Qualitative identification method of cuttlefish juice powder based on microstructure analysis

Abstract

This application discloses a qualitative identification method for squid ink powder based on microstructure analysis, belonging to the field of food testing technology. The method includes the following steps: taking a squid ink powder sample mixed with a water-soluble carrier, placing it in water to fully dissolve the carrier, obtaining a sample suspension; centrifuging the sample suspension, discarding the supernatant, eluting soluble excipients, and freeze-drying the insoluble precipitate to obtain a dried sample; sputter-coating the dried sample with gold, and observing the microstructure and surface morphology differences under a scanning electron microscope. This method overcomes the technical limitations of traditional detection methods due to the insolubility of large molecules and complex matrices, achieving unique and highly efficient accurate identification by utilizing the irreversible microscopic morphological differences in the physical genesis of substances.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, and in particular to a qualitative identification method for squid ink powder based on microstructure analysis. Background Technology

[0002] Cuttlefish ink powder, as a natural pigment and functional ingredient, has a high extraction cost. In recent years, some unscrupulous merchants have used inorganic colorants—vegetable carbon black (a colorant permitted by GB 2760)—which is much cheaper than cuttlefish ink powder, to impersonate or adulterate it.

[0003] In routine quality control checks targeting such adulteration, traditional physicochemical testing methods face significant challenges. Solubility difference tests, protein content characteristic tests, or ICP-MS multi-element analysis all prove ineffective.

[0004] Meanwhile, the chromatograms analyzed by liquid chromatography-mass spectrometry (LC-MS) showed no significant differences. This is because natural squid ink melanin has the characteristics of a highly polymerized, poorly soluble macromolecule, while plant carbon black is an extremely inert inorganic carbon. Both are difficult to effectively dissociate in conventional mobile phases and produce characteristic ion fragments with significant differences, resulting in a lack of discriminative power in the LC-MS chromatograms. Summary of the Invention

[0005] The purpose of this invention is to provide a qualitative identification method for cuttlefish ink powder based on microstructure analysis.

[0006] To achieve the above objectives, this invention provides a qualitative identification method for cuttlefish ink powder based on microstructure analysis, comprising the following steps:

[0007] Step S1: Take a sample of squid ink powder mixed with a water-soluble carrier, place it in water to fully dissolve the water-soluble carrier, and obtain a sample suspension with a concentration of 5 g / mL-15 g / mL.

[0008] Step S2: Centrifuge the sample suspension and discard the supernatant, elute the soluble excipients, and freeze-dry the insoluble precipitate to obtain a dried sample for later use;

[0009] Step S3: Sputter gold onto the dried, ready-to-use sample and place it in a scanning electron microscope to observe the differences in microstructure and surface morphology.

[0010] In one embodiment of the present invention, in step S1, the water-soluble carrier includes one or more of maltodextrin and gum arabic.

[0011] In one embodiment of the present invention, step S1, the step of placing the water-soluble carrier in water to fully dissolve it includes: performing ultrasonication at a power of 450W-550W for more than 5 minutes to fully dissolve the water-soluble carrier.

[0012] In one embodiment of the present invention, step S2, which involves centrifuging the sample suspension and discarding the supernatant to elute the soluble excipients, includes: centrifuging at 4500 r / min-5500 r / min for more than 10 minutes, discarding the supernatant, and repeating the steps of adding water and centrifuging to elute the soluble excipients.

[0013] In one embodiment of the present invention, in step S3, after gold sputtering, the surface of the sample is dried to obtain a conductive gold film of 5nm-15nm.

[0014] In one embodiment of the present invention, step S3, the gold sputtering process includes: at a temperature below 5 × 10⁻⁶. -4 Under a vacuum of Pa, the dried sample was sputtered with gold using a sputtering instrument. The working distance of the sputtering instrument to the dried sample was 5mm-10mm, and the sputtering angle was 30°-45°.

[0015] In one embodiment of the present invention, the sputtering current of the sputtering instrument is 10mA-15mA, the gold sputtering time is 40s-90s, and the deposition rate is 0.1 nm / s-0.5 nm / s.

[0016] In one embodiment of the present invention, in step S3, the differences in microstructure and surface morphology are observed at magnifications of 500x, 1000x, 2000x and 10000x respectively.

[0017] In one embodiment of the present invention, in step S3, the differences in microstructure and surface morphology are observed at magnifications of 200x, 500x, 1000x, 3000x and 5000x respectively.

[0018] In one embodiment of the present invention, in step S3, when the sample is observed to have a rounded spherical shape or a regular structure resembling a shell at any magnification from 500x to 10000x, and the particle surface has a smooth and structurally complete surface morphology, it is preliminarily determined to be squid ink powder.

[0019] In one embodiment of the present invention, in step S3, if the sample is observed to have an irregular angular and flaky fragment structure at any magnification from 500x to 10000x, it is preliminarily determined to be plant carbon black.

[0020] In one embodiment of the present invention, in step S3, if a rounded spherical structure and an irregular angular debris structure are observed to coexist at any magnification from 500x to 10000x, it is preliminarily determined that the squid ink powder sample is doped with plant carbon black.

[0021] Compared with existing technologies, the qualitative identification method for squid ink powder based on microstructure analysis according to the present invention effectively removes water-soluble excipients such as maltodextrin through pretreatment including washing, centrifugation, and freeze-drying, preventing them from masking the true morphology of the squid ink powder. To address the issue of poor sample conductivity, gold sputtering is used to eliminate the charging effect, ensuring clear imaging under scanning electron microscopy. The determination is based on differences in micromorphology: rounded spheres or shell-like structures with smooth surfaces are squid ink powder, while irregularly shaped or large fragments are vegetable carbon black; the latter, due to its inherent inorganic form, is difficult to process into nanoscale regular particles at low cost, making counterfeiting impractical. This method overcomes the technical blind spot of traditional detection methods due to matrix interference, achieving accurate and efficient identification by utilizing the irreversible differences in the physical morphology of substances. Attached Figure Description

[0022] Figure 1 This is a scanning electron microscope (SEM) image of the pure cuttlefish ink powder from Zhongda Hengyuan according to Example 1 of the present invention, under a scanning electron microscope at 500x magnification (200μm).

[0023] Figure 2 This is a scanning electron microscope (SEM) image of the pure cuttlefish ink powder from Zhongda Hengyuan according to Example 1 of the present invention, under a scanning electron microscope at 1000x magnification (100μm).

[0024] Figure 3 This is a scanning electron microscope (SEM) image of the pure cuttlefish ink powder from Zhongda Hengyuan according to Example 1 of the present invention, under a scanning electron microscope at 2000x magnification (50μm).

[0025] Figure 4 This is a scanning electron microscope (SEM) image of the pure cuttlefish ink powder from Zhongda Hengyuan according to Example 1 of the present invention, under a scanning electron microscope at 10,000x magnification (10μm).

[0026] Figure 5 This is a scanning electron microscope image of the pure plant carbon black product according to Example 2 of the present invention at 1000x magnification (100μm);

[0027] Figure 6 This is a scanning electron microscope image of the pure plant carbon black product according to Example 2 of the present invention at 2000x magnification (50μm);

[0028] Figure 7This is a scanning electron microscope (SEM) image of the pure plant carbon black product according to Example 2 of the present invention at 10,000x magnification (10 μm).

[0029] Figure 8 This is a scanning electron microscope (SEM) image of a compound sample containing 75% cuttlefish ink powder and 25% vegetable carbon black according to Example 3 of the present invention, under a scanning electron microscope at 1000x magnification (100μm).

[0030] Figure 9 This is a scanning electron microscope (SEM) image of a compound sample containing 75% cuttlefish ink powder and 25% vegetable carbon black according to Example 3 of the present invention, under a scanning electron microscope at 2000x magnification (50μm).

[0031] Figure 10 This is a scanning electron microscope (SEM) image of a compound sample containing 75% cuttlefish ink powder and 25% vegetable carbon black according to Example 3 of the present invention, under a scanning electron microscope at 10,000x magnification (10 μm).

[0032] Figure 11 This is a scanning electron microscope (SEM) image of a compound sample containing 97.5% cuttlefish ink powder and 2.5% vegetable carbon black according to Example 4 of the present invention, under a scanning electron microscope at 1000x magnification (100μm).

[0033] Figure 12 This is a scanning electron microscope (SEM) image of a compound sample containing 97.5% cuttlefish ink powder and 2.5% vegetable carbon black according to Example 4 of the present invention, under a scanning electron microscope at 2000x magnification (50μm).

[0034] Figure 13 This is a scanning electron microscope (SEM) image of a compound sample containing 97.5% cuttlefish ink powder and 2.5% vegetable carbon black according to Example 4 of the present invention, under a scanning electron microscope at 10,000x magnification (10 μm). Detailed Implementation

[0035] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0036] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated components without excluding other components.

[0037] like Figure 1 As shown, a qualitative identification method for cuttlefish ink powder based on microstructure analysis according to a preferred embodiment of the present invention includes the following steps:

[0038] Step S1: Take a sample of squid ink powder mixed with a water-soluble carrier, place it in water to fully dissolve the water-soluble carrier, and obtain a sample suspension with a concentration of 5 g / mL-15 g / mL.

[0039] Step S2: Centrifuge the sample suspension and discard the supernatant, elute the soluble excipients, and freeze-dry the insoluble precipitate to obtain a dried sample for later use;

[0040] Step S3: Sputter gold onto the dried, ready-to-use sample and place it in a scanning electron microscope to observe the differences in microstructure and surface morphology.

[0041] Specifically, water-soluble carriers or excipients such as maltodextrin, soluble starch, sucrose, glucose, salt, and cyclodextrin are often added during the processing of squid ink powder to improve its flowability, dispersibility, moisture resistance, or as fillers. When placed in water, these water-soluble components can selectively dissolve, allowing the insoluble characteristic particles in the squid ink powder to be released and dispersed from the carrier matrix.

[0042] In this embodiment, the pretreatment steps of washing, centrifugation, and freeze-drying achieve preliminary separation of the water-soluble carrier and the target particles, effectively eliminating interference caused by water-soluble excipients such as maltodextrin embedding the core pigment. This successfully constructs a microscopic model of the sample, preventing the formation of continuous films, clumps, or crystals by the water-soluble excipients during subsequent observation, which could obscure the true morphology of the squid ink particles. Squid ink powder mainly contains organic components and has poor conductivity. In a scanning electron microscope, when an electron beam irradiates a non-conductive sample, negative charges accumulate on the surface, resulting in a charging effect that causes the image to brighten, drift, and decrease in resolution, or even become unobservable. Sputtering the sample with gold or palladium can form a uniform conductive film on the particle surface, effectively conducting away electrons and reducing the charging phenomenon. Subsequently, the sample is placed under a scanning electron microscope, allowing observation of the particle's shape, size, surface texture, pore structure, layered or sheet-like structure, and aggregation state at different magnifications. If the sample exhibits a rounded spherical or shell-like regular structure with a smooth and structurally complete surface, it is preliminarily identified as containing squid ink powder. If it has an irregular, angular, or large, fragmented microstructure, it is preliminarily identified as containing vegetable carbon black. Vegetable carbon black is an inorganic material that naturally exhibits irregular, angular shapes. To transform it into a nanoscale, rounded, and regular shape like squid ink powder through physical processing requires special treatment, the cost of which is far higher than that of food additive-grade squid ink powder. From a market counterfeiting perspective, this is not feasible, further solidifying the accuracy of the identification. This method overcomes the technical blind spots of traditional detection methods that fail due to the poor solubility of large molecules and complex matrices. By utilizing the irreversible differences in the microscopic morphology of substances in their physical genesis, it achieves unique and highly efficient precise identification.

[0043] The concentration of the sample suspension is 5 g / mL-15 g / mL, which ensures a balance between the complete dissolution of the water-soluble carrier and the acquisition of sufficient insoluble squid ink powder. This ensures the water-soluble carrier dissolves completely, reducing surface residue of insoluble particles; avoids incomplete dissolution due to carrier oversaturation or excessive viscosity; and creates favorable conditions for subsequent centrifugation to remove soluble excipients. It also facilitates ultrasonic cavitation and particle dispersion, resulting in more complete release of characteristic squid ink particles; uniform particle dispersion in the solution without clumping; and allows for relatively shorter ultrasonic time, reducing potential damage to the particle microstructure. Furthermore, it ensures effective subsequent centrifugation, with clear separation of the supernatant and precipitate after centrifugation; complete particle sedimentation with minimal target loss; easy redispersibility of the precipitate during washing; and more thorough removal of soluble excipients. Finally, it enriches sufficient insoluble particles for SEM observation, with ample precipitate after centrifugation to meet the requirements for freeze-drying and SEM sample preparation; allows for the observation of more characteristic particles under SEM, improving identification accuracy; and reduces the need for repeated experiments due to insufficient sample volume. It balances ease of operation and repeatability, making the experiment convenient and highly reproducible; the conditions of each step are easy to control, which is conducive to method standardization; and it reduces errors caused by differences in operation.

[0044] In one embodiment of the present invention, in step S1, the water-soluble carrier includes one or more of maltodextrin and gum arabic.

[0045] In this embodiment, both maltodextrin and gum arabic have good water solubility, but their dissolution behaviors differ slightly: maltodextrin is a mixture of oligosaccharides / polysaccharides, readily soluble in water, forming a transparent solution with low or medium viscosity. Gum arabic is a hydrophilic polymer colloid that dissolves in water to form a viscous, transparent liquid with strong film-forming and thickening properties.

[0046] In this embodiment, maltodextrin and gum arabic gradually dissociate from the surface and interparticle spaces of the squid ink powder particles, initially disrupting the encapsulation structure between the carrier and the squid ink powder. They dissolve into the aqueous phase, releasing the insoluble squid ink powder particles that were previously encapsulated or adhered to, thus creating conditions for subsequent centrifugation. This prevents the formation of continuous films, transparent hard shells, or colloidal agglomerations during subsequent drying or observation due to undissolved carrier, which could obscure the true morphology of the squid ink powder.

[0047] In one embodiment of the present invention, step S1, the step of placing the water-soluble carrier in water to fully dissolve it includes: performing ultrasonication at a power of 450W-550W for more than 5 minutes to fully dissolve the water-soluble carrier.

[0048] Specifically, a power range of 450W-550W provides sufficient ultrasonic energy to achieve a significant cavitation effect without being excessively high. Too low a power may result in insufficient cavitation and incomplete dissolution of the carrier; too high a power may generate excessive shear or localized overheating, damaging the original microstructure of the insoluble squid ink particles.

[0049] Complete hydration and dissolution of colloids such as gum arabic requires a certain amount of time; maltodextrin may also require a long time to disperse fully at high concentrations. Sonication for more than 5 minutes ensures complete dissolution of the carrier, especially for samples containing gum arabic, ensuring sufficient swelling and dissociation of the colloid and preventing residue.

[0050] In this embodiment, the cavitation effect, mechanical vibration, and micro-jet action of ultrasound are used to quickly and fully dissolve the water-soluble carrier and peel it off from the surface of the squid ink powder. The insoluble characteristic particles in the squid ink powder are effectively released and dispersed in the solution. This reduces carrier residue and particle agglomeration, creating favorable conditions for subsequent centrifugation. While ensuring dissolution efficiency, the microstructure of the squid ink powder particles is minimized.

[0051] In one embodiment of the present invention, step S2, which involves centrifuging the sample suspension and discarding the supernatant to elute the soluble excipients, includes: centrifuging at 4500 r / min-5500 r / min for more than 10 minutes, discarding the supernatant, and repeating the steps of adding water and centrifuging to elute the soluble excipients.

[0052] Specifically, centrifugation at 4500 r / min-5500 r / min, within the rotor radius of a conventional benchtop centrifuge, generates sufficient centrifugal force to ensure that insoluble particles in squid ink settle completely within 10 minutes. If the speed is too low, small or low-density squid ink particles may not settle completely and may remain in the supernatant, resulting in the loss of the target component. Simultaneously, the supernatant will become turbid, affecting the removal of the carrier. If the speed is too high, the particles will be excessively compacted, resulting in tightly packed precipitates that are difficult to redisperse with water, potentially affecting the washing effect. Furthermore, excessive shear force may also damage the surface structure of the particles.

[0053] The insoluble particles of squid ink powder may have a wide particle size distribution, and some fine particles settle slowly. Centrifugation for more than 10 minutes can ensure that the vast majority of target particles settle to the bottom of the tube, reducing sample loss due to insufficient centrifugation time.

[0054] After centrifugation, the dissolved maltodextrin, gum arabic, and other soluble excipients are mainly found in the supernatant. Discarding the supernatant removes most of the water-soluble impurities. After the first centrifugation and discarding of the supernatant, a small amount of soluble excipients will still remain in the precipitate. Repeatedly adding water and centrifuging is equivalent to washing, diluting, and settling the precipitate multiple times. Each time water is added, the remaining soluble excipients in the precipitate are redissolved and diluted. After another centrifugation, these soluble excipients are discarded with the supernatant. After repeating this process 2-3 times, the content of soluble excipients decreases significantly with increasing dilution factor, and the purity of the precipitate is significantly improved.

[0055] In this embodiment, the density difference between insoluble squid ink particles and dissolved excipients is utilized. Centrifugation causes the particles to settle, and the supernatant is discarded to effectively remove water-soluble carriers (maltodextrin, gum arabic, etc.) and soluble salts, sugars, and other excipients from the squid ink powder. This yields highly pure insoluble squid ink powder particles; it also prevents the carrier from forming continuous films, glassy shells, or amorphous lumps during subsequent drying and SEM observation, thus preventing them from obscuring the true morphology of the squid ink powder particles; and the centrifugation conditions are gentle, ensuring separation efficiency while preserving the original particle size, shape, and surface structure of the squid ink powder particles to the maximum extent.

[0056] In one embodiment of the present invention, in step S1

[0057] In one embodiment of the present invention, in step S3, after gold sputtering, the surface of the sample is dried to obtain a conductive gold film of 5nm-15nm.

[0058] In this embodiment, while improving sample conductivity and eliminating the charging effect, the true microstructure of the squid ink particle surface is preserved as much as possible, avoiding the obscuring of details or the generation of artifacts due to excessively thick metal films. It provides a uniform conductive path, eliminates the charging effect, and results in uniform and stable image brightness without drift or discharge streaks; clear images can be obtained under higher accelerating voltages and larger beam currents, improving resolution and signal-to-noise ratio. A thickness of 5nm-15nm is sufficient to form a continuous conductive film, creating a substantially continuous conductive layer on the sample surface; conductivity meets SEM observation requirements; the gold film is less prone to cracking or peeling, allowing for stable image imaging. It avoids the gold film being too thick and obscuring microscopic morphological details, preserving the original microstructure of the squid ink particle surface as much as possible; fine textures and pores can still be distinguished at high magnification; morphological distortion caused by excessively thick gold films is avoided, improving the accuracy of qualitative identification. It avoids insufficient conductivity or image instability caused by excessively thin gold films; the image is stable and can be repeatedly observed; higher resolution conditions can be used to obtain clearer particle morphology; and image artifacts caused by charging are reduced. It facilitates secondary electron emission, improves image quality, and enhances the image signal-to-noise ratio; it also makes the surface morphology contrast of particles more obvious; and it facilitates the observation and recording of microstructural differences in squid ink particles. Under conditions of no carrier interference, no charge interference, and no morphological distortion, characteristic SEM images of squid ink particles are obtained; these images can be compared with standard samples or common adulterants for qualitative identification; the method has good repeatability and reliable results.

[0059] In one embodiment of the present invention, step S3, the gold sputtering process includes: at a temperature below 5 × 10⁻⁶. -4 Under a vacuum of Pa, the dried sample was sputtered with gold using a sputtering instrument. The working distance of the sputtering instrument to the dried sample was 5mm-10mm, and the sputtering angle was 30°-45°.

[0060] In this embodiment, a uniform, continuous, well-adhesive, and controllable-thickness conductive gold film is formed on the surface of squid ink particles, while avoiding damage to the heat-sensitive organic particles or obscuring their microstructure due to the gold spraying process.

[0061] Vacuum conditions below 5×10 -4 Pa, squid ink particles are organic samples, and their surfaces may adsorb small amounts of moisture or gas. High vacuum can reduce residual oxygen and water vapor, eliminate oxygen, water vapor, and impurity gases, and prevent gold atoms from being oxidized or contaminated during sputtering, thus improving the purity and conductivity of the gold film. The higher the vacuum level, the fewer gas molecules there are, and the lower the probability of gold atoms colliding with residual gas as they fly from the target to the sample. This extends the mean free path of the sputtered particles, maintaining higher energy and directionality, which is beneficial for forming a uniform and dense gold film. At low vacuum levels, residual gas may participate in the discharge, affecting the sputtering rate and film quality. Reaching 5 × 10⁻⁶ -4 A pressure below 1 Pa ensures stable sputtering conditions and a stable, repeatable working atmosphere when argon gas is subsequently introduced for sputtering.

[0062] The working distance refers to the distance between the gold target and the sample surface, controlled at 5-10 mm. The closer the distance, the greater the density of sputtered gold atoms reaching the sample surface, resulting in a higher deposition rate. A short distance of 5-10 mm allows for obtaining the required thickness in a shorter time, reducing thermal damage to the sample from prolonged sputtering and ensuring a high deposition rate. At an appropriate distance, gold atoms are sputtered from the target and distributed at a certain angle to the sample surface. Too far a distance leads to a greater difference in thickness between the edge and the center; too close a distance may cause localized over-thickness due to uneven target etching. 5-10 mm is usually combined with sample stage rotation or tilting to ensure more uniform coverage of all parts of the particles, improving film uniformity. Squid ink powder is an organic substance and is not heat-resistant. Too close a working distance will subject the sample to the radiation heat from the target and the kinetic energy of the sputtered particles, leading to particle deformation, shrinkage, or surface damage. 5-10 mm is a safe distance that balances deposition efficiency and thermal impact.

[0063] Sputtering angles of 30°-45° are used. Cuttlefish ink insoluble particles have irregular shapes and surface pores, depressions, and layered structures. If sputtered perpendicularly, gold atoms mainly deposit on the top and convex parts of the particles, while depressions, sidewalls, and back surfaces may be insufficiently covered due to "shadowing," leading to localized non-conductivity. An angle of 30°-45° allows gold atoms to reach the surface from multiple directions, improving coverage of sidewalls and depressions and reducing the shadowing effect. A suitable angle, combined with sample stage rotation, ensures a more uniform distribution of gold atoms on the particle surface, avoiding the problem of excessively thick deposition in hilly areas and insufficiently thin deposition in depressions caused by perpendicular incident sputtering. During tilted sputtering, gold atoms are incident at a certain angle, resulting in a relatively milder direct impact on the sample surface, reducing damage from high-energy particles and helping to protect the fine structure of the organic particles.

[0064] In one embodiment of the present invention, the sputtering current of the sputtering instrument is 10mA-15mA, the gold sputtering time is 40s-90s, and the deposition rate is 0.1 nm / s-0.5 nm / s.

[0065] In this embodiment, a conductive gold film with moderate thickness, uniformity, good adhesion, and fine particles is formed on the surface of squid ink particles, while minimizing the damage to heat-sensitive organic particles during the gold spraying process and avoiding obscuring their microscopic morphological details.

[0066] Sputtering current is the current intensity of argon ions bombarding a gold target in a sputtering instrument, directly determining the sputtering yield and deposition rate of gold atoms. When the current is too low (<10 mA), the sputtering rate is slow, requiring a longer time to reach the target thickness. Prolonged sputtering can lead to continuous heating and particle bombardment of the sample, potentially causing squid ink powder particles to shrink, deform, or suffer surface damage. Simultaneously, the gold film may have a loose structure and poor adhesion due to the low deposition rate. When the current is too high (>15 mA), the sputtering rate is too fast, and gold atoms do not have enough time to migrate and align upon reaching the sample surface, easily forming coarse grains or island-like aggregates. This results in an uneven gold film with many pores, even exhibiting a grainy appearance that obscures sample surface details. High current also intensifies target heating and increases thermal radiation, causing thermal damage to organic samples.

[0067] A moderate current of 10-15 mA can provide a stable and moderate gold atom sputtering rate, allowing gold atoms sufficient time to migrate and form a film on the sample surface, resulting in a dense, continuous, and fine-grained gold film. It controls the deposition rate within a reasonable range, avoiding problems caused by being too fast or too slow, and reduces damage to squid ink particles from thermal radiation and particle bombardment, protecting their original microstructure.

[0068] The gold sputtering time is 40-90 s, and this time determines the total deposition thickness of the gold film. At a constant deposition rate, a longer time results in a thicker film. A sputtering time of 40-90 s ensures the gold film reaches a sufficient thickness (approximately 5-15 nm), forming a continuous conductive layer; avoids island-like discontinuities and localized charging caused by too short a time; and avoids excessively thick gold films and blurred surface details caused by too long a time. While ensuring conductivity, it also minimizes the sample's heating time, protecting organic particles.

[0069] The deposition rate is 0.1-0.5 nm / s, and it is the result of the combined effects of parameters such as sputtering current, vacuum level, working distance, and target condition. This rate range is considered low-speed sputtering. At rates too low (<0.1 nm / s), sputtering efficiency is low, requiring a long time to reach the target thickness. Prolonged exposure of the sample to plasma and thermal radiation may cause thermal damage, and at low rates, gold atoms have poor migration ability, potentially resulting in a porous film. At rates too high (>0.5 nm / s), gold atoms reach the surface too quickly, lacking sufficient time for migration and orderly arrangement, easily forming coarse grains, columnar structures, or island-like aggregates. This leads to a rough, uneven gold film, even obscuring the fine textures and pores of the sample surface. High rates are also accompanied by stronger thermal effects and particle impact. A moderate deposition rate of 0.1-0.5 nm / s allows gold atoms sufficient time to diffuse and migrate on the sample surface, forming a uniform, dense, and smooth gold film; avoids artifacts caused by coarse gold particles and preserves the microscopic details of the squid ink particle surface; reduces heat accumulation and particle bombardment damage; and, when combined with a time of 40-90 s, stably obtains an ideal film thickness of 5-15 nm.

[0070] In one embodiment of the present invention, in step S3, the differences in microstructure and surface morphology are observed at magnifications of 500x, 1000x, 2000x and 10000x respectively.

[0071] In this embodiment, scanning electron microscopy is used at a series of magnifications from low to high, namely 500x, 1000x, 2000x and 10000x, to observe the morphological information of squid ink particles step by step from the whole to the part, from the macroscopic distribution to the microscopic details, so as to make more comprehensive and reliable qualitative identification.

[0072] In one embodiment of the present invention, in step S3, the differences in microstructure and surface morphology are observed at magnifications of 200x, 500x, 1000x, 3000x and 5000x respectively.

[0073] In one embodiment of the present invention, step S3, the step of observing the differences in microstructure and surface morphology includes: if it has a regular structure that presents a rounded spherical shape or a shell-like shape and a smooth and structurally complete surface morphology, it is initially determined to be squid ink powder.

[0074] In one embodiment of the present invention, in step S3, if the sample is observed to have an irregular angular and flaky fragment structure at any magnification from 500x to 10000x, it is preliminarily determined to be plant carbon black.

[0075] In one embodiment of the present invention, in step S3, if a rounded spherical structure and an irregular angular debris structure are observed to coexist at any magnification from 500x to 10000x, it is preliminarily determined that the squid ink powder sample is doped with plant carbon black.

[0076] Specifically, if the microstructure exhibits a rounded spherical shape or a regular, shell-like structure, with a relatively smooth surface and highly intact structure, it is identified as natural squid ink powder. If the microstructure mainly shows irregular angular shapes and large, flaky fragments, it is identified as vegetable carbon black. If a mixture of spherical and fragmented structures is observed in the field of view, it is determined that the squid ink powder sample is adulterated with vegetable carbon black.

[0077] In this embodiment, plant carbon black is an inorganic material that naturally has irregular angular shapes. If it is to be transformed into a rounded and regular shape at the nanoscale like squid ink powder through physical processing, special treatment is required. Its cost is much higher than that of food additive grade squid ink powder, which is not feasible from the perspective of market counterfeiting logic, further consolidating the accuracy of the judgment.

[0078] Example 1

[0079] 1. Take 3g of pure cuttlefish ink powder produced by Henan Zhongda Hengyuan Biotechnology Co., Ltd. into a 50mL centrifuge tube, add 25mL of water, and sonicate at 500W power for 5min. Manually shake once every 1min to fully dissolve and obtain a sample suspension.

[0080] 2. Centrifuge the sample suspension at 5000 r / min for 10 min, discard the supernatant, repeat the steps of adding water and centrifugation 3 times, take the insoluble precipitate and freeze-dry it to obtain the dried sample for later use;

[0081] 3. Below 5×10 -4 Under a vacuum of Pa, a gold sputtering process was performed on the dried sample. The working distance of the sputtering instrument to the dried sample was 8 mm, the sputtering angle was 40°, the sputtering current was 12 mA, and the gold sputtering time was 60 s, resulting in a deposition rate of 0.3 nm / s.

[0082] Placed in a scanning electron microscope, with an accelerating voltage of 5.00 kV, the microstructure and surface morphology differences were observed at magnifications of 500x (200 μm), 1000x (100 μm), 2000x (50 μm), and 10000x (10 μm). Figures 1 to 4 As shown, the squid ink powder exhibits a rounded, spherical shape, a shell-like structure, and a complete structure under different magnifications.

[0083] Example 2

[0084] 1. Take 3g of pure plant carbon black into a 50mL centrifuge tube, add 25mL of water, and sonicate at 500W for 5min, manually shaking once every 1min to fully dissolve and obtain a sample suspension.

[0085] 2. Centrifuge the sample suspension at 5000 r / min for 10 min, discard the supernatant, repeat the steps of adding water and centrifugation 3 times, take the insoluble precipitate and freeze-dry it to obtain the dried sample for later use;

[0086] 3. Below 5×10 -4 Under a vacuum of Pa, a gold sputtering process was performed on the dried sample. The working distance of the sputtering instrument to the dried sample was 8 mm, the sputtering angle was 40°, the sputtering current was 12 mA, and the gold sputtering time was 60 s, resulting in a deposition rate of 0.3 nm / s.

[0087] Placed in a scanning electron microscope, with an accelerating voltage of 5.00 kV, the microstructure and surface morphology differences were observed at magnifications of 1000x (100 μm), 2000x (50 μm), and 10000x (10 μm), respectively. Figures 5 to 7 As shown, even when magnified to the 10-micrometer level, plant carbon black still appears as irregular angular and large flakes with significant angular features on the cut surface, which is a fundamental difference in microscopic morphology from the pure cuttlefish ink powder from Zhongda Hengyuan in Example 1.

[0088] Example 3

[0089] 1. By weight fraction, prepare 5g of a compound sample containing 75% cuttlefish ink powder and 25% vegetable carbon black in a 50mL centrifuge tube, add 30mL of water, and sonicate at 450W for 5min, manually shaking once every 1min to ensure complete dissolution and obtain a sample suspension.

[0090] 2. Centrifuge the sample suspension at 4500 r / min for 10 min, discard the supernatant, repeat the steps of adding water and centrifugation 3 times, take the insoluble precipitate and freeze-dry it to obtain the dried sample for later use.

[0091] 3. Below 5×10 -4 Under a vacuum of Pa, a gold sputtering process was performed on the dried sample. The working distance of the sputtering instrument to the dried sample was 5 mm, the sputtering angle was 30°, the sputtering current was 10 mA, and the gold sputtering time was 40 s, resulting in a deposition rate of 0.1 nm / s.

[0092] Placed in a scanning electron microscope, with an accelerating voltage of 5.00 kV, the microstructure and surface morphology differences were observed at magnifications of 1000x (100 μm), 2000x (50 μm), and 10000x (10 μm), respectively. Figures 8 to 10 As shown, the physical mixing characteristics of the two substances were clearly observed under an electron microscope. Within the field of view, round spherical and irregular carbon black debris coexisted stably, effectively constructing a microscopic comparison model of adulterated positive samples.

[0093] Example 4

[0094] 1. By weight fraction, prepare 2g of a compound sample containing 97.5% cuttlefish ink powder and 2.5% vegetable carbon black in a 50mL centrifuge tube, add 25mL of water, and sonicate at 550W for 5min, manually shaking once every 1min to ensure complete dissolution and obtain a sample suspension.

[0095] 2. Centrifuge the sample suspension at 5500 r / min for 10 min, discard the supernatant, repeat the steps of adding water and centrifugation 3 times, take the insoluble precipitate and freeze-dry it to obtain the dried sample for later use.

[0096] 3. Below 5×10 -4 Under a vacuum of Pa, a gold sputtering process was performed on the dried sample. The working distance of the sputtering instrument to the dried sample was 10 mm, the sputtering angle was 45°, the sputtering current was 15 mA, and the gold sputtering time was 90 s, resulting in a deposition rate of 0.5 nm / s.

[0097] Placed in a scanning electron microscope, with an accelerating voltage of 5.00 kV, the microstructure and surface morphology differences were observed at magnifications of 1000x (100 μm), 2000x (50 μm), and 10000x (10 μm), respectively. Figures 11 to 13 As shown, the physical mixing characteristics of the two substances were clearly observed under an electron microscope. Within the field of view, round spherical and irregular carbon black debris coexisted stably, effectively constructing a microscopic comparison model of adulterated positive samples.

[0098] The description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A qualitative identification method for cuttlefish ink powder based on microstructure analysis, characterized in that, Includes the following steps: Step S1: Take a sample of squid ink powder mixed with a water-soluble carrier, place it in water to fully dissolve the water-soluble carrier, and obtain a sample suspension with a concentration of 5 g / mL to 15 g / mL. Step S2: Centrifuge the sample suspension and discard the supernatant, wash off the soluble excipients, and freeze-dry the insoluble precipitate to obtain a dried sample for later use; Step S3: Spray gold onto the dried sample and place it in a scanning electron microscope to observe the differences in microstructure and surface morphology.

2. The qualitative identification method for cuttlefish ink powder based on microstructure analysis as described in claim 1, characterized in that, In step S1, the water-soluble carrier includes one or more of maltodextrin and gum arabic.

3. The qualitative identification method for cuttlefish ink powder based on microstructure analysis as described in claim 1, characterized in that, In step S1, the step of placing the water-soluble carrier in water to fully dissolve it includes: performing ultrasound at a power of 450W-550W for more than 5 minutes to fully dissolve the water-soluble carrier.

4. The qualitative identification method for cuttlefish ink powder based on microstructure analysis as described in claim 1, characterized in that, In step S2, the step of centrifuging the sample suspension to discard the supernatant and elute the soluble excipients includes: centrifuging at 4500 r / min-5500 r / min for more than 10 minutes, discarding the supernatant, and repeating the steps of adding water and centrifuging to elute the soluble excipients.

5. The qualitative identification method for cuttlefish ink powder based on microstructure analysis as described in claim 1, characterized in that, In step S3, after the gold sputtering treatment, a 5nm-15nm conductive gold film is obtained on the surface of the dried standby sample.

6. The qualitative identification method for cuttlefish ink powder based on microstructure analysis as described in claim 5, characterized in that, In step S3, the gold sputtering process includes: at a temperature below 5×10 -4 Under a vacuum of Pa, the dried sample is sputtered with gold using a sputtering instrument. The working distance of the sputtering instrument to the dried sample is 5mm-10mm, and the sputtering angle is 30°-45°.

7. The qualitative identification method for cuttlefish ink powder based on microstructure analysis as described in claim 6, characterized in that, The sputtering current of the sputtering instrument is 10mA-15mA, and the gold sputtering time is 40s-90s, resulting in a deposition rate of 0.1 nm / s-0.5 nm / s.

8. The qualitative identification method for cuttlefish ink powder based on microstructure analysis as described in claim 1, characterized in that, In step S3, the differences in microstructure and surface morphology are observed at magnifications of 500x, 1000x, 2000x and 10000x respectively.

9. The qualitative identification method for cuttlefish ink powder based on microstructure analysis as described in claim 1, characterized in that, In step S3, the differences in microstructure and surface morphology are observed at magnifications of 200x, 500x, 1000x, 3000x and 5000x respectively.

10. The qualitative identification method for cuttlefish ink powder based on microstructure analysis as described in claim 1, characterized in that, In step S3, if the sample is observed to have a rounded spherical shape or a regular structure resembling a shell at any magnification from 500x to 10000x, and the particle surface has a smooth and structurally complete surface morphology, it is preliminarily determined to be squid ink powder.

11. The qualitative identification method for cuttlefish ink powder based on microstructure analysis as described in claim 1, characterized in that, In step S3, if the sample is observed to have an irregular angular and flaky fragment structure at any magnification from 500x to 10000x, it is preliminarily determined to be plant carbon black.

12. The qualitative identification method for cuttlefish ink powder based on microstructure analysis as described in claim 1, characterized in that, In step S3, if a rounded spherical structure and an irregular angular debris structure are observed to coexist at any magnification from 500x to 10000x, it is preliminarily determined that the squid ink powder sample is doped with plant carbon black.

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