A microbial combination, fermented camel milk and their applications

Abstract

The present invention belongs to the field of application of microbial technology, and relates to a microbial combination and fermented milk and its application, specifically to a multi-strain microbial combination, fermented milk prepared by using the microbial combination, and its application in fermented camel milk. The present invention also provides a microbial composition for fermenting camel dairy products, and the microbial composition contains at least two kinds of lactic acid bacteria and yeasts derived from traditional fermented dairy products in Xinjiang Uygur Autonomous Region, including Leuconostoc pseudomesenteroides, Lactobacillus rhamnosus, Debaryomyces hansenii, and Kluyveromyces marxianus. The microbial composition provided by the present invention ensures that industrialized fermented camel milk products have the flavor and nutritional functions of naturally fermented sour camel milk, but with higher quality, safety level, significantly extended shelf life, higher acidification efficiency, protein hydrolysis ability, and lactose decomposition ability, and can improve the bad flavor of industrialized fermented camel dairy products using conventional strains, and has broad application prospects in industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology applications, and relates to a microbial combination and fermented camel milk and its application, specifically to a multi-strain microbial combination and fermented camel milk prepared by using the microbial combination and its application. Background Art

[0002] Camel milk is rich in nutrients. Traditional fermented camel milk, also known as naturally fermented sour camel milk, is a common traditional drink / food among pastoral residents and has certain dietary therapy effects and medical values. The camel milk product market has great potential and high development and utilization value. Due to the large quality differences and short shelf life of naturally fermented sour camel milk in different regions, it cannot be circulated in the long-distance market.

[0003] The fermented camel milk sold on the market is currently produced by two techniques: the traditional natural fermentation process technology or the technology of using different strains of lactic acid bacteria and imitating the fermentation process of cow and sheep milk to produce fermented camel milk. The former is the traditional process. After the fresh camel milk is sterilized, it is fermented by naturally passed-down strains to produce products. The advantages are that the types of strains are rich, containing rich natural lactic acid bacteria, yeasts and other different types of strains, the product flavors are diverse, and the nutritional value is high. The disadvantages are that it is brewed by herdsmen, and the preparation conditions and hygiene conditions of each household are different, resulting in large quality differences, instability of the products, and difficult guarantee of hygiene and safety, there are certain quality risks, and the product shelf life is short.

[0004] The latter is produced by some food enterprises according to the GB19302-2010 technology. The products are hygienic, safe and of stable quality. Due to the use of commercially available general lactic acid bacteria strains and processes, the advantages of the diversity of strains of naturally fermented camel milk are lost, and the products also lose the characteristics of naturally fermented camel milk. Camel, horse and donkey milk are special types of milk, and their chemical compositions and physical and chemical properties are quite different from those of cow's milk. The strains used for fermentation are also different from those of cow's milk. Using commercially available strains to imitate the fermentation process of cow's milk has a worse flavor than naturally fermented products, with a strong smell of mutton and a tendency to produce a rancid smell, and the acceptance by consumers is not high. There is no mature process and specific strains for the preparation method of fermented camel milk products.

[0005] Chinese Patent Application No. 202010546811.7 discloses a probiotic fermented camel milk for relieving constipation and its preparation method. The raw materials are taken in the following weights: 120-125 grams of camel milk powder, 10-80 grams of granulated sugar, 5-20 grams of Siraitia grosvenorii concentrated juice, 0.03-0.08 grams of yogurt starter, and 775-865 grams of purified water. The yogurt starter is one or more combinations of Bifidobacterium longum BL-G301, Bifidobacterium breve BB-G95, Bifidobacterium animalis subsp. lactis BL-G101, Bifidobacterium bifidum BB-G90, Bifidobacterium infantis BI-G201, and Bifidobacterium adolescentis BQ-G66.

[0006] Chinese Patent Application No. 201510553213.1 discloses a method for preparing high-quality fermented camel milk with auxiliary hypoglycemic function. This method uses Lactobacillus plantarum Grx16 and combines other strains as starter cultures to prepare fermented camel milk with auxiliary hypoglycemic function. In particular, Lactobacillus plantarum Grx16, Lactobacillus casei grx12, Lactobacillus delbrueckii subsp. bulgaricus grx33, and Lactobacillus fermentum grx07 are mixed in a ratio of 1:1:1:1 (v / v) as starter cultures. The fermented camel milk prepared by fermentation not only has a good flavor, but also has weak post-acidification. At the same time, it can reduce the blood sugar of diabetic rats, improve the blood lipid level of diabetic rats, and enhance its antioxidant capacity.

[0007] At present, most of the existing technologies for fermenting camel milk use basic strains such as lactic acid bacteria and their probiotics, imitating the process of fermenting cow's milk. Simply using a combination of bacteria may meet the product requirements in terms of function, but the fermentation flavor is inferior to that of naturally fermented products and cannot meet the needs of consumers. Therefore, there is an urgent need for new strains suitable for fermenting camel milk and industrial preparation methods. Summary of the Invention

[0008] In order to overcome the defects of the prior art, based on the principle of natural strain mixed fermentation of traditional fermented foods, the present invention discovers that the natural strains screened from traditional fermented dairy products in Xinjiang Uygur Autonomous Region are rich in categories, and the flavor of the fermented camel milk products is superior to that of the products fermented by commercially available strains. Therefore, a multi-strain microbial composition is hereby proposed.

[0009] The technical solutions for achieving the above objectives are as follows:

[0010] In the first aspect, the present invention provides a Leuconostoc pseudomesenteroides, which is deposited in the China General Microbiological Culture Collection Center (CGMCC), and its deposit number is CGMCC No. 19779.

[0011] In the second aspect, the present invention provides a Lactobacillus rhamnosus, which is deposited in the China General Microbiological Culture Collection Center (CGMCC), and its deposit number is CGMCC No. 26601.

[0012] In the third aspect, the present invention provides a Lactobacillus paracasei, which is deposited in the China General Microbiological Culture Collection Center (CGMCC), and its deposit number is CGMCC No. 20803;

[0013] Fourth aspect, the present invention provides a Debaryomyces hansenii, which is deposited in the China General Microbiological Culture Collection Center (CGMCC), and its deposit number is CGMCC No. 26604.

[0014] Fifth aspect, the present invention provides a Kluyveromyces marxianus, which is deposited in the China General Microbiological Culture Collection Center (CGMCC), and its deposit number is CGMCC No. 19778.

[0015] Sixth aspect, the present invention provides a multi-strain microbial composition for fermenting camel milk, and the microbial composition includes at least two of Leuconostoc pseudomesenteroides, Lactobacillus rhamnosus, Lactobacillus paracasei, Debaryomyces hansenii and Kluyveromyces marxianus.

[0016] In one embodiment, the microbial composition can be Leuconostoc pseudomesenteroides, Lactobacillus paracasei and Debaryomyces hansenii.

[0017] In one embodiment, the microbial composition can be Lactobacillus rhamnosus, Kluyveromyces marxianus and Leuconostoc pseudomesenteroides.

[0018] In one embodiment, the microbial composition can be Lactobacillus paracasei, Leuconostoc pseudomesenteroides and Debaryomyces hansenii.

[0019] In one embodiment, the microbial composition can be Leuconostoc pseudomesenteroides, Lactobacillus rhamnosus and Kluyveromyces marxianus.

[0020] In one embodiment, the microbial composition can be Lactobacillus rhamnosus, Leuconostoc pseudomesenteroides and Debaryomyces hansenii.

[0021] In one embodiment, the microbial composition can be Lactobacillus rhamnosus, Leuconostoc pseudomesenteroides and Kluyveromyces marxianus.

[0022] In one embodiment, the microbial composition can be Leuconostoc pseudomesenteroides, Debaryomyces hansenii and Kluyveromyces marxianus.

[0023] In one embodiment, the microbial composition can be Lactobacillus rhamnosus, Leuconostoc pseudomesenteroides, Debaryomyces hansenii and Kluyveromyces marxianus.

[0024] In a preferred embodiment, the viable cell numbers of Lactobacillus rhamnosus, Leuconostoc pseudomesenteroides, Debaryomyces hansenii and Kluyveromyces marxianus are 3:3:1:1.

[0025] In a preferred embodiment, the Lactobacillus rhamnosus is deposited in the China General Microbiological Culture Collection Center (CGMCC), and its deposit number is CGMCC No. 26601.

[0026] In a preferred embodiment, the Leuconostoc pseudomesenteroides is deposited in the China General Microbiological Culture Collection Center (CGMCC), and its deposit number is CGMCC No. 19779.

[0027] In a preferred embodiment, the Debaryomyces hansenii is deposited in the China General Microbiological Culture Collection Center (CGMCC), and its deposit number is CGMCC No. 26604.

[0028] In a preferred embodiment, the Kluyveromyces marxianus is deposited in the China General Microbiological Culture Collection Center (CGMCC), and its deposit number is CGMCC No. 19778.

[0029] In one embodiment, the microbial composition further comprises Lactobacillus paracasei.

[0030] In a preferred embodiment, the Lactobacillus paracasei is deposited in the China General Microbiological Culture Collection Center (CGMCC), and its deposit number is CGMCC No. 20803.

[0031] In one embodiment, the viable cell numbers of the Lactobacillus rhamnosus, Leuconostoc pseudomesenteroides, Lactobacillus paracasei, Debaryomyces hansenii and Kluyveromyces marxianus are 3:3:3:1:1.

[0032] In a fifth aspect, the present invention provides a culture medium for culturing the Lactobacillus rhamnosus described in the first aspect, the Leuconostoc pseudomesenteroides described in the second aspect, the Debaryomyces hansenii described in the third aspect and / or the microbial composition described in the fourth aspect. The culture medium comprises 14.5% camel milk powder, 2% anhydrous glucose and 83.5% purified water.

[0033] In one embodiment, the pH of the culture medium is 4.5 - 5.5.

[0034] In a sixth aspect, the present invention provides a microbial inoculant, which comprises the Leuconostoc pseudomesenteroides described in the first aspect, the Lactobacillus rhamnosus described in the second aspect, the Debaryomyces hansenii described in the third aspect or the microbial composition described in the fourth aspect, or their culture solutions, culture solution extracts, whole bacteria, whole bacteria extracts, fermentation broths and / or fermentation broth extracts.

[0035] In one embodiment, the microbial inoculant can be a solid preparation, such as freeze-dried bacterial powder; or a liquid preparation, such as liquid bacterial strain.

[0036] In a seventh aspect, there is provided the use of the Lactobacillus rhamnosus described in the first aspect, the Leuconostoc pseudomesenteroides described in the second aspect, the Debaryomyces hansenii described in the third aspect, the microbial composition described in the fourth aspect or the microbial inoculant described in the sixth aspect in the production of fermented milk.

[0037] In an eighth aspect, the present invention provides a method for producing fermented milk, the method comprising the following steps:

[0038] (1) Culturing the Lactobacillus rhamnosus described in the first aspect, the Leuconostoc pseudomesenteroides described in the second aspect, the Debaryomyces hansenii described in the third aspect or the microbial composition described in the fourth aspect with the culture medium described in the fifth aspect until the viable count reaches 5.0×10 8 -1.0×10 9 CFU / mL to obtain a primary culture;

[0039] (2) Inoculating the primary culture into the culture medium described in the fifth aspect for scale-up culture to obtain a secondary culture;

[0040] (3) Subjecting the secondary culture to low-temperature centrifugation, concentration, washing with sterile distilled water at 0-4°C, adding a cryoprotectant in a volume ratio of 1:1, maintaining at -45°C to -40°C for 1 h, and sublimating to 32-35°C to obtain dry powder bacterial strains;

[0041] (4) Adding the dry powder bacterial strains to sterilized milk for fermentation, and stopping fermentation until the acidity reaches 75-80°T to obtain fermented milk.

[0042] In one embodiment, the cryoprotectant comprises 12-15% skim camel milk powder, 30-40% glycerol, 3-5% glucose, and 40-55% distilled water.

[0043] In one embodiment, the viable count of the dry powder bacterial strains is 4.0×10 8 -9×10 8 CFU / g.

[0044] In a ninth aspect, the present invention provides a fermented milk prepared by the method described in the eighth aspect.

[0045] In one embodiment, the fermented milk includes fermented camel milk, fermented horse milk, fermented donkey milk, etc.

[0046] In a preferred embodiment, the fermented milk can be fermented camel milk.

[0047] Tenth aspect, the present invention provides a dairy product, which comprises the fermented milk described in the eighth aspect and at least one food-acceptable excipient.

[0048] In one embodiment, the dairy product includes, but is not limited to, fermented milk powder, fermented milk tablets, probiotic powder, fermented milk candies, milk cheese, etc.

[0049] The present invention uses multiple strains in combination for fermenting camel milk, ensuring that the industrialized fermented camel milk products have the flavor and nutritional functions of naturally fermented sour camel milk, but with higher quality, safety level, significantly extended shelf life, and can form commercial products for large-scale production and circulation. Compared with the prior art, through the sensory evaluation of the fermented products, Leuconostoc pseudomesenteroides FY.Chen.GL-9 selected can eliminate the unpleasant forage odor of camel milk during the fermentation process, Lactobacillus rhamnosus WY.FH-19 produces acid quickly and has many flavor-producing substances, which can reduce the muttony smell of camel milk, Debaryomyces hansenii WY.FH-22 has anti-lipid oxidation ability and can reduce the rancid smell during the shelf life of fermented camel milk, Kluyveromyces marxianus FY.Chen.GL-8 ferments to produce fruit aroma, which can enhance the aromatic taste of fermented camel milk, and Lactobacillus paracasei FW.Chen.GL-13 ferments camel milk with delicate texture and mild acidity. Using a combination of 5 strains to directly produce fermented camel milk, or using different genera among them as starter cultures and adding other commercially available starter cultures to produce fermented camel milk, the products overcome the defects of the existing production technology, presenting the unique taste and fragrance of naturally fermented camel milk. The microbial composition provided by the present invention has high acidification efficiency, protein hydrolysis ability, and lactose decomposition ability, and can improve the flavor of industrial fermented camel milk, showing broad application prospects in industrial fermentation.

[0050] Biological preservation information:

[0051] Lactobacillus rhamnosus:

[0052] Biological material: WY.FH-19

[0053] Taxonomic name: Lactobacillus rhamnosus Lacticaseibacillus rhamnosus

[0054] Preservation number: CGMCC No. 26601

[0055] Preservation time: February 21, 2023

[0056] Preservation unit: China General Microbiological Culture Collection Center

[0057] Preservation address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0058] Lactobacillus paracasei:

[0059] Biological material: FW.Chen.GL-13

[0060] Classification name: Lactobacillus paracasei Lactobacillus paracasei

[0061] Deposit number: CGMCC No. 20803

[0062] Deposit date: September 22, 2020

[0063] Depositary institution: China General Microbiological Culture Collection Center

[0064] Deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0065] Leuconostoc pseudomesenteroides:

[0066] Biological material: FY.Chen.GL-9

[0067] Classification name: Leuconostoc pseudomesenteroides Leuconostoc pseudomesenteroides

[0068] Deposit number: CGMCC No. 19779

[0069] Deposit time: May 6, 2020

[0070] Depositary institution: China General Microbiological Culture Collection Center

[0071] Deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0072] Kluyveromyces marxianus:

[0073] Biological material: FY.Chen.GL-8

[0074] Classification name: Kluyveromyces marxianus Kluyveromyces maxianus

[0075] Deposit number: CGMCC No. 19778

[0076] Deposit time: May 6, 2020

[0077] Depositary institution: China General Microbiological Culture Collection Center

[0078] Deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0079] Debaryomyces hansenii:

[0080] Biological material: WY.FH-22

[0081] Classification name: Debaryomyces hansenii Debaryomyces hansenii

[0082] Deposit number: CGMCC No. 26604

[0083] Deposit date: February 21, 2023

[0084] Depositary institution: China General Microbiological Culture Collection Center

[0085] Deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Description of the drawings

[0086] Figure 1 It is the determination result of the total viable bacteria count at the beginning and end of the strain during fermentation.

[0087] Figure 2 It is the determination result of the acidity value of fermented milk at different fermentation times.

[0088] Figure 3 It is the determination result of the degree of proteolysis of fermented milk during fermentation.

[0089] Figure 4 It is the determination result of the lactose content of fermented milk during fermentation.

[0090] Figure 5 It is the determination result of the scavenging effect of fermented milk on DPPH free radicals. Detailed implementation manners

[0091] Unless otherwise defined, all technical terms and scientific and technical terms used in the present invention have the same meanings as those commonly used in the field to which the present invention belongs. For the purpose of explaining this specification, the following definitions will be applied, and where appropriate, terms used in the singular form will also include the plural form, and vice versa.

[0092] Unless the context clearly indicates otherwise, the expressions "a" and "an" used herein include plural referents. For example, referring to "a cell" includes multiple such cells and equivalents known to those skilled in the art, and so on.

[0093] The term "about" used herein represents a range of ±20% of the value following it. In some embodiments, the term "about" represents a range of ±10% of the value following it. In some embodiments, the term "about" represents a range of ±5% of the value following it.

[0094] The materials or reagents involved in the present invention are as follows:

[0095] Fresh camel milk is produced from the Wangyuan Camel Breeding Base in Xinjiang Uygur Autonomous Region, skimmed camel milk is obtained by centrifuging fresh camel milk to remove fat, and camel milk powder is provided by Wangyuan Camel Milk Industry Co., Ltd.;

[0096] Raw materials such as white granulated sugar or glucose are all commercially available;

[0097] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with embodiments. For those conditions not specified in the embodiments, they shall be carried out according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments without indicating the manufacturer can be conventional products purchased on the market. In order to better illustrate the present invention, numerous specific details are given in the following specific implementation manners. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present invention.

[0098] Example 1 Treatment of Camel Milk

[0099] 1. Acceptance of raw camel milk: Inspection and purchase are carried out in accordance with the raw milk purchase standards formulated by the enterprise. The raw milk indicators meet the following requirements:

[0100] (1) Acidity (°T): The raw camel milk used for preparing fermented camel milk products is required to be fresh, and the natural acidity is 16 - 24;

[0101] (2) Relative density (20°C / 4°C): ≥1.028;

[0102] (3) Protein content (g / 100 g): ≥3.5%;

[0103] (4) Fat content (g / 100 g): ≥4.0;

[0104] (5) Non-fat milk solid content (g / 100 g): ≥8.5;

[0105] (6) The pollutant limit and mycotoxin limit comply with the national requirements for dairy products

[0106] 2. Clarification of milk: Select appropriate milk clarification equipment to remove spore bacteria, macromolecular fungi and impurities in the milk;

[0107] 3. Blending: Auxiliary materials such as sweeteners can be added according to product requirements, and hydration and blending are carried out in a certain proportion;

[0108] 4. Homogenization: The prepared liquid is heated to 70 - 75°C and homogenized at a pressure of 20 ± 2 Mpa;

[0109] 5. Sterilization: The homogenized liquid is heated to 95°C and kept warm for 5 minutes for sterilization;

[0110] 6. Cooling: The sterilized liquid is cooled to 30 ± 2°C.

[0111] Example 2 Preparation of Bacterial Strains

[0112] The lactic acid bacteria (Leuconostoc pseudomesenteroides, Lactobacillus rhamnosus, Lactobacillus paracasei) and yeasts (Debaryomyces hansenii, Kluyveromyces marxianus) in the microbial composition of the present invention are separately cultured and then mixed in a ratio of 3:1.

[0113] (1) Preparation of liquid medium: 90% fresh camel milk, 5% white granulated sugar, 5% pure water diluted lactic acid, adjust the pH to 4.5 - 5.5, and sterilize at 115°C for 15 min or 90 - 95°C for 30 min before use;

[0114] (2) Take out the pure strains stored in the refrigerator, pick an appropriate amount and add it to a test tube or Erlenmeyer flask containing sterilized stock solution, activate according to the requirements of the strain, and when the viable count reaches 5.0×10 8 -1.0×10 9 CFU / mL, and perform an enlarged culture according to an inoculation amount of 3 - 5%;

[0115] (3) The cultured liquid bacterial solution is centrifuged at low temperature of 0 - 4°C, concentrated, washed with sterile distilled water, and then made into a protective agent with 14.5% skim camel milk powder, 2% glucose, and 83.5% distilled water, and sterilized at 115°C for 15 min or 90 - 95°C for 30 min before use. Embed according to a ratio of 1:1 of the protective agent and the bacterial solution, and through a freezing process of maintaining at -45°C to -40°C for 1 h and sublimating to 32 - 35°C, a dry powder bacterial strain with a viable count reaching 4.0×10 8 -9.0×10 8 CFU / g is obtained.

[0116] (4) Mix the lactic acid bacteria and yeasts in a ratio of 3:1 to prepare a mixed bacterial agent.

[0117] Example 3 Preparation of dairy products by fermenting camel milk

[0118] Add the mixed bacterial agent obtained in Example 2 to the camel milk obtained in Example 1 for fermentation at a volume ratio of 4%. According to the product requirements, segmented fermentation or one-stage fermentation can be adopted, and the culture temperature is maintained at 32 - 36°C. Start detecting the acidity after 8 h of fermentation. When the acidity reaches 65 - 70°T, stop the fermentation.

[0119] Inactivation of bacterial strains: The fermented camel milk can be treated at 75°C - 80°C for 1 s according to the requirements of the product to reduce or lose the activity of the bacterial strains.

[0120] Homogenization again: The inactivated fermented camel milk can be homogenized at a pressure of 20 ± 2 Mpa while maintaining the inactivation temperature.

[0121] Filling: The fermented camel milk after homogenization again is filled as required, and then stored in a cold storage at 0 - 4°C.

[0122] 3.1 Activation of Strains and Preparation of Fermented Milk

[0123] The strains stored at -80 °C were cultured in MRS medium at 37 °C for 24 h to activate the strains for two generations. The strains after two generations of activation were washed twice with sterile normal saline, and their OD values were adjusted respectively. Then they were inoculated into sterilized milk at an inoculation amount of 4% to make the initial inoculation concentration 10 7 CFU / mL, and cultured at 37 °C for 24 h and in milk for three generations to prepare the mother starter.

[0124] Dissolve 14.5% (w / v) skim milk powder in water, sterilize at 105 °C for 15 min, cool to the inoculation temperature, inoculate 4% (v / v) mother starter into the sterilized skim milk respectively, and culture at 33 °C for 18 h.

[0125] 3.2 Sensory Evaluation

[0126] The sensory experience of the product directly affects the quality of fermented milk and people's acceptance of it. In order to further explore the market acceptability of camel milk fermented with mixed strains, the finished products produced under the optimal formula determined in the above experiment were subjected to sensory evaluation analysis, and the strains were optimized; after determining the strains and fermentation process, the camel milk fermented with multi-strain mixture was compared with traditional fermented camel milk in terms of sensory evaluation. Sensory evaluation indicators: color, aroma, taste, texture, and acceptability.

[0127] Sensory evaluation method: Adopt the group evaluation method. Select 10 professional sensory evaluation personnel to form an evaluation group to conduct sensory evaluation on the fermented milk. Comprehensive evaluation is carried out from five aspects: color, smell, taste, texture, and acceptability. The sensory evaluation criteria are shown in Table 1, with a full score of 100 points, and the score is the average value of 10 people.

[0128] Table 1 Sensory Scoring Table of Fermented Milk

[0129]

[0130] In this invention, traditional fermented camel milk with the highest flavor, taste, and acceptability and stable quality was selected from multiple regions in Xinjiang Uygur Autonomous Region as a reference for sensory evaluation, hoping to obtain fermented camel milk with a taste and flavor similar to that of traditional camel milk in Xinjiang Uygur Autonomous Region. The experimental groups are shown in Table 2.

[0131] Table 2

[0132]

[0133] The results are as Figure 1As shown, it can be seen from the radar chart of the sensory evaluation diagram that the differences in color and texture state between different fermented camel milks are relatively small, while the differences in flavor, taste and acceptability are relatively large. Because the fermented camel milk prepared with camel milk commercial starter has a special taste, resulting in a relatively low sensory acceptability. The multi-strain fermented milk prepared by the present invention has higher scores in terms of texture state, color, flavor and taste, and better acceptability. In addition, if the yeast addition amount is high, such as exceeding 50% of the total starter, the resulting alcohol flavor is relatively strong (not shown due to a large amount of data), reducing the acceptability. However, if the amount of lactic acid bacteria in the added fermented milk is relatively large (exceeding 90%), the sour taste of camel milk is stronger. And if the ratio of bacteria:yeast is between 1.5:1 and 4:1, the sour taste is mixed with the alcohol flavor produced by the yeast starter, resulting in a better flavor.

[0134] Through this sensory evaluation, it is verified that the fermented camel milk with 0.5% Kluyveromyces marxianus + 0.5% Debaryomyces hansenii + 1% Lactobacillus paracasei + 1% Leuconostoc pseudomesenteroides + 1% Lactobacillus rhamnosus is closer to the target traditional camel milk in terms of taste and flavor. Therefore, this combination and ratio are used as the experimental group, and the commercial starter (Streptococcus thermophilus subsp. and Lactobacillus bulgaricus) is used as the control group to compare the properties of other fermented milks.

[0135] The combinations of ingredients and strains used in this example are shown in Table 3:

[0136] Table 3 Combinations of strains and ingredients

[0137]

[0138] Example 4 Determination of viable cell count and acid production ability of fermented milk

[0139] Samples of fermented milk at 0, 6, 12, and 18 h during the fermentation process were taken to measure their titratable acidity to determine the acid production ability at the end of fermentation. For the viable cell type, the titratable acidity changes and viable cell count changes after fermentation for 18 h (i.e., stored for 0 days) and after storage at 4°C for 1, 7, 14, and 21 days were analyzed. The acidity was measured using a T5 automatic potentiometric titrator (Yu J, et al .Milk fermented by combined starter cultures comprising three Lactobacillusstrains exerts an alleviating effect on loperamide-induced constipation inBALB / c mice[J]. Food&Function, 2023, 14(11): 5264-5276.) The viable count was determined with reference to GB 4789.35-2016. The fermented milk sample was serially diluted with 0.85% sterile NaCl solution. Using the pour plate method, 100 μL of the diluted bacterial solution was poured onto MRS plates and incubated at 37°C for 48 h, and the viable count was determined.

[0140] The TA value is a key indicator of the acidity of fermented milk and reflects the characteristics of the acid production dynamics of fermented milk (Dan T, et al . Volatile flavor compounds profile and fermentation characteristics of milk fermented by Lactobacillus delbrueckii subsp. bulgaricus[J]. Frontiers in Microbiology , 2019, 10: 2183.). As shown in Table 4, when the five experimental strains were inoculated into camel milk, the acidity value of camel milk increased continuously with the increase of fermentation time. Among them, the acidity of fermented milk increased significantly from 12 h to 18 h, and the fermentation was completed at 18 h, and the acidity reached 65.80°T, which was regarded as the fermentation end point.

[0141] The results are shown in Table 4. During storage, the titratable acidity of the control group continued to increase and reached 127.178°T after 21 days of storage. Although the titratable acidity of the experimental group increased, the upward trend was slow and only reached 98.35°T after 21 days, indicating that the degree of post-acidification was acceptable. Post-acidification is an important quality characteristic of fermented milk. Donkor et al. believed that a titratable acidity exceeding 110 would affect the taste and flavor of fermented milk, and a titratable acidity between 70-110°T was the most ideal. The titratable acidity of the experimental group was lower than 100°T after 21 days of storage, indicating that the storage could still be extended, and the commercial shelf life was expected to reach 28 days. (Donkor O N, Henriksson A, Vasiljevic T, et al. Effect of acidification on the activity of probiotics in yoghurt during cold storage[J]. International Dairy Journal, 2006, 16(10): 1181-1189.)

[0142] The viable count is an important indicator of probiotic products. The group standard T / CNFIA 131-2021 stipulates that the viable count in probiotic products should not be less than 1×10 7CFU / mL. The results are shown in Table 4. The viable cell counts inoculated in the experimental group and the control group were both at 7×10 7 CFU / mL. The total viable cell counts of the experimental group after 18 h of fermentation and post-ripening (storage for 1 day) were 122.89×10 7 CFU / mL and 177.17×10 7 CFU / mL, which were significantly lower than those of the control group. However, during the storage test, the viable cell count of the control group decreased sharply, and the viable cell count decreased to 8.89×10 7 CFU / mL after 21 days of storage. Although the viable cell count of the experimental group also decreased after 21 days of storage, it was one order of magnitude higher than that of the control group, being 82.90×10 7 CFU / mL, far higher than 1×10 7 CFU / mL. The relatively high viable cell count also indicates that the interaction among Lactobacillus rhamnosus, Lactobacillus paracasei, Leuconostoc pseudomesenteroides, Debaryomyces hansenii, and Kluyveromyces marxianus can effectively inhibit post-acidification, which is beneficial to the taste, flavor, and long-term storage of camel milk. In addition, in the single-strain screening experiment, Leuconostoc pseudomesenteroides produces carbon dioxide during the fermentation process, which can effectively reduce the generation of off-flavors in fermented camel milk.

[0143] Table 4 Titratable acidity and viable cell counts of fermented camel milk stored at 4°C for different times

[0144]

[0145] Example 5 Determination of the proteolytic ability of the microbial composition

[0146] The method for determining the free amino acid content is the o-phthalaldehyde (OPA) derivatization colorimetric method (Laroque D, etal. Kinetic study on the Maillard reaction Consideration of sugar reactivity[J]. Food chemistry, 2008, 111(4): 1032-1042.).

[0147] The proteolytic activity of the starter culture plays an important role in both acid production and flavor formation. In addition, lactic acid bacteria decompose proteins and release amino acids, which can also be used for their own growth and development (Nezhad S J E, et al.Technological characteristics of Lactobacillus spp.Isolated from Iranian raw milk Motal cheese[J]. LWT, 2020, 133: 110070.). The change in the degree of protein hydrolysis was manifested as an overall increase in the concentration of free amino acids. The results showed that the content of free amino acids in camel milk fermented by the mixed fermentation of 5 strains was significantly higher than that of the control group ( P < 0.05), indicating that the proteolytic ability of the 5 strains in co-fermentation was significantly higher than that of the blank group and the control group ( Figure 2 ). Both Kluyveromyces marxianus and Debaryomyces hansenii in the starter culture screened in the present invention have strong proteolytic abilities, and Lactobacillus paracasei among lactic acid bacteria also has good proteolytic ability. The hydrolysis of proteins is beneficial to the digestion and absorption of proteins in fermented camel milk in the human body, especially for people in special medical conditions. The hydrolysis of proteins produces various products, including functional peptides that can improve the functional activity of camel milk, flavor peptides that can improve the taste of fermented camel milk, and small molecular volatile components that can enhance the flavor of fermented camel milk. These proteolytic products can even mask the sour taste produced by a large amount of lactic acid, which is beneficial to improving the quality and functional activity of fermented camel milk.

[0148] 5.1 Determination of lactose content

[0149] The lactose content of fermented milk was determined by the high performance liquid chromatography method of GB 5009.8-2016. Samples were taken at 0, 6, 12, and 18 h of fermentation to determine the lactose content.

[0150] Lactic acid bacteria have β-galactosidase to decompose lactose for their growth and promote the production of galactooligosaccharides during fermentation (Hikmetoglu M, et al. Changes in carbohydrate profile in kefir fermentation[J]. Bioactive Carbohydrates and Dietary Fibr e, 2020, 23:100220.). The results showed that the lactose content of the fermented camel milk (experimental group) obtained by the 5 composite strains involved in the present invention and the control group were both significantly lower than that of the blank group ( P < 0.05), which was consistent with the result of the increase in the titratable acidity after fermentation ( Figure 3 ). In the single-strain screening experiment, Lactobacillus rhamnosus was the strain with the strongest ability to utilize lactose and the fastest acid production among the 5 strains, followed by Lactobacillus paracasei.

[0151] 5.2 Determination of free amino acids

[0152] The content of free amino acids was analyzed by chromatography, using 18 amino acids as standard substances (aspartic acid, glutamic acid, serine, arginine, glycine, threonine, proline, alanine, valine, methionine, leucine, isoleucine, tryptophan, histidine, phenylalanine, cysteine, lysine, tyrosine).

[0153] The results are shown in Table 5. Four free amino acids were detected from fermented camel milk, namely serine, threonine, tryptophan and histidine. Among them, the content of tryptophan in the experimental group was significantly higher than that in the control group, while the differences in the other three amino acids were relatively small. The increase in the content of tryptophan indicates that multi-strain fermentation strengthened the metabolic pathway of tryptophan. Tryptophan is an essential amino acid for the human body and a limiting amino acid in common grains and vegetables. Tryptophan helps to improve immunity, regulate the intestinal flora, restore the intestinal barrier and other functional activities.

[0154] Table 5 Content of free amino acids in fermented camel milk

[0155]

[0156] Example 6 Determination of DPPH free radical scavenging ability

[0157] Take 1 g of the fermented camel milk sample and dissolve it in 9 mL of 95% ethanol solution. Take 2 mL of the sample mixed solution. 16 mmol / L DPPH solution, heat it in a water bath at 25 °C for 30 min, measure the absorbance of the sample (Ai) at 517 nm, measure the blank absorbance (A0) by replacing the sample mixed solution in the above system with distilled water, and replace the DPPH solution in the above system with 95% ethanol. Measure the background absorbance of the sample (Aj), and make 3 parallels for each sample. Calculate the elimination rate according to the following formula:

[0158] .

[0159] DPPH is a classic screening experiment for antioxidant activity. The results show that the DPPH free radical scavenging ability of the experimental group is significantly higher than that of the blank group and the control group ( P < 0.05), indicating that the co-fermentation of 5 strains has a better effect on scavenging DPPH free radicals in camel milk. It may be that the growth and metabolism of multiple strains produce various metabolites with antioxidant activity. The peptides produced by proteolysis mentioned above also contain antioxidant peptides. The DPPH results can reflect that the camel milk fermented by 5 strains has better antioxidant activity, and may have functional activities in relieving inflammation, delaying aging and improving immunity ( Figure 4 ).

[0160] 6.1 Determination of flavor diversity

[0161] The volatile components of fermented camel milk were detected using an electronic nose system (E-nose), referring to the method of Bai X et al. (Bai X, et al. Effects of Pretreatment on the Volatile Composition, Amino Acid, and Fatty Acid Content of Oat Bran[J]. Foods , 2022, 11(19): 3070.) with some modifications, as well as pattern recognition software (WinMuster 1.6.2).

[0162] A precisely weighed 30 g sample of fermented camel milk was used for electronic nose analysis, in triplicate. The sensors were cleaned with clean air for 120 s, then the gas was sampled into the electronic nose using a vacuum pump at an intake rate of 0.3 L / min for a detection time of 150 s. The substances corresponding to each sensor of the electronic nose are shown in Table 6.

[0163] Table 6 Performance of each sensor

[0164]

[0165] Flavor is an important sensory characteristic of food, which determines the acceptance and preference of consumers (Shi Z, et al. Comparison of changes in fermented milk quality due to differences in the proteolytic system between Lactobacillus helveticus R0052 and Lactococcus lactis subsp . lactis JCM5805[J]. Food Bioscience , 2023, 51: 102271.). The volatile flavor substances of the experimental group containing methyl components, alcohols, aldehydes, ketones, and aromatic components were significantly higher than those of the control group and the blank group ( P < 0.05). The trend of the electronic nose detection results of the experimental group was completely similar to that of traditional fermented camel milk, but the content of each component was lower than that of traditional fermented milk. This result indicates that in terms of the flavor components detectable by the electronic nose, the camel milk fermented by the mixed culture of 5 strains of bacteria is similar to traditional fermented camel milk. This result is consistent with the sensory evaluation results and also shows that the compound fermentation of lactic acid bacteria and yeast in camel milk can achieve the modern controllable production of traditional camel milk. It is worth mentioning that the flavor components of the experimental group were lower than those of traditional fermented milk ( P＜0.05), which may be due to the fact that pure strains were used as the starter culture in this experiment, while traditional fermented camel milk uses the previous batch of fermented camel milk as the starter culture. During the addition of the starter culture, the metabolites and flavor components of the previous batch of camel milk have been introduced. That is, the flavor components of traditional fermented milk are higher than those of the experimental group at the starting point of fermentation. The Lactobacillus paracasei involved in the experimental group is the strain that produces the most 2,3-butanedione and 3-hydroxy-2-butanone among the 5 strains, contributing milky and buttery aromas to fermented camel milk; Lactobacillus rhamnosus is the strain that produces the highest content of organic acids among the 5 strains; while in the single-strain screening experiment, Leuconostoc pseudomesenteroides, Debaryomyces hansenii, and Kluyveromyces marxianus mainly contribute to the production of alcohols, esters, sulfides, nitrogen oxides, and aldehydes in fermented camel milk, giving fermented camel milk a fresh and fragrant aroma and a fatty aroma, making the flavor of fermented milk richer ( Figure 5 ).

[0166] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement made by those of ordinary skill in the art to the technical solution of the present invention shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A fermented milk, characterized in that, The fermented milk is composed of 96% camel milk, 0.5% Kluyveromyces marxianus, 0.5% Debaryomyces hansenii, 1% Lactobacillus paracasei, 1% Leuconostoc pseudomesenteroides and 1% Lactobacillus rhamnosus. The viable cell numbers of Lactobacillus rhamnosus, Leuconostoc pseudomesenteroides, Lactobacillus paracasei, Debaryomyces hansenii and Kluyveromyces marxianus are 3:3:3:1:

1. The Lactobacillus rhamnosus is deposited in the China General Microbiological Culture Collection Center, and its deposit number is CGMCC No. 26601. The Leuconostoc pseudomesenteroides is deposited in the China General Microbiological Culture Collection Center, and its deposit number is CGMCC No. 19779. The Debaryomyces hansenii is deposited in the China General Microbiological Culture Collection Center, and its deposit number is CGMCC No. 26604. The Kluyveromyces marxianus is deposited in the China General Microbiological Culture Collection Center, and its deposit number is CGMCC No. 19778. The Lactobacillus paracasei is deposited in the China General Microbiological Culture Collection Center, and its deposit number is CGMCC No. 20803.

2. A production method of fermented milk as described in claim 1, characterized in that, The method comprises the following steps: (1) Cultivate the Kluyveromyces marxianus, Debaryomyces hansenii, Lactobacillus paracasei, Leuconostoc pseudomesenteroides, and Lactobacillus rhamnosus as described in claim 1 until the viable cell count reaches 5.0×10 8 -1.0×10 9 CFU / mL to obtain a primary culture; (2) Inoculating the primary culture into a medium for subculture to obtain a secondary culture. (3) Centrifuging the secondary culture at a low temperature of 0-4°C, concentrating it, washing it with sterile distilled water, adding a cryoprotectant in a volume ratio of 1:1, maintaining it at -45°C to -40°C for 1 h, and sublimating it to 32-35°C to obtain dry powder bacterial strains. (4) Adding the dry powder bacterial strains into sterilized camel milk for fermentation, and stopping the fermentation until the acidity reaches 70-80°T to obtain fermented milk.

3. The method according to claim 2, wherein The cryoprotectant comprises 12-15% defatted camel milk powder, 30-40% glycerol, 3-5% glucose and 40-55% distilled water. and / or, the viable count of the dry powder strain is 4.0×10 8 -9×10 8 CFU / g.

4. A dairy product, characterized in that, The dairy product comprises the fermented milk according to claim 1 and at least one food-acceptable auxiliary material.

5. The dairy product according to claim 4, wherein, The dairy product comprises fermented milk powder, fermented milk tablets, probiotic powder, fermented milk candies and milk cheese.

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