Extraction process of high-stability hydroxysafflor yellow A and application thereof in fat-burning metabolism

Abstract

The application discloses a high-stability HYSA extraction method and application thereof in fat-burning metabolism, and belongs to the field of natural product extraction technology and healthy nutrition. The extraction steps comprise raw material pretreatment, combined fermentation bacteria liquid preparation, fermentation pretreatment, 60 DEG C hot water ultrasonic extraction, solid-liquid separation, concentration and drying. The application adjusts the system to be acidic by organic acid generated by combined fermentation, significantly improves the stability of HYSA, and promotes dissolution of effective components by using hydrolytic enzymes secreted by yeast to destroy safflower cell walls. The 60 DEG C ultrasonic mild extraction avoids high-temperature degradation, and further guarantees activity. The method has high extraction efficiency, green and environment-friendly process, low cost, and is easy to industrialize, solves the problems of poor HYSA extraction stability, low extraction content and high cost in the prior art, explores the influence of the HYSA on fat-burning metabolism of mice in resting and exercise states, and is suitable for the fields of medicines, foodstuffs, health care products and the like.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction technology and natural product health nutrition. Specifically, it relates to an extraction process for highly stable hydroxysaffron yellow pigment A based on the synergistic effect of combined fermentation and mild extraction. It is particularly suitable for the efficient and stable extraction of hydroxysaffron yellow pigment A from safflower. It also relates to the application of hydroxysaffron yellow pigment A in promoting fat burning metabolism and can be widely used in the fields of medicine, food, and sports nutrition. Background Technology

[0002] The sports fat-burning product market has experienced rapid growth in recent years. However, beneath this prosperity lies a deep-seated problem of ingredient homogenization. Most mainstream products on the market are minor tweaks to "classic" ingredients such as white kidney bean extract, caffeine, and L-carnitine, lacking systematic exploration and application of novel, naturally derived functional factors. This lack of innovation not only weakens brand differentiation but also limits the upgrade potential of the entire category. Against this backdrop, developing novel natural active substances with clear scientific evidence is both a breakthrough to fill market gaps and a key path to building a differentiated technological moat.

[0003] Safflower is the dried flower of *Carthamus tinctorius* L., a plant in the Asteraceae family. Hydroxysafflor Yellow A (HSYA) is its signature active ingredient. HSYA has good water solubility and, in addition to being used as a natural food coloring, is also added as a functional ingredient to pharmaceuticals and health products. Numerous studies have shown that HSYA exhibits multi-target regulatory effects in metabolic diseases such as diabetes, abnormal lipid metabolism, hyperlipidemia, and cardiovascular diseases, demonstrating its important application value in pharmaceutical preparations and functional foods. However, HSYA has poor stability and is highly sensitive to factors such as light, temperature, pH, and metal ions. It is easily degraded during processing and storage, leading to a decrease in content and changes in color, which severely limits its practical application and product quality stability.

[0004] Currently, extraction methods for HSYA mainly include hot water extraction, ethanol extraction, ultrasound-assisted extraction, and microwave-assisted extraction. Among these, hot water extraction is one of the most widely used methods in industrial production due to its simplicity, low cost, and absence of organic solvent residue. However, traditional hot water extraction has significant shortcomings: firstly, the extraction system is often neutral or weakly alkaline, and in a pH environment unfavorable to HSYA stability, some active ingredients will degrade during extraction; secondly, hot water extraction alone is insufficient to fully disrupt the dense structure of safflower cells, resulting in limited HSYA dissolution efficiency and the presence of numerous impurities in the extract, increasing the difficulty of subsequent separation and purification. While physical aids such as ultrasound and microwaves can enhance cell disruption and increase dissolution rates to some extent, they cannot fundamentally improve the pH environment of the extraction system, and the stability problem of HSYA during extraction remains unresolved.

[0005] To further improve extraction efficiency, microbial fermentation-assisted extraction technology has been gradually introduced into the field of natural product extraction. The core of this technology lies in utilizing various hydrolytic enzymes produced by microbial metabolism to degrade the cell wall structure of plants, thereby promoting the release of intracellular active ingredients. Existing technologies have reported the use of microbial fermentation for the extraction of flavonoids from safflower, primarily using yeast or a combination of Escherichia coli and yeast. These methods utilize cellulase and pectinase secreted by the microorganisms to disrupt the safflower cell wall, which has a certain effect on improving the extraction rate of the target components. However, the existing microbial fermentation-assisted extraction methods still have the following drawbacks: First, yeast and other microorganisms have limited acid production capacity during fermentation, making it difficult to form and maintain an acidic environment conducive to HSYA stability in the extraction system, and the active ingredients may still be significantly degraded during extraction; second, if strains such as Escherichia coli are introduced, there are safety risks such as endotoxin residues, which limits their applicability in the production of food and health food raw materials; third, the enzyme production spectrum of existing single or simple combination microbial systems is relatively limited, and the cell wall breaking strength of safflower is insufficient, which restricts the potential for improving the HSYA extraction rate.

[0006] In summary, existing physical aids and reported microbial fermentation methods in the extraction and preparation of HSYA cannot simultaneously resolve the two core contradictions of "stabilizing and protecting active ingredients during extraction" and "efficiently breaking down cell walls and promoting solubility." How to simultaneously improve HSYA extraction efficiency and protect structural stability during extraction, while ensuring safety and environmental friendliness, thereby obtaining high-quality HSYA extracts and expanding their applications in functional foods and other fields, remains a pressing technical problem to be solved in this field. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing HYSA extraction technologies, such as poor stability and low extraction efficiency, and to provide a rapid and highly stable HYSA extraction process. This process leverages the greater stability of HYSA under acidic conditions, combined with 60℃ hot water ultrasonic-assisted extraction, and utilizes the synergistic advantages of acid and enzyme production from the co-fermentation of Lactobacillus and yeast to achieve efficient dissolution and stable retention of HYSA, thereby improving the product extraction rate. This invention also explores the potential of HYSA in fat-burning metabolism, laying a solid foundation for its subsequent industrial production.

[0008] To achieve the above objectives, the present invention first provides an extraction process for highly stable hydroxysaffron yellow pigment A, comprising the following steps: (1) Raw material pretreatment: Take dried safflower filaments, remove impurities and crush them, then pass them through a 40-80 mesh sieve to obtain safflower powder; (2) Preparation of co-fermentation culture: The activated Lactobacillus plantarum was inoculated into MRS medium and cultured statically at 30-37℃ for 12-18 h to obtain Lactobacillus plantarum seed culture; the activated Saccharomyces cerevisiae was inoculated into YPD medium and cultured with shaking at 28-32℃ for 10-14 h to obtain yeast seed culture; the Lactobacillus plantarum seed culture and Saccharomyces cerevisiae seed culture were mixed to obtain co-fermentation culture; (3) Fermentation pretreatment: The safflower powder pretreated in step (1) is mixed with deionized water and fermented with co-fermentation bacteria. (4) Hot water extraction: The fermented material obtained in step (3) is transferred to an extraction tank and extracted under hot water bath conditions; (5) Solid-liquid separation: After extraction, the extract is cooled to room temperature, centrifuged, and the supernatant is collected. The precipitate is washed with deionized water 2-3 times, and the washing liquid is combined with the supernatant to obtain HYSA extract.

[0009] In one embodiment of the present invention, in step (2), the volume ratio of the Lactobacillus plantarum seed liquid and the Saccharomyces cerevisiae seed liquid is 1:1 to 3, preferably 1:2. At this volume ratio, the synergistic effect of the strains in producing acid and enzymes is optimal.

[0010] In one embodiment of the present invention, in step (2), the conditions for activating *Lactobacillus plantarum* are: inoculating *Lactobacillus plantarum* freeze-dried powder into MRS medium and incubating at 37°C for 16 h; the conditions for activating *Saccharomyces cerevisiae* are: inoculating *Saccharomyces cerevisiae* freeze-dried powder into YPD medium and incubating at 30°C and 180 r / min for 12 h; wherein, preferably, *Lactobacillus plantarum* is deposited at the China Industrial Microbial Culture Collection Center (CICC) with accession number CICC20261; preferably, *Saccharomyces cerevisiae* is deposited at the China Industrial Microbial Culture Collection Center (CICC) with accession number CICC1002.

[0011] In one embodiment of the present invention, the viable counts of both *Lactobacillus plantarum* and *Saccharomyces cerevisiae* in the co-fermentation broth are not less than 1 × 10⁻⁶. 8 CFU / mL.

[0012] In one embodiment of the present invention, in step (3), the mass-to-volume ratio of safflower powder to deionized water is 1:15~25 (g / mL), preferably 1:20 (g / mL).

[0013] In one embodiment of the present invention, in step (3), the inoculation amount of the combined fermentation broth is 3-8% of the total mass of the mixture of safflower powder and deionized water, preferably 5%. Before fermentation, the initial pH of the system needs to be adjusted to 5.0-6.0, the fermentation temperature is 28-32℃, preferably 30℃, and the fermentation time is 8-12h, preferably 10h. During fermentation, *Lactobacillus plantarum* produces organic acids, causing the pH of the system to decrease naturally. *Saccharomyces cerevisiae* secretes hydrolytic enzymes such as cellulase and pectinase, which destroy the cell wall structure of safflower filaments. Under the preferred fermentation conditions, the pH of the fermentation system can be stabilized at around 4.0, and the enzyme activity reaches its peak, thus achieving both acid production and enzyme production.

[0014] In one embodiment of the present invention, in step (4), the temperature of the hot water bath is 60°C and the extraction time is 0.5~2h.

[0015] In one embodiment of the present invention, in step (4), ultrasonic-assisted extraction can also be performed during hot water bath extraction. The ultrasonic power is 100~200W. Preferably, ultrasonic-assisted extraction is performed under constant temperature of 60℃ for 1 hour. The ultrasonic frequency is 200W. Mild extraction at 60℃ can avoid the degradation of HYSA caused by excessively high temperature. Ultrasound promotes the full dissolution of HYSA, and the acidic system can improve the stability of HYSA. The preferred extraction conditions can ensure the full dissolution of HYSA and avoid component degradation caused by excessive stirring.

[0016] In one embodiment of the present invention, in step (5), the rotation speed during centrifugation is 3000-5000 r / min, and the centrifugation time is 10-15 min.

[0017] The present invention also provides the use of hydroxysaffron yellow pigment A or a composition containing hydroxysaffron yellow pigment A in the preparation of functional foods, pharmaceuticals, health products or exercise simulators for promoting fat burning metabolism in the body under resting and exercise conditions.

[0018] In one embodiment of the present invention, promoting fat-burning metabolism in the body at rest and during exercise includes reducing the fat content in the soleus muscle, gastrocnemius muscle, and tibialis anterior muscle, as well as the content of free fatty acids in the blood plasma, in both resting and exercise states.

[0019] In one embodiment of the present invention, the functional food includes: functional beverages, functional powders, functional effervescent tablets, functional biscuits, functional capsules, etc.

[0020] In one embodiment of the present invention, the composition further includes pharmaceutically acceptable excipients, including solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc.

[0021] Beneficial effects: 1. This invention uses a mixed strain of *Lactobacillus plantarum* and *Saccharomyces cerevisiae* to pre-treat safflower through fermentation, followed by hot water extraction to extract hydroxysaffron yellow pigment (HYSA). The lactic acid and other organic acids produced by the co-fermentation of *Lactobacillus plantarum* and *Saccharomyces cerevisiae* adjust the system to an acidic state, ensuring high stability of HYSA during extraction. Simultaneously, the gentle extraction at 60℃ promotes the full dissolution of HYSA from safflower while avoiding degradation caused by high temperatures, further guaranteeing the activity and stability of HYSA and solving the core problem of easy degradation of active ingredients in existing extraction techniques. The co-fermentation of *Lactobacillus plantarum* and *Saccharomyces cerevisiae* in this invention exhibits significant synergistic advantages in acid and enzyme production. The cellulase and pectinase secreted by *Saccharomyces cerevisiae* effectively break down the cellulose and pectin structures of safflower cell walls, resulting in efficient dissolution of active ingredients. The organic acids produced by *Lactobacillus plantarum* soften the cell walls, further promoting HYSA dissolution. Simultaneously, the acidic environment enhances the water solubility of HYSA. The synergistic effect of these two methods significantly improves the extraction rate of HYSA compared to the traditional 60℃ hot water extraction method.

[0022] 2. The average HSYA content in the crude extract obtained by the process provided by this invention is 25.7 mg / g, which is significantly higher than that of conventional hot water extraction and single-strain assisted extraction processes. Furthermore, the promoting effect of the compound strain on HSYA content in this invention is far greater than the sum of the promoting effects of the two single strains, indicating that *Lactobacillus plantarum* and *Saccharomyces cerevisiae* have a significant synergistic effect in promoting HSYA content enhancement. In addition, this invention organically combines 60℃ hot water extraction with *Lactobacillus plantarum*-*Saccharomyces cerevisiae* co-fermentation, achieving a dual synergistic effect of "acid production for stability + enzyme production for extraction." This solves the problems of poor stability and low extraction rate in traditional hot water extraction, and overcomes the shortcomings of insufficient acid or enzyme production capacity of single fermentation strains. The overall process exhibits strong synergy and outstanding comprehensive benefits.

[0023] 3. Ultrasonic-assisted hot water extraction can further increase the average extraction content of HSYA. Appropriate ultrasonic technology can increase the average extraction content of HSYA in crude extract to 29.6 mg / g.

[0024] 4. This invention uses 60℃ hot water extraction, eliminating the need for organic solvents and leaving no organic residues, making it environmentally friendly. The *Lactobacillus plantarum* and *Saccharomyces cerevisiae* used in the co-fermentation are safe and non-toxic, balancing extraction efficiency with reducing potential risks such as endotoxin residues associated with *E. coli*. Furthermore, the fermentation process requires no additional acid regulators or enzymes, reducing production costs. The process is simple to operate, with clear steps, requiring no complex equipment, and is easily scalable for large-scale industrial production.

[0025] 5. This invention administers HYSA to mice via gavage. Experimental results show that, in both resting and active states, the skeletal muscle fat content of the treated mice was significantly reduced, while the free fatty acid content in their plasma increased. Attached Figure Description

[0026] Figure 1 The chemical structural formula of hydroxysaffron yellow pigment A is given.

[0027] Figure 2 The effect of co-fermentation of *Lactobacillus plantarum* and *Saccharomyces cerevisiae* on HYSA extraction was investigated. A represents the standard curve of HYSA standard under HPLC detection, and B represents the effect of Examples 1 & Comparative Examples 1-5 on HYSA extraction. Data are expressed as mean ± standard error, with each group having three replicates. Compared with the control, *, P < 0.05; **, P < 0.01; ***, P < 0.001 (T-test).

[0028] Figure 3 The effect of different ultrasonic power and ultrasonic time on HYSA extraction is shown. Data are expressed as mean ± standard error, with each group having three replicates. Compared with control, *, P < 0.05; **, P < 0.01; ***, P < 0.001 (T-test).

[0029] Figure 4This study demonstrates the effects of HYSA supplementation on the percentage of fat in skeletal muscle and the level of free fatty acids in serum in mice under resting and exercise conditions. (A) & (B) represent the effects of HYSA on the percentage of fat in the soleus muscle of mice under resting and exercise conditions. (C) & (D) represent the effects of HYSA on the percentage of fat in the gastrocnemius muscle of mice under resting and exercise conditions. (E) & (F) represent the effects of HYSA on the percentage of fat in the soleus muscle of mice under resting and exercise conditions. (G) & (H) represent the effects of HSYA on the level of free fatty acids in serum in mice under resting and exercise conditions. Data are expressed as mean ± standard error, with three replicates per group. Compared with the control, *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way ANOVA). Detailed Implementation

[0030] Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0031] The *Lactobacillus plantarum* freeze-dried powder and *Saccharomyces cerevisiae* freeze-dried powder used in the embodiments and comparative examples of this invention were obtained from the Shanghai Biotechnology Center for Collection and Research. The corresponding live *Lactobacillus plantarum* strains are deposited at the China Industrial Microbial Culture Collection Center (CICC), accession number CICC20261; the live *Saccharomyces cerevisiae* strains are deposited at the China Industrial Microbial Culture Collection Center (CICC), accession number CICC1002.

[0032] Example 1 A method for extracting highly stable HYSA includes the following steps: 1. Raw material pretreatment: Take 50 g of dried safflower filaments, remove impurities, pulverize, and pass through a 60-mesh sieve to obtain safflower powder; place the safflower powder in a 60℃ oven to dry for 1.5 h to remove moisture, and set aside for later use; 2. Preparation of co-fermentation broth: Lyophilized *Lactobacillus plantarum* powder was inoculated into MRS medium and incubated at 37°C for 16 h to obtain *Lactobacillus plantarum* seed culture (viable count 1.2 × 10⁻⁶). 8 CFU / mL); The freeze-dried Saccharomyces cerevisiae powder was inoculated into YPD medium and cultured at 30℃ and 180 r / min for 12 h with shaking to obtain a Saccharomyces cerevisiae seed culture (1.5 × 10⁻⁶ CFU / mL). 8 (CFU / mL); mix Lactobacillus plantarum seed culture and Saccharomyces cerevisiae seed culture at a volume ratio of 1:2 to obtain a co-fermentation culture; 3. Fermentation pretreatment: The safflower powder pretreated in step 1 was mixed with deionized water at a mass-to-volume ratio of 1:20 (g / mL), and the co-fermentation broth was added. The inoculation amount of the fermentation broth was 5% of the total mass of the mixed system. The initial pH of the system was adjusted to 5.5 with 0.1 mol / L citric acid solution, and the mixture was placed at 30℃ for constant temperature fermentation for 10 h to obtain the fermented material. 4. 60℃ hot water extraction: Transfer the fermented material obtained in step 3 to an extraction tank, heat it in a water bath to 60℃, set the extraction time to 2 h, and the stirring rate to 100 rpm / min; 5. Solid-liquid separation: After extraction, the extract was cooled to room temperature and centrifuged at 4000 r / min for 15 min. The supernatant was collected. The precipitate was washed three times with deionized water. The washing liquid and the supernatant were combined to obtain the crude extract.

[0033] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that steps 2 and 3 are omitted, and the raw material in step 1 is directly mixed with deionized water at a mass-volume ratio of 1:20 (g / mL). The mixture is then transferred to an extraction tank and extracted by water bath heating.

[0034] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the fermentation broth in step 2 contains only Lactobacillus plantarum seed broth, while the inoculation amount of the fermentation broth in step 3 is the same as that in Example 1.

[0035] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the fermentation liquid in step 2 contains only brewer's yeast seed liquid, and the inoculation amount of the fermentation liquid in step 3 is the same as that in Example 1.

[0036] Comparative Example 4: The difference between Comparative Example 4 and Example 1 is that the volume ratio of Lactobacillus plantarum seed liquid to Saccharomyces cerevisiae seed liquid in step 2 is 1:1.

[0037] Comparative Example 5: The difference between Comparative Example 5 and Example 1 is that the volume ratio of Lactobacillus plantarum seed liquid to Saccharomyces cerevisiae seed liquid in step 2 is 1:3.

[0038] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that Lactobacillus plantarum in step 2 is replaced with Escherichia coli.

[0039] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is that the temperature of the hot water extraction in step 4 is changed to 30°C.

[0040] Example 2: The difference between Example 2 and Example 1 is that step 4 uses hot water combined with ultrasound-assisted extraction, that is, after heating the water bath to 60°C, extraction is assisted under 200W ultrasound conditions for 2 minutes.

[0041] Comparative Example 8: The difference between Comparative Example 8 and Example 2 is that the ultrasound time in step 4 is 1 minute.

[0042] Comparative Example 9: The difference between Comparative Example 9 and Example 2 is that the ultrasound time in step 4 is 3 minutes.

[0043] Comparative Example 10: The difference between Comparative Example 10 and Example 2 is that the ultrasonic power in step 4 is 100W.

[0044] Comparative Example 11: The difference between Comparative Example 11 and Example 2 is that the ultrasonic power in step 4 is 300W.

[0045] The crude extracts prepared in the examples and comparative examples were filtered through a 0.45 μm filter membrane, and the content of HSYA in the crude extracts was determined by HPLC. The structural formula of HSYA is shown below. Figure 1 As shown, the chromatographic conditions were as follows: a C18 column (4.6 × 250 mm, 5 μm) was used for detection; the column temperature was 35℃; and the injection volume was 10 μL. The mobile phase conditions are shown in Table 1. The standard curve for the HYSA standard was: y = 7325.4x + 191911, R² = 0.9999.

[0046] Table 1. High Performance Liquid Chromatography Separation Conditions

[0047] from Figure 2 As shown in Figure B, compared with Comparative Example 1, the participation of different strains can increase the extraction content of HYSA to varying degrees. Furthermore, the effect of compound strains is more significant than that of single strains. Through screening different ratios of *Lactobacillus plantarum* and *Saccharomyces cerevisiae*, it was ultimately determined that when the ratio of lactic acid bacteria to yeast is 1:2, the extraction yield of HYSA is the highest, with a content increase of nearly 60% compared to Comparative Example 1.

[0048] Specifically, the average HSYA extraction content obtained in Example 1 was 25.7 mg / g, significantly higher than that in Comparative Example 1 (16.6 mg / g). Example 1 showed an increase of approximately 54.8% compared to Comparative Example 1, and the difference was statistically significant (p<0.001). Comparative Example 2 used only *Lactobacillus plantarum* for fermentation, and its average HSYA extraction content was 18.5 mg / g, only an increase of 11.4% compared to Comparative Example 1. Comparative Example 3 used only *Saccharomyces cerevisiae* for fermentation, and its average HSYA extraction content was 19.45 mg / g, only an increase of 17.2% compared to Comparative Example 1. However, Example 1, using a combination of two strains for fermentation, showed a much greater increase in the average HSYA extraction content than the sum of the increases in the average HSYA extraction content of Comparative Examples 2 and 3 compared to Comparative Example 1. This indicates that the two strains had a synergistic effect during the combined fermentation, jointly promoting the dissolution of HSYA.

[0049] Further comparison of different strain ratios showed that when the volume ratio of Lactobacillus plantarum seed liquid to Saccharomyces cerevisiae seed liquid was 1:1 or 1:3, the average HSYA extraction content of Comparative Examples 4 and 5 was 22.5 mg / g and 22.47 mg / g, respectively, both significantly lower than that of Example 1. This indicates that a 1:2 ratio of Lactobacillus plantarum to Saccharomyces cerevisiae can achieve better extraction results.

[0050] Furthermore, the average extraction content of Comparative Example 6, in which *Lactobacillus plantarum* was replaced with *Escherichia coli*, was 21.53 mg / g, which was not only lower than that of Example 1, but also posed potential safety risks such as endotoxin residues, making it unsuitable for applications in the food and health product fields. The average extraction content of Comparative Example 7, in which the extraction temperature was lowered to 30°C, was 23.43 mg / g, also lower than that of Example 1, indicating that the mild hot water extraction conditions at 60°C are more conducive to the full dissolution of HSYA while maintaining its stability. These results fully demonstrate that Example 1 of this invention, through the co-fermentation of *Lactobacillus plantarum* and *Saccharomyces cerevisiae* in a preferred ratio, utilizes the acid produced by *Lactobacillus plantarum* metabolism to create an acidic environment conducive to the stable existence of HSYA, while simultaneously utilizing the cellulase, pectinase, and other hydrolytic enzymes produced by *Saccharomyces cerevisiae* to disrupt the cell wall structure of safflower, thereby significantly improving the extraction efficiency and stability of HSYA. This demonstrates the comprehensive advantages compared to traditional hot water extraction, single-strain fermentation, non-preferred strain ratios, and low-temperature extraction processes.

[0051] from Figure 3As can be seen, compared with Example 1, the use of ultrasound assistance (Example 2) significantly improved the extraction content of HYSA. Furthermore, by comparing the effects of different ultrasound powers and durations, it is easy to find that there is a certain positive correlation between ultrasound power and duration and the extraction content, but after reaching a certain level, there is no longer a significant change. Considering factors such as energy saving and environmental protection, 200W for 2 minutes is considered most suitable. The extraction content of HYSA was 29.6 mg / g.

[0052] Example 3: Verification of the ability of hydroxysaffron yellow pigment A to promote fat burning metabolism in mouse skeletal muscle under resting and exercise conditions. 1 Experimental Methods 1.1 Animal Experiments Five-week-old male mice purchased from the Model Animal Research Center of Nanjing University were housed in a specific pathogen-free animal room. During this period, the mice had free access to food and water. The animal room maintained a 12-hour dark / light cycle, with temperature and humidity maintained at 23±2°C and 50%±5%, respectively. All procedures and protocols in this experiment were approved by the Animal Ethics Committee of Jiangnan University.

[0053] After two weeks of acclimatization, mice were randomly divided into four groups: a control group, an HSYA treatment group (administered via gavage, 50 mg / kg / day), an exercise group, and an exercise-HSYA treatment group (administered via gavage, 50 mg / kg / day). The control and exercise groups were administered physiological saline via gavage for three weeks at a dose of 10 μL per mouse body weight (g). HSYA was dissolved in physiological saline to prepare a 5 mg / mL HSYA solution, which was administered via gavage to the HSYA treatment and exercise-HSYA treatment groups for three weeks at a dose of 10 μL per mouse body weight (g). After three weeks of gavage, the exercise and exercise-HSYA treatment groups were placed on a treadmill and a fatigue rotundus system for acclimatization training for three consecutive days. Three days later, the same instruments were used to perform running and fatigue rotundus tests on the mice in the exercise and exercise-HSYA treatment groups. Finally, all mice were euthanized with CO2, and serum, gastrocnemius, tibialis anterior, and soleus muscles were collected. A portion of the muscle was immersed in 4% paraformaldehyde for later histological morphology analysis. The serum and the remaining tissues were immediately frozen in liquid nitrogen and then stored at -80°C.

[0054] Mouse skeletal muscle sections were stained with Oil Red O to further observe the effect of HSYA on the fat content of muscle tissue. Figure 4As shown, the fat content in the soleus, gastrocnemius, and tibialis anterior muscles of mice in the HSYA and exercise groups was significantly lower than that in the blank control group. Furthermore, compared with the blank control group, both exercise and HSYA significantly reduced the content of free fatty acids in mouse plasma. These results collectively indicate that HSYA can promote fat burning in the skeletal muscle of mice during both resting and exercise.

[0055] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. An extraction process for highly stable hydroxysaffron yellow pigment A, characterized in that, Includes the following steps: (1) Raw material pretreatment: Take dried safflower filaments, remove impurities and crush them, then pass them through a 40-80 mesh sieve to obtain safflower powder; (2) Preparation of combined fermentation culture: The activated Lactobacillus plantarum was inoculated into MRS medium and cultured statically at 30-37℃ for 12-18 h to obtain Lactobacillus plantarum seed culture; the activated Saccharomyces cerevisiae was inoculated into YPD medium and cultured with shaking at 28-32℃ for 10-14 h to obtain yeast seed culture; The seed culture of Lactobacillus plantarum and the seed culture of Saccharomyces cerevisiae were mixed to obtain a co-fermentation culture. (3) Fermentation pretreatment: The safflower powder pretreated in step (1) is mixed with deionized water and fermented with co-fermentation bacteria. (4) Hot water extraction: The fermented material obtained in step (3) is transferred to an extraction tank and extracted under hot water bath conditions; (5) Solid-liquid separation: After extraction, the extract is cooled to room temperature, centrifuged, and the supernatant is collected. The precipitate is washed with deionized water 2-3 times, and the washing liquid is combined with the supernatant to obtain HYSA extract.

2. The extraction process according to claim 1, characterized in that, In step (2), the volume ratio of the Lactobacillus plantarum seed liquid and the Saccharomyces cerevisiae seed liquid is 1:1~3, and the viable count of both Lactobacillus plantarum and Saccharomyces cerevisiae in the co-fermentation liquid is not less than 1×10⁻⁶. 8 CFU / mL.

3. The extraction process according to claim 1, characterized in that, In step (3), the mass-to-volume ratio of safflower powder to deionized water is 1g:(15~25)mL.

4. The extraction process according to claim 1, characterized in that, In step (3), the inoculation amount of the combined fermentation liquid is 3-8% of the total mass of the mixture of safflower powder and deionized water; before fermentation, the initial pH value of the system needs to be adjusted to 5.0-6.0, the fermentation temperature is 28-32℃, and the fermentation time is 8-12h.

5. The extraction process according to claim 1, characterized in that, In step (4), the temperature of the hot water bath is 60℃ and the extraction time is 0.5~2h.

6. The extraction process according to claim 1, characterized in that, In step (4), ultrasonic extraction can also be performed during hot water bath extraction. The ultrasonic power is 100~200W, and ultrasonic extraction is performed under constant temperature of 60℃ for 1 hour.

7. Use of hydroxysaffron yellow A or a composition containing hydroxysaffron yellow A in the preparation of functional foods, medicines, health products or exercise simulators for promoting fat burning metabolism in the body at rest and during exercise.

8. The use according to claim 7, characterized in that, The promotion of fat-burning metabolism in the body at rest and during exercise includes reducing the fat content in the soleus, gastrocnemius, and tibialis anterior muscles, as well as the content of free fatty acids in the blood plasma, in both resting and exercise states.

9. The use according to claim 7, characterized in that, The functional foods include: functional beverages, functional powders, functional effervescent tablets, functional biscuits, functional capsules, etc.

10. The use according to claim 7, characterized in that, The composition further includes pharmaceutically acceptable excipients, including solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and release inhibitors.

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