A slowly-digesting high-satiety self-assembled whey protein and a preparation method thereof

Abstract

The present application relates to a kind of slow digestion high satiety self-assembled whey protein and its preparation method, with whey protein isolate as raw material, after pulse electric field treatment, glutamine transaminase (TG enzyme) is added and crosslinked, by microwave freeze-drying technology, obtain low water content, whey protein powder easy to store and transport, it is self-assembled into colloid after reconstitution, and with slow digestion, high satiety and the protein supplement that is helpful to the weight management of consumer.

Description

Technical Field

[0001] This invention relates to a slow-digesting, high-satiety self-assembled whey protein and its preparation method, belonging to the field of food processing. Background Technology

[0002] Whey protein isolate (WPI) is a high-purity whey protein obtained through purification processes such as ultrafiltration based on whey protein concentrate, achieving a purity of over 90%. It boasts a high content of high-quality protein and is easily digested and absorbed, making it popular among certain groups with specific needs, such as fitness enthusiasts engaged in heavy resistance training, athletes undergoing high-intensity training, and patients requiring high-quality protein for recovery.

[0003] In addition, more and more people are paying attention to the benefits of high-protein diets for weight management, especially for obese individuals. High-protein diets can increase the secretion of satiety hormones such as PYY and GLP-1, while reducing the level of hunger hormones, thereby reducing appetite and increasing satiety; increase basal metabolic rate (BMR) and resting metabolic rate (RMR), helping the body burn more calories after eating and during sleep; protect muscle mass and prevent a decrease in metabolic rate; and protein has a high thermic effect, meaning that digesting and metabolizing protein requires more energy.

[0004] WPI contains a large number of bioactive peptide fragments. After digestion in the gastrointestinal tract, these bioactive peptides are released, stimulating the release of satiety hormones. WPI also contains up to 25% of three branched-chain amino acids (BCAAs): leucine, isoleucine, and valine. These can stimulate muscle growth and reduce body fat percentage. In particular, leucine can inhibit SESN2, thereby activating the mTORC1 signaling pathway and promoting muscle protein synthesis.

[0005] However, due to its excellent water solubility, WPI empties from the stomach very quickly, usually within 30 minutes. This rapid reduction in stomach volume can affect the feeling of fullness. Furthermore, the main components of WPI, β-lactoglobulin and α-lactalbumin, can cause allergies in some individuals, leading to gastrointestinal discomfort such as diarrhea. Therefore, there is an urgent need for a slow-digesting protein that, after a single dose, is slowly digested in the body, prolonging its residence time in the gastrointestinal tract and slowly releasing active peptides and amino acids to maintain a prolonged feeling of fullness, reduce appetite, and achieve weight management and control.

[0006] Currently available slow-digesting proteins primarily consist of casein, or a combination of casein and WPI (wheat protein ingestion). Casein encounters calcium phosphate in the stomach, forming calcium bridges and polymerizing to form micellar casein, resulting in slow digestion. Alternatively, they are made into protein gels, where the protein binds with water to form a relatively stable three-dimensional structure, slowing down gastric emptying and reducing the contact area with pepsin, thus achieving slow digestion.

[0007] However, existing slow-digesting proteins have the following problems:

[0008] (1) In the compound slow-digesting protein, the protein that plays a slow-digesting role is mainly casein, but its BCAA content is much lower than that of WPI, so it is weaker in stimulating muscle growth and cannot achieve the same effect as WPI.

[0009] (2) The β-lactoglobulin and α-lactalbumin contained in commonly used WPIs have certain sensitizing properties;

[0010] (3) Although protein gels can achieve slow digestion, they are prone to deterioration due to their high water content, making their storage requirements more stringent and increasing storage costs. At the same time, the large volume of the gel also leads to increased transportation costs.

[0011] To address the drawbacks of WPIs, which are digested relatively quickly and may not be efficiently absorbed by the human body, thus failing to maintain adequate amino acid levels during prolonged periods of fasting, CN99812255.6 discloses slowly digestible proteins and their uses. However, this patent still has the following issues:

[0012] (1) This product mainly enters the human body through the intestinal route and cannot be taken orally. The intake threshold is high, so it cannot be used as a daily protein supplement and has a relatively narrow target audience.

[0013] (2) The protein content is low, with only 5% protein in the product. To meet the protein requirements, a large amount of protein needs to be consumed, which puts a burden on the gastrointestinal tract.

[0014] (3) By adding alginate to bind with protein, protein is digested slowly, which has a certain impact on protein absorption in the body.

[0015] To address the drawbacks of WPI containing allergenic components mentioned above, CN113016930A discloses a method for preparing low-allergenic whey protein using extreme-condition assisted glycosylation modification combined with enzymatic methods, as well as the prepared low-allergenic whey protein. However, the following problems still exist:

[0016] (1) By glycosylating WPI with glucose, the reaction cannot be completely completed and some glucose will still remain, which will cause the human body to ingest excessive sugar while ingesting protein.

[0017] (2) Enzymatic hydrolysis can reduce the allergenicity of β-lactoglobulin, but the hydrolyzed product contains a large number of polypeptides, which increases the bitterness.

[0018] (3) The ultrasonic treatment uses an ultrasonic disruptor, but the sample size is small and it is difficult to achieve industrialization.

[0019] To address the drawbacks of difficult storage and high transportation costs associated with protein gels, CN201922257731.5 discloses a collagen drying device, but the following problems still exist:

[0020] (1) It is essentially still a spray drying process, but the protein gel has high viscosity and a stable gel structure, which makes feeding difficult and can easily clog the nozzle.

[0021] (2) If the gel is pretreated by high-speed dispersion and then spray-dried, although powder is formed, the protein gel structure is severely damaged and the gel cannot be formed again after reconstitution. Summary of the Invention

[0022] The purpose of this invention is to provide a slow-digesting, high-satiety, self-assembling whey protein and its preparation method. Specifically, it involves developing a slow-digesting whey protein isolate (SDW) with high BCAA content and spontaneous gelation after dissolution. First, the WPI is pretreated with a pulsed electric field (PEF) to promote the unfolding of its three-dimensional structure and increase the binding sites of TG enzyme. Then, TG enzyme is added to cross-link it, increasing the degree of polymerization and forming a tightly packed and stable three-dimensional network gel. Finally, microwave-assisted drying yields SDW protein powder.

[0023] The technical solution adopted by this invention to solve the problem is:

[0024] The first objective of this invention is to provide a slow-digesting, high-satiety, self-assembling whey protein, wherein the slow-digesting, high-satiety, self-assembling whey protein spontaneously forms a gel upon dissolving in water, and the gel elastic modulus of the slow-digesting, high-satiety, self-assembling whey protein is ≥200 Pa.

[0025] The slow-digesting, high-satiety, self-assembled whey protein has a digestibility of less than 50% after 60 minutes.

[0026] The slow-digesting, high-satiety self-assembled whey protein has a digestibility of less than 85% at 120 minutes.

[0027] Optionally, in one embodiment of the present invention, the slow-digesting, high-satiety self-assembled whey protein has a water content ≤6%, the BCAA content of the slow-digesting, high-satiety self-assembled whey protein is ≥25%, and the slow-digesting, high-satiety self-assembled whey protein does not contain β-lactoglobulin or α-lactalbumin.

[0028] Further optionally, in one embodiment of the present invention, the water content of the slow-digesting, high-satiety self-assembled whey protein is 5%-6%. Specifically, in one embodiment, the water content of the slow-digesting, high-satiety self-assembled whey protein is 5.45%. It should be noted that all the above water contents are in parts by weight.

[0029] Optionally, in one embodiment of the present invention, the BCAA content of the slow-digesting, high-satiety self-assembled whey protein is 25%-28%. Specifically, in one embodiment, the BCAA content of the slow-digesting, high-satiety self-assembled whey protein is 26.5%. It should be noted that the above BCAA content is in parts by weight.

[0030] Optionally, in one embodiment of the present invention, the slow-digesting, high-satiety self-assembling whey protein spontaneously forms a gel after dissolving in water.

[0031] Optionally, in one embodiment of the present invention, the gel elastic modulus of the slow-digesting, high-satiety self-assembled whey protein is 200-240 Pa.

[0032] The second objective of this invention is to provide a method for preparing a slow-digesting, high-satiety, self-assembling whey protein, comprising the following steps:

[0033] Step (1): Treat whey protein isolate (WPI) solution with pulsed electric field (PEF) to open its three-dimensional structure, expose the hidden hydrophobic groups inside, increase the binding sites for subsequent enzyme cross-linking, and at the same time moderately modify β-lactoglobulin and α-lactalbumin.

[0034] Step (2): The protein solution treated with the pulsed electric field is cross-linked with transglutaminase (TG enzyme) to form a protein gel.

[0035] Step (3): The protein gel is subjected to microwave freeze-drying (MFD).

[0036] The preparation method of the present invention mainly includes the following points:

[0037] (1) First, a pulsed electric field is used to change the tertiary structure of WPI, increase the cross-linking sites of TG enzyme, and improve the cross-linking efficiency. At the same time, the content of β-sheet and β-turn in the secondary structure of the protein decreases, while the random coil structure increases, which causes mild denaturation of sensitizing factors such as β-lactoglobulin and reduces the sensitization of WPI.

[0038] (2) Adding TG enzyme to the WPI solution after pulsed electric field treatment crosslinks the protein. TG enzyme catalyzes the binding reaction between the ε-amino group on the lysine residue and the γ-hydroxyamide group on the glutamine residue in the protein molecule, forming ε-(γ-glutamyl)lysine peptide bonds within and between the protein molecules. Therefore, WPI polymerizes into large protein molecules and forms a stable protein gel through non-covalent bonding.

[0039] (3) Microwave-assisted freeze drying (MFD) turns gels into powders, making them easier to store and transport. Microwaves provide more heat, resulting in faster, more uniform drying with lower energy consumption and less nutrient loss. It also reduces the loss of volatile substances, allowing the material to retain its shape and aroma more closely resemble the original. This advantage makes MFD more suitable for food drying. Another advantage is that MFD dehydration is more thorough, making the material less prone to oxidation and thus extending its shelf life. Furthermore, during MFD, microwaves can inactivate TG enzymes, eliminating the need for heating to inactivate the enzyme, improving processing efficiency, and reducing production costs. Finally, easily soluble and slowly digestible WPI powder is obtained.

[0040] Optionally, in one embodiment of the present invention, the concentration of the initial whey protein solution is 15%-25% (w / v).

[0041] Optionally, in one embodiment of the present invention, in the preparation method described in step (1), the pulse electric field treatment intensity is 14-20 kV / cm, and the treatment time is 5-20 min.

[0042] Optionally, in one embodiment of the present invention, in the preparation method described in step (2), 10-40 U / g of TG enzyme is added during cross-linking. The TG enzyme is purchased from Novozymes.

[0043] Optionally, in one embodiment of the present invention, in the preparation method described in step (2), the temperature of the protein solution crosslinking reaction is 50-60°C and the crosslinking time is 30-40 min.

[0044] Optionally, in one embodiment of the present invention, in the preparation method described in step (3), the microwave power of the microwave freeze-drying MFD is 500-700w and the vacuum degree is 120-200pa.

[0045] Optionally, in one embodiment of the present invention, in the preparation method described in step (3), the drying temperature of the protein gel is below -40°C and the drying time is 5-8 hours.

[0046] A third objective of this invention is to provide the use of a slow-digesting, high-satiety, self-assembling whey protein (SDW) in food or pharmaceuticals.

[0047] Optionally, in one embodiment of the present invention, slow-digesting, high-satiety self-assembling whey protein (SDW) is used as a food supplement in food. The food includes meal replacement products for increasing satiety and / or controlling weight.

[0048] Optionally, in one embodiment of the invention, the slow-digesting, high-satiety self-assembling whey protein is used to delay the gastric emptying rate.

[0049] Further, optionally, in one embodiment of the invention, the slow-digesting, high-satiety self-assembling whey protein is used to delay the gastric emptying rate by at least 120 minutes.

[0050] The present invention provides a slow-digesting, high-satiety, self-assembling whey protein (SDW) that can be added to food or pharmaceuticals, especially food. When dissolved in water, it can spontaneously form a gel, delaying gastric emptying rate within 120 minutes, increasing satiety, and reducing weight.

[0051] The advantages and positive effects of this invention are:

[0052] (1) The present invention uses pulsed electric field treatment to modify whey protein, which can not only improve the cross-linking efficiency of TG enzyme, but also greatly reduce the sensitization of WPI.

[0053] (2) The pulsed electric field modification of WPI in the previous step can improve the cross-linking efficiency of TG enzyme and cross-link it to produce proteins with large molecular weight and form a gel.

[0054] (3) Microwave freeze drying can not only dry gels efficiently and obtain protein powders that are easy to transport and store, but also inactivate enzymes through microwaves, thereby increasing processing efficiency.

[0055] (4) After WPI powder is redissolved, it will self-assemble into a protein gel and remain stable under acidic conditions and 25-40℃ conditions, thus achieving slow digestion.

[0056] (5) This invention can increase satiety, suppress appetite, effectively reduce people's craving for food when controlling their diet to lose weight, and is beneficial for people with high body weight to manage their weight. Attached Figure Description

[0057] Figure 1This is a graph showing the effect of temperature on the energy storage modulus and loss modulus of the SDW according to the present invention.

[0058] Figure 2 This is an in vitro digestion diagram of WPI, SDW, and casein according to the present invention.

[0059] Figure 3 This is a graph showing the intestinal propulsion rate of mice after gavage administration of deionized water (DW), WPI, and SDW samples, respectively, according to the present invention.

[0060] Figure 4 This is a graph showing the changes over time in (a) cholecystokinin (CCK) and (b) glucagon-like peptide-1 in the gastrointestinal tract of mice after oral administration of DW, WPI, and SDW samples, respectively.

[0061] Figure 5 This is a graph showing (a) food intake and (b) body weight of mice that have undergone long-term DW, WPI and SDW dietary supplementation according to the present invention. Detailed Implementation

[0062] This invention aims to provide a method for preparing a slow-digesting, high-satiety, self-assembled whey protein, comprising the following steps:

[0063] Step (1): Treat whey protein isolate (WPI) solution with pulsed electric field (PEF) to open its three-dimensional structure, expose the hidden hydrophobic groups inside, increase the binding sites for subsequent enzyme cross-linking, and at the same time moderately modify β-lactoglobulin and α-lactalbumin.

[0064] Step (2): The protein solution treated with the pulsed electric field is cross-linked with transglutaminase (TG enzyme) to form a protein gel.

[0065] Step (3): The protein gel is subjected to microwave freeze-drying (MFD).

[0066] Specifically, in some examples, the concentration of the initial whey protein solution is 15%-25% (w / v).

[0067] Specifically, in some examples, in the preparation method described in step (1), the pulsed electric field treatment intensity is 14-20 kV / cm. Specifically, in some examples, the pulsed electric field treatment intensity can be selected as 14, 16 and 20 kV / cm, and the treatment time of the protein solution by each intensity of the electric field is 5-20 min independently.

[0068] Specifically, in some examples, during the preparation method described in step (2), 10-40 U / g of TG enzyme is added during cross-linking. The TG enzyme was purchased from Novozymes.

[0069] Specifically, in some examples, in the preparation method described in step (2), the temperature of the protein solution crosslinking reaction is 50-60℃ and the crosslinking time is 30-40min.

[0070] Specifically, in some examples, in the preparation method described in step (3), the microwave power of the microwave freeze-drying MFD is 500-700w and the vacuum degree is 120-200pa.

[0071] Specifically, in some examples, in the preparation method described in step (3), the drying temperature of the protein gel is below -40°C and the drying time is 5-8 hours.

[0072] The present invention will be further described in detail below through specific embodiments. The following embodiments are merely descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0073] Example 1

[0074] (1) Stir to dissolve 10L of 20% (w / v) WPI solution and remove the precipitate by ultrafiltration to ensure complete dissolution.

[0075] (2) Treat it with a pulsed electric field of 14 kV / cm for 15 min.

[0076] (3) Heat the treated protein solution to 50°C, add 40 U / g protein TG enzyme, and continue cross-linking for 40 min to obtain a stable protein gel.

[0077] (4) The gel was dried using MFD. After the temperature was below -40℃, the drying was started. The microwave power was 600W, the vacuum degree was 200pa, and the drying time was 6h. The resulting protein powder had a water content as low as 5.45wt%, of which the BCAA content was 26.5wt%.

[0078] Comparative Example 1

[0079] The difference between this comparative example and Example 1 is the absence of step (2) pulse processing, intended to explore the effect of pulse processing on WPI. Specifically, it includes the following steps:

[0080] (1) Stir to dissolve 10L of 20% (w / v) WPI solution and remove the precipitate by ultrafiltration to ensure complete dissolution.

[0081] (2) Heat the protein solution to 50°C, add 40 U / g protein TG enzyme, and continue cross-linking for 180 min to obtain a protein solution that cannot form a stable protein gel.

[0082] This is because the TG enzyme reaction in WPI has fewer cross-linking sites, resulting in low cross-linking efficiency. Even with prolonged cross-linking treatment, a stable gel cannot be formed.

[0083] Comparative Example 2

[0084] The difference between this comparative example and Example 1 is the drying method in step (3), which aims to explore the effect of microwave freeze-drying on WPI. Specifically, it includes the following steps:

[0085] (1) Stir to dissolve 10L of 20% (w / v) WPI solution and remove the precipitate by ultrafiltration to ensure complete dissolution.

[0086] (2) Treat it with a pulsed electric field of 20 kV / cm for 10 min.

[0087] (3) Heat the treated protein solution to 50°C, add 40 U / g protein TG enzyme, and continue cross-linking for 40 min to obtain a stable protein gel.

[0088] (4) The gel was dried using a low-temperature hot air drying method: the drying temperature was 50°C and the drying time was 20 hours, which yielded a protein powder with a water content as low as 4.3%.

[0089] The poor solubility of protein samples after hot air drying may be due to excessive polymerization and denaturation of proteins during the drying process, making them unable to redissolve and form a gel.

[0090] Test case

[0091] Physicochemical properties: The rheological properties of the protein gel were detected using a Discovery Hybrid Rheometer-3. A simulated gastric environment was used, with the pH adjusted to 3 and the test temperature ranging from 25-40℃. Changes in the elastic modulus and loss modulus of the gel were measured. Figure 1 ).from Figure 1 It can be seen that SDW can maintain a stable gel state in the simulated gastric environment (37℃, pH 3), that is, G' is greater than G", and the gel elastic modulus G' reaches more than 200 Pa, with a maximum value of 234 Pa.

[0092] Static in vitro digestion experiment: The digestion rates of WPI, casein, and SDW were evaluated using the INFOGEST static in vitro simulated digestion model. Digestion time was 4 hours, with samples taken every 30 minutes. Precipitation was performed using 20% ​​trichloroacetic acid, and the precipitate was considered undigested protein. The protein content in the precipitate was measured using the Kjeldahl method, and the digestibility was calculated using the following formula:

[0093]

[0094] from Figure 2As can be seen, in the 4-hour in vitro digestion experiment, WPI had the fastest digestion rate, reaching 89.15%, but SDW could achieve a digestion rate similar to casein, at 38.14% and 37.82%, respectively.

[0095] Short-term in vivo digestion experiment: C57BL / 6J mice were used as experimental subjects for in vivo digestion testing. After fasting for 12 hours, mice were administered an equal volume of deionized water containing rhodamine B, 10% (w / v) WPI, and 10% (w / v) SDW samples by gavage. Mice were euthanized by carbon dioxide gas anesthesia within 0-120 minutes, and their gastrointestinal tracts were collected for fluorescence imaging. Figure 3 ). Figure 3 In group a, after 30 minutes of gavage, fluorescence was detected in most locations of the intestines in both the DW and WPI groups, with the propulsion rate in the DW group approaching 100%, significantly higher than the 75.59% in the WPI group. However, in the SDW group, most of the fluorescence intensity was concentrated in the stomach, with a propulsion rate of only 33.72%, indicating that at 30 minutes of digestion, the SDW sample was still primarily in the gastric digestion stage and had not emptied significantly into the small intestine. Figure 3 As shown in Figures b and c, after 60 and 120 minutes of digestion, the fluorescent area in the intestine of the DW group gradually decreased, possibly due to the rapid water transport rate in the intestine, causing much of the fluorescent indicator to be excreted in the urine. In the WPI group, after 60 minutes of digestion, more than half of the intestinal tissue showed fluorescence (72.62%). With further increases in digestion time to 120 minutes, fluorescence was distributed throughout the entire intestine, and its intestinal propulsion rate reached nearly 100%, showing no significant difference from the DW group, indicating that it had been almost completely digested through intestinal peristalsis. However, the propulsion rate of the SDW group only increased to 42.57% at 60 minutes, significantly lower than the other two groups at the same time. At 120 minutes, the propulsion rate was 82.69%, still below 100%, indicating that its intestinal digestion was still lower than that of the WPI group during the 120-minute in vivo digestion period.

[0096] Collect their serum and test the relevant hormone levels. Figure 4Following gavage administration of both WPI and SDW protein samples, serum CCK concentrations showed an increasing trend, with the SDW group slightly higher than the WPI group, both reaching peak levels at 60 min, at 56.10 ng / L and 61.28 ng / L, respectively. At 120 min, the CCK level in the WPI group decreased to 32.35 ng / L, while the CCK level in the SDW group remained relatively high at 66.63 ng / L. Serum GLP-1 levels in both the WPI and SDW groups continued to increase at 30 and 60 min, reaching peak levels at 60 min, at 6.35 pmol / L and 7.50 pmol / L, respectively. Although both groups showed a decrease at 120 min, the SDW group only decreased to 6.85 pmol / L, while the WPI group experienced a significant decrease to 3.02 pmol / L.

[0097] Long-term feeding experiment: Obese C57BL / 6J mice (weight > 40g) were used as experimental subjects for long-term feeding tests. The mice were divided into 3 groups and, while having free access to water and food (high-fat diet), were administered isoenzyme deionized water, 10% (w / v) WPI, and 10% (w / v) SDW samples by gavage daily for 8 weeks. The average food intake was recorded and calculated daily, and the weight change was recorded weekly.

[0098] result Figure 5 As shown, the food intake of mice in the DW group and WPI group was 4.12 g / day and 4.07 g / day, respectively. However, the food intake of mice in the SDW group was significantly reduced to only 3.36 g / day. The average body weight of mice in the DW and WPI groups steadily increased over time, reaching 45.2 g and 44.3 g, respectively. In contrast, the average body weight of mice in the SDW group continuously decreased to 36.7 g, indicating that dietary supplementation with SDW has an appetite-suppressing effect and is beneficial for weight management.

[0099] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several modifications and improvements can be made without departing from the inventive concept, and these all fall within the protection scope of the present invention.

Claims

1. A method for preparing a slowly digestible, satiating self-assembled whey protein, characterized by, The method comprises the following steps: Step (1): treating an initial whey protein isolate solution with a pulsed electric field; The intensity of the pulsed electric field treatment is 14 kV / cm, and the treatment time of the protein solution is 5-20 min; Step (2): adding transglutaminase for cross-linking to form a protein gel; the added TG enzyme is 10-40 U / g of protein, the cross-linking reaction temperature is 50-60 ℃, and the cross-linking time is 30-40 min, Step (3): microwave freeze-drying the protein gel.

2. The method of claim 1, wherein, The concentration of the initial whey protein isolate solution is 15%-25% w / v.

3. The method of claim 1, wherein, In the preparation method of step (3), the microwave power of microwave freeze-drying is 500-700 w, and the vacuum degree is 120-200 pa.

4. The method of claim 1, wherein, In the preparation method of step (3), the drying temperature of the protein gel is lower than -40 ℃, and the drying time is 5-8 h.

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