CN116925974B - Bacterial cellulose-producing strain and application thereof - Google Patents
Bacterial cellulose-producing strain and application thereof Download PDFInfo
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- CN116925974B CN116925974B CN202311043726.9A CN202311043726A CN116925974B CN 116925974 B CN116925974 B CN 116925974B CN 202311043726 A CN202311043726 A CN 202311043726A CN 116925974 B CN116925974 B CN 116925974B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of microorganisms, and relates to bacteria for producing bacterial cellulose and application thereof. The bacteria for producing bacterial cellulose are preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.22145, classified and named Komagataeibacter xylinus and the strain number of mm01 in the year 04 and 09 of 2021, and can produce bacterial cellulose with high yield and furfural resistance in the fermentation process. The bacterial strain can utilize cheap and easily available agricultural and forestry waste as a fermentation raw material, and efficiently produce bacterial cellulose through static or dynamic culture, so that the production efficiency of the bacterial cellulose is effectively improved, the production cost of the bacterial cellulose is reduced, and the bacterial strain is applicable to industrial large-scale production of the bacterial cellulose.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacteria for producing bacterial cellulose and application thereof.
Background
It has now been found that microorganisms capable of synthesizing bacterial cellulose are mostly concentrated in nine genera of aerobic bacteria, acetobacter, rhizobium, pseudomonas, sarcina, achromobacter, aerobacter, alcaligenes, azotobacter and Agrobacterium, respectively. These genera all have an intact system that synthesizes bacterial cellulose. Acetobacter is the first strain to find the bacterial cellulose to be synthesized, and is also the strain known to have the highest ability to synthesize bacterial cellulose.
The bacterial cellulose has the advantages of high chemical purity, high polymerization degree, high crystallinity, high mechanical strength, young's modulus, good tensile strength, hydrophilic water retention, biocompatibility and the like. The unique properties are widely applied to the fields of foods, textiles, cosmetics, medicines and the like, particularly in biomedicine, drug carriers, thickeners, food stabilizers and the like, the market demand is extremely high, however, the problems of high synthesis cost, low yield and the like of bacterial cellulose are bottlenecks of large-scale production and popularization and application, and researches show that the cost of a culture medium accounts for 50-65% of the total cost of the microbial fermentation production process. Therefore, the use of a cellulose synthesis medium with low cost and wide sources and the screening of bacterial species with high bacterial cellulose production are more critical in solving the bacterial cellulose production. At present, acetobacter has the characteristics of wide raw materials, strong fiber production capacity and the like, and is the strain which has the potential to be applied to industrialized mass production.
In recent years, along with the increasing global population and low-cost biosynthesis demands, it is becoming more and more important to convert wastes rich in cellulose and lignin as raw materials into carbon sources for microbial growth and fermentation, however, fermentation inhibitors such as furfural and the like can be generated in the processes of acid hydrolysis, steam explosion and high-temperature digestion pretreatment of lignin and cellulose, so that microbial growth is influenced, and the production efficiency is reduced, so that it is very important to obtain bacterial strains capable of efficiently producing bacterial cellulose in hydrolysate containing the inhibitors.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a bacterium which can produce bacterial cellulose with high yield and can resist furfural.
The invention also solves the technical problem of providing the application of the bacteria in bacterial cellulose production.
In order to solve the technical problems, the invention adopts the following technical scheme:
the bacterial strain for producing bacterial cellulose is classified and named Komagataeibacter xylinus, the strain number is mm01, and is preserved in China general microbiological culture Collection center (CGMCC) No.22145 in the year 2021 and 09 in the year 04, and the preservation address is North Chen West Lu No.1 and No. 3 in the Korean region of Beijing city.
Wherein, the nucleotide sequence of the 16S rDNA of the bacterial cellulose-producing bacterium is shown as SEQ ID NO. 1.
Wherein the bacteria producing bacterial cellulose is tolerant to pH 3.0-7.0 and tolerant to furfural concentration of 0-2.0 g/L.
The use of the strain Komagataeibacter xylinus mm01 described above for the fermentative production of bacterial cellulose is also within the scope of the invention.
Specifically, seed liquid containing the strain Komagataeibacter xylinus mm01 is inoculated into a fermentation medium for dynamic fermentation or static fermentation, and fermentation liquid containing bacterial cellulose is obtained.
Wherein, the inoculation amount is 8-12% v/v, and the preferable inoculation amount is 10% v/v.
Wherein, the dynamic fermentation or static fermentation is carried out under the following fermentation conditions: the initial pH is 4.5-6.0 at 26-32 ℃, and the fermentation culture is carried out for 5-9 days, wherein the preferred fermentation conditions are as follows: and (3) carrying out stationary fermentation culture for 5 days at the initial pH of 5.5 and the constant temperature of 30 ℃.
Wherein, the formula of the fermentation medium is as follows: 10-30g/L of carbon source, 10-25g/L of nitrogen source, 1.0-2.5g/L of inorganic salt and 1.15g/L of ethanol;
further, the carbon source is any one of glucose, sucrose, fructose and mannitol, preferably glucose, and the preferred glucose concentration is 15g/L;
further, the nitrogen source is any one of casein peptone, tryptone, soybean peptone, fish meal peptone, yeast powder and beef extract, preferably soybean peptone, and the preferable soybean protein concentration is 15g/L;
further, the inorganic salt is MgSO 4 、KH 2 PO 4 、Na 2 HPO 4 、ZnCl 2 Any of them, preferably Na 2 HPO 4 Preferred Na 2 HPO 4 The concentration was 2.5g/L.
Low cost green manufacture of bacterial cellulose is key to achieving its wide application. The agricultural and forestry waste with lower cost is used as a substrate of a fermentation product, is a sustainable development way, is beneficial to realizing full and efficient utilization of waste, realizes resource utilization, and reduces the cost of bacterial cellulose produced by fermentation.
Furfural materials are produced under high temperature conditions, and these materials inhibit microbial growth and metabolism. Therefore, the hydrolysis liquid of the agricultural and forestry waste is used as the raw material of the fermentation product and is subjected to detoxification treatment before fermentation, however, the detoxification process not only can cause a great loss of the total sugar content in the hydrolysis liquid, but also has complicated steps, consumes time and also increases the production cost. Therefore, the bacterial strain which can tolerate furfural and can efficiently produce the bacterial cellulose is screened and applied to actual production, and has positive significance for reducing the production cost of the bacterial cellulose by using agricultural and forestry waste as a raw material.
Therefore, the application of the strain Komagataeibacter xylinus mm01 in the fermentation production of bacterial cellulose by taking agricultural and forestry waste as a raw material is also within the scope of the invention.
Specifically, the hydrolysate of the agricultural and forestry waste is used as a fermentation raw material to perform dynamic or static fermentation to obtain fermentation liquor containing bacterial cellulose.
The agricultural and forestry waste is any one or a combination of more than one of bagasse, rice hulls, wheat straw, corn straw, sorghum straw and bean dregs, and the corn straw is preferred. Wherein the hydrolysis time of the agriculture and forestry waste hydrolysis liquid is 30-100min, and the hydrolysis temperature is 140-180 ℃.
Wherein, the fermentation medium taking the agriculture and forestry waste hydrolysate as the fermentation raw material comprises the following formula: and (3) adjusting the pH value of the agricultural and forestry waste hydrolysate to 4.5-6.0 by using calcium hydroxide, and directly using the pH value as a fermentation medium for fermentation.
Wherein, the fermentation conditions are as follows: fermenting at 26-32deg.C and initial pH of 4.5-6.0 for 5-9d. Preferred fermentation conditions are: the pH is adjusted to 4.5-6.0 by calcium hydroxide, and then the culture is carried out for 5 days at 30 ℃.
The bacterial cellulose produced by the strain Komagataeibacter xylinus mm01 is also used as a food regulator in the field of food processing and is within the scope of the invention.
The beneficial effects are that:
1. the strain Komagataeibacter xylinus mm01 provided by the invention has a certain furfural resistance, can tolerate the concentration of 2g/L furfural, realizes the production of bacterial cellulose by taking agricultural and forestry waste as a raw material when taking the hydrolysate of the agricultural and forestry waste as a fermentation raw material, is a low-cost green production, is a sustainable development way, is beneficial to realizing full and efficient utilization of waste, realizes resource utilization, and reduces the cost of bacterial cellulose produced by fermentation.
2. The bacterial strain Komagataeibacter xylinus mm01 for producing bacterial cellulose is obtained through separation, and compared with other bacterial strains, the bacterial strain can produce bacterial cellulose at high yield, improves fermentation efficiency and shortens fermentation period of bacterial cellulose.
3. The invention optimizes the fermentation medium for producing bacterial cellulose, so that the strain Komagataeibacter xylinus mm01 can produce bacterial cellulose in high yield through the fermentation medium with low cost.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
Figure 1Komagataeibacter xylinus mm01 chromosome map;
FIG. 2Komagataeibacter xylinus mm01 related enzyme family;
FIG. 3GO database three major class statistics;
FIG. 4COG annotation classification statistics;
FIG. 5KEGG database annotation statistics;
FIG. 6 effects of different carbon sources on bacterial cellulose production by different strains;
FIG. 7 effect of glucose concentration on Komagataeibacter xylinus mm01 bacterial cellulose production;
FIG. 8 effect of different nitrogen sources on Komagataeibacter xylinus mm01 bacterial cellulose production;
FIG. 9 effect of different concentrations of nitrogen source soy peptone on Komagataeibacter xylinus mm bacterial cellulose production;
FIG. 10 effect of different inorganic salts on Komagataeibacter xylinus mm01 bacterial cellulose production;
FIG. 11 inorganic salt Na 2 HPO 4 Different concentrations affect Komagataeibacter xylinus mm bacterial cellulose yield;
FIG. 12 effect of different fermentation temperatures on Komagataeibacter xylinus mm01 bacterial cellulose yield;
FIG. 13 effect of different initial pH on Komagataeibacter xylinus mm01 bacterial cellulose production;
FIG. 14 effect of different fermentation times on Komagataeibacter xylinus mm01 bacterial cellulose yield;
FIG. 15 growth ability of different bacterial cellulose-producing strains in fermentation medium without furfural;
FIG. 16 test of the Furfural tolerance of different bacterial cellulose producing strains at 0.5 g/L;
FIG. 17 test of the Furfural tolerance of different bacterial cellulose producing strains at 1.0 g/L;
FIG. 18 test of the Furfural tolerance of different bacterial cellulose-producing strains at 1.5 g/L;
FIG. 19 test of the Furfural tolerance of different bacterial cellulose producing strains at 2.0 g/L;
FIG. 20 test of the Furfural tolerance of different bacterial cellulose-producing strains by 2.5 g/L;
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
In the following examples, the medium formulation was as follows:
(1) Acetic acid bacteria solid culture medium: glucose 20g/L, yeast powder 5g/L, peptone 5g/L, na 2 HPO 4 2.7g/L, 1.15g/L ethanol and 20g/L agar.
(2) Acetic acid bacteria liquid seed culture medium: glucose 20g/L, yeast powder 5g/L, peptone 5g/L, na 2 HPO 4 2.7g/L and 1.15g/L ethanol.
(3) Fermentation medium: 5-35g/L of carbon source (glucose, sucrose, fructose or mannitol), 5-35g/L of nitrogen source (casein peptone, tryptone, soybean peptone, fish meal peptone, yeast powder or beef extract), and inorganic salt (MgSO) 4 、KH 2 PO 4 、Na 2 HPO 4 、ZnCl 2 ) 0.5-3.0g/L, and 1.15g/L of ethanol.
In the examples described below, the bacterial cellulose-producing acetic acid bacteria strains AS1, NLQ, CY-1, CE11 were derived from the laboratory deposit.
Example 1 isolation and identification of strains and Whole genome sequencing thereof
Test materials: black tea fungus (a co-organism of yeast, lactic acid bacteria and acetic acid bacteria, 19 days from Shandong Jining City, 4 months of 2019) preserved in the laboratory;
1. isolation and identification of strains
(1) Separation of strains: sucking 1mL of black tea fungus liquid, placing into a centrifuge tube filled with 9mL of physiological saline, immediately sucking 1mL of suspension after vortex shaking is sufficient, adding into the centrifuge tube filled with 9mL of physiological saline, and repeating the steps to obtain 10 -5 ，10 -6 ，10 -7 Three dilution gradients. 100 μl of each of the three dilutions was applied to acetic acid bacteria solid medium, and the plates were transferred to a 28℃incubator for 2 days. Single colonies with different forms and sizes are picked and numbered, inoculated into acetic acid bacteria liquid seed culture medium by an inoculating loop, cultured for 48 hours at 28 ℃, and then preserved in glycerol.
(2) Identification of strain mm 01: single-cell genomic DNA designated as strain mm01 isolated from acetic acid bacteria solid medium was extracted by a HeroGen kit (Injuries, MDP) and PCR amplified using the same as a template. The PCR unpurified product was sent to the Probiotechnological engineering (Shanghai) Co., ltd for sequencing, and the sequencing result (16S rDNA sequence of strain mm01 as shown in SEQ ID NO. 1) was aligned in the NCBI' S Genbank database (https:// blast. NCBI. Lm. Nih. Gov/blast. Cgi), and the strain with higher homology pattern was selected as the 16S rDNA sequence, which indicated that the strain mm01 was the strain with highest homology was Komagataeibacter xylinus.
The strain mm01 is sent to a collection center for collection, the classification is Komagataeibacter xylinus, the strain number is mm01, and the strain is collected in China general microbiological culture collection center (CGMCC) No.22145 in the year 2021 and the month 04 and 09 are collected in China general microbiological culture collection center (China general microbiological culture collection center), and the collection number is CGMCC No. 22145: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
Wherein, primer pair for PCR amplification: 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R (5'-GGTTACCTTGTTACGACTT-3'); PCR amplification reaction system: the total reaction system was 20. Mu.L, including 2 XHeroGen Direct-PCR Mix 10. Mu.L, primer F (10. Mu.M) 0.5. Mu.L, primer R (10. Mu.M) 0.5. Mu.L, genomic DNA 0.5. Mu.L, ddH 2 O was made up to 20. Mu.L; PCR amplification procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 52℃for 30s, extension at 72℃for 90s,30 cycles; after the completion, the mixture was extended at 72℃for 10min.
2. Whole genome sequencing of Komagataeibacter xylinus mm01
1. Experimental method
(1) Extraction of DNA: culturing Komagataeibacter xylinus mm01 in acetic acid bacteria liquid culture medium at 28deg.C for 48 hr, centrifuging at 10000rpm for 10min, collecting bacterial precipitate, and fully grinding with liquid nitrogen; cracking by using SDS lysate, adding a proper amount of proteinase K and mercaptoethanol, gently reversing and mixing uniformly, cooling to room temperature, centrifuging at 10000rpm for 3min to obtain supernatant, adding chloroform/isoamyl alcohol (24:1 v/v) into the supernatant for extraction, and extracting for 2 times; precipitating DNA with isopropanol, slightly reversing and mixing, and centrifuging at 10000rpm for 3min; the precipitate was washed with 75% ethanol for 2 times, and 100. Mu.L of nuclease-free water was added after the ethanol was completely evaporated to give a DNA sample of Komagataeibacter xylinus mm.
(2) Whole genome sequencing and splicing: breaking the DNA sample qualified by Nanodrop, qbuit and electrophoresis into fragments of about 350bp, capturing sequences at two sides of the circularized primer by using a probe, and constructing a subsequent DNA library. Splicing genomes by adopting a second generation and third generation sequencing technology, respectively constructing an Illumina Novaseq 6000 library and a Nanopore whole-gene library, performing quality control on the obtained sequencing data, and completing Komagataeibacter xylinus mm01 whole-genome completion map drawing by utilizing a bioinformatics analysis means. The raw data was filtered using fastp software (https:// github. Com/OpenGene/fastp) with the following filtering criteria: (1) excision of the sequencing adapter and primer sequences in Reads; (2) filtering reads having a mean homogeneity value less than Q5; (3) filtering out N reads greater than 5. The high quality Reads obtained after the series of quality control is clear Data. Whole genome sequencing was done by south-Beijing Jisi Huiyuan biotechnology limited.
(3) BLAST alignment of predicted genes and functional databases, including GO (http:// geneontology. Org /), COG (http:// www.ncbi.nlm.nih.gov/COG /), KEGG (http:// www.genome.jp/KEGG /) and CAZy, etc., was performed, and BLAST results were filtered, and BLAST results for each sequence were annotated with the highest scoring alignment (default identity > =40%, coverage > =40%).
2. Experimental results
(1) Whole genome sequencing: the full genome effective sequence length of Komagataeibacter xylinus mm01 is 3640771bp and GC content is 63.25% obtained by high throughput sequencing of the Illumina Hiseq platform, and 3185 open reading frames are predicted to be 15 and 58 rRNA and tRNA respectively. Furthermore, komagataeibacter xylinus mm01 contained 6 plasmids. Komagataeibacter xylinus mm01 chromosome map is shown in FIG. 1.
(2) CAZy annotation results: hydrolysis of carbohydrates by microorganisms is a combination of biochemical processes responsible for the formation, degradation and conversion of carbohydrates. In such an environment, the metabolic pathways used and the metabolites produced are entirely determined by the enzymatic mechanism of the microbial community. The relevant enzyme family that catalyzes carbohydrate degradation, modification and biosynthesis was analyzed Komagataeibacter xylinus mm (fig. 2) by the annotated results obtained by the professional database CAZy of carbohydrate enzymes. It was found that up to 129 genes related to Glycoside Hydrolase (GH) are enzymes that cleave glycosidic linkages in glycosides, glucosides and glycoconjugates. The number of genes involved annotated to glycosyltransferases is 87, an enzyme that catalyzes the synthesis of the glycosyl chain, through transfer of glycosyl residues from a donor substrate to an acceptor, which plays a key role in the biosynthetic pathways of oligosaccharides and polysaccharides as well as protein glycosylation and the formation of valuable natural products. In addition, there are 22 coenzyme-related genes, 17 carbohydrate lipolytic enzyme (CE) -related genes.
(3) GO, COG, KEGG database annotation results: according to the GO database annotation results, komagataeibacter xylinus mm01 has 2455 genes annotated and is classified into three major categories according to their functions: is a cellular component (cellular component), a biological process (biological process) and a molecular function (molecular function). The three classification statistics of the GO database are shown in FIG. 3. In COG notes, the most abundant are general functional predictive genes (General function prediction only), followed by amino acid transport and metabolism (Amino acid transport and metabolism), replication, recombination and repair (Replication, recombination and repair) and carbohydrate transport and metabolism (carbohydrate transport and metabolism) (fig. 4). The most numerous of the KEGG database notes are carbohydrate metabolism (carbohydrate metabolism) (fig. 5).
The results show that Komagataeibacter xylinus mm01 gene functions are closely related to cell metabolism, participate in the transformation of various substance metabolic pathways and contribute to the formation of bacterial cellulose.
Example 2 analysis of bacterial cellulose production ability of different strains
1. The experimental method comprises the following steps:
(1) Seed activation: the bacterial cellulose-producing acetic acid bacteria strains AS1, NLQ, CY-1 and CE11 stored at the temperature of 4 ℃ in the refrigerator and Komagataeibacter xylinus mm01 obtained by separation and identification in the example 1 are respectively inoculated on an acetic acid bacteria solid culture medium, and are cultured at the constant temperature of 28 ℃ for 24 hours until new bacterial colonies grow on a flat plate.
(2) And (3) performing expansion culture: the strains which are successfully activated by the seeds in the step (1) are respectively inoculated with the strains which are picked up by an inoculating loop and subjected to flat plate culture, and are transferred to a liquid seed culture medium filled with acetic acid bacteria, and are cultured in a shaking table of 150r/min for about 12 hours at the temperature of 28 ℃; the OD value is measured at 600nm wavelength to be about 3.5, and the seed liquid is prepared for standby.
(3) Fermentation experiment: the carbon source is one of the main components constituting the medium, and has mainly two functions: firstly, providing energy and necessary carbon components for the growth and propagation of microorganism strains; and secondly, providing a required carbon component for synthesizing a target product of the thallus. Most microorganisms can use saccharides as carbon sources, but the kind of carbon source that can be used by each microorganism varies depending on the physiological characteristics of each microorganism. Komagataeibacter xylinus cellulose can be synthesized using a variety of carbon sources. Therefore, in the embodiment, 20g/L of glucose, sucrose, fructose and mannitol are respectively selected as the only carbon sources for static fermentation in the fermentation experiment.
Inoculating seed solution of each strain into sterilized fermentation medium (fermentation medium formula: carbon source 20g/L, yeast powder 5g/L, peptone 5g/L, na) according to 10% inoculum size (v/v) 2 HPO 4 2.7g/L and 1.15g/L of ethanol), and shaking uniformly to uniformly distribute the seed liquid in the fermentation medium. The culture vessel was allowed to stand still at 28℃for 7 days. Bacterial cellulose films formed in the medium after 7 days of culture were then extracted, treated and assayed.
The extraction, treatment and measurement methods of the bacterial cellulose membrane are as follows: taking out bacterial cellulose membrane in the culture medium with forceps, and washing with distilled water for several times to remove surface culture medium and impurities. Soaking the membrane in distilled water for 2 days, boiling in 0.1mol/L NaOH solution for 40min, removing thallus and residual culture medium, making the membrane in milky semitransparent state, washing with deionized water for several times, and measuring pH value to neutrality. After the bacterial cellulose was completely dried in an oven at 60 ℃, the film became a white film, and the resulting film was defined as a bacterial cellulose dry film. The bacterial cellulose membrane was weighed with an electronic balance and bacterial cellulose yield was calculated as follows:
2. experimental results
In the embodiment, 20g/L of glucose, sucrose, fructose and mannitol are respectively selected as unique carbon sources for static fermentation in a fermentation experiment, and the bacterial cellulose membrane after 7 days of fermentation culture is light yellow in color, has certain flexibility and is rich in moisture. Bacterial cellulose production after 7 days of fermentation is shown in figure 6.
As can be seen from FIG. 6, all of the five strains (AS 1, NLQ, CY-1, CE11 and mm 01) were able to utilize all of the carbon sources selected in the experiment, and the trend of the results of the fermentative preparation of bacterial cellulose at different carbon sources was, although consistent, different strains were different in the carbon source utilization and bacterial cellulose production capacity, and the bacterial cellulose yields were greatly different. Bacterial cellulose yield of the strain mm01 is 11.68g/L at the highest; the bacterial cellulose produced by other carbon sources is lower than 3g/L, so the bacterial cellulose producing capacity of the strain mm01 is obviously higher than that of other strains by about 3 times. In addition, glucose was found to be the best carbon source for bacterial cellulose production by the five strains from the figure.
Example 3 Komagataeibacter xylinus mm01 fermentation preparation of bacterial cellulose Condition optimization
1. Experimental method optimization
(1) Fermentation medium optimization: respectively configuring different glucose concentrations (5, 10, 15, 20, 25, 30, 35 g/L), different nitrogen sources (casein peptone, tryptone, soybean peptone, fish meal peptone, yeast powder, beef extract), different nitrogen source concentrations (5, 10, 15, 20, 25, 30, 35 g/L), and different inorganic salts (MgSO) 4 、KH 2 PO 4 、Na 2 HPO 4 、ZnCl 2 ) Optimized fermentation medium was prepared from different inorganic salt concentrations (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 g/L) and 1.15g/L ethanol, inoculated at 10%The seed liquid is inoculated into the optimized fermentation culture medium after sterilization treatment, and the seed liquid is fully and evenly shaken after inoculation, so that the seed liquid is evenly distributed in the culture medium. The culture vessel was allowed to stand still at 28℃for 7 days.
(2) Fermentation temperature optimization: the seed liquid is inoculated into the sterilized fermentation medium according to the inoculation amount (v/v) of 10 percent, and the seed liquid is sufficiently and evenly shaken after inoculation, so that the seed liquid is evenly distributed in the medium. The culture vessel was allowed to stand for 7 days under constant temperature conditions at different temperatures (24, 26, 28, 30, 32, 34 ℃).
(3) Initial pH optimization: seed liquid is inoculated into fermentation media with different initial pH values (3.0, 4.0, 4.5, 5.0, 5.5, 6.0 and 7.0) after sterilization treatment according to the inoculation amount (v/v) of 10 percent, and the seed liquid is sufficiently and evenly shaken after inoculation, so that the seed liquid is evenly distributed in the media. The culture vessel was allowed to stand still at a constant temperature of 30℃for 7 days.
(4) Fermentation time optimization: the seed solution was inoculated in 10% inoculum size (v/v) into the sterilized fermentation medium having an initial pH of 6.0, and the seed solution was thoroughly shaken after inoculation to uniformly distribute the seed in the medium. The culture vessel was left to stand at a constant temperature of 30℃for different times (3, 4, 5, 6, 7, 8, 9 d).
Bacterial cellulose membranes prepared by fermentation culture were extracted, treated and assayed in the same manner as in example 2.
2. Experimental results
(1) Fermentation medium optimization results:
1) Glucose concentration optimization: as a result, as shown in fig. 7, bacterial cellulose production increases with increasing glucose concentration, but when the concentration increases to some extent, the production decreases, and this phenomenon may occur because saccharides as a carbon source and an energy source promote the growth metabolism of the cells, and cellulose as a secondary metabolite may be synthesized into cellulose only when the cells grow to around the stationary phase, and thus the bacterial cellulose production increases with increasing carbon source concentration; when the glucose concentration is high, a large amount of sugar is decomposed to produce metabolites such as gluconic acid, which significantly lowers the pH of the culture solution, thereby inhibiting bacterial cellulose membrane production by the cells. At a glucose concentration of 15g/L, the bacterial cellulose yield reached a maximum of 12.70g/L. When the glucose concentration is more than 15g/L, the yield thereof gradually decreases. Thus, the optimal glucose concentration is 15g/L.
2) Nitrogen source type optimization: biological fermentation generally requires a nitrogen source as an energy source to provide nutrients necessary for growth, and when inorganic nitrogen and organic nitrogen are in a certain range, the yield of cellulose is greatly related to the growth amount of bacteria, and when the growth amount of bacteria is high, the yield of cellulose is also high. The nitrogen sources required for the growth of different microorganisms are different, so that the experiment is to obtain high-yield results, and the nitrogen sources suitable for the growth of the strain Komagataeibacter xylinus mm01 are required to be selected for the synthesis of bacterial cellulose. As is clear from FIG. 8, when soybean peptone, yeast powder, casein peptone, tryptone and beef extract are used as nitrogen sources, the bacterial cellulose yield is high and reaches 5g/L or more, and especially when soybean peptone is used as nitrogen source, the bacterial cellulose yield is 13.65g/L or more. Because the production cost of yeast powder, casein peptone and tryptone is high, the prices of the yeast powder, the casein peptone and the tryptone are respectively up to 1000 yuan/kg, 600 yuan/kg and 500 yuan/kg, and more economic and reasonable nitrogen source substances are needed to be selected in actual production; taking the industrial cost into consideration, soybean peptone is selected as a nitrogen source for later experiments.
3) Nitrogen source concentration optimization: as shown in FIG. 9, with soybean peptone as a nitrogen source, bacterial cellulose production gradually increased and decreased as the nitrogen source concentration increased. When the concentration of soybean peptone is 15g/L, the highest cellulose yield is 14.21g/L.
4) Inorganic salt type optimization: inorganic salts are an important influencing factor for microbial growth, so that research on inorganic salts is of great importance. Inorganic salts are added into the optimized fermentation medium determined by carbon and nitrogen source tests. The experimental results are shown in FIG. 10. Inorganic salt ZnCl 2 、MgSO 4 、KH 2 PO 4 、Na 2 HPO 4 Promoting cellulose yield, wherein Na 2 HPO 4 Has the strongest promoting effect, and further selects Na 2 HPO 4 And carrying out subsequent optimization.
5) Optimizing the concentration of inorganic salt: by Na 2 HPO 4 As a result of optimizing the concentration of the preferable inorganic salt, as shown in FIG. 11, it is clear from the fermentation result that Na 2 HPO 4 The concentration is 2.5g/L, and the bacterial cellulose yield reaches the maximum of 14.89g/L.
(2) Fermentation temperature optimization: the temperature is critical for the growth and metabolism of the bacterial cells, and the proper temperature is an important guarantee for ensuring that bacterial cells normally produce bacterial cellulose. As can be seen from FIG. 12, the temperature greatly affects the film formation of bacterial cellulose, the optimum film formation temperature of the strain is 30℃and when the temperature is lower than 30℃the film formation amount increases with an increase in temperature, but when the temperature exceeds this value, the film formation amount significantly decreases. At a temperature of 30 ℃, the bacterial cellulose yield is 15.41g/L at maximum.
(3) Initial pH optimization: the pH is an important factor in the growth of microorganisms and the synthesis of bacterial cellulose, which has a great influence on the growth of the cells and the accumulation of the products. In this experiment, the initial pH was adjusted to the desired pH with NaOH and HCl, respectively, and the experimental results are shown in FIG. 13, in which strain Komagataeibacter xylinus mm01 grew well in the pH range of 4.5-6.0. The optimal initial pH for membrane production is 5.5, at this time, the maximum bacterial cellulose yield is 18.67g/L, and when the bacterial cellulose yield is lower or higher than the value, the membrane yield is obviously reduced, probably because the strain Komagataeibacter xylinus mm01 tends to produce acid during growth metabolism, and the strain adapts to acidic conditions through long-term evolution, so that the acidic conditions are more favorable for the growth and membrane production, and the alkaline conditions can make the strain difficult to grow.
(4) Fermentation time optimization: the fermentation time is also critical to the growth and metabolism of the thalli, and the proper fermentation time can not only improve the efficiency of producing bacterial cellulose, but also save the cost and increase the yield. As can be seen from FIG. 14, the bacterial cellulose production gradually increased with the increase in fermentation time, and an equilibrium of 19.85g/L was reached on day 5, so that fermentation time was selected to be more appropriate for 5d.
To sum up, bacterial cellulose is prepared by fermenting the strain Komagataeibacter xylinus mm, and the optimized fermentation medium is as follows: 15g/L glucose, dazhangSoytone 15g/L, na 2 HPO 4 2.5g/L and 1.15g/L ethanol, the initial pH is 5.5, the fermentation temperature is 30 ℃, and the fermentation time is 5d.
Furfural tolerance of example 4 Komagataeibacter xylinus mm01
Low cost green manufacture of bacterial cellulose is key to achieving its wide application. The agricultural and forestry waste with lower cost is used as a substrate of a fermentation product, is a sustainable development way, is beneficial to realizing full and efficient utilization of waste, realizes resource utilization, and reduces the cost of bacterial cellulose produced by fermentation.
Furfural materials are produced under high temperature conditions, and these materials inhibit microbial growth and metabolism. Therefore, the hydrolysis liquid of the agricultural and forestry waste is used as the raw material of the fermentation product and is subjected to detoxification treatment before fermentation, however, the detoxification process not only can cause a great loss of the total sugar content in the hydrolysis liquid, but also has complicated steps, consumes time and also increases the production cost. Therefore, the screening of the bacterial strain which can tolerate furfural and can efficiently produce the bacterial cellulose has positive significance for reducing the production cost of the bacterial cellulose by using the agricultural and forestry waste as the raw material.
1. Furfural resistance test of strain
The bacterial cellulose-producing acetic acid bacteria strains AS1, NLQ, CY-1, CE11 and the strain Komagataeibacter xylinus mm01 isolated and identified in example 1 were compared for their furfural resistance. Inoculating the above five strains stored at 4deg.C in acetic acid bacteria solid culture medium, culturing at 26deg.C for 24 hr until new colony grows on the plate, selecting single colony, inoculating into 5mL acetic acid bacteria liquid seed culture medium, culturing and activating to logarithmic phase, and regulating OD with sterilized distilled water 600 At 3.0, 40mL of fermentation medium (fermentation medium is optimized in example 3) containing 0, 0.5, 1.0, 1.5, 2.0 and 2.5g/L furfural gradient is respectively inoculated according to the inoculation amount of 3% (v/v), the temperature is 30 ℃, the rpm is 150, and samples are taken after every few hours to measure OD 600 Values.
As shown in FIG. 15, the growth of the five strains was not significant in the fermentation medium without furfuralThe difference, the inoculation is faster to enter the logarithmic growth phase, and reaches the stationary phase at about 24h, the maximum OD 600 The value was about 3.9. Under the stress of furfural, the five strains need a certain time to reduce the furfural into substances such as furfuryl alcohol with smaller toxicity, so that the growth delay period is increased to a certain extent, but the specific growth rate can be restored to a normal level after entering the logarithmic phase. As can be seen from FIGS. 16-18, in the fermentation medium containing 0.5g/L (FIG. 16), 1.0g/L (FIG. 17) and 1.5g/L (FIG. 18) of furfural concentration, the growth delay of the five strains was correspondingly prolonged with the increase of furfural concentration, and at OD 600 Reaching a stationary phase of about 3.6, and a maximum OD in a fermentation medium without furfural 600 The value is reduced compared with the other strains, and the growth delay of the strain Komagataeibacter xylinus mm01 is shortened by about 4 hours, 8 hours and 4 hours under the concentration of 0.5g/L, 1.0g/L and 1.5g/L furfural. As shown in FIG. 19, in the fermentation medium with the furfural concentration of 2.0g/L, the growth capacity of the strain Komagataeibacter xylinus mm01 has obvious advantages over other strains, and the OD is 32h 600 The value was 3.41, while other strains had weaker growth ability. As shown in FIG. 20, when the furfural concentration reached 2.5g/L, the growth of each strain was greatly restricted, but the OD of strain Komagataeibacter xylinus mm01 was greatly restricted 600 The value is still the highest of the five strains, which shows that the strain Komagataeibacter xylinus mm01 has stronger furfural tolerance and can bring more economic value for subsequent production and utilization.
2. Preparation of corn stalk hydrolysate at different hydrolysis time and temperature
The cost of producing bacterial cellulose by adopting a fermentation medium is high, and agricultural and forestry waste sources such as agricultural straws (corn, wheat, rice, sorghum and the like) and bagasse and the like are wide, and the cost is low. The agricultural straw has high cellulose content, and the fermented monosaccharide can be obtained through the technologies of hydrolysis, purification, enrichment and the like, so that the fermented monosaccharide becomes a carbon source raw material for producing bacterial cellulose. However, since substances such as furfural, etc., which inhibit the growth of microorganisms are produced during the hydrolysis of agricultural stalks, detoxification treatment of the hydrolysate is required. The detoxification methods such as activated carbon adsorption, ion exchange and laccase treatment can cause the problems of serious loss of carbon sources in the hydrolysate, high cost and the like, so that the screening of microorganisms resistant to inhibitors such as furfural and the like provides possibility for low-cost preparation of bacterial cellulose.
In the embodiment, the corn stalk of agricultural and forestry waste is used as a raw material, and the preparation method of the corn stalk hydrolysate comprises the following steps: corn stalks were crushed to 200 mesh with a crusher, and then soaked with deionized water and washed three times to remove contaminants. And (5) placing the dried product in a ventilation place for airing. The influence of different temperatures and time on total sugar and furfural in the hydrolysis liquid of corn straw by acetic acid hydrolysis is studied, straw powder with certain quality is taken and put into a reaction kettle, 3% (w/v) acetic acid is added, and the solid-liquid ratio is 1:10. Respectively hydrolyzing the reaction solution at 140 deg.C, 160 deg.C and 180 deg.C for 30min, 60min and 100min, and removing the residue to obtain hydrolyzed solution.
The results are shown in Table 1. The hydrolysis reaction becomes severe with the increase of temperature and time, cellulose and hemicellulose in the corn stalks can be degraded to form monosaccharides, and furfural substances can be formed at the same time. As the hydrolysis temperature and time increases, the total sugar and furfural content increases. When the hydrolysis temperature is 180 ℃ and the hydrolysis time is 60min, the total sugar content is 31.29g/L, and the furfural content also reaches 2.47g/L.
TABLE 1 influence of temperature and time on total sugar and Furfural content in corn straw hydrolysate
3. Bacterial cellulose production of strain Komagataeibacter xylinus mm01 in corn stalk hydrolysate
Komagataeibacter xylinus mm 01% (v/v) was added to the above corn stalk hydrolysates at different hydrolysis temperatures and hydrolysis times, pH was adjusted to 4.5-6.0 with calcium hydroxide, and cultured at 30℃for 5 days, and the bacterial cellulose yields in the different corn stalk hydrolysates were as shown in Table 2. When the hydrolysis temperature of the corn straw is 180 ℃ and the hydrolysis time is 100min, the furfural content reaches 3.89g/L although the hydrolysate contains abundant sugar, and at the moment, the growth of mm01 is completely inhibited, and bacterial cellulose cannot be produced. When the furfural content in the hydrolysate is 0.56-1.84g/L, the mm01 can normally produce bacterial cellulose, and the yield is 3.12-7.06g/L.
TABLE 2 Effect of corn straw hydrolysate on bacterial cellulose production for hydrolysis at different temperatures and times
Note that: ND indicates that no bacterial cellulose production was detected.
Example 5 Komagataeibacter xylinus mm01 bacterial cellulose production for sausage processing
Bacterial cellulose sausage formula: (in the following experimental groups,% represents mass percent)
Experiment group 1: 7.5% of fat meat, 7.5% of bacterial cellulose, 0.2% of monosodium glutamate, 0.1% of spice powder, 33.6% of water, 40% of lean pork, 5% of soybean protein isolate, 3% of salt, 0.1% of pepper powder and 3% of white sugar.
Experiment group 2: 15% of fat meat, 0% of bacterial cellulose, 0.2% of monosodium glutamate, 0.1% of spice powder, 33.6% of water, 40% of lean pork, 5% of soybean protein isolate, 3% of salt, 0.1% of pepper powder and 3% of white sugar.
Experiment group 3: 0% of fat meat, 15% of bacterial cellulose, 0.2% of monosodium glutamate, 0.1% of spice powder, 33.6% of water, 40% of lean pork, 5% of soybean protein isolate, 3% of salt, 0.1% of pepper powder and 3% of white sugar.
According to GB/T22210-2008 'meat and meat product sensory evaluation Specification', 15 persons trained in food sensory profession are selected to perform sensory evaluation on the sausage, and evaluation is performed on aspects of color, smell, taste, tissue state and the like of the sausage. By sensory evaluation of the sausage added with bacterial cellulose (see table 3), it was found that the sausage added with 7.5% bacterial cellulose (experimental group 1) was not significantly different from the sausage of experimental group 2 in terms of color, smell, taste, tissue state. The sausage (test group 3) added with 15% bacterial cellulose was not significantly different from the sausage of test group 2 in terms of color, smell, taste, but was slightly different in terms of tissue state, such as slightly loose slices. The results show that the bacterial cellulose can be partially replaced, even completely replace fat meat in sausage, so that the heat of the sausage is reduced to the greatest extent. The sausage has no defect in tissue state caused by fat, and can be improved by using carrageenan and other colloids together with bacterial cellulose in the later period, so that the sausage has better taste.
Table 3 meat sausage sensory evaluation
| Experiment group 1 | Experiment group 2 | Experiment group 3 | |
| Chewing feeling | Strong strength | Strong strength | Strong strength |
| Elastic body of intestine | In general | In general | In general |
| Slice cavity | Void-free | Void-free | Slightly hollow |
| Degree of slice loosening | Compact form | Compact form | Slightly loose |
| Slicing property | The section is neat | The section is neat | Slightly break |
| Smell of | Meat flavor, no foreign flavor | Meat flavor | Meat flavor, no foreign flavor |
| Color | Bluish white | Bluish white | Bluish white |
The invention provides a bacterial strain for producing bacterial cellulose, and an application idea and a method thereof, and a method and a way for realizing the technical scheme are numerous, the above description is only a preferred embodiment of the invention, and it should be noted that, for a person skilled in the art, a plurality of improvements and modifications can be made without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (9)
1. Bacterial cellulose-producing strain, classified and namedKomagataeibacter xylinusThe strain number is mm01, and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.22145 in 2021, 04 and 09.
2. Use of the bacterium of claim 1 for the fermentative production of bacterial cellulose.
3. The use of the bacterium of claim 1 for producing bacterial cellulose by fermentation using an agricultural and forestry waste hydrolysate as a raw material; the agricultural and forestry waste is any one or a combination of more than one of bagasse, rice hulls, wheat straw, corn straw, sorghum straw and bean dregs.
4. The use according to claim 2, characterized in that the bacterial-containing seed liquor is inoculated into a fermentation medium for static fermentation to obtain a bacterial cellulose-containing fermentation liquor.
5. The use according to claim 4, wherein the inoculation amount is 8-12% v/v; the static fermentation is carried out under the following fermentation conditions: fermenting and culturing at 26-32deg.C and initial pH of 4.5-6.0 at 5-9d.
6. The use according to claim 5, wherein the fermentation conditions are: the initial pH is 5.5, and the culture is kept stand and fermented at a constant temperature of 30 ℃ for 5d.
7. The use according to claim 4, wherein the fermentation medium comprises the following formula: 10-30g/L of carbon source, 10-25g/L of nitrogen source, 1.0-2.5g/L of inorganic salt and 1.15g/L of ethanol;
wherein the carbon source is any one of glucose, sucrose, fructose and mannitol;
the nitrogen source is any one of casein peptone, tryptone, soybean peptone, fish meal peptone, yeast powder and beef extract;
the inorganic salt is MgSO 4 、KH 2 PO 4 、Na 2 HPO 4 、ZnCl 2 Any one of the following.
8. The use according to claim 7 ofCharacterized in that the carbon source is glucose, and the concentration is 15g/L; the nitrogen source is soybean peptone, and the concentration of the nitrogen source is 15g/L; the inorganic salt is Na 2 HPO 4 The concentration thereof was 2.5. 2.5g/L.
9. The use according to claim 3, wherein the hydrolysis time of the agricultural and forestry waste is 30-100min and the hydrolysis temperature is 140-180 ℃.
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