A method for making pet attractant with mandarin fish by-product

Abstract

The application discloses a method for preparing pet food attractant with the flavor of stink mandarin fish by using mandarin fish by-products, and comprises the following steps: subjecting stink mandarin fish fermentation liquor to microfiltration concentration-in-situ embedding treatment to obtain high-abundance liquid microsphere leavening agent; subjecting stink mandarin fish by-product raw materials to step-by-step enzymatic hydrolysis process treatment to obtain high-hydrolysis-degree fermentation substrate; mixing the fermentation substrate with the liquid microsphere leavening agent to perform anaerobic fermentation, and then removing bacteria by microfiltration to obtain liquid food attractant with the flavor of stink mandarin fish; using hesperidin / xanthan gum / corn starch to prepare microcapsules for encapsulating the liquid food attractant, and obtaining pet food attractant with the flavor of stink mandarin fish after freeze-drying. The method is green and environmentally friendly throughout, improves the utilization rate and bioavailability of mandarin fish by-products, realizes the customized production of the characteristic flavor of stink mandarin fish, and realizes the long-period stable release of flavor compounds, and the introduction of hesperidin through embedding effectively enhances the functional activity of the product, and meets the diversified needs of pet food at present.

Description

Technical Field

[0001] This invention belongs to the technical field of pet food, specifically relating to a method for producing a pet palatability enhancer with a stinky mandarin fish flavor using mandarin fish by-products. Background Technology

[0002] Fishery by-products typically account for more than 20% of total fishery output, representing a vast and exploitable resource. Among these, by-products, primarily fish scales, gills, and viscera, are rich in protein, lipids, and trace elements. After modern processing and extraction, they can become important sources of active enzymes, active peptides, unsaturated fatty acids, and trace elements such as calcium, iron, and zinc.

[0003] Pet food is the core of the pet market, accounting for over 50% of the market share. In 2024, my country's total pet food production reached 1.613 million tons, with an annual growth rate exceeding 10%. As pets' status in modern living environments continues to rise, the pet food industry is experiencing explosive growth. In 2023, the pet food industry market size reached 190 billion yuan. Driven by this huge market, refinement is a crucial direction for the current development of pet food, mainly reflected in the nutritional characteristics, digestibility, palatability, and portability of the feed.

[0004] Mandarin fish is one of my country's important and high-quality freshwater fish, with the Huangshan region of Anhui Province alone producing 20,000 tons of by-products annually. These by-products provide rich nutrients to improve pets' growth and development, and are important raw materials for pet food and palatability enhancers. Therefore, fully utilizing my country's vast mandarin fish by-product resources can meet the growing needs of the pet food industry. Furthermore, fermented mandarin fish, a traditional Chinese fermented fish product, not only appeals to consumers with its unique aroma but also satisfies pets' natural attraction to strong smells, giving it a natural appeal. Therefore, enhancing the flavor of fermented mandarin fish in pet food preparation can not only provide pets with rich nutrition but also stimulate their appetite, strengthening the intake and absorption of nutrients.

[0005] In the process of preparing pet food using fish viscera, the raw materials first need to be pretreated to promote the release of their functional components and improve the digestibility and utilization rate after pets ingest them. Enzyme catalysis has the advantages of high efficiency and being environmentally friendly, and is currently the main technical method used in viscera processing.

[0006] Chinese patent CN111743107A discloses a method for rapidly fermenting mandarin fish using fermented mandarin fish broth. However, this method only collects and ultrafilters the fermented broth, which is rich in microbial communities. It is difficult to maintain the activity and abundance of the microbial community in subsequent processing steps, and the fermentation stability is also difficult to guarantee.

[0007] Chinese patent CN102349594A discloses a comprehensive processing method for fish viscera. This method uses acidic protease in conjunction with yeast and lactic acid bacteria fermentation to produce functional peptides from fish viscera and prepare protein feed. However, this patent only explores the possibility of separating fish viscera components from a technical perspective and does not consider the practicality of its prepared products in pet food applications.

[0008] Chinese patent CN119014489A discloses a method for preparing pet palatability enhancers using freshwater fish viscera. This method combines one-step enzymatic hydrolysis with exogenous flavor enhancers. However, the patent focuses on the one-step enzymatic hydrolysis method to degrade the fish viscera. This process generates a significant amount of bitter amino acids, and the amount of volatile compounds that contribute to palatability is low, limiting its effectiveness in attracting pets. Furthermore, the patent uses monosodium glutamate, cyclamate, and acidic substances as flavor enhancers. These additives only improve the product's taste (e.g., masking the bitterness of amino acids) without improving the volatile flavor profile of the palatability enhancer. Additionally, these additives pose certain health risks.

[0009] This invention starts with the preparation of highly active fermenting agents and fermentation substrates with high degradation levels, and ends with the directional induction of flavor in stinky mandarin fish through anaerobic fermentation. The two are different in purpose and innovative means.

[0010] Chinese patent CN103005167A discloses a fishbone pet palatability enhancer and its preparation method, while Chinese patent CN110547358A discloses a special enzyme for pet palatability enhancers, enzymatic hydrolysates, and pet palatability enhancers and their preparation methods. Both existing patents disclose the application of flavor proteases in the production of palatability enhancers. Flavor proteases can improve the taste of enzymatic hydrolysates to some extent. However, relying solely on flavor proteases in conjunction with other proteases to process raw materials has drawbacks: firstly, the degree of hydrolysis is generally low, resulting in low raw material utilization; secondly, the hydrolysates are homogeneous, making it impossible to achieve customized characteristic flavors or tastes, thus failing to meet the market's demand for diversified pet food. Although Chinese patent CN103005167A discloses the preparation of palatability enhancers with special aromas via the Maillard reaction, the Maillard reaction relies on high-temperature and high-pressure production conditions, resulting in high energy consumption and difficult process control, which is detrimental to the green development of the pet food industry. Summary of the Invention

[0011] The purpose of this invention is to provide a method for making a stinky mandarin fish flavored pet attractant using mandarin fish by-products, in order to solve the current problems of low utilization of mandarin fish by-products and scarcity of high-quality pet attractants.

[0012] Enhancing the flavor characteristics of palatability enhancers is the primary problem this invention aims to solve. Simultaneously, considering practical application scenarios, products typically require prolonged exposure to the environment, and their palatability-enhancing effects are easily diminished under the stimulation of light, heat, and other factors. Therefore, the stable release and persistence of volatile flavor compounds, which are the main contributors to flavor characteristics, is another issue that should be addressed. Thus, this invention innovates in the sustained-release of odors from palatability enhancers. This innovation involves the design of microencapsulation technology. The microencapsulation material uses xanthan gum and corn starch to achieve efficient encapsulation of volatile compounds. Simultaneously, hesperidin, a natural plant extract, is widely available, odorless, tasteless, and possesses excellent antioxidant and antibacterial properties. By using microencapsulation technology to blend it with the fermentation broth, the overall antioxidant capacity of the microencapsulation can be improved, maintaining the stability of its palatability-enhancing properties during environmental exposure.

[0013] The objective of this invention can be achieved through the following technical solutions:

[0014] A method for producing a stinky mandarin fish flavored pet attractant using mandarin fish byproducts, the method comprising the following steps:

[0015] Step 1: Microfiltration concentration and in-situ encapsulation treatment of fermented mandarin fish broth to obtain liquid microsphere fermentation agent;

[0016] Step 2: The homogenized liquid of mandarin fish by-products is subjected to a stepwise enzymatic hydrolysis process to obtain the fermentation substrate;

[0017] Step 3: Mix the fermentation substrate with the liquid microsphere fermentation agent for anaerobic fermentation, and then sterilize by microfiltration to obtain a liquid attractant with the flavor of stinky mandarin fish;

[0018] Step 4: Microencapsulate the liquid attractant and freeze-dry it to obtain a pet attractant with the flavor of stinky mandarin fish that has good sustained-release properties and excellent antioxidant effects.

[0019] Furthermore, in step one, the fermented liquid of stinky mandarin fish is the liquid that seeps out of the mandarin fish during the natural fermentation process. After collecting the liquid and filtering it, the filtrate is concentrated by microfiltration and in-situ encapsulation.

[0020] Furthermore, the microfiltration concentration method is as follows: the microfiltration membrane has a pore size of 1.4µm, and microfiltration is carried out to 20% of the original volume. The retentate is the fermentation concentrate rich in microorganisms. The in-situ embedding process is as follows: low melting point modified agarose is mixed with the fermentation concentrate at a mass ratio of 1:50, the temperature is raised to 37℃ and stirred for 10 min to promote complete dissolution of agarose, 10 times the volume of polyethylene glycol 10000 is added, and the mixture is stirred evenly to obtain a liquid microsphere fermentation agent containing high abundance and high activity of mandarin fish bacteria.

[0021] Furthermore, the value of the high abundance is 10. 6 Approximately CFU / mL.

[0022] Furthermore, the homogenization process in step two is as follows: 1.5 times the mass of deionized water is added to the mandarin fish by-product raw material for homogenization. The homogenization parameters are: 5000 r / min, 4 min, and the homogenized liquid is collected.

[0023] Furthermore, the stepwise enzymatic hydrolysis process in step two is a two-step enzymatic hydrolysis method, namely: the first enzyme addition is one of neutral protease or alkaline protease, with an enzyme addition amount of 1.2%, pH of 7.5-8.0, temperature of 50℃, and enzymatic hydrolysis time of 2 hours; the second enzyme addition is flavor protease, with an enzyme addition amount of 0.8%-1.2%, pH of 7.0, temperature of 50℃, and enzymatic hydrolysis time of 2 hours. After enzymatic hydrolysis, the hydrolysate is heated to 95℃ for 20 minutes to inactivate the enzyme, thereby obtaining the fermentation substrate, which has a high degree of hydrolysis of 22%-26%.

[0024] Furthermore, in step three, the volume of liquid microsphere fermentation agent added is 20%-40% of the fermentation substrate volume, the mixed fermentation temperature is 15-20℃, and the fermentation time is 3-5 days; the microfiltration membrane pore size is selected as 0.8µm, and the permeate is a liquid attractant with the flavor of stinky mandarin fish.

[0025] Further, the microencapsulation method in step four is as follows: prepare a sodium hydroxide solution with a pH of 12, dissolve hesperidin in the sodium hydroxide solution by stirring, and adjust the concentration of hesperidin solution to 1 mg / mL; mix xanthan gum and corn starch evenly at a mass ratio of 1:10 to prepare a powder; add 10 times the mass of liquid attractant and 0.5 times the mass of hesperidin solution to the powder, heat in a water bath to 85°C, stir for 30 min, cool to room temperature, freeze-dry for 24 h, and then pulverize through a 100-mesh sieve to obtain a pet attractant with the flavor of stinky mandarin fish.

[0026] Furthermore, the bitterness value of the pet palatability enhancer is 1.61-1.81; the second enzyme addition is a flavor protease, and when the enzyme content in the enzymatic hydrolysis system is 1%, the bitterness value of the pet palatability enhancer is 1.66; the volume of liquid microsphere fermentation agent added is 30% of the fermentation substrate volume.

[0027] Furthermore, this pet palatability enhancer is a dry powder, which is easy to store and convenient to use. It can be added to the pet food formula in proportion or reconstituted and sprayed onto the surface of the pet food in appropriate amounts.

[0028] The pet palatability enhancer is used by adding it to the pet food formula in the specified proportion or by spraying it onto the surface of the pet food after reconstitution.

[0029] The procedure for adding it to pet food is as follows: Pet palatability enhancer powder is added as a supplement to the pet food formula at a rate of 0.2%-0.3% of the total weight of the pet food formula ingredients;

[0030] The procedure for reconstituted pet food and spraying it onto the surface is as follows: reconstitute the dry powder of pet palatability enhancer to a concentration of 10 mg / mL, and spray it onto the surface of the extruded cat food at a rate of 1 mL / 100g.

[0031] The beneficial effects of this invention are:

[0032] This invention aims to comprehensively utilize mandarin fish by-products. Leveraging the odor characteristics of traditionally fermented stinky mandarin fish, it develops pet food or palatability enhancers with unique appealing properties. The process avoids acid and heat treatments in the processing of mandarin fish by-products, employing a multi-step enzymatic catalytic synergistic fermentation process to promote the extensive degradation of visceral components, reducing the digestive and absorptive stress on pets after consumption. Simultaneously, the fermentation effect of the stinky mandarin fish fermentation liquid enhances the odor characteristics of the enzymatic hydrolysate, resulting in a product with high palatability.

[0033] This invention discloses a method for producing a stinky mandarin fish flavored pet attractant using mandarin fish by-products. The method involves stepwise enzymatic hydrolysis of the mandarin fish by-products using endopeptidases and exopeptidases. The first enzyme addition is either a neutral protease or an alkaline protease; this endopeptidase can rapidly degrade large protein molecules into polypeptides. The second enzyme addition is a flavor protease, which possesses both endopeptidase and exopeptidase activity, enabling it to degrade proteins and polypeptides to a higher degree and reduce bitterness in the hydrolysis system. This yields a low-bitterness, high-degradation-level hydrolysate. The hydrolysate and unhydrolyzed substrate are retained for further fermentation, significantly improving the utilization rate of the offal and providing a green and safe product.

[0034] The present invention discloses a method for producing a pet palatability enhancer with the flavor of stinky mandarin fish using mandarin fish by-products. The prepared by-product product (liquid palatability enhancer) has the traditional flavor characteristics of stinky mandarin fish and can be directly used as a pet palatability enhancer or pet food component to improve the nutritional characteristics and palatability of pet food.

[0035] This invention discloses a method for producing a pet attractant with the flavor of stinky mandarin fish using mandarin fish by-products. This method allows for the recycling and reuse of the fermentation liquid after the traditional fermentation of stinky mandarin fish. It fully utilizes the rich natural microbial community and some endogenous enzymes derived from the muscle tissue of mandarin fish in the fermentation liquid. Furthermore, the method uses in-situ encapsulation of agarose microspheres to maintain the high activity of the microbial community and endogenous enzymes, thereby achieving the targeted induction of flavor from mandarin fish by-products. This reduces environmental pollution pressure, improves the efficiency of viscera processing, and enhances the flavor characteristics of the product.

[0036] This invention achieves sustained release of the special flavor of palatability enhancers based on gel encapsulation, improves the antioxidant properties of products, prolongs the duration of palatability enhancement, and greatly expands practical application scenarios.

[0037] This invention utilizes modern enzyme technology and fermentation technology for mandarin fish to achieve comprehensive utilization of mandarin fish by-products. It has low production costs, is easy to operate continuously, and is suitable for industrial production.

[0038] This attractant enables the long-term stable release of flavor compounds, and the introduction of hesperidin through encapsulation effectively enhances the functional activity of the product, while also improving the utilization rate and bioavailability of mandarin fish by-products.

[0039] The preparation method of this attractant is green and environmentally friendly throughout the entire process, which improves the utilization rate and bioavailability of mandarin fish by-products, realizes the customized production of the characteristic flavor of stinky mandarin fish, and the long-term stable release of flavor compounds. Furthermore, the introduction of hesperidin through encapsulation effectively enhances the functional activity of the product, meets the diversified needs of current pet food, and is suitable for industrial production. Attached Figure Description

[0040] The invention will now be further described with reference to the accompanying drawings.

[0041] Figure 1 This is a flowchart of the process for producing a pet attractant with the flavor of stinky mandarin fish according to the present invention. Detailed Implementation

[0042] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0043] Example 1

[0044] A method for producing a stinky mandarin fish flavored pet attractant using mandarin fish byproducts, see [link to relevant documentation]. Figure 1 The flowchart shown includes the following steps:

[0045] Step 1: Microfiltration concentration and in-situ encapsulation treatment of fermented mandarin fish broth to obtain liquid microsphere fermentation agent with high abundance and high activity of bacteria;

[0046] The liquid in the container after the fermentation of stinky mandarin fish is completed (the liquid that seeps out of the mandarin fish during the natural fermentation process, i.e., the fermented liquid of stinky mandarin fish) is collected, filtered, and then the filtrate is concentrated by microfiltration and in-situ encapsulation. The microfiltration concentration method is as follows: the pore size of the microfiltration membrane is 1.4µm, and microfiltration is carried out to 20% of the original volume. The retentate is the fermentation concentrate rich in microorganisms.

[0047] The filtration method is as follows: collect the filtrate using a 100-mesh filter.

[0048] The in-situ encapsulation process is as follows: Low-melting-point modified agarose and fermentation concentrate are mixed at a mass ratio of 1:50, heated to 37℃ and stirred for 10 min to promote complete dissolution of the agarose; 10 times the volume of the above mixture (the combined volume of agarose and fermentation concentrate) of polyethylene glycol 10000 is added, and the mixture is stirred until homogeneous to obtain a liquid microsphere fermentation agent containing high abundance, with a high abundance value of 10. 6 Approximately CFU / mL; low melting point modified agarose (ST105, Shanghai Beyotime Biotechnology Co., Ltd.).

[0049] Step 2: Process the mandarin fish by-products using a stepwise enzymatic hydrolysis process to obtain a fermentation substrate with a high degree of hydrolysis;

[0050] Pretreatment: Add 1.5 times the mass of deionized water to the mandarin fish by-product raw material and homogenize it. The homogenization parameters are: 5000 r / min, 4 min. Collect the homogenized liquid. The above mandarin fish by-product raw material mainly consists of a mixture of fish scales, gills and viscera.

[0051] Homogenizing additives are added during the homogenization process. The amount of homogenizing additives added is 0.6% of the mass of the mandarin fish by-product raw material. The homogenizing additives include 1 part citric acid, 1 part sodium chloride, 1 part lecithin, 1 part nanocellulose and 2 parts β-cyclodextrin by mass.

[0052] Among them, the multiple carboxyl groups in citric acid can bind to the peptide chains in collagen, which can play a swelling role, initially destroying the collagen structure in fish scales and making it easier to be enzymatically hydrolyzed.

[0053] Sodium chloride accounts for 1 / 6 of the homogenizing additive, and the actual amount used is 0.1% of the mass of mandarin fish by-product raw material. Trace amounts of sodium chloride can activate the endogenous enzymes in mandarin fish by-product raw material and the proteases added in subsequent stepwise enzymatic hydrolysis.

[0054] Lecithin can reduce the interfacial tension between lipids and proteins in mandarin fish by-products, which helps to promote the dispersion of lipids and the dissolution of proteins, thereby improving the efficiency of subsequent enzymatic hydrolysis.

[0055] Nanocellulose and β-cyclodextrin act as thickeners, synergistically improving the dispersibility and suspension stability of the homogenized liquid.

[0056] During the homogenization process, a grinding aid is added to improve the cell breakage rate and protein release rate of the mandarin fish by-product raw material. The amount of grinding aid added is 3 times the mass of the mandarin fish by-product raw material, and the grinding aid is zirconia beads with a particle size of 1-2 mm.

[0057] Stepwise enzymatic hydrolysis: For the first addition of enzyme, choose one of neutral protease or alkaline protease. The amount of enzyme added to the hydrolysis system is 1.2% (the ratio of the mass of protease to the mass of homogenate), the pH value is 7.5-8.0, the temperature is 50℃, and the hydrolysis time is 2 hours.

[0058] For the second enzyme addition, flavor protease (F768979, Shanghai Maclean Biochemical Technology Co., Ltd.) was selected. The enzyme dosage in the enzymatic hydrolysis system was 0.8% (the ratio of protease mass to homogenate mass), the pH was 7.0, the temperature was 50℃, and the hydrolysis time was 2 hours. After the enzymatic hydrolysis was completed, the hydrolysate was heated to 95℃ for 20 minutes to inactivate the enzyme, yielding the fermentation substrate. The degree of hydrolysis of this fermentation substrate was 22%-26%.

[0059] Step 3: Mix the fermentation substrate with the liquid microsphere fermentation agent for anaerobic fermentation, and then sterilize by microfiltration to obtain a liquid attractant with the flavor of stinky mandarin fish;

[0060] The volume of liquid microsphere fermentation agent added is 20% of the fermentation substrate volume. The mixed fermentation temperature is 15-20℃, and the fermentation time is 3 days. After fermentation, microfiltration sterilization is performed. The pore size of the microfiltration membrane is selected as 0.8µm. The permeate is the by-product fermentation liquid (liquid attractant) with the flavor of stinky mandarin fish.

[0061] Step 4: Microencapsulate the liquid attractant and freeze-dry it to obtain a pet attractant with the flavor of stinky mandarin fish;

[0062] Prepare a sodium hydroxide solution with a pH of 12. Dissolve hesperidin in the sodium hydroxide solution by stirring, and adjust the concentration of hesperidin solution to 1 mg / mL.

[0063] Xanthan gum and corn starch were mixed evenly at a mass ratio of 1:10 to prepare a powder mixture.

[0064] Add 10 times the mass of by-product fermentation broth (liquid palatability enhancer) and 0.5 times the mass of hesperidin solution to the mixed powder, heat in a water bath to 85°C, stir for 30 min, cool to room temperature, freeze dry for 24 h, and then pulverize through a 100-mesh sieve to obtain a pet palatability enhancer (dry powder) with the flavor of stinky mandarin fish. The pet palatability enhancer (dry powder) was reconstituted to a concentration of 10 mg / mL and the following tests were performed: (1) Electronic tongue test. (2) Palatability test: The palatability enhancer was sprayed onto the surface of commercially available extruded cat food at a spraying amount of 1 mL / 100 g (palatability enhancer solution / cat food mass). Fifteen cats of the same breed, aged 1-1.5 years, cage-housed, were selected. The palatability of the cat food was analyzed using the double-bowl method, and the first-choice rate and the feeding rate were recorded.

[0065] Example 2

[0066] The difference between this embodiment and Example 1 is that the amount of flavor protease added in step two is adjusted from 0.8% to 1%, while the remaining raw materials and preparation process remain the same as in Example 1.

[0067] Example 3

[0068] The difference between this embodiment and Example 1 is that the amount of flavor protease added in step two is adjusted from 0.8% to 1.2%, while the other raw materials and preparation process remain the same as in Example 1.

[0069] Example 4

[0070] The difference between this embodiment and embodiment 2 is that the volume of liquid microsphere fermentation agent added in step 3 is adjusted to 30% of the volume of the fermentation substrate, while the remaining raw materials and preparation process remain the same as in embodiment 2.

[0071] Example 5

[0072] The difference between this embodiment and embodiment 2 is that the volume of the liquid microsphere fermentation agent added in step 3 is adjusted to 40% of the volume of the fermentation substrate, while the remaining raw materials and preparation process remain the same as in embodiment 2.

[0073] Example 6

[0074] Compared to Example 4, this embodiment omits steps one and three. In step two, the fermentation substrate is concentrated by microfiltration (0.8µm) and then freeze-dried. Electronic tongue and palatability analysis are performed.

[0075] Example 7

[0076] Compared with Example 4, this embodiment omits step four and directly collects the fermentation broth, a byproduct from step three, freeze-dries it, and then reconstitutes it at a concentration of 10 mg / mL. This broth is then sprayed onto the surface of commercially available extruded cat food at a spraying amount of 1 mL / 100g (attractant solution / cat food mass). Example 4 is used as a control, and volatile compounds are detected at 0h, 2h, and 4h.

[0077] Example 8

[0078] The difference between this embodiment and Example 4 is that only xanthan gum and corn starch were used in the microencapsulation step four, without the addition of hesperidin solution. After freeze-drying, the solution was reconstituted at a concentration of 10 mg / mL and sprayed onto the surface of commercially available extruded cat food at a rate of 1 mL / 100 g (attractant solution / cat food mass). Example 4 was used as a control, and volatile compound detection was performed after 0 h, 2 h, and 4 h. To evaluate the antioxidant effect of the hesperidin gel, the reconstituted attractant was injected into disposable petri dishes and incubated at 35°C for 0 h, 2 h, and 4 h before antioxidant activity testing. Antioxidant activity was evaluated using the ABTS free radical scavenging test and the DPPH free radical scavenging test.

[0079] The results corresponding to Examples 1-6 are recorded in Table 1:

[0080] Table 1. Results of Electronic Tongue Detection

[0081]

[0082] As shown in Table 1, there are certain differences in the taste characteristics of the palatability enhancers in Example 1 and Example 6. Comparing the bitterness values, the bitterness values ​​of Examples 1, 2, and 3 decreased with increasing amounts of flavor protease, which is consistent with expectations. This indicates that flavor protease makes a significant contribution to reducing bitterness. Specifically, the bitterness value in Example 2 decreased by 8.3% compared to Example 1, while the bitterness value in Example 3 decreased by only 3% compared to Example 2. This suggests that although flavor protease can reduce bitterness, its effect is limited. Therefore, considering processing costs, the 1% flavor protease addition in Example 2 is preferred for the enzymatic hydrolysis process. Comparing the fermentation broth addition amounts (Examples 4 and 5), it was found that the bitterness value decreased significantly with increasing fermentation broth proportion. However, the saltiness value also increased with increasing fermentation broth content. This may be due to the salt content in the fermentation broth; excessive salt may have adverse effects on pet health. Therefore, the 30% fermentation broth proportion in Example 4 is preferred for fermentation. Furthermore, the saltiness and richness were both at high levels in Example 5, indicating that moderate enzymatic hydrolysis and fermentation can produce more flavor molecules such as umami peptides and richness peptides. In Example 6, where fermentation was not incorporated into the production of the palatability enhancer, the bitterness value was significantly higher than in other examples, while the umami and richness were significantly lower. This result demonstrates the significant contribution of fermentation to reducing bitterness and enhancing umami and richness.

[0083] Table 2 Palatability Test

[0084] Palatability testing reflects pets' preference for the flavor of the palatability enhancer. Table 2 shows that the preferred selection rate for Examples 1-5 was higher than that for the unfermented Example 6, demonstrating the improvement effect of fermentation with fermented mandarin fish broth on the palatability enhancer flavor. Examples 4 and 5 had the highest preferred selection rates, indicating that increasing the proportion of fermented broth further enhances the flavor of the fermented mandarin fish, thereby increasing pets' preference. Regarding consumption rate, pets in Examples 4 and 5 showed a significant increase in intake, indicating that the flavor substances in the palatability enhancer and the flavor of the fermented mandarin fish can stimulate pets' appetite and increase food intake.

[0085] Table 3 Flavor Stability Test

[0086]

[0087] Table 4 Antioxidant Activity Test

[0088]

[0089] Flavor stability testing reflects the release process of volatile compounds in attractants, thus assessing the duration of their attractant effect. Table 3 lists the main volatile compounds of the stinky mandarin fish flavor and their relative content at different attractant durations. Example 7, prepared using a direct freeze-drying method without encapsulation, exhibited the highest concentration of volatile flavor compounds at the start of spraying, exceeding that of Example 4 after encapsulation and freeze-drying. However, with prolonged attractant time, the concentration of flavor compounds decreased sharply, reaching only about 25% of the initial concentration after 4 hours of attractant treatment. In contrast, the concentration of compounds in Example 4 remained above 75% of the initial concentration even after 4 hours of continuous exposure, indicating that corn starch / xanthan gum encapsulation significantly enhances the duration of the attractant's effect.

[0090] Antioxidant activity testing can reflect the preservation quality stability of palatability enhancers in real-world application scenarios, especially in resisting environmental factors such as light and heat. This test is used to demonstrate whether the introduction of hesperidin can improve the antioxidant performance of palatability enhancers. Table 4 lists the antioxidant activity test values ​​of Examples 4, 7, and 8 under environmental exposure at 0h, 2h, and 4h, respectively. Compared with Example 4, Example 7 did not encapsulate the palatability enhancer, and Example 8 did not add hesperidin to the encapsulation system. Their initial antioxidant activity was significantly lower than that of the hesperidin-encapsulated group (Example 4), and their antioxidant retention effect after 4h was significantly lower than that of Example 4. This indicates that the hesperidin / xanthan gum / corn starch microcapsules effectively improved the antioxidant activity and stability of the palatability enhancer. A comparison between Examples 7 and 8 revealed no significant difference in initial antioxidant values. However, in terms of activity loss, Example 7 showed a faster rate of activity loss, with antioxidant activity values ​​after 4 hours only reaching approximately 18% (ABTS test) and 22% (DPPH test) of the initial values. In contrast, Example 8 retained approximately 59% (ABTS test) and 28% (DPPH test) of the initial values, indicating that microencapsulation enhances the retention of the palatability enhancer's activity. In conclusion, the introduction of hesperidin during microencapsulation is essential for improving the antioxidant properties and stability of palatability enhancers.

[0091] In summary, the present invention proposes a method for producing a pet attractant with a stinky mandarin fish flavor using mandarin fish by-products. The pet attractant with a stinky mandarin fish flavor is prepared by stepwise enzymatic hydrolysis and synergistic fermentation of mandarin fish by-products. It does not require excessive processing and can be directly used in pet food or as an attractant. It is green and safe, reduces production costs, is convenient for continuous operation, and is suitable for industrial production.

[0092] The attractant prepared by this method carries the flavor of stinky mandarin fish, making it more appealing to pets. It achieves long-term stable release of flavor compounds, and the introduction of hesperidin through encapsulation effectively improves the functional activity of the product. At the same time, it improves the utilization rate and bioavailability of stinky mandarin fish by-products.

[0093] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0094] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for producing a pet attractant with a stinky mandarin fish flavor using by-products of mandarin fish, characterized in that, The method includes the following steps: Step 1: Microfiltration concentration and in-situ encapsulation treatment of fermented mandarin fish broth to obtain liquid microsphere fermentation agent; The fermented liquid of stinky mandarin fish is the liquid that seeps out of the mandarin fish during the natural fermentation process. The liquid is collected, filtered, and then the filtrate is concentrated by microfiltration and in-situ encapsulation. The microfiltration concentration method is as follows: the microfiltration membrane has a pore size of 1.4µm, and the microfiltration is carried out to 20% of the original volume. The retentate is the fermentation concentrate rich in microorganisms. The in-situ embedding process is as follows: low melting point modified agarose and fermentation concentrate are mixed at a mass ratio of 1:50, heated to 37℃ and stirred for 10 min to promote complete dissolution of agarose, 10 times the volume of polyethylene glycol 10000 is added, and the mixture is stirred evenly to obtain liquid microsphere fermentation agent. Step 2: The homogenized liquid of mandarin fish by-products is subjected to a stepwise enzymatic hydrolysis process to obtain the fermentation substrate; The stepwise enzymatic hydrolysis process is a two-step enzymatic hydrolysis method, namely: the first enzyme addition is selected from neutral protease and alkaline protease, the enzyme amount in the enzymatic hydrolysis system is 1.2%, the pH is 7.5-8.0, the temperature is 50℃, and the enzymatic hydrolysis time is 2 hours; For the second enzyme addition, flavor protease was selected. The enzyme dosage in the enzymatic hydrolysis system was 1%-1.2%, the pH was 7.0, the temperature was 50℃, and the hydrolysis time was 2 hours. After the enzymatic hydrolysis was completed, the hydrolysate was heated to 95℃ and subjected to enzyme inactivation treatment for 20 minutes to obtain the fermentation substrate. Step 3: Mix the fermentation substrate with the liquid microsphere starter culture for anaerobic fermentation, and then sterilize by microfiltration to obtain a liquid attractant with the flavor of stinky mandarin fish; the volume of the liquid microsphere starter culture added is 20%-40% of the volume of the fermentation substrate; Step 4: Microencapsulate the liquid attractant and freeze-dry it to obtain a pet attractant with the flavor of stinky mandarin fish; The microencapsulation method is as follows: prepare a sodium hydroxide solution with a pH of 12, stir and dissolve hesperidin in the sodium hydroxide solution, and adjust the concentration of hesperidin solution to 1 mg / mL; Xanthan gum and corn starch were mixed evenly at a mass ratio of 1:10 to prepare a powder mixture. Add 10 times the mass of liquid attractant and 0.5 times the mass of hesperidin solution to the mixed powder, heat in a water bath to 85°C, stir for 30 min, cool to room temperature, freeze dry for 24 h, and then pulverize and pass through a 100-mesh sieve to obtain a pet attractant with the flavor of stinky mandarin fish.

2. The method for producing a stinky mandarin fish flavored pet attractant using mandarin fish by-products according to claim 1, characterized in that, The homogenization process in step two is as follows: 1.5 times the mass of deionized water is added to the mandarin fish by-product raw material for homogenization. The homogenization parameters are: 5000 r / min, 4 min, and the homogenized liquid is collected.

3. The method for producing a stinky mandarin fish flavored pet attractant using mandarin fish by-products according to claim 1, characterized in that, The degree of hydrolysis of this fermentation substrate is 22%-26%.

4. A method for preparing a stinky mandarin fish flavored pet attractant using mandarin fish by-products according to claim 1, characterized in that, In step three, the mixed fermentation temperature is 15-20℃, and the fermentation time is 3-5 days; the microfiltration membrane pore size is selected as 0.8µm, and the permeate is a liquid attractant with the flavor of stinky mandarin fish.

5. A method for preparing a stinky mandarin fish flavored pet attractant using mandarin fish by-products according to claim 1, characterized in that, This pet palatability enhancer is a dry powder. The method of use for this pet palatability enhancer is to add it to the pet food formula in proportion or to reconstitute it and then spray it on the surface of the pet food. The procedure for adding it to pet food is as follows: the amount added is 0.2%-0.3% of the total weight of the pet food formula ingredients; The procedure for reconstituted pet food and spraying it onto the surface is as follows: reconstitute the dry powder of pet palatability enhancer to a concentration of 10 mg / mL, and spray it onto the surface of the extruded cat food at a rate of 1 mL / 100g.

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