤(2)培养所得含有Gluconacetobacter xylinus菌体的基础培养基Ⅱ中，先进行动态培养，再进行静态培养，培养结束后，收集菌液，提取细菌纤维素/PHB复合产物。(3) Take the Ralstonia eutropha bacterial liquid obtained from the expanded culture in the step (1) and add it to the basal medium II containing the Gluconacetobacter xylinus cells obtained from the culture in the step (2), first carry out dynamic culture, and then carry out static culture. After the culture is completed, collect Bacterial liquid to extract bacterial cellulose/PHB composite products.
上述方案中，步骤(1)所述Ralstonia eutropha菌株为Ralstonia eutropha H16(DSM428)；步骤(2)所述葡糖醋酸杆菌Gluconacetobacter xylinus为Gluconacetobacterxylinus ATCC700178。In the above scheme, the Ralstonia eutropha strain described in step (1) is Ralstonia eutropha H16 (DSM428); the Gluconacetobacter xylinus described in step (2) is Gluconacetobacterxylinus ATCC700178.
上述方案中，步骤(1)中所述种子培养基的配方为：胰蛋白胨17g/L，大豆蛋白胨3g/L，NaCl 5.0g/L，K2HPO4 2.5g/L，pH值为6.7。In the above scheme, the formula of the seed medium in step (1) is: tryptone 17g/L, soybean peptone 3g/L, NaCl 5.0g/L, K2 HPO4 2.5g/L, and pH value is 6.7.
上述方案中，步骤(1)中所述基础培养基I的配方为：果糖20g/L，NH4Cl 1g/L，NaH2PO4·2H2O 5.20g/L，Na2HPO4·12H2O 11.6g/L，K2SO4 0.45g/L，MgSO4·7H2O 0.799g/L，CaCl2·2H2O 0.082g/L，微量元素溶液1mL，pH值为6.7；所述微量元素溶液的组分为：FeSO4·7H2O 15g/L，MnSO4·H2O 2.4g/L，ZnSO4·7H2O 2.4g/L，CuSO4·5H2O 0.48g/L，HCl0.1M。In the above scheme, the formula of basal medium I described in step (1) is: fructose 20g/L, NH4 Cl 1g/L, NaH2 PO4 .2H2 O 5.20g/L, Na2 HPO4 .12H2 O 11.6g/L, K2 SO4 0.45g/L, MgSO4 ·7H2 O 0.799g/L, CaCl2 ·2H2 O 0.082g/L, trace element solution 1mL, pH value 6.7; The components of the trace element solution are: FeSO4 ·7H2 O 15g/L, MnSO4 ·H2 O 2.4g/L, ZnSO4 ·7H2 O 2.4g/L, CuSO4 ·5H2 O 0.48g/L , HCl 0.1M.
上述方案中，步骤(2)中所述基础培养基II的配方为：葡萄糖50g/L，酵母提取物5g/L，胰蛋白胨5g/L，柠檬酸1g/L，KH2PO4 1g/L，Na2HPO4 2g/L，pH值5.0。In the above scheme, the formula of the basal medium II in step (2) is: glucose 50g/L, yeast extract 5g/L, tryptone 5g/L, citric acid 1g/L, KH2 PO4 1g/L , Na2 HPO4 2g/L, pH value 5.0.
上述方案中，步骤(3)中所述动态培养的条件为：温度30℃，摇床转速200r/min，培养时间24h～36h；所述静态培养的条件为：温度30℃，培养时间72h～600h。In the above scheme, the conditions of the dynamic culture in step (3) are: temperature 30°C, shaker rotation speed 200r/min, and culture time 24h～36h; the static culture conditions are: temperature 30°C, culture time 72h～ 600h.
上述方案中，步骤(3)中所述Ralstonia eutropha菌液的加入量为5％～8％。In the above scheme, the addition amount of the Ralstonia eutropha bacterial solution in step (3) is 5% to 8%.
本发明的有益效果如下：本发明提出了一种快速简单的生产细菌纤维素和PHB复合材料的方法，只需在适宜的条件下混合培养Ralstonia eutropha菌体和Gluconacetobacter xylinus菌体，则可得到细菌纤维素和PHB的复合材料，制备所得PHB/细菌纤维素复合材料在具有高拉伸强度和弹性模量的同时，还有具有生物相容性和可降解性的特点，本发明所述方法具有操作简单，成本低廉，条件温和，环境友好等优点。The beneficial effects of the present invention are as follows: the present invention proposes a fast and simple method for producing bacterial cellulose and PHB composite materials, and only needs to mix and cultivate Ralstonia eutropha thalline and Gluconacetobacter xylinus thalline under suitable conditions, then bacteria can be obtained The composite material of cellulose and PHB, the obtained PHB/bacterial cellulose composite material has the characteristics of high tensile strength and elastic modulus, as well as biocompatibility and degradability, and the method of the present invention has the advantages of high tensile strength and elastic modulus. It has the advantages of simple operation, low cost, mild conditions and environmental friendliness.
附图说明Description of drawings
图1为实施例1制备所得PHB/BC复合材料的FTIR谱图。1 is the FTIR spectrum of the PHB/BC composite material prepared in Example 1.
图2为实施例2制备所得PHB/BC复合材料的FTIR谱图。FIG. 2 is the FTIR spectrum of the PHB/BC composite material prepared in Example 2. FIG.
图3为实施例3制备所得PHB/BC复合材料的FTIR谱图。FIG. 3 is the FTIR spectrum of the PHB/BC composite material prepared in Example 3. FIG.
具体实施方式Detailed ways
为了更好地理解本发明，下面结合实施例进一步阐明本发明的内容，但本发明的内容不仅仅局限于下面的实施例。In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with the embodiments, but the content of the present invention is not limited to the following embodiments.
以下实施例中，所述种子培养基的组分配方为：胰蛋白胨17g/L，大豆蛋白胨3g/L，NaCl5.0g/L，K2HPO4 2.5g/L，pH值为6.7。In the following examples, the component formula of the seed medium is: tryptone 17g/L, soybean peptone 3g/L, NaCl 5.0g/L, K2 HPO4 2.5g/L, and pH 6.7.
所述基础培养基I的配方为：果糖20g/L，NH4Cl 1g/L，NaH2PO4·2H2O 5.20g/L，Na2HPO4·12H2O 11.6g/L，K2SO4 0.45g/L，MgSO4·7H2O 0.799g/L，CaCl2·2H2O 0.082g/L，微量元素溶液1mL，pH值为6.7；所述微量元素溶液的组分为：FeSO4·7H2O 15g/L，MnSO4·H2O 2.4g/L，ZnSO4·7H2O 2.4g/L，CuSO4·5H2O 0.48g/L，HCl 0.1M。The formula of the basal medium I is: fructose 20g/L, NH4 Cl 1g/L, NaH2 PO4 2H2 O 5.20g/L, Na2 HPO4 12H2 O 11.6g/L, K2 SO4 0.45g/L, MgSO4 ·7H2 O 0.799g/L, CaCl2 ·2H2 O 0.082g/L, trace element solution 1mL, pH value 6.7; the composition of the trace element solution: FeSO4 ·7H2 O 15g/L, MnSO4 ·H2 O 2.4g/L, ZnSO4 ·7H2 O 2.4g/L, CuSO4 ·5H2 O 0.48g/L, HCl 0.1M.
所述基础培养基II的配方为：葡萄糖50g/L，酵母提取物5g/L，胰蛋白胨5g/L，柠檬酸1g/L，KH2PO4 1g/L，Na2HPO4 2g/L，pH值5.0。The formula of the basal medium II is: glucose 50g/L, yeast extract 5g/L, tryptone 5g/L, citric acid 1g/L, KH2 PO4 1g/L, Na2 HPO4 2g/L, pH 5.0.
实施例1Example 1
一种简单快速生产PHB/细菌纤维素复合材料的方法，包括如下步骤：A simple and rapid method for producing PHB/bacterial cellulose composite material, comprising the following steps:
(1)微生物培养：将保存在冷冻干燥管里的Ralstonia eutropha H16(DSN428)菌株在种子培养基中活化，待生长24h后，以5％的接种量，将所得菌液接种至基础培养基Ⅰ中进行扩大培养至OD600值为1，以相同接种量、将菌液再次接种至基础培养基Ⅰ中进行重复扩大培养至其OD600值为1；(1) Microbial culture: the Ralstonia eutropha H16 (DSN428) strain stored in the freeze-dried tube is activated in the seed medium, and after 24 hours of growth, with 5% of the inoculum, the obtained bacterial liquid is inoculated into the basal medium I Carry out the expansion culture to the OD600 value of 1 in the same inoculum amount, and inoculate the bacterial liquid into the basal medium I again to repeat the expansion culture to its OD600 value of 1;
(2)将保存在冷冻干燥管里的Gluconacetobacter xylinus ATCC700178菌株在基础培养基II中活化，待生长48h后，将所得菌液接种至基础培养基II中进行扩大培养24h，接种量为5％，取相同接种量的菌液再次接种至基础培养基II中进行重复扩大培养24h，再次取相同接种量的菌液接种至两组基础培养基II中，培养24h；(2) the Gluconacetobacter xylinus ATCC700178 strain stored in the freeze-drying tube is activated in the basal medium II, after 48h of growth, the obtained bacterial liquid is inoculated into the basal medium II and expanded for 24h, and the inoculation amount is 5%, Take the bacterial liquid of the same inoculum amount and inoculate it into the basal medium II again for repeated expansion for 24h, then take the bacterial liquid of the same inoculum amount again and inoculate it into the two groups of basic medium II, and cultivate for 24h;
(3)微生物混合培养：取步骤(2)培养了24h后的两组基础培养基II，向其中一组中加入1ml步骤(1)已经扩大培养、OD600值为1的Ralstonia eutropha菌株培养液，另外一组什么都不加；将这两组放置在相同的条件下生长，每一组的基础培养基体积是100ml，生长条件是在30℃，200转/每分钟，摇床动态培养24h，再同时放入30℃恒温箱静态培养120h；收集菌液，提取细菌纤维素/PHB复合产物。(3) Mixed cultivation of microorganisms: take two groups of basal medium II after culturing in step (2) for 24 hours, and add 1 ml of the Ralstonia eutropha strain culture liquid that has been expanded in step (1) and has an OD600 value of 1 to one group. , the other group did not add anything; the two groups were placed under the same conditions for growth, the basal medium volume of each group was 100ml, and the growth conditions were at 30°C, 200 rpm, and dynamic culture on a shaker for 24h. , and then placed in a 30°C incubator for static culture for 120h; the bacterial liquid was collected, and the bacterial cellulose/PHB composite product was extracted.
复合材料(细菌纤维素/PHB复合产物)的性能测试：培养结束后，取培养菌液的气-固液面上的PHB/BC膜，测量其厚度。将PHB/BC(细菌纤维素/PHB复合产物)放入超纯水中煮沸2h，取出，冷冻干燥，进行傅里叶红外光谱测试和拉伸测试分析。Performance test of the composite material (bacterial cellulose/PHB composite product): after the cultivation, take the PHB/BC film on the gas-solid liquid surface of the cultured bacterial solution, and measure its thickness. The PHB/BC (bacterial cellulose/PHB composite product) was put into ultrapure water and boiled for 2 hours, taken out, freeze-dried, and analyzed by Fourier transform infrared spectroscopy and tensile test.
图1为PHB/BC复合材料的红外光谱，红外测试表明，PHB/BC复合材料的红外峰具有BC和PHB各自的特征峰，表明其PHB/BC复合材料是BC和PHB的复合材料。表1为拉力分析测试结果，结果表明，PHB/BC复合膜的拉伸测试与纯BC膜相比，前者的拉伸强度和弹性模量明显高于后者。Figure 1 shows the infrared spectrum of the PHB/BC composite material. The infrared test shows that the infrared peak of the PHB/BC composite material has the respective characteristic peaks of BC and PHB, indicating that the PHB/BC composite material is a composite material of BC and PHB. Table 1 shows the test results of tensile analysis. The results show that the tensile strength and elastic modulus of the PHB/BC composite film are significantly higher than those of the pure BC film.
表1拉力测试分析Table 1 Tensile test analysis
实施例2Example 2
一种简单快速生产PHB/细菌纤维素复合材料的方法，包括如下步骤：A simple and rapid method for producing PHB/bacterial cellulose composite material, comprising the following steps:
(1)微生物培养：将保存在冷冻干燥管里的Ralstonia eutropha H16(DSM428)菌株在种子培养基中活化，待生长24h后，以5％的接种量，将所得菌液接种至基础培养基Ⅰ中进行扩大培养至OD600值为1，以相同接种量、将菌液再次接种至基础培养基Ⅰ中进行重复扩大培养至其OD600值为1；(1) Microbial culture: the Ralstonia eutropha H16 (DSM428) strain stored in the freeze-dried tube was activated in the seed medium, and after 24 hours of growth, with 5% of the inoculum, the obtained bacterial liquid was inoculated into the basal medium I Carry out the expansion culture to the OD600 value of 1 in the same inoculum amount, and inoculate the bacterial liquid into the basal medium I again to repeat the expansion culture to its OD600 value of 1;
(2)将保存在冷冻干燥管里的Gluconacetobacter xylinus ATCC700178菌株在基础培养基II中活化，待生长48h后，将所得菌液接种至基础培养基II中进行扩大培养24h，接种量为5％，取相同接种量的菌液再次接种至基础培养基II中进行重复扩大培养24h，再次取相同接种量的菌液接种至两组基础培养基II中，培养24h；(2) the Gluconacetobacter xylinus ATCC700178 strain stored in the freeze-drying tube is activated in the basal medium II, after 48h of growth, the obtained bacterial liquid is inoculated into the basal medium II and expanded for 24h, and the inoculation amount is 5%, Take the bacterial liquid of the same inoculum amount and inoculate it into the basal medium II again for repeated expansion for 24h, then take the bacterial liquid of the same inoculum amount again and inoculate it into the two groups of basic medium II, and cultivate for 24h;
(3)微生物混合培养：取步骤(2)培养了24h后的两组基础培养基II，向其中一组中加入3ml步骤(1)已经扩大培养、OD600值为1的Ralstonia eutropha菌株培养液，另外一组什么都不加；将这两组放置在相同的条件下生长，每一组的基础培养基体积是100ml，生长条件是在30℃，200转/每分钟，摇床动态培养24h，再同时放入30℃恒温箱静态培养72h；收集菌液，提取细菌纤维素/PHB复合产物。(3) Mixed cultivation of microorganisms: take two groups of basal medium II after culturing for 24 hours in step (2), add 3 ml of the Ralstonia eutropha strain culture liquid that has been expanded in step (1) and has an OD600 value of 1 to one group of them , the other group did not add anything; the two groups were placed under the same conditions for growth, the basal medium volume of each group was 100ml, and the growth conditions were at 30°C, 200 rpm, and dynamic culture on a shaker for 24h. , and then placed in a 30°C incubator for static culture for 72 hours; the bacterial liquid was collected, and the bacterial cellulose/PHB composite product was extracted.
复合材料(细菌纤维素/PHB复合产物)的性能测试：培养结束后，取培养菌液的气-固液面上的PHB/BC膜，测量其厚度。将PHB/BC(细菌纤维素/PHB复合产物)放入超纯水中煮沸2h，取出，冷冻干燥，进行傅里叶红外光谱测试和拉伸测试分析。Performance test of the composite material (bacterial cellulose/PHB composite product): after the cultivation, take the PHB/BC film on the gas-solid liquid surface of the cultured bacterial solution, and measure its thickness. The PHB/BC (bacterial cellulose/PHB composite product) was boiled in ultrapure water for 2 hours, taken out, freeze-dried, and analyzed by Fourier transform infrared spectroscopy and tensile test.
图1为PHB/BC复合材料的红外光谱，红外测试表明，PHB/BC复合材料的红外峰具有BC和PHB各自的特征峰，表明其PHB/BC复合材料是BC和PHB的复合材料。Figure 1 shows the infrared spectrum of the PHB/BC composite material. The infrared test shows that the infrared peak of the PHB/BC composite material has the respective characteristic peaks of BC and PHB, indicating that the PHB/BC composite material is a composite material of BC and PHB.
表2为拉力分析测试结果，结果表明，PHB/BC复合膜的拉伸测试与纯BC膜相比，前者的拉伸强度和弹性模量明显高于后者。Table 2 shows the results of the tensile analysis test. The results show that the tensile strength and elastic modulus of the PHB/BC composite film are significantly higher than those of the pure BC film in the tensile test of the former.
表2拉力测试分析Table 2 Tensile test analysis
实施例3Example 3
一种简单快速生产PHB/细菌纤维素复合材料的方法，包括如下步骤：A simple and rapid method for producing PHB/bacterial cellulose composite material, comprising the following steps:
(1)微生物培养：将保存在冷冻干燥管里的Ralstonia eutropha H16(DSM428)菌株在种子培养基中活化，待生长24h后，以5％的接种量，将所得菌液接种至基础培养基Ⅰ中进行扩大培养至OD600值为1，以相同接种量、将菌液再次接种至基础培养基Ⅰ中进行重复扩大培养至其OD600值为1；(1) Microbial culture: the Ralstonia eutropha H16 (DSM428) strain stored in the freeze-dried tube was activated in the seed medium, and after 24 hours of growth, with 5% of the inoculum, the obtained bacterial liquid was inoculated into the basal medium I Carry out the expansion culture to the OD600 value of 1 in the same inoculum amount, and inoculate the bacterial liquid into the basal medium I again to repeat the expansion culture to its OD600 value of 1;
(2)将保存在冷冻干燥管里的Gluconacetobacter xylinus ATCC700178菌株在基础培养基II中活化，待生长48h后，将所得菌液接种至基础培养基II中进行扩大培养24h，接种量为5％，取相同接种量的菌液再次接种至基础培养基II中进行重复扩大培养24h，再次取相同接种量的菌液接种至两组基础培养基II中，培养24h；(2) the Gluconacetobacter xylinus ATCC700178 strain stored in the freeze-drying tube is activated in the basal medium II, after 48h of growth, the obtained bacterial liquid is inoculated into the basal medium II and expanded for 24h, and the inoculation amount is 5%, Take the bacterial liquid of the same inoculum amount and inoculate it into the basal medium II again for repeated expansion for 24h, and again take the bacterial liquid of the same inoculum amount and inoculate it into the two groups of basic medium II, and cultivate for 24h;
(3)微生物混合培养：取步骤(2)培养了24h后的两组基础培养基II，向其中一组中加入5ml步骤(1)已经扩大培养、OD600值为1的Ralstonia eutropha菌株培养液，另外一组什么都不加；将这两组放置在相同的条件下生长，每一组的基础培养基体积是100ml，生长条件是在30℃，200转/每分钟，摇床动态培养24h，再同时放入30℃恒温箱静态培养600h；收集菌液，提取细菌纤维素/PHB复合产物。(3) Mixed culture of microorganisms: take two groups of basal medium II after culturing for 24 hours in step (2), add 5 ml of the Ralstonia eutropha strain culture liquid that has been expanded in step (1) and has an OD600 value of 1 to one group. , the other group did not add anything; the two groups were placed under the same conditions for growth, the basal medium volume of each group was 100ml, and the growth conditions were at 30°C, 200 rpm, and dynamic culture on a shaker for 24h. , and then placed in a 30°C incubator for static culture for 600h; the bacterial liquid was collected, and the bacterial cellulose/PHB composite product was extracted.
复合材料(细菌纤维素/PHB复合产物)的性能测试：培养结束后，取培养菌液的气-固液面上的PHB/BC膜，测量其厚度。将PHB/BC(细菌纤维素/PHB复合产物)放入超纯水中煮沸2h，取出，冷冻干燥，进行傅里叶红外光谱测试和拉伸测试分析。Performance test of the composite material (bacterial cellulose/PHB composite product): after the cultivation, take the PHB/BC film on the gas-solid liquid surface of the cultured bacterial solution, and measure its thickness. The PHB/BC (bacterial cellulose/PHB composite product) was boiled in ultrapure water for 2 hours, taken out, freeze-dried, and analyzed by Fourier transform infrared spectroscopy and tensile test.
图3为PHB/BC复合材料的红外光谱，红外测试表明，PHB/BC复合材料的红外峰具有BC和PHB各自的特征峰，表明其PHB/BC复合材料是BC和PHB的复合材料。表3为拉力分析测试结果，结果表明，PHB/BC复合膜的拉伸测试与纯BC膜相比，前者的拉伸强度和弹性模量明显高于后者。Figure 3 shows the infrared spectrum of the PHB/BC composite material. The infrared test shows that the infrared peak of the PHB/BC composite material has the respective characteristic peaks of BC and PHB, indicating that the PHB/BC composite material is a composite material of BC and PHB. Table 3 shows the test results of tensile analysis. The results show that the tensile strength and elastic modulus of the PHB/BC composite film are significantly higher than those of the pure BC film.
表3拉力测试分析Table 3 Tensile test analysis
显然，上述实施例仅仅是为清楚地说明所作的实例，而并非对实施方式的限制。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而因此所引申的显而易见的变化或变动仍处于本发明创造的保护范围之内。Obviously, the above-mentioned embodiments are only examples for clear illustration, rather than limiting the implementation. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. However, the obvious changes or changes derived therefrom still fall within the protection scope of the present invention.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101509026A (en)* | 2009-03-27 | 2009-08-19 | 上海应用技术学院 | Bacteria cellulose compound film, preparation and uses thereof |
| CN102816810A (en)* | 2012-09-10 | 2012-12-12 | 广东省微生物研究所 | A kind of microbial preparation method of bacterial cellulose which controls the degree of polymerization of bacterial cellulose |
| CN104312966A (en)* | 2008-03-03 | 2015-01-28 | 焦耳无限科技公司 | CO2 fixing engineered microorganisms producing carbon-based target products |
| CN107245232A (en)* | 2017-07-10 | 2017-10-13 | 东莞市联洲知识产权运营管理有限公司 | A kind of PHBV composites being modified based on bacteria cellulose |
| WO2018050092A1 (en)* | 2016-09-14 | 2018-03-22 | 四川蓝光英诺生物科技股份有限公司 | Artificial tissue precursor and preparation method therefor |
| CN109055445A (en)* | 2018-08-31 | 2018-12-21 | 武汉理工大学 | A method of alcaligenes eutrophus, which is improved, using optical drive produces PHB yield |
- 2018
- 2018-12-29CNCN201811639140.8Apatent/CN109735593B/enactiveActive
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104312966A (en)* | 2008-03-03 | 2015-01-28 | 焦耳无限科技公司 | CO2 fixing engineered microorganisms producing carbon-based target products |
| CN101509026A (en)* | 2009-03-27 | 2009-08-19 | 上海应用技术学院 | Bacteria cellulose compound film, preparation and uses thereof |
| CN102816810A (en)* | 2012-09-10 | 2012-12-12 | 广东省微生物研究所 | A kind of microbial preparation method of bacterial cellulose which controls the degree of polymerization of bacterial cellulose |
| WO2018050092A1 (en)* | 2016-09-14 | 2018-03-22 | 四川蓝光英诺生物科技股份有限公司 | Artificial tissue precursor and preparation method therefor |
| CN107245232A (en)* | 2017-07-10 | 2017-10-13 | 东莞市联洲知识产权运营管理有限公司 | A kind of PHBV composites being modified based on bacteria cellulose |
| CN109055445A (en)* | 2018-08-31 | 2018-12-21 | 武汉理工大学 | A method of alcaligenes eutrophus, which is improved, using optical drive produces PHB yield |
Non-Patent Citations (3)
| Title |
|---|
| Direct incorporation of glucosamine and N-acetylglucosamine into exopolymers by Gluconacetobacter xylinus (=Acetobacter xylinum) ATCC 10245: production of chitosan-cellulose and chitin-cellulose exopolymers;Lee J W等;《Applied and Environmental Microbiology》;20011231;第67卷(第9期);3970-3975* |
| In situ modifications to bacterial cellulose with the water insoluble polymer poly-3-hydroxybutyrate;Dianne R. Ruka等;《Carbohydrate Polymers》;20121109;第92卷;1717-1723* |
| 细菌纤维素增强聚羟基丁酸酯复合材料的制备及性能;蔡志江等;《高分子材料科学与工程》;20131231;第29卷(第12期);176-184* |
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