Method for promoting dissolution of calcium in fishbone by adopting probiotic fermentation

Abstract

The invention is applicable to the technical field of food processing, and provides a method for promoting dissolution of calcium in fishbone by adopting probiotic fermentation, which comprises the following steps: step 1, preparing an improved MRS culture medium containing fishbone, and sterilizing for later use; 2, preferably selected lactobacillus rhamnosus ATCC53103 is adopted to be inoculated into the improved MRS culture medium in the step 1 for fermentation; 3, after fermentation is finished, solid-liquid separation is conducted, and fermentation liquor rich in soluble calcium and probiotics is collected; the aquatic product waste fishbone can be recycled, the process is simple, the cost is low, dissolution of calcium in the fishbone can be effectively promoted, the obtained product is rich in probiotics and soluble calcium and can be used for developing a novel dietary supplement with the double-effect functions of a probiotics agent and soluble calcium, and support is provided for high-value utilization of the sea eel bone resource.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, in particular to a method for promoting calcium dissolution in fish bones by fermenting with probiotics. Background Art

[0002] Moray eel ( Muraenesox cinereus Conger eel bones are a byproduct of conger eel processing and are often simply processed or discarded, causing environmental pollution and wasting resources. Conger eel bones account for 10-20% of the total mass of the conger eel and are rich in calcium, phosphorus, and magnesium, with the highest calcium content at approximately 7.56 mg / g. -1 Conger eel bone has a calcium-to-phosphorus ratio of approximately 2:1, making it a potential high-quality calcium source for children, pregnant and lactating women, and the elderly. Therefore, conger eel bone calcium is a natural, high-quality calcium source suitable for the development of dietary calcium supplements.

[0003] Previous research has typically processed fish bones into products such as fish bone meal, fish bone collagen, and snack foods, resulting in low added value and a failure to fully utilize the calcium resources found in fish bones. In recent years, some research on fish bone calcium has promoted the development of technologies and theories for deep processing of fish bone calcium, but this process is complex and costly.

[0004] Therefore, in view of the above situation, there is an urgent need to provide a method for utilizing calcium in fish bones with simple process, low cost, high calcium bioavailability, and multiple biological effects. The method of promoting the dissolution of calcium in fish bones by probiotic fermentation can overcome the pain points and difficulties of high-value utilization of fish bones. Summary of the Invention

[0005] The object of the present invention is to provide a method for promoting the dissolution of calcium in fish bones by fermenting probiotics. The method comprises the following steps: using conger eel bones as raw materials, selecting a strain of probiotics that promotes the dissolution of calcium in fish bones from three probiotics, determining the fermentation probiotics, designing a process optimization experiment for fermenting conger eel bones with Lactobacillus rhamnosus by using the response surface methodology, determining the optimal mathematical model by using JMP software, obtaining the optimal fermentation parameters, and performing surface analysis by scanning the fermented fish bones using SEM to determine the production process of soluble calcium from conger eel bones fermented with probiotics. The method can recycle and increase the value of conger eel bone waste, produce probiotic composite soluble calcium, and effectively solve the problems in the above-mentioned background technology.

[0006] The present invention is achieved by providing a method for promoting calcium dissolution in fish bones by fermentation with probiotics, the method comprising the following steps: Step 1: Prepare modified MRS medium and sterilize it for later use; Step 2: Select Lactobacillus rhamnosus ATCC53103 ( Lactobacillus rhamnosus ATCC53103) was inoculated into the modified MRS medium described in step 1 for fermentation; Step 3: After the fermentation is completed, the solid and liquid are separated and the fermentation liquid rich in soluble calcium is collected.

[0007] As a further solution of the present invention: in step 1, the preparation method of the improved MRS medium is: Take 35.0 g of fish bone powder, 5.0 g of yeast powder, 20.0 g of glucose, 2.0 g of K2HPO4, 2.0 g of triammonium citrate, 5.0 g of CH3COONa·3H2O, 0.58 g of MgSO4·7H2O, 0.25 g of MnSO4·H2O, 1.0 mL of Tween 80, and 1000 mL of distilled water, mix, adjust the pH to 6.2-6.4, and sterilize at 121°C for 15 min and then cool.

[0008] As a further solution of the present invention: the fish bone powder is derived from sea eel bones.

[0009] As a further solution of the present invention: in step 2, Lactobacillus rhamnosus ATCC53103 was added at 1 mL, 10 7 The inoculum volume of CFU·mL⁻¹ was inoculated into the modified MRS medium.

[0010] As a further solution of the present invention: in step 2, the fermentation temperature is 35°C.

[0011] As a further solution of the present invention: in step 2, the fermentation time is 41.3 h.

[0012] As a further solution of the present invention: during the fermentation process of step 2, the stirring speed is 1000 rpm to ensure that the bacteria are fully in contact with the culture medium and promote uniform distribution of metabolites.

[0013] As a further solution of the present invention: in step 3, the solid-liquid separation is centrifugal separation.

[0014] As a further embodiment of the present invention, the method further comprises the step of detecting the soluble calcium concentration in the fish bone fermentation broth: 50 mL of fermentation broth was centrifuged at 750 rpm and 35°C for 1 min. 2 mL of supernatant was taken and diluted to 25 mL with ultrapure water. 2 mL each of 30% H2O2, 12 mol·L⁻¹ HCl, and 18 mol·L⁻¹ HNO3 were added. The mixture was digested at 50°C for 5 min, 100°C for 5 min, and 190°C for 30 min. The calcium ion concentration was detected by ICP-MS, and the soluble calcium concentration in the fermentation broth was calculated.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The invention uses sea eel bones, a by-product of aquatic product processing, as raw materials, thereby realizing resource utilization of waste, reducing environmental pollution and lowering raw material costs.

[0016] Lactobacillus rhamnosus was selected as the optimal fermentation strain. This strain can produce organic acids and enzymes through metabolism, synergistically promoting the dissolution of calcium from sea eel bones. The soluble calcium concentration can reach 285.25±2.29 mg・L⁻¹, which is significantly higher than the calcium dissolution rate from fish bones processed by existing simple processing methods such as boiling and dissolving.

[0017] The experiment was designed through response surface methodology, and the mathematical model was optimized using JMP software to determine the optimal fermentation process. The process parameters are clear and stable, and the operation is simple, without the need for complex equipment, which is conducive to industrial large-scale production.

[0018] The resulting fermentation product is a complex of probiotics and soluble calcium, combining the dual functions of probiotics and calcium supplementation, expanding the product categories of dietary supplements and possessing high market value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 Standard curve for calculation of calcium concentration.

[0021] Figure 2 Schematic diagram of the effect of fermentation time on calcium dissolution.

[0022] Figure 3 Schematic diagram of the effect of temperature on calcium dissolution.

[0023] Figure 4 Schematic diagram of the effect of bacterial inoculum size on calcium dissolution.

[0024] Figure 5 A plot of the relationship between the predicted values ​​and the actual values ​​of the fitted least squares model.

[0025] Figure 6 Schematic diagram of the prediction profiler for the prediction model.

[0026] Figure 7Response surface and contour plots; (a) (b) are the response surface plot and contour plot of the interaction effect of temperature and inoculum size on calcium concentration, respectively; (c) (d) are the response surface plot and contour plot of the interaction effect of temperature and fermentation time on calcium concentration, respectively; (e) (f) are the response surface plot and contour plot of the interaction effect of inoculum size and fermentation time on calcium concentration, respectively.

[0027] Figure 8 SEM scans of the freeze-dried samples of the fermentation group, control group, and blank group; (a), (b), and (c) are the fermentation sample, control sample, and blank sample observed under a scanning electron microscope at 10,000 times magnification, respectively. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0029] The present invention will be further explained below with reference to specific embodiments.

[0030] See also Figures 1-8 The embodiment of the present invention provides a method for promoting calcium dissolution in fish bones by fermentation with probiotics, the method comprising the following steps: Step 1: Prepare a modified MRS medium and sterilize it for later use; the preparation method of the modified MRS medium is as follows: Take 35.0 g fish bone powder, 5.0 g yeast powder, 20.0 g glucose, 2.0 g K2HPO4, 2.0 g ammonium citrate tribasic, 5.0 g CH3COONa·3H2O, 0.58 g MgSO4·7H2O, 0.25 g MnSO4·H2O, 1.0 mL Tween 80, and 1000 mL distilled water, mix, adjust the pH to 6.2-6.4, and sterilize at 121°C for 15 min before cooling. Step 2: Select Lactobacillus rhamnosus ATCC53103 and press 1mL, 10 7 The inoculum size of CFU·mL⁻¹ was inoculated into the modified MRS medium described in step 1 for fermentation at a temperature of 35°C and a fermentation time of 41.3 h. Step 3: After the fermentation is completed, the solid and liquid are separated and the fermentation liquid rich in soluble calcium is collected.

[0031] The method further comprises the step of detecting the calcium concentration in the fermentation broth: Take 50 mL of fermentation broth and centrifuge it at 750 rpm and 35°C for 1 min. Take 2 mL of supernatant and dilute to 25 mL with ultrapure water. Add 2 mL each of 30% H2O2, 12 mol・L⁻¹HCl, and 18 mol・L⁻¹HNO3. Digest the mixture at 50°C for 5 min, 100°C for 5 min, and 190°C for 30 min. Detect the calcium ion concentration by ICP-MS, and calculate the calcium concentration in the fermentation broth.

[0032] In this embodiment, the present invention uses sea eel bones, a by-product of aquatic product processing, as raw materials, thereby realizing resource utilization of waste, reducing environmental pollution, and lowering raw material costs; Lactobacillus rhamnosus was selected as the optimal fermentation strain. This strain can produce organic acids and enzymes through metabolism, synergistically promoting the dissolution of calcium from sea eel bones. The calcium concentration in the fermentation broth can reach 285.25±2.29mg・L⁻¹, significantly higher than the soluble calcium dissolution rate achieved by existing simple processing methods. The optimal fermentation process was determined through response surface methodology optimization. The process parameters are clear and stable, and the operation is simple, without the need for complex equipment, which is conducive to industrial large-scale production.

[0033] The following experiments are used to further demonstrate the above technical solution: 1. Experiment 1.1 Experimental methods 1.1.1 Comparative study on the effect of probiotics on calcium dissolution in conger eel bones Take 1mL, 10 6 CFU·mL -1 Bifidobacterium animalis ATCC27536 (BB), Lactobacillus rhamnosus ATCC53103 (LR), and Lactococcus lactis ATCC19435 (LL) were inoculated into 1 L of modified MRS medium (35 g·L fish bone -1 Fermentation was performed under the following conditions: 37°C, 24 hours, and 1000 rpm agitation. The fermentation broth was centrifuged at 750 rpm for 1 minute, and the supernatant was collected for analysis.

[0034] In addition, 1 mL 10 6 CFU·mL -1 The LR was inoculated into MRS medium, and other fermentation conditions remained unchanged as a blank control.

[0035] 1.1.2 Determination and calculation of calcium concentration According to the "Determination of Calcium in Food" (GB5009.92-2016), 50 mL of fermentation broth was centrifuged at 750 rpm and 35°C for 1 min, 2 mL of supernatant was taken and the volume was made up to 25 mL with ultrapure water, mixed, and 30% H2O2 and 12 mol·L -1 HCl, 18 mol·L -1 2mL of HNO3 was used for each digestion procedure, and the digestion procedure was 50℃, 5min, 100℃, 5min, and 190℃, 30min. The concentration of calcium ions was detected by ICP-MS, and a standard curve was drawn as shown below. Figure 1 shown.

[0036] 1.1.3 Single-factor experiment The calcium dissolution test of LR fermented sea eel bone was carried out at 35℃, 37℃, 39℃, 41℃ and 43℃ respectively; the inoculum size was n=4, 5, 6, 7, 8 (10 n CFU·mL -1 ), 1mL; fermentation time 24h, 30h, 36h, 42h, 48h, three factors were tested for single factor to obtain the optimal range of each factor.

[0037] 1.1.4 Response surface experiment Temperature (A), bacterial inoculum size (B), and fermentation time (C) were used as independent variables, and calcium concentration in the fermentation broth (mg·L -1 ) as the response value, a factor level coding table was designed (Table 1), and a response surface test was performed based on the Box-Behnken test.

[0038] Table 1 Box-Behnken test factor coding and levels

[0039] 1.1.5 Sample processing and SEM examination Take 50 mL of fermentation broth and centrifuge at 6000 rpm for 5 min, discard the supernatant, take the precipitate and place it in a 10 mL test tube, add 2 mL of 2.5% glutaraldehyde, vortex for 5 min, fix in the dark at 4 ℃ for 4 h, centrifuge at 6000 rpm for 5 min, discard the supernatant. Add 25 mL of 0.1 mol·L -1The cells were then immersed in pH 7.4 phosphate buffer, vortexed for 5 minutes, and centrifuged at 6000 rpm for 5 minutes. The supernatant was discarded and rinsed three times to remove excess glutaraldehyde. The cells were then dehydrated using a gradient of 30%, 50%, 70%, and 90% ethanol, followed by 100% ethanol. The ethanol was discarded, and the cells were allowed to evaporate for 2 hours before being freeze-dried. Scanning electron microscopy (SEM) was used to scan the following samples: fermentation group (fermentation broth from LR fermentation in modified MRS medium); control group (fermentation broth from LR fermentation in MRS medium); and blank group (culture broth from modified MRS medium). SEM parameters were an acceleration voltage of 5 kV and a magnification of 10,000.

[0040] 1.2 Data Processing All samples were tested in triplicate, and data are presented as mean ± standard deviation (x ± s). Response surface design, mathematical modeling, and analysis were performed using JMP Pro 17 (trial version), and graphing was performed using Origin Pro 2024.

[0041] 2. Results 2.1 Comparison of probiotic fermentation-induced calcium dissolution in conger eel bones BB, LR, and LL were used to ferment conger eel bones, and the calcium concentrations in the fermentation broths of conger eel bones fermented with probiotics BB, LR, and LL were compared, as shown in Table 2.

[0042] Table 2 Calcium dissolution from sea eel bones fermented by different strains

[0043] Probiotic fermentation not only produces large amounts of organic acids but also secretes extracellular enzymes such as proteases, lipases, and peptidases. The synergistic effect of these organic acids and extracellular enzymes promotes calcium dissolution from sea eel bones. Experiments have shown that the three probiotic strains have different calcium dissolution efficiencies: LR > LL > BB. LR fermentation produces relatively high concentrations of organic acids such as lactic acid and acetic acid, which promote the release of acidifiable calcium ions from fish bones. The extracellular enzymes secreted by probiotics promote the breakdown of proteins, fats, and peptides in fish bones, facilitating the release of some bound calcium. They also promote the formation of stable soluble complexes between peptides and calcium ions, reducing secondary precipitation of calcium from fish bones, increasing the calcium concentration in the fermentation broth and enhancing its stability. This study selected LR as the preferred strain for sea eel bone fermentation to achieve optimal process conditions for calcium precipitation from sea eel bones.

[0044] 2.2 Single-factor experiment on LR fermented conger eel bone 2.2.1 Effect of fermentation time on calcium dissolution promoted by LR fermentation The study found that from 24 to 36 hours, as the fermentation time extended, LR entered a rapid growth phase, capable of rapid reproduction and metabolism. Enzyme and organic acid content continued to accumulate, promoting the dissolution of calcium from sea eel bones, and the soluble calcium content showed a significant upward trend. From 36 to 42 hours, the microorganisms entered a stable phase, with the accumulation of metabolites such as enzymes and organic acids reaching saturation. The calcium concentration in the fermentation broth did not change significantly. After 42 hours, excessive microorganisms may begin to digest the calcium in the fermentation broth for growth and metabolic activities, causing the calcium concentration in the fermentation broth to decrease. When the fermentation time was 36 hours, the highest calcium content was 132.74±2.86 mg·L -1 , which was significantly higher than the calcium concentration in the fermentation broth at 33 h (68.16±4.22 mg·L -1 ), the study determined that the optimal temperature range for LR fermentation of conger eel bones to promote calcium dissolution is 30~42 h.

[0045] 2.2.2 Effect of temperature on calcium dissolution from LR fermented conger eel bones Different temperatures significantly affected the calcium dissolution from conger eel bones during LR fermentation. With increasing fermentation temperature (33°C-41°C), the calcium concentration in the fermentation broth first increased and then decreased, which was consistent with the optimum temperature for LR fermentation. At the optimum fermentation temperature for LR, microbial metabolism was vigorous, metabolites accumulated, and the enzyme and organic acid contents continued to increase, promoting the dissolution of calcium from conger eel bones. At lower and higher temperatures, the reproduction and metabolism of LR itself were inhibited, which inevitably affected the calcium concentration in the fermentation broth. At 35°C, the highest calcium concentration was 423.73±1.25 mg·L -1 , which was significantly higher than the calcium concentration in the fermentation broth at 41 °C (62.97 ± 6.49 mg·L -1） The study determined that the optimal temperature range for LR fermented conger eel bones to promote calcium dissolution is 33~37℃.

[0046] 2.2.3 Analysis of LR inoculation volume to promote calcium dissolution from sea eel bones The LR inoculum size had a greater effect on the dissolution of sea eel bone calcium. When the fermentation time (36 h) was low, the inoculum size (10 4 CFU·mL -1 The cumulative number, reproduction and metabolic rate, and metabolite accumulation of LR in the fermentation broth with a low inoculum size (10 6 CFU·mL -1 and 10 7 CFU·mL -1 ) in the fermentation broth, the calcium concentration in the low inoculum fermentation broth (298.56±15.23mg·L -1 ) was lower than the calcium concentration in the high inoculum fermentation broth (467.45±1.79 mg·L-1 ); With the increase of initial inoculum size, the concentration of calcium in the fermentation broth also increases; when the inoculum size is high (10 6 CFU·mL -1 and 10 7 CFU·mL -1 ), the microorganisms in the fermentation broth quickly reach the equilibrium period, and the cumulative number of microorganisms and the amount of metabolites remain basically unchanged. Therefore, at high inoculation concentrations, the calcium concentrations in the fermentation broth are not much different, namely: 467.28±6.4 mg·L -1 (10 6 CFU·mL -1 ) and 467.45±1.79 mg·L -1 (10 7 CFU·mL -1 ), the study determined that the optimal inoculation range of LR for promoting calcium dissolution of sea eel bones is 10 6 ~10 8 CFU·mL -1 (1 mL).

[0047] 2.3 Response surface design and prediction model optimization for LR fermentation process The response surface experimental design is shown in Table 3.

[0048] Table 3 Response surface experimental design

[0049] JMP Pro 17 (trial version) software was used to fit the experimental results in Table 3 and screen the prediction model for the process conditions of LR fermentation of conger eel bone calcium, as shown in Table 4.

[0050] Table 4 Model screening

[0051] The fitting models of least squares method, support vector machine, generalized regression, neural network, Bootstrap forest method, stepwise fitting and K nearest neighbor method were compared and analyzed respectively. 2 and RASE values ​​as the measure of model fitting effect, R 2 The larger the value and the smaller the RASE value, the better the model fitting effect. Model screening found that the R of the least squares method was 2 The value is the largest, the RASE value is relatively small, and the lack of fit is not obvious. Therefore, the study selected the fitting least squares method to establish a fitting model for LR fermentation to promote the dissolution of calcium from sea eel bones, and the optimal fermentation conditions can be obtained.

[0052] 2.4 Establishment and analysis of the least squares fitting model 2.4.1 Predicted Values ​​and Actual Values Relationship diagram between predicted value and actual value after model fitting ( Figure 5 ) shows that there is good consistency between the actual values ​​and predicted values ​​of the model. The distribution of points in the figure is relatively concentrated, with no obvious outliers. The shaded area represents the uncertainty range of the predicted values. This indicates that the least squares fitting model predicts that the process conditions for calcium dissolution from sea eel bones promoted by LR fermentation are relatively stable, without significant deviations. However, as the predicted values ​​increase, the uncertainty range also increases. This may be due to a decrease in the model's predictive ability in areas with high predicted values ​​or an increase in data variability. Therefore, further model optimization and data analysis may improve the overall performance of the model.

[0053] 2.4.2 Prediction Profiler A predictive profiler was established based on the fitted least squares model to obtain the calcium concentration in the fermentation broth of LR fermented sea eel bones at different temperatures, LR inoculum amounts and fermentation times. Figure 6 This is a screenshot of the prediction profiler when the calcium content in the sea eel bone fermentation liquid is the highest. At this time, the temperature is 35.087992°C and the LR inoculum size is 107.113049 CFU·mL -1 The fermentation time was 41.284484 h, and the calcium concentration in the fermentation broth was predicted to be 281.4054~299.4516 mg·L -1 The mean value is 290.4285 mg·L -1 , the willingness value is 0.88409, indicating that the prediction effect is good. To facilitate verification, the process parameters of the LR fermentation sea eel bone verification experiment were determined as follows: temperature 35 ℃, bacterial inoculation volume 107 CFU·mL -1 , fermentation time 41 h.

[0054] 2.4.3 Verification Experiment The optimal process parameters were obtained according to 2.4.2 and the calcium concentration in the fermentation broth was detected to be 285.25±2.29 mg·L -1 , which is within the prediction interval obtained based on the fitted least squares model (281.4054~299.4516 mg·L -1 ), indicating that the model prediction is relatively accurate. This model can well predict the effect of LR fermentation of conger eel bones in promoting calcium dissolution under different conditions.

[0055] 2.4.4 Analysis of response surface experimental results The response surface and contour plots of the response surface experiment results are shown in Figure 7 (af).

[0056] The interaction surfaces for temperature and inoculum size (Figures (a) and (b)) show a vertex and are relatively smooth, indicating an extreme value for the interaction between temperature and inoculum size on the calcium concentration in the fermentation broth. The contour lines are elliptical, consistent with the surface analysis results. The interaction surfaces for temperature and fermentation time (Figures (c) and (d)) show a ridge-like shape, indicating that the interaction between fermentation time and temperature may lead to an extreme value for calcium concentration in the fermentation broth. Based on the contour plots, the center of the elliptical contour lines lies within the plots, confirming that the interaction between temperature and fermentation time leads to an extreme value for calcium concentration in the fermentation broth. Similarly, the interaction surfaces for inoculum size and fermentation time (Figures (e) and (f)) also show an extreme value for calcium concentration in the fermentation broth. In summary, the response surface design experiment can predict the optimal process conditions for calcium concentration in sea eel bone fermentation broth using LR.

[0057] 2.4.5 Prediction Regression Equation The study obtained the prediction regression equation of calcium concentration in LR fermented conger eel bone fermentation liquid: Y=(-5129.4)+8.56479166666672*fermentation time+120.489583333333*inoculation size+274.424791666667*temperature+(-0.0953240740740744)*fermentation time^2+(-10.3741666666667)*inoculation size^2+(-4.01979166666667)*temperature^2+(-0.178750000000001)*fermentation time*inoculation size+0.0164583333333326*fermentation time*temperature+0.982499999999998*inoculation size*temperature.

[0058] 2.5 SEM analysis of LR attached to sea eel bones According to the optimal fermentation conditions for LR to promote calcium dissolution from sea eel bones confirmed by the validation experiment, SEM scanning was performed on the sediment in the fermentation matrix. The results are as follows: Figure 8 As shown in the figure, SEM scanning shows that bacteria are clearly attached to the surface of the fermentation sample sediment. They are rod-shaped, well-developed, and slightly longer than those in the control sample, with obvious aggregation. The control sample also contains a large number of bacteria, but their rod-shaped characteristics are inferior to those in the fermentation sample. The SEM image of the blank sample shows a matrix (fish bone particles) without bacterial growth, indicating that the bacteria in the fermentation sample sediment are attached to the matrix. LR thrives in the culture medium containing sea eel bones, showing good results.

[0059] 3. Conclusion This study evaluated the effectiveness of three probiotics (Bifidobacterium animalis, Lactobacillus rhamnosus, and Lactococcus lactis) in promoting calcium dissolution from sea eel bones. The results showed that Lactobacillus rhamnosus was the optimal probiotic fermentation bacterium. A least-squares screening and fitting method was used to establish a predictive model for the effect of Lactobacillus rhamnosus on calcium dissolution from sea eel bones. The model was evaluated and the optimal fermentation conditions were obtained: a temperature of 35°C and an inoculum size of 107 CFU·mL. -1 (1 mL) and fermentation time of 41.3 h. Under these conditions, the calcium concentration in the fermentation broth was 285.25 ± 2.29 mg·L -1 Scanning electron microscopy (SEM) analysis further confirmed that Lactobacillus rhamnosus grew optimally in a fermentation matrix containing conger eel bones. This invention establishes a method for producing probiotics by fermenting fish bones to promote calcium dissolution from conger eel bones, opening up a new avenue for the resourceful utilization of fish bones.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0061] The present invention discloses a method for promoting calcium dissolution from fish bones using probiotic fermentation. It should be understood that the expression "one or more of" individually includes each of the items recited after the expression, as well as various combinations of two or more of the recited items, unless otherwise indicated by the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise indicated by the context.

[0062] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0063] It should be understood that the order of steps or the order in which certain actions are performed are not important as long as the present invention remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0064] The use of any and all examples or exemplary language, such as "such as" or "including," herein is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0065] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant numerical values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently contains standard deviations caused by individual testing methods.

Claims

1. A method for promoting calcium dissolution in fish bones by fermentation with probiotics, characterized in that: The method comprises the following steps: Step 1: Prepare modified MRS medium and sterilize it for later use; Step 2: Select Lactobacillus rhamnosus ATCC53103 ( Lactobacillus rhamnosus ATCC53103) was inoculated into the modified MRS medium described in step 1 for fermentation; Step 3: After the fermentation is completed, the solid and liquid are separated and the fermentation liquid rich in soluble calcium is collected; The preparation method of the improved MRS culture medium is: Take 35.0 g fish bone powder, 5.0 g yeast powder, 20.0 g glucose, 2.0 g K2HPO4, 2.0 g ammonium citrate tribasic, 5.0 g CH3COONa·3H2O, 0.58 g MgSO4·7H2O, 0.25 g MnSO4·H2O, 1.0 mL Tween 80, and 1000 mL distilled water, mix, adjust the pH to 6.2-6.4, and sterilize at 121°C for 15 min before cooling. In step 2, Lactobacillus rhamnosus ATCC53103 was added in 1 mL, 10 7 The inoculum volume of CFU·mL⁻¹ was inoculated into the modified MRS medium.

2. The method according to claim 1, characterized in that The fish bone meal is derived from sea eel bones.

3. The method according to claim 1, characterized in that In step 2, the fermentation temperature was 35°C.

4. The method according to claim 1, wherein In step 2, the fermentation time was 41.3 h.

5. The method according to claim 1, wherein During the fermentation process in step 2, the stirring speed was 1000 rpm to ensure full contact between the bacteria and the culture medium and promote uniform distribution of metabolites.

6. The method according to claim 1, characterized in that In step 3, the solid-liquid separation is centrifugal separation.

7. The method according to claim 1, characterized in that The method further comprises the step of detecting the concentration of soluble calcium in the fermentation broth: 50 mL of fermentation broth was centrifuged at 750 rpm and 35°C for 1 min. 2 mL of supernatant was taken and diluted to 25 mL with ultrapure water. 2 mL each of 30% H2O2, 12 mol·L⁻¹ HCl, and 18 mol·L⁻¹ HNO3 were added. The mixture was digested at 50°C for 5 min, 100°C for 5 min, and 190°C for 30 min. The calcium ion concentration was detected by ICP-MS, and the soluble calcium concentration in the fermentation broth was calculated.

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