Plant protein fiber, its preparation method and application

Abstract

The present application relates to plant protein fiber and its preparation method and application. The plant protein fiber is flexible and worm-like. The plant protein fiber of the present application can inhibit the digestion and absorption of part of oil and fat, and has certain nutritional and health care functions. The method of the present application can improve the application value of the original plant protein, and is simple to operate and does not need to use various complex instruments, and can be conveniently and quickly operated.

Description

Technical Field

[0001] This invention relates to the processing of plant protein fibers. Specifically, this invention relates to plant protein fibers, their preparation methods, and their applications. Background Technology

[0002] Protein is an essential macronutrient, not only a crucial building block of all living organisms in nature but also a key structural component of many foods. Plant protein is widely available, highly renewable, and virtually pollution-free, making it one of the ideal primary sources of food raw materials.

[0003] Plant-derived proteins have attracted significant attention due to their sustainable sources, low economic cost, and positive health benefits. However, previous research has primarily focused on legume and cereal proteins, with limited exploration of other plant proteins. Furthermore, the difficulty in isolating and purifying most plant proteins, their low water solubility, and low digestibility limit their applications. Therefore, it is necessary to improve the physicochemical properties of plant proteins from various sources and modify them to enhance their bioactivity and bioavailability, thereby diversifying their applications. Commonly used protein modification methods include physical methods (such as heat treatment and high-pressure treatment), chemical methods (such as phosphorylation and glycosylation), biological methods (such as enzyme modification and fermentation), and other methods (such as fibrosis). Among these, protein fibrosis is a highly efficient and ideal method for protein modification and transformation. After acid-heat treatment, proteins can yield protein aggregates rich in β-sheet structures, which possess properties such as high Young's modulus, high tensile strength, high hydrophobicity, excellent stability and interfacial properties, antioxidant activity, and antibacterial activity. Therefore, using plant protein as raw material to process protein into fibers can enhance its utilization value, broaden the application of plant protein in the food field, and thus promote the industrialization of plant-based food protein and realize its high-value utilization, which has important practical significance. Summary of the Invention

[0004] The present invention aims to develop new plant protein fibers and expand their applications.

[0005] Therefore, according to a first aspect of the present invention, a plant protein fiber is provided, characterized in that it is in the form of a flexible worm and has a length in the range of 100-400 nm.

[0006] According to a second aspect of the present invention, a method for preparing plant protein fibers is provided, characterized by comprising the following steps:

[0007] I) Dissolve the plant protein in an acidic solution with a pH range of 2.0-3.0 to obtain plant protein solution A;

[0008] II) Centrifuge the plant protein solution A at a temperature within the range of 4-6℃ to obtain supernatant A;

[0009] III) Adjust the pH of supernatant A to the range of 1.90-2.10;

[0010] IV) Filter the supernatant A with a pH range of 1.90-2.10 to obtain plant protein solution B;

[0011] V) Heat and stir the plant protein solution B at a temperature in the range of 80-95℃ to make the plant protein fibrotic, then cool it to room temperature and centrifuge to obtain supernatant B;

[0012] VI) Dialyze the supernatant B at a temperature ranging from 4 to 10°C to obtain the dialyzed solution C; and

[0013] VII) The solution C was concentrated to obtain plant protein fiber.

[0014] According to a third aspect of the invention, the use of plant protein fiber according to the first aspect of the invention for inhibiting the digestion and absorption of fats and oils is provided.

[0015] The plant protein fiber of this invention can inhibit the digestion and absorption of some oils and has certain nutritional and health benefits.

[0016] The method of the present invention can enhance the application value of the original plant protein. It is simple to operate and does not require the use of various complex instruments, and can be operated conveniently and quickly. Attached Figure Description

[0017] The present invention will now be described and explained in more detail with reference to the accompanying drawings, wherein:

[0018] Figure 1 The test results of the papain and bromelain fibers prepared in Example 1 are shown, where a: circular dichroism spectroscopy results of papain fiber; b: circular dichroism spectroscopy results of bromelain fiber; c: atomic force map of papain fiber; d: atomic force map of bromelain fiber; e: electrophoresis diagram of molecular weight change of papain during fiberization process; f: electrophoresis diagram of molecular weight change of bromelain during fiberization process.

[0019] Figure 2 The microstructures of the high internal phase emulsion stabilized by papain fibers or bromelain fibers in Example 2 are shown, where a: stabilized by papain fibers; b: stabilized by bromelain fibers.

[0020] Figure 3The figures show the body weight, fecal fat content, and plasma triglyceride content of different groups of experimental mice during the experiment in Example 3, where a: body weight; b: fecal fat content; c: plasma triglyceride content; experimental results are expressed as mean ± standard deviation (Mean ± SD). The data were analyzed using IBM SPSS Statistics software. p < 0.05 was considered statistically significant, * indicates p < 0.05; ** indicates p < 0.01.

[0021] Figure 4 The images show the weights of epididymal fat, mesenteric fat, and perirenal fat in different groups of experimental mice in Example 3, where a: epididymal fat weight; b: mesenteric fat weight; c: perirenal fat weight.

[0022] Figure 5 The differences in the breakdown of the high internal phase emulsion stabilized by papain fiber and bromelain fiber in Example 4 are shown in a simulated digestion experiment, where a: bile salt digestion; b: release of free fatty acids.

[0023] Figure 6 The differences in chylomicron particle size in mouse plasma after gavage with high internal phase emulsions stabilized by papain fiber and bromelain fiber in Example 4 are shown. Among them, a: electron micrograph of chylomicrons in mice after gavage with papain fiber high internal phase emulsion (PE); b: electron micrograph of chylomicrons in mice after gavage with bromelain fiber high internal phase emulsion (BE); c: particle size distribution of chylomicrons in plasma of mice in different groups of experimental mice.

[0024] Figure 7 The table shows the levels of triglycerides, free fat, and glycerol in the plasma of mice after gavage with a high-inner-phase emulsion stabilized by papain fiber and bromelain fiber in Example 4, where a: triglyceride content; b: free fat content; c: glycerol content. Detailed Implementation

[0025] The various aspects of the invention, as well as its further objects, features and advantages, will be more fully demonstrated below in conjunction with some specific embodiments.

[0026] The terms "comprising" and "including" as used in this application cover situations where other elements not explicitly mentioned are also included, as well as situations where the elements mentioned are constituted.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any discrepancy between the definitions of terms in this specification and their commonly understood meaning by one of ordinary skill in the art to which this invention pertains, the definitions set forth herein shall prevail.

[0028] Unless otherwise stated, all numerical values ​​for the amount of an ingredient, temperature, etc., used in the specification and claims are to be understood as being modified by the term "about". Therefore, unless otherwise indicated, the numerical parameters set forth herein are approximate values ​​that can be varied to obtain the desired performance.

[0029] Plant protein fiber

[0030] According to a first aspect of the present invention, a plant protein fiber is provided, characterized in that it is in the form of a flexible worm and has a length in the range of 100-400 nm.

[0031] In the plant protein fiber of the present invention, the plant protein is not particularly limited.

[0032] In some embodiments, the plant protein is selected from bromelain, soy protein, mung bean protein, and cowpea protein, etc.

[0033] Preferably, the plant protein is bromelain.

[0034] Bromelain is a plant protease derived from fruit, primarily from the stem or fruit of the pineapple.

[0035] The plant protein fiber of this invention can inhibit the digestion and absorption of some oils and has certain nutritional and health-promoting properties.

[0036] In some embodiments, the oil is an edible vegetable oil, such as sunflower seed oil, olive oil, rapeseed oil, etc.

[0037] Preparation method of plant protein fiber

[0038] According to a second aspect of the present invention, a method for preparing the above-mentioned plant protein fiber is provided, characterized by comprising the following steps:

[0039] I) Dissolve the plant protein in an acidic solution with a pH range of 2.0-3.0 to obtain plant protein solution A;

[0040] II) Centrifuge the plant protein solution A at a temperature within the range of 4-6℃ to obtain supernatant A;

[0041] III) Adjust the pH of supernatant A to the range of 1.90-2.10;

[0042] IV) Filter the supernatant A with a pH range of 1.90-2.10 to obtain plant protein solution B;

[0043] V) Heat and stir the plant protein solution B at a temperature in the range of 80-95℃ to make the plant protein fibrotic, then cool it to room temperature and centrifuge to obtain supernatant B;

[0044] VI) Dialyze the supernatant B at a temperature ranging from 4 to 10°C to obtain the dialyzed solution C; and

[0045] VII) The solution C was concentrated to obtain plant protein fiber.

[0046] The solvent in step I) is an acidic aqueous solution, such as an acidic aqueous solution with a pH of 2.00, like a dilute hydrochloric acid solution.

[0047] Preferably, the concentration of the resulting plant protein solution A is in the range of 20-50 mg / ml, more preferably 35-45 mg / ml.

[0048] Preferably, the food is placed at a temperature in the range of 4-6°C for a period of time before step II), for example, overnight at 4°C, to ensure sufficient hydration.

[0049] Those skilled in the art can adjust the rotation speed and time during centrifugation in step II) as needed.

[0050] For example, the centrifugation in step II) is performed at 8000-10000 rpm and 4°C for 15-30 min.

[0051] Preferably, in step III), the pH of supernatant A is adjusted to the range of 1.95-2.05, for example, by using an acidic aqueous solution, such as HCl solution, to adjust the pH of supernatant A to 2.0.

[0052] The filtration in step IV) can be performed in any suitable manner, such as using a filter membrane. Preferably, the pore size of the filter membrane is in the range of 0.4-0.5 μm.

[0053] Preferably, fiberization is carried out in step V) at a temperature in the range of 85-90°C.

[0054] Cooling can be done in any suitable way, such as by placing the object in an ice water bath.

[0055] Those skilled in the art can adjust the rotation speed and time during centrifugation in step V) as needed.

[0056] For example, the centrifugation in step V) is performed at 5000 rpm and 4°C for 15-30 min.

[0057] Those skilled in the art can adjust the dialysis conditions in step VI) as needed.

[0058] Preferably, in step VI), the supernatant B is dialyzed at a temperature in the range of 4-6°C.

[0059] For example, dialysis is performed at 4°C using dialysis bags with a molecular weight cutoff of 7000D for a period of time (e.g., 70-80 hours), with water changes performed every so often (e.g., every 8 hours).

[0060] Those skilled in the art can adjust the concentration conditions in step VII) as needed.

[0061] For example, solution C can be concentrated at temperatures ranging from 35 to 45°C.

[0062] In some implementations, rotary evaporation concentration is carried out at 40°C.

[0063] In some embodiments, the concentrate is also dried.

[0064] In the method of the present invention, the plant protein is not particularly limited.

[0065] In some embodiments, the plant protein is selected from bromelain, papain, soy protein, mung bean protein, and cowpea protein, etc.

[0066] In some embodiments, bromelain fiber is prepared by the following:

[0067] Bromelain was dissolved in a dilute hydrochloric acid solution with a pH of 2.0 to prepare a 40 mg / ml bromelain aqueous solution, and placed at 4°C overnight to allow the bromelain to fully hydrate and dissolve.

[0068] The obtained protease solution was centrifuged at 8000 rpm and 4℃ for 15 min, the precipitate was discarded, and the supernatant was retained. The pH of the two supernatants was adjusted to 2.0 using 2M HCl solution, and then the solution was filtered through a 0.45 μm filter. After processing at 90℃ and 300 rpm for 24 h, the solution was removed and immediately cooled to room temperature in an ice-water bath.

[0069] The obtained solution was centrifuged at 5000 rpm and 4℃ for 20 min, the precipitate was discarded, and the supernatant was retained. The supernatant was dialyzed at 4℃ using a dialysis bag with a molecular cutoff of 7000D for 72 h, with water changed every 8 h. The dialyzed solution was collected and concentrated by rotary evaporation at 40℃ to obtain bromelain fiber solution.

[0070] The resulting bromelain fibers are short, flexible, and worm-like.

[0071] In some embodiments, papain fiber is prepared by:

[0072] Papain was dissolved in a dilute hydrochloric acid solution with a pH of 2.0 to prepare a 20 mg / ml papain aqueous solution, and placed at 4°C overnight to allow it to fully hydrate and dissolve.

[0073] The obtained protease solution was centrifuged at 8000 rpm and 4℃ for 15 min, the precipitate was discarded, and the supernatant was retained. The pH of the supernatant was adjusted to 2.0 using 2M HCl solution, and then filtered through a 0.45 μm filter. After processing at 90℃ and 300 rpm for 24 h, the solution was removed and immediately cooled to room temperature in an ice-water bath.

[0074] The obtained solution was centrifuged at 5000 rpm and 4℃ for 20 min, the precipitate was discarded, and the supernatant was retained. The supernatant was dialyzed at 4℃ using a dialysis bag with a molecular cutoff of 7000D for 72 h, with water changed every 8 h. The dialyzed solution was collected and concentrated by rotary evaporation at 40℃ to obtain papain fiber solution.

[0075] Papain is also a plant protease derived from fruits, primarily from unripe papaya fruits.

[0076] The resulting papain fibers are mainly composed of slender, rigid protofibrils.

[0077] The method of the present invention has the advantages of wide availability of experimental raw materials, simple and easy experimental operation steps, and low equipment requirements.

[0078] use

[0079] According to a third aspect of the invention, the use of plant protein fiber according to the first aspect of the invention for inhibiting the digestion and absorption of fats and oils is provided.

[0080] The inventors have discovered that plant protein fibers according to the first aspect of the present invention can inhibit the digestion and absorption of fats by humans and animals, thereby reducing the accumulation of adipose tissue and promoting a healthy diet.

[0081] In some embodiments, the oil is an edible vegetable oil, such as sunflower seed oil, olive oil, rapeseed oil, etc.

[0082] Example

[0083] The following will further illustrate the concept and technical effects of the present invention with reference to embodiments and accompanying drawings, so that those skilled in the art can fully understand the purpose, features, and effects of the present invention. Those skilled in the art will understand that the embodiments described herein are merely illustrative, and the scope of the present invention is not limited thereto.

[0084] Example 1

[0085] Papain fiber and bromelain fiber were prepared as follows.

[0086] Papain and bromelain were dissolved in dilute hydrochloric acid solution at pH 2.0 to prepare protein solutions of 20 mg / ml and 40 mg / ml, respectively, and incubated overnight at 4°C.

[0087] The two protease solutions were centrifuged at 8000 rpm for 15 min at 4 °C, the precipitate was discarded, and the supernatant was retained. The pH of the two supernatants was readjusted to 2.0 using 2M HCl solution. The solutions were then filtered through a 0.45 μm filter and treated at 90 °C and 300 rpm for 24 h to induce fibrosis. Finally, the solutions were cooled to room temperature in an ice-water bath.

[0088] The obtained protease solution was centrifuged at 5000 rpm for 20 min at 4 °C. The supernatant was collected and then dialyzed for 72 h using a dialysis bag with a molecular weight cutoff of 7000 D, with the water changed every 8 h. The protein solution after dialysis was collected and concentrated under vacuum at 40 °C to obtain a protein cellulose solution.

[0089] Characterization

[0090] (1) Circular dichroism (CD) determination

[0091] The protein cellulose solution, after rotary evaporation concentration, was diluted to a concentration of 0.25 mg / ml with dilute hydrochloric acid solution at pH 2.0, and the untreated protease solution was diluted to the same concentration. The scanning wavelength was set to 190 nm to 270 nm, the optical path of the sample cell was 2.00 nm, and the measurement temperature was set to 25 °C. The average of three measurements was used as the final numerical result.

[0092] The results of circular dichroism spectroscopy determination of papain and bromelain before and after fibrosis treatment are as follows: Figure 1 As shown in a and 1b.

[0093] from Figure 1 As can be seen, papain transforms from a secondary structure dominated by α-helices to a secondary structure dominated by β-sheets after undergoing fibrillation treatment.

[0094] from Figure 1 b shows that after fibrosis, bromelain changes from a secondary structure dominated by random coils to a secondary structure dominated by β-sheets.

[0095] (2) Atomic force microscope (AFM)

[0096] Samples treated with papain and bromelain for 24 hours were dialyzed. The dialyzed fiber samples were diluted to a concentration of 0.01 wt% with dilute hydrochloric acid solution at pH 2.0. 20 μL of the diluted fiber sample was deposited onto a lysed mica sheet, incubated for 2 min, immediately rinsed with deionized water, dried with compressed air, and placed on a stage. AFM measurements were performed using a scanning probe microscope in a soundproof environment to minimize the impact of vibration and noise.

[0097] The atomic force diagrams of papain fiber and bromelain fiber are as follows: Figure 1 As shown in c and 1d.

[0098] from Figure 1 c shows that papain forms protein fibers mainly composed of slender, rigid fibrils.

[0099] from Figure 1 c shows that bromelain forms short, flexible, worm-like protein fibers with a length ranging from 100 to 400 nm.

[0100] (3) Polyacrylamide gel electrophoresis (SDS-PAGE)

[0101] Samples (v1) from different time points during protein fibrillation were mixed with 4x loading buffer (v2) (v1:v2 = 1:3) and placed in a boiling water bath for 5 min. After centrifugation, the supernatant was collected to obtain electrophoresis samples. 10 μL of Maker and an equal volume of electrophoresis samples were added to different pore sizes of a pre-prepared stacking gel. Electrophoresis was first performed at 80V for 40 min, and then the voltage was switched to 110V for another 80 min. When the bromophenol blue was about 1 cm from the bottom of the gel, the voltage was cut off, the gel was removed, and the gel was stained with staining solution. The gel was then repeatedly destained until the bands were clear and the background color was removed. The bands were then photographed using a gel imaging system.

[0102] Electrophoresis diagrams showing the molecular weight changes of papain and bromelain during fibrosis are shown below. Figure 1 As shown in e and 1f.

[0103] from Figure 1 As can be seen from e and 1f, at 0h of fibrosis treatment, the molecular weights of different subunits of papain and bromelain are mainly concentrated between 10 and 15KD and around 25KD. During the fibrosis process, the color of the large molecular subunit bands of the proteases becomes lighter and disappears, while the color of the small molecular subunit bands becomes darker. At 24h, almost all papain and bromelain are hydrolyzed into small molecular peptides, indicating that the protease molecules undergo deconstruction and recombination during the treatment process, and the large molecular subunits are hydrolyzed into small molecular polypeptide fragments.

[0104] Example 2

[0105] Prepare a high internal phase emulsion stabilized by papain cellulose or bromelain cellulose as follows.

[0106] A high internal phase emulsion with an oil phase volume fraction of 86% was prepared by mixing and dispersing 1 wt% protease cellulose as the aqueous phase and sunflower seed oil as the oil phase in a homogenizer at 5000 rpm for 2 min. The emulsion was then allowed to stand at room temperature for 1 h to stabilize. Unless otherwise explicitly stated, the high internal phase emulsion referred to below is the high internal phase emulsion obtained herein.

[0107] Characterization

[0108] (1) Cryo-scanning electron microscopy of fiber-stabilized high internal phase emulsion

[0109] A small amount of the prepared emulsion sample was dropped onto a copper support, flash-frozen in liquid nitrogen, etched at ultra-low temperature (-140℃), sublimated at this temperature for about 10 min, and then coated at -175℃ to prepare a film. A layer of sputtered gold (10mA, 90s) was applied, and the sample was imaged by scanning electron microscopy using an Everhart Thornley detector (ETD) and a solid-state detector (SSD).

[0110] Cryo-scanning electron microscopy images of high internal phase emulsions stabilized by papain fibers or bromelain fibers are shown below. Figure 2 As shown in a (left) or 2b (left).

[0111] from Figure 2 As can be seen from left a, the oil droplets in the high internal phase emulsion stabilized by papain fibers are uneven in diameter and relatively dispersed, and the oil droplets are round.

[0112] from Figure 2 As can be seen in b (left), the oil droplets in the high internal phase emulsion stabilized by bromelain fibers are densely distributed, and the oil droplets are squeezed against each other, thus presenting a typical polygonal structure of the high internal phase emulsion.

[0113] (2) Laser confocal microscopy of fiber-stabilized high internal phase emulsions

[0114] Quantitative amounts of Nilelan (0.8 mg / ml) and Nile Red (0.375 mg / ml) were dissolved in protein cellulose and sunflower seed oil, respectively. After stirring overnight, the mixture was centrifuged at 5000g for 20 min. The supernatant was collected to remove insoluble impurities. The protein cellulose containing Nilelan was used as the aqueous phase, and the sunflower seed oil containing Nile Red was used as the oil phase. The mixture was dispersed in a homogenizer at 5000 rpm for 2 min to prepare a high internal phase emulsion with an oil phase volume fraction of 86%. A small amount of the emulsion was placed on a glass slide, and a coverslip was gently placed on top of the slide. The slide was then inverted and placed under a laser confocal microscope. Nile Red and Nilelan were excited under lasers at 488 nm and 633 nm, respectively. The emulsion structure was observed and photographed under a 20x dry microscope.

[0115] Laser confocal microscopy images of high internal phase emulsions stabilized by papain fibers or bromelain fibers are shown below. Figure 2 As shown in a (right) or 2b (right).

[0116] Figure 2 a (right) further confirms that the oil droplets in the high internal phase emulsion stabilized by papain fibers are uneven in diameter and relatively dispersed, and the oil droplets are round.

[0117] Figure 2 b (right) further confirms that the oil droplets in the high internal phase emulsion stabilized by bromelain fibers are densely distributed and squeezed together, thus exhibiting the typical polygonal structure of the high internal phase emulsion.

[0118] Example 3

[0119] This study investigated the effects of long-term gavage administration of a high-internal-phase emulsion stabilized by papain or bromelain fibers on mice.

[0120] Animal experiments were conducted by the Animal Experiment Center of Nanjing Agricultural University (License No.: SYXK-(Su)-

[0121] (2011-0037) Reviewed and approved and complies with the National Guidelines for Laboratory Animal Welfare and the Ethics Standards for Animal Experimentation.

[0122] The lighting conditions in the rearing room were controlled, and the temperature and humidity were suitable for the mice's activity. Thirty-six SPF-grade C57BL / 6J mice were randomly divided into three groups according to body weight after seven days of free-range acclimatization: the NC group, the PE group, and the BE group, with 12 mice in each group. After grouping, each group was gavaged with 400 μL of the corresponding sample every morning. The NC, PE, and BE groups were gavaged with sterile water, a high internal phase emulsion stabilized by papain fiber (c = 1 wt%), and a PE group, respectively. 5k rpm) and a high internal phase emulsion stabilized by bromelain fiber (c = 1wt%) Mice were gavaged at 500 rpm for 19 weeks. The weight of the experimental animals was measured and recorded every 3 days. Feces from different groups of mice were collected every month and the fat content in the feces was measured. The animal experiment was terminated after 132 days of gavage treatment.

[0123] One day before the end of the rearing period, fresh feces from each mouse were collected and immediately placed in liquid nitrogen, then transferred to a -80°C freezer for later analysis. The mice were then fasted for 12 hours. After fasting, blood was collected from the mice via ocular sampling, and the mice were euthanized by cervical dislocation. The collected blood was immediately placed in EP tubes containing anticoagulant and allowed to stand at room temperature for 4 hours before centrifugation to obtain plasma for biochemical analysis. The epididymis, mesentery, and perirenal adipose tissue of the mice were removed, and their weights were accurately recorded.

[0124] Characterization

[0125] (1) Monitoring of mouse weight change

[0126] During the experiment of gavage feeding mice with high internal phase emulsions stabilized by papain fiber and bromelain fiber, the body weight of mice in each group was measured and recorded every 3 days. The results are as follows: Figure 3 As shown in a.

[0127] from Figure 3 It can be seen that the body weight of mice fed with PE (papain fiber high internal phase emulsion) was significantly higher than that of mice fed with BE (bromelain fiber high internal phase emulsion) after about one month of feeding.

[0128] (2) Determination of lipid content in mouse feces

[0129] Take an appropriate amount of freshly collected mouse feces and place it in a centrifuge tube. Add 5 times the volume of physiological saline and soak overnight at 4°C. The next morning, remove the tube and vortex for 10 minutes to fully disperse the feces. Add 5 ml of chloroform-methanol (V:V:methanol = 1:2) mixture and vortex for another 10 minutes. Centrifuge at 3500 rpm for 10 minutes. At this point, the two liquid phases are separated by a solid phase. The upper liquid phase is aqueous, the middle solid phase is insoluble matter from the feces, and the lower liquid phase contains the extracted lipids. Carefully aspirate the lower liquid phase using a 1 ml syringe and transfer it to a pre-weighed clean glass culture dish. Add an appropriate amount of chloroform-methanol mixture to the centrifuge tube and repeat the extraction process until no more lower liquid phase precipitates. Allow the culture dish containing the collected lipids to evaporate freely in a ventilated and dry place for 3-4 days until all liquid has evaporated. After evaporation, reweigh the culture dish and subtract the initial weight of the culture dish to obtain the lipid content in the mouse feces.

[0130] The results of fecal fat content determination in mice gavaged in the NC, PE, and BE groups are as follows: Figure 3 As shown in b.

[0131] Depend on Figure 3 b shows that the fat content in the feces of mice fed with PE (papain fiber high internal phase emulsion) was lower than that in mice fed with BE (bromelain fiber high internal phase emulsion).

[0132] (3) Measurement of plasma biochemical indicators

[0133] The triglyceride levels in mouse plasma were determined according to the instructions of the corresponding kit.

[0134] The triglyceride content in the plasma of mice in the NC, PE, and BE groups was as follows: Figure 3 As shown in c.

[0135] from Figure 3 c shows that the triglyceride content in the plasma of mice gavaged with PE (papain fiber high internal phase emulsion) was significantly higher than that of mice gavaged with BE (bromelain fiber high internal phase emulsion) and NC (sterile water).

[0136] (4) Weighing of mouse adipose tissue

[0137] After euthanizing the mice by dislocation and collecting their blood, the epididymis, mesentery, and perirenal adipose tissue of the mice were removed and their weights were accurately recorded.

[0138] The weights of epididymal fat, mesenteric fat, and perirenal adipose tissue in mice gavaged in the NC, PE, and BE groups are as follows: Figure 4 As shown in a, 4b, and 4c.

[0139] from Figure 4 As shown in a, 4b, and 4c, the weights of epididymal, mesenteric, and perirenal adipose tissue in mice gavaged with the PE (papain fiber high inner phase emulsion) group were all higher than those in mice gavaged with the BE (bromelain fiber high inner phase emulsion) group.

[0140] Example 4

[0141] This study investigated the effect of a high-internal-phase emulsion stabilized by papain and bromelain fibers on lipid absorption in mice.

[0142] Effects of papain- and bromelain-stabilized high-inner-phase emulsions on lipid metabolism in mice after gavage ring

[0143] The animal experiments were reviewed and approved by the Animal Experiment Center of Nanjing Agricultural University (License No.: SYXK-(Su)-2011-0037) and complied with the National Guidelines for Laboratory Animal Welfare and the Ethical Standards for Animal Experimentation.

[0144] The lighting conditions in the housing were controlled, and the temperature and humidity were suitable for the mice's activity. Sixty SPF-grade male C57BL / 6 mice (20.0±0.5g) were randomly divided into two groups: PE-C and BE-C (n=30 in each group). All mice were allowed free access to water and food for one week for acclimatization, and were fasted overnight with free access to water the day before sacrifice. On day 8, the PE-C and BE-C groups were each gavaged with 250μL of a high internal phase emulsion stabilized by papain fiber (c=1wt%). 5k rpm) and a high internal phase emulsion stabilized by bromelain (c = 1wt%) Mice were sacrificed at 5k rpm at corresponding time points (0h, 1h, 2h, 3h, 4h) (6 mice were sacrificed at each time point within the 5 time points). The collected blood was immediately placed in EP tubes containing anticoagulant and centrifuged at room temperature for 4h to obtain plasma for biochemical analysis.

[0145] The levels of triglycerides, glycerol, and free fatty acids in the plasma of mice sacrificed at different treatment groups and time points were determined using a kit. Freshly collected mouse plasma was diluted with physiological saline and then ultracentrifuged. The supernatant plasma sample was collected and the morphology and size of chylolipoglobules were observed using TEM transmission electron microscopy.

[0146] Characterization

[0147] (1) Simulated digestion of a high internal phase emulsion stabilized by papain and bromelain fibers

[0148] The simulated gastric juice contains 2 g / L NaCl and 3.2 g / L pepsin. Before use, adjust the pH of the solution to 1.2 with 1.0 M hydrochloric acid and preheat it in a 37°C water bath.

[0149] Weigh 0.5 g of a high internal phase emulsion stabilized by different protease celluloses, add 15 ml of PBS (0.01 M, pH 7.0) solution, mix, and place in a 37°C water bath with stirring for 10 min. Then add 12 ml of simulated gastric juice, adjust the pH to 2.0 with 1.0 M hydrochloric acid, and simulate gastric digestion in a 37°C water bath for 2 h.

[0150] The pH of the mixture obtained from simulated gastric digestion was adjusted to 7.5. Then, 4 ml of PBS (0.01 M, pH 7.0) solution containing 375 g of porcine bile salts, 2.5 ml of PBS (0.01 M, pH 7.0) solution containing 60 mg of lipase, and 7 ml of PBS (0.01 M, pH 7.0) solution were added sequentially. Then, 2.5 ml of PBS (0.01 M, pH 7.0) solution containing 60 mg of trypsin was quickly added, and the pH of the solution was quickly adjusted to 7.5 before digestion began. Digestion was carried out in a 37°C water bath for 3 hours. During this time, the mixed solution was rapidly titrated with 0.05 M NaOH solution to stabilize the pH at 7.5. The volume of NaOH solution consumed to maintain the pH at 7.5 was recorded at different time points. The amount of free fatty acids released during simulated intestinal digestion was calculated using the following formula.

[0151]

[0152] Among them, C NaOH V represents the concentration of NaOH. NaOH M represents the volume of NaOH consumed. Lipid This indicates the molecular weight of sunflower seed oil, m. Lipid This indicates the weight of sunflower seed oil in the sample.

[0153] The substitution effect of bile acids on protein fibrils at the interface of HIPEs oil droplets was investigated using a simulated intestinal digestive fluid without pancreatic enzymes. Small samples were collected at the beginning and end of 3 hours of incubation and centrifuged (5000 rpm, 5 min) to separate the oil droplets. The supernatant was collected, and the concentration of protein fibrils was determined using the thioflavin T fluorescence method.

[0154] The results of bile salt digestion and free fatty acid release rates during simulated digestion of high internal phase emulsions stabilized by papain fiber and high internal phase emulsions stabilized by bromelain fiber are as follows: Figure 5 As shown in a and 5b.

[0155] like Figure 5 As shown in Figure a, the protein fiber content of PE emulsion in solution increased significantly before and after bile salt digestion, while the content of BE emulsion in solution did not change significantly.

[0156] like Figure 5 As shown in b, the oil release rate of PE was consistently significantly higher than that of BE, exhibiting a higher lipid release rate.

[0157] (2) Transmission electron microscopy observation of plasma chylomicron particles

[0158] Two hours before blood collection, mice were gavaged with 0.25 ml of PE and BE, respectively. For the PE-K and BE-K groups, plasma was collected from three mice in each group and diluted with physiological saline at a 1:3 ratio to 200 μL. The plasma was centrifuged at 117000 g and 4℃ for 3 hours. Using a clean 1 ml syringe, 5 μL of the supernatant was carefully drawn and spread onto a copper grid. The grid was stained with uranyl acetate for 15 min, dried at room temperature for 10 min, and then observed under an electron microscope. Image J was used to quantitatively analyze the chylomicron size in different groups.

[0159] Electron micrographs of chylomicrons in mice after gavage with PE and BE are shown below. Figure 6 As shown in a and 6b, the particle size distribution of mouse chylomicrons is as follows: Figure 6 As shown in c.

[0160] from Figure 6 As can be seen from a, 6b, and 6c, the particle size of chylomicrons in the plasma of mice gavaged by PE is significantly larger than that in the plasma of mice gavaged by BE.

[0161] (3) Measurement of plasma biochemical indicators

[0162] The levels of triglycerides, free fatty acids, and glycerol in mouse plasma were determined according to the respective kit instructions. The results of the determination of triglyceride, free fatty acid, and glycerol levels in mouse plasma after PE and BE gavage are shown below. Figure 7 As shown in a, 7b, and 7c.

[0163] from Figure 7 As can be seen from a, 7b, and 7c, the levels of triglycerides, free fatty acids, and glycerol in the plasma of mice gavaged via PE were higher than those in mice gavaged via BE.

[0164] The foregoing descriptions are merely exemplary embodiments or examples of the present invention and are not intended to limit the invention. Those skilled in the art will recognize that the present invention can be modified and varied in many ways. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention are included within the scope of the claims of this application.

Claims

1. A plant protein fiber, characterized in that, It is worm-like in shape, with a length ranging from 100 to 400 nm. The plant protein is bromelain. The method for preparing the plant protein fiber includes the following steps: I) Dissolve the plant protein in an acidic solution with a pH range of 2.0-3.0 to obtain plant protein solution A; II) Centrifuge the plant protein solution A at a temperature within the range of 4-6℃ to obtain supernatant A; III) Adjust the pH of supernatant A to the range of 1.90-2.10; IV) Filter the supernatant A with a pH range of 1.90-2.10 to obtain plant protein solution B; V) Heat and stir the plant protein solution B at a temperature in the range of 80-95℃ to make the plant protein fibrotic, then cool it to room temperature and centrifuge to obtain supernatant B; VI) Dialyze the supernatant B at a temperature ranging from 4 to 10°C to obtain the dialyzed solution C; and VII) The solution C was concentrated to obtain plant protein fiber.

2. A method for preparing plant protein fiber, wherein the plant protein is bromelain, characterized in that, Includes the following steps: I) Dissolve the plant protein in an acidic solution with a pH range of 2.0-3.0 to obtain plant protein solution A; II) Centrifuge the plant protein solution A at a temperature within the range of 4-6℃ to obtain supernatant A; III) Adjust the pH of supernatant A to the range of 1.90-2.10; IV) Filter the supernatant A with a pH range of 1.90-2.10 to obtain plant protein solution B; V) Heat and stir the plant protein solution B at a temperature in the range of 80-95℃ to make the plant protein fibrotic, then cool it to room temperature and centrifuge to obtain supernatant B; VI) Dialyze the supernatant B at a temperature in the range of 4-10℃ to obtain the dialyzed solution C; and VII) The solution C was concentrated to obtain plant protein fiber.

3. The preparation method according to claim 2, characterized in that, The solvent in step I) is a dilute hydrochloric acid solution.

4. The preparation method according to claim 2, characterized in that, The concentration of the resulting plant protein solution A is in the range of 20-50 mg / ml or 35-45 mg / ml.

5. The preparation method according to claim 2, characterized in that, Before step II), place the container at a temperature within the range of 4-6°C for a period of time to allow for complete hydration.

6. The preparation method according to claim 2, characterized in that, In step III), the pH of supernatant A is adjusted to the range of 1.95-2.

05.

7. The preparation method according to claim 2, characterized in that, In step IV), filtration is performed using a filter membrane.

8. The preparation method according to claim 7, characterized in that, The pore size of the filter membrane is in the range of 0.4-0.5 μm.

9. The preparation method according to claim 2, characterized in that, In step V), fiberization is carried out at a temperature in the range of 85-90°C.

10. The use of the plant protein fiber according to claim 1 for inhibiting the digestion and absorption of oils.

11. The use according to claim 10, characterized in that, The oil is an edible vegetable oil.

12. The use according to claim 10, characterized in that, The oils are selected from sunflower oil, olive oil, and rapeseed oil.

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