Preparation method and application of vitamin nano-particles with high bioavailability

Abstract

The invention discloses a preparation method and application of vitamin nanoparticles with high bioavailability, and belongs to the technical field of children food processing. The biological availability of the vitamin D3 in the yoghurt product prepared by the invention can reach 60.25%, and the yoghurt product is vitamin D3 fortified yoghurt meeting the self development and nutritional requirements of children. The soybean protein isolate is pretreated on the basis of a dynamic high-pressure micro-jet technology, the vitamin D3 nanoparticles are further constructed, the soybean protein isolate-vitamin D3 nanoparticles, fresh milk, mixed fermentation strains and the like are compounded to prepare the yoghourt, the digestion and slow release characteristics of vitamin D3 are effectively guaranteed, and the nutritional value of the yoghourt is improved. The biological availability rate of the vitamin D3 in the yoghourt is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of children's food processing, and in particular relates to a preparation method and application of vitamin nanoparticles with high bioavailability. Background Art

[0002] Children, especially those under 3 years old, still have developing intestinal flora, and the digestive enzyme concentration and pH conditions are very different from those of adults. For example, children lack amylase in their small intestines, the concentration of pepsin in gastric digestive fluid is 8 times lower than that of adults, the concentration of bile salts in duodenal digestive mixture is 4 times lower than that of adults, and the secretion of pancreatic enzymes and bile salts is only about 10% of that of adults. Children aged 3 and under cannot absorb nutrients well because their intestinal flora is not fully developed until they are close to 5 years old.

[0003] Vitamin D 3 (Cholecalciferol) is mainly synthesized by the human body. The human skin contains a kind of cholesterol, which turns into vitamin D after being exposed to sunlight. 3 Therefore, if children can fully receive direct sunlight on their skin for more than 4-6 hours, their own synthesis of vitamin D 3 , it can basically meet the needs. However, due to the increase in skin cancer caused by ultraviolet radiation, air pollution, and weather changes in various regions, the time people in various countries receive sunlight is decreasing, and many countries have clear regulations to limit the time of receiving sunlight. Therefore, vitamin D deficiency is widespread around the world. But vitamin D 3 Supplements are not just about increasing vitamin D 3 The content of vitamin D is not enough, but it needs to be slowly released in the human body to fully exert its effect, so the relevant vitamin D 3 Supplements must form a suitable combination of vitamins and carriers to meet the low enzyme activity characteristics of children's digestive system and achieve a sustained release effect.

[0004] Therefore, it is necessary to prepare nutrient carrier dosage forms suitable for children's digestion in a targeted manner based on the children's own developmental characteristics and nutritional needs, so as to meet the recommended intake in the National Food Safety Standard for Children's Formula Foods GB10767-2021 and the National Food Safety Standard for the Use of Food Nutrient Fortifiers GB14880-2012.

[0005] Chinese invention patent CN1015959177A discloses a method for preparing vitamin-fortified children's yogurt, but the vitamin-fortified yogurt product described in the invention is only to directly compound and ferment the optimized vitamins with premixes such as fresh milk, stabilizers, and fermented bacteria, and then fill them. The research on the bioavailability of nutrients in the product is not involved, so the nutritional effect of the yogurt product on children's bodies is still unclear. Chinese invention patent CN109198013A discloses a quinoa yogurt for infants and young children, which belongs to the field of infant food. However, the quinoa yogurt product described in the invention is only to physically mix and stir quinoa porridge with fermented yogurt and then fill it, and the research on the gastrointestinal digestion and absorption function of infants and young children is not involved, so the applicability of the yogurt product to infants and young children is still unclear.

[0006] Nutrients in food usually have problems such as poor solubility, poor processing and storage stability, and low bioavailability, which limit their absorption and utilization in the body. In order to improve the stability of nutrients in the gastrointestinal environment and their bioavailability, the food processing industry has applied many food-grade delivery systems, including emulsions, gels (nanogels, microgels and emulsion gels), nanoparticles and liposomes, but it should be pointed out that few applications are specifically tested and designed for children's low enzyme activity digestive systems. At the same time, with the rise of the "clean label" concept, higher process requirements are placed on the preparation of these delivery systems. Therefore, how to reasonably design the digestive system of children and reduce the use of food additives is an urgent problem that needs to be solved. Summary of the invention

[0007] Technical issues

[0008] At present, dynamic high-pressure microfluidization technology has been widely used in the modification of protein mono-systems. This technology can improve the functional properties of proteins such as solubility, emulsification, antioxidant properties and environmental stability, and give them the ability to carry nutrients. Therefore, protein nanoparticles pretreated with microfluidization will effectively load fat-soluble vitamins and slowly release them in the gastrointestinal environment. Their application in yogurt products will meet children's own digestion and absorption characteristics and nutritional needs. However, there are still few existing technologies for using dynamic high-pressure microfluidization technology to prepare foods specifically for children.

[0009] Technical content

[0010] In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing soy protein isolate-vitamin nanoparticles with high bioavailability, the method comprising the following steps:

[0011] S1. Pretreatment of soy protein isolate: dissolving soy protein isolate powder in water, stirring to obtain a protein solution, and then subjecting the protein solution to dynamic high-pressure microfluidization treatment at 10-140 MPa, and then drying to obtain pretreated soy protein isolate;

[0012] S2. Preparation of soy protein isolate-vitamin nanoparticles: dissolve vitamins in anhydrous ethanol to obtain a vitamin solution, add the pretreated soy protein isolate into water, stir to obtain a pretreated soy protein isolate solution, then add the vitamin solution, continue stirring, and freeze-dry to obtain soy protein isolate-vitamin nanoparticles with high bioavailability.

[0013] Furthermore, dynamic high-pressure microfluidization pretreatment was performed for 2-4 cycles in S1.

[0014] Furthermore, the pressure of the dynamic high-pressure microfluidic pretreatment in S1 is 40 to 120 MPa.

[0015] Specifically, optionally, the pressure of the dynamic high-pressure microjet pretreatment in S1 is 80 MPa.

[0016] Furthermore, the vitamins in S2 are one or more of vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, and vitamin K.

[0017] Preferably, the vitamin in S2 is vitamin D.

[0018] Specifically, the vitamin in S2 is vitamin D 3 .

[0019] Furthermore, the concentration of the vitamin solution in S2 is 3 to 8 mg / mL.

[0020] Preferably, the concentration of the vitamin solution in S2 is 3 to 5 mg / mL.

[0021] Furthermore, the concentration of the pretreated soy protein isolate solution in S2 is 0.3-0.8% (w / v).

[0022] Preferably, the concentration of the pretreated soy protein isolate solution in S2 is 0.5% (w / v).

[0023] Furthermore, the volume ratio of the vitamin solution to the pretreated soy protein isolate solution in S2 is (1-5):(95-99).

[0024] Preferably, the volume ratio of the vitamin solution to the pretreated soy protein isolate solution in S2 is 1:49.

[0025] The method of the present invention is applied in the technical field of preparing children's food processing.

[0026] The invention provides soybean protein isolate-vitamin nanoparticles with high bioavailability prepared by the method.

[0027] Application of the soybean protein isolate-vitamin nanoparticles with high bioavailability prepared by the invention in the preparation of children's food.

[0028] The present invention provides a method for preparing a vitamin-fortified children's yogurt product, comprising the following steps:

[0029] Step 1: heat-treating the fresh milk, adding white sugar and highly bioavailable soy protein isolate-vitamin nanoparticles, and cooling the milk to 38-42° C. to obtain cooled fresh milk;

[0030] Step 2: Add the bacteria to the cooled fresh milk for fermentation, then cool and mature it, and finally package and store it to obtain vitamin-fortified yogurt.

[0031] Furthermore, the heat treatment conditions in step 1 are: heat treatment at 80-100° C. for 10-15 min.

[0032] Furthermore, the added weight of the white sugar in step 1 is 8-12% of the weight of the fresh milk.

[0033] Furthermore, the addition amount of the highly bioavailable soy protein isolate-vitamin nanoparticles described in step 1 is 0.001-0.01% by weight of fresh milk.

[0034] Preferably, the amount of soy protein isolate-vitamin nanoparticles added should meet the requirement of vitamin D in the prepared product. 3 The amount added should not exceed 40μg / kg.

[0035] Furthermore, the bacterial species described in step 2 are thermophilic Streptococcus and bulgaricus Lactobacillus, and the mass ratio of thermophilic Streptococcus to bulgaricus Lactobacillus is 0.5-1.5:1.

[0036] Furthermore, the weight of the thermophilic streptococci and bulgaricus added in step 2 is 0.1-0.2% of the weight of the cooled fresh milk.

[0037] Furthermore, the fermentation conditions in step 2 are fermentation in a constant temperature incubator at 40-42° C. for 6-10 hours.

[0038] Furthermore, the cooling and ripening time in step 2 is 20-28 hours.

[0039] Beneficial Effects

[0040] (1) The present invention uses dynamic high-pressure microfluidization, which is a new environmentally friendly and advanced high-pressure homogenization processing technology that can effectively modify the structure of proteins.

[0041] (2) The present invention utilizes dynamic high-pressure microfluidization to process soybean protein isolate so that it reacts with vitamin D 3 There is a stronger binding force between the nanoparticles, and the nanoparticles have a more uniform size distribution, thus giving soy protein isolate-vitamin D 3 Nanoparticles have good dispersion. This increases the amount of vitamin D 3 The encapsulation rate, loading capacity, stability and other characteristics of the product enable it to better protect vitamin D during digestion. 3 Protected from chemical degradation, thus improving its digestive stability and bioavailability.

[0042] (3) In practical applications, vitamin D is loaded into soy protein isolate after microfluidization pretreatment. 3 The nanoparticles can be used as food nutrient enhancers in yogurt to improve its bioavailability after digestion. The fat-soluble nutrients in the children's yogurt of the present invention have good digestion stability and high bioavailability in the gastrointestinal tract, which is in line with the developmental characteristics of children and is suitable for children to eat. In summary, the food-grade vitamin D prepared by the present invention is 3 The addition of nanoparticles can improve the nutritional efficacy and edible quality of yogurt. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a comparison of Fourier infrared spectra of soy protein isolate-vitamin D3 nanoparticles and their preparation materials;

[0044] Figure 2 It is a comparison chart of X-diffraction spectra of soy protein isolate-vitamin D3 nanoparticles and their preparation raw materials;

[0045] Figure 3 It is the fluorescence spectrum of soy protein isolate-vitamin D3 nanoparticles and their preparation raw materials;

[0046] Figure 4 It is the surface hydrophobicity of soy protein isolate-vitamin D3 nanoparticles;

[0047] Figure 5 is the loading capacity and encapsulation efficiency of soy protein isolate-vitamin D3 nanoparticles;

[0048] Figure 6 It is the bioavailability of vitamin D3 from soy protein isolate-vitamin D3 nanoparticles;

[0049] Figure 7 is the bioavailable rate of vitamin D3 in vitamin D3 fortified yogurt;

[0050] Figure 8 It is a physical visual image of vitamin D3 fortified yogurt. DETAILED DESCRIPTION

[0051] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0052] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0053] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0054] Source of raw materials:

[0055] Soy protein isolate was purchased from Shandong Shansong Bioengineering Group Co., Ltd. (Shandong, China). 3 (purity greater than 98%) was purchased from Maclean (Shanghai, China); Streptococcus thermophilus and Lactobacillus bulgaricus were purchased from China Industrial Microbiological Culture Collection Center (CICC) with the collection numbers 6048 and 6037, respectively; other raw materials used in the examples were purchased commercially unless otherwise specified.

[0056] Testing process:

[0057] Surface hydrophobicity detection:

[0058] The surface hydrophobicity of the nanoparticles was determined using 8-aniline-1-naphthalenesulfonic acid (ANS) as a fluorescent probe. The sample solution (2 mg / mL) was diluted to 0.0375, 0.075, 0.1125, 0.15, and 0.1875 mg / mL, respectively. Then 4 mL of the sample solution was mixed with 20 μL of 8 mM ANS solution, and the fluorescence intensity was measured. The excitation wavelength was set to 390 nm and the emission wavelength was set to 470 nm. The linear slope of the fluorescence intensity-protein concentration (mg / mL) was used as the hydrophobicity index H 0 .

[0059] Encapsulation efficiency and loading capacity detection:

[0060] Total vitamin D was obtained by dispersing 10 mg of freeze-dried nanoparticle powder in 10 mL of ethanol and sonicating in an ice bath for 10 min. 3 Another batch of the same dispersion was vortexed (30 s) at room temperature to obtain free vitamin D3 Afterwards, the samples were centrifuged at 10,000 g for 10 min at 4 °C, and the absorbance of the supernatant was measured at 265 nm using a UV-visible spectrophotometer. Preparation of Vitamin D 3 -Standard curve of ethanol Y = 0.0419x + 0.0241 (R 2 =0.9991, Y = absorbance, X = vitamin D 3 Concentration (μg / mL) to determine vitamin D 3 The encapsulation efficiency and loading capacity are calculated by the following formula:

[0061]

[0062]

[0063] Bioavailability determination:

[0064] Measuring vitamin D in digesta and mixed micelles 3 concentration to calculate the bioavailability of vitamins. The digestate refers to the sample collected from the gastrointestinal tract after the digestion process, which contains unabsorbed residues, undigested food residues, and digested and absorbed substances. The mixed micelle refers to a structure formed in the gastrointestinal tract, which is composed of fat-soluble nutrients and bile acids and other substances. During the gastrointestinal digestion process, fat-soluble nutrients such as fat and fat-soluble vitamins combine with bile acids to form mixed micelles. The formation of mixed micelles helps the digestion and absorption of fat-soluble nutrients.

[0065] The micellar layer obtained by ultracentrifugation separation in the present invention is the layer where the mixed micelles are located, and the micellar layer contains a complex of fat-soluble nutrients and bile acids, etc. By extracting the micellar phase, the bioavailability of fat-soluble nutrients in the digestion process can be further studied and measured.

[0066] To extract vitamin D 3 , an aliquot of 3 mL of the sample was mixed with 3 mL of a hexane:ethanol (1:1, v / v) mixture. The resulting system was then centrifuged at 4000 rpm for 2 minutes and the supernatant layer was collected. After three extraction processes, the combined extracts were mixed with a saturated sodium chloride solution and then centrifuged again to remove any excess aqueous phase. The supernatant was collected and dried under nitrogen. An aliquot of ethanol was added to dissolve the vitamin D 3 , the absorbance of the solution was measured at 265 nm.

[0067] Vitamin D 3 The bioavailability was calculated using the following formula:

[0068]

[0069] Simulated gastrointestinal digestion in children:

[0070] Nanoparticles and yogurt were mixed with simulated gastric fluid (SGF) at a ratio of 2:1 (v / v) in an oscillating water bath preheated to 37 °C and at a speed of 95 rpm, and then CaCl was added. 2 and pepsin to a final concentration of 0.075 mM and 268 U / mL, respectively. The pH was adjusted to 4.2 with hydrochloric acid (1 M). The mixture was incubated at 37 °C for 120 min to simulate gastric digestion. Finally, simulated intestinal fluid (SIF) was added to the gastric digestion fluid at a ratio of 1:1 (v / v) to the initial sample. CaCl was then added to the mixture. 2 , bile salts and trypsin to a final concentration of 2.25 mM, 3.1 mM and 90 U / mL, respectively. The pH was adjusted to 6.5 with NaOH (1 M). The mixture was incubated at 37°C for 120 min to simulate small intestinal digestion.

[0071] Simulated gastrointestinal digestion in adults:

[0072] Simulated gastric fluid consists of 3.2 mg / mL pepsin, 0.7% (v / v) hydrochloric acid, and 2 mg / mL NaCl. The sample was mixed with gastric fluid in a 1:1 ratio, the pH of the mixture was always kept at 2.0 with 0.1 M HCl, and incubated at 37°C with continuous shaking (95 r / min) for 2 hours to simulate gastric digestion. Simulated intestinal fluid consists of 5 mg / mL bile salts, 0.4 mg / mL pancreatic enzymes, and 3.2 mg / mL lipase. The gastric digesta were mixed with intestinal fluid in a 1:1 ratio, the pH of the mixture was kept at 7.0 with 0.1 M NaOH, and incubated at 37°C with continuous shaking (100 r / min) for 2 hours to simulate small intestinal digestion.

[0073] Example 1

[0074] Preparation of soy protein isolate-vitamin D in this example 3 The method of producing nanoparticles comprises the following steps:

[0075] S1. Pretreatment of soy protein isolate: dissolve soy protein isolate powder in deionized water, stir magnetically at room temperature for 4 h, treat the protein solution at a concentration of 1% (w / v) at a pressure of 40 MPa for 3 cycles, and then freeze-dry the sample in vacuum for 56 h to obtain soy protein isolate treated by dynamic high-pressure microfluidization;

[0076] S2, Soy Protein Isolate-Vitamin D 3 Preparation of Nanoparticles:

[0077] First, vitamin D 3 Dissolve in anhydrous ethanol to obtain a concentration of 5 mg / mL of vitamin D 3 Solution.

[0078] Secondly, the dynamic high-pressure microfluidization treated soy protein isolate powder was dispersed in PBS (0.01 mol / L, pH 7) to obtain a dynamic high-pressure microfluidization treated soy protein isolate solution with a concentration of 0.5% (w / v), and the solution was stirred continuously at room temperature for 1 h.

[0079] Then, under stirring, 1 mL of vitamin D 3 The solution was continuously added to 49 mL of dynamic high-pressure microfluidization treated soy protein isolate solution.

[0080] Finally, the obtained solution was stirred at 500 rpm for 5 h at 25 °C, and all samples were freeze-dried for 48 h to obtain soy protein isolate-vitamin D 3 Nanoparticles were stored at -20°C.

[0081] Example 2

[0082] Preparation of soy protein isolate-vitamin D in this example 3 The method of producing nanoparticles comprises the following steps:

[0083] S1. Pretreatment of soy protein isolate: dissolve soy protein isolate powder in deionized water, stir magnetically at room temperature for 4 h, treat the protein solution at a concentration of 1% (w / v) at a pressure of 80 MPa for 3 cycles, and then freeze-dry the sample in vacuum for 56 h to obtain soy protein isolate treated by dynamic high-pressure microfluidization;

[0084] S2, Soy Protein Isolate-Vitamin D 3 Preparation of nanoparticles: First, vitamin D 3 Dissolve in anhydrous ethanol to obtain a concentration of 5 mg / mL of vitamin D 3 Solution.

[0085] Secondly, the dynamic high-pressure microfluidization treated soy protein isolate powder was dispersed in PBS (0.01 mol / L, pH 7) to obtain a dynamic high-pressure microfluidization treated soy protein isolate solution with a concentration of 0.5% (w / v), and the solution was stirred continuously at room temperature for 1 h.

[0086] Then, under stirring, 1 mL of vitamin D 3 The solution was continuously added to 49 mL of dynamic high-pressure microfluidization treated soy protein isolate solution.

[0087] Finally, the obtained solution was stirred at 500 rpm for 5 h at 25 °C, and all samples were freeze-dried for 48 h to obtain soy protein isolate-vitamin D 3 Nanoparticles were stored at -20°C.

[0088] Example 3

[0089] Preparation of soy protein isolate-vitamin D in this example 3 The method of producing nanoparticles comprises the following steps:

[0090] S1. Pretreatment of soy protein isolate: dissolve soy protein isolate powder in deionized water, stir magnetically at room temperature for 4 h, treat the protein solution at a concentration of 1% (w / v) at a pressure of 120 MPa for 3 cycles, and then freeze-dry the sample in vacuum for 56 h to obtain soy protein isolate treated by dynamic high-pressure microfluidization;

[0091] S2, Soy Protein Isolate-Vitamin D 3 Preparation of Nanoparticles:

[0092] First, vitamin D 3 Dissolve in anhydrous ethanol to obtain a concentration of 5 mg / mL of vitamin D 3 Solution.

[0093] Secondly, the dynamic high-pressure microfluidization treated soy protein isolate powder was dispersed in PBS (0.01 mol / L, pH 7) to obtain a dynamic high-pressure microfluidization treated soy protein isolate solution with a concentration of 0.5% (w / v), and the solution was stirred continuously at room temperature for 1 h.

[0094] Then, under stirring, 1 mL of vitamin D 3 The solution was continuously added to 49 mL of dynamic high-pressure microfluidization treated soy protein isolate solution.

[0095] Finally, the obtained solution was stirred at 500 rpm for 5 h at 25 °C, and all samples were freeze-dried for 48 h to obtain soy protein isolate-vitamin D 3 Nanoparticles were stored at -20°C.

[0096] Example 4

[0097] The soy protein isolate-vitamin D obtained in Example 1 3 Nanoparticles for Vitamin D 3 Fortified yogurt preparation.

[0098] According to GB 14880-94 "Hygienic Standards for the Use of Food Nutrition Fortifiers", vitamin D 3 The maximum addition amount in milk and milk beverages is 40μg / kg, and the vitamin D nanoparticles obtained by 40MPa microfluidization treatment 3 The loading amount was 1.93 μg / mg, which determined that soy protein isolate-vitamin D 3 The amount of nanoparticles added was 0.00168%, of which vitamin D3 The added amount is 32.424μg / kg, and the content meets the national standards.

[0099] The specific steps are as follows: the fresh milk is heat treated at 90°C for 10 minutes, white sugar is added to 9% of the weight of the fresh milk, and the soy protein isolate-vitamin D is processed by dynamic high-pressure microfluidization. 3 The weight of the nanoparticles added was 0.00168% of the weight of the fresh milk, and the milk was cooled to 40°C to obtain cooled fresh milk; thermophilic streptococci and bulgaricus lactobacillus were added to the cooled fresh milk, and the added weight was 0.1% of the weight of the cooled fresh milk, and then the milk was fermented in a constant temperature incubator at 40°C for 8 hours, and then cooled and matured for 24 hours, and then packaged and stored to obtain vitamin D 3 Fortified yogurt.

[0100] Example 5

[0101] The soy protein isolate-vitamin D obtained in Example 2 3 Nanoparticles for Vitamin D 3 Fortified yogurt preparation.

[0102] According to GB 14880-94 "Hygienic Standards for the Use of Food Nutrition Fortifiers", vitamin D 3 The maximum addition amount in milk and milk beverages is 40 μg / kg, and the vitamin D nanoparticles obtained by 80MPa microfluidization treatment 3 The loading amount was 2.31 μg / mg, which determined that soy protein isolate-vitamin D 3 The amount of nanoparticles added was 0.00141%, of which vitamin D 3 The added amount is 32.571μg / kg, and the content meets the national standards.

[0103] The specific steps are as follows: the fresh milk is heat treated at 90°C for 10 minutes, white sugar is added to 9% of the weight of the fresh milk, and the soy protein isolate-vitamin D is processed by dynamic high-pressure microfluidization. 3 The weight of the nanoparticles added was 0.00141% of the weight of the fresh milk, and the milk was cooled to 40°C to obtain cooled fresh milk; thermophilic streptococci and bulgaricus lactobacillus were added to the cooled fresh milk, and the added weight was 0.1% of the weight of the cooled fresh milk, and then the milk was fermented in a constant temperature incubator at 40°C for 8 hours, and then cooled and matured for 24 hours, and then packaged and stored to obtain vitamin D 3 Fortified yogurt.

[0104] Example 6

[0105] The soy protein isolate-vitamin D obtained in Example 3 3 Nanoparticles for Vitamin D 3 Fortified yogurt preparation.

[0106] According to GB 14880-94 "Hygienic Standards for the Use of Food Nutrition Fortifiers", vitamin D 3 The maximum addition amount in milk and milk beverages is 40 μg / kg, and the vitamin D nanoparticles obtained by 120MPa microfluidization treatment 3 The loading amount was 1.96 μg / mg, which determined that soy protein isolate-vitamin D 3 The amount of nanoparticles added was 0.00166%, of which vitamin D 3 The added amount is 32.536μg / kg, and the content meets the national standards.

[0107] The specific steps are as follows: the fresh milk is heat treated at 90°C for 10 minutes, white sugar is added to 9% of the weight of the fresh milk, and the soy protein isolate-vitamin D is processed by dynamic high-pressure microfluidization. 3 The weight of the nanoparticles added was 0.00166% of the weight of the fresh milk, and the milk was cooled to 40°C to obtain cooled fresh milk; thermophilic streptococci and bulgaricus lactobacillus were added to the cooled fresh milk, and the added weight was 0.1% of the weight of the cooled fresh milk, and then the milk was fermented in a constant temperature incubator at 40°C for 8 hours, and then cooled and matured for 24 hours, and then packaged and stored to obtain vitamin D 3 Fortified yogurt.

[0108] Comparative Example 1

[0109] Preparation of Soy Protein Isolate-Vitamin D in this Comparative Example 3 The method of producing nanoparticles comprises the following steps:

[0110] S1. Pretreatment of soy protein isolate: dissolve soy protein isolate powder in deionized water, stir magnetically at room temperature for 4 h, treat the protein solution at a concentration of 1% (w / v) at a pressure of 10 MPa for 3 cycles, and then freeze-dry the sample in vacuum for 56 h to obtain soy protein isolate treated by dynamic high-pressure microfluidization;

[0111] S2, Soy Protein Isolate-Vitamin D 3 Preparation of Nanoparticles:

[0112] First, vitamin D 3 Dissolve in anhydrous ethanol to obtain a concentration of 5 mg / mL of vitamin D 3 Solution.

[0113] Secondly, the soy protein isolate powder was dispersed in PBS (0.01 mol / L, pH 7) to obtain a soy protein isolate solution with a concentration of 0.5% (w / v), and the solution was stirred continuously at room temperature for 1 h.

[0114] Then, under stirring, 1 mL of vitamin D 3 The solution was continuously added to 49 mL of soy protein isolate solution.

[0115] Finally, the obtained solution was stirred at 500 rpm for 5 h at 25 °C, and all samples were freeze-dried for 48 h to obtain soy protein isolate-vitamin D 3 Nanoparticles were stored at -20°C.

[0116] The difference between this comparative example and Examples 1, 2 and 3 is that the working pressure of the dynamic high-pressure microfluidization treatment is 10 MPa, and the soy protein isolate-vitamin D 3 Nanoparticles, according to GB 14880-94 "Food Nutrition Fortifier Use Hygiene Standard" Vitamin D 3 The maximum addition amount in milk and milk beverages is 40 μg / kg, and the vitamin D nanoparticles obtained by 10MPa microfluidization treatment 3 The loading amount was 0.71 μg / mg, which determined that soy protein isolate-vitamin D 3 The amount of nanoparticles added was 0.00458%, of which vitamin D 3 The added amount is 32.518μg / kg, and the content meets the national standards.

[0117] The remaining yogurt operation process and parameter settings are the same as those in Examples 4, 5, and 6.

[0118] Comparative Example 2

[0119] The method for preparing soy protein isolate-vitamin D3 nanoparticles in this comparative example comprises the following steps:

[0120] S1. Pretreatment of soy protein isolate: dissolve soy protein isolate powder in deionized water, stir magnetically at room temperature for 4 h, treat the protein solution at a concentration of 1% (w / v) at a pressure of 140 MPa for 3 cycles, and then freeze-dry the sample in vacuum for 56 h to obtain soy protein isolate treated by dynamic high-pressure microfluidization;

[0121] S2, Soy Protein Isolate-Vitamin D 3 Preparation of Nanoparticles:

[0122] First, vitamin D 3 Dissolve in anhydrous ethanol to obtain a concentration of 5 mg / mL of vitamin D 3 Solution.

[0123] Secondly, the soy protein isolate powder was dispersed in PBS (0.01 mol / L, pH 7) to obtain a soy protein isolate solution with a concentration of 0.5% (w / v), and the solution was stirred continuously at room temperature for 1 h.

[0124] Then, under stirring, 1 mL of vitamin D 3 The solution was continuously added to 80-100 mL of soy protein isolate solution.

[0125] Finally, the obtained solution was stirred at 500 rpm for 5 h at 25 °C, and all samples were freeze-dried for 48 h to obtain soy protein isolate-vitamin D 3 Nanoparticles were stored at -20°C.

[0126] The difference between this comparative example and Examples 1, 2 and 3 is that the working pressure of the dynamic high-pressure microfluidization treatment is 140 MPa, and the soy protein isolate-vitamin D 3 Nanoparticles, according to GB 14880-94 "Food Nutrition Fortifier Use Hygiene Standard" Vitamin D 3 The maximum addition amount in milk and milk beverages is 40 μg / kg, and the vitamin D nanoparticles obtained by 140MPa microfluidization treatment 3 The loading amount was 1.655 μg / mg, which determined that soy protein isolate-vitamin D 3 The amount of nanoparticles added was 0.00196%, of which vitamin D 3 The added amount is 32.438μg / kg, and the content meets the national standards.

[0127] The remaining yogurt operation process and parameter settings are the same as those in Examples 4, 5, and 6.

[0128] Comparative Example 3

[0129] This comparative example is the preparation of yogurt, which is different from Examples 4, 5, and 6 in that vitamin D is directly added during the preparation of yogurt. 3 The remaining operation process and parameter settings are the same as those in Examples 1, 2, and 3.

[0130] According to GB 14880-94 "Hygienic Standards for the Use of Food Nutrition Fortifiers", vitamin D 3 The maximum amount of vitamin D added to milk and milk beverages is 40 μg / kg. 3 The monomer addition amount is 0.00000325%, of which vitamin D 3 The added amount is 32.5μg / kg, and the content meets the national standards.

[0131] Comparative Example 4

[0132] This comparative example is prepared according to Example 2 3 The operation process and parameter settings of the nanoparticles and yogurt are the same as those in Example 2.

[0133] The difference between this comparative example and Example 2 is that this comparative example is a digestion test based on an in vitro simulated digestion model for adults.

[0134] Test results

[0135] (1) Figure 1 The soy protein isolate-vitamin D prepared in Examples 1 to 3 and Comparative Examples 1 to 2 3 Nanoparticles and Vitamin D of Comparative Example 3 3 Fourier transform infrared spectrum comparison of monomers, Figure 2 The X-ray diffraction spectrum comparison chart of the above samples.

[0136] Figure 1 The dynamic high-pressure microfluidization treatment had little effect on the CH stretching vibration zone of protein. The dynamic high-pressure microfluidization treatment did not affect the soy protein isolate and vitamin D 3 New chemical structures or groups are generated, but the changes in peak wave number indicate that the functional group content in the protein has changed. These changes in amide I and II bands indicate that soy protein isolate and vitamin D 3 There is an intermolecular electrostatic force, but the displacement is not obvious, indicating that the electrostatic interaction exists but is not the key force.

[0137] Depend on Figure 2 It can be seen that when soy protein isolate and vitamin D 3 When formed into nanoparticles, its X-ray diffraction spectrum is similar to that of natural soy protein isolate, and vitamin D 3 The diffraction peak of vitamin D disappeared. This result shows that 3 Effectively embedded in the matrix formed by soy protein isolate, forming an amorphous form of vitamin D 3 . Soy Protein Isolate - Vitamin D 3 The diffraction peak height of the nanoparticles first increases and then decreases. Soy protein isolate-vitamin D 3 The height variation of the diffraction peaks of the nanoparticles represents the different amounts of amorphous structures formed. Soy protein isolate-vitamin D nanoparticles were prepared under microfluidization pretreatment at an intensity of 80 MPa. 3 The diffraction peak intensity of nanoparticles was the highest, indicating that more amorphous structures were formed.

[0138] (2) Figure 3 The soy protein isolate-vitamin D prepared in Examples 1 to 3 and Comparative Examples 1 to 2 3The fluorescence spectrum comparison of nanoparticles shows that after dynamic high-pressure microfluidization treatment, the fluorescence intensity of soy protein isolate increases first and then decreases with the increase of treatment intensity. The reason for the decrease may be that the dynamic high-pressure microfluidization treatment exposes the tryptophan residues inside the protein, and the high pressure induces the fluorescence quenching of the protein, which leads to a decrease in the fluorescence intensity of soy protein isolate. When the microfluidization intensity continues to increase to 120MPa, the fluorescence intensity increases. This is because the excessive energy causes the protein molecules to physically aggregate, allowing the originally unfolded protein structure to repolymerize and the amino acid residues to be re-embedded, thereby increasing the fluorescence intensity. Compared with soy protein isolate, soy protein isolate-vitamin D 3 The fluorescence intensity of nanoparticles decreased significantly, which is due to the interaction of soy protein isolate with vitamin D 3 After binding, it can produce a quenching effect on soy protein isolate, exposing the internal tryptophan residues to a polar environment, thereby causing fluorescence quenching of the soy protein isolate and a decrease in fluorescence intensity.

[0139] (3) Figure 4 Soy protein isolate-vitamin D prepared in Examples 1 to 3 and Comparative Examples 1 to 2 3 Surface hydrophobicity analysis of nanoparticles, soy protein isolate and vitamin D 3 After binding, it showed hydrophobicity. This may be because the soy protein isolate-vitamin D 3 Nanoparticles are formed by hydrophobic bonding. Soy protein isolate-vitamin D with increasing treatment pressure 3 The surface hydrophobicity of the nanoparticles first decreases and then increases. Soy protein isolate-vitamin D under 80MPa treatment 3 The surface hydrophobicity of natural soy protein isolate is the highest. The hydrophobic area of ​​natural soy protein isolate is buried inside the molecule. Appropriate microfluidization treatment will expose some hydrophobic areas of soy protein isolate to the surface of the molecule, which is beneficial to the hydrophobic effect of vitamin D 3 Non-covalent binding with proteins. With the increasing intensity of microfluidization treatment, the surface hydrophobicity of nanoparticles increases. Under 80MPa treatment, microfluidization can change the tertiary structure of soy protein isolate, leading to protein unfolding and extension of non-covalent bonds of protein molecules, increasing the number of hydrophobic groups and regions in the molecules exposed to polar environments. However, excessive treatment will also lead to a decrease in protein flexibility and an increase in protein aggregation, thereby reducing its surface hydrophobicity.

[0140] Comparative Example 1 Example 1 Example 2 Example 3 Comparative Example 2 Surface hydrophobicity 1254.8 1902.7 3494.0 2775.8 2004.0

[0141] Table 1 Soy protein isolate-vitamin D 3 Analysis of Surface Hydrophobicity of Nanoparticles

[0142] (4) Figure 5The soy protein isolate-vitamin D prepared in Examples 1 to 3 and Comparative Examples 1 to 2 3 Analysis of the loading capacity and encapsulation efficiency of nanoparticles; Soy protein isolate for vitamin D with increasing treatment intensity 3 The encapsulation efficiency and loading capacity first increased and then decreased. The hydrophobic region in soy protein is the binding site for vitamin D 3 However, due to its spherical structure, the hydrophobic area is wrapped inside, resulting in the natural soy protein isolate and vitamin D 3 The binding rate of soy protein isolate is low. Microfluidization can unfold the structure of the protein and expose the hidden hydrophobic groups in the soy protein isolate molecules to the external environment. Therefore, soy protein isolate has a low binding rate to vitamin D. 3 The adsorption capacity of soy protein isolate was enhanced, resulting in an increase in encapsulation efficiency and loading capacity. When the microfluidization intensity was 80MPa, the encapsulation efficiency (67.41%) and loading capacity (2.31μg / mg) reached the highest. The encapsulation efficiency and loading capacity decreased as the microfluidization pretreatment intensity increased from 10MPa to 120MPa, indicating that pretreatment of soy protein isolate with too high pressure would cause protein aggregation reaction, bury the hydrophobic area, and cause the binding with vitamin D. 3 The binding area is reduced, resulting in a decrease in encapsulation efficiency and loading capacity. This shows that appropriate microfluidization pretreatment can effectively improve the encapsulation efficiency of soy protein isolate for vitamin D 3 carrying capacity.

[0143] Comparative Example 1 Example 1 Example 2 Example 3 Comparative Example 2 Loading amount / μg / mg 0.710 1.930 2.310 1.960 1.655 Encapsulation rate / % 12.990 54.230 67.400 56.060 31.104

[0144] Table 2 Soy protein isolate-vitamin D 3 Analysis of nanoparticle loading and encapsulation efficiency

[0145] (5) Figure 6 The soy protein isolate-vitamin D prepared in Examples 1 to 3 and Comparative Examples 1 to 2 3 Vitamin D in Nanoparticles 3 The results of the bioavailability analysis and the in vitro simulated digestion test of adults in comparative example 4 show that high-pressure dynamic microfluidization pretreatment can inhibit the 3 Release of vitamin D in the stomach 3 After combining with soy protein isolate, the protective effect of soy protein isolate inhibits the activity of oxidase in the gastrointestinal tract, making vitamin D 3 It is able to be transported smoothly through the upper gastrointestinal tract to the small intestine, thereby reducing its release in the stomach but promoting its release in the small intestine. 3 The reason why the bioavailability of vitamin D increased first and then decreased with the intensity of microfluidization treatment may be that appropriate microfluidization pretreatment can open the protein structure, increase the flexibility of protein molecules, and expose more trypsin recognition sites. 3Induced protein conformational changes facilitate trypsin hydrolysis of soy protein isolate-vitamin D 3 Nanoparticles to boost vitamin D 3 However, excessive microfluidization pretreatment will cause protein aggregation and form a tight and orderly structure, resulting in a decrease in protein digestibility. This is not conducive to the digestion of soy protein isolate-vitamin D by intestinal trypsin. 3 Enzymatic hydrolysis of nanoparticles. Therefore, pretreatment of soy protein isolate with appropriate treatment intensity can better protect vitamin D 3 Protects against degradation caused by gastric acid corrosion, promotes its large-scale release in the small intestine, thereby increasing vitamin D 3 Compared with the digestion model of adults, the bioavailability of soy protein isolate-vitamin D treated by microfluidization 3 Vitamin D in a digestive model in children 3 The higher bioavailability of vitamin D in the adult digestive model may be due to the higher concentration of digestive enzymes and lower pH, which promotes the 3 Release in the stomach results in less release in the small intestine.

[0146] Comparative Example 1 Example 1 Example 2 Example 3 Comparative Example 2 Comparative Example 4 Bioavailability / % 41.88 49.60 74.40 61.80 43.00 53.00

[0147] Table 3 Soy protein isolate-vitamin D 3 Bioavailability analysis of nanoparticles

[0148] (6) Figure 7 Vitamin D prepared in Examples 4 to 6 and Comparative Examples 1 to 4 3 Vitamin D-fortified yogurt 3 The results of bioavailability analysis showed that the prepared vitamin D 3 Vitamin D in fortified yogurt after simulated in vitro digestion 3 Bioavailability and corresponding soy protein isolate-vitamin D 3 Vitamin D in nanoparticles 3 The change trend of bioavailability was the same, indicating that soy protein isolate-vitamin D 3 Vitamin D prepared from nanoparticles 3 Fortified yogurt can effectively increase vitamin D 3 Bioavailability and more suitable for children's digestion.

[0149] Comparative Example 1 Example 1 Example 2 Example 3 Comparative Example 2 Comparative Example 3 Comparative Example 4 Bioavailability / % 36.55 42.83 60.25 56.83 50.17 17.33 48.56

[0150] Table 4 Vitamin D 3 Bioavailability Analysis of Fortified Yogurt

[0151] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing soy protein isolate-vitamin nanoparticles with high bioavailability, characterized in that: The following steps are involved: S1. Pretreatment of soy protein isolate: dissolving soy protein isolate powder in water, stirring to obtain a protein solution, and then subjecting the protein solution to dynamic high-pressure microfluidization treatment at 10-140 MPa, and then drying to obtain pretreated soy protein isolate; S2. Preparation of soy protein isolate-vitamin nanoparticles: dissolve vitamins in anhydrous ethanol to obtain a vitamin solution, add the pretreated soy protein isolate into water, stir to obtain a pretreated soy protein isolate solution, then add the vitamin solution, continue stirring, and freeze-dry to obtain soy protein isolate-vitamin nanoparticles with high bioavailability.

2. The method according to claim 1, characterized in that In S1, the dynamic high-pressure microfluidic pretreatment is performed for 2-4 cycles; the pressure of the dynamic high-pressure microfluidic pretreatment is 40-120 MPa.

3. The method according to claim 1, characterized in that The vitamins in S2 are one or more of vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, and vitamin K.

4. The method according to claim 1, characterized in that In S2, the concentration of the vitamin solution is 3-8 mg / mL; the concentration of the pretreated soy protein isolate solution is 0.3-0.8%; and the volume ratio of the vitamin solution to the pretreated soy protein isolate solution in S2 is (1-5):(95-99).

5. Application of the method according to any one of claims 1 to 4 in the technical field of preparing children's food processing.

6. Soy protein isolate-vitamin nanoparticles with high bioavailability prepared according to the method of any one of claims 1 to 4.

7. Use of the highly bioavailable soy protein isolate-vitamin nanoparticles of claim 6 in the field of children's food preparation.

8. A method for preparing a vitamin-fortified children's yogurt product, characterized in that: The following steps are involved: Step 1: heat-treating fresh milk, adding white sugar and the highly bioavailable soy protein isolate-vitamin nanoparticles described in claim 6, and cooling the milk to 38-42° C. to obtain cooled fresh milk; Step 2: Add the bacteria to the cooled fresh milk for fermentation, then cool and mature it, and finally package and store it to obtain vitamin-fortified yogurt.

9. The method according to claim 8, characterized in that In step 1, the heat treatment condition is 80-100° C. for 10-15 min; the added weight of the white sugar is 8-12% of the weight of fresh milk; and the added amount of the highly bioavailable soy protein isolate-vitamin nanoparticles is 0.001-0.01% of the weight of fresh milk.

10. The method according to claim 8, characterized in that In step 2, the bacteria are thermophilic streptococcus and bulgaricus lactobacillus; the mass ratio of thermophilic streptococcus to bulgaricus lactobacillus is 0.5-1.5:1; the added weight of thermophilic streptococcus and bulgaricus lactobacillus is 0.1-0.2% of the weight of the cooled fresh milk.

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