Preparation method and application of camel milk with hypoglycemic activity

Abstract

The invention discloses a preparation method and application of camel milk with hypoglycemic activity, and belongs to the technical field of animal product processing. According to the research, lactobacillus bulgaricus, streptococcus thermophilus and kluyveromyces marxianus are used for carrying out microbial fermentation treatment on camel milk, and the optimal camel milk fermentation process is screened out by detecting various indexes such as texture characteristics, water holding stability, sensory evaluation, enzyme inhibition activity and protein hydrolysis degree; the fermented camel milk with potential hypoglycemic activity is prepared, the inhibitory activity of the camel milk to DPP-IV enzyme, alpha-amylase and alpha-glucosidase before and after digestion is detected in combination with an in-vitro simulated digestion experiment, and the prepared camel milk has high inhibitory activity to DPP-IV enzyme, alpha-amylase and alpha-glucosidase.

Description

Technical Field

[0001] This invention relates to a method for preparing camel milk with hypoglycemic activity and its application, belonging to the field of livestock product processing technology. Background Technology

[0002] Diabetes is one of the leading diseases affecting human health worldwide. Approximately one-quarter of the world's diabetes cases are in China, and the number of people with diabetes in my country is projected to reach 174 million by 2045. Furthermore, diabetes can lead to cardiovascular disease, kidney failure, and other problems, increasing patient mortality and the burden on social healthcare. Therefore, the prevention and treatment of diabetes has become a pressing global issue. DPP-IV enzyme inhibitors are among the main drugs used clinically to treat diabetes. Compared to other clinical drugs, they have relatively fewer side effects, but they can still cause gastrointestinal adverse reactions and skin problems in some individuals. Therefore, it is necessary to obtain DPP-IV enzyme inhibitors with good activity and low side effects from natural foods. Among various food proteins, milk protein is an important source of DPP-IV enzyme-inhibiting peptides.

[0003] Camel milk, secreted by Bactrian and dromedary camels, is known as "desert gold" due to its rich nutritional content. It has long been renowned for its medicinal value and has a long history of use in treating various diseases and in traditional medicine. Existing research has confirmed its effects on lowering blood sugar and blood pressure, as well as its antioxidant and anti-cancer properties, with its anti-diabetic effects being particularly well-established clinically. Milk-derived bioactive peptides are naturally occurring or hydrolyzed peptides with specific biological activities found in milk proteins and are also important components that contribute to the physiological functions of camel milk.

[0004] Existing research mainly utilizes methods such as chemical synthesis, hydrolysis (enzymatic hydrolysis, acid hydrolysis, and alkaline hydrolysis), and microbial fermentation to prepare bioactive peptides. Chemical synthesis yields peptides with accurate sequences and high purity, but may leave chemical residues, and the uncontrollable reaction direction during synthesis may produce toxic substances or impurities. Hydrolysis is a mature process, but it is costly, and acid and alkaline hydrolysis can damage bioactive peptides. Therefore, both of these methods have their drawbacks, and the resulting products are not directly edible. Microbial fermentation, however, offers significant advantages, producing peptides with a richer and more diverse range of activities. The complex enzyme systems produced by microorganisms during metabolism can enzymatically hydrolyze proteins, releasing specific high concentrations of bioactive peptides. Microorganisms also synthesize and secrete small peptides during their growth and metabolism, and products fermented using microorganisms have better flavor and texture. Therefore, this study utilizes microbial fermentation to prepare peptides with specific bioactivities, simultaneously obtaining fermented camel milk products with excellent flavor and texture.

[0005] Therefore, research into how to prepare fermented camel milk with potential hypoglycemic activity, and how to combine in vitro simulated digestion experiments to detect the inhibitory activity of camel milk on DPP-IV enzyme, α-amylase and α-glucosidase before and after digestion, and explore the antidigestive properties of bioactive peptides in fermented camel milk, will help to further explore the hypoglycemic effect of fermented camel milk and expand its application prospects as a diabetes alternative therapy. Summary of the Invention

[0006] This study utilized *Lactobacillus bulgaricus*, *Streptococcus thermophilus*, and *Kluyveromyces martensii* to ferment camel milk. By assessing textural properties, water-holding stability, sensory evaluation, enzyme inhibitory activity, and degree of protein hydrolysis, the optimal fermentation process was selected to produce fermented camel milk with potential hypoglycemic activity. Furthermore, in vitro simulated digestion experiments were conducted to detect the inhibitory activities of camel milk on DPP-IV enzymes, α-amylase, and α-glucosidase before and after digestion, exploring the anti-digestive properties of bioactive peptides in the fermented camel milk. This research contributes to further exploring the hypoglycemic effect of fermented camel milk and expanding its application prospects as a diabetes alternative therapy.

[0007] The first objective of this invention is to provide a method for preparing fermented camel milk, comprising the steps of: (1) Camel milk powder is mixed with water, dissolved, and refrigerated; then heated, homogenized, and sterilized to obtain reconstituted camel milk; (2) Inoculate Lactobacillus bulgaricus, Streptococcus thermophilus and Kluyveromyces martensii at a live count ratio of 0.5-50:0.5-50:0-1 into reconstituted camel milk, ferment at 35-45℃ for 8-16 h, ripen, and sterilize to obtain fermented camel milk; In step (2), the preservation number of Lactobacillus bulgaricus is CICC®6103; the preservation number of Streptococcus thermophilus is CICC®20380; and the preservation number of Kluyveromyces martensii is CGMCC 2.1977. Based on the volume of reconstituted camel milk, the total inoculum of Lactobacillus bulgaricus, Streptococcus thermophilus, and Kluyveromyces martensii was 10. 9 ~10 10 CFU / L.

[0008] In one embodiment, in step (1), camel milk powder and water are mixed at a mass ratio of 1~2:4~6; the homogenization is performed at 6500~8500 r / min for 15~25 min; and the sterilization is performed by heating at 90~100℃ for 8~15 min.

[0009] In one embodiment, the post-ripening in step (2) is refrigerated at 0~8℃ for 22~26 h; the sterilization is heated at 90~100℃ for 5~10 min.

[0010] In one embodiment, Lactobacillus bulgaricus, Streptococcus thermophilus, and Kluyveromyces martensii are inoculated into reconstituted camel milk at a live count ratio of 5-50:5-50:1.

[0011] In one embodiment, the camel milk powder was purchased from Wusu Gaoquan Tiantian Dairy Co., Ltd.

[0012] A second objective of this invention is to provide fermented camel milk prepared by any of the methods described above; A third objective of this invention is to provide a product containing the aforementioned fermented camel milk; the product includes pharmaceuticals and health products.

[0013] The fourth objective of this invention is to provide a method for improving the hypoglycemic activity of fermented camel milk, comprising the following steps: (1) Camel milk powder is mixed with water, dissolved, and refrigerated; then heated, homogenized, and sterilized to obtain reconstituted camel milk; (2) Inoculate Lactobacillus bulgaricus, Streptococcus thermophilus and Kluyveromyces martensii at a live count ratio of 0.5-50:0.5-50:0-1 into reconstituted camel milk, ferment at 35-45℃ for 8-16 h, ripen, and sterilize to obtain fermented camel milk; In step (2), the preservation number of Lactobacillus bulgaricus is CICC®6103; the preservation number of Streptococcus thermophilus is CICC®20380; and the preservation number of Kluyveromyces martensii is CGMCC 2.1977. Based on the volume of reconstituted camel milk, the total inoculum of Lactobacillus bulgaricus, Streptococcus thermophilus, and Kluyveromyces martensii was 10. 9 ~10 10 CFU / L.

[0014] In one embodiment, in step (1), camel milk powder and water are mixed at a mass ratio of 1~2:4~6; the homogenization is performed at 6500~8500 r / min for 15~25 min; and the sterilization is performed by heating at 90~100℃ for 8~15 min. The post-ripening in step (2) is refrigerated at 0~8℃ for 22~26 h; the sterilization is heated at 90~100℃ for 5~10 min.

[0015] In one embodiment, Lactobacillus bulgaricus, Streptococcus thermophilus, and Kluyveromyces martensii are inoculated into reconstituted camel milk at a live count ratio of 5-50:5-50:1.

[0016] In one embodiment, the camel milk powder was purchased from Wusu Gaoquan Tiantian Dairy Co., Ltd.

[0017] The fifth objective of this invention is to provide a method for simultaneously improving the water-holding capacity, hardness, consistency, and cohesive properties of fermented camel milk, comprising the following steps: (1) Camel milk powder is mixed with water, dissolved, and refrigerated; then heated, homogenized, and sterilized to obtain reconstituted camel milk; (2) Inoculate Lactobacillus bulgaricus, Streptococcus thermophilus and Kluyveromyces martensii at a live count ratio of 0.5-50:0.5-50:0-1 into reconstituted camel milk, ferment at 35-45℃ for 8-16 h, ripen, and sterilize to obtain fermented camel milk; In step (2), the preservation number of Lactobacillus bulgaricus is CICC®6103; the preservation number of Streptococcus thermophilus is CICC®20380; and the preservation number of Kluyveromyces martensii is CGMCC 2.1977. Based on the volume of reconstituted camel milk, the total inoculum of Lactobacillus bulgaricus, Streptococcus thermophilus, and Kluyveromyces martensii was 10. 9 ~10 10 CFU / L.

[0018] In one embodiment, in step (1), camel milk powder and water are mixed at a mass ratio of 1~2:4~6; the homogenization is performed at 6500~8500 r / min for 15~25 min; and the sterilization is performed by heating at 90~100℃ for 8~15 min. The post-ripening in step (2) is refrigerated at 0~8℃ for 22~26 h; the sterilization is heated at 90~100℃ for 5~10 min.

[0019] In one embodiment, Lactobacillus bulgaricus, Streptococcus thermophilus, and Kluyveromyces martensii are inoculated into reconstituted camel milk at a live count ratio of 5-50:5-50:1.

[0020] In one embodiment, the camel milk powder was purchased from Wusu Gaoquan Tiantian Dairy Co., Ltd.

[0021] The sixth objective of this invention is to provide the application of the above-mentioned fermented camel milk in health foods and pharmaceuticals.

[0022] Beneficial effects This invention utilizes *Lactobacillus bulgaricus*, *Streptococcus thermophilus*, and *Kluyveromyces martensii* to ferment camel milk. By testing various indicators such as textural properties, water-holding stability, sensory evaluation, enzyme inhibitory activity, and protein hydrolysis degree, the optimal camel milk fermentation process was screened to prepare fermented camel milk with potential activity. Specifically: (1) The fermented camel milk prepared by the present invention has stable texture, moderate hardness and viscosity, low fluidity, close to commercial yogurt products, and good water retention stability, reaching 57.07%.

[0023] (2) The fermented camel milk prepared by the present invention has strong inhibitory activity against DPP-IV enzyme, α-amylase and α-glucosidase. Specifically, the inhibition rates of DPP-IV enzyme, α-amylase and α-glucosidase of the active peptides of fermented camel milk are 65.02%, 33.44% and 34.47%, respectively. After in vitro digestion simulation, the inhibition rate of DPP-IV of fermented camel milk increased from 65.02% to 85.74%, the inhibition rate of α-amylase increased from 33.44% to 47.91%, and the inhibition rate of α-glucosidase increased from 34.47% to 66.24%. Attached Figure Description

[0024] Figure 1 The changes in pH and titratable acidity of fermented camel milk over time; where (a) and (c) represent the changes in pH and titratable acidity of fermented camel milk with an inoculum size of 10. 9 Changes in pH and titration acidity at CFU / L; (b) and (d) are respectively for inoculum amounts of 10 10 Changes in pH and titration acidity at CFU / L; Figure 2 It is in the form of fermented camel milk; Figure 3 Water-holding capacity for fermenting camel milk; Figure 4 Sensory radar image of fermented camel milk; Figure 5 The degree of protein hydrolysis in fermented camel milk; Figure 6 To investigate the DPP-IV enzyme inhibitory activity of fermented camel milk with different inoculation ratios; Figure 7 The enzyme inhibitory activity of fermented camel milk is denoted as (a) DPP-Ⅳ inhibition rate; (b) α-amylase inhibition rate; and (c) α-glucosidase inhibition rate.

[0025] In the figure, A is camel milk A prepared in Example 1; B is camel milk B prepared in Example 2; C is camel milk C prepared in Example 3; D is camel milk D prepared in Example 4; E is camel milk E prepared in Example 5; F is camel milk F prepared in Example 6; A1 is camel milk A1 prepared in Example 7; D1 is camel milk D1 prepared in Example 8; F1 is camel milk F1 prepared in Example 9; and T is unfermented camel milk prepared in Comparative Example 1. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, where specific conditions are not specified, are generally performed under conventional conditions in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art.

[0027] Raw material source: The whole milk powder was purchased from Wusu Gaoquan Tiantian Dairy Co., Ltd. Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus delbrueckii subsp. bulgaricus The accession number is CICC®6103; Streptococcus salivarius thermophilus subsp. Streptococcus thermophilus Accession number CICC®20380, China Industrial Microbial Culture Collection Center; Max Kluwer yeast Kluyveromyces marxianus The accession number is CGMCC 2.1977, China General Microbiological Culture Collection Center; MRS broth and M17 broth were purchased from Haibo Biotechnology Co., Ltd. YPD liquid culture medium was purchased from Beijing Solarbio Technology Co., Ltd. Agar powder, cobalt sulfate heptahydrate (CoSO4·7H2O), p-nitrobenzene-α-D-glucoside, and soluble starch were purchased from Shanghai Maclean Biochemical Technology Co., Ltd. DPP-IV inhibitor screening kit, α-amylase inhibitor screening kit, and α-glucosidase inhibitor screening kit were purchased from Wuhan Elite Biotechnology Co., Ltd. Disposable syringe filters, 0.45 μm, were purchased from Zhejiang Orsace Technology Co., Ltd. Trichloroacetic acid, sodium hydroxide, ethanol, concentrated sulfuric acid, and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0028] The measurement methods involved in the examples are as follows: 1. Determination of pH value and titratable acidity pH value was determined using a pHS-3TC high-precision acidity meter. Titrable acidity was determined according to GB 5009.239-2016 "National Food Safety Standard - Determination of Acidity in Food", using a 0.1 mol / L NaOH standard solution for titration.

[0029] 2. Ethanol content determination Ethanol content was determined using a GC-2030AF gas chromatograph. Chromatographic conditions: column, DB-23 (0.32 mm × 60 m × 0.25 μm); carrier gas flow rate, N2 - 30 mL / min, H2 - 40 mL / min, air - 400 mL / min; FID detector temperature 250℃; injector temperature 200℃; column temperature 180℃; injection volume 1 μL.

[0030] 3. Determination of textural properties The textural properties of fermented camel milk were evaluated using a TA-XT plus physical property analyzer with the following parameters: probe P25, 25 mm; distance into fermented camel milk 10 mm; trigger point load 10 g; deformation rate 60%; speed in pre-test 2 mm / s; speed in test 1 mm / s; return speed 1 mm / s.

[0031] 4. Water-holding stability test Measure 5 mL of fermented camel milk and weigh it. Centrifuge at 4℃ and 4000 r / min for 10 min. Discard the supernatant and weigh the precipitate. Calculate the water-holding capacity of the fermented camel milk using the following formula:

[0032] In the formula: m0 is the mass of the blank centrifuge tube (g); m1 is the total mass of fermented camel milk and centrifuge tube (g); m2 is the total mass of the precipitate after centrifugation and centrifuge tube (g).

[0033] 5. Sensory evaluation The sensory evaluation method for fermented camel milk was based on the requirements of RHB 104—2020 "Sensory Evaluation Rules for Fermented Milk", with slight modifications to Liao Yimo's scoring criteria. The evaluation panel consisted of 7 trained evaluators, and the detailed scoring rules for each sensory evaluation indicator are shown in Table 1.

[0034] Table 1

[0035] 6. Enzyme inhibitory activity assay Take an appropriate amount of fermented camel milk for pretreatment, and centrifuge at 4℃ and 8000 r / min for 15 min. Collect the supernatant, filter it through a 0.45 μm organic membrane, and store it at -20℃ for subsequent activity determination.

[0036] The enzyme inhibitory activity of the samples was determined using a DPP-IV inhibitor screening kit, an α-amylase inhibitor screening kit, and an α-glucosidase inhibitor screening kit.

[0037] 7. Determination of protein hydrolysis degree The degree of protein hydrolysis was determined using the trichloroacetic acid (TCA) method.

[0038] Accurately weigh 2.0 g of fermented camel milk, bring the volume to 25 mL with 5% (w / v) TCA, mix well, sonicate at room temperature for 20 min, and let stand for 2 h. Take the supernatant and centrifuge at 4℃ and 8000 r / min for 10 min to obtain the supernatant. Free amino nitrogen content was determined using an Agilent 1100 high-performance liquid chromatograph. The chromatographic conditions were as follows: Agilent Hypersil ODS column (5 μm, 4.00 × 250 mm); column temperature 40℃; mobile phase A was 27.6 mmol / L sodium acetate-triethylamine-tetrahydrofuran (v / v 500:0.11:2.5, pH 7.2); mobile phase B was 80.9 mmol / L sodium acetate-methanol-acetonitrile (v / v 1:2:2, pH 7.2); mobile phase flow rate 1.0 mL / min; gradient elution program: 0 min, 8% B → 17 min, 50% B → 20.1 min, 100% B → 24 min, 0% B; UV detector wavelength 338 nm (proline 262 nm).

[0039] The determination of total nitrogen content was performed according to Method I of GB 5009.5-2025 "National Food Safety Standard - Determination of Protein in Food", Kjeldahl method.

[0040] The formula for calculating the degree of protein hydrolysis is as follows:

[0041] 8. In vitro simulated gastrointestinal digestion method To prepare a simulated gastric juice (SGF) with a pepsin concentration of 2000 U / mL, pepsin was dissolved in a 0.15 mol / L NaCl solution at pH 2.0. To prepare a simulated intestinal juice (SIF) with a trypsin concentration of 100 U / mL, trypsin was dissolved in a mixed solution of 50 mmol / L KH₂PO₄, 20 mmol / L CaCl₂, and 0.15 mol / L NaCl (pH 6.5).

[0042] Take an appropriate amount of fermented camel milk, add 10% (w / v) TCA to a final concentration of 1% in the sample, and centrifuge at 4℃ and 12000 r / min for 10 min. Take the supernatant, filter it through a 0.45 μm organic membrane, and then centrifuge it in a 10 kDa ultrafiltration centrifuge tube at 4℃ and 8000 r / min for 15 min. Collect the filtrate. After vacuum freeze-drying, obtain the polypeptide component of fermented camel milk. Take an appropriate amount of freeze-dried sample, mix it with SGF at a ratio of 1:10 (w / v), and react at 37℃ for 2 h. After gastric digestion, adjust the pH of the solution to 6.5 with 1 mol / L NaOH to inactivate pepsin. Add an equal volume of SIF, and continue the reaction at 37℃ for 2 h.

[0043] Examples 1-9: Preparation of Fermented Camel Milk A method for preparing fermented camel milk, comprising the following steps: 1. Raw material preparation (1) Preparation of reconstituted camel milk: Camel milk powder and water were mixed at a mass ratio of 1:5, heated to 65°C to dissolve, and then hydrated overnight at 4°C for 12 h. The reconstituted camel milk was heated to 60°C and homogenized (7500 r / min, 18 min), and then sterilized by heating at 95°C for 10 min.

[0044] (2) Activation of strains: Lactobacillus bulgaricus (accession number CICC®6103), Streptococcus thermophilus (accession number CICC®20380), and Kluyveromyces martensii (accession number CGMCC 2.1977) were inoculated into liquid culture medium at an inoculation rate of 2% v / v and cultured for 2 generations to activate the strains.

[0045] The culture conditions for Lactobacillus bulgaricus were as follows: in MRS broth medium, at 37°C and 160 r / min, for 16 h.

[0046] The culture conditions for *Streptococcus thermophilus* were 37°C, 160 r / min, and constant temperature culture for 16 h in M17 broth medium.

[0047] The culture conditions for *Kluyveromyces martensii* were as follows: cultured in YPD medium at 28°C and 160 r / min for 16 h.

[0048] 2. Preparation of Fermented Camel Milk Activated Lactobacillus bulgaricus, Streptococcus thermophilus, and Kluyveromyces macrocephala were mixed in a 1:1:0 ratio and 10 9After mixing the total inoculum of CFU / L reconstituted camel milk, centrifuge at 6000 r / min for 15 min to precipitate the bacterial cells. Wash twice with 0.9% physiological saline and then inoculate into the reconstituted camel milk. Ferment at 40℃ for 12 h (pH≤4.6), then refrigerate at 4℃ for 24 h for post-ripening and heat at 95℃ for 5 min for secondary sterilization to obtain fermented camel milk, named Camel Milk A.

[0049] The difference between Examples 1-9 lies in the inoculation amount and ratio of Lactobacillus bulgaricus, Streptococcus thermophilus, and Kluyveromyces martensii. They are named camel milk B, camel milk C, camel milk D, camel milk E, camel milk F, camel milk A1, camel milk D1, and camel milk F1, as shown in Table 2. Table 2

[0050] Comparative Example 1: Unfermented Camel Milk The specific implementation method is the same as the example, except that no bacteria are added for fermentation, and the reconstituted camel milk in Example 1 is used directly; it is named unfermented camel milk T.

[0051] Example 10: Screening of Fermented Camel Milk Inoculum 1. Determination of pH value and titratable acidity of fermented camel milk During fermentation, the pH and titratable acidity of the fermented camel milk in Examples 1, 4, 6, and Examples 7-9 were measured every 1 hour. The results are as follows: Figure 1 As shown.

[0052] The results showed that with prolonged fermentation time, the pH value of fermented camel milk in all groups continuously decreased, while the titratable acidity value continuously increased. In the early stages of fermentation, the acid production rate of the camel milk system was slow, due to the low number and metabolic activity of the initial bacterial population. As fermentation continued, the bacterial strains gradually adapted to the environment and entered a rapid proliferation phase, with a significant increase in both bacterial population number and metabolic activity. During this stage, *Lactobacillus bulgaricus* and *Streptococcus thermophilus* rapidly grew and multiplied, increasing the efficiency of lactose decomposition into lactic acid. The accumulated lactic acid metabolite in the system accelerated the decrease in pH and the increase in titratable acidity.

[0053] The isoelectric point of casein, pH 4.6, was selected as the fermentation endpoint. Figure 1 It can be seen that the acidity value of each fermented camel milk exceeded 70°T at the end of fermentation, meeting the acidity requirements for fermented milk. Overall, the total inoculum size was 10. 10At a concentration of CFU / L, the pH decrease was more significant, leading to a faster fermentation endpoint (pH ≤ 4.6) and a higher titratable acidity. This indicates that increasing the inoculum size promotes bacterial reproduction and acid production rate, further shortening the fermentation time. Under the same inoculum size, a 5:5:1 inoculum ratio reached the fermentation endpoint fastest and had a higher titratable acidity, while a 1:1:0 inoculum ratio reached the endpoint slowest. This phenomenon suggests that a higher proportion of *Kluyveromyces martensii* in the inoculum results in a faster acid production and fermentation rate in the camel milk system. Since the lactic acid in the system is mainly produced by the metabolism of the two lactic acid bacteria, this phenomenon is attributed to the synergistic effect of the yeast and lactic acid bacteria. The metabolites of *Kluyveromyces martensii* promote the growth and reproduction of both lactic acid bacteria, thus further increasing the acid production and fermentation rates.

[0054] 2. Determination of alcohol content The alcohol content of fermented camel milk prepared in Examples 1, 4, 6 and Examples 7-9 was determined.

[0055] According to the national standard GB 19301-2010 "National Food Safety Standard for Raw Milk", fermented milk with added alcohol or alcoholic fermentation products (such as flavored fermented milk) must meet the food additive standards, and the alcohol content is usually limited to less than 0.5%. The alcohol in fermented camel milk is mainly produced by the metabolism of Kluyveromyces martensii. The cow milk prepared in Examples 1 and 7 did not contain added yeast, so no alcohol was produced during fermentation. The alcohol content of the fermented camel milk in the other groups is shown in Table 2.

[0056] Table 2. Alcohol content of fermented camel milk with different inoculation amounts

[0057] As can be seen from the table, 10 9 Fermented camel milk at CFU / L inoculum levels (Examples 4 and 6) had an alcohol content of less than 0.5%, meeting national standards. Meanwhile, 10... 10 The alcohol content of fermented camel milk at CFU / L inoculum levels (Examples 8 and 9) exceeded the national standard limit, with the former being significantly lower than the latter. However, at the same inoculum level, camel milk D (Example 4) and camel milk D1 (Example 8), with a higher proportion of *Kluyveromyces martensii*, had higher alcohol content than camel milk F (Example 6) and camel milk F1 (Example 9). These experimental results indicate that reducing the inoculum level and the proportion of yeast inoculated is beneficial for reducing the alcohol content of fermented camel milk. Therefore, a concentration of 10 CFU / L should be selected in this experiment. 9 The CFU / L vaccination dose meets national standards.

[0058] 3. Texture characteristics of fermented camel milk The appearance and textural properties of the fermented camel milk prepared in Examples 1, 4, 5, 6 and Examples 7-9 are as follows: Figure 2As shown in Table 3.

[0059] Table 3. Texture properties of fermented camel milk

[0060] Note: Data are in the form of "mean ± standard deviation". Different letters in the same column indicate significant differences (p < 0.05).

[0061] As shown in Table 3, the viscosity and viscosity index values ​​of the fermented camel milk in each group were similar. Only the camel milk D1 group (Example 8) exhibited the lowest viscosity, at -37.13 ± 28.02 g. This may be because the camel milk D1 group had a lower pH and higher titratable acidity during fermentation, leading to water release and whey separation. In addition, the large amount of CO2 gas produced during yeast fermentation with high inoculum size and high inoculum ratio may also have damaged the texture of the fermented camel milk.

[0062] As fluids, all fermented camel milks exhibited relatively low consistency, with group D (Example 4) showing the highest consistency at 28.76 ± 3.84 g. The differences in consistency among the groups were not particularly significant, but overall, 10 9 Fermented camel milk (groups A, D, E, and F) at CFU / L inoculum levels had a consistency value higher than 10. 10 Fermented camel milk at CFU / L inoculum levels (camel milk groups A1, D1, and F1) indicates that 10 9 The fermented camel milk in the CFU / L group exhibited lower fluidity and greater stability. Therefore, Examples 1, 4, 5, and 6 possessed more stable textural properties.

[0063] The overall textural properties of fermented camel milk differ from those of everyday fermented cow's milk products. This is because cow's milk has a higher solids content than camel milk, and the protein compositions of the two milk sources are fundamentally different. Cow's main proteins, β-lactoglobulin and α-lactalbumin, can form specific interactions on the micelle surface, while camel milk lacks β-lactoglobulin, and its α-lactalbumin can only bind weakly to casein micelles. Although camel milk has a higher total protein content, its casein is difficult to separate effectively. This is the main reason why the rheological properties of fermented camel milk products, such as hardness and viscosity, are significantly lower than those of fermented cow's milk products.

[0064] 4. Water-holding stability of fermented camel milk Water-holding capacity is an important indicator for evaluating the quality and stability of dairy products; yogurt with high water-holding capacity is less prone to whey separation. The water-holding capacity of the fermented camel milk prepared in Examples 1, 4, 5, 6, and Examples 7-9 is as follows: Figure 3As shown, the camel milk F1 group (Example 9) had the highest water-holding capacity (66.23%), significantly higher than the other groups (p<0.05), while the D1 group (Example 8) had the lowest water-holding capacity (52.76%), consistent with the cohesiveness results. Under the same inoculum size, the inoculum ratio of 50:50:1 (camel milk F and F1 groups) had the best water-holding capacity, while 5:5:1 (camel milk D and D1 groups) had the worst. This indicates that the addition of yeast can improve the water-holding capacity of fermented camel milk to some extent, but an excessive proportion of yeast will reduce the water-holding capacity again. Figure 3 It was found that, under the same inoculation ratio, there were no significant differences between the camel milk groups A and A1, and between groups D and D1 (p > 0.05). This indicates that water-holding capacity is mainly affected by the inoculation ratio, while the inoculation amount has a smaller impact on water-holding capacity. The results show that Examples 1, 5, 6, 7, and 8 exhibited good water-holding stability.

[0065] 5. Sensory evaluation Sensory evaluation was performed on the fermented camel milk prepared in Examples 1, 4, 6, and Examples 7-9. The results are shown in Table 4. Figure 4 As shown in the figure. The results showed that there was no significant difference in the total sensory evaluation scores of the fermented camel milk in each group (p>0.05). However, the total score of group A (Example 1) was the highest at 62.71±11.30. This may be because group A did not add Kluyveromyces martensii, and its texture and flavor were closest to traditionally fermented yogurt. In addition, the inoculation amount was appropriate, the fermented camel milk had a uniform texture, and it had a good taste and suitable acidity.

[0066] Figure 4 This study more intuitively illustrates the score distribution of each group of fermented camel milk in terms of aroma, taste, color, and texture. Groups D1 and F1 showed an advantage in aroma, but scored significantly lower than other groups in texture and color. This may be related to the role of *Kluyveromyces martensii*. Yeast metabolism produces alcohol, giving fermented camel milk a rich, wine-like aroma. However, yeast metabolites promote the growth and reproduction of lactic acid bacteria, resulting in a more acidic taste and varying degrees of whey separation and clumping, which damages the texture and color of the fermented camel milk. In contrast, an inoculum size of 10... 9 The camel milk samples (groups D and F, CFU / L) showed good texture and color, with no whey separation or stratification, a more delicate mouthfeel, and a pleasant fermented aroma thanks to the yeast. Therefore, 10 CFU / L was selected. 9 An inoculation rate of CFU / L can yield fermented camel milk with better sensory quality.

[0067] Table 4 Sensory evaluation scores of fermented camel milk

[0068] Note: Data are in the form of "mean ± standard deviation". Different letters in the same column indicate significant differences (p < 0.05).

[0069] In summary, 10 10 The fermented camel milk groups with CFU / L exhibited a faster acid production rate; however, the low pH and high acidity environment led to whey separation and clumping, severely damaging the textural properties and sensory quality of the fermented camel milk. Furthermore, the excessive yeast proliferation in the D1 and F1 groups resulted in high alcohol content, exceeding the national standard of 0.5%. 9 Although the fermented camel milk group with CFU / L has a slower fermentation rate, it has a compliant alcohol content, better textural properties, and good sensory quality. The fermented camel milk system has a higher viscosity and is therefore more stable.

[0070] Example 11 Determination of the inoculation ratio for fermented camel milk 1. Determination of alcohol content in fermented camel milk with different inoculation ratios The alcohol content of fermented camel milk prepared in Examples 2-6 was determined, and the results are shown in Table 5.

[0071] Table 5. Alcohol content of fermented camel milk with different inoculation ratios

[0072] 2. Degree of protein hydrolysis in fermented camel milk The degree of protein hydrolysis of fermented camel milk prepared in Examples 1-6 and Comparative Example 1 was determined, and the results are as follows: Figure 5 As shown in the figure. The results indicated that the degree of protein hydrolysis in all groups was significantly higher than that in unfermented camel milk (7.62%) (p<0.05), suggesting that fermentation helps release peptides from camel milk. The degree of hydrolysis in group A of camel milk without added yeast was significantly higher than that in the other groups, reaching 15.42%. Increasing the proportion of yeast in the system reduced the degree of protein hydrolysis, indicating that lactic acid bacteria play a more important role in hydrolyzing camel milk proteins and releasing peptides.

[0073] 3. DPP-IV inhibitory activity of fermented camel milk Fermented camel milk prepared in Examples 1-6 and Comparative Example 1 was used to determine its DPP-IV inhibitory activity. The results are as follows: Figure 6 As shown.

[0074] The results showed that after microbial fermentation, the DPP-IV inhibition rate of all groups was significantly higher than that of unfermented camel milk (23.96%) (p<0.05), which is related to the hydrolysis and release of bioactive peptides in the protein during microbial fermentation. The DPP-IV inhibition rate of the BF group with added yeast was 34.35% higher than that of the camel milk A group without yeast, and all groups except the B group showed significant differences (p<0.05). This indicates that the addition of yeast is beneficial to the generation of bioactive peptides, possibly because the metabolites of yeast promote the growth and reproduction of lactic acid bacteria. However, increasing the proportion of yeast in the system showed a trend of first increasing and then decreasing the DPP-IV inhibition rate. Among them, the DPP-IV inhibition rate of group E (25:25:1) reached the maximum value of 49.44%, which was twice that of unfermented camel milk. This indicates that an appropriate proportion of yeast is beneficial to the degradation of proteins by microorganisms, while an excessively high proportion of yeast has an adverse effect.

[0075] 4. Verification of enzyme inhibitory activity in simulated in vitro digestion and fermentation of camel milk Fermented camel milk prepared in Examples 4-6 was used to determine its inhibitory activity against DPP-IV enzyme, α-amylase, and α-glucosidase. The results are as follows: Figure 7 As shown in the figure. The results showed that both unfermented and fermented camel milk exhibited certain DPP-IV enzyme inhibitory activity. The DPP-IV enzyme inhibition rate of unfermented camel milk was 18.30%, while the DPP-IV enzyme inhibition rates of fermented camel milk D, E, and F were 57.72%, 65.02%, and 61.69%, respectively, which were significantly higher than those of unfermented camel milk (p < 0.05).

[0076] Depend on Figure 7 As shown in b and 7c, the inhibition rates of α-amylase and α-glucosidase in unfermented camel milk were close to zero, while the inhibition rates of α-amylase and α-glucosidase in fermented camel milk increased significantly (p < 0.05). The inhibition rates of α-amylase in fermented camel milk D, E, and F were 29.40%, 33.44%, and 30.56%, respectively, and the inhibition rates of α-glucosidase were 30.39%, 34.47%, and 32.12%, respectively. These experimental results indicate that microbial fermentation can, to some extent, increase the inhibition rates of DPP-IV enzyme, α-amylase, and α-glucosidase in camel milk, thereby enhancing its hypoglycemic effect. This is because microbial fermentation hydrolyzes potentially bioactive proteins in the camel milk system, releasing more bioactive peptides.

[0077] Figure 7 The study also revealed the effect of in vitro simulated gastrointestinal digestion on the inhibition rate of fermented camel milk enzymes. Figure 7It was found that after in vitro simulated digestion, the inhibition rates of both unfermented and fermented camel milk for the three enzymes were significantly increased (p < 0.05). After in vitro simulated digestion, the inhibition rate of DPP-IV in camel milk increased by 1.2 to 2 times. Specifically, the inhibition rate of DPP-IV in unfermented camel milk increased from 18.30% to 35.91%, while the inhibition rate of DPP-IV in group D increased from 57.72% to 78.50%, in group E from 65.02% to 85.74%, and in group F from 61.69% to 81.75%. The inhibition rates of α-amylase and α-glucosidase also showed similar results. After simulated digestion in vitro, the α-amylase inhibition rate of fermented camel milk increased from 29.40% to 42.55% in group D, from 33.44% to 47.91% in group E, and from 30.56% to 45.40% in group F. Similarly, the α-glucosidase inhibition rate increased from 30.39% to 61.23% in group D, from 34.47% to 66.24% in group E, and from 32.12% to 62.99% in group F. These results indicate that digestion further hydrolyzes fermented camel milk, generating more bioactive peptides.

[0078] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing fermented camel milk, characterized in that, Including the following steps: (1) Camel milk powder is mixed with water, dissolved, and refrigerated; then heated, homogenized, and sterilized to obtain reconstituted camel milk; (2) Inoculate Lactobacillus bulgaricus, Streptococcus thermophilus and Kluyveromyces martensii at a live count ratio of 0.5-50:0.5-50:0-1 into reconstituted camel milk, ferment at 35-45℃ for 8-16 h, ripen, and sterilize to obtain fermented camel milk; In step (2), the preservation number of Lactobacillus bulgaricus is CICC®6103; the preservation number of Streptococcus thermophilus is CICC®20380; and the preservation number of Kluyveromyces martensii is CGMCC 2.1977. Based on the volume of reconstituted camel milk, the total inoculum of Lactobacillus bulgaricus, Streptococcus thermophilus, and Kluyveromyces martensii was 10. 9 ~10 10 CFU / L.

2. The method according to claim 1, characterized in that, In step (1), camel milk powder and water are mixed at a mass ratio of 1~2:4~6; the homogenization is carried out at 6500~8500 r / min for 15~25 min; the sterilization is carried out at 90~100℃ for 8~15 min.

3. The method according to claim 1, characterized in that, The post-ripening in step (2) is refrigerated at 0~8℃ for 22~26 hours; the sterilization is heated at 90~100℃ for 5~10 minutes.

4. Fermented camel milk prepared by any of the methods described in claims 1 to 3.

5. A product characterized in that, The product contains the fermented camel milk as described in claim 4; the product includes pharmaceuticals and health products.

6. A method for improving the hypoglycemic activity of fermented camel milk, characterized in that, Including the following steps: (1) Camel milk powder is mixed with water, dissolved, and refrigerated; then heated, homogenized, and sterilized to obtain reconstituted camel milk; (2) Inoculate Lactobacillus bulgaricus, Streptococcus thermophilus and Kluyveromyces martensii at a live count ratio of 0.5-50:0.5-50:0-1 into reconstituted camel milk, ferment at 35-45℃ for 8-16 h, ripen, and sterilize to obtain fermented camel milk; In step (2), the preservation number of Lactobacillus bulgaricus is CICC®6103; the preservation number of Streptococcus thermophilus is CICC®20380; and the preservation number of Kluyveromyces martensii is CGMCC 2.1977. Based on the volume of reconstituted camel milk, the total inoculum of Lactobacillus bulgaricus, Streptococcus thermophilus, and Kluyveromyces martensii was 10. 9 ~10 10 CFU / L.

7. The method according to claim 6, characterized in that, In step (1), camel milk powder and water are mixed at a mass ratio of 1~2:4~6; the homogenization is carried out at 6500~8500 r / min for 15~25 min; the sterilization is carried out at 90~100℃ for 8~15 min. The post-ripening in step (2) is refrigerated at 0~8℃ for 22~26 h; the sterilization is heated at 90~100℃ for 5~10 min.

8. A method for simultaneously improving the water-holding capacity, hardness, consistency, and cohesive properties of fermented camel milk, characterized in that, Including the following steps: (1) Camel milk powder is mixed with water, dissolved, and refrigerated; then heated, homogenized, and sterilized to obtain reconstituted camel milk; (2) Inoculate Lactobacillus bulgaricus, Streptococcus thermophilus and Kluyveromyces martensii at a live count ratio of 0.5-50:0.5-50:0-1 into reconstituted camel milk, ferment at 35-45℃ for 8-16 h, ripen, and sterilize to obtain fermented camel milk; In step (2), the preservation number of Lactobacillus bulgaricus is CICC®6103; the preservation number of Streptococcus thermophilus is CICC®20380; and the preservation number of Kluyveromyces martensii is CGMCC 2.1977. Based on the volume of reconstituted camel milk, the total inoculum of Lactobacillus bulgaricus, Streptococcus thermophilus, and Kluyveromyces martensii was 10. 9 ~10 10 CFU / L.

9. The method according to claim 8, characterized in that, In step (1), camel milk powder and water are mixed at a mass ratio of 1~2:4~6; the homogenization is carried out at 6500~8500 r / min for 15~25 min; the sterilization is carried out at 90~100℃ for 8~15 min. The post-ripening in step (2) is refrigerated at 0~8℃ for 22~26 h; the sterilization is heated at 90~100℃ for 5~10 min.

10. The application of the fermented camel milk according to claim 4 in health foods and pharmaceuticals.

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