A nano-dietary polyphenol drink and its production method and application

Abstract

This invention relates to the field of sleep-aiding health food technology, and discloses a nano-dietary polyphenol beverage, its production method, and its application. The production method is as follows: Cyclodextrin, skim milk powder, and sodium caseinate are added to hot water at a temperature of 50-60°C and stirred until dissolved to obtain liquid 1; oligosaccharides and dietary plant polyphenols are added to hot water at a temperature of 60-80°C until fully dissolved, and the pH is adjusted to 6.2-6.8 to obtain liquid 2; while stirring, liquid 1 is heated to 85-95°C and kept at this temperature for 10-20 minutes. Then, while still stirring, liquid 2 and liquid 3 (formed from pre-dissolved ascorbic acid, sodium citrate, and concentrated fruit juice) are successively added to the kept-temperature liquid 1. Water is added to make up the difference, stirring is maintained, and after cooling, filtration, bottling, and sterilization, the dietary polyphenol beverage is obtained. This application solves the problem of bitter taste in high-polyphenol content beverages, improves the bioavailability of polyphenols, and has the effect of improving sleep quality.

Description

[0001] This invention is a divisional application based on the application filed on June 25, 2023, with application number 202310746521.0 and entitled "A dietary polyphenol beverage with the effect of improving sleep quality and its production method". Technical Field

[0002] This invention relates to the field of sleep-aiding health food technology, and in particular to a nano-dietary polyphenol beverage, its production method, and its application in improving sleep quality. Background Technology

[0003] Insomnia has become a major health problem affecting one-third of the world's population. To date, no effective solution to insomnia has been found, either domestically or internationally. Modern medicine primarily uses sedative-hypnotic drugs for treatment. While these drugs are effective in the short term, they also have a range of side effects, including drug tolerance, addiction, rebound insomnia, difficulty in withdrawal, and cognitive impairment. Therefore, an increasing number of people are turning to safer sleep-aiding health products to address their sleep disorders.

[0004] Currently, the main sleep-aiding ingredients on the sleep health food market are melatonin, L-theanine, and gamma-aminobutyric acid (GABA). Melatonin is generally considered to have the best sleep-aiding effect, but its safety remains controversial. Melatonin is an important sleep hormone that regulates the body's circadian rhythm. A decrease in its secretion disrupts this rhythm, leading to difficulty falling asleep. Besides insufficient melatonin secretion, many other causes of insomnia include physical illness, metabolic disorders, and mental stress, the latter of which are beyond the reach of melatonin. Furthermore, long-term excessive use of melatonin may cause hormonal imbalances and cerebral vasoconstriction. In some countries, products containing melatonin are even banned for minors. GABA is currently the second most common sleep-aiding ingredient on the market, primarily inhibiting neurotransmitters in the central nervous system of mammals. Low GABA levels in the brain are one of the causes of insomnia. A 2021 animal study showed that mice with insomnia who consumed GABA-rich yogurt experienced a significant increase in GABA levels in their brain tissue, and their insomnia symptoms also improved. However, the true effectiveness of GABA for people with insomnia remains highly controversial. Currently, it is believed that GABA improves sleep by soothing emotions and alleviating sleep disorders. However, the acceptable daily intake of GABA is ≤500 mg. Whether GABA can effectively cross the blood-brain barrier and reach the brain to exert its effects at such a dosage remains unknown.

[0005] Plant polyphenols are superior natural antioxidants and free radical scavengers. After ingestion, they can directly eliminate free radicals (or oxidative substances) in the body, enhance the activity of the body's antioxidant enzyme system, protect lipids from oxidative damage, and maintain the integrity of biological membrane structure and function. Therefore, these active substances are hailed as an indispensable "golden nutrient" for modern people. Furthermore, plant polyphenols that are not directly absorbed can enter the colon, where they can regulate the colonic flora. They can also be transformed by microbial enzymes, producing metabolites that are reabsorbed by the body. This latter mechanism is even considered a treatment option for some chronic and mental illnesses (such as diabetes and depression). Recent studies have found that the synergistic effect of plant polyphenols and oligosaccharides can achieve optimal results in improving colonic flora structure and even overall health. In light of this, the efficacy of dietary plant-derived polyphenols in improving sleep disorders has received increasing attention in recent years. With in-depth scientific exploration, numerous scientists have discovered that polyphenols can not only effectively improve sleep quality by regulating circadian rhythms, but also by improving gut health and increasing the body's antioxidant levels (J. Agric. Food Chem., 2019, 67:8735; Food & Funct., 2021, 12:6136). However, plant polyphenols typically suffer from poor solubility, a strong bitter taste, and poor absorption, which significantly limits the development of related sleep-aiding health foods.

[0006] A review of domestic and international patents and products reveals that, aside from tea beverages and some fruit juices (such as cranberry juice and mulberry juice), there are very few (almost none) liquid drinks rich in polyphenols on the market. Patent CN102511551A discloses a liquid dairy product and its production method that helps improve menopausal insomnia. This liquid dairy product contains nutrients including soy isoflavones (a type of polyphenolic phytoestrogen), vitamin D, and ginseng. Soy isoflavones account for 0.1-0.3% of the total weight of the dairy product (optimized). A 10-day trial confirmed a good effect on improving insomnia in menopausal women (efficacy rate exceeding 90%). CN113439784A discloses a production method for polyphenol-containing dairy products. Through direct mixing, homogenization, and sterilization of polyphenols with liquid milk, a non-bitter polyphenol milk is obtained. Volunteer trials have shown that this dairy product has certain health benefits such as improving sleep, whitening and clearing acne, and improving blood lipids. The aforementioned patents require a minimum human consumption of 200 ml for the dairy products described in the trials, and they have not undergone storage stability assessments. Considering that most common beverages on the current health food market are available in 50 or 30 ml sizes, it is essential to develop a dietary polyphenol beverage with high polyphenol content, good flavor, and sleep-improving effects. This beverage would contain polyphenols in nanoparticle form to meet the urgent needs of people with insomnia and sleep disorders who wish to improve their sleep through diet. Summary of the Invention

[0007] The purpose of this invention is to provide a nano-dietary polyphenol beverage with the effect of improving sleep quality and its production method, so as to solve the problems existing in the prior art.

[0008] The dietary polyphenol beverage produced by this invention has the advantages of high polyphenol content ("high polyphenol content" means that the crude polyphenol content in every 500 ml of beverage is at least 600 mg (dry weight)), good flavor (no bitterness) and industrial applicability.

[0009] This invention's dietary polyphenol beverage differs from current sleep aids on the market based on GABA and theanine. This invention's dietary polyphenol beverage effectively improves sleep quality, supported not only by substantial scientific evidence (polyphenols regulate circadian rhythms or biological clocks and improve gut microbiota), but also by animal studies and extensive human trials. Furthermore, this invention's dietary polyphenol beverage incorporates oligosaccharides, further enhancing the health benefits of improving sleep quality through gut health enhancement.

[0010] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is a nano-dietary polyphenol beverage with the effect of improving sleep quality, comprising the following raw materials by mass percentage: 6-12% skim milk powder, 3-10% oligosaccharides, 0.5-3% sodium caseinate, 1-2% concentrated fruit juice, 0.5-2% dietary plant polyphenols, 0.05-2% cyclodextrin, 0.02-0.05% ascorbic acid, 0.025% sodium citrate, and the balance being water.

[0011] Furthermore, the nano-dietary polyphenol beverage with the effect of improving sleep quality comprises, by weight percentage, the following ingredients: 8-10% skim milk powder, 5-8% oligosaccharides, 1-3% sodium caseinate, 1-2% concentrated fruit juice, 1-1.5% dietary plant polyphenols, 0.05-2% cyclodextrin, 0.02-0.05% ascorbic acid, 0.025% sodium citrate, with the balance being water.

[0012] Furthermore, the nano-dietary polyphenol beverage that is said to improve sleep quality also includes flavoring (to adjust the flavor of the beverage).

[0013] Furthermore, the dietary plant polyphenols include one or more of the following as food or new resource food (raw material that meets food production license standards) with high polyphenol content: tea powder, grape powder, grape seed powder, apple powder, blueberry powder, cranberry powder, citrus powder, turmeric powder, vine tea powder, and sugarcane polyphenols.

[0014] The selection of dietary plant polyphenols and their dosage mainly depend on their solubility in hot water and the polyphenol content of the raw materials.

[0015] Furthermore, the tea powder has a polyphenol content higher than 80 wt.%; the grape seed powder has a proanthocyanidin content higher than 90 wt.%; the apple powder has a polyphenol content higher than 30 wt.%; and the blueberry powder, cranberry powder, and citrus powder all have a polyphenol content higher than 20 wt.%.

[0016] Furthermore, the dietary plant polyphenols mentioned are tea powder.

[0017] Unlike GABA and theanine, the mechanism by which polyphenols improve sleep quality is more clearly defined and recognized by the academic community. Numerous studies have found that polyphenols can improve sleep quality by regulating circadian rhythms and improving gut health.

[0018] Furthermore, the oligosaccharides include one or more of xylooligosaccharides, isomaltooligosaccharides, fructooligosaccharides, galactooligosaccharides, raffinose, gentiosaccharides, lactulose, soybean oligosaccharides, and stachyose; the concentrated fruit juice includes one or more of concentrated peach juice, concentrated peach juice, concentrated pear juice, and concentrated cranberry juice.

[0019] Furthermore, the oligosaccharide includes one or more of fructooligosaccharides, isomaltooligosaccharides, and soybean oligosaccharides.

[0020] Furthermore, the cyclodextrin is β-cyclodextrin and / or γ-cyclodextrin.

[0021] High-protein-polyphenol blended liquid systems are highly unstable, prone to precipitation and phase separation. Ensuring the maximum amount of dietary polyphenols introduced into systems with high protein concentrations (e.g., weight ratios exceeding 4%), enabling them to withstand subsequent high-intensity sterilization treatments (e.g., 115°C for over half an hour) and maintain long-term storage stability, remains a technical challenge with no literature reports on this topic. Based on previous in-depth understanding of protein assembly and encapsulation of hydrophobic bioactive substances (see the inventor's series of review papers published in authoritative international journals: Food Hydrocolloids, 2021, 112:106344; Food Hydrocolloids, 2021, 117:106710; Adv. Colloid Interface Sci., 2021, 292:102432), this invention proposes a polyphenol nano-encapsulation technology based on the principle of milk protein assembly and encapsulation, suitable for industrial production. This technology has successfully enabled the industrial production of polyphenol beverages with high polyphenol content and high stability. Using skim milk powder as the milk protein raw material, this invention achieves efficient protein-polyphenol complexation at relatively high temperatures (e.g., 85–95°C) by controlling the temperature-mediated protein structure unfolding and reassembly process. Subsequently, a cooling process further assembles the relevant protein-polyphenol complexes, forming stable nanocomposites. The production method of this invention for high-polyphenol content and stable polyphenol beverages is not only simple but also easily industrialized, and can be carried out according to the following process: Skim milk powder (sodium caseinate) → hot water reconstitution → preheating treatment → protein-polyphenol compounding → cooling (under stirring conditions) → filtration → filling → sterilization → labeling and packaging → finished nano dietary polyphenol beverage.

[0022] The specific steps are as follows: The second technical solution of the present invention: a method for producing the above-mentioned nano-dietary polyphenol beverage, comprising the following steps: Specifically, the following steps are included: (1) Add cyclodextrin, skim milk powder, and sodium caseinate to hot water at a temperature of 50-60℃ and stir to dissolve (redissolve in hot water, and no milk particles should appear) to obtain solution 1; add oligosaccharides and dietary plant polyphenols to hot water at a temperature of 60-80℃ until fully dissolved, and adjust the pH to 6.2-6.8 to obtain solution 2. When dietary plant polyphenols dissolve in water, the resulting solution is acidic. Therefore, it is necessary to adjust the pH of the solution to be similar to that of the reconstituted milk powder dispersion (solution 1). Otherwise, flocculation or even precipitation may easily occur.

[0023] (2) Heat liquid 1 under stirring (preheating treatment), and then slowly add liquid 2 under stirring (mix liquid 1 and liquid 2 to form a protein-polyphenol complex at high temperature). Then add pre-dissolved ascorbic acid, sodium citrate and concentrated fruit juice in sequence, add water to make up the volume, maintain stirring, cool to ≤40℃, filter (treated with 100-mesh silk cloth), fill (≤40℃, glass bottle), and sterilize to obtain the dietary polyphenol beverage.

[0024] Filtration can be performed simultaneously with filling.

[0025] Furthermore, the sterilization process also includes labeling and packaging steps.

[0026] Furthermore, the heat treatment temperature is 85–95°C, and the holding time is 10–20 min.

[0027] Reconstituted milk protein or milk protein nanoparticles (milk protein or milk protein nanoparticles in skim milk powder) will undergo certain protein denaturation and structural dissociation when treated at 85-95℃ for a certain time (10-20 min), which is the guarantee for achieving efficient complexation of protein and polyphenols (polyphenols in dietary plant polyphenols).

[0028] The role of the cooling process: Once the protein and polyphenols are fully compounded at 85-95°C, a sufficient cooling process is necessary before filling and sterilization to allow the protein-polyphenol complex to form a more compact nanostructure, making it less prone to dissociation during the vigorous sterilization process, which could lead to system instability (precipitation).

[0029] Furthermore, the sterilization method is selected from any of the following: Method 1: High-temperature sterilization; the high-temperature sterilization temperature is 115-125℃, and the time is 20-40 minutes; Method 2: First, perform ultra-high temperature instantaneous sterilization, then perform pasteurization; the ultra-high temperature instantaneous sterilization temperature is 120-150℃, and the time is 2-8s; the pasteurization temperature is 72-95℃, and the time is 15-30min.

[0030] This invention chooses to introduce oligosaccharides into a pre-prepared polyphenol solution, rather than adding them directly during the reconstitution of skim milk powder. This is primarily due to the scientific fact that the presence of a certain concentration of sugar induces protein molecules to form a more compact structure. Adding oligosaccharides during the reconstitution of skim milk powder is not conducive to the subsequent heating-induced protein structure unfolding; while adding them to dietary plant polyphenols is beneficial for the further assembly of nanocomplexes, forming more stable nanostructures. Of course, the addition of oligosaccharides is also primarily for the purpose of enhancing the ability of related polyphenol beverages to regulate gut microbiota health.

[0031] This invention provides a dietary polyphenol beverage that is not only high in polyphenols but also has a pleasant flavor. In addition to the main skim milk powder (milk protein), it also contains sodium caseinate. Extensive preliminary research has demonstrated that adding a certain amount of sodium caseinate not only helps mask the bitterness of the beverage but also significantly improves its storage stability after high-temperature sterilization (sodium caseinate can optimize the stability of the protein-polyphenol composite nanostructure). Adding a certain amount of cyclodextrin further enhances the masking of the bitterness of polyphenols, while adding a certain amount of ascorbic acid (which has antioxidant properties) can produce a synergistic antioxidant effect with polyphenols.

[0032] This invention solves the technical limitations of polyphenol beverages with high polyphenol content, such as bitterness and poor stability, by further assembling milk protein and polyphenol nanocomposites into nanoparticles that are stable for a long time (and thermally stable).

[0033] Furthermore, a certain amount of sweetener or flavoring can be added to the product formula of this invention as needed to prepare a polyphenol beverage with a better flavor. The selection of the types and amounts of the relevant substances can be carried out in accordance with conventional techniques in the art, provided that the stability and safety of the beverage are not affected.

[0034] The nano-sized dietary polyphenol beverage of this invention has been verified through animal experiments and large-scale human trials to improve sleep quality, including shortening sleep onset time, increasing total sleep time and deep sleep time, and providing a significant health benefit for insomnia caused by metabolic disorders. Analysis of various biochemical indicators in experimental insomnia mice revealed that taking the dietary polyphenol beverage of this invention significantly increased the release of sleep-related inhibitory neurotransmitters (including serotonin, melatonin, and GABA) in the mouse brain, improved the antioxidant capacity of brain tissue, and reduced the release of inflammatory factors (including interleukin-1 and tumor necrosis factor-α) caused by insomnia.

[0035] The present invention discloses the following technical effects: (1) The nano-dietary polyphenol beverage of the present invention has the advantages of high polyphenol content ("high polyphenol content" means that the crude polyphenol content in each 500 ml beverage is at least 600 mg (dry weight)), good flavor (no bitter taste) and industrial applicability.

[0036] (2) The method for producing nano-dietary polyphenol beverages of the present invention is an innovative polyphenol encapsulation technology based on the latest protein nano-assembly principle in the field of food colloids. It overcomes the current technical limitations restricting the application of polyphenols in beverages both domestically and internationally, such as severe bitterness and low bioavailability. The method of the present invention is simple, feasible, and easy to industrialize. More importantly, the raw materials involved in the dietary polyphenol beverages of the present invention can all be selected from approved "new resource foods" (meeting the requirements of food production licenses), which has significant economic and social implications for creating a completely new type of plant-based health beverage.

[0037] (3) One of the most important understandings in modern nutrition is that many bioactive substances from dietary plants play an important role in maintaining human health and preventing chronic diseases. Numerous scientific studies have confirmed that polyphenols, as the most important bioactive substances from dietary plants, not only have superior antioxidant, anti-inflammatory, and free radical scavenging effects, but also have a wide range of effects in promoting human health and preventing modern chronic diseases, including preventing cardiovascular diseases, anti-cancer, and tumor inhibition. However, due to their poor water solubility, strong bitter taste, and low bioavailability, the application of dietary polyphenols in health foods has been greatly limited. This invention perfectly integrates important scientific discoveries in modern nutrition with the latest protein nano-encapsulation technology, solving the problems of poor stability and low bioavailability of polyphenols, and significantly increasing the polyphenol content in beverages.

[0038] (4) The dietary polyphenol beverages of the present invention have the effect of improving sleep quality (or aiding sleep), and have been verified by animal experiments and large-scale human trials.

[0039] (5) The shelf life of the dietary polyphenol beverage produced in the form of glass bottle according to the present invention is more than 12 months.

[0040] (6) The present invention produces a polyphenol beverage with high polyphenol content and good stability by heating a solution containing skim milk powder (and sodium caseinate) (85-95°C), protein-polyphenol complexing at high temperature, and cooling-induced assembly. The preparation process of the present invention is simple and easy to industrialize.

[0041] (7) Animal experiments have confirmed that the nano-polyphenol beverage produced by this invention has a significant effect on improving the sleep quality of insomniac mice, mainly manifested in: increasing the incidence of sleep, shortening the sleep latency, and increasing the sleep duration. Its mechanism of action is related to improving liver function, regulating the release of neurotransmitters in brain tissue, enhancing the body's antioxidant capacity, and reducing inflammatory factors in brain cells. Further human trials have verified that the polyphenol beverage of this invention has a good effect on improving sleep quality.

[0042] (8) Based on the principle of protein-polyphenol nanocomposite and assembly, the production method of polyphenol beverage provided by the present invention not only solves the technical problem of the strong bitter taste of beverages with high polyphenol content, but also improves the bioavailability and health experience of polyphenols. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 This is a particle size distribution diagram of the dietary polyphenol beverage produced in Example 1 of the present invention before and after sterilization; Figure 2 This is a particle size distribution diagram of the dietary polyphenol beverage produced in Example 2 of the present invention before and after sterilization; Figure 3 This is a particle size distribution diagram of the dietary polyphenol beverage produced in Comparative Example 1 of the present invention before and after sterilization; Figure 4 The particle size distribution diagrams of the series of dietary polyphenol beverages produced in Example 2 and Comparative Example 3 of this invention before sterilization are shown. Figure 5 The particle size distribution of the sterilized dietary polyphenol beverages produced in Example 1 and Comparative Example 1 of the present invention after storage for 0, 3 and 6 months is shown. Figure 6 The sensory evaluation results of the dietary polyphenol beverages produced in Examples 1, 2 and Comparative Example 2 of the present invention are shown in the figure. Figure 7 The effects of different treatment groups on liver injury indicators in insomnia mice were investigated, where ALT was alanine aminotransferase and AST was aspartate aminotransferase. Figure 8 The effects of different treatment groups on the antioxidant properties of mouse brains were investigated, where a) was the content of superoxide dismutase (SOD), b) was the content of malondialdehyde (MDA), and c) was the content of glutathione peroxidase (GSH-Px). Figure 9 The effects of different treatment groups on mouse cytokines are shown, where a) is 1L-1β and b) is TNF-α. Detailed Implementation

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0048] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0049] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0050] In the following examples, "parts" refers to "parts by weight".

[0051] Example 1 A method for producing a nano-sized dietary polyphenol beverage that improves sleep quality: This dietary polyphenol beverage, which is effective in improving sleep quality, is composed of the following ingredients by weight percentage: 10.0% skim milk powder, 7.8% fructooligosaccharides, 0.1% soybean oligosaccharides, 0.5% sodium caseinate, 1.0% concentrated peach juice (55 degrees Brix), 1.6% dietary plant polyphenols (tea powder, grape seed powder, and blueberry powder in a ratio of 2:2:1, with a total crude polyphenol content of approximately 80 wt.%), 0.1% β-cyclodextrin, 0.025% ascorbic acid, 0.025% sodium citrate, and the remainder being water.

[0052] The production method is as follows: (1) Take 70 / 100 volume of water into the first dissolving tank, heat it to 50°C, add β-cyclodextrin first under stirring, and then gradually add skim milk powder and sodium caseinate. After it is fully dissolved, liquid 1 is obtained (the volume of liquid 1 accounts for about 70% of the total volume of the final beverage).

[0053] (2) Take 20 / 100 volume of water into the second dissolving tank, heat it to 80°C, and add fructooligosaccharides, soybean oligosaccharides, trehalose and dietary plant polyphenols under stirring. After they are fully dissolved, adjust the pH of the solution to 6.2 with 4M sodium hydroxide solution to obtain liquid 2 (the volume of liquid 2 accounts for about 20% of the total volume of the final beverage).

[0054] (3) Take 6 / 100 volume of water into the third dissolving tank, heat it to 50°C, add concentrated peach juice, ascorbic acid and sodium citrate, and continue stirring until completely dissolved. Adjust the pH of the solution to 6.2 using 4M sodium hydroxide solution to obtain liquid 3 (the volume of liquid 3 accounts for about 6% of the total volume of the final beverage).

[0055] (4) Before mixing all the liquids, heat the temperature of the above liquid 1 to 90°C under stirring and keep it warm for 15 minutes. Then, immediately under stirring, slowly add liquid 2 and liquid 3 to the kept-warm liquid 1, adjust the volume to the required total volume, and then continue to stir slowly for 30 minutes to obtain the premixed composition.

[0056] (5) The premixed composition is naturally cooled to about 40°C under slow stirring, then filtered through 100-mesh silk, and then filled (filling temperature is 36-38°C, glass bottle) and sterilized at high temperature (sterilization, 115°C, 30 min) to obtain dietary polyphenol beverage.

[0057] The quality indicators of the dietary polyphenol beverage produced in this embodiment are as follows: light purple color, solid content of 21%, pH of 6.3; silky texture, slightly sweet. The particle size distribution and average particle size (D) of the above polyphenol beverage before and after sterilization (115℃, 30 min) were determined using a nanoparticle size analyzer based on the dynamic laser scattering principle (model Zetasizer Nano ZS; detection conditions: temperature 25℃, fixed scattering angle 173°). z ), the results are shown Figure 1 .

[0058] The beverages were filtered through a 0.22 μm filter membrane and diluted with a pH 7.0, 5 mM phosphate buffer to a solids content of approximately 2 mg / mL.

[0059] from Figure 1As can be seen from the data, the particle size of the polyphenol beverage (sterilized at high temperature) produced in this embodiment is mainly distributed between 100 and 300 nm, and its D... z The particle size is approximately 174 nm, indicating that all the polyphenols and proteins are assembled to form a type of nanoparticle with uniform particle size; the particle size of the beverage before and after high-temperature sterilization is basically the same, indicating that the beverage produced in this embodiment is extremely heat-stable.

[0060] Example 2 A method for producing a nano-sized dietary polyphenol beverage that improves sleep quality: This dietary polyphenol beverage, which is effective in improving sleep quality, is composed of the following ingredients by weight percentage: 8.0% skim milk powder, 9.0% isomaltooligosaccharide, 2.5% sodium caseinate, 1% concentrated apple juice (sweetness 45-50 degrees), 1.2% dietary plant polyphenols (grape seed powder, apple powder, and cranberry powder; the ratio of the three is 2:2:1, and the total crude polyphenol content is about 60-70 wt.%), 1.0% γ-cyclodextrin, 0.05% ascorbic acid, 0.025% sodium citrate, and the balance being water.

[0061] The production method is as follows: (1) Take 65 / 100 volume of water into the first dissolving tank, heat it to 60°C, add γ-cyclodextrin first under stirring, and then gradually add skim milk powder and sodium caseinate. After it is fully dissolved, liquid 1 is obtained (the volume of liquid 1 accounts for about 65% of the total volume of the final beverage).

[0062] (2) Take 15 / 100 volume of water into the second dissolving tank, heat it to 70°C, and add isomaltooligosaccharide and dietary plant polyphenols under stirring. After they are fully dissolved, adjust the pH of the solution to 6.5 with 4M sodium hydroxide solution to obtain liquid 2 (the volume of liquid 2 accounts for about 15% of the total volume of the final beverage).

[0063] (3) Take 3 / 100 volume of water into the third dissolving tank, heat it to 70°C, add concentrated apple juice, ascorbic acid and sodium citrate, and continue stirring until completely dissolved. Adjust the pH of the solution to 6.5 using 4M sodium hydroxide solution to obtain liquid 3 (the volume of liquid 3 accounts for about 3% of the total volume of the final beverage).

[0064] (4) Before mixing all the liquids, heat the temperature of the above liquid 1 to 90°C under stirring and keep it warm for 10 minutes. Then, immediately under stirring, slowly add liquid 2 and liquid 3 to the kept-warm liquid 1, make up to the required total volume, and then continue to stir slowly for 30 minutes to obtain the premixed composition.

[0065] (5) The premixed composition is naturally cooled to 40°C under slow stirring, then filtered through 100-mesh silk, and then filled (filling temperature is about 38°C, glass bottle) and sterilized at high temperature (sterilization, 115°C, 30 min) to obtain dietary polyphenol beverage.

[0066] The quality indicators of the dietary polyphenol beverage produced in this embodiment are as follows: It has a light purple color, a solid content of approximately 22%, a pH value of approximately 6.4-6.5, a silky texture, and a certain degree of sweetness. The particle size distribution and average particle size (D) of the polyphenol beverage produced in this embodiment before sterilization were detected using a nanoparticle size analyzer (same as above). z The result is as follows Figure 2 As shown.

[0067] from Figure 2 As can be seen, the particle size of the polyphenol beverage (without high-temperature sterilization) produced in this embodiment is mainly distributed between 100 and 300 nm, and its D... z The particle size is approximately 181 nm, indicating that all the polyphenols and proteins are assembled into a class of uniformly sized nanoparticles. The polyphenol beverage produced in this example has a D... z Slightly larger than the unsterilized beverage of Example 1 of the present invention. From Figure 2 It can also be seen that the sterilization treatment has no significant effect on the particle size of the related polyphenol beverages, indicating that the polyphenol beverages produced in this embodiment are extremely heat-stable. Besides the difference in formulation, another difference is that the liquid 1 in this embodiment is dissolved in water at 60°C, while the liquid 1 in Example 1 of this invention is dissolved at 50°C. Therefore, it can be considered that the dissolution temperature of the skim milk powder in liquid 1 has a significant impact on the quality of the final beverage.

[0068] Example 3 A method for producing a nano-sized dietary polyphenol beverage that improves sleep quality: This dietary polyphenol beverage, which is effective in improving sleep quality, is composed of the following ingredients by weight percentage: 6.0% skim milk powder, 10.0% oligosaccharides (fructooligosaccharides, isomaltooligosaccharides, and soybean oligosaccharides in a 1:1:1 weight ratio), 0.5% sodium caseinate, 2.0% concentrated fruit juice (concentrated peach juice and concentrated pear juice in a 1:1 weight ratio, with a sugar content of 55 degrees Brix), 0.5% dietary plant polyphenols (tea powder, with a crude polyphenol content of approximately 80%), 2.0% β-cyclodextrin, 0.02% ascorbic acid, 0.025% sodium citrate, and the remainder being water.

[0069] The production method is as follows: (1) Take 70 / 100 volume of water into the first dissolving tank, heat it to 55°C, add β-cyclodextrin first under stirring, and then gradually add skim milk powder and sodium caseinate. After it is fully dissolved, liquid 1 is obtained (the volume of liquid 1 accounts for about 70% of the total volume of the final beverage).

[0070] (2) Take 20 / 100 volume of water into the second dissolving tank, heat it to 90°C, add oligosaccharides and dietary plant polyphenols under stirring, and after they are fully dissolved, adjust the pH of the solution to 6.8 with 4M sodium hydroxide solution to obtain liquid 2 (the volume of liquid 2 accounts for about 20% of the total volume of the final beverage).

[0071] (3) Take 6 / 100 volume of water into the third dissolving tank, heat it to 90°C, add concentrated fruit juice, ascorbic acid and sodium citrate, and continue stirring until completely dissolved. Adjust the pH of the solution to 6.8 using 4M sodium hydroxide solution to obtain liquid 3 (the volume of liquid 3 accounts for about 6% of the total volume of the final beverage).

[0072] (4) Before mixing all the liquids, heat the temperature of the above liquid 1 to 85°C under stirring and keep it warm for 20 minutes. Then, immediately under stirring, slowly add liquid 2 and liquid 3 to the kept-warm liquid 1, adjust the volume to the required total volume, and then continue to stir slowly for 30 minutes to obtain the premixed composition.

[0073] (5) The premixed composition is naturally cooled to 40°C under slow stirring, then filtered through 100-mesh silk, and then filled (filling temperature is 40°C, glass bottle), sterilized (first ultra-high temperature instantaneous sterilization, then pasteurization; ultra-high temperature instantaneous sterilization temperature is 130°C, time is 6s; pasteurization temperature is 85°C, time is 25min) to obtain dietary polyphenol beverage.

[0074] The quality indicators of the dietary polyphenol beverage produced in this embodiment are as follows: the color is light yellow, the solid content is about 22%, the pH value is about 6.4 to 6.5, the taste is silky and has a certain sweetness.

[0075] Example 4 A method for producing a nano-sized dietary polyphenol beverage that improves sleep quality: This dietary polyphenol beverage, which is effective in improving sleep quality, is composed of the following ingredients by weight percentage: 12.0% skim milk powder, 3.0% oligosaccharides (gentiosaccharides), 3.0% sodium caseinate, 1.0% concentrated fruit juice (concentrated cranberry juice, sugar content 55 degrees Brix), 2.0% dietary plant polyphenols (tea powder, grape seed powder, and blueberry powder in a ratio of 2:2:1, with a crude polyphenol content of approximately 80%), 0.05% β-cyclodextrin, 0.05% ascorbic acid, 0.025% sodium citrate, and the remainder is water.

[0076] The production method is as follows: (1) Take 70 / 100 volume of water into the first dissolving tank, heat it to 60°C, add β-cyclodextrin first under stirring, and then gradually add skim milk powder and sodium caseinate. After it is fully dissolved, liquid 1 is obtained (the volume of liquid 1 accounts for about 70% of the total volume of the final beverage).

[0077] (2) Take 20 / 100 volume of water into the second dissolving tank, heat it to 70°C, and add oligosaccharides and dietary plant polyphenols under stirring. After they are fully dissolved, liquid 2 is obtained (the volume of liquid 2 accounts for about 20% of the total volume of the final beverage).

[0078] (3) Take 6 / 100 volume of water into the third dissolving tank, heat it to 70°C, add concentrated fruit juice, ascorbic acid and sodium citrate, and continue stirring until completely dissolved. Adjust the pH of the solution to 6.2 using 4M sodium hydroxide solution to obtain liquid 3 (the volume of liquid 3 accounts for about 6% of the total volume of the final beverage).

[0079] (4) Before mixing all the liquids, heat the temperature of the above liquid 1 to 95°C under stirring and keep it warm for 10 minutes. Then, immediately under stirring, slowly add liquid 2 and liquid 3 to the kept-warm liquid 1, adjust the volume to the required total volume, and then continue to stir slowly for 30 minutes to obtain the premixed composition.

[0080] (5) The premixed composition is naturally cooled to 40°C under slow stirring, then filtered through 100-mesh silk, and then filled (filling temperature is 40°C, glass bottle) and sterilized at high temperature (sterilization, 115°C, 30 min) to obtain dietary polyphenol beverage.

[0081] The quality of the dietary polyphenol beverage produced in this embodiment is roughly equivalent to that of the dietary beverage produced in Embodiment 1.

[0082] Comparative Example 1 Same as in Example 1, except that the temperature of the above-mentioned liquid 1 was not heated to 90°C and kept at that temperature for 15 minutes in step (4); The natural cooling process in step (5) was not performed; the other steps were the same.

[0083] The particle size distribution and average particle size (D) of the polyphenol beverage before and after sterilization (115℃, 30 minutes) were determined using a nanoparticle size analyzer (as above). z The result is as follows Figure 3 As shown.

[0084] from Figure 3 As can be seen, the particle size distribution of the polyphenol beverage (without high-temperature sterilization) produced in this comparative example is similar to that of Example 1 of the present invention, with its particle size mainly between 100 and 300 nm and its Dz estimated to be 166 nm, indicating that the protein and polyphenols in this example can still effectively form nanoparticles with uniform particle size. However, once subjected to high-temperature sterilization, the Dz of the polyphenol beverage in this comparative example significantly increased to 229 nm, and the related particle size distribution also became more diversified, with a clear flocculation distribution peak (particle size of approximately 5-8 micrometers) appearing. Figure 3 This indicates that the polyphenol beverage produced in this comparative embodiment is unstable under high-temperature heat treatment (115°C, 30 minutes). Compared with Example 1 of the present invention, the difference in this comparative embodiment is the absence of the preheating treatment (90°C, 15 minutes) of liquid 1 before mixing, and the subsequent cooling process. It is evident that preheating liquid 1 (skim milk powder + sodium caseinate) is a crucial step in forming a polyphenol beverage with high thermal stability. This is because heating induces the structural expansion of milk proteins (and protein particles) in the system, which is more conducive to the composite assembly with polyphenols to form more stable protein-polyphenol composite nanoparticles.

[0085] Comparative Example 2 Same as Example 1, except that the ingredients of the dietary polyphenol beverage that improves sleep quality do not contain skim milk powder and sodium caseinate.

[0086] The production method is as follows: (1) Take 20 / 100 volume of water into the first dissolving tank, heat it to 80°C, and add fructooligosaccharides, soybean oligosaccharides, dietary plant polyphenols and β-cyclodextrin under stirring. After it is fully dissolved, liquid 1 is obtained (the volume of liquid 1 accounts for about 20% of the total volume of the final beverage).

[0087] (2) Take 6 / 100 volume of water into the second dissolving tank, heat it to 50°C, add concentrated peach juice, ascorbic acid and sodium citrate under stirring, and continue stirring until completely dissolved. Adjust the pH of the solution to 6.2 using 4M sodium hydroxide solution to obtain liquid 2 (the volume of liquid 2 accounts for about 6% of the total volume of the final beverage).

[0088] (3) Add liquid 2 slowly to liquid 1 under stirring, then bring the volume to the required total volume and continue stirring for 30 minutes to obtain the premixed composition.

[0089] (4) The premixed composition is naturally cooled to 40°C under slow stirring, then filtered through 100-mesh silk, and then filled (filling temperature is 40°C, glass bottle) and sterilized at high temperature (sterilization, 115°C, 30 min) to obtain dietary polyphenol beverage.

[0090] Comparative Example 3 Same as Example 2, except that the heating and melting temperature in step (1) is 50, 70, 80 or 90°C, while the other steps are the same as in Example 2.

[0091] Comparative Example 4 Same as Example 1, except that the natural cooling to below 40°C in step (5) was not performed, but instead the temperature was cooled to 60°C. The other steps are the same.

[0092] Comparative Example 5 Same as Example 1, except that the fructooligosaccharides and soybean oligosaccharides from step (2) are added to liquid 1.

[0093] (1) Take 80 / 100 volume of water into the first dissolving tank, heat it to 50°C, add β-cyclodextrin first under stirring, and then gradually add skim milk powder, sodium caseinate, fructooligosaccharide and soybean oligosaccharide. After it is fully dissolved, liquid 1 is obtained (the volume of liquid 1 accounts for about 70% of the total volume of the final beverage).

[0094] (2) Take 10 / 100 volume of water into the second dissolving tank, heat it to 80°C, add dietary plant polyphenols under stirring, and after it is fully dissolved, adjust the pH of the solution to 6.2 with 4M sodium hydroxide solution to obtain liquid 2 (the volume of liquid 2 is about 20% of the total volume of the final beverage).

[0095] Comparative Example 6 Same as Example 1, except that sodium caseinate was not added; all other steps are the same as in Example 1.

[0096] Example of effect 1 The particle size distribution, storage stability, sensory quality, bioavailability, sleep-improving effects (animal experiments), and sleep quality improvement effects of the nano-dietary polyphenol beverages produced in the embodiments and comparative embodiments of the present invention were determined.

[0097] (I) Particle size distribution (before beverage sterilization) The particle size distribution (D) of a series of dietary polyphenol beverages produced in Example 2 and Comparative Example 3 of the present invention before sterilization was compared.z ), the results are shown Figure 4 .

[0098] from Figure 4 As can be seen, step (1) of the production method of this invention, namely the initial temperature for dissolving skim milk powder, has a significant impact on the particle size distribution of the final dietary polyphenol beverage before sterilization. As the dissolution temperature increases from 50°C to 90°C, the main particle size distribution peak of the produced polyphenol beverage before sterilization becomes increasingly broad and shows a trend towards larger particle sizes. Correspondingly, the relevant D... z The particle size increased continuously from an initial 166 nm to 223 nm. It was clearly observed that the beverage produced at a dissolution temperature of 90°C contained flocculation peaks with particle sizes as high as several micrometers. Therefore, controlling the dissolution temperature of skim milk powder has a significant impact on the quality of the dietary polyphenol beverage provided by this invention. The optimal dissolution temperature is 50–60°C.

[0099] (II) Storage stability The sterilized nano-dietary polyphenol beverages (in intact packaging) produced in Example 1 and Comparative Example 1 of this invention were stored at room temperature for 3 to 6 months. The particle size distribution (Dsize) of the newly produced dietary polyphenol beverages and those stored for 3 and 6 months were then measured. z ), the results are shown Figure 5 .

[0100] from Figure 5 As can be seen from the above, the nano-dietary polyphenol beverage (in intact packaging) produced in Example 1 of this invention, after 6 months of room temperature storage, exhibits a particle size distribution (and D... z The particle size showed almost no significant change, indicating that the dietary polyphenol beverage produced in Example 1 has good storage stability. Correspondingly, the particle size of the dietary polyphenol beverage (sterilized at high temperature) produced in Comparative Example 1 showed a continuous increasing trend with increasing storage time (3-6 months); after 6 months of storage, the D... z The nm wavelength increased from 229 nm to 337 nm. The results further clarify that preheating the feed solution 1 (fat-soluble milk powder + sodium caseinate) before the protein and polyphenols are combined is a key production step to ensure the superior storage stability of the dietary polyphenol beverage produced by this invention.

[0101] (III) Sensory Quality Sensory evaluations were performed on the dietary polyphenol beverages produced in Examples 1, 2, and Comparative Example 2 of this invention. The results are shown in [the table below]. Figure 6 .

[0102] The evaluation method is as follows: 20 mL of each beverage was placed in a transparent plastic dish with a random 3-digit code and presented to 25 evaluators in a random order. The 25 evaluators scored the beverages from 0 to 7 points, with each sample repeated once. The beverages were smelled first, then tasted. In accordance with GB / T 10220-2012, the sensory evaluation room was quiet, comfortable, odorless, well-ventilated, and well-lit. The temperature was controlled at approximately 21±1℃, and the humidity at approximately 65%±5%. The sensory descriptions and quantitative descriptive analysis forms are shown in Table 1, and the sensory evaluation results are shown in [Table 1]. Figure 5 .

[0103] Table 1. Definitions and Reference Samples of Sensory Properties

[0104] from Figure 6 As can be seen from the above, the dietary polyphenol beverages produced in Examples 1-2 of this invention all have certain fruity aroma, milky flavor, and sweetness, while the sourness and astringency are relatively weak. Figure 6 Therefore, it can be considered a novel polyphenol beverage with a good flavor and appealing to the general public. However, the beverage made by simply dissolving and mixing all the raw materials (Comparative Example 2) has an extremely strong astringent taste and a relatively strong sour taste. It is evident that the production method of a high-polyphenol beverage based on protein assembly nanotechnology provided by this invention effectively solves the biggest limitation of polyphenols in beverage applications, such as a strong bitter and astringent taste.

[0105] This invention also compared the sensory qualities, particularly the astringency, of the dietary polyphenol beverages of Example 1 and Comparative Example 4. The results showed that the dietary polyphenol beverage of Comparative Example 4 (after sterilization) exhibited significant precipitation after one week of storage, and its astringency was also stronger than that of Example 1. Compared to Example 1, the production of the dietary polyphenol beverage of Comparative Example 4 only lacked the natural cooling process to below 40°C in step (5). Therefore, the relevant natural cooling process plays a crucial role in the structural tolerance of the protein-polyphenol nanocomposite particles to subsequent high-temperature (115°C) sterilization treatment.

[0106] This invention also compared the sensory qualities of the dietary polyphenol beverages of Example 1 and Comparative Example 5. Compared with Example 1, the production of the dietary polyphenol beverage of Comparative Example 5 adjusted the order of adding oligosaccharides, adding them to the pre-dissolved liquid 1 (skim milk and sodium caseinate). The results showed that the sensory qualities of the two were almost identical.

[0107] Furthermore, this invention further evaluated the effect of not adding sodium caseinate on the quality of the final produced dietary polyphenol beverage. Compared with Example 1 of this invention, the dietary polyphenol beverage of Comparative Example 6 exhibited more severe flocculation after one month of storage, and the product also had a more pronounced astringent taste. The results indicate that adding a certain amount of sodium caseinate plays an important role in improving the flavor (astringency) and stability of the polyphenol beverage.

[0108] (iv) Bioavailability of polyphenols Using a simulated in vitro gastrointestinal digestion model, the antioxidant capacity of the polyphenol beverages produced in Example 1 and Comparative Example 2 after simulated in vitro gastrointestinal digestion was measured, with Trolox (water-soluble vitamin E) as the equivalent. The specific determination method is as follows: Take 20 mL of the beverage produced in Example 1 and Comparative Example 3 into an Erlenmeyer flask, add 5 mL of 10 mM phosphate buffer, adjust the pH of each solution to 1.5 with 4 M HCl, and place them in a shaker. Preheat at 37°C and 95 rpm for 10 min. Add 20 mg of pepsin to each Erlenmeyer flask to simulate gastric digestion for 1 h. Then, adjust the pH of the dispersion to 7.0 with 2 M NaOH, mix and disperse in a shaker for 10 min, add 40 mg of pancreatin, and start simulating intestinal digestion for 2 h. At the end of digestion, centrifuge the samples (10000 g, 30 min) and collect the supernatant. Add 20 μL of supernatant, phosphate buffer or Trolox standard solution sequentially to each well of a 96-well plate. Add 120 μL of sodium fluorescein solution to each well, vortex to mix, and incubate at 37°C for 20 min. Then, quickly add 60 μL of 2,2′ amidopropyl hydrochloride (AAPH) solution, vortex, and immediately measure the fluorescence intensity using a microplate reader (excitation wavelength 485 nm, emission wavelength 528 nm). Record the values ​​every 4 min for a total reaction time of 140 min. AUC represents the area under the fluorescence curve. AUC = 2 + 4(f2 / f1 + f3 / f1 + ... + f 34 / f1)-2f 35 f1: First fluorescence reading, f i The fluorescence reading in the i-th round, NetAUC = AUC 样品 -AUC 空白 .

[0109] The antioxidant capacity index (ORAC) of the polyphenol beverage produced in Example 1 of this invention is 9328.4±96.6 μM / g, which is significantly higher than that of the beverage in Comparative Example 2 (4365.61±78.42 μM / g). This result further demonstrates that the production method of polyphenol beverage based on protein assembly nanotechnology provided by this invention significantly improves the bioavailability of polyphenols, thus resulting in a better health experience.

[0110] (V) Verification Experiment on Improving Insomnia in Animals Insomnia Model Establishment: Sixty healthy, specific pathogen-free (SPF) grade male ICR mice (average body weight approximately 20g) aged 4 weeks were selected for the experiment. The animal housing temperature was maintained at 24–26℃ and humidity at 40–60%. After arrival, the animals underwent an acclimatization period of one week, with free access to food and water. The bedding, feed, and drinking water were changed regularly by the experimenter. The 60 mice were randomly divided into 6 groups according to body weight: normal group, model group, positive group (using the insomnia drug estazolam as a control), low-dose group, medium-dose group, and high-dose group (see Table 2). Except for the normal control group, which received an intraperitoneal injection of an equal volume of physiological saline, the other groups received a 350mg / kg solution of p-chlorophenylalanine (PCPA) in physiological saline solution. The solution was thoroughly shaken before each injection, and administered intraperitoneally once daily for 2 days. Compared with the normal control group, the initial model establishment was considered successful when the mice exhibited restlessness, continuous activity, loss of diurnal rhythm, decreased appetite, and significant weight loss. The specific experimental steps for inducing sleep in mice with sodium pentobarbital, and the methods for measuring sleep latency and sleep duration are as follows: Table 2. Grouping of 60 mice (insomnia modeling and polyphenol drink intervention)

[0111] The beverages listed in Table 2 are dietary polyphenol beverages produced in Example 1 of this invention.

[0112] (1) Effect of beverage (produced in Example 1) on sodium pentobarbital-induced sleep in mice A. Effects of subthreshold doses of sodium pentobarbital on sleep onset rate in mice After successful modeling, starting on day 3, each group was administered the corresponding drug via gavage once daily. The normal group and the model group were administered an equal volume of distilled water via gavage. This administration was continued for 10 days. Sixty minutes after the last administration, each group of mice received an intraperitoneal injection of 30 mg / kg sodium pentobarbital. The time from the start of the sodium pentobarbital injection to the disappearance of the righting reflex was defined as the sleep latency, and the time from the disappearance of the righting reflex to its recovery was defined as the sleep duration. In the experiment, the disappearance of the righting reflex for 1 minute was used as the indicator of sleep onset, and the number of times the animal turned over 3 times within 30 seconds was used as the indicator of sleep termination.

[0113] B. Effects of suprathreshold dose sodium pentobarbital on sleep latency and sleep duration in mice The mouse grouping, modeling, and administration methods were the same as described above. Sixty minutes after the last administration, each group of mice received an intraperitoneal injection of 45 mg / kg sodium pentobarbital. The time of disappearance and reappearance of the righting reflex were recorded for each group of mice. Sleep was defined as the period from the disappearance of the righting reflex to its reappearance. Finally, the sleep latency and sleep duration for each group of mice were calculated.

[0114] (2) Observation Records The diet and water intake of mice, as well as their mental state, activity and behavior, hair condition and sleep before and after drug administration, were observed and recorded. Their weight was measured daily.

[0115] (3) Measurement of mouse serum markers After the final gavage (drink), all mice were fasted and deprived of water overnight. Blood was collected from the eyeballs under mild anesthesia with ether and placed in 2 mL centrifuge tubes. The serum was obtained by centrifugation and stored at -80°C for later use. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured.

[0116] (4) Examination of mouse brain tissue pathological sections After blood was drawn from the eyeballs, the mice were euthanized by dislocation and placed prone on an ice pack. The neck skin was first horizontally cut open, then a "T"-shaped cut was made along the midline of the head to the tip of the nose. The skin was then pulled apart from the center to both sides with forceps. The occipital bone was cut open with tissue scissors, and the parietal bone was similarly cut open in a "T" shape and broken to both sides to expose the entire brain tissue. The skull was removed, the brain tissue was extracted, and the hypothalamus was isolated and homogenized. The levels of 5-hydroxytryptamine (5-HT), dopamine (DA), glutamate (Glu), and γ-aminobutyric acid (GABA) were measured. The remaining brain tissue was also homogenized, and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and the lipid peroxidation metabolite malondialdehyde (MDA), as well as the levels of cellular inflammatory factors IL-1β and TNF-α, were measured.

[0117] Table 3 Effects on sleep quality in insomnia mice

[0118] Note: Different letters A through D represent significant differences at the p<0.05 level between different groups of data in the same column.

[0119] Sodium pentobarbital is a drug that acts on the central nervous system and is metabolized by liver enzymes. To eliminate the possibility of false positives due to the inhibitory effect of the sample on liver enzymes, a subthreshold dose of sodium pentobarbital was used for hypnosis. The results showed that compared with the control group and the model group, the number of mice falling asleep in the drug-positive group and the polyphenol drink group was significantly increased, especially in the medium- and high-dose drink groups, where the sleep rate reached 100% (Table 3). The speed of falling asleep is expressed as sleep latency. Rapid sleep onset is an important indicator of sleep quality, which is expressed as the duration of sleep. As shown in Table 3, compared with the model group, the sleep time in each dose group (low, medium, and high) of the polyphenol drink was significantly increased, and the sleep latency was significantly shortened. The polyphenol drink improved the sleep quality of insomniac mice comparable to the positive drug, and in shortening sleep latency, the polyphenol drink was even superior to the drug-positive group (Table 3). These results verify that the polyphenol drink produced in Example 1 of this invention has an efficacy comparable to hypnotic drugs in improving insomnia symptoms. Based on the changes in mouse body weight, it can be concluded that the polyphenol beverage of the present invention has a greater advantage in improving the health of insomniac mice.

[0120] Changes in body weight reflect, to some extent, the growth and health of mice. Table 4 shows the results of body weight changes in each group of mice during the 12-day experimental period (2 days of modeling + 10 days of drug administration).

[0121] Table 4. Effects of polyphenol drinks on mouse body weight (g)

[0122] Inducing insomnia in mice via intraperitoneal injection of PCPA for two days revealed varying degrees of weight loss in all model groups compared to the control group. Mice exhibited reduced food intake but increased agitation, dry and dull fur, and eventually became lethargic and unable to sleep, indicating successful model establishment. On day 3, gavage administration of the drug showed a slight increase in weight in all gavage groups. Compared to the model group, mice in the positive control group and polyphenol drink groups (at various doses) achieved near-normal weight gain and resumed normal activity, actively feeding again. This demonstrates that both the positive control drug and the polyphenol drink effectively improved the mental state of mice, regulated their circadian rhythms, and had no toxic side effects.

[0123] The results of the measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice are shown in the figure. Figure 7 .

[0124] Figure 7 In comparison with the blank control group, *p<0.05, **p<0.01, ***p<0.001; compared with the model group, # p<0.05, ##p<0.01, ### p<0.001.

[0125] Serum ALT and AST levels are two important indicators for assessing liver function. Generally, higher serum ALT and AST levels indicate more severe hepatocellular damage. Figure 7 It was found that, compared with the control group, the serum ALT and AST levels in the model group mice were almost doubled, indicating severe hepatocellular damage in the insomnia mice. Compared with the model group, the ALT and AST levels in the drug-treated (positive group) and polyphenol beverage (each dose group) groups were significantly reduced, indicating that the administration of the insomnia drug and polyphenol beverage reduced hepatocellular damage in the insomnia mice. In particular, the AST level in the medium-dose beverage group was almost equivalent to that in the control group. Figure 7 The results indicate that the liver function indicators of mice given the appropriate dose were close to normal levels, which means that it has a significant protective effect against liver damage.

[0126] Sleep occurs in the brain, regulated by the interaction of neural circuits. Neurotransmitters are involved in this process, generally divided into two types: excitatory neurotransmitters related to wakefulness, primarily dopamine (DA), norepinephrine (NE), and glutamate (Glu); and inhibitory neurotransmitters related to sleep, primarily serotonin (5-HT), melatonin (MT), and gamma-aminobutyric acid (GABA). Increased levels of excitatory neurotransmitters in brain tissue promote wakefulness, while increased levels of inhibitory neurotransmitters promote sleep. Sleep disorders are often associated with imbalances in neurotransmitter release.

[0127] Table 5. Effects of polyphenol beverages on neurotransmitter release in mice.

[0128] Table 5 shows that the levels of 5-HT and GABA in the model group mice decreased to some extent, while the levels of DA and Glu increased, proving the successful establishment of the model. Compared with the model group, the levels of excitatory neurotransmitters in the gavage groups (drug and polyphenol beverage) were increased, while the levels of inhibitory neurotransmitters were significantly decreased (Table 5). The levels of inhibitory neurotransmitters 5-HT and GABA in the medium-dose beverage group were even higher than those in the drug-positive group, while the levels of excitatory neurotransmitters DA and Glu were the opposite. This indicates that taking polyphenol beverages can significantly promote the production of inhibitory neurotransmitters in insomnia mice while reducing the release of excitatory neurotransmitters. These results suggest that the mechanism by which polyphenols improve sleep quality in insomnia mice is closely related to their regulation of neurotransmitters in the hypothalamus.

[0129] Numerous studies have indicated that insomnia is closely related to oxidative stress, which leads to the excessive production of reactive oxygen species (ROS), causing toxic damage to brain nerve cells. The levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA), a lipid peroxidation metabolite, were measured in the brain tissue of mice in each group. The results are as follows: Figure 8 As shown.

[0130] Figure 8 In the figures, a) represents the content of superoxide dismutase (SOD), b) represents the content of malondialdehyde (MDA), a lipid peroxidation metabolite, and c) represents the content of glutathione peroxidase (GSH-Px). (Note: Compared with the blank control group, *p<0.05, **p<0.01, ***p<0.001; compared with the model group, # p<0.05, ## p<0.01, ### p<0.001).

[0131] Superoxide dismutase (SOD) is the primary substance for scavenging free radicals in the body. It can resist the attack of free radicals on tissues, cells, and nucleic acids, reduce the damage caused by free radicals to the body, and repair damaged cells in a timely manner. GSH-Px is an important peroxidase that participates in catalyzing the reaction of reduced glutathione (GSH) with peroxides to produce non-toxic hydroxyl compounds and oxidized glutathione (GSSG). GSSG is reduced to GSH by H+ ions in the liver or red blood cells to maintain the continuous scavenging of peroxides. SOD and GSH-Px are important components and evaluation indicators of the body's antioxidant defense. Malondialdehyde (MDA) is one of the most important products of lipid peroxidation. Its excessive production can damage the membrane systems of human organs and cells, affecting the body's normal metabolism. MDA can be considered an important indicator for evaluating the degree of lipid peroxidation in the body.

[0132] from Figure 8 As can be seen, after PCPA-induced insomnia in the model group mice, all oxidative stress indicators changed significantly, with SOD and GSH-Px levels decreasing significantly and MDA levels increasing significantly. Figure 8 This indicates that the insomniac mice in the model group suffered severe oxidative damage to their brains. Mice treated with the drug or polyphenol drink showed significant improvement in various oxidative indicators, especially the medium-dose polyphenol drink group, where all indicators were almost equivalent to the control group. This demonstrates that PCPA-induced insomnia in mice resulted in significant oxidative damage to the brain, while the polyphenol drink significantly reversed sleep deprivation-induced oxidative damage, thus exhibiting superior neuroprotective effects.

[0133] Cytokines are signals involved in the neuro-immune-endocrine interplay and are important regulators of sleep. Interleukin-1 (IL-1) is a sleep-promoting cytokine produced by various cells and, under physiological conditions, regulates appetite, sleep, and endocrine function. Among them, IL-1β, which regulates sleep, is the most extensively studied cytokine. It is generally believed that IL-1β levels are positively correlated with insomnia duration. Tumor necrosis factor (TNF) exists in two forms: TNF-α and TNF-β. As a pro-inflammatory cytokine produced by macrophages, TNF participates in immune and inflammatory responses. The effects of polyphenolic beverages on inflammatory factors in brain cells of insomniac mice were investigated, and the results are as follows... Figure 9 As shown.

[0134] Figure 9 In the study, a) was 1L-1β, and b) was TNF-α. (Note: Compared to the blank control group, *p<0.05, **p<0.01, ***p<0.001; compared to the model group, ...) # p<0.05, ## p<0.01, ### p<0.001).

[0135] from Figure 9 The results showed that, compared with the control group, the levels of IL-1β and TNF-α in the model group mice were significantly increased, indicating that insomnia, while causing oxidative stress, also greatly promoted the release of inflammatory factors. Compared with the model group, the administration of drugs or polyphenol drinks (at various doses) significantly reduced the levels of IL-1β and TNF-α. In particular, the levels of IL-1β and TNF-α in the medium-dose group mice were almost equivalent to those in the control group. These results suggest that the mechanism by which polyphenol drinks improve sleep quality in insomniac mice is related to reducing inflammatory factors in brain tissue.

[0136] (V) Human trial to verify that polyphenols improve sleep quality The polyphenol beverage from Example 1 of this invention was used as the test product to conduct a verification experiment on its ability to improve sleep quality in a population. A total of 60 participants (30 men and 30 women) were divided into three groups of 20 each (10 men and 10 women). Group 1 consumed 30 ml of the beverage, Group 2 consumed 50 ml, and Group 3 consumed 50 ml of the beverage without added polyphenols (except for polyphenols, all other raw materials and production processes were the same as in Example 1). The beverage was taken on an empty stomach before breakfast daily for one week. After one week of continuous use, the participants recorded their ease of falling asleep, sleep duration, and overall feelings to evaluate the product's efficacy in improving sleep quality.

[0137] The results showed that in the first group, after one week of trial use, 16 people experienced improved sleep quality, with 5 (4 women and 1 man) experiencing complete relief from insomnia. In the second group, 18 people showed significant effects, with 8 experiencing complete relief from insomnia on the second or third day of use (sleep became very good), and they also had excellent daytime energy. In the third group, only 3 people reported improved sleep, and most insomnia symptoms did not show significant improvement. Therefore, the polyphenol beverage of Example 1 provided by this invention has a good effect on improving sleep quality (especially in improving insomnia symptoms). This verification experiment shows that consuming 50 ml of the beverage daily is sufficient to improve sleep quality.

[0138] Examples of improving insomnia: Mr. A, a 47-year-old male, suffered from chronic insomnia and often woke up in the middle of the night and could not fall back asleep. He took 50 ml of the polyphenol drink of Example 1 of this invention every day. After a week, his sleep quality improved significantly and he no longer woke up in the middle of the night.

[0139] Ms. Yi, 20 years old, had long suffered from poor sleep quality and had been taking sleeping pills for 4 months. After taking 50 ml of the polyphenol drink of Example 1 of this invention, she did not need to take sleeping pills to fall asleep on the first day (and has not taken sleeping pills since). Moreover, she fell asleep quickly after waking up in the middle of the night. After taking it for 1 week, her sleep quality improved significantly. She could fall asleep as soon as she lay down and had good energy during the day.

[0140] Ms. Bing, 34 years old, experienced a decline in sleep quality after the pandemic, waking up in the middle of the night and finding it difficult to fall back asleep. She also became lethargic during the day. After taking 50 ml of the polyphenol drink from Example 1 of this invention for one week, her sleep quality improved significantly. She no longer woke up in the middle of the night and her mental state was good during the day.

[0141] Ms. Ding, 70 years old, had long suffered from poor sleep quality, difficulty falling asleep, and was easily awakened, only sleeping for 3-4 hours a night. After taking 50 ml of the polyphenol drink of Example 1 of this invention, she was able to fall asleep on the first day. After taking it continuously for 1 week, she was able to fall asleep every day, and her sleep quality improved significantly.

[0142] Ms. Wu, 36 years old, had long-term poor sleep quality. She used to go to bed at 10 pm, but would wake up at 12 am, and then wake up every 2 hours. She took 50 ml of the polyphenol drink of Example 1 of this invention every day. After taking it for less than a week, she no longer woke up easily at night. Even if she woke up once, she would fall asleep again quickly until 6-7 am.

[0143] Ms. Ji, a 56-year-old woman, suffers from the "three highs" (hyperlipidemia, hypertension, and hyperuricemia) and severe insomnia. She took 50 ml of the polyphenol drink from Example 1 of this invention daily for one week, and her sleep quality significantly improved, as did her overall energy and spirit. She reported that during the period of taking the product, she felt energetic during the day and did not feel tired.

[0144] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for producing a nano-sized dietary polyphenol beverage, characterized in that, Includes the following steps: (1) Add cyclodextrin, skim milk powder and sodium caseinate to hot water at a temperature of 50-60℃ and stir to dissolve to obtain liquid 1; (2) Add oligosaccharides and dietary plant polyphenols to hot water at a temperature of 60-80℃ until fully dissolved, adjust the pH to 6.2-6.8, and obtain solution 2; (3) Under stirring, heat liquid 1 to 85-95℃ and keep it warm for 10-20 min. Then, immediately under stirring, add liquid 2 and liquid 3 formed by pre-dissolved ascorbic acid, sodium citrate and concentrated fruit juice to liquid 1 after keeping it warm, add water to make up the difference, and keep stirring to obtain a premixed composition. (4) Cool the premixed composition to a temperature ≤40°C, filter, fill and sterilize to obtain the dietary polyphenol beverage.

2. The method for producing nano-dietary polyphenol beverage according to claim 1, characterized in that, The feed solution 3 is prepared by adding ascorbic acid, sodium citrate and concentrated fruit juice to hot water at a temperature of 50-90℃ until fully dissolved, and adjusting the pH to 6.2-6.8 to obtain feed solution 3.

3. The method for producing nano-dietary polyphenol beverage according to claim 1, characterized in that, The sterilization process employs high-temperature sterilization. The high-temperature sterilization temperature is 115-125℃, and the time is 20-40 minutes.

4. The method for producing nano-dietary polyphenol beverage according to claim 1, characterized in that, The sterilization process involves first performing ultra-high temperature instantaneous sterilization, followed by pasteurization. The ultra-high temperature instantaneous sterilization temperature is 120-150℃ and the time is 2-8s; the pasteurization temperature is 72-95℃ and the time is 15-30min.

5. The method for producing nano-dietary polyphenol beverage according to any one of claims 1-4, characterized in that, The contents of each ingredient by mass percentage are as follows: skim milk powder 6-12%, oligosaccharides 3-10%, sodium caseinate 0.5-3%, concentrated fruit juice 1-2%, dietary plant polyphenols 0.5-2%, cyclodextrin 0.05-2%, ascorbic acid 0.02-0.05%, sodium citrate 0.025%, and the balance is water.

6. The method for producing nano-dietary polyphenol beverage according to claim 5, characterized in that, The contents of each ingredient by mass percentage are as follows: skim milk powder 8-10%, oligosaccharides 5-8%, sodium caseinate 1-3%, concentrated fruit juice 1-2%, dietary plant polyphenols 1-1.5%, cyclodextrin 0.05-2%, ascorbic acid 0.02-0.05%, sodium citrate 0.025%, and the balance is water.

7. The method for producing nano-dietary polyphenol beverage according to claim 5, characterized in that, The dietary plant polyphenols include one or more of the following: tea powder, grape powder, grape seed powder, apple powder, blueberry powder, cranberry powder, citrus powder, and turmeric powder. The oligosaccharides include one or more of xylooligosaccharides, isomaltooligosaccharides, fructooligosaccharides, galactooligosaccharides, raffinose, gentiosaccharides, lactulose, soybean oligosaccharides, and stachyose. The concentrated fruit juice includes one or more of the following: concentrated peach juice, concentrated peach juice, concentrated pear juice, and concentrated cranberry juice. The cyclodextrin is β-cyclodextrin and / or γ-cyclodextrin.

8. The method for producing nano-dietary polyphenol beverage according to claim 7, characterized in that, The tea powder has a polyphenol content higher than 80 wt.%; the grape seed powder has a proanthocyanidin content higher than 90 wt.%; the apple powder has a polyphenol content higher than 30 wt.%; and the blueberry powder, cranberry powder, and citrus powder all have a polyphenol content higher than 20 wt.%.

9. A nano-dietary polyphenol beverage produced by the method of any one of claims 1-8.

10. The application of a nano-dietary polyphenol beverage produced by the method of any one of claims 1-8 in a beverage for improving sleep quality, characterized in that, The beverage contains more than 1.0 wt% polyphenols, and the polyphenols exist in the form of nanoparticles.

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