Method for identifying mulberry leaf silkworm rearing and feed silkworm rearing raw silk based on HPLC (High Performance Liquid Chromatography) fingerprint spectrum

Abstract

The invention discloses a method for identifying mulberry leaf silkworm rearing and feed silkworm rearing raw silk based on an HPLC (High Performance Liquid Chromatography) fingerprint spectrum. The method comprises the following steps: sample collection: respectively collecting a mulberry leaf silkworm rearing raw silk sample and a feed silkworm rearing raw silk sample; pre-treating the sample; performing HPLC (high performance liquid chromatography) analysis; constructing a fingerprint spectrum; and evaluating and identifying the similarity. The invention belongs to the technical field of silk identification, and particularly provides a mulberry leaf silkworm rearing and feed silkworm rearing raw silk identification method based on an HPLC (High Performance Liquid Chromatography) fingerprint spectrum so as to solve the problem that mulberry leaf silkworm rearing and feed silkworm rearing raw silk are difficult to accurately distinguish by an existing raw silk identification method. And the problems of incomplete analysis, poor result repeatability and the like of the existing raw silk identification method based on the HPLC technology are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of silk identification, and specifically refers to a method for identifying raw silk from silkworms raised on mulberry leaves and silkworms raised on feed based on HPLC fingerprint. Background Art

[0002] In the silk production industry, raw silk comes from a variety of sources. Silk produced by silkworms raised on mulberry leaves and silkworms raised on artificial feeds coexist in the market. Due to their similar appearance, the two are difficult to accurately distinguish through simple visual inspection and touch. Currently, there is an urgent need for a method to effectively and accurately distinguish these two types of raw silk.

[0003] Traditional methods for identifying raw silk have numerous limitations. For example, some methods rely on physical properties, such as measuring the diameter and strength of raw silk. These physical properties are affected by a variety of factors, including the silkworm species and the environment in which they were grown. Even raw silk from the same source can exhibit significant variations in physical properties, making it difficult to accurately distinguish between silk from mulberry-leaf-reared silk and silk from feed-reared silk. While chemical analysis methods, such as combustion and dissolution, can determine the type of raw silk to a certain extent, they are complex and destructive to the sample, making non-destructive testing impossible. Furthermore, these traditional methods fail to fully reflect the complex chemical composition of raw silk, making it difficult to achieve stable and accurate identification results for raw silk products from complex sources.

[0004] With the development of science and technology, high-performance liquid chromatography (HPLC) technology has gradually been applied to the field of material composition analysis. However, in terms of raw silk identification, especially for the identification of raw silk from mulberry leaf-reared silkworms and silkworms raised on feed, existing HPLC-based methods also have shortcomings. On the one hand, most existing methods focus on the analysis of only a few known components in raw silk, failing to fully consider the complexity and diversity of the overall chemical composition of raw silk, and are unable to form a comprehensive and accurate spectrum reflecting the characteristics of raw silk. On the other hand, existing related studies have differences in sample pretreatment and optimization of chromatographic conditions, resulting in poor reproducibility and comparability of experimental results, making it difficult to establish a unified and reliable identification standard. Summary of the Invention

[0005] The present invention aims to provide a method for distinguishing raw silk from silkworms raised on mulberry leaves and silkworms raised on feed based on HPLC fingerprint, so as to solve the problems that the existing raw silk identification methods are difficult to accurately distinguish raw silk from silkworms raised on mulberry leaves and silkworms raised on feed, and the existing raw silk identification methods based on HPLC technology have incomplete analysis and poor result reproducibility.

[0006] In order to solve the above problems, the technical solutions adopted by the present invention are as follows:

[0007] The method for distinguishing raw silk from silkworms raised on mulberry leaves and silkworms raised on feed based on HPLC fingerprints proposed in the present invention comprises the following steps:

[0008] S1. Sample collection: Collect raw silk samples from mulberry leaf-reared silkworms and raw silk samples from feed-reared silkworms respectively;

[0009] S2. Sample pretreatment: Cut the raw silk sample into small pieces, soak it in deionized water at a bath ratio of 1:25, extract it in a 60°C water bath for 2.5 h, perform ultrasonic-assisted extraction, filter it, concentrate it by rotary evaporation, redissolve it, and filter it through a 0.22 μm filter membrane to obtain the sample solution to be tested;

[0010] S3. HPLC analysis: Using a C18 column, gradient elution was performed with methanol as phase A and an aqueous solution containing 0.3% acetic acid as phase B. The flow rate, detection wavelength, column temperature, and injection volume were set, and the HPLC spectrum of the raw silk sample was recorded.

[0011] S4. Fingerprint construction: The HPLC spectra of the mulberry leaf-raised silkworm raw silk sample and the feed-raised silkworm raw silk sample were respectively imported into the similarity evaluation system, and chromatographic peak correction and matching were performed, characteristic peak information was calibrated, reference peaks were selected and relative peak areas were calculated, and by analyzing the relative peak areas and relative standard deviations of retention times, respective HPLC consensus fingerprint spectra were generated, namely, the mulberry leaf-raised silkworm common fingerprint spectra and the feed-raised silkworm common fingerprint spectra;

[0012] S5. Similarity evaluation and identification: Using the common fingerprint of each group of raw silk as a reference, calculate the similarity between the HPLC spectrum of the sample to be tested and the common fingerprint spectrum, and determine the source of the raw silk based on the similarity.

[0013] Preferably, the S2 specifically comprises: ultrasonic extraction at 40° C. and 300 W for 30 min, filtration, and concentration to dryness by rotary evaporation at 50° C., and addition of 1 mL of deionized water for ultrasonic dissolution.

[0014] Preferably, in the HPLC analysis of S3, the gradient elution specifically comprises:

[0015] 0-15min, A increased from 10% to 50%; 15-18min, A increased from 50% to 100%; 18-21min, A decreased from 100% to 10%;

[0016] The flow rate was 1.0 mL / min, the detection wavelength was 280 nm, the column temperature was 30 °C, the injection volume was 20 μL, and the HPLC spectrum was recorded.

[0017] Preferably, in S4, the peak with retention time of 1.62 min is used as the reference peak for raising silkworms with mulberry leaves, and the peak with retention time of 1.57 min is used as the reference peak for raising silkworms with feed, and multi-point calibration and chromatographic peak matching are performed.

[0018] Preferably, when determining the source of raw silk, if the similarity between the raw silk sample to be tested and the fingerprint of raw silk from mulberry leaves is greater than 0.92, it is determined to be raw silk from mulberry leaves; if the similarity with the fingerprint of raw silk from feed is greater than 0.95, it is determined to be raw silk from feed.

[0019] Preferably, in said S5, a system cluster analysis is used to assist in identification, and a threshold value of Euclidean distance squared ≥ 3 is used to distinguish between raw silk produced by silkworms raised with mulberry leaves and raw silk produced by silkworms raised with feed.

[0020] The beneficial effects achieved by the present invention using the above scheme are as follows:

[0021] 1. The HPLC fingerprint constructed by this invention comprehensively reflects the chemical composition characteristics of raw silk, accurately distinguishing raw silk from silk raised on mulberry leaves and silk raised on feed, overcoming the drawback of traditional identification methods that are subject to interference from multiple factors and cannot accurately distinguish. The fingerprint covers information on the various chemical components of raw silk and comprehensively considers the overall chemical composition of raw silk, making the identification results more reliable. For example, when distinguishing raw silk raised on mulberry leaves from raw silk raised on feed from different production areas, the differences in the fingerprint's characteristic peaks can accurately determine the raw silk's origin with an accuracy rate of over 95%.

[0022] 2. From sample pretreatment to fingerprint construction and evaluation, this invention establishes clear, unified standards and operational procedures, significantly improving the reproducibility and comparability of experimental results. Different researchers following this method can achieve similar identification results, laying the foundation for establishing a unified raw silk identification standard. Multiple experiments have verified that fingerprints obtained from the same batch of raw silk samples processed and analyzed by different researchers using this method have a similarity of over 90%.

[0023] 3. Sample pretreatment causes minimal damage to the raw silk sample. It only requires cutting the raw silk into pieces and performing simple extraction. Compared with traditional destructive chemical analysis methods, it has the potential for non-destructive testing, which is conducive to identification without affecting the subsequent use value of the raw silk.

[0024] 4. HPLC fingerprints not only reveal differences in known components in raw silk but also reveal information on unknown components, providing rich data support for in-depth research on the differences between raw silk produced by mulberry leaf-reared silkworms and silkworms raised on feed, as well as for raw silk quality control. Analysis of the characteristic peaks in the fingerprints reveals the relative content and distribution of different chemical components in raw silk, providing comprehensive information for related research. For example, fingerprint analysis revealed higher levels of certain polyphenolic compounds in raw silk produced by mulberry leaf-reared silkworms than in raw silk produced by feed, providing clues for further research into the relationship between silk quality and raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 HPLC chromatograms of various fresh cocoon and raw silk samples of mulberry leaf-raised silkworms / feed-raised silkworms provided in Example 1;

[0026] Figure 2 HPLC chromatograms of fresh cocoon raw silk samples from mulberry leaf-reared silkworms at different cocooning stages;

[0027] Figure 3 This is a graph showing the relative contents of trace components in fresh silk from mulberry leaf-reared silkworms at different cocooning stages;

[0028] Figure 4 HPLC profiles of fresh cocoons and raw silk from mulberry leaves grown in different locations;

[0029] Figure 5 This is a chart showing the relative contents of trace components in fresh silk cocoons grown from mulberry leaves from different origins.

[0030] Figure 6 HPLC spectrum of fresh / dried cocoons of silkworms raised on mulberry leaves;

[0031] Figure 7 HPLC spectrum of fresh / dried cocoons and raw silk from feed silkworms;

[0032] Figure 8 The relative contents of trace components in fresh cocoons / dried cocoons of silkworms raised with mulberry leaves / feed;

[0033] Figure 9 This is the systematic cluster analysis diagram of fresh cocoon and raw silk samples of mulberry leaf-reared silkworms and feed-reared silkworms;

[0034] Figure 10 HPLC fingerprint of fresh cocoon and raw silk of mulberry leaf-raised silkworms / feed-raised silkworms;

[0035] Figure 11 This is the HPLC spectrum of commercial raw silk samples;

[0036] Figure 12 This is a systematic cluster analysis diagram of commercial raw silk.

[0037] in:

[0038] Figure 3 (a) shows the relative contents of trace components in raw silk from spring fresh cocoons, and (b) shows the relative contents of trace components in raw silk from autumn fresh cocoons.

[0039] Figure 8 Figure (a) shows the relative contents of trace components in fresh / dried silk from silkworms raised on mulberry leaves, and Figure (b) shows the relative contents of trace components in fresh / dried silk from silkworms raised on feed.

[0040] Figure 10 Figure (a) shows the fingerprint S of fresh cocoon silk produced by silkworms raised on mulberry leaves, and Figure (b) shows the fingerprint L of fresh cocoon silk produced by silkworms raised on feed.

[0041] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0043] Example 1

[0044] The method for identifying raw silk from silkworms raised on mulberry leaves and silkworms raised on feed based on HPLC fingerprints comprises the following steps:

[0045] S1. Experimental Materials: The mulberry leaf-reared silk and feed-reared silk silk used in the experiment were provided by Zhejiang Institute of Inspection and Quarantine Science and Technology and Huzhou Institute of Quality and Technical Supervision, respectively. A total of 12 groups of mulberry leaf-reared fresh cocoon raw silk were provided, and the production areas were: Hangzhou, Zhejiang (S1), Jinhua, Zhejiang (S2), Hechi, Guangxi (S3-S6), Baise, Guangxi (S7), Nanning, Guangxi (S8), Xiangyun, Yunnan (S9), Guizhou (S10), Hechi, Guangxi (S11), and Baise, Guangxi (S12). The feed-reared silk was produced by artificial feed breeding of the Babe Group. Specific information is shown in Table 1:

[0046] Table 1 Origin, cocoon stage, feeding method and reeling process of raw silk samples

[0047]

[0048]

[0049] S2. Sample Pretreatment: Cut each raw silk sample into smaller pieces (less than 1 mm). Weigh 2.0 g using an electronic balance and place in a beaker. Add deionized water at a bath ratio of 1:25 and stir until the silk is fully absorbed. Extract in a 60°C waterbath for 2.5 hours. Place in an ultrasonic cleaner and perform assisted ultrasonic extraction at 40°C, 300W for 30 minutes. Filter. Transfer the filtrate to a rotary evaporator at a vacuum of 0.1 MPa and a temperature of 50°C. Evaporate to dryness, then redissolve in 1 mL of deionized water. Filter through a 0.22 μm filter membrane and transfer to an HPLC sample vial for analysis.

[0050] S3, HPLC analysis: LC-100 high performance liquid chromatograph was used to analyze the sample, the chromatographic column was Sharpsil-UC18 (4.6 mm x 250 mm, 5 μm), the mobile phase A was methanol, the mobile phase B was water solution containing 0.3% acetic acid for gradient elution, the elution program was as follows: 0-15 min, the concentration of A increased from 10% to 50%. 15-21 min, 50%-100% A; 18-21 min, 100%-10% A; the flow rate was 1.0 mL / min, the injection volume was 20 L; the detection wavelength was 280 nm, and the column temperature was 30 °C.

[0051] It should be noted that the experimental instruments in the embodiment are shown in Table 2 as follows:

[0052] Table 2 Instruments used

[0053] Instrument name Manufacturer QHX-250B artificial climate incubator Shanghai Lichen Bangxi Instrument Technology Co., Ltd. AR124CN electronic analytical balance Ohaus Instruments Shanghai Co., Ltd. DHG-9055A Blast Drying Oven Shanghai Yiheng Scientific Instrument Co., Ltd. HH-12468 Digital constant temperature water bath Changzhou Hongze Experimental Technology Co., Ltd. JP-100S ultrasonic cleaning machine Shenzhen Jiemeng Ultrasonic Cleaning Machine Co., Ltd. R201D Rotary Evaporator Shanghai Yarong Biochemical Instrument Factory LC-100 High Performance Liquid Chromatograph Shanghai Wufeng Chromatography Instrument Co., Ltd.

[0054] S4, Fingerprint construction: the HPLC chromatogram of the mulberry leaf silkworm rearing / silkworm rearing feed silk sample was introduced into the similarity evaluation system, the reference chromatogram was selected, and the time window was set to 0.1 min. The chromatographic peaks were corrected at multiple points, and the chromatographic peaks were automatically matched, and the retention time and peak area of each characteristic peak were recorded. Select the common characteristic peaks with high separation degree and peak intensity as reference peaks, and set their peak area to 1, and calculate the relative peak area of other chromatographic peaks. Finally, the average method was used to generate the HPLC common mode spectrum of mulberry leaf silkworm rearing / silkworm rearing feed silk, and the construction of the fingerprint was completed.

[0055] 4.1 HPLC chromatogram characteristics of mulberry leaf silkworm rearing / silkworm rearing feed silk

[0056] HPLC detection was performed on the trace components of mulberry leaf silkworm rearing / silkworm rearing feed silk from different sources in Table 1, and the HPLC chromatograms of various mulberry leaf silkworm rearing / silkworm rearing feed fresh cocoon silk samples obtained are shown in Table 1. Figure 1

[0057] ​At a retention time of 1.70 min, all samples of fresh cocoon raw silk from mulberry leaves showed a significant strong characteristic peak. At 5.38 min, all samples showed characteristic peaks with lower intensity and less obvious fluctuations. By 9.05 min, among the fresh cocoon raw silk samples from mulberry leaves, samples S1 and S2 showed characteristic peaks with medium intensity and good separation, showing a clearer component distribution feature; in contrast, the corresponding characteristic peaks in the other samples were weaker and the peak shape was not obvious, reflecting the difference in component content or lower response signal. At 10.31 min, samples S1 and S2 showed obvious characteristic peaks. After 12 min, the retention times of the characteristic peaks of all samples were close, but the intensities were different. Among them, the characteristic peak signals of samples S1 and S2 were stronger, while the characteristic peaks of samples S3 to S9 had similar intensities and peak shapes, indicating that differences in origin may have an impact on the content of trace components in raw silk. The signal intensity of S10~S13 was greatly reduced during the entire retention time, and there were differences in trace components between spring cocoon silk and autumn cocoon silk, indicating that the cocoon stage would have a significant impact on the trace components of raw silk.

[0058] Samples L1 to L3 of fresh silk from feed-reared silkworms all exhibited distinct and intense characteristic peaks at a retention time of 1.70 minutes. At 5.38 minutes, all samples displayed characteristic peaks of low intensity and minimal fluctuation. At 9.05 minutes, the samples exhibited characteristic peaks of moderate intensity and good resolution. After 12 minutes, the characteristic peaks of the fresh silk from feed-reared silkworms showed similar retention times and consistent intensities, indicating that the trace components of feed-reared silk are highly stable. Because it grows in a constant temperature and humidity environment, factors such as its origin and cocooning stage have minimal impact on it. Therefore, feed-reared silk was not considered when exploring the influence of factors such as cocooning stage and origin.

[0059] 4.2 HPLC chromatogram characteristics of raw silk from mulberry leaves at different cocoon stages:

[0060] The specific information of raw silk produced by mulberry leaf-reared silkworms at different cocoon stages is shown in Table 3 below:

[0061] Table 3 Origin, cocoon stage, feeding method and type of raw silk samples

[0062] serial number Origin Cocoon period Feeding method category S3 Hechi, Guangxi Spring Cocoon Mulberry leaf feeding Fresh silk cocoons S4 Hechi, Guangxi Spring Cocoon Mulberry leaf feeding Fresh silk cocoons S5 Hechi, Guangxi Spring Cocoon Mulberry leaf feeding Fresh silk cocoons S6 Hechi, Guangxi Spring Cocoon Mulberry leaf feeding Fresh silk cocoons S10 Guizhou Autumn Cocoon Mulberry leaf feeding Fresh silk cocoons S11 Hechi, Guangxi Autumn Cocoon Mulberry leaf feeding Fresh silk cocoon S12 Baise, Guangxi Autumn Cocoon Mulberry leaf feeding Fresh silk cocoons

[0063] Combine Figure 2Figure 2 shows HPLC chromatograms of raw silk samples from fresh cocoons raised on mulberry leaves at different cocooning stages. At a retention time of 1.70 min, a distinct and intense characteristic peak signal was detected for all raw silk samples; however, at 5.38 min, all samples exhibited a characteristic peak with a weaker signal and minimal fluctuation. At a retention time of 9.05 min, samples S3 to S6 displayed a characteristic peak with moderate intensity and good separation. In contrast, the corresponding peak signals for samples S10 to S13 were relatively weak. After 12 min, characteristic peaks with similar retention times appeared in samples S3 to S6, and with the exception of sample S6, the peak shapes and intensities of the remaining samples were essentially consistent. Compared to samples S3 to S6, the signal intensities of samples S10 to S13 were significantly reduced throughout the retention time, and characteristic peaks of varying intensities appeared after 12 min.

[0064] Depend on Figure 3 Figure 1 shows the relative contents of trace components in raw silk from fresh cocoons raised on mulberry leaves at different cocooning stages. Figure a shows the relative contents of trace components in spring fresh cocoon silk, and Figure b shows the relative contents of trace components in autumn fresh cocoon silk. Significant differences in trace components are evident between spring and autumn silk, indicating that the cocooning stage significantly influences the trace composition of raw silk. The lower temperatures and large diurnal temperature differences in autumn affect the growth quality and nutritional content of mulberry leaves, resulting in a relative decrease in the nutritional intake of silkworms. Furthermore, the lower temperatures in autumn, the longer growth cycle of silkworms, and the reduced metabolic activity of the silkworms lead to insufficient accumulation of trace components in raw silk, resulting in lower trace component content in autumn silk compared to spring silk.

[0065] 4.3 HPLC chromatogram characteristics of mulberry leaf-raised silk from different origins:

[0066] The detailed information of raw silk samples from mulberry leaves raised on silkworms of different origins, the same cocoon stage and type is shown in Table 4 below:

[0067] Table 4 Origin, cocoon stage, feeding method and type of raw silk samples

[0068] serial number Origin Cocoon period Feeding method category S1 Hangzhou, Zhejiang Spring Cocoon Mulberry leaf feeding Fresh silk cocoon S2 Jinhua, Zhejiang Spring Cocoon Mulberry leaf feeding Fresh silk cocoons S3 Hechi, Guangxi Spring Cocoon Mulberry leaf feeding Fresh silk cocoon S7 Baise, Guangxi Spring Cocoon Mulberry leaf feeding Fresh silk cocoons S8 Nanning, Guangxi Spring Cocoon Mulberry leaf feeding Fresh silk cocoons S9 Yunnan Xiangyun Spring Cocoon Mulberry leaf feeding Fresh silk cocoons

[0069] Combine Figure 4 As shown, Figure 4HPLC chromatograms of raw silk from fresh cocoons raised on mulberry leaves from different origins are presented. At retention time 1.70 min, all samples exhibit a characteristic peak with high intensity and clear peak shape. Notably, the peak intensity of sample S9 is significantly higher than that of samples S3, S7, and S8, indicating a relatively higher content of this component. At 5.38 min, while characteristic peaks were detected in all samples, the peak intensity was weak and the fluctuations were not obvious. At 9.05 min, all samples exhibited a characteristic peak with moderate intensity and good resolution. At 10.34 min, only samples S1 and S2 showed a characteristic peak with moderate intensity and good peak shape. After 12 min, samples S1 and S2 exhibited multiple characteristic peaks with essentially identical retention times, peak shapes, and peak heights, indicating a high degree of similarity in their composition. Similar trends were observed in samples S3, S7, S8, and S9, but these samples still exhibited differences from S1 and S2, reflecting possible differences in their origins.

[0070] like Figure 5 As shown in the figure, a relative content diagram of trace components in fresh silk cocoons from mulberry leaves raised from different origins is provided. Figure 5 Analysis revealed that the contents of various trace components varied between raw silk samples from different origins. The relative content differences between raw silk samples from Hangzhou, Zhejiang, and Jinhua, Zhejiang, were minimal, while those from Hechi, Guangxi, Baise, Guangxi, and Nanning, Guangxi, showed the smallest differences. The two raw silk samples from Zhejiang differed significantly from the three raw silk samples from Guangxi and the raw silk sample from Yunnan. The three raw silk samples from Guangxi and the raw silk sample from Yunnan showed minor but similar relative contents. The study indicates that raw silk samples from different origins exhibit similarities in trace component composition, but their relative contents exhibit distinct origin-specific differences. Further analysis revealed that the degree of variation in trace component content between origins was positively correlated with geographic distance.

[0071] 4.4 HPLC chromatogram characteristics of raw silk from mulberry leaf-reared silkworms and feed-reared silkworms using different reeling techniques:

[0072] The detailed information of raw silk samples from mulberry leaf-reared silkworms and feed-reared silkworms using different silk-making processes is shown in Table 5 below:

[0073] Table 5 Number, origin, cocoon stage, feeding method and category of raw silk samples

[0074]

[0075]

[0076] Combine Figure 6-Figure 8Figure 2 shows the HPLC chromatograms of raw silk samples obtained through different reeling processes. At a retention time of 1.70 min, all raw silk samples exhibited strong and clear characteristic signals; at 5.38 min, a characteristic peak with a weaker signal and insignificant fluctuation was detected in all samples. By 9.05 min, all samples showed chromatographic peaks of medium intensity and good separation; at 10.34 min, sample S1 independently exhibited a characteristic peak of medium intensity and good separation. After 12 min, the characteristic peaks exhibited by different raw silk samples differed in retention time, peak shape, and signal intensity.

[0077] Overall, the overall peak intensity of raw silk from feed-reared silkworms is lower than that from mulberry leaf-reared silkworms, indicating that the content of soluble compounds in the silk from feed-reared silkworms is lower. Whether it is raw silk from mulberry leaf-reared silkworms or raw silk from feed-reared silkworms, the characteristic peak intensity of fresh cocoon raw silk is significantly higher than that of dried cocoon raw silk, and this is particularly obvious in the case of cocoon silk from feed-reared silkworms. This shows that different reeling methods will have a significant impact on the trace components of raw silk. The drying treatment of silk cocoons will change the content of trace components inside them. Drying treatment will cause the content of soluble components to decrease, and after reeling treatment, the content of trace components will decrease again. The peak intensity of both groups of dry cocoon silk samples decreased, but the peak intensity of the raw silk sample from feed-reared silkworms decreased more significantly, indicating that the loss of components in the cocoon silk from feed-reared silk is more serious after drying and silk making.

[0078] S5. Similarity evaluation and identification:

[0079] 5.1 Similarity Analysis

[0080] Correlation analysis reveals the degree of relationship between multiple variables and is unaffected by interactions between factors. Therefore, it can be used as a criterion for assessing sample similarity. The HPLC chromatographic data of the raw silk samples were imported into the SPSS 26.0 system to generate a consensus HPLC fingerprint for raw silk from mulberry leaf-reared silkworms and feed-reared silkworms. Using this consensus fingerprint as a reference, intra-group correlations were calculated for each sample, thereby completing the similarity assessment of raw silk samples from mulberry leaf-reared silkworms and feed-reared silkworms.

[0081] The similarity evaluation was performed on all pairs of raw silk samples raised on mulberry leaves / raised on feed in S4, and the results are shown in Tables 6 and 7, respectively.

[0082] Table 6 Evaluation results of leaf-reared silkworm raw silk and mulberry similarity

[0083] serial number S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S1 1.00 0.96 0.88 0.87 0.86 0.88 0.87 0.88 0.88 0.78 0.79 0.79 S2 0.96 1.00 0.87 0.86 0.86 0.87 0.86 0.87 0.87 0.77 0.78 0.77 S3 0.88 0.87 1.00 0.98 0.97 0.98 0.99 0.99 0.98 0.78 0.80 0.79 S4 0.87 0.86 0.98 1.00 0.99 0.96 0.98 0.98 0.97 0.83 0.81 0.80 S5 0.86 0.86 0.97 0.99 1.00 0.93 0.94 0.92 0.92 0.75 0.78 0.76 S6 0.88 0.87 0.98 0.96 0.93 1.00 0.99 0.98 0.98 0.74 0.78 0.77 S7 0.87 0.86 0.99 0.98 0.94 0.99 1.00 0.99 0.98 0.79 0.77 0.78 S8 0.88 0.87 0.99 0.98 0.92 0.98 0.99 1.00 0.99 0.79 0.78 0.76 S9 0.88 0.87 0.98 0.97 0.92 0.98 0.98 0.99 1.00 0.76 0.78 0.77 S11 0.78 0.77 0.78 0.83 0.75 0.74 0.79 0.79 0.76 1.00 0.96 0.92 S12 0.79 0.78 0.80 0.81 0.78 0.78 0.77 0.78 0.78 0.96 1.00 0.93 S13 0.79 0.77 0.79 0.80 0.76 0.77 0.78 0.76 0.77 0.92 0.93 1.00

[0084] Table 6 shows that the compositional diversity among raw silk samples from mulberry leaf-reared silkworms is significant, indicating distinct differences in the distribution of trace components and their relative content. However, samples from the same cocoon stage and origin exhibit high similarity values, indicating that under the same environmental and harvesting conditions, the composition and relative content of trace components are highly consistent across raw silk samples.

[0085] Table 7 Similarity evaluation results of raw silk produced by silkworm rearing

[0086]

[0087]

[0088] As shown in Table 7, the similarities of raw silk samples from feed-reared silkworms are all greater than 0.96, indicating that their trace components are similar. This is because they are obtained under year-round, all-weather rearing conditions in a constant temperature and humidity environment, and are not affected by factors such as cocoon stage and production area.

[0089] 5.2 Systematic Cluster Analysis

[0090] Cluster analysis can intuitively display the relationships between samples. Therefore, this method was used to differentiate raw silk from mulberry leaf-reared and feed-reared silk. HPLC chromatographic data of raw silk samples were imported into SPSS 26.0. Chromatographic peak areas of raw silk were selected as the target for cluster analysis. Squared Euclidean distance was used as the metric, and cluster analysis of peak area variables was performed using the inter-group linkage method. Finally, the effectiveness of cluster analysis in differentiating raw silk samples was evaluated based on the analysis results.

[0091] Based on the above analysis method, a systematic cluster analysis was conducted on the fresh cocoon and raw silk samples obtained from mulberry leaf-reared silkworms and feed-reared silkworms. The results are as follows: Figure 9The results show that when the square of the Euclidean distance is 3, the fresh cocoon raw silk samples of mulberry leaf-reared silkworms / feed silkworms are clustered into 6 categories: L1 to L3 fresh cocoon raw silk samples of feed silkworms are clustered into one category, at this time, the fresh cocoon raw silk samples of mulberry leaf-reared silkworms / feed silkworms can be distinguished; S10 to S12 fresh autumn cocoon raw silk samples of mulberry leaf-reared silkworms are clustered into one category, indicating that the cocoon stage has an obvious effect on the trace components of mulberry leaf-reared silk; S3 to S5 and S7 to S8 samples from Guangxi are clustered into one category, and S1 and S2 fresh cocoon raw silk samples of mulberry leaf-reared silkworms from Hangzhou, Zhejiang and Jinhua, Zhejiang are clustered into one category. The results showed that the geographical origin significantly affected the composition of trace components of fresh cocoon raw silk, and samples from different origins showed obvious differences in the types and contents of components; the S6 mulberry leaf-reared fresh cocoon raw silk sample from Hechi, Guangxi was classified into one category; the S9 mulberry leaf-reared fresh cocoon raw silk sample from Xiangyun, Yunnan was classified into one category; when the square of the Euclidean distance was greater than 12, all the mulberry leaf-reared fresh cocoon raw silk samples were clustered into one category, and all the feed-reared fresh cocoon raw silk samples were clustered into one category. The systematic cluster analysis has good recognition ability and can be used to accurately distinguish raw silk from different sources or feeding methods.

[0092] 5.3 Construction of fingerprint

[0093] The fingerprints of each raw silk sample were imported into the similarity evaluation system, and the fingerprint of sample S1 was selected as the reference fingerprint of fresh cocoon raw silk from mulberry leaf-reared silkworms, and the fingerprint of sample L1 was selected as the reference fingerprint of fresh cocoon raw silk from feed-reared silkworms. According to the above analysis process, each sample was processed, and finally the HPLC common fingerprint of fresh cocoon raw silk from mulberry leaf-reared silkworms and feed-reared silkworms was constructed and derived as follows: Figure 10 shown.

[0094] Depend on Figure 10 (a) It can be seen that in the HPLC common fingerprint of the fresh cocoon raw silk sample raised on mulberry leaves, a well-resolved high-intensity chromatographic peak can be seen at the retention time of 1.62 min; characteristic peaks with strong signals are also observed at 2.24, 19.03, and 19.58 min. In addition, multiple medium-intensity chromatographic characteristic peaks appear at 5.38, 8.79, 15.23, 16.13, and 17.39 min, showing a relatively stable fingerprint spectrum. Figure 10(b) The HPLC fingerprint of the fresh cocoon raw silk sample from feed-reared silkworms shows a well-resolved, strong peak at retention time 1.57 min; strong peaks are visible at 2.32 min and 17.83 min; and several weaker peaks appear at 4.60, 9.16, 14.62, 16.58, and 19.45 min. With the exception of a relatively distinct peak at 17.83 min, the overall peak shape becomes flatter after retention time 6.05 min, with minimal signal fluctuation, demonstrating the relative stability of the chromatographic characteristics of the feed-reared silk sample. The peaks appearing after retention time 6.05 min primarily represent polyphenolic compounds attached to the sericin surface. During the reeling process, especially during the cocoon cooking step, these polyphenolic compounds are susceptible to thermal decomposition and dissolution. Due to the differences in the composition and content of polyphenolic compounds in raw silk from mulberry leaves and feed-reared silk, the HPLC fingerprints of the two exhibit significant differences in the number and shape of characteristic peaks. Therefore, based on the differences in these characteristic components of raw silk, raw silk produced by silkworms raised with mulberry leaves and raw silk produced by silkworms raised with feed can be effectively distinguished and identified.

[0095] S6. Test of HPLC fingerprint identification effect:

[0096] The information of commercial raw silk samples is shown in Table 8 below, and its HPLC spectrum is shown in Figure 11 shown.

[0097] Table 8 Identity information of commercial raw silk

[0098] Sample number Feeding method Origin category W1 Mulberry leaf feeding Huzhou Fresh silk cocoons W2 Mulberry leaf feeding Huzhou Fresh silk cocoons W3 Mulberry leaf feeding Huzhou Fresh silk cocoons W4 Mulberry leaf feeding Huzhou Fresh silk cocoons M1 Feeding Zhejiang Inspection Fresh silk cocoons M2 Feeding Zhejiang Inspection Fresh silk cocoons M3 Feed feeding Zhejiang Inspection Fresh silk cocoons M4 Feeding Hai'an, Jiangsu Fresh silk cocoons

[0099] The HPLC fingerprints of fresh silk cocoons from mulberry leaves and those from feed-reared silkworms differed significantly. Mulberry leaf-reared silk exhibited more abundant and more intense characteristic peaks in the 6-21 min interval, while feed-reared silk exhibited fewer and less intense peaks, indicating a lower content of soluble trace components. This suggests that fresh silk cocoons from mulberry leaves are richer in characteristic compounds and exhibit significant chemical composition differences from feed-reared silk, providing a basis for distinguishing the two.

[0100] The collected commercial raw silk samples were evaluated for similarity based on the HPLC common fingerprint of fresh cocoon raw silk samples from mulberry leaf-reared silkworms / feed-reared silkworms to test the identification effect of HPLC fingerprint on raw silk from mulberry leaf-reared silkworms / feed-reared silkworms. The results are shown in Table 9.

[0101] Table 9 Similarity evaluation results of commercial raw silk samples

[0102] Refer to the atlas category A1 A2 A3 A4 B1 B2 B3 B4 Mulberry leaf silkworm production 0.95 0.96 0.95 0.92 Feed silkworm raw silk 0.99 0.97 0.97 0.95

[0103] Combined with Table 9 and Figure 11As shown in the data analysis, the similarity between fresh silk cocoons from mulberry leaf-reared silkworms was high, ranging from 0.92 to 0.96. This indicates that the HPLC fingerprints of fresh silk cocoons from mulberry leaf-reared silkworms are highly consistent, indicating that the composition and content of their trace components are relatively stable and have little variation. The similarity between fresh silk cocoons from feed-reared silkworms was even higher, ranging from 0.95 to 0.99. Compared with mulberry leaf-reared silk, the HPLC fingerprints of feed-reared silkworms are more consistent, indicating that the composition and content of their trace components vary less. This is related to the standardized formula of the feed.

[0104] Overall, the raw silk from mulberry leaves and the raw silk from feed silkworms formed their own high similarity groups, which showed that the HPLC fingerprint can effectively distinguish the silk from the silk from feed silkworms. Figure 12 As shown, when the squared Euclidean distance is greater than 6, the shared fingerprints S of fresh cocoon silk from mulberry leaf-reared silkworms A1-A4 and fresh cocoon silk from mulberry leaf-reared silkworms are clustered into one category, and the shared fingerprints L of fresh cocoon silk from feed-reared silkworms B1-B4 and fresh cocoon silk from feed-reared silkworms are clustered into one category. Cluster analysis results indicate that the constructed HPLC fingerprints can be used to identify commercial raw silk species. The results show that the use of HPLC fingerprints of fresh cocoon silk from mulberry leaf-reared silkworms and feed-reared silkworms is feasible and reliable for identifying raw silk species.

[0105] This example analyzed HPLC fingerprints of raw silk from mulberry leaf-reared and feed-reared silkworms from different sources. The primary sources of differences in soluble trace components were identified by analyzing the HPLC fingerprints of the raw silk samples. The results showed that these differences primarily stemmed from differences in the content of polyphenols and other trace components between raw silk from mulberry leaf-reared and feed-reared silkworms. Based on these differences, HPLC fingerprints of raw silk from mulberry leaf-reared and feed-reared silkworms were successfully established, and through comparative analysis, effective differentiation between the two types of raw silk was achieved.

[0106] HPLC chromatograms of raw silk from mulberry leaf-reared silkworms and feed-reared silkworms at different cocooning stages, origins, and silk-refining processes were obtained. The results showed that the types of trace components in fresh silk from mulberry leaf-reared silkworms at different cocooning stages were essentially the same, but the content of each component varied with the cocooning stage. Trace components in fresh silk from mulberry leaf-reared silkworms from different origins differed, and this increased with increasing distance between the origins. The silk-refining processes significantly affected the trace components of raw silk from mulberry leaf-reared silkworms and feed-reared silkworms. The characteristic peak intensities of fresh silk from both mulberry leaf-reared and feed-reared silkworms were significantly higher than those of dry silk from dried silkworms, and the reeling treatment of dry cocoons had a greater impact on the silk from feed-reared silkworms.

[0107] Based on the similarity evaluation results of fresh silk from mulberry leaf-reared and feed-reared silkworms, the following conclusions can be drawn: The similarity of fresh silk from mulberry leaf-reared silkworms of the same cocooning stage was greater than 0.86, while the similarity of fresh silk from feed-reared silkworms was greater than 0.96. This indicates that there is good similarity and consistency between the same types of raw silk, especially in feed-reared silk, which has less variation in trace components and exhibits higher stability.

[0108] Cluster analysis of fresh silk cocoons from silkworms raised on mulberry leaves and those raised on feed revealed that samples raised on mulberry leaves with the same cocooning stage were grouped together, indicating that cocooning stage significantly influences the types and content distribution of trace components. Furthermore, samples raised on mulberry leaves from the same origin also showed good cluster consistency, suggesting that origin also plays a key role in the composition of trace components. When the squared Euclidean distance was greater than 12, fresh silk cocoons from silkworms raised on mulberry leaves and those raised on feed could be completely distinguished. This demonstrates that the systematic cluster analysis method is capable of distinguishing between the two.

[0109] By developing a common HPLC fingerprint of fresh silk cocoons from mulberry leaf-reared silkworms and those from feed-reared silkworms, and using this as a reference for similarity evaluation and phylogenetic cluster analysis, the researchers determined that the similarity between raw silk samples from mulberry leaf-reared silkworms exceeded 0.92, while the similarity between raw silk samples from feed-reared silkworms exceeded 0.95, confirming that the chemical characteristics of the same raw silk were highly consistent. The phylogenetic cluster analysis further demonstrated that commercial raw silk samples from mulberry leaf-reared silkworms and those from feed-reared silkworms could be completely distinguished. This study confirms the reliable accuracy and reproducibility of the HPLC fingerprint-based raw silk identification method.

[0110] The present invention and its embodiments are described above. This description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if a person skilled in the art is inspired by this and, without departing from the purpose of the present invention, designs structures and embodiments similar to this technical solution without inventiveness, they shall fall within the scope of protection of the present invention.

Claims

1. A method for distinguishing raw silk from silkworms raised on mulberry leaves and silkworms raised on feed based on HPLC fingerprint, characterized in that: The following steps are involved: S1. Sample collection: Collect raw silk samples from mulberry leaf-reared silkworms and raw silk samples from feed-reared silkworms respectively; S2. Sample pretreatment: Cut the raw silk sample into small pieces, soak it in deionized water at a bath ratio of 1:25, extract it in a 60°C water bath for 2.5 h, perform ultrasonic-assisted extraction, filter it, concentrate it by rotary evaporation, redissolve it, and filter it through a 0.22 μm filter membrane to obtain the sample solution to be tested; S3. HPLC analysis: Using a C18 column, gradient elution was performed with methanol as phase A and an aqueous solution containing 0.3% acetic acid as phase B. The flow rate, detection wavelength, column temperature, and injection volume were set, and the HPLC spectrum of the raw silk sample was recorded. S4. Fingerprint construction: The HPLC spectra of the mulberry leaf-raised silkworm raw silk sample and the feed-raised silkworm raw silk sample were respectively imported into the similarity evaluation system, and chromatographic peak correction and matching were performed, characteristic peak information was calibrated, reference peaks were selected and relative peak areas were calculated, and by analyzing the relative peak areas and relative standard deviations of retention times, respective HPLC consensus fingerprint spectra were generated, namely, the mulberry leaf-raised silkworm common fingerprint spectra and the feed-raised silkworm common fingerprint spectra; S5. Similarity evaluation and identification: Using the common fingerprint of each group of raw silk as a reference, calculate the similarity between the HPLC spectrum of the sample to be tested and the common fingerprint spectrum, and determine the source of the raw silk based on the similarity.

2. The method for distinguishing raw silk from silkworms raised with mulberry leaves and silkworms raised with feed based on HPLC fingerprint according to claim 1, characterized in that: The S2 specifically includes: ultrasonic extraction at 40° C. and 300 W for 30 min, filtration, and concentration to dryness by rotary evaporation at 50° C., and addition of 1 mL of deionized water for ultrasonic dissolution.

3. The method for distinguishing raw silk from silkworms raised with mulberry leaves and silkworms raised with feed based on HPLC fingerprint according to claim 1, characterized in that: In the HPLC analysis of S3, the gradient elution specifically includes: From 0 to 15 minutes, the concentration of phase A increased from 10% to 50%; from 15 to 18 minutes, the concentration of phase A increased from 50% to 100%; from 18 to 21 minutes, the concentration of phase A decreased from 100% to 10%; The flow rate was 1.0 mL / min, the detection wavelength was 280 nm, the column temperature was 30 °C, the injection volume was 20 μL, and the HPLC spectrum was recorded.

4. The method for distinguishing raw silk from silkworms raised with mulberry leaves and silkworms raised with feed based on HPLC fingerprint according to claim 1, characterized in that: In the S4, the peak with retention time of 1.62 min is used as the reference peak for raising silkworms using mulberry leaves, and the peak with retention time of 1.57 min is used as the reference peak for raising silkworms using feed, and multi-point calibration and chromatographic peak matching are performed.

5. The method for distinguishing raw silk from silkworms raised with mulberry leaves and silkworms raised with feed based on HPLC fingerprint according to claim 4, characterized in that: When determining the source of raw silk, if the similarity between the raw silk sample to be tested and the fingerprint of raw silk from mulberry leaves is greater than 0.92, it is determined to be raw silk from mulberry leaves; if the similarity with the fingerprint of raw silk from feed is greater than 0.95, it is determined to be raw silk from feed.

6. The method for distinguishing raw silk from silkworms raised with mulberry leaves and silkworms raised with feed based on HPLC fingerprints according to claim 5, characterized in that: In the S5, systematic cluster analysis is used to assist in identification, and the threshold value of Euclidean distance squared ≥ 3 is used to distinguish between raw silk produced by silkworms raised with mulberry leaves and raw silk produced by silkworms raised with feed.

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