CN101012442A - Method of preparing culture medium carbon source for producing bacteria cellulose - Google Patents

Abstract

The invention relates to a method for preparing a medium carbon source for producing bacterial cellulose, comprising (1) dilute acid hydrolysis of konjac flour: the solid/liquid ratio of konjac flour and dilute acid solution is 1:30-1:100 , hydrolyzed at 50-121°C for 0.5-8 hours; (2) Detoxification of the hydrolyzate: use NaOH, lime or Ca(OH) ₂ to detoxify the hydrolyzate, including adjusting the pH value of the konjac powder acid hydrolyzate to 4.5-5.5, or first adjusted to 9.5-11 and then back to 4.5-5.5, or combined with changing the detoxification time and activated carbon adsorption detoxification methods to improve the detoxification effect; (3) Preparation of bacterial cellulose: add 0.1-1 % yeast extract and 0.1-0.5% tryptone or peptone, make a culture medium, insert bacteria such as Acetobacter or glucose oxidizing bacteria with an inoculum of 6%-10% at 26-30°C, 160-250 rpm Cultivate in a shaker for 8-23 days or culture statically in an incubator at 26-30°C for 8-23 days. The medium carbon source prepared by the method has good quality and low price, and can be used in industrial production.

Description

A kind of preparation method of medium carbon source for producing bacterial cellulose

technical field

The invention relates to the field of microbial fermentation, in particular to a method for preparing a medium carbon source for producing bacterial cellulose,

Background technique

Konjac powder (Konjac powder) is a white powder product, which is derived from a monocotyledonous plant Konjac (Amorphophallus rerieri) and processed. Our country is rich in konjac resources, and 25 varieties have been found, which are cheap and good. The main component of konjac flour is glucomannan. In addition, it also contains a small amount of protein, 18 kinds of amino acids, vitamins, iron, calcium, phosphorus and other nutrients. It is now generally believed that the main component of konjac flour, glucomannan, is a polysaccharide linked by multiple β-1,4 glycosidic bonds by D-glucose and D-mannose at a molecular ratio of 1:1.6, and its molecular weight ranges from tens of Tens of thousands to several million belong to polymer compounds.

Bacterial Cellulose (BC for short), also known as Microbial Cellulose, is not only another type of natural inert material synthesized by bacteria besides plant cellulose on the earth, but also a recognized performance in the world. Excellent new biological material. Bacteria capable of producing cellulose mainly include Acetobacter, Rhizobium, Agrobacterium, and Sarcina, among which Acetobacter xylinum is the most researched and the most productive. From the molecular composition of cellulose, BC, like plant fiber, is a straight chain formed by β-D-glucose through β-1,4-glucosidic bonds. The straight chains are parallel to each other, not in a helical conformation, and without branching structure. , also known as β-1,4-glucan. But from the perspective of physical, chemical, and mechanical properties, it has its own unique properties. It is a new type of nano-biological material that has been widely used in various fields such as food, papermaking, medical materials, and sound diaphragms. hotspot.

In order to promote the application of this new type of natural nano-biological material, the cellulose must be produced on a large scale to reduce production costs. Among them, raw materials, especially carbon sources, account for a large proportion of production costs, so finding cheap and high-quality raw materials is a key. Since D-glucose and D-mannose are two carbon sources of culture medium for bacterial cellulose production, and konjac flour is rich in glucomannan, dilute acid is used to hydrolyze glucomannan in konjac flour to obtain two A complex sugar solution of monosaccharides, thereby obtaining a low-cost and high-quality medium carbon source that can be used for industrial production of bacterial cellulose. Since the konjac flour acid hydrolyzate contains some microbial growth inhibitors produced during the hydrolysis process, it is directly used as a fermentation carbon source, and the microorganisms do not grow or grow slowly, so it must be detoxified.

Contents of the invention

The purpose of this invention is to provide a kind of preparation method of the culture medium carbon source that is used for producing bacterial cellulose, the culture medium carbon source prepared by this method is of good quality, low price, can be used for industrial production.

A kind of preparation method for the medium carbon source that is used to produce bacterial cellulose provided by the invention comprises the following steps:

1. Dilute acid hydrolysis of konjac flour

Use 0.5mol/L sulfuric acid or 1mol/L hydrochloric acid, the solid/liquid ratio of konjac flour and dilute acid solution is 1:30-1:100, hydrolyze at 50-100°C for 1-8 hours, or higher temperature Shorten the hydrolysis time under certain conditions, such as hydrolysis at 121°C for 30 minutes, and the concentration of reducing sugar in the hydrolyzate is about 20 g/L.

2. Detoxification of hydrolyzate

Method 1: Use NaOH to adjust the pH value of the konjac flour acid hydrolyzate to 4.5-5.5.

Method 2: Use NaOH to adjust the pH value of the konjac flour acid hydrolyzate to 4.5-5.5, immediately add 2% (mass percentage) activated carbon for adsorption (5-10min at room temperature), filter/centrifuge to remove the activated carbon and then fine-tune the pH value to 4.5-5.5.

Method 3: Use NaOH to adjust the pH value of the konjac flour acid hydrolyzate to 9.5-11, immediately add activated carbon with 1-6% of the hydrolyzate mass for adsorption, filter out the activated carbon and readjust the pH value to 4.5-5.5.

Method 4: Use NaOH to adjust the pH value of the konjac flour acid hydrolyzate to 9.5-11, react overnight in a water bath at 25-60° C., and then adjust the pH value to 4.5-5.5.

Method 5: Use NaOH to adjust the pH value of the konjac flour acid hydrolyzate to 9.5-11, react overnight in a 30°C water bath at 25-60°C, then adjust the pH value to 4.5-5.5, add activated carbon with a mass of 1-6% of the hydrolyzate for adsorption , filter out the charcoal and re-fine-tune the pH to 4.5-5.5.

Method 6: Using Ca(OH) ₂ to adjust the pH value of the acid hydrolyzate to 4.5-5.5.

Method 7: Use Ca(OH) ₂ to adjust the pH value of the acid hydrolyzate to 4.5-5.5, immediately add activated carbon with 1-6% of the hydrolyzate mass for adsorption, filter out the activated carbon and fine-tune the pH value to 4.5-5.5 again.

Method 8: Use Ca(OH) to adjust the pH value of the acid hydrolyzate to 9.5-11, _immediately add 1-6% (mass percentage) activated carbon for adsorption, filter out the activated carbon and readjust the pH value to 4.5-5.5.

Method 9: Use Ca(OH) ₂ to adjust the pH value of the acid hydrolyzate to 9.5-11, react overnight in a water bath at 25-60° C., and re-adjust the pH value to 4.5-5.5.

Method 10: Use Ca(OH) ₂ to adjust the pH value of the acid hydrolyzate to 9.5-11, react overnight in a water bath at 25-60°C, adjust the pH value to 5.5, then add 2% activated carbon to absorb the hydrolyzate, and filter Remove the charcoal and re-fine-tune the pH to 4.5-5.5.

The activated carbon is 1-6% (mass percentage) activated carbon and treated at room temperature for 5-10 minutes.

3. Preparation of Bacterial Cellulose

Take the carbon source of the above-mentioned medium, add 0.1-1% yeast extract and 0.1-0.5% tryptone to make a medium, and insert bacteria such as Acetobacter or Glucobacterium oxidizing bacteria at an inoculation amount of 6%-10%. 26-30°C, 160-250 rpm in a shaking table or static culture in a 26-30°C incubator, after a period of time (8-23 days), you can get a more ideal bacterial cellulose product or a richer Bacterial cellulose membrane.

However, the detoxification effects of the first five methods are not as good as those of the last five, and the yield and yield of bacterial cellulose are far lower than those of the last five methods. However, in the culture medium prepared by the carbon source detoxified by the 6-10 method, a relatively ideal bacterial cellulose film can be formed in about 8 days.

Because the hydrolyzate contains certain toxic substances, Acetobacter or Gluconobacter Oxydans cannot grow and synthesize bacterial cellulose in the medium with the hydrolyzate as the carbon source, so detoxification of the hydrolyzate is required.

The main advantage of the present invention is to use konjac flour, a common and cheap raw material, to carry out dilute acid hydrolysis and detoxification, and produce a kind of medium carbon source that can be used for bacterial cellulose cultivation, which is an industrialized large-scale production of bacterial cellulose This emerging material, Su, provides new ideas and approaches. The experimental data showed that under the same conditions, the yield of bacterial cellulose produced by the culture medium prepared with detoxified konjac flour hydrolyzate was higher than that of the culture medium prepared with pure glucose or pure mannose. After testing, the medium carbon source produced by the invention can also be used for the cultivation of other industrial microorganisms, and is a low-cost and high-quality carbon source.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

Use sulfuric acid to hydrolyze konjac flour, and then use Ca(OH) ₂ to adjust the pH value of the acid hydrolyzate to 4.5-5.5 for detoxification.

Prepare concentrated sulfuric acid into a dilute acid solution of 0.5mol/L, mix it with konjac powder at a solid/liquid ratio of 1:30-1:100, and stir with a glass rod while mixing to make the konjac powder swell as much as possible. Gel-like. Seal with a film, hydrolyze at 50-100°C for 1-8 hours, or at 121°C for 30 minutes, and stir several times in the middle to make the hydrolysis even. After hydrolysis, use filter paper to filter out the hydrolysis residue to obtain a hydrolysis clear liquid.

Add Ca(OH) ₂ to adjust the pH value of the hydrolyzed liquid to about 5.5, filter the precipitate with filter paper to obtain a detoxified hydrolyzed liquid, and then fine-tune the pH value to 5.5. The detoxified konjac flour hydrolyzate is tested for sugar and used as the carbon source of the medium, and then 0.1%-1% of yeast extract and 0.1%-0.5% tryptone or peptone are added to make konjac flour hydrolyzate medium ,spare.

Example 2

Use hydrochloric acid to hydrolyze konjac flour, then adjust the pH value of the acid hydrolyzate to 5.5 with Ca(OH) ₂ , immediately add 2% (mass percentage) activated carbon adsorption, filter the activated carbon and fine-tune the pH value to 5.5 again.

Concentrated hydrochloric acid is diluted to make a dilute acid solution of 1.0 mol/L, mixed with konjac fine powder at a solid/liquid ratio of 1:30-1:100, and stirred with a glass rod while mixing to make the konjac powder moisten as much as possible into a gel. Seal with a film, hydrolyze at 50-100°C for 1-8 hours, or at 121°C for 30 minutes, and stir several times in the middle to make the hydrolysis even. After hydrolysis, use filter paper to filter out the hydrolysis residue to obtain a hydrolysis clear liquid.

Add Ca(OH) ₂ to adjust the pH value of the hydrolyzed liquid to about 5.5, filter the precipitate with filter paper to obtain a detoxified hydrolyzed liquid, and then fine-tune the pH value to 5.5. Then add 2% (mass percentage) of activated carbon, stir (5-10min at room temperature) and filter out the activated carbon with filter paper to obtain a detoxified hydrolyzed clear solution, then fine-tune the pH value to 5.5 with dilute hydrochloric acid. The detoxified hydrolyzate was tested for sugar and used as the carbon source of the culture medium, and 0.1%-1% of yeast extract and 0.1%-0.5% tryptone were added therein to prepare the konjac flour hydrolyzate medium for future use.

Example 3

Use sulfuric acid to hydrolyze konjac flour, then use Ca(OH) ₂ to adjust the pH value of the acid hydrolyzate to 10, immediately add 2% (mass percentage) activated carbon adsorption, filter the activated carbon and readjust the pH value to 5.5.

Prepare concentrated sulfuric acid into a dilute acid solution of 0.5mol/L, mix it with konjac powder at a solid/liquid ratio of 1:30-1:100, and stir with a glass rod while mixing to make the konjac powder swell as much as possible. Gel-like. Seal with a film, hydrolyze at 50-100°C for 1-8 hours, or at 121°C for 30 minutes, and stir several times in the middle to make the hydrolysis even. After hydrolysis, use filter paper to filter out the hydrolysis residue to obtain a hydrolysis clear liquid.

Add Ca(OH) ₂ to adjust the pH value of the hydrolyzed liquid to about 10.0, filter the precipitate with filter paper to obtain the treated hydrolyzed liquid, and then fine-tune the pH value to 10.0. Then add 2% (mass percentage) activated carbon, stir (at room temperature for 5-10 min), filter out the activated carbon with filter paper to obtain a detoxified hydrolyzate, and fine-tune the pH value to 4.5-5.5 with dilute sulfuric acid. The detoxified hydrolyzate was tested for sugar and used as the carbon source of the culture medium, and 0.1%-1% of yeast extract and 0.1%-0.5% tryptone were added therein to prepare the konjac flour hydrolyzate medium for future use.

Example 4

Use hydrochloric acid to hydrolyze konjac flour, then use Ca(OH) ₂ to adjust the pH value of the acid hydrolyzate to 10, react overnight in a water bath at 25-60°C, and readjust the pH value to 5.5.

Concentrated hydrochloric acid is diluted to make a dilute acid solution of 1.0 mol/L, mixed with konjac fine powder at a solid/liquid ratio of 1:30-1:100, and stirred with a glass rod while mixing to make the konjac powder moisten as much as possible into a gel. Seal with a film, hydrolyze at 50-100°C for 1-8 hours, or at 121°C for 30 minutes, and stir several times in the middle to make the hydrolysis even. After hydrolysis, use filter paper to filter out the hydrolysis residue to obtain a hydrolysis clear liquid.

Add Ca(OH) ₂ to adjust the pH value of the hydrolyzed liquid to about 10.0, filter the precipitate with filter paper to obtain the treated hydrolyzed liquid, and then fine-tune the pH value to 10.0. Then seal it with a film, place it in a water bath at 25-60° C. to react overnight, and finally adjust the pH value of the hydrolyzed solution to 5.5 with dilute hydrochloric acid. The detoxified hydrolyzate was tested for sugar and used as the carbon source of the culture medium, and 0.1%-1% of yeast extract and 0.1%-0.5% tryptone were added therein to prepare the konjac flour hydrolyzate medium for future use.

Example 5

Use sulfuric acid to hydrolyze konjac flour, then use Ca(OH) ₂ to adjust the pH value of the acid hydrolyzate to 10, react overnight in a water bath at 25-60°C, adjust the pH value back to 5.5, and then add 2% (mass percentage) of activated carbon For adsorption, filter out the charcoal and re-fine-tune the pH to 5.5.

Prepare concentrated sulfuric acid into a dilute acid solution of 0.5mol/L, mix it with konjac powder at a solid/liquid ratio of 1:30-1:100, and stir with a glass rod while mixing to make the konjac powder swell as much as possible. Gel-like. Seal with a film, hydrolyze at 0-100°C for 1-8 hours, or at 121°C for 30 minutes, and stir several times in the middle to make the hydrolysis even. After hydrolysis, use filter paper to filter out the hydrolysis residue to obtain a hydrolysis clear liquid.

Add Ca(OH) ₂ to adjust the pH value of the hydrolyzed liquid to about 10, filter the precipitate with filter paper to obtain the treated hydrolyzed liquid, and then fine-tune the pH value to 10.0. Then seal it with a film, place it in a water bath at 25-60°C to react overnight, and finally adjust the pH value of the hydrolyzed solution to 5.5 with dilute acid.

Then add 2% (mass percentage) activated carbon, stir (at room temperature for 5-10 min), filter out the activated carbon with filter paper to obtain a detoxified hydrolyzate, and fine-tune the pH value to 4.5-5.5 with dilute sulfuric acid. The detoxified hydrolyzate was tested for sugar and used as the carbon source of the culture medium, and 0.1%-1% of yeast extract and 0.1%-0.5% tryptone were added therein to prepare the konjac flour hydrolyzate medium for future use.

Example 6

Use above-mentioned various methods to detoxify konjaku flour hydrolyzate, and regulate hydrolyzate sugar concentration to be 15g/L, prepare D-glucose, D-mannose and glucose-mannose mixed solution of same concentration respectively simultaneously (molar ratio 1 : 1.6), then add 0.1%-1% yeast extract and 0.1%-0.5% tryptone to make 50mL konjac flour hydrolyzate medium, glucose medium, mannose medium, and glucose- Mannose mixed media. Inoculate acetic acid bacteria or glucose oxidizing bacteria with 6-10% inoculum into the konjac flour hydrolyzate medium and culture it statically in a 30°C incubator for 8-23 days to obtain a more ideal bacterial cellulose product or more abundant bacteria Cellulose membrane, the experimental results are shown in Table 1.

Table 1 Absolute dry weight of bacterial cellulose produced by different types of medium (unit: g)

Carbon source type Absolute dry weight after 8 days 23 days absolute dry weight D-glucose 0.069±0.013 - D-Mannose 0.033±0.013 - Glucose-Mannose 0.041±0.014 - method 1 Unfilmed 0.077±0.006 Method 2 Unfilmed 0.164±0.023 Method 3 Unfilmed 0.134±0.028 Method 4 Unfilmed 0.098±0.023 Method 5 Unfilmed 0.130±0.001 Method 6 0.101±0.004 - Method 7 0.123±0.007 - Method 8 0.143±0.023 - Method 9 0.172±0.017 -

Method 10 0.212±0.047 -

It can be seen from Table 1 that the detoxification methods using Ca(OH) ₂ and Ca(OH) ₂ combined with activated carbon are far superior to those using NaOH and NaOH combined with activated carbon in terms of bacterial cellulose yield and yield. The carbon source prepared by the detoxification method of Ca(OH) ₂ and Ca(OH) ₂ combined with activated carbon can form a more ideal bacterial cellulose film in about 8 days, while the detoxification method of NaOH or NaOH combined with activated carbon It takes about 20 days for the detoxified carbon source to form a satisfactory bacterial cellulose film.

It can be seen from the table that the order of production of bacterial cellulose prepared from carbon sources detoxified by different methods is: method 10>method 9>method 8>method 7>method 6>D-glucose carbon source>glucose-mannose Mixed carbon source > D-mannose carbon source. The above sequence shows that under the same conditions, the bacterial cellulose yield produced by the culture medium prepared by using the detoxified konjac flour hydrolyzate is higher than that of the culture medium prepared by pure glucose or pure mannose. The preparation of the culture medium carbon source for bacterial cellulose production and the detoxification method have high practical application value and obvious advantages.

Claims (5)

1. a preparation method for producing the medium carbon source of bacterial cellulose, it is characterized in that comprising the following steps: (1) dilute acid hydrolysis of konjac flour Using 0.5mol/L sulfuric acid or 1mol/L hydrochloric acid, the solid/liquid ratio of konjac flour and dilute acid solution is 1:30-1:100, and hydrolyze at 50-121°C for 0.5-8 hours; (2) Detoxification of hydrolyzate Use Ca(OH) ₂ , lime or NaOH to detoxify the hydrolyzate, change the pH value and time of detoxification, and combine with activated carbon to adsorb and detoxify; (3) Preparation of bacterial cellulose Take the carbon source of the above-mentioned medium, add 0.1-1% yeast extract and 0.1-0.5% tryptone to make a medium, insert Acetobacter or Glucobacterium oxidans with an inoculum size of 6%-10%, and in 26 -30°C, 160-250 rpm shaker culture or static culture in 26-30°C incubator, after 8-23 days to produce bacterial cellulose. 2. A method for preparing a medium carbon source for producing bacterial cellulose according to claim 1, characterized in that: said step (1) is hydrolyzed at a temperature of 50-121° C. for 0.5-8 hours. 3. a kind of preparation method for the medium carbon source that is used to produce bacterial cellulose according to claim 1, is characterized in that: the detoxification of the hydrolyzate of described step (2) NaOH is to select following method: (1) NaOH adjusts the pH value to 4.5-5.5; (2) Adjust the pH value to 4.5-5.5 with NaOH, add activated carbon for adsorption, filter out the activated carbon and fine-tune the pH value to 4.5-5.5; (3) Adjust the pH value to 9.5-11 with NaOH, add activated carbon for adsorption, filter out the activated carbon and re-adjust the pH value to 4.5-5.5; (4) Adjust the pH value to 9.5-11 with NaOH, react overnight in a warm water bath at 25-60°C, and re-adjust the pH value to 4.5-5.5; 5) Adjust the pH value to 9.5-11 with NaOH, react overnight in a warm water bath at 25-60°C, adjust the pH value back to 4.5-5.5, then add activated carbon for adsorption, filter out the activated carbon and fine-tune the pH value to 4.5- 5.5. 4. a kind of preparation method that is used to produce the culture medium carbon source of bacterial cellulose according to claim 1, is characterized in that: described step (2) Ca (OH) The detoxification of _the hydrolyzate is selected The following methods: (1) Ca(OH) ₂ adjusts the pH value to 4.5-5.5; (2) Ca(OH) ₂ adjust the pH value to 4.5-5.5, add activated carbon for adsorption, filter out the activated carbon and fine-tune the pH value to 4.5-5.5; (3) Ca(OH) ₂ adjust the pH value to 9.5-11, add activated carbon for adsorption, filter out the activated carbon and re-adjust the pH value to 4.5-5.5; (4) Ca(OH) ₂ adjusted the pH value to 9.5-11, reacted overnight in a warm water bath at 25-60°C, and re-adjusted the pH value to 4.5-5.5; (5) Ca(OH) ₂ Adjust the pH value to 9.5-11, react overnight in a warm water bath at 25-60°C, adjust the pH value back to 4.5-5.5, add activated carbon for adsorption, filter out the activated carbon and fine-tune the pH value again to 4.5-5.5. 5. a kind of preparation method that is used to produce the medium carbon source of bacterial cellulose according to claim 4, is characterized in that: described gac is the gac of 1-6% (mass percentage) and processes 5 at room temperature -10min.