Preparation method of zeaxanthin-dietary fiber complex, nutritional preparation containing zeaxanthin capable of enhancing skeletal muscle function, and preparation method thereof

Abstract

The present invention discloses a preparation method of a zeaxanthin dietary fiber complex, characterized in that the steps include: grinding dietary fiber and sieving it, adding dietary fiber to deionized water, adding a zeaxanthin ethanol solution after stirring, performing vortex oscillation, discarding the supernatant after the shock ends, and freeze-drying the bottom sediment in cold hydrazine, and then storing it at low temperatures to obtain a zeaxanthin dietary fiber complex. A nutritional preparation containing zeaxanthin that can enhance skeletal muscle function, comprising: 0.05% to 0.6% of a zeaxanthin dietary fiber complex, 1% to 9% of sucrose, 0.01% to 0.08% of malic acid, and 0.05% to 0.30% of a composite stabilizer. By adjusting the ratio of each group, the formula is optimized, and the nutritional preparation obtained has strong stability and good water solubility, can enhance skeletal muscle energy metabolism, and alleviates handgrip loss. The present invention also provides a method for preparing a nutritional preparation containing zeaxanthin that can enhance skeletal muscle function.

Description

Technical Field

[0001] The present invention relates to a preparation method of a zeaxanthin-dietary fiber complex, a nutritional preparation containing zeaxanthin capable of enhancing skeletal muscle function and a preparation method thereof, and belongs to the field of functional foods. Background Art

[0002] Population aging is a universal trend in global demographic development and a major challenge facing human society in the 21st century. Data shows that by the end of 2021, my country's population aged 60 and over had reached 267 million. It is projected that by around 2035, the elderly population will exceed 400 million, accounting for over 30% of the total population, marking the beginning of a severely aging society. In this aging society, sarcopenia is receiving increasing public health attention. In 2016, the World Health Organization (WHO) recognized sarcopenia as a separate disease, with the International Classification of Diseases, 10th Revision, Clinical Modification (ICD-10-CM) code M62.84. While the dietary nutrition status of the elderly has improved significantly in recent years, there are still pressing issues with nutritional balance and overall health that need to be addressed.

[0003] Sarcopenia increases the risk of hospitalization, the cost of nursing care during hospitalization, and the cost of hospitalization for the elderly. Therefore, developing and optimizing current therapies for sarcopenia in the elderly has significant social value. Currently, the main treatments for sarcopenia are strength training, medication, and nutritional support. However, exercise carries certain risks for patients with sarcopenia and the elderly, and medications and hormones often have certain toxic side effects. Therefore, the search for exogenous natural products that can serve as human nutrients is of great significance for improving skeletal muscle function in aging and preventing and treating sarcopenia. Studies have shown that a healthy diet and balanced nutritional supplements have a positive effect on promoting skeletal muscle health and improving physical function. The International Sarcopenia Clinical Practice Guidelines (ICFSR) and the Asian Working Group on Sarcopenia (AWGS) consensus specifically recommend dietary and nutritional interventions in primary care settings to prevent and treat frailty and sarcopenia.

[0004] Corn is the world's and my country's largest grain crop and a cereal rich in carotenoids. Lutein, a dihydroxy derivative of β-carotene, is the primary carotenoid in yellow corn and one of the six most important carotenoids in the human body. Its molecule contains nine conjugated double bonds and two hydroxyionone rings, and possesses multiple physiological functions, including antioxidant and vision-protecting properties. Previous research by our research team has shown that lutein can significantly improve mitochondrial function in adipose tissue, alleviate oxidative stress, enhance mitochondrial biogenesis, improve energy metabolism, and thus reduce lipid accumulation. Furthermore, some researchers have found that lutein supplementation increases bone morphogenetic protein-2 (BMP2) mRNA expression in osteoblasts and inhibits sclerostin mRNA expression. In a 12-year follow-up study of 2,452 elderly individuals (aged 52 to 70 years), SAHNI et al. found that increasing intake of natural antioxidants, including total carotenoids, lutein, zeaxanthin, and lycopene, can alleviate handgrip strength loss in the elderly, a key indicator of sarcopenia. However, the molecular mechanism by which zeaxanthin inhibits myofiber atrophy and enhances skeletal muscle energy metabolism remains largely unreported. Zeaxanthin's poor stability and water solubility severely restrict its application in food processing. Dietary supplementation with natural antioxidants has significant potential for improving skeletal muscle health in the elderly. Dietary supplements offer broad clinical application prospects due to their wide availability, safety, effectiveness, minimal toxicity, and high patient acceptability. Summary of the Invention

[0005] The present invention designs and develops a method for preparing a zeaxanthin-dietary fiber complex. The zeaxanthin-dietary fiber complex is prepared by an anti-solvent method, thereby improving the water solubility of zeaxanthin.

[0006] The present invention also designs and develops a nutritional preparation containing lutein that can enhance skeletal muscle function. By adjusting the distribution ratio of each group and optimizing the formula, the obtained nutritional preparation has strong stability and good water solubility, can enhance skeletal muscle energy metabolism, and relieve grip strength loss.

[0007] The present invention also designs and develops a method for preparing a nutritional preparation containing lutein that can enhance skeletal muscle function. The preparation formula is optimized through response surface methodology to increase the content of lutein, so that the nutritional preparation has higher stability and can have a stronger inhibitory effect on aging skeletal muscle cells.

[0008] The technical solution provided by the present invention is:

[0009] A method for preparing a zeaxanthin-dietary fiber complex comprises:

[0010] The dietary fiber is ground and sieved, and the dietary fiber is added to deionized water. After stirring, a lutein ethanol solution is added and vortex-oscillated. After the shaking is completed, the supernatant is discarded, and the bottom precipitate is freeze-dried in cold hydrazine and stored at low temperature to obtain a lutein-dietary fiber complex.

[0011] Preferably, the dietary fiber is ground and passed through a 200-mesh sieve, the volume of deionized water is 5 mL, the stirring time is 30 min, the volume of the lutein ethanol solution is 1 mL, the concentration is 5 mg / mL, the vortex shaking time is 5 min, the freeze-drying temperature in cold hydrazine is -80°C, and the low-temperature storage temperature is -80°C.

[0012] Preferably, the dietary fiber is resistant starch, and the zeaxanthin-dietary fiber complex is a zeaxanthin-resistant starch complex.

[0013] A nutritional preparation containing zeaxanthin and capable of enhancing skeletal muscle function, comprising the following components in percentage by mass:

[0014] Lutein-resistant starch complex 0.05%-0.6%, sucrose 1%-9%, malic acid 0.01%-0.08%, composite stabilizer 0.05%-0.30%, and the balance is water.

[0015] Preferably, it includes:

[0016] Lutein-resistant starch complex 0.20%, sucrose 5.00%, malic acid 0.04%, complex stabilizer 0.225%.

[0017] A method for preparing a nutritional preparation containing zeaxanthin and capable of enhancing skeletal muscle function comprises:

[0018] 0.05% to 0.6% of the freeze-dried zeaxanthin-resistant starch complex is ground into a fine powder, 0.05% to 0.30% of a composite stabilizer and 1% to 9% of sucrose are stirred and evenly mixed, and then warm water is added to prepare a stabilizer solution. The zeaxanthin-resistant starch complex powder is added to the stabilizer solution and mixed evenly, and then 0.01% to 0.08% of malic acid is added, and the mixture is vortexed at 1500 rpm for 15 minutes to obtain a mixed solution.

[0019] The sieved mixed solution is poured into a sterile bottle and sterilized at high temperature in a high-pressure steam sterilizer. After sterilization, the obtained product is cooled and allowed to stand to obtain a finished nutritional preparation.

[0020] Preferably, it includes:

[0021] 0.20% of the freeze-dried zeaxanthin-resistant starch complex was ground into fine powder, 0.225% of the composite stabilizer and 5.00% of sucrose were stirred and mixed, and then warm water was added to prepare a stabilizer solution. The zeaxanthin-resistant starch complex powder was added to the stabilizer solution and mixed, and then 0.04% of malic acid was added, and the mixture was vortexed at 1500 rpm for 15 minutes to obtain a mixed solution;

[0022] The mixed solution was passed through a 100-mesh sieve, and the sieved solution was poured into a sterile bottle and sterilized at high temperature in a high-pressure steam autoclave. After sterilization, the obtained product was cooled and allowed to stand for 24 hours to obtain a finished nutritional preparation.

[0023] The beneficial effects of the present invention are:

[0024] 1. This patent clarifies the safe range of action of zeaxanthin on skeletal muscle cells, and intuitively demonstrates under microscopic conditions that zeaxanthin significantly inhibits skeletal muscle fiber atrophy caused by aging. It also clarifies that zeaxanthin significantly increases the decreased ATP production in skeletal muscle cells caused by aging.

[0025] 2. The preparation method of the nutritional preparation containing lutein that can enhance skeletal muscle function provided by the present invention utilizes the anti-solvent precipitation method to prepare the lutein-resistant starch complex to improve the solubility of lutein in water, thereby improving the current situation in which its application in the food field is limited; at the same time, the response surface method is used to optimize the product formula, and the obtained nutritional preparation has strong stability and high lutein content, providing a method for improving the water solubility and stability of lutein for the functional food market, thereby improving the nutritional intervention for sarcopenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1(a) is a schematic diagram of the changes in C2C12 cell viability according to the present invention. Note: Sample number n = 3. Compared with the blank group * p<0.05, ** p<0.01 *** p<0.001, **** p<0.0001.

[0027] FIG1( b ) is a diagram showing the effect of corn on myotube differentiation of C2C12 cells according to the present invention, with a scale bar of 200 μm.

[0028] FIG. 2( a ) is a diagram showing the effect of zeaxanthin on the activity level of β-galactosidase in skeletal muscle cells according to the present invention, with a scale bar of 100 μm.

[0029] Figure 2(b) is a comparative diagram showing the effect of zeaxanthin on the activity level of β-galactosidase in skeletal muscle cells. Note: Sample number n = 3. Compared with the control group * p<0.05, **p<0.01 *** p<0.001, **** p<0.0001.

[0030] FIG3( a ) is a standard curve of ATP production according to the present invention.

[0031] Figure 3(b) is a schematic diagram of the changes in ATP production in aging skeletal muscle according to the present invention. Note: Sample number n = 3. Compared with the control group * p<0.05, ** p<0.01 *** p<0.001, **** p<0.0001.

[0032] FIG4( a ) is a schematic diagram showing the effect of the dietary fiber addition amount on the water solubility of zeaxanthin in the zeaxanthin complex according to the present invention.

[0033] FIG4( b ) is a schematic diagram showing the effect of the ratio of anti-solvent (water) to solvent (ethanol) on the water solubility of zeaxanthin in the zeaxanthin complex according to the present invention.

[0034] FIG4( c ) shows the effect of the standing time on the water solubility of zeaxanthin in the zeaxanthin complex according to the present invention.

[0035] FIG5( a ) is a schematic diagram showing the effect of the added amount of the zeaxanthin-resistant starch complex of the present invention on the sensory perception of the oral solution.

[0036] FIG5( b ) is a schematic diagram showing the effect of the amount of sucrose added on the sensory perception of the oral solution according to the present invention.

[0037] FIG5( c ) is a schematic diagram showing the effect of the addition amount of the composite stabilizer of the present invention on the sensory properties of the oral solution.

[0038] FIG5( d ) is a schematic diagram showing the effect of the amount of malic acid added on the sensory perception of the oral solution according to the present invention.

[0039] FIG6( a ) shows the effect of the interaction between the addition amount of the zeaxanthin-resistant starch complex and the addition amount of sucrose on the sensory quality of the zeaxanthin oral solution.

[0040] FIG6( b ) shows the effect of the interaction between the addition amount of the zeaxanthin-resistant starch complex and the addition amount of the composite stabilizer on the sensory quality of the zeaxanthin oral solution described in the present invention.

[0041] FIG6( c ) shows the effect of the interaction between the addition amount of the zeaxanthin-resistant starch complex and the addition amount of citric acid on the sensory quality of the zeaxanthin oral solution.

[0042] FIG6( d ) shows the effect of the interaction between the amount of sucrose added and the amount of the composite stabilizer added on the sensory quality of the zeaxanthin oral solution according to the present invention.

[0043] FIG6(e) shows the effect of the interaction between the amount of sucrose added and the amount of citric acid added on the sensory quality of the zeaxanthin oral solution according to the present invention.

[0044] FIG6( f ) shows the effect of the interaction between the addition amount of the composite stabilizer and the addition amount of citric acid on the sensory quality of the zeaxanthin oral solution according to the present invention.

[0045] Figure 7 This is a picture of the nutritional preparation product described in the present invention. DETAILED DESCRIPTION

[0046] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0047] As shown in Figures 1-7, the present invention provides a method for preparing a zeaxanthin-dietary fiber complex, comprising:

[0048] The dietary fiber is ground and sieved, and the dietary fiber is added to deionized water. After stirring, a lutein ethanol solution is added and vortex-oscillated. After the shaking is completed, the supernatant is discarded, and the bottom precipitate is freeze-dried in cold hydrazine and stored at low temperature to obtain a lutein-dietary fiber complex.

[0049] Among them, the dietary fiber was ground and passed through a 200-mesh sieve, the volume of deionized water was 5 mL, the stirring time was 30 min, the volume of the lutein ethanol solution was 1 mL, the concentration was 5 mg / mL, the vortex shaking time was 5 min, the freeze-drying temperature in cold hydrazine was -80°C, and the low-temperature storage temperature was -80°C.

[0050] Effects of different dietary fiber addition amounts on the water solubility of lutein in the complex:

[0051] 5.0 mg of zeaxanthin was accurately weighed and dissolved in 1.0 mL of 95% ethanol solution. Zeaxanthin complexes with different dietary fiber contents (0.0, 4.0, 8.0, 12.0, 16.0, 20.0, and 30.0 mg / mL) were prepared for two dietary fibers (resistant starch and resistant dextrin). The volume ratio of antisolvent (water) to solvent (ethanol) was 5:1. After standing for 24 hours, the water solubility of zeaxanthin in the two zeaxanthin-dietary fiber complex aqueous solutions was measured. The results are shown in Figure 4(a): With increasing dietary fiber content in the system, the water solubility of zeaxanthin continued to increase. When the dietary fiber content was 12.0 mg / mL, the water solubility of zeaxanthin reached its maximum. When the dietary fiber content was higher than 12.0 mg / mL, the water solubility of zeaxanthin decreased. This result also shows that the water solubility of zeaxanthin in the zeaxanthin-resistant starch complex was greater than that in the zeaxanthin-resistant dextrin complex.

[0052] Effect of different volume ratios of antisolvent and solvent on the water solubility of lutein in the complex:

[0053] Accurately weigh 5.0 mg of zeaxanthin and dissolve it in 1.0 mL of 95% ethanol solution. Based on the optimal results obtained in the above experiments, an appropriate amount of solution with a dietary fiber content of 12.0 mg / mL was prepared. Mixture systems with different volume ratios of antisolvent to solvent (1:1, 2:1, 3:1, 4:1, 5:1, and 6:1) were prepared. After standing for 24 hours, the water solubility of zeaxanthin in the two zeaxanthin-dietary fiber complex aqueous solutions was measured. The results are shown in Figure 4(b): As the ratio of antisolvent to solvent increases, the water solubility of zeaxanthin in the complex continues to increase. When the ratio of the two is 5:1, the water solubility of zeaxanthin is the highest. Based on the above results, the system with a volume ratio of 5:1 was selected as the optimal system ratio for subsequent experiments.

[0054] Therefore, it is determined that in the present invention, the zeaxanthin-dietary fiber complex is a zeaxanthin-resistant starch complex.

[0055] Effect of different standing times on the water solubility of lutein in the complex:

[0056] Accurately weigh 5.0 mg of lutein and dissolve it in 1.0 mL of 95% ethanol solution, prepare an appropriate amount of solution with a dietary fiber content of 12.0 mg / mL, prepare a mixture system with a volume ratio of anti-solvent to solvent of 5:1, and measure the water solubility of lutein in the two lutein-dietary fiber complex aqueous solutions after standing for 6, 12, 24, 36, and 48 hours. The results are shown in Figure 4 (c): As the standing time of the lutein-resistant starch complex increases from 6 hours to 24 hours, the water solubility of lutein in the complex continues to increase. After reaching 24 hours, the water solubility of lutein fluctuates only in a small range. The lutein-resistant starch complex is allowed to stand for 24 hours for subsequent experiments

[0057] The optimal compounding process conditions were: a 5 mg / mL ethanolic zeaxanthin solution concentration, a 12 mg / mL dietary fiber content, a 5:1 antisolvent to solvent volume ratio, a 24-hour resting time for the zeaxanthin-resistant starch complex, and a 36-hour resting time for the zeaxanthin-resistant dextrin complex. Zeaxanthin-dietary fiber complexes were prepared under these conditions. Three parallel tests revealed that the water solubility of zeaxanthin in the zeaxanthin-resistant dextrin complex was 100.0 μg / mL, 97.0 μg / mL, and 94.9 μg / mL, and in the zeaxanthin-resistant starch complex was 128.8 μg / mL, 124.4 μg / mL, and 127.2 μg / mL. However, the water solubility of lutein in the lutein-resistant dextrin complex is lower than that in the lutein-resistant starch complex, and the standing time is too long. Therefore, lutein-resistant starch complex is used as raw material in subsequent product development. The preparation process is as follows: the concentration of lutein ethanol solution is 5 mg / mL, the dietary fiber content is 12 mg / mL, the volume ratio of antisolvent to solvent is 5:1, and the standing time is 24 hours.

[0058] The present invention provides a nutritional preparation containing zeaxanthin that can enhance skeletal muscle function. By adjusting the distribution ratio of each component and optimizing the formula, the obtained nutritional preparation has strong stability and good water solubility, can enhance skeletal muscle energy metabolism, and alleviate grip strength loss. The nutritional preparation is composed of the following components in the following mass percentages:

[0059] Lutein-resistant starch complex, 0.05%-0.6%, sucrose 1%-9%, malic acid 0.01%-0.08%, composite stabilizer 0.05%-0.30%, and the balance is water.

[0060] The present invention also provides a method for preparing a nutritional preparation containing zeaxanthin that can enhance skeletal muscle function, comprising:

[0061] 0.05% to 0.60% of the freeze-dried zeaxanthin-resistant starch complex is ground into a fine powder, 0.05% to 0.30% of a composite stabilizer and 1.00% to 9.00% of sucrose are stirred and evenly mixed, and then warm water is added to prepare a stabilizer solution. The zeaxanthin-resistant starch complex powder is added to the stabilizer solution and mixed evenly, and then 0.01% to 0.08% of malic acid is added, and the mixture is vortexed at 1500 rpm for 15 minutes to obtain a mixed solution;

[0062] The sieved mixed solution is poured into a sterile bottle and sterilized at high temperature in a high-pressure steam sterilizer. After sterilization, the obtained product is cooled and allowed to stand to obtain a finished nutritional preparation.

[0063] The product formula was optimized through the following experiments:

[0064] (1) Addition amount of lutein-resistant starch complex: 5.00% sucrose and 0.10% composite stabilizer (gelatin: CMC = 1:1) were stirred and evenly mixed, and then warm water was added to prepare a stabilizer solution. Accurately weighed 0.05%, 0.10%, 0.20%, 0.40%, and 0.60% lutein-resistant starch complex were added to the stabilizer solution, and then 0.04% malic acid was added. The mixture was vortexed at 1500 r / min for 15 min, and then filled, sterilized, cooled, and allowed to stand for 24 h for sensory evaluation.

[0065] (2) Amount of sucrose added: 1.00%, 3.00%, 5.00%, 7.00%, and 9.00% sucrose were mixed with 0.10% composite stabilizer (gelatin: CMC = 1:1) and then warm water was added to prepare a stabilizer solution. 0.20% lutein-resistant starch complex was accurately weighed and added to the stabilizer solution. 0.04% malic acid was then added. The solution was vortexed at 1500 rpm for 15 min, then filled, sterilized, cooled, and allowed to stand for 24 h before sensory evaluation.

[0066] (3) Amount of malic acid added: 5.00% sucrose and 0.10% composite stabilizer (gelatin: CMC = 1:1) were stirred and evenly mixed, and then warm water was added to prepare a stabilizer solution. 0.20% lutein-resistant starch complex was accurately weighed and added to the stabilizer solution. 0.01%, 0.02%, 0.04%, 0.06%, and 0.08% malic acid were added, respectively. The mixture was vortexed at 1500 rpm for 15 min, and then the mixture was filled, sterilized, cooled, and allowed to stand for 24 h for sensory evaluation.

[0067] (4) Amount of compound stabilizer added: 5.00% sucrose was mixed with 0.05%, 0.10%, 0.15%, 0.20%, and 0.30% of compound stabilizer, respectively, and then warm water was added to prepare a stabilizer solution. 0.20% of lutein-resistant starch complex was accurately weighed and added to the stabilizer solution. Then, 0.04% malic acid was added. The solution was vortexed at 1500 rpm for 15 min, and then the solution was filled, sterilized, cooled, and allowed to stand for 24 h for sensory evaluation.

[0068] The addition amount of lutein complex (A), sucrose (B), composite stabilizer (C), and malic acid (D) were selected as the four factors for the response surface experiment in this study. Three optimal addition levels were selected for each factor. The response surface methodology was used to analyze and optimize the test results, and the response value was the sensory score of the oral liquid.

[0069] Among them, the optimal concentration range was selected based on single-factor experiments, including:

[0070] 0.05% to 0.6% of the freeze-dried zeaxanthin-resistant starch complex was ground into fine powder, 0.05% to 0.30% of the composite stabilizer and 1% to 9% of sucrose were stirred and evenly mixed, and then warm water was added to prepare a stabilizer solution. The zeaxanthin-resistant starch complex powder was added to the stabilizer solution and mixed evenly, and then 0.01% to 0.08% of malic acid was added and mixed evenly at 1500 r / min vortex for 15 minutes to obtain a mixed solution. Sensory evaluation was used as an indicator. The sensory scoring standards are shown in Table 1-2 and the sensory evaluation standards are shown in Table 1-3. The sensory scoring results of the selected zeaxanthin complex addition amount are shown in Figure 5(a). As shown in the results, when the addition amount of the complex increased from 0.05% to 0.20%, the sensory score increased with the increase of the addition amount. The sensory score was the highest when the addition amount of the complex was 0.20%. The solution of this concentration was still uniform and stable after 24 hours of standing, without any impurities such as precipitation visible to the naked eye, and showed the orange-yellow color that a lutein solution should have. When the addition amount of the complex was less than 0.20%, the color of the solution was lighter than the orange-yellow color that a lutein solution should have after 24 hours of standing, and it was slightly unnatural. When the addition amount of the complex was greater than 0.40%, the solution had a slight precipitation after 24 hours of standing, indicating that the solution of this concentration was not very stable. Therefore, the addition amount of lutein-resistant starch complex was selected as 0.10%~0.30% as the optimal level of response surface; the sensory score results of sucrose addition are shown in Figure 5(b): when the sucrose addition amount increased from 1.00% to 5.00%, the sensory score also increased. The sensory score was the highest when the sucrose addition amount was 5.00%. After 24 hours of precipitation, the solution of this concentration had no obvious precipitation and had a suitable taste without sweet or sour taste. When the sucrose addition amount was lower than 5%, the sour taste of the oral solution was more obvious and there was basically no sweetness. When the sucrose addition amount was higher than 5.00, the sweetness of the oral solution was too obvious and the sensory score gradually decreased. Therefore, the sucrose addition amount was selected as 3.00%~7.00 as the optimal level of response surface; the sensory score of the composite stabilizer addition amount was the highest. The scoring results are shown in Figure 5(c): When the compound stabilizer addition amount is less than 0.20%, a small amount of visible complex precipitation will appear in the solution after standing for 24 hours. When the compound stabilizer addition amount is higher than 0.20%, the mouthfeel is sticky and the solution texture is uneven. When the compound stabilizer addition amount is 0.20%, the solution is uniform, there is no precipitation after standing, and the mouthfeel is natural and the texture is delicate. Therefore, 0.15% to 0.30% of the compound stabilizer is selected as the optimal level range of the response surface. The results of the four factors of malic acid addition amount are shown in Figure 5(d): When the malic acid addition amount increases from 0.01% to 0.04%, the sensory score gradually increases, and the sour taste in the oral solution becomes more obvious. The sour taste is most suitable at the addition amount of 0.04%, and the sensory score is the highest. Between 0.04% and 0.08%, as the malic acid addition amount increases, the sour taste in the oral solution becomes more prominent, and even bitterness appears, and the sensory score decreases significantly.Therefore, the malic acid addition amount of 0.02% to 0.06% was selected as the optimal level of the response surface. The response surface factor level table is shown in Table 1-1.

[0071] Table 1-1 Response surface factor level design table

[0072]

[0073] In the present invention, as a preferred embodiment, the quality standards of nutritional preparation products refer to the 2015 edition of the Chinese Pharmacopoeia and GB 16740-2014.

[0074] (1) Sensory evaluation standards and evaluation methods

[0075] A sensory evaluation panel comprised of 10 students majoring in food science evaluated the oral liquids' color, aroma, flavor, and form using the same sensory scoring criteria for beverages, assigning corresponding scores. (They rinsed their mouths with warm water before tasting each sample to prevent cross-contamination.) Based on the role of each evaluation item in the criteria, the weights for color, aroma, flavor, and form were 0.3, 0.4, and 0.3, respectively.

[0076] Table 1-2 Sensory evaluation standards for lutein oral solution

[0077]

[0078] (2) Sensory standards for oral solutions are shown in Table 1-3

[0079] Table 1-3 Sensory evaluation standards for oral liquids

[0080]

[0081] (3) Physical and chemical indicators of oral solution are shown in Table 1-4

[0082] Table 1-4 Physical and chemical indicators of oral solution

[0083]

[0084] (3) Oral solution microbial indicators are shown in Table 1-5

[0085] Table 1-5 Microbial indicators of oral solution

[0086]

[0087] Based on single-factor experiments, this method optimized the zeaxanthin oral liquid formulation using response surface methodology (RSM) with zeaxanthin-resistant starch, sucrose addition, composite stabilizer addition, and malic acid addition as variables and sensory scores as responses to determine the optimal process parameters. The results of the response surface experiment are shown in Table 1-6, and the results of the variance analysis of the quadratic response surface model are shown in Table 1-7.

[0088] Table 1-6 Response surface design scheme and results of lutein complex oral solution

[0089]

[0090]

[0091] Table 1-7 Analysis of variance of response surface quadratic model

[0092]

[0093]

[0094] Use Design-Expert 13 software to perform regression fitting on the data in Table 5-8 and obtain the regression equation:

[0095] Sensory evaluation = 89.40-3.42A+3.17B+0.25B+0.25C-4.83D+0.50AB+1.25AC+2.50AD-2.25BC-1.25BD+1.25CD-11.82A 2 -8.70B 2 -11.82C 2 -13.95D 2

[0096] The results of variance analysis of the response surface quadratic model are shown in Table 5-9: P < 0.0001 indicates that the model is significant, and the lack of fit coefficient is 0.9657 > 0.05, indicating that there is no lack of fit factor and the fit is good. 2 =0.9985, indicating that the equation can explain 99.85% of the sample size and has a good fit. The measured CV% value reached 0.8108, indicating that the method has good accuracy. The linear terms A, B, and D had a significant impact on the response, while the interaction terms AC, AD, BC, BD, and CD had a highly significant impact on the response. Based on the F value, the order of influence on sensory scores was malic acid addition > zeaxanthin complex addition > sucrose addition > composite stabilizer addition.

[0097] Effect of two factors' interaction on sensory quality of lutein oral solution

[0098] Design-Expert 13 software was used to construct and analyze response surface plots. The plots show highly significant interaction effects between the amount of zeaxanthin complex and sucrose, the amount of zeaxanthin complex and compound stabilizer, the amount of zeaxanthin complex and malic acid, the amount of sucrose and compound stabilizer, and the amount of malic acid and compound stabilizer. This result is consistent with the results of the analysis of variance analysis. The optimal formulation parameters for the nutritional preparation are: 0.20% zeaxanthin-resistant starch complex, 0.04% malic acid, 5.00% sucrose, and 0.225% compound stabilizer, achieving a maximum sensory score of 91. The response surface and contour lines for the interaction effects of various factors on the sensory evaluation of the zeaxanthin oral solution are shown in Figures 5-7. Three parallel experiments were conducted using this process to validate the established model. The oral solution prepared using the optimal process exhibited excellent color, aroma, taste, form, and texture, resulting in superior overall sensory quality. The results showed that the optimized formulation process of lutein oral solution based on response surface methodology proposed in this study is feasible, and its results are reliable and have practical application value.

[0099] Inhibition of zeaxanthin on senescence of C2C12 skeletal muscle cells:

[0100] C2C12 cells were grown in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2. The MTT method was used to screen the safe concentration of zeaxanthin for C2C12 cells. The cells were cultured in differentiation medium (DMEM medium containing 2% horse serum) for 6 days to induce differentiation of C2C12 cells. C2-ceramide was used to construct a senescent cell model. Jenner-Giemsa staining was used to characterize skeletal muscle

[0101] Cell group setting: Control group: cultured in DMEM high-glucose medium containing 2% HS for 12 hours.

[0102] Model group: Cultured in DMEM high-glucose medium containing 50 μM C2-ceramide and 2% HS for 12 h.

[0103] ZEA5: Cultured in DMEM high-glucose medium containing 50 μM C2-ceramide, 5 μM zeaxanthin and 2% HS for 12 h.

[0104] ZEA10: Cultured in DMEM high-glucose medium containing 50 μM C2-ceramide, 10 μM zeaxanthin, and 2% HS for 12 h.

[0105] ZEA15: Cultured in DMEM high-glucose medium containing 50 μM C2-ceramide, 15 μM zeaxanthin and 2% HS for 12 h.

[0106] To determine the safe dose range of zeaxanthin for C2C12 skeletal muscle cells, the relative cell viability was measured by MTT assay. The results are shown in Figure 1(a). Compared with the blank group (0μM), there was no significant difference in cell viability between the control group (0.6% DMSO solvent control group) and the 5μM, 10μM, and 15μM zeaxanthin intervention groups. However, after intervention with 20μM zeaxanthin, cell viability decreased to 90% of the blank group (P<0.001), indicating toxicity. Based on these results, zeaxanthin concentrations of 5-15μM were selected for subsequent experiments. In order to determine the effect of zeaxanthin on skeletal muscle myotube differentiation, C2C12 cells were stained with Jenner-Gimsa to observe myotube differentiation and structural changes. The results are shown in Figure 1(b). Compared with the control group, the myotubes in the model group were significantly atrophied into spherical shapes, the number of myotubes was significantly reduced, and the length and width of the myotubes were significantly decreased, indicating that the aging model was successfully established. Compared with the model group, the number of myotubes in the ZEA5, ZEA10 and ZEA15 groups increased in a dose-dependent manner, the length and width of the myotubes showed a significant increasing trend, and the number of cells atrophied into spherical shapes was significantly reduced, as shown in Figure 1(b). This shows that zeaxanthin has an inhibitory effect on C2-ceramide-induced myotube atrophy, and it is dose-dependent.

[0107] Inhibition of zeaxanthin on the production of β-galactosidase, a marker of aging

[0108] C2C12 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2. Differentiation was induced in DMEM supplemented with 2% horse serum for 6 days. A senescent cell model was established using C2-ceramide. β-galactosidase staining was used to characterize skeletal muscle cell senescence and to determine the inhibitory effect of zeaxanthin on C2C12 skeletal muscle cell senescence.

[0109] During cellular senescence, the expression level of β-galactosidase, a biomarker of senescent cells, increases with the degree of aging. β-galactosidase staining was used to determine the inhibitory effect of zeaxanthin on C2-ceramide-induced skeletal muscle cell senescence. The results are shown in Figures 2(a) and (b). Compared with the control group, the model group showed a significant increase in dark blue product, indicating a significant increase in β-galactosidase expression. Compared with the model group, with increasing zeaxanthin concentration, a significant decrease in dark blue product was observed. β-galactosidase expression levels in the ZEA5, ZEA10, and ZEA15 groups decreased to 0.78-, 0.70-, and 0.68-fold, respectively, compared with those in the MOD group. This result indicates that zeaxanthin inhibits C2-ceramide-induced skeletal muscle cell senescence in a dose-dependent manner.

[0110] Lutein enhances ATP production in aging skeletal muscle

[0111] ATP is a high-energy phosphate compound in cells. When cells enter a senescent state, their ATP levels decrease. To evaluate the effect of zeaxanthin on mitochondrial number in skeletal muscle cells, after induction of C2C12 skeletal muscle cell differentiation, cells were treated with 5μM, 10μM, and 15μM zeaxanthin for 12 hours. Cell senescence was also induced using C2-ceramide. Following intervention, ATP was measured using an ATP assay kit. As shown in Figure 3(b), ATP production in ZEA15 increased 1.45-fold compared to the model group, indicating that zeaxanthin enhances mitochondrial-based energy production in skeletal muscle cells. This result suggests that zeaxanthin enhances energy production in aging skeletal muscle cells in a dose-dependent manner.

[0112] (1) Preparation of ATP concentration standard curve

[0113] Melt the reagents to be used in a 4°C metal bath. Take an appropriate amount of ATP assay reagent and dilute it with diluent at a ratio of 1:9 to prepare the ATP assay working solution. This solution can be temporarily stored at low temperatures. Melt the reagents to be used in an ice bath and dilute the ATP standard solution to concentrations of 0.01, 0.03, 0.10, 0.30, 1.00, 3.00, and 10.00 μM. Prepare the appropriate amount of ATP assay working solution.

[0114] To completely consume the background ATP and thus reduce the background, 100 μL of ATP assay working solution was added to each test well at room temperature and allowed to stand for 5 min. Then, 20 μL of standard diluent was added to each test well and the mixture was thoroughly mixed by rapid pipetting. The RLU value was measured using a chemiluminescence analyzer and a standard curve was plotted. The results are shown in Figure 3(a). The obtained standard curve is y = 1.4133x + 0.1284, (R 2 =0.9993). The ATP levels of each cell group were calculated based on this standard curve.

[0115] (2) Detection of intracellular ATP levels

[0116] Twelve hours after cell administration, the cell culture medium was removed and the cells were washed three times with PBS. 200 μL of lysis buffer was added to each well of a 6-well plate to lyse the cells. The cells were shaken at low speed on ice for 15 minutes to allow the lysis buffer to fully contact and lyse the cells. After lysis, the cells were centrifuged at 10,000 rpm for 5 minutes at 4°C, and the supernatant was collected for subsequent analysis. 100 μL of ATP assay working solution was added to each well at room temperature and allowed to stand for 5 minutes. Then, 20 μL of sample diluent was added to the well and mixed thoroughly with a pipette. The RLU values ​​were measured using a chemiluminescence analyzer, and the ATP concentration in the samples was calculated based on the standard curve. As shown in Figure 3(b), ZEA15 increased ATP production by 1.45-fold compared to the MOD group, indicating that zeaxanthin enhances mitochondrial-based energy production in skeletal muscle cells. This result indicates that zeaxanthin enhances energy production in aged skeletal muscle cells in a dose-dependent manner.

[0117] In the present invention, as a preference, GraphPad Prism 9.0 software is used for graphing and statistical analysis, and one-way ANOVA is used to compare differences among groups.

[0118] In the present invention, as a preference, p < 0.05 is considered to be statistically significant, p < 0.01 is considered to be a significant statistical difference, p < 0.001 is considered to be a very significant statistical difference, and p < 0.0001 is considered to be an extremely strong statistically significant difference. Among them, the number of samples n = 3; compared with the model group, * p<0.05, ** p<0.01 *** p<0.001, **** p<0.0001.

[0119] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing a zeaxanthin-dietary fiber complex, characterized in that: include: The dietary fiber is ground and sieved, and the dietary fiber is added to deionized water. After stirring, a zeaxanthin ethanol solution is added and vortexed. After the shaking is completed, the supernatant is discarded, and the bottom precipitate is freeze-dried in cold hydrazine and stored at low temperature to obtain a zeaxanthin-dietary fiber complex; The dietary fiber was ground and passed through a 200-mesh sieve. The volume of deionized water was 5 mL and the stirring time was 30 min. The volume of the zeaxanthin ethanol solution was 1 mL and the concentration was 5 mg / mL. The vortex shaking time was 5 min. The temperature of freeze drying in cold hydrazine was -80°C and the temperature of low-temperature storage was -80°C. The dietary fiber is resistant starch, and the zeaxanthin-dietary fiber complex is a zeaxanthin-resistant starch complex.

2. A nutritional preparation containing zeaxanthin that can enhance skeletal muscle function, characterized in that: The zeaxanthin-dietary fiber complex prepared according to claim 1 is used, and the nutritional preparation consists of the following components in percentage by mass: Lutein-resistant starch complex 0.05%~0.6%, sucrose 1%~9%, malic acid 0.01%~0.08%, complex stabilizer 0.05%~0.30%, and the balance is water.

3. The nutritional preparation of zeaxanthin capable of enhancing skeletal muscle function according to claim 2, characterized in that: include: Lutein-resistant starch complex 0.20%, sucrose 5.00%, malic acid 0.04%, complex stabilizer 0.225%.

4. A method for preparing a nutritional preparation containing zeaxanthin that can enhance skeletal muscle function, characterized in that: The nutritional preparation for preparing the zeaxanthin-containing nutritional preparation for enhancing skeletal muscle function according to claim 2 comprises: 0.05%-0.6% of the freeze-dried zeaxanthin-resistant starch complex was ground into a fine powder, 0.05%-0.30% of the composite stabilizer and 1%-9% of sucrose were stirred and mixed, and then warm water was added to prepare a stabilizer solution. The zeaxanthin-resistant starch complex powder was added to the stabilizer solution and mixed, and then 0.01%-0.08% of malic acid was added, and the mixture was vortexed at 1500 rpm for 15 minutes to obtain a mixed solution; The sieved mixed solution is poured into a sterile bottle and sterilized at high temperature in a high-pressure steam sterilizer. After sterilization, the obtained product is cooled and allowed to stand to obtain a finished nutritional preparation.

5. The method for preparing the nutritional preparation containing zeaxanthin for enhancing skeletal muscle function according to claim 4, characterized in that: include: 0.20% of the freeze-dried zeaxanthin-resistant starch complex was ground into fine powder, 0.225% of the composite stabilizer and 5.00% of sucrose were stirred and mixed, and then warm water was added to prepare a stabilizer solution. The zeaxanthin-resistant starch complex powder was added to the stabilizer solution and mixed, and then 0.04% of malic acid was added, and the mixture was vortexed at 1500 rpm for 15 minutes to obtain a mixed solution; The mixed solution was passed through a 100-mesh sieve, and the sieved solution was poured into a sterile bottle and sterilized at high temperature in a high-pressure steam autoclave. After sterilization, the obtained product was cooled and allowed to stand for 24 hours to obtain a finished nutritional preparation.

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