Combined fresh-keeping method for improving quality of raw milk and application of combined fresh-keeping method

Abstract

The invention belongs to the technical field of food science, and particularly relates to a combined fresh-keeping method for improving the quality of raw milk and application of the combined fresh-keeping method. The total number of bacterial colonies, coliforms and psychrophilic bacteria are reduced through the high-density carbon dioxide and 75 MPa dynamic high-pressure microjet treatment; furthermore, near-freezing-point storage is more beneficial to slowing down the growth of microorganisms in the raw goat milk and the raw camel milk, the synergistic treatment effect of the two technologies is superior to that of the single technology, and the effect on the raw goat milk and the raw camel milk is superior to that of the raw cow milk. The combined technology can effectively inhibit the growth of putrefying bacteria and delay the spoilage of the raw milk, can maintain the protein structure to the maximum extent, and has a remarkable industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of food science and technology, specifically relating to a combined preservation method for improving the quality of raw milk and its application. Background Technology

[0002] Goat milk and camel milk, as distinctive dairy products with highly differentiated advantages, have seen a continuous increase in demand in recent years for their application in infant formula, high-end dairy products, and health foods, thanks to their unique product attributes and market positioning. This has become an important direction for promoting the diversified development of the dairy industry. However, due to their inherent compositional characteristics, these two types of raw milk are highly susceptible to microbial contamination and biochemical changes during storage and transportation after milking. This leads to quality deterioration issues such as off-flavors, layering, and sedimentation, weakening their core market competitiveness and significantly shortening shelf life, severely hindering the industrial-scale promotion of these specialty milk products from production areas to end markets. Therefore, developing preservation technologies that can effectively kill microorganisms and ensure food safety while maximizing the preservation of the natural quality characteristics of raw goat milk and raw camel milk has become a key requirement for overcoming the bottlenecks in the development of the specialty dairy industry.

[0003] Currently, conventional heat sterilization technologies in the dairy industry (such as pasteurization and ultra-high temperature sterilization), while capable of inactivating most pathogenic and spoilage microorganisms through high temperatures and meeting basic safety standards, are particularly detrimental to the quality of goat milk and camel milk. For example, high temperatures cause the volatilization of natural milk fat flavor substances and the denaturation of whey proteins in goat milk, undermining its core advantages of easy digestibility and low allergenicity; at the same time, they also cause denaturation of camel milk proteins, with the secondary structure changing from ordered to disordered. Dynamic high-pressure microfluidics (DHPM), as a novel non-thermal physical sterilization technology, uses high pressure to drive fluid through microchannels to generate high-speed shearing, impact, and cavitation effects, which can destroy the microbial cell structure at lower temperatures, while also offering the advantages of high sterilization efficiency and better preservation of the natural color, aroma, flavor, and nutrients of food. However, single dynamic high-pressure microjet treatment has technical bottlenecks. If the treatment intensity is too low, its effect on killing pressure-resistant bacteria and spores in milk is limited, making it difficult to meet the safety requirements for long-term storage. If the treatment intensity is increased to improve the sterilization effect, it can easily lead to irreversible denaturation and aggregation of milk proteins, weakening the inherent processing adaptability and product quality of milk, thus limiting its application in high-end dairy products. In addition, high-density carbon dioxide (DPCD) is another highly promising non-thermal sterilization technology. It achieves sterilization by destroying the cell membrane structure and enzyme system activity of microorganisms through the molecular permeation of carbon dioxide in gaseous, liquid, or supercritical states. Combining lower-intensity dynamic high-pressure microjet with DPCD can overcome the limitations of single technologies through their synergistic effect. For example, the lower DHPM intensity significantly reduces the risk of milk protein denaturation, while DPCD itself has a relatively mild effect on milk protein structure. The synergy between the two can maximize the protection of the natural quality characteristics of specialty milks such as goat milk and camel milk while achieving efficient sterilization. In addition, storage temperature is another key factor affecting the shelf life of specialty milk products. Near-freezing storage can significantly inhibit the growth and reproduction rate of microorganisms and enzyme activity, delaying biochemical reactions such as fat oxidation in goat milk and protein hydrolysis in camel milk, thereby further reducing the rate of quality deterioration. Storing goat milk and camel milk that have undergone DHPM and DPCD co-treatment under near-freezing conditions can further synergistically inhibit the resurgence and growth of residual microorganisms after treatment, thus more effectively maintaining the freshness and food safety of specialty milk products, providing a guarantee for extending product shelf life and expanding cross-regional distribution. In summary, developing a preservation technology for specialty milks (goat milk and camel milk) based on synergistic sterilization using low-intensity DHPM and DPCD, supplemented by near-freezing storage, is an effective way to solve problems such as the degradation of specialty milk quality by traditional heat sterilization, insufficient sterilization effect of single non-thermal technologies, or damage to milk protein structure by high-intensity treatment. This technology is expected to maximize the preservation of the natural flavor, physicochemical stability, and processing compatibility of goat milk and camel milk while efficiently ensuring their microbial safety, providing key technical support for the industrial development and market promotion of high-quality goat milk and camel milk products. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a method for preserving newborn milk, and the technical solution is as follows: In a first aspect, the present invention provides a method for preserving raw milk, characterized by comprising the following steps: (1) Treat raw milk samples under high-density carbon dioxide (DPCD) conditions of 5-10 MPa for 5-15 min; (2) The raw milk sample after step (1) was treated with dynamic high pressure microjet (DHPM) at 60-180 MPa. (3) Store the raw milk sample processed in step (2) at a near-freezing temperature.

[0005] Furthermore, the DPCD process is performed at a pressure of 8 MPa for 10 minutes.

[0006] Furthermore, the low pressure of the DHPM is 75 MPa, while the high pressure is 150 MPa.

[0007] Furthermore, the storage temperature is -0.27℃ to 4℃.

[0008] Furthermore, the raw milk sample is one or more of raw cow's milk, raw camel's milk, or raw goat's milk.

[0009] Furthermore, the raw milk sample is one or both of raw sheep milk or raw camel milk.

[0010] The second aspect of the present invention provides the application of the method described in the first aspect in the preparation of liquid milk, fermented dairy products, functional dairy products, and health foods.

[0011] Furthermore, the raw milk sample is one or more of raw cow's milk, raw camel's milk, or raw goat's milk.

[0012] Furthermore, the raw milk sample is one or both of raw camel milk or raw goat milk.

[0013] Thirdly, the present invention provides a method for inhibiting the proliferation of microorganisms in raw milk samples, characterized in that the method comprises the following steps: (1) Treat raw milk samples under high-density carbon dioxide (DPCD) conditions of 5-10 MPa for 5-15 min; (2) The raw milk sample after step (1) was treated with dynamic high pressure microjet (DHPM) at 60-180 MPa. (3) Store the raw milk sample processed in step (2) at a near-freezing temperature.

[0014] Furthermore, the DPCD process is performed at a pressure of 8 MPa for 10 minutes.

[0015] Furthermore, the low pressure of the DHPM is 75 MPa, while the high pressure is 150 MPa.

[0016] Furthermore, the storage temperature is -0.27℃ to 4℃.

[0017] Furthermore, the raw milk sample is one or more of raw cow's milk, raw camel's milk, or raw goat's milk.

[0018] Furthermore, the raw milk sample is one or both of raw sheep milk or raw camel milk.

[0019] The beneficial effects of this invention include: (1) The present invention combines DPCD with lower pressure DHPM, which is more effective than single treatment. It can be seen that, in terms of inhibiting microbial growth, this method has a better inhibitory effect on total bacterial count and coliforms in sheep milk and camel milk than cow milk, indicating that this method is more suitable for the preservation of sheep milk and camel milk.

[0020] (2) The present invention combines DPCD with lower pressure DHPM. Compared with DHPM treatment under high pressure, it can better preserve the secondary structure of milk-like proteins and maintain the stability of milk storage.

[0021] (3) When the processed sheep milk and camel milk are stored near freezing point, the pH, acidity and microbial changes are better improved than those of samples stored at 4°C, indicating that optimizing the storage temperature can further improve their quality characteristics. Attached Figure Description

[0022] Figure 1 Effects of DPCD (8 MPa), DHPM (75 MPa), and DPCD combined with DHPM treatment on the physicochemical properties of raw milk stored at 4℃. A: pH; B: titratable acidity.

[0023] Figure 2 Effects of DPCD (8 MPa), DHPM (75 MPa), and DPCD combined with DHPM treatment on the physicochemical properties of raw sheep milk stored at 4℃. A: pH; B: titratable acidity.

[0024] Figure 3 Effects of DPCD (8 MPa), DHPM (75 MPa), and DPCD combined with DHPM treatment on the physicochemical properties of raw camel milk during storage at 4℃. A: pH; B: titratable acidity.

[0025] Figure 4Effects of DPCD (8 MPa), DHPM (75 MPa), and DPCD combined with DHPM treatment on the microbial characteristics of raw milk stored at 4°C. A: Total bacterial count; B: Coliforms; C: Psychrophilic bacteria.

[0026] Figure 5 Effects of DPCD (8 MPa), DHPM (75 MPa), and DPCD combined with DHPM treatment on the microbial characteristics of raw sheep milk stored at 4℃. A: Total bacterial count; B: Coliforms; C: Psychrophilic bacteria.

[0027] Figure 6 Effects of DPCD (8 MPa), DHPM (75 MPa), and DPCD combined with DHPM treatment on the microbial characteristics of raw camel milk stored at 4℃. A: Total bacterial count; B: Coliforms; C: Psychrophilic bacteria.

[0028] Figure 7 Effects of DPCD (8 MPa) combined with DHPM (75 MPa) treatment on the physicochemical properties of raw cow milk, raw goat milk, and raw camel milk during storage at 4℃. A: pH; B: titratable acidity.

[0029] Figure 8 Effects of DPCD (8 MPa) combined with DHPM (75 MPa) treatment on the microbial characteristics of raw cow's milk, raw goat's milk, and raw camel's milk during storage at 4°C. A: Total bacterial count; B: Coliforms; C: Psychrophilic bacteria.

[0030] Figure 9 Effects of DPCD (8 MPa) combined with DHPM (75 MPa) treatment on the physicochemical properties of raw sheep milk and raw camel milk during near-freezing storage. A: pH; B: titratable acidity.

[0031] Figure 10 Effects of DPCD (8 MPa) combined with DHPM (75 MPa) treatment on the microbial characteristics of raw sheep milk and raw camel milk during near-freezing storage. A: Total bacterial count; B: Coliforms; C: Psychrophilic bacteria.

[0032] Figure 11 Effects of DPCD (8 MPa) combined with DHPM (75 MPa) treatment on the proportion of protein secondary structures in raw sheep milk and raw camel milk.

[0033] Figure 12 Effects of different treatments adjusting DPCD (6, 8, and 10 MPa) on the physicochemical properties of raw sheep milk and raw camel milk during storage at 4°C. A: pH; B: titratable acidity.

[0034] Figure 13Effects of different treatments adjusting DPCD (6, 8, and 10 MPa) on the microbial characteristics of raw sheep milk and raw camel milk during storage at 4°C. A: Total bacterial count; B: Coliforms; C: Psychrophilic bacteria.

[0035] Figure 14 Effects of different treatment conditions (DHPM, 32.5, 75, and 150 MPa) on the physicochemical properties of raw sheep milk and raw camel milk at 4°C. A: pH; B: titratable acidity.

[0036] Figure 15 Effects of different treatment conditions (DHPM, 32.5, 75, and 150 MPa) on the microbial characteristics of raw sheep milk and raw camel milk during storage at 4°C. A: Total bacterial count; B: Coliforms; C: Psychrophilic bacteria.

[0037] Figure 16 Effects of different treatment conditions (DHPM, 32.5, 75 and 150 MPa) on the proportion of protein secondary structure during storage at 4℃ in raw sheep milk and raw camel milk. Detailed Implementation

[0038] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0039] Example 1: pH adjustment of raw milk samples treated with a combination of DPCD8 and DHPM75 and frozen storage. Raw cow's milk, raw goat's milk, and raw camel's milk samples were placed into multiple 50 mL sterile centrifuge tubes, sealed with sealing film, and placed in two different batches into a high-density CO2 sterilizer. The samples were processed at 8 MPa for 10 min. For microfluidic processing, the samples were processed at 75 MPa. After processing, all samples were refrigerated at 4°C. Samples were taken every 5 days from day 0 until day 10.

[0040] After the milk samples were brought to room temperature, the pH value was measured using the three-point calibration method of the pH meter, according to GB 5009.237-2016 "National Food Safety Standard - Determination of pH Value in Food". Each sample was measured three times, and the readings were recorded. After the milk samples were brought to room temperature, the titration acidity was determined according to GB 5009.239-2016 "National Food Safety Standard - Determination of Acidity in Food". For 10 mL of milk sample, the amount of sodium hydroxide standard titration solution consumed was recorded, and the titration acidity of the milk was calculated in milliliters.

[0041] like Figure 1 As shown in Figures A and B, the pH values ​​of the control group and the 75 MPa high-pressure microjet (DHPM75) treatment group were relatively stable and relatively high. The pH value of the high-density carbon dioxide (DPCD8) treatment group was significantly lower than that of the first two groups. The pH change of the DPCD8 and DHPM75 combined treatment group was between that of the DPCD8 group, the control group, and the DHPM75 group, combining the effects of both. With the extension of storage time, the titratable acidity of raw milk in each group changed. The titratable acidity of the control group and the DHPM75 treatment group increased relatively slowly. The titratable acidity of the DPCD8 group and the DPCD9 and DHPM75 combined treatment group was significantly higher than that of the first two groups and remained at a high level during storage.

[0042] like Figure 2 As shown in Figures A and 2B, the pH of raw goat milk under different treatments exhibited different change characteristics with prolonged storage at 4℃. The pH of the control group and the 75 MPa and DHPM75 treatment groups remained relatively stable and relatively high. The pH of the DPCD8 treatment group was significantly lower than the other two groups. The pH change of the DPCD8+DHPM75 treatment group was between that of the DPCD8 group, the control group, and the DHPM75 group, combining the effects of both. The titratable acidity of raw goat milk in all groups changed with prolonged storage. The titratable acidity of the control group and the DHPM75 treatment group increased relatively slowly. The titratable acidity of the DPCD8 group and the combined DPCD8 and DHPM75 treatment group was significantly higher than the other two groups and remained at a high level during storage.

[0043] like Figure 3 As shown in Figures A and 3B, the pH of raw camel milk under different treatments exhibited different characteristics with prolonged storage at 4℃. The pH of the control group and the 75 MPa and DHPM75 treatment groups remained relatively stable and relatively high. The pH of the DPCD8 treatment group was significantly lower than the other two groups. The pH change of the DPCD8+DHPM75 treatment group was between that of the DPCD8 group, the control group, and the DHPM75 group, combining the effects of both. With prolonged storage, the titratable acidity of raw camel milk in each group changed: the titratable acidity of the control group and the DHPM75 treatment group increased relatively slowly; the titratable acidity of the DPCD8 group and the combined DPCD8 and DHPM75 treatment group was significantly higher than that of the other two groups and remained at a high level during storage.

[0044] Example 2: Microbial treatment of raw milk samples by combined DPCD8 and DHPM75 treatment and freezing point storage. The total bacterial count, Escherichia coli, and psychrophilic bacteria in raw cow's milk, raw goat's milk, and raw camel's milk stored at 4℃ were tested according to three standards: GB 4789.2-2022 "National Food Safety Standard - Microbiological Examination of Food: Determination of Total Colony Count", GB 4789.3-2016 "National Food Safety Standard - Microbiological Examination of Food: Coliform Count Plates", and NY / T1331-2007 "Determination of Psychrophilic Bacteria, Aerobic Spores and Thermophilic Aerobic Spores in Milk and Dairy Products".

[0045] like Figure 4 As shown in A, B, and C, the total bacterial count in raw milk generally increased with prolonged storage at 4℃, but the rate of increase varied significantly among different treatment groups. During storage, the control group consistently had the highest total bacterial count, while the DPCD8 and DHPM75 treatment groups had significantly lower counts. The DPCD8+DHPM75 group had the lowest total bacterial count, indicating that the combined treatment was more effective at inhibiting the total bacterial count than a single treatment, and could more effectively delay the proliferation of microorganisms in raw milk. During storage at 4℃, the number of coliform bacteria in raw milk gradually increased over time. At each storage time point, the number of coliform bacteria in the control group was significantly higher than in other treatment groups. The number of coliform bacteria in the DPCD8 and DHPM75 groups was lower than in the control group but higher than in the combined treatment group. The combined treatment group consistently had the lowest number of coliform bacteria, indicating that the combined treatment had a more prominent inhibitory effect on coliform bacteria and could better control the proliferation of coliform bacteria in raw milk. With prolonged storage at 4℃, the number of psychrophilic bacteria in raw milk showed a gradual increasing trend. During storage, the number of psychrophilic bacteria in the control group was consistently the highest, while the number of psychrophilic bacteria in the DPCD8 and DHPM75 groups was significantly lower than that in the control group. The number of psychrophilic bacteria in the DPCD8+DHPM75 group was the lowest, indicating that the combined treatment had the best inhibitory effect on psychrophilic bacteria and could more effectively delay the growth of psychrophilic bacteria in raw milk.

[0046] To further demonstrate the synergistic effect of the two technologies, the reduction in total bacterial count, coliform bacteria, and psychrophilic bacteria after treating raw milk with DPCD8 and DHPM75 alone was calculated, summed, and compared with the reduction in bacterial count after synergistic treatment of the two technologies, further proving the synergistic effect. As shown in Tables 1, 2, and 3, compared with the control group, the sum of the reductions in total bacterial count, coliform bacteria, and psychrophilic bacteria after treating the two technologies alone was less than the reduction in the synergistic treatment, indicating that the synergistic treatment resulted in a more significant reduction in microorganisms and had better sterilization efficiency.

[0047] Table 1. Effects of treatments on the reduction of total bacterial count in raw milk during storage.

[0048] Table 2. Effects of treatments on the reduction of coliform bacteria during raw milk storage.

[0049] Table 3. Effects of treatments on the reduction of psychrophilic bacteria during raw milk storage.

[0050] like Figure 5 As shown in Figures A, B, and C, the total bacterial count in raw goat milk generally increased with prolonged storage at 4℃, but the rate of increase varied significantly among different treatment groups. During storage, the control group consistently had the highest total bacterial count, while the DPCD8 and DHPM75 treatment groups had significantly lower total bacterial counts than the control group. The DPCD8+DHPM75 combined treatment group had the lowest total bacterial count, indicating that the combined treatment was more effective at inhibiting the total bacterial count than a single treatment, and could more effectively delay the proliferation of microorganisms in raw goat milk. During storage at 4℃, the number of coliform bacteria in raw goat milk gradually increased with time. At each storage time point, the number of coliform bacteria in the control group was significantly higher than in other treatment groups. The number of coliform bacteria in the DPCD8 and DHPM75 groups was lower than in the control group but higher than in the combined treatment group; the combined treatment group consistently had the lowest number of coliform bacteria, indicating that the combined treatment had a more prominent inhibitory effect on coliform bacteria and could better control the proliferation of coliform bacteria in raw goat milk. With prolonged storage at 4℃, the number of psychrophilic bacteria in raw goat milk showed a gradual increasing trend. During storage, the number of psychrophilic bacteria in the control group was consistently the highest; the number of psychrophilic bacteria in the DPCD8 group and DHPM75 group was significantly lower than that in the control group, and the number of psychrophilic bacteria in the combined treatment group was the lowest, indicating that the combined treatment had the best inhibitory effect on psychrophilic bacteria and could more effectively delay the growth of psychrophilic bacteria in raw goat milk.

[0051] To further demonstrate the synergistic effect of the two technologies, the reduction in total bacterial count, coliform bacteria, and psychrophilic bacteria after treating raw sheep milk with DPCD8 and DHPM75 alone was calculated, summed, and compared with the reduction in bacterial count after synergistic treatment of the two technologies, further proving the synergistic effect. As shown in Tables 4, 5, and 6, compared with the control group, the sum of the reductions in total bacterial count, coliform bacteria, and psychrophilic bacteria after treating the two technologies alone was less than the reduction in the synergistic treatment, indicating that the synergistic treatment resulted in a more significant reduction in microorganisms and had better bactericidal efficiency.

[0052] Table 4. Effects of treatments on the reduction of total bacterial count in raw goat milk during storage.

[0053] Table 5. Effects of treatments on the reduction of coliform bacteria in raw sheep milk during storage.

[0054] Table 6. Effects of treatments on the reduction of psychrophilic bacteria during the storage period of raw sheep milk.

[0055] like Figure 6 As shown in A, B, and C, the total bacterial count of raw camel milk generally increased with prolonged storage at 4℃, but the rate of increase varied significantly among different treatment groups. During storage, the control group consistently had the highest total bacterial count; the DPCD8 and DHPM75 treatment groups had significantly lower total bacterial counts than the control group. The DPCD8+DHPM75 combined treatment group had the lowest total bacterial count, indicating that the combined treatment was more effective at inhibiting the total bacterial count than a single treatment, and could more effectively delay the proliferation of microorganisms in raw camel milk. During storage at 4℃, the number of coliform bacteria in raw camel milk gradually increased over time. At each storage time point, the number of coliform bacteria in the control group was significantly higher than in other treatment groups. The number of coliform bacteria in the DPCD8 and DHPM75 groups was lower than the control group but higher than the combined treatment group; the combined treatment group consistently had the lowest number of coliform bacteria, indicating that the combined treatment had a more prominent inhibitory effect on coliform bacteria and could better control the proliferation of coliform bacteria in raw camel milk. With prolonged storage at 4℃, the number of psychrophilic bacteria in raw goat milk gradually increased. During storage, the number of psychrophilic bacteria in the control group was consistently the highest; the number of psychrophilic bacteria in the DPCD8 group and DHPM75 group was significantly lower than that in the control group, and the number of psychrophilic bacteria in the combined treatment group was the lowest, indicating that the combined treatment had the best inhibitory effect on psychrophilic bacteria and could more effectively delay the growth of psychrophilic bacteria in raw camel milk.

[0056] To further demonstrate the synergistic effect of the two technologies, the reduction in total bacterial count, coliform bacteria, and psychrophilic bacteria after treating raw camel milk with DPCD8 and DHPM75 alone was calculated, summed, and compared with the reduction in bacterial count after synergistic treatment of the two technologies, further proving the synergistic effect. As shown in Tables 7, 8, and 9, compared with the control group, the sum of the reductions in total bacterial count, coliform bacteria, and psychrophilic bacteria after treating the two technologies alone was less than the reduction in the synergistic treatment, indicating that the synergistic treatment resulted in a more significant reduction in microorganisms and had better bactericidal efficiency.

[0057] Table 7. Effects of treatments on the reduction of total bacterial count during the storage period of raw camel milk.

[0058] Table 8. Effects of treatments on the reduction of coliform bacteria during the storage period of raw camel milk.

[0059] Table 9. Effects of treatments on the reduction of psychrophilic bacteria during the storage period of raw camel milk.

[0060] In summary, when testing the total bacterial count, coliform count, and number of psychrophilic bacteria in raw cow's milk, raw goat's milk, and raw camel's milk stored at 4℃, the DPCD8 and DHPM75 synergistic treatment group consistently showed the lowest levels of these microbial indicators. Its inhibitory effect on various microorganisms was significantly better than that of DPCD8 treatment alone, DHPM75 treatment alone, and the control group, demonstrating a synergistic effect.

[0061] Previous studies have confirmed that single treatments of DPCD and DHPM improve milk quality, and that combined treatments are more effective than single treatments. Therefore, this study further investigates the differences in the effects of 8 MPa DPCD treatment combined with 150 MPa DHPM treatment on the storage characteristics of cow's milk, goat's milk, and camel's milk. Figure 7 As shown in Figures A and B, the pH of cow's milk, goat's milk, and camel's milk all decreased significantly after this combined treatment. This is because a large amount of carbon dioxide entered the milk, forming carbonic acid and producing H₂O. + This process significantly lowers the pH of the milk, with camel milk exhibiting the lowest pH. During the later stages of storage, as CO2 escapes, the pH of all three types of milk rises again. Titration acidity analysis shows that camel milk has a higher titration acidity compared to cow milk. In the early stages of storage, the initial titration acidity of cow milk, goat milk, and camel milk treated with DPCD was significantly higher than that of the untreated milk samples. As the storage period lengthens, the titration acidity of the samples treated with this combined treatment gradually decreases due to CO2 escape during storage, with camel milk showing a faster rate of decrease.

[0062] like Figure 8 As shown in Figures A, B, and C, throughout the storage process, the total bacterial count and coliform count in cow's milk, goat's milk, and camel's milk treated with the combined DPCD and DHPM were significantly lower than their respective untreated control groups. Further observation revealed that, compared to untreated raw milk, this combined treatment significantly inhibited the total bacterial count and coliform count in goat's milk and camel's milk more effectively throughout the storage process. This indicates that this combined treatment method is more advantageous for preserving goat's milk and camel's milk, with a better effect in camel's milk. This may be related to the smaller fat globules in camel's milk; smaller fat globules reduce CO2 diffusion resistance and increase its diffusion efficiency in the system, thus producing a better inhibitory effect on microorganisms. Furthermore, regarding the changes in the number of psychrophilic bacteria, the DPCD combined with DHPM treatment also had a certain inhibitory effect on psychrophilic bacteria, while the difference in inhibitory effect between goat's milk and camel's milk was not significant.

[0063] Example 3: Microbial proliferation after treatment of raw goat milk and raw camel milk samples Raw goat milk and raw camel milk samples were placed into multiple 50 mL sterile centrifuge tubes, sealed with sealing film, and placed in two different batches in a high-density CO2 sterilizer. The samples were processed at 8 MPa for 10 min. For microfluidic processing, the samples were processed at 75 MPa. After processing, all samples were refrigerated at near-freezing temperature (-0.27℃). Samples were taken every 5 days from day 0 to day 10.

[0064] After raw goat milk and camel milk were brought to room temperature, the pH value of the camel milk samples was determined using the three-point calibration method of a pH meter, according to GB 5009.237-2016 "National Food Safety Standard - Determination of pH Value in Food". Each sample was measured three times, and the readings were recorded. After the camel milk was brought to room temperature, the titration acidity was determined according to GB 5009.239-2016 "National Food Safety Standard - Determination of Acidity in Food". For 10 mL of sample milk, the amount of sodium hydroxide standard titration solution consumed was recorded, and the titration acidity of the milk was calculated in milliliters.

[0065] like Figure 9 As shown in Figure A, after treatment with 8 MPa DPCD combined with 75 MPa DHPM, the pH of both sheep milk and camel milk showed a trend of first decreasing and then rising again in the later stage of storage as CO2 escaped, when stored near freezing point and at 4°C. Moreover, under near freezing point storage, the pH change of sheep milk and camel milk was more gradual, and the pH stability was better maintained compared to storage at 4°C.

[0066] like Figure 9 As shown in Figure B, in terms of titratable acidity, the initial titratable acidity of the combined-treated sheep milk and camel milk stored at near-freezing point and 4℃ was relatively similar in the early stage of storage. However, as the storage period lengthened, the titratable acidity of sheep milk and camel milk stored at near-freezing point decreased at a significantly slower rate than that of the samples stored at 4℃, indicating that near-freezing point storage is more conducive to delaying the decrease in titratable acidity of sheep milk and camel milk.

[0067] Example 4: Microbial proliferation of raw cow's milk and raw camel's milk after treatment The total bacterial count, Escherichia coli, and psychrophilic bacteria in raw cow's milk and raw camel's milk stored near freezing point were tested according to three standards: GB 4789.2-2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count", GB 4789.3-2016 "National Food Safety Standard - Microbiological Examination of Food - Coliform Count Plates", and NY / T1331-2007 "Determination of Psychrophilic Bacteria, Aerobic Spores and Thermoaerobic Spores in Milk and Dairy Products".

[0068] like Figure 10As shown in A, B, and C, in terms of microbial characteristics, the growth rate of microorganisms (total colony count, coliforms, and psychrophilic bacteria) in both the untreated control group and the combined-treated goat milk and camel milk stored near freezing point was significantly lower than that in the samples stored at 4℃. During storage, the number of microorganisms in goat milk and camel milk stored near freezing point was consistently lower than that in samples stored at 4℃, indicating that near freezing point storage can more effectively inhibit the proliferation of microorganisms in goat milk and camel milk.

[0069] Example 5: Effects of DPCD8 synergistic with DHPM75 treatment on the secondary structure of milk proteins The combined-processed cow, sheep, and camel milk samples were freeze-dried and preserved. Subsequently, the freeze-dried samples were thoroughly mixed with potassium bromide at a ratio of 1:100, ground, and pressed into tablets. Fourier transform infrared spectroscopy was used in the scanning range of 4000–4000 cm⁻¹. -1 8 cm resolution -1 Three parallel scans were performed under the specified conditions. PeakFit Version 4.12 software was used to smooth and normalize the bands, focusing on the amide I band (1700–1600 cm⁻¹). -1 Baseline correction and deconvolution processing were performed sequentially within the specified range. Gaussian curve fitting was then performed on the spectral bands using the second derivative. Finally, the relative abundance of the four protein secondary structures—α-helix, β-sheet, β-turn, and random coil—was calculated based on the peak area. Figure 11 As shown, after treatment with DPCD8 in combination with DHPM75, the relative contents of the secondary structures of the four proteins in cow's milk, sheep's milk, and camel's milk did not change significantly, indicating that the combined treatment did not have a significant impact on the secondary structure of proteins in cow's milk, sheep's milk, and camel's milk, and could maintain the stability of the protein structure in the three types of milk well.

[0070] Comparative Example 1 Fresh raw sheep milk and raw camel milk were used as raw materials, and three treatment schemes were set up: control group (no DPCD treatment), 6 MPa DPCD treatment group (DPCD6), 8 MPa DPCD treatment group (DPCD8), and 10 MPa DPCD treatment group (DPCD10). After DPCD treatment at the corresponding pressures, all three groups of raw materials were refrigerated at 4℃. Starting from day 0, samples were taken every 5 days until day 10. The changes in pH, titratable acidity, total bacterial count, coliform bacteria, and psychrophilic bacteria were measured and analyzed.

[0071] like Figure 12 A and Figure 12As shown in Figure B, the pH of goat milk and camel milk in the DPCD6 treatment group was significantly lower than that of the control group in the early stage of storage, while the titratable acidity was significantly higher than that of the control group. The pH of goat milk and camel milk in the DPCD8 and DPCD10 treatment groups was significantly lower than that of the DPCD6 group and the control group, while the titratable acidity was significantly higher than that of the control group and DPCD6. These differences remained stable in both the early stage (days 0 and 5) and the later stage (day 10) of storage. However, there were no significant differences in pH and titratable acidity between the DPCD8 and DPCD10 treatment groups at any storage time point, indicating that 8 MPa and 10 MPa had similar regulatory effects on the physicochemical properties of the two milks, and that the regulatory effect was stronger than that of the 6 MPa treatment group.

[0072] Comparative Example 2 The total bacterial count, Escherichia coli, and psychrophilic bacteria in raw goat milk and raw camel milk during storage were tested according to three standards: GB 4789.2-2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count", GB 4789.3-2016 "National Food Safety Standard - Microbiological Examination of Food - Coliform Count Plates", and NY / T1331-2007 "Determination of Psychrophilic Bacteria, Aerobic Spores and Thermoaerobic Spores in Milk and Dairy Products".

[0073] like Figure 13 As shown in AC, with prolonged storage at 4℃, the total bacterial count, coliform count, and psychrophilic bacteria count of both goat milk and camel milk increased, with the control group showing the fastest microbial growth rate. The 6 MPa DPCD treatment group showed no significant difference in total bacterial count, coliform count, and psychrophilic bacteria count compared to the control group at any storage time point, indicating no effective microbial inhibition. The 8 MPa and 10 MPa DPCD treatment groups showed significantly lower total bacterial count, coliform count, and psychrophilic bacteria count compared to the control group, effectively delaying microbial proliferation; furthermore, there was no significant difference in total bacterial count, coliform count, and psychrophilic bacteria count between the 8 MPa and 10 MPa treatment groups, indicating no significant difference in the antibacterial effect of the two pressures.

[0074] Comparative Example 3 Fresh raw sheep milk and raw camel milk were used as raw materials, and four treatment groups were set up: control group (no DHPM treatment), 32.5 MPa DHPM treatment group (DHPM32.5), 75 MPa DHPM treatment group (DHPM75), and 150 MPa DHPM treatment group (DHPM150). After DHPM treatment at the corresponding pressures, the raw milk was filled into well-sealed containers, and all samples were stored at 4℃. During storage, samples were collected on days 0, 5, and 10, and the changes in pH and titratable acidity were measured and analyzed.

[0075] like Figure 14 As shown in Figure A, the pH values ​​of all treatment groups decreased over time during storage. The pH decrease was more significant in the control group, while treatment with different pressures of DHPM helped to slow down the pH decrease. The effect of storage time on the titratable acidity of the samples is shown in Figure A. Figure 14 As shown in Figure B, the titration acidity of all groups increased with increasing storage time. The acidity increase was slower in the DHPM-treated groups, especially the high-pressure treated group (DHPM150). This indicates that DHPM treatment can effectively inhibit the formation or accumulation of acidic substances, thereby delaying the increase in sample acidity.

[0076] Comparative Example 4 The total bacterial count, Escherichia coli, and psychrophilic bacteria in raw goat milk and raw camel milk during storage were tested according to three standards: GB 4789.2-2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count", GB 4789.3-2016 "National Food Safety Standard - Microbiological Examination of Food - Coliform Count Plates", and NY / T1331-2007 "Determination of Psychrophilic Bacteria, Aerobic Spores and Thermoaerobic Spores in Milk and Dairy Products".

[0077] like Figure 15 As shown in Figure A, the total bacterial count of both goat milk and camel milk generally increased with prolonged storage time. DHPM treatment at 32.5 MPa had no significant inhibitory effect on the total bacterial count, while treatment pressures greater than 75 MPa effectively inhibited the growth of the total bacterial count. In the early stages of storage, the total bacterial count in the DHPM treatment group was significantly lower than that in the control group, and the total bacterial count in the 150 MPa group was slightly lower than that in the 75 MPa group, indicating that the 150 MPa treatment was more effective than the 75 MPa treatment. By day 10, the later stage of storage, the DHPM treatment groups at 75 and 150 MPa were still lower than the control group and the 32.5 MPa treatment group, but the difference between the 75 MPa and 150 MPa groups narrowed, indicating that the antibacterial effect of pressure differences gradually weakened with prolonged storage time.

[0078] like Figure 15As shown in Figure B, the overall coliform counts in both goat milk and camel milk showed a trend of initial increase followed by stabilization. DHPM treatment at 32.5 MPa had no significant inhibitory effect on coliforms. However, treatment pressures exceeding 75 MPa significantly inhibited coliform growth. During the early storage period, the coliform counts in the DHPM-treated groups (75 MPa and 150 MPa) were significantly lower than those in the control group, with the 150 MPa group showing a lower count than the 75 MPa group, indicating that the 150 MPa treatment was more effective than the 75 MPa treatment. On day 10, the DHPM-treated groups remained lower than the control group, while the difference between the 75 MPa and 150 MPa groups decreased, suggesting that the effect of pressure differences gradually weakened with prolonged storage.

[0079] like Figure 15 As shown in Figure C, the overall number of psychrophilic bacteria in both goat and camel milk increased with prolonged storage time. DHPM treatment at 32.5 MPa had no significant inhibitory effect on psychrophilic bacteria, while DHPM treatment at 75 and 150 MPa effectively inhibited their growth. In the early stages of storage, the number of psychrophilic bacteria in the DHPM-treated group was significantly lower than that in the control group, and the number in the 150 MPa group was lower than that in the 75 MPa group, indicating that the 150 MPa treatment was more effective than the 75 MPa treatment. On day 10, the number in the DHPM-treated group remained lower than that in the control group, while the difference between the 75 MPa and 150 MPa groups narrowed. Therefore, DHPM treatment inhibited the total bacterial count, coliform bacteria, and psychrophilic bacteria in both goat and camel milk, and there were no significant differences after treatment at 75 MPa and 150 MPa, indicating good efficacy in both treatments.

[0080] Comparative Example 5 Camel milk samples treated with dynamic high-pressure microfluidics were freeze-dried for preservation. The freeze-dried samples were then thoroughly mixed with potassium bromide at a ratio of 1:100, ground, and compressed into tablets. Fourier transform infrared spectroscopy was used to scan the samples within the range of 4000–400 cm⁻¹. -1 8 cm resolution -1 Three parallel scans were performed. PeakFitVersion 4.12 software was used to smooth and normalize the bands. Baseline correction and deconvolution were performed on the amide I band (1700–1600 cm⁻¹), and Gaussian curves were fitted to the bands using the second derivative. Finally, the relative abundance of various structures (α-helices, β-sheets, β-turns, and random coils) was calculated based on the peak area. Figure 16As shown, in the control groups of goat milk and camel milk, ordered structures such as α-helices and β-sheets accounted for a higher proportion, while the proportion of relatively disordered structures such as β-turns and random coils remained relatively stable. After treatment with DHPM at 32.5 and 75 MPa, the proportions of various secondary structures in goat milk and camel milk were not significantly different from those in the control group, indicating that the effects of 32.5 and 75 MPa treatment on the secondary structure of the proteins were limited.

[0081] After treatment with DHPM at 150 MPa, the proportions of α-helices and β-sheets in goat milk decreased significantly, while the proportions of β-turns and random coils increased significantly. Similarly, the proportions of α-helices and β-sheets in camel milk also decreased significantly, while the proportions of β-turns and random coils increased substantially. In summary, DHPM treatments at 32.5 and 75 MPa had minimal impact on the secondary structure of proteins in both goat and camel milk, with no significant difference in the proportions of each structure compared to the control group. However, treatment at 150 MPa significantly affected secondary structure transformation, reducing the proportions of ordered structures such as α-helices and β-sheets, while increasing the proportions of relatively disordered structures such as β-turns and random coils.

[0082] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A method for preserving raw milk, characterized in that, Includes the following steps: (1) Treat raw milk samples under high-density carbon dioxide (DPCD) conditions of 5-10 MPa for 5-15 min; (2) The raw milk sample after step (1) was treated with dynamic high pressure microjet (DHPM) at 75-160 MPa. (3) Store the raw milk sample processed in step (2) at a near-freezing temperature.

2. The method according to claim 1, characterized in that, The DPCD is processed at a pressure of 8 MPa for 10 min.

3. The method according to claim 2, characterized in that, The pressure of the DHPM is 75 MPa.

4. The method according to claim 3, characterized in that, The storage temperature is -0.27℃ to 4℃.

5. The method according to any one of claims 1 to 4, characterized in that, The raw milk sample is one or more of raw cow's milk, raw camel's milk, or raw goat's milk.

6. The method according to claim 5, characterized in that, The raw milk sample is one or both of raw sheep milk or raw camel milk.

7. The application of the method according to any one of claims 1 to 4 in the preparation of liquid milk, fermented dairy products, functional dairy products, and health foods.

8. The application according to claim 7, characterized in that, The raw milk sample is one or more of raw cow's milk, raw camel's milk, or raw goat's milk.

9. The application according to claim 8, characterized in that, The raw milk sample is one or both of raw camel milk or raw goat milk.

10. A method for inhibiting the proliferation of microorganisms in raw milk samples, characterized in that, The method includes the following steps: (1) Treat raw milk samples under high-density carbon dioxide (DPCD) conditions of 5-10 MPa for 5-15 min; (2) The raw milk sample after step (1) was treated with dynamic high pressure microjet (DHPM) at 60-180 MPa. (3) Store the raw milk sample after step (2) at a near-freezing temperature.

Images