Preparation method of zeaxanthine-dietary fiber compound, nutrient preparation containing zeaxanthine and capable of enhancing skeletal muscle function and preparation method of nutrient preparation

Abstract

The invention discloses a preparation method of a zeaxanthine-dietary fiber compound, which is characterized by comprising the following steps: grinding and sieving dietary fibers, adding the dietary fibers into deionized water, stirring, adding a zeaxanthine ethanol solution, carrying out eddy current oscillation, discarding supernate after oscillation is finished, and freeze-drying bottom precipitates in cold hydrazine to obtain the zeaxanthine-dietary fiber compound. And preserving at low temperature to obtain the zeaxanthine-dietary fiber compound. The invention discloses a zeaxanthine-containing nutritional preparation capable of enhancing skeletal muscle functions. The zeaxanthine-containing nutritional preparation comprises 0.05%-0.6% of a zeaxanthine-dietary fiber compound, 1%-9% of cane sugar, 0.01%-0.08% of malic acid and 0.05%-0.30% of a compound stabilizer. By adjusting the proportion of all the components and optimizing the formula, the obtained nutritional preparation is high in stability and good in water solubility, and can enhance skeletal muscle energy metabolism and relieve grip strength decline. The invention also provides a preparation method of the zeaxanthine-containing nutrition preparation capable of enhancing skeletal muscle functions.

Description

Technical Field

[0001] The invention relates to a method for preparing a zeaxanthin-dietary fiber complex, a nutritional preparation containing zeaxanthin and 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 general trend in global population development and a major problem facing human society in the 21st century. Data show that by the end of 2021, the number of elderly people aged 60 and above in my country has reached 267 million. It is estimated that by around 2035, the number of elderly people in my country will exceed 400 million, accounting for more than 30% of the total population, entering a severe aging stage in an aging society. In an aging society, public health attention to sarcopenia is increasing. In 2016, the World Health Organization (WHO) recognized sarcopenia as an independent disease, and its International Classification of Diseases, 10th Revision, Clinical Modification (ICD-10-CM) code is M62.84. In recent years, the dietary nutrition status of the elderly has been significantly improved, but there are still some problems such as nutritional balance and physical health of the elderly that need to be solved urgently.

[0003] Sarcopenia increases the risk of hospitalization, nursing costs during hospitalization, and hospitalization costs for the elderly. Therefore, the development and optimization of current treatments for sarcopenia in the elderly has great social value. At present, the main treatments for sarcopenia are strength training, drug therapy, and nutritional support. However, exercise has certain risks for patients with sarcopenia and the elderly, and drugs and hormones often have certain toxic side effects. Therefore, seeking exogenous natural products that can serve as human nutrients is of great significance for improving aging skeletal muscle function and preventing and treating sarcopenia. Studies have shown that healthy dietary patterns 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 Sarcopenia Working Group (AWGS) consensus specifically recommend the prevention and treatment of physical frailty and sarcopenia through dietary nutritional interventions in primary care institutions.

[0004] Corn is the largest food crop in the world and my country, and is also a cereal food containing a large amount of carotenoids. Lutein is a dihydroxy derivative of β-carotene. As the main carotenoid in yellow corn, it is also one of the six most important carotenoids in the human body. The molecule contains nine conjugated double bonds and two hydroxyl ionone rings, and has multiple physiological functions such as anti-oxidation and vision protection. The research team's previous studies have found that lutein can significantly improve the mitochondrial function of adipose tissue, relieve oxidative stress, enhance mitochondrial biosynthesis, improve energy metabolism and thus reduce lipid accumulation. In addition, some scholars have found that the addition of lutein increases the expression of bone morphogenetic protein-2 (BMP2) mRNA in osteoblasts and inhibits the expression of sclerostin mRNA. SAHNI et al. conducted a 12-year follow-up survey of 2,452 elderly people (52 to 70 years old) and found that increasing the intake of natural antioxidants such as total carotenoids, lutein, zeaxanthin and lycopene can alleviate the decline in grip strength in the elderly, and grip strength decline is an important reference indicator for sarcopenia. However, there are no reports on the molecular mechanism of zeaxanthin in inhibiting muscle fiber atrophy and enhancing skeletal muscle energy metabolism. Zeaxanthin has problems such as poor stability and poor water solubility, which seriously restricts its application in the field of food processing. Dietary supplementation of natural antioxidants has a positive significance for improving the health of elderly skeletal muscles. Dietary supplements have broad clinical application prospects due to their wide sources, safety and effectiveness, low toxicity and side effects, and strong patient acceptability. Summary of the invention

[0005] The 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, the lutein ethanol solution is added and vortexed. After the shaking, 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 zeaxanthin ethanol solution is 1 mL, the concentration is 5 mg / mL, the vortex oscillation 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, using the prepared zeaxanthin-dietary fiber complex, the nutritional preparation consists of the following components in percentage by mass:

[0014] The invention comprises 0.05% to 0.6% zeaxanthin-resistant starch complex, 1% to 9% sucrose, 0.01% to 0.08% malic acid, 0.05% to 0.30% composite stabilizer, 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, comprising:

[0018] Grind 0.05% to 0.6% of the freeze-dried zeaxanthin-resistant starch complex into fine powder, stir and mix 0.05% to 0.30% of the composite stabilizer and 1% to 9% of sucrose, then add warm water to prepare a stabilizer solution, add the zeaxanthin-resistant starch complex powder to the stabilizer solution, mix well, add 0.01% to 0.08% of malic acid, vortex and shake at 1500 r / min 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] Grind 0.20% of the freeze-dried zeaxanthin-resistant starch complex into fine powder, stir and mix 0.225% of the composite stabilizer and 5.00% of sucrose, add warm water to prepare a stabilizer solution, add the zeaxanthin-resistant starch complex powder to the stabilizer solution, mix well, add 0.04% malic acid, vortex and shake at 1500 r / min 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 sterilizer. 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 shows under microscopic conditions that zeaxanthin significantly inhibits skeletal muscle fiber atrophy caused by aging, while also clarifying that zeaxanthin significantly increases the decrease in ATP production in skeletal muscle cells caused by aging.

[0025] 2. The method for preparing a nutritional preparation containing zeaxanthin that can enhance skeletal muscle function provided by the present invention utilizes an anti-solvent precipitation method to prepare a zeaxanthin-resistant starch complex to improve the solubility of zeaxanthin 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 a high zeaxanthin content, thereby providing a method for improving the water solubility and stability of zeaxanthin for the functional food market and 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] FIG. 1( b ) is a diagram showing the effect of corn on myotube differentiation of C2C12 cells according to the present invention, with scale bar = 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 of 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] FIG. 3( 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 aged 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] FIG. 4( 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 described in the present invention.

[0034] FIG. 4( c ) shows the effect of the standing time on the water solubility of zeaxanthin in the zeaxanthin complex described in the present invention.

[0035] FIG. 5( a ) is a schematic diagram showing the effect of the addition 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 amount of the composite stabilizer added according to the present invention on the sensory perception 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 liquid described in the present invention.

[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 liquid described in the present invention.

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

[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 described in 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 described in 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 described in 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 is further described in 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, the lutein ethanol solution is added and vortexed. After the shaking, 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 oscillation time was 5 min, the freeze-drying temperature in cold hydrazine was -80°C, and the low-temperature storage temperature was -80°C.

[0050] Effect of different dietary fiber addition amounts on the water solubility of zeaxanthin 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, 30.0 mg / mL) were prepared for two dietary fibers (resistant starch and resistant dextrin) respectively. 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 the increase of 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 was the highest. When the dietary fiber content was higher than 12.0 mg / mL, the water solubility of zeaxanthin decreased. It can also be found from this result that the water solubility of zeaxanthin in the zeaxanthin-resistant starch complex was greater than that in the zeaxanthin-resistant dextrin.

[0052] Effect of different volume ratios of antisolvent to solvent on the water solubility of zeaxanthin 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 test, prepare an appropriate amount of a solution with a dietary fiber content of 12.0 mg / mL, prepare a mixture system with different volume ratios of antisolvent and solvent (1:1, 2:1, 3:1, 4:1, 5:1, 6:1), and measure the water solubility of zeaxanthin in two zeaxanthin-dietary fiber complex aqueous solutions after standing for 24 hours. The results are shown in Figure 4 (b): As the ratio of antisolvent to solvent increases, the water solubility of zeaxanthin in the complex increases continuously. When the ratio of the two is 5:1, the water solubility of zeaxanthin is the largest. Based on the above results, the system with a volume ratio of 5:1 is 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 time on the water solubility of zeaxanthin in the complex:

[0056] Accurately weigh 5.0 mg of zeaxanthin 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 zeaxanthin in the two zeaxanthin-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 zeaxanthin-resistant starch complex increases from 6 hours to 24 hours, the water solubility of zeaxanthin in the complex continues to increase. After 24 hours, the water solubility of zeaxanthin fluctuates only in a small range. The zeaxanthin-resistant starch complex was left to stand for 24 hours for subsequent experiments.

[0057] The optimal compounding process conditions were as follows: when the concentration of zeaxanthin ethanol solution was 5 mg / mL, the dietary fiber content was 12 mg / mL, the volume ratio of antisolvent to solvent was 5:1, the standing time of zeaxanthin-resistant starch complex was 24 hours, and the standing time of zeaxanthin-resistant dextrin complex was 36 hours. Under these conditions, zeaxanthin-dietary fiber complex was prepared. The water solubility of zeaxanthin in zeaxanthin-resistant dextrin complex was 100.0 μg / mL, 97.0 μg / mL, and 94.9 μg / mL, and the water solubility of zeaxanthin in zeaxanthin-resistant starch complex was 128.8 μg / mL, 124.4 μg / mL, and 127.2 μg / mL, respectively, after three parallel tests. However, the water solubility of zeaxanthin in the zeaxanthin-resistant dextrin complex is lower than that in the zeaxanthin-resistant starch complex, and the standing time is too long. Therefore, zeaxanthin-resistant starch complex is used as raw material in subsequent product development. The preparation process is as follows: the concentration of zeaxanthin ethanol solution is 5 mg / mL, the dietary fiber content is 12 mg / mL, the volume ratio of anti-solvent 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. The formula is optimized by adjusting the distribution ratio of each group. The obtained nutritional preparation has strong stability and good water solubility, can enhance skeletal muscle energy metabolism, and relieve grip loss. The nutritional preparation is composed of the following components in percentage by mass:

[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 and capable of enhancing skeletal muscle function, comprising:

[0061] Grind 0.05% to 0.60% of the freeze-dried zeaxanthin-resistant starch complex into fine powder, stir and mix 0.05% to 0.30% of the composite stabilizer and 1.00% to 9.00% of sucrose, add warm water to prepare a stabilizer solution, add the zeaxanthin-resistant starch complex powder to the stabilizer solution, mix well, add 0.01% to 0.08% of malic acid, vortex and shake at 1500 r / min 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) Amount of zeaxanthin-resistant starch complex added: 5.00% sucrose and 0.10% composite stabilizer (gelatin: CMC=1:1) were stirred and 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% of zeaxanthin-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) respectively, 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. Then 0.04% malic acid was added, and the mixture was vortexed at 1500 r / min for 15 min. Then, the mixture was 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 mixed, and then warm water was added to prepare a stabilizer solution. 0.20% zeaxanthin-resistant starch complex was accurately weighed and added to the stabilizer solution. 0.01%, 0.02%, 0.04%, 0.06%, and 0.08% of malic acid were added respectively. The mixture was vortexed at 1500 r / min 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 composite stabilizer added: 5.00% sucrose was mixed with 0.05%, 0.10%, 0.15%, 0.20%, and 0.30% of composite stabilizer, respectively, and then warm water was added to prepare a stabilizer solution. 0.20% zeaxanthin-resistant starch complex was accurately weighed and added to the stabilizer solution. Then 0.04% malic acid was added, and the mixture was vortexed at 1500 r / min for 15 min. Then, the mixture 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 of 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 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% to 0.08% of malic acid was added, and the mixture was vortexed at 1500 r / min for 15 minutes to obtain a mixed solution. The sensory evaluation was used as an indicator. The sensory evaluation standards are shown in Table 1-2, and the sensory evaluation standards are shown in Table 1-3. The sensory evaluation results of the selected zeaxanthin complex addition amount are shown in Figure 5(a). As shown: when the amount of the complex added increased from 0.05% to 0.20%, the sensory score increased with the increase of the amount of addition, and the sensory score was the highest when the amount of the complex added was 0.20%. The solution of this concentration was still uniform and stable after 24 hours of standing, without impurities such as precipitation visible to the naked eye, and showed the orange-yellow color that the lutein solution should have; when the amount of the complex added was less than 0.20%, the color of the solution was lighter than the orange-yellow color that the lutein solution should have after 24 hours of standing, and it was slightly unnatural; when the amount of the complex added 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 scoring results are shown in Figure 5(c): When the amount of the composite stabilizer added is less than 0.20%, a small amount of visible composite precipitation will appear in the solution after standing for 24 hours. When the amount of the composite stabilizer added is higher than 0.20%, the taste is sticky and the texture of the solution is uneven. When the amount of the composite stabilizer added is 0.20%, the solution is uniform, there is no precipitation after standing, and the taste is natural and the texture is delicate. Therefore, 0.15% to 0.30% of the composite stabilizer is selected as the optimal level range of the response surface; the four factors of malic acid addition, the results are shown in Figure 5(d): When the amount of malic acid added increases from 0.01% to 0.04%, the sensory score gradually increases, and the sour taste in the oral solution gradually becomes obvious. When the amount of malic acid added is 0.04%, the sour taste is most suitable and the sensory score is the highest. Between 0.04% and 0.08%, with the increase of malic acid addition, the sour taste in the oral solution becomes more prominent, and even bitter taste appears, and the sensory score decreases significantly.Therefore, the addition amount of malic acid was selected as 0.02% to 0.06% 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 general rules of the 2015 edition of the Chinese Pharmacopoeia and GB 16740-2014.

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

[0075] A sensory evaluation team consisting of 10 students majoring in food evaluated the color, aroma, taste and shape of the oral liquid according to the sensory scoring standards for beverages, and gave corresponding scores (they rinsed their mouths with warm water before tasting each sample to prevent interference between samples). According to the role of each evaluation item in the evaluation standard, the weights of color, aroma, and shape in this evaluation standard are 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) Microbiological indicators of oral solution are shown in Table 1-5

[0085] Table 1-5 Microbiological indicators of oral liquid

[0086]

[0087] On the basis of single factor experiment, this method uses zeaxanthin-resistant starch, sucrose addition, composite stabilizer addition and malic acid addition as variables, sensory score as response value, and optimizes the formula of zeaxanthin oral liquid through response surface methodology to determine the optimal process parameters. The results of response surface test are shown in Table 1-6, and the results of variance analysis of response surface quadratic model are shown in Table 1-7.

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

[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, R 2 =0.9985, indicating that the equation can explain 99.85% of the sample size and has a good fitting effect. The measured value of CV% reached 0.8108, indicating that the method has good accuracy. The linear terms A, B, and D have a significant effect on the response, while the interaction terms AC, AD, BC, BD, and CD have a very significant effect on the response. From the perspective of the size of the F value, the order of the effect on the sensory score is the amount of malic acid added> the amount of zeaxanthin complex added> the amount of sucrose added> the amount of composite stabilizer added.

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

[0098] The response surface diagram was drawn and analyzed using Design-Expert 13 software. It can be seen from the figure that there is a very significant interaction effect between the addition amount of zeaxanthin complex and the addition amount of sucrose, the addition amount of zeaxanthin complex and the addition amount of composite stabilizer, the addition amount of zeaxanthin complex and the addition amount of malic acid, the addition amount of sucrose and the addition amount of composite stabilizer, and the addition amount of malic acid and the addition amount of composite stabilizer. This result is the same as the result of variance analysis. The optimal formula parameters of the nutritional preparation are: the addition amount of zeaxanthin-resistant starch complex is 0.20%, the addition amount of malic acid is 0.04%, the addition amount of sucrose is 5.00%, the addition amount of composite stabilizer is 0.225%, and the highest sensory score is 91 points. The response surface and contour lines of the interaction of various factors on the sensory evaluation of zeaxanthin oral liquid are shown in Figures 5-7. Three groups of parallel experiments were prepared using this process, and the established model was verified. The oral liquid prepared by the optimal process has good color, aroma, taste, shape, texture, and overall sensory quality. The results showed that the optimized formulation process of zeaxanthin oral solution based on response surface methodology proposed in this study was feasible, and its results were reliable and had practical application value.

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

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

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

[0102] Model group: The cells were 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] In order to determine the safe dose range of zeaxanthin for C2C12 skeletal muscle cells, the relative cell viability was determined by MTT test. 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, while the cell viability after 20μM zeaxanthin intervention decreased to 90% of the blank group (P<0.001), indicating toxicity. Based on the above results, 5-15μM zeaxanthin concentrations 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 constructed; 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), indicating 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] The cells were incubated at 37°C and 5% CO 2 C2C12 cells were grown in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin under the same conditions, and differentiation medium (DMEM medium containing 2% horse serum) was used to culture for 6 days to induce differentiation. C2-ceramide was used to construct a senescent cell model. β-galactosidase staining was used to characterize the senescence of skeletal muscle cells, and the inhibitory effect of zeaxanthin on the senescence of C2C12 skeletal muscle cells was determined.

[0109] During cell 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 dark blue product in the model group increased significantly, that is, the expression level of β-galactosidase increased significantly. Compared with the model group, as the concentration of zeaxanthin increased, a significant decrease in dark blue products was observed, and the expression levels of β-galactosidase in the ZEA5, ZEA10 and ZEA15 groups decreased to 0.78 times, 0.70 times and 0.68 times that of the MOD group, respectively. This result shows that zeaxanthin inhibits C2-ceramide-induced skeletal muscle cell senescence in a dose-dependent manner.

[0110] Effect of zeaxanthin on the enhancement of ATP production in aged skeletal muscle

[0111] ATP is a high-energy phosphate compound in cells. When cells are in a state of aging, the ATP level of cells will decrease. In order to evaluate the effect of zeaxanthin on the number of mitochondria in skeletal muscle cells, after the induction of C2C12 skeletal muscle cell differentiation, 5μM, 10μM and 15μM zeaxanthin were used for intervention for 12 hours, and C2-ceramide was used to induce cell senescence. After intervention, the ATP detection kit was used for detection. As shown in Figure 3(b), compared with the model group, the ATP production of ZEA15 increased by 1.45 times, indicating that zeaxanthin enhanced the energy production of mitochondria-based skeletal muscle cells. This result shows that zeaxanthin dose-dependently enhances the energy production of aged skeletal muscle cells.

[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 detection reagent, dilute the ATP determination reagent with diluent at a ratio of 1:9 to form an ATP detection working solution, which can be temporarily stored at low temperature. Melt the reagents to be used in an ice bath, dilute the ATP standard solution to several concentrations of 0.01, 0.03, 0.10, 0.30, 1.00, 3.00 and 10.00 μM, and prepare an appropriate amount of ATP detection working solution.

[0114] In order to consume all the background ATP and reduce the background, 100 μL of ATP detection working solution was added to each detection well at room temperature and allowed to stand for 5 min. Then 20 μL of standard diluent was added to each detection well, and the mixture was mixed thoroughly by rapid suction and blowing with a pipette. The RLU value was measured with a chemiluminescence instrument and a standard curve was drawn. The results are shown in FIG3(a). The obtained standard curve is y=1.4133x+0.1284, (R 2 =0.9993). The ATP level of each cell group was calculated based on this standard curve.

[0115] (2) Detection of intracellular ATP levels

[0116] 12 hours after cell administration, the cell culture medium was removed, the cells were washed three times with PBS, 200 μL of lysis solution was added to each well of the 6-well plate to lyse the cells, and the cells were shaken at low speed in an ice bath for 15 minutes to allow the lysis solution to fully contact and lyse the cells. After lysis, the cells were centrifuged at 10000 rpm for 5 minutes at 4°C, and the supernatant was taken for subsequent determination; 100 μL of ATP detection working solution was added to each detection well at room temperature and allowed to stand for 5 minutes, and then 20 μL of sample diluent was added to the detection well, and the pipette was quickly sucked and blown to mix it thoroughly, and the RLU value was measured by a chemiluminometer, and the ATP concentration in the sample was calculated according to the standard curve. As shown in Figure 3(b), compared with the MOD group, the ATP production of ZEA15 increased by 1.45 times, indicating that zeaxanthin enhanced the energy production of mitochondria-based skeletal muscle cells. This result shows that zeaxanthin enhances the energy production of 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 preferred value, 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 as above, they are not limited to the applications listed in the specification and the implementation modes, and 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 the 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, the lutein ethanol solution is added and vortexed. After the shaking, 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.

2. The method for preparing the zeaxanthin-dietary fiber complex according to claim 1, characterized in that: 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 oscillation time is 5 min, the freeze-drying temperature in cold hydrazine is -80°C, and the low-temperature storage temperature is -80°C.

3. The method for preparing the zeaxanthin-dietary fiber complex according to claim 2, characterized in that: The dietary fiber is resistant starch, and the zeaxanthin-dietary fiber complex is a zeaxanthin-resistant starch complex.

4. A nutritional preparation containing zeaxanthin that can enhance skeletal muscle function, characterized in that: The zeaxanthin-dietary fiber complex prepared using any one of claims 1 to 3, wherein the nutritional preparation consists of the following components in percentage by mass: The invention comprises 0.05% to 0.6% zeaxanthin-resistant starch complex, 1% to 9% sucrose, 0.01% to 0.08% malic acid, 0.05% to 0.30% composite stabilizer, and the balance is water.

5. The nutritional preparation of zeaxanthin capable of enhancing skeletal muscle function according to claim 4, characterized in that: include: Lutein-resistant starch complex 0.20%, sucrose 5.00%, malic acid 0.04%, complex stabilizer 0.225%.

6. A method for preparing a nutritional preparation containing zeaxanthin for enhancing skeletal muscle function, characterized in that: include: Grind 0.05% to 0.6% of the freeze-dried zeaxanthin-resistant starch complex into fine powder, stir and mix 0.05% to 0.30% of the composite stabilizer and 1% to 9% of sucrose, then add warm water to prepare a stabilizer solution, add the zeaxanthin-resistant starch complex powder to the stabilizer solution, mix well, add 0.01% to 0.08% of malic acid, vortex and shake at 1500 r / min 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.

7. The method for preparing a nutritional preparation containing zeaxanthin for enhancing skeletal muscle function according to claim 6, characterized in that: include: Grind 0.20% of the freeze-dried zeaxanthin-resistant starch complex into fine powder, stir and mix 0.225% of the composite stabilizer and 5.00% of sucrose, add warm water to prepare a stabilizer solution, add the zeaxanthin-resistant starch complex powder to the stabilizer solution, mix well, add 0.04% malic acid, vortex and shake at 1500 r / min 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 sterilizer. After sterilization, the obtained product was cooled and allowed to stand for 24 hours to obtain a finished nutritional preparation.

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