CN113186104B - Aspergillus niger and application and method thereof - Google Patents
Aspergillus niger and application and method thereof Download PDFInfo
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Abstract
The invention discloses an Aspergillus niger and application and a method thereof, wherein the Aspergillus niger PW-2 is preserved in China center for type culture Collection in 2020, 10, 21 months, with the biological preservation number as follows: CCTCC NO: m2020618, the Aspergillus niger is applied to the processing of black tea which takes steamed hard-tipped tea as raw material, which can improve the quality of the black tea, promote the conversion of polyphenol substances and improve the color of tea soup. The processing method of the black tea is simple and easy to operate, low in cost and good in finished product quality, and can solve the problem that the existing black tea is poor in quality, so that the black tea processed by the method is high in quality, high in polyphenol substance conversion rate and good in tea soup color.
Description
Technical Field
The invention relates to a processing method of black tea, and particularly relates to aspergillus niger and application and a method thereof.
Background
The dark tea belongs to post-fermented tea, has various health care effects of reducing fat and losing weight, resisting oxidation, inhibiting bacteria, resisting tumors, regulating immunity and the like, and is widely loved by consumers. Microbial fermentation is an important process for processing the black tea, and has obvious influence on the quality of the black tea. In the microbial fermentation process, aspergillus niger plays an important role, can secrete various extracellular enzymes including cellulase, hemicellulase, protease, amylase, tannase and the like, and can promote the material conversion in the tea fermentation process.
The raw materials of the dark tea are mostly coarse and old leaves, and the tea stalk content is high, so that the quality improvement of the dark tea has great limitation.
Disclosure of Invention
The invention aims to provide an Aspergillus niger and application and a method thereof, which solve the problem of poor quality of the existing black tea, and the Aspergillus niger PW-2 is inoculated in the black tea for fermentation, so that the degradation of tea polyphenol substances is promoted, the color of the black tea soup is improved, and the quality of the black tea is improved.
In order to achieve the aim, the invention provides an Aspergillus niger PW-2, wherein the Aspergillus niger PW-2 is preserved in China center for type culture Collection in 2020 within 10 months and 21 days, and the biological preservation number is as follows: CCTCC NO: m2020618.
Preferably, the Aspergillus niger PW-2 is used for reducing the content of total phenols in the dark tea and improving the color of the tea soup.
Preferably, the aspergillus niger PW-2 is used for reducing the content of catechin, epigallocatechin gallate and epicatechin gallate in the dark tea.
Preferably, the Aspergillus niger PW-2 reduces the content of gallic acid, epigallocatechin and epicatechin after 6 days of inoculation in the black tea fermentation.
Another object of the present invention is to provide a processing method of dark tea, which comprises: inoculating the bacterial suspension containing the aspergillus niger spores into tea leaves, and fermenting.
Preferably, the fermentation time is 6 days or more.
Preferably, the bacterial suspension of the aspergillus niger spores is inoculated into steamed needle tea for fermentation.
The aspergillus niger and the application and the method thereof solve the problem of poor quality of the existing black tea, and have the following advantages:
according to the method, the steamed black tea is used as a raw material, and aspergillus niger PW-2 is used for artificial inoculation and fermentation to produce the black tea, so that the degradation of tea polyphenol substances is promoted, the color of the black tea soup is improved, and the quality of the black tea is improved.
The method is simple and easy to operate, has low cost, and improves the quality of the dark green tea by inoculating the bacterial suspension containing the Aspergillus niger PW-2 to the steamed soft-shelled tea.
Drawings
FIG. 1 is a morphological feature diagram of Aspergillus niger PW-2 strain provided by the present invention.
FIG. 2 is a diagram of the construction of ITS phylogenetic tree of Aspergillus niger PW-2 strain provided by the present invention.
FIG. 3 is a graph of the percent total phenol content of inventive samples 1-9.
FIG. 4 is a graph showing the mass percentage of catechins in samples 1 to 9 according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EXAMPLE 1 isolation and purification of Aspergillus niger PW-2
Aspergillus niger PW-2 is obtained by separation and purification in the following way:
inoculating tea leaves or tea stalks of different parts of the Pingwu Wufu brick tea to a PDA culture medium, culturing at constant temperature of 28 ℃, selecting bacterial colonies when the bacterial colonies grow out, streaking and purifying the bacterial colonies in a new PDA culture medium until a single bacterial colony is obtained, and storing the bacterial colony.
The colony obtained by the separation is aspergillus niger which can secrete a plurality of extracellular enzymes, can promote the degradation of protein in tea leaves, can catalyze the oxidation of polyphenol compounds to convert the polyphenol compounds into substances beneficial to human bodies, and improves the taste and the efficacy of the tea soup.
The Aspergillus Niger PW-2 (Aspergillus Niger PW-2) is preserved in China center for type culture Collection, CCTCC NO: m2020618.
EXAMPLE 2 morphological Observation of Aspergillus niger PW-2
Morphological observation is carried out on the Aspergillus niger PW-2, specifically, the separated Aspergillus niger PW-2 is cultured in a PDA culture medium, an MEA culture medium, a CDA culture medium and a CYA culture medium for 7d (constant temperature culture at 25 ℃) respectively by adopting a single-point inoculation method, and the growth condition is observed. The preparation method of the PDA culture medium comprises the following steps: mixing 3g potato extract powder, 20g glucose and 14g agar with 1L water. The preparation method of the MEA culture medium comprises the following steps: 130g of malt extract powder, 0.1g of chloramphenicol and 15g of agar are mixed with 1L of water, and the pH of the culture system is adjusted to 6.0 +/-0.2. The preparation method of the CDA culture medium comprises the following steps: 3g of sodium nitrate, 1g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate, 0.5g of potassium chloride, 0.01g of ferrous sulfate, 30g of sucrose and 15g of agar were mixed with 1L of water. The preparation method of the CYA culture medium comprises the following steps: 3g of sodium nitrate, 1g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate, 0.5g of potassium chloride, 0.01g of ferrous sulfate, 30g of sucrose, 5g of yeast extract and 15g of agar were mixed with 1L of water.
Referring to A and B in FIG. 2, wherein A is a reverse morphological picture of a colony of Aspergillus niger PW-2 cultured in PDA medium for 4d, and B is a forward morphological picture of the colony of Aspergillus niger PW-2 cultured in PDA medium for 4 d. Growth of Aspergillus niger PW-2 in PDA medium was as follows: when the culture is carried out for 1d, a bacterial colony grows in the culture medium; the diameter of the colony is 42-52 mm at 4d, the shape is nearly circular, the center is flat, a circle of white velvet-shaped mycelium is arranged on the periphery of the colony, the structure of conidium is large, the surface is brownish black, and no exudate exists; the reverse side of the bacterial colony is dark yellow brown.
Referring to C and D in FIG. 2, wherein C is a reverse morphological picture of the bacterial colony when Aspergillus niger PW-2 is cultured in MEA medium for 4D, and D is a forward morphological picture of the bacterial colony when Aspergillus niger PW-2 is cultured in MEA medium for 4D. Growth of A.niger PW-2 in MEA medium was as follows: the bacterial colony grows fast; when the culture is carried out for 1d, a bacterial colony grows out of the culture medium, and when the culture is carried out for 4d, the diameter of the bacterial colony is 51-69 mm; the colony is nearly circular, the center is raised, and no radial groove is formed; the velvet hyphae at the periphery are white, white mycelium can be seen in the center, the conidium structure is abundant, the conidium structure is charcoal black, and the back surface of the colony is dark yellow brown.
Reference is made to E and F in FIG. 2, where E is a reverse morphological map of the colonies when Aspergillus niger PW-2 is cultured in CDA medium for 7d, and F is a forward morphological map of the colonies when Aspergillus niger PW-2 is cultured in CDA medium for 7 d. Growth of A.niger PW-2 in CDA medium is as follows: when the culture is carried out for 2d, bacterial colonies grow out in the culture medium; the diameter of the colony at 4d is 7-18mm, and the diameter of the colony at 7d is 47-50 mm; the colony is nearly circular, the center is protruded, the periphery velvet-shaped hyphae are white, and a small amount of conidium structures are generated on the surface of the colony along with the increase of the culture time and are black; the reverse side of the bacterial colony is colorless.
Referring to G and H in FIG. 2, wherein G is a reverse morphological picture of a colony when Aspergillus niger PW-2 is cultured in CYA medium for 4d, and H is a positive morphological picture of the colony when Aspergillus niger PW-2 is cultured in CYA medium for 4 d. Growth of A.niger PW-2 in CYA medium is as follows: the bacterial colony grows fast; when the culture is carried out for 1d, a bacterial colony grows out of the culture medium, when the culture is carried out for 4d, the diameter of the bacterial colony is 32-52mm, and when the culture is carried out for 7d, the diameter of the bacterial colony is 67-80 mm; the colony is nearly circular and has radial grooves; the periphery velvet-shaped hyphae are white, the center is light yellow, the color of the bacterial colony is deepened along with the increase of the culture time, no conidium is seen, and the surface of the bacterial colony is compact; the reverse side of the colony is yellow.
Further, in order to effectively observe the morphology of the Aspergillus niger PW-2, the isolated Aspergillus niger PW-2 was cultured in PDA medium for 7 days (incubated at 25 ℃ C.) and observed for growth. Preferably, the strain with the culture time of 2d to 4d is taken for microscopic observation under an optical microscope. The specific operation is as follows:
dropping 1-2 drops of lactic acid phenol cotton blue staining solution on a clean glass slide, taking a small amount of hyphae with spores from the edge of a bacterial colony by using an inoculating ring, placing the hyphae in the staining solution, then picking up the hyphae, covering a cover glass, observing the hyphae from a low-power lens to a high-power lens under a microscope, and taking a picture and recording.
The results are shown in FIG. 2, I-K, which represents the meristematic peduncle, hyphae and ascospore patterns (. Times.40) of A.niger PW-2, respectively, under light microscopy. The morphology is as follows: the conidiophores of the Aspergillus niger PW-2 occur in the matrix, the stalk stem is longer and has smooth wall, the conidiophores head is spherical to radial type, the top capsule is spherical or nearly spherical, and the diameter is 20-32 mu m; hyphae have septa and are asymmetrically branched; conidiophore is spherical, and after aging, the conidiophore becomes flat in transverse direction and has a diameter of 2.5-3.3 μm.
EXAMPLE 3 molecular biological characterization of Aspergillus niger PW-2
The ITS molecular biology identification of the Aspergillus niger PW-2 strain specifically comprises the following steps:
selecting primers ITS1 and ITS4 for amplification, carrying out agarose gel electrophoresis detection on the product, and taking a picture by an ultraviolet imaging system; and (3) performing sequence determination on the purified PCR product, performing homology comparison with an NCBI database, selecting a sequence with higher homology, analyzing the sequence by using MEGA analysis software, and constructing a phylogenetic tree, wherein the result is shown in figure 3.
The nucleotide sequence of the primer ITS1 is as follows:
5’-TCCGTAGGTGAACCTGCGG-3’(SEQ ID NO.1)；
the nucleotide sequence of the primer ITS4 is as follows:
5’-TCCTCCGCTTATTGATATGC-3’(SEQ ID NO.2)。
the aspergillus niger PW-2 strain has the highest homology with aspergillus niger, and the similarity is 100 percent. And determining the strain to be aspergillus niger according to the morphological characteristics and molecular biological identification results, and naming the strain to be aspergillus niger PW-2.
Experimental example 4 Aspergillus niger PW-2 fermentation steamed soft tea
Inoculating the bacterial suspension containing the Aspergillus niger PW-2 spores to the sterilized steamed needle tea raw material (needle tea produced in Meitan City, guizhou province is produced by adopting a steam fixation process), and fermenting. Wherein the raw material of the steamed needle tea is sterilized by high-pressure steam at 121 ℃ for 20min. The bacterial suspension is prepared by mixing sterile water with spores of Aspergillus niger PW-2, and contains 1.44 × 10 bacteria per mL 3 380mL of the bacterial suspension is inoculated to each kilogram of spores of Aspergillus niger PW-2 in the raw material of the steamed green needle tea. Inoculating the bacterial suspension, and fermenting at 28 deg.C for 21d.
The raw materials can make the black tea soft, soft in taste and better in quality; the processing method can improve the conversion rate of polyphenol and improve the color of tea soup.
And (3) setting 1-8 sample groups, wherein the sample 1 is a tea sample inoculated with the Aspergillus niger PW-2 bacterial suspension, and the samples 2-8 are black tea inoculated with the fermented 3d, 6d, 9d, 12d, 15d, 18d and 21d. Mass percent (%) of total phenols and catechins in the Fuzhuan tea of comparative samples 1 to 8.
The content of total phenols is determined according to GB/T8313-2008 detection of tea polyphenol and catechin content in tea. The catechin content is measured according to the following method, and the sample pretreatment and high performance liquid chromatography analysis conditions in the measuring method are as follows:
(1) Pretreatment of samples
Extracting a sample by using 70wt% of methanol water solution, wherein the material-liquid ratio is 1:40, ultrasonic extraction is carried out for 15min, centrifugation is carried out for 15min at 10000g at the temperature of 20 ℃, 10mL of supernatant is absorbed, and the supernatant is diluted by 2 times and then passes through a filter membrane of 0.22 mu m to be detected.
(2) Conditions of HPLC analysis
ODS-4 column (4.6X 250mm,5 μm); mobile phase A: water (0.1 wt% formic acid); and (3) mobile phase B: methanol; flow rate: 1mL/min; column temperature: 30 ℃; sample introduction amount: 10 mu L of the solution; ultraviolet detection wavelength: 280nm. Time elution procedure, as shown in table 1.
Table 1 shows HPLC elution procedure
The results of the measurement are shown in FIGS. 3 and 4, in which GA represents gallic acid, EGC represents epigallocatechin, C represents catechin, EGCG represents epigallocatechin gallate, EC represents epicatechin, and ECG represents epicatechin gallate. As can be seen from FIG. 3, the inoculation of Aspergillus niger PW-2 bacterial suspension can reduce the total phenol content of steamed coniferous tea by 59.63%. As can be seen from FIG. 4, inoculation of Aspergillus niger PW-2 bacterial suspension can decrease the total amount of catechin in dark green tea prepared from steamed soft tea, and the total amount of catechin in No. 8 black tea is decreased by 84.15% compared with steamed soft tea. GA. The content of EGC and EC rises first and then falls in the whole fermentation process, and the content of EGCG and ECG is in a descending trend as a whole. The content of EGCG was higher than the other substances and decreased to undetectable level on day 9 of fermentation (sample No. 4). The ECG content is second to EGCG, and the content of the ECG content is reduced by 97.18 percent after the fermentation is finished. The content of EGC is reduced by 39.64%. Other GA, C and EC with lower content are also in a downward trend finally. From the results, it was found that catechins were continuously degraded and the content was decreased as the degree of fermentation was increased, and that aspergillus niger PW-2 was able to promote the degradation of catechins. The result may be related to that the microorganism secretes a large amount of extracellular enzyme in the fermentation process, and the enzymatic oxidation of catechins is promoted, so that the content of the catechins is reduced.
according to the material-liquid ratio of 1:100 adding boiling water to the sample for brewing for 10min. Carrying out vacuum filtration on the tea soup, fixing the volume for standby, preheating a color difference meter, preparing a blank sample, correcting, taking the tea soup with the volume of 2/3 cuvette, measuring the color difference value, repeating each sample for three times by adopting a Hunter Lab color system, taking an average value, and measuring the values of L, a and b of the tea soup obtained by different samples, wherein L is a brightness value, a is a red value, b is a yellow value, and the measurement results are shown in Table 2.
Table 2 shows the color and luster results of the tea soup
Where Δ E is the total color difference value. As can be seen from the table 2, the degree of reddish yellow of the black tea soup after the black tea is brewed increases with the increase of the fermentation time, which shows that the black tea soup has the excellent quality of transparent and bright clear red after being fermented by the Aspergillus niger PW-2, and the color difference of the black tea soup is larger and larger as the fermentation degree is deepened and is clearly distinguished by naked eyes.
In conclusion, according to the processing method of the dark green tea taking the steamed soft-shelled turtle tea as the raw material, the bacterial suspension containing the aspergillus niger PW-2 is inoculated into the steamed soft-shelled turtle tea raw material, so that on one hand, a new application method can be provided for the steamed soft-shelled turtle tea, and the quality of the dark green tea is improved; on the other hand, by inoculating bacterial suspension containing Aspergillus niger PW-2, the conversion of polyphenol substances in the black tea can be promoted, and the color of the tea soup is improved.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
Sequence listing
<110> Sichuan university
<120> Aspergillus niger and application and method thereof
<141> 2021-03-16
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tccgtaggtg aacctgcgg 19
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcctccgctt attgatatgc 20
Claims (6)
1. A new kind of Aspergillus nigerAspergillus Niger) PW-2, characterized in that, this aspergillus niger PW-2 is preserved in China center for type culture Collection, and the biological preservation number is: CCTCC NO: m2020618.
2. The application of the aspergillus niger PW-2 to the black tea, as claimed in claim 1, wherein the aspergillus niger PW-2 is used for reducing the content of total phenols in the black tea and improving the color of tea soup; the raw material of the dark tea is steamed oolong tea.
3. The use according to claim 2, wherein aspergillus niger PW-2 is used to reduce the total amount of catechins in dark tea.
4. The use according to claim 2, wherein aspergillus niger PW-2 is used in black tea fermentation to reduce the content of epigallocatechin, epigallocatechin gallate, epicatechin gallate.
5. A processing method of dark tea is characterized by comprising the following steps:
inoculating a bacterial suspension containing the spores of Aspergillus niger according to claim 1 into steamed soft-shelled tea, and fermenting.
6. The process of claim 5, wherein the fermentation time is 6 days or more.
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| CN108112715A (en) * | 2016-11-28 | 2018-06-05 | 勐海茶业有限责任公司 | The method for preparing fermented tea, the fermented tea prepared with this method and its application |
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