A Bacillus velezensis strain and a method for accelerating the aging of citrus peel by fermenting the strain

Abstract

The present application provides a strain of Bacillus velezensis TMCC10152 with a deposit number of CGMCC No. 22652. It is a common dominant bacterium discovered by the applicant in tangerine peels of different years. Morphological, physiological and biochemical, and molecular identification experiments indicate that this bacterium is Bacillus velezensis. The present application also provides a method for accelerating the aging of tangerine peels by fermenting with this strain, including steps such as preparation of tangerine peel raw materials for fermentation, preparation of bacterial agents, inoculation and fermentation, and control of fermentation conditions. This method can rapidly transform the soup color, aroma, and taste of tangerine peels, obtaining the taste, flavor, and active ingredients of tangerine peels with a certain number of years. Compared with the traditional natural aging process, this method can greatly save economic and time costs.

Description

Technical Field

[0001] This application belongs to the field of microbial fermentation, and specifically relates to a method for preparing a bacterial agent by using dominant microorganisms isolated from naturally aged tangerine peels and fermenting tangerine peels to accelerate the aging of tangerine peels. Background Art

[0002] Tangerine peel is the dried and mature pericarp of the citrus reticulata Blanco and its cultivated varieties of the Rutaceae family. As a medicinal and edible material, it has the effects of regulating qi and strengthening the spleen, and drying dampness and resolving phlegm. The medicinal materials are divided into "tangerine peel" and "Guangdong tangerine peel". Among them, tangerine peel is mainly produced in Xinhui, Guangdong.

[0003] Newly harvested tangerine peels need to be placed for 3 years to become tangerine peel for medicinal use, and the longer the aging time, the better the medicinal effect. The main active ingredients of tangerine peel, such as flavonoids, hesperidin, and volatile oils, are continuously transformed with the aging time, and the content of flavonoids shows an increasing trend.

[0004] At present, there are literature studies using high-throughput sequencing and pure culture methods to analyze the microorganisms on the surface of fresh fruits and tangerine peels of different aging years, and ascomycetes, aspergillus, penicillium, yeast, bacteria, etc. have been detected. For other medicinal plants, there are endophytic fungi that can increase the content of active ingredients. In recent years, it has become a trend to ferment Chinese herbal medicines by biological fermentation methods to improve the taste and increase their active ingredients.

[0005] In recent years, scientists at home and abroad have successively isolated a large number of Bacillus velezensis from plant rhizosphere soil, aquaculture ponds, deep sea, and daqu. This strain has the good characteristics of rapid growth and stability, is easy to isolate and culture, is harmless to humans and animals, does not pollute the environment, has rich metabolites, has broad-spectrum antibacterial activity and strong anti-stress ability. It has functions in the fields of agriculture, environment, and fermentation industry such as promoting plant growth, antagonizing pathogenic bacteria, and brewing white wine.

[0006] Currently, the patents on Bacillus velezensis mainly focus on the preparation of bacterial agents, the field of plant biological control and fermentation applications. For example, CN202010064158.0, a biocontrol Bacillus velezensis SF259 and its application; CN201811266027.X, a Bacillus velezensis L1-21 and its application in controlling citrus huanglongbing; CN201710279437.7, a Bacillus velezensis strain producing nattokinase and its application; CN201911084267.2, a method for improving the quality of tobacco leaves by fermenting with Bacillus velezensis. However, the research on using Bacillus velezensis for tangerine peel fermentation to accelerate its aging is still blank. Summary of the Invention

[0007] Based on this, the present application provides a Bacillus velezensis strain and a method for accelerating the aging of citrus peel by fermenting with this strain.

[0008] Specifically, the present application first isolates a Bacillus velezensis strain TMCC10152 from old tangerine peel, and the preservation number of the strain is CGMCC No. 22652. The strain was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on June 2, 2021. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the postal code is 100101.

[0009] According to the physiological and biochemical characteristics of this strain, the present application applies this strain to the aging process of citrus peel.

[0010] Furthermore, the present application provides a method for accelerating the aging of citrus peel, including the following steps:

[0011] (1) Screening of citrus peel raw materials;

[0012] (2) Treatment of citrus peel raw materials: passing through steam treatment and cooling to room temperature;

[0013] (3) Preparation of inoculum and inoculation on citrus peel: inoculating the Bacillus velezensis strain TMCC10152 on the citrus peel raw materials cooled to room temperature;

[0014] (4) Fermentation control: placing the inoculated citrus peel into a storage box; fermenting for 60 days or more, controlling the temperature, humidity, oxygen content, and controlling the moisture content of the citrus peel raw materials;

[0015] (5) Finished product: taking out and drying after fermentation, controlling the moisture content to obtain fermented citrus peel.

[0016] Even further, the method for accelerating the aging of citrus peel includes the following steps:

[0017] (1) Screening of citrus peel raw materials;

[0018] (2) Treatment of citrus peel raw materials: passing through steam treatment for 5 - 60 seconds and cooling to room temperature;

[0019] (3) Preparation of inoculum and inoculation on citrus peel: inoculating the Bacillus velezensis strain on the citrus peel raw materials cooled to room temperature;

[0020] (4) Fermentation control: placing the inoculated citrus peel into a 50 - liter storage box with 2 holes on all 6 sides and equipped with breathable silica gel plugs; fermenting for 60 days or more under the condition of 20°C - 55°C, controlling the moisture content at 13% - 25%, and the oxygen content greater than 50%;

[0021] (5) Finished product: After the fermentation is completed, take it out and dry it, and control the moisture content at 9.5%-13% to obtain fermented tangerine peel.

[0022] Furthermore, the tangerine peel raw material is preferably the tangerine peel from Xinhui District.

[0023] Furthermore, the steam treatment time is preferably 10-30 s.

[0024] The specific operation of inoculating the tangerine peel with the bacterial agent is as follows: inoculate the Bacillus velezensis strain in the exponential growth phase into the tangerine peel for fermentation. Preferably, the inoculation amount of the Bacillus velezensis strain is 10 4 -10 5 bacteria per gram of tangerine peel; the bacterial agent is the strain cultured in the culture medium or the strain in the form of bacterial powder; the water content of the tangerine peel is less than 25% of the weight of the water-containing tangerine peel, preferably 15%-18%.

[0025] According to some embodiments of the present application, the fermentation temperature is preferably 25-40 °C; more preferably, the fermentation temperature is 28-37 °C.

[0026] According to some embodiments of the present application, the fermentation control method is preferably to put the inoculated tangerine peel into a 50-liter storage box with 2 holes on all 6 sides and breathable silica gel plugs, place it in an incubator at 35 °C for temperature control and ferment at 45% humidity for 60 days, and turn it over once a week to increase ventilation and heat balance.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] The present application discovers that compared with the strains and other bacterial species isolated from old tangerine peel, this strain can accelerate the aging of tangerine peel, making the aroma and taste of the aged tangerine peel reach the aroma and taste of tangerine peel with a certain number of years. The sour taste of fresh fruit peel is removed, the sweetness and smoothness are increased, the internal substances have undergone great changes, and the color of the soup changes from light yellow to dark yellow.

[0029] The present application explores and optimizes the fermentation conditions when the Bacillus velezensis CGMCC No.22652 strain is applied to the aging process of tangerine peel, further improving the above-mentioned beneficial effects and making the fermentation quality more stable.

[0030] Through the above method, the present application obtains a fermented citrus peel obtained by fermenting new citrus peel with Bacillus velezensis CGMCC No. 22652 as a microbial agent. The contained substances in the new citrus peel are effectively transformed and enhanced by this strain. At the same time, under the comprehensive influence of the fermentation method and conditions of the present application, the obtained citrus peel has a bright yellow soup color, a sweet and smooth taste, has the aroma of tangerine peel, and the active ingredients such as hesperidin and total flavonoids are significantly increased compared with the citrus peel stored naturally in the same year, and the antioxidant activity is significantly improved. Detailed implementation manners

[0031] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present application.

[0032] The experimental methods in the following embodiments are all conventional methods unless otherwise specified.

[0033] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0034] The CGMCC No. 22652 strain of the present application has particularly good effects in promoting the accelerated aging fermentation and processing of citrus peel. The present application discovers that this Bacillus velezensis can degrade limonin, produce antibacterial peptides to inhibit the growth of Aspergillus, so that the fresh fruit taste of citrus peel is quickly transformed and gradually presents the aroma and taste of old tangerine peel of a certain year. The soup color is darker than that of the new peel in the same year, the taste is rich, sweet, thick and smooth, with the aroma of old tangerine peel, and the active ingredients such as hesperidin and total flavonoids increase significantly, and the antioxidant activity is enhanced. Compared with the traditional process, this method can greatly save economic and time costs.

[0035] Another aspect of the present application provides a process method for accelerating the aging of citrus peel, including inoculating the microbial agent prepared from the Bacillus velezensis CGMCC No. 22652 strain on the citrus peel of the current year, controlling temperature, oxygen, and moisture, and performing fermentation culture.

[0036] The present application provides a strain of Bacillus velezensis and a method for accelerating the aging of citrus peel by using this microorganism for fermentation, which mainly consists of the following steps: isolation, screening, identification and preservation of microorganisms; preparation of microbial agents; preparation of raw materials for fermenting citrus peel; inoculation and fermentation; detection of physical and chemical components and activities of fermented citrus peel.

[0037] In a specific implementation manner of the present application, the method for aging citrus peel includes:

[0038] (1) Screening of tangerine peel raw materials;

[0039] (2) Treatment of tangerine peel raw materials: Treat with steam for 5 - 60 seconds and cool to room temperature;

[0040] (3) Preparation of inoculant and inoculation on tangerine peel: Inoculate the tangerine peel raw materials cooled to room temperature with Bacillus velezensis CGMCC No.22652 strain;

[0041] (4) Fermentation control: The inoculated tangerine peel is placed in a 50 - liter storage box with 2 holes on all 6 sides and breathable silica gel plugs; Ferment at 25°C to less than 40°C for 60 days or more, control the moisture content at 13% - 25%, and the oxygen content is greater than 50%;

[0042] (5) Finished product: After the fermentation is completed, take out and dry, control the moisture content at 9.5 - 13%, and use a 20 - mesh sieve to screen out the white inner pulp in the tangerine peel to obtain fermented tangerine peel.

[0043] This application discovers that fermenting Xinhui tangerine peel with the said CGMCC No.22652 can rapidly transform the soup color, aroma and taste, obtaining the taste and flavor and active ingredients of tangerine peel with a certain age. The aroma of the naturally aged tangerine peel without fermentation by this strain remains unchanged, and the taste and flavor are those of fresh peel.

[0044] This application also discovers that other strains such as Lactobacillus casei and Lactobacillus plantarum, etc. when fermenting tangerine peel raw materials alone, do not grow under aerobic conditions, and grow under the condition of reducing the oxygen content, but the fermented tangerine peel is extremely bitter. This application discovers that compared with other varieties of tangerine peel, the dried peel of Citrus reticulata 'Chachiensis' in Xinhui area can produce obvious changes in aroma and taste after fermentation, and the slightly red peel is superior to the big red peel and the green peel.

[0045] Preferably, the raw material used in this application is the tangerine peel from Xinhui area.

[0046] Before fermentation inoculation, steam treatment for 5 - 60S (preferably 10 - 30S) is adopted. On the one hand, it can kill the microorganisms carried by the tangerine peel itself, and on the other hand, it can kill the insect eggs carried by the tangerine peel raw materials. At the same time, the dried peel is easy to break due to low moisture content during the inoculation and mixing process, damaging the integrity of the tangerine peel. Through steam treatment, the tangerine peel quickly absorbs heat and moisture and becomes soft, greatly reducing the loss rate during the inoculation process. This application discovers that after the raw materials are treated with steam, the aroma and taste of the tangerine peel are transformed faster. When the treatment time is less than 5S, the surface temperature of the tangerine peel has not risen, and the effect of enhancing the aroma and killing microorganisms cannot be achieved. When the steam treatment time is higher than 60S, the tangerine peel absorbs a large amount of water and swells due to long - term high - temperature treatment, and the internal substances ooze out, which is not conducive to subsequent moisture and humidity control and is prone to mildew.

[0047] At the time of inoculation, the water content of the tangerine peel is usually less than 35% (preferably 15%-18%) of the weight of the hydrated tangerine peel.

[0048] Preferably, the strain of CGMCC No. 22652 of the present application in the exponential growth phase is inoculated into the tangerine peel for fermentation. More preferably, the inoculation amount of the Bacillus velezensis strain is 10 4 -10 5 strains per gram of tangerine peel.

[0049] The strain in the form of bacterial powder can be inoculated, or the strain can be directly taken from the culture medium for inoculation.

[0050] The exponential growth phase can be determined by depicting the growth curve of the Bacillus velezensis by conventional methods.

[0051] The inventors of the present application found that when the strain of CGMCC No. 22652 derived from aged tangerine peel is inoculated into the tangerine peel for fermentation, when fermented for more than 60 days under the conditions of 20°C - 55°C (preferably 28 - 37°C), the aroma, taste and texture can be transformed to be close to the flavor of aged tangerine peel. When the fermentation time is extended to 180 days under this condition, the taste and flavor of aged tangerine peel can be gradually formed. When the temperature is lower than 20°C, the strain hardly grows in the tangerine peel and cannot ferment and transform the tangerine peel; when the temperature is higher than 55°C, the water in the fermentation system is lost quickly, the growth of the strain is hindered, and the tangerine peel has a stuffy smell at high temperature, the color of the soup is deep but the taste becomes dull and the sour taste is heavy. Preferably, the fermentation culture is carried out under the conditions of 25°C to 40°C. Under this fermentation condition, the strain grows rapidly and degrades limonin, and transforms volatile oil components such as limonene. Most preferably, the fermentation temperature is 28 - 37°C.

[0052] The content of the present invention is further explained or illustrated by the following examples, but these examples should not be construed as limiting the protection scope of the present invention.

[0053] Isolation, screening, identification and preservation of microorganisms

[0054] 1. Isolation: Different-year tangerine peel samples sold on the market are collected. After the samples are crushed, a sample suspension is prepared with sterile water at a ratio of 1:9. 200 μl is taken and spread on an LB plate, and cultured at 30 degrees Celsius for 3 days. Strains with morphological differences are selected for purification, culture and preservation in glycerol tubes.

[0055] 2. Screening: The growth and aroma transformation of 143 tangerine peel strains fermenting tangerine peel at 37°C are screened. Among them, strains similar in morphology to Bacillus velezensis TMCC10152 are almost present in all samples, and are the main microorganisms in each sample, and can grow and transform the aroma under the screening conditions.

[0056] 3. Identification and evaluation: Select Bacillus velezensis TMCC10152 for morphological, physiological and biochemical, and conventional 16S molecular identification, and gyrB gene sequencing identification.

[0057] (1) Culture and morphological identification: Bacillus velezensis TMCC10152 can be revived, activated, and cultured using various media such as conventional LB medium, PDA medium, PCA medium, MRS medium, YPD medium, etc. The culture characteristics of the strain are as follows: on TSA medium, cultured at 37°C for 24 h, the colonies are light yellow, nearly round, flat, opaque, and the edges are irregular. On TSA medium, cultured at 37°C for 24 h, the microscopic characteristics of the strain are as follows: the cells are rod-shaped, 0.6 - 0.7 μm × 1.5 - 3.5 μm, arranged singly or in pairs, the spores are oval, central or subterminal, the sporangium does not swell, and it is Gram-positive.

[0058] (2) Physiological and biochemical characteristics:

[0059] a. The experimental results of the utilization of the sole carbon source or sole nitrogen source by the strain are as follows:

[0060] This strain can utilize D-mannose, pyruvate, D-glucose, D-mannitol, and archaic sugar. It cannot utilize cyclodextrin, inositol, maltotriose, D-melezitose, L-rhamnose, inulin, D-galactose, glycogen, D-tagatose, D-trehalose, methyl-D-xyloside, and D-ribose.

[0061] b. The experimental results of the enzyme activities of the strain are as follows:

[0062] This strain can produce β-xylosidase, phenylalanine arylamidase, α-galactosidase, alanine arylamidase, alanine-phenylalanine-proline arylamidase, β-glucosidase, L-pyrrolidone arylamidase, and tyrosine arylamidase; it cannot produce L-lysine arylamidase, L-proline arylamidase, α-mannosidase, β-glucosidase, L-aspartate arylamidase, leucine arylamidase, β-galactosidase, Β-N-acetylglucosaminidase, glycine arylamidase, and β-mannosidase.

[0063] c. The experimental results of the tolerance of the strain are as follows:

[0064] Drug resistance experimental results: The kanamycin resistance result is negative, the polymyxin B resistance result is negative, and the oleandomycin resistance result is negative.

[0065] Salt tolerance experimental results: It can grow in 6.5% sodium chloride.

[0066] d. Molecular identification of the strain:

[0067] The gene sequence of the gyrB gene segment of the strain is shown as SEQ ID NO.1 in the sequence listing, and the identification result shows that the strain is Bacillus velezensis.

[0068] e. Toxicity evaluation of the strain: The fermentation broth of Bacillus velezensis TMCC10152 was used to feed male and female mice, and the changes in body weight and death of the mice were observed. The results showed that the strain had no effect on the body weight of the mice, and no toxic reactions or deaths were observed in the tested mice.

[0069] Table 1 Effects of TMCC10152 culture on the body weight of male mice (Mean±SD)

[0070]

[0071] Table 2 Effects of TMCC10152 culture on the body weight of female mice (Mean±SD)

[0072]

[0073]

[0074] Table 3 Acute toxic effects of TMCC10152 culture on mice

[0075]

[0076] 4. Preservation: The strain was preserved in the General Microbiological Center of the China National Culture Collection of Microorganisms on June 2, 2021. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101, and the preservation number is CGMCC No. 22652.

[0077] Example 1 Fermentation of slightly reddish pericarp of Xinhui tangerine peel with Bacillus velezensis

[0078] 1. Preparation of fermentation raw materials: The slightly reddish pericarp raw materials of Xinhui tangerines in 2020 were turned over and dried in the sun, and then dried at 105°C until constant weight, and the moisture content of the raw materials was measured. The pericarp raw materials were treated with steam from a steam generator for 20 seconds, placed flat in an inoculation tank to cool down and remove the surface heat, and samples were taken and dried at 105°C until constant weight, and the moisture content of the raw materials was measured.

[0079] 2. Preparation of microbial inoculum:

[0080] (1). Activation of the strain: The preserved Bacillus velezensis CGMCC No. 22652 was streaked on a YPD solid culture plate (2% glucose, 2% yeast powder, 1% peptone, 1% agar) and cultured at 20 - 55°C for 24 - 72 hours.

[0081] (2) Shake flask scale-up culture: Inoculate the activated strain into 200 ml of YPD liquid medium without agar, and culture it at a constant temperature of 100 rpm on a shaker at 20 - 55 °C for 24 - 36 hours until OD600 ≥ 2.5. Then raise the shaker temperature by 5 °C and culture for 8 hours to promote the transformation of bacteria into spores.

[0082] (3) Collect bacterial sludge: Centrifuge at 4000 - 7500 rpm for 10 - 20 min to collect Bacillus velezensis CGMCC No. 22652 with a spore conversion rate of ≥ 85%. Add a composite protective agent (6% skim milk powder, 5% trehalose, 8% inulin, 5% maltodextrin) at a ratio of 1:1, stir and mix well, and load it into a sterile freeze-drying tray.

[0083] (4) Freeze-drying: Pre-freeze at -50 °C for 1.5 hours, perform freeze-drying in a vacuum freeze-dryer for 20 hours, and perform analytical drying at 25 - 50 °C for 3 hours to obtain a direct-inoculum bacterial agent of Bacillus velezensis.

[0084] 3. Inoculate the bacterial agent: Weigh the above-mentioned Bacillus velezensis agent according to an inoculation amount of 0.2%, dissolve it in sterile water at 40 °C, stir and mix well to obtain a bacterial suspension, and inoculate it on the surface of slightly red-skin raw materials by pressure spraying in 3 times. During this period, let the water absorb for 5 - 10 min and then turn and mix well to further reduce the loss of fermented tangerine peel. The final moisture content is 19%, and the initial strain is 10 4 per gram of tangerine peel.

[0085] 4. Fermentation culture: Divide the inoculated tangerine peel into 50-liter perforated storage boxes, with 5 kg of raw materials in each box. After covering, place it in a culture room for fermentation at a controlled temperature of 35 °C and a humidity of 45% for 60 days, and turn and mix it once a week to increase ventilation and heat balance.

[0086] 5. Finished product: After the fermentation is completed, take it out and dry it in the air, control the moisture content at 9.5 - 13%, and use a 20-mesh sieve to sieve out the white inner pulp in the tangerine peel to obtain fermented tangerine peel.

[0087] 6. Evaluation: Compare the color of the outer skin and inner skin of the fermented tangerine peel with that of the unfermented tangerine peel; take 2 g of the fermented tangerine peel, tear it up and add it to 100 ml of boiling water and boil for 10 - 20 minutes to evaluate the taste; take 2 g of the fermented tangerine peel, tear it up and place it in a 250-ml evaluation cup, pour boiling water and steep for 5 min to evaluate the aroma.

[0088] Example 2

[0089] The steps are the same as those in Example 1, except that the fermentation raw material is the Sanjiang Dahong peel of Xinhui tangerines in 2020.

[0090] Example 3

[0091] The steps are the same as those in Example 1, except that the fermentation raw material is the green peel of Xinhui Sanjiang in 2020.

[0092] Example 4

[0093] The steps are the same as those in Example 1, except that the raw material is treated with steam for 5 seconds.

[0094] Example 5

[0095] The steps are the same as those in Example 1, except that the raw material is treated with steam for 10 seconds.

[0096] Example 6

[0097] The steps are the same as those in Example 1, except that the raw material is treated with steam for 30 seconds.

[0098] Example 7

[0099] The steps are the same as those in Example 1, except that the raw material is treated with steam for 60 seconds.

[0100] Example 8

[0101] The steps are the same as those in Example 1, except that the initial inoculated strain is 10 5 per gram of tangerine peel.

[0102] Example 9

[0103] The steps are the same as those in Example 1, except that the fermentation temperature is 20°C.

[0104] Example 10

[0105] The steps are the same as those in Example 1, except that the fermentation temperature is 25°C.

[0106] Example 11

[0107] The steps are the same as those in Example 1, except that the fermentation temperature is 28°C.

[0108] Example 12

[0109] The steps are the same as those in Example 1, except that the fermentation temperature is 37°C.

[0110] Example 13

[0111] The steps are the same as those in Example 1, except that the fermentation temperature is 55°C.

[0112] Example 14

[0113] The steps are the same as those in Example 1, except that the humidity of the fermentation environment is controlled at 40%.

[0114] Example 15

[0115] The steps are the same as those in Example 1, except that the humidity of the fermentation environment is controlled at 65%.

[0116] Comparative Example 1

[0117] The steps are the same as those in Example 1, except that the fermentation raw material is the citrus peel from Guangxi region.

[0118] It was found that during the fermentation process, the aroma change of the citrus peel during turning was not as good as that of the Xinhui citrus peel.

[0119] Comparative Example 2

[0120] The steps are the same as those in Example 1, except that the raw material is treated with steam for 4 seconds.

[0121] It was found that the treatment time was short, and the integrity of the citrus peel was damaged during turning with moistening.

[0122] Comparative Example 3

[0123] The steps are the same as those in Example 1, except that the raw material is treated with steam for 65 seconds.

[0124] It was found that some citrus peels absorbed more water and showed swelling phenomenon, and it was easy to get moldy during the subsequent fermentation process.

[0125] Comparative Example 4

[0126] The steps are the same as those in Example 1, except that the fermentation temperature is 15 °C.

[0127] It was found that due to the low temperature, the microorganisms did not grow. Under the same fermentation time, there was almost no difference compared with the unfermented control citrus peel.

[0128] Comparative Example 5

[0129] The steps are the same as those in Example 1, except that the fermentation temperature is 60 °C.

[0130] It was found that due to the high temperature, the microorganisms did not grow. At the same time, the moisture of the moistened citrus peel was quickly lost in a short period, and it was necessary to replenish water frequently, increasing the complexity of the operation. At the same time, the volatile oil of the citrus peel decreased at high temperature and there was no aroma of the citrus peel.

[0131] Comparative Example 6

[0132] The steps are the same as those in Example 1, except that the humidity is controlled at 20%.

[0133] It was found that the environmental humidity was too low, and the moisture in the fermentation box was extremely easy to lose. It was necessary to replenish water regularly, greatly increasing the complexity of the fermentation process.

[0134] Comparative Example 7

[0135] The steps are the same as those in Example 1, except that the humidity is controlled to 80%.

[0136] The results showed that due to the high humidity in the environment, the moisture content of the tangerine peel was too high, causing the growth of miscellaneous bacteria, resulting in obvious mold and strange odors.

[0137] Comparative Example 8

[0138] The steps are the same as those in Example 1, except that the strain used for fermentation is Lactobacillus plantarum.

[0139] The results showed that no live bacteria could be detected on the tangerine peel just one week after inoculation. After 60 days of fermentation, the color, aroma and taste of the tangerine peel did not change significantly. Moreover, due to the sealing and isolation of oxygen during the fermentation process, the substances in the tangerine peel were transformed in a direction that was not conducive to the taste under the influence of temperature and humidity, and the bitterness was extremely strong.

[0140] Comparative Example 9

[0141] The steps are the same as those in Example 1, except that the strain used for fermentation is Lactobacillus casei.

[0142] The results showed that the Lactobacillus casei grew slightly on the tangerine peel. After 60 days of fermentation, samples were taken for evaluation. The color, aroma and taste of the fermented tangerine peel did not change significantly. Due to the low oxygen content and poor material conversion during the fermentation process, the tangerine peel tasted very bitter.

[0143] Experimental Example 1 Evaluation of the taste and aroma of tangerine peel after fermentation under different conditions

[0144] According to the review in the embodiment, the results shown in Table 4 are obtained:

[0145] Table 4 Evaluation of taste and aroma of tangerine peel after fermentation under different conditions

[0146]

[0147]

[0148] It can be seen that by using the method of accelerating aging of tangerine peel by fermenting with Bacillus Velez subtilis in the present application, the color of the outer and inner surfaces of the new peel can be deepened, and the aroma and taste can be transformed to achieve an aroma and taste similar to tangerine peel.

[0149] Compared with other varieties of tangerine peels, the dried tangerine peels from Xinhui area can produce obvious changes in aroma and taste after fermentation, and the slightly red peel is better than the dark red peel and better than the green peel.

[0150] When lactic acid bacteria such as Lactobacillus casei and Lactobacillus plantarum are used to ferment tangerine peel alone, the strains do not grow under aerobic conditions. The strains grow for a period of time under conditions of reduced oxygen content, but the fermented tangerine peel tastes extremely bitter.

[0151] Experimental Example 2 Detection of Physicochemical Components of Fermented Tangerine Peel

[0152] Take 140 g of the fermented tangerine peel sample, crush it, and determine the moisture content according to the second drying method of the moisture determination method 0832 in the fourth part of the Chinese Pharmacopoeia 2015. Determine the water extract content according to the hot extraction method of the water-soluble extract determination method 1 in the extract determination method 2201 in the fourth part of the Chinese Pharmacopoeia 2015. Determine the total flavonoid content according to NY / T 2010-2011 Determination of Total Flavonoid Content in Citrus Fruits and Their Products. Determine the hesperidin content according to the hesperidin content determination method in the first part of the Chinese Pharmacopoeia 2015. Determine the volatile oil content according to the first method of the volatile oil determination method 2204 in the fourth part of the Chinese Pharmacopoeia 2015. As shown in Table 5, it is the physicochemical components of the naturally aged tangerine peel of different years from the commercially available Ganzhilin. Table 6 is the detection data of the physicochemical components of the tangerine peel in each example and comparative example of the present application.

[0153] Table 5 Determination of Physicochemical Components of Naturally Aged Tangerine Peel of Different Years from Commercially Available Ganzhilin

[0154]

[0155] Table 6 Determination of Physicochemical Components of Fermented Tangerine Peel by CGMCC No.22652

[0156]

[0157]

[0158] It can be seen from the above detection results of physicochemical components that after the tangerine peel is fermented by Bacillus velezensis CGMCC No.22652 for 60 days, the volatile oil content decreases, and the hesperidin and total flavonoid contents increase, approaching the material components of the 3-year tangerine peel.

[0159] Experimental Example 3 Detection of Antioxidant Activity of Fermented Tangerine Peel

[0160] Crush the fermented tangerine peel sample, perform ultrasonic treatment at 1:20 with 70% ethanol solution for 15 min, centrifuge at 4500 rpm for 10 min, filter to obtain the supernatant, and determine the antioxidant activity of the tangerine peel before and after fermentation by DPPH free radical scavenging experiment and ABTS free radical scavenging experiment.

[0161] Table 7 Determination of DPPH Free Radical Scavenging Rate of Fermented Tangerine Peel by CGMCC No.22652

[0162]

[0163]

[0164] Table 8 Determination of ABTS radical scavenging rate of fermented citrus peel using CGMCC No.22652

[0165]

[0166] From the above antioxidant activity results, it can be seen that after 60 days of fermentation with Bacillus velezensis CGMCC No.22652, the DPPH radical scavenging rate and ABTS radical scavenging rate of slightly red peel and big red peel were both significantly increased, and the antioxidant activity was enhanced. Among them, the DPPH radical scavenging rate and ABTS radical scavenging rate of slightly red peel increased by 37% and 10.8% respectively; the DPPH radical scavenging rate and ABTS radical scavenging rate of big red peel increased by 19.4% and 8.6% respectively. The effect of this strain on enhancing the antioxidant activity of slightly red peel was more significant than that of big red peel, which was related to the differences in the substance components of the citrus peel raw materials themselves.

[0167] In summary, the present invention uses a strain isolated from old tangerine peel, which is identified as Bacillus velezensis CGMCC No.22652. Combining the fermentation process of the present invention can accelerate the transformation of citrus peel, form the unique aroma during the aging process of tangerine peel, enhance the sweet taste, and significantly increase the contents of active ingredients such as total flavonoids and hesperidin and the antioxidant activity.

[0168] The above has introduced the embodiments of the present application in detail. Specific examples are used in this article to elaborate on the principle and implementation manner of the present application. The description of the above embodiments is only used to help understand the method and its core idea of the present application. At the same time, any changes or deformations made by those skilled in the art based on the idea of the present application, within the specific implementation manner and application scope of the present application, all belong to the protection scope of the present application. In summary, the content of this specification should not be construed as a limitation to the present application. Sequence Listing <110> Yunnan Dayi Tea Industry Group Co., Ltd. <120> A Bacillus velezensis and a method for accelerating the aging of citrus peel by using this microorganism for fermentation <130> 210516CI <140> 2021-8-27 <210> 1 <211> 1121 <212> DNA <213> SEQ ID NO.1 <214> gyrB gene segment of Bacillus velezensis 1 GGTGTAGGGG CATCTGTCGT AAACGCCTTG TCGACCACTC TTGACGTTAC 51 GGTTCATCGT GACGGAAAAA TCCACTATCA GGCGTACGAG CGCGGTGTAC 101 CTGTGGCCGA TCTTGAAGTG ATCGGTGATA CTGATAAGAC CGGAACGATT 151 ACCCACTTCG TTCCGGATCC GGAAATCTTC AAAGAAACAA CCGTATACGA 201 CTATGATCTG CTTTCAAACC GTGTCCGGGA ATTGGCCTTC CTGACAAAAG 251 GCGTAAACAT CACGATTGAA GACAAACGTG AAGGACAAGA ACGGAAAAAC 301 GAGTACCACT ACGAAGGCGG AATCAAAAGC TATGTTGAGT ACTTAAACCG 351 TTCCAAAGAA GTCGTTCATG AAGAGCCGAT TTATATCGAA GGCGAGAAAG 401 ACGGCATAAC GGTTGAAGTT GCATTGCAAT ACAACGACAG CTATACAAGC 451 AATATTTATT CTTTCACGAA TAATATCAAC ACATACGAAG GCGGCACGCA 501 CGAGGCCGGA TTTAAAACCG GTCTGACCCG TGTCATAAAC GACTATGCAA 551 GAAGAAAAGG GATTTTCAAA GAAAATGATC CGAATTTAAG CGGGGATGAT 601 GTGAGAGAAG GGCTGACTGC CATTATTTCA ATTAAGCACC CTGATCCGCA 651 ATTCGAAGGG CAGACGAAAA CGAAGCTCGG CAACTCCGAA GCGAGAACGA 701 TCACTGATAC GCTGTTTTCT TCTGCGCTGG AAACATTCCT TCTTGAAAAT 751 CCGGACTCAG CCCGCAAAAT CGTTGAAAAA GGTTTAATGG CCGCAAGAGC 801 GCGGATGGCA GCGAAAAAAG CACGGGAATT GACCCGGCGC AAAAGTGCGC 851 TTGAGATTTC CAATCTGCCG GGCAAACTGG CGGACTGTTC TTCTAAAGAT 901 CCGAGCATTT CCGAGCTGTA TATCGTAGAG GGTGACTCTG CGGGCGGATC 951 AGCGAAACAG GGACGGGACC GTCATTTCCA AGCTATTCTG CCGCTGCGCG 1001 GTAAGATTCT GAACGTTGAG AAAGCCAGAC TTGATAAGAT TCTCTCAAAC 1051 AATGAGGTCA GATCAATGAT CACGGCCCTC GGAACAGGAA TCGGAGAAGA 1101 TTTTAATCTT GAAAAAGCGC G <210> 1 <211> 1381 <212> DNA <213> SEQ ID NO.2 <214> Bacillus velezensis 16S rDNA segment 1 ATGGGAGCTT GCTCCCTGAT GTTAGCGGCG GACGGGTGAG TAACACGTGG 51 GTAACCTGCC TGTAAGACTG GGATAACTCC GGGAAACCGG GGCTAATACC 101 GGATGGTTGT TTGAACCGCA TGGTTCAGAC ATAAAAGGTG GCTTCGGCTA 151 CCACTTACAG ATGGACCCGC GGCGCATTAG CTAGTTGGTG AGGTAACGGC 201 TCACCAAGGC GACGATGCGT AGCCGACCTG AGAGGGTGAT CGGCCACACT 251 GGGACTGAGA CACGGCCCAG ACTCCTACGG GAGGCAGCAG TAGGGAATCT 301 TCCGCAATGG ACGAAAGTCT GACGGAGCAA CGCCGCGTGA GTGATGAAGG 351 TTTTCGGATC GTAAAGCTCT GTTGTTAGGG AAGAACAAGT GCCGTTCAAA 401 TAGGGCGGCA CCTTGACGGT ACCTAACCAG AAAGCCACGG CTAACTACGT 451 GCCAGCAGCC GCGGTAATAC GTAGGTGGCA AGCGTTGTCC GGAATTATTG 501 GGCGTAAAGG GCTCGCAGGC GGTTTCTTAA GTCTGATGTG AAAGCCCCCG 551 GCTCAACCGG GGAGGGTCAT TGGAAACTGG GGAACTTGAG TGCAGAAGAG 601 GAGAGTGGAA TTCCACGTGT AGCGGTGAAA TGCGTAGAGA TGTGGAGGAA 651 CACCAGTGGC GAAGGCGACT CTCTGGTCTG TAACTGACGC TGAGGAGCGA 701 AAGCGTGGGG AGCGAACAGG ATTAGATACC CTGGTAGTCC ACGCCGTAAA 751 CGATGAGTGC TAAGTGTTAG GGGGTTTCCG CCCCTTAGTG CTGCAGCTAA 801 CGCATTAAGC ACTCCGCCTG GGGAGTACGG TCGCAAGACT GAAACTCAAA 851 GGAATTGACG GGGGCCCGCA CAAGCGGTGG AGCATGTGGT TTAATTCGAA 901 GCAACGCGAA GAACCTTACC AGGTCTTGAC ATCCTCTGAC AATCCTAGAG 951 ATAGGACGTC CCCTTCGGGG GCAGAGTGAC AGGTGGTGCA TGGTTGTCGT 1001 CAGCTCGTGT CGTGAGATGT TGGGTTAAGT CCCGCAACGA GCGCAACCCT 1051 TGATCTTAGT TGCCAGCATT CAGTTGGGCA CTCTAAGGTG ACTGCCGGTG 1101 ACAAACCGGA GGAAGGTGGG GATGACGTCA AATCATCATG CCCCTTATGA 1151 CCTGGGCTAC ACACGTGCTA CAATGGACAG AACAAAGGGC AGCGAAACCG 1201 CGAGGTTAAG CCAATCCCAC AAATCTGTTC TCAGTTCGGA TCGCAGTCTG 1251 CAACTCGACT GCGTGAAGCT GGAATCGCTA GTAATCGCGG ATCAGCATGC 1301 CGCGGTGAAT ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCACGA 1351 GAGTTTGTAA CACCCGAAGT CGGTGAGGTA A

Claims

1. A strain of Bacillus velezensis ( Bacillus velezensis ) strain TMCC10152, with a preservation number of CGMCC No. 22652.

2. Use of the strain according to claim 1 in the aging process of citrus peel.

3. A method for accelerating the aging of citrus peel, characterized in that it comprises the following steps: (1) Screening of citrus peel raw materials; (2) Treatment of citrus peel raw materials: passing through steam treatment and cooling to room temperature; (3) Preparation of inoculum and inoculation on citrus peel: inoculating the strain according to claim 1 on the citrus peel raw materials cooled to room temperature; (4) Fermentation control: placing the inoculated citrus peel into a storage box; fermenting for 60 days or more, controlling the temperature, environmental humidity, oxygen content, and controlling the moisture content of the citrus peel raw materials; (5) Finished product: taking out and drying after fermentation, controlling the moisture content to obtain fermented citrus peel.

4. The method according to claim 3, characterized in that it comprises the following steps: (1) Screening of citrus peel raw materials; (2) Treatment of citrus peel raw materials: passing through steam treatment for 5 - 60 seconds and cooling to room temperature; (3) Preparation of inoculum and inoculation on citrus peel: inoculating the strain according to claim 1 on the citrus peel raw materials cooled to room temperature; (4) Fermentation control: placing the inoculated citrus peel into a 50 - liter storage box with 2 holes on each of the 6 sides and equipped with breathable silica gel plugs; fermenting for 60 days or more under the conditions of 20°C - 55°C, controlling the environmental humidity at 40% - 65%, controlling the moisture content of the citrus peel raw materials at 13% - 25%, and the oxygen content greater than 50%; (5) Finished product: taking out and drying after fermentation, controlling the moisture content at 9.5% - 13% to obtain fermented citrus peel.

5. The method according to claim 4, characterized in that the citrus peel raw materials are preferably citrus peel from Xinhui District.

6. The method according to claim 4, characterized in that the time of passing through steam treatment is preferably 10 - 30 s.

7. The method according to claim 4, characterized in that the preparation of inoculum and inoculation on citrus peel is as follows: inoculating the Bacillus velezensis strain in the exponential growth phase into the citrus peel for fermentation, the inoculum is the strain cultured in a medium or the strain in the form of bacterial powder; the moisture content of the citrus peel is less than 25% of the weight of the water - containing citrus peel.

8. The method according to claim 7, characterized in that The inoculation amount of the Bacillus velezensis strain is 10 4 -10 5 colony-forming units per gram of citrus peel.

9. The method according to claim 7, characterized in that the moisture content of the citrus peel is 15% - 18% of the weight of the water - containing citrus peel.

10. The method according to claim 4, characterized in that the temperature for fermentation control is 25 - 40°C.

11. The method according to claim 4, characterized in that the temperature for fermentation control is 28 - 37°C.

12. The method according to claim 4, characterized in that the fermentation control method is preferably placing the inoculated citrus peel into a 50 - liter storage box with 2 holes on each of the 6 sides and equipped with breathable silica gel plugs, placing it in an incubator at 35°C for temperature control and 45% humidity for fermentation for 60 days, and turning it over once a week to increase ventilation and heat balance.

13. A microbial inoculum, characterized in that: its active ingredient is the Bacillus velezensis strain TMCC10152; its deposit number is CGMCC No.22652.

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