Microbial combination and fermented camel milk and use thereof

Abstract

The present invention relates to a microbial combination and a fermented milk and the use thereof, in particular to a multi-strain microbial combination, a fermented milk prepared from the microbial combination and the use of the microbial combination in the fermentation of a camel milk. The microbial composition contains at least two of lactic acid bacteria and yeasts derived from traditional fermented milk products in Xinjiang, including Leuconostoc pseudomesenteroides, Lactobacillus rhamnosus, Debaryomyces hansenii and Kluyveromyces marxianus. The microbial composition ensures that industrially fermented camel milk products have the flavor and nutritional functions of naturally fermented sour camel milk, but with higher quality, better safety and significantly prolonged shelf life. The microbial composition has relatively high acidification efficiency, proteolysis capacity and lactose decomposition capability, can improve the unpleasant flavor of camel milk products industrially fermented by using conventional strains, and has wide application prospects in industrial production.

Description

A microbial combination and fermented camel milk and its application

[0001] This application claims priority to Chinese patent application CN202410172744.5 filed with the State Intellectual Property Office of China on February 6, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of microbial technology application, relates to a microbial combination and fermented camel milk and applications thereof, and specifically relates to a multi-species microbial combination and fermented camel milk prepared by using the microbial combination and applications thereof. Background Art

[0003] Camel milk is rich in nutrients. Traditionally fermented camel milk, also known as naturally fermented sour camel milk, is a common traditional beverage and food for pastoral residents and boasts therapeutic and medicinal properties. Camel milk products have significant market potential and are highly valued for development and utilization. However, naturally fermented sour camel milk, due to significant regional quality variations and a short shelf life, cannot be readily distributed across long-distance markets.

[0004] Fermented camel milk currently sold on the market is produced using either traditional natural fermentation techniques or by using different strains of lactic acid bacteria, or by mimicking the fermentation of cow and goat milk. The traditional process involves sterilizing fresh camel milk and then fermenting it with naturally cultured bacteria. Its advantages include a rich variety of bacterial strains, including naturally occurring lactic acid bacteria and yeasts, resulting in diverse flavors and high nutritional value. However, its disadvantages are that it is home-brewed by herders, and preparation conditions and hygiene vary from household to household, leading to significant product quality variations, instability, and difficulty ensuring hygienic safety. This poses certain quality risks and results in a short shelf life.

[0005] The latter is produced by some food companies according to GB19302-2010 technology. While the product is hygienic, safe, and of stable quality, it uses commercially available lactic acid bacteria strains and processes, which negate the advantages of naturally fermented camel milk's bacterial diversity and characteristics. Camel, horse, and donkey milk are specialty milks with chemical composition and physical and chemical properties that differ significantly from cow's milk. The fermentation bacteria used also differ from cow's milk. Using commercially available bacteria to mimic the fermentation process for cow's milk results in a flavor inferior to naturally fermented products, with a strong muttony odor and a tendency to produce a rancid taste, which is not widely accepted by consumers. Fermented camel milk products lack mature processes and specific bacterial strains for their preparation.

[0006] Chinese patent application 202010546811.7 discloses a laxative probiotic-fermented camel milk and its preparation method. The ingredients are as follows: 120-125 grams of camel milk powder, 10-80 grams of white sugar, 5-20 grams of monk fruit juice concentrate, 0.03-0.08 grams of yogurt starter, and 775-865 grams of purified water. The yogurt starter is one or a combination of more than one 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.

[0007] Chinese patent application 201510553213.1 discloses a method for preparing high-quality fermented camel milk with hypoglycemic properties. This method utilizes Lactobacillus plantarum Grx16 (Lactobacillus plantarum Grx16) in combination with other bacterial strains as starter cultures to produce fermented camel milk with hypoglycemic properties. Specifically, a mixture of Lactobacillus plantarum Grx16, Lactobacillus casei Grx12, Lactobacillus delbrueckii subsp. bulgaricus Grx33, and Lactobacillus fermentum Grx07 (Lactobacillus fermentum Grx07) in a 1:1:1:1 (v / v) ratio is used as the starter culture. The fermented camel milk produced not only has a good flavor and low post-acidification, but also lowers blood sugar in diabetic rats, improves blood lipid levels, and enhances antioxidant capacity.

[0008] Currently, camel milk fermentation technologies mostly use basic strains such as lactic acid bacteria and probiotics, mimicking the process of fermenting cow milk. While using a single bacterial combination may meet product requirements, the fermented flavor is inferior to naturally fermented products and fails to meet consumer demand. Therefore, new strains suitable for camel milk fermentation and industrial production methods are urgently needed.

[0009] Summary of the Invention

[0010] In order to overcome the defects of the existing technology, the present invention is based on the principle of mixed fermentation of natural strains of traditional fermented foods. It is found that the natural strains screened from traditional fermented dairy products in Xinjiang are rich in categories, and the fermented camel milk products have a better flavor than products fermented by commercially available strains. Therefore, a multi-species microbial composition is specially proposed.

[0011] The technical solutions to achieve the above objectives are as follows:

[0012] In a first aspect, the present invention provides a Leuconostoc pseudomesenteroides, which is deposited in the China General Microbiology Center (CGMCC) of the China Culture Collection Administration of Microorganisms, with a deposit number of CGMCC No. 19779.

[0013] In a second aspect, the present invention provides a Lactobacillus rhamnosus, which is deposited in the China General Microbiology Center (CGMCC) of the China Culture Collection Administration of Microorganisms, and its deposit number is CGMCC No. 26601.

[0014] In a third aspect, the present invention provides a Lactobacillus paracasei, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) with a deposit number of CGMCC No. 20803.

[0015] In a fourth aspect, the present invention provides a Debaryomyces hansenii yeast, which is deposited in the China General Microbiology Center (CGMCC) of the China Culture Collection Administration of Microorganisms, and its deposit number is CGMCC No. 26604.

[0016] In a fifth aspect, the present invention provides a Kluyveromyces marxianus yeast, which is deposited in the China General Microbiology Center (CGMCC) of the China Culture Collection Administration of Microorganisms, and its deposit number is CGMCC No. 19778.

[0017] In a sixth aspect, the present invention provides a multi-species microbial composition for fermenting camel milk, wherein the microbial composition comprises at least two of Leuconostoc pseudomesenteroides, Lactobacillus rhamnosus, Lactobacillus paracasei, Debaryomyces hansenii and Kluyveromyces marxianus.

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

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

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

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

[0022] In one embodiment, the microbial composition may be Lactobacillus rhamnosus, Leuconostoc pseudomesenteroides, and Debaryomyces hansenii.

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

[0024] In one embodiment, the microbial composition may be Leuconostoc pseudomesenteroides, Debaryomyces hansenii, and Kluyveromyces marxianus.

[0025] In one embodiment, the microbial composition may be Lactobacillus rhamnosus, Leuconostoc pseudomesenteroides, Debaryomyces hansenii, and Kluyveromyces marxianus.

[0026] In a preferred embodiment, the number of live bacteria of Lactobacillus rhamnosus, Leuconostoc pseudomesenteroides, Debaryomyces hansenii and Kluyveromyces marxianus is 3:3:1:1.

[0027] In a preferred embodiment, the Lactobacillus rhamnosus is deposited in the China General Microbiology Center (CGMCC) of the China Culture Collection Administration of Microorganisms, and its deposit number is CGMCC No. 26601.

[0028] In a preferred embodiment, the Leuconostoc pseudomesenteroides is deposited in the China General Microbiology Center (CGMCC) under the China Culture Collection Administration, and its deposit number is CGMCC No. 19779.

[0029] In a preferred embodiment, the Debaryomyces hansenii is deposited in the China General Microbiology Center (CGMCC) with a deposit number of CGMCC No. 26604.

[0030] In a preferred embodiment, the Kluyveromyces marxianus is deposited in the China General Microbiology Center (CGMCC) with a deposit number of CGMCC No. 19778.

[0031] In one embodiment, the microbial composition further comprises Lactobacillus paracasei.

[0032] In a preferred embodiment, the Lactobacillus paracasei is deposited in the China General Microbiology Center (CGMCC) of the China Culture Collection Administration of Microorganisms, and its deposit number is CGMCC No. 20803.

[0033] In one embodiment, the number of viable bacteria of Lactobacillus rhamnosus, Leuconostoc pseudomesenteroides, Lactobacillus paracasei, Debaryomyces hansenii and Kluyveromyces marxianus is 3:3:3:1:1.

[0034] 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 comprising 14.5% camel milk powder, 2% anhydrous glucose and 83.5% purified water.

[0035] In one embodiment, the pH of the culture medium is 4.5-5.5.

[0036] In a sixth aspect, the present invention provides a microbial agent, comprising 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 a culture fluid, a culture fluid extract, a whole bacterium, a whole bacterium extract, a fermentation fluid, and / or a fermentation fluid extract thereof.

[0037] In one embodiment, the microbial agent can be a solid preparation, such as freeze-dried bacterial powder; or a liquid preparation, such as liquid bacterial strain.

[0038] In the seventh aspect, 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 agent described in the sixth aspect in the production of fermented milk.

[0039] In an eighth aspect, the present invention provides a method for producing fermented milk, the method comprising the following steps:

[0040] (1) Using the culture medium described in the fifth aspect, 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 is cultured until the viable count reaches 5.0×10 8 -1.0×10 9 CFU / mL, obtained primary culture;

[0041] (2) inoculating the primary culture into the culture medium described in the fifth aspect for expansion culture to obtain a secondary culture;

[0042] (3) The secondary culture was centrifuged at 0-4°C, concentrated, and washed with sterile distilled water. A lyophilization protective agent was added at a volume ratio of 1:1, maintained at -45°C to -40°C for 1 hour, and sublimated to 32-35°C to obtain a dry powder of the culture;

[0043] (4) adding the dry powdered bacteria to sterilized milk for fermentation until the acidity reaches 75-80°T, thereby obtaining fermented milk.

[0044] In one embodiment, the lyoprotectant comprises 12-15% skim camel milk powder, 30-40% glycerol, 3-5% glucose, and 40-55% distilled water.

[0045] In one embodiment, the viable count of the dry powder strain is 4.0×10 8 -9×10 8CFU / g.

[0046] In a ninth aspect, the present invention provides a fermented milk prepared by the method described in the eighth aspect.

[0047] In one embodiment, the fermented milk includes fermented camel milk, fermented horse milk, fermented donkey milk, etc.

[0048] In a preferred embodiment, the fermented milk may be fermented camel milk.

[0049] In a tenth aspect, the present invention provides a dairy product comprising the fermented milk according to the eighth aspect and at least one food-acceptable auxiliary material.

[0050] In one embodiment, the dairy products include, but are not limited to, fermented milk powder, fermented milk flakes, probiotic powder, fermented milk candy, milk cheese, and the like.

[0051] The present invention uses multiple strains of bacteria to ferment camel milk, ensuring that industrially fermented camel milk products have the flavor and nutritional functions of naturally fermented sour camel milk, but with higher quality and safety, significantly extended shelf life, and can form large-scale production and circulation products. Compared with the existing technology, after sensory evaluation of the fermented products, the selected pseudoenteroides FY.Chen.GL-9 can eliminate the unpleasant forage odor of camel milk during the fermentation process, rhamnosus Lactobacillus WY.FH-19 has fast acid production and produces many aromatic substances, which can reduce the mutton smell of camel milk, hansen Debaryomyces WY.FH-22 resists fat oxidation and can reduce the rancid smell of fermented camel milk during the shelf life, Kluyveromyces marxianus FY.Chen.GL-8 produces a fruity aroma that can enhance the aromatic taste of fermented camel milk, and Lactobacillus paracasei F The camel milk fermented by W.Chen.GL-13 has a delicate texture and a mild sour taste. The fermented camel milk is directly produced by using a combination of 5 bacterial strains, or different species are used as basic starters and other commercially available starters are added to produce fermented camel milk. The products overcome the defects of existing production technologies and present the unique taste and aroma 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 industrially fermented camel milk, and has broad application prospects in industrial fermentation.

[0052] Biodeposit Information:

[0053] Lactobacillus rhamnosus:

[0054] Biomaterial: WY.FH-19

[0055] Classification name: Lacticaseibacillus rhamnosus

[0056] Accession number: CGMCC No.26601

[0057] Preservation time: February 21, 2023

[0058] Depository: General Microbiology Center of China Culture Collection Administration

[0059] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0060] Lactobacillus paracasei:

[0061] Biomaterial: FW.Chen.GL-13

[0062] Classification name: Lactobacillus paracasei

[0063] Deposit number: CGMCC No.20803

[0064] Date of deposit: September 22, 2020

[0065] Depository: General Microbiology Center of China Culture Collection Administration

[0066] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0067] Leuconostoc pseudomesenteroides:

[0068] Biomaterial: FY.Chen.GL-9

[0069] Classification name: Leuconostoc pseudomesenteroides

[0070] Accession number: CGMCC No.19779

[0071] Storage time: May 6, 2020

[0072] Depository: General Microbiology Center of China Culture Collection Administration

[0073] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0074] Kluyveromyces marxianus:

[0075] Biomaterial: FY.Chen.GL-8

[0076] Classification name: Kluyveromyces maxianus

[0077] Deposit number: CGMCC No.19778

[0078] Storage time: May 6, 2020

[0079] Depository: General Microbiology Center of China Culture Collection Administration

[0080] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0081] Debaryomyces hansenii:

[0082] Biomaterial: WY.FH-22

[0083] Classification name: Debaryomyces hansenii

[0084] Accession number: CGMCC No.26604

[0085] Preservation time: February 21, 2023

[0086] Depository: General Microbiology Center of China Culture Collection Administration

[0087] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 shows the results of determination of the initial and final total viable counts of the strain during the fermentation process.

[0089] Figure 2 shows the results of determining the acidity of fermented milk at different fermentation times.

[0090] FIG3 shows the results of measuring the degree of protein hydrolysis of fermented milk during the fermentation process.

[0091] FIG4 shows the results of measuring the lactose content of fermented milk during the fermentation process.

[0092] FIG5 shows the results of the determination of the DPPH free radical scavenging effect of fermented milk. DETAILED DESCRIPTION

[0093] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0094] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0095] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0096] The materials or reagents involved in the present invention are as follows:

[0097] Fresh camel milk is produced from Xinjiang Wangyuan Wantuoyuan Breeding Base, skimmed camel milk is produced by centrifugation and skimming of fresh camel milk, and camel milk powder is prepared and provided by Wangyuan Camel Milk Industrial Co., Ltd.

[0098] Raw materials such as white sugar or glucose are commercially available;

[0099] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the examples. In the examples, if no specific conditions are specified, the conditions according to normal conditions or manufacturer's recommendations are carried out. If all reagents or instruments are not specified by the manufacturer, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0100] Example 1 Processing of camel milk

[0101] 1. Acceptance of raw camel milk: The raw milk is inspected and purchased according to the raw milk purchase standards formulated by the company. The raw milk indicators meet the following requirements:

[0102] (1) Acidity (°T): The raw camel milk used to prepare fermented camel milk products must be fresh and have a natural acidity of 16-24;

[0103] (2) Relative density (20℃ / 4℃): ≥1.028;

[0104] (3) Protein content (g / 100g): ≥3.5%;

[0105] (4) Fat content (g / 100g): ≥4.0;

[0106] (5) Non-fat milk solids content (g / 100g): ≥8.5;

[0107] (6) The pollutant limits and mycotoxin limits meet the national requirements for dairy products

[0108] 2. Milk purification: Select appropriate milk purification equipment to remove spores, macromolecular fungi and impurities in milk;

[0109] 3. Ingredients: Sweeteners and other auxiliary materials can be added according to product needs, and hydration and ingredients can be carried out in a certain proportion;

[0110] 4. Homogenization: Heat the prepared liquid to 70-75℃ and homogenize at a pressure of 20±2Mpa;

[0111] 5. Sterilization: Heat the homogenized liquid to 95°C and keep it warm for 5 minutes for sterilization;

[0112] 6. Cooling: After sterilization, the liquid is cooled to 30±2℃.

[0113] Example 2 Preparation of bacterial strains

[0114] 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 cultured separately and then mixed in a ratio of 3:1.

[0115] (1) Preparation of liquid culture medium: 90% fresh camel milk, 5% white sugar, 5% purified water to dilute lactic acid, adjust pH to 4.5-5.5, and sterilize at 115°C for 15 min or 90-95°C for 30 min before use;

[0116] (2) Take out the pure strain stored in the refrigerator, pick out an appropriate amount and add it to the test tube or triangular flask containing the sterilized liquid to activate it according to the requirements of the strain until the number of viable bacteria reaches 5.0×10 8 -1.0×10 9 CFU / mL, expand the culture according to 3-5% inoculum size;

[0117] (3) The cultured liquid bacterial solution was centrifuged at 0-4℃, concentrated, and washed with sterile distilled water. A protective agent was prepared using 14.5% skim camel milk powder, 2% glucose, and 83.5% distilled water. Before use, it was sterilized at 115℃ for 15 minutes or 90-95℃ for 30 minutes. The bacterial solution was embedded in a ratio of 1:1, kept at -45℃ to -40℃ for 1 hour, and sublimated to 32-35℃ for freezing. The number of viable bacteria reached 4.0×10 8 -9.0×10 8 CFU / g dry powder bacteria.

[0118] (4) Lactic acid bacteria and yeast were mixed in a ratio of 3:1 to prepare a mixed bacterial agent.

[0119] Example 3: Preparation of dairy products by fermentation of camel milk

[0120] The mixed bacterial agent obtained in Example 2 was added to the camel milk obtained in Example 1 at a volume ratio of 4% for fermentation. According to product requirements, staged fermentation or single-stage fermentation was adopted. The culture temperature was maintained at 32-36°C. The acidity was detected after 8 hours of fermentation. Fermentation was stopped when the acidity reached 65-70°T.

[0121] Inactivation of bacteria: Fermented camel milk can be heated at 75℃-80℃ for 1 second to reduce or inactivate the bacteria activity according to product requirements.

[0122] Homogenize again: the fermented camel milk after inactivation can be homogenized at a pressure of 20±2Mpa while maintaining the inactivation temperature.

[0123] Filling: The fermented camel milk after homogenization is filled as required and stored in a cold storage at 0-4℃.

[0124] 3.1 Activation of strains and preparation of fermented milk

[0125] The strain stored at -80℃ was cultured in MRS medium at 37℃ for 24h to activate the strain for two generations. The strain after two generations of activation was washed twice with sterile saline, and its OD value was adjusted respectively. The strain was inoculated into sterilized milk at a 4% inoculation volume to make the initial inoculation concentration 10 7 CFU / mL, cultured at 37 °C for 24 h, and cultured in milk for three generations to prepare the mother starter culture.

[0126] 14.5% (w / v) skim milk powder was dissolved in water, sterilized at 105°C for 15 min, cooled to the inoculation temperature, and 4% (v / v) mother starter culture was inoculated into the sterilized skim milk and cultured at 33°C for 18 h.

[0127] 3.2 Sensory evaluation

[0128] The sensory experience of a product directly impacts the quality of fermented milk and its acceptance. To further explore the market acceptability of mixed-strain camel milk fermented with this product, sensory evaluation and analysis were conducted on finished products produced using the optimal formula determined in the previous experiment. The strains were then selected. Once the strains and fermentation process were determined, sensory evaluations of the mixed-strain fermented camel milk were compared with those of traditionally fermented camel milk. Sensory evaluation criteria included color, aroma, mouthfeel, texture, and acceptability.

[0129] Sensory evaluation method: A group evaluation method was adopted. Ten professional sensory evaluators were selected to form an evaluation panel to conduct sensory evaluation of fermented milk. A comprehensive evaluation was conducted from five aspects: color, smell, taste, texture, and acceptability. The sensory evaluation standards are shown in Table 1. The full score is 100 points, and the average score of the ten people was taken.

[0130] Table 1 Sensory score table of fermented milk

[0131] This study screened traditional fermented camel milk from various regions of Xinjiang for the most flavorful, palatable, and stable fermented camel milk, using it as a reference for sensory evaluation. The goal was to obtain fermented camel milk with a taste and flavor similar to traditional Xinjiang camel milk. The experimental groups are shown in Table 2.

[0132] Table 2

[0133] The results, as shown in Figure 1, show that the sensory evaluation radar chart shows that the differences in color and texture between the different fermented camel milks are small, while the flavor, mouthfeel, and acceptability vary significantly. This is because fermented camel milk prepared with commercial camel milk starters has a unique flavor, resulting in lower sensory acceptance. The multi-strain fermented milk produced according to the present invention scored higher in texture, color, flavor, and mouthfeel, indicating better acceptance. Furthermore, if the yeast addition level is high, such as exceeding 50% of the total starter, the resulting mellow flavor is stronger (data not shown due to the large volume), reducing acceptability. However, if the fermented milk contains a high concentration of lactic acid bacteria (over 90%), the camel milk tastes more sour. A bacteria:yeast ratio between 1.5:1 and 4:1 results in a more flavorful blend of the sour flavor and the mellow flavor produced by the yeast starter.

[0134] This sensory evaluation confirmed that camel milk fermented with 0.5% Kluyveromyces marxianus + 0.5% Debaryomyces hansen + 1% Lactobacillus paracasei + 1% Leuconostoc pseudomesenteroides + 1% Lactobacillus rhamnosus was closer in taste and flavor to the target traditional camel milk. Therefore, this combination and ratio served as the experimental group, while commercial starter cultures (Streptococcus salivarius subsp. thermophilicus and Lactobacillus bulgaricus) served as the control group to compare the properties of other fermented milks.

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

[0136] Table 3 Combination of strains and ingredients

[0137] Example 4 Determination of viable cell count and acid production capacity of fermented milk

[0138] The fermented milk was sampled at 0, 6, 12, and 18 hours during the fermentation process, and its titratable acidity was measured to determine the acid production capacity at the fermentation endpoint. For the viable bacteria type, the titrated acidity changes and viable bacteria count changes were analyzed after 18 h of fermentation (i.e., storage for 0 days) and storage at 4°C for 1, 7, 14, and 21 days, respectively. The acidity was determined using a T5 automatic potentiometric titrator (Yu J, et al. Milk fermented by combined starter cultures comprising three Lactobacillus strains exerts an alleviating effect on loperamide-induced constipation in BALB / c mice[J]. Food & Function, 2023, 14(11): 5264-5276.). Viable bacteria were counted according to GB 4789.35-2016. The fermented milk sample was gradiently diluted with 0.85% sterile NaCl solution. 100 μL of the diluted bacterial solution was poured into an MRS plate using the pour plate method, and the plate was incubated at 37°C for 48 h to count the viable bacteria.

[0139] The TA value is a key indicator of fermented milk acidity, reflecting the dynamic characteristics of fermented milk acid production (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, the acidity of camel milk increased with increasing fermentation time after the five experimental strains were inoculated into the milk. The acidity of the fermented milk increased significantly between 12 and 18 hours, and fermentation was complete at 18 hours, reaching an acidity of 65.80°T, considered the end point of fermentation.

[0140] The results are shown in Table 4. During storage, the titrated acidity of the control group continued to increase, reaching 127.178°T after 21 days of storage. Although the titrated acidity of the experimental group increased, the upward trend was slow, reaching only 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. believe that a titrated acidity exceeding 110 will affect the taste and flavor of fermented milk, and a titrated acidity between 70-110°T is most ideal. The titrated acidity of the experimental group after 21 days of storage was less than 100°T, indicating that storage can still be extended, and the commercial shelf life is expected to reach 28 days. (Donkor ON, Henriksson A, Vasiljevic T, et al. Effect of acidification on the activity of probiotics in yogurt during cold storage [J]. International Dairy Journal, 2006, 16(10): 1181-1189.)

[0141] The number of viable bacteria is an important indicator of probiotic products. The group standard T / CNFIA 131-2021 stipulates that the number of viable bacteria in probiotic products cannot be less than 1×10 7 CFU / mL. The results are shown in Table 4. The number of viable bacteria inoculated in the experimental group and the control group was 7×10 7 The total number of viable bacteria in the experimental group after fermentation for 18 h and post-ripening (storage for 1 day) was 122.89×10 7 CFU / mL and 177.17×10 7 CFU / mL was significantly lower than that of the control group. However, the number of viable bacteria in the control group decreased sharply during the storage test, and after 21 days of storage, the number of viable bacteria decreased to 8.89×10 7 CFU / mL, while the number of viable bacteria in the experimental group also decreased after 21 days of storage, but it was one order of magnitude higher than that in the control group, which was 82.90×10 7 CFU / mL, much higher than 1×10 7 CFU / mL. The high viable count also suggests that the interaction between Lactobacillus rhamnosus, Lactobacillus paracasei, Leuconostoc pseudomesenteroides, Debaryomyces hansenii, and Kluyveromyces marxianus can effectively inhibit post-acidification, benefiting the taste, flavor, and long-term storage of camel milk. Furthermore, in single-strain screening experiments, the production of carbon dioxide during the fermentation of Leuconostoc pseudomesenteroides can effectively reduce the occurrence of unpleasant flavors in fermented camel milk.

[0142] Table 4 Titrated acidity and viable bacterial count of fermented camel milk stored at 4°C for different times

[0143] Example 5 Determination of proteolytic capacity of microbial compositions

[0144] The free amino acid content was determined by o-phthalaldehyde (OPA) derivatization colorimetry (Laroque D, et al. Kinetic study on the Maillard reaction Consideration of sugar reactivity [J]. Food chemistry, 2008, 111 (4): 1032-1042.).

[0145] The proteolytic activity of the starter 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 SJE, 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 is 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 with a mixed fermentation of 5 strains of bacteria was significantly higher than that of the control group (P < 0.05), indicating that the proteolytic ability of the co-fermentation of the 5 strains of bacteria was significantly higher than that of the blank group and the control group (Figure 2). The proteins of Kluyveromyces marxianus and Debaryomyces hansenii in the starter screened by the present invention have strong proteolytic ability, and Lactobacillus paracasei among the lactic acid bacteria also has good proteolytic ability. Protein hydrolysis is beneficial to the digestion and absorption of protein in fermented camel milk in the human body, especially for people with special medical conditions. Protein hydrolysis produces a variety of 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 molecule volatile components that can enhance the flavor of fermented camel milk. These protein hydrolysis 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.

[0146] 5.1 Lactose content determination

[0147] The lactose content of fermented milk was determined by high-performance liquid chromatography (HPLC) according to GB 5009.8-2016. Samples were taken at 0, 6, 12, and 18 h of fermentation to determine the lactose content.

[0148] Lactic acid bacteria have β-galactosidase to break down lactose for their growth and promote the production of oligosaccharides during the fermentation process (Hikmetoglu M, et al. Changes in carbohydrate profile in kefir fermentation [J]. Bioactive Carbohydrates and Dietary Fiber, 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 was significantly lower than that of the blank group (P < 0.05), which is consistent with the result of the increase in titrated acidity after fermentation (Figure 3). In the single bacteria 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.

[0149] 5.2 Determination of free amino acids

[0150] The free amino acid content was analyzed by chromatography using 18 amino acids as standards (aspartic acid, glutamic acid, serine, arginine, glycine, threonine, proline, alanine, valine, methionine, leucine, isoleucine, tryptophan, histidine, phenylalanine, cysteine, lysine, and tyrosine).

[0151] The results, shown in Table 5, showed that four free amino acids were detected in fermented camel milk: serine, threonine, tryptophan, and histidine. The tryptophan content 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 tryptophan indicates that multi-strain fermentation enhanced the tryptophan metabolic pathway. Tryptophan is an essential amino acid for humans and a limiting amino acid in common grains and vegetables. It contributes to various functional activities, including boosting immunity, regulating intestinal flora, and restoring the intestinal barrier.

[0152] Table 5 Free amino acid content in fermented camel milk

[0153] Example 6 Determination of DPPH free radical scavenging ability

[0154] Dissolve 1g of fermented camel milk sample in 9mL of 95% ethanol solution, and take 2mL of the sample mixture. Add 16mmol / L DPPH solution and heat in a 25℃ water bath for 30min. Measure the sample absorbance (Ai) at 517nm. Replace the sample mixture in the above system with distilled water to measure the blank absorbance (A0). Replace the DPPH solution in the above system with 95% ethanol. Measure the sample background absorbance (Aj). Perform three replicates for each sample. Calculate the elimination rate according to the following formula:

[0155] Elimination rate (%) = A0-(Ai -A j ) / A0×100%.

[0156] DPPH is a classic screening test for antioxidant activity. Results showed that the experimental group had significantly higher DPPH free radical scavenging capacity than the blank and control groups (P < 0.05), indicating that the co-fermentation of the five bacterial strains was more effective in scavenging DPPH free radicals in camel milk. This is likely due to the multi-strain growth and metabolism producing a variety of metabolites with antioxidant activity. The peptides produced by protein hydrolysis mentioned above also include antioxidant peptides. The DPPH results suggest that camel milk fermented with the five bacterial strains has superior antioxidant activity, potentially contributing to its potential in alleviating inflammation, delaying aging, and enhancing immunity (Figure 4).

[0157] 6.1 Flavor diversity determination

[0158] The volatile components of fermented 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, and pattern recognition software (Win Muster 1.6.2).

[0159] Accurately weighed 30g of fermented milk sample was analyzed by the electronic nose in triplicate. The sensor was purged with clean air for 120 seconds. The sample gas was then drawn into the electronic nose using a vacuum pump at an intake rate of 0.3 L / min for 150 seconds. The substances corresponding to each sensor in the electronic nose are shown in Table 6.

[0160] Table 6 Performance of each sensor

[0161] Flavor is an important sensory property of food that determines consumer acceptance and preference (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 experimental group showed significantly higher levels of methyl-containing components, alcohols, aldehydes, ketones, and aromatic volatile flavor compounds than the control and blank groups (P < 0.05). The trends in the electronic nose detection results for the experimental group and traditional fermented camel milk were completely similar, but the content of each component was lower in traditional fermented milk. This result indicates that the flavor components detected by the electronic nose are similar between camel milk fermented with the five bacterial strains and traditional fermented camel milk. This result is consistent with the sensory evaluation results and also indicates that the combined fermentation of camel milk with lactic acid bacteria and yeast can achieve modern and controllable production of traditional camel milk. It's worth noting that the flavor components of the experimental group were lower than those of the traditional fermented milk (P < 0.05). This may be because this experiment used a pure bacterial strain as the starter, while the traditional fermented camel milk used a previous batch of fermented camel milk as the starter. During the addition of the starter, metabolites and flavor components from the previous batch of camel milk were introduced. This resulted in a higher flavor component in the traditional fermented milk than in the experimental group at the start of fermentation. Lactobacillus paracasei, the strain involved in the experimental group, produced the highest levels of 2,3-butanedione and 3-hydroxy-2-butanone among the five strains, contributing to the milky and buttery aromas of the fermented camel milk. Lactobacillus rhamnosus produced the highest levels of organic acids among the five strains. In the single-bacteria screening experiment, Monilinia pseudomesenteroides, Debaryomyces hansenii, and Kluyveromyces marxianus primarily contributed to the production of alcohols, esters, sulfides, nitrogen oxides, and aldehydes, imparting a refreshing aroma and fatty flavor to the fermented camel milk, enriching the flavor of the fermented milk (Figure 5).

[0162] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A Leuconostoc pseudomesenteroides, characterized in that The Leuconostoc pseudomesenteroides is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, and its deposit number is CGMCC No.19779.

2. A Lactobacillus rhamnosus, characterized in that The Lactobacillus rhamnosus is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, and its deposit number is CGMCC No.26601.

3. A Debaryomyces hansenii yeast, characterized in that The Debaryomyces hansenii yeast is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, and its deposit number is CGMCC No.26604.

4. A microbial composition for fermenting camel milk, characterized in that: The microbial composition includes Leuconostoc pseudomesenteroides, Lactobacillus rhamnosus, Debaryomyces hansenii and Kluyveromyces marxianus.

5. The microbial composition according to claim 4, characterized in that The live bacterial counts of the Lactobacillus rhamnosus, Leuconostoc pseudomesenteroides, Debaryomyces hansenii and Kluyveromyces marxianus are 3:3:1:

1.

6. The microbial composition according to claim 4, characterized in that The Lactobacillus rhamnosus is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, and its deposit number is CGMCC No. 26601; and / or The Leuconostoc pseudomesenteroides is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number being CGMCC No. 19779; and / or The Debaryomyces hansenii yeast is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, and its deposit number is CGMCC No. 26604; and / or The Kluyveromyces marxianus is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, and its deposit number is CGMCC No.19778.

7. The microbial composition according to any one of claims 4 to 6, characterized in that The microbial composition also includes Lactobacillus paracasei.

8. The microbial composition according to claim 7, characterized in that The Lactobacillus paracasei is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 20803. And / or, the number of viable bacteria of Lactobacillus rhamnosus, Leuconostoc pseudomesenteroides, Lactobacillus paracasei, Debaryomyces hansenii and Kluyveromyces marxianus is 3:3:3:1:

1.

9. A culture medium, characterized in that The culture medium is used to culture the Leuconostoc pseudomesenteroides according to claim 1, the Lactobacillus rhamnosus according to claim 2, the Debaryomyces hansenii according to claim 3 and / or the microbial composition according to any one of claims 4 to 8, and the culture medium comprises 14.5% camel milk powder, 2% anhydrous glucose and 83.5% purified water.

10. A microbial agent, characterized in that: The microbial agent includes the Leuconostoc pseudomesenteroides according to claim 1, the Lactobacillus rhamnosus according to claim 2, the Debaryomyces hansenii according to claim 3 and / or the microbial composition according to any one of claims 4 to 8, or a culture or isolate thereof.

11. Use of the Leuconostoc pseudomesenteroides according to claim 1, the Lactobacillus rhamnosus according to claim 2, the Debaryomyces hansenii according to claim 3, or the microbial composition according to any one of claims 4 to 8 in producing fermented milk.

12. A method for producing fermented milk, characterized in that: The method comprises the following steps: (1) Using the culture medium according to claim 9, the Leuconostoc pseudomesenteroides according to claim 1, the Lactobacillus rhamnosus according to claim 2, the Debaryomyces hansenii according to claim 3, or the microbial composition according to any one of claims 4 to 8 is cultured until the viable cell count reaches 5.0×10 8 -1.0×10 9 CFU / mL, obtained primary culture; (2) inoculating the primary culture into the culture medium of claim 9 for expansion culture to obtain a secondary culture; (3) The secondary culture was centrifuged at 0-4°C, concentrated, and washed with sterile distilled water. A lyophilization protective agent was added at a volume ratio of 1:1, maintained at -45°C to -40°C for 1 hour, and sublimated to 32-35°C to obtain a dry powder of the culture; (4) adding the dry powdered bacteria to sterilized milk for fermentation until the acidity reaches 70-80°T, thereby obtaining fermented milk.

13. The method according to claim 12, characterized in that The freeze-drying protective agent comprises 12-15% skim camel milk powder, 30-40% glycerol, 3-5% glucose, and 40-55% distilled water; And / or, the viable bacterial count of the dry powder strain is 4.0×10 8 -9×10 8 CFU / g.

14. Fermented milk produced according to the method according to any one of claims 12 to 13.

15. A dairy product, characterized in that The dairy product comprises the fermented milk according to claim 14 and at least one food-acceptable auxiliary material.

16. The dairy product according to claim 15, characterized in that The dairy products include fermented milk powder, fermented milk slices, probiotic powder, fermented milk candy and milk cheese.

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