A kind of squid paste and its preparation method and application

Abstract

The present invention relates to the field of microbial fermentation, and in particular to a squid paste and a preparation method and application thereof. The squid paste is prepared by fermenting squid viscera and seaweed raw materials with bacterial enzymes, is rich in nutrition, can be widely used in aquatic feed, significantly improves the growth performance of aquatic animals, and enhances their immune performance and disease resistance, with remarkable effects.

Description

Technical Field

[0001] The invention relates to the field of microbial fermentation, and in particular to squid paste and a preparation method and application thereof. Background Art

[0002] Squid paste, as a unique type of feed, has attracted much attention in the feed market in recent years. It is made of squid as the main raw material and processed through a special process. It is rich in various nutrients such as protein, fat, vitamins and minerals. Among them, protein is the basic substance that constitutes the tissue of organisms and is essential for the growth and development of farmed organisms. Fat is an important source of energy and is also the main component of cell membranes. In addition, the vitamins and minerals in squid paste also play an important role in the metabolism and immune function of farmed organisms. Since squid paste is rich in nutrients and easy to digest and absorb, the application of squid paste in feed can significantly improve the growth rate and health of farmed organisms.

[0003] In the field of aquaculture, squid paste is widely used in feeds for aquatic animals such as fish, shrimp, and crabs. Practice has shown that feeds with squid paste can significantly increase the food intake and growth rate of aquatic animals, while improving their meat quality and taste. In freshwater aquaculture, squid paste can increase the feeding enthusiasm of aquatic animals such as fish and shrimp, promote nutrient absorption, and thus accelerate their growth rate; in the field of marine aquaculture, squid paste also plays an important role. It is particularly suitable for the breeding of marine fish and shellfish, which can help them better adapt to the marine environment and improve their survival rate and growth efficiency. For some special aquaculture organisms, such as ornamental fish and rare fish, squid paste also has a good application effect. It not only provides rich nutrition, but also improves the color and body shape of aquatic organisms, enhancing their ornamental value.

[0004] As a type of high-protein, high-fat feed, squid paste has rich nutrients and unique feed properties and has broad application prospects. Summary of the invention

[0005] The purpose of the present invention is to provide a squid paste and a preparation method and application thereof. The squid paste is prepared by fermenting squid viscera and seaweed raw materials with bacterial enzymes, is rich in nutrition, can be widely used in aquatic feed, significantly improves the growth performance of aquatic animals, enhances their immune performance and disease resistance, and has a significant effect.

[0006] On one hand, the invention provides a squid paste, which is prepared by fermenting squid viscera and seaweed enzymatic hydrolysate with Bacillus lentus.

[0007] The Bacillus lentus is Bacillus lentus SK001, which was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on September 2, 2024, and its deposit number is CCTCC NO:

[0008] M20241882.

[0009] The seaweed is selected from any one or more of Spirulina, Sargassum, Laminaria, Chlorella and Schizochytrium.

[0010] The preparation method of the squid paste comprises the following steps:

[0011] (1) Beat the squid viscera into a slurry, pass it through a 60-mesh sieve, mix it evenly with seaweed powder in a weight ratio of 1-3:1, and sterilize it at 121° C. for 15 minutes to obtain a squid seaweed slurry;

[0012] (2) After cooling to 50° C., adding protease to the squid seaweed slurry at a mass ratio of 0.3-0.5%, performing enzymolysis at 50° C.-60° C. for 2-6 hours, inactivating the enzyme, and obtaining an enzymolysis solution;

[0013] (3) adding 0.5% by weight of Bacillus lentus powder to the enzymatic hydrolyzate, and fermenting at 35° C.-40° C. for 24-36 hours to obtain a fermentation liquid;

[0014] (4) Concentrating the fermented liquid at a low temperature of 40°C-60°C and packaging it to obtain squid paste.

[0015] In the step (2), the protease is selected from any one or two of alkaline protease, flavor protease and neutral protease.

[0016] The amount of live bacteria in the Bacillus lentus powder in step (3) is not less than 10 9 CFU / g.

[0017] The invention also provides application of the squid paste in the production of aquatic feed.

[0018] The squid paste is added in an amount of 20-30 kg / ton in aquatic feed.

[0019] The total live bacteria content of the squid paste provided by the present invention exceeds 10 10 CFU / g, the protein content exceeds 90g / 100g, of which the polypeptide content exceeds 80%, and most of them are small molecule peptides with a relative molecular mass ≤2000U, which are more easily absorbed; in addition, the content of heavy metal pollutants in squid paste is low and within the safe range.

[0020] The squid paste can significantly improve the growth and biochemical indexes of yellow catfish, enhance its immunity, and improve its survival rate. Compared with the control group, the weight gain rate, survival rate and specific growth rate of the yellow catfish in the experimental group fed with the squid paste described in Examples 6 and 7 were increased by 22.84%-35.24%, 8.89%-10.00%, and 22.73%-26.36%, respectively, and the feed coefficient was reduced by 12.18%-22.44%.

[0021] The squid paste can effectively improve the antioxidant capacity of farmed animals, enhance immunity, and help reduce the occurrence of diseases. Compared with the control group, the malondialdehyde content in the serum of the yellow catfish in the experimental group fed with the squid paste described in Examples 6 and 7 was reduced by 62.44%-71.80%, and the total superoxide dismutase, alkaline phosphatase and lysozyme activities were increased by 12.11%-16.47%, 41.56%-65.63% and 32.72%-61.22%, respectively, with significant effects.

[0022] In summary, the squid paste prepared by the synergistic fermentation of bacteria and enzymes in the present invention can be widely used in aquatic feed, can significantly improve the utilization rate of feed by aquatic animals, improve the growth performance of aquatic animals, enhance their immune performance and disease resistance, reduce the occurrence of diseases, and thus help improve the economic benefits of aquaculture. In addition, the development of squid paste realizes the effective utilization of squid viscera resources, reduces environmental pollution, and increases its product added value, and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the colony morphology of Bacillus lentus SK001. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The Bacillus lentus SK001 selected in the embodiment of the present invention was isolated from fresh squid viscera at Manyuwan Farmers' Market in Licang District, Qingdao City, and has strong protease production ability. It was deposited in the China Type Culture Collection Center of Wuhan University, Wuhan, China on September 2, 2024, and its preservation number is CCTCCNO: M20241882.

[0026] The present invention will be further described below in conjunction with specific embodiments.

[0027] Example 1 Screening, identification and functional analysis of Bacillus lentus SK001

[0028] A strain with the strongest protease production ability was screened from fresh squid viscera purchased from Manyuwan Farmers' Market in Licang District, Qingdao City. It was identified as Bacillus lentus and named Bacillus lentus SK001. It was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on September 2, 2024, and its preservation number is CCTCC NO: M20241882.

[0029] The colony morphology of Bacillus lentus SK001 on nutrient agar medium is as follows Figure 1 As shown, the colony is 1-2mm in diameter, round, with neat edges, opaque, grayish white on the front, convex in the middle, smooth, bright on the surface, moist in texture, and easy to pick up.

[0030] Bacillus lentus SK001 has good biosafety and is sensitive to common antibiotics such as ampicillin, furazolidone, chloramphenicol, tetracycline, penicillin G, and gentamicin.

[0031] The Bacillus lentus SK001 can produce a high amount of protease, and the protease activity in the fermentation supernatant reaches 389.1 U / mL, which is significantly higher than that of the commercially available Bacillus lentus in the control group, achieving an unexpected technical effect.

[0032] Bacillus lentus SK001 has a strong tolerance to the digestive tract environment. After being treated in artificial gastric juice and artificial intestinal juice for 3 hours, its survival rate is still as high as over 85%. It can better pass through the stomach into the intestine, exert its prebiotic effect, and achieve unexpected technical results.

[0033] Example 2 Analysis of the self-aggregation and hydrophobicity of Bacillus lentus

[0034] The self-aggregation and surface hydrophobicity of probiotics are important factors that reflect the strength of their adhesion ability and whether they can colonize in the intestines and play a probiotic role. The greater the hydrophobicity of the cell surface, the stronger its adhesion. The self-aggregation of probiotics refers to the phenomenon that the bacteria aggregate into clusters due to large-scale and rapid reproduction during the cultivation process. The stronger the surface hydrophobicity and self-aggregation, the stronger its intestinal adhesion.

[0035] Inoculate Bacillus in LB liquid culture medium and culture at 37℃ for 18h. Centrifuge the cultured bacterial solution at 4℃ and 12000r / min for 5min to collect the bacteria, and wash twice with sterile saline to remove the residual liquid of the culture medium. Adjust the OD600 of the bacterial suspension to 0.5 (A0). Take 2mL of the bacterial suspension and let it stand at room temperature for 3h, take 200μL of the upper bacterial solution, and measure the OD value at 600nm (A1); take another 2mL of the bacterial suspension and mix it with an equal volume of xylene for 2min, let it stand for 30min, and take the aqueous phase to measure the OD at 600nm (A2). Use saline as a blank.

[0036] The calculation formula of self-aggregation and hydrophobicity is:

[0037] Self-aggregation (%) = (1-A1 / A0) x 100%.

[0038] Hydrophobicity (%) = [(A0-A2) / A0] x 100%.

[0039] Where: A0 is the OD value of the initial bacterial solution at 600nm; A1 is the OD value of the supernatant at 600nm after standing for 3 hours; A2 is the OD value of the aqueous phase at 600nm after mixing with xylene.

[0040] Table 1 Self-aggregation and hydrophobicity of Bacillus lentus SK001

[0041]

[0042] As can be seen from Table 1, the self-aggregation and hydrophobicity of Bacillus lentus SK001 are as high as 43.1% and 56.1%, respectively, which are higher than those of commercially available strains, thus facilitating its better colonization in the intestinal tract of aquatic organisms.

[0043] Example 3 Analysis of antibacterial properties of Bacillus lentus SK001

[0044] Centrifuge the 18-20h lentus SK001 nutrient broth culture at 12000r / min for 5min, and place the supernatant in a 4℃ refrigerator for later use.

[0045] The concentration is 1×10 6 100 μL of indicator bacteria solution with CFU / mL was evenly spread on the LB solid plate, an Oxford cup was placed, 200 μL of fermentation supernatant of Bacillus lentus SK001 was added, and the plate was cultured at 37°C for 24 h to observe whether an inhibition zone was produced and measure the size of the inhibition zone.

[0046] At the same time, commercially available Bacillus lentus was used as a control.

[0047] Table 2 Antibacterial activity of Bacillus lentus SK001

[0048]

[0049] From the data in Table 2, it can be seen that the Bacillus lentus SK001 provided by the present invention can effectively inhibit various pathogens such as Staphylococcus aureus, Escherichia coli, Bacillus cereus, Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas fluorescens, Escherichia coli, etc., especially the strongest antibacterial effect on Staphylococcus aureus and Escherichia coli, with the diameter of the inhibition zone exceeding 22 mm, which is significantly better than the commercially available Bacillus lentus.

[0050] Example 4 Application of Bacillus lentus SK001 in the cultivation of Penaeus vannamei

[0051] 1. Preparation of bacterial powder

[0052] The Bacillus lentus SK001 was activated, expanded, and fermented in liquid form, centrifuged, concentrated, and freeze-dried to produce a culture medium with a live bacterial content of about 10 10 CFU / g of bacterial powder.

[0053] 2. Experimental Design

[0054] The experiment set up a control group and a probiotic group. The control group was fed with basic feed. The probiotic group added the above-mentioned Bacillus lentus SK001 powder to the basic feed at a weight ratio of 3‰, mixed evenly and fed. Three parallels were set up in each group, with 50 shrimps in each parallel. The breeding experiment lasted for 8 weeks, and the daily feeding amount was 3% of the total weight of the shrimp. The shrimps were fed twice a day, and the shrimps were weighed every 2 weeks to adjust the daily feeding amount. The dry matter weight of the feed fed to the control group and the probiotic group was kept consistent. The breeding water was replaced once a week, and the water change amount was 1 / 4 of the total water volume. During the experiment, the dissolved oxygen was ≥8.3mg / l, the temperature was 25-29℃, the salinity was 6-7‰, and the pH was 7.5-7.9. After the breeding experiment, the shrimps were counted and weighed, and fasted for one day before weighing. Its survival rate, specific growth rate, weight gain rate, etc. were calculated. Hepatopancreatic T-AOC, superoxide dismutase, and lysozyme were detected using the Nanjing Jiancheng Bioengineering Institute kit. The specific results are shown in Tables 3 and 4.

[0055] Table 3 Effects of Bacillus lentus SK001 on growth and survival of Litopenaeus vannamei

[0056] Control group Probiotics group Initial body weight / g 5.41±0.12 5.37±0.23 Final body weight / g 14.42±0.56 16.21±0.27 Survival rate / % <![CDATA[74.34±3.33 a ]]> <![CDATA[93.12±3.54 b ]]> Weight gain rate / % <![CDATA[166.54±20.25 a ]]> <![CDATA[201.86±15.92 b ]]> Specific growth rate / % <![CDATA[3.50±0.11 a ]]> <![CDATA[3.95±0.14 b <!-- 4 -->]]> Feed coefficient <![CDATA[1.73±0.21 a ]]> <![CDATA[1.41±0.35 b ]]>

[0057] Note: Different letters indicate significant differences (P<0.05).

[0058] Table 4 Effects of Bacillus lentus SK001 on immunity of Litopenaeus vannamei

[0059] Project (U / mg prot) Control group Probiotics group T-AOC 2.12±0.13 2.47±0.15 Superoxide dismutase 43.52±2.10 43.46±2.43 Lysozyme activity <![CDATA[101.30±7.67 b ]]> <![CDATA[143.82±5.32 a ]]>

[0060] Note: Different letters indicate significant differences (P<0.05).

[0061] From the results of Table 3 and Table 4, it can be seen that compared with the control group, the survival rate, weight gain rate, and specific growth rate of the probiotic group of shrimp fed with Bacillus lentus SK001 powder increased by 25.3%, 21.2%, and 12.9%, respectively, and the feed coefficient decreased by 18.5%; and the hepatopancreatic lysozyme activity of the probiotic group increased by 42.0%. This shows that the Bacillus lentus SK001 provided by the present invention can better induce the production of immune factors, actively participate in the body's immune defense, thereby improving the body's immune defense, and achieving unexpected technical effects.

[0062] Example 5 Application of Bacillus lentus SK001 in freshwater grouper farming

[0063] Freshwater grouper with an average weight of about 60g was selected, and a control group and a probiotic group were set. The control group was fed with basic bait, and the probiotic group was fed with the lentus SK001 powder described in Example 4 at a weight ratio of 5‰ on the basis of the basic bait, and the mixture was evenly mixed before feeding. Three parallels were set for each group, with 40 freshwater groupers in each parallel, and the water temperature was controlled to be 22-25°C. The feeding time was 9:00 and 17:00, respectively. The feeding time at 9:00 should be appropriate. After 0.5 h, the feeding situation of the fish was observed and recorded. The feeding amount was adjusted appropriately according to the feeding situation, and the remaining bait was removed after 0.5 h of feeding. The bottom of the breeding barrel was sucked every morning and evening, and 1 / 3 of the breeding water was replaced. The replaced water was disinfected, aerated, and tap water of the same temperature. Feeding and breeding for 28 days.

[0064] On the 28th day of the experiment, 3 freshwater groupers were selected from each barrel, weighed, and measured in length. The weight gain rate, specific growth rate, survival rate, fatness, liver-body ratio, organ-body ratio, and feed coefficient were calculated. The fish were starved for 24 hours the day before blood collection, and blood was collected from the tail vein. The blood samples were centrifuged at 4°C and 4000 r / min for 10 min, and the supernatant was taken to determine the antioxidant index, which was determined using the kit of Nanjing Jiancheng Bioengineering Research Institute. The results are shown in Tables 5-7.

[0065] Table 5 Effects of Bacillus lentus SK001 on the growth performance of freshwater grouper

[0066] Group Control group Probiotics group Weight gain rate / % <![CDATA[57.61±0.15 a ]]> <![CDATA[98.42±0.74 b ]]> Specific growth rate / (% / d) <![CDATA[2.31±0.36 a ]]> <![CDATA[3.53±0.41 b ]]> Liver to body ratio / % 1.53±0.18 1.64±0.31 Viscera to body ratio / % 3.32±0.26 3.73±0.22 <![CDATA[Condition factor / (g / cm 3 )]]> 1.25±0.29 1.42±0.27 Feed coefficient 1.62±0.24 1.13±0.24 Survival rate / % 95 100

[0067] Table 6 Effects of Bacillus lentus SK001 on the immune capacity of freshwater grouper

[0068] index Control group Probiotics group Total superoxide dismutase / (U / mL) <![CDATA[55.42±0.91 a ]]> <![CDATA[68.83±1.32 a ]]> Catalase / (U / mL) 9.75±0.22 <![CDATA[4.30±0.69 b ]]> Malondialdehyde / (μmol / L) <![CDATA[140.27±1.06 a ]]> <![CDATA[128.05±2.44 b <!-- 5 -->]]> Glutathione peroxidase GSH / (μmol / L) <![CDATA[103.53±3.41 a ]]> <![CDATA[117.50±3.42 b ]]>

[0069] Table 7 Effects of Bacillus lentus SK001 on serum liver function indicators of freshwater grouper

[0070] index Control group Probiotics group Aspartate aminotransferase / (U / L) <![CDATA[21.32±1.47 a ]]> <![CDATA[15.73±0.54 b ]]> Alanine aminotransferase / (U / L) <![CDATA[21.07±1.22 a ]]> <![CDATA[15.62±1.42 a ]]>

[0071] From the data in Table 5, it can be seen that compared with the control group, the weight gain rate of the freshwater grouper in the probiotic group fed with Bacillus lentus SK001 powder increased by 70.8% (P<0.05), the specific growth rate increased by 52.8% (P<0.05), and the growth performance was significantly improved.

[0072] From the data in Table 6, it can be seen that compared with the control group, the activities of total superoxide dismutase and glutathione peroxidase in the serum of freshwater grouper in the probiotic group fed with Bacillus lentus SK001 powder increased by 24.2% and 13.5%, respectively, and the contents of catalase and malondialdehyde decreased by 126.7% and 9.5%, respectively.

[0073] As shown in Table 7, compared with the control group, the levels of aspartate aminotransferase and alanine aminotransferase in the serum of freshwater grouper in the probiotic group fed with Bacillus lentus SK001 powder decreased by 35.5% and 34.9%, respectively.

[0074] The above results show that the Bacillus lentus SK001 provided by the present invention can significantly improve the antioxidant capacity and immune level in the body of freshwater grouper, reduce liver damage in aquatic animals, and effectively promote the growth of farmed animals.

[0075] Example 6 A squid paste and its preparation method

[0076] 1. Beat the squid viscera into a pulp, pass it through a 60-mesh sieve, mix it evenly with spirulina powder in a weight ratio of 1:1, and sterilize it at 121℃ for 15 minutes to obtain squid seaweed pulp;

[0077] 2. After cooling to 50°C, add flavor protease with an enzyme activity of 50,000 U / g to the squid seaweed slurry at a mass ratio of 0.3%, perform enzymolysis at 60°C for 2 hours, inactivate the enzyme, and obtain an enzymolysis solution;

[0078] 3. Add 0.5% by weight of Bacillus lentus SK001 powder to the enzymatic hydrolysate, and ferment at 35° C. for 36 hours to obtain a fermentation liquid;

[0079] 4. Concentrate the fermentation liquid at 40°C and package it to obtain squid paste.

[0080] Example 7 A squid paste and its preparation method

[0081] 1. Beat the squid viscera into a paste, pass it through a 60-mesh sieve, mix it evenly with chlorella powder in a weight ratio of 3:1, sterilize it at 121℃ for 15 minutes, and obtain squid seaweed paste;

[0082] 2. After cooling to 50° C., add 30,000 U / g neutral protease to the squid seaweed slurry at a mass ratio of 0.5%, perform enzymolysis at 50° C. for 6 h, inactivate the enzyme, and obtain an enzymolysis solution;

[0083] 3. Add 0.5% by weight of Bacillus lentus SK001 powder to the enzymatic hydrolyzate, and ferment at 40° C. for 24 hours to obtain a fermentation liquid;

[0084] 4. Concentrate the fermentation liquid at 60°C and package it to obtain squid paste.

[0085] Example 8 Analysis of physical and chemical properties of squid paste

[0086] The squid paste prepared in Examples 6 and 7 was subjected to physical and chemical testing, and the results are shown in Table 8.

[0087] Table 8 Analysis of physical and chemical properties of squid paste

[0088] index Example 6 Example 7 Live bacteria content / (CFU / g) <![CDATA[2.3×10 10 ]]> <![CDATA[4.7×10 10 ]]> Protein content / (g / 100g) 90.7 92.3 Peptide content / % 83.7 80.7 Relative molecular weight <1000U / % 91.8 89.0 Relative molecular weight 1000-2000U proportion / % 6.7 8.3 Relative molecular weight <2000-5000U / % 1.5 2.7 Inorganic arsenic / (mg / kg) ≤0.5 ≤0.5 Methylmercury / (mg / kg) ≤0.1 ≤0.1 Lead / (mg / kg) ≤1.0 ≤1.0 Chromium / (mg / kg) ≤1.0 ≤1.0 Water content / (g / 100g) ≤8.0 ≤8.0

[0089] From the results in Table 8, it can be seen that the total viable bacteria content of the squid paste provided by the present invention exceeds 10 10 CFU / g, the protein content exceeds 90g / 100g, of which the polypeptide content exceeds 80%, and most of them are small molecule peptides with a relative molecular mass ≤2000U, which are more easily absorbed; in addition, the content of heavy metal pollutants in squid paste is low and within the safe range.

[0090] Example 8 Application of squid paste in yellow catfish breeding

[0091] The squid paste prepared in Examples 6 and 7 was used to feed Pelteobagrus fulvidraco, and the growth performance and immune ability of Pelteobagrus fulvidraco were tested.

[0092] Experimental group: The squid paste prepared in Example 6 or 7 was added to the basic bait at a mass ratio of 3%.

[0093] Control group: basic bait.

[0094] (I) Experimental fish farming management

[0095] The purchased yellow catfish were temporarily kept in the breeding pond for 2 weeks, during which they were fed with basic feed and fasted for 24 hours before the experiment. Yellow catfish in good condition were selected for the experiment and randomly assigned to circulating water breeding tanks, with 30 fish in each breeding tank, 3 replicates in each experimental group, and a breeding cycle of 42 days. Feed and observe until fullness, and feed twice a day (08:00 and 17:30). During the experiment, the water temperature was maintained at 25.0-30.0℃, pH was 7.8-8.0, dissolved oxygen>5.0 mg / L, ammonia nitrogen<0.05 mg / L, and nitrite<0.1 mg / L. During the breeding period, 50% of the water was changed every day.

[0096] After the breeding experiment, the fish were fasted for 24 hours, and the total weight of the fish was weighed, the body length was measured and counted, and the growth performance was calculated. Six fish were randomly selected from each tank, and tail vein blood was collected. The blood was centrifuged at 5000r / min for 10 minutes, and the supernatant was taken and stored in an ultra-low temperature refrigerator at -80℃ for the subsequent determination of serum biochemical indicators, antioxidant enzyme activity and immune indicators.

[0097] 1. Growth performance

[0098] Table 9 Effects of squid paste on growth and survival of Pelteobagrus fulvidraco

[0099] project Control group Example 6 Example 7 Weight gain rate (%) 58.59±6.81a 79.24±4.90b 71.97±5.27a Survival rate (%) 90±0.31a 99±0.34b 98±0.21b Feed coefficient 1.56±0.25a 1.21±0.14b 1.37±0.18a Specific growth rate (%) 1.10±0.06a 1.39±0.07b 1.35±0.09ab

[0100] As shown in Table 9, compared with the control group, the weight gain rate, survival rate and specific growth rate of the yellow catfish in the experimental group fed with the squid paste described in Examples 6 and 7 increased by 22.84%-35.24%, 8.89%-10.00%, and 22.73%-26.36%, respectively, and the feed coefficient decreased by 12.18%-22.44%. It can be seen that the squid paste provided by the present invention can effectively promote the growth of farmed animals and improve their growth performance, with significant effects.

[0101] 2. Biochemical indicators

[0102] The biochemical indicators such as total superoxide dismutase (T-SOD), catalase (CAT), malondialdehyde (MDA), alkaline phosphatase (ALP) and lysozyme (LZM) in the serum of yellow catfish are shown in Table 10.

[0103] Table 10 Effects of squid paste on antioxidant and immune indicators of yellow catfish

[0104]

[0105] From the data in Table 10, it can be seen that compared with the control group, the malondialdehyde content in the serum of the yellow catfish in the experimental group fed with the squid paste described in Examples 6 and 7 was reduced by 62.44%-71.80%, and the activities of total superoxide dismutase, alkaline phosphatase and lysozyme were increased by 12.11%-16.47%, 41.56%-65.63% and 32.72%-61.22%, respectively, with significant effects. It can be shown that the squid paste provided by the present invention can effectively improve the antioxidant capacity of farmed animals, enhance immunity, and help reduce the occurrence of diseases.

[0106] In summary, the squid paste prepared by the synergistic fermentation of Bacillus lentus SK001 and protease in the present invention can be widely used in aquatic feed, can significantly improve the growth performance of aquatic animals, increase feed utilization, enhance the immune performance and disease resistance of aquatic animals, and reduce the occurrence of diseases, and the effect is very significant.

Claims

1. A squid paste, characterized in that: By Bacillus lentus ( Bacillus lentus ) is made from fermented squid viscera and seaweed.

2. The squid paste according to claim 1, characterized in that: The deposit number of the Bacillus lentus is CCTCC NO: M20241882.

3. The squid paste according to claim 2, characterized in that: The seaweed is selected from any one or more of Spirulina, Sargassum, Laminaria, Chlorella and Schizochytrium.

4. Application of the squid paste described in any one of claims 1 to 3 in aquatic feed production.

5. The use according to claim 4, characterized in that The squid paste is added in an amount of 20-30 kg / ton in aquatic feed.

6. A method for preparing squid paste, characterized in that: The steps include: (1) Beat the squid viscera into a pulp, pass it through a 60-mesh sieve, mix it evenly with seaweed powder in a weight ratio of 1-3:1, and sterilize it at 121° C. for 15 min to obtain squid seaweed pulp; (2) After cooling to 50°C, adding protease to the squid seaweed slurry at a mass ratio of 0.3-0.5%, performing enzymolysis at 50°C-60°C for 2-6 hours, inactivating the enzyme, and obtaining an enzymolysis solution; (3) adding 0.5% by weight of Bacillus lentus powder to the enzymatic hydrolysate, and fermenting at 35°C-40°C for 24-36 hours to obtain a fermentation liquid; (4) Concentrate the fermentation liquid at a low temperature of 40°C-60°C and package it to obtain squid paste.

7. The preparation method according to claim 6, characterized in that: In the step (2), the protease is selected from any one or two of alkaline protease, flavor protease and neutral protease.

8. The preparation method according to claim 6 or 7, characterized in that: The amount of live bacteria in the Bacillus lentus powder in step (3) is not less than 10 9 CFU / g.

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