Preparation method and application of yellow wine and wine roasted female and male swelling hippocampus composition

Abstract

The invention discloses a preparation method and application of a composition of male and female swelling hippocampus japonicus roasted with yellow rice wine, and the preparation method comprises the following steps: (1) mixing female swelling hippocampus japonicus and male swelling hippocampus japonicus according to the mass ratio of (0.8-1.2): (0.8-1.2), and the female swelling hippocampus japonicus and the male swelling hippocampus japonicus are both dry swelling hippocampus japonicus products; (2) preheating the material obtained in the step (1), moistening for 50-60 minutes by using yellow wine of which the weight is 14-16% of the dry weight of the material, and baking for 1.8-2.2 hours at 85-90 DEG C; and (3) crushing the material obtained in the step (2) into powder, so as to obtain the male and female distension hippocampus composition with the total protein content of more than or equal to 50%. Through scientific raw material selection, proportion optimization and a processing technology, the defects of insufficient reservation of nutritional ingredients, incomplete safety evaluation and unstable technology in a traditional technology are overcome, the nutritional value, safety and production efficiency of the hippocampus japonicus composition are remarkably improved, and the hippocampus japonicus composition is suitable for industrial production in the field of health-care foods and has wide market prospects. And the market prospect is wide.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine raw materials, and particularly relates to a preparation method and application of a yellow rice wine processed Hippocampus kelloggi and Hippocampus histrix composition. BACKGROUND

[0002] Hippocampus is a precious marine medicinal material in Asia, especially in China, and has a long history of medicinal use. As early as in the Shennong Bencao Jingjiuzhu, there is a record about Hippocampus. It is warm in nature and sweet in taste, and belongs to the liver and kidney channels, and has the effects of tonifying the kidney and strengthening yang, warming blood vessels, and relaxing the tendons and activating the collaterals. Modern studies have shown that Hippocampus is rich in various bioactive components, such as amino acids, polypeptides, fatty acids, etc. These components endow Hippocampus with various pharmacological effects such as antioxidant, anti-inflammatory, anti-fatigue and anti-tumor effects. Among them, amino acids play a crucial role in regulating metabolism, growth and development, EPA and DHA fatty acids help reduce the risk of cardiovascular disease, and active polypeptides can exert anti-hypertensive, anti-inflammatory, antioxidant and immunomodulatory functions.

[0003] The Pharmacopoeia of the People's Republic of China (2020 edition) has included five species of Hippocampus, including Hippocampus kelloggi, Hippocampus histrix, Hippocampus kuda, Hippocampus trimaculatus and Hippocampus japonicus. These species are widely used in traditional medicine for the treatment of impotence, sprains and strains, carbuncles, boils and abscesses. However, as one of the most traded marine animals in the world, Hippocampus is facing a continuous decline in wild resources due to overfishing and habitat destruction. All Hippocampus species have been listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), and international trade is strictly regulated.

[0004] In order to alleviate the problem of lack of wild resources, China has attempted to artificially breed Hippocampus since the 1950s. However, the artificial breeding of the five species of Hippocampus included in the Pharmacopoeia has not yet been achieved, and is facing difficulties such as diseases, pests and low reproductive capacity, leading to an imbalance between supply and demand of Hippocampus, and fluctuations in market prices.

[0005] Among the 15 species of medicinal seahorses in the world, Hippocampus abdominalis is attracting attention due to its unique advantages in cultivation. H. abdominalis is originally from the coastal waters near Australia and New Zealand, and has also been used in traditional medicine in places such as Korea. In 2017, Xia'men Xiaoliang Aquatic Technology Co., Ltd. and Xiamen En Ci Health Co., Ltd. successfully introduced H. abdominalis and, through technological innovation, overcame key problems in artificial cultivation, such as disease and pest control, and achieved large-scale artificial breeding in places such as Fujian and Shandong. Studies have shown that H. abdominalis has high similarity to the seahorse species included in the pharmacopoeia in terms of chemical composition and pharmacological activity, including antioxidant, anti-inflammatory and anti-fatigue properties, and is therefore considered an ideal substitute for the pharmacopoeia seahorse.

[0006] Although the cultivation of H. abdominalis has been successful, the industrial development of H. abdominalis still faces challenges. Traditional preparation processes mostly use single gender or unoptimized ratio, resulting in insufficient content of key amino acids such as isoleucine and methionine in the product, which cannot fully exert the nutritional value and medicinal potential of H. abdominalis. Amino acids are the basic units that make up proteins, and many amino acids in H. abdominalis play a key role in maintaining normal physiological functions, promoting metabolism, and enhancing immunity in the human body. In particular, essential amino acids cannot be synthesized by the human body and must be obtained from food. Therefore, optimizing the preparation process to improve the content and quality of amino acids in the product is of great significance to enhance the market competitiveness of H. abdominalis products. SUMMARY

[0007] The present application aims to overcome the defects of the prior art and provide a preparation method of a yellow rice wine processed H. abdominalis composition.

[0008] Another object of the present application is to provide the application of the H. abdominalis composition prepared by the above preparation method.

[0009] The preparation method of the H. abdominalis composition.

[0010] The technical solution of the present application is as follows:

[0011] A preparation method of a yellow rice wine processed H. abdominalis composition, comprising the following steps:

[0012] (1) mixing female H. abdominalis and male H. abdominalis at a mass ratio of 0.8-1.2:0.8-1.2, wherein the female H. abdominalis and the male H. abdominalis are both H. abdominalis dried products;

[0013] (2) preheating the material obtained in step (1), then moistening it with 14-16% of the dry weight of yellow rice for 50-60 min, and then baking it at 85-90℃ for 1.8-2.2 h;

[0014] (3) crushing the material obtained in step (2) into powder, to obtain the female-male H. paeoniocircus composition with total protein content ≥ 50%.

[0015] In a preferred embodiment of the present application, in step (1), the mass ratio of the female H. paeoniocircus and the male H. paeoniocircus is 1:1.

[0016] In a preferred embodiment of the present application, step (2) is: moistening 15% of the dry weight of the material obtained in step (1) with yellow rice for 52 min, and then baking at 87℃ for 2 h.

[0017] In a preferred embodiment of the present application, the size of the powder in step (3) is ≤ 2000±70 μm.

[0018] In a preferred embodiment of the present application, in step (1), the mass ratio of the female H. paeoniocircus and the male H. paeoniocircus is 1:1; in step (2), 15% of the dry weight of the material obtained in step (1) is moistened with yellow rice for 52 min, and then baked at 87℃ for 2 h; and in step (3), the size of the powder is ≤ 2000±70 μm.

[0019] In a preferred embodiment of the present application, the yellow rice is refreshing type secondary yellow rice (according to GB / T13662).

[0020] The use of the female-male H. paeoniocircus composition prepared by the above preparation method in preparing an oral health care composition.

[0021] An oral health care composition, the effective component of which comprises the female-male H. paeoniocircus composition prepared by the above preparation method.

[0022] The present application has the following advantages:

[0023] 1. The present application selects H. paeoniocircus of 8-12 months old (dry weight 2-3 g), to ensure the highest content of core nutritional components such as dipeptides, carboxylic acids and amino acids. This selection is based on advanced chemical isotope labeling and liquid chromatography-mass spectrometry analysis technology (Rapid LC-MS Analysis for DeepMarker MT), which determines 8-12 months old as the best raw material by analyzing the chemical composition differences of H. paeoniocircus of different ages. Compared with the traditional process which lacks scientific basis and randomly selects raw materials, the present application significantly improves the nutritional quality of the raw material, laying a solid foundation for subsequent processing.

[0024] 2、The application adopts scientifically determined 1:1 male and female sea horse mass ratio, and optimizes the complementary effect of essential amino acids through an amino acid nutrition evaluation model (essential amino acid index EAAI and amino acid proportion coefficient score SRCAA). The experimental results show that the EAAI of the 1:1 ratio is 2.671, and the SRCAA is 83.56, which is significantly higher than that of other ratios (such as 2.356 and 78.23 of 1:0, and 2.389 and 79.01 of 0:1). Compared with the insufficient content of key amino acids (such as isoleucine and methionine) caused by single gender or non-optimized ratio in the traditional process, the application realizes higher nutritional balance, and meets the market demand for high-quality protein.

[0025] 3、The application optimizes the wine steaming process through the response surface method (Box-Behnken design), and determines the optimal processing conditions: 15% yellow wine addition amount, 52 min soaking time and 87℃ baking temperature. Under the conditions, the amino acid retention rate reaches 57.92g / 100g, and the relative standard deviation (RSD) is only 1.47%. Compared with the traditional process which lacks systematic optimization, the amino acid retention rate is low, and is easily affected by factors such as alcohol content, soaking time and baking temperature. The optimized process of the application significantly improves the retention rate of amino acids, and ensures the maximization of the nutritional efficacy of the product.

[0026] 4、The application confirms the edible safety of the product through comprehensive toxicology tests. The acute oral toxicity test shows that LD50≥10g / kg, which belongs to the actual non-toxic level. In addition, the results of three genetic toxicity tests (bacterial reverse mutation test, mammalian erythrocyte micronucleus test, and male mouse spermatocyte chromosome aberration test) are all negative, indicating that the product has no mutagenic potential. Compared with the defects of the traditional process which lacks comprehensive safety evaluation, the application significantly improves the market applicability of the product.

[0027] 5、The final product prepared by the application has a particle size of ≤2000±70μm, and a total protein content of ≥50%, and can be used as a source of high-quality nutrients and bioactive substances. The excellent nutritional properties and safety of the product lay a foundation for the industrial application of the sea horse. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The total ion flow chart of different month-old female sea horses in Example 1 of the application is shown in the figure. Among them: black is PC1 group, red is PC 2 group, green is PC 3 group, blue is PC 4 group, and purple is PC 5 group.

[0029] Figure 2 The total ion flow chart of different month-old male sea horses in Example 1 of the application is shown in the figure. Among them: black is PX1 group, red is PX 2 group, green is PX 3 group, blue is PX 4 group, and purple is PX 5 group.

[0030] Figure 3 Compound class proportion heat map of different age female P. v. in Example 1 of the present application. Among them: PC 1: female P. v. 0.5-1 g (3-5 months old), PC 2: female P. v. 1-2 g (5-8 months old), PC 3: female P. v. 2-3 g (8-12 months old), PC 4: female P. v. 3-4 g (12-16 months old), PC 5: female P. v. 4-5 g (16-20 months old).

[0031] Figure 4 Compound class proportion heat map of different age male P. v. in Example 1 of the present application. Among them: PX 1: male P. v. 0.5-1 g (3-5 months old), PX 2: male P. v. 1-2 g (5-8 months old), PX 3: male P. v. 2-3 g (8-12 months old), PX 4: male P. v. 3-4 g (12-16 months old), PX 5: male P. v. 4-5 g (16-20 months old).

[0032] Figure 5 Compound class proportion heat map of different age female and male P. v. in Example 1 of the present application. Among them: PC 1: female P. v. 0.5-1 g (3-5 months old), PC 2: female P. v. 1-2 g (5-8 months old), PC 3: female P. v. 2-3 g (8-12 months old), PC 4: female P. v. 3-4 g (12-16 months old), PC 5: female P. v. 4-5 g (16-20 months old), PX 1: male P. v. 0.5-1 g (3-5 months old), PX 2: male P. v. 1-2 g (5-8 months old), PX 3: male P. v. 2-3 g (8-12 months old), PX 4: male P. v. 3-4 g (12-16 months old), PX 5: male P. v. 4-5 g (16-20 months old).

[0033] Figure 6Comparison of chemical components between different months of female and male P. v. in Example 1 of the present application. Among them: PC1: female P. v. 0.5-1 g (3-5 months old), PC 2: female P. v. 1-2 g (5-8 months old), PC 3: female P. v. 2-3 g (8-12 months old), PC 4: female P. v. 3-4 g (12-16 months old), PC 5: female P. v. 4-5 g (16-20 months old), PX 1: male P. v. 0.5-1 g (3-5 months old), PX 2: male P. v. 1-2 g (5-8 months old), PX 3: male P. v. 2-3 g (8-12 months old), PX 4: male P. v. 3-4 g (12-16 months old), PX 5: male P. v. 4-5 g (16-20 months old).

[0034] Figure 7 Comparison of chemical components between different months of female P. v. in Example 1 of the present application. Among them: PC1: female P. v. 0.5-1 g (3-5 months old), PC 2: female P. v. 1-2 g (5-8 months old), PC 3: female P. v. 2-3 g (8-12 months old), PC 4: female P. v. 3-4 g (12-16 months old), PC 5: female P. v. 4-5 g (16-20 months old).

[0035] Figure 8 Comparison of chemical components between different months of male P. v. in Example 1 of the present application. Among them: PX1: male P. v. 0.5-1 g (3-5 months old), PX 2: male P. v. 1-2 g (5-8 months old), PX 3: male P. v. 2-3 g (8-12 months old), PX 4: male P. v. 3-4 g (12-16 months old), PX 5: male P. v. 4-5 g (16-20 months old).

[0036] Figure 9 Principal component analysis of female P. v. of different months in Example 1 of the present application. Among them: PC 1: female P. v. 0.5-1 g (3-5 months old), PC 2: female P. v. 1-2 g (5-8 months old), PC 3: female P. v. 2-3 g (8-12 months old), PC 4: female P. v. 3-4 g (12-16 months old), PC 5: female P. v. 4-5 g (16-20 months old).

[0037] Figure 10Principal component analysis of different age male P. v. in Example 1 of the present application is shown. Among them: PX 1: male P. v. 0.5-1 g (3-5 months old), PX 2: male P. v. 1-2 g (5-8 months old), PX 3: male P. v. 2-3 g (8-12 months old), PX 4: male P. v. 3-4 g (12-16 months old), PX 5: male P. v. 4-5 g (16-20 months old).

[0038] Figure 11 OPLS-DA analysis of different age female P. v. in Example 1 of the present application is shown. Among them: PC 1: female P. v. 0.5-1 g (3-5 months old), PC 2: female P. v. 1-2 g (5-8 months old), PC 3: female P. v. 2-3 g (8-12 months old), PC 4: female P. v. 3-4 g (12-16 months old), PC 5: female P. v. 4-5 g (16-20 months old).

[0039] Figure 12 OPLS-DA analysis of different age male P. v. in Example 1 of the present application is shown. Among them: PX 1: male P. v. 0.5-1 g (3-5 months old), PX 2: male P. v. 1-2 g (5-8 months old), PX 3: male P. v. 2-3 g (8-12 months old), PX 4: male P. v. 3-4 g (12-16 months old), PX 5: male P. v. 4-5 g (16-20 months old).

[0040] Figure 13 Permutation test of different age female P. v. in Example 1 of the present application is shown.

[0041] Figure 14 Permutation test of different age male P. v. in Example 1 of the present application is shown.

[0042] Figure 15 Volcano plot analysis of different age female P. v. in Example 1 of the present application is shown. Among them: PC 1: female P. v. 0.5-1 g (3-5 months old), PC 2: female P. v. 1-2 g (5-8 months old), PC 3: female P. v. 2-3 g (8-12 months old), PC 4: female P. v. 3-4 g (12-16 months old), PC 5: female P. v. 4-5 g (16-20 months old).

[0043] Figure 16The volcano plot analysis of different age male Hippocampus kuda in Example 1 of the present application is shown. Among them: PX 1: male Hippocampus kuda 0.5-1 g (3-5 months old), PX 2: male Hippocampus kuda 1-2 g (5-8 months old), PX 3: male Hippocampus kuda 2-3 g (8-12 months old), PX 4: male Hippocampus kuda 3-4 g (12-16 months old), PX 5: male Hippocampus kuda 4-5 g (16-20 months old).

[0044] Figure 17 The high performance liquid chromatogram for amino acid determination of Hippocampus in Example 2 of the present application is shown. Among them: A: blank solution derivatization chromatogram, B: 17 kinds of amino acid control product derivatization chromatogram, C: sample solution derivatization chromatogram, 1: Asp 2: Glu 3: Hypro 4: Ser 5: Gly 6: His 7: Arg 8: Thr 9: Ala 10: Pro 11: Tyr 12: Val 13: Met 14: Ile 15: Leu 16: Phe 17: Lys.

[0045] Figure 18 The effect of wine addition amount on amino acid content in Example 3 of the present application is shown.

[0046] Figure 19 The effect of moistening time on Hippocampus amino acid content in Example 3 of the present application is shown.

[0047] Figure 20 The effect of baking temperature on amino acid content in Example 3 of the present application is shown.

[0048] Figure 21 The 3D graph and contour graph of the effect of the interaction between wine addition amount (A) and moistening time (B) on amino acid content in Example 3 of the present application is shown.

[0049] Figure 22 The 3D graph and contour graph of the effect of the interaction between wine addition amount (A) and baking temperature (C) on amino acid content in Example 3 of the present application is shown.

[0050] Figure 23 The 3D graph and contour graph of the effect of the interaction between moistening time (B) and baking temperature (C) on amino acid content in Example 3 of the present application is shown.

[0051] Figure 24 The finally determined process flow chart in Example 3 of the present application is shown. DETAILED DESCRIPTION

[0052] The technical solutions of the present application are further described and explained by specific embodiments in combination with the accompanying drawings.

[0053] Example 1

[0054] In order to screen the best quality of the inflated hippocampus, the present embodiment adopts high-efficiency chemical isotope labeling combined with liquid chromatography-mass spectrometry (Rapid LC-MS Analysis for Deep Marker MT) to systematically analyze the female and male inflated hippocampus samples at different growth stages, to explore the influence of different months of age on the chemical composition of female and male inflated hippocampus individuals, so as to provide a scientific basis for selecting the best quality of medicinal inflated hippocampus in production.

[0055] Firstly, the experimental method adopted in the present embodiment is as follows:

[0056] (1) Sample preparation and extraction

[0057] Precisely weigh the hippocampus powder through a No. 5 sieve, soak it in 50% ethanol with a solid-liquid ratio of 1:25 for 1 hour, then connect the reflux in a condenser tube, heat to boiling, and keep it slightly boiling for 1 hour. After cooling, remove the conical flask, tightly seal it, shake it well, filter it with a dry filter, and transfer the filtrate to a dry evaporation dish. After evaporating to dryness on a water bath, place it in a 55°C oven to dry to constant weight. Remove the sample and store it in a sealed bag at -20°C for future use.

[0058] Transfer the sample with known weight to a grinding centrifuge tube. Add methanol / water mixed solution (1:1, v / v) to each sample according to the sample weight at a ratio of 400 μL / 10 mg. Use a bead mill tissue grinder to grind the sample for 20 s. After grinding, vortex the mixture and centrifuge the sample at high speed (12000 rpm, 10 min) at 4°C. Transfer the supernatant to a new centrifuge tube for sample labeling.

[0059] According to the analysis channel, divide the supernatant obtained in the previous step. Each sample is divided into 6 parts for four-channel analysis (40 μL / channel), backup sample, and mixed sample preparation. For mixed sample preparation, take 230 μL of supernatant from each sample and combine them into one sample. After careful vortex mixing, form a mixed sample, and after labeling, use it as a reference sample.

[0060] (2) Sample labeling

[0061] In this embodiment, the sample is analyzed by deep metabolomics. Four channels of amine / phenol, carboxyl, hydroxyl, and carbonyl secondary compounds need to be labeled and analyzed. These compounds basically include 95% of the compound content in the sample. Therefore, the sample is labeled using the following method.

[0062] Labeling of amine / phenolic secondary compounds: For aliquots of samples intended for amine / phenolic secondary compound analysis, reconstitute the samples with 25 μL of mass spectrometry-grade water and label the samples according to the kit instructions. First, add 12.5 μL of buffer reagent A and 37.5 μL of [unspecified reagent] to the sample. 12 C-labeled reagent B.12 (for labeling individual and pooled samples) or 13 C-labeled reagent B.13 (for mixed sample labeling only) was vortexed and incubated at 40°C for 45 min. After incubation, 7.5 μL of reagent C was added to quench excess labeling reagent, and the mixture was incubated at 40°C for 10 min. Finally, 30 μL of pH adjustment reagent D was added.

[0063] Labeling of carboxyl secondary compounds: For aliquots of samples intended for carboxyl secondary compound analysis, reconstitute the sample with 25 μL of mass spectrometry-grade acetonitrile / water (3:1, v / v) and label the samples according to the kit instructions. First, add 10 μL of reaction catalyst A and 25 μL of... 12 C-labeled reagent B.12 or 13 Reagent C was labeled with reagent B.13 and vortexed to mix thoroughly before incubation at 80°C for 60 min. After incubation, 40 μL of reagent C was added to quench excess labeling reagent, and the mixture was incubated at 80°C for 30 min.

[0064] Labeling of hydroxyl secondary compounds: For aliquots of samples intended for hydroxyl secondary compound analysis, reconstitute the sample with 25 μL of mass spectrometry-grade acetonitrile / water (3:1, v / v) and label the samples according to the kit instructions. First, add 25 μL of reaction activator A and 40 μL of [unspecified reagent / concentrate] to the sample. 12 C-labeled reagent B.12 or 13 Reagent C was labeled with reagent B.13 and vortexed to mix thoroughly before incubation at 60°C for 60 min. After incubation, 5 μL of reagent C was added to quench excess labeling reagent, and the mixture was incubated at 60°C for 10 min. Finally, 25 μL of pH adjustment reagent D was added.

[0065] Labeling of carbonyl secondary compounds: For aliquots of samples intended for carbonyl secondary compound analysis, add 25 μL of mass spectrometry-grade water to reconstitute the sample and label the samples according to the kit instructions. First, add 25 μL of pH adjustment reagent A and 25 μL of [reagent name missing] to the sample. 12 C-labeled reagent B.12 or 13C-labeled reagent B.13, after vortex mixing, was incubated at 40°C for 60 min. After incubation, the sample was transferred to -80°C for 10 min to terminate the reaction. After the sample was dried by nitrogen blowing, 100 μL of acetonitrile / water mixed solution (50:50, v / v) was added for reconstitution.

[0066] Sample mixing: the labeled amine / phenol secondary compounds were quantified by LC-UV according to the standard operation procedure. According to the quantitative results, equal amounts of 13 C-labeled mixed samples were added to 12 C-labeled single samples for liquid chromatography-mass spectrometry analysis. Before liquid chromatography-mass spectrometry analysis, quality control samples were prepared at the same time, i.e., equal volumes of 13 C-labeled mixed samples and 12 C-labeled mixed samples were mixed thoroughly and used as quality control samples. After all sample preparation was completed, liquid chromatography-mass spectrometry analysis was performed.

[0067] (3) Liquid chromatography-mass spectrometry analysis conditions: liquid chromatography-mass spectrometry analysis was performed according to the specific analysis conditions (see Table 3), and quality control samples and retention time calibration samples were analyzed once every 10 samples to monitor the stability of the instrument.

[0068] Table 3

[0069]

[0070] (4) Analysis method: in this example, according to the month as the division basis, the female and male H. imponcatus samples from Xiamen Xiaoliang Aquatic Technology Co., Ltd. were divided into 5 groups (see Table 4), which were PC 1 to PC 5 (female samples) and PX 1 to PX 5 (male samples). Excel was used to complete data processing and preliminary statistical analysis to ensure data integrity and consistency; then simca 18.0 software was used for multivariate statistical analysis of the data, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), aiming to reflect the differences between groups. The visualization part was completed by GraphPad Prism 9.5 software, and the specified parameters were set to draw volcano plots, heat maps, etc., to present the distribution characteristics of chemical components in a more intuitive way; the production of Venn diagrams was carried out by using the microbio website.

[0071] Table 4 Grouping of female and male H. imponcatus samples

[0072]

[0073] Second, the results and analysis of this example

[0074] (1) Analysis of total ion chromatograms of female and male H. imponcatus of different months:

[0075] The samples of the Hippocampus kuda were separated and identified by four channels (amine / phenol, carboxyl, hydroxyl, and carbonyl) using Rapid LC-MS Analysis for DeepMarker MT technology, and the total ion chromatograms of the female and male Hippocampus kuda of different months were obtained (as shown in FIGS. 1 and 2). Figure 1 and Figure 2 It can be seen that the number of chromatographic peaks of the PC and PX sample solutions of different months is basically consistent, and the overlapping is good.

[0076] (2) Metabolite identification results of female and male Hippocampus kuda of different months:

[0077] Among the metabolite peak detection results, 6096 characteristic peaks were obtained, of which as high as 93.9% (5722 peaks) could be accurately identified or reasonably inferred. Specifically, 538 metabolites were successfully identified by comparing with the derivatization standard database (CIL Library); 87 metabolites were identified by using the associated metabolite database (LI Library). For the remaining unidentifiable metabolites, this embodiment matched and analyzed by means of the MyCompoundID (MCID) database, and according to the identification level division, 1147, 2566 and 598 metabolites were matched in the 0th, 1st and 2nd databases, respectively.

[0078] (3) Difference analysis of chemical components of female and male Hippocampus kuda of different months

[0079] A, descriptive analysis of female and male Hippocampus kuda of different months:

[0080] As shown in Table 5, the female Hippocampus kuda sample contained 37 types of compounds, of which dipeptides (26.23% ± 0.35%) and carboxylic acids (14.54% ± 1.13%) accounted for a large proportion, and the data distribution was relatively concentrated. The average content of amino acids (10.13% ± 2.38%) and their derivatives (8.37% ± 2.75%) was relatively high, but the data dispersion degree was relatively large (the standard deviations were 2.38% and 2.75%, respectively). It is indicated that amino acids and their derivatives may be more susceptible to the influence of month factors.

[0081] Table 5 Descriptive analysis of female Hippocampus kuda of different months

[0082]

[0083]

[0084] As shown in Table 6, among the 37 compounds contained in male bloated seahorse samples, dipeptides (mean 23.30% ± 1.45%), amino acids (13.58% ± 0.30%), and carboxylic acids (13.39% ± 0.68%) ranked in the top three in terms of average content. Compared with female bloated seahorses, these three compounds were less affected by age.

[0085] Table 6. Descriptive analysis of male bloated seahorses of different ages.

[0086]

[0087]

[0088] B. Compositional analysis of male and female bloated seahorses of different ages:

[0089] The proportions of 37 compounds in samples from female bloated hippocampuses were analyzed across five different age gradients (3-5 months to 16-20 months). Results are as follows: Figure 3 As shown, dipeptides (25.90%–26.64%), carboxylic acids (13.28%–16.05%), and amino acids (8.12%–13.65%) are the top three compound categories, accounting for over 55% combined, and these three categories can be classified as the core component group. Among them, amino acids show a significant increasing trend (an increase of 68.1%), while the proportion of amino acid derivatives decreased from 11.34% to 4.73% (a decrease of 58.3%). These data suggest that these compounds may exhibit periodic activity, with higher proportions in specific age groups.

[0090] The proportions of 37 compounds in male bloated hippocampal samples were analyzed across five different age gradients (3-5 months to 16-20 months). Results are as follows: Figure 4 As shown, dipeptides (ranging from 21.61% to 24.90%), carboxylic acids (12.85% to 14.38%), and amino acids (13.11% to 13.96%) consistently ranked as the top three components across all age groups, accounting for approximately 50% to 55% of the total composition. Similar to the female bloated seahorse samples, these three compounds were classified as core components. Specifically, dipeptide content peaked at 24.90% in the 8-12 month age group, subsequently showing a continuous downward trend (a decrease of 12.4%), while the proportion of carboxylic acids showed a slight upward trend during this period (an increase of 10.3%). Comparative analysis with female bloated seahorse samples revealed that male bloated seahorse samples had a higher proportion of amino acids with smaller fluctuations (the increase was 68.1% in female samples and only 6.5% in male samples), suggesting potential differences in metabolic stability between individuals of different sexes.

[0091] To further analyze the information presented by the heat map, this embodiment will further use bar charts and line charts to visualize the top 10 and top 3 compound categories in female and male P. volitans (see Figure 5 ). The main purpose of the analysis is to better reflect the proportion characteristics of the compounds in female and male P. volitans during different growth months, and to provide visual support for subsequent determination of the best quality of female and male P. volitans.

[0092] As shown in Figure 5 , it is found through visual analysis that the proportion of dipeptides, carboxylic acids and amino acid derivatives in the PC sample group is significantly higher than that in the PX sample group. Among them, dipeptides and carboxylic acids reach the peak in PC 3 group, and amino acid derivatives show a higher proportion in PC 1 group and PC 3 group. The advantage of amino acids and fatty acids in the PC group is not obvious. The values of amine, aldehyde, steroid, amide and nucleoside in the two types of samples are relatively low, among which the values of aldehyde and steroid in PX group are slightly lower than those in PC group. In PX sample group, dipeptide compounds reach the peak in PX 3 group, and carboxylic acid and amino acid and their derivatives have a high proportion in PX 4 and PX 5 groups, but the content of dipeptide is low. It shows that different compounds exist dynamic changes in different months and genders.

[0093] C. Comparative analysis of components of female and male P. volitans at different months

[0094] Through analysis and identification, the number of components in different month intervals of female P. volitans is: PC 1 contains 1367 kinds, PC 2 contains 1333 kinds, PC 3 contains 1329 kinds, PC 4 contains 1296 kinds, and PC 5 contains 1332 kinds; while the number of components in different month intervals of male P. volitans is: PX 1 contains 1281 kinds, PX 2 contains 1214 kinds, PX 3 contains 1289 kinds, PX 4 contains 1217 kinds, and PX 5 contains 1189 kinds.

[0095] In different growth stages (3-5 to 16-20 months), the number of chemical components detected in female P. volitans (1296-1367) is more than that in male P. volitans (1189-1289). Among them, the number of components in female P. volitans reaches the peak (1367) in the 3-5 month growth stage, which is 6.7% higher than that of male P. volitans (1281) in the same stage. The number of components in male individuals increases to the peak (1289) in the 8-12 month growth stage. With the increase of growth stage, the number of components of female and male P. volitans shows a downward trend, but the decrease of male in the 16-20 month growth stage is more obvious (1189).

[0096] The common components and compound types of female and male P. volitans are analyzed (as shown in Figure 6The proportion of common components showed a decreasing trend with the increase of growth stage (3-5 months: 90.4%→ 16-20 months: 84.0%), indicating that the similarity in compound composition between the two decreased; while the number of unique components increased significantly (female: 109 species in 3-5 months→ 181 species in 16-20 months; male: 24 species in 3-5 months→ 38 species in 16-20 months), which reflected that with growth, the pufferfish gradually developed its own unique characteristics in compound synthesis or metabolism, which may be related to individual development, growth needs, and gender-specific physiological functions, etc.

[0097] In the growth stage of 3-5 months, the unique components of female pufferfish were mainly tripeptides (0.22%), while those of male pufferfish were concentrated in phenylpropanoids (0.31%) and amino acid-like compounds (0.16%). This indicates that there may be differences in the physiological metabolism of the two in the early growth stage.

[0098] D. Analysis of the differences in unique chemical components of female and male pufferfish at different ages

[0099] The data were analyzed by Venn diagram analysis of unique components (see Figure 7 、 8 ), and there were 1170 common components in the 5 groups of female pufferfish and 1069 in the male pufferfish. The distribution characteristics of unique components in female pufferfish at different ages are shown in Table 7. The number of unique components in female pufferfish in the rapid growth period of 3-5 months reached a peak (5 species), including phenols, nucleosides, amines, and dipeptides. With the increase of age, the number of unique components in the intervals of 5-8 months, 8-12 months, and 12-16 months decreased to 1, 1, and 2, respectively, and there were no unique components in the 16-20 month interval. This phenomenon may be related to the attenuation of key synthesis pathways in the sexual maturation stage and the maturation of metabolic homeostatic mechanisms.

[0100] Table 7 Unique components of different age groups of female pufferfish

[0101]

[0102] Note: PC 1: 0.5-1 g of female pufferfish (3-5 months old), PC 2: 1-2 g of female pufferfish (5-8 months old), PC 3: 2-3 g of female pufferfish (8-12 months old), PC 4: 3-4 g of female pufferfish (12-16 months old), PC 5: 4-5 g of female pufferfish (16-20 months old).

[0103] The male pufferfish showed a relatively rich composition of compounds at different ages (see Figure 8and Table 8), including dipeptides, amides, nucleosides, fatty acids, steroids, and other various categories. Among them, male pufferfish in March to May contained 10 unique components, which decreased to 4 in May to August, but reached a peak in August to December, containing 19 kinds of compounds and a relatively rich compound category, including 8 kinds of compound categories, and the unique compounds tended to be stable in December to February and February to April, containing 5 and 7 kinds, respectively. It can be seen that the number of unique components changes periodically with the age stage.

[0104] Table 8 Unique components of each group in male pufferfish of different months

[0105]

[0106]

[0107] Note: PX 1: male pufferfish 0.5-1 g (3-5 months old), PX 2: male pufferfish 1-2 g (5-8 months old), PX 3: male pufferfish 2-3 g (8-12 months old), PX 4: male pufferfish 3-4 g (12-16 months old), PX 5: male pufferfish 4-5 g (16-20 months old).

[0108] (4) Difference analysis of metabolomics of female and male pufferfish of different months

[0109] Principal component analysis (PCA) is a multivariate statistical analysis method that selects a few important variables through linear transformation of multiple variables. By classifying the characteristic variables of metabolites after dimensionality reduction according to the similarity of principal components, the metabolic differences between samples and the difference size between samples in the same group can be reflected overall. The PC 1-PC 5 and PX 1-PX 5 data were standardized and normalized before PCA analysis (see Figure 9 、 10 ).

[0110] The samples of female and male pufferfish groups were strictly distributed in the 95% confidence ellipse interval (see Figure 9 、 10 ), and the 5-month-old groups were significantly separated. In the female group, the principal component distribution of the samples in PC 1-PC 5 showed high aggregation, reflecting good consistency of the metabolic characteristics within the group; the distance between the samples in PC 2 and PC 5 was relatively close, indicating that the metabolic profile difference was relatively small. The male group also showed similar phenomena, and the principal component distribution of the samples in PX 1-PX 5 also showed high aggregation, and the distance between the samples in PX 1, PX 2 and PX 5 was relatively close, indicating that the difference between the three groups was small. Since PCA is an unsupervised discriminant analysis statistical method, OPLS-DA, a supervised classification model, will be continued.

[0111] OPLS-DA is a supervised technique based on partial least squares method, which can be used to classify 2 or more groups of data under multiple commonalities and identify variables that have significant impact on classification. The data in 10 groups of samples were imported into simca 18.0 software, and the OPLS-DA model was automatically fitted (see Figure 11 , 12 and Table 9). The results show that the samples are all located within the 95% confidence interval, and the model cumulative explanation ability parameters R 2 X of female and male P. v. are 0.807 and 0.768, respectively; R 2 Y are 1 and 0.999, respectively, and the prediction ability parameters (Q 2 ) are 0.977 and 0.96, respectively, all of which are >0.5, indicating that the model is stable and reliable, and has good prediction ability.

[0112] The established OPLS-DA model was detected by permutation for 200 times (see Table 9), and the model Q 2 values were all less than the original model Q 2 values, and the regression line slopes of R 2 and Q 2 were larger (see Figure 13 , 14 ), the Y-axis intercept of the fitting straight line of R 2 was less than 0.5, and the Y-axis intercept of the fitting straight line of Q 2 was negative, and the R 2 and Q 2 obtained by left random arrangement were all less than the original values on the right, suggesting that the OPLS-DA model does not exist over-fitting, and can be used for further data analysis.

[0113] Table 9 OPLS-DA model parameters and permutation test

[0114]

[0115] Note: PC 1: female P. v. 0.5-1 g (3-5 months old), PC 2: female P. v. 1-2 g (5-8 months old), PC 3: female P. v. 2-3 g (8-12 months old), PC 4: female P. v. 3-4 g (12-16 months old), PC 5: female P. v. 4-5 g (16-20 months old), PX 1: male P. v. 0.5-1 g (3-5 months old), PX 2: male P. v. 1-2 g (5-8 months old), PX 3: male P. v. 2-3 g (8-12 months old), PX 4: male P. v. 3-4 g (12-16 months old), PX 5: male P. v. 4-5 g (16-20 months old).

[0116] (5) Difference metabolic analysis of female and male P. v. at different months

[0117] In this example, female and male P. v. at different months were divided into 8 groups for comparative analysis (PC 2 vs PC 1, PC 3 vs PC 2, PC 4 vs PC 3, PC 5 vs PC 4, PX 2 vs PX 1, PX 3 vs PX 2, PX 4 vs PX 3, PX 5 vs PX 4). All volcano plots represent the fold change of metabolite abundance (X-axis) as log2 Fold Change, and the statistical significance (Y-axis) as -Log 10 P-value. The higher the vertical coordinate (-log 10 P-value), the stronger the significant difference; the horizontal coordinate (log2 Fold Change) reflects the difference fold. The volcano plot was drawn by the difference fold (FC) and p value. The difference fold was calculated by the average value ratio of the analysis results between each two groups, and the p value was from Welch's t-test. The screening criteria for differential metabolites were defined as: |log2(fold change)|>0.26 (corresponding to linear fold>1.2 or <0.83) and p-value<0.05 (Welch's t-test). The results directly show the log2 Fold Change and statistical significance (-log 10 P-value) of metabolite expression, and the significantly up-regulated (red), significantly down-regulated (blue) and non-significant (gray) metabolites are distinguished by color (see Figure 15 , 16 ).

[0118] In the volcano plot analysis of female P. v. samples, 8 metabolite markers with significant and non-significant differences were screened (see Figure 15 ). Through group comparison, it was found that compared with PC 1 group, C-T2909, C-T2869 and other metabolites in PC 2 group were significantly up-regulated, and K-15, A-330 and other metabolites were significantly down-regulated; compared with PC 2 group, A-T199, A-T1273 and other metabolites in PC 3 group were significantly up-regulated, and C-T2888, C-T2858 and other metabolites were significantly down-regulated; compared with PC 3 group, C-T2888, C-T2927 and other metabolites in PC 4 group were significantly up-regulated, and A-T199, A-330 and other metabolites were significantly down-regulated; compared with PC 4 group, A-T1673, C-T2620 and other metabolites in PC 5 group were significantly up-regulated, and A-T329, C-T2910 and other metabolites were significantly down-regulated. These results show that there are significant differences in the expression of metabolites between different groups.

[0119] In the volcano plot analysis of female P. v. v. samples of different groups, it was found that some metabolites showed different changes in multiple group comparisons. For example, metabolite A-T199 was significantly up-regulated in the comparison between PC 3 and PC 2, and significantly down-regulated in the comparison between PC 4 and PC 3; metabolite C-T2888 and its opposite, were significantly down-regulated in the comparison between PC 3 and PC 2, and significantly up-regulated in the comparison between PC 4 and PC 3, etc., indicating that the significance of the same metabolite in female P. v. v. at different ages was different.

[0120] The volcano plot results of male P. v. v. samples of different ages showed (see Figure 16 ) that compared with PX 1 group, C-T2318, A-T2594 and other metabolites were significantly up-regulated in PX 2 group, and A-T1509, A-T1673 and other metabolites were significantly down-regulated; compared with PX 2 group, A-T1273, A-T1509 and other metabolites were significantly up-regulated in PX 3 group, and C-T308, A-T1306 and other metabolites were significantly down-regulated; in the comparison between PX 4 group and PX 3 group, H-1802, A-T329 and other metabolites were significantly down-regulated, and K-182, C-T1499 and other metabolites were significantly up-regulated; in the comparison between PX 5 group and PX 4 group, A-59, A-T1155 and other metabolites were significantly up-regulated, and C-T2931, A-330 and other metabolites were significantly down-regulated. The results showed that there were significant differences in metabolites at different ages of male P. v. v.

[0121] In the volcano plot analysis of PX group, it was found that there were many common metabolites with significant expression differences in the group, and the dynamic changes of these metabolites under different age conditions revealed the potential biological regulation rules. For example, the expression level of A-T1509 was significantly down-regulated in PX 2 vs PX 1, and significantly up-regulated in PX 3 vs PX 2; A-330 was significantly down-regulated in PX 2 vs PX 1, and significantly up-regulated in PX 5 vs PX 4.

[0122] In the comparative analysis of the changes of metabolite expression between the female group (PX group) and the male group (PC group), some common metabolites in the sample group were found to have significant changes in both groups. The common metabolites showed a trend of down-regulation followed by up-regulation or vice versa in different groups, such as C-T2888 and A-T1673, suggesting that they may be related to age growth stage and gender specificity.

[0123] The number of significantly up-regulated, down-regulated and non-significantly different metabolites in each group (see Table 10). Taking the PC 3 and PC 2 groups as an example, the number of significantly up-regulated metabolites reached 795; in contrast, the number of significantly down-regulated metabolites increased to 699 in the PX 4 and PX 3 groups, respectively, and the number of significantly down-regulated metabolites was greater. The number of different metabolites between each group was different, and was related to the growth stage, and the greater the number of non-significantly different metabolites indicated that the similarity between the two groups was higher.

[0124] Table 10 Number of different metabolites in female and male P. v. at different ages

[0125]

[0126] Note: PC 1: female P. v. 0.5-1 g (3-5 months old), PC 2: female P. v. 1-2 g (5-8 months old), PC 3: female P. v. 2-3 g (8-12 months old), PC 4: female P. v. 3-4 g (12-16 months old), PC 5: female P. v. 4-5 g (16-20 months old), PX 1: male P. v. 0.5-1 g (3-5 months old), PX 2: male P. v. 1-2 g (5-8 months old), PX 3: male P. v. 2-3 g (8-12 months old), PX 4: male P. v. 3-4 g (12-16 months old), PX 5: male P. v. 4-5 g (16-20 months old).

[0127] III. Conclusion of the present embodiment

[0128] According to the above analysis results, the optimal quality ranking of female P. v. is 8-12 months > 16-20 months > 12-16 months > 5-8 months > 3-5 months, and the optimal quality ranking of male P. v. is 8-12 months > 3-5 months > 5-8 months > 12-16 months > 16-20 months. Therefore, in the present embodiment, female and male P. v. of 8-12 months of age (i.e. dry product quality of 2-3 g) are selected for subsequent processing.

[0129] Embodiment 2

[0130] In traditional applications of P. v., the processing technology is mostly based on experience, and lacks scientific basis for proportioning, resulting in that the nutritional value and medicinal efficacy of P. v. cannot be fully utilized, and it is urgent to innovate the traditional technology. The amino acid complementation effect, as an important theory in nutrition, provides a new idea for solving this problem. Different sources of proteins differ in the types and contents of amino acids, and reasonable collocation of them can make the amino acid composition closer to the human demand pattern, and improve the nutritional value of proteins.

[0131] Based on this theory, the present embodiment focuses on the inflated hippocampus, aiming to screen the best combination ratio of female and male inflated hippocampus compositions through the amino acid nutrition evaluation model. There may be differences in physiological characteristics and nutritional ingredients between female and male inflated hippocampus. By determining the best combination ratio of female and male, it is expected to improve the overall nutritional value and medicinal efficacy of the inflated hippocampus, provide a scientific basis for the development and utilization of the inflated hippocampus, and promote its application in the fields of nutrition and health care and medicine.

[0132] Based on the results of Example 1, the present embodiment selects female and male inflated hippocampus with dry matter quality of 2-3 g (i.e. 8-12 months old) for experiments.

[0133] Firstly, the experimental design and methods used in the present embodiment are as follows:

[0134] (1) Ratio setting scheme

[0135] The ratio setting of female and male inflated hippocampus compositions is shown in Table 11.

[0136] Table 11 Ratio setting of female and male inflated hippocampus compositions

[0137]

[0138] (2) Nutrition evaluation method

[0139] In the present embodiment, two nutrition evaluation methods, EAAI and SRCAA, are selected to evaluate the amino acid nutrition of the inflated hippocampus.

[0140] EAAI (essential amino acid index) can evaluate the comprehensive proportion of essential amino acids, which is obtained by calculating the geometric mean of 7 essential amino acids, and can reflect the overall quality of protein.

[0141] SRCAA (amino acid ratio coefficient) focuses on analyzing the balance of amino acids. All three models take the reference value proposed by FAO / WHO as the benchmark, compare the sample data with it, and accurately evaluate the amino acid nutritional value of the inflated hippocampus.

[0142] (3) Data collection and preprocessing

[0143] A. Chromatographic conditions

[0144] Mobile phase: A phase is 0.1 mol·L -1 sodium acetate (pH 6.5)-acetonitrile = 93:7, B phase is 80% acetonitrile; elution mode: gradient elution (proportion see Table 12 below); flow rate: 1 mL·min -1 ; column temperature: 40℃; detection wavelength: 254nm; injection volume: 2μL.

[0145] Table 12 Elution proportion of different hippocampus amino acid determination mobile phase

[0146]

[0147]

[0148] B, solution preparation

[0149] 1) 6 mol·L -1 Hydrochloric acid solution: take the appropriate amount of water, and accurately pipette hydrochloric acid (mass fraction 37-38%) 50 mL, add water to constant volume in 100 mL volumetric flask, shake well, and get.

[0150] 2) 0.1 mol·L -1 PITC-acetonitrile solution: accurately pipette PITC 0.12 mL, add acetonitrile to constant volume in 10 mL volumetric flask, shake well, and get.

[0151] 3) 1 mol·L -1 Triethylamine-acetonitrile solution accurately pipette triethylamine 1.4 mL, add acetonitrile to constant volume in 10 mL volumetric flask, shake well, and get.

[0152] 4) mixed reference solution: accurately weigh the appropriate amount of 17 kinds of amino acid reference (see Table 13 below), add water, ultrasonic treatment for about 5 min, and add water to constant volume in 25 mL volumetric flask, shake well, and make mixed reference stock solution, store at 4℃, for future use.

[0153] Table 13 hippocampus amino acid determination standard amount

[0154]

[0155] 5) test solution: take female and male inflated hippocampus granules or powder, crush, pass through No. 5 sieve, accurately weigh about 0.04 g of each sample powder, place in 25 mL hydrolysis tube, accurately add 6 mol·L -1 hydrochloric acid solution 2 mL, place in 150℃ oven for 1 h, cool down, filter, add water 2 ml to rinse the hydrolysis tube, repeat 3 times, and combine the hydrolysis solution and washing solution into an evaporating dish, evaporate in water bath, dissolve the residue with water and transfer to 25 mL volumetric flask, add water to constant volume, shake well, and make test sample stock solution, store at 4℃, for future use. Another 2 mL 6 mol·L -1 hydrochloric acid solution is used as blank control solution according to the above method.

[0156] 6) PITC derivatization: accurately pipette 2 mL of amino acid mixed reference solution and test sample solution into 10 mL stoppered test tube, accurately add 1 mol·L -1 triethylamine-acetonitrile solution 1 mL, shake well, and accurately add 0.1 mol·L -1PITC-acetonitrile solution 1 mL, mixed, 50 °C water bath reaction for 45 min, cool, precise addition of n-hexane 1 mL, oscillation extraction, static 30 min, discard the upper, take the lower solution, 0.45 μm microporous filter membrane filter, obtained. The blank solution is simultaneously derivatized according to this method as a blank solution derivatization solution.

[0157] C, methodological investigation

[0158] 1) System suitability test: take the blank, mixed reference substance and test sample derivative solution, according to the chromatographic conditions in the chromatographic conditions part, the separation degree of the 17 amino acid derivatives is high, which indicates that the peaks can be well separated, the theoretical plate number is greater than 5 x 10 3 , the separation degree is greater than 1.8, which indicates that the column efficiency is high, and the high performance liquid chromatogram is shown in the following Figure 17 .

[0159] 2) Linear relationship test: precisely transfer the 4) mixed reference substance stock solution in the solution preparation part to a 25 mL volumetric flask, dilute with water to the mark, shake well, and derivatize according to the method in "2.2.6", and determine the chromatographic conditions in the chromatographic conditions part. The linear curve is drawn with the amino acid reference substance peak area (Y) as the vertical coordinate and the sample concentration (X, mg / mL) as the horizontal coordinate, the regression equation and the correlation coefficient r are calculated, and the results are shown in Table 14. As can be seen from the experiment, the linear relationship of the 17 kinds of amino acids is good, and the correlation coefficient is greater than 0.9990, and the correlation is high.

[0160] Table 14 Linear relationship of 17 kinds of amino acids in hippocampus

[0161]

[0162] 3) Precision test: precisely weigh 1 portion of sample powder, about 0.04 g, prepare and derivatize the solution according to the methods in 5) and 6) in the solution preparation part, and continuously inject 6 times according to the chromatographic conditions in the chromatographic conditions part, and determine the peak area of the 17 kinds of amino acids in the sample, the results are shown in Table 14, and the RSD of each peak area is less than 1.23%, which indicates that the instrument precision is good.

[0163] 4) Reproducibility test: precisely weigh 6 portions of sample powder, about 0.04 g, prepare and derivatize the solution according to the methods in the solution preparation part, and inject to determine the content of the 17 kinds of amino acids in the sample according to the chromatographic conditions in the chromatographic conditions part, the results are shown in Table 5, and the RSD is less than 2.77%, which indicates that the experimental method has good reproducibility.

[0164] 5) Stability test: 1 portion of sample powder, about 0.04 g, was precisely weighed, and the solution was prepared and derivatized according to the method of 5) and 6) in the solution preparation section above. The peak areas of 17 amino acids in the sample were determined at 0, 2, 4, 6, 8, 24 h according to the chromatographic conditions in the chromatographic conditions section above. The results are shown in Table 15. The RSD of each peak area was less than 2.86%, indicating that the sample was stable within 24 h after derivatization.

[0165] Table 15 RSD of the determination method of amino acids in hippocampus

[0166]

[0167] 6) Recovery test: 9 portions of the first sample were precisely transferred, 1 mL each. The amino acid mixed control substance was precisely added in an amount of 1.2, 1, 0.8 times the content of the amino acids to be determined in the sample. The solution was prepared and derivatized according to the method of 5) and 6) in the solution preparation section above. The recovery rate of each amino acid was calculated. The average recovery rate of the 17 amino acids was 85.88% to 106.35%, and the RSD was less than 2.49%.

[0168] Second, the experimental results of this embodiment

[0169] (1) Sample determination results

[0170] 0.04 g of hippocampus powder of different varieties was precisely weighed, two portions each. The solution was prepared and derivatized according to the method of 5) and 6) in the solution preparation section above. The peak areas of each amino acid were determined by injection according to the chromatographic conditions in the chromatographic conditions section above. The content of each amino acid was calculated by substituting into the regression equation. The results are shown in Table 16. The composition and characteristics of amino acids in female and male inflated hippocampus are shown in Table 17.

[0171] Table 16 Content of 17 amino acids in female and male inflated hippocampus (mg / g)

[0172]

[0173]

[0174] Note: TAA: total amount of hydrolyzed amino acids; * indicates essential amino acids for the human body; 1 indicates umami amino acids; 2 indicates branched-chain amino acids; 3 indicates aromatic amino acids; 4 indicates antioxidant amino acids; 5 indicates medicinal amino acids.

[0175] (2) Nutritional evaluation of compositions of female and male inflated hippocampus with different proportions

[0176] From Table 17, it can be seen that the EAAI value is the highest at a ratio of 1:1, indicating that the comprehensive proportion of essential amino acids is the most ideal at this ratio, and the overall quality of the protein is higher; the SRCAA value also presents a similar trend, and the balance coefficient is the highest at a ratio of 1:1, meaning that the balance of amino acids is the best. With the increase of the ratio of male Hippocampus, each index first increases and then decreases, and the optimal state is at a ratio of 1:1, which may be because there is a complementary effect between female and male Hippocampus in nutritional components, and this complementary effect reaches the maximum when the ratio is 1:1.

[0177] Table 17 Nutritional evaluation of 7 essential amino acids in compositions of female and male Hippocampus kuda at different ratios

[0178]

[0179] Note: (n is the number of amino acids participating in comparison; aai is the proportion of a certain EAA in the sample; AAi is the proportion corresponding to aai in the WHO / FAO model spectrum); SRCAA = 100-CVx100 (CV is the coefficient of variation of RCAA).

[0180] Third, the conclusion of the present embodiment:

[0181] The composition of female and male Hippocampus kuda at different ratios has a significant advantage in the complementation of essential amino acids at a ratio of 1:1.

[0182] Example 3

[0183] In the present embodiment, Hippocampus kuda (dry product, from Xiaomie Aquatic Technology Co., Ltd. in Xiamen) is used as raw material, and PITC pre-column derivatization-HPLC method is used to determine the content of 17 kinds of amino acids, and the total content of 17 kinds of amino acids is used as an evaluation index. Single factor test is carried out on the factors affecting the processing technology of wine-processed Hippocampus kuda (amount of wine added, soaking time, drying temperature), and the wine-processed Hippocampus kuda processing technology is optimized by response surface on the basis of single factor test. The wine added in the wine-processed technology of the present embodiment is yellow rice wine (refreshing type, second grade, purchased from Fengyuanchun Wine Industry Co., Ltd. in Xiamen, product standard number: GB / T13662, raw materials: water, glutinous rice, millet, food additives: caramel color, alcohol content ≥12.0%vol, total sugar ≤15.0g / L).

[0184] First, the experimental method of the present embodiment

[0185] (1) Preparation of derivatization reagent

[0186] A. Preparation of 6 mol / L hydrochloric acid solution: weigh 50 mL of hydrochloric acid with a burette, add ultrapure water to a 100 mL volumetric flask, and shake.

[0187] B. Preparation of 1 mol / L triethylamine-acetonitrile solution: precisely weigh 7 mL of triethylamine with a pipette into a 50 mL volumetric flask, and then add acetonitrile to constant volume and shake.

[0188] C. Preparation of 0.1 mol / L PITC-acetonitrile solution: precisely weigh 0.6 mL of phenyl isothiocyanate (PITC) with a pipette into a 50 mL volumetric flask, and then add acetonitrile to constant volume and shake.

[0189] D. Preparation of control solution: precisely weigh an appropriate amount of 17 kinds of amino acid control samples, and then add water to constant volume in a 25 mL volumetric flask, and shake well. Store in a refrigerator at 4°C for subsequent experiments.

[0190] Table 18 Amino acid standard sample weighing

[0191]

[0192] (2) Preparation of test solution

[0193] A. Sample hydrolysis: precisely weigh about 0.1 g of powder (No. 5 sieve) into a 25 ml hydrolysis tube. Precisely add 5 ml of 6 mol / L hydrochloric acid solution, and place in a 150°C oven for 1 h. Filter, add 2 ml of rinse water to the hydrolysis tube, repeat 3 times, transfer to a 25 ml volumetric flask, add water to constant volume, and prepare a test sample stock solution for use.

[0194] B. Sample derivatization: take 0.8 ml of 2.2 test sample stock solution, add 1.2 ml of distilled water, and then add 1 ml of 1 mol / L triethylamine-acetonitrile solution to a 50 ml centrifuge tube, respectively, and then shake well. Then add 1 ml of 0.1 mol / L PITC-acetonitrile solution, mix well, and then place in a 50°C water bath for 45 min, and then cool down. Add 1 ml of n-hexane, shake, and stand for 30 min. Take the lower layer solution with a needle, filter with a 0.45 μm filter membrane, and obtain a sample solution for use.

[0195] (3) Preparation of blank solution test solution: take 5 ml of 6 ml / L hydrochloric acid solution, and hydrolyze and derivatize according to the conditions in the test solution preparation section to obtain a blank group test solution.

[0196] (4) Preparation of control group test solution: dilute the above control solution by the number of times to prepare 5 gradient mixed standards, and then derivatize according to the conditions in the sample derivatization section.

[0197] (5) Chromatographic conditions

[0198] Mobile phase: A was 0.1 mol / L sodium acetate (pH 6.5) - acetonitrile = 93:7, B was 80% acetonitrile; elution method: gradient elution; flow rate: 1 mL / min; column temperature: 40℃; detection wavelength: 254 nm; injection volume: 2 μL. The HPLC mobile phase elution ratios are shown in Table 19.

[0199] Table 19 Liquidity Washout Ratio

[0200]

[0201] (6) Single-factor experiment

[0202] A. Investigation of alcohol addition amount: Five parallel samples of the same mass (50g) were weighed, with a fixed soaking time of 60min and a drying temperature of 90℃. The alcohol addition amounts were investigated at (10%, 15%, 20%, 25%, and 30%). The drying time was 2h.

[0203] B. Investigation of soaking time: Five identical samples (50g each) were weighed in parallel, with a fixed alcohol content of 20%, and the drying temperature was 90℃. Different soaking times (10, 30, 60, 120, 240 min) were investigated. The drying time was 2 hours.

[0204] C. Investigation of drying temperature: Five identical 50g samples were weighed in parallel, with a fixed alcohol content of 20% and a soaking time of 60min. Different drying temperatures (80, 90, 100, 110, 120℃) were investigated. The drying time was 2h.

[0205] II. Experimental Results of this Example

[0206] (1) Linear relationship

[0207] A standard curve was plotted using the detection results of the previously prepared reference solution. The regression equation and correlation coefficient are shown in Table 20. The fitted regression equation showed good linearity, and the correlation coefficient r was [value missing]. 2 All are ≥0.99.

[0208] Table 20 Linear ranges of 17 amino acids

[0209]

[0210]

[0211] (2) Results of single-factor experiments

[0212] A. The effect of alcohol content on amino acid content: such as Figure 18As shown in the table, the amino acid content gradually increased with the increase of the amount of wine, and the amino acid content was the highest when the amount of wine was 15%, and then the overall amino acid content showed a significant downward trend. The possible reason is that ethanol in yellow rice wine can change the permeability of cells and promote the release of hippocampal amino acids. Excessive ethanol may accelerate the esterification of amino acids, resulting in a decrease in the content of amino acids.

[0213] B, the effect of moistening time on the content of amino acids: as shown in the table, Figure 19 The moistening time showed an up and down fluctuation, which may be due to different moistening time and different duration of moistening temperature change, affecting the change of amino acid content. However, the amino acid content was the highest at 60 min, so 60 min was selected as the optimal moistening time.

[0214] C, the effect of drying temperature on the content of amino acids: as shown in the table, Figure 20 With the gradual increase of temperature, the amino acid content was the highest at 90℃, and then decreased with the increase of drying temperature. The possible reason is that a certain temperature can accelerate the molecular diffusion rate, and the leaching of amino acids increases. However, when the temperature rises to a certain extent, the amino acids, peptides and proteins in hippocampus may undergo polymerization or condensation reaction with carbohydrates, generating other substances, and the higher the temperature, the faster the reaction, resulting in a decrease in the content of amino acids.

[0215] (3) Response surface test results and analysis

[0216] A, response surface test design: according to the analysis results of the above single factor test, the Design-Expert 13 software was used to design a 3-factor 3-level Box-Behnken experiment to optimize the processing technology of wine-baked swollen abdomen hippocampus. The test factors and levels are shown in Table 21. The test results are shown in Table 22.

[0217] Table 21 Factors and levels of response surface optimization test

[0218]

[0219] Table 22 Optimization results of response surface test

[0220]

[0221] B, model fitting and variance analysis

[0222] Table 21 Data of Design-Expert 13 regression fitting of the regression equation Y = 57.86-0.6375A-0.975B-0.9125C-1.70AB-0.8750AC-0.3500BC-2.74A 2 -1.57B 2-1.44C 2 Wherein: A, B, C represent liquor addition (%), steaming time (min), drying temperature (℃), respectively.

[0223] Through the multiple regression model verification (Table 23), the response surface model shows excellent statistical performance, overall significance test F value = 35.01, p value < 0.0001, p = 0.2023 (F = 2.46) of the lack of fit, confirming that the model has no systematic bias, the model is suitable. The correlation coefficient R 2 = 0.9783, R Adj 2 = 0.9503 obtained by software analysis, indicating that the model has good fitting degree, small error, and can explain 95.03% of the change of response value. CV = 0.9617%, the confidence is considerable, the error is small. Therefore, this model can be used to predict the trend of amino acid content with factors. Steaming time (B), C (drying temperature), quadratic term (A 2 , B 2 , C 2 ) has extremely significant effect on amino acid content (p < 0.01). Liquor addition (A) has statistical significance (p < 0.05), the interaction term AB has statistical extremely significant (p < 0.01), the interaction term AC has statistical significance (p < 0.05). Through F value, the order of factors affecting hippocampal amino acid content is: B (steaming time) > C (drying temperature) > A (liquor addition).

[0224] Table 23 Analysis of variance and significance analysis results of amino acid regression model

[0225]

[0226] Note: P < 0.01 is extremely significant, represented by **, P < 0.05 is significant, represented by *.

[0227] C, the influence of each factor interaction on amino acid content

[0228] Using Design-Expert 13 software to get response surface and contour map, analyze the influence law of each factor on amino acid content and the strength of interaction between each factor. The steepness of the response surface is positively correlated with the significance of each factor on amino acid content, the shape of the contour and the density of the axis change are related to the strength of the interaction between each factor.

[0229] Figure 21For the AB response surface plot, when the wine addition is at a low level, the amino acid content increases first and then decreases with the increase of the moistening time, and under the condition of high wine addition, the trend is the same. The curvature of the AB interaction area in the 3D surface plot is significant, confirming that the interaction of the two has a significant effect on the amino acid content. By positioning the spatial consistency of the surface vertex and the red dot center of the contour line, the optimal interaction parameters are determined as wine addition of 14-16% and moistening time of 50-60 min, at which the amino acid content reaches the peak value. The ellipticity in the contour line is large, proving that the interaction of the two factors has a significant effect on the amino acid content. As shown in Table 9, the p value of the AB interaction is less than 0.01, which is extremely significant.

[0230] Figure 22 For the AC response surface plot, when the wine addition is at a low level, the amino acid content increases first and then decreases with the increase of the baking temperature; under the condition of high wine addition, the parabolic trend is the same. The AC interaction area in the 3D surface plot presents steep curvature, confirming that the interaction of the two has statistical significance (p<0.05). By positioning the spatial consistency of the surface vertex and the red dot center of the contour line, the optimal wine addition interval is determined as 14-16%, and the baking temperature interval is 85-90℃, at which the amino acid content reaches the peak value.

[0231] Figure 23 For the BC response surface plot, when the moistening time is short, the amino acid content increases first and then decreases with the increase of the baking temperature. Under the condition of long moistening time, the trend is the same. Similarly, when the baking temperature is low, the amino acid content increases first and then decreases with the increase of the moistening time, and under the condition of high baking temperature, the trend is the same. The 3D surface curvature of the BC interaction is small and the contour line is nearly circular, confirming that the interaction of the two does not have statistical significance (p<0.05). By positioning the spatial consistency of the surface vertex and the red dot center of the contour line, the current optimal optimization interval is determined as moistening time of 40-60 min and baking temperature of 85-90℃, at which the amino acid content reaches the optimal level.

[0232] In summary, the significance of the interaction between AB, AC and BC on the amino acid content is consistent with the analysis results of the interaction term p value in the variance analysis table. In summary, the interaction between the factors affecting the amino acid content is AB>AC>BC.

[0233] D. Process verification: The optimal process conditions of this example were obtained by Design-Expert 13 software. The optimal process parameters of wine-fried T. inflatulus are as follows: wine addition amount 15.100%, soaking time 51.304 min, drying temperature 87.132℃, and predicted amino acid content 58.126 g / 100 g. For convenience of testing, the parameters were modified as follows: wine addition amount 15%, soaking time 52 min, and drying temperature 87℃. Under these conditions, the amino acid content of the processed T. inflatulus was determined three times. The average amino acid content was 57.92 g / 100 g, and the RSD was 1.47%. This indicates that the wine-frying process of T. inflatulus optimized by the response surface method in this example is reliable and has practical application value.

[0234] E. Final production process:

[0235] The final production process of this example is shown in Figure 24 and includes the following steps:

[0236] (1) Mix female T. inflatulus and male T. inflatulus in a mass ratio of 1:1. The female and male T. inflatulus are both T. inflatulus dry products with a single dry weight of 2-3 g and a water content of ≤12%;

[0237] (2) Soak 15% of the dry weight of the material obtained in step (1) in yellow rice wine for 50 min, and then dry at 87-90℃ for 2 h. Specifically, the process includes the following steps:

[0238] A. Preheating: Place the material obtained in step (1) in a hot air circulation oven, set the temperature to 87-90℃, and preheat for 30 min to ensure uniform heating;

[0239] B. Yellow rice wine spraying: Transfer the preheated material to a trough-type mixer quickly, turn on the stirring function, and simultaneously spray yellow rice wine (addition ratio: 15 kg of yellow rice wine per 100 kg of material obtained in step (1)) uniformly. Keep the stirring state during the spraying process to ensure that the yellow rice wine and the material are in full contact;

[0240] C. Soaking: After the yellow rice wine spraying is completed, cover the trough-type mixer tightly, and soak at room temperature for 50 min. Stir every 10 min for 2 min during the soaking period to promote the penetration and absorption of the yellow rice wine;

[0241] D. Drying and cooling: After the soaking is completed, transfer the material back to the hot air circulation oven, and dry at 87-90℃ for 2 h. After drying is completed, turn off the heating function, turn on the circulating air cooling, and cool the oven to a temperature below 40℃. Then, take out the material for use.

[0242] (3) Crush the material obtained in step (2) into powder to obtain the female-male T. inflatulus composition with a total protein content of ≥50%. Specifically, the process includes the following steps:

[0243] A, coarse grinding (granular level): the material obtained in step (2) is put into a universal grinder, and a 10-mesh screen (pore size about 2 mm) is used to prepare coarse granular products (particle size 1-2 mm) suitable for wine or medicinal food products, etc.

[0244] B, fine grinding (powder level): the coarse granular products are further ground using an 80-mesh screen (pore size about 0.18 mm) to obtain loose, uniform, and consistent color fine granular products (particle size ≤0.18 mm) suitable for formula food or powder products.

[0245] (4) According to the product specification requirements, the material obtained in step (3) is packaged into 50 g / bag, 500 g / bag and 1000 g / bag specifications, immediately sealed (aluminum-plastic composite bag or food-grade PE bag) after packaging, and labeled with production date, batch number, specification, etc. information to ensure product traceability.

[0246] Example 4

[0247] Toxicological evaluation is an important means to assess the safety of substances. The conventional toxicological evaluation path usually adopts the combination of acute toxicity test and mutagenicity test. Acute toxicity test can quickly evaluate the damage degree of the test substance to the organism in a short period of time, and determine its toxicity classification; mutagenicity test focuses on whether the test substance can cause changes in the genetic material of the organism, which is an important link for evaluating the potential health risks of long-term use of substances. Through the combination of these two tests, the safety of the female and male P. scaber composition can be comprehensively and systematically evaluated from different aspects and different time scales, providing a scientific basis for its reasonable application in the field of nutrition and health.

[0248] I. Materials and methods of this example

[0249] (1) Test sample and animals

[0250] The sample of this test is a 1:1 composition of female and male P. scaber, which is in the form of solid particles or powder and has no odor, and can be stored at room temperature.

[0251] SPF level KM mice were selected as experimental animals, with body weight ranging from 18 to 35 g, half male and half female. The mice were purchased from Sperofou (Beijing) Biotechnology Co., Ltd. During the feeding process, the mice were placed in an environment with temperature maintained at 20-26℃ and humidity maintained at 30-70% to ensure stable living environment and reduce external factors interference on the experimental results.

[0252] (2) Design of acute oral toxicity test

[0253] The acute oral toxicity test was performed by the limited test method, and the gavage dose was set to 10000 mg / kg BW. During the test, the mice were observed for signs of poisoning, the number of deaths, body weight changes, and gross autopsy. The body weight of the mice was accurately measured using an electronic balance IE-5836. Before the test, the H. ventricosus powder was suspended in a 1.0% sodium carboxymethylcellulose solution to ensure that the sample could be uniformly ingested by the mice, thereby more accurately assessing its acute oral toxicity.

[0254] (3) Design of mutagenicity test

[0255] The test design is shown in Table 24. The mutagenicity test of this embodiment includes three items, namely, bacterial reverse mutation test, mammalian erythrocyte micronucleus test, and mouse spermatocyte chromosome aberration test. The bacterial reverse mutation test uses 5 strains of TA97a, etc., and the dose range is 5-0.3125 mg / dish; the micronucleus test sets 3 dose groups, with doses of 2.5-10 g / kg BW; the chromosome aberration test also sets 3 dose groups, with doses of 1.25-5 g / kg BW. The three tests all use S9 mixed solution as the metabolic activation system, and use Enecarbam, cyclophosphamide, etc. as positive controls to verify the effectiveness and accuracy of the test.

[0256] Table 24 Design of mutagenicity test

[0257]

[0258] Second, the experimental results of this embodiment

[0259] (1) Acute oral toxicity test results: In the acute oral toxicity test, the LD50 values of male and female mice were both greater than 10000 mg / kg BW. During the entire test, no deaths occurred in each group of 10 mice. The body weight of the mice was observed, and by the 14th day, the body weight of the female mice was between 29.5-44.5 g, and the body weight of the male mice was between 38.5-42.5 g. Gross autopsy results showed that all mice were normal, marked as "N". This indicates that H. ventricosus powder did not produce significant acute toxicity to mice at the dose of this acute oral toxicity test.

[0260] Table 25. Results of acute oral toxicity test

[0261]

[0262] (2) Bacterial reverse mutation test results: The bacterial reverse mutation test results (Tables 26 and 27) showed that the number of revertant bacteria of each dose of the test substance group did not reach more than twice that of the solvent control, and there was no dose-response relationship. However, the positive control, dicamba, significantly increased the number of revertant bacteria, verifying the effectiveness of the test. This indicates that the Tridacna crocea powder did not show mutagenic activity in this bacterial reverse mutation test.

[0263] Table 26 First bacterial reverse mutation test results Unit: pieces / dish

[0264]

[0265] Note: The above results are the mean ± standard deviation of three plates.

[0266] Table 27 Second bacterial reverse mutation test results Unit: pieces / dish

[0267]

[0268] (3) Mammalian erythrocyte micronucleus test results: In the mammalian erythrocyte micronucleus test, the micronucleus cell rate of female mice was 0.9-1.2‰, and that of male mice was 0.8-1.7‰. Compared with the solvent control group, the difference was not statistically significant (P>0.05). The PCE / RBC ratio was between 0.50 and 0.63. This suggests that the Tridacna crocea powder did not cause a significant increase in the micronucleus rate of mouse red blood cells at the dose of this test, and did not show mutagenic effects (see Tables 28 and 29)

[0269] Table 28 Erythrocyte micronucleus test results in female mice

[0270]

[0271] **: There was a very significant difference (P<0.01) between the positive control group and the solvent control group

[0272] Table 29 Erythrocyte micronucleus test results in male mice

[0273]

[0274] **: There was a very significant difference (P<0.01) between the positive control group and the solvent control group

[0275] (4) The results of mouse spermatocyte chromosome aberration test: As shown in Tables 30 to 32, the aberration cell rates of the test groups were 1.4-2.2%, and the univalent rates were 0.8-2.2%. Compared with the solvent control group, the differences were not statistically significant (P>0.05). The aberration cell rate of the positive control group reached 10.0%, which verified the effectiveness of the test. This indicates that the H. tuberculata powder did not cause significant chromosome aberration of mouse spermatocytes in this test, and did not show mutagenic activity.

[0276] Table 30 Results of mouse spermatocyte chromosome aberration test

[0277]

[0278] # : Significant difference compared with the solvent control group (P<0.01)

[0279] Table 31 Results of mouse spermatocyte chromosome aberration test

[0280]

[0281] # : Significant difference compared with the solvent control group (P<0.01)

[0282] Table 32 Results of mouse spermatocyte chromosome aberration test

[0283]

[0284] ii : Significant difference compared with the solvent control group (P<0.01)

[0285] III. Conclusion of the present example

[0286] It was found in the present example that the acute oral toxicity of the female and male H. tuberculata composition was actually non-toxic, with LD50 values greater than 10000 mg / kg BW in both female and male mice, and no deaths occurred during the test. No abnormalities were observed in gross dissection. The results of the three mutagenicity tests (bacterial reverse mutation test, mammalian erythrocyte micronucleus test, and mouse spermatocyte chromosome aberration test) were all negative, indicating that the H. tuberculata powder had no mutagenic activity within the tested dose range. The high consistency of the results of these three tests enhances the reliability of the results and indicates that the H. tuberculata powder has good safety in terms of genetic toxicity.

[0287] The present example provides a solid toxicological basis for the application of the female and male H. tuberculata composition in the field of nutrition and health, which is helpful for further development and rational use.

[0288] The above merely describes preferred embodiments of the present application, and therefore cannot limit the scope of the present application, i.e. equivalent changes and modifications made according to the patent scope and content of the present application should still fall within the scope of the present application.

Claims

1. A method for preparing a composition of yellow rice wine and fried male and female Halicampus sp., characterized by comprising the following steps: The preparation method comprises the following steps: ​ (1) mixing female and male P. scaber with a mass ratio of 0.8-1.2:0.8-1.2, wherein the female and male P. scaber are both P. scaber dry products; (2) preheating the material obtained in step (1), then moistening the material with 14-16% of the dry weight of the material of yellow rice for 50-60 min, and then baking the material at 85-90℃ for 1.8-2.2 h; (3) crushing the material obtained in step (2) into powder, thereby obtaining the female and male P. scaber composition with a total protein content of ≥50%.

2. The production method according to claim 1, characterized by: In step (1), the mass ratio of the female and male P. scaber is 1:

1.

3. The production method according to claim 1, wherein: In step (2), 15% of the dry weight of the material obtained in step (1) of yellow rice is moistened for 52 min, and then the material is baked at 87℃ for 2 h.

4. The production method according to claim 1, wherein: The specification of the powder in step (3) is ≤2000±70 μm.

5. The production method according to claim 1, wherein: In step (1), the mass ratio of the female and male P. scaber is 1:1; in step (2), 15% of the dry weight of the material obtained in step (1) of yellow rice is moistened for 52 min, and then the material is baked at 87℃ for 2 h; and the specification of the powder in step (3) is ≤2000±70 μm.

6. The production method according to any one of claims 1 to 5, characterized by: The yellow rice is a refreshing type of secondary yellow rice.

7. Use of the female and male P. scaber composition prepared by the preparation method in any of claims 1 to 6 in the preparation of an oral health care composition.

8. An oral health care composition characterized in that: The effective component of the oral health care composition comprises the female and male P. scaber composition prepared by the preparation method in any of claims 1 to 6.

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