Application of native DHA raw milk in improvement of DHA bioaccessibility of cow milk

Abstract

The invention provides an application of native DHA raw milk in improvement of DHA bioaccessibility of cow milk. The adopted native DHA raw milk is natural raw milk containing DHA, the bioaccessibility of the prepared cow milk DHA can be remarkably improved through the raw milk, the phospholipid type DHA of the raw milk is various, the raw milk contains specific phosphatidylinositol type DHA (PI-DHA) and cardiolipin type DHA (CLs-DHA), the proportion of phosphatidylserine type DHA (PS-DHA) is remarkably higher than that of DHA milk from fish oil and algae oil, and the DHA milk is rich in DHA, rich in DHA, rich in DHA, rich in DHA, rich in DHA, rich in DHA, rich in DHA, rich in DHA, rich in DHA, rich in DHA, rich in DHA, rich in DHA and rich in DHA. The proportion of the total phospholipid type DHA in the total DHA is obviously higher than that of other fish oil and algae oil DHA milk, the variety and content of the phospholipid type DHA of cow milk can be increased by adopting the original DHA raw milk to prepare the cow milk, and the bioaccessibility of the DHA of the cow milk is improved.

Description

Technical Field

[0001] The invention relates to application of native DHA raw milk in improving the biological accessibility of DHA in cow's milk, and belongs to the technical field of cow's milk preparation. Background Art

[0002] Docosahexaenoic acid (DHA) belongs to the n-3 long-chain polyunsaturated fatty acids (LCPUFAs) and plays an important role in fetal development and brain and retinal development in the first two years of life.

[0003] DHA is one of the most important n-3 LCPUFAs in breast milk. Depending on the differences in dietary habits around the world, the DHA content varies from 0.15% to 1.4% of the total fatty acids in breast milk (Brenna et al., 2007; Stam et al., 2013). For both mothers and infants, the dietary sources of DHA are limited and the accessibility is poor. The efficiency of linolenic acid conversion to DHA in the body is low, so it is often necessary to fortify foods to ensure adequate DHA dietary intake. Based on the importance of DHA to the visual and cognitive development of infants and young children, the EU Directive EU2016 / 127 stipulates that infant formula powder must be supplemented with DHA, with an addition range of 20-50 mg / 100 kcal, which accounts for 0.36%-1.23% of the total fatty acids. The effective start and end time of this regulation is from February 2020 to February 2025.

[0004] Because DHA is a polyunsaturated fatty acid, it is easy to oxidize when added in high amounts, causing the quality of infant formula to deteriorate. Therefore, the DHA fortified in infant formula currently only accounts for about 0.1%-0.3% of the total fatty acids, and the main sources of DHA are algae (Schizochytrium, Okenichthys and Crypthecodinium cohnii) and tuna oil. The key to DHA's biological activity is oral bioavailability, which involves the release of DHA from food matrix or gastrointestinal fluid, the dissolution of DHA and its interaction with other components of gastrointestinal fluid. These digestion and absorption processes will greatly affect the biological activity of DHA.

[0005] At present, algae oil and fish oil raw materials rich in DHA are widely added to various food-based carriers such as milk-based, oil-based, and gum-based to meet the nutritional needs of infants and young children through nutritional fortification. DHA has recognized health benefits, but to play this role, DHA needs to be released from the food matrix and absorbed into the mixed micelles (Shani-Levi et al., 2017; M'enard et al., 2018).

[0006] The determination of in vitro bioaccessibility has no ethical restrictions or requirements and can be used to assess the amount of easily absorbable DHA. Bioaccessibility refers to the ease with which a substance enters the body and is utilized. The sample digestion is performed using an infant in vitro simulated digestion model (M′enard et al. 2018.), and then the amount of DHA available for absorption in the micelle component is measured as a proportion of the total DHA in the sample to evaluate the bioaccessibility of the sample DHA. In the prior art background document, the same test method was used, and the bioaccessibility of DHA in two infant formula powders was about 9% (MCMarques et al. 2021), and the bioaccessibility was low.

[0007] Therefore, it is urgent to develop a milk source of DHA that is more bioaccessible and easily absorbed to improve the health effects of DHA on infants and young children. Summary of the invention

[0008] In order to solve the above technical problems, the purpose of the present invention is to provide a method for improving the bioaccessibility of DHA in bovine milk, which uses native DHA raw milk as a raw material to prepare bovine milk, thereby significantly improving the bioaccessibility of DHA in bovine milk.

[0009] In order to achieve the above object, the present invention first provides the use of native DHA raw milk in improving the bioaccessibility of DHA in cow's milk.

[0010] According to a specific embodiment of the present invention, preferably, the native DHA raw milk is prepared according to the feeding and management method of dairy cows in CN 105660520B; the native DHA raw milk is used as a raw material for cow's milk.

[0011] According to a specific embodiment of the present invention, preferably, the DHA content of the cow's milk is above 10 mg / 100 ml.

[0012] According to a specific embodiment of the present invention, preferably, the cow's milk contains phospholipid DHA. Compared with the common technical solutions in the prior art that use fish oil, algae oil, etc. as raw materials to increase the DHA content of cow's milk by adding them externally, the present invention can significantly increase the type and content of phospholipid DHA in the prepared cow's milk product by using native DHA raw milk, thereby improving the bioaccessibility of cow's milk DHA.

[0013] The present invention can provide rich phospholipid DHA to cow's milk by adopting native DHA raw milk, which far exceeds the content that can be provided by fish oil and algae oil.

[0014] According to a specific embodiment of the present invention, preferably, based on the total DHA content of the cow's milk, the content of the phospholipid DHA is 8%-35%; more preferably, it is 20.95%±0.65%.

[0015] The present invention can provide PI type DHA and CLs type DHA to cow's milk by adopting native DHA raw milk, which are unable to be provided by fish oil and algae oil.

[0016] The present invention can provide rich PS-type DHA to cow's milk by adopting native DHA raw milk, which far exceeds the content that can be provided by fish oil and algae oil.

[0017] According to a specific embodiment of the present invention, preferably, the bovine milk contains one or a combination of two or more of PI-type DHA, CLs-type DHA and PS-type DHA.

[0018] According to a specific embodiment of the present invention, preferably, based on the total phospholipid DHA content of the cow's milk, the content of the PI DHA is 1%-10%; more preferably 3.56%±0.07%.

[0019] According to a specific embodiment of the present invention, preferably, the content of CLs-type DHA is 10%-20%; more preferably, 14.17%±0.10%.

[0020] According to a specific embodiment of the present invention, preferably, based on the total phospholipid DHA content of the milk, the content of the PS DHA is 10%-20%; more preferably, 15.43%±0.02%.

[0021] The present invention can provide cow milk with one or a combination of two or more phospholipid DHAs such as PE DHA, PC DHA and LPLs DHA by using native DHA raw milk, which is a content range that fish oil and algae oil cannot provide.

[0022] According to a specific embodiment of the present invention, preferably, the bovine milk contains one or a combination of two or more of PE-type DHA, PC-type DHA and LPLs-type DHA.

[0023] According to a specific embodiment of the present invention, preferably, based on the total phospholipid DHA content of the cow's milk, the content of the PE DHA is -40%-50%; more preferably, it is 46.79%±0.18%.

[0024] According to a specific embodiment of the present invention, preferably, based on the total phospholipid DHA content of the cow's milk, the content of the PC-type DHA is 5%-20%; more preferably, it is 12.86%±0.03%.

[0025] According to a specific embodiment of the present invention, preferably, based on the total phospholipid DHA content of the bovine milk, the content of the LPLs DHA is 5.5%-7.5%; more preferably 7.19%±0.07%.

[0026] The cow's milk provided by the present invention can be prepared by using a pure milk preparation process commonly used in the art, for example, according to the following process steps:

[0027] 1) Collection and storage of raw milk rich in DHA: Collect the milk according to the raw milk standard, the temperature of the milk collection is 1-8°C, and after the milk is collected, it is filtered with a 60-80 mesh single or double filter, and then cooled to 1-6°C for storage;

[0028] 2) Milk separation: The milk in step 1) is sterilized and impurities removed by a high-speed centrifuge, the separation temperature is 50-65° C., and the separation speed is 4500-5000 rpm / min;

[0029] 3) Pre-sterilization: homogenize and pre-sterilize the sterilized and impurity-removed milk in step 2), with a homogenization temperature of 50-65°C and a homogenization pressure of 160-200 bar for primary homogenization and 20-40 bar for secondary homogenization; pre-sterilization requires heating the milk to 75-85°C, keeping it warm for 10-25s (preferably 15s), and then cooling it to 1-8°C for storage;

[0030] 4) Ultra-high temperature sterilization: vacuum degas the milk in step 3), then heat the milk to 70-75°C and homogenize it at a homogenization pressure of 200-300 bar for primary homogenization and 40-60 bar for secondary homogenization; heat the homogenized milk to 137-140°C and keep it for 3-8 seconds (preferably 4 seconds), then cool it to 20-30°C to obtain sterilized milk, i.e. native DHA milk.

[0031] The native DHA raw milk used in the present invention is raw milk that naturally contains DHA. The raw milk can significantly improve the bioaccessibility of DHA in the prepared cow's milk. In addition, the raw milk has various types of phospholipid DHA, including unique phosphatidylinositol DHA (PI-DHA) and cardiolipin DHA (CLs-DHA). The proportion of phosphatidylserine DHA (PS-DHA) is significantly higher than that of DHA milk derived from fish oil and algae oil, and the proportion of total phospholipid DHA in total DHA is significantly higher than that of other fish oil and algae oil DHA milks. Using the native DHA raw milk to prepare cow's milk can increase the types and contents of phospholipid DHA in cow's milk and improve the bioaccessibility of DHA in cow's milk. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a graph comparing the bioaccessibility of DHA from different sources in an in vitro digestion model for infants.

[0033] Figure 2 This is the FFA release kinetics curve per unit volume of milk during the intestinal digestion stage. DETAILED DESCRIPTION

[0034] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.

[0035] Preparation of native DHA raw milk:

[0036] The raw milk material rich in phospholipid DHA used in the embodiment contains natural native DHA raw milk obtained by feed regulation technology. The preparation method thereof is described in Example 2 of patent CN201410673240.8, which is as follows:

[0037] A dairy cow breeding management method, which is used for the breeding of dairy cows for the production of natural DHA raw milk, with a breeding scale of 100 dairy cows;

[0038] The composition of the functional premix used includes (based on 1 kg of premix):

[0039]

[0040] The amount of conventional feed used was 45 kg / day / head, of which 78% was roughage and 22% was concentrated feed.

[0041] The composition and mixing ratio of the roughage are shown in Table 1.

[0042] Table 1 (by weight)

[0043] Alfalfa Beet meal Cottonseed Silage water DDGS Flake corn Honey 35 17 15 220 28 5 20 10

[0044] In terms of weight percentage, the concentrate feed includes the following ingredients:

[0045] 5.5% expanded soybeans, 50% corn, 10% bran, 0.5% vitamins, 10% soybean meal, 5% carrots, 1% salt, 2% beets, 2% bone meal, 2% distiller's grains, 2% tofu dregs, and 10% water.

[0046] Specific feeding process:

[0047] Use high-efficiency mixing equipment to prepare functional premixes. In the first week, the functional premixes replace concentrate feeds with a replacement amount of 4% of the total concentrate feed (400 g / day, divided into two meals per head). In the second week, the functional premixes replace concentrate feeds with a replacement amount of 5% of the total concentrate feed (500 g / day, divided into two meals per head). In the third week, the functional premixes replace concentrate feeds with a replacement amount of 6% of the total concentrate feed (600 g / day, divided into two meals per head). In the fourth week, the functional premixes replace concentrate feeds with a replacement amount of 10% of the total concentrate feed (600 g / day, divided into two meals per head). In the fifth week, the functional premix replaces the concentrate feed, and the replacement amount is 8% of the total concentrate feed (800 g / day·head fed in two meals). In the sixth week, the functional premix replaces the concentrate feed, and the replacement amount is 9% of the total concentrate feed (900 g / day·head fed in two meals). In the seventh week, the functional premix replaces the concentrate feed, and the replacement amount is 10% of the total concentrate feed (1000 g / day·head fed in two meals). The prefeeding period ends.

[0048] After the pre-feeding period, the DHA content of the raw milk was continuously tested, once every 10 days, for a total of three tests.

[0049] The results of the DHA content test of raw milk are shown in Table 2.

[0050] Table 2

[0051] Time after the pre-feeding period 10 days 20 days 30 days 40 days DHA content of samples 14.4mg / 100g 15.2mg / 100g 16.7mg / 100g 17.8mg / 100g

[0052] It can be seen from the contents of Table 2 that the three test results all reached above 10 mg / 100 g, and this feeding amount can be maintained until the end of lactation.

[0053] Example 1 Native DHA milk

[0054] This embodiment provides a native DHA cow's milk, the raw material of which contains the above-mentioned native DHA raw milk, and the raw milk naturally and stably contains DHA with a content of more than 10 mg / 100 ml.

[0055] The process of producing native DHA milk using DHA-rich raw milk as raw material is as follows:

[0056] 1) Collection and storage of DHA-rich raw milk: Collect milk according to raw milk standards, with a collection temperature of 1-8°C. After collecting the milk, filter it with a 60-80 mesh single or double filter, and then cool it to 1-6°C for storage.

[0057] 2) Milk separation: The milk in step 1) is sterilized and impurities removed by a high-speed centrifuge, with a separation temperature of 50-65° C. and a separation speed of 4500-5000 rpm / min.

[0058] 3) Pre-sterilization: homogenize and pre-sterilize the sterilized and impurity-free milk in step 2) at a homogenization temperature of 50-65°C and a two-stage homogenization of 30 / 180±5bar (first adjust the second stage so that the pressure gauge indicates 30bar, and then adjust the first stage so that the pressure gauge indicates 180±5bar); pre-sterilization requires heating the milk to 80°C, keeping it warm for 15s, and then cooling it to 1-8°C for storage.

[0059] 4) Ultra-high temperature sterilization: vacuum degas the milk from step 3), then heat the milk to 70-75°C and homogenize it at a homogenization pressure of 250±5 bar for the first homogenization and 50 bar for the second homogenization; heat the homogenized milk to 137-140°C and maintain for 4 seconds, then cool it to 20-30°C to obtain sterilized milk, i.e. native DHA milk.

[0060] 5) Filling: The sterilized milk is filled and stored in an ultra-clean or aseptic environment.

[0061] Example 2 Algae Oil DHA Milk

[0062] This embodiment provides an algae oil DHA milk, which is prepared by adding 0.057% algae oil raw material to DHA-free milk and processing it through a conventional homogenization process, the process being as follows:

[0063] 1) Ordinary raw milk collection and storage: Collect milk according to raw milk standards, the collection temperature is 1-8℃, after collecting the milk, use a 60-80 mesh single or double filter to filter, and then cool to 1-6℃ for storage.

[0064] 2) Milk separation: The milk in step 1) is sterilized and impurities removed by a high-speed centrifuge, with a separation temperature of 50-65° C. and a separation speed of 4500-5000 rpm / min.

[0065] 3) Mixing and pre-sterilization: The algae oil extract containing DHA and the sterilized and impurity-free milk in step 2) are mixed, homogenized and pre-sterilized at a homogenization temperature of 50-65°C, using a two-stage homogenization of 30 / 180±5bar (first adjust the second stage so that the pressure gauge indicates 30bar, and then adjust the first stage so that the pressure gauge indicates 180±5bar); pre-sterilization requires heating the milk to 80°C, keeping it warm for 15s, and then cooling it to 1-8°C for storage.

[0066] 4) Ultra-high temperature sterilization: vacuum degas the milk from step 3), heat the milk to 70-75°C, and homogenize it at a homogenization pressure of 250±5 bar for the first homogenization and 50 bar for the second homogenization. The homogenized milk is heated to 137-140°C and maintained for 4 seconds, then cooled to 20-30°C to obtain sterilized milk, i.e., algae oil DHA milk.

[0067] 5) Filling: The sterilized milk is filled and stored in an ultra-clean or aseptic environment.

[0068] Example 3 Fish Oil DHA Milk

[0069] This embodiment provides a fish oil DHA milk, which is prepared by adding 0.16% of fish oil raw material to DHA-free milk and processing it through a conventional homogenization process, the process is as follows:

[0070] 1) Ordinary raw milk collection and storage: Collect milk according to raw milk standards, the collection temperature is 1-8℃, after collecting the milk, use a 60-80 mesh single or double filter to filter, and then cool to 1-6℃ for storage.

[0071] 2) Milk separation: The milk in step 1) is sterilized and impurities removed by a high-speed centrifuge, with a separation temperature of 50-65° C. and a separation speed of 4500-5000 rpm / min.

[0072] 3) Mixing and pre-sterilization: Mix, homogenize and pre-sterilize the fish oil extract containing DHA and the sterilized and impurity-free milk in step 2) at a homogenization temperature of 50-65°C and use a two-stage homogenization of 30 / 180±5bar (first adjust the second stage so that the pressure gauge indicates 30bar, then adjust the first stage so that the pressure gauge indicates 180±5bar). Pre-sterilization requires heating the milk to 80°C, keeping it warm for 15s, and then cooling it to 1-8°C for storage.

[0073] 4) Ultra-high temperature sterilization: vacuum degas the milk from step 3), heat the milk to 70-75°C, and homogenize it at a homogenization pressure of 250±5 bar for the first homogenization and 50 bar for the second homogenization. The homogenized milk is heated to 137-140°C and maintained for 4 seconds, then cooled to 20-30°C to obtain sterilized milk, i.e., fish oil DHA milk.

[0074] 5) Filling: The sterilized milk is filled and stored in an ultra-clean or aseptic environment.

[0075] Comparative Example 1 Pure milk + algae oil

[0076] This comparative example provides milk with added algae oil extract, which is obtained by adding 0.057% of algae oil extract accounting for the drinking amount of milk to the prepared pure milk (no DHA detected), and the milk simulates the scenarios of drinking pure milk alone and taking algae oil DHA dietary supplements, wherein the algae oil content in the algae oil extract is ≥350 mg / g.

[0077] Comparative Example 2 Pure milk + fish oil

[0078] This comparative example provides milk with added fish oil extract, which is obtained by adding 0.16% of the fish oil extract accounting for the drinking amount of milk to the prepared pure milk (no DHA detected), and the milk simulates the scenarios of drinking pure milk alone and taking fish oil DHA dietary supplements, wherein the fish oil content in the fish oil extract is ≥125 mg / g.

[0079] Experimental test:

[0080] 1. Determination of the bioaccessibility of DHA from different sources in an in vitro digestion model for infants

[0081] Experimental methods:

[0082] Take 63mL of milk sample and add it to a 250mL beaker, preheat to 37℃. Take 37mL of simulated gastric fluid (94mM NaCl and 13mM KCl, pH adjusted to 5.3) preheated to 37℃ and add it to the beaker containing milk. Then add pepsin to make the final enzyme activity of the system reach 268U / mL, and add gastric lipase to make the final enzyme activity of the system reach 19U / mL. Immediately adjust the pH to 5.3 and start timing. After magnetic stirring for 120min at 37℃ and 95r / min, take out the sample and immediately adjust the pH to 6.6 with NaOH. The digestion system with pH adjusted to 6.6 was placed in a 37°C water bath, and 40 mL of simulated intestinal fluid (164 mM NaCl, 10 mM KCl and 85 mM NaHCO3, pH adjusted to 7.0, and CaCl2 was added to the volume of intestinal fluid at a concentration of 3 mM before the start of the intestinal phase) and 1.4 mL of 127 mg / mL pig bile salt solution were added, and then pig pancreatic lipase was added to make the final enzyme activity of the system reach 90 U / mL, and trypsin was added to make the final enzyme activity of the system reach 16 U / mL. The pH of the system was adjusted to 6.6, and the pH was maintained at 6.6 with a potentiometric titrator. After magnetic stirring at 37°C and 95 r / min for 120 minutes, the sample was taken out and the enzyme was immediately inactivated to terminate the digestion.

[0083] After centrifuging the digested chyme at 4000g for 5 minutes, it was then centrifuged at 21000g for 30 minutes, and the upper fat layer and the lower sediment were discarded. The clear liquid in the middle was the micelle part. The micelle part sample was freeze-dried, and all the dry powder was weighed and recorded after freeze-drying. The DHA content in the freeze-dried powder was detected. The calculation formula for the bioaccessibility of DHA in the sample is as follows:

[0084] Bioaccessibility (%) = DHA content in digested chyme micelles / DHA content before sample digestion × 100

[0085] 2. Test of total DHA content in freeze-dried powder

[0086] Weigh about 0.5g of freeze-dried powder into a 15mL dry screw-capped glass tube and add 5mL of toluene. Add 6mL of 10% acetyl chloride-methanol solution to the sample, fill with nitrogen, and tighten the screw cap. After oscillation and mixing, place in a 80℃±1℃ water bath for 2h, take out and shake once every 20min, take out after the water bath and cool to room temperature. Transfer the reacted sample solution to a 50mL centrifuge tube, wash the glass tube with 3mL of sodium carbonate solution 3 times, combine the sodium carbonate solution in a 50mL centrifuge tube, and mix well. Centrifuge at 5000r / min for 5min. Take the supernatant as the test solution and measure it by gas chromatograph.

[0087] Take the DHA methyl ester standard series working solution and sample solution and inject them into the gas chromatograph respectively. The chromatographic conditions are as follows. Capillary column: polydicyanopropylsiloxane strong polar stationary phase, column length 100m, inner diameter 0.25mm, film thickness 0.2μm. Injector temperature: 270℃, detector temperature: 260℃. Program temperature rise: initial temperature 130℃, last 5min; 130℃-240℃, heating rate 4℃ / min, hold 20min. Carrier gas: nitrogen, split ratio: 100:1. Injection volume: 1.0μL.

[0088] Draw a linear regression standard curve with the chromatographic peak area of ​​DHA methyl ester standard as the ordinate and the concentration as the abscissa. Substitute the chromatographic peak area of ​​the target substance in the sample into the linear equation of the standard curve to calculate the concentration of the sample to be tested, and calculate the actual content of the target substance in the sample based on the sampling amount. The calculation formula is as follows:

[0089] X=c×V×f / m

[0090] Where:

[0091] X—total DHA content in freeze-dried powder (mg / g);

[0092] c—Concentration of DHA methyl ester in the lyophilized powder dilution, mg / mL;

[0093] V—total volume of lyophilized powder diluent, mL;

[0094] m—mass of freeze-dried powder sampling, g;

[0095] f—Conversion coefficient of DHA methyl ester to DHA, 0.9590.

[0096] 3. Lipolysis rate determination

[0097] The consumption of NaOH during the 2-h simulated small intestinal digestion was recorded using a pH-stat automatic potentiometric titrator, and the amount of FFA (free fatty acids) produced could be calculated using the following formula.

[0098]

[0099] Where:

[0100] M FFA —The amount of FFA released per unit volume of milk, μmol / mL;

[0101] V NaOH —The volume of NaOH consumed during the digestion process, mL;

[0102] C NaOH —The molar concentration of NaOH consumed during the digestion process, 0.1 M;

[0103] V milk —The volume of milk digested in the small intestine, 30 mL.

[0104] 4. Statistics

[0105] Software was used for statistical analysis of data and plotting of experimental results. The mean and standard deviation of each group of data were calculated using relevant software. ANOVA analysis and Tukey test were used to statistically analyze the significance of the differences between the groups of data. The experimental results are expressed as mean ± standard deviation (Means ± SD). Different letters indicate that there is statistical significance between the groups, P < 0.05.

[0106] 5. Experimental results

[0107] The DHA contents of the embodiments and comparative examples are shown in Table 3.

[0108] Table 3

[0109]

[0110] The embodiments and comparative examples provided by the present invention describe the differences in bioaccessibility in products with different DHA sources and processing techniques. The DHA content in all embodiments and comparative examples is about 20 mg / 100 ml, maintaining the consistency of the DHA content.

[0111] Table 4 shows the comparison of the bioaccessibility of DHA from different sources in an in vitro digestion model for infants. Figure 1 Comparison of bioaccessibility of DHA from different sources in an in vitro digestion model for infants. Note: n = 3. Different superscript letters indicate statistical differences between data from different sample groups (lowercase letters indicate p ≤ 0.05).

[0112] Table 4 Comparison of bioaccessibility of DHA from different sources in an in vitro digestion model for infants

[0113]

[0114] The amount of NaOH consumed by different milks during the intestinal digestion stage was monitored by the pH-stat method, and the amount of FFA (free fatty acids) released per unit volume of milk was calculated (Table 5), and then the digestion kinetics curve of milk samples during the intestinal digestion stage was drawn ( Figure 2 ), through which the stage hydrolysis rate and final hydrolysis degree of different cow's milk can be determined.

[0115] Table 5 FFA release per unit volume of milk during intestinal digestion over time

[0116]

[0117]

[0118] Note: Data are expressed as mean ± standard deviation (Mean ± SD) (n = 3). Different superscript letters in each row indicate statistical differences between data of different sample groups under the same digestion system and digestion time (uppercase letters indicate p ≤ 0.01, lowercase letters indicate p ≤ 0.05).

[0119] Fatty acid hydrolysis rate

[0120] In the intestinal digestive system of infants with insufficient bile salt and lipase content, the hydrolysis rates of the four milk samples showed a continuous decrease at different levels starting from 5 minutes of intestinal digestion. Figure 2 From the change in the slope of the FFA release kinetic curve of cow's milk in the infant intestinal digestive system shown, it can be seen that compared with other cow's milk, the hydrolysis rate of native DHA cow's milk (Example 1) decreases the slowest and can maintain a relatively higher fatty acid hydrolysis rate for a longer time, indicating that native DHA cow's milk (Example 1) is easier to be digested by lipase in the infant intestinal digestive system.

[0121] Final release of fatty acids

[0122] The final degree of hydrolysis of milk is quantified by the amount of FFA released per unit volume of milk after 2 h of intestinal digestion. Figure 2 As shown in the data of Table 5, in the intestinal digestive system of infants, the final release of fatty acids from native DHA milk was significantly higher than that from other milk samples (p≤0.01), indicating that native DHA milk is easier to digest.

[0123] The difference between Example 1 and Example 2 is that the sources of DHA are different, which proves that native DHA milk naturally rich in DHA has significantly higher bioaccessibility in infants and young children, and is more conducive to absorption by infants and young children.

[0124] The difference between Example 2 and Comparative Example 1 is whether there is a homogenization process for dairy processing. The present invention innovatively discovered that the homogenization process will cause significant differences in DHA from the same source in the in vitro digestion system of infants with weaker enzyme activity. The bioaccessibility of the algae oil DHA milk of Example 2 is significantly better than that of an ordinary mixture of pure milk and algae oil without homogenization, representing a product that uses cow's milk as a matrix carrier and is fortified with DHA through a homogenization process, which can be better absorbed by infants and young children.

[0125] The difference between Comparative Example 1 and Comparative Example 2 is that the DHA sources are different, which proves that whether algae oil extract or fish oil extract is taken alone without other processing techniques, there is no difference in their bioaccessibility.

[0126] Bioaccessibility

[0127] Figure 1 Table 4 shows the comparative results of the bioaccessibility of DHA from different sources in the infant in vitro digestion model.

[0128] By comparing Example 1 with Comparative Examples 1 and 2, it was found that the bioaccessibility of the native DHA milk of the present invention can reach about 1.5 times that of Comparative Examples 1 and 2. Combined with the difference data between Example 2 and Comparative Example 1, this shows that the bioaccessibility of raw milk rich in DHA is significantly better than DHA from other sources.

[0129] 6. Determination of the proportion of phospholipid DHA in the sample

[0130] After freeze-drying the milk sample, weigh about 0.4g of freeze-dried powder in a 15mL centrifuge tube, add 8mL of dichloromethane-methanol solution, vortex and centrifuge at 9000r / min for 5min, and take the supernatant. Add 4mL of dichloromethane-petroleum ether solution to the precipitate, vortex and centrifuge at 9000r / min for 5min, take the supernatant, repeat twice, combine the supernatants, blow dry with nitrogen, and obtain the lipid components in milk.

[0131] The prepared lipid solution was injected into the LC-MS instrument for analysis. The analysis conditions were as follows: chromatographic column (BEH-C18, 1.6 μm, 2.1×100 mm), the mobile phase was a solution prepared by mixing water, acetonitrile and isopropanol, wherein phase A: water: acetonitrile: isopropanol = 5:3:2, containing 10 mM ammonium formate, phase B: water: acetonitrile: isopropanol = 1:9:90, containing 10 mM ammonium formate. The initial mobile phase ratio was 10% phase B, at 1 min, the phase B ratio increased to 40%, at 3 min, the phase B ratio increased to 75%, to 6 min, the phase B ratio increased to 100%, maintained for 1 min, at 7 min, the phase B recovered to 10%, and the total running time was 10 min. The mass spectrometry ion source was an electrospray ion source, positive ion mode, capillary voltage 4.2 KV, ion source temperature 350 ° C. Mass spectrometry acquisition adopted dd-MS2 mode, and loop count was set to 5.

[0132] 7. Statistics

[0133] Lipidomics analysis software was used to process the collected data, identify the lipid types in each sample, and screen out DHA-related lipids for analysis.

[0134] 8. Experimental results

[0135] (1) Total DHA content in the sample

[0136] The test results of total DHA content in different samples are shown in Table 6.

[0137] Table 6 Total DHA content in samples

[0138] Sample name Total DHA content Raw DHA milk 0.214mg / mL Algae Oil DHA Milk 0.170mg / mL Fish Oil DHA Milk 0.211mg / mL

[0139] (2) Percentage of phospholipid DHA in the samples

[0140] The test results of the relative percentage of multifunctional phospholipid DHA in the samples of Examples and Comparative Examples 1-2 are shown in Table 7. The data show that native DHA milk contains unique phosphatidylinositol DHA (PI-DHA) (the content can account for 3.56% of the total phospholipid DHA) and cardiolipin DHA (CLs-DHA) (the content can account for 14.17% of the total phospholipid DHA), and the proportion of phosphatidylserine DHA (PS-DHA) is significantly higher than that of DHA milk from other sources, and the proportion of total phospholipid DHA in total DHA is as high as 20.95%, which is significantly higher than that of DHA milk from other sources.

[0141] Table 7 Forms and relative percentages of phospholipid DHA in different milks (area normalization method)

[0142]

[0143] According to the experimental results in Table 6 and Table 7, it can be seen that:

[0144] The embodiments of the present invention use products from different DHA sources, and the types and contents of the phospholipid DHA in each product are significantly different.

[0145] The difference between Example 1 and Example 2 and Example 3 is that the sources of DHA are different. The detection and analysis of the sample phospholipid DHA found that: the native DHA milk contains unique phosphatidylinositol DHA (PI-DHA) (the content can account for 3.56% of the total phospholipid DHA) and cardiolipin DHA (CLs-DHA) (the content can account for 14.17% of the total phospholipid DHA), while fish oil DHA milk and algae oil DHA milk do not contain PI-DHA and CLs-DHA; the phosphatidylserine in the native DHA milk is 1.34% higher than that in the Acid-type DHA (PS-DHA) accounts for as much as 15% of phospholipid DHA, while in fish oil DHA milk, PS-DHA accounts for only about 5% of phospholipid DHA, and in algae oil DHA milk, PS-DHA accounts for only about 8% of phospholipid DHA; in native DHA milk, total phospholipid DHA accounts for as much as 20.95% of total DHA, while in algae oil DHA milk, total phospholipid DHA accounts for only about 6% of total DHA, and in fish oil DHA milk, total phospholipid DHA accounts for only about 5% of total DHA.

[0146] By comparing the differences in the subdivisions of phospholipid DHA in the samples of Example 1, Example 2 and Example 3, it can be found that the proportions of PI DHA and CLs DHA in native DHA milk are 3.56%±0.07% and 14.17%±0.10%, respectively, while the proportions in algae oil DHA milk and fish oil DHA milk are both 0.

[0147] The proportion of PS DHA in native DHA milk is 15.43%±0.02%, which is 1.8 times that of algae oil DHA milk and 3.3 times that of fish oil DHA milk.

[0148] Based on the above content, it can be seen that using raw milk rich in DHA can enable cow's milk to obtain more types and contents of phospholipid DHA.

Claims

1. Application of native DHA raw milk in improving the bioaccessibility of DHA in bovine milk.

2. The use according to claim 1, wherein: The native DHA raw milk is prepared according to the feeding and management method of dairy cows in CN 105660520B.

3. The use according to claim 1, wherein: The native DHA raw milk is used as the raw material for making cow's milk.

4. The use according to claim 1, wherein: The DHA content of the milk is above 10 mg / 100 ml.

5. The use according to claim 1, wherein: The milk contains phospholipid-type DHA; Preferably, based on the total DHA content of the cow's milk, the content of the phospholipid DHA is 8%-35%; more preferably, it is 20.95%±0.65%.

6. The use according to claim 1, wherein: The cow's milk contains one or a combination of two or more of PI-type DHA, CLs-type DHA and PS-type DHA.

7. The use according to claim 6, wherein: Calculated based on the total phospholipid DHA content of the milk, the content of PI DHA is 1%-10%; preferably 3.56%±0.07%; Based on the total phospholipid DHA content of the milk, the content of the CLs DHA is 10%-20%; preferably 14.17%±0.10%; Calculated based on the total phospholipid DHA content of the milk, the content of the PS DHA is 10%-20%, preferably 15.43%±0.02%.

8. The use according to claim 5, wherein: The cow's milk contains one or a combination of two or more of PE-type DHA, PC-type DHA and LPLs-type DHA.

9. The use according to claim 8, wherein: Based on the total phospholipid DHA content of the milk, the content of the PE DHA is 40%-50%; preferably 46.79%±0.18%; Based on the total phospholipid DHA content of the milk, the content of PC DHA is 5%-20%; preferably 12.86%±0.03%; Calculated based on the total phospholipid DHA content of the milk, the content of LPLs DHA is 5.5%-7.5%, preferably 7.19%±0.07%.

10. The use according to claim 1, wherein: The milk is prepared according to the following steps: 1) Collection and storage of raw milk rich in DHA: Collect the milk according to the raw milk standard, the temperature of the milk collection is 1-8°C, and after the milk is collected, it is filtered with a 60-80 mesh single or double filter, and then cooled to 1-6°C for storage; 2) Milk separation: The milk in step 1) is sterilized and impurities removed by a high-speed centrifuge, the separation temperature is 50-65° C., and the separation speed is 4500-5000 rpm / min; 3) Pre-sterilization: homogenize and pre-sterilize the sterilized and impurity-removed milk in step 2), with a homogenization temperature of 50-65°C, a first-stage homogenization of 160-200 bar, and a second-stage homogenization of 20-40 bar; pre-sterilization requires heating the milk to 75-85°C, keeping it warm for 10-25 seconds, and then cooling it to 1-8°C for storage; 4) Ultra-high temperature sterilization: vacuum degas the milk from step 3), then heat the milk to 70-75°C and homogenize it at a homogenization pressure of 200-300 bar for primary homogenization and 40-60 bar for secondary homogenization; heat the homogenized milk to 137-140°C and maintain for 3-8 seconds, then cool it to 20-30°C to obtain sterilized milk, i.e. native DHA milk.

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