Tussah preservation method

Abstract

The invention provides an antheraea pernyi fresh-keeping method which comprises the following steps: S1, dividing a first group and a second group with different freezing response characteristics according to development biological characteristics of antheraea pernyi; the freezing response characteristic is represented by at least one parameter of tissue moisture content, methylene blue penetration depth and body wall elasticity modulus; s2, differential preprocessing is applied to the first group and the second group, so that the difference of the first group and the second group on any characterization parameter does not exceed a preset threshold value; s3, combining the two groups of tussah and freezing; in the first cooling stage, cooling from 0-1 DEG C to-10 DEG C at the speed of 0.8-1.2 DEG C / min; in the second cooling stage, cooling is conducted from-10 DEG C to-30 DEG C at the speed of 0.45-0.55 DEG C / min; in the third cooling stage, cooling from-30 DEG C to-45 DEG C at the speed of 1.8-2 DEG C / min; and S4, after freezing is completed, vacuum packaging is performed, and storage is performed at minus 45-minus 40 DEG C. According to the fresh-keeping method for the tussah, the integrity of a cell structure is maintained, juice loss is reduced, and abnormal release of endogenous enzyme is inhibited, so that nutritional ingredients are better reserved in the long-term freezing storage process, and the sensory characteristics of compact meat, uniform color and luster and no peculiar smell are maintained.

Description

Technical Field

[0001] This application relates to the field of food preservation technology, and in particular to a method for preserving tussah silkworms. Background Technology

[0002] Tussah silkworm larvae are a unique insect resource, high in protein, low in fat, rich in essential amino acids, and abundant in various minerals. Fresh 5th instar larvae can contain over 28 g / 100g of crude protein and over 25 g / 100g of total amino acids, with extremely low fat content, making them an excellent edible insect with high nutritional value. In recent years, with the large-scale promotion of *Artemisia annua*, a new feed resource for tussah silkworms, in western Liaoning and eastern Inner Mongolia, the annual yield of 5th instar larvae has continued to increase. However, the harvesting of 5th instar larvae is highly concentrated around September each year, resulting in vigorous post-harvest physiological metabolism and a shelf life of less than 48 hours at room temperature, severely restricting their cross-regional distribution and off-peak sales capabilities.

[0003] In existing technologies, a quick-freezing process is used to freeze 5th instar larvae of tussah silkworms: after harvesting, the 5th instar larvae are simply washed and drained, then directly placed in a freezing device at -18 to -22°C for one-time freezing, and subsequently transferred to a cold storage at the same temperature. While this method is simple to operate, the lack of precise control during the freezing process easily leads to the formation of large, unevenly distributed ice crystals in the tissue, causing severe damage to cell membranes and tissue structures. This results in significant juice loss after thawing, accelerated degradation of nutrients (such as proteins and amino acids), increased risk of microbial growth, and a marked deterioration in sensory quality (including texture, color, and flavor). Due to these limitations, the actual shelf life of this method is usually no more than 3 months, making it difficult to meet the requirements for long-term storage and high-quality preservation.

[0004] Therefore, existing freezing and preservation technologies for tussah silkworm larvae cannot effectively maintain the cellular structure integrity of tussah silkworms while achieving long-term (more than 12 months) freezing and storage, resulting in the loss of nutrients, increased risk of microbial safety, and deterioration of sensory quality and taste, thus reducing their value. Summary of the Invention

[0005] The purpose of this application is to provide a method for preserving tussah silkworms, so as to effectively maintain the cellular structure integrity of tussah silkworms while achieving long-term (more than 12 months) frozen storage, thereby ensuring the retention rate of its nutrients, microbial safety and sensory quality.

[0006] To address the aforementioned technical problems, this application provides the following technical solutions: The first aspect of this application provides a method for preserving tussah silkworms, comprising the following steps: Based on the developmental biological characteristics of the tussah silkworm, S1 divides the tussah silkworm into a first group and a second group with different freezing response characteristics; the freezing response characteristics are defined as the degree of ice crystal damage to the cellular tissue structure of the tussah silkworm under the same freezing conditions; the freezing response characteristics are characterized by at least one of the following parameters: tissue water content, methylene blue penetration depth, and body wall elastic modulus. S2 applies differential preprocessing to the first group and the second group respectively, so that the difference between the two on any characterization parameter does not exceed a preset threshold; S3 combines the two groups of silkworms that have undergone the differentiated pretreatment and freezes them using the same three-stage gradient freezing procedure; wherein... First cooling stage: Cooling from 0~1℃ to -10℃ at a cooling rate of 0.8~1.2℃ / min; The second cooling stage: cooling from -10℃ to -30℃ at a cooling rate of 0.45~0.55℃ / min; The third cooling stage: cooling from -30℃ to -45℃ at a cooling rate of 1.8~2℃ / min; After S4 is frozen, it is vacuum-packed and stored at -45~-40℃.

[0007] In some modified embodiments of the first aspect of this application, the freezing response characteristics of the first group are higher than those of the second group; The pretreatment of the first group includes: air drying at 2-4°C for 8-12 min, followed by soaking in a first preservation solution at 2-4°C with a pH of 5.0-5.5 for 10-20 min; The pretreatment of the second group includes: wetting with 0.3-0.7% citric acid solution in an environment of 2-5℃, and then soaking in a second preservation solution with pH 4.0-4.5 at 2-5℃ for 25-35 min; The first preservative solution, by weight, includes: 0.15-0.25 parts chitosan, 0.08-0.12 parts tea polyphenols, 0.03-0.06 parts ascorbic acid, and 100 parts sterile water; The second preservative solution, by weight, includes: 0.05-0.12 parts chitosan, 0.12-0.18 parts tea polyphenols, 0.05-0.07 parts ascorbic acid, 0.02-0.04 parts citric acid, and 100 parts sterile water.

[0008] In some embodiments, a standardization preprocessing step is included prior to step S1: Select live tussah silkworms, rinse them with sterile water at 3-5℃, and then drain off the surface moisture. After draining, place the tussah silkworms in an environment of -2℃ for 30~60 minutes to pre-cool them until the surface temperature of the silkworms drops to 0~1℃.

[0009] In some embodiments, the preset threshold satisfies at least one of the following: The absolute value of the difference in tissue moisture content between the two groups of tussah silkworms is ≤3%; The absolute value of the difference in methylene blue penetration depth between the two groups of silkworms was ≤20 μm; The absolute value of the difference in body wall elastic modulus between the two groups of tussah silkworms is ≤0.05 MPa.

[0010] In some embodiments, the three-stage gradient freezing procedure further includes: After cooling to -10°C in the first cooling stage, maintain the temperature at -10°C for 20~40 min; After cooling to -30°C in the second cooling stage, maintain the temperature at -30°C for 45~75 minutes. After cooling to -45°C in the third cooling stage, the temperature is maintained at -45°C for 75~105 min.

[0011] In some embodiments, the method further includes the following steps prior to step S1: Multiple tussah silkworm samples were collected, and the developmental biological characteristics and corresponding freezing response characteristic parameters of each tussah silkworm sample were measured. The developmental biological characteristics included body length, head shell width, and body wall transmittance, and the freezing response characteristic parameters included tissue water content, methylene blue penetration depth, and body wall elastic modulus. Based on the measured data, a mapping relationship is established between the developmental biological characteristics and the freezing response characteristic characterization parameters to obtain a prediction model; the prediction model is used to predict the freezing response characteristics of the tussah silkworms to be classified according to the developmental biological characteristics, and to classify the tussah silkworms to be classified into the first group or the second group accordingly.

[0012] In some embodiments, step S1 includes: The body length, head shell width, and body wall transmittance of the tussah silkworms to be classified were measured. The prediction model was used to predict the tissue water content, methylene blue penetration depth, and body wall elastic modulus of the silkworm to be divided. If the predicted value meets any of the following conditions: tissue water content ≥ 84%, methylene blue penetration depth ≥ 150 μm, body wall elastic modulus ≤ 0.25 MPa, then its freezing response characteristics are considered high and it is classified as the first group. Otherwise, it is determined that its freezing response characteristics are low and it is classified into the second group.

[0013] In some embodiments, before step S1 and 6-12 hours before the differential pretreatment, the silkworms are placed in a closed environment and fumigated with a gas mixture. The gas mixture contains methyl jasmonate or salicylic acid, the volume concentration of methyl jasmonate or salicylic acid is 10~50 ppm, the fumigation temperature is 15~25℃, and the fumigation time is 1~2 h.

[0014] In some embodiments, the vacuum packaging is performed in an environment not exceeding -10°C.

[0015] In some embodiments, after step S4 is completed and before consumption, a three-stage gradient reheating process is further included: First rewarming stage: Heat from -45~-40℃ to -1℃ at a heating rate of 0.8~1.2℃ / min, and hold at -1℃ for 25~35 min; The second rewarming stage: the temperature is increased from -1℃ to 4℃ at a heating rate of 0.05~0.1℃ / min, and held at 4℃ for 45~55 min; The third rewarming stage: the temperature is increased from 4℃ to 12℃ at a heating rate of 0.3~0.6℃ / min, and held at 12℃ for 10~15 min.

[0016] Compared with existing technologies, the tussah silkworm preservation method provided in this application firstly groups the raw materials efficiently based on the rapidly observable external developmental characteristics of tussah silkworms (such as body length, head shell width, body wall light transmittance, etc.) and their known correlation with freezing response characteristics, providing a reliable basis for subsequent differentiated pretreatment.

[0017] Subsequently, differentiated pretreatments were applied to different groups to proactively adjust the key physiological states of each group (characterized by water content, methylene blue penetration depth, and / or body wall elastic modulus parameters) to a convergent state (differences not exceeding a preset threshold). This "identification first, convergence later" pre-processing eliminated the inherent heterogeneity of the materials at the source, enabling the subsequent uniform three-stage gradient freezing procedure to apply appropriate freezing stress to groups with consistent states—avoiding both the compression of extracellular ice crystals in highly sensitive individuals due to slow freezing and the puncture of intracellular ice crystals in low-sensitivity individuals due to rapid freezing. This not only significantly improved the quality uniformity among individuals within a batch but also ensured that each individual reached the optimal protection state under this freezing process, thereby effectively maintaining the integrity of the tissue structure after thawing.

[0018] The three-stage gradient freezing program is a refined design based on the physical laws of ice crystal formation and growth in different temperature ranges: The first cooling stage uses a moderately fast rate (0.8~1.2℃ / min) to promote the simultaneous freezing of intracellular and extracellular water and inhibit the formation of large extracellular ice crystals; the second cooling stage significantly slows down the rate (0.45~0.55℃ / min), allowing ice crystals to grow in an orderly manner in the cryogenic transition zone (-10~-30℃), alleviating the mechanical stress caused by ice crystal expansion and tissue contraction, making ice crystal growth more orderly, and avoiding the accumulation of microcracks; the third cooling stage accelerates the cooling (1.8~2.0℃ / min) after the tissue has basically completed freezing, efficiently completing the final cooling and shortening the overall freezing time. This gradient strategy, which dynamically matches the phase transition process, works in conjunction with the homogenization of materials brought about by pretreatment to guide the formation of small and evenly distributed ice crystals. This minimizes ice crystal puncture and compression damage at the cellular and subcellular levels, effectively maintaining the integrity of cell membranes and body wall muscle tissue structures, significantly reducing juice loss, and ensuring firm texture and good flavor after thawing. This allows the product to maintain sensory quality close to that of fresh tussah silkworms even after long-term frozen storage.

[0019] The highly intact cell structure and uniform freezing state achieved through the aforementioned synergistic effects, combined with vacuum packaging and stable low-temperature storage at -45 to -40°C in step S4, create ideal conditions for long-term preservation: maintaining cell integrity significantly reduces juice loss and slows down the rate of endogenous enzymatic reactions; the vacuum environment effectively isolates oxygen, inhibiting lipid oxidation and flavor deterioration; and the stable deep low temperature maximally inhibits microbial proliferation and residual biochemical reactions. These measures work synergistically to delay quality deterioration from three dimensions: physical structure, chemical stability, and biological safety, significantly extending the product's shelf life and stability.

[0020] The tussah silkworm preservation method described in this application effectively maintains cell structure integrity, significantly reduces juice loss, and inhibits the abnormal release of endogenous enzymes. This allows for better preservation of nutrients such as proteins and amino acids during long-term (over 12 months) frozen storage, while maintaining the firm texture, uniform color, and odor-free sensory characteristics. The method features a standardized technical process, with controllable key process parameters (such as grouping criteria, pretreatment conditions, gradient cooling rate, and final storage temperature). It is suitable for industrial-scale mass production and can significantly reduce quality loss and spoilage risks during storage and transportation, providing reliable technical support for cross-regional distribution and off-peak sales. Attached Figure Description

[0021] The above and other objects, features, and advantages of exemplary embodiments of this application will become readily understood by reading the following detailed description with reference to the accompanying drawings. In the drawings, several embodiments of this application are illustrated by way of example and not limitation, with the same or corresponding reference numerals denoteing the same or corresponding parts, wherein: Figure 1 A flowchart illustrating a method for preserving tussah silkworms according to this application is shown schematically. Detailed Implementation

[0022] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0023] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application shall have the ordinary meaning as understood by one of ordinary skill in the art to which this application pertains.

[0024] like Figure 1 As shown, this application provides a method for preserving tussah silkworms, including the following steps: Based on the developmental biological characteristics of the tussah silkworm, S1 divides the tussah silkworm into a first group and a second group with different freezing response characteristics; the freezing response characteristics are defined as the degree of ice crystal damage to the cellular tissue structure of the tussah silkworm under the same freezing conditions; the freezing response characteristics are characterized by at least one of the following parameters: tissue water content, methylene blue penetration depth, and body wall elastic modulus.

[0025] Specifically, developmental biological characteristics refer to observable or measurable indicators that stably reflect the physiological state of tussah silkworms and are significantly correlated with their freezing response characteristics. Developmental biological characteristics have the advantages of being non-invasive, easy to operate, and quick to identify, and can be obtained rapidly without damaging the tussah silkworms (e.g., through image analysis), making them more suitable for online grouping operations in large-scale production.

[0026] Developmental biological characteristics can include instar stage, etc. Among them, the late fifth instar tussah silkworm is at the critical period of physiological maturity, with relatively high tissue water content and relatively loose body wall structure. Under the same freezing conditions, the methylene blue penetration depth is relatively deep, and the ice crystal sensitivity is strong (i.e., high freezing response characteristics). On the other hand, the fourth instar or early fifth instar tussah silkworm has relatively low tissue water content and relatively dense body wall structure, with relatively shallow methylene blue penetration depth and stronger freezing resistance (i.e., lower freezing response characteristics).

[0027] In some embodiments, a quick and simple method can be used to determine the instar of tussah silkworms for efficient grouping. Operators can quickly determine the instar by observing the external morphological characteristics of the silkworms: individuals at the end of the fifth instar have a bright jade-white body color, a semi-transparent body wall, and significantly enlarged abdominal segments, and move slowly or remain still; while individuals at the fourth or early fifth instar have a bluish-white body color, an opaque body wall, compact body segments, and are active in crawling. This method can be mastered with short-term training and is suitable for manual sorting lines. For highly automated production lines, machine vision systems can be used to analyze features such as body color, transparency, and body segment outlines to achieve automatic instar determination.

[0028] Freezing response characteristics can be quantified by one, two, or three of the following parameters: Tissue moisture content: The higher the tissue moisture content, the greater the proportion of free water, and the easier it is to form large, destructive ice crystals when frozen; Methylene blue penetration depth: The deeper the dye penetrates after thawing, the more severe the damage to the cell membrane and body wall integrity. Body wall elastic modulus: The lower the modulus, the looser the tissue structure and the weaker the resistance to ice crystal damage.

[0029] To achieve efficient, accurate, and non-destructive prediction of the freezing response characteristics of silkworms, in some embodiments, the following method is included before step S1: Multiple tussah silkworm samples were collected, and the developmental biological characteristics and corresponding freezing response characteristic parameters of each tussah silkworm sample were measured. The developmental biological characteristics included body length, head shell width, and body wall transmittance, and the freezing response characteristic parameters included tissue water content, methylene blue penetration depth, and body wall elastic modulus. Based on the measured data, a mapping relationship is established between the developmental biological characteristics and the freezing response characteristic characterization parameters to obtain a prediction model; the prediction model is used to predict the freezing response characteristics of the tussah silkworms to be classified according to the developmental biological characteristics, and to classify the tussah silkworms to be classified into the first group or the second group accordingly.

[0030] Specifically, this step involves building and validating an offline prediction model before performing step S1 (i.e., grouping the tussah silkworms to be divided). This process does not involve the actual grouping of tussah silkworms in production; it only provides a decision-making tool for subsequent online division.

[0031] For ease of distinction, the terminology used in this invention is as follows: Tussah silkworms: a general term for raw materials that need to be preserved; Tussah silkworm samples: refer to individual tussah silkworms used only for offline construction of prediction models. These samples require the measurement of three developmental biological characteristics and three freezing response characteristic characterization parameters and do not enter the formal preservation process.

[0032] Tussah silkworms to be divided: refers to fresh tussah silkworm individuals that need to be grouped in actual production, and the grouping is based on the output results of the prediction model.

[0033] First, a representative sample of tussah silkworms (e.g., 100-200 individuals) can be collected, covering different developmental biological characteristics. The developmental biological characteristics of each silkworm sample are measured to obtain corresponding developmental biological characteristic data. Specifically, body length can be measured along the midline of the back using a digital caliper; head shell width can be measured using a high-powered stereomicroscope with image calibration; and body wall transmittance can be obtained by placing the silkworm under a standard backlight, photographing it with an industrial camera, and calculating the image grayscale value. During the measurement process, each silkworm sample is non-destructively marked for subsequent identification.

[0034] Then, the three-stage gradient freezing procedure of this application was used to freeze the samples under the same freezing process parameters. After freezing, a uniform thawing process was used to thaw the samples, and the freezing response characteristics of each tussah silkworm sample were measured: tissue moisture content was measured by drying, methylene blue penetration depth was measured by soaking the sample in a methylene blue aqueous solution (0.1% by volume) for 30 min followed by transverse sectioning, and body wall elastic modulus was determined by microindentation. Thus, developmental biological characteristics data and corresponding freezing response characteristics data for each tussah silkworm sample were obtained.

[0035] Next, the paired data are input into a machine learning algorithm (such as multiple linear regression, random forest, or neural network) to construct a prediction model, which is used to predict the corresponding freezing response characteristic parameters based on developmental biological characteristics. In some embodiments, prediction sub-models for each characteristic parameter can be established separately (i.e., tissue water content prediction sub-model, methylene blue penetration depth prediction sub-model, and body wall elastic modulus prediction sub-model).

[0036] In some embodiments, the final determination of freezing response characteristics can be based on the prediction results of three characterization parameters. For example, a multi-parameter fusion strategy (such as weighted scoring or logical rules) can be used to comprehensively judge the level of freezing response characteristics, and then groups can be divided based on the level of freezing response characteristics. This division can be achieved manually or automatically. After the model training is completed and its effectiveness is verified, it is deployed in the production system. In actual preservation operations, when the live tussah silkworms to be divided arrive: their body length, head shell width, and body wall light transmittance are quickly measured (without freezing); the measured data are input into each trained prediction sub-model, which outputs the predicted values ​​of their tissue moisture content, methylene blue penetration depth, and body wall elastic modulus, respectively. Then, based on the predicted values ​​of the three parameters, a preset fusion strategy is used to determine the freezing response characteristics and perform the final group division.

[0037] In some embodiments, an end-to-end classification model can be used to directly output grouping results. The end-to-end classification model needs to be trained based on tussah silkworm samples with labeled grouping tags. Its input is developmental biological characteristics, and its output is the classification result of the first group or the second group.

[0038] To improve decision-making speed and reduce computational complexity, thereby enhancing automated production efficiency, in some embodiments, step S1 includes: The body length, head shell width, and body wall transmittance of the tussah silkworms to be classified were measured. The prediction model was used to predict the tissue water content, methylene blue penetration depth, and body wall elastic modulus of the silkworm to be divided. If the predicted value meets any of the following conditions: tissue water content ≥ 84%, methylene blue penetration depth ≥ 150 μm, body wall elastic modulus ≤ 0.25 MPa, then its freezing response characteristics are considered high and it is classified as the first group. Otherwise, it is determined that its freezing response characteristics are low and it is classified into the second group.

[0039] Specifically, since this judgment logic only involves numerical comparison and Boolean operations, it does not require complex model reasoning and can achieve microsecond-level response on ordinary industrial control equipment. Its computational overhead is far lower than methods such as multi-parameter weighting or neural network classification. At the same time, this judgment strategy prioritizes safety, effectively preventing highly sensitive individuals from being misclassified as low-sensitive, thereby ensuring the overall effectiveness of group preservation.

[0040] In actual production, to meet the needs of automated production and improve sorting efficiency, in some embodiments, the silkworms to be sorted can be arranged in a single row using a vibrating plate or guide trough and transported by a uniform-speed conveyor belt. Two imaging stations are set up along the conveying path. The first station is a backlit side-view imaging system: a highly uniform cold white backlight is set below the conveyor belt, and an industrial camera is set above the conveyor belt to capture a vertically downward image of the tussah silkworm; based on the image, the outline of the worm is extracted by an image processing algorithm to calculate the body length, and the average gray value of the worm area is analyzed to obtain the body wall transmittance. The second station is the head close-up imaging system: a high-magnification telecentric lens is set above the conveyor belt in conjunction with a ring LED light source, and an industrial camera is aimed at the head area of ​​the tussah silkworm to collect magnified images of the local area; the head shell position is automatically located by the target detection model, and the distance between its left and right edges is measured to obtain the head shell width data.

[0041] The measurement data from the two workstations are synchronously linked to the same individual tussah silkworm by time or location, forming a complete dataset of developmental biological characteristics for subsequent prediction and grouping of freezing response characteristics. Based on the grouping results output by the model, the control system can drive pneumatic actuators (such as pneumatic push rods or air nozzles) in real time to accurately guide the tussah silkworms into the first or second group of collection channels at the diversion point, achieving efficient and non-destructive automated sorting.

[0042] In some embodiments, a standardization preprocessing step is included prior to step S1: Select live tussah silkworms, rinse them with sterile water at 3-5℃, and then drain off the surface moisture. After draining, place the tussah silkworms in an environment of -2℃ for 30~60 minutes to pre-cool them until the surface temperature of the silkworms drops to 0~1℃.

[0043] Specifically, select live, undamaged silkworms, remove surface impurities and residual mulberry leaves, rinse them 2-3 times with sterile water at 3-5℃, and then drain the surface moisture. Place the drained silkworms in a -2℃ environment for 30-60 minutes to pre-cool them, so that the surface temperature of the silkworms drops to 0-1℃, thereby reducing their metabolic rate.

[0044] To ensure the stability of the measurement of developmental biological characteristics (especially body wall transmittance), the subsequent transport, imaging and grouping operations in step S1 can be carried out in a low temperature environment of 0~4℃ to prevent the body temperature of the silkworm from rising significantly.

[0045] S2 applies differential preprocessing to the first group and the second group respectively, so that the difference between the two in any characterization parameter does not exceed a preset threshold.

[0046] Specifically, any characterization parameter refers to a parameter used in actual operation to evaluate the freezing response characteristics; if a parameter is not measured or used for group determination, there is no need to set convergence requirements for it. For example, when grouping is based solely on tissue moisture content, it is only necessary to reduce the difference in tissue moisture content between the first and second groups to within a preset threshold through differential pretreatment; while methylene blue penetration depth and body wall elastic modulus do not participate in grouping decisions and therefore do not require convergence control. Furthermore, in embodiments employing a three-parameter fusion determination, it is necessary to coordinately regulate pretreatment conditions to ensure that both groups meet their respective convergence threshold requirements for the three parameters of tissue moisture content, methylene blue penetration depth, and body wall elastic modulus, thereby achieving consistency in the pre-freezing state and laying the foundation for a unified freezing process.

[0047] In some embodiments, the preset threshold satisfies one, two, or three of the following: The absolute value of the difference in tissue moisture content between the two groups of tussah silkworms is ≤3%; The absolute value of the difference in methylene blue penetration depth between the two groups of silkworms was ≤20 μm; The absolute value of the difference in body wall elastic modulus between the two groups of tussah silkworms is ≤0.05 MPa.

[0048] Preferably, the preset threshold satisfies all three of the above conditions.

[0049] Specifically, similar tissue water content (≤3% difference) ensures that the nucleation rate and growth kinetics of ice crystals are highly consistent between the two groups during freezing, avoiding localized overcooling or the formation of large ice crystals due to differences in free water content; similar methylene blue penetration depth (≤20μm difference) indicates that the permeability of cell membranes and body walls is similar, reflecting their comparable barrier function against freeze-thaw stress, thereby reducing juice loss after thawing and ensuring that the two groups respond consistently to the same freezing process; similar body wall elastic modulus (≤0.05 MPa difference) means that the mechanical strength and structural toughness of the two groups are at the same level, and they can resist the mechanical stress generated by ice crystal expansion in a similar way, preventing tissue tearing or ulceration.

[0050] When the above three parameters converge synchronously, the overall physiological and mechanical states of the first group (high sensitivity) and the second group (low sensitivity) before freezing reach a high degree of equilibrium. This not only significantly improves the consistency of their response to the same three-stage gradient freezing process, but also reduces the inter-group coefficient of variation of quality indicators (such as juice loss rate, tissue integrity, and nutrient retention rate) after thawing to below 5%. This not only ensures high product uniformity and meets the stringent standards of high-end food raw materials, but also completely eliminates quality fluctuations caused by individual sensitivity differences, making fully automated continuous production possible and significantly reducing the need for manual intervention and raw material loss rate.

[0051] In some embodiments, before step S1 and 6-12 hours before the differential pretreatment, the silkworms are placed in a closed environment and fumigated with a gas mixture. The gas mixture contains methyl jasmonate or salicylic acid, the volume concentration of methyl jasmonate or salicylic acid is 10~50 ppm, the fumigation temperature is 15~25℃, and the fumigation time is 1~2 h.

[0052] Specifically, to ensure that methyl jasmonate or salicylic acid effectively induces an anti-cold physiological response, fumigation treatment must be carried out when the silkworms are in a state of normal metabolic activity, and therefore it is arranged before the standardized pretreatment.

[0053] After fumigation, the silkworms were removed and left to stand for 6–11 hours, ensuring that the time interval between the end of fumigation and the start of step S1 was within 6–12 hours to guarantee that signaling molecules fully activated cold-resistance-related pathways. Subsequently, the silkworms underwent standardized pretreatment (including sterile water rinsing, draining, and pre-cooling at -2°C to lower the body surface temperature to 0–1°C), and step S1 (including developmental biological characteristic determination and grouping) was performed. Differential pretreatment was then applied to the first and second groups respectively.

[0054] This fumigation treatment can induce the expression of cold-resistance-related genes in silkworms, enhance cell membrane stability and antioxidant capacity, and moderately regulate their freezing response characteristics without altering their developmental biological characteristics, thus providing a better physiological basis for subsequent differentiated pretreatment.

[0055] In some embodiments, the freezing response characteristics of the first group are higher than those of the second group; The pretreatment of the first group includes: air drying at 2-4°C for 8-12 min, followed by soaking in a first preservation solution at 2-4°C with a pH of 5.0-5.5 for 10-20 min; The pretreatment of the second group includes: wetting with 0.3-0.7% citric acid solution in an environment of 2-5℃, and then soaking in a second preservation solution with pH 4.0-4.5 at 2-5℃ for 25-35 min; The first preservative solution, by weight, includes: 0.15-0.25 parts chitosan, 0.08-0.12 parts tea polyphenols, 0.03-0.06 parts ascorbic acid, and 100 parts sterile water; The second preservative solution, by weight, includes: 0.05-0.12 parts chitosan, 0.12-0.18 parts tea polyphenols, 0.05-0.07 parts ascorbic acid, 0.02-0.04 parts citric acid, and 100 parts sterile water.

[0056] Specifically, the freezing response characteristics of the first group were higher than those of the second group, indicating that the first group had higher tissue water content, looser body wall structure and / or weaker membrane integrity, and was more sensitive to ice crystal damage.

[0057] For the first group: Place the silkworms in an environment of 2-4℃ (e.g., 2℃, 3℃, or 4℃) and air dry for 8-12 minutes (e.g., 8 min, 9 min, 10 min, 11 min, or 12 min) to moderately reduce the free water content on the body surface and decrease the sites for ice nucleation during the initial freezing stage. Then, at 2-4℃ (e.g., 2℃, 3℃, or 4℃), completely immerse them in a first preservative solution with a pH of 5.0-5.5 (e.g., 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5) for 10-20 minutes (e.g., 10 min, 14 min, 18 min, or 20 min). After soaking, remove the first group of silkworms and gently blot away any remaining preservative solution with sterile gauze.

[0058] For the second group: First, in an environment of 2-5℃ (e.g., 2℃, 3℃, 4℃, or 5℃), evenly moisten the surface of the silkworms with a 0.3-0.7% (w / v) citric acid solution (e.g., 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%) to slightly acidify the body wall and enhance the permeability of the subsequent preservative solution. Then, at 2-5℃ (e.g., 2℃, 3℃, 4℃, or 5℃), completely immerse them in a second preservative solution with a pH of 4.0-4.5 (e.g., 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5) for 25-35 minutes (e.g., 25 minutes, 28 minutes, 31 minutes, or 35 minutes). After soaking, remove the second group of silkworms and gently blot away any remaining preservative solution with sterile gauze.

[0059] The first preservative solution, by weight, includes: 0.15-0.25 parts chitosan (e.g., 0.15, 0.17, 0.20, 0.23, or 0.25 parts), 0.08-0.12 parts tea polyphenols (e.g., 0.08, 0.10, or 0.12 parts), 0.03-0.06 parts ascorbic acid (e.g., 0.03, 0.04, 0.05, or 0.06 parts), and 100 parts sterile water. The second preservative solution, by weight, includes: 0.05-0.12 parts chitosan (e.g., 0.05, 0.07, 0.10, or 0.12 parts), 0.12-0.18 parts tea polyphenols (e.g., 0.12, 0.14, 0.16, or 0.18 parts), 0.05-0.07 parts ascorbic acid (e.g., 0.05, 0.06, or 0.07 parts), 0.02-0.04 parts citric acid (e.g., 0.02, 0.03, or 0.04 parts), and 100 parts sterile water.

[0060] The pH of the first preservative solution can be adjusted to 5.0-5.5 by adding food-grade acetic acid to dissolve chitosan and then adjusting it with dilute sodium hydroxide solution. The pH of the second preservative solution can be adjusted by citric acid in the formula and then finely adjusted to 4.0-4.5 with trace amounts of citric acid or sodium bicarbonate, and finally confirmed by a pH meter.

[0061] The first preservative solution has a higher chitosan concentration than the second preservative solution, which forms a dense protective film on the surface of the highly sensitive individuals in the first group, thus strengthening the physical barrier. The second preservative solution has a lower chitosan concentration, which avoids excessive film formation on the surface of the individuals in the second group, thus hindering the penetration of active ingredients into the dense body wall.

[0062] The concentration of tea polyphenols in the second preservative solution can be slightly higher than that in the first preservative solution to compensate for the insufficient internal antioxidant capacity of the second group of silkworms due to the dense cuticle; the concentration of ascorbic acid in the second preservative solution can be slightly higher than that in the first preservative solution to compensate for the potential loss of activity due to the longer soaking time, and to ensure that an effective level of antioxidant protection is maintained throughout the entire treatment process.

[0063] In addition, citric acid is added to the second preservation solution to regulate the osmotic pressure of the system and to synergistically promote the transmembrane absorption of active ingredients (tea polyphenols, ascorbic acid).

[0064] The preservatives used in this invention (tea polyphenols, chitosan, ascorbic acid, and citric acid) are all food-grade additives permitted under the National Food Safety Standard for the Use of Food Additives (GB 2760), and are safe and reliable, suitable for the preservation of edible silkworms.

[0065] By specifically regulating the surface condition and internal antioxidant capacity of two groups of tussah silkworms, and combining the synergistic effect of natural preservatives (chitosan, tea polyphenols, ascorbic acid, etc.), the activity of endogenous enzymes (such as proteases and lipoxygenases) and the growth of microorganisms (bacteria and molds) were effectively inhibited. Under the same subsequent freezing conditions, the treated tussah silkworms maintained good commercial characteristics after storage for more than 12 months, with bright body color and intact tissue after thawing; the total loss rate of key nutrients (such as crude protein and free amino acids) was ≤3%.

[0066] Furthermore, the differentiated pretreatment has achieved "convergent regulation" of the freezing response characteristics of the two groups: the first group reduces the risk of ice crystal damage by enhancing film formation, while the second group compensates for the limited absorption of active ingredients due to its dense body wall and the oxidation risk caused by prolonged weak acid treatment by increasing the concentration of antioxidants and adjusting osmotic pressure. This maintains its inherent low-sensitivity characteristics and ensures that it achieves a balance in response characteristics with the first group before freezing, enabling it to adapt to the same three-stage gradient freezing process. This not only simplifies the control logic of the freezing equipment but also avoids the increased production line complexity and energy consumption caused by setting separate freezing curves for different groups, improving the efficiency of automated production while ensuring quality.

[0067] S3 combines the two groups of silkworms that have undergone the differentiated pretreatment and freezes them using the same three-stage gradient freezing procedure; wherein... First cooling stage: Cooling from 0~1℃ to -10℃ at a cooling rate of 0.8~1.2℃ / min; The second cooling stage: cooling from -10℃ to -30℃ at a cooling rate of 0.45~0.55℃ / min; The third cooling stage: cooling from -30℃ to -45℃ at a cooling rate of 1.8~2℃ / min.

[0068] Specifically, the two groups of silkworms with the surface residual preservative removed are laid flat on a freezing tray (single layer, avoid stacking) and placed in the freezing equipment for three-stage gradient freezing.

[0069] The cooling rate in the first cooling stage can be 0.8℃ / min, 0.9℃ / min, 1.0℃ / min, 1.1℃ / min, or 1.2℃ / min, etc. Using a moderately fast cooling rate in the first cooling stage can promote the simultaneous freezing of water inside and outside the cell, avoid the large extracellular ice crystals that would compress the cell due to slow cooling, and also prevent the explosive formation of intracellular ice crystals or thermal stress cracking caused by excessively rapid cooling.

[0070] The cooling rate in the second cooling stage can be 0.45℃ / min, 0.47℃ / min, 0.50℃ / min, 0.53℃ / min, or 0.55℃ / min, etc. This second cooling stage is the cryogenic transition zone, where the cooling rate slows significantly to alleviate the mechanical stress caused by tissue contraction and ice crystal expansion. The slower cooling allows ice crystal growth to become more orderly, preventing the accumulation of microcracks.

[0071] The cooling rate in the third cooling stage can be 1.8℃ / min, 1.9℃ / min, or 2.0℃ / min, etc. The third cooling stage is carried out in the deep freezing zone, at which point the tissue has been basically frozen. Using a faster cooling rate can efficiently complete the final cooling, shorten the overall freezing time, and reduce energy consumption.

[0072] In some embodiments, the three-stage gradient freezing procedure further includes: After cooling to -10°C in the first cooling stage, the temperature is maintained at -10°C for 20 to 40 minutes, such as 20 minutes, 30 minutes, or 40 minutes, to facilitate the full migration and phase transition of unfrozen water molecules, reduce residual liquid water in the subsequent cryogenic stage, and lower the risk of recrystallization.

[0073] After cooling to -30°C in the second cooling stage, the system is held at -30°C for 45 to 75 minutes, for example, 45 minutes, 60 minutes, or 75 minutes, to ensure that the thermodynamic state of the system is fully balanced and that all individual components reach a stable freezing endpoint, thus laying a uniform foundation for the final deep cryogenic treatment.

[0074] After cooling to -45°C in the third cooling stage, the temperature is maintained at -45°C for 75~105 min, for example, 75 min, 90 min or 105 min, etc. This not only ensures that the core temperature is completely uniform, but also allows the tiny ice crystals to fuse into a more stable form, further suppressing recrystallization during storage.

[0075] After S4 is frozen, it is vacuum-packed and stored at -45~-40℃.

[0076] In some embodiments, the vacuum packaging is carried out in an environment not exceeding -10°C to prevent the ice crystals on the surface of the silkworm from melting locally due to temperature rise, effectively inhibiting lipid oxidation and residual enzymatic reactions (such as browning), and preventing the formation of frost or droplets on the inner wall of the packaging due to water vapor condensation. This low-temperature packaging operation ensures that the frozen quality does not deteriorate during the sealing process, providing a guarantee for long-term storage stability. Vacuum degree ≤ 0.09 MPa.

[0077] After sealing, the packaging bags are tested for airtightness to ensure there are no leaks, damages, or sealing defects. Then, the qualified sealed packaging products are transferred to a low-temperature cold storage at -45 to -40°C. During storage, the temperature stability is strictly controlled, with fluctuations not exceeding ±2°C, to minimize ice crystal recrystallization and nutrient degradation, maintaining product quality uniformity.

[0078] Before consumption, the food can be naturally thawed at 4°C until it reaches a temperature of 0-2°C. Alternatively, in some embodiments, after step S4 but before consumption, a three-stage gradient rewarming process is included. The first rewarming stage involves heating from -45 to -40°C to -1°C at a rate of 0.8 to 1.2°C / min. For example, the heating rate could be 0.8°C / min, 0.9°C / min, 1.0°C / min, 1.1°C / min, or 1.2°C / min, etc., and then holding at -1°C for 25 to 35 minutes, such as 25 minutes, 30 minutes, or 35 minutes. This moderately fast heating rate allows the product to quickly pass through the high-risk low-temperature range (approximately -40°C to -5°C), thereby minimizing ice crystal rearrangement and coarsening that may occur due to prolonged stagnation in the unstable ice crystal temperature zone, and preventing the slow recovery of microorganisms. Holding at -1°C aims to fully and uniformly melt any remaining tiny ice nuclei within the tissue and initiate an initial balance of intracellular and extracellular osmotic pressure, laying a stable foundation for the subsequent slow rewarming in the critical phase transition zone.

[0079] The second rewarming stage involves heating from -1°C to 4°C at a rate of 0.05–0.1°C / min. For example, the heating rate could be 0.05°C / min, 0.06°C / min, 0.07°C / min, 0.09°C / min, or 0.1°C / min, etc., and then holding at 4°C for 45–55 minutes, such as 45 minutes, 50 minutes, or 55 minutes. This slower heating, occurring within the phase transition temperature range where ice crystals melt significantly, allows sufficient time for the orderly absorption of water by the cell structure. This minimizes internal mechanical stress caused by volume changes and thermal expansion differences during the ice-water phase transition, effectively preventing the already fragile cell membranes from rupturing during rapid water absorption. Holding at 4°C (close to refrigeration temperature) for an extended period promotes the gentle restart and functional recovery of cellular metabolic systems (such as ion pumps and basic enzyme systems), while ensuring sufficient reabsorption of free water, significantly reducing the loss of thawed juice.

[0080] The third rewarming stage involves heating from 4°C to 12°C at a rate of 0.3–0.6°C / min (e.g., 0.3°C / min, 0.4°C / min, 0.5°C / min, or 0.6°C / min), and holding at 12°C for 10–15 minutes (e.g., 10, 12, or 15 minutes). This stage is for final temperature adjustment before consumption. This heating rate is significantly higher than the second rewarming stage, quickly traversing the mesophilic danger zone of 4–12°C where microorganisms can proliferate rapidly, efficiently achieving the final temperature increase and reducing microbial safety risks. After reaching 12°C, the holding period aims to homogenize the temperature of the entire batch of product, achieving the ideal starting state for immediate consumption or subsequent cooking.

[0081] Compared with existing technologies, the tussah silkworm preservation method provided in this application firstly groups the raw materials efficiently based on the rapidly observable external developmental characteristics of tussah silkworms (such as body length, head shell width, body wall light transmittance, etc.) and their known correlation with freezing response characteristics, providing a reliable basis for subsequent differentiated pretreatment.

[0082] Subsequently, differentiated pretreatments were applied to different groups to proactively adjust the key physiological states of each group (characterized by water content, methylene blue penetration depth, and / or body wall elastic modulus parameters) to a convergent state (differences not exceeding a preset threshold). This "identification first, convergence later" pre-processing eliminated the inherent heterogeneity of the materials at the source, enabling the subsequent uniform three-stage gradient freezing procedure to apply appropriate freezing stress to groups with consistent states—avoiding both the compression of extracellular ice crystals in highly sensitive individuals due to slow freezing and the puncture of intracellular ice crystals in low-sensitivity individuals due to rapid freezing. This not only significantly improved the quality uniformity among individuals within a batch but also ensured that each individual reached the optimal protection state under this freezing process, thereby effectively maintaining the integrity of the tissue structure after thawing.

[0083] The three-stage gradient freezing program is a refined design based on the physical laws of ice crystal formation and growth in different temperature ranges: The first cooling stage uses a moderately fast rate (0.8~1.2℃ / min) to promote the simultaneous freezing of intracellular and extracellular water and inhibit the formation of large extracellular ice crystals; the second cooling stage significantly slows down the rate (0.45~0.55℃ / min), allowing ice crystals to grow in an orderly manner in the cryogenic transition zone (-10~-30℃), alleviating the mechanical stress caused by ice crystal expansion and tissue contraction, making ice crystal growth more orderly, and avoiding the accumulation of microcracks; the third cooling stage accelerates the cooling (1.8~2.0℃ / min) after the tissue has basically completed freezing, efficiently completing the final cooling and shortening the overall freezing time. This gradient strategy, which dynamically matches the phase transition process, works in conjunction with the homogenization of materials brought about by pretreatment to guide the formation of small and evenly distributed ice crystals. This minimizes ice crystal puncture and compression damage at the cellular and subcellular levels, effectively maintaining the integrity of cell membranes and body wall muscle tissue structures, significantly reducing juice loss, and ensuring firm texture and good flavor after thawing. This allows the product to maintain sensory quality close to that of fresh tussah silkworms even after long-term frozen storage.

[0084] The highly intact cell structure and uniform freezing state achieved through the aforementioned synergistic effects, combined with vacuum packaging and stable low-temperature storage at -45 to -40°C in step S4, create ideal conditions for long-term preservation: maintaining cell integrity significantly reduces juice loss and slows down the rate of endogenous enzymatic reactions; the vacuum environment effectively isolates oxygen, inhibiting lipid oxidation and flavor deterioration; and the stable deep low temperature maximally inhibits microbial proliferation and residual biochemical reactions. These measures work synergistically to delay quality deterioration from three dimensions: physical structure, chemical stability, and biological safety, significantly extending the product's shelf life and stability.

[0085] The tussah silkworm preservation method described in this application effectively maintains cell structure integrity, significantly reduces juice loss, and inhibits the abnormal release of endogenous enzymes. This allows for better preservation of nutrients such as proteins and amino acids during long-term (over 12 months) frozen storage, while maintaining the firm texture, uniform color, and odor-free sensory characteristics. The method features a standardized technical process, with controllable key process parameters (such as grouping criteria, pretreatment conditions, gradient cooling rate, and final storage temperature). It is suitable for industrial-scale mass production and can significantly reduce quality loss and spoilage risks during storage and transportation, providing reliable technical support for cross-regional distribution and off-peak sales.

[0086] Example 1

[0087] Healthy and fresh tussah silkworms from Zhangwu, Liaoning Province were selected and subjected to standardized pretreatment. Their freezing response characteristics were evaluated based on their body length, head shell width, and body wall light transmittance. Individuals with high freezing response characteristics were assigned to the first group, and individuals with low freezing response characteristics were assigned to the second group.

[0088] Differential pretreatment was applied to the first and second groups respectively: Group 1: Air-dry at 3°C ​​for 10 min, then soak in a first preservative solution at pH 5.3 at 3°C ​​for 15 min. The first preservative solution, by weight, includes: 0.2 parts chitosan, 0.1 parts tea polyphenols, 0.05 parts ascorbic acid, and 100 parts sterile water.

[0089] Group 2: After being moistened with 0.5% citric acid solution at 3℃, the samples were soaked in a second preservative solution at pH 4.3 at 3℃ for 30 min. The second preservative solution, by weight, includes: 0.09 parts chitosan, 0.14 parts tea polyphenols, 0.06 parts ascorbic acid, 0.03 parts citric acid, and 100 parts sterile water.

[0090] The two groups of silkworms that underwent differentiated pretreatment were combined and frozen using the same three-stage gradient freezing procedure: First cooling stage: Cool from 0~1℃ to -10℃ at a cooling rate of 1.0℃ / min, and hold for 30 min; Second cooling stage: Cool from -10℃ to -30℃ at a cooling rate of 0.5℃ / min, and hold for 60 min; The third cooling stage: cooling from -30℃ to -45℃ at a cooling rate of 2℃ / min, and holding at that temperature for 90 min; After freezing, the product is divided into three portions and vacuum-packed (vacuum degree ≤ 0.09 MPa). After checking for any damage or leakage, it is transferred to a -42℃ low-temperature cold storage for stable storage, with temperature fluctuations controlled within ±2℃.

[0091] After being frozen for 3 months, 6 months, and 12 months, a sample of vacuum-packed tussah silkworms was taken and thawed naturally at 4°C until the core temperature reached 0-2°C for testing.

[0092] Comparative Example 1 Healthy and fresh tussah silkworms were selected from Zhangwu area of ​​Liaoning Province. After cleaning and draining, they were placed in a -20℃ cold storage and frozen for 4 hours (temperature fluctuation ±3℃). After the core temperature of the silkworms dropped to -18℃, they were divided into three portions and vacuum-packed (vacuum degree ≤0.09MPa) and then transferred to a -18℃ cold storage for frozen storage.

[0093] After being frozen for 3 months, 6 months, and 12 months, a sample of vacuum-packed tussah silkworms was taken and thawed naturally at 4°C until the core temperature reached 0–2°C for testing.

[0094] The samples obtained in Examples 1 and 2 were tested after being frozen for 3 months, 6 months, and 12 months, respectively, using the following methods: I. Cell structure indicators Ice crystal morphology: The structure of muscle cells of silkworms after freezing and thawing was observed using an optical microscope.

[0095] Cell damage rate: The body wall muscle tissue of thawed silkworms was taken and cut into 1 mm pieces. 3 Small pieces were fixed in 4% paraformaldehyde for 24 h, dehydrated in a gradient manner, and then embedded to prepare paraffin sections (5 μm thick). The sections were stained with hematoxylin-eosin (HE) and the morphology of muscle cells was observed under an optical microscope (400×). The cell damage rate was statistically analyzed from 20 randomly selected fields of view. Juice loss rate: Weigh the product before and after thawing, and calculate the juice loss rate = (weight before thawing - weight after thawing) / weight before thawing × 100%.

[0096] Test results after 3 months of frozen storage: Silkworms treated with the preservation method of Example 1 formed uniformly distributed tiny ice crystals in their muscle cells, with a cell breakage rate of only 6%±2% and a juice loss rate as low as 4%±1%.

[0097] In contrast, the tussah silkworm in Comparative Example 1 formed large, irregular ice crystals inside its cells, with a cell damage rate of 35% ± 5% and a juice loss rate as high as 18% ± 3%.

[0098] The above results demonstrate that the present invention, through differentiated pretreatment based on freezing response characteristics and a three-stage gradient freezing process dynamically matched with the phase change process, effectively regulates ice crystal nucleation and growth behavior, significantly reduces mechanical damage caused by ice crystal compression and puncture during freezing, and thus establishes a good tissue structure foundation in the early stage of frozen storage, providing a key guarantee for long-term quality stability.

[0099] II. Sensory quality and nutritional indicators Sensory quality: Five professional judges conducted a blind evaluation based on three dimensions: color, texture, and odor.

[0100] Nutritional indicators: Protein content was determined by the Kjeldahl method, and total amino acid content was determined by high performance liquid chromatography. The average value was taken from three parallel tests.

[0101] Test results: Example 1: Silkworms treated with preservation methods: After 3 months of frozen storage, the meat was firm, uniform in color, and odorless, with a protein content of 28.3 g / 100g; after 6 months of frozen storage, the meat was relatively firm and odorless, with a protein content of 27.2 g / 100g and a total amino acid content of 25.1 g / 100g; after 12 months of frozen storage, the meat remained basically firm and odorless, with a stable protein content of 26.5 g / 100g, showing no signs of spoilage and meeting edible standards.

[0102] In contrast, the tussah silkworms in Comparative Example 1 showed slightly loose texture and a slight off-odor after 3 months of frozen storage, with a protein content of 25.1 g / 100g; after 6 months of frozen storage, the texture was loose and the off-odor was obvious, with the protein content dropping to 22.0 g / 100g; and after 12 months of frozen storage, the sample was severely spoiled (moldy on the surface and soft and rotten in texture), with a protein content of less than 18 g / 100g, making it inedible.

[0103] The above results show that the method of the present invention can effectively delay sensory deterioration and nutrient loss, and achieve high-quality frozen storage for up to 12 months.

[0104] In summary, the present invention features a standardized and convenient technical process, enabling batch processing (single batch processing volume ≥ 50 kg). The energy consumption of the freezing process is comparable to that of existing technologies, and the storage period is significantly extended to more than 12 months. It effectively solves the problem of unsold and spoiled tussah silkworms during the peak harvest season, providing solid technical support for long-distance transportation, off-peak sales, and high-value utilization, and possesses outstanding industrial application value.

[0105] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preserving tussah silkworms, characterized in that, Includes the following steps: Based on the developmental biological characteristics of the tussah silkworm, S1 divides the tussah silkworm into a first group and a second group with different freezing response characteristics; the freezing response characteristics are defined as the degree of ice crystal damage to the cellular tissue structure of the tussah silkworm under the same freezing conditions; the freezing response characteristics are characterized by at least one of the following parameters: tissue water content, methylene blue penetration depth, and body wall elastic modulus. S2 applies differential preprocessing to the first group and the second group respectively, so that the difference between the two on any characterization parameter does not exceed a preset threshold; S3 combines the two groups of silkworms that have undergone the differentiated pretreatment and freezes them using the same three-stage gradient freezing procedure; wherein... First cooling stage: Cooling from 0~1℃ to -10℃ at a cooling rate of 0.8~1.2℃ / min; The second cooling stage: cooling from -10℃ to -30℃ at a cooling rate of 0.45~0.55℃ / min; The third cooling stage: cooling from -30℃ to -45℃ at a cooling rate of 1.8~2℃ / min; After S4 is frozen, it is vacuum-packed and stored at -45~-40℃.

2. The method for preserving tussah silkworms according to claim 1, characterized in that, The freezing response characteristics of the first group are higher than those of the second group; The pretreatment of the first group includes: air drying at 2-4°C for 8-12 min, followed by soaking in a first preservation solution at 2-4°C with pH 5.0-5.5 for 10-20 min; The pretreatment of the second group includes: wetting with 0.3-0.7% citric acid solution in an environment of 2-5℃, and then soaking in a second preservation solution with pH 4.0-4.5 at 2-5℃ for 25-35 min; The first preservative solution, by weight, includes: 0.15-0.25 parts chitosan, 0.08-0.12 parts tea polyphenols, 0.03-0.06 parts ascorbic acid, and 100 parts sterile water; The second preservative solution, by weight, includes: 0.05-0.12 parts chitosan, 0.12-0.18 parts tea polyphenols, 0.05-0.07 parts ascorbic acid, 0.02-0.04 parts citric acid, and 100 parts sterile water.

3. The method for preserving tussah silkworms according to claim 2, characterized in that, The S1 step is preceded by a standardized preprocessing step: Select live tussah silkworms, rinse them with sterile water at 3-5℃, and then drain off the surface moisture. After draining, place the tussah silkworms in an environment of -2℃ for 30~60 minutes to pre-cool them, so that the surface temperature of the silkworms drops to 0~1℃.

4. The method for preserving tussah silkworms according to claim 1, characterized in that, The preset threshold satisfies at least one of the following: The absolute value of the difference in tissue moisture content between the two groups of tussah silkworms is ≤3%; The absolute value of the difference in methylene blue penetration depth between the two groups of silkworms was ≤20 μm; The absolute value of the difference in body wall elastic modulus between the two groups of tussah silkworms is ≤0.05 MPa.

5. The method for preserving tussah silkworms according to claim 1, characterized in that, The three-stage gradient freezing procedure also includes: After cooling to -10°C in the first cooling stage, maintain the temperature at -10°C for 20~40 min; After cooling to -30°C in the second cooling stage, maintain the temperature at -30°C for 45~75 minutes. After cooling to -45°C in the third cooling stage, the temperature is maintained at -45°C for 75~105 min.

6. The method for preserving tussah silkworms according to claim 1, characterized in that, The steps preceding step S1 also include: Multiple tussah silkworm samples were collected, and the developmental biological characteristics and corresponding freezing response characteristic parameters of each tussah silkworm sample were measured. The developmental biological characteristics included body length, head shell width, and body wall transmittance, and the freezing response characteristic parameters included tissue water content, methylene blue penetration depth, and body wall elastic modulus. Based on the measured data, a mapping relationship is established between the developmental biological characteristics and the freezing response characteristic characterization parameters to obtain a prediction model; the prediction model is used to predict the freezing response characteristics of the tussah silkworms to be classified according to the developmental biological characteristics, and to classify the tussah silkworms to be classified into the first group or the second group accordingly.

7. The method for preserving tussah silkworms according to claim 6, characterized in that, Step S1 includes: The body length, head shell width, and body wall transmittance of the tussah silkworms to be classified were measured. The prediction model was used to predict the tissue water content, methylene blue penetration depth, and body wall elastic modulus of the silkworm to be divided. If the predicted value meets any of the following conditions: tissue water content ≥ 84%, methylene blue penetration depth ≥ 150 μm, body wall elastic modulus ≤ 0.25 MPa, then its freezing response characteristics are considered high and it is classified as the first group. Otherwise, it is determined that its freezing response characteristics are low and it is classified into the second group.

8. The method for preserving tussah silkworms according to claim 1, characterized in that, Before step S1 and 6-12 hours before the differential pretreatment, the silkworms are placed in a closed environment and fumigated with a gas mixture. The gas mixture contains methyl jasmonate or salicylic acid, the volume concentration of methyl jasmonate or salicylic acid is 10~50 ppm, the fumigation temperature is 15~25℃, and the fumigation time is 1~2 h.

9. The method for preserving tussah silkworms according to claim 1, characterized in that, The vacuum packaging is carried out in an environment not higher than -10°C.

10. The method for preserving tussah silkworms according to claim 1, characterized in that, After step S4 is completed and before consumption, a three-stage gradient reheating process is also included: First rewarming stage: Heat from -45~-40℃ to -1℃ at a heating rate of 0.8~1.2℃ / min, and hold at -1℃ for 25~35 min; The second rewarming stage: the temperature is increased from -1℃ to 4℃ at a heating rate of 0.05~0.1℃ / min, and held at 4℃ for 45~55min; The third rewarming stage: the temperature is increased from 4℃ to 12℃ at a heating rate of 0.3~0.6℃ / min, and then held at 12℃ for 10~15min.

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