WO2021087738A1 - 一种利用酶法大豆水解液制备细菌纤维素膜的方法 - Google Patents
一种利用酶法大豆水解液制备细菌纤维素膜的方法 Download PDFInfo
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- WO2021087738A1 WO2021087738A1 PCT/CN2019/115679 CN2019115679W WO2021087738A1 WO 2021087738 A1 WO2021087738 A1 WO 2021087738A1 CN 2019115679 W CN2019115679 W CN 2019115679W WO 2021087738 A1 WO2021087738 A1 WO 2021087738A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/14—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
- A23J1/148—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds by treatment involving enzymes or microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D21/00—Separation of suspended solid particles from liquids by sedimentation
- B01D21/26—Separation of sediment aided by centrifugal force or centripetal force
- B01D21/262—Separation of sediment aided by centrifugal force or centripetal force by using a centrifuge
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
- C08J7/02—Chemical treatment or coating of shaped articles made of macromolecular substances with solvents, e.g. swelling agents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L1/00—Compositions of cellulose, modified cellulose or cellulose derivatives
- C08L1/02—Cellulose; Modified cellulose
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- the invention belongs to the technical field of preparation of bacterial cellulose membranes, and specifically relates to a method for preparing bacterial cellulose membranes by using an enzymatic soybean hydrolysate.
- Bacterial cellulose refers to certain types of bacteria from Acetobacter, Agrobacterium, Rhizobium and Sarcina under different conditions. A general term for cellulose synthesized by microorganisms. Bacterial cellulose forms a unique texture structure, and due to the "nano effect", it has high water absorption and high water retention, high permeability to liquids and gases, high wet strength, and can be processed in situ, especially in wet conditions. And other characteristics. High purity and excellent performance make the bacterial cellulose fiber can be widely used in special fields.
- Enzymatic extraction of vegetable oil is an emerging oil extraction technology developed in the 1970s. As an emerging vegetable oil extraction technology, it is based on mechanical crushing of oil tissues and complexes such as lipoproteins and lipopolysaccharides. Carry out enzymatic hydrolysis to free the oil. The remaining water phase of the enzymatic extraction of vegetable oil still contains many valuable components. Direct discarding can not avoid the waste of resources, so you can try to use them.
- the present invention provides a method for preparing a bacterial cellulose membrane by using an enzymatic soybean hydrolysate. Specific steps are as follows:
- the waste liquid in step (1) is prepared by the following method: the soybeans are crushed and then pressed into tablets, water is added to modulate the soybean powder mucilage, and then subjected to extrusion treatment, and the obtained soybean powder and water are mixed to obtain soybeans.
- the powder liquid, soybean powder liquid is then hydrolyzed by complex enzymes, and the hydrolyzed liquid is obtained by three-phase separation of the enzyme hydrolysate.
- the impurity removal in step (1) is to use a 300-400 mesh filter to filter the original liquid for impurity removal.
- the fermentation strain in step (2) is Kombucha.
- the inoculation concentration of the fermentation strain in step (2) is 6 ⁇ 10 6 -6 ⁇ 10 8 cfu/ml.
- the temperature conditions for the extrusion expansion are 40-60°C in one stage, 60-80°C in the second stage, 80-95°C in the third stage, and 95-105°C in the fourth stage.
- the temperature conditions for the extrusion expansion are 48°C in one stage, 72°C in the second stage, 86°C in the third stage, and 102°C in the fourth stage.
- the compound enzymatic hydrolysis refers to the use of a mixed enzyme soybean powder liquid of alkaline protease with a mass fraction of soybean powder of 4 ⁇ -8 ⁇ and a flavor protease with a mass fraction of soybean powder of 1 ⁇ -4 ⁇ . solution.
- the compound enzymatic hydrolysis is a mixed enzyme of alkaline protease with a mass fraction of soybean powder of 5 ⁇ and a flavor protease with a mass fraction of soybean powder of 2 ⁇ to perform enzymatic hydrolysis of the soybean powder liquid.
- the three-phase separation is performed under the conditions of a three-phase horizontal separator, a centrifugal rotation speed of 4300-4500r, and a centrifugation for 10-20s.
- the method of the present invention makes reasonable use of the waste liquid remaining from the enzymatic preparation of soybean oil without acidic water treatment of the culture medium.
- the bacterial strain directly uses the enzymatic soybean hydrolysate to synthesize the amount of bacterial cellulose, and the microfiber More dense, the maximum thermal degradation temperature is higher.
- FIG. 1 Three-phase horizontal separation results, a is the separation result of Example 1, b is the separation result of Example 2, and c is the separation result of Example 3;
- the materials, reagents, methods and instruments used in the following examples are all conventional materials, reagents, methods and instruments in the art without special instructions, and can be obtained by those skilled in the art through commercial channels.
- Kombucha is cultivated and produced by the Probiotic Family Kombucha Biological Cultivation Co., Ltd.
- Example 1 A method for preparing a bacterial cellulose membrane using an enzymatic soybean hydrolysate.
- Enzymatic soybean hydrolyzate is the waste liquid from the preparation of soybean oil by the enzymatic method. It is the hydrolysate obtained by crushing, tableting, conditioning, extruding, crushing and sieving, compound enzymatic hydrolysis, and three-phase separation of soybeans. The specific obtaining steps are as follows:
- step 3 The soybean flakes obtained in step 2) are mixed with water, and the mass fraction of the water is 10.5% to obtain the soybean powder slime;
- the puffing temperature is 48°C in one stage, 72°C in the second stage, 86°C in the third stage, and 102°C in the fourth stage;
- step 5) pulverize the extruded soybean powder in step 4) through a 70-mesh sieve, and mix the sieved soybean powder and water in a ratio of 1g:6.5ml to prepare a soybean powder liquid;
- FIG. 1 a Using a three-phase horizontal separator, 4400rad/min, centrifugation for 16s to obtain the hydrolyzate, the three-phase horizontal separation result is shown in Figure 1 a, where the bottom layer is the enzymatic soybean hydrolysate, and the hydrolysate is subjected to 121°C Sterilize for 19 minutes, and filter the sterilized hydrolysate with a 300-mesh filter to remove the precipitated impurities to obtain the enzymatic soybean hydrolysate.
- the pH of the enzymatic soybean hydrolysate obtained in the above process is adjusted to 4.5, the impurities are filtered with a 300-mesh filter, and the filtrate is sterilized to obtain an enzymatic soybean hydrolysate liquid medium.
- step 2) Inoculate the HS culture medium of step 1) with a volume fraction of 6% Kombucha, culture at a temperature of 28°C, and cultivate for 24 hours to complete the activation and rejuvenation of the strain;
- the components of the seed liquid are glucose with a mass concentration of 40g/L and black tea with a mass concentration of 5g/L;
- step 2) Inoculate the activated kombucha in the above step 2) into the seed liquid, the inoculation volume is 6% of the volume of the seed liquid, and culture it at 28° C. for 24 hours until the strain concentration is 10 7 cfu/ml.
- strains are inoculated into the enzymatic soybean hydrolysate liquid medium obtained in step (1) for fermentation, the inoculation volume fraction is 6%, the fermentation temperature is 28° C., and the fermentation time is 13 days to obtain a crude bacterial cellulose membrane.
- the cellulose membrane obtained in step (2) is boiled in water for 20 minutes, then boiled with 0.2M sodium hydroxide for 40 minutes, soaked in water to adjust the pH to neutral, and washed with water until the cellulose membrane is transparent.
- Example 2 A method for preparing a bacterial cellulose membrane using an enzymatic soybean hydrolysate.
- Enzymatic soybean hydrolyzate is the waste liquid from the preparation of soybean oil by the enzymatic method. It is the hydrolysate obtained by crushing, tableting, conditioning, extruding, crushing and sieving, compound enzymatic hydrolysis, and three-phase separation of soybeans. The specific obtaining steps are as follows:
- step 2) The soybean flakes obtained in step 2) are mixed with water, and the mass fraction of the water is 10% to obtain the soybean powder slime;
- the puffing temperature is 40°C in one stage, 60°C in the second stage, 80°C in the third stage, and 95°C in the fourth stage;
- step 5) pulverize the extruded soybean powder in step 4) through a 60-mesh sieve, and mix the sieved soybean powder with water in a ratio of 1g:6ml to prepare a soybean powder liquid;
- FIG. 7 Use a three-phase horizontal separator, 4300rad/min, centrifugation for 10s to obtain the hydrolysate.
- the three-phase horizontal separation result is shown in Figure 1 b.
- the bottom layer is the enzymatic soybean hydrolysate, and the hydrolysate is subjected to 121°C Sterilize for 18 minutes, and filter the sterilized hydrolysate with a 300-mesh filter to remove the precipitated impurities to obtain the enzymatic soybean hydrolysate.
- step 2) Inoculate the HS culture medium of step 1) with a volume fraction of 6% Kombucha, and cultivate at a temperature of 25°C for 20 hours to complete the activation and rejuvenation of the strain;
- the components of the seed liquid are glucose with a mass concentration of 20g/L and black tea with a mass concentration of 3g/L;
- step 2) Inoculate the activated kombucha in the above step 2) into the seed liquid, the inoculum volume is 6% of the volume of the seed liquid, and cultivate at 25° C. for 20 hours until the strain concentration is 10 6 cfu/ml.
- strains are inoculated into the enzymatic soybean hydrolysate liquid medium obtained in step (1) for fermentation, the inoculation volume fraction is 6%, the fermentation temperature is 25° C., and the fermentation time is 8 days to obtain a crude bacterial cellulose membrane.
- the cellulose membrane obtained in step (4) is boiled in water for 10 minutes, then boiled with 0.1M sodium hydroxide for 20 minutes, soaked in water to adjust the pH to neutral, and washed with water until the cellulose membrane is transparent.
- Example 3 A method for preparing a bacterial cellulose membrane using an enzymatic soybean hydrolysate.
- Enzymatic soybean hydrolyzate is the waste liquid from the preparation of soybean oil by the enzymatic method. It is the hydrolysate obtained by crushing, tableting, conditioning, extruding, crushing and sieving, compound enzymatic hydrolysis, and three-phase separation of soybeans. The specific obtaining steps are as follows:
- step 2) The soybean flakes obtained in step 2) are mixed with water, and the mass fraction of the water is 11% to obtain the soybean powder slime;
- the puffing temperature is 60°C in one stage, 80°C in the second stage, 95°C in the third stage, and 105°C in the fourth stage;
- step 4) The soybean powder extruded in step 4) is crushed and passed through an 80-mesh sieve, and the sieved soybean powder is mixed with water in a ratio of 1g:7ml to prepare a soybean powder liquid;
- FIG. 7 Using a three-phase horizontal separator, 4500rad/min, centrifugation for 20s to obtain the hydrolysate, the three-phase horizontal separation result is shown in Figure 1 c, where the bottom layer is the enzymatic soybean hydrolysate, and the hydrolysate is subjected to 121°C Sterilize for 20 minutes, and filter the sterilized hydrolysate with a 400-mesh filter to remove the precipitated impurities to obtain the enzymatic soybean hydrolysate.
- step 2) Inoculate the HS culture medium of step 1) with a volume fraction of 6% Kombucha, culture at a temperature of 32°C, and cultivate for 48 hours to complete the activation and rejuvenation of the strain;
- the components of the seed liquid are glucose with a mass concentration of 60g/L and black tea with a mass concentration of 7g/L;
- step 2) Inoculate the activated kombucha in the above step 2) into the seed liquid, the inoculation volume is 6% of the volume of the seed liquid, and cultivate at 32° C. for 48 hours until the strain concentration is 10 8 cfu/ml.
- strains are inoculated into the enzymatic soybean hydrolysate liquid medium obtained in step (1) for fermentation, the inoculation volume fraction is 6%, the fermentation temperature is 32° C., and the fermentation time is 15 days to obtain a crude bacterial cellulose membrane.
- the cellulose membrane obtained in step (4) was boiled for 30 minutes, then boiled with 1M sodium hydroxide for 60 minutes, soaked in water to adjust the pH to neutral, and washed with water until the cellulose membrane was transparent.
- Example 4 Comparative Example of Example 1.
- Example 2 It is the same as Example 1, except that in the step (2) of the crude bacterial cellulose membrane preparation in this example, the fermentation medium used in 4) also adopts the standard HS medium to prepare the bacterial cellulose membrane.
- Example 4 Comparing the bacterial cellulose synthesized in Example 1 and Example 4, the yield of cellulose obtained from the enzymatic soybean hydrolysate medium in Example 1 is 1.78 g/L, which is 1.25 g/L as compared with that of the standard HS medium in Example 4. Compared with g/L, the output has increased by 30.0%;
- the surface morphology of freeze-dried bacterial cellulose was observed by SEM scanning electron microscope (SEM, SU8010, HITACHI), and the results showed that the bacterial cellulose was composed of rod-shaped nanofibers and formed a porous three-dimensional network structure.
- the results obtained by the scanning electron microscope of Example 1 are shown in Figure 2, and the results of Example 4 are shown in Figure 3. Comparing the two figures, it can be seen that the fibers produced by the standard HS medium of Example 4 are slightly thicker, while the results of Example 4 1
- the bacterial fiber produced by the enzymatic soybean hydrolysate medium is slightly finer. The type of surface medium will affect the morphological properties of the fibers in the bacterial cellulose.
- Example 1 and Example 4 The cellulose diameters obtained in Example 1 and Example 4 were counted.
- the statistical results of Example 1 are shown in Fig. 4, the statistical results of Example 4 are shown in Fig. 5, and the cellulose in Figs. 4 and 5
- the diameter of the fiber is between 40nm-200nm. Compare the diameter of the bacterial cellulose fiber.
- the fiber diameter of the enzymatic soybean hydrolysate medium in Example 1 is 100nm, and the average fiber diameter of the standard HS medium in Example 4 is 114nm.
- the microfibers of bacterial cellulose obtained from the enzymatic soybean hydrolysate medium in Example 1 are finer and denser.
- the atomic force microscope can be used to observe the microscopic morphology and microscopic details of the bacterial cellulose surface, and to observe the typical dense and aggregated structure of freeze-dried bacterial cellulose more clearly.
- the atomic force microscope observation shows a nano-level network structure and the bacterial cellulose microfibers are compact. Stacked and arranged irregularly.
- the three-dimensional structure of the cellulose obtained in Example 1 was observed by AFM atomic force microscope (Bruker, Germany). The result is shown in Figure 6, and the three-dimensional structure of the cellulose obtained in Example 4 is shown in Figure 7.
- Example 1 The maximum thermal degradation temperature was analyzed by TGA thermogravimetric analyzer (Pyris 6 TGA, Perkin Elmer Co., Ltd. USA). The results of Example 1 are shown in Figure 8 and the results of Example 4 are shown in Figure 9.
- Bacterial cellulose exhibits two different thermal degradation stages. The first thermal degradation stage occurs at 90°C to 100°C. It is mainly the moisture content absorbed on the surface and the loss of coordinated water molecules between layers. The second thermal degradation stage occurs at 300°C to 400°C, which is the thermal degradation and cracking of the bacterial cellulose skeleton. Finally, the bacterial cellulose is decomposed into water, carbon dioxide and so on.
- the thermal degradation rate temperatures of the bacterial cellulose produced by the standard HS medium of Example 4 and the enzymatic soybean hydrolysate medium of Example 1 were 331.67°C and 338.89°C, respectively.
- the thermal degradation rate temperature increased by 7.22°C.
- Example 2 The yield, morphology, diameter and thermal degradation degree of cellulose obtained in Example 2 and Example 3 are similar to those in Example 1.
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Abstract
本发明提供了一种利用酶法大豆水解液制备细菌纤维素膜的方法,包括如下步骤(1)酶法大豆水解液培养基的制备;(2)细菌纤维素膜的粗品制备;(3)细菌纤维素膜的纯化。本发明的方法对酶法制备大豆油剩余的废液进行了合理利用,无需对培养基进行酸水处理,菌株直接利用酶法大豆水解液合成的细菌纤维素的量更高,微纤维更细密,最大热降解温度更高。
Description
本发明属于细菌纤维素膜制备的技术领域,具体涉及一种利用酶法大豆水解液制备细菌纤维素膜的方法。
细菌纤维素(Bacterial cellulose,BC)是指在不同条件下,由醋酸菌属(Acetobacter)、土壤杆菌属(Agrobacterium)、根瘤菌属(Rhizobium)和八叠球菌属(Sarcina)等中的某种微生物合成的纤维素的统称。细菌纤维素形成独特的织态结构,并因“纳米效应”而具有高吸水性和高保水性、对液体和气体的高透过率、高湿态强度、尤其在湿态下可原位加工成型等特性。高纯度和优异的性能使细菌纤维素纤维可在特殊领域广泛应用。
目前,有利用多种工农业废弃物作为制备细菌纤维素培养基的研究,如加工浓缩蛋白产生的大豆糖蜜、加工蔗糖或甜菜产生的糖蜜,因合成细菌纤维素的菌株在纤维素的合成过程利用单一葡萄糖,因此糖蜜作为培养基前需要进行高强度酸水解(盐酸、硝酸、硫酸等),从而提高菌株的发酵速率。另外,也有利用生产大豆分离蛋白产生的乳清水(清蛋白和白蛋白),不利于菌株的利用。
酶法提取植物油是在20世纪70年代发展起来的一项新兴提油技术,作为一种新兴的植物油脂提取技术,是在机械破碎的基础上,对油料组织以及脂蛋白、脂多糖等复合体进行酶解,从而使油脂游离出来。酶法提取植物油剩余的水相中依旧含有许多有价值的成分,直接丢弃避免不了对资源的浪费,因此可以尝试对其加以利用。
发明内容
为了解决酶法制备大豆油剩余的废液得不到合理利用的问题,本发明提供了一种利用酶法大豆水解液制备细菌纤维素膜的方法。具体步骤如下:
(1)酶法大豆水解液培养基的制备:以酶法制备大豆油剩余的废液作为原液,调节pH至4.3-4.6,除杂后作为培养基;
(2)细菌纤维素膜的粗品制备:将发酵菌种接种至步骤(1)获得的酶法大豆水解液液体培养基中进行发酵,发酵温度为25-32℃,发酵时间为8-15天,获得细菌纤维素膜粗品;
(3)细菌纤维素膜的纯化:将步骤(2)得到的细菌纤维素膜粗品沸水煮10-30min,再用0.1-1M氢氧化钠煮沸20-60min,水中浸泡调节pH至中性,换水冲洗直至纤维素膜呈透明。
优选的,步骤(1)所述废液通过如下方法制备获得:大豆破碎后制成压片,加水调质成大豆粉粘液,然后进行挤压膨化处理,将得到的大豆粉与水混合得大豆粉液,大豆粉液再经复合酶酶解,酶解液经三相分离得到的水解液。
优选的,步骤(1)所述的除杂是采用300-400目滤网过滤原液进行除杂。
优选的,步骤(2)所述发酵菌种为红茶菌。
优选的,步骤(2)所述发酵菌种接种浓度为6×10
6-6×10
8cfu/ml。
优选的,所述挤压膨化的温度条件是一段为40-60℃,二段为60-80℃,三段为80-95℃,四段为95-105℃。
优选的,所述挤压膨化的温度条件是一段为48℃,二段为72℃,三段为86℃,四段为102℃。
优选的,所述复合酶酶解是指用占大豆粉质量分数为4‰-8‰的碱性蛋白酶与占大豆粉质量分数为1‰-4‰的风味蛋白酶的混合酶大豆粉液进行酶解。
优选的,所述复合酶酶解是占大豆粉质量分数为5‰的碱性蛋白酶与占大豆粉质量分数为2‰的风味蛋白酶的混合酶对大豆粉液进行酶解。
优选的,所述三相分离是采用三项卧式分离机,离心转速为4300-4500r,离心10-20s的条件下进行的分离。
本发明所述的方法对酶法制备大豆油剩余的废液进行了合理利用,无需对培养基进行酸水处理,菌株直接利用酶法大豆水解液合成的细菌纤维素的量更高,微纤维更细密,最大热降解温度更高。
图1.三相卧式分离结果,a为实施例1分离结果,b为实施例2分离结果,c为实施例3分离结果;
图2.实施例1获得的细菌细菌纤维素扫描电子显微镜观察结果;
图3.实施例4获得的细菌细菌纤维素扫描电子显微镜观察结果;
图4.对实施例1获得的细菌细菌纤维素直径统计结果;
图5.对实施例4获得的细菌细菌纤维素直径统计结果;
图6.实施例1获得的细菌细菌纤维素原子力显微镜观察结果;
图7.实施例4获得的细菌细菌纤维素原子力显微镜观察结果;
图8.实施例1获得的细菌纤维素热降解温度结果;
图9.实施例4获得的细菌纤维素热降解温度结果。
下面结合具体实施例对本发明做进一步说明,但本发明不受实施例的限制。
以下实施例中所用材料、试剂、方法和仪器,未经特殊说明,均为本领域常规材料、试剂、方法和仪器,本领域技术人员均可通过商业渠道获得。红茶菌为益菌世家红茶菌生物培育有限公司培育生产。
实施例1.一种利用酶法大豆水解液制备细菌纤维素膜的方法。
本实施例所述的利用酶法大豆水解液制备细菌纤维素膜的方法,具体步骤如下:
(1)酶法大豆水解液培养基的制备:
酶法大豆水解液是酶法制备大豆油剩余的废液,是大豆经过破碎,压片,调质,挤压膨化,粉碎过筛,复合酶解,三相分离得到的水解液,具体获取步骤如下:
1)将大豆破碎成5瓣;
2)压成3mm厚的豆片;
3)步骤2)所得的豆片与水混合,水所占质量分数为10.5%,获得大豆粉粘液;
4)对步骤3)所得的获得大豆粉粘液进行挤压膨化,膨化温度一段为48℃,二段为72℃,三段为86℃,四段为102℃;
5)对步骤4)挤压膨化的大豆粉进行粉碎过70目筛,过筛后的大豆粉与水按照1g:6.5ml比例混合制成大豆粉液;
6)将占大豆粉质量分数为5‰的碱性蛋白酶与占大豆粉质量分数为2‰风味蛋白酶与制备的大豆粉液混合,55℃,水解5h,94℃灭酶3min。
7)采用三项卧式分离机,4400rad/min,离心16s得到水解液,三相卧式分离结果如图1中a所示,其中最下层为酶法大豆水解液,对水解液进行121℃灭菌19min,灭菌后的水解液采用300目过滤网过滤除去析出的杂质,获得酶法大豆水解液。
对上述过程获得的酶法大豆水解液调节pH至4.5,采用300目过滤网过滤除去杂质,对滤液进行灭菌,获得酶法大豆水解液液体培养基。
(2)细菌纤维素膜的粗品制备:
1)配制标准HS培养基,培养基成分为葡萄糖(20g/L)、酵母浸粉(5g/L)、蛋白胨(5g/L)、柠檬酸(1.15g/L)及磷酸氢二钠(2.7g/L),pH 6.0;
2)向步骤1)的HS培养基中接种体积分数为6%的红茶菌,培养温度为28℃,培养24h,完成菌株的活化复壮;
3)配制种子液,种子液成分为葡萄糖质量浓度为40g/L,红茶质量浓度为5g/L;
4)将上述步骤2)活化后的红茶菌接种入种子液中,接种体积为种子液体积的6%,在28℃下,培养24h,培养至菌种浓度为10
7cfu/ml。
将上述菌种接种至步骤(1)获得的酶法大豆水解液液体培养基中进行发酵,接种体积分数为6%,发酵温度为28℃,发酵时间为13天,获得细菌纤维素膜粗品。
(3)细菌纤维素膜的纯化:
将步骤(2)得到的纤维素膜沸水煮20min,再用0.2M氢氧化钠煮沸40min,水中浸泡调节pH至中性,换水冲洗直至纤维素膜呈透明。
实施例2.一种利用酶法大豆水解液制备细菌纤维素膜的方法。
本实施例所述的利用酶法大豆水解液制备细菌纤维素膜的方法,具体步骤如下:
(1)酶法大豆水解液培养基的制备:
酶法大豆水解液是酶法制备大豆油剩余的废液,是大豆经过破碎,压片,调质,挤压膨化,粉碎过筛,复合酶解,三相分离得到的水解液,具体获取步骤如下:
1)将大豆破碎成4瓣;
2)压成2mm厚的豆片;
3)步骤2)所得的豆片与水混合,水所占质量分数为10%,获得大豆粉粘液;
4)对步骤3)所得的获得大豆粉粘液进行挤压膨化,膨化温度一段为40℃,二段为60℃,三段为80℃,四段为95℃;
5)对步骤4)挤压膨化的大豆粉进行粉碎过60目筛,过筛后的大豆粉与水按照1g:6ml比例混合制成大豆粉液;
6)将占大豆粉质量分数为4‰的碱性蛋白酶与占大豆粉质量分数为1‰风味蛋白酶与制备的大豆粉液混合,50℃,水解2h,90℃灭酶3min。
7)采用三项卧式分离机,4300rad/min,离心10s得到水解液,三相卧式分离结果如图1中b所示,其中最下层为酶法大豆水解液,对水解液进行121℃灭菌18min,灭菌后的水解液采用300目过滤网过滤除去析出的杂质,获得酶法大豆水解液。
对上述过程获得的酶法大豆水解液调节pH至4.3,采用300目过滤网过滤除去析出的杂质,对滤液进行灭菌,获得酶法大豆水解液液体培养基。
(2)细菌纤维素膜的粗品制备:
1)配制标准HS培养基,培养基成分为葡萄糖(20g/L)、酵母浸粉(5g/L)、蛋白胨(5g/L)、柠檬酸(1.15g/L)及磷酸氢二钠(2.7g/L),pH 6.0;
2)向步骤1)的HS培养基中接种体积分数为6%的红茶菌,培养温度为25℃,培养20h,完成菌株的活化复壮;
3)配制种子液,种子液成分为葡萄糖质量浓度为20g/L,红茶质量浓度为3g/L;
4)将上述步骤2)活化后的红茶菌接种入种子液中,接种体积为种子液体积的6%,在25℃下,培养20h,培养至菌种浓度为10
6cfu/ml。
将上述菌种接种至步骤(1)获得的酶法大豆水解液液体培养基中进行发酵,接种体积分数为6%,发酵温度为25℃,发酵时间为8天,获得细菌纤维素膜粗品。
(3)细菌纤维素膜的纯化:
将步骤(4)得到的纤维素膜沸水煮10min,再用0.1M氢氧化钠煮沸20min,水中浸泡调节pH至中性,换水冲洗直至纤维素膜呈透明。
实施例3.一种利用酶法大豆水解液制备细菌纤维素膜的方法。
本实施例所述的利用酶法大豆水解液制备细菌纤维素膜的方法,具体步骤如下:
(1)酶法大豆水解液培养基的制备:
酶法大豆水解液是酶法制备大豆油剩余的废液,是大豆经过破碎,压片,调质,挤压膨化,粉碎过筛,复合酶解,三相分离得到的水解液,具体获取步骤如下:
1)将大豆破碎成6瓣;
2)压成4mm厚的豆片;
3)步骤2)所得的豆片与水混合,水所占质量分数为11%,获得大豆粉粘液;
4)对步骤3)所得的获得大豆粉粘液进行挤压膨化,膨化温度一段为60℃,二段为80℃,三段为95℃,四段为105℃;
5)对步骤4)挤压膨化的大豆粉进行粉碎过80目筛,过筛后的大豆粉与水按照1g:7ml比例混合制成大豆粉液;
6)将占大豆粉质量分数为8‰的碱性蛋白酶与占大豆粉质量分数为4‰风味蛋白酶与制备的大豆粉液混合,60℃,水解6h,90℃灭酶3min。
7)采用三项卧式分离机,4500rad/min,离心20s得到水解液,三相卧式分离结果如图1中c所示,其中最下层为酶法大豆水解液,对水解液进行121℃灭菌20min,灭菌后的水解液采用400目过滤网过滤除去析出的杂质,获得酶法大豆水解液。
对上述过程获得的酶法大豆水解液调节pH至4.6,采用400目过滤网过滤除去析出的杂质,对滤液进行灭菌,获得酶法大豆水解液液体培养基。
(2)细菌纤维素膜的粗品制备:
1)配制标准HS培养基,培养基成分为葡萄糖(20g/L)、酵母浸粉(5g/L)、蛋白胨(5g/L)、柠檬酸(1.15g/L)及磷酸氢二钠(2.7g/L),pH 6.0;
2)向步骤1)的HS培养基中接种体积分数为6%的红茶菌,培养温度为32℃,培养48h,完成菌株的活化复壮;
3)配制种子液,种子液成分为葡萄糖质量浓度为60g/L,红茶质量浓度为7g/L;
4)将上述步骤2)活化后的红茶菌接种入种子液中,接种体积为种子液体积的6%,在32℃下,培养48h,培养至菌种浓度为10
8cfu/ml。
将上述菌种接种至步骤(1)获得的酶法大豆水解液液体培养基中进行发酵,接种体积分数为6%,发酵温度为32℃,发酵时间为15天,获得细菌纤维素膜粗品。
(3)细菌纤维素膜的纯化:
将步骤(4)得到的纤维素膜沸水煮30min,再用1M氢氧化钠煮沸60min,水中浸泡调节pH至中性, 换水冲洗直至纤维素膜呈透明。
实施例4.实施例1的对比例。
与实施例1相同,不同之处在于本实施例中步骤(2)细菌纤维素膜的粗品制备中,4)中所用的发酵培养基也是采用标准HS培养基,制备细菌纤维素膜。
经实施例1与实施例4合成的细菌纤维素进行比对,实施例1酶法大豆水解液培养基制得的纤维素产量为1.78g/L,与实施例4标准HS培养基产量为1.25g/L相比,产量提高了30.0%;
通过SEM扫描电子显微镜(SEM,SU8010,HITACHI)观察冻干细菌纤维素的表面形态,结果显示了细菌纤维素由杆状的纳米纤维组成,并形成了多孔的三维网络结构。实施例1的扫描电子显微镜获得的结果如图2所示,实施例4的结果如图3所示,对比两幅图可知,由实施例4标准HS培养基生产的纤维略粗,而实施例1酶法大豆水解液培养基生产的细菌纤维略细。表面培养基种类会影响细菌纤维素中纤维的形态性能。
对实施例1与实施例4中所获得的纤维素直径进行统计,实施例1的统计结果如图4所示,实施例4的统计结果如图5所示,图4与图5中纤维素的纤维直径分布在40nm-200nm之间,比较细菌纤维素纤维直径,实施例1中酶法大豆水解液培养基获得的纤维直径为100nm,实施例4中标准HS培养基的平均纤维直径为114nm,实施例1中酶法大豆水解液培养基获得的细菌纤维素的微纤维更细密。
原子力显微镜可用于观察细菌纤维素表面的微观形态和微观细节,更加清晰的观察冻干细菌纤维素致密且聚集的典型结构,原子力显微镜观察显示出了纳米级的网络结构,细菌纤维素微纤维紧密堆积,呈不规则排列。通过AFM原子力显微镜(Bruker,Germany)观察实施例1获得的纤维素的三维结构,结果如图6所示,实施例4获得的纤维素的三维结构,结果如图7所示,比较标准HS培养基和实施例1中酶法大豆水解液培养基产生的细菌纤维素的微纤维直径宽度有所不同,在标准HS培养基形成的微纤维直径更宽,与SEM电镜观察的结果一致。
通过TGA热重分析仪(Pyris 6 TGA,Perkin Elmer Co.,Ltd.USA)分析最大热降解温度。实施例1的结果如图8所示,实施例4的结果如图9所示,细菌纤维素表现出了两个不同的热降解阶段,第一个热降解阶段发生在90℃至100℃,主要是表面上吸收的水分含量及层间配位水分子的损失。第二个热降解阶段发生在300℃至400℃,是细菌纤维素骨架的热降解和开裂,最终,细菌纤维素被分解为水、二氧化碳等。实施例4标准HS培养基与实施例1中酶法大豆水解液培养基产生的细菌纤维素的热降解速率温度分别为331.67℃和338.89℃。热降解速率温度提高了7.22℃。
实施例2与实施例3所获得的纤维素产量、形态、直径与热降解度均与实施例1相类似。
虽然本发明已以较佳的实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可以做各种改动和修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
- 一种利用酶法大豆水解液制备细菌纤维素膜的方法,其特征在于,由如下步骤制成:(1)酶法大豆水解液培养基的制备:以酶法制备大豆油剩余的废液作为原液,调节pH至4.3-4.6,除杂后作为培养基;(2)细菌纤维素膜的粗品制备:将发酵菌种接种至步骤(1)获得的酶法大豆水解液液体培养基中进行发酵,发酵温度为25-32℃,发酵时间为8-15天,获得细菌纤维素膜粗品;(3)细菌纤维素膜的纯化:将步骤(2)得到的细菌纤维素膜粗品沸水煮10-30min,再用0.1-1M氢氧化钠煮沸20-60min,水中浸泡调节pH至中性,换水冲洗直至纤维素膜呈透明。
- 根据权利要求1所述的利用酶法大豆水解液制备细菌纤维素膜的方法,其特征在于,步骤(1)所述废液通过如下方法制备获得:大豆破碎后制成压片,加水调质成大豆粉粘液,然后进行挤压膨化处理,将得到的大豆粉与水混合得大豆粉液,大豆粉液再经复合酶酶解,酶解液经三相分离得到的水解液。
- 根据权利要求1所述的利用酶法大豆水解液制备细菌纤维素膜的方法,其特征在于,步骤(1)所述的除杂是采用300-400目滤网过滤原液进行除杂。
- 根据权利要求1所述的利用酶法大豆水解液制备细菌纤维素膜的方法,其特征在于,步骤(2)所述发酵菌种为红茶菌。
- 根据权利要求1所述的利用酶法大豆水解液制备细菌纤维素膜的方法,其特征在于,步骤(2)所述发酵菌种接种浓度为6×10 6-6×10 8cfu/ml。
- 根据权利要求2所述的利用酶法大豆水解液制备细菌纤维素膜的方法,其特征在于,所述挤压膨化的温度条件是一段为40-60℃,二段为60-80℃,三段为80-95℃,四段为95-105℃。
- 根据权利要求6所述的利用酶法大豆水解液制备细菌纤维素膜的方法,其特征在于,所述挤压膨化的温度条件是一段为48℃,二段为72℃,三段为86℃,四段为102℃。
- 根据权利要求2所述的利用酶法大豆水解液制备细菌纤维素膜的方法,其特征在于,所述复合酶酶解是指用占大豆粉质量分数为4‰-8‰的碱性蛋白酶与占大豆粉质量分数为1‰-4‰的风味蛋白酶的混合酶大豆粉液进行酶解。
- 根据权利要求8所述的利用酶法大豆水解液制备细菌纤维素膜的方法,其特征在于,所述复合酶酶解是占大豆粉质量分数为5‰的碱性蛋白酶与占大豆粉质量分数为2‰的风味蛋白酶的混合酶对大豆粉液进行酶解。
- 根据权利要求2所述的利用酶法大豆水解液制备细菌纤维素膜的方法,其特征在于,所述三相分离是采用三项卧式分离机,离心转速为4300-4500r,离心10-20s的条件下进行的分离。
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