Spanish mackerel high-calcium fishbone freeze-dried powder as well as preparation method and application thereof

Abstract

The invention provides mackerel high-calcium fish bone freeze-dried powder as well as a preparation method and application thereof. Mackerel fish bones are treated step by step through compound protease enzymolysis and plant lactobacillus fermentation, the mackerel fish bones are crushed and then subjected to enzymolysis with compound enzyme of alkaline protease and neutral protease, enzyme deactivation and filtration are performed, and mackerel enzymolysis filtrate is obtained; and fermenting the fishbone by adopting plant lactic acid bacteria, and freeze-drying to obtain the mackerel high-calcium fishbone freeze-dried powder. The fish bone freeze-dried powder prepared by the method provided by the invention is high in calcium ion content and rich in flavor amino acid, the utilization rate of protein is improved, and meanwhile, the fishy smell removal effect is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and particularly relates to a high-calcium mackerel fish bone freeze-dried powder, a preparation method and an application thereof. Background Art

[0002] The pet food market is gradually shifting from traditional pet food to functional, specialized products, and consumers are demanding higher functionality and nutritional value from pet food. Pet health and longevity are a growing concern for pet owners. The intestinal tract, the primary organ for nutrient digestion and absorption, determines the animal's overall health. Therefore, providing a safe, nutritionally complete, and balanced diet is crucial for maintaining pet health. A pet's intestinal health is closely related to the type and content of nutrients in their diet. High-calcium fish bone freeze-dried powder, a product rich in calcium and other nutrients, meets the market demand for healthy pet food.

[0003] Spanish mackerel, a widely distributed marine fish, also produces calcium-rich bones. In recent years, high-calcium fermented fish bone powder, produced through microbial fermentation, has not only improved the bioavailability of calcium from fish bones but also imparted a unique flavor and functionality. This new calcium supplement shows great potential for improving bone health. Fish bones, a resource rich in nutrients such as protein and minerals, are gaining increasing attention. Compared to other marine fish, Spanish mackerel boasts a high calcium content, rich nutritional profile, and abundant resources, making them relatively easy to obtain. However, their strong fishy odor makes processing more challenging. Fish bone fermentation utilizes lactic acid bacteria to metabolize and degrade fish bones, thereby altering their properties. Because lactic acid bacteria secrete proteases that metabolize and produce aroma, they can alleviate the unpleasant odor of fish bone powder, offering a novel method for processing fish bone powder.

[0004] Patent document CN113072616B discloses a functional peptide microcapsule that promotes the proliferation of osteoblast precursor cells and a preparation method thereof. The invention uses citric acid and acid protease to enzymatically hydrolyze tuna bones, and then prepares functional peptide microcapsules through mixed fermentation of lactic acid bacteria and glucose; Patent document CN107495184A discloses a fish bone powder and protein peptide mixture chewable tablet. The application first uses glucose and lactic acid bacteria to ferment the fish bone powder, and then prepares the fish bone powder and protein peptide mixture chewable tablets by step-by-step enzymatic hydrolysis, isoelectric precipitation and freeze-drying. Although the technical solutions disclosed in the above applications all utilize a combination of enzymatic hydrolysis and lactic acid bacteria fermentation to process fish bones, the processing difficulty of fish is relatively small, while the acquisition cost of tuna is high, and the nutritional content of the prepared products is limited.

[0005] Therefore, there is an urgent need to provide a new fish bone meal with high nutritional content and a processing method thereof to meet the market demand for high-quality pet food. Summary of the Invention

[0006] The present invention aims to provide a high-calcium mackerel bone freeze-dried powder, a preparation method and an application thereof. The mackerel bones are enzymatically hydrolyzed and fermented using a complex enzyme and lactic acid bacteria, so that the substances metabolized by the lactic acid bacteria mask the bitterness and fishy smell of the bones and improve the utilization rate of protein, thereby obtaining a high-calcium fish bone freeze-dried powder with a good flavor.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] On the one hand, the present invention provides a method for preparing high-calcium mackerel bone freeze-dried powder. The preparation method first uses a complex enzyme to hydrolyze the mackerel bones, and then uses lactic acid bacteria to ferment them.

[0009] The calcium in fish bones mainly exists in the form of hydroxyapatite, which has poor solubility and low direct extraction efficiency. However, hydroxyapatite has high solubility in acidic conditions (pH < 4.5), but traditional acid hydrolysis may destroy other nutrients (such as protein). Therefore, in order to effectively promote the calcium dissolution rate in fish bones, gently dissolve calcium, and reduce the destruction of beneficial substances such as flavor amino acids in fish bones, the present invention creatively utilizes a combined process of enzymatic hydrolysis of fish bones with a complex enzyme and then fermentation with lactic acid bacteria to treat Spanish mackerel bones. The protease species of the enzymatic hydrolysis process and the lactic acid bacteria species of the fermentation process are compared and studied, and the optimal preparation process for effectively improving the calcium ion content in Spanish mackerel fish bone freeze-dried powder is screened out, while also increasing the content of flavor amino acids in the fish bones.

[0010] Furthermore, the complex enzyme includes any one or more of acidic protease (pepsin), neutral protease, and alkaline protease.

[0011] In some embodiments, the present invention uses a variety of enzymatic hydrolysis methods such as acidic protease, neutral protease, alkaline protease, a combination of acidic protease and neutral protease, and a combination of alkaline protease and neutral protease for comparison. The results show that only the composite enzymatic hydrolysis method using a combination of alkaline protease and neutral protease can effectively promote the conversion of calcium in fish bones from the macromolecular state of hydroxyapatite to the free state, significantly reduce the particle size of fish bone powder, improve the bioavailability of calcium, and at the same time avoid the loss of flavor amino acids in fish bones, giving it a unique fermented flavor. After using acidic protease or a composite enzyme of acidic protease and neutral protease, the content of calcium ions and flavor amino acids in the fermentation broth is low. The reason is that acidic protease prefers to cleave the amino termini of aromatic amino acids (phenylalanine, tyrosine), producing peptides rich in hydrophobic amino acids. These hydrophobic peptides easily form bitter peptides, inhibit microbial activity or mask flavor, and are difficult to be further converted into flavor amino acids (such as glutamic acid and aspartic acid).

[0012] Furthermore, the lactic acid bacteria include any one or more of Lactobacillus plantarum and Lactobacillus acidophilus, and the fermentation process further includes adding glucose for mixed fermentation.

[0013] Different microbial strains differ significantly in the raw materials they utilize and the products they metabolize during fermentation. To obtain high-calcium fish bone freeze-dried powder, acid-producing microorganisms should be used to ferment and dissolve hydroxyapatite, thereby promoting calcium ion dissolution. Therefore, in some embodiments, the present invention compared the fermentation differences of several different acid-producing microorganisms. The results showed that the calcium ion content and flavor amino acid content of fish bone fermentation broths fermented with Lactobacillus plantarum were significantly higher than those of other groups, and the particle size of the fish bone powder was significantly reduced. This is because, compared with other acid-producing strains, the lactic acid produced by Lactobacillus plantarum metabolism lowers the pH, promoting the dissolution of hydroxyapatite in the fish bones and releasing soluble calcium. Organic acids such as lactic acid form stable chelates with calcium, increasing calcium bioaccessibility. Furthermore, in the later stages of fermentation, microorganisms autolyze and release intracellular amino acids, further increasing the content of flavor compounds. Furthermore, because glucose is a fast-acting carbon source for microorganisms, rapidly generating ATP through glycolysis to support bacterial growth and proliferation, adding a certain amount of glucose during the fermentation process promotes acid production by Lactobacillus plantarum, further promoting the dissolution of hydroxyapatite in the fish bones and increasing calcium ion content.

[0014] Furthermore, the complex enzyme includes alkaline protease and neutral protease, and the lactic acid bacteria includes plant lactobacillus.

[0015] Furthermore, the ratio of the alkaline protease to the neutral protease is (1-5):1, the inoculation amount of the plant lactobacillus is 2%-6%, and the addition amount of the glucose is 10-30 g / L.

[0016] In some embodiments, the present invention is based on the results of a single-factor experiment, with the independent variables selected as glucose addition (A), bacterial inoculum size (B), and fermentation time (C), and the response value selected as the calcium ion content of the fish bone fermentation liquid, and a response surface experiment is performed. The F test and lack-of-fit test results of the quadratic regression equation obtained show that there is a significant interaction relationship between the free calcium ion content in the fish bone fermentation liquid and the fermentation time, inoculum size, and glucose addition. Further software is used to analyze the response relationship between the three factors. The results of the three-dimensional surface plot and contour map show that the inoculum size of plant lactobacillus is preferably 4.27%, the amount of glucose added is preferably 17.11 g / L, and the fermentation time is preferably 24.7 h.

[0017] Furthermore, the method comprises the following steps:

[0018] S1: preparing mackerel bone powder;

[0019] S2: hydrolyzing the Spanish mackerel bone powder obtained in step S1 with alkaline protease and neutral protease to obtain a Spanish mackerel bone powder hydrolyzate;

[0020] S3: performing enzyme inactivation treatment on the mackerel bone powder enzymatic hydrolyzate obtained in step S2;

[0021] S4: filtering the Spanish mackerel bone powder enzymatic hydrolyzate after the enzyme inactivation in step S3 to obtain a filtrate;

[0022] S5: inoculating Lactobacillus plantarum into the filtrate obtained in step S4;

[0023] S6: mixing the filtrate obtained after the inoculation in step S5 with glucose and fermenting the mixture to obtain a fermented mackerel bone liquid;

[0024] S7: freeze-drying the Spanish mackerel bone fermentation liquid from step S6 to obtain Spanish mackerel high-calcium fish bone freeze-dried powder.

[0025] On the other hand, the present invention provides Spanish mackerel high calcium fish bone freeze-dried powder prepared by the preparation method of the Spanish mackerel high calcium fish bone freeze-dried powder as described above.

[0026] In another aspect, the present invention provides a use of a complex enzyme for preparing a reagent for increasing the calcium ion content in freeze-dried mackerel bone powder, wherein the complex enzyme comprises any one or more of an acidic protease, a neutral protease, and an alkaline protease.

[0027] Furthermore, the complex enzyme includes alkaline protease and neutral protease.

[0028] In another aspect, the present invention provides use of Lactobacillus plantarum for increasing the calcium ion content in freeze-dried mackerel bone powder.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The present invention uses a combination of alkaline protease and neutral protease enzymatic hydrolysis and plant lactic acid bacteria fermentation to treat mackerel bones, significantly increasing the calcium ion content and flavor amino acid content in the freeze-dried fish bone powder while also achieving the effect of removing fishy smell, giving the product a unique fermented flavor while fully retaining nutrients.

[0031] 2. The Spanish mackerel high-calcium fish bone freeze-dried powder obtained by the preparation method provided by the present invention has a small average molecular weight, strong activity, high bioavailability, and is more easily absorbed. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the solutions in this application, a brief introduction will be given below to the drawings required for use in the description of the embodiments of this application. Obviously, the drawings described below are some embodiments of this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0033] Figure 1 These are the response surface diagram and contour diagram of the effects of bacterial inoculum size and glucose addition amount on the calcium ion content of fish bone fermentation broth when other variables are not affected.

[0034] Figure 2 It is a response surface diagram and contour diagram showing the effects of fermentation time and glucose addition on the calcium ion content of fish bone fermentation liquid when no other variables are affected.

[0035] Figure 3 These are the response surface diagram and contour diagram of the effects of bacterial inoculum size and fermentation time on the calcium ion content of fish bone fermentation broth when other variables are not affected.

[0036] Figure 4 The scanning electron microscope was used to observe the microstructural changes of the fermentation liquid of Spanish mackerel bones before and after fermentation (left: before fermentation; right: after fermentation).

[0037] Figure 5 This is the particle size distribution diagram of the fermented liquid of Spanish mackerel bones before and after fermentation (top: fish bone powder, bottom: fermented fish bone powder). DETAILED DESCRIPTION

[0038] In order to make the technical personnel of this technical field better understand the present application scheme, the technical scheme of the present invention is clearly and completely described below in conjunction with the examples and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0039] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0040] Example 1: Optimal preparation method of Spanish mackerel high calcium fish bone freeze-dried powder provided by the present invention

[0041] The preparation process of Spanish mackerel high calcium fish bone freeze-dried powder is as follows:

[0042] (1) Preparation of swordfish bone powder: fresh fish bones and water were boiled in boiling water at a volume ratio of 1:1 for 10 minutes, non-bone components such as fish meat were removed, dried at 60°C for 6 hours, crushed and passed through an 80-mesh sieve, and then the fish bone powder was mixed with anhydrous ethanol (bone powder: anhydrous ethanol = 1:10), stirred for 20 hours, and centrifuged at 3000 r / min for 15 minutes. The precipitate was rinsed clean, dried and stored at -20°C for later use.

[0043] (2) Enzymatic hydrolysis: After the Spanish mackerel bones of step (1) are crushed and dried, a composite enzyme (alkaline protease (purchased from Beijing Solebow Technology Co., Ltd., 200,000 U / g): neutral protease (purchased from Beijing Solebow Technology Co., Ltd., 200,000 U / g) = 2:1) with a mass fraction of 3% of the fish bone powder is added for enzymatic hydrolysis at 55° C. for 5 h to obtain a Spanish mackerel bone powder enzymatic hydrolyzate with a high degree of protein hydrolysis.

[0044] (3) Inactivation of enzyme: The mackerel bone powder enzymatic hydrolysate obtained in step (2) was inactivated in a 100° C. water bath for 15 min, cooled to room temperature, and refrigerated at 4° C. for later use.

[0045] (4) Filtration: The Spanish mackerel bone powder enzymatic hydrolyzate after the enzyme inactivation in step (3) is filtered through a 180-240 mesh sieve to obtain a filtrate.

[0046] (5) Inoculation: 2% to 6% of Lactobacillus plantarum (preferably 4.27% inoculation in this embodiment, Lactobacillus plantarum, strain accession number ATCC8014) is inoculated into the filtrate obtained in step (4);

[0047] (6) Fermentation: The filtrate of (5) is mixed with 10-30 g / L glucose (preferably 17.11 g / L in this embodiment) and fermented for 12-36 h (preferably 24.7 h in this embodiment) to obtain a Spanish mackerel bone fermentation liquid;

[0048] (7) Freeze-drying: After the fermentation treatment is completed, filter the mixture through a 180-240 mesh sieve, and then homogenize the obtained filtrate to obtain a homogenous liquid; finally, pre-freeze the prepared homogenous liquid at a temperature of -80°C for 6-10 hours; after pre-freezing, vacuumize the mixture at 90kPa-110kPa; and then freeze-dry the mixture into powder at -30-40°C for 46-50 hours to obtain high-calcium fish bone freeze-dried powder.

[0049] Example 2: Protease screening for enzymatic hydrolysis

[0050] Because marine life lives in an environment completely different from that of terrestrial life, characterized by extremes such as high pressure, low temperature, high temperature, and high salinity, the proteins produced by marine life are significantly different from those produced on land in order to adapt to these extremes. Therefore, in order to fully utilize the protein resources in marine life, a series of research on extraction processes is required.

[0051] Based on this, the present invention conducted a comparative verification on the enzymatic hydrolysis step in the optimal preparation method of the Spanish mackerel high calcium fish bone freeze-dried powder in Example 1, and explored the quality differences of the Spanish mackerel fish bone freeze-dried powder prepared by the five enzymatic hydrolysis processes shown in Table 1. Compared with the preparation method in Example 1, only the type of enzyme used was different, and the remaining steps were the same as in Example 1. The calcium ion content in the fish bone powder was then measured, specifically in the following steps:

[0052] Add 20 μl of standard or sample to each well of a 96-well plate and bring to 50 μl with deionized water. Record the sample volume as V. Add 150 μl of assay working solution to each well and mix thoroughly. Incubate at room temperature in the dark for 5–10 minutes. Measure the absorbance at 575 nm using a microplate reader and calculate the calcium ion content of the mackerel bone fermentation broth based on the standard curve.

[0053] The flavor amino acid content (including glutamic acid, glycine, alanine, serine, and aspartic acid) in the fermentation broth was determined by the acid hydrolysis method: 1 mL of fermentation broth sample was added with 9 mL of 1% sulfosalicylic acid to make up to 10 mL. The filtrate was centrifuged at 10,000 rpm for 10 min and filtered through a 0.45 μm microporous membrane. The filtrate was assayed according to the working conditions of the amino acid analyzer.

[0054] Bone meal particle size was measured using a laser particle size distribution analyzer. The average particle size of bone meal is expressed as the median particle size (D50). Particle size is a key factor in the properties of fermented bone meal. The smaller the particle size, the higher the bioavailability of the fermented fish bone meal. The results are shown in the table below.

[0055] Table 1 Effects of different proteases on the quality of freeze-dried mackerel bone powder

[0056]

[0057]

[0058] As shown in the calculation results in Table 1, compared with the control group without enzymatic hydrolysis, the enzymatic hydrolysis of fish bones with protease can increase the content of calcium ions and flavor amino acids in the fermentation liquid, and the average particle size of fish bone powder is reduced. However, the calcium ion content and flavor amino acid content in the fermentation liquid after the Spanish mackerel bones are hydrolyzed with different types of proteases are quite different. Among them, the calcium ion content and flavor amino acid content obtained by the composite enzymatic hydrolysis method composed of alkaline protease and neutral protease were the highest, and the average particle size was the smallest, which was 2.34μm. The effect of alkaline protease or neutral protease alone in promoting calcium dissolution was limited, and the particle size changed little compared with the control, indicating that the combination of alkaline protease and neutral protease can effectively improve the bioavailability of Spanish mackerel bones and promote the conversion of calcium substances in fish bones from bound state to free state; the calcium ion content obtained by enzymatic hydrolysis of Spanish mackerel bones by acidic protease alone or the composite enzyme combination of acidic protease and neutral protease was also significantly lower than that by the combination of alkaline protease and neutral protease. The reason is that the calcium in fish bones mainly exists in the form of hydroxyapatite, whose solubility is highly dependent on pH. Excessive acidification will cause free Ca 2+ It combines with anions (such as Cl-) in an acidic environment and reprecipitates as CaCl2·2H2O. Therefore, only the use of a complex enzyme of alkaline protease and neutral protease can effectively increase the calcium ion content and flavor amino acid content in the freeze-dried mackerel bone powder, giving the freeze-dried fish bone powder an excellent flavor.

[0059] Example 3: Screening of microbial strains for fermentation process

[0060] According to existing research, microbial fermentation technology can significantly promote the dissolution rate of calcium in fish bones, improve protein hydrolysis efficiency, and give the product special functional activity (such as prebiotic properties). Different strains play a role through metabolic acid production, secretion of enzymes or biotransformation. Therefore, it is necessary to select suitable fermentation strains in combination with target product characteristics and process adaptability. In view of the fermentation demand after enzymatic hydrolysis of Spanish mackerel fish bones and the high calcium content requirement of the obtained freeze-dried powder, the present invention has made further comparative screening of the strains in the fermentation process, and inoculated fermentation is carried out using several fermentation strains shown in Table 2. Other preparation processes and steps are the same as in Example 1. The content of calcium ions and flavor amino acids in the Spanish mackerel fish bone fermentation liquid and the particle size of the fish bone powder are determined using the same method as in Example 2. The results are shown in the table below.

[0061] Table 2 Effects of different fermentation strains on the quality of mackerel bone freeze-dried powder

[0062]

[0063] The results in Table 2 show that fish bones without microbial fermentation after enzymatic hydrolysis have a low calcium dissolution rate, low calcium ion content, and low flavor amino acid content in the fermentation broth. However, fermentation with different strains of bacteria can increase the calcium ion content and flavor amino acid content of the fish bone fermentation broth to varying degrees, while also narrowing the particle size distribution of the fish bone powder. Fermentation with Lactobacillus plantarum yields the best results, with the highest calcium ion content in the fermentation broth at 27.55 mg / 100 mL, a flavor amino acid content of 69.025 g / 100 g, and an average particle size of 2.36 μm.

[0064] The fermentation effect of Lactobacillus acidophilus or Bacillus subtilis is significantly inferior to that of Lactobacillus plantarum. This may be because Lactobacillus plantarum, as a homolactic fermentation bacterium, can efficiently metabolize glucose to produce large amounts of lactic acid, rapidly lowering the pH of the fermentation system. This acidic environment not only directly dissolves the hydroxyapatite in the fish bones, releasing soluble calcium ions, but also forms a stable calcium lactate complex through the chelation of lactic acid and calcium. Although Lactobacillus acidophilus is also a lactic acid bacterium, its acid production rate and total amount are generally lower, and the pH drop is limited, resulting in insufficient calcium dissolution efficiency. Bacillus subtilis, on the other hand, mainly metabolizes aerobically, and its metabolites are mainly short-chain fatty acids such as acetic acid and butyric acid. These organic acids have weak calcium chelation ability and cannot effectively dissolve minerals.

[0065] The calcium ion content and flavor amino acid content in the fish bone fermentation liquid after adopting plant lactobacillus and lactobacillus acidophilus composite fermentation simultaneously are lower than plant lactobacillus, and the average particle size of fish bone powder is also larger. It may be because after adding lactobacillus acidophilus, the competition of two bacteria for carbon source (such as glucose) may cause overall metabolic efficiency to decline: lactobacillus acidophilus acid production rate is slow and total amount is limited, and continuous low pH environment cannot be maintained, resulting in incomplete dissolution of hydroxyapatite. Therefore, the present invention is preferably prepared by plant lactobacillus fermentation of Spanish mackerel high calcium fish bone freeze-dried powder, which can significantly improve the local flavor and quality of fish bone after fermentation.

[0066] Example 4: Carbon source selection for fermentation process

[0067] Because plant lactobacillus is a heterotrophic organism, it needs to rely on a carbon source for energy metabolism and fermentation during its fermentation process. Therefore, the present invention further compares the carbon source material that effectively promotes plant lactobacillus fermentation, to screen out the carbon source that can significantly improve the quality of fish bone freeze-dried powder. Maltose and / or glucose are used as carbon sources to ferment together with plant lactobacillus, other processes and steps are the same as in Example 1, and the content of calcium ions and flavor amino acids in Spanish mackerel fish bone fermentation liquid and the particle size of fish bone powder are measured using the same method as in Example 2. The results are shown in Table 3.

[0068] Table 3 Effects of different fermentation carbon sources on the quality of mackerel bone freeze-dried powder

[0069]

[0070] According to the results of the effects of different fermentation carbon sources on the quality of mackerel bone freeze-dried powder in Table 3, different carbon sources have a significant effect on the quality of mackerel bone freeze-dried powder. + The best results were achieved in terms of content and flavor amino acid content, Ca + The content reached 28.77 mg / 100 mL, which was significantly higher than that of the groups with maltose, glucose + maltose as carbon source and no carbon source inoculation; the flavor amino acid content of the glucose group reached 68.312 g / 100 g, which was significantly higher than that of other groups. This may be because different carbon sources will affect the types and contents of organic acids produced by microorganisms. When glucose is used as a carbon source, lactic acid bacteria will produce a large amount of lactic acid; and when maltose is used as a carbon source, in addition to lactic acid, a certain amount of other organic acids such as propionic acid may also be produced. The type and content of organic acids will not only affect the pH value of the fermentation liquid, and thus affect the stability of the fermentation system and the growth of microorganisms, but will also have an important impact on the flavor and quality of the final product. In addition, when glucose is used as a fermentation carbon source, the average particle size of the fish bone powder is also smaller. Therefore, the present invention preferably uses glucose as a carbon source for fermenting and preparing Spanish mackerel high-calcium fish bone freeze-dried powder, which can significantly improve the flavor and quality of the fish bones after fermentation.

[0071] Example 5: Optimization of bacterial fermentation conditions

[0072] In the step of utilizing microbial strains to ferment, factors such as microbial activity, strain inoculum size will also significantly affect calcium ion content in the finished product. In order to further determine the appropriate conditions for microbial fermentation, the present invention utilizes Design-Expert 13 to carry out Box-Behnken design response surface experiment, based on the single factor test result, independent variable selection glucose addition (A), bacteria inoculum size (B) and fermentation time (C), response value selection fish bone fermentation liquid calcium ion content, carry out response surface experiment, carry out 17 groups of tests according to each condition level designed by software, each test is repeated three times and average value is taken as final result. Response surface analysis test design and result are as shown in Table 4, and regression equation variance analysis result is as shown in Table 5.

[0073] Table 4 Response surface experimental design and results

[0074]

[0075] Table 5 Analysis of variance of response surface test regression model

[0076] Source of variation sum of squares degrees of freedom mean square F-number P-value Significance Model 123.52932 9 13.73 68.16 <0.0001 ** A-Fermentation time 0.98 1 0.98 4.87 0.0632 — B-bacteria inoculum amount 0.15125 1 0.1513 0.7512 0.4148 — C-glucose addition 5.95125 1 5.95 29.56 0.001 ** AB 1.3225 1 1.32 6.57 0.0374 * AC 0.1225 1 0.1225 0.6084 0.461 — BC 0.49 1 0.49 2.43 0.1627 — <![CDATA[A 2 ]]> 56.711158 1 56.71 281.64 <0.0001 ** <![CDATA[B 2 ]]> 45.713789 1 45.71 227.03 <0.0001 ** <![CDATA[C 2 ]]> 3.0064211 1 3.01 14.93 0.0062 ** residual 1.4095 7 0.2014 — — — Lack of Fit 0.7375 3 0.2458 1.46 0.3508 — Pure error 0.672 4 0.168 — — — Corrected Sum 124.93882 16 — — — —

[0077] Note: R 2 =0.9887, R 2 Adj=0.9742; “**” indicates extremely significant effect (p < 0.01); “*” indicates significant effect (p < 0.05).

[0078] The results shown in Table 4 were subjected to quadratic multivariate fitting using Design-Expert 13. The quadratic multivariate regression equation for the free calcium ion content (Y) in the fish bone fermentation broth versus the fermentation time (A), bacterial inoculum size (B), and glucose addition amount (C) was obtained as follows:

[0079] As shown in Table 5, the F test and lack-of-fit test results of the quadratic regression equation obtained from 17 groups of test points show that the model P value is less than 0.0001, which is highly significant, indicating that it fits the actual situation well; the lack-of-fit term P = 0.7375> 0.05, which is not significant, indicating that the unknown factors have little interference with the test results and the model selection is appropriate. At the same time, the R 2 =0.9887, close to 1, indicating that the model has good reliability. CV <10%, indicating high reliability and precision. Overall, the equation fits the actual situation well and well reflects the interaction between free calcium ion content in fish bone fermentation broth and fermentation time, inoculum size, and glucose addition. Therefore, the model is suitable. Analysis of the results shows that the response value Y (calcium ion content) is highly significant. The linear term C (glucose addition amount) reaches an extremely significant level (P < 0.01), while A (fermentation time) and B (inoculum size) are not significant. Among the quadratic terms, A2, B2, and C2 all reach extremely significant levels (P < 0.01). Among the interaction terms, only AB reaches a significant level (P < 0.01), while the other interaction terms are not significant.

[0080] The three-dimensional spatial surface and contour map between the three factors were obtained by response surface software ( Figure 1-3 ), can more intuitively express the interaction between factors. The elliptical contour line indicates that the interaction is significant, and the steeper the slope of the three-dimensional surface graph, the more significant the impact. Figure 1-3 As can be seen, the contour lines are elliptical, and the parabola of the response surface plot points downward, indicating that the model has a maximum value and is well established. Software analysis determined that the optimal process is a fermentation time of 24.7 hours, an inoculum size of 4.27%, and a glucose addition of 17.11 g / L. Under these conditions, the calcium ion content in the fish bone fermentation broth was predicted to be 27.27 mg / mL. Three parallel experiments were conducted under these conditions, and the resulting calcium ion content was 27.55 mg / mL, which is consistent with the predicted value. This demonstrates that the model predicts the experimental results well and is reliable.

[0081] The scanning electron microscopy image of the Spanish mackerel bone fermentation broth optimized based on response surface methodology is shown in the figure. Figure 4As shown in the particle size distribution diagram Figure 5 As shown. Figure 4 It can be seen that the fermented fish bone meal has more pores and a looser structure than the original bone meal, while the original bone meal has a denser surface and fewer pores. Under the 300μm field of view, it can be clearly seen that the fermented bone meal particles are smaller, while the original bone meal particles are larger. This shows that some substances in the bone of the fermented bone meal have been released, which is conducive to the full release of nutrients in the bone, thereby increasing the nutritional value of the bone meal and making it easier for the human body to digest and absorb. Figure 5 The average particle size of bone powder after simple crushing and drying was 3.08 μm, with a relatively wide particle size distribution, consistent with the results from scanning electron microscopy. However, the average particle size of bone powder treated under optimal fermentation conditions was 2.35 μm, a significantly smaller particle size. This is because the combined use of enzymatic hydrolysis and fermentation technology breaks down macromolecular binding compounds in fish bones, converting them into a free state and narrowing the particle size distribution, resulting in a more uniform particle size. This demonstrates the advantages and role of fermentation technology in the utilization of livestock and poultry bones.

[0082] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing Spanish mackerel high-calcium fish bone freeze-dried powder, characterized in that: The preparation method comprises the following steps: firstly utilizing a composite enzyme to enzymatically hydrolyze mackerel bones, and then utilizing lactic acid bacteria to perform fermentation treatment.

2. The preparation method according to claim 1, wherein The complex enzyme includes any one or more of acidic protease, neutral protease and alkaline protease.

3. The preparation method according to claim 2, wherein The lactic acid bacteria include any one or more of Lactobacillus plantarum and Lactobacillus acidophilus, and the fermentation process further includes adding glucose for mixed fermentation.

4. The preparation method according to claim 3, wherein The complex enzyme comprises alkaline protease and neutral protease, and the lactic acid bacteria is plant lactobacillus.

5. The preparation method according to claim 4, wherein The ratio of the alkaline protease to the neutral protease is (1-5):1, the inoculation amount of the plant lactobacillus is 2%-6%, and the addition amount of the glucose is 10-30 g / L.

6. The preparation method according to claim 5, wherein The following steps are involved: S1: preparing mackerel bone powder; S2: hydrolyzing the Spanish mackerel bone powder obtained in step S1 with alkaline protease and neutral protease to obtain a Spanish mackerel bone powder hydrolysate; S3: performing enzyme inactivation treatment on the mackerel bone powder enzymatic hydrolyzate obtained in step S2; S4: filtering the Spanish mackerel bone powder enzymatic hydrolyzate after the enzyme inactivation in step S3 to obtain a filtrate; S5: inoculating Lactobacillus plantarum into the filtrate obtained in step S4; S6: mixing the filtrate obtained after the inoculation in step S5 with glucose and fermenting the mixture to obtain a fermented mackerel bone liquid; S7: freeze-drying the Spanish mackerel bone fermentation liquid from step S6 to obtain Spanish mackerel high-calcium fish bone freeze-dried powder.

7. The Spanish mackerel high calcium fish bone freeze-dried powder prepared by the method for preparing the Spanish mackerel high calcium fish bone freeze-dried powder according to any one of claims 1 to 6.

8. Use of a complex enzyme for preparing a reagent for increasing the calcium ion content in freeze-dried mackerel bone powder, characterized in that: The complex enzyme includes any one or more of acidic protease, neutral protease and alkaline protease.

9. The use according to claim 8, characterized in that The complex enzyme comprises alkaline protease and neutral protease.

10. Use of plant lactobacillus for increasing the calcium ion content in freeze-dried mackerel bone powder.

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