CN118185792A - Bacterial strain for improving flavonoid compounds in pericarpium citri reticulatae and application of bacterial strain - Google Patents
Bacterial strain for improving flavonoid compounds in pericarpium citri reticulatae and application of bacterial strain Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a bacterial strain for improving the content of flavonoid compounds in pericarpium citri reticulatae and application thereof. The invention separates a strain of Siamese Bacillus N1 from pericarpium Citri Reticulatae Chachiensis, which can promote the accumulation of flavonoid compounds in pericarpium Citri Reticulatae Chachiensis and shorten the aging time of pericarpium Citri Reticulatae, and the strain is preserved in the microorganism strain collection in Guangdong province at 11/23 of 2023 with the preservation number of GDMCC NO:64058. According to the invention, the research shows that the N1 strain can improve the content of medicinal components such as luteolin, apigenin, nornobiletin and the like in the aging raw materials, and promote the accumulation of flavonoid compounds in pericarpium citri reticulatae; meanwhile, the strain is used for ageing fermentation, so that the flavonoid content in the pericarpium citri reticulatae can be improved in a short period, the pericarpium citri reticulatae ageing time is shortened, not only are excellent strain resources for pericarpium citri reticulatae ageing enriched, but also a new method for accelerating pericarpium citri reticulatae ageing is provided.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation. More particularly, relates to a bacterial strain for improving flavonoid compounds in pericarpium citri reticulatae and application thereof.
Background
The pericarpium Citri Tangerinae is mature pericarp of Citrus reticulata Blanco of Rutaceae and its cultivar, and is obtained by air drying and aging, and has effects of relieving asthma and cough, resisting oxidation, and resisting tumor, wherein pericarpium Citri Tangerinae (Citri Reticulatae 'chachi') is a genuine medicinal material in pericarpium Citri Tangerinae, and is derived from Citrus reticulata Blanco (Citrus reticulata 'CHACHIENSIS'), and can be made into pericarpium Citri Tangerinae after more than 3 years for normal aging, which is best known as pericarpium Citri Tangerinae of New Congress of Guangdong province. The term "Chen Jiuzhe good" of dried orange peel means that the longer the aging period is, the more remarkable the medicinal efficacy of dried orange peel is. In the ageing process of the dried orange peel, the content of medicinal substances such as flavonoid compounds in the dried orange peel is gradually accumulated and increased, and volatile oil substances are slowly converted, so that unique pleasant aroma and efficacy of the dried orange peel are finally formed. However, the prior wide orange peel aging process is mainly based on natural aging, namely, the wide orange peel aging process is stored in an aging bin with good ventilation for three to five years, the aging time is too long, the yield of the orange peel is not easy to improve, the natural storage environment is complex and uncontrollable, the standardization is difficult to realize, and the wide orange peel is easy to mildew, rot or worm-eaten.
The process of ageing pericarpium Citri Reticulatae Chachiensis is critical for its quality. The prior researches show that the composition and the content of flavonoid substances can be changed in the ageing process of the pericarpium citri reticulatae, and a large amount of microbial metabolism activities exist on the surface. In the orange peel aging process, microorganisms play an important role in influencing accumulation of active substances in the orange peel and formation of flavor, and the microorganisms can be utilized to realize artificial aging, shorten aging time, reduce mildew and the like. In the prior art, the microbial functional flora in the citrus reticulation process is researched, and three microbial strains capable of promoting the aging function are obtained through screening: siamese bacillus (Bacillus siamensis) BY-1, amyloliquefaciens bacillus (Bacillus amyloliquefaciens) BY-1 and Schmidt bacillus (Meyerozyma smithsonii) FY-1 can promote accumulation of active substances phenolic acid, hesperidin and synephrine in orange peels respectively (Zhang Sha. Research on microbial functions and residual peel and residue recycling in orange peel aging [ D ]. Chongqing university, 2022.DOI:10.27670/d.cnki. Gcqdu.2022.001809). However, the existing microbial strains for accelerating the ageing of the pericarpium citri reticulatae and improving the content of flavonoids as main active substances of the pericarpium citri reticulatae are fewer, and the excellent strain resources for promoting the ageing of the pericarpium citri reticulatae are still lacking.
Disclosure of Invention
The invention aims to overcome the defects of the existing strain for promoting the accumulation of flavonoid substances in pericarpium citri reticulatae and provides a strain for improving the flavonoid compounds in pericarpium citri reticulatae and application thereof.
The first object of the invention is to provide a Siamese Bacillus sp N1 strain for increasing the flavonoid compound content in pericarpium citri reticulatae.
A second object of the invention is to provide the use of a strain of Bacillus siamensis (Bacillus sp.) N1.
The third purpose of the invention is to provide a preparation for improving the content of flavonoid compounds in pericarpium citri reticulatae.
The fourth object of the invention is to provide a method for shortening the ageing time of dried orange peel.
The fifth object of the invention is to provide a method for increasing the flavonoid content in pericarpium citri reticulatae.
The above object of the present invention is achieved by the following technical scheme:
The invention separates a strain of Siamese Bacillus N1 which can promote the accumulation of flavonoid compounds in pericarpium citri reticulatae and shorten the aging time from pericarpium citri reticulatae, and the strain is preserved in the microorganism strain preservation center of Guangdong province at 11/23 of 2023 with the preservation number of GDMCC NO:64058. The research of the invention shows that the bacterial colony of the N1 bacterial strain is white, round, serrated at the edge, larger in bacterial colony, about 2-3 cm in diameter, rough in surface and provided with wrinkles; the strain is used for ageing and fermenting pericarpium citri reticulatae, can improve the content of medicinal components such as luteolin, apigenin, nornobiletin and the like in pericarpium citri reticulatae, promote the accumulation of flavonoid compounds in pericarpium citri reticulatae, achieve and are superior to the ageing effect of natural ageing for 1 year in a short time, and accelerate the ageing of pericarpium citri reticulatae; meanwhile, the N1 strain is used for aging fermentation, so that the contents of luteolin, apigenin and demethyl nobiletin in the pericarpium citri reticulatae can be improved in a short period, the pericarpium citri reticulatae aging time is shortened, the pericarpium citri reticulatae aging can be accelerated, the large-scale and enlarged production is facilitated, the excellent strain resource strain for the pericarpium citri reticulatae aging is enriched, and a new method is provided for accelerating the pericarpium citri reticulatae aging and improving the content of main active substances of the pericarpium citri reticulatae.
Therefore, the invention provides application of the Siamese bacillus N1 strain in shortening the ageing time of the dried orange peel.
The invention provides an application of a Siamese bacillus N1 strain in preparing a product for shortening ageing time of dried orange peel.
Preferably, the pericarpium Citri Tangerinae is pericarpium Citri Tangerinae.
The invention provides application of a Siamese bacillus N1 strain in improving flavonoid content in pericarpium citri reticulatae.
The invention provides application of a Siamese bacillus N1 strain in preparation of a preparation for improving flavonoid compound content in pericarpium citri reticulatae.
Preferably, the flavonoid compound in the pericarpium citri reticulatae is one or more of luteolin, apigenin and nornobiletin.
The invention provides a preparation for improving the content of flavonoid compounds in pericarpium citri reticulatae, which contains a Siamese bacillus N1 strain or bacterial liquid thereof.
The invention provides a method for shortening the ageing time of dried orange peel, which adopts a Siamese bacillus N1 strain or bacterial liquid thereof to ferment and treat ageing raw materials. Particularly, the N1 strain or bacterial liquid thereof is adopted for fermentation treatment, so that new skin with low cost can be added before aging, and aging is accelerated; or adding N1 strain or its bacterial liquid into pericarpium Citri Tangerinae aged for a certain time for fermentation to promote aging.
Preferably, the activated N1 strain or bacterial liquid thereof is evenly mixed with the aging raw material in a weight ratio of 2-4 percent and then is fermented.
More preferably, the aging raw materials include new skin raw materials for preparing dried orange peel, or dried orange peel raw materials in aging, i.e. dried orange peel raw materials aged for a certain time.
As a preferred embodiment, the present invention provides a specific fermentation process: uniformly mixing the fresh strain activated by the N1 strain with an ageing raw material in a weight ratio of 2-4%, then placing the mixture in a sterile container for fermentation, wherein the fermentation temperature is 25-28 ℃, the humidity is 70-80%, the fermentation time is not less than 90 days, and periodically turning and ventilating during the fermentation process.
In the scheme provided by the invention, the fermentation time is not less than 90 days, and the research shows that the effect of natural aging for 1 year can be achieved by adopting the N1 strain to age for at least 3 months, and the aging effect of one year can be achieved in a short time, so that the aging is accelerated. Therefore, according to the actual aging requirement, the fermentation time can be automatically adjusted so as to prepare the dried orange peel with different aging years: to prepare short-term aging samples for fermentation mechanism research, or to prepare dried orange peel products aged for three to five years. Based on the method provided by the invention, the person skilled in the art can further improve the quality of the dried orange peel by optimizing the condition of the aging method or combining other fermentation methods.
Preferably, the aging raw material adopts new tea branch citrus peel.
Based on the method for shortening the ageing time of the dried orange peel, which is provided by the invention, the preparation of the dried orange peel which is the subsequent ageing product can be carried out by referring to the conventional process method in the field.
The invention also provides a method for improving the content of flavonoid compounds in the pericarpium citri reticulatae, which adopts the Siamese bacillus N1 strain or bacterial liquid thereof to treat the sample.
The invention has the following beneficial effects:
The invention provides a Siamese bacillus (Bacillus sp.) N1 strain for improving flavonoid compounds in pericarpium citri reticulatae, which is separated from pericarpium citri reticulatae, can better adapt to physiological environment of pericarpium citri reticulatae, has mild fermentation conditions, can improve the contents of luteolin, apigenin and normethyl pericarpium citri reticulatae in a short time through fermentation, promotes the accumulation of flavonoid compounds in pericarpium citri reticulatae, achieves and is superior to the ageing effect of natural ageing for 1 year in a short time, and can accelerate the ageing of pericarpium citri reticulatae; meanwhile, the strain is used for ageing fermentation, so that the ageing time of the dried orange peel can be shortened, the accumulation of main medicinal components of the dried orange peel can be accelerated, the ageing efficiency can be improved, the production cost can be saved, the strain resources for the microbial ageing of the dried orange peel can be enriched, and the dried orange peel strain has a good application prospect.
Drawings
FIG. 1 is a colony morphology of Bacillus SIAMENSIS N strain.
FIG. 2 is a process flow diagram of a method for accelerating the aging of pericarpium Citri Reticulatae Chachiensis by microbial fermentation.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The raw material tea branch oranges used in the following examples are all the same batch of products picked from the same orchard in the new meeting area of the river, the Guangdong province.
NB solid medium: 10.0g of peptone, 3.0g of beef powder, 5.0g of sodium chloride, 15.0g of agar and 1L of deionized water; final pH 7.3±0.1 (25 ℃); autoclaving at 121℃for 15 minutes.
Example 1 isolation and identification of strains
1. Isolation and morphological characterization of strains
The method comprises the steps of preparing a sample from commercial high-quality Xinhui dried orange peel aged for more than three years, crushing the dried orange peel sample, preparing a sample suspension by using sterile water according to the dosage ratio of 1:9, carrying out gradient dilution, respectively taking 200 mu L of diluted sample suspension to be coated on NB solid culture medium, culturing for 3 days at 30 ℃, selecting strains with different forms for purification, and finally storing the strains obtained by separation in glycerol tubes. The morphological characteristics of the strain are shown in figure 1, the cultured colony is white, round, sawtooth-shaped at the edge, larger in colony, about 2-3 cm in diameter, rough in surface and provided with wrinkles.
2. Sequencing and identification of 16S rRNA sequence
After the purified strain is activated for 24 hours in NB culture medium, 1mL of bacterial liquid is taken as a DNA template, and PCR amplification is carried out by adopting universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), and the PCR amplification product is sent to sample sequencing. The sequence of about 1500bp of the total length of 16S rDNA of the strain is obtained through Sanger sequencing analysis, and the specific sequence is shown as SEQ ID NO. 1; and uploading the sequence to NCBI, and performing BLAST comparison and analysis to identify the species, wherein the result shows that the similarity of the strain and the strain of the bacillus siamensis (Bacillus siamensis) reaches 100%. The taxonomy of the isolated and purified strain was assigned to the strain of Bacillus (Bacillus siamensis) of the species Bacillus siamensis, designated N1, and deposited at the collection of microorganisms in the cantonese province at 11/23 of 2023 under the deposit number GDMCC NO, in combination with the morphological characteristics analysis described above: 64058, deposit address: building 5, guangzhou city martyr, road 100, college 59.
EXAMPLE 2 preparation of pericarpium Citri Reticulatae Chachiensis by Strain fermentation
1. Selecting materials
And (3) taking the tea branch citrus with good color and no obvious plant diseases and insect pests, cleaning and peeling the tea branch citrus, naturally drying until the moisture content is lower than 13%, and obtaining new tea branch citrus peel, and storing at room temperature and 45% humidity for standby.
2. Pretreatment of raw materials
Drying dried fresh tea branch mandarin orange peel at 60deg.C for 30min, pulverizing into fine powder (adjustable in preparation of different aged products, without pulverizing), sieving with 60 mesh sieve, and mixing.
3. Strain activation
Preparation of the culture medium: weighing 18g of NB culture medium, adding distilled water or deionized water 1L, stirring, heating and boiling to dissolve completely, packaging into triangular flask, sterilizing at 121deg.C under high pressure for 15min, and cooling to normal temperature for use. Inoculating Bacillus SIAMENSIS N strain preserved at-80 deg.c into culture medium for resuscitating culture at 25 deg.c for 24-48 hr. After streak culture on a flat plate, the uniform thalli, no mixed bacterial pollution and good activity are observed, namely normal; if the strain is contaminated, it needs to be re-streaked for purification. After confirming the purity and activity of the strain, the strain is subjected to subculture until generation 2 for later use.
4. Bacterial preparation
Inoculating the activated N1 strain into NB culture medium, shake culturing at 30deg.C for 2-3 days, centrifuging to remove culture solution, leaving precipitate, washing the precipitate with sterile deionized water for 2-3 times, removing liquid, and retaining precipitate to obtain fresh strain.
5. Inoculation and fermentation
The fresh strain of the N1 strain is uniformly mixed with the new skin powder of the citrus reticulata in a weight ratio of 2-4%, and then the mixture is placed in a sterile container for fermentation, wherein the fermentation temperature is 25-28 ℃, the humidity is 70-80%, the fermentation time is 90 days (dried orange peel with different years can be prepared by adjusting the fermentation time), and the mixture is periodically turned and ventilated in the fermentation process.
6. Post fermentation treatment
And (5) drying the tea branch orange peel powder at 35 ℃ for 24 hours after fermentation to obtain a finished product.
Comparative example 1
1. Selecting materials
And (3) taking the tea branch citrus with good color and no obvious plant diseases and insect pests, cleaning and peeling the tea branch citrus, naturally drying until the moisture content is lower than 13%, and obtaining new tea branch citrus peel, and storing at room temperature and 45% humidity for standby.
2. Pretreatment of raw materials
Drying natural dried new skin of Citrus reticulata at 60deg.C for 30 min, pulverizing into fine powder, sieving with 60 mesh sieve, and mixing.
3. Inoculation and fermentation
The tea branch citrus new peel powder with equal quantity is put into a sterile container for fermentation without inoculating fermentation strain, the fermentation temperature is 25-28 ℃, the humidity is 70-80%, the fermentation time is 90 days, and the tea branch citrus new peel powder is periodically turned and ventilated in the fermentation process.
4. Post fermentation treatment
And (5) drying the tea branch orange peel powder at 35 ℃ for 24 hours after fermentation to obtain a finished product.
Comparative example 2
The process was identical to comparative example 1, the only difference being that fermentation was performed using a tea branch citrus aging sample of the same variety as example 1 and having been naturally aged for 1 year.
Comparative example 3
The process was identical to comparative example 1, the only difference being that the fermentation was carried out using wide orange peel of the same variety as in example 1 and which had been naturally aged for 3 years.
EXAMPLE 3 variation of active substance content in pericarpium Citri Reticulatae Chachiensis after fermentation
The test was carried out by measuring the contents of luteolin, apigenin and desmethyl pericarpium Citri Tangerinae in the aged samples of example 1 and comparative examples 1 to 3 by ultra-high performance liquid chromatography. And measuring the flavonoid content by using ultra-high performance liquid chromatography (UPLC). The column was subjected to gradient elution using Poroshell SB-C18 column (4.6X105 mm i.d.,2.7 μm) with acetonitrile as mobile phase A and 0.1% formic acid as phase B. The flow rate is set to be 0.8mL/min, the column temperature is 40 ℃, and the sample injection amount is 2 mu L; the detection wavelength for luteolin and apigenin is 283nm, and the detection wavelength for demethyl pericarpium Citri Tangerinae is 330nm, and the test result is counted.
The test results are shown in the following table 1, and it can be seen that the luteolin, apigenin and nornobiletin content in the dried orange peel naturally aged for 3 years in the comparative example is higher than that of the aged sample naturally aged for 1 year; the new skin is fermented by inoculating the strain N1, so that the content of medicinal ingredients such as luteolin, apigenin, nornobiletin and the like is improved, the effect of the preparation is higher than that of an aged sample which is aged for 1 year naturally, the effect of the wide dried orange peel which is aged for 3 years naturally is not similar, but the ageing can be accelerated after the strain is fermented, the ageing is carried out on the basis, the ageing time of the dried orange peel can be greatly shortened, and the content of the medicinal ingredients of the preparation is improved.
TABLE 1 variation of the active substance content of different aged samples
| Sample of | Luteolin μg/g | Apigenin μg/g | Demethyl nobiletin μg/g |
| Example 1 | 302.96 | 56.38 | 445.39 |
| Comparative example 1 | 260.61 | 51.19 | 420.67 |
| Comparative example 2 | 271.82 | 43.77 | 434.96 |
| Comparative example 3 | 365.42 | 94.41 | 576.25 |
According to the invention, the effect of naturally aging the sample for 1 year can be achieved by using the N1 strain for at least 3 months, the aging time is shortened, and on the basis, the dried orange peel with different aging years can be prepared by continuing the aging fermentation or adjusting the fermentation time: preparing a short-term aging sample for researching fermentation mechanism, or continuing aging fermentation on the basis of the invention to prepare the dried orange peel product aged for three to five years. The N1 strain can be used for ageing and fermenting new peels, and can be applied to dried orange peels with different ageing years to promote ageing. Based on the method provided by the invention, the quality of the dried orange peel can be further improved by optimizing the condition of the aging method or combining other fermentation methods, and the subsequent aging preparation can be performed by referring to the conventional process method in the art.
In conclusion, the invention separates a strain N1 of the Siamese bacillus (Bacillus siamensis) which can promote the accumulation of flavonoid compounds in the pericarpium citri reticulatae and shorten the ageing time of the pericarpium citri reticulatae from the pericarpium citri reticulatae, and the strain is preserved in the microorganism strain collection in Guangdong province at 11 and 23 days of 2023, with the preservation number of GDMCCNO:64058. Researches show that the N1 strain can improve the content of medicinal components such as luteolin, apigenin, nornobiletin and the like in the aging raw materials, promote the accumulation of flavonoid compounds in the pericarpium citri reticulatae, achieve and are superior to the aging effect of natural aging for 1 year in a short time, and can accelerate the aging of the pericarpium citri reticulatae; meanwhile, the invention also provides a method for shortening the ageing time of the dried orange peel, the process flow chart of the ageing method is shown in figure 2, the ageing fermentation of the dried orange peel is realized by utilizing the microorganism N1 strain, the ageing time of the dried orange peel can be shortened, the accumulation of main medicinal components of the dried orange peel can be accelerated, the ageing efficiency can be improved, the production cost is saved, the strain resources for the microorganism ageing of the dried orange peel are enriched, and the method has a good application prospect.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A strain of Bacillus siamensis (Bacillus sp.) N1, wherein the strain has been deposited at the cantonese province microorganism strain deposit at month 11 and 23 of 2023 under accession number GDMCC NO:64058.
2. The use of the strain N1 of bacillus siamensis as claimed in claim 1 for shortening the ageing time of dried orange peel.
3. The use of the strain N1 of bacillus siamensis as claimed in claim 1 for the preparation of a product for reducing ageing time of dried orange peel.
4. The use of the strain N1 of bacillus siamensis as claimed in claim 1 for increasing the flavonoid content of pericarpium Citri Reticulatae Chachiensis.
5. Use of the strain N1 of bacillus siamensis according to claim 1 for the preparation of a preparation for increasing the content of flavonoids in pericarpium Citri Reticulatae Chachiensis.
6. The use according to claim 5 or 6, wherein the flavonoid in pericarpium Citri Reticulatae Chachiensis is one or more of luteolin, apigenin and nornobiletin.
7. A preparation for increasing the content of flavonoid compounds in pericarpium citri reticulatae is characterized by comprising the Siamese bacillus N1 strain or bacterial liquid thereof according to claim 1.
8. A method for shortening the ageing time of dried orange peel is characterized in that the Siamese bacillus N1 strain or bacterial liquid thereof in claim 1 is adopted to ferment the ageing raw material.
9. The method according to claim 8, wherein the activated N1 strain or a bacterial liquid thereof is uniformly mixed with the aging raw material in a weight ratio of 2-4% and then subjected to fermentation treatment.
10. A method for improving the content of flavonoid compounds in pericarpium citri reticulatae is characterized in that a sample is treated by adopting the Siamese bacillus N1 strain or bacterial liquid thereof according to claim 1.
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