Compound microbial agent fermented roxburgh rose black beer and application of compound microbial agent fermented roxburgh rose black beer to sophorolipid production

Abstract

The invention discloses rosa roxburghii tratt fruit black beer fermented by a compound microbial agent and application of the rosa roxburghii tratt fruit black beer to sophorolipid production. A synthetic microbial agent composed of two isolated and screened Wickerhamomyces anomalus strains and Candida bumblebee strains is utilized to ferment the roxburgh rose black beer, the roxburgh rose black beer with strong roxburgh rose flavor and mellow aftertaste is produced, one strain of the microbial Wickerhamomyces anomalus L251 is preserved in the China Center for Type Culture Collection, the preservation date is March 14, 2024, and the preservation number is CCTCC NO: M 2024491, and the other strain of the microbial Wickerhamomyces anomalus L251 is preserved in the China Center for Type Culture Collection (CCTCC). The invention relates to a method for preparing a bacillus bombycis strain L252, which is characterized in that the bacillus bombycis strain L252 is preserved in the China Center for Type Culture Collection on March 18, 2024, and the preservation number is CCTCC NO: M 2024521. The bacillus bombycis strain L252 has been preserved in the China Center for Type Culture Collection on March 18, 2024.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a compound microbial inoculum for fermenting Rosa roxburghii Tratt black beer and its use for producing sophorolipids. Background Art

[0002] Rosa roxburghii Tratt is a wild shrub growing in areas such as Guizhou, western Hunan, Liangshan in Sichuan, and Lichuan in Hubei. It belongs to the Rosaceae family and has high nutritional value. The vitamin C content in every 100g of Rosa roxburghii Tratt pulp can reach 2075 - 2725mg. In addition, Rosa roxburghii Tratt also contains various other vitamins and tannin compounds, such as vitamin P, B vitamins, vitamin E, folic acid, and carotenoids. It also contains 18 kinds of amino acids, including 8 essential amino acids for the human body, as well as various trace elements beneficial to the human body, such as iron, zinc, selenium, manganese, copper, cobalt, nickel, fluorine, aluminum, vanadium, and silicon. At present, the types of deep - processed Rosa roxburghii Tratt products are rich, covering multiple fields such as food, beverages, health products, and cosmetics. Food products mainly include: dried Rosa roxburghii Tratt fruits, Rosa roxburghii Tratt preserves, Rosa roxburghii Tratt cakes, Rosa roxburghii Tratt fresh fruit C jelly, and Rosa roxburghii Tratt fruit cakes; beverage products mainly include: Rosa roxburghii Tratt juice, Rosa roxburghii Tratt wine, Rosa roxburghii Tratt fruit vinegar, and Rosa roxburghii Tratt lactic acid bacteria beverage; health products mainly include: Rosa roxburghii Tratt oral liquid, Rosa roxburghii Tratt soft capsules, Rosa roxburghii Tratt chewable tablets, and Rosa roxburghii Tratt fine powder, etc. The diversified development of deep - processed Rosa roxburghii Tratt products not only meets the needs of different consumers but also provides strong support for the sustainable development of the Rosa roxburghii Tratt industry.

[0003] Currently, the wines developed from Rosa roxburghii Tratt mainly include Rosa roxburghii Tratt raw wine, Rosa roxburghii Tratt fruit wine, Rosa roxburghii Tratt brandy, and Rosa roxburghii Tratt glutinous rice wine. There are relatively few Rosa roxburghii Tratt beer products, especially Rosa roxburghii Tratt black beer, developed using Rosa roxburghii Tratt. At present, commercial fermenting agents are mostly used for fermenting Rosa roxburghii Tratt rice wine, fruit wine, and beer, and there are few reports on fermenting Rosa roxburghii Tratt black beer using the compound microorganism Wickerhamomyces anomalus combined with Candida bombicola. The types of fermenting microorganisms in the beer brewing process have an important impact on the quality and flavor of the fermented wine. At the same time, there are few reports on directly producing high - yield sophorolipids by fermenting beer with Candida bombicola that has not been genetically modified. Due to its environmental protection, high efficiency, and multi - functionality, sophorolipids have become a popular research object in the field of biosurfactants and have been widely used in multiple industries. Therefore, fermenting Rosa roxburghii Tratt black beer using the compound microorganism Wickerhamomyces anomalus combined with Candida bombicola is of great significance for increasing the types of beer products and deep - processed Rosa roxburghii Tratt products, improving the nutritional value of Rosa roxburghii Tratt black beer, the deep - processing value of Rosa roxburghii Tratt raw materials, and the production, extraction, and wide application of sophorolipids. Summary of the Invention

[0004] The purpose of the present invention is to provide a compound microbial inoculum composed of 2 microbial strains, namely the isolated and screened Wickerhamomyces anomalus and Candida bombicola strains. The content of sophorolipids in Rosa roxburghii Tratt black beer is increased through the biological fermentation method of the microbial inoculum, and at the same time, the flavor substances of Rosa roxburghii Tratt black beer are enriched.

[0005] Two strains of isolated and screened Wickerhamomyces anomalus and Candida bombicola are used to ferment Rosa roxburghii stout, producing Rosa roxburghii stout with a strong Rosa roxburghii flavor and a mellow aftertaste. One of the microorganisms, Wickerhamomyces anomalus (L251), has been deposited in the China Center for Type Culture Collection. The deposit date is March 14, 2024, and the deposit number is CCTCC NO: M 2024491;

[0006] The other microorganism, Candida bombicola (L252), has been deposited in the China Center for Type Culture Collection. The deposit date is March 18, 2024, and the deposit number is CCTCC NO: M 2024521.

[0007] The steps for making the Rosa roxburghii stout of the present invention are as follows:

[0008] (1) Malt crushing: 650 g of pale malt, 250 g of dark caramel malt, and 100 g of black malt are ground and crushed using a grinder;

[0009] (2) Feeding: 1 kg of crushed malt is added to the saccharification pot, and 3.5 - 4 L of clear water is heated to 55 °C to complete the feeding;

[0010] (3) Saccharification: After feeding, the infusion saccharification method is used for saccharification. The temperature increase steps of the mash are as follows: heated to 50 - 55 °C, held for 30 min, stirred appropriately, heated to 65 - 68 °C, held for 50 min, iodine solution is measured every 30 min and once every 10 min until the iodine solution reaction is complete, stirred appropriately, heated to 76 - 78 °C, and held for 10 min;

[0011] (4) Wort filtration: Before wort filtration, hot water is topped up just to cover the sieve plate. On the one hand, it can preheat the filtration tank and keep the temperature of the saccharified mash after transfer. On the other hand, it can wash away the small particles in the space below the sieve plate to ensure smooth filtration. During reflux, when the wort becomes clear, filtration can be carried out. Reflux generally lasts for 15 - 20 min. The filtration speed is from slow to fast. After filtration, two mash washes are carried out. The temperature of the two mash wash waters is 76 - 78 °C. The wort concentration before boiling is controlled at 13 - 13.3 °P;

[0012] (5) Wort boiling: The final gravity of the wort for the craft black beer is 14 °P. After boiling, the wort gravity should be controlled between 13.7 and 14.2 °P. During boiling, hops are added in three portions. The first addition is made at the 10th minute of boiling, with the amount being 30% of the total hop addition; the second addition is made at the 30th minute of boiling, accounting for 40% of the total addition; the third addition is made at the 60th minute of boiling, accounting for 30% of the total addition.

[0013] (6) Whirlpool sedimentation and wort cooling: After the wort boiling is completed, it is transferred to a whirlpool sedimentation tank to precipitate and separate the hot coagulants in the wort. During the wort cooling process, pay attention to sterile air to ensure the normal growth and reproduction of yeast and guarantee the normal progress of fermentation. The wort inlet temperature to the tank is maintained at 20 - 24 °C, and at the same time, prickly pear juice is added at a ratio of 4% - 8%.

[0014] (7) Strain activation: The slant-cultured Wickerhamomyces anomalus L251 and Starmerella bombicol L252 are respectively activated on malt extract agar medium and cultured at 28 °C - 30 °C

[0015] for 36h - 48h. The medium formula is: malt extract powder 130g / L, chloramphenicol 0.1g / L, agar 15g / L, pH 5.8 - 6.2,

[0016] sterilized at 115 °C for 20 min. The purchased commercial ale yeast is mixed evenly with 100 mL of warm water according to 3 g of ale yeast per 500 g of malt to complete the activation of ale yeast.

[0017]

[0018] (8) Seed liquor preparation: Prepare 600 ml of food-grade malt extract liquid medium, divide it into three 500 ml Erlenmeyer flasks, sterilize at 115 °C for 20 min, and after cooling, inoculate the activated Wickerhamomyces anomalus L251, Starmerella bombicol L252 and the activated commercial ale yeast respectively, and culture them on a shaker at 25 °C - 30 °C, 150 r / min - 160 r / min for 36h - 48h.

[0019] (9) Primary fermentation: 2% - 6% of Wickerhamomyces anomalus L251 and Starmerella bombicol L252 are inoculated into the cooled wort, and the inoculation ratio is 1:1. The control group is inoculated with 2% - 6% of commercial ale yeast. After inoculation, it is placed in an 18 °C fermentation for 15 d. As the aerobic fermentation progresses, the sugar content of the black beer gradually decreases. When the detected sugar content drops to the range of 4.3 - 5.2 °Brix, the exhaust valve of the fermentation tank can be closed, and the fermentation tank is sealed for anaerobic fermentation. After sealing, yeast produces alcohol under anaerobic conditions.

[0020] (10) Second fermentation: Bottled fermentation. After the first fermentation, the fermented liquid is sub-packed into 500 ml brown glass bottles, and a small amount of granulated sugar is added respectively. The feeding ratio is 1 L: 6 - 8 g. The fermentation temperature for generating sufficient carbon dioxide is 5 °C, and the fermentation time is 14 - 21 d.

[0021] 3. Determination of physical and chemical indexes of Rosa roxburghii black beer

[0022] The physical and chemical indexes of Rosa roxburghii black beer were determined and analyzed. For the fermentation group, the alcohol content was 4.3 ± 1.02% vol, the original wort concentration was 13.8 ± 2.88 °P, the diacetyl content was 0.04 ± 0.07 mg / L, the total acid was 1.63 ± 2.71 mL / 100 mL, and the colority was 62 ± 4.07 EBC, all of which met the black beer standard in GB / T 4927 - 2008 "Beer". In addition, the trehalose lipid content of Rosa roxburghii black beer in the fermentation group was detected to be 62.76 ± 3.49 g / L, and no trehalose lipid was detected in the control group using commercial fermentation agents.

[0023] Secondly, the headspace solid-phase microextraction-gas chromatography-mass spectrometry was used to analyze the volatile components in the fermentation group of Rosa roxburghii black beer. The main volatile components of Rosa roxburghii black beer samples were esters, alcohols, alkenes, aldehydes, other pyrazines, acids, etc. Among them, esters accounted for about 67%, alcohols accounted for 21%, alkanes and aldehydes each accounted for 2%, and acids, pyrazines, etc. accounted for 8%.

[0024] The odor activity value (OAV) of aroma compounds is used to determine whether they are important aroma compounds in the wine body. Generally, compounds with an OAV of not less than 1 contribute to the overall aroma of the wine body. The larger the OAV of an aroma compound, the greater its contribution to the overall aroma. After querying its threshold value and calculating the OAV value, it can be seen that ethyl butyrate, isoamyl acetate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl decanoate, isoamyl acetate, and ethyl cinnamate have an OAV value greater than 1, providing the main ester aroma for the Rosa roxburghii stout. Among them, ethyl butyrate usually presents the aroma of tropical fruits in beer, such as the flavors of bananas and pineapples. When its concentration is appropriate, it adds a pleasant fruity aroma, especially apple aroma, to the beer, thus enhancing the overall flavor experience of the beer. Isoamyl acetate usually presents the smell of bananas and sweet aroma in beer, making a significant contribution to the flavor of beer. Its appropriate presence can increase the aroma complexity of beer and enhance the overall flavor, especially in some beer styles with prominent fruit flavors. Ethyl hexanoate usually presents the aroma characteristics of apples and pears in beer. The appropriate presence of ethyl heptanoate can bring a pleasant fruity aroma to beer and is one of the important flavor compounds in beer. Ethyl octanoate usually presents the special aroma of esters in beer, including apple aroma, pear aroma, and candy aroma. Ethyl decanoate usually presents the aroma of coconut, fruit flavor, and apple flavor in beer. Isoamyl acetate usually presents the smell of bananas and sweet aroma in beer. Ethyl cinnamate usually presents the flavors of sweet oranges and grapes in beer.

[0025] After querying the threshold values of alcohols and calculating the OAV value, the results show that the OAV values of n-hexanol, leaf alcohol, linalool, and phenethyl alcohol in the Rosa roxburghii stout are greater than 1, providing the main volatile alcohol aroma for the Rosa roxburghii stout. Among them, n-hexanol usually presents a faint floral and fruity aroma in beer, sometimes also with a slight green plant smell. Leaf alcohol usually presents the aroma of lilac, lily of the valley, and rose in beer, and also has the smell of wood and fruit. Leaf alcohol is one of the important components in hop essential oil and makes a great contribution to the hop aroma of beer. Its concentration is considered to be used to predict the flavor intensity of beer. Adding hops at different stages of beer brewing makes a significant difference in the contribution to the hop aroma. Linalool is a key aroma contributor in beer, presenting the aroma characteristics of floral and fruity, similar to the aromas of citrus, lilac, lily of the valley, and rose. Phenethyl alcohol usually presents an aroma similar to that of roses in beer. It is one of the most important aromatic alcohols in beer. Although the sensory threshold of phenethyl alcohol is higher than its normal content in beer, it has an additive effect and can combine with other flavor components to form the beer flavor, and other higher alcohols can also enhance the special aroma of phenethyl alcohol.

[0026] The preparation of the above compound microbial inoculum-fermented Rosa roxburghii stout, in which the fermentation strains are Wickerhamomyces anomalus L251 and Starmerella bombicol L252, both isolated from Guizhou Maotai-flavor liquor fermented grains samples. The isolation and culture methods and colony characteristics are as follows:

[0027] (1) Isolation of Wickerhamomyces anomalus L251 and Starmerella bombicol L252:

[0028] Take 10 g of fermented grains sample from the fermented grains during the brewing process of Guizhou Maotai-flavor liquor, add it to a triangular flask containing 90 ml of sterile normal saline and glass beads, shake it on a shaker at 28 °C for 30 min, mix well, and suck 1 mL of the bacterial suspension into a test tube containing 9 mL of sterile normal saline in a laminar flow hood for gradient dilution. Take 10 -1 , 10 -2 ,10 -3 ,10 -4 ,10 -5 and 10 -6 gradients respectively for pour plating and spread plating; count after inverted culture at 28 °C for 48 h, select single colonies with typical yeast colony morphology, inoculate and streak them onto malt extract agar medium, repeat 3 - 4 times until pure strains are obtained, then number each strain respectively, and finally streak the slant at 4 °C for low-temperature preservation.

[0029] (2) Culture

[0030] The media used for Wickerhamomyces anomalus L251 and Starmerella bombicol L252:

[0031] ①1① Identification medium:

[0032] Malt extract agar medium, malt extract powder 130 g / L, chloramphenicol 0.1 g / L, agar 15 g / L, pH 5.8 - 6.2, sterilized at 115 °C for 20 min.

[0033] ②2② Preservation medium:

[0034] Malt extract agar medium, malt extract powder 130 g / L, chloramphenicol 0.1 g / L, agar 15 g / L, pH 5.8 - 6.2, sterilized at 115 °C for 20 min.

[0035] (3) Preservation and subculture of the strains:

[0036] ①Strain preservation medium:

[0037] Malt extract agar medium: malt extract powder 130 g / L, chloramphenicol 0.1 g / L, agar 15 g / L, pH 5.8 - 6.2, sterilized at 115 °C for 20 min.

[0038] ②Preservation method

[0039] Glycerol strain preservation method: Inoculate yeast on the malt extract agar slant medium, incubate in an incubator at 28 °C for 2 d, then inoculate into the malt extract liquid medium for culture. After 48 h of culture of the bacterial liquid, perform ultra-low temperature preservation with 30% sterilized glycerol and the cultured bacterial liquid at a volume ratio of 1:1. The preservation temperature is -80 °C. Transplant regularly every two years to check the yeast viability and whether it is contaminated.

[0040] (4)Ecological characteristics of the strain:

[0041] The colonies formed by Wickerhamomyces anomalus L251 on the malt extract medium, incubated at 28 °C for 48 h, have a colony diameter of 0.82 - 0.98 mm. The colonies are small and round, with a bulge in the middle, white in color, opaque, shiny, covered with white powder on the colony surface, with regular colony edges, and are easy to pick up. The single colony has a prominent wine fragrance and sweet fragrance; microscopic examination shows that the cells are long oval, with a cell length of 4.06 - 5.22 μm and a width of 2.05 - 3.02 μm.

[0042] The colonies formed by Starmerella bombicol L252 on the malt extract medium, incubated at 28 °C for 48 h, have a colony diameter of 0.64 - 0.76 mm. The colonies are round, with a bulge in the middle, white in color, opaque, without luster, with regular colony edges, and are easy to pick up. The single colony has a prominent sweet fragrance. Microscopic examination shows that the cells are oval, with a cell length of 5.35 - 6.65 μm and a width of 2.30 - 3.55 μm.

[0043] (5)Culture characteristics of the strain

[0044] The culture temperature is 13 °C - 30 °C, and the optimal temperature is 15 °C - 25 °C;

[0045] The culture pH is 4.5 - 6.8, and the optimal range is 5.2 - 6.2.

[0046] The use of the above-mentioned composite microbial inoculant for fermenting Rosa roxburghii stout and producing sophorolipids, Wickerhamomyces anomalus

[0047] (Wickerhamomyces anomalus) L251 is a strain with obvious wine and aroma production; Starmerella bombicol L252 is a strain with obvious aroma and sophorolipid production; compared with the prior art, the present invention has obvious feasibility. From the above technical solutions, it can be seen that using composite functional microbial strains to replace commercial yeast strains for beer fermentation can increase the flavor substances and their contents in Rosa roxburghii stout, and at the same time significantly increase the content of sophorolipid in Rosa roxburghii stout through the microbial fermentation method. The operation method of this process is simple, the microbial fermentation has good stability, the process parameters are easy to control, the alcohol content is controllable and moderate, and the Rosa roxburghii flavor and stout flavor are strong. It is of great significance for increasing the variety of beer products and deep-processed Rosa roxburghii products, improving the nutritional value of Rosa roxburghii stout, the deep-processing value of Rosa roxburghii raw materials, and the production, extraction and wide application of sophorolipid. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a colony morphology diagram of Wickerhamomyces anomalus L251 of the present invention on the culture medium;

[0049] Figure 2 It is a colony morphology diagram of Starmerella bombicol L252 of the present invention on the culture medium;

[0050] Figure 3 It is a microscopic examination diagram of Wickerhamomyces anomalus L251 of the present invention;

[0051] Figure 4 It is a microscopic examination diagram of Starmerella bombicol L252 of the present invention;

[0052] Figure 5 Sequencing nucleotide sequence of Wickerhamomyces anomalus L251;

[0053] Figure 6 Sequencing nucleotide sequence of Starmerella bombicol L252;

[0054] Figure 7 It is a phylogenetic tree of Wickerhamomyces anomalus L251 and Starmerella bombicol L252 of the present invention;

[0055] Figure 8 It is a pie chart distribution diagram of volatile components in the Rosa roxburghii stout fermentation group of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0056] Example 1

[0057] 1. Fermentation strains: Wickerhamomyces anomalus L251 and Starmerella bombicol L252, and their isolation, culture methods and colony characteristics:

[0058] (1) Isolation of Wickerhamomyces anomalus L251 and Starmerella bombicol L252;

[0059] From the fermented grains during the brewing process of Guizhou Maotai-flavor liquor, take 10 g of the fermented grains sample and add it to a triangular flask containing 90 ml of sterile normal saline and glass beads. Shake it on a shaker at 28 °C for 30 min to mix evenly; In the ultra-clean workbench, pipette 1 mL of the bacterial suspension into a test tube containing 9 mL of sterile normal saline for gradient dilution. Take 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 gradients for pour plating and spread plating respectively. Incubate the plates upside down at 28 °C for 48 h and then count. Select single colonies with typical yeast colony morphology, inoculate and streak them onto malt extract agar medium, repeat 3 - 4 times until pure strains are obtained, then number each strain respectively, and finally streak the slant at 4 °C for low-temperature preservation.

[0060] (2) Cultivation

[0061] Media used for Wickerhamomyces anomalus L251 and Starmerella bombicol L252:

[0062] Identification medium:

[0063] Malt extract agar medium, malt extract powder 130 g / L, chloramphenicol 0.1 g / L, agar 15 g / L, pH 5.8 - 6.2, sterilized at 115 °C for 20 min;

[0064] Preservation medium:

[0065] Malt extract agar medium, malt extract powder 130 g / L, chloramphenicol 0.1 g / L, agar 15 g / L, pH 5.8 - 6.2, sterilized at 115 °C for 20 min.

[0066] (3) Preservation and subculture of strains:

[0067] Strain preservation medium:

[0068] Malt extract agar medium: malt extract powder 130 g / L, chloramphenicol 0.1 g / L, agar 15 g / L, pH 5.8 - 6.2, sterilized at 115 °C for 20 min;

[0069] Preservation method

[0070] Glycerol stock culture method: Inoculate yeast on a wort agar slant medium, incubate in an incubator at 28 °C for 2 days, then inoculate into a wort liquid medium for culture. After 48 hours of culture of the bacterial liquid, perform ultra-low temperature preservation with 30% sterilized glycerol and the cultured bacterial liquid at a volume ratio of 1:1. The preservation temperature is -80 °C. Transplantation should be carried out regularly every two years to check the yeast viability and whether it is contaminated with bacteria.

[0071] (4) Ecological characteristics of the strains:

[0072] The colonies formed by Wickerhamomyces anomalus L251 on the wort medium, incubated at 28 °C for 48 hours, have a colony diameter of 0.82 - 0.98 mm. The colonies are small and round, with a bulge in the middle, white in color, opaque, shiny, covered with white powder on the colony surface, with regular colony edges, easy to pick up. The single colony has a prominent wine fragrance and sweet fragrance. Microscopic examination shows that the cells are long oval, with a cell length of 4.06 - 5.22 μm and a width of 2.05 - 3.02 μm.

[0073] The colonies formed by Starmerella bombicol L252 on the wort medium, incubated at 28 °C for 48 hours, have a colony diameter of 0.64 - 0.76 mm. The colonies are round, with a bulge in the middle, white in color, opaque, without luster, with regular colony edges, easy to pick up. The single colony has a prominent sweet fragrance. Microscopic examination shows that the cells are oval, with a cell length of 5.35 - 6.65 μm and a width of 2.30 - 3.55 μm.

[0074] (5) Culture characteristics of the strains

[0075] The culture temperature is 13 °C - 30 °C, and the optimal temperature is 15 °C - 25 °C;

[0076] The culture pH is 4.5 - 6.8, and the optimal range is pH 5.2 - 6.2.

[0077] 2. Fermentation of Rosa roxburghii stout with a compound bacterial agent and application implementation

[0078] (1) Malt crushing: Crush 650 g of pale malt, 250 g of dark caramel malt, and 100 g of black malt using a grinder;

[0079] (2) Feeding: Add 1 kg of crushed malt to the saccharification pot, add 3.5 - 4 L of clear water, and heat to 55 °C to complete the feeding.

[0080] (3) Saccharification: After feeding, the infusion saccharification method is used for saccharification. The temperature-rising steps experienced by the mash are as follows: Heat to 50 - 55°C, hold for 30 min with proper stirring, then heat to 65 - 68°C, hold for 50 min. When holding for 30 min, perform iodine solution determination, measure every 10 min until the iodine solution reaction is complete, and stir properly. Then heat to 76 - 78°C and hold for 10 min;

[0081] (4) Wort filtration: Before wort filtration, pay attention to topping up with hot water just enough to cover the sieve plate. On the one hand, it can preheat the filter tank and keep the temperature of the saccharified mash after transfer. On the other hand, it can wash away small particles in the space below the sieve plate to ensure smooth filtration. During recirculation, filtration can be carried out only after the wort becomes clear. Recirculation generally lasts for 15 - 20 min, and the filtration speed increases from slow to fast gradually. After filtration, perform two washings of the grains. The temperature of the two wash waters is 76 - 78°C. It is best to control the wort concentration at 13 - 13.3°P before boiling;

[0082] (5) Wort boiling: The final wort concentration of the craft black beer is generally 14°P. After boiling, the wort concentration should be controlled between 13.7 - 14.2°P. During boiling, the hops are added in three portions. Add the first portion of hops at the 10th min of boiling, with the addition amount being 30% of the total hop addition amount; add the second portion of hops at the 30th min of boiling, accounting for 40% of the total addition amount; add the third portion of hops at the 60th min of boiling, accounting for 30% of the total addition amount;

[0083] (6) Whirlpool sedimentation and wort cooling: After the wort boiling is completed, transfer it to the whirlpool sedimentation tank to precipitate and separate the hot coagulants in the wort. During the wort cooling process, pay attention to sterile air to ensure the normal growth and reproduction of yeast and the normal progress of fermentation. The wort inlet temperature of the tank is maintained at about 22°C, and at the same time, add Rosa roxburghii Tratt original juice at a ratio of 4% - 8%;

[0084] (7) Strain activation: Activate the Wickerhamomyces anomalus L251 and Starmerella bombicol L252 cultured on the slant in the malt extract agar medium, and culture at 28°C - 30°C

[0085] for 36 h - 48 h;

[0086] The medium formula is: malt extract powder 130 g / L, chloramphenicol 0.1 g / L, agar 15 g / L, pH 5.8 - 6.2, sterilize at 115°C for 20 min. Mix the purchased commercial ale yeast evenly with 100 mL of warm water at a ratio of 3 g of ale yeast per 500 g of malt to complete the activation of the ale yeast;

[0087] (8) Preparation of seed liquid: Prepare 600 ml of food-grade malt extract liquid medium and dispense it into three 500-ml Erlenmeyer flasks. Sterilize at 115 °C for 20 min. After cooling, inoculate the activated Wickerhamomyces anomalus L251, Candida bombicola L252, and the activated commercial ale yeast respectively. Incubate them on a shaker at 25 °C - 30 °C and 150 r / min - 160 r / min for 36 h - 48 h;

[0088] (9) First fermentation, i.e., main fermentation: Inoculate 2% - 6% of Wickerhamomyces anomalus L251 and Candida bombicola L252 into the cooled wort, with an inoculation ratio of 1:1. In the control group, inoculate 2% - 6% of commercial ale yeast. After inoculation, ferment at 18 °C for 15 d. During the fermentation process, with aerobic fermentation, the sugar content of the black beer gradually decreases. When the detected sugar content drops to the range of 4.3 - 5.2 °Brix, close the exhaust valve of the fermentation tank and seal the fermentation tank for anaerobic fermentation. After sealing, the yeast produces alcohol under anaerobic conditions;

[0089] (10) Second fermentation, i.e., post-fermentation: Ferment in bottles. Dispense the fermented liquid after the first fermentation into 500-ml brown glass bottles, and then add a small amount of granulated sugar respectively, with a feeding ratio of 1 L: 6 - 8 g, to produce sufficient carbon dioxide. The fermentation temperature is 5 °C, and the fermentation time is 14 - 21 d.

[0090] 3. Determination of physicochemical indexes of Rosa roxburghii Tratt black beer

[0091] Determine and analyze the physicochemical indexes of Rosa roxburghii Tratt black beer. For the fermentation group, the alcohol content is 4.3 ± 1.02% vol, the original wort concentration is 13.8 ± 2.88 °P, the diacetyl content is 0.04 ± 0.07 mg / L, the total acid is 1.63 ± 2.71 mL / 100 mL, and the colority is 62 ± 4.07 EBC, all of which meet the black beer standards in GB / T 4927 - 2008 "Beer". In addition, the trehalose lipid content in the Rosa roxburghii Tratt black beer of the fermentation group is detected to be 62.76 ± 3.49 g / L, while the trehalose lipid is not detected in the control group using commercial fermentation agents.

[0092] Secondly, analyze the volatile components in the fermentation group of Rosa roxburghii Tratt black beer using headspace solid-phase microextraction-gas chromatography-mass spectrometry. The main volatile components of the Rosa roxburghii Tratt black beer sample are esters, alcohols, alkenes, aldehydes, other pyrazines, and acids, etc. Among them, esters account for about 67%, alcohols account for 21%, alkanes and aldehydes each account for 2%, and acids, pyrazines, etc. account for 8%.

[0093] The odor activity value (OAV) of aroma compounds is used to determine whether they are important aroma compounds in the wine body. Generally, compounds with an OAV of not less than 1 contribute to the overall aroma of the wine body; the larger the OAV of an aroma compound, the greater its contribution to the overall aroma. After querying its threshold value and calculating the OAV value, it can be seen that ethyl butyrate, isoamyl acetate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl decanoate, isoamyl acetate, and ethyl cinnamate have an OAV value greater than 1, providing the main ester aroma in Rosa roxburghii Tratt black beer. Among them, ethyl butyrate usually presents the aroma of tropical fruits in beer, such as the flavors of bananas and pineapples. When its concentration is appropriate, it adds a pleasant fruity aroma, especially apple aroma, to the beer, thus enhancing the overall flavor experience of the beer; isoamyl acetate usually presents the smell of bananas and sweet aroma in beer, making a significant contribution to the flavor of the beer. Its appropriate presence can increase the aroma complexity of the beer and enhance the overall flavor, especially in some beer styles with prominent fruity flavors. Ethyl hexanoate usually presents the aroma characteristics of apples and pears in beer. The appropriate presence of ethyl heptanoate can bring a pleasant fruity aroma to the beer and is one of the important flavor compounds in beer. Ethyl octanoate usually presents the special aroma of esters in beer, including apple aroma, pear aroma, and candy aroma. Ethyl decanoate usually presents the aroma of coconut, fruit flavor, and apple flavor in beer. Isoamyl acetate usually presents the smell of bananas and sweet aroma in beer. Ethyl cinnamate usually presents the flavors of sweet oranges and grapes in beer.

[0094] After querying the threshold values of alcohols and calculating the OAV value, the results show that the OAV values of n-hexanol, leaf alcohol, linalool, and phenethyl alcohol in Rosa roxburghii Tratt black beer are greater than 1, providing the main volatile alcohol aroma in Rosa roxburghii Tratt black beer; among them, n-hexanol usually presents a faint floral and fruity aroma in beer, sometimes also with a slight green plant smell; leaf alcohol usually presents the aroma of lilac, lily of the valley, and rose in beer, and also has the smell of wood and fruit. Leaf alcohol is one of the important components in hop essential oil and makes a great contribution to the hop aroma of beer. Its concentration is considered to be used to predict the flavor intensity of beer. Adding hops at different stages of beer brewing makes a significant difference in the contribution to hop aroma. Linalool is a key aroma contributor in beer, presenting the aroma characteristics of floral and fruity aromas, similar to the aromas of citrus, lilac, lily of the valley, and rose. Phenethyl alcohol usually presents an aroma similar to that of roses in beer. It is one of the most important aromatic alcohols in beer. Although the sensory threshold of phenethyl alcohol is higher than its normal content in beer, it has an additive effect and can combine with other flavor components to form the beer flavor, and other higher alcohols can also enhance the special aroma of phenethyl alcohol.

[0095] The above-mentioned compound microbial inoculum-fermented Rosa roxburghii stout and its use for producing sophorolipids. Wickerhamomyces anomalus L251 is a strain with obvious wine and aroma production, and Starmerella bombicol L252 is a strain with obvious aroma and sophorolipid production. Compared with the prior art, the present invention has obvious feasibility. From the above technical solutions, it can be seen that compound functional microbial strains are used to replace commercial yeast strains for beer fermentation, increasing the flavor substances and their contents in Rosa roxburghii stout. At the same time, the content of sophorolipids in Rosa roxburghii stout is significantly increased by the microbial fermentation method. The process operation method is simple, the microbial fermentation stability is good, the process parameters are easy to control, the alcohol content is controllable and moderate, and the Rosa roxburghii flavor and stout flavor are strong. It is of great significance for increasing the variety of beer products and deep-processed Rosa roxburghii products, improving the nutritional value of Rosa roxburghii stout, the deep-processing value of Rosa roxburghii raw materials, and the production, extraction and wide application of sophorolipids.

[0096] Of course, the present invention can also have many other embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and deformations according to the present invention, but these corresponding changes and deformations should all fall within the protection scope of the appended claims of the present invention.

[0097] Appendix

[0098] Table 1 Determination of Physicochemical Indexes of Rosa roxburghii Stout

[0099]

[0100]

[0101] Table 2 Main Ester Volatile Components and OAV Values in the Fermentation Group

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[0103] Table 3 Main Alcohol Volatile Components and OAV Values in the Fermentation Group

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[0105]

Claims

1. An abnormal Wickham yeast ( Wickerhamomyces anomalus ) L251, characterized in that, The deposit number is CCTCC NO: M 2024491.

2. A Starmerella bombicol L252, characterized in that: The deposit number is CCTCCNO: M 2024521.

3. Use of abnormal Wickham yeast in producing sophorolipids as claimed in claim 1.

4. Use of Candida bombus for producing sophorolipids as claimed in claim 2.

5. Use of a composite microbial agent composed of the microorganism Abnormal Wickham's yeast according to claim 1 and the microorganism Candida bombus according to claim 2 for producing sophorolipids.

6. A method for making roxburghii black beer, characterized in that: The steps include: (1) Malt grinding: 650 g of barley malt, 250 g of dark caramel malt, and 100 g of black malt are ground using a grinder; (2) Adding materials: Add 1 kg of crushed malt into the saccharification pot and add 3.5-4 L of clean water and heat to 55 °C to complete the feeding; (3) Saccharification: After the feeding is completed, the saccharification is carried out by the leaching saccharification method. The temperature rising steps of the mash are as follows: heating to 50-55 °C, keeping warm for 30 min, stirring appropriately, heating to 65-68 °C, keeping warm for 50 min, measuring the iodine solution every 30 min, once every 10 min until the iodine solution reacts completely, stirring appropriately, heating to 76-78 °C, and keeping warm for 10 min; (4) Wort filtration; (5) Boil the wort. After the boiling is completed, the wort concentration should be controlled between 13.7 and 14.2 °P. Hops are added three times during the boiling period. The first hop is added at the 10th minute of boiling, accounting for 30% of the total hop addition; the second hop is added at the 30th minute of boiling, accounting for 40% of the total addition; the third hop is added at the 60th minute of boiling, accounting for 30% of the total addition; (6) Cyclotonic sedimentation and wort cooling After the wort is boiled, it is transferred to the cyclotron sedimentation tank to precipitate and separate the heat coagulants in the wort. During the wort cooling process, the wort inlet temperature is maintained at 20-24 ℃, and at the same time, the original prickly pear juice is added at a ratio of 4%-8%; (7) Activation of bacterial strains: The cultured Abnormal Wickham yeast L251 and Candida bombus L252 were activated on malt extract agar medium, and the purchased commercial ale yeast was activated; (8) Seed liquid preparation: Prepare 600 ml of food-grade malt extract liquid medium and divide it into three 500 ml Erlenmeyer flasks. Sterilize at 115 °C for 20 min, then cool and inoculate activated abnormal Wickham yeast L251, bumblebee Candida albicans L252 and activated commercial ale yeast respectively. Incubate at 25 °C-30 °C and 150 r / min-160 r / min on a shaking incubator for 36 h-48 h. (9) First fermentation: 2%-6% abnormal Wickham yeast L251 and Bombus candida L252 were inoculated into the cooled wort at a ratio of 1:

1. The control group was inoculated with 2%-6% commercial ale yeast. After inoculation, the wort was fermented at 18 °C for 15 days. As the aerobic fermentation progressed, the sugar content of the dark beer gradually decreased. When the sugar content dropped to the range of 4.3-5.2 °Brix, the exhaust valve of the fermentation tank was closed and the fermentation tank was sealed for anaerobic fermentation. (10) Second fermentation: Fermentation in separate bottles. After the first fermentation, the fermentation liquid is divided into 500 ml brown glass bottles. Then a small amount of white sugar is added to each bottle. The feed ratio is 1 L: 6-8 g. The fermentation temperature is 5 °C and the fermentation time is 14-21 days.

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