A method for identifying, mining and evaluating the function of components of a muscle-building and fat-reducing nutritional solid beverage composition

Abstract

The present application belongs to the field of biological medicine, food science and functional evaluation technology, and specifically relates to a component identification, activity mining and functional evaluation method of a muscle-increasing and fat-reducing nutritional solid beverage composition. The present application organically combines non-targeted metabolite analysis, fingerprint quality control and nematode model functional evaluation, and constructs a "component mining-quality guarantee-function verification" three-in-one technical system. On the one hand, high-resolution mass spectrometry is used to reveal the active substance basis related to fat metabolism and muscle synthesis in the composition; on the other hand, the nematode model is used to directly verify the muscle-increasing and fat-reducing function from the phenotype to the molecular level. The integrated scheme overcomes the defect that the component analysis and functional evaluation are disconnected in the prior art, and upgrades the food chemical analysis from simple "component confirmation" to a new height of "activity mining" and "mechanism verification", thereby providing a complete scientific evidence chain for the efficacy claim of functional food.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine, food science and functional evaluation technology, and specifically relates to a method for component identification, activity exploration and functional evaluation of a muscle-building and fat-reducing nutritional solid beverage composition. Background Technology

[0002] With the continued rise in global incidence of obesity, metabolic syndrome, and sarcopenia, the market for nutritional solid beverages focused on muscle gain and fat loss (such as the "Lipid20" combination) centered on "weight management" and "muscle health" has experienced explosive growth. These products are typically formulated with a variety of ingredients, including but not limited to whey protein concentrate, soy protein isolate, resistant dextrin, plant extracts, and fortified amino acid and vitamin / mineral complexes. Their compositional systems are complex and contain various trace functional factors. However, the core challenge currently facing the food industry and regulatory agencies lies in how to scientifically verify the efficacy claims of such compounded compositions at the molecular level, especially how to simultaneously evaluate their dual functions of "fat loss" and "muscle gain," and construct a scientific chain of evidence linking ingredients and efficacy.

[0003] In the existing technology, the analysis and evaluation system for such muscle-building and fat-loss functional foods is not yet perfect, and it is difficult to meet the requirements of industrial development and supervision. The main shortcomings are as follows: (1) Component analysis methods are insufficient for in-depth discovery of trace active ingredients in complex matrices. Currently, the mainstream method for food quality control is still high-performance liquid chromatography (HPLC) combined with ultraviolet (UV) or diode array detectors (DAD). However, for complex matrices like "Lipid 20" which are composed of multiple raw materials, detectors based on the principle of light absorption have inherent physical defects: multiple compounds may co-elute at a single retention time point, and UV spectra lack sufficient structural information to distinguish these overlapping chromatographic peaks, making qualitative analysis prone to errors and unable to effectively identify trace functional isomers or unknown active ingredients. Although triple quadrupole mass spectrometry (MS / MS) has high detection sensitivity, its detection process depends on preset target compounds, which can only achieve targeted screening and cannot complete non-targeted full-spectrum component analysis. Furthermore, it lacks "retrospective analysis" and data mining capabilities, making it difficult to discover new active substances related to muscle gain and fat loss from complex samples, and failing to meet the needs of in-depth component mechanism research.

[0004] (2) Lack of targeted multi-wavelength fingerprint standards for product quality consistency evaluation. For compound nutritional solid beverages, the consistency of their chemical composition directly determines the stability and repeatability of product efficacy. Existing quality standards often only test a few conventional indicator components (such as protein content, individual vitamin or mineral content), which cannot comprehensively reflect the overall chemical composition characteristics of the product and make it difficult to achieve a comprehensive evaluation of product quality. Although fingerprint technology has been widely used in the quality control of traditional Chinese medicine, in the fields of sports nutrition foods and special medical foods, a standardized fingerprint construction method for compound compositions for muscle building and fat reduction has not yet been established. In particular, there is a lack of a systematic scheme for simultaneously monitoring multiple substances such as organic acids, amino acids, short peptides, vitamins, alkaloids and polyphenols at multiple characteristic wavelengths such as 210nm, 254nm and 280nm, which makes it impossible to provide a rapid, objective and quantifiable means of quality consistency evaluation for such products.

[0005] (3) Functional evaluation models are difficult to simultaneously and accurately evaluate the dual effects of "fat reduction" and "muscle gain". Obesity is no longer just a single pathological state of excessive fat accumulation, but has gradually evolved into a complex metabolic syndrome. In aging societies and among people who lose weight improperly, the comorbidity of obesity and sarcopenia is becoming increasingly prominent, manifested as a significant decline in skeletal muscle mass and function accompanied by excessive body fat percentage. Traditional evaluation methods for weight loss products often unilaterally pursue weight reduction or fat reduction, while neglecting the maintenance and improvement of muscle tissue. If muscle loss occurs during the fat reduction process, it will not only reduce the body's basal metabolic rate and cause weight rebound, but also increase the risk of falls, fractures and metabolic disorders. Existing mammalian evaluation models (such as rats and mice) can provide experimental data that are closer to those of humans, but they have problems such as long experimental cycles, high culture costs, and strict ethical restrictions, and it is difficult to simultaneously take into account the bidirectional regulatory mechanism of "fat metabolism" and "muscle function". Caenorhabditis elegans, as a classic model organism, has advantages such as a short lifespan, low culture cost, and clear genetic background. Furthermore, its lipid metabolism pathways and myosarcoma structure are highly conserved compared to mammals, making it an ideal tool for nutritional metabolism research. However, current evaluation methods based on nematode models mostly focus on the lifespan or lipid metabolism of single compounds or simple plant extracts. There is a lack of standardized pretreatment protocols for complex compound formulations like "Lipid 20" and standardized swimming motion modeling methods. Even fewer studies comprehensively evaluate muscle function (such as motor behavior, muscle structural integrity, and muscle-specific gene expression) and fat loss effects as equally important endpoints.

[0006] (4) The components analysis and functional evaluation are disconnected, lacking a complete chain of scientific evidence. In existing technologies, the chemical analysis of the components and the evaluation of the biological functions of functional foods are often two separate processes: chemical analysis can only report "what components are contained in the sample," while functional evaluation can only visually observe "what effects are produced," lacking an organic connection and verification between the two. For example, whether the trace active ingredients identified by mass spectrometry (such as specific fatty acid amides, amino acid derivatives, and signal lipids) truly mediate the muscle-building and fat-reducing effects observed in experiments has not yet been established as a complete technical chain from "component discovery" to "efficacy verification" and then to "mechanism analysis." This results in the lack of solid molecular-level scientific basis for the efficacy claims of products, making it difficult to meet the strict requirements of regulatory authorities for evidence-based evidence for functional foods, and also restricting the standardized and scientific development of the functional food industry.

[0007] In summary, there is an urgent need to establish a comprehensive technical solution that integrates in-depth exploration of active ingredients, consistency evaluation of product quality, and multi-dimensional verification of the dual effects of muscle building and fat reduction. This solution would enable the full-chain analysis of muscle-building and fat-reducing nutritional solid beverage compositions (such as "Lipid 20") from "ingredient identification" to "activity exploration" and then to "mechanism verification." This would overcome many shortcomings of existing technologies, provide a complete and reliable chain of scientific evidence for the efficacy claims of such functional foods, and promote the standardization and scientific development of analysis and evaluation technologies in the entire functional food industry. Summary of the Invention

[0008] The purpose of this invention is to address existing problems by providing a method for component identification, activity discovery, and functional evaluation of a muscle-building and fat-reducing nutritional solid beverage composition.

[0009] This invention is achieved through the following technical solution: A method for verifying the efficacy of a muscle-building and fat-reducing nutritional solid beverage composition includes a step of evaluating the function of the composition using a Caenorhabditis elegans model; the functional evaluation includes a comprehensive assessment from at least two dimensions: exercise behavior, body fat content, muscle structure, and gene expression level.

[0010] Furthermore, the functional evaluation includes the following four dimensions: (a) Evaluation of motor behavior: Determine the head oscillation frequency and / or the number of sinusoidal body movements of the nematode; (b) Evaluation of body fat content: The accumulation of lipid droplets in nematodes was determined by Oil Red O staining. (c) Evaluation of muscle structure: Using transgenic nematodes expressing muscle-specific fluorescent proteins, the morphology, width, or fluorescence intensity of the body wall muscles were observed and analyzed; (d) Evaluation of gene expression: The expression levels of lipid metabolism-related genes and / or muscle development-related genes in nematodes were detected.

[0011] Furthermore, the Caenorhabditis elegans model was pre-trained in swimming exercises, which included placing the nematodes in an M9 buffer solution for intermittent swimming exercises. The intermittent swimming exercise consists of swimming for 1 minute followed by resting for 5 minutes, repeated 10 times in total.

[0012] Furthermore, the method also includes a step of active ingredient discovery in the composition; the active ingredient discovery includes: preparing a sample solution of the composition using an ultrasound-assisted extraction method, and then performing non-targeted metabolite analysis using ultra-high performance liquid chromatography-linear ion trap-electrostatic field orbital trap high-resolution mass spectrometry (UPLC-LTQ-Orbitrap MS / MS), and screening for active ingredients related to lipid metabolism and / or muscle synthesis by comparing with a high-resolution mass spectrometry database.

[0013] Furthermore, the chromatographic conditions for the analysis of the non-targeted metabolites are as follows: a reversed-phase C18 column is used, and the mobile phase is formic acid aqueous solution and formic acid acetonitrile solution, with gradient elution; the mass spectrometry conditions are: electrospray ionization source, positive ion mode, and data-dependent secondary mass spectrometry scan.

[0014] Furthermore, the method also includes the step of establishing a standard fingerprint spectrum of the composition using high performance liquid chromatography: detecting more than 10 different batches of the composition, with a sample concentration of 1 mg / mL, recording the chromatograms, extracting common characteristic peaks after normalization, recording the retention time and peak area, calculating the batch similarity, and establishing a standard fingerprint spectrum.

[0015] Furthermore, the detection wavelength of the high-performance liquid chromatography includes at least one of 210 nm, 254 nm, and 280 nm.

[0016] Furthermore, the muscle-building and fat-reducing nutritional solid beverage composition is a lipid 20 composition, the raw materials of which include concentrated whey protein powder, soy protein isolate, purple sweet potato powder, konjac powder, complex minerals, citrus fiber, hydrolyzed casein peptides, complex vitamins, steviol glycosides, and betaine.

[0017] Furthermore, the lipid metabolism-related genes include at least one of nhr-49, fat-6, and acs-2; the muscle development-related genes include at least one of hlh-1, unc-54, and myo-3; and the expression of the genes is detected by conventional PCR and / or real-time quantitative PCR.

[0018] Furthermore, the method includes a step of measuring the level of reactive oxygen species (ROS) in nematodes to evaluate the antioxidant capacity of the composition; the measurement is performed using the fluorescent probe H2DCF-DA, with an excitation wavelength of 485 nm and an emission wavelength of 525 nm.

[0019] The method described herein can be applied to the quality control, efficacy verification, or new product development of functional foods, sports nutrition foods, special medical foods, or health products.

[0020] The present invention has the following advantages over the prior art: 1. This invention organically combines non-targeted metabolite analysis, fingerprinting quality control, and nematode model functional evaluation to construct a three-in-one technical system of "component discovery—quality assurance—efficacy verification." On the one hand, high-resolution mass spectrometry reveals the bioactive substances in the composition related to fat metabolism and muscle synthesis; on the other hand, the nematode model directly verifies the muscle-building and fat-reducing functions from the phenotypic to the molecular level. This integrated approach overcomes the shortcomings of existing technologies where component analysis and functional evaluation are disconnected, elevating food chemical analysis from simple "component identification" to a new level of "activity discovery" and "mechanism verification," providing a complete chain of scientific evidence for the efficacy claims of functional foods.

[0021] 2. This invention employs ultra-high performance liquid chromatography-linear ion trap-electrostatic field orbital trap high-resolution mass spectrometry (UPLC-LTQ-Orbitrap MS / MS) for non-targeted metabolite analysis. Combined with high-resolution database comparison, it can screen functional factors with low content but significant physiological activity from complex matrices. Examples have successfully identified five major classes of active ingredients: amino acid derivatives, glycosides, medium- and long-chain fatty acids and their amide / ester derivatives, arachidonic acid and bile acid pathway signal lipids, polar small-molecule organic acids, and inorganic anionic derivatives. This provides a molecular-level scientific basis for understanding the muscle-building and fat-reducing mechanisms of the compositions. Compared with traditional targeted detection or ultraviolet detection methods, this invention possesses retrospective data mining capabilities, enabling the discovery of unknown active substances and overcoming the limitation of traditional detection methods in identifying trace functional isomers.

[0022] 3. This invention employs high-performance liquid chromatography (HPLC) to detect more than 10 batches of samples at three characteristic wavelengths: 210 nm, 254 nm, and 280 nm. A standard fingerprint spectrum is established by extracting common characteristic peaks and calculating similarity. Experimental results show that the main chromatographic peaks of each batch of samples have high consistency, indicating a stable production process. This method provides a rapid, objective, and quantifiable technical means for the quality control of muscle-building and fat-reducing nutritional solid beverages, overcoming the shortcomings of existing quality standards that only detect individual indicator components and cannot comprehensively evaluate product quality consistency.

[0023] 4. This invention utilizes the *Caenorhabditis elegans* model to comprehensively evaluate multiple dimensions, including kinetic behavior (head swaying frequency, number of sinusoidal movements), body fat content (Oil Red O staining), muscle structure (muscle-specific fluorescent protein labeling), and gene expression (genes related to lipid metabolism and muscle development). Compared to traditional methods that only focus on weight or fat reduction, this invention can distinguish between "healthy fat loss" and "pathological emaciation accompanied by muscle loss," making it particularly suitable for evaluating functional products that claim both muscle gain and fat loss. Experimental results show that the composition significantly reduces lipid droplet accumulation in nematodes while increasing muscle width, maintaining muscle fiber integrity, and upregulating the expression of muscle structural protein genes such as hlh-1, unc-54, and myo-3, achieving synergistic validation of muscle gain and fat loss.

[0024] 5. *C. elegans* possesses advantages such as a short lifespan (approximately 3 days), low culture cost, clear genetic background, and high conservation with mammalian lipid metabolism and muscle development pathways. The standardized swimming model and multi-dimensional phenotypic detection system constructed in this invention can complete the functional evaluation of multiple candidate samples in a short time, significantly reducing the cost and time required for animal experiments. It also avoids the ethical limitations of mammalian experiments, making it suitable for early efficacy screening and mechanism research in functional foods, sports nutrition foods, and special medical foods, thus improving R&D efficiency.

[0025] 6. This invention further elucidates the mechanism of action of the composition through reactive oxygen species (ROS) fluorescent probe detection and real-time quantitative PCR analysis. Experimental results show that the composition can significantly reduce ROS levels in nematodes at effective concentrations, protecting muscle cells from oxidative damage; simultaneously, it upregulates the expression of lipid metabolism-related genes (nhr-49, fat-6, acs-2), activates the fatty acid β-oxidation pathway, reduces lipid deposition, and improves the body's energy metabolism homeostasis, providing efficient energy substrates and neural regulatory signals for muscle contraction, thereby achieving a synergistic effect of muscle building and fat reduction from multiple dimensions of antioxidation and energy metabolism regulation.

[0026] 7. The integrated analysis and verification method established in this invention, encompassing "ingredient discovery—quality control—efficacy verification—mechanism analysis," can be widely applied to quality control, efficacy verification, and new product development in sports nutrition foods, special medical foods, health products, and traditional Chinese medicine compound preparations. This method combines food chemical analysis with molecular nutritional mechanism analysis, providing a standardized technical reference for the entire functional food industry to transition from "nutrient analysis" to "molecular mechanism analysis," and possesses strong promotional value and industrial applicability. Attached Figure Description

[0027] Figure 1 The ion flow chromatograms are for three parallel samples in Example 1. Figure 2 The results of non-targeted metabolomics analysis of the lipid 20 composition in Example 1; Figure 3 The flow chromatogram of non-free phospholipid ions in the lipid 20 composition of Example 1; Figure 4 The ion chromatogram of glycosides in the lipid 20 composition in Example 1 is shown. Figure 5 The ion chromatogram of ester derivatives in the lipid 20 composition of Example 1 is shown. Figure 6 The ion chromatogram of amino acid derivatives in the lipid 20 composition in Example 1 is shown. Figure 7 The chromatograms of 10 batches of lipid 20 composition at 210 nm are superimposed. Figure 8 The chromatograms of 10 batches of lipid 20 composition at 254 nm are superimposed. Figure 9 The chromatograms of 10 batches of lipid 20 composition at 280 nm are superimposed. Figure 10 These are the primer sequences used for conventional PCR and RT-qPCR; Figure 11 The nematode's head swing frequency and the number of sinusoidal motions; Figure 12 The results of ROS staining in nematodes were obtained by intervening with different concentrations of lipid 20 compositions; Figure 13 Results of Oil Red O staining for nematodes; Figure 14 Fluorescence results of nematode muscle fibers; Figure 15 These are the results of routine PCR expression in nematodes; Figure 16 The qPCR amplification curve and qPCR quantification results of nematodes are shown. Detailed Implementation

[0028] To further explain the present invention, the following specific embodiments are described.

[0029] Example 1: Identification of components and discovery of active ingredients in the "Lipid 20" composition 1. Analysis of non-targeted metabolites of the "Lipid 20" composition This invention uses UPLC-LTQ-Orbitrap MS / MS to analyze non-targeted metabolites in lipid 20 samples.

[0030] Lipid 20 sample: Weigh 30g of "Lipid 20" sample, add water at a material-to-liquid ratio of 1:30 (g:mL), and perform ultrasonic-assisted extraction at 50℃ for 30min, repeating 3 times to obtain a homogenized sample solution. Concentrate the homogenate using a rotary evaporator (50℃). After ultrasonic extraction with water and methanol for 30min respectively, the sample is filtered through a membrane for analysis.

[0031] Chromatographic conditions: A C18 ACQUITY BEH column was used to separate lipid 20 samples. The injection volume was 20 µL, the flow rate was 0.4 mL / min, and the mobile phases were 0.1% formic acid-water solution (A) and 0.1% formic acid-acetonitrile (B). Gradient elution was used: 0–4 min, 95%–65% B; 4–9 min, 65%–50% B; 9–11 min, 50%–10% B; 11–12 min, 10% B; 12–13 min, 10%–95% B; 13–15 min, 95% B.

[0032] Mass spectrometry conditions: Data-dependent scanning (DDA) was performed on lipid 20 samples in positive ion mode within the m / z range of 70-2000. The capillary temperature and aux gas heater temperature were 300℃ and 350℃, respectively; the sheath gas flow rate and Aux gas flow rate were 40 Arb and 20 Arb, respectively; the sweep gas flow rate was 0 Arb; and the spray voltage was 3.2 kV. Furthermore, the dynamic exclusion parameters were set as follows: repeat count, 2 times; duration, 10 s; exclusion list size, 50; and exclusion every 20 s. Data acquisition and processing were performed using Xcalibur 4.1 software (Thermo Fisher Scientific).

[0033] Data were analyzed using the Compound Discover 3.3 database. The search results were then analyzed, with p-values ​​less than or equal to 0.05 and VIP values ​​greater than 2. After screening the samples, they were compared with the HMBD database and relevant literature was consulted. The database descriptions and references were combined to identify substances related to muscle and fat metabolism. Substances with high content and related to fat and muscle metabolism were screened and their characteristic peaks were identified.

[0034] Metabolomics characterization analysis showed that ( Figures 1-6The "Lipid 20" composition samples exhibited a significant banded aggregation distribution in the log10 intensity range of 5-6, constituting the most representative group of moderately abundant metabolites in this system. Compound structural annotations show that they are mainly composed of four classes of small molecules. The first class consists of amino acids, dipeptides, and their guanidinolated derivatives, including various modifications or condensates related to arginine, ornithine, and lysine. These may originate from protein hydrolysates or amino acid fortification raw materials and participate in nitrogen metabolism, amino acid cycling, and energy generation processes. The second class consists of carbohydrate glycosides, representing the background metabolic characteristics of carbohydrate metabolism, nucleotide turnover, and redox processes. The chemical composition of this portion of metabolites mainly consists of long-chain lipids, arachidonic acid-like signaling molecules, and amino acids / peptides, presenting a complex lipid-amino acid-organic acid interaction metabolic landscape. This provides an important chemical basis for understanding the raw material properties of meal replacement powders and their potential nutritional metabolic characteristics. The third category consists of medium- and long-chain fatty acids and their amide / ester derivatives, including C10–C22 fatty acids (docosapentaenoic acid, docosatrienoic acid) and various fatty amides and phosphate acyl compounds. This reflects the significant presence of polyunsaturated lipids and long-chain fatty acids in the samples and is associated with potential lipid remodeling or β-oxidation. The fourth category comprises signal lipids derived from the arachidonic acid and bile acid pathways, including thromboxane, prostaglandin F / E derivatives, and some steroidal structures. Their presence suggests the presence of a certain number of bioactive lipids in the samples, which play important roles in inflammation regulation, oxidative stress response, and lipid homeostasis.

[0035] Table 7.1 Substances in the active ingredients of the Lipid 20 composition that have a significant impact on fat and muscle ; 2. Establishment of fingerprint spectrum for "Lipid 20" composition High-performance liquid chromatography (HPLC) was used with a C18 reversed-phase column (4.6 mm × 250 mm, 5 μm). Phase A consisted of water + 0.1% TFA, and phase B consisted of acetonitrile + 0.1% TFA. The gradient elution program was set as follows: 0-10 min, 10%-30% B; 10.01-20 min, 40% B; 20.01-30 min, 55% B; 30.01-40 min, 90% B; 40.01-50 min, 10% B.

[0036] The flow rate was 1.0 mL / min, the column temperature was 30℃, the injection volume was 10 μL, and the detection wavelengths were 210 nm, 254 nm, and 280 nm. More than 10 batches of "Lipid 20" samples were tested, with a loading concentration of 1 mg / mL. After normalization, common characteristic peaks were extracted, their retention times and peak areas were recorded, the similarity between batches of samples was calculated, and a standard spectrum was established.

[0037] The Lipid 20 sample was scanned at 210 nm to monitor organic acids, amino acids, short peptides, and some additives. Visually, the main chromatographic peaks of the Lipid 20 samples from different batches showed high agreement, with only batch 1 and batch 2 showing relatively higher relative contents of chromatographic peaks at retention times of 27 min and 47 min, respectively.

[0038] The Lipid 20 sample was scanned at 254 nm to monitor substances such as vitamins and alkaloids. Visually, the main chromatographic peaks of the Lipid 20 samples from different batches showed high agreement, with only the sample from batch 1 showing a relatively high content of the chromatographic peak at the 27 min retention time.

[0039] The lipid 20 sample was scanned at 280 nm to monitor proteins and polyphenols. Visually, the main chromatographic peaks of the different batches of lipid 20 samples showed high agreement, with only batch 1 showing a relatively high content of the chromatographic peak at the 27 min retention time. Figures 7-9 ).

[0040] In summary, this study elucidated the material basis of lipids through non-targeted metabolomics. Lipid 20 constructed a unique "lipid-amino acid-organic acid" interactive metabolic network, rich in medium- and long-chain fatty acids, arachidonic acid pathway signaling lipids, and functional amino acids, providing efficient energy substrates and neural regulatory signals for muscle contraction. Compound structure annotations suggest that this metabolic band is mainly composed of the above five major classes of small molecules. The first class consists of medium- and long-chain fatty acids and their amide / ester derivatives; the second class consists of signaling lipids derived from arachidonic acid and bile acid pathways; the third class consists of amino acids, dipeptides, and their guanidine derivatives, including various modifications or condensates related to arginine, ornithine, and lysine; and the fourth class consists of polar small-molecule organic acids and inorganic anionic derivatives.

[0041] Ten batches of "Zhi20" samples from different production dates were analyzed by high performance liquid chromatography (HPLC). The samples were scanned at 210 nm, 254 nm, and 280 nm to monitor organic acids, amino acids, short peptides, some additives, vitamins, alkaloids, proteins, and polyphenols. Visually, the main chromatographic peaks of the different batches of Zhi20 samples showed high agreement, except for batch 1 sample, which had a relatively high content of the chromatographic peak at the 27 min retention time, indicating that the production process of "Zhi20" was relatively stable.

[0042] Example 2: Evaluation of the muscle-building and fat-reducing functions of the "Lipid 20" composition using a nematode model 1. Culture and modeling of Caenorhabditis elegans First, accurately weigh 6.8g of agar powder, 0.8g of tryptone, 1.2g of sodium chloride, and 0.08g of streptomycin, and dissolve them in 400mL of ultrapure water. Then, place this mixture, along with 1mol / L CaCl2, MgSO4, and K2HPO4-KH2PO4 buffer solution, into an autoclave and sterilize at 121℃ for 20min. After sterilization, allow the solution to cool naturally to 80-90℃. In a clean bench, sequentially add 0.4mL of CaCl2, 0.4mL of MgSO4, 10mL of K2HPO4-KH2PO4 buffer solution, and 0.4mL of 5mg / mL cholesterol ethanol solution filtered through a 0.22μm membrane, and mix thoroughly. Finally, transfer the well-mixed culture medium to pre-sterilized petri dishes, allow it to solidify, and then refrigerate to obtain solid nematode growth medium (NGM). Uracil-deficient *E. coli* OP50 was streaked onto solid agar plates and incubated for 1-2 days. Single colonies were then picked and transferred to 100 mL of sterilized LB (Luria-Bertani) liquid medium. *E. coli* OP50 was incubated at 37°C and 170 rpm for 12 hours using a shaker. When OD600 = 0.4, it was inoculated onto NGM plates and incubated for 1 day. The bacterial growth was observed; the absence of contaminating bacteria indicated successful preparation of the culture, which was then ready for feeding nematodes. After preparing the NGM, it was air-dried in a fume hood for one day to prevent excessive moisture in the culture environment. *E. coli* OP50 was then spread onto the NGM plates, and nematodes were subsequently transferred to the plates containing *E. coli* OP50. All nematodes were incubated in a 20°C constant temperature and humidity incubator.

[0043] Swimming experiments were used to model nematodes. Nematodes of the same age were obtained using a synchronous culture method. The nematodes were grouped and placed on NGM plates containing different concentrations of "Lipid 20" (optimal concentration 300 μg / mL), a positive control of 100 μg / mL metformin, and an OP50 blank control. At different stages, the nematodes were aspirated into M9 buffer for swimming experiments, with a 1-minute swimming interval of 5 minutes, repeated 10 times. Finally, L4 stage nematodes were collected for measurement or frozen at -80℃ for later use.

[0044] 2. Screening of the optimal concentration of the "Lipid 20" composition Using the N2 strain of *Caenorhabditis elegans*, synchronous nematodes were first obtained by rinsing and cultured to adulthood. The nematodes were then grouped and placed on NGM plates containing different concentrations of "Lipid 20" formulation (1, 3, 5, 8, 10, 20 mg / mL) for 24 h. After treatment, the head-wagging frequency and movement speed of the nematodes were measured. Furthermore, the oxidative stress (hydrogen peroxide) method was used to evaluate the stress resistance of the nematodes, and the survival rate was recorded. Each group contained at least 30 nematodes, and the experiment was repeated three times. Data were analyzed using ANOVA to screen for the optimal concentration of "Lipid 20" formulation.

[0045] Reactive oxygen species (ROS) levels were measured using the fluorescent probe 2,7-dichlorofluorescein diacetate (H2DCF-DA; Molecular Probes, Invitrogen). Nematodes exposed to IL for 12 h were transferred to centrifuge tubes and washed three times with phosphate-buffered saline (PBS, 10 mM). The nematodes were then sonicated in 1 mL PBS (500 W, 5 min, 30 °C). After centrifugation, 100 μL of each supernatant was added to the wells of a 96-well microtiter plate containing H2DCF-DA (final concentration 50 μM in PBS) and incubated at 22 °C for 20 min. ROS production in the nematodes was visualized under an inverted fluorescence microscope after staining with the H2DCF-DA fluorescent probe; the excitation wavelength was 485 nm and the emission wavelength was 525 nm.

[0046] In nematode behavioral experiments, sinusoidal motion and head-swing frequency are measured, primarily based on the sinusoidal motion of the body and the head-swing frequency within 60 seconds during three phases of the nematode lifespan test. A sinusoidal motion is defined as the worm completing a full 180° sinusoidal curve cycle during its bending motion, while a head-swing is defined as the worm swinging back and forth once near the pharynx.

[0047] 3. Effects of the "Lipid 20" composition on muscle gain Transgenic nematodes of the RW1596 strain expressing a muscle-specific GFP marker were cultured and modeled. GFP fluorescence signals in the nematode body wall muscle were observed using a fluorescence microscope under identical parameter settings, with a focus on assessing the integrity and regularity of muscle fiber arrangement. The proportion of muscle fiber breakage, deformation, or irregular distribution was recorded through image acquisition and quantitative analysis. At least 30 nematodes were observed in each group, and the experiment was repeated three times. Statistical analysis was used to evaluate the effect of the "Lipid 20" formulation on muscle growth.

[0048] 4. Effects of the "Lipid 20" composition on body fat distribution RW1596 transgenic nematodes expressing a muscle-specific GFP marker were cultured and modeled. The nematodes were washed three times with M9 buffer, fixed in 4% paraformaldehyde for 20 min, frozen at -80℃ for 20 min, thawed under running water, and subjected to three freeze-thaw cycles before finally thawing on ice. The nematodes were dehydrated with 60% isopropanol for 20 min, the supernatant was discarded, and the nematodes were stained with 60% Oil Red O solution at 25℃ in the dark for 4 h on a shaker. The distribution and accumulation of lipid droplets were observed and photographed under a microscope.

[0049] 5. Evaluation of the mechanism of "Lipid 20" in promoting muscle gain and reducing fat in nematodes based on conventional PCR and RT-qPCR. L4 stage nematode samples were collected 48 h after intervention with the "Lipid 20" combination. Total RNA was extracted using TRIzol reagent and cDNA was synthesized via reverse transcription. Genes related to lipid metabolism, including nhr-49 (a PPAR-like transcription factor), fat-6 (Δ9 fatty acid desaturase), and acs-2 (acyl-CoA synthase), as well as genes related to muscle development and contraction, including hlh-1 (muscle precursor cell differentiation regulator), unc-54 (myosin heavy chain), and myo-3 (muscle structural protein), were selected as target genes. Real-time quantitative PCR was performed using the SYBR Green dye method. The internal reference genes were act-1 (actin-1) or cdc-42 (a homolog of cell division regulator protein 42) to correct for expression differences (primer sequences are shown in the image). Figure 10 (As shown). The relative expression level changes were calculated using the ΔΔCt method, and the differences between the intervention group and the blank control group were compared to clarify the molecular regulatory effect of the composition in the lipid metabolism and muscle maintenance pathways.

[0050] 6. Results 6.1 Effects of Lipid 20 on Nematode Behavior like Figure 11 As shown, lipid 20 can increase the head-waving frequency of nematodes, with the 3 mg / mL and 5 mg / mL concentrations showing the most significant increases, both reaching highly significant differences compared to the control group. The 5 mg / mL concentration had the most pronounced promoting effect, nearly doubling the number of head-wavings. While the 1 mg / mL group did not reach the effect of the high dose, it still showed a certain upward trend. The number of sinusoidal movements showed a similar trend: the 3 mg / mL and 5 mg / mL groups showed significantly enhanced motor output, both significantly higher than the control group, with 5 mg / mL remaining the most effective. High doses resulted in varying degrees of decrease, with no significant difference compared to the control group.

[0051] Overall, lipid 20 exhibited the most stable and significant exercise-promoting effect in the 3-5 mg / mL range, while higher doses no longer produced an increase but instead showed an inhibitory trend, consistent with a typical inverted U-shaped dose-response effect.

[0052] From the perspectives of neuromuscular regulation and energy metabolism, the exercise-promoting effects of lipid-20 at concentrations of 3-5 mg / mL may be related to its combined influence on nerve conduction efficiency, muscle contractility, and metabolic substrate supply. In *C. elegans*, head swing is driven by the rhythmic firing of cholinergic motor neurons and the synchronous contraction of body wall muscles. Therefore, this result suggests that lipid-20 may enhance the excitability of the neuromuscular junction, thereby improving motor performance. From a metabolic perspective, the medium- and long-chain fatty acids (including some unsaturated fatty acids and their amide derivatives) contained in lipid-20 can directly enter mitochondria for β-oxidation, providing a relatively efficient and sustained ATP supply for muscle activity. Unsaturated fatty acids have also been reported to affect energy sensing pathways such as AMPK and mTOR, improving metabolic efficiency and helping to maintain muscle function. Therefore, at appropriate doses, lipid-20 may optimize energy supply and metabolic homeostasis, leading to higher output levels in short-term exercise tests in nematodes. At higher doses, the head swing showed a decreasing trend, possibly because: 4. Nematodes have a small metabolic volume and are more sensitive to high concentrations of lipids. The size and metabolic capacity of Caenorhabditis elegans are limited. Increased lipids in the culture medium may increase the absorption pressure in the nematode's gastrointestinal tract. The accumulation of lipids in the body cavity can lead to excessively high local concentrations, affecting the balance of movement-related muscle rhythms and energy supply.

[0053] Figure 11 In the middle: (a) the frequency of the nematode's head swing; (b) the number of sinusoidal motions.

[0054] 6.2 Effects of lipid 20 on nematode oxidation This invention utilizes a ROS fluorescent probe to evaluate the effect of different concentrations of lipid 20 on oxidative stress in *C. elegans*. Overall fluorescence intensity showed that the ROS level in the 3 mg / mL group was significantly lower than that in the control group and other treatment groups, as evidenced by a significant decrease in fluorescence intensity in the whole worm, suggesting that this concentration has the strongest ability to scavenge free radicals or inhibit oxidative stress. In the remaining gradients of 1-2 mg / mL, fluorescence intensity showed varying degrees of increase or maintenance, and no significant decreasing trend similar to that observed in the 3 mg / mL group was observed compared to the control group. Figure 12 This indicates that its antioxidant effect has a clear dose window.

[0055] 6.3 Effects of Lipid 20 on Lipid Accumulation in Nematodes Oil Red O lipid staining results showed that ( Figure 13Compared with the blank control group, the number of lipid droplets in the body cavity of nematodes treated with Lipid 20 was significantly reduced, and the overall staining was significantly lighter, indicating a lower level of lipid accumulation. The figure shows a decrease in lipid droplet distribution density in the body wall and intestinal region of the sample group, indicating a reduced degree of lipid aggregation in the body cavity. Quantitative analysis further confirmed this trend: the proportion of lipid droplets in the sample group was significantly lower than that in the control group (P<0.0001). Although the reduction in lipid droplet content was weaker than that in the positive control group, it still showed a stable lipid-lowering effect overall. The results indicate that Lipid 20 effectively inhibits lipid deposition in nematodes, reduces the number of observable fat particles in the body cavity, and significantly reduces the overall lipid accumulation level.

[0056] Staining results suggest that lipid 20 may exert its lipid-lowering effect by regulating energy metabolism and lipid droplet dynamics. Metabolically, it is speculated that the medium- and long-chain fatty acids, ω-3 / ω-6 PUFAs, amino acids, and small peptides abundant in lipid 20 may collectively participate in the regulation of lipid homeostasis in nematodes. Studies have shown that some unsaturated fatty acids can promote mitochondrial β-oxidation, increase the rate of fatty acid consumption, and thus reduce the re-esterification and accumulation of lipid droplets. Secondly, fatty acid amides and bile acid-like structures may affect the expression of lipid metabolism genes through receptors (NHR-49, DAF-12 nuclear receptors), promoting fat mobilization and inhibiting fat storage. Furthermore, small peptides and guanidine-containing amino acid derivatives may indirectly affect the fatty acid oxidation pathway by improving nitrogen metabolism and energy supply, thereby reducing the deposition of excess lipids.

[0057] In summary, lipid20 significantly reduces lipid accumulation in nematodes by acting on fatty acid oxidation, lipid droplet metabolism, and energy homeostasis through multiple nutrients.

[0058] 6.4 Effects of Lipid 20 on the width and length of nematode muscles Experimental results show that ( Figure 14 The sample showed significant protective and enhancing effects on the body wall muscles of nematodes: compared with the control group, the muscle fibers in the sample group appeared thicker, more continuous, and more tightly packed in microscopic images, with clear muscle contours, and the overall condition was close to or even slightly better than the positive control group. Quantitative results showed that the sample significantly increased muscle width (P<0.0001) and significantly enhanced muscle fluorescence intensity (P=0.0236), suggesting that it not only improved muscle morphology but may also increase the expression of muscle structural proteins or related muscle markers. From a functional and mechanistic perspective, it is speculated that the lipid 20 sample may delay nematode muscle aging and improve muscle quality by slowing down muscle protein degradation, maintaining the integrity of muscle fiber structure, or improving energy and antioxidant status.

[0059] 6.5 Routine PCR detection of gene expression in the muscle fat metabolism of Lipid 20 nematodes Conventional PCR results showed ( Figure 15In the CK, Z20, and Y groups, the expression levels of six key genes related to muscle structure and lipid metabolism (fat-6, myo-3, acs-2, unc-54, nhr-49, and hlh-1) all showed a consistent upward trend. The Y group had the highest overall expression level, followed by Z20, while CK had the lowest. In gel electrophoresis images, the bands of each gene in the Z20 group showed greater brightness and thicker band width than those in the CK group, perfectly consistent with the quantitative data in the bar chart. This indicates that the lipid 20 sample treatment significantly increased the expression levels of muscle structural proteins (myo-3, unc-54) and muscle regulatory factors (hlh-1), while also promoting the upregulation of fatty acid metabolism (fat-6) and energy metabolism genes (acs-2). In particular, the strong enhancement of ACS-2 and FAT-6 suggests that the sample may improve the lipid metabolism efficiency of nematodes, providing energy support for muscle maintenance. The simultaneous upregulation of Myo-3, Unc-54, and HLH-1 indicates that the sample helps improve muscle fiber integrity and muscle generation capacity. The significant enhancement of NHR-49 further suggests that the sample may regulate overall energy status through the lipid metabolism nuclear receptor pathway. In summary, the Lipid20 sample not only increased the expression of muscle structure-related genes but also improved lipid and nematode energy metabolism pathways, enabling muscles to obtain better energy supply and structural maintenance capabilities.

[0060] 6.6 qPCR detection of lipid metabolism gene expression levels in Lipid 20 nematodes qPCR amplification curves showed ( Figure 16 Each gene reaction exhibited good amplification efficiency and a typical "S-shaped" growth trend. The basal fluorescence was stable, and the exponential amplification phase was clear, indicating that the experimental system was reliable and reproducible. In the quantitative results, sample treatment group Z20 showed varying degrees of upregulation of six key genes (unc-54, fat-6, myo-3, acs-2, nhr-49, and hlh-1), with overall expression levels generally higher than the control group CK. Specifically: structural proteins unc-54 and myo-3 showed slight increases, suggesting improved basic muscle fiber structure; upregulation of fat-6 and acs-2 reflected enhanced lipid metabolism and fatty acid activation, contributing to energy supply for muscle maintenance and contraction; the increase in nhr-49 further supports the possibility that the sample may activate energy metabolism-related pathways; and the most significant change was in the transcription factor hlh-1, which is related to muscle development and repair, indicating that the sample may have a stronger promoting effect on muscle regeneration and fiber repair. In summary, the qPCR data showed a consistent trend in band intensity with conventional PCR, indicating that the sample can simultaneously regulate the expression of muscle structural proteins and energy metabolism pathway genes.

[0061] In summary, this study verified the muscle-building and fat-reducing functions of the "Lipid 20" sample through a multi-dimensional experimental approach, including behavioral experiments, ROS fluorescence imaging, Oil Red O staining, muscle fluorescence labeling observation, microscopic morphological observation, and PCR / qPCR gene expression analysis.

[0062] Behavioral experiments and ROS fluorescence imaging clarified the dose-response relationship. Lipid 20 showed the best effect in the concentration range of 3-5 mg / mL, significantly improving the head swing and body bending rhythm of nematodes, enhancing the excitability of the neuromuscular junction, and scavenging ROS, significantly reducing oxidative stress in vivo, protecting muscle fibers from oxidative damage, and demonstrating a significant ability to maintain cell homeostasis, indicating that lipid 20 may contain a direct free radical scavenger.

[0063] The lipid redistribution and lipid-reducing effects were evaluated using Oil Red O staining and microscopic morphological observation. Despite the presence of fatty acids in the samples, the number of lipid droplets in the nematode body cavity was significantly reduced after treatment. This suggests that lipid20 may inhibit lipid deposition by activating NHR-49-mediated high expression of ACS-2, thereby accelerating fatty acid oxidation for energy.

[0064] The efficacy of lipid 20 in delaying muscle fiber aging was evaluated by observing muscle fluorescence labeling and microscopic morphology. Lipid 20 significantly upregulated the expression of hlh-1 and structural proteins myo-3 and unc-54, indicating that lipid 20 can improve muscle fiber morphology and delay the muscle aging process.

[0065] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for verifying the efficacy of a muscle-building and fat-reducing nutritional solid beverage composition, characterized in that, The method includes a step of evaluating the function of the composition using a Caenorhabditis elegans model; the functional evaluation includes a comprehensive assessment from at least two dimensions of kinetic behavior, body fat content, muscle structure and gene expression level.

2. The method according to claim 1, characterized in that, The functional evaluation includes the following four dimensions: (a) Evaluation of motor behavior: Determine the head oscillation frequency and / or the number of sinusoidal body movements of the nematode; (b) Evaluation of body fat content: The accumulation of lipid droplets in nematodes was determined by Oil Red O staining. (c) Evaluation of muscle structure: Using transgenic nematodes expressing muscle-specific fluorescent proteins, the morphology, width, or fluorescence intensity of the body wall muscles were observed and analyzed; (d) Evaluation of gene expression: The expression levels of lipid metabolism-related genes and / or muscle development-related genes in nematodes were detected.

3. The method according to claim 1, characterized in that, The Caenorhabditis elegans model was pre-trained in swimming exercises, which included placing the nematodes in an M9 buffer solution for intermittent swimming exercises. The intermittent swimming exercise consists of swimming for 1 minute followed by resting for 5 minutes, repeated 10 times in total.

4. The method according to claim 1, characterized in that, The method also includes a step of active ingredient discovery in the composition; the active ingredient discovery includes: preparing a sample solution of the composition using an ultrasound-assisted extraction method, and then performing non-targeted metabolite analysis using ultra-high performance liquid chromatography-linear ion trap-electrostatic field orbital trap high-resolution mass spectrometry, combined with high-resolution mass spectrometry database comparison, to screen out active ingredients related to lipid metabolism and / or muscle synthesis.

5. The method according to claim 4, characterized in that, The chromatographic conditions for the analysis of the non-targeted metabolites were as follows: reversed-phase C18 column, mobile phase of formic acid aqueous solution and formic acid acetonitrile solution, gradient elution; and mass spectrometry conditions: electrospray ionization source, positive ion mode, data-dependent secondary mass spectrometry scan.

6. The method according to claim 1, characterized in that, The method also includes the step of establishing a standard fingerprint spectrum of the composition using high performance liquid chromatography: detecting more than 10 different batches of the composition, with a sample concentration of 1 mg / mL, recording the chromatogram, extracting common characteristic peaks after normalization, recording the retention time and peak area, calculating the batch similarity, and establishing a standard fingerprint spectrum.

7. The method according to claim 6, characterized in that, The detection wavelength of the high-performance liquid chromatography includes at least one of 210 nm, 254 nm and 280 nm.

8. The method according to claim 1, characterized in that, The muscle-building and fat-reducing nutritional solid beverage composition is a lipid 20 composition, the raw materials of which include concentrated whey protein powder, soy protein isolate, purple sweet potato powder, konjac powder, compound minerals, citrus fiber, hydrolyzed casein peptides, compound vitamins, steviol glycosides, and betaine.

9. The method according to any one of claims 1-8, characterized in that, The method also includes a step of measuring the level of reactive oxygen species in nematodes to evaluate the antioxidant capacity of the composition; the measurement is performed using the fluorescent probe H2DCF-DA, with an excitation wavelength of 485 nm and an emission wavelength of 525 nm.

10. The application of the method according to any one of claims 1-9 in the quality control, efficacy verification, or new product development of functional foods, sports nutrition foods, special medical foods, or health products.

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