A torulopsis tsukubaensis ts-73 and its use in fermented fruit juice

Abstract

The application provides a Torulopsis glbrata TS-73 and application thereof in fermented fruit juice, and belongs to the technical field of microbial fermentation.The application adopts a composite mutagenesis method combining ARTP mutagenesis and ultraviolet mutagenesis to obtain a Torulopsis glbrata TS-73 capable of high yield of ethyl hexanoate;when the application is used for preparing fermented cherry juice and raspberry juice, the content of ethyl hexanoate in the cherry juice and the raspberry juice is increased by 44.01% and 54.19% respectively compared with the original strain Torulopsis glbrata G4.The Torulopsis glbrata TS-73 has the advantages of increasing the content of aroma substance ethyl hexanoate and stable performance.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial fermentation, and particularly relates to a Rhodotorula mucilaginosa TS-73 and application thereof in fermented fruit juice. BACKGROUND

[0002] Ethyl hexanoate, an important aroma substance in fruit juice, has fruity flavor, and ethyl hexanoate is mainly produced by fermentation of glucose by yeast. In the fermentation process of fruit juice, the characteristic aroma of fruit juice and the content of substances beneficial to human health are particularly important. The characteristic aroma of fruit juice mainly comes from raw material aroma, fermentation aroma and aging aroma.

[0003] ARTP mutagenesis technology is a new mutagenesis method for treating microbial cells by plasma, aiming to induce mutations in the genome of microorganisms, thereby creating new genetic variations and providing new ways for the improvement and application of microorganisms. Ultraviolet mutagenesis technology is a commonly used genetic improvement method. In the mutagenesis process, ultraviolet energy acts on the DNA molecules of organisms, causing chemical bond breakage and cross-linking between bases. These damages trigger the DNA repair mechanism of organisms, but the repair process is not always perfect, and may cause erroneous repair, which causes changes in DNA sequences and produces a large number of random mutations, thereby increasing the genetic diversity of organisms and providing more options and variation opportunities for breeding, gene research and evolution research. Compound mutagenesis refers to a strategy of combining multiple mutagenesis technologies for gene mutation. Compound mutagenesis can improve mutation efficiency and diversity, and accelerate the breeding of new varieties and gene function research. By reasonably designing and combining different mutagenesis technologies, more effective genetic modification can be achieved, and more possibilities can be brought to the research and application of biological fields. There is currently a lack of research on obtaining a new ethyl hexanoate-producing strain by combining ARTP mutagenesis and ultraviolet mutagenesis. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a new ethyl hexanoate-producing strain of Rhodotorula mucilaginosa TS-73 obtained by a compound mutagenesis method combining ARTP mutagenesis and ultraviolet mutagenesis and application thereof.

[0005] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions.

[0006] The present application provides a Rhodotorula mucilaginosa TS-73, which is classified as Rhodotorula mucilaginosa and preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 30360.

[0007] The application also provides the application of the above-mentioned Torulopsis glbrata TS-73 in fermenting fruit juice.

[0008] The application also provides the application of the above-mentioned Torulopsis glbrata TS-73 in producing high-ethyl hexanoate fruit juice products.

[0009] Preferably, the fruit juice comprises cherry juice or raspberry juice.

[0010] The application also provides a method for increasing the content of ethyl hexanoate in fermented fruit juice, comprising the following steps: inoculating the above-mentioned Torulopsis glbrata TS-73 into fermentation raw materials to perform fermentation.

[0011] Preferably, the fermentation raw materials comprise cherry or raspberry.

[0012] Preferably, the fermentation is aerobic fermentation.

[0013] Preferably, the temperature of the aerobic fermentation is 26-30℃, and the time is 4.5-5.5 days.

[0014] The application also provides fermented fruit juice fermented by the above-mentioned Torulopsis glbrata TS-73.

[0015] The application has the following advantages:

[0016] The application first adopts a composite mutagenesis method combining ARTP mutagenesis and ultraviolet mutagenesis to obtain Torulopsis glbrata TS-73 capable of high-yield ethyl hexanoate. When the strain provided by the application is used to prepare fermented cherry juice and raspberry juice, the content of ethyl hexanoate in the cherry juice and raspberry juice is increased by 44.01% and 54.19% respectively compared with the original strain Torulopsis glbrata G4. The Torulopsis glbrata TS-73 provided by the application has the advantages of increasing the content of aroma substance ethyl hexanoate, and stable performance. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a phylogenetic tree of the original strain Torulopsis glbrata G4;

[0018] Figure 2 It is a growth curve of the original strain Torulopsis glbrata G4;

[0019] Figure 3 It is the screening result of ARTP mutagenesis, wherein the abscissa represents the number of the original Torulopsis glbrata G4 strain and each screening mutagenesis strain;

[0020] Figure 4 It is the screening result of ultraviolet mutagenesis, wherein the abscissa represents the number of the original Torulopsis glbrata A35 strain and each screening mutagenesis strain;

[0021] Figure 5The GC-MS total ion chromatogram results of fermentation of cherry juice by the original strain of Rhodotorula mucilaginosa G4 in Example 4 and the mutant strain of Rhodotorula mucilaginosa TS-73;

[0022] Figure 6 The GC-MS total ion chromatogram results of fermentation of cherry juice by the original strain of Rhodotorula mucilaginosa G4 in Comparative Example 1;

[0023] Figure 7 The GC-MS total ion chromatogram results of fermentation of cherry juice by the mutant strain of Rhodotorula mucilaginosa TS-73 in Example 2;

[0024] Figure 8 The GC-MS total ion chromatogram results of fermentation of raspberry juice by the original strain of Rhodotorula mucilaginosa G4 in Comparative Example 2;

[0025] Figure 9 The GC-MS total ion chromatogram results of fermentation of raspberry juice by the mutant strain of Rhodotorula mucilaginosa TS-73 in Example 3.

[0026] DEPOSIT DESCRIPTION

[0027] The classification name of the Rhodotorula mucilaginosa TS-73 is Rhodotorula mucilaginosa, which is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Yard 3, Beijing Chaoyang District, Institute of Microbiology of Chinese Academy of Sciences, and has a preservation number of CGMCC No. 30360 and a preservation date of April 16, 2024. DETAILED DESCRIPTION

[0028] The present application provides a Rhodotorula mucilaginosa TS-73, which has a classification name of Rhodotorula mucilaginosa and is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 30360.

[0029] The Rhodotorula mucilaginosa TS-73 is obtained by a combined mutagenesis method of ARTP mutagenesis and ultraviolet mutagenesis of the Rhodotorula mucilaginosa G4, specifically, the Rhodotorula mucilaginosa G4 is subjected to ARTP mutagenesis, and the single colonies with obvious transparent circles after the mutagenesis treatment are inoculated on an ester-producing screening medium, the diameters d of the yeast colonies and the diameters D of the transparent circles are measured every day using a vernier caliper, and the ratio D / d is calculated, and the strain with the largest transparent circle is selected as the starting strain for the next ultraviolet mutagenesis. The ultraviolet mutagenesis strain is again screened by the transparent circle method, and finally a Rhodotorula mucilaginosa TS-73 with the strongest ester-producing capacity is obtained. The original strain of Rhodotorula mucilaginosa G4 is improved by the combined mutagenesis method of ARTP mutagenesis and ultraviolet mutagenesis, and the purpose of increasing aroma substances is finally achieved.

[0030] The present invention also provides the use of the above-mentioned Rhodotorula TS-73 in fermenting fruit juice. The present invention also provides the use of the above-mentioned Rhodotorula TS-73 in producing high-ethyl hexanoate fruit juice products.

[0031] In the present invention, the juice preferably includes cherry juice or raspberry juice.

[0032] The present invention also provides a method for increasing the content of ethyl hexanoate in fermented juice, comprising the following steps: inoculating the above-mentioned Rhodotorula TS-73 into fermentation raw materials for fermentation.

[0033] In the present invention, the fermentation raw materials preferably include cherries or raspberries. The present invention has no particular limitation on the specific sources of cherries and raspberries. In the present invention, the fermentation is preferably aerobic fermentation, the temperature of the aerobic fermentation is preferably 26°C-30°C, more preferably 28°C, and the time of the aerobic fermentation is preferably 4.5-5.5 days, more preferably 5 days. In the present invention, preferably, the red yeast TS-73 bacterial solution is inoculated into the juice for fermentation, the volume ratio of the red yeast TS-73 bacterial solution to the juice is 5:100, and the concentration of the red yeast TS-73 bacterial solution is preferably 1×10 7 CFU / mL.

[0034] The present invention also provides a fermented juice, wherein the fermented juice is fermented using the above-mentioned Rhodotorula TS-73 as a fermentation bacterium. The specific fermentation method is the same as above and will not be repeated here.

[0035] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] In the following examples, unless otherwise specified, all methods are conventional.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0038] Example 1

[0039] 1. Determination of the Growth Curve of Rhodotorula G4

[0040] The phylogenetic tree of the red yeast G4 strain (from the Culture Collection Center of Taishan University) is shown in Figure 2. Figure 1 After activation on YPD medium (as shown), 4% of the bacterial solution was inoculated into YPD liquid medium. Culture was carried out at 28°C and 150 rpm. From the 4th hour after culture, samples were taken every 2 hours to measure the OD value at 600 nm until the end of culture. The OD value was plotted on the horizontal axis with culture time as the horizontal axis. 600 The value is used as the vertical axis to draw the growth curve of the red yeast G4 strain.Figure 2 As shown, the subsequent Figure 2 The growth curve shown is used to determine the culture time required for mutagenesis.

[0041] 2. ARTP mutagenesis

[0042] The Rhodotorula G4 strain (from the Culture Collection Center of Taishan University) was activated on YPD medium, and then a loop of bacterial slurry was taken and placed in YPD liquid medium and cultured at 28°C and 150 rpm until the logarithmic growth phase (according to Figure 2 Next, the cells were centrifuged at 6000 rpm for 10 minutes to collect the cells. The cells were then washed three times with sterile water to remove impurities. The cells were resuspended and prepared to a cell concentration of 10 8 cfu / mL bacterial suspension. Before the mutagenesis treatment, adjust the gas flow rate of the plasma mutagenizer to 10L / min and the power supply to 120W, take 10μL of the prepared bacterial suspension and drop it on the sterilized metal slide, set the distance between the plasma emission source and the sample to 2mm, and use helium as the working gas for irradiation. The mutagenized samples were mutated for 0, 3, 6, 9, 12, 15, and 18 minutes respectively. The mutagenized samples were appropriately diluted and spread on the ester-producing medium (2% potato, 2% glucose, 2% agar, 0.4% tributyrin). The culture medium was placed in a constant temperature incubator at 28°C and cultured for 2 to 3 days. The single colonies with more obvious transparent circles after mutagenesis were point-to-point inoculated on the ester-producing medium. The yeast colony diameter d and the transparent circle diameter D were measured every day with a vernier caliper, and the ratio D / d was calculated. The strain with the largest D / d was selected as the starting strain for the next step of ultraviolet mutagenesis. The results are as follows Figure 3 As shown, Rhodotorula a35 was selected as the starting strain for UV mutagenesis.

[0043] 3. UV mutagenesis

[0044] The mutant strain Rhodotorula A35 screened by ARTP mutagenesis was used as the starting strain. The strain was activated on YPD medium, and then a loop of bacterial slurry was taken and placed in YPD liquid medium. The culture was carried out at 28°C and 150 r / min until the logarithmic growth phase (according to Figure 2 (determined by the growth curve). Under dark conditions, take 10ml of bacterial solution and put it into a sterile glass dish. Place a rotor inside and irradiate with ultraviolet light while rotating. The ultraviolet irradiation conditions are 0, 3, 6, 9, 12, 15, and 18 minutes respectively. After the irradiation, the mutagenized sample is appropriately diluted and spread on the ester-producing culture medium. Place the culture medium in a constant temperature incubator at 28°C and culture it in the dark for 2 to 3 days. Use the transparent circle method to screen the ultraviolet mutagenized strain again (the screening method is the same as 2. ARTP mutagenesis) to select the strain with the largest D / d. The results are as followsFigure 4 As shown in the table, the TS-73 strain of Rhodotorula mucilaginosa is the optimal strain.

[0045] Example 2

[0046] Preparation of cherry juice: cherry (purchased from Shandong Quanling Winery Co., Ltd.) was juiced, and the soluble solids of the cherry juice were adjusted to Brix 12° with white granulated sugar. The cherry juice was sterilized at 88°C for 15 min, and was ready for use.

[0047] Preparation of TS-73 strain of Rhodotorula mucilaginosa: a ring of yeast paste (TS-73 strain of Rhodotorula mucilaginosa obtained in Example 1) was inoculated into purified water containing 2% white granulated sugar, and was cultured at 28°C and 150 r / min for 16 h. The cultured yeast liquid was inoculated again into purified water containing 2% white granulated sugar, and was cultured until the bacterial concentration reached 1 x 10 7 CFU / mL, to obtain the TS-73 strain of Rhodotorula mucilaginosa liquid.

[0048] Fermentation: the TS-73 strain of Rhodotorula mucilaginosa liquid was inoculated into cherry juice for fermentation. The inoculation amount of the TS-73 strain of Rhodotorula mucilaginosa liquid was 5 mL per 100 mL of cherry juice, the fermentation temperature was 28°C, and the fermentation time was 5 days, to obtain fermented cherry juice.

[0049] Example 3

[0050] Preparation of raspberry juice: raspberry (purchased from Linxia City Shanhe Agricultural Science and Technology Development Co., Ltd.) was juiced, and the soluble solids of the raspberry juice were adjusted to Brix 12° with white granulated sugar. The raspberry juice was sterilized at 88°C for 15 min, and was ready for use. The preparation of the TS-73 strain of Rhodotorula mucilaginosa liquid was the same as in Example 2.

[0051] Fermentation: the prepared TS-73 strain of Rhodotorula mucilaginosa liquid was inoculated into raspberry juice for fermentation. The inoculation amount of the TS-73 strain of Rhodotorula mucilaginosa liquid was 5 mL per 100 mL of raspberry juice, the fermentation temperature was 28°C, and the fermentation time was 5 days, to obtain fermented raspberry juice.

[0052] Comparative Example 1

[0053] The difference from Example 2 is that the fermentation strain used for fermentation is Rhodotorula mucilaginosa G4, and the rest is the same as in Example 2.

[0054] Comparative Example 2

[0055] The difference from Example 3 is that the fermentation strain used for fermentation is Rhodotorula mucilaginosa G4, and the rest is the same as in Example 3.

[0056] Example 4

[0057] The fermented liquid (i.e. fermented fruit juice) 8 mL of Example 2-3 and Comparative Example 1-2 respectively was subjected to GC-MS headspace solid phase microextraction to determine the content of aroma substances, and the determination method was as follows:

[0058] Sample treatment: 8 mL of the fermented fruit juice sample was placed in a 20 mL sample bottle, 3 g of NaCl was added, and ultrasonic treatment was performed for 15 min, and GC-MS headspace solid phase microextraction was performed.

[0059] GC conditions: InertCap capillary column (30 m x 0.25 mm x 0.25 um), initial temperature 40℃ for 3 min, temperature increased to 220℃ at 2℃ / min, and held for 4 min, no split injection, helium as carrier gas, flow rate: 1 mL / min, injection port temperature 250℃.

[0060] MS conditions: Chromatograph-mass spectrometer interface temperature 250℃, ion source temperature 230℃, electron impact (EI) source, electron energy 70 eV.

[0061] Each sample was repeated three times, and the results were expressed as mean ± standard deviation, and are shown in Table 1 and Figures 5-9 .

[0062] Table 1 Ethyl hexanoate content (mg / L) of different groups

[0063]

[0064] From Table 1 and Figure 5 it can be seen that the ethyl hexanoate content of the mutagenized strain of Rhodotorula TS-73 fermented cherry juice and raspberry juice was increased by 44.01% and 54.19% respectively compared with the original strain of Rhodotorula G4.

[0065] From Figures 6-9 it can be seen that the aroma substances of the original strain G4 and the mutagenized strain TS-73 fermented cherry juice and raspberry juice were changed.

[0066] From the above results, it can be seen that the new strain of Rhodotorula TS-73 is used for the fermentation of cherry juice and raspberry juice, and high ethyl hexanoate content of cherry juice and raspberry juice can be obtained, i.e. the aroma-fermented fruit juice is obtained.

[0067] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. An application of Rhodotorula TS-73 in the production of high ethyl hexanoate juice products, characterized in that: The classification of the red yeast TS-73 is named red yeast Rhodotorula mucilaginosa , deposited in the General Microbiology Center of China Culture Collection Administration Committee of Microorganisms, with the deposit number CGMCC No.30360; the juice is cherry juice or raspberry juice.

2. A method for increasing the content of ethyl caproate in fermented fruit juice, characterized in that: The method comprises the following steps: inoculating red yeast TS-73 into fermentation raw materials for fermentation; the red yeast TS-73 is classified as red yeast Rhodotorula mucilaginosa , deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with the deposit number CGMCC No.30360; The fermentation raw material is cherry juice or raspberry juice.

3. The method according to claim 2, characterized in that The fermentation is aerobic fermentation.

4. The method according to claim 3, characterized in that The temperature of the aerobic fermentation is 26° C.-30° C., and the time is 4.5-5.5 days.

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