Method for bioresource utilization of seafood waste

Abstract

The application belongs to the technical field of biological resource utilization, and more particularly relates to a method for biological resource utilization of seafood waste. The method specifically comprises the following steps: 1) drying and crushing seafood waste for standby; 2) inoculating lactic acid bacteria into a fermentation medium, adding the seafood waste obtained in step 1) in an amount of 10-30 g / 100 mL, at a temperature of 40-45 DEG C, a rotation speed of 50-200 rpm, and fermenting for 36-72 h; when the sugar concentration of the fermentation broth is reduced to 10 g / L or less, start sugar supplementing, and the total sugar consumption in the fermentation process is not more than 150 g / L; 3) when the pH value of the fermentation broth is reduced to 4.5-5.0, the fermentation is completed, and the obtained precipitate is used for preparing chitin; and the filtered fermentation broth is used for extracting lactic acid or lactate. The lactic acid fermentation coupled with the seafood waste decalcification and deproteinization method established in the application does not need to add other organic nitrogen sources in the lactic acid fermentation process, and the fermentation medium and crab shells can not be sterilized, thereby effectively saving the treatment cost.

Description

Technical Field

[0001] This invention belongs to the field of biological resource utilization technology, and more specifically relates to a method for the biological resource utilization of seafood waste. Background Technology

[0002] Chitin, also known as chitosan, is a natural high-molecular-weight polysaccharide widely found in the shells of crustaceans such as shrimp, crabs, and insects. Chitin and its derivatives have wide applications in agriculture, medicine, food, and chemical industries, and it is the second largest natural polysaccharide product in terms of annual production, second only to cellulose.

[0003] Currently, traditional processes for producing chitin from shrimp and crab shells, both domestically and internationally, often employ strong acids and alkalis to remove calcium carbonate and protein from the shells. While this process is simple and cost-effective, it consumes large amounts of strong acids and alkalis, causing significant environmental pollution. Furthermore, the harsh reaction conditions result in unstable chitin products with inconsistent properties, such as difficulty in controlling the degree of deacetylation, molecular weight, and acetyl group position. CN201710165181.7 discloses a method for preparing chitin, protein, and organic calcium acid from shrimp shells using ionic liquids. This method involves removing calcium carbonate from shrimp shell powder using a eutectic ionic liquid or its aqueous solution to obtain calcium-depleted shrimp shell powder; then removing protein from the calcium-depleted shrimp powder using a phosphate ester ionic liquid to obtain insoluble matter, i.e., the chitin product; collecting the supernatant, adding a back-extraction agent, and regenerating the resulting products are organic calcium acid and protein, respectively. Simultaneously, the supernatant containing the back-extraction agent from both the eutectic ionic liquid and the phosphate ester ionic liquid is collected. This method can utilize waste shrimp shells to produce high-purity chitin, organic calcium acids, and proteins, achieving comprehensive utilization of shrimp shells. Furthermore, the resulting ionic liquid is pollution-free and recyclable. However, this method has a small processing capacity and a low degree of industrialization.

[0004] Compared to chemical methods, bio-fermentation for chitin production offers milder reaction conditions, less environmental pollution, and yields various byproducts such as feed protein, calcium salts, and organic acids. CN201910427758.6 discloses a method for extracting chitin from waste shrimp and crab shells using Bacillus cereus. This method utilizes Bacillus cereus to ferment the waste shrimp and crab shells, removing proteins and minerals, achieving a protein removal rate of over 95% and an ash removal rate of over 92%. However, this method has a long processing cycle, with fermentation time approaching 14 days, severely limiting the processing efficiency of the shrimp and crab shells. Summary of the Invention

[0005] To address the problems of existing technologies, the purpose of this invention is to propose a method for the biological resource utilization of seafood waste. This method uses shrimp and crab shells as a neutralizing agent in lactic acid fermentation, obtaining calcium lactate while removing proteins and minerals from the shrimp and crab shells, ultimately yielding a variety of products including chitin, calcium lactate, and protein feed.

[0006] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution:

[0007] A method for the biological resource utilization of seafood waste, the method specifically includes the following steps:

[0008] 1) Dry and pulverize the seafood waste for later use;

[0009] 2) Inoculate lactic acid bacteria into the fermentation medium, and add the seafood waste obtained in step 1) at a rate of 10-30g / 100mL. Ferment at a temperature of 40-45℃ and a rotation speed of 50-200rpm for 36-72h. When the sugar concentration of the fermentation liquid drops below 10g / L, start sugar supplementation. The total sugar consumption during the fermentation process should not exceed 150g / L.

[0010] The fermentation medium consists of the following components: sugar 60-150 g / L, sodium chloride 5-15 g / L, sodium acetate 1-5 g / L, and ammonium citrate 0.1-0.5 g / L; the lactic acid bacteria are anaerobic or facultative anaerobic fermenting bacteria.

[0011] 3) When the pH of the fermentation broth drops to 4.5-5.0, the fermentation ends, and the broth is filtered. The resulting precipitate is used to prepare chitin; the filtered fermentation broth is used to extract lactic acid or lactate.

[0012] Preferably, the lactic acid bacteria are Lactobacillus acidophilus or Lactobacillus plantarum.

[0013] Preferably, the process of using the precipitate to prepare chitin includes the following steps:

[0014] 1) Wash and dry the obtained precipitate, and treat it in 0.05-0.5 mol / L hydrochloric acid solution at a mass-volume ratio of 1:5-10 for 20-300 min to remove residual mineral elements;

[0015] 2) Solid-liquid separation: The precipitate is placed in a 0.1-0.5 mol / L sodium hydroxide solution at a mass-to-volume ratio of 1:5-10 and treated for 0.5-12 hours to remove residual protein. The treatment temperature is 60-90℃.

[0016] 3) Clean the precipitate and dry it to obtain chitin raw material.

[0017] More preferably, the process of using the precipitate to prepare chitin includes the following steps:

[0018] 1) Wash and dry the obtained precipitate, and treat it in 0.1-0.2 mol / L hydrochloric acid at a mass-to-volume ratio of 1:5 for 60-90 min;

[0019] 2) Solid-liquid separation: The precipitate is placed in 0.1-0.25 mol / L sodium hydroxide at a mass-to-volume ratio of 1:10 and treated for 0.5-4 hours at a temperature of 80-90℃.

[0020] 3) Wash the precipitate and dry it to obtain chitin raw material; the chitin content in the chitin raw material is more than 95%.

[0021] Preferably, no organic nitrogen source is added to the fermentation medium; the organic nitrogen source is any one or a mixture of two or more of the following in any proportion: peptone, yeast powder, corn steep liquor, corn hydrolysate, beef extract, and soybean meal hydrolysate.

[0022] Preferably, no acid-base neutralizing agent is used in step 2); the acid-base neutralizing agent is any one or a mixture of two or more of sodium hydroxide, potassium hydroxide, calcium carbonate, calcium hydroxide, hydrochloric acid, lactic acid, and sulfuric acid in any proportion.

[0023] Preferably, step 2) employs batch-continuous fermentation: when the sugar concentration of the fermentation broth drops to 5±3 g / L, the liquid component is taken and transferred to a new fermentation medium at a volume ratio of 1:5-10. 10-30 g / 100 mL of seafood waste is added, and fermentation is restarted. When the sugar concentration drops to 5±3 g / L, the transfer is repeated again. This transfer is repeated 1-3 times. After fermentation, the fermentation broth from multiple batches is combined and filtered. The resulting precipitate is used to prepare chitin, and the filtered fermentation broth is used to extract lactic acid and / or feed protein.

[0024] Preferably, the filtered fermentation broth is first heated and then filtered by plate and frame press. The resulting filtrate is cooled to obtain crude calcium lactate, and the resulting filter residue is granulated by spraying to obtain feed protein.

[0025] Preferably, the seafood waste is crab shells; the crab shells are crushed and sieved, with a particle size of 0.17-5mm.

[0026] This invention utilizes *Lactobacillus acidophilus* for lactic acid fermentation coupled with decalcification and deproteinization of crab shells. After condition optimization, this strain synthesizes 133.88 g / L of lactic acid after 65 hours of fermentation, achieving a calcium removal rate exceeding 80% and a protein removal rate exceeding 60% in the crab shells. Because the fermentation temperature exceeds 40℃ and the fermentation process is anaerobic, multiple batches of semi-continuous fermentation can be carried out under non-sterilized conditions, effectively improving the efficiency and volume of crab shell processing while reducing processing costs. This invention also establishes an endpoint determination criterion for lactic acid fermentation coupled with crab shell decalcification and deproteinization. When the pH of the fermentation broth reaches approximately 4.6, the decalcification rate of the crab shells exceeds 80%, indicating a linear relationship between pH and the decalcification rate. The decalcification rate of the crab shells can be estimated by detecting the pH during the fermentation process, thus determining the fermentation endpoint. To verify the applicability of this method, lactic acid fermentation was carried out using the facultative anaerobic strain *Lactobacillus plantarum* (CICC21793), which was purchased from CICC and domesticated. The results showed that the strain synthesized 97.22 g / L of lactic acid within 68 h, the final pH value of fermentation was 4.8, the decalcification rate of crab shell was 78.25%, and the protein removal rate was 68.25%, indicating that the above method is also applicable to lactic acid fermentation by *Lactobacillus plantarum*.

[0027] Compared with the prior art, the present invention has the following significant advantages:

[0028] 1) The lactic acid fermentation coupled with seafood waste decalcification and deproteinization method established in this invention does not require the addition of other organic nitrogen sources during the lactic acid fermentation process, and the fermentation culture medium and crab shells do not need to be sterilized, which effectively saves processing costs.

[0029] 2) Short fermentation time and high processing efficiency. Using the method of this invention, up to 30g / 100mL of crab shell can be processed within 72 hours, achieving a calcium removal rate of 80% and a protein removal rate of over 60%.

[0030] 3) A coupled production process of lactic acid fermentation and crab shell processing was established. After simple post-processing, chitin, lactic acid or lactate, and feed protein with a purity of over 95% can be obtained. Attached Figure Description

[0031] Figure 1 Comparison of pH change trends during fermentation in Example 2;

[0032] Figure 2 Example 4: Comparison of pH changes after washing crab shells using different washing methods;

[0033] Figure 3 Example 7: Linear relationship between pH value of fermentation broth and decalcification rate of crab shells;

[0034] Figure 4 Example 8: Comparison of pH changes after treatment with hydrochloric acid solution for different time periods;

[0035] Figure 5 Example 8: Comparison of pH changes after treatment with sodium hydroxide solution for different time periods. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to specific embodiments.

[0037] 1. Materials

[0038] (1) Reagents: peptone, yeast extract (OXOID), other reagents were commercially available domestic analytical grade;

[0039] (2) Bacterial strain: Lactobacillus acidophilus was directly sourced from the Microbiology Laboratory of Shandong University, and the original strain was purchased from CICC;

[0040] (3) Culture medium:

[0041] Liquid seed culture medium (g / L): glucose 20, yeast extract 5, peptone 10, beef extract 10, sodium chloride 10, sodium acetate 5, ammonium citrate 2, magnesium sulfate 0.2, manganese sulfate 0.05, pH natural, sterilized at 115°C for 20 min;

[0042] Fermentation medium (g / L): glucose 20-150, corn steep liquor 15mL, yeast extract 5, beef extract 10, sodium chloride 10, sodium acetate 2.5, ammonium citrate 0.2, pH 6.5, sterilized at 115℃ for 20min;

[0043] (4) Crab shell: After removing the meat from the Alaskan snow crab and drying the shell, crush it and sieve it through 30 mesh, 60 mesh and 90 mesh sieves respectively for later use.

[0044] 2. Experimental Methods

[0045] After activation via slant culture, the inoculum is inoculated into liquid seed culture medium and activated at 40-45℃ for 8-20 hours to obtain seed culture. Lactic acid fermentation is then carried out in fermentation medium at an inoculation rate of 10-20% (v / v) at 40-45℃ and a rotation speed of 50-200 rpm. Crab shells of varying particle sizes are added at the beginning of fermentation, and no other neutralizing agents are added during fermentation. When feed is required during fermentation, a concentrated sugar solution with a concentration of 800 g / L is added using a fed-batch or intermittent feeding method.

[0046] 3. Analytical Methods

[0047] (1) Decalcification rate (DM): Before fermentation, the crab shells were washed, dried to constant weight, and their mass was measured as M0 (g). After complete calcination in a box-type resistance furnace, their ash content was measured and recorded as S0 (%). After fermentation, the crab shells with a mass of M1 (g) were treated in the same way, and their ash content was measured as S1 (%).

[0048] The formula for calculating the decalcification rate is as follows:

[0049]

[0050] (2) Determination of chitin: Take a quantitative amount of crab shell M0, soak it in acid solution until the minerals are completely removed; wash and dry; then soak it in alkaline solution by heating until the protein is completely removed, wash and dry, and weigh it as M1;

[0051]

[0052] (3) Protein removal rate (DP): Determined using the Kjeldahl method. The total nitrogen content of the crab shell before treatment and at the end of fermentation were recorded as N0 and N1, respectively. The nitrogen content in chitin was determined using the same method and recorded as N0'. The formula for calculating the protein removal rate is shown below:

[0053]

[0054] Where M0 and M1 are the total weight of crab shells before treatment and after fermentation, respectively, and M0' is the mass of chitin in the crab shells before treatment;

[0055] (4) Determination of total lactic acid: Total lactic acid was determined by liquid chromatography. The chromatographic column was SB-Aq (4.5×250mm, 5μm), the injection volume was 5μL, the detection wavelength was 215nm, and the mobile phase was 0.02mol / L potassium dihydrogen phosphate and acetonitrile;

[0056] (5) L-lactic acid and glucose: measured using an SBA-40C biosensor.

[0057] Example 1

[0058] Analysis revealed that the crab shells used contained approximately 47.03% calcium salts, 18.19% protein, 19.33% chitin, 10.07% lipids, and 5.38% other components. Based on previous research on this strain, an initial sugar concentration of 20 g / L and a residual sugar concentration below 10 g / L were considered optimal conditions for fed-batch fermentation. During fermentation, 8% (w / v) calcium carbonate was added as a neutralizing agent. Based on the calcium carbonate content in the crab shells, the theoretical amount of crab shells replacing calcium carbonate was approximately 17%. However, considering that the availability of calcium carbonate in crab shells is lower than that of pure calcium carbonate, crab shells were added at a weight-to-volume ratio (w / v) of 20%.

[0059] Fermentation was carried out with initial sugar concentrations of 20 g / L, 40 g / L, 60 g / L and 80 g / L respectively. Sugar supplementation was started when the glucose concentration of the fermentation broth dropped to 10 g / L. The lactic acid concentration and crab shell treatment after 60 h of fermentation are shown in the table below.

[0060] Table 1 Comparison of lactic acid fermentation and crab shell treatment under different initial sugar concentrations

[0061]

[0062] Fermentation results showed that the acid production rate was faster at initial sugar concentrations of 60 g / L and 80 g / L, but the final lactic acid concentration and sugar-acid conversion rate were higher at an initial sugar concentration of 60 g / L. Combined with fermentation process parameters, it was found that at an initial sugar concentration of 20 g / L, the strain produced acid rapidly in the early stages of fermentation, but with the addition of more glucose solution, the strain stopped producing acid at 38 hours, and at an initial sugar concentration of 40 g / L, acid production also stopped at 44 hours. This indicates that excessively low initial sugar concentrations lead to a decrease or cessation of acid production rate in the later stages of fermentation, affecting the final lactic acid concentration and the efficiency of crab shell processing.

[0063] Example 2

[0064] The effect of crab shell particle size on lactic acid fermentation was investigated under the conditions of initial sugar content of 60 g / L and crab shell addition (w / v) of 20%. Concentrated sugar solution was added twice during fermentation. The crab shells were ground using a grinder and then passed through 30-mesh, 60-mesh, and 90-mesh sieves, resulting in four particle sizes: larger than 30 mesh, 30-60 mesh, 60-90 mesh, and smaller than 90 mesh. Based on the sieve aperture and particle size analyzer measurements, the obtained crab shell particles were 0.6-5 mm, 0.25-0.6 mm, 0.17-0.25 mm, and <0.17 mm, respectively. Lactic acid fermentation was carried out at an 18% inoculum for 65 h. The comparison of lactic acid synthesis with the crab shell treatment effect is shown in Table 2. The pH changes during fermentation are shown in [Table 2]. Figure 1 ;

[0065] Table 2 Comparison of lactic acid fermentation results with the addition of crab shells of different particle sizes.

[0066]

[0067] The results showed that as the size of the crab shell particles decreased, the decalcification rate of the crab shells at the end of fermentation increased from 79.20% to 88.72%, while the protein removal rate increased from 42.32% to 84.82%. However, in terms of acid production, the 0.17-0.25 mm particles exhibited the fastest acid production rate and the highest final lactic acid concentration. Regarding pH changes during fermentation, smaller crab shell particles resulted in a smoother pH change curve. However, when the crab shell particles were smaller than 0.17 mm, the pH value of the fermentation system remained above 7.5 for 0-24 hours, which was unfavorable for the growth of lactic acid bacteria and lactic acid synthesis. Consequently, the acid production rate of the bacteria under this condition was actually lower than that under the 0.17-0.25 mm condition. To address this, the treatment time was extended, with the crab shells soaked in the fermentation system for 72 hours, and the final decalcification rate of the crab shells was measured. The results showed that the final decalcification rates of crab shells under the conditions of 0.6-5 mm, 0.25-0.6 mm, 0.17-0.25 mm, and <0.17 mm were 82.15%, 84.04%, 85.92%, and 88.83%, respectively.

[0068] The above results indicate that the final decalcification rate of crab shells is related to the lactic acid concentration in the fermentation system, and reducing the size of the crab shells is beneficial to improving the decalcification efficiency per unit time. However, excessively small crab shell particles can cause the pH value of the fermentation broth to be too high, thus affecting the acid production rate of the bacteria. Considering that excessively small crab shell particles are also not conducive to solid-liquid separation at the end of fermentation, especially causing difficulties in separating the crab shells from the bacteria, subsequent experiments will use crab shells in the range of 0.17-5 mm for process optimization.

[0069] Example 3

[0070] In the above embodiments, the organic nitrogen sources used in the fermentation culture medium were yeast powder, peptone, corn steep liquor, and beef extract. Based on the component analysis results, crab shells contain a certain amount of organic nitrogen. Whether this organic nitrogen can be utilized by the microorganisms during fermentation is the objective of this embodiment. Yeast powder, peptone, beef extract, and corn steep liquor were selected as the factors of investigation, and a four-factor, three-level orthogonal experiment was designed, as shown in Table 3.

[0071] Table 3. Orthogonal experimental design for organic nitrogen sources

[0072]

[0073] The results showed that the four factors had no significant effect on lactic acid production after 48 hours of fermentation. Under the condition of no added organic nitrogen source, the final lactic acid concentration was 80.22 g / L, which was not significantly different from the optimal condition of 80.50 g / L. This indicates that the organic nitrogen source in crab shells can be utilized by the microorganisms during lactic acid fermentation. Comparing the crab shell protein removal rate, under the condition of highest acid production, the protein removal rate was 20.38%, while the protein removal rate without added organic nitrogen source was 32.98%. This shows that the organic nitrogen source in crab shells can not only be utilized by the microorganisms, but the utilization by the microorganisms also promotes the removal of protein from the crab shell. Therefore, lactic acid fermentation using crab shells as a neutralizing agent can be carried out without adding organic nitrogen sources to the culture medium.

[0074] Example 4

[0075] Based on preliminary experimental results, when the initial crab shell addition reached 30g / 100mL (w / v), the pH value was higher than 10 in the early stages of fermentation. This was especially true when crab shell particles <0.17mm were added, where the initial pH value reached 10.98, preventing normal lactic acid fermentation. Further testing revealed that the pH value of the fermentation broth gradually increased with increasing crab shell addition. This indicates that crab shells contain a relatively high amount of alkaline substances, and excessive addition leads to an excessively high pH value in the early stages of fermentation, thereby inhibiting the activity of the lactic acid fermenting bacteria.

[0076] To eliminate the influence of alkaline substances in the crab shells on fermentation, the crab shells were cleaned using different washing methods. Figure 2 A) Measure the change in pH value of the supernatant. Figure 2 B represents the pH measurement results under the corresponding conditions. As can be seen from the figure, after washing with different methods, the pH value of the washing liquid from crab shells of different particle sizes not only did not decrease, but actually increased to varying degrees. This indicates that the alkaline substances in the crab shells are not soluble in water and cannot be removed by methods such as ultrasonic washing. The inventors attempted to lower the initial pH value of the fermentation liquid by adding acid to eliminate the influence of alkaline substances in the crab shells during the initial fermentation stage, but this was unsuccessful. Considering that the strain used in this invention mainly synthesizes D-lactic acid at 32–37℃ and mainly synthesizes L-lactic acid at 40℃, in order to lower the initial pH value of fermentation without affecting the L-lactic acid yield of the strain, different concentrations of analytical grade L-lactic acid were added during the initial fermentation stage for comparison. The fermentation results with different amounts of L-lactic acid added are shown in Table 4.

[0077] Table 4 Comparison of lactic acid fermentation results after adding different concentrations of L-lactic acid

[0078]

[0079] The results in Table 4 show that adding L-lactic acid can mitigate the impact of alkaline substances in crab shells on lactic acid fermentation and promote lactic acid synthesis. With increasing L-lactic acid addition, the total lactic acid concentration and sugar-acid conversion rate at the end of fermentation both increased, and the corresponding decalcification rate of the crab shell also gradually increased. However, from the perspective of L-lactic acid synthesis, increasing the amount of pre-added L-lactic acid leads to a decrease in the optical purity of L-lactic acid at the end of fermentation.

[0080] In previous examples, the average culture time of the seed culture was 11 hours, and the L-lactic acid concentration in the seed culture at inoculation was approximately 10 g / L. Theoretically, appropriately extending the seed culture culture time can increase the lactic acid concentration in the seed culture at inoculation and decrease the initial pH value of the fermentation broth. Seed cultures with different activation times were inoculated and fermented for 54 hours; the fermentation results are shown in Table 5.

[0081] Table 5 Effect of activation time on lactic acid fermentation

[0082]

[0083] It can be seen that, compared to the 10-12h culture conditions, the lactic acid concentration was highest at the end of fermentation after 14h of seed culture, and the corresponding decalcification rate of crab shells was also highest under these conditions. However, longer seed culture time is not necessarily better; the lactic acid concentration at the end of fermentation after 16h was lower than that under the 14h conditions. Measurements showed that the pH of the seed culture dropped to 4.0 after 16h of culture, which may be the reason for the decreased acid-producing capacity of the strain.

[0084] Comparing the data in Tables 4 and 5, it can be seen that appropriately extending the seed culture time helps in the synthesis of L-lactic acid. Inoculation 14 hours after strain activation resulted in an optical purity of 92.37% for L-lactic acid at the end of fermentation, significantly higher than that achieved with pre-added analytical grade L-lactic acid.

[0085] Example 5

[0086] The process of lactic acid fermentation coupled with decalcification of crab shells essentially utilizes the lactic acid produced during fermentation to neutralize the calcium carbonate in the crab shells, thereby removing calcium. Theoretically, the higher the sugar concentration, the higher the concentration of lactic acid produced during fermentation, and the greater the amount of crab shells that can be processed. However, the actual fermentation process is not a simple acid-base neutralization reaction, but rather a process in which the activity of the microbial strain is influenced and regulated by environmental factors.

[0087] The aforementioned experimental results indicate that both sugar concentration and crab shell addition amount affect the fermentation outcome. To determine the appropriate addition ratio, a central composite design (CCD) was first used in Design-Expert v8.0 to conduct a response surface methodology analysis on the initial sugar concentration and crab shell addition amount (Table 6). The independent variables were the initial sugar concentration and crab shell addition amount, and the variable under investigation was the sugar-acid conversion rate. The fermentation process adopted an intermittent feeding method to ensure that the total sugar concentration was the same under all conditions. The sugar-acid conversion rate was chosen as the variable under investigation because it better reflects the activity of the microorganisms and the acid production efficiency in lactic acid fermentation. At the same time, the higher the sugar-acid conversion rate, the higher the amount of lactic acid synthesized per unit mass of sugar, the more calcium carbonate can be removed, and the higher the economic efficiency of crab shell treatment.

[0088] Table 6 Experimental Factors and Levels in CCD Design

[0089]

[0090] The experimental results designed using CCD were subjected to regression fitting using analysis software to obtain a quadratic equation in two variables:

[0091] Y=92.00-13.76*A+14.89*B+5.78*A*B-4.58*A 2 -10.80*B 2

[0092] Y represents the predicted sugar-acid conversion rate, A represents the initial sugar concentration, and B represents the amount of crab shell added. Analysis of variance results show that A, B, and B... 2 The effect of [specific ingredient name] was significant, while the others were not. The F-value of the regression equation was 35.85, and the p-value was 0.0006 < 0.05, indicating that the regression equation had a good fit. The lack of fit term was 0.0894 > 0.05, indicating that the regression equation had a small error. According to response surface methodology, the theoretical sugar-acid conversion rate could reach 100% when the initial sugar concentration was predicted to be 57.25 g / L and the crab shell addition was 24.45 g / 100 mL.

[0093] Next, with an initial sugar concentration of 60 g / L, a single-factor experiment was conducted to investigate the interaction between total sugar and the amount of crab shell added. The experimental design is shown in Table 7.

[0094] Table 7. Single-factor experimental design of the relationship between total sugar and crab shell addition.

[0095]

[0096]

[0097] The results showed that when the crab shell addition was 10 g / 100 mL, the highest lactic acid concentration (54.15 g / L) was achieved at a total sugar concentration of 60 g / L. When the crab shell addition was 20 g / 100 mL, the highest lactic acid concentration (102.54 g / L) was achieved at a total sugar concentration of 120 g / L; while when the crab shell addition was 30 g / 100 mL, the highest acid concentration (133.88 g / L) was achieved at a total sugar concentration of 150 g / L. These results indicate that an appropriate ratio of sugar to crab shell can maintain a balance between bacterial growth and acid production, thus ensuring the effectiveness of the crab shell treatment.

[0098] Since the calcium lactate formed when the lactic acid concentration in the fermentation broth exceeds 130 g / L is easily crystallized below 40°C, the fermentation broth solidifies, affecting the separation of the crab shell from the fermentation broth at the end of fermentation. Therefore, a total sugar concentration of no more than 150 g / L is the appropriate amount of total sugar to be added for decalcification of crab shells using lactic acid fermentation.

[0099] Example 6

[0100] In the production process of treating crab shells using lactic acid fermentation, high-temperature sterilization can cause the degradation of chitin in the crab shells, leading to protein denaturation and increasing energy consumption during production. Since the lactic acid bacteria used are anaerobic fermentation bacteria and the fermentation temperature is relatively high, the fermentation effects under non-sterilization conditions (neither the fermentation broth nor the crab shells are sterilized) and sterilized conditions were investigated. The parameter measurement results are shown in Table 8.

[0101] Table 8 Comparison of fermentation results between sterilized and non-sterilized fermentation

[0102]

[0103] Table 8 shows that the lactic acid fermentation coupled with decalcification and deproteinization of crab shells carried out without sterilization resulted in a higher rate of lactic acid synthesis and a higher rate of calcium and protein removal from the crab shells compared to sterilization.

[0104] Under non-sterilized conditions, the inventors conducted multiple batches of semi-continuous fermentation to investigate the effects of lactic acid synthesis and calcium and protein removal from crab shells during semi-continuous fermentation under natural conditions. The fermentation medium was prepared according to the optimized nitrogen source conditions: initial sugar concentration 60 g / L, NaCl 10 g / L, anhydrous sodium acetate 2.5 g / L, and ammonium citrate 0.2 g / L. Crab shells were added at a rate of 30 g / 100 mL, and a seed culture activated for 14 h was inoculated. Fermentation began at 42℃ and 100 rpm. When the residual sugar concentration dropped below 10 g / L, feeding was initiated, with concentrated sugar solution added in three batches, for a total sugar addition of 90 g / L. The fermentation endpoint was defined as the residual sugar concentration dropping below 5 g / L. The supernatant was immediately aspirated and inoculated at a volume ratio of 10% into freshly prepared fermentation medium (containing crab shells) for fermentation, with three consecutive inoculations. The acid production concentration and crab shell treatment effect of each batch of semi-continuous fermentation were measured, and the results are compared in Table 9.

[0105] Table 9 Comparison of acid production concentration and crab shell treatment results for each batch in semi-continuous fermentation.

[0106]

[0107] Because the seed culture used in the semi-continuous fermentation was the fermentation broth from the previous batch, the initial lactic acid concentrations in the second and third batches were high, and the final decalcification rate of the crab shells gradually increased, with the second batch showing the highest lactic acid yield and acid production efficiency. However, when transferred to the third batch, i.e., the fourth batch of continuous fermentation, the acid production capacity of the lactic acid bacteria decreased after continuous subculturing, and contaminating bacteria appeared in the system, leading to a decrease in both the sugar-acid conversion rate and the crab shell processing efficiency during the fermentation process. These results indicate that the lactic acid fermentation system using crab shells as a neutralizing agent has a certain resistance to contamination by contaminating bacteria, and it is feasible to conduct 3-4 batches of semi-continuous fermentation under this system.

[0108] Example 7

[0109] Based on the aforementioned experimental results, when using lactic acid fermentation to remove calcium and protein from crab shells, the amount of lactic acid synthesized directly affects the removal efficiency of mineral elements from the crab shells. Experimental verification showed that this strain removes protein and mineral elements simultaneously. Therefore, theoretically, monitoring lactic acid synthesis during fermentation can predict the decalcification and deproteinization rates of crab shells, thus determining the treatment endpoint. pH value is the most direct monitoring indicator for the acid production process. Data on the pH value at the end of fermentation under different conditions and the corresponding decalcification rate of crab shells were collected, and a linear correlation analysis was performed. The results are shown in […]. Figure 3 .

[0110] It can be seen that there is a certain linear relationship between the pH value of the fermentation broth and the decalcification rate of crab shells, and this linear relationship is consistent under different crab shell addition amounts and different initial sugar concentrations. Since the neutralization reaction of lactic acid with calcium in crab shells requires a certain amount of time, predicting the decalcification rate based on pH value has a certain lag. Theoretically, the size of crab shell particles may affect the parameters of the linear relationship between the two. Multiple batches of experiments have verified that when the crab shell particle size is within the range of 0.17-5mm, and the pH value in the fermentation broth drops to around 4.6, the decalcification rate of crab shells can reach over 80%, which is basically in line with expectations. Figure 3 The relationship between y = 173.89794 - 20.40949x.

[0111] Example 8

[0112] Data from Examples 1-7 show that by optimizing the use of lactic acid fermentation to treat crab shells, approximately 80% of the calcium and 60% of the protein can be removed. The remaining calcium and protein require further processing to obtain crude chitin.

[0113] After fermentation, washing, and drying, crab shells were treated with hydrochloric acid at concentration gradients of 0.05 mol / L to 0.5 mol / L for 20-300 min at a mass-to-volume ratio of 1:10 (w / v). The shells were then washed with water until pH neutral, dried, and the calcium content was determined. The results showed that soaking in 0.05 mol / L hydrochloric acid for 90 min completely removed residual calcium salts from the crab shells, while soaking in 0.1 mol / L hydrochloric acid for 60 min completely removed calcium. Treatment with 0.2 mol / L and 0.5 mol / L hydrochloric acid took even less time. The pH value of the treated solution was also measured. Figure 4 After decalcifying crab shells with a 0.05 mol / L hydrochloric acid solution, the residual liquid had a pH of approximately 5.10, which is more environmentally friendly. Next, the treatment effects of equal amounts of hydrochloric acid were compared: decalcification was performed using 0.1 mol / L hydrochloric acid solution at a mass-to-volume ratio of 1:5 and 0.05 mol / L hydrochloric acid solution at a mass-to-volume ratio of 1:10, respectively. The results showed that there was no significant difference in the effectiveness of the two methods in removing residual calcium, but excessively low mass-to-volume ratios may lead to problems in subsequent wastewater treatment.

[0114] After fermentation, the washed and dried crab shells were added to NaOH solutions with concentration gradients ranging from 0.1 mol / L to 1 mol / L at a solid-liquid ratio of 1:10 (w / v). The treatment temperature was 40-60℃, and the treatment time was 0.5-12 h. After treatment, the shells were washed with water until the pH was neutral, dried, and the protein content was measured. The amount of protein removed was calculated, and the results are shown below. Figure 5 It can be seen that temperature has a significant effect on protein removal. At 40℃ and 50℃, the removal of protein was incomplete with four concentration gradients of NaOH within 9 hours. At 60℃, treatment with 0.25 mol / L NaOH for 7 hours completely removed residual protein, and the higher the NaOH concentration, the shorter the treatment time. Further increasing the treatment temperature, the results showed that at 80℃, treatment with 0.25 mol / L NaOH for 2 hours completely removed residual protein, and at 90℃, treatment with 0.1 mol / L NaOH for 2 hours completely removed residual protein. The results of the residual protein removal experiments indicate that suitable temperature and concentration are beneficial for protein removal, while extending the treatment time does not remove protein from crab shells under low concentration or low temperature conditions. Furthermore, extending the treatment time leads to denaturation of the removed protein or deacetylation of chitin, resulting in a decrease in protein recovery rate and a reduction in chitin quality.

[0115] The results showed that the crude product was first treated with 0.1 mol / L hydrochloric acid solution at a mass-to-volume ratio of 1:5 for 90 min, filtered to collect the precipitate, and then treated with 0.25 mol / L NaOH at a mass-to-volume ratio of 1:10 at 80℃ for 2 h. After washing and drying, the chitin content in the crude product was 95.88%.

Claims

1. A method for the biological resource utilization of seafood waste, characterized in that, The method includes the following steps: 1) Dry and crush the seafood waste for later use; the seafood waste is crab shells; the crushed crab shells are sieved to a particle size of 0.17-5 mm; 2) Inoculate the lactic acid bacteria activated for 8-20 h into the fermentation medium at a volume ratio of 1:5-10, and simultaneously add the seafood waste obtained in step 1) at a rate of 10-30 g / 100 mL. Ferment at a temperature of 40-45℃ and a rotation speed of 50-200 rpm for 36-72 h. When the sugar concentration of the fermentation liquid drops below 10 g / L, start sugar supplementation. The total sugar consumption during the fermentation process should not exceed 150 g / L. The fermentation medium consists of the following components: glucose 60-150 g / L, sodium chloride 5-15 g / L, sodium acetate 1-5 g / L, and ammonium citrate 0.1-0.5 g / L; the lactic acid bacteria are Lactobacillus acidophilus or Lactobacillus plantarum. 3) When the pH of the fermentation broth drops to 4.5-5.0, fermentation ends. After filtration, the precipitate is used to prepare chitin; the filtered fermentation broth is used to extract lactic acid or lactate. No organic nitrogen source is added to the fermentation medium; no acid-base neutralizer is used during the fermentation process.

2. The method for utilizing biological resources as described in claim 1, characterized in that, The process of using the precipitate to prepare chitin includes the following steps: 1) Wash and dry the obtained precipitate, and treat it in 0.05-0.5 mol / L hydrochloric acid solution at a mass-to-volume ratio of 1:5-10 for 20-300 min; 2) Solid-liquid separation: The precipitate is placed in a 0.1-0.5 mol / L sodium hydroxide solution at a mass-to-volume ratio of 1:5-10 and treated at 60-90 ℃ for 0.5-12 h. 3) Clean the precipitate and dry it to obtain chitin raw material.

3. The method for utilizing biological resources as described in claim 2, characterized in that: The chitin raw material contains more than 95% chitin.

4. The method for utilizing biological resources as described in claim 1, characterized in that, Step 2) adopts batch continuous fermentation: when the sugar concentration of the fermentation liquid drops to 5±3 g / L, take the liquid component and transfer it to a new fermentation medium at a volume ratio of 1:5-10. Add 10-30 g / 100mL of seafood waste and start fermentation again; when the sugar concentration drops to 5±3 g / L, transfer it again. Repeat the transfer 1-3 times.

5. The method for utilizing biological resources as described in any one of claims 1-4, characterized in that: The filtered fermentation broth is first heated and then filtered by plate and frame press. The resulting filtrate is cooled to obtain crude calcium lactate. The resulting filter residue is then sprayed and granulated to obtain feed protein.

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