Functional probiotic sheep milk and method for its preparation

Abstract

The application discloses a kind of functional probiotic sheep milk and preparation method thereof, belong to the field of fermentation engineering.The application uses the high-yield probiotic of fermented sheep milk gamma-aminobutyric acid (gamma-aminobutyric acid, GABA) as fermentation strain, and prepares freeze-dried bacteria powder, studies the influence of single and compound bacteria powder on the GABA production of sheep milk fermentation, determines that the compound probiotic composed of fermented Lactobacillus mucus, rhamnose lactobacillus and paracasein lactobacillus is the optimal fermenting agent, and the application is fermented using the fermenting agent, and the sweetener and stabilizer are optimized, and the functional probiotic sheep milk with sleep aid function is prepared, and the probiotic viable count and GABA yield are high, and the stability is good.Therefore, the fermented milk prepared in the application lays a foundation for developing sleep aid functional sheep milk products, and provides a new idea for the development of Shaanxi sheep milk industry.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fermentation engineering, in particular to a sleep-aiding probiotic sheep milk developed by fermenting sheep milk with probiotics and a preparation method thereof. BACKGROUND

[0002] According to the statistics of the Food and Agriculture Organization of the United Nations, the total global sheep milk production in 2019 was 10,587,900 tons, accounting for 1.15% of the total milk production and 34.43% of the total goat milk production. Sheep milk is the leader of special milk, and sheep milk belongs to a new industry. In western provinces of China, sheep are bred on a large scale, and the milk production is also rapidly increasing. Compared with cow milk and goat milk, sheep milk has its unique advantages. At present, the products mainly include infant formula sheep milk powder, large bag powder and liquid sheep milk products. The product technology content is low, the homogeneity is serious, and the competition is fierce. Therefore, it is necessary to develop functional sheep milk products with high technology content.

[0003] According to the survey results announced by the China Sleep Research Association, the insomnia rate of domestic adults is as high as 32.8%, which means that there are more than 400 million people in China who have sleep disorders. The China Sleep Research Report 2023 also shows that the sleep index of Chinese people in 2022 is 67.8 points (on a 100-point scale), and the sleep time is basically concentrated between 23:00 and 1:00. Compared with 2021, the concentrated sleep time of Chinese people has been pushed back by one hour, and there is a serious sleep delay behavior. The large population base of sleep problems in China has increased the demand for sleep-aiding products, creating business opportunities for sleep-aiding products. The "sleep economy" has emerged. At present, many dairy companies have noticed the "sleep economy" and have made corresponding arrangements to launch related products, such as the ultimate good night pure milk of Sanyuan Food and the Mongolian Milk Good Night Sleep Milk with Chamomile Flavor.

[0004] The common sleep-aiding ingredients on the domestic market are γ-aminobutyric acid (GABA), L-theanine, spina date and glycine. Among them, GABA is a naturally occurring non-protein amino acid that exists in plants. It can relax the body and mind through nerve regulation and promote sleep. Its application in dairy products is mainly exogenous addition. There is no dairy product that directly converts free glutamic acid and protein hydrolysis glutamic acid in dairy products into GABA through probiotic fermentation. SUMMARY

[0005] The present application aims to provide a probiotic sheep milk with sleep-aiding function and a preparation method thereof, so as to solve the problems of the prior art. The functional probiotic ferment with high production of gamma-aminobutyric acid and exopolysaccharide is screened, and the sheep milk is fermented by using the ferment, so as to enrich the types of functional probiotic fermented sheep milk and provide a theoretical basis for the development of Shaanxi sheep milk industry.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0007] The present application provides a functional probiotic sheep milk, which comprises a complex probiotic bacteria composed of Limosilactobacillus fermentum KD6, Lacticaseibacillus rhamnosus KD5 and Lacticaseibacillus paracasei KD11. The preservation number of the Limosilactobacillus fermentum KD6 is CCTCC NO: M20231642, and the preservation time is September 7, 2023. The preservation number of the Lacticaseibacillus rhamnosus KD5 is CCTCC NO: M20231641, and the preservation time is September 7, 2023. The preservation number of the Lacticaseibacillus paracasei KD11 is CCTCC NO: M2024605, and the preservation time is April 1, 2024. The preservation units of the above three strains are all China Center for Type Culture Collection, and the preservation address is Wuhan, China, Wuhan University.

[0008] Preferably, the mass ratio of the Limosilactobacillus fermentum KD6, the Lacticaseibacillus rhamnosus KD5 and the Lacticaseibacillus paracasei KD11 is 1:(1-3):(1-3).

[0009] The present application also provides a preparation method of the functional probiotic sheep milk, which comprises the step of fermenting sheep milk by using a complex probiotic bacteria. The complex probiotic bacteria is composed of Limosilactobacillus fermentum KD6, Lacticaseibacillus rhamnosus KD5 and Lacticaseibacillus paracasei KD11. The preservation number of the Limosilactobacillus fermentum KD6 is CCTCC NO: M20231642, the preservation number of the Lacticaseibacillus rhamnosus KD5 is CCTCC NO: M20231641, and the preservation number of the Lacticaseibacillus paracasei KD11 is CCTCC NO: M2024605.

[0010] Preferably, the mass ratio of the Limosilactobacillus fermentum KD6, the Lacticaseibacillus rhamnosus KD5 and the Lacticaseibacillus paracasei KD11 is 1:(1-3):(1-3).

[0011] Preferably, the preparation method comprises the following steps:

[0012] mixing the sheep milk with water, sterilizing, preparing sterilized reconstituted sheep milk, cooling;

[0013] inoculating the compound probiotics into the cooled sterilized reconstituted sheep milk, fermenting, adding sweeteners and thickening agents, preparing the functional probiotic sheep milk.

[0014] Preferably, the mass percentage of the sheep milk in the sterilized reconstituted sheep milk is 18% (w / v).

[0015] Preferably, the fermentation conditions are as follows: the inoculation amount is 0.005%-0.025%, the fermentation temperature is 33-41℃, and the fermentation time is 17-25h.

[0016] Preferably, the fermentation conditions are as follows: the inoculation amount is 0.010%, the fermentation temperature is 37℃, and the fermentation time is 21h.

[0017] Preferably, the sweeteners include erythritol, acesulfame potassium and neotame, and the mass ratio of erythritol, acesulfame potassium and neotame is (30-45):(0.1-0.3):(0.003-0.007).

[0018] The thickening agents include modified tapioca starch, hydroxypropyl distarch phosphate and agar, and the mass ratio of the modified tapioca starch, hydroxypropyl distarch phosphate and agar is 1:1:1.

[0019] The application further provides the use of the sheep milk in improving sleep quality or preparing sheep milk products for improving sleep quality.

[0020] The application discloses the following technical effects:

[0021] The present application finds that the viable cell count of single bacteria fermentation of L.mucosus KD5, L.rhamnosus KD6 or L.paracasei KD11 fermented goat milk is significantly higher than that of compound bacteria powder, but the selenium conversion rate, GABA and EPS yield of three-strain compound bacteria powder fermented sheep milk are significantly higher than that of single bacteria powder, therefore, the KD5+KD6+KD11 bacteria powder with a ratio of 1:1:1 is determined as the fermenting agent, with the increase of inoculation amount (0.005%-0.025%) or the increase of fermentation temperature (33-41℃), the water holding capacity, sensory evaluation and GABA yield of the fermented sheep milk all increase first and then decrease; with the extension of fermentation time (17-25h), the GABA yield of the fermented sheep milk increases, the water holding capacity and sensory evaluation still show the trend of first increasing and then decreasing, the GABA yield of the prepared sheep milk is significantly improved, reaching 0.981g / L. 7 During 28 days of cold storage at 4℃, the fermentation acidity, pH of the probiotic fermented sheep milk are basically stable, the taste is good, the viable cell count decreases, but at the end of the cold storage, it is still higher than 7.5x10 7 The present application screens and obtains functional probiotic fermenting agent with high GABA yield, sweetener and thickening agent, develops functional probiotic fermented sheep milk, studies the changes during the cold storage, provides reference for the research and development of functional probiotic fermented sheep milk, and provides a new direction for the development of Shaanxi sheep milk industry. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below only show some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.

[0023] Figure 1 Effect of probiotic strains on GABA production during sheep milk fermentation;

[0024] Figure 2 Antagonistic test of probiotics; A: KD6 as the background bacteria, B: KD11 as the background bacteria, C: KD5 as the background bacteria;

[0025] Figure 3 Effect of single and compound probiotic bacteria powder on the viable cell count of sheep milk;

[0026] Figure 4 The effects of single and compound bacterial cultures on the acidity and pH of fermented sheep milk; A: pH value, B: acidity;

[0027] Figure 5 The effects of single and compound probiotic powders on the water-holding capacity of fermented goat milk;

[0028] Figure 6 The effects of single and compound probiotic powders on GABA production in goat milk;

[0029] Figure 7 The effect of inoculum size on pH and acidity of fermented goat milk; A: pH value, B: acidity;

[0030] Figure 8 The effect of inoculum size on the water-holding capacity, GABA content, and sensory evaluation of probiotic-fermented goat milk;

[0031] Figure 9 The effect of fermentation temperature on the pH and acidity of fermented goat milk; A: pH value, B: acidity;

[0032] Figure 10 The effect of fermentation temperature on the water-holding capacity, GABA content, and sensory evaluation of probiotic-fermented goat milk;

[0033] Figure 11 The effects of fermentation time on pH, acidity (A), water-holding capacity, GABA and sensory evaluation (B) of probiotic fermented goat milk;

[0034] Figure 12 The effect of thickeners on the water-holding capacity of fermented goat milk;

[0035] Figure 13 The effect of sweeteners on the sensory scores of fermented goat milk;

[0036] Figure 14 The effects of probiotic fermentation on pH, acidity, water-holding capacity, GABA concentration (A), sensory score, viable count (B), and texture (C) of goat milk during refrigeration were investigated.

[0037] Figure 15 The effects of probiotic fermentation of goat milk with added sweeteners and thickeners on pH, acidity, water holding capacity (A), sensory score, viable count (B), and texture (C) during refrigeration;

[0038] Figure 16 T2 inversion map of sheep milk fermented with probiotics to aid sleep. Detailed Implementation

[0039] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of certain aspects, features and embodiments of the present application, but not a limitation thereof.

[0040] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, a parameter, an intermediate value of the parameter is encompassed if the intermediate value is within the range of values along with the upper and lower limits of the range. Any smaller range within a larger range is also encompassed.

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in further detail the methods and / or materials associated with the documents. In the event of conflict between the present specification and any incorporated document, the present specification controls.

[0042] Many modifications and variations of the present application described in the detailed description of the application can be made by those skilled in the art, without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given should be considered exemplary only.

[0043] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", and the like are open-ended terms that are intended to denote the inclusion of elements or steps, but not to exclude any other elements or steps.

[0044] Example 1

[0045] 1. Strains and raw materials

[0046] The 6 probiotic bacteria used in the present study - Limosilactobacillus fermentum KD6, Lacticaseibacillus rhamnosus KD5, Lacticaseibacillus paracasei KD11, Limosilactobacillus fermentum 38, Lactiplantibacillus plantarum 7830, Lacticaseibacillus rhamnosus L20;

[0047] Sheep milk powder was purchased from Xi'an Baiyue Sheep Milk Group.

[0048] Lacticaseibacillus rhamnosus KD5 was isolated from kefir grains. After repeated isolation using MRS agar medium to obtain pure colonies formed by a single strain, mass spectrometry identification by a full-automatic microbial mass spectrometer (VITEK MS, Mérieux, France) and biochemical identification by a full-automatic microbial identification instrument (VITEK 2, Mérieux, France), and comparison using Launch pad software and AES8.01 database of VITEK-MS mass spectrometer research library (RUO), the strain KD5 was identified as Lacticaseibacillus rhamnosus.

[0049] Limosilactobacillus fermentum KD6 was isolated from kefir grains. After fermentation of sheep milk, the lactic acid bacteria were obtained by series dilution and streaking on MRS agar plates, and a strain of Lactobacillus with strong ability to produce gamma-aminobutyric acid was obtained after repeated primary screening and rescreening. The strain was characterized by homology analysis of 16S rDNA partial gene fragments, and the measured sequence was compared and analyzed with the base sequence in the NCBI database, confirming that the strain was Limosilactobacillus fermentum. The strain was named Limosilactobacillus fermentum KD6.

[0050] Isolation and identification of Lacticaseibacillus paracasei KD11: After the fermentation of goat milk by Kefir granules, the milk was crushed and then serially diluted and streaked on MRS agar. After repeated purification, a strain of lactic acid bacteria was obtained. The strain was identified by 16S rDNA. After amplification and purification of the target fragment, the strain was characterized according to the homology analysis of the partial gene fragment of 16S rDNA. The measured sequence was compared and analyzed with the base sequence in the NCBI database, and the strain was identified as Lacticaseibacillus paracasei, named Lacticaseibacillus paracasei KD11, with the base pair sequence (SEQ IDNO: 1) as follows: GACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCGCTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGCCGACAACAGTTACTCTGCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAAGCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATACGCCGCGGGTCCATCCAAAAGCGATAGCTTACGCCATCTTTCAGCCAAGAACCATGCGGTTCTTGGATCTATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCCCACTTAAGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCGTTCCATGTTGAATCTCGGTGCAAGCACCGATCATCAACGAGAACTCGTTCGACT.

[0051] The preservation number of the above-mentioned fermented L. muci-de KD6 is CCTCC NO: M20231642, and the preservation time is September 7, 2023; the preservation number of L. rhamnosus KD5 is CCTCC NO: M20231641, and the preservation time is September 7, 2023; the preservation number of L. paracasei KD11 is CCTCC NO: M2024605, and the preservation time is April 1, 2024; the preservation unit of the above-mentioned three strains is China Center for Type Culture Collection, and the preservation address is Wuhan, China, Wuhan University.

[0052] 2. Experimental method

[0053] 2.1 Strain activation

[0054] The freeze-dried bacteria powder of the 6 strains of probiotics was inoculated into MRS broth medium, incubated at 37°C for 24h, and activated once. Then, it was inoculated into new MRS broth medium at an inoculation amount of 5% (v / v), incubated at 37°C for 24h, and the above operation was repeated twice, and the third generation was activated for standby.

[0055] 2.2 Screening of GABA-producing probiotic bacteria

[0056] The activated strains of the third generation were inoculated into sheep milk at a 5% inoculation rate. After 24 h of fermentation at 37°C, the whey was separated and the GABA content was determined using the Berthlot colorimetric method.

[0057] 2.3 Probiotic strain antagonism test

[0058] The activated probiotic bacteria were grouped and subjected to antagonism test using Oxford cups. After 24 h of incubation at 37°C, the presence of inhibition zones around the Oxford cups was observed, which served as the basis for determining whether antagonism existed between the background bacteria and the inoculated strains.

[0059] 2.4 Preparation of probiotic bacterial powder

[0060] The functional probiotic bacteria obtained through screening were inoculated into MRS broth medium and incubated at 37°C for 24 h. The bacterial cells were collected by centrifugation (8000 r, 10 min, 4°C), washed with sterile physiological saline, centrifuged again to obtain the bacterial cells, and washed once more. Then, an equal amount of freeze-drying protectant was added to the bacterial slurry, which was pre-frozen at -20°C for 5 h and then freeze-dried in a freeze-drying machine for 24 h to obtain the probiotic bacterial powder.

[0061] 2.5 Preparation of sheep milk fermented by probiotic bacteria

[0062] The sheep milk powder was reconstituted with purified water to obtain 18% (m / v) reconstituted milk, which was sterilized at 90°C for 15 min and then cooled. The probiotic bacteria were inoculated into the reconstituted sheep milk and shaken thoroughly, and the fermentation was terminated when the pH reached 4.5, resulting in fermented sheep milk.

[0063] In addition, the effects of probiotic bacterial powder and its compound powder on sheep milk fermentation were studied, and the viable cell count, γ-aminobutyric acid production, water holding capacity, sensory evaluation, acidity, and pH were determined.

[0064] (1) Viable cell count determination

[0065] Fermented milk viable cell count determination: Refer to Gong Junhui et al. "Method of counting viable cells by dilution and spreading plate method".

[0066] Bacterial powder viable cell count determination: Under sterile conditions, 0.1 g of target bacterial powder was added to 0.9% sterile physiological saline to 10 mL, diluted to 10 -2 concentration, and then diluted and spread for counting according to the same method. Finally, the unit viable cell count (CFU / g) was calculated.

[0067] (2) γ-aminobutyric acid determination

[0068] The content of γ-aminobutyric acid was determined according to the method of Yang Shengyuan et al. "Research on GABA colorimetric determination method in glutamate decarboxylase activity determination". The standard curve equation was y = 2.0686x + 0.0804, and the regression equation coefficient R was 0.9968 when the concentration of γ-aminobutyric acid was in the range of 0-1.0 μg / mL, indicating that the linear relationship between absorbance and γ-aminobutyric acid concentration was good. Within the linear relationship range, it can be used for the determination of γ-aminobutyric acid concentration. 2 The content of γ-aminobutyric acid was determined according to the method of Yang Shengyuan et al. "Research on GABA colorimetric determination method in glutamate decarboxylase activity determination". The standard curve equation was y = 2.0686x + 0.0804, and the regression equation coefficient R was 0.9968 when the concentration of γ-aminobutyric acid was in the range of 0-1.0 μg / mL, indicating that the linear relationship between absorbance and γ-aminobutyric acid concentration was good. Within the linear relationship range, it can be used for the determination of γ-aminobutyric acid concentration.

[0069] (3) Water holding capacity determination

[0070] According to Huang Wenqi et al. "Effect of Tremella polysaccharide on immune and intestinal mucosal barrier function of mice fermented by probiotic fermented camel milk", the following formula was used for calculation:

[0071] Water holding capacity (%) = m / M x 100%

[0072] In the formula: m - the mass of the precipitate (g); M - the mass of the fermented milk (g).

[0073] (4) Sensory evaluation of fermented goat milk

[0074] According to Zhang Qiuhong's method "Optimization and purification of ACE-inhibitory peptides produced by Lactobacillus plantarum fermented goat milk", the fermented goat milk was evaluated by 5 or more people after tasting, and the result was in percentage.

[0075] (5) Acidity determination: reference GB 5009.239-2016, determined by phenolphthalein indicator method.

[0076] (6) pH determination: determined directly at room temperature using pHs-3c acidity meter.

[0077] 2.7 Single factor test of fermentation conditions

[0078] The bacteria powder was inoculated in the reconstituted sheep milk, and the effects of fermentation time (17 h, 19 h, 21 h, 23 h, 25 h), fermentation temperature (33℃, 35℃, 37℃, 39℃, 41℃), inoculation amount (0.005%, 0.010%, 0.015%, 0.020%, 0.025%) on the probiotic fermented sheep milk were studied, wherein selenium (79g / mol) was added according to the maximum addition amount (280μg / kg) in the national standard (GB14880-2012), and the maximum addition amount of sodium selenite (173g / mol) (0.08μg / mL) was calculated according to the addition amount of reconstituted milk (12.5g / 100mL). The pH value, acidity, sensory evaluation, water holding capacity, selenium enrichment ability, γ-aminobutyric acid yield, and exopolysaccharide yield of the fermented milk were determined. The basic experimental conditions were fermentation time 18h, fermentation temperature 37℃, and inoculation amount 0.005%, and then the suitable conditions of each single factor were obtained, which were combined as the suitable fermentation conditions.

[0079] 2.8 Thickening agent and sweetener screening

[0080] The bacteria powder was inoculated in the reconstituted 18% (m / v) sheep milk, and the effects of starch (corn modified starch, cassava modified starch, hydroxypropyl distarch phosphate), agar, and pectin on the probiotic bacteria powder fermented sheep milk were studied. The whey separation amount, water holding capacity, and sensory evaluation of the fermented milk were determined, and the basic addition amount of each component was (0.25%, 0.5%, 0.75%, 1%, 1.25%, 1.5%, w / v). The suitable type and addition amount of starch were determined.

[0081] The effects of single and compound sweeteners (erythritol, acesulfame potassium, neotame, aspartame) and sucrose on the probiotic bacteria powder fermented sheep milk were also studied. The sensory evaluation of the fermented milk was determined, and the basic addition amount of each component was (sweetness coefficient addition amount 6%, 9%, 12%, 15%, 18%). The suitable type and addition amount of sweetener were determined.

[0082] 2.9 Cold storage study of functional probiotic fermented sheep milk

[0083] The functional probiotic fermented sheep milk prepared in the above "2.7 Fermentation condition single factor test" was placed in a refrigerator at 4℃ for cold storage, and the changes of GABA, sensory evaluation, water holding capacity, texture, viable count, pH value, and acidity were determined at 0d, 1d, 3d, 7d, 14d, 21d, and 28d. The changes of pH value, acidity, sensory evaluation, water holding capacity, texture, viable count, and water activity of the probiotic fermented sheep milk with added sweetener and thickening agent prepared in "2.8 Thickening agent and sweetener screening" were determined during the 4℃ cold storage for 28d.

[0084] 3, Results and analysis

[0085] 3.1 Effects of probiotic strains on the production of γ-aminobutyric acid (GABA) from goat milk fermentation

[0086] like Figure 1 As shown, without the addition of monosodium glutamate (MSG) to sheep milk, all six probiotic strains produced high concentrations of GABA. This is likely due to the high activity of glutamate decarboxylase in the probiotics, which utilizes the amino acids in sheep milk to convert them into GABA. After fermentation at 37℃ for 24 hours, the GABA yield in sheep milk ranged from 0.106 to 0.321 g / L. Among these, KD5, KD6, and KD11 showed significantly higher GABA yields in sheep milk than other strains (P < 0.05). KD5, KD6, and KD11 were selected for subsequent preparation of the bacterial powder.

[0087] 3.2 Research and Development of Functional Probiotic Fermented Goat Milk

[0088] 3.2.1 Probiotic antagonism test

[0089] The results of antagonistic experiments using KD6, KD11, and KD5 as background bacteria are as follows: Figure 2 As shown in AC. Figure 2 As shown in section A, using KD6 as the background bacterium and 0.9% NaCl as the blank control, overlapping colonies were observed around the Oxford cups of KD5, KD11, and KD5+KD11. Therefore, it was determined that KD6 does not exhibit antagonism with KD5 or KD11. Figure 2 The results from BC were consistent. Therefore, it can be inferred that there was no antagonistic effect between any of the three functional probiotic strains.

[0090] 3.2.2 Effects of single and compound bacterial cultures on sheep milk fermentation

[0091] Freeze-drying was used to prepare freeze-dried probiotic powders of three strains of bacteria. Four different compound probiotic powders were then prepared by blending the powders: KD5+KD11, KD5+KD6, KD6+KD11, and KD5+KD6+KD11 (all compound probiotic powders were prepared at a mass ratio of 1:1 or 1:1:1 for each strain). These were then used for sheep milk fermentation, and their viable cell count, γ-aminobutyric acid (GABA) production, water-holding capacity, acidity, and pH were determined. The results are shown in [Figure missing]. Figures 3 to 6 .

[0092] Viable bacteria were counted using the dilution plating method. Figure 3 The results show that the viable bacteria count in the fermented milk produced by the starter culture was 1×10⁻⁶. 9 The CFU / mL level was above 3.13 × 10⁻⁶, with the highest live bacteria count in KD5 maturing sheep milk reaching 3.13 × 10⁻⁶. 9 CFU / mL; viable bacterial count in sheep milk ranges from 1.83 to 3.0 × 10⁻⁶. 9 CFU / mL; KD5 had the highest viable count (P < 0.05).

[0093] From Table 2A, it can be seen that the pH of the bacteria powder fermented milk gradually decreased with the increase of fermentation time; the fermentation endpoint of the fermented goat milk generally appeared around 18 h, among which the three-strain fermented milk reached the fermentation endpoint the fastest. Figure 4 From Table 2B, it can be seen that the acidity of the bacteria powder fermented milk gradually increased with the increase of fermentation time, and there was little difference between the strains; the fermentation endpoint of the fermented goat milk generally appeared around 17 h, among which the KD5+KD11 fermented milk reached the fermentation endpoint the fastest. Figure 4 From Table 2C, it can be seen that the water holding capacity of the fermented goat milk ranged from 30.16% to 34.87%, among which the water holding capacity of the three-strain fermented goat milk was the highest (P<0.05), and after ripening, it was 38.78%. The water holding capacity of the fermented goat milk after ripening showed an upward trend, which may be due to the interaction between whey protein and casein during cold storage to form a stable colloidal structure, thereby improving the water holding capacity.

[0094] Figure 5 From Table 2D, it can be seen that the GABA production of the goat milk ranged from 0.294 to 0.498 g / L, and the GABA production of the three-strain compound fermentation in goat milk was the highest (P<0.05).

[0095] From Table 2E, it can be seen that the GABA production of the goat milk ranged from 0.294 to 0.498 g / L, and the GABA production of the three-strain compound fermentation in goat milk was the highest (P<0.05). Figure 6 By comparing the GABA production of the goat milk fermented by the probiotic bacteria powder and its compound bacteria powder, and combining the live bacteria count, acidity, pH, and water holding capacity of the fermented goat milk, it was determined that the fermentation agent with a ratio of 1:1:1 of KD5+KD6+KD11 probiotic bacteria powder compound had the best effect, which was used for subsequent fermentation process optimization.

[0096] 3.2.3 Effect of fermentation conditions on probiotic fermented goat milk

[0097] From Table 3A, it can be seen that as the inoculum increased, the pH of the fermented milk decreased significantly, the acidity of the fermented milk increased significantly, and the fermentation time gradually shortened; when the inoculum increased from 0.005% to 0.025%, the fermentation time of the goat milk gradually shortened from 18 h to 15 h.

[0098] Figure 7 The water holding capacity and GABA production of the subsequent fermented milk were measured at their respective fermentation endpoints, and the sensory evaluation of the fermented milk was measured after ripening after fermentation and cold storage, and the results are shown in Table 3B.

[0099] The water holding capacity and GABA production of the subsequent fermented milk were measured at their respective fermentation endpoints, and the sensory evaluation of the fermented milk was measured after ripening after fermentation and cold storage, and the results are shown in Table 3B. Figure 8 ​​As shown, the water-holding capacity of fermented sheep milk initially increased and then decreased with increasing inoculum amount. This may be because, with increasing inoculum amount, the efficiency of probiotics in utilizing casein in sheep milk increases, resulting in enhanced coagulation. However, when the inoculum amount is too large, the bacteria produce acid too quickly, reaching the fermentation endpoint in a shorter time, leading to incomplete coagulation of the fermented milk. The water-holding capacity of sheep milk reached its maximum value of 28.32% when the inoculum amount was 0.010%. The GABA production in fermented sheep milk initially increased and then decreased with increasing inoculum amount. This may be because, with increasing inoculum amount, the bacteria grow faster within the same time frame, increasing GABA production. However, when the inoculum amount is too large, the bacteria produce acid too quickly, leading to reaching the fermentation endpoint in a shorter time. Furthermore, excessively high bacterial concentrations prevented effective binding to the substrate, and competition between strains inhibited GAD conversion efficiency. Simultaneously, the limited nutrients in sheep milk were insufficient to support the metabolic activities of all bacteria, ultimately leading to reduced GABA production. The GABA production in fermented sheep milk generally reached its maximum value of 0.517 g / L at an inoculum size of 0.010%. The sensory scores of fermented sheep milk initially increased and then decreased with increasing inoculum size; the highest sensory score of 72 was achieved at an inoculum size of 0.010%. Considering all indicators, an inoculum size of 0.010% was recommended for fermented sheep milk.

[0100] Depend on Figure 9 It can be seen that as the temperature increases, the rate of pH decrease in fermented milk increases significantly, the acidity of fermented milk increases significantly, and the fermentation time gradually shortens; when the temperature increases from 33℃ to 41℃, the fermentation time of sheep milk gradually decreases from 27h to 18h.

[0101] The water-holding capacity and γ-aminobutyric acid (GABA) yield of the fermented milk were measured at their respective fermentation endpoints. Sensory evaluation of the fermented milk was performed after fermentation was completed and the milk was refrigerated and ripened. The results are as follows: Figure 10 As shown. By Figure 10 It was observed that the water-holding capacity of fermented sheep milk initially increased and then decreased with rising temperature. This may be because higher temperatures lead to more vigorous metabolism of probiotics, resulting in enhanced curdling; however, excessively high temperatures can lead to insufficient curdling by probiotics. The water-holding capacity of sheep milk reached its maximum of 27.96% at 37℃. The GABA production in fermented sheep milk initially increased and then decreased with rising temperature. This may be because higher temperatures allow for faster growth of the bacteria within the same time frame, increasing GABA production. However, excessively high temperatures cause rapid acid production by the bacteria, reaching the fermentation endpoint in a shorter time, thus reducing the time available for GABA conversion. The GABA production in sheep milk reached its maximum of 0.54 g / L at 37℃. The sensory score of fermented sheep milk initially increased and then decreased with rising temperature; the sensory score reached its highest point of 73 at 37℃. Considering all indicators, the optimal fermentation temperature for sheep milk is 37℃.

[0102] The effects of inoculum size and fermentation temperature on different sheep milk fermentations have been studied above. Subsequently, sheep milk fermentation was conducted using the optimal inoculum size and fermentation temperature, and the effect of different fermentation times on probiotic powder fermentation of sheep milk was investigated. The results are as follows: Figure 11 As shown. By Figure 11 As shown in section A, the pH of fermented sheep milk gradually decreases with increasing fermentation time; and the rate of decrease slows down, possibly because as fermentation time increases, the reduction of nutrients and the accumulation of acid lead to a decrease in the rate of bacterial growth and metabolism. Conversely, the acidity of fermented sheep milk gradually increases with increasing fermentation time. Figure 11 As shown in Figure B, the water-holding capacity of fermented sheep milk initially increases and then stabilizes with increasing fermentation time. This may be because probiotics ferment fully in the sheep milk with increasing fermentation time. However, as fermentation exceeds the endpoint, over-fermentation leads to whey separation, affecting the quality of the fermented milk. The water-holding capacity of sheep milk reaches its maximum of 28.91% after 23 hours of fermentation. The GABA content of sheep milk increases with increasing fermentation time, reaching a maximum of 0.641 g / L at 25 hours. The sensory score of fermented sheep milk initially increases and then decreases with increasing fermentation time. Before 19 hours, fermentation is incomplete, and curdling is not yet complete. After 23 hours, over-fermentation results in an overly sour taste, leading to a decrease in the sensory score. The sensory score of fermented sheep milk reaches its highest point of 73 points at 21 hours of fermentation. Considering all indicators, the optimal fermentation time for sheep milk is 21 hours.

[0103] By combining the optimal conditions for inoculation amount, fermentation temperature, and fermentation time, the suitable fermentation conditions for fermented sheep milk were obtained: sheep milk inoculation amount 0.010%, fermentation time 21h, and fermentation temperature 37℃.

[0104] 3.3.4 Effects of Thickeners and Sweeteners on Probiotic Fermented Goat Milk

[0105] The water-holding capacity of fermented milk can be significantly improved by adding modified starch and pectin. Among them, pectin has the most significant effect on improving the water-holding capacity of sheep fermented milk. As the amount of pectin added increases, the water-holding capacity of sheep fermented milk gradually increases, and the water-holding capacity of sheep fermented milk reaches its maximum value of 59.46% when the amount of pectin added is 1.5%. The water-holding capacity of other thickeners generally shows a trend of first increasing and then decreasing with the increase of the amount added, see [see figure]. Figure 12 .

[0106] Taking into account both water-holding capacity and the sensory properties of goat milk, a thickener addition of 0.5% was selected for the fermentation test. Since high temperature (pasteurization) will gelatinize starch and increase the overall viscosity of the system, this invention adopts the method of adding thickener before sterilization, with water-holding capacity and sensory evaluation as evaluation indicators. The results are shown in Table 1.

[0107] Table 1 Effect of thickening agent on water holding capacity and sensory evaluation of probiotic fermented goat milk

[0108]

[0109] Single thickening agent has great limitations in improving curd properties, which may have poor curd effect and poor stability. Compound thickening agent can effectively avoid some problems and achieve synergistic thickening effect. Therefore, agar, hydroxypropyl distarch phosphate and modified tapioca starch were selected for subsequent compounding test, and the total addition amount of thickening agent was still 0.5%. The test results are shown in Table 2.

[0110] Table 2 Effect of thickening agent combination on water holding capacity and sensory evaluation of probiotic fermented goat milk

[0111]

[0112] From Table 2, the compounding scheme of the three thickening agents has good effect on fermented goat milk, so the modified tapioca starch + hydroxypropyl distarch phosphate + agar is selected as the most suitable scheme, and the addition amount is 1 / 3 of the best addition amount of each single fermentation, then the thickening agent addition scheme of goat milk is obtained: the ratio of modified tapioca starch, hydroxypropyl distarch phosphate and agar added in goat milk is 0.2%:0.2%:0.2%.

[0113] After sterilizing the three reconstituted milks, different doses of sweeteners were added before inoculating the bacteria powder: sucrose (sweetness coefficient 1), erythritol (sweetness coefficient 1), aspartame (sweetness coefficient 150), acesulfame (sweetness coefficient 200), neotame (sweetness coefficient 8000); the basic addition amount of each component is (sweetness coefficient addition amount 6%, 9%, 12%, 15%, 18%), and the results are shown in Table 3. Figure 13 The results show that the addition of sweeteners can significantly improve the sensory score of fermented milk. With the increase of the addition amount, the sensory score generally presents the trend of first increasing and then decreasing. For different addition amounts, the sensory score of sucrose is generally higher than that of other sweeteners. With the increase of the addition amount, the unnatural taste of sweeteners gradually appears, including bitterness and grassy odor, etc. At the same time, the acidity of goat milk is also affected. Therefore, it is judged that sweeteners may have an impact on probiotic fermented goat milk. Therefore, the effect of adding sweeteners before and after fermentation on the sensory evaluation of fermented goat milk is studied, and the sweetness coefficient addition amount is 9%, and the results are shown in Table 3.

[0114] Table 3 Effect of sweetener addition time on sensory evaluation of probiotic fermented goat milk

[0115] Sweetener Before fermentation of sheep milk After fermentation of sheep milk Sucrose (90 g / L) 78 81 Erythritol (90 g / L) 70 75 Acesulfame-K (0.45 g / L) 73 80 Neotame (0.01125 g / L) 71 78 Aspartame (0.6 g / L) 63 68

[0116] From Table 3, it can be seen that the sensory scores of sheep milk after fermentation with the addition of sterilized sweeteners are generally higher than those before fermentation, so the way of adding sweeteners after fermentation is adopted subsequently. For sheep milk, sucrose, neotame and erythritol all have good effects. Aspartame has insufficient sweetness, resulting in a lower sensory score; although the sensory score of fermented milk with added sucrose is high, sucrose can easily cause dental caries and other problems, so sugar substitutes with higher sweetness and lower calorie content are selected: erythritol, acesulfame and neotame.

[0117] Single sweeteners have problems such as weak sweetness or instability, for example, the sweetness of acesulfame is fast, and the aftertaste is insufficient, high-concentration single use will have a slight bitter aftertaste, aspartame has insufficient sweetness, and excessive sugar alcohol addition can cause diarrhea and other problems, so sweetener compounding experiments are carried out to achieve the goal of improving taste and enhancing flavor. The addition amount of sweetness coefficient is 9%, and the results are shown in Table 4.

[0118] Table 4 Effect of sweetener compounding on sensory evaluation of probiotic fermented sheep milk

[0119]

[0120]

[0121] As can be seen from Table 4, the compounding scheme of the three sweeteners has good effect on fermented sheep milk, so erythritol + acesulfame + neotame is selected as the most suitable scheme, and the addition amount is 1 / 3 of the best addition amount of each single fermentation, then the addition scheme of sweeteners for sheep milk is obtained: the ratio of erythritol, acesulfame and neotame added in sheep milk is 40:0.3:0.00625.

[0122] 3.3.5 Study on the refrigeration of probiotic fermented sheep milk

[0123] According to the method of 2.9, the functional probiotic fermented sheep milk prepared by the single factor test of 2.7 fermentation conditions is placed in the refrigerator at 4℃, and the results are shown in Figure 14 .

[0124] From Figure 14 A in the table, the pH of fermented milk changes rapidly from 0d to 7d of refrigeration, mainly due to the growth inertia of probiotics, which causes the bacteria to still maintain vigorous metabolic activity even in a 4℃ environment. The average decrease is 0.1 units per day in the first 3 days, and the speed gradually slows down after 7 days, with an average decrease of 0.1 units per week. The pH of sheep milk after 28 days of refrigeration is 3.97.

[0125] The acidity of fermented milk increased rapidly from 0 d to 7 d of refrigeration, with an average increase of 4 °T per day in the first 3 d, and an average increase of 3-5 °T per week after 7 d. The acidity of sheep milk was 112 °T after 28 d of refrigeration. Overall, the acidity of sheep milk changed greatly, which may be because there was a lot of nutrients left in the sheep milk after fermentation, making the metabolism of the bacteria thrive.

[0126] The water holding capacity of sheep milk increased during refrigeration, and the highest water holding capacity of sheep milk was 44.3% after 28 d of refrigeration. The water holding capacity of sheep milk was relatively high because the solid content of sheep milk was relatively high, which could better wrap water.

[0127] The GABA concentration in sheep milk decreased during refrigeration. It was considered that the activity of GAD was low at low temperature, which led to the inability of probiotics to convert and prepare GABA. The GABA concentration decreased rapidly in the first 7 d, which was speculated to be due to the metabolic activity of probiotics. With the consumption of nutrients in sheep milk, probiotics converted and utilized part of GABA, but as probiotics gradually stopped metabolic activity, the GABA reduction rate gradually flattened. The GABA concentration in sheep milk after 28 d of refrigeration was 0.588 g / L.

[0128] From Figure 14 It can be seen from Table 5 that during refrigeration, the sensory evaluation value of sheep milk first increased and then decreased, and the best taste appeared on the 7th day. The organization of sheep milk was not good at the end of fermentation, and there was a little odor. With the extension of refrigeration time, the curd became more complete and the odor disappeared, but with the increase of refrigeration time, the increase of acidity led to the decrease of taste. The taste of sheep milk after 28 d of refrigeration was still acceptable, and the sensory evaluation value was 63.

[0129] The viable count of fermented sheep milk first increased and then slowly decreased with the refrigeration time. The viable count of sheep milk after fermentation was about 1.9 × 10 9 CFU / mL, and the maximum viable count of sheep milk was reached after 1 d of refrigeration, which was caused by growth inertia. With the extension of time, the bacteria basically did not grow, and then a certain number of bacteria began to die. The viable count of fermented sheep milk decreased to 2.1 × 10 8 CFU / mL after 28 d of refrigeration, which decreased by an order of magnitude, but was still more than 10 6 CFU / mL. This result met the standard that the number of lactic acid bacteria in fermented milk should be greater than or equal to 1 × 10 6 CFU / mL according to the national standard (GB19302-2010).

[0130] From Figure 14As shown in Figure C, the hardness of sheep milk first increases and then decreases during refrigeration, reaching a maximum of 25.39g on day 1. After 28 days of refrigeration, the hardness of sheep milk is 15.87g.

[0131] Regarding consistency, the consistency of sheep milk first increased and then decreased during refrigeration, reaching a maximum of 257.27 gs on day 1. After 28 days of fermentation, the consistency of fermented sheep milk was 236.27 gs.

[0132] In terms of cohesiveness, the cohesiveness of sheep milk first increased and then decreased with the extension of refrigeration time, reaching a maximum of 13.51g at 1 day; after refrigeration for 28 days, the cohesiveness of sheep milk showed a decreasing trend compared with the fermentation endpoint.

[0133] Regarding viscosity index, the viscosity index of sheep milk increases from the end of fermentation to 1 day of refrigeration, and then decreases continuously during the subsequent storage process, with a maximum value of 69.58 gs. After 28 days of refrigeration, the viscosity index of sheep milk is relatively high, mainly due to the high solids content, which is 44.18 gs.

[0134] Because sheep milk has a high solids content, its texture only reflects the physical changes of sheep milk during refrigeration and cannot be used as the sole basis for evaluating the quality of sheep milk.

[0135] Following the method in 2.9, the functional probiotic fermented goat milk prepared in the "2.8 Screening of Thickeners and Sweeteners" experiment was refrigerated at 4°C. The results are shown in [Figure 2]. Figure 15 .

[0136] Depend on Figure 15 As can be seen from A, the addition of sweeteners and thickeners to fermented milk has little effect on the changes in pH and acidity. The pH and acidity changes of fermented milk before and after the addition are consistent during refrigeration. The pH of sheep milk after 28 days of refrigeration is 3.96 and the acidity is 109°T.

[0137] The addition of thickeners significantly increased the water-holding capacity of sheep milk, and no whey separation occurred after 28 days of refrigeration. After 28 days of refrigeration, the water-holding capacity of the sheep milk reached a maximum of 54.7%, an increase of 10.4% compared to before optimization.

[0138] Depend on Figure 15 As shown in section B, the viable bacterial count in fermented sheep milk was minimally affected by thickeners and sweeteners, with the viable bacterial count remaining at 2.1 × 10⁻⁶ after fermentation. 9 The maximum viable count in sheep milk was around CFU / mL, reaching its peak after 3 days of refrigeration. This is likely because sweeteners and thickeners provide some nutrients to the bacteria and encapsulate them, leading to a lower bacterial mortality rate. By day 28 of refrigeration, the viable count in sheep milk had decreased to 1.3 × 10⁻⁶. 8CFU / mL, still in line with the national standard requirements.

[0139] The addition of sweeteners significantly improved the sensory evaluation value of sheep milk, reaching the best taste after 1 day of refrigeration, i.e. after ripening, with the highest value being 5 points higher than before optimization. The sensory evaluation value of sheep milk after 28 days of refrigeration was 70 points.

[0140] From Figure 15 As can be seen from Table 6, the addition of sweeteners and thickeners has a significant effect on the texture of fermented sheep milk. During the refrigeration process, the hardness of sheep milk first increases and then decreases, with the maximum value of 31.39 g appearing on the 7th day. After 28 days of refrigeration, the hardness of sheep milk is 21.87 g, which is higher than before optimization. The consistency of sheep milk first increases and then decreases during the refrigeration process, with the maximum value of 294.87 g.s appearing on the 7th day. After 28 days of fermentation, the consistency of sheep milk is 256.88 g.s, which is higher than before optimization. In terms of cohesiveness, the cohesiveness of fermented sheep milk first increases and then decreases with the extension of refrigeration time, reaching the maximum value of 16.51 g on the 7th day. After 28 days of refrigeration, the cohesiveness of fermented sheep milk is 10.88 g, which is lower than at the end of fermentation. The viscosity index of sheep milk continuously decreases during the refrigeration process, with the maximum value of 78.58 g.s, which is higher than before optimization. After 28 days of refrigeration, the viscosity index of fermented sheep milk is 43.78 g.s.

[0141] The T2 inversion spectrum of the three fermented milks can be obtained using low-field nuclear magnetic resonance technology. The relaxation time represents the state of water, and the peak area represents the content of water. During the refrigeration process of fermented sheep milk, the bound water content first increases and then decreases, reaching the maximum value of 0.02 on the 7th day. After 28 days of refrigeration, the bound water content of fermented sheep milk is 0.01, which is lower than at the end of fermentation. The content of water not easy to flow first increases and then decreases, reaching the maximum value of 0.22 on the 7th day. After 28 days of refrigeration, the content of water not easy to flow of fermented sheep milk is 0.18, which is lower than at the end of fermentation. The content of free water first increases and then decreases, reaching the maximum value of 0.76 on the 7th day. After 28 days of refrigeration, the content of free water of fermented sheep milk is 0.71, which is lower than at the end of fermentation. Figure 16 As can be seen from the inversion spectrum, there are mainly three peaks. The shorter the transverse relaxation time T2, the tighter the combination with the material, i.e. T21 (0.02-0.2 ms) represents bound water, T22 (3-10 ms) represents water not easy to flow, and T23 (10-1000 ms) represents free water. The content of bound water is represented as A1, the content of water not easy to flow is represented as A2, the content of free water is represented as A3, and the total water content is represented as A2, then A2=A1+A2+A3. 21 , the content of water not easy to flow is represented as A 22 , the content of free water is represented as A 23 , and the total water content is represented as A2, then A2=A1+A2+A3. 21 +A 22 +A 23 .

[0142] From Table 5, it can be seen that the largest change in moisture content of the probiotic fermented sheep milk during cold storage is T23 free water, which significantly increases compared to the starting point. It is speculated that this is due to the gradual post-acidification of the fermented milk during cold storage, with the increase in the acidity of the fermented milk, part of the not easily flowing water is transferred to free water, resulting in a small amount of whey separation, and T22 not easily flowing water also changes to a certain extent during cold storage, and the T21 bound water remains basically unchanged.

[0143] Table 5 Inverse spectrum peak area A2 statistical table

[0144]

[0145] The above-described embodiments are only to describe the preferred modes of the present application, and not to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A functional probiotic sheep milk, characterized in that, The sheep milk comprises a compound probiotic consisting of *Limosilactobacillus fermentum* KD6, *Lacticaseibacillus rhamnosus* KD5, and *Lacticaseibacillus paracasei* KD11. The preservation number of *Limosilactobacillus fermentum* KD6 is CCTCC NO: M20231642, the preservation number of *Lacticaseibacillus rhamnosus* KD5 is CCTCC NO: M20231641, and the preservation number of *Lacticaseibacillus paracasei* KD11 is CCTCC NO: M2024605.

2. The functional probiotic sheep milk as described in claim 1, characterized in that, The mass ratio of the fermenting *Lactobacillus mucinus* KD6, the *Lactobacillus rhamnosus* KD5, and the *Lactobacillus paracasei* KD11 is 1:(1-3):(1-3).

3. A method for preparing functional probiotic sheep milk, characterized in that, The method includes the step of fermenting sheep milk using a compound probiotic, wherein the compound probiotic is composed of *Lactobacillus fermentum* KD6, *Lactobacillus rhamnosus* KD5, and *Lactobacillus paracasei* KD11. The preservation number of *Lactobacillus fermentum* KD6 is CCTCC NO: M20231642, the preservation number of *Lactobacillus rhamnosus* KD5 is CCTCC NO: M20231641, and the preservation number of *Lactobacillus paracasei* KD11 is CCTCC NO: M2024605.

4. The preparation method according to claim 3, characterized in that, The mass ratio of the fermenting *Lactobacillus mucinus* KD6, the *Lactobacillus rhamnosus* KD5, and the *Lactobacillus paracasei* KD11 is 1:(1-3):(1-3).

5. The preparation method according to claim 3, characterized in that, The preparation method includes the following steps: Mix sheep milk with water, sterilize, prepare sterilized reconstituted sheep milk, and cool. The compound probiotics were inoculated into the cooled, sterilized, and reconstituted sheep milk, fermented, and sweeteners and thickeners were added to obtain functional probiotic sheep milk.

6. The preparation method according to claim 5, characterized in that, The sterilized and reconstituted sheep milk contains 18% sheep milk by weight (w / v).

7. The preparation method according to claim 3, characterized in that, The fermentation conditions are as follows: inoculum size of 0.005%-0.025%, fermentation temperature of 33-41℃, and fermentation time of 17-25h.

8. The preparation method according to claim 7, characterized in that, The fermentation conditions were as follows: inoculum size of 0.010%, fermentation temperature of 37°C, and fermentation time of 21 hours.

9. The preparation method according to claim 5, characterized in that, The sweeteners include erythritol, acesulfame potassium, and neotame, wherein the mass ratio of erythritol, acesulfame potassium, and neotame is (30-45):(0.1-0.3):(0.003-0.007). The thickener comprises modified cassava starch, hydroxypropyl distarch phosphate, and agar, wherein the mass ratio of the modified cassava starch, hydroxypropyl distarch phosphate, and agar is 1:1:

1.

10. The use of the functional probiotic sheep milk as described in claim 1 or 2 in the preparation of sheep milk products that improve sleep quality.

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