A natural milk flavoring base and a preparation method and application thereof

Abstract

The application discloses a natural milk flavoring base and a preparation method and application thereof. Butter or beef tallow is used as a base to prepare a culture medium, and the culture medium is fermented by mixed strains of Yarrowia lipolytica and Candida viswanathii, and then is subjected to steps such as blending, high-pressure homogenization and spray drying to prepare a powdery natural milk flavoring base. The natural milk flavoring base has sufficient aroma intensity, and the aroma is full and realistic, and no oil-water separation phenomenon occurs in an aqueous solution.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a natural milk flavoring base material, its preparation method, and its application. Background Technology

[0002] Milk flavorings or fragrances are widely used in the production of dairy products, beverages, confectionery, baked goods, and animal feed, enhancing the main aroma of products or masking unpleasant odors. With increasing consumer demand for nutritious, healthy, and diverse flavors in food, natural milk flavoring bases with natural, realistic, mellow, and full-bodied aromas are becoming increasingly popular among producers and consumers. Currently, the main methods for producing natural milk flavoring bases using milk fat include blending, enzymatic hydrolysis, and fermentation. Blending primarily improves the emulsification properties and aroma richness of milk flavoring bases, but its aroma-enhancing effect is not significant. Enzymatic hydrolysis and fermentation can promote the release of specific flavor substances, resulting in a more pronounced aroma-enhancing effect; however, differences in lipase preparations, fermentation strains, and processes often lead to significant variations in product flavor, and defects such as low aroma realism and insufficient richness exist. Optimizing fermentation strains and processes is a crucial way to address the problems existing in current fermented milk flavoring bases. Summary of the Invention

[0003] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing a natural milk flavoring base.

[0004] Another object of the present invention is to provide a natural milk flavoring base obtained by the above preparation method.

[0005] Another object of the present invention is to provide the application of the above-mentioned natural milk flavoring base.

[0006] The objective of this invention is achieved through the following technical solution: a method for preparing a natural milk flavoring base, comprising the following steps: (1) Preparation of fermentation medium: Mix whole milk powder and water, put it into a fermentation tank, add flavor protease for enzymatic hydrolysis; then add animal oil, glucose, sodium citrate, maltodextrin, malt extract and potassium dihydrogen phosphate, stir to dissolve, sterilize, cool to room temperature to obtain fermentation medium; animal oil is at least one of butter and tallow; (2) Fermentation of fermentation medium: Inoculate the fermentation medium with seed liquid of Yersinia lipolytica and seed liquid of Candida viresis, and ferment under stirring and aeration conditions; (3) Spray drying of fermentation liquid: Boil the fermentation liquid obtained in step (2), cool it to 50~60℃, separate the solid and liquid, add maltodextrin to the obtained liquid, stir to dissolve, homogenize, and then use spray drying method to make dry powder to obtain natural milk flavor base.

[0007] In step (1): The whole milk powder mentioned is a commercially available product, and the preferred usage is calculated at 26~30 g / L.

[0008] The preferred dosage of the flavor protease is calculated as 5000-6000 U of flavor protease per gram of whole milk powder.

[0009] The preferred enzymatic hydrolysis conditions for the flavor protease are: temperature 50~55℃, pH 6.0~6.5, and time 6~8 h.

[0010] The preferred amount of butter used is 60-70 g / L.

[0011] The amount of glucose used is preferably calculated at 20~24 g / L.

[0012] The preferred dosage of sodium citrate is 10-12 g / L.

[0013] The preferred dosage of the maltodextrin is calculated at 20-24 g / L.

[0014] The preferred dosage of the malt extract is 10-12 g / L.

[0015] The preferred dosage of potassium dihydrogen phosphate is calculated at 1.0~1.2 g / L.

[0016] The preferred sterilization conditions are 115-121℃ for 15-30 minutes.

[0017] In step (2): The aforementioned Yersinia lipolytica seed culture is prepared by the following steps: Yersinia lipolytica is inoculated into a sterile shake flask culture medium and cultured by shaking to obtain the Yersinia lipolytica seed culture.

[0018] The preferred Yersinia lipolyticis yeast is Yersinia lipolyticis (Yersinia lipolyticis yeast). Yarrowia lipolytica YD19, with accession number GDMCC No:60782, was deposited on September 20, 2019, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and has been published under ZL201911001480.2.

[0019] The preferred inoculation amount is one loop of slant bacterial growth per 100 mL of shake flask culture medium.

[0020] The shake flask culture medium has the following composition: glucose 30-40 g / L, casein peptone 8-12 g / L, malt extract 6-10 g / L, potassium dihydrogen phosphate 1.8-2.2 g / L, pH 5.5-6.0.

[0021] The preferred sterilization conditions are 115-121℃ for 15-30 minutes.

[0022] The preferred conditions for the oscillation culture are: temperature 26~30℃, rotation speed 180~200 r / min, and time 18~20 h.

[0023] The Candida vesicularis seed culture is prepared by the following steps: Candida vesicularis is inoculated into a sterile shake flask culture medium and cultured by shaking to obtain the Candida vesicularis seed culture.

[0024] The preferred form of Candida viride is Candida viride (C. viride). Candida viswanathii TB07, with accession number GDMCC NO:60785, was deposited on September 20, 2019, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and has been published under ZL201911001462.4.

[0025] The preferred inoculation amount is one loop of slant bacterial growth per 100 mL of shake flask culture medium.

[0026] The shake flask culture medium has the following composition: glucose 30-40 g / L, casein peptone 8-12 g / L, malt extract 6-10 g / L, potassium dihydrogen phosphate 1.8-2.2 g / L, pH 5.5-6.0.

[0027] The preferred sterilization conditions are 115-121℃ for 15-30 minutes.

[0028] The preferred conditions for the oscillation culture are: temperature 26~30℃, rotation speed 180~200 r / min, and time 18~20 h.

[0029] The preferred inoculation amount of the Yersinia lipolytica seed culture into the fermenter is 2% (v / v) to 4% (v / v).

[0030] The preferred inoculation amount of the *Candida viride* seed culture into the fermenter is 2% (v / v) to 4% (v / v).

[0031] The fermentation temperature is preferably 26~30℃.

[0032] The fermentation speed is preferably 220~260 r / min.

[0033] The preferred aeration ratio for fermentation is 0.28 vvm for 0-12 h, 0.36 vvm for 13-36 h, and 0.32 vvm after 36 h.

[0034] The fermentation time is preferably 72-96 h.

[0035] In step (3): The preferred method for solid-liquid separation is centrifugation.

[0036] The preferred centrifugation conditions are: rotation speed 5000~6000 r / min, time 15~20 min.

[0037] The preferred amount of maltodextrin used is equal to that of animal oil.

[0038] The preferred pressure for homogenization is 30-40 MPa.

[0039] The homogenization process is preferably performed once.

[0040] The preferred conditions for spray drying are: inlet air temperature 160~170℃ and outlet air temperature 40~50℃.

[0041] A natural milk flavoring base is obtained by the above preparation method.

[0042] The aforementioned natural milk flavoring base is particularly suitable for use in the preparation of beverages in the food industry.

[0043] The present invention has the following advantages and effects compared with the prior art: (1) Compared with several commercially available natural milk flavor powder products, the aroma intensity of the natural milk flavor base material made by the present invention is more than 140% of that of commercially available products, and the aroma realism and richness are significantly better than those of commercially available products.

[0044] (2) The Candida viscera used in this invention has the ability to synthesize biological emulsifiers. Although butter or tallow is used as the base material, no emulsifier needs to be added. The resulting natural milk flavor base material is used in beverage production without precipitation or oil-water separation. Its appearance quality in solution is superior to commercially available products. The Yersinia lipolytica used has a strong ability to decompose fats and oils, resulting in a natural milk flavor base material with a strong aroma. The inventors of this invention have discovered that combining Candida viscera and Yersinia lipolytica in the preparation of natural milk flavor base materials has a synergistic effect.

[0045] (3) Compared with commercially available natural milk flavor powder, the present invention does not use lactic acid producing bacteria, and the natural milk flavor base produced has no lactic acid taste. It is used in the production of other beverages besides lactic acid beverages and has no adverse effects caused by lactic acid. Detailed Implementation

[0046] The present invention will now be described in further detail, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0047] Example 1

[0048] (1) Weigh 560 g of whole milk powder, mix it evenly with 20 L of water, and put it into a 30 L fully automatic fermentation tank. Adjust the pH of the solution to 6.2. Calculate the addition of 5500 U of flavor protease per gram of milk powder, add flavor protease, control the stirring speed at 50 r / min, and hydrolyze at 52℃ for 7 h. Add 1300 g of butter, 440 g of glucose, 220 g of sodium citrate, 440 g of maltodextrin, 220 g of malt extract, and 22 g of potassium dihydrogen phosphate (food grade) in sequence, stir evenly, sterilize at 121℃ for 15 min, cool to room temperature, and set aside.

[0049] (2) Weigh 42 g glucose, 12 g casein peptone, 9.6 g malt extract, and 2.4 g potassium dihydrogen phosphate (food grade), mix with 1200 mL water, adjust the pH to 5.8, and dispense 200 mL into six 1000 mL Erlenmeyer flasks. Sterilize at 121℃ for 15 min. After cooling, inoculate with an inoculation loop using *Yersinia lipophila* (…). Yarrowia lipolytica YD19 (accession number GDMCC No:60782, deposited on September 20, 2019, at the Guangdong Provincial Microbial Culture Collection Center, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, and published under ZL201911001480.2) slant culture was inoculated into three Erlenmeyer flasks, and *Candida virescens* (*Candida virescens*) was added using an inoculation loop. Candida viswanathii The slant culture of TB07 (accession number GDMCC NO:60785, deposited on September 20, 2019, at the Guangdong Provincial Microbial Culture Collection Center, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and published under ZL201911001462.4) was inoculated into three other Erlenmeyer flasks, with two loops of bacterial growth inoculated into each flask. The culture media from the six Erlenmeyer flasks were incubated in a shaking incubator at 28℃ and 190 r / min for 19 h. Then, the yeast culture from the six flasks was transferred to a fermenter. During fermentation, the stirring speed was controlled at 240 r / min, the temperature at 28℃, the aeration ratio was controlled at 0.28 vvm from 0 to 12 h, 0.36 vvm from 13 to 36 h, and 0.32 vvm after 36 h. Fermentation was completed after 84 hours.

[0050] (3) The fermentation broth was boiled and then cooled to 55°C. It was centrifuged at 5500 r / min for 18 min to obtain 19.8 L of supernatant. 1300 g of maltodextrin was added to the supernatant and stirred evenly. The mixture was homogenized once at 35 MPa pressure and then dried using a spray drying tower. The inlet air temperature during the drying process was controlled at 160~170°C and the outlet air temperature was controlled at 40~50°C. 1.99 kg of dry powder was collected, which is the natural milk flavoring base. An evaluation group composed of 9 people with rich aroma evaluation experience conducted the evaluation. The aroma threshold of the flavoring base in water was 2.20 mg / L. Compared with the minimum aroma threshold (3.30 mg / L) of several commercially available natural milk flavorings, its aroma intensity is 150% of that of commercially available products. The natural milk flavoring base was prepared into a 3 g / L solution with water. The solution had a distinct aroma, with a rich and realistic aroma, and no precipitation or oil-water separation.

[0051] The natural milk flavoring base and commercially available natural milk flavoring were analyzed by solid-phase microextraction (SPE) (the SPE method is referenced in: Li Ning, Sun Baoguo, Liang Menglan. Analysis of volatile components in a milk flavoring by solid-phase microextraction combined with gas chromatography-mass spectrometry [J]. Food Science, 2011, 32(22):221-225), and the aroma components were analyzed by GC-MS. For components with a relative content greater than 3%, the main components and their relative contents of the natural milk flavoring base included: butyric acid 10.42%, hexanoic acid 4.16%, ethyl caprylate 10.47%, lauric acid 3.38%, ethyl laurate 5.25%, ethyl myristate 7.41%, ethyl palmitate 15.13%, and ethyl oleate 12.44%. The main components and their relative contents of commercially available natural milk flavorings include: butyric acid 38.16%, valeric acid 22.82%, caprylic acid 13.14%, capric acid 6.85%, and lauric acid 3.22%. It is evident that the natural milk flavoring base provided by this invention differs significantly from the main components and their relative contents of commercially available natural milk flavorings.

[0052] Example 2

[0053] (1) Weigh 520 g of whole milk powder, mix it evenly with 20 L of water, and put it into a 30 L fully automatic fermentation tank. Adjust the pH of the solution to 6.5. Calculate the addition of 6000 U of flavor protease per gram of milk powder, add flavor protease, control the stirring speed at 50 r / min, and hydrolyze at 55℃ for 6 h. Add 1200 g of butter, 400 g of glucose, 200 g of sodium citrate, 400 g of maltodextrin, 200 g of malt extract, and 20 g of potassium dihydrogen phosphate (food grade) in sequence, stir evenly, sterilize at 121℃ for 15 min, cool to room temperature, and set aside.

[0054] (2) Weigh 24 g glucose, 6.4 g casein peptone, 4.8 g malt extract, and 1.44 g potassium dihydrogen phosphate (food grade), mix with 800 mL water, adjust the pH to 5.5, and dispense 200 mL of the solution into four 1000 mL Erlenmeyer flasks. Sterilize at 121°C for 15 min. After cooling, inoculate with an inoculation loop using *Yersinia lipophila* (…). Yarrowia lipolytica YD19 slant culture was inoculated into two Erlenmeyer flasks, and Candida virescens was added using an inoculation loop. Candida viswanathii The TB07 slant culture was inoculated into two more Erlenmeyer flasks, with two loops of bacterial growth added to each flask. The four Erlenmeyer flasks were incubated in a shaking incubator at 26°C and 180 rpm for 20 h. Then, the four yeast cultures were transferred to a fermenter. During fermentation, the stirring speed was controlled at 220 rpm, the temperature at 26°C, the aeration ratio was controlled at 0.28 vvm from 0 to 12 h, 0.36 vvm from 13 to 36 h, and 0.32 vvm after 36 h. Fermentation was completed after 96 hours.

[0055] (3) The fermentation broth was boiled and then cooled to 50°C. It was centrifuged at 5000 r / min for 20 min to obtain 19.2 L of supernatant. 1200 g of maltodextrin was added to the supernatant and stirred evenly. The mixture was homogenized once at 30 MPa pressure and then dried using a spray drying tower. The inlet air temperature was controlled at 160~170°C and the outlet air temperature was controlled at 40~50°C. 1.82 kg of dry powder was collected, which is the natural milk flavoring base. An evaluation group composed of 9 people with rich aroma evaluation experience conducted the evaluation. The aroma threshold of the flavoring base in water was 2.35 mg / L. Compared with the minimum aroma threshold (3.30 mg / L) of several commercially available natural milk flavorings, its aroma intensity is 140% of that of commercially available products. The natural milk flavoring base was prepared into a 3 g / L solution with water. The solution had a distinct aroma, and the aroma was rich and realistic.

[0056] The natural milk flavoring base and commercially available natural milk flavoring were analyzed by solid-phase microextraction (SPE) (the SPE method is referenced in: Li Ning, Sun Baoguo, Liang Menglan. Analysis of volatile components in a milk flavoring by solid-phase microextraction combined with gas chromatography-mass spectrometry [J]. Food Science, 2011, 32(22):221-225), and the aroma components were analyzed by GC-MS. For components with a relative content greater than 3%, the main components and their relative contents of the natural milk flavoring base included: butyric acid 10.61%, hexanoic acid 4.05%, ethyl caprylate 10.32%, lauric acid 3.51%, ethyl laurate 5.48%, ethyl myristate 7.29%, ethyl palmitate 15.26%, and ethyl oleate 12.23%. The main components and their relative contents of commercially available natural milk flavorings include: butyric acid 38.16%, valeric acid 22.82%, caprylic acid 13.14%, capric acid 6.85%, and lauric acid 3.22%. It is evident that the natural milk flavoring base provided by this invention differs significantly from the main components and their relative contents of commercially available natural milk flavorings.

[0057] Example 3

[0058] (1) Weigh 600 g of whole milk powder, mix it evenly with 20 L of water, and put it into a 30 L fully automatic fermentation tank. Adjust the pH of the solution to 6.0. Calculate the addition of 5000 U of flavor protease per gram of milk powder, add flavor protease, control the stirring speed at 50 r / min, and hydrolyze at 50℃ for 8 h. Add 1400 g of butter, 480 g of glucose, 240 g of sodium citrate, 480 g of maltodextrin, 240 g of malt extract, and 24 g of potassium dihydrogen phosphate (food grade) in sequence, stir evenly, sterilize at 121℃ for 15 min, cool to room temperature, and set aside.

[0059] (2) Weigh 64 g glucose, 19.2 g casein peptone, 16 g malt extract, and 3.52 g potassium dihydrogen phosphate (food grade), mix with 1600 mL water, adjust the pH to 6.0, and dispense 200 mL of the solution into eight 1000 mL Erlenmeyer flasks. Sterilize at 121°C for 15 min. After cooling, inoculate with an inoculation loop using *Yersinia lipophila* (…). Yarrowia lipolytica YD19 slant culture was inoculated into four Erlenmeyer flasks, and Candida virescens was added using an inoculation loop. Candida viswanathii The slant culture of TB07 was inoculated into four more Erlenmeyer flasks, with two loops of bacterial growth added to each flask. The eight Erlenmeyer flasks were incubated in a shaking incubator at 30°C and 200 rpm for 18 hours. Then, the eight yeast cultures were transferred to a fermenter. During fermentation, the stirring speed was controlled at 240 rpm, the temperature at 30°C, the aeration ratio was controlled at 0.28 vvm from 0 to 12 hours, 0.36 vvm from 13 to 36 hours, and 0.32 vvm after 36 hours. Fermentation was completed after 72 hours.

[0060] (3) The fermentation broth was boiled and then cooled to 60°C. It was centrifuged at 6000 r / min for 15 min to obtain 20.1 L of supernatant. 1400 g of maltodextrin was added to the supernatant and stirred evenly. The mixture was homogenized once at 40 MPa pressure and then dried using a spray drying tower. The inlet air temperature during the drying process was controlled at 160~170°C and the outlet air temperature was controlled at 40~50°C. 2.21 kg of dry powder was collected, which is the natural milk flavoring base. An evaluation group composed of 9 people with rich aroma evaluation experience conducted the evaluation. The aroma threshold of the flavoring base in water was 2.30 mg / L. Compared with the minimum aroma threshold (3.30 mg / L) of several commercially available natural milk flavorings, its aroma intensity was 143% of that of commercially available products. The natural milk flavoring base was prepared into a 3 g / L solution with water. The solution had a distinct aroma, and the aroma was rich and realistic.

[0061] The natural milk flavoring base and commercially available natural milk flavoring were analyzed by solid-phase microextraction (SPE) (the SPE method is referenced in: Li Ning, Sun Baoguo, Liang Menglan. Analysis of volatile components in a milk flavoring by solid-phase microextraction combined with gas chromatography-mass spectrometry [J]. Food Science, 2011, 32(22):221-225), and the aroma components were analyzed by GC-MS. For components with a relative content greater than 3%, the main components and their relative contents of the natural milk flavoring base included: butyric acid 10.73%, hexanoic acid 4.02%, ethyl caprylate 10.49%, lauric acid 3.34%, ethyl laurate 5.28%, ethyl myristate 7.15%, ethyl palmitate 15.43%, and ethyl oleate 12.19%. The main components and their relative contents of commercially available natural milk flavorings include: butyric acid 38.16%, valeric acid 22.82%, caprylic acid 13.14%, capric acid 6.85%, and lauric acid 3.22%. It is evident that the natural milk flavoring base provided by this invention differs significantly from the main components and their relative contents of commercially available natural milk flavorings.

[0062] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that this comparative example does not use Yersinia lipolyticis and Candida viresis to ferment the butter-based culture medium.

[0063] (1) Weigh 560 g of whole milk powder, mix it evenly with 20 L of water, and put it into a 30 L fully automatic fermentation tank. Adjust the pH of the solution to 6.2. Calculate the addition of 5500 U of flavor protease per gram of milk powder, add flavor protease, control the stirring speed at 50 r / min, and hydrolyze at 52℃ for 7 h. Add 1300 g of butter, 440 g of glucose, 220 g of sodium citrate, 440 g of maltodextrin, 220 g of malt extract, and 22 g of potassium dihydrogen phosphate (food grade) in sequence, stir evenly, sterilize at 121℃ for 15 min, cool to room temperature, and set aside.

[0064] (2) Weigh 42 g glucose, 12 g casein peptone, 9.6 g malt extract, and 2.4 g potassium dihydrogen phosphate (food grade), mix with 1200 mL water, adjust the pH to 5.8, sterilize at 121℃ for 15 min, cool, pour into a fermenter, and stir at 240 r / min for 1 h. Then, boil the material, cool to 55℃, and centrifuge at 5500 r / min for 18 min to obtain 20.9 L of supernatant. Add 1300 g maltodextrin to the supernatant, stir evenly, homogenize once at 35 MPa, and then dry using a spray drying tower. The inlet air temperature during the drying process is controlled at 160~170℃, and the outlet air temperature is 40~50℃. Collect 3.28 kg of dry powder, which is the natural milk flavoring base. An evaluation panel of nine experienced fragrance tasters conducted the test. The aroma threshold of this fragrance base in water was 6.45 mg / L, which is 51% of the minimum aroma threshold (3.30 mg / L) of several commercially available natural milk flavorings. When the flavoring was prepared into a 3 g / L solution with water, the solution lacked aroma and exhibited oil-water separation.

[0065] The natural milk flavoring base was analyzed by solid-phase microextraction (SPE) (see reference: Li Ning, Sun Baoguo, Liang Menglan. Analysis of volatile components in a milk flavoring by solid-phase microextraction combined with gas chromatography-mass spectrometry [J]. Food Science, 2011, 32(22):221-225), and the aroma components were analyzed by GC-MS. For components with a relative content greater than 3%, the main components and their relative contents of this natural milk flavoring include: nonanal 17.25%, capric acid 3.56%, lauric acid 7.36%, myristic acid 12.45%, palmitic acid 26.62%, and linoleic acid 3.78%.

[0066] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that this comparative example only uses a single strain of Yersinia lipolyticis to ferment the culture medium of butter base.

[0067] (1) Weigh 560 g of whole milk powder, mix it evenly with 20 L of water, and put it into a 30 L fully automatic fermentation tank. Adjust the pH of the solution to 6.2. Calculate the addition of 5500 U of flavor protease per gram of milk powder, add flavor protease, control the stirring speed at 50 r / min, and hydrolyze at 52℃ for 7 h. Add 1300 g of butter, 440 g of glucose, 220 g of sodium citrate, 440 g of maltodextrin, 220 g of malt extract, and 22 g of potassium dihydrogen phosphate (food grade) in sequence, stir evenly, sterilize at 121℃ for 15 min, cool to room temperature, and set aside.

[0068] (2) Weigh 42 g glucose, 12 g casein peptone, 9.6 g malt extract, and 2.4 g potassium dihydrogen phosphate (food grade), mix with 1200 mL water, adjust the pH to 5.8, and dispense 200 mL into six 1000 mL Erlenmeyer flasks. Sterilize at 121℃ for 15 min. After cooling, inoculate with an inoculation loop using *Yersinia lipophila* (…). Yarrowia lipolytica YD19 slant culture was inoculated into six Erlenmeyer flasks, with two loops of bacterial culture added to each flask. The six flasks were incubated in a shaking incubator at 28℃ and 190 r / min for 19 h. Then, the yeast culture was transferred to a fermenter. During fermentation, the stirring speed was controlled at 240 r / min, the temperature at 28℃, the aeration ratio was controlled at 0.28 vvm from 0 to 12 h, 0.36 vvm from 13 to 36 h, and 0.32 vvm after 36 h. Fermentation was completed after 84 hours.

[0069] (3) The fermentation broth was boiled and then cooled to 55°C. It was centrifuged at 5500 r / min for 18 min to obtain 19.3 L of supernatant. 1300 g of maltodextrin was added to the supernatant and stirred evenly. The mixture was homogenized once at 35 MPa pressure and then dried using a spray drying tower. The inlet air temperature during the drying process was controlled at 160~170°C and the outlet air temperature was controlled at 40~50°C. 2.06 kg of dry powder was collected, which is the natural milk flavoring base. An evaluation group composed of 9 experienced flavor tasters conducted the evaluation. The aroma threshold of the flavoring base in water was 2.45 mg / L. Compared with the minimum aroma threshold (3.30 mg / L) of several commercially available natural milk flavorings, its aroma intensity was 135% of that of commercially available products. The flavoring was prepared into a 3 g / L solution with water. The solution had a distinct aroma, with a rich and realistic fragrance, but oil-water separation occurred.

[0070] The natural milk flavoring base was analyzed by solid-phase microextraction (solid-phase microextraction method reference: Li Ning, Sun Baoguo, Liang Menglan. Analysis of volatile components in a milk flavoring by solid-phase microextraction combined with gas chromatography-mass spectrometry [J]. Food Science, 2011, 32(22):221-225), and the aroma components were analyzed by GC-MS. For components with a relative content greater than 3%, the main components of the natural milk flavoring and their relative contents are as follows: butyric acid 10.17%, hexanoic acid 3.92%, ethyl caprylate 9.78%, lauric acid 3.54%, ethyl laurate 4.06%, ethyl myristate 5.82%, ethyl palmitate 16.13%, and ethyl oleate 10.75%.

[0071] Comparative Example 3 The difference between Comparative Example 2 and Example 1 is that this comparative example only uses a single strain of Candida virescens to ferment the butter-based culture medium.

[0072] (1) Weigh 560 g of whole milk powder, mix it evenly with 20 L of water, and put it into a 30 L fully automatic fermentation tank. Adjust the pH of the solution to 6.2. Calculate the addition of 5500 U of flavor protease per gram of milk powder, add flavor protease, control the stirring speed at 50 r / min, and hydrolyze at 52℃ for 7 h. Add 1300 g of butter, 440 g of glucose, 220 g of sodium citrate, 440 g of maltodextrin, 220 g of malt extract, and 22 g of potassium dihydrogen phosphate (food grade) in sequence, stir evenly, sterilize at 121℃ for 15 min, cool to room temperature, and set aside.

[0073] (2) Weigh 42 g glucose, 12 g casein peptone, 9.6 g malt extract, and 2.4 g potassium dihydrogen phosphate (food grade), mix with 1200 mL water, adjust the pH to 5.8, and dispense 200 mL of the solution into six 1000 mL Erlenmeyer flasks. Sterilize at 121°C for 15 min. After cooling, inoculate with an inoculation loop using *Candida viride* (…). Candida viswanathii The TB07 slant culture was inoculated into six Erlenmeyer flasks, with two loops of bacterial growth added to each flask. The six flasks were incubated in a shaking incubator at 28°C and 190 rpm for 19 h. Then, the yeast culture was transferred to a fermenter. During fermentation, the stirring speed was controlled at 240 rpm, the temperature at 28°C, the aeration ratio was controlled at 0.28 vvm from 0 to 12 h, 0.36 vvm from 13 to 36 h, and 0.32 vvm after 36 h. Fermentation was completed after 84 hours.

[0074] (3) The fermentation broth was boiled and then cooled to 55°C. It was centrifuged at 5500 r / min for 18 min to obtain 19.5 L of supernatant. 1300 g of maltodextrin was added to the supernatant and stirred evenly. The mixture was homogenized once at 35 MPa pressure and then dried using a spray drying tower. The inlet air temperature during the drying process was controlled at 160~170°C and the outlet air temperature was controlled at 40~50°C. 2.65 kg of dry powder was collected, which is the natural milk flavoring base. An evaluation group composed of 9 people with rich aroma evaluation experience conducted the evaluation. The aroma threshold of the flavoring base in water was 3.55 mg / L. Compared with the minimum aroma threshold (3.30 mg / L) of several commercially available natural milk flavorings, its aroma intensity was 93% of that of commercially available products. The flavoring was prepared into a 3 g / L solution with water. The solution had a distinct aroma, a rich and realistic aroma, and no oil-water separation.

[0075] The natural milk flavoring base was analyzed by solid-phase microextraction (SPE) (the SPE method is referenced in: Li Ning, Sun Baoguo, Liang Menglan. Analysis of volatile components in a milk flavoring by solid-phase microextraction combined with gas chromatography-mass spectrometry [J]. Food Science, 2011, 32(22):221-225), and the aroma components were analyzed by GC-MS. For components with a relative content greater than 3%, the main components and their relative contents of this natural milk flavoring include: butyric acid 5.27%, nonanal 9.43%, caprylic acid 3.12%, lauric acid 4.21%, myristic acid 4.15%, ethyl caprylate 4.31%, ethyl laurate 4.25%, ethyl myristate 3.07%, ethyl palmitate 6.38%, and ethyl oleate 5.14%.

[0076] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a natural milky flavoring base, characterized in that... Includes the following steps: (1) Preparation of fermentation medium: Mix whole milk powder and water, put it into a fermentation tank, add flavor protease for enzymatic hydrolysis; then add animal oil, glucose, sodium citrate, maltodextrin, malt extract and potassium dihydrogen phosphate, stir to dissolve, sterilize, cool to room temperature to obtain fermentation medium; animal oil is at least one of butter and tallow; (2) Fermentation of fermentation medium: Inoculate the fermentation medium with seed liquid of Yersinia lipolytica and seed liquid of Candida viresis, and ferment under stirring and aeration conditions; (3) Spray drying of fermentation liquid: Boil the fermentation liquid obtained in step (2), cool it to 50~60℃, separate the solid and liquid, add maltodextrin to the obtained liquid, stir to dissolve, homogenize, and then use spray drying method to make dry powder to obtain natural milk flavor base material. In step (1): The dosage of the whole milk powder is calculated at 26~30 g / L; The amount of flavor protease used is calculated based on adding 5000~6000 U of flavor protease per gram of whole milk powder; The enzymatic hydrolysis conditions for the flavor protease are: temperature 50~55℃, pH 6.0~6.5, and time 6~8 h; The amount of butter used is calculated at 60~70 g / L; The amount of glucose used is calculated at 20~24 g / L; The dosage of sodium citrate is calculated at 10~12 g / L; The dosage of the maltodextrin is calculated at 20~24 g / L; The dosage of the malt extract is calculated at 10~12 g / L; The dosage of potassium dihydrogen phosphate is calculated at 1.0~1.2 g / L; The *Yersinia lipolyticis* mentioned in step (2) is *Yersinia lipolyticis* ( Yarrowia lipolytica YD19, with accession number GDMCC No:60782; The Candida viscera mentioned in step (2) is Candida viscera ( Candida viswanathii ) TB07, accession number GDMCC NO:60785; In step (2): The fermentation temperature is 26~30℃; The fermentation speed is 220~260 r / min; The aeration ratio for fermentation is controlled as follows: 0.28 vvm for 0~12 h, 0.36 vvm for 13~36 h, and 0.32 vvm after 36 h. The fermentation time is 72-96 hours.

2. The method for preparing the natural milk flavoring base according to claim 1, characterized in that: The seed culture of Yersinia lipolytica described in step (2) is prepared by the following steps: Yersinia lipolytica is inoculated into a sterile shake flask culture medium and cultured by shaking to obtain the seed culture of Yersinia lipolytica. The Candida vesiculosus seed culture described in step (2) is prepared by the following steps: Candida vesiculosus is inoculated into a sterile shake flask culture medium and cultured by shaking to obtain the Candida vesiculosus seed culture.

3. The method for preparing the natural milk flavoring base according to claim 2, characterized in that: The shake flask culture medium has the following composition: glucose 30-40 g / L, casein peptone 8-12 g / L, malt extract 6-10 g / L, potassium dihydrogen phosphate 1.8-2.2 g / L, pH 5.5-6.0; The conditions for the shaking culture are: temperature 26~30℃, rotation speed 180~200 r / min, and time 18~20 h.

4. The method for preparing the natural milk flavoring base according to claim 1, characterized in that: In step (3): The solid-liquid separation method is centrifugation; The amount of maltodextrin used is equal to that of animal fat; The pressure for homogenization is 30~40 MPa; The spray drying conditions are: inlet air temperature 160~170℃, outlet air temperature 40~50℃.

5. The method for preparing the natural milk flavoring base according to claim 4, characterized in that: The centrifugation conditions are: rotation speed 5000~6000 r / min, time 15~20 min; The homogenization process is performed once.

6. A natural milk-flavored base material, characterized in that: It is obtained by the preparation method according to any one of claims 1 to 5.

7. The application of the natural milk flavoring base material according to claim 6 in the food industry.