CN117363522A - Enterobacter cholerae BZE-5 capable of producing high-activity feruloyl esterase and application thereof in preparation of bacterial bran koji - Google Patents
Enterobacter cholerae BZE-5 capable of producing high-activity feruloyl esterase and application thereof in preparation of bacterial bran koji Download PDFInfo
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- 101001065065 Aspergillus awamori Feruloyl esterase A Proteins 0.000 title claims abstract description 21
- 230000000694 effects Effects 0.000 title claims abstract description 21
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
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- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
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- WKOLLVMJNQIZCI-UHFFFAOYSA-N vanillic acid Chemical compound COC1=CC(C(O)=O)=CC=C1O WKOLLVMJNQIZCI-UHFFFAOYSA-N 0.000 description 1
- TUUBOHWZSQXCSW-UHFFFAOYSA-N vanillic acid Natural products COC1=CC(O)=CC(C(O)=O)=C1 TUUBOHWZSQXCSW-UHFFFAOYSA-N 0.000 description 1
- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 description 1
- FGQOOHJZONJGDT-UHFFFAOYSA-N vanillin Natural products COC1=CC(O)=CC(C=O)=C1 FGQOOHJZONJGDT-UHFFFAOYSA-N 0.000 description 1
- 235000012141 vanillin Nutrition 0.000 description 1
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01073—Feruloyl esterase (3.1.1.73)
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- 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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Abstract
An enterobacter cholerae (Enterobacter hormaechei) BZE-5 for producing high-activity feruloyl esterase and application thereof in preparing bacterial bran koji relate to the field of microorganisms. The strain BZE-5 is preserved in China Center for Type Culture Collection (CCTCC) at the preservation number of NO: m2023976. The strain BZE-5 is inoculated into wheat bran through expansion culture to prepare bacterial bran koji; the bacterial bran koji is applied to sesame-flavor white spirit production in a material mixing mode, so that the quality of the sesame-flavor white spirit is improved, and the bacterial bran koji is characterized in that: compared with the contrast bran koji ferulic acid esterase activity, the sesame-flavor wine base has improved alcohol content, 4-vinyl guaiacol content and ethyl acetate content.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to enterobacter cholerae (Enterobacter hormaechei) BZE-5 for producing high-activity feruloyl esterase and application thereof in preparation of bacterial moldy bran.
Background
The use of bran koji gives higher technological content to sesame-flavor white spirit production process, and is beneficial to the process stability and the product quality improvement. The bacterial bran koji is prepared by culturing bacteria through bran, and has certain liquefying, saccharifying, protein decomposing, fat decomposing and other capabilities.
Feruloyl esterase (Feruloyl esterase, FAE, EC 3.1.1.73), also known as cinnamoyl esterase, is a carboxylic acid hydrolase that hydrolyzes ester bonds between ferulic acid and polysaccharide in plant cell walls, breaking the plant cell walls, releasing free ferulic acid. Feruloyl esterase can also cooperate with xylanase, beta-glucosidase, cellulose lactonase and the like to degrade plant cell walls more thoroughly.
Ferulic Acid (FA) is an effective natural polyphenol antioxidant, whose structure contains hydroxyl groups and phenoxy groups, and can rapidly scavenge free radicals and protect cells, thus having remarkable antioxidant activity. Ferulic acid has been shown to have good therapeutic effects on various diseases such as skin diseases, cancer, cardiovascular and cerebrovascular diseases, obesity, etc. The anti-inflammatory effect can be exerted by regulating cell signal paths, controlling the expression of certain stress response genes, increasing the number of anti-inflammatory cytokines and the like.
Ferulic acid forms the skeleton of plant cell wall mainly by crosslinking with lignin or polysaccharide via ester bond. It is usually present in the cell wall of cereals, such as rice, wheat bran, beans, etc. The main raw materials and auxiliary materials for brewing sesame-flavor white spirit comprise sorghum, wheat, bran, rice hulls and the like, wherein the raw materials and auxiliary materials are rich in ferulic acid, but exist in a cell wall mainly in an esterified form, and ferulic acid esterase secreted by fermented grain microorganisms can hydrolyze arabinoxylans and ester bonds connected between pectin and ferulic acid in the plant cell wall to release the ferulic acid.
Ferulic acid is degraded into 4-vinylguaiacol (4-VG), vanillin, vanillic acid, dihydroferulic acid and the like through a non-oxidative decarboxylation pathway, a non-beta-oxidation pathway dependent on coenzyme A, a beta-oxidation pathway dependent on coenzyme A and a side chain reduction pathway. Wherein 4-VG can endow the white spirit with sweet fragrance, flower fragrance and fumigating flavor, and is an important flavor substance in the white spirit. The ferulic acid is decarboxylated under the action of ferulic acid decarboxylase to obtain 4-VG.
Up to the present, the research on the preparation of sesame-flavor white spirit bacterial bran koji by using enterobacter cholerae (Enterobacter hormaechei) has not been reported yet.
Disclosure of Invention
The invention aims to provide enterobacter cholerae (Enterobacter hormaechei) BZE-5 for producing high-activity feruloyl esterase and application thereof in preparation of bacterial moldy bran.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the invention relates to a strain of enterobacter cholerae (Enterobacter hormaechei) BZE-5 for producing high-activity feruloyl esterase, which is preserved in China Center for Type Culture Collection (CCTCC) in the year 2023, month 6 and day 9, and has the preservation number of CCTCC NO: m2023976.
The invention relates to an application of enterobacter cholerae (Enterobacter hormaechei) BZE-5 for producing high-activity feruloyl esterase in preparation of bacterial bran koji.
The invention relates to an application of enterobacter cholerae (Enterobacter hormaechei) BZE-5 for producing high-activity feruloyl esterase in preparation of bacterial bran koji, which comprises the following steps:
inoculating the strain BZE-5 on an LB culture medium plate, picking a bacterial colony of the strain BZE-5 from the LB culture medium plate, inoculating into an LB liquid culture medium, and performing stationary culture at a constant temperature of 37 ℃ for 24 hours; transferring the obtained culture into LB liquid medium to prepare seed liquid, and shake culturing at 37deg.C and 180r/min for 24 hr; transferring the culture into a seed culture medium, and culturing at 37 ℃ and 180r/min under shaking for 24 hours; transferring the culture into a cassette type tank containing a seed culture medium, and culturing at 37 ℃ for 24 hours to obtain a seed solution; stirring bran and rice husk uniformly, adding caustic soda, adding distilled water while stirring, stirring uniformly, scattering pimples by using a bran lifter, moistening and steaming, taking out of a steamer, cooling by using a cooling lifter, cooling to 45 ℃, inoculating to a bran koji culture medium in a proportion of 8% (v/w), and culturing for 74 hours at 37 ℃ in a room to obtain the bacterial bran koji.
Preferably, 100% of bran and 5.7% (w/w) of rice hull are stirred evenly and 0.22% (w/w) of caustic soda is added.
Preferably, the bacterial bran koji is added in the sesame-flavor liquor production to replace part of the traditional bran koji usage amount, so as to prepare the high-quality sesame-flavor liquor.
Preferably, the mass ratio of the traditional bran koji to the bacterial bran koji is 5:1.
The beneficial effects of the invention are as follows:
the invention provides a strain of enterobacter cholerae BZE-5 for producing high-activity feruloyl esterase and application thereof in preparation of bacterial bran koji; inoculating the strain BZE-5 into wheat bran through expansion culture to prepare bacterial bran koji; the bacterial bran koji is applied to sesame-flavor white spirit production in a material mixing mode, so that the quality of the sesame-flavor white spirit is improved, and the bacterial bran koji is characterized in that: compared with the contrast bran koji ferulic acid esterase activity, the sesame-flavor wine base has improved alcohol content, 4-vinyl guaiacol content and ethyl acetate content.
Drawings
FIG. 1 is a photograph of a colony of E.cholerae BZE-5.
FIG. 2 is an optical micrograph of E.cholerae BZE-5.
FIG. 3 is a phylogenetic tree of the gene sequences of E.cholerae BZE-5.
FIG. 4 shows the transparent circles produced by E.cholerae BZE-5 enzyme solution on plates of medium containing ethyl ferulate alone.
Fig. 5 is a diagram of the sensory analysis radar of the base wine samples of the control group and the test group.
FIG. 6 is a graph showing the flavor profile of the base wines of the control and test groups.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 isolation, identification and preservation of strains
1. Isolation of strains
The strain is separated from sesame-flavor stacked fermented grains of Baotu Jiuquan brewing Limited liability company. Grinding the sample, diluting the sample in a gradient way, inoculating the sample on a culture medium flat plate containing ethyl ferulate, culturing the sample for 3d at 37 ℃, screening out a strain with stronger ferulate esterase producing ability according to the size of a transparent ring around a colony, and naming the strain as BZE-5.
2. Identification of strains
(1) Morphological identification
Bacterial strain BZE-5 is inoculated on LB solid medium, cultured for 24 hours in a 37 ℃ incubator, subjected to colony photographing, and subjected to bacterial microscopic observation, and specific morphological characteristics are shown in figures 1 and 2.
As shown in fig. 1 and 2, the morphology of strain BZE-5 is characterized by: bacterial colony of strain BZE-5 on LB solid culture medium is round, milky white, and smooth and moist on the surface of microprotrusions; gram staining is red, is gram negative bacteria, and the thallus is in a small sphere shape.
(2) Molecular biological identification
Obtaining the 16S rDNA sequence (SEQ ID NO: 1) of the strain BZE-5, and identifying the strain to be tested by performing homology alignment analysis on the sequence information of the known strain in the GenBank library by the BLAST program; the genus classification is carried out by taking the 16S rDNA sequence homology of more than 99% as a standard, and then the 16S rDNA sequences of the strain to be detected and the model strain are used for constructing a phylogenetic tree by utilizing a Neighbor-Joing method in MEGA4.0 software. The phylogenetic tree is shown in detail in figure 3. Strain BZE-5 was identified to species accurately by phylogenetic analysis of the 16SrDNA gene sequences, and strain BZE-5 of the present invention was identified to its taxonomic position accurately by morphological characterization and phylogenetic analysis of the nucleic acid sequences.
Based on the above identification, strain BZE-5 was identified as Enterobacter cholerae (Enterobacter hormaechei).
3. Preservation of strains
The enterobacter cholerae (Enterobacter hormaechei) BZE-5 of the invention is preserved in China center for type culture collection, abbreviated as CCTCC, and has the following addresses: in the Jiuqiu No. 299 university of Wuhan in Wuchang district of Wuhan, hubei province (Wuhan university collection), the preservation number is: cctccc NO: m2023976.
EXAMPLE 2 study of Strain BZE-5 production of feruloyl esterase
(1) Qualitative detection of enzyme activity
The strain BZE-5 isolated in example 1 was inoculated onto a medium plate containing ethyl ferulate containing 0.3g NaCl, 1.3g (NH) 4 ) 2 SO 4 、0.3g MgSO 4 ·7H 2 O、0.3gK 2 HPO 4 18g of agar powder, 5 ten thousand U of nystatin, 15mL of ethyl ferulate (10% V/V dissolved in dimethylformamide) and 1000mL of distilled water, pH7.0, and sterilizing at 121 ℃ for 20 min; culturing in an incubator at 37 ℃ for 24 hours, if transparent circles appear around the colonies, the strains can generate feruloyl esterase, and judging the enzyme production capacity of the strains according to the sizes of the transparent circles. As a result, as shown in FIG. 4, the diameter of the enzyme solution transparent ring was 18.5mm.
(2) Quantitative detection of enzyme activity
The strain BZE-5 obtained by separation in the example 1 is inoculated in an LB culture medium to obtain seed bacterial liquid, and the OD value of the quantitative bacterial liquid at 600nm is 0.5, so that the quantitative bacterial liquid is used for wheat bran solid-state culture. 200g of bran is weighed, 160mL of distilled water is added, the mixture is stirred uniformly, the mixture is kept stand for 30min, the bran is fully wetted, and then the bran is packaged in triangular bottles (18 g per bottle) and sterilized at 121 ℃ for 40min. After the triangular flask is cooled in a sterile room, 3 percent of seed bacterial liquid is inoculated into the bran, and the bran is uniformly shaken and cultivated for 48 hours at the constant temperature of 37 ℃. Accurately weighing 5g (converted into absolute weight, DW), placing in a 250mL sterilization triangular flask, adding 60mL acetic acid-sodium acetate buffer solution (pH 4.6, 100 mM), placing in a shaking table with constant temperature of 30 ℃ for 180r/min for leaching for 1h,10000r/min, centrifuging at 4 ℃ for 15min, and taking the supernatant as crude enzyme liquid to be tested. Taking 1mL of diluted crude enzyme solution to be detected, preserving heat for 5min in a water bath at 30 ℃, adding 1mL of MFA solution (pH 4.6), reacting for 10min at 30 ℃, adding 2mL of 10% glacial acetic acid (V/V) to stop the reaction, centrifuging for 20min at 8000r/min at 4 ℃, and filtering with a 0.22 mu m filter membrane in a sample injection bottle to perform high performance liquid chromatography. Wherein feruloyl esterase enzyme activity is defined as: the amount of enzyme required for 1. Mu. Mol ferulic acid production by 1min ester hydrolysis of methyl ferulate at 30deg.C and pH4.6 is 1 enzyme activity unit (U). The enzyme activity of the strain BZE-5 solid state fermentation feruloyl esterase is determined as follows: 1024+ -43.63 mU/g DW.
Example 3 test of enhanced addition of bacterial moldy bran of Strain BZE-5
(1) Preparation of bacterial bran koji using enterobacter cholerae BZE-5
The strain BZE-5 isolated in example 1 was inoculated on an LB medium plate, bacterial colonies of the strain BZE-5 were picked from the LB medium plate and inoculated in 10mL of LB liquid medium, and the culture was allowed to stand at 37℃for 24 hours. 2mL of the culture was transferred to 100mL of LB liquid medium to prepare a seed solution, and the seed solution was subjected to shake culture at 37℃and 180r/min for 24 hours. Next, 100mL of the culture was transferred to 1000mL of seed medium, and the culture was subjected to shaking culture at 37℃and 180r/min for 24 hours. Subsequently, 1L of the culture was transferred to a 15L cassette pot containing 9L of seed medium, and cultured at 37℃for 24 hours to obtain a seed solution. Mixing bran 100%, rice hull 5.7% (w/w), adding caustic soda 0.22% (w/w), adding distilled water while stirring, scattering pimple with bran lifter, moistening for 30min, and steaming for 60min. After steaming, the materials can be discharged out of the steamer and cooled by a cooling machine, and the materials can be inoculated into a bran koji culture medium according to the proportion of 8% (v/w) after the temperature of the materials is reduced to 45 ℃, and then the materials are put into a room for culture for 74 hours at 37 ℃. The bacterial bran koji prepared above was weighed and counted in accordance with GB4789.2 (total colony count measurement), and the number of viable colonies thereof was 1X 10 7 Above CFU/g, the number of mixed bacteria should be lower than 1%.
(2) Preparation of sesame-flavor white spirit by using escherichia coli BZE-5 to prepare bacterial bran koji
The strengthening mode of the enterobacter cholerae bacterial bran koji is a mode of mixing materials. The method comprises the following steps: the prepared bacterial bran koji is added in the sesame flavor white spirit production to replace part of the usage amount of the traditional bran koji, wherein the mass ratio of the traditional bran koji to the bacterial bran koji is as follows: traditional bran koji bacterial bran koji = 5:1, w/w. The total bran koji amounts of the test group and the control group were kept consistent. And analyzing the content of ferulic acid and 4-VG and evaluating the sensory flavor of the obtained wine base sample.
(3) Analysis of ferulic acid and 4-VG content of wine base sample
The bacterial bran koji prepared by utilizing the enterobacter cholerae BZE-5 is characterized in that: the viable count of the enterobacter cholerae is 1 multiplied by 10 7 ～1×10 9 CFU/g; the feruloyl esterase activity is high and reaches 543.29mU/kg; compared with the control group, the alcohol content of the reinforced bran koji fermentation wine of the test group is improved by 8.3%, the content of 4-vinyl guaiacol is improved by 223.76%, and the content of ethyl acetate is improved by 48.09%.
(4) Sensory flavor evaluation of wine base samples
After the fermentation is finished, the fermented grains of the test group and the control group are distilled in a steamer, the obtained raw wine samples are used for sensory flavor evaluation, and different flavor profiles of the two raw wines on a radar chart (figure 5) are drawn. The wine base obtained by the test group shows better aroma release strength and typical sesame-flavor white spirit aroma (burnt aroma), and the obtained wine base has higher scores on aroma release strength, burnt aroma, sweet aroma, harmony degree and fullness; the raw wine obtained by the control group has higher score in the aspect of bitter taste, and the result shows that the quality of the raw wine is greatly improved after the bacterial bran koji prepared by the invention is added. FIG. 6 is a graph showing the content of flavor substances in the base wine of the control group and the test group, wherein 4-VG is an important aroma substance in the white wine, and the 4-VG in the base wine is improved by 223.76% after the bacterial bran koji prepared by the invention is added. From FIG. 6, it can be seen that other important flavor components such as ethyl acetate, n-propanol, etc. in the wine base obtained from the test group are also significantly increased. In addition, the added sourness of the wine base after the bacterial bran koji prepared by the invention has a certain contribution to the improvement of the sweetness and fullness of the wine body. Therefore, after the bacterial bran koji is added, the sensory quality and the flavor quality of the sesame-flavor white spirit are obviously improved.
The invention discloses a strain of enterobacter cholerae (Enterobacter hormaechei) BZE-5 for producing high-activity feruloyl esterase and application thereof in preparing bacterial bran koji, and a person skilled in the art can properly improve process parameters by referring to the content of the disclosure. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the invention has been described with reference to preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the invention described herein without departing from the spirit or scope of the invention.
Sequence listing
<120> A strain of Escherichia coli BZE-5 producing high activity feruloyl esterase and application thereof in preparation of bacterial moldy bran
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<211>1431
<212>DNA
<213>Artificial sequence (Artificial sequence)
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Claims (6)
1. The escherichia coli (Enterobacter hormaechei) BZE-5 for producing the high-activity feruloyl esterase is characterized in that the strain BZE-5 is preserved in China Center for Type Culture Collection (CCTCC) at the preservation number of CCTCC NO: m2023976.
2. Use of a strain of enterobacter cholerae (Enterobacter hormaechei) BZE-5 producing high activity feruloyl esterase according to claim 1 for the preparation of bacterial moldy bran.
3. The use according to claim 2, characterized by the steps of:
inoculating the strain BZE-5 on an LB culture medium plate, picking a bacterial colony of the strain BZE-5 from the LB culture medium plate, inoculating into an LB liquid culture medium, and performing stationary culture at a constant temperature of 37 ℃ for 24 hours; transferring the obtained culture into LB liquid medium to prepare seed liquid, and shake culturing at 37deg.C and 180r/min for 24 hr; transferring the culture into a seed culture medium, and culturing at 37 ℃ and 180r/min under shaking for 24 hours; transferring the culture into a cassette type tank containing a seed culture medium, and culturing at 37 ℃ for 24 hours to obtain a seed solution; stirring bran and rice husk uniformly, adding caustic soda, adding distilled water while stirring, stirring uniformly, scattering pimples by using a bran lifter, moistening and steaming, taking out of a steamer, cooling by using a cooling lifter, cooling to 45 ℃, inoculating to a bran koji culture medium in a proportion of 8% (v/w), and culturing for 74 hours at 37 ℃ in a room to obtain the bacterial bran koji.
4. Use according to claim 3, wherein 100% of bran and 5.7% (w/w) of rice husk are mixed and caustic 0.22% (w/w) is added.
5. The use according to claim 3, wherein the bacterial bran koji is added in the production of sesame-flavor liquor to replace part of the traditional bran koji usage amount to prepare high quality sesame-flavor liquor.
6. The use according to claim 5, characterized in that the mass ratio of traditional bran koji to bacterial bran koji is 5:1.
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