Preparation and application of anti-vomiting protein emulsion

Abstract

The invention discloses preparation and application of an anti-vomiting protein emulsion, and belongs to the field of food processing. High-hydrolysis-degree low-bitterness protein is obtained through composite enzymolysis, then fermentation is conducted through lactobacillus casei and saccharomyces cerevisiae, bitterness and free amino acid are further removed, osmotic pressure is reduced, and finally oligosaccharide and medium-chain and long-chain diglyceride are added for high-pressure homogenization to form stable emulsion. The prepared protein emulsion is low in digestion burden, low in sensitization, free of bitter taste, easy to accept and capable of preventing vomiting.

Description

TECHNICAL FIELD

[0001] The present application relates to the preparation and application of a vomit-preventing protein emulsion, belonging to the field of food processing. BACKGROUND

[0002] With the acceleration of the aging process of Chinese society, irregular life rhythm and the increasing prevalence of unbalanced dietary structure, residents are generally suffering from health problems such as skeletal muscle loss, decreased immunity and increased obesity rate. At the same time, the public health awareness is gradually improving, and protein dietary supplements are increasingly widely used in sports nutrition, weight management, elderly nutrition fortification and special population recovery period nutrition support. However, in the actual application process of existing liquid high-protein nutritional preparations, especially in high-protein intake or weak gastrointestinal function populations, gastrointestinal discomfort reactions such as nausea, stomach upset and even vomiting often occur, which seriously affects the compliance and application effect of the product. The existing technology generally believes that hydrolyzing protein can reduce the molecular weight of protein, reduce the digestive burden, and thus improve the gastrointestinal tolerance. However, practice shows that simply increasing the degree of protein hydrolysis cannot effectively avoid gastrointestinal discomfort, but may form a transient high-stimulation environment in the gastrointestinal tract due to the massive release of free amino acids and low-molecular peptides in a short time, which may trigger adverse reactions.

[0003] The main mechanism of vomiting is that mechanical stimulation, chemical stimulation and osmotic pressure change can induce the Ca 2+ dependent release of emetic neurotransmitters by enterochromaffin cells in the gastrointestinal tract, activate the vagal afferent pathway and brainstem vomiting center, and trigger the vomiting reflex characterized by calcium signal cascade amplification. In the digestion process of existing liquid high-protein nutritional preparations, high degree of hydrolysis protein carries a large amount of free amino acids or bitter peptides, significantly increasing the osmotic pressure of the gastrointestinal tract. The body may reduce the load through reverse peristalsis and other ways to adjust the osmotic pressure, which may induce abnormal activation of enterochromaffin cells and easily induce vomiting; low degree of hydrolysis protein cannot effectively relieve the digestive burden, and protein cannot be fully digested and absorbed in the stomach and small intestine, which may cause intestinal flora disorder, abnormal fermentation and gas production after excessive entry into the large intestine, leading to abdominal distension and increasing the risk of nausea or vomiting; in addition, some proteins have poor water solubility and dispersibility, and the prepared liquid protein supplements often have a sticky taste, a strong oral retention feeling and poor palatability, which affects the drinking experience.

[0004] Patent CN202110786367.0 discloses a kind of anti-vomiting infant yak formula milk powder, only for infantile vomiting caused by reflux, but does not consider the high molecular weight casein in dairy products causes vomiting caused by digestion and metabolic burden;CN202511376418.7 discloses a kind of preparation method of hydrolyzed whey protein, although the bitterness of hydrolyzed protein is reduced by high pressure and other methods, but the specific value of the degree of hydrolysis is not specified;Patent CN202511366512.4 discloses a kind of control method, device, equipment and storage medium for preparing low-osmotic pressure protein nutritional product, but does not mention easy-to-digest, low-allergenic hydrolyzed protein product.

[0005] Therefore, there is still a lack of a protein supplement product in the prior art that can provide high-quality protein nutrition, easy digestion and absorption, and effectively avoid vomiting caused by gastrointestinal discomfort. SUMMARY

[0006] In view of the problem that existing liquid high-protein nutritional preparations are prone to form an acute stimulating environment in the gastrointestinal tract, induce abnormal activation of enterochromaffin cells, and thus cause nausea or vomiting, the purpose of the present application is to provide a preparation and application of anti-vomiting protein emulsion. The protein emulsion regulates the stimulation intensity, stimulation rate and stimulation peak distribution of hydrolyzed protein in the gastrointestinal tract, reduces the probability of the formation of emetic nerve signals under the premise of ensuring protein supply efficiency, and improves the gastrointestinal tolerance of the preparation, so as to be less likely to cause nausea or vomiting. The present application provides a kind of protein emulsion with low digestive burden, weak gastrointestinal stimulation, low allergenicity, no bitterness and easy acceptance, which can be used for preparing protein nutritional supplements. First, low-bitterness oligopeptides are obtained by heat-induced unfolding pretreatment combined with double enzymolysis; then free amino acids are removed by Lactobacillus casei fermentation and Saccharomyces cerevisiae fermentation, and the osmotic pressure is reduced; finally, low oligosaccharides and medium-long chain diglycerides are added to form a stable emulsion by high-pressure homogenization.

[0007] The first object of the present application is to provide a method for preparing a protein emulsion, comprising the steps of: (1) mixing protein with water, adjusting pH, heat-induced unfolding, cooling, then adding Lactobacillus helveticus cell wall protease for enzymolysis, and then adding soybean sprout protease for enzymolysis to obtain a hydrolyzed protein solution; (2) adding Lactobacillus casei fermentation to the hydrolyzed protein solution, and then adding Saccharomyces cerevisiae fermentation; after fermentation is completed, sterilization and enzyme inactivation are performed to obtain a hydrolyzed protein fermentation liquor; (3) adding low oligosaccharides and medium-long chain glycerides to the hydrolyzed protein fermentation liquor, mixing and homogenizing to obtain a protein emulsion; In step (1), the protein is one or more of whey protein, soy protein and pea protein. The preparation method of Lactobacillus helveticus cell wall protease is as follows: after culturing and collecting Lactobacillus helveticus cells, add buffer solution, mix well, break up, centrifuge to collect supernatant, purify and dry to obtain Lactobacillus helveticus cell wall protease; The preparation method of soybean sprout protease is as follows: soybean seeds are cultured in the dark until seedlings emerge; the seedlings are collected, freeze-dried into powder, mixed with salt solution, homogenized, incubated, and centrifuged to collect the supernatant; the supernatant is mixed with ammonium sulfate, allowed to stand, centrifuged to collect the protein precipitate; the protein precipitate is reconstituted, dialyzed, purified, and dried to obtain soybean sprout protease; In step (2), Lactobacillus casei was purchased from Shandong Pingao Pharmaceutical Co., Ltd., with product number / model number PG-LCG139 and preservation number CICC 23185; Saccharomyces cerevisiae was purchased from Jinan Jinyuyuan Biotechnology Co., Ltd., with product number / model number SC-324 and preservation number CICC 1001.

[0008] In one embodiment, the extraction method of Lactobacillus helveticus cell wall protease is as follows: culturing and collecting the strain, washing with CaCl2 buffer, gentle elution with ultrasonic-assisted EDTA, and chromatographic purification.

[0009] In one embodiment, the Lactobacillus helveticus was purchased from Jiuhe Biotechnology Co., Ltd., with product number / model number BL-012 and accession number ATCC 15009.

[0010] In one embodiment, the concentration of *Lactobacillus helveticus* cells after mixing with the buffer solution is 4 × 10⁻⁶. 8 ~10 9 CFU / mL.

[0011] In one embodiment, the ratio of protein to water in step (1) is 5-10 kg: 50-100 L; the pH adjustment is adjusted to 7.0-7.5; the heat-induced development is heated at 80-90℃ for 30-60 min; the enzymatic hydrolysis of Lactobacillus helveticus cell wall protease is performed by adding 1-2% (w / w) of Lactobacillus helveticus cell wall protease at 30-35℃ and 120-180 rpm for 1-2 h; the enzymatic hydrolysis of soybean sprouts is performed by adding 1-2% (w / w) of soybean sprout protease at 30-35℃ and 120-180 rpm for 3-5 h.

[0012] In one embodiment, the Lactobacillus casei fermentation in step (2) is Lactobacillus casei with 1000~1500 CFU / g protein added, fermented at 35~40℃ for 6~8 h; the Saccharomyces cerevisiae fermentation is Saccharomyces cerevisiae with 1500~2000 CFU / g protein added, fermented at 35~40℃ for 3~5 h.

[0013] In one embodiment, the mass ratio of protein: oligosaccharide: medium- and long-chain diglycerides in step (3) is 50~100:2~3:2~5.

[0014] In one embodiment, the homogenization in the preparation of soybean sprout protease is performed at 10-20 MPa for 5-10 min.

[0015] In one embodiment, the centrifugation in the preparation of soybean sprout protease is 8000~9000 rpm for 10~15 min.

[0016] In one embodiment, the ratio of supernatant to ammonium sulfate in the preparation of soybean sprout protease is 10-20 mL: 0.2-0.5 g.

[0017] A second objective of this invention is to provide a protein emulsion prepared by any of the methods described above.

[0018] A third objective of this invention is to provide a product containing the aforementioned protein emulsion, wherein the product is a pharmaceutical, health product, or food.

[0019] The fourth objective of this invention is to provide a method for improving the anti-vomiting and hypoallergenic properties of protein emulsions, comprising the following steps: (1) After mixing the protein with water, adjust the pH, heat-induced expansion, and cooling, first add cell wall protease for enzymatic hydrolysis, then add bean sprout protease for enzymatic hydrolysis to obtain hydrolyzed protein solution; (2) After adding Lactobacillus casei to the hydrolyzed protein solution for fermentation, Saccharomyces cerevisiae is added for fermentation; after fermentation, the solution is sterilized and the enzymes are inactivated to obtain the hydrolyzed protein fermentation broth. (3) Add fructooligosaccharides and medium- and long-chain glycerides to the hydrolyzed protein fermentation broth, mix and homogenize to obtain a protein nutritional supplement; The protein mentioned in step (1) is one or more of whey protein, soy protein, and pea protein; The preparation method of Lactobacillus helveticus cell wall protease is as follows: after culturing and collecting Lactobacillus helveticus cells, add buffer solution, mix well, break up, centrifuge to collect supernatant, purify and dry to obtain Lactobacillus helveticus cell wall protease; The preparation method of soybean sprout protease is as follows: soybean seeds are cultured in the dark until seedlings emerge; the seedlings are collected, freeze-dried into powder, mixed with salt solution, homogenized, incubated, and centrifuged to collect the supernatant; the supernatant is mixed with ammonium sulfate, allowed to stand, centrifuged to collect the protein precipitate; the protein precipitate is reconstituted, dialyzed, purified, and dried to obtain soybean sprout protease; In step (2), Lactobacillus casei was purchased from Shandong Pingao Pharmaceutical Co., Ltd., with product number / model number PG-LCG139 and preservation number CICC 23185; Saccharomyces cerevisiae was purchased from Jinan Jinyuyuan Biotechnology Co., Ltd., with product number / model number SC-324 and preservation number CICC 1001.

[0020] In one embodiment, the Lactobacillus helveticus was purchased from Jiuhe Biotechnology Co., Ltd., with product number / model number BL-012 and accession number ATCC 15009.

[0021] In one embodiment, the concentration of *Lactobacillus helveticus* cells after mixing with the buffer solution is 4 × 10⁻⁶. 8 ~10 9 CFU / mL.

[0022] In one embodiment, the ratio of protein to water in step (1) is 5-10 kg: 50-100 L; the pH adjustment is adjusted to 7.0-7.5; the heat-induced development is heated at 80-90℃ for 30-60 min; the enzymatic hydrolysis of Lactobacillus helveticus cell wall protease is performed by adding 1-2% (w / w) of Lactobacillus helveticus cell wall protease at 30-35℃ and 120-180 rpm for 1-2 h; the enzymatic hydrolysis of soybean sprouts is performed by adding 1-2% (w / w) of soybean sprout protease at 30-35℃ and 120-180 rpm for 3-5 h.

[0023] In one embodiment, the Lactobacillus casei fermentation in step (2) is Lactobacillus casei with 1000~1500 CFU / g protein added, fermented at 35~40℃ for 6~8 h; the Saccharomyces cerevisiae fermentation is Saccharomyces cerevisiae with 1500~2000 CFU / g protein added, fermented at 35~40℃ for 3~5 h; the mass ratio of protein: oligosaccharide: medium- and long-chain diglycerides in step (3) is 50~100:2~3:2~5.

[0024] The fifth objective of this invention is to provide the application of the protein emulsion described above in the preparation of anti-vomiting, hypoallergenic formula foods.

[0025] Beneficial effects The protein emulsion prepared by this invention has the following advantages: (1) Reducing the digestive burden and adverse sensory stimulation of protein, and improving the basic tolerance of liquid high-protein emulsions: This invention uses a two-stage enzymatic hydrolysis process to regulate the structure of proteins, which not only reduces the high digestive and metabolic burden of proteins in the gastrointestinal tract, but also effectively reduces protein sensitization and partially inhibits the formation of bitter peptides. The initial hydrolysis can significantly improve protein solubility, reduce the retention of undigested protein in the stomach, and reduce gastric emptying pressure; at the same time, by destroying the original conformation of the protein, it reduces the exposure of sensitizing epitopes, thereby reducing the risk of sensitization. On this basis, the secondary hydrolysis introduces soybean seedling protease to directionally cleave the hydrophobic amino acid residues at the C-terminus of bitter peptides to form free amino acids, which not only significantly reduces the bitterness of hydrolyzed proteins, but also further improves the degree of hydrolysis and improves the sensory quality of protein emulsions.

[0026] (2) By regulating fermentation and system structure, the osmotic pressure and local stimulation intensity are reduced, and the formation of acute stimulation peaks is avoided: This invention uses a two-stage fermentation process to synergistically regulate the peptide composition and osmotic pressure of the hydrolyzed protein system. First, Lactobacillus casei is used for fermentation, and the prolyl dipeptidyl peptidase produced can further hydrolyze the residual bitter peptides, achieving deep debittering of the hydrolyzed protein. Then, Saccharomyces cerevisiae is introduced for mild fermentation, consuming the free amino acids in the system, thereby effectively balancing the osmotic pressure of the solution and reducing the stimulation of the hyperosmotic environment on the gastrointestinal tract. In addition, by adding fructooligosaccharides to the system, it can not only act as a prebiotic to regulate the intestinal flora structure, inhibit the excessive proliferation of harmful bacteria, and reduce the risk of flatulence, but also homogenize with medium- and long-chain diglycerides and hydrolyzed proteins to form a Pickering emulsion structure. This structure can further reduce the osmotic pressure of the protein emulsion and improve the dispersion and transport state of the system in the gastrointestinal tract without affecting the protein digestion and absorption efficiency.

[0027] (3) Reducing enterochromaffin cell stimulation and vomiting signal formation, and improving the gastrointestinal tolerance of liquid high-protein emulsion: Through the synergistic regulation of the above-mentioned enzymatic hydrolysis, fermentation and system structure, the present invention effectively reduces the chemical stimulation intensity, osmotic pressure stimulation and digestion rate fluctuation of hydrolyzed protein in the gastrointestinal tract, reduces abnormal activation of enterochromaffin cells, and thus reduces the probability of the release of 5-hydroxytryptamine, a vomiting neurotransmitter. At the same time, by avoiding the concentrated generation of free amino acids and low molecular weight peptides in a short period of time, it prevents the formation of acute stimulation peaks in the gastrointestinal tract, thereby weakening the excessive activation of the vagus nerve 5-HT pathway and delaying the formation of vomiting reflex signals. Under the premise of ensuring protein supply efficiency and nutritional value, the liquid hydrolyzed high-protein emulsion of the present invention is less likely to induce nausea or vomiting, and significantly improves gastrointestinal tolerance and long-term use compliance. Attached Figure Description

[0028] Figure 1 The degree of hydrolysis of the sample; Figure 2 The in vitro digestibility of the sample; Figure 3 This refers to the emptying rate of the sample in the gastrointestinal tract. Figure 4 The bitterness score of the sample in the sensory evaluation; Figure 5 For the determination of the content of hydrophobic short peptides in the sample; Figure 6 For the determination of sample sensitization; Figure 7 This represents the percentage of beneficial and harmful bacteria in the gut after the sample was ingested. Figure 8 For sample osmotic pressure measurement; Figure 9 The sample's effect on gastrointestinal irritation. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, where specific conditions are not specified, are generally performed under conventional conditions in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art.

[0030] Raw material source: Lactobacillus casei was purchased from Shandong Pingao Pharmaceutical Co., Ltd., with product number / model number PG-LCG139 and accession number CICC 23185.

[0031] The brewing yeast was purchased from Jinan Jinyuyuan Biotechnology Co., Ltd., with product number / model number SC-324 and preservation number CICC 1001.

[0032] Lactobacillus helveticus R0052 was purchased from Jiuhe Biotechnology Co., Ltd., with product number / model number BL-012 and accession number ATCC 15009.

[0033] Lactobacillus LA85 was purchased from WeCon Probiotics (Suzhou) Co., Ltd. Kluyveromycin was purchased from Shanghai Ruichu Biotechnology Co., Ltd., model number SMHCC (SHBCC) D21832.

[0034] MRS broth culture medium: purchased from Qingdao Haibo Biotechnology Co., Ltd., weigh 52.4 g of powder, heat to dissolve in 1 L of distilled water, autoclave at 118℃ for 15 min, and set aside.

[0035] YPD culture medium: purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd. Weigh 50.0 g of powder, heat and dissolve in 1 L of distilled water, autoclave at 121℃ for 15 min, and set aside.

[0036] The IgE kit was purchased from Shanghai Abogen Biosciences Co., Ltd.; soybean seeds were purchased from Cangzhou Xiangyun Food Technology Co., Ltd.; fructooligosaccharides were purchased from Anhui Pinqing Food Industry Co., Ltd.; and medium- and long-chain diglycerides were purchased from Shaanxi Angxu Biotechnology Co., Ltd.

[0037] The measurement methods involved in the examples are as follows: 1. Degree of hydrolysis and digestion characteristics (1) The degree of protein hydrolysis was detected by the ortho-phthalaldehyde (OPA) method.

[0038] (2) The digestion rate of protein was evaluated using the INFOOGEST 2.0 static in vitro digestion model.

[0039] 2. Bitterness detection (1) Sensory evaluation A 10-person evaluation panel was established. Quinine hydrochloride at concentrations ranging from 0.001 to 0.01 mmol / L was used as the bitterness standard. A nine-point scale scoring method was employed, with 1 point indicating no bitterness, 5 points indicating moderate bitterness, and 9 points indicating extremely bitterness. Each evaluator independently tasted the product and scored it, and the average score was taken. (2) Hydrophobic short peptides were measured by HPLC-MS / MS. Take the hydrolyzed protein solution, centrifuge it using a 10 kDa ultrafiltration membrane (12000 r / min, 20 min), and collect the retentate; A C18 reversed-phase column (4.6 mm × 250 mm, 5 μm) was used with mobile phases A (0.1% trifluoroacetic acid aqueous solution) and B (0.1% trifluoroacetic acid acetonitrile solution), gradient elution (0–30 min, B phase 10%–40%), column temperature 30 °C, and flow rate 1 mL / min. Electrospray ionization was performed in the range of m / z 100–1000. The short peptide structure was identified by fragment ion matching using secondary mass spectrometry. A standard curve was plotted using the synthesized standard bitter peptide, and the total content of the target bitter peptide in the sample was calculated using the external standard method.

[0040] 3. Solution osmotic pressure measurement Using a freezing point depression osmometer, with 100, 290, and 500 mOsm / kg NaCl solutions as standards, the osmotic pressure of the samples was measured after instrument calibration.

[0041] 4. Allergenicity testing The protein sensitization of the samples was tested using an IgE kit.

[0042] 5. Gastric emptying test C57 mice were divided into groups of eight, and each group was given an equal amount of deionized water or test sample by gavage. Rhodamine B was added to all samples. The emptying rate of the samples in the gastrointestinal tract was then measured using small animal in vivo imaging.

[0043] 6. Intestinal flora assay C57 mice were divided into groups of eight, and each group was given an equal amount of deionized water or test sample by gavage once a day for one month. The mice were then dissected to obtain colon contents and the abundance of key bacterial strains was determined.

[0044] 7. Vomiting signal detection An in vitro cell model exhibiting the functional characteristics of enterochromaffin cells was selected for experiments. Cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 until they reached the logarithmic growth phase. Samples from the examples and comparative studies were prepared into solutions with equal protein concentrations using serum-free medium and sterilized through a 0.22 μm filter. Cells were seeded in 24-well plates and cultured to approximately 80% confluence. Then, the cells were starved with serum-free medium for 12 h to synchronize their state. Subsequently, equal volumes of sample solution were added to each group, with a stimulation time of 60 min. After stimulation, the culture supernatant was collected, and the 5-HT content in the supernatant was determined using a kit for subsequent assays.

[0045] Example 1: Preparation of anti-vomiting whey protein emulsion An anti-emetic liquid hydrolyzed high-protein nutritional supplement and its preparation method, comprising the following steps: 1. Raw material preparation (1) Preparation and purification of Lactobacillus helveticus cell wall protease Freeze-dried *Lactobacillus helveticus* R0052 was inoculated into 50 mL of liquid MRS medium and cultured at 37°C under anaerobic conditions for 18–24 h until the bacteria reached the late logarithmic growth phase (OD50). 600 ≈1.0); The bacteria were centrifuged, washed with 50 mmol / L Tris-HCl (pH 7.10) and 30 mmol / L CaCl2, centrifuged again, and the bacterial cells were collected. Buffer solution (50 mmol / L Tris-HCl (pH 6.50) + 45 mmol / L EDTA-Na2) was added and mixed to achieve a bacterial concentration of 4 × 10⁻⁶. 8 The concentration was CFU / mL, and the mixture was sonicated at 50 W for 2 seconds with an 8-second interval, for a total processing time of 8 minutes, all in an ice bath. After sonication, the mixture was centrifuged at 4°C and 6000 r / min for 15 minutes, and the supernatant was the crude enzyme solution. Finally, the Lactobacillus helveticus cell wall protease was obtained by DEAE-Sepharose CL-6B ion exchange chromatography and dried.

[0046] (2) Preparation and purification of soybean sprout protease Soybean seeds were sown on vermiculite with a moisture content of 75% and cultured at 25°C in the dark for 7 days until seedlings emerged. The seedlings were collected, washed, and then freeze-dried in liquid nitrogen to form a powder. The seedling powder was mixed with 0.15 M NaCl solution at a ratio of 1 g: 20 mL, homogenized at 10 MPa for 5 min, and incubated at 4°C for 1 hour. After incubation, the supernatant was collected by centrifugation at 6000 g for 10 min.

[0047] 10 mL of the supernatant was mixed with 0.2 g of ammonium sulfate and centrifuged at 8000 rpm for 10 min to obtain a protein precipitate. 0.5 g of the protein precipitate was dissolved in 10 g of distilled water and dialyzed against a 3.5 kDa regenerated cellulose membrane for 3 h. Purification was performed using DEAE-Sepharose FF (1.6 × 35 cm) column chromatography, eluting sequentially with distilled water and 0.05, 0.1, 0.3, 0.5, and 1.0 M NaCl solutions, collecting the precipitate, dialyzing, and freeze-drying to obtain the bean sprout protease.

[0048] (2) Preparation of strains Preparation of *Lactobacillus casei*: *Lactobacillus casei* was inoculated into MRS medium and cultured at 37℃ and 180 rpm for 20 h to obtain *Lactobacillus casei* seed culture. The *Lactobacillus casei* seed culture was then inoculated into MRS medium at an inoculation rate of 2% v / v and cultured at 37℃ and 180 rpm for 12 h to obtain a highly active bacterial culture. The bacterial culture was centrifuged at 5000 rpm for 10 min to collect the bacterial cells. The bacterial cells were washed twice with sterile water to remove residual metabolites. After washing, the cells were resuspended in sterile water and the bacterial concentration was adjusted to 10. 6 CFU / mL.

[0049] Preparation of Saccharomyces cerevisiae: Saccharomyces cerevisiae was inoculated into YPD medium and cultured at 28℃ and 180 rpm for 12 h to obtain a Saccharomyces cerevisiae seed culture. The Saccharomyces cerevisiae seed culture was then inoculated into YPD medium at a 4% v / v inoculation rate and cultured at 28℃ and 180 rpm for 12 h to obtain a highly active bacterial culture. The bacterial culture was centrifuged at 5000 rpm for 8 min to collect the bacterial cells. The bacterial cells were washed twice with sterile water to remove residual metabolites. After washing, the cells were resuspended in sterile water and the bacterial concentration was adjusted to 10. 6 CFU / mL.

[0050] 2. Preparation of anti-vomiting protein emulsion (1) 5 kg of whey protein was dispersed in 50 L of water to obtain a dispersion. The pH of the dispersion was adjusted to 7.0 and subjected to heat-induced development pretreatment at 80℃ for 30 min. The heat-induced development pretreated dispersion was then cooled to 35℃, and 1% (w / w) of whey protein cell wall protease was added. The mixture was then enzymatically hydrolyzed at 33℃ and 150 rpm for 2 h. Subsequently, 1% (w / w) of whey protein bean sprout protease was added and the mixture was enzymatically hydrolyzed at 33℃ and 150 rpm for 3 h to obtain a hydrolyzed protein solution.

[0051] (2) Add 1000 CFU / g whey protein of Lactobacillus casei to the hydrolyzed protein solution and ferment at 35℃ for 8 h; then add 1500 CFU / g whey protein of Saccharomyces cerevisiae and ferment at 35℃ for 5 h. After fermentation, sterilize by treating at 80℃ for 30 min to inactivate enzymes and obtain hydrolyzed protein fermentation broth.

[0052] (3) Add fructooligosaccharides and medium- and long-chain diglycerides to the hydrolyzed protein fermentation broth to obtain a mixture, wherein the mass ratio of whey protein: oligosaccharides: medium- and long-chain diglycerides is 100:2:2; the mixture is homogenized at 60 MPa for 5 min to emulsify it, so that the system forms a stable Pickering emulsion, and finally obtains a protein emulsion.

[0053] Example 2: Changing the amount of bacterial strain added and the fermentation temperature The specific implementation method is the same as in Example 1, except that the amount of *Lactobacillus casei* and *Saccharomyces cerevisiae* added and the fermentation temperature are changed in step (2) of the preparation of the anti-vomiting protein emulsion. Specifically, 1500 CFU / g of whey protein of *Lactobacillus casei* is added to the hydrolyzed protein solution and fermented at 40℃ for 8 h; then 2000 CFU / g of *Saccharomyces cerevisiae* is added and fermented at 40℃ for 5 h. After fermentation, the solution is treated at 80℃ for 30 min to inactivate enzymes and sterilize, thereby obtaining the hydrolyzed protein fermentation broth; the remaining steps remain the same to prepare the protein emulsion.

[0054] Example 3: Changing the amount of galactooligosaccharides and medium- and long-chain diglycerides added The specific implementation method is the same as in Example 1, except that the amount of galactooligosaccharide and medium- and long-chain diglycerides added in step (3) of the preparation of the anti-vomiting protein emulsion is changed so that the mass ratio of whey protein: oligosaccharide: medium- and long-chain diglycerides is 100:3:4; the remaining steps remain the same, and the protein emulsion is prepared.

[0055] The protein emulsions prepared in Examples 2 and 3 were tested, and the results showed that there were no significant differences in performance in terms of degree of hydrolysis, digestibility, and 5-HT inhibition, indicating that they had the same anti-vomiting effect.

[0056] Comparative Example 1: No thermally induced development pretreatment performed The specific implementation method is the same as in Example 1, except that in the preparation step (1) of the anti-vomiting protein emulsion, no heat-induced development pretreatment is performed, and protease hydrolysis is directly added. The remaining steps are the same, and the protein emulsion is prepared.

[0057] Comparative Example 2: No bean sprout protease added The specific implementation method is the same as in Example 1, except that in the preparation step (1) of the anti-vomiting protein emulsion, soybean sprout protease is not used for hydrolysis, but only cell wall protease is used for hydrolysis for 5 h. The remaining steps are the same to prepare the protein emulsion.

[0058] Comparative Example 3: No protease digestion used The specific implementation method is the same as in Example 1, except that cell wall protease and bean sprout protease are not used for enzymatic hydrolysis in step (1) of the preparation of anti-vomiting protein emulsion, while the other steps remain the same to prepare protein emulsion.

[0059] Comparative Example 4: Fermentation without Lactobacillus casei The specific implementation method is the same as in Example 1, except that Lactobacillus casei fermentation is not used in step (2) of the preparation of the anti-vomiting protein emulsion, while the other steps remain the same to prepare the protein emulsion.

[0060] Comparative Example 5: Fermentation without using brewer's yeast The specific implementation method is the same as in Example 1, except that in step (2) of the preparation of the anti-vomiting protein emulsion, brewer's yeast is not used for fermentation, while the other steps remain the same to prepare the protein emulsion.

[0061] Comparative Example 6: No oligosaccharides added The specific implementation method is the same as in Example 1, except that oligosaccharides are not added in step (3) of the preparation of the anti-vomiting protein emulsion, while the other steps remain the same, and the protein emulsion is prepared.

[0062] Comparative Example 7: No medium- or long-chain diglycerides added The specific implementation method is the same as in Example 1, except that medium- and long-chain diglycerides are not added in step (3) of the preparation of the anti-vomiting protein emulsion, while the other steps remain the same, and the protein emulsion is prepared.

[0063] Comparative Example 8: Replacing cell wall protease with alkaline protease The specific implementation method is the same as in Example 1, except that in the preparation step (1) of the anti-vomiting protein emulsion, the cell wall protease is replaced with alkaline protease. First, alkaline protease is used for enzymatic hydrolysis, and then bean sprout protease is used for enzymatic hydrolysis. The remaining steps are the same to prepare the protein emulsion.

[0064] Comparative Example 9: Replacing Lactobacillus casei with Lactobacillus acidophilus The specific implementation method is the same as in Example 1, except that in step (2) of the preparation of the anti-vomiting protein emulsion, Lactobacillus casei is replaced with Lactobacillus acidophilus LA85 (purchased from Weikang Probiotics (Suzhou) Co., Ltd.), while the other steps remain the same, and the protein emulsion is prepared.

[0065] Comparative Example 10: Replacing the brewer's yeast with Kluyveromyces cerevisiae The specific implementation method is the same as in Example 1, except that in step (2) of the preparation of the anti-vomiting protein emulsion, the brewer's yeast is replaced with Kluyveromyces kluyveromyces (purchased from Shanghai Ruichu Biotechnology Co., Ltd., model SMHCC(SHBCC)D21832), and the other steps are kept the same to prepare the protein emulsion.

[0066] Comparative Example 11: One-step enzymatic hydrolysis The specific implementation method is the same as in Example 1, except that in the preparation step (1) of the anti-vomiting protein emulsion, cell wall protease and bean sprout protease are added simultaneously for 5 hours of enzymatic hydrolysis, while the other steps remain the same to prepare the protein emulsion.

[0067] Comparative Example 12: One-step fermentation The specific implementation method is the same as in Example 1, except that in step (2) of the preparation of the anti-vomiting protein emulsion, brewer's yeast and Lactobacillus casei are simultaneously introduced into the hydrolyzed protein solution and fermented at 35°C for 8 hours. The remaining steps are kept the same to prepare the protein emulsion.

[0068] Comparative Example 13: Add brewer's yeast first, then add Lactobacillus casei. The specific implementation method is the same as in Example 1, except that in the preparation step (2) of the anti-vomiting protein emulsion, 1500 CFU / g of brewer's yeast is added first and fermented at 33°C for 5 h, and then 1000 CFU / g of Lactobacillus casei is added and fermented at 33°C for 8 h. The remaining steps are kept the same to prepare the protein emulsion.

[0069] Example 4: Performance determination of protein emulsion 1. Determination of degree of hydrolysis The protein emulsions prepared in Example 1 and Comparative Examples 2 and 3 were used to determine their degree of hydrolysis. The results are as follows: Figure 1 As shown.

[0070] The results showed that when no protease was used for hydrolysis, the degree of hydrolysis was only about 1% (Comparative Example 3); when only cell wall protease was used for hydrolysis (Comparative Example 1), the degree of hydrolysis was only 12.41%; while after two-step enzymatic hydrolysis using cell wall protein and bean sprout protease, the degree of hydrolysis was 23.58%.

[0071] 2. Determination of in vitro digestion The protein emulsions prepared in Example 1 and Comparative Example 3 were used to determine their in vitro digestibility. The results are as follows:Figure 2 As shown.

[0072] The results showed that the in vitro digestibility of Comparative Example 3 was 61.24%, while the in vitro digestibility of Example 1 was close to 100%, indicating that single enzymatic hydrolysis or no enzymatic hydrolysis resulted in low digestibility and a high digestive burden.

[0073] 3. Determination of gastrointestinal emptying rate The protein emulsions prepared in Example 1 and Comparative Examples 2 and 3 were administered to mice by gavage to determine their gastrointestinal emptying rates. The results are as follows: Figure 3 As shown.

[0074] The results showed that both unhydrolyzed samples and samples with low degree of hydrolysis resulted in slower gastrointestinal emptying rates, indicating a greater digestive burden.

[0075] 4. Bitterness determination The protein nutritional supplements prepared in Example 1 and Comparative Examples 2 and 8 were tested for bitterness score and content of hydrophobic short peptides. The results are as follows: Figure 4~5 As shown.

[0076] The results showed that the samples had a higher bitterness without treatment with bean sprout protease. Although the bitterness decreased when the samples were first hydrolyzed with alkaline protease and then hydrolyzed with bean sprout protease, the bitterness score and the content of hydrophobic short peptides were still higher than those in Example 1.

[0077] 5. Sensitization test The protein nutritional supplements prepared in Example 1 and Comparative Example 3 were tested for sensitization, and the results are as follows: Figure 6 As shown.

[0078] The results showed that the unhydrolyzed protein sample had a certain potential for sensitization, while the sensitization of Example 1 decreased significantly after double-enzyme hydrolysis.

[0079] 6. Intestinal flora assay The protein nutritional supplements prepared in Example 1 and Comparative Example 6 were used to determine the abundance of key bacterial strains in the colonic contents of mice after gavage. The results are as follows: Figure 7 As shown.

[0080] The results showed that Comparative Example 6, which did not contain added oligosaccharides, led to an increase in the proportion of harmful bacteria and a decrease in the proportion of beneficial bacteria in the gut. The harmful bacteria fermented and produced gas, resulting in intestinal discomfort such as bloating.

[0081] 7. Osmotic pressure measurement The protein nutritional supplements prepared in Example 1 and Comparative Examples 4, 5, 7, 9, 10, 12 and 13 were used to measure their osmotic pressure, and the results are as follows. Figure 8 As shown.

[0082] The results showed that Example 1 had the lowest osmotic pressure, at only 290.13 mOm / kg. While single fermentation with *Lactobacillus casei* or yeast to remove free amino acids reduced the osmotic pressure to 402.38 mOm / kg and 378.26 mOm / kg respectively, it remained very high. Without encapsulation with medium- and long-chain diglycerides, the osmotic pressure was 347.63 mOm / kg, still very high. Changing the fermentation order and combination of *Lactobacillus casei* and yeast resulted in osmotic pressures of 361.85 mOm / kg and 368.26 mOm / kg respectively, but none of these methods effectively reduced the osmotic pressure.

[0083] 9. Gastrointestinal irritation measurement The protein nutritional supplements prepared in Example 1 and Comparative Examples 1-13 were used to determine their gastrointestinal irritation level, i.e., the amount of 5-HT released. The results are as follows: Figure 9 As shown.

[0084] The results showed that, compared with the comparative sample, the sample of the example induced the lowest 5-hydroxytryptamine release, only 0.294 μmol / L, which was 75.52% lower than the most irritating comparative sample 2 (1.201 μmol / L). Moreover, the release process was more gradual, indicating that it had a lower stimulating effect on enterochromaffin cells in the gastrointestinal tract, which helped to delay the formation of nausea-inducing signals.

[0085] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a protein emulsion, characterized in that, Including the following steps: (1) After mixing the protein with water, adjust the pH, heat-induced development, and cooling, first add Lactobacillus helveticus cell wall protease for enzymatic hydrolysis, then add soybean sprout protease for enzymatic hydrolysis to obtain hydrolyzed protein solution; (2) After adding Lactobacillus casei to the hydrolyzed protein solution for fermentation, Saccharomyces cerevisiae is added for fermentation; after fermentation, the solution is sterilized and the enzymes are inactivated to obtain the hydrolyzed protein fermentation broth. (3) Add fructooligosaccharides and medium- and long-chain glycerides to the hydrolyzed protein fermentation broth, mix and homogenize to obtain a protein emulsion; In step (1), the protein is one or more of whey protein, soy protein, and pea protein; The preparation method of Lactobacillus helveticus cell wall protease is as follows: after culturing and collecting Lactobacillus helveticus cells, add buffer solution, mix well, break up, centrifuge to collect supernatant, purify and dry to obtain Lactobacillus helveticus cell wall protease; The preparation method of soybean sprout protease is as follows: soybean seeds are cultured in the dark until seedlings emerge; the seedlings are collected, freeze-dried into powder, mixed with salt solution, homogenized, incubated, and centrifuged to collect the supernatant; the supernatant is mixed with ammonium sulfate, allowed to stand, centrifuged to collect the protein precipitate; the protein precipitate is reconstituted, dialyzed, purified, and dried to obtain soybean sprout protease; In step (2), Lactobacillus casei was purchased from Shandong Pingao Pharmaceutical Co., Ltd., with product number / model number PG-LCG139 and preservation number CICC 23185; Saccharomyces cerevisiae was purchased from Jinan Jinyuyuan Biotechnology Co., Ltd., with product number / model number SC-324 and preservation number CICC 1001.

2. The method according to claim 1, characterized in that, In step (1), the ratio of protein to water is 5-10 kg: 50-100 L; the pH adjustment is to adjust the pH to 7.0-7.5; the heat-induced development is to heat at 80-90℃ for 30-60 min; the enzymatic hydrolysis of Lactobacillus helveticus cell wall protease is to add 1-2% (w / w) of Lactobacillus helveticus cell wall protease at 30-35℃ and 120-180 rpm for 1-2 h; the enzymatic hydrolysis of soybean sprouts is to add 1-2% (w / w) of soybean sprout protease at 30-35℃ and 120-180 rpm for 3-5 h.

3. The method according to claim 1, characterized in that, The Lactobacillus casei fermentation in step (2) is Lactobacillus casei with 1000~1500 CFU / g protein added, fermented at 35~40℃ for 6~8 h; the Saccharomyces cerevisiae fermentation is Saccharomyces cerevisiae with 1500~2000 CFU / g protein added, fermented at 35~40℃ for 3~5 h.

4. The method according to claim 1, characterized in that, In step (3), the mass ratio of protein: oligosaccharide: medium- and long-chain diglycerides is 50~100:2~3:2~5.

5. The protein emulsion prepared by any one of claims 1 to 4.

6. A product characterized in that, The product contains the protein emulsion as described in claim 5, and is a pharmaceutical, health product, or food.

7. A method for improving the anti-vomiting and hypoallergenic properties of protein emulsions, characterized in that, Including the following steps: (1) After mixing the protein with water, adjust the pH, heat-induced development, and cooling, first add Lactobacillus helveticus cell wall protease for enzymatic hydrolysis, then add soybean sprout protease for enzymatic hydrolysis to obtain hydrolyzed protein solution; (2) After adding Lactobacillus casei to the hydrolyzed protein solution for fermentation, Saccharomyces cerevisiae is added for fermentation; after fermentation, the solution is sterilized and the enzymes are inactivated to obtain the hydrolyzed protein fermentation broth. (3) Add fructooligosaccharides and medium- and long-chain glycerides to the hydrolyzed protein fermentation broth, mix and homogenize to obtain a protein nutritional supplement; In step (1), the protein is one or more of whey protein, soy protein, and pea protein; The preparation method of Lactobacillus helveticus cell wall protease is as follows: after culturing and collecting Lactobacillus helveticus cells, add buffer solution, mix well, break up, centrifuge to collect supernatant, purify and dry to obtain Lactobacillus helveticus cell wall protease; The preparation method of soybean sprout protease is as follows: soybean seeds are cultured in the dark until seedlings emerge; the seedlings are collected, freeze-dried into powder, mixed with salt solution, homogenized, incubated, and centrifuged to collect the supernatant; the supernatant is mixed with ammonium sulfate, allowed to stand, centrifuged to collect the protein precipitate; the protein precipitate is reconstituted, dialyzed, purified, and dried to obtain soybean sprout protease; In step (2), Lactobacillus casei was purchased from Shandong Pingao Pharmaceutical Co., Ltd., with product number / model number PG-LCG139 and preservation number CICC 23185; Saccharomyces cerevisiae was purchased from Jinan Jinyuyuan Biotechnology Co., Ltd., with product number / model number SC-324 and preservation number CICC 1001.

8. The method according to claim 7, characterized in that, In step (1), the ratio of protein to water is 5-10 kg: 50-100 L; the pH adjustment is to adjust the pH to 7.0-7.5; the heat-induced development is to heat at 80-90℃ for 30-60 min; the enzymatic hydrolysis of Lactobacillus helveticus cell wall protease is to add 1-2% (w / w) of Lactobacillus helveticus cell wall protease at 30-35℃ and 120-180 rpm for 1-2 h; the enzymatic hydrolysis of soybean sprouts is to add 1-2% (w / w) of soybean sprout protease at 30-35℃ and 120-180 rpm for 3-5 h.

9. The method according to claim 7, characterized in that, In step (2), the Lactobacillus casei fermentation is Lactobacillus casei with 1000~1500 CFU / g protein added, fermented at 35~40℃ for 6~8 h; the Saccharomyces cerevisiae fermentation is Saccharomyces cerevisiae with 1500~2000 CFU / g protein added, fermented at 35~40℃ for 3~5 h; in step (3), the mass ratio of protein: oligosaccharide: medium- and long-chain diglycerides is 50~100:2~3:2~5.

10. The use of the protein emulsion of claim 5 in the preparation of anti-vomiting, hypoallergenic formula foods.

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