Special ecological fermented feed for Procambarus clarkii and production method thereof

Abstract

The invention discloses a special ecological fermented feed for Procambarus clarkii and a production method thereof, and relates to the technical field of fermented feed for aquaculture. The fermented feed is prepared by fermenting a mixed bacterial liquid with crayfish feed as a fermentation matrix; wherein the mixed bacterial liquid is prepared by adding cutinase, phytase, sodium butyrate, a fermentation promoter, and a mixed bacterial species consisting of lactic acid bacteria, bacillus, yeast, and Aspergillus oryzae into water. When the ecological fermented feed provided by the invention is applied to feeding Procambarus clarkii, it can ensure that Procambarus clarkii has good growth performance while promoting the improvement of the quality of Procambarus clarkii. From the aspect of shrimp shell, the soft shell rate of Procambarus clarkii is reduced while the hardness of the shrimp shell is improved. From the aspect of shrimp meat quality, the shrimp meat is tender, elastic and chewy, the meat is good, and the edible quality is good, which helps to improve its market preference and improve the breeding efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture fermented feed, and particularly relates to a special ecological fermented feed for Procambarus clarkii and a production method thereof. Background Art

[0002] Procambarus clarkii is commonly known as crayfish and is loved by the market and consumers for its fresh, smooth and delicious meat. Its meat is delicious and it is widely cultured in China. With the expansion of the Procambarus clarkii farming scale, the feed demand has also increased accordingly. Crayfish are omnivorous animals with a rather extensive diet. However, currently, crayfish in freshwater aquaculture mainly feed on artificial compound feeds, and their food types are relatively single. This is mainly because there is still a lack of research on the evaluation of the application effects of different types of feeds in crayfish farming.

[0003] As a new type of environment-friendly feed in recent years, biological fermented feed is gradually being recognized by people. Biological fermented feed can effectively improve the immunity and disease resistance of aquatic animals, which is beneficial to the production of green aquatic products and the protection of the aquaculture ecological environment. Since these microorganisms are rich in protein and can also produce various nutrients such as amino acids and vitamins, or secrete highly active decomposition enzymes and various digestive enzymes, appropriate addition of fermented feed can improve the animal intestinal flora, and improve the feed utilization rate and animal immunity, thereby promoting animal growth.

[0004] Currently, there is little research on the effects of feeding biological fermented feed on the growth and meat quality of crayfish. In order to improve the aquaculture effect of crayfish, promote its growth, and improve its product quality, the present invention provides a special ecological fermented feed for Procambarus clarkii and a production method thereof. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a special ecological fermented feed for Procambarus clarkii and a production method thereof.

[0006] The present invention is achieved through the following technical solutions. The present invention includes the following steps:

[0007] In the first aspect of the present invention, a special ecological fermented feed for Procambarus clarkii is provided. The fermented feed is prepared by fermenting a crayfish feed as a fermentation substrate with a mixed bacterial liquid; wherein, the mixed bacterial liquid is prepared by adding cutinase, phytase, sodium butyrate, a fermentation promoter and a mixed bacterial strain composed of lactic acid bacteria, bacillus, yeast and aspergillus oryzae into water.

[0008] As a further optimized scheme of the present invention, the addition amounts of the cutinase, phytase, sodium butyrate and the fermentation promoter respectively account for 0.1-0.5% of the total mass of the mixed bacterial strain.

[0009] As a further optimization scheme of the present invention, the fermentation promoter is linseed gum or guar gum.

[0010] As a further optimization scheme of the present invention, the viable count of the mixed strains is 2.5 - 3.0×10 8 cfu / g, and the viable component ratio of lactic acid bacteria, bacillus, yeast and aspergillus oryzae is 1:2:1:0.05.

[0011] In the second aspect of the present invention, there is also provided a production method of the special ecological fermentation feed for procambarus clarkii as described in any one of the above, including the following steps:

[0012] (1) Select a mixed strain composed of lactic acid bacteria, bacillus, yeast and aspergillus oryzae, stir evenly and then let it stand. Put the standing mixed strain into water at 20 - 25°C, add cutinase and a fermentation promoter, and stir evenly to form a mixed bacterial liquid;

[0013] (2) Mix the crayfish feed with the mixed bacterial liquid obtained in step (1), adjust the water content to 60 - 70%, and ferment to obtain the special ecological fermentation feed for procambarus clarkii.

[0014] As a further optimization scheme of the present invention, in step (2), the fermentation process is divided into two stages. The fermentation conditions in the first stage are 25 - 30°C, pH 3.5 - 5.0, and ferment for 72 h. The fermentation conditions in the second stage are 28 - 32°C, pH 6.5 - 7.5, and ferment for 48 h.

[0015] The present invention has the following advantages compared with the prior art:

[0016] (1) The present invention proposes an ecological fermentation feed for the cultivation of procambarus clarkii, which is prepared by fermenting a crayfish feed as a fermentation substrate with a mixed bacterial liquid. The mixed bacterial liquid is prepared by adding cutinase, phytase and sodium butyrate, a fermentation promoter and a mixed strain composed of lactic acid bacteria, bacillus, yeast and aspergillus oryzae into water. The prepared ecological fermentation feed is beneficial to improving the activity of digestive enzymes in the shrimp body, promoting growth and weight gain, and enhancing the economic benefits of procambarus clarkii cultivation.

[0017] (2) The quality of procambarus clarkii fed with the ecological fermentation feed provided by the present invention is improved, which is specifically reflected in the shrimp shell and shrimp meat. In terms of the shrimp shell quality, the soft shell rate of procambarus clarkii decreases while the hardness of the shrimp shell increases. In terms of the shrimp meat quality, the shrimp meat is tender, the elasticity and chewiness of the shrimp meat are improved, and the edible quality is good, which helps to improve its market preference.

[0018] (3) The Procambarus clarkii fed with the ecological fermentation feed provided by the present invention has a shortened time required for the new shell to harden completely after molting, resulting in a shortened single molting cycle. To a certain extent, this will increase the number of molts of the shrimp during the breeding period, contribute to the weight gain of the shrimp, and improve the breeding efficiency. In addition, the shortened time for the new shell to harden after molting of the shrimp body can, to a certain extent, reduce the risk of invasion by germs, toxins or other organisms brought about by a long molting period, which also has a certain positive significance for improving the survival rate of the shrimp. Detailed implementation mode

[0019] The present invention will be further described in detail below. It is necessary to point out here that the following specific implementation modes are only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0020] I. Reagents and materials

[0021] The following reagents and materials used are all commercially available products unless otherwise specified.

[0022] The following strains are all purchased from Mingzhou Biology:

[0023] Lactobacillus, product number BMZ133551; Bacillus, product number BMZ339683; Saccharomyces, product number BMZ126481; Aspergillus oryzae, product number BMZ135265; Aspergillus niger, product number BMZ135480.

[0024] Cutinase, with an enzyme activity of 5000 U / mL, is purchased from Jiangsu Yihaotian Biotechnology.

[0025] II. Methods

[0026] The following methods used are all conventional methods known to those skilled in the art unless otherwise specified.

[0027] 1. Preparation of mixed bacterial solution

[0028] Mixed bacterial solution A: Select a mixed strain composed of Lactobacillus, Bacillus, Saccharomyces and Aspergillus oryzae, stir evenly and then let stand. The viable bacteria count is 2.5×10 8cfu / g, and the viable bacteria component ratio of the lactic acid bacteria, bacillus, yeast and aspergillus oryzae is 1:2:1:0.05. The mixed bacteria after standing are put into water at 20-25 °C, and cutinase, phytase, sodium butyrate and a fermentation promoter are added, and stirred evenly to form a mixed bacterial liquid. Among them, the addition amounts of the cutinase and the fermentation promoter respectively account for 0.5% of the total mass of the mixed bacteria, and the fermentation promoter is sesame gum.

[0029] Mixed bacterial liquid B: Different from mixed bacterial liquid A, the fermentation promoter is guar gum.

[0030] Mixed bacterial liquid C: Different from mixed bacterial liquid A, the fermentation promoter is soybean polysaccharide.

[0031] Mixed bacterial liquid D: Different from mixed bacterial liquid A, aspergillus niger is used instead of aspergillus oryzae.

[0032] Mixed bacterial liquid E: Different from mixed bacterial liquid A, the mixed bacteria selected are a mixed bacteria of lactic acid bacteria, bacillus and yeast. After stirring evenly, it is left standing, and the viable bacteria count is 2.5×10 8 cfu / g, and the viable bacteria component ratio of the lactic acid bacteria, bacillus and yeast is 1:2:1.

[0033] Mixed bacterial liquid F: Different from mixed bacterial liquid C, aspergillus niger is used instead of aspergillus oryzae.

[0034] Mixed bacterial liquid G: Different from mixed bacterial liquid A, cutinase is not added.

[0035] 2. Preparation of special ecological fermentation feed for Procambarus clarkii

[0036] The mixed bacterial liquids A-G are respectively used for fermenting crayfish feed at the same addition amount. The specific fermentation process is as follows: the crayfish feed is mixed with the mixed bacterial liquid, the water content is adjusted to 60-70%, and fermentation is carried out. The fermentation is divided into two stages: the first stage fermentation conditions are 25-30 °C, the pH value is 3.5-5.0, and the fermentation is carried out for 72 h; the second stage fermentation conditions are 28-32 °C, the pH value is 6.5-7.5, and the fermentation is carried out for 48 h. After fermentation, the fermented feeds A-G are obtained.

[0037] Composition of crayfish feed: fish meal 16%, soybean meal 12%, rapeseed meal 15%, corn starch 28%, wheat flour 19%, fish oil 3%, soybean oil 4%, calcium dihydrogen phosphate 1.4%, premix 1.5% and mold inhibitor 0.1%; the premix provides 7.2 mg of copper, 59.5 mg of zinc, 71.5 mg of iron, 0.35 mg of iodine, 55.5 mg of manganese, 0.8 mg of selenium, 7300 IU of vitamin A, 32750 IU of vitamin D3, 30 mg of vitamin E, vitamin B 11.8 mg, vitamin B 2 2.8 mg, vitamin B 6 2.4 mg, vitamin B 12 0.01 mg, vitamin K 32.5 mg, folic acid 0.72 mg, biotin 1.45 mg.

[0038] The nutritional levels of the crayfish feed: crude protein 32%, crude fat 6%, ash 18%, crude fiber 8%, lysine 1.5% and moisture 10%.

[0039] In order to illustrate the feeding and breeding effects of the fermented feed provided by the present invention on Procambarus clarkii, in addition, a commercially available lobster fermented feed was used as control group 1, and a crayfish feed without fermentation treatment was used as control group 2.

[0040] The formula of the commercially available lobster fermented feed is 70% soybean meal, 10% corn starch, 18.13% bacterial liquid (Bacillus licheniformis, Enterococcus faecalis, Saccharomyces cerevisiae, Clostridium butyricum), 0.00125% vitamin A, 0.00035% vitamin D, 0.0175% vitamin E, 0.0004% vitamin K, vitamin B 1 0.00075%, vitamin B 2 0.002%, niacin 0.006%, pantothenic acid 0.00375%, vitamin B 6 0.001%, vitamin B 12 0.00025%, folic acid 0.00025%, biotin 0.00375%, copper sulfate 0.0005%, ferrous sulfate 0.0085%, zinc sulfate 0.003%, manganese sulfate 0.0032%, 0.0003% sodium selenite 1%, 0.0055% calcium iodate 1%, 0.0025% cobalt chloride 1% and 1.8% calcium dihydrogen phosphate. The lobster fermented feed can be fermented at 30 °C for 72 hours with a pH value lower than 4, and has an obvious alcoholic fragrance.

[0041] 3. Test animals and breeding management

[0042] Procambarus clarkii with sound appendages and good vitality were selected for breeding. The initial weight of Procambarus clarkii was 5 ± 0.5 g, and they were taken from the breeding base of Quanjiao Procambarus clarkii Breeding Cooperative. They were all bred in the crayfish ponds of the Fisheries Research Institute of Anhui Academy of Agricultural Sciences in June. Nine test ponds with the same specifications and an area of about 0.20 hm 2 were selected. The water sources were kept the same, and Procambarus clarkii were randomly allocated at a stocking density of 60,000 tails / hm 2 . The breeding period was 60 days. The feed was fed once at 7:00 and 18:00 every day, and the daily feeding amount was about 4% of the shrimp body weight. At the same time, daily water quality management was carried out.

[0043] 4. Effects on the growth performance of Procambarus clarkii

[0044] After the end of the breeding period, 10 individuals were randomly selected from each of the 9 experimental ponds, and 3 replicates were taken from each experimental treatment group. An electronic balance was used to weigh the body weight to calculate the weight gain rate and specific growth rate. The results were averaged and rounded to two decimal places. The specific calculation formulas are as follows:

[0045] Weight gain rate (WGR, %) = [(m t - m 0 ) / m 0 × 100%;

[0046] Specific growth rate (SGR, %·d -1 ) = [(lnm t - lnm 0 ) / d] × 100%;

[0047] Where: m 0 is the initial body weight (g) at the start of breeding; m t is the final body weight (g) at the end of breeding; d is the number of feeding days.

[0048] The results are shown in Table 1.

[0049] Table 1 Effects of different feeds on the growth performance of Procambarus clarkii

[0050]

[0051] As can be seen from Table 1, compared with feeding Procambarus clarkii with unfermented crayfish feed, feeding with fermented feed can improve the weight gain rate and specific growth rate of the shrimp to a certain extent. Because fermented feed can stimulate intestinal peristalsis, and the increase in various digestive enzymes promotes the decomposition and absorption of feed, improving the utilization rate of feed, which is beneficial to the growth and weight gain of shrimp. In addition, it can be seen from the table that compared with commercially available fermented lobster feed, feeding Procambarus clarkii with the fermented feed provided by the present invention can improve the weight gain rate and specific growth rate of Procambarus clarkii, and thus is beneficial to improving the breeding efficiency of Procambarus clarkii.

[0052] 5. Effects on the digestive enzyme activity of Procambarus clarkii

[0053] After the end of the breeding period, 10 individuals were randomly selected from each of the 9 experimental ponds, and 3 replicates were taken from each experimental treatment group. The hepatopancreas and intestine were collected and stored in ice, and immediately taken back to the laboratory to measure the digestive enzyme activity. Strictly in accordance with the instructions of the commercially available kit, after mixing all samples with an appropriate amount of physiological saline, they were homogenized at 4°C and the supernatant was separated and collected to measure the activities of trypsin, amylase, lipase and cellulase. The results were averaged and rounded to two decimal places.

[0054] The results are shown in Table 2.

[0055] Table 2 Effects of different feeds on the digestive enzyme activities of Procambarus clarkii (unit: U / mg)

[0056]

[0057]

[0058] As can be seen from Table 2, compared with the unfermented crayfish feed, feeding Procambarus clarkii with fermented feed can improve the digestive enzyme activities in its hepatopancreas and intestine to a certain extent, which has a promoting effect on the growth of Procambarus clarkii. In addition, it can be seen from the table that different fermented feeds have different effects on the digestive enzyme activities in the shrimp hepatopancreas and intestine. Compared with the commercially available fermented lobster feed, feeding Procambarus clarkii with the fermented feed obtained by fermenting crayfish feed with the mixed bacterial liquid provided by the present invention can improve the digestive enzyme activity index of Procambarus clarkii.

[0059] 6. Effects on the shell quality of Procambarus clarkii

[0060] After the end of the breeding period, 1000 shrimps were randomly taken from each of the 9 experimental ponds, and the shrimps with soft shell problems were picked out and the quantity was recorded. The soft shell rate was calculated. 10 shrimps with normal shrimp shell hardness were randomly taken from each of the 9 experimental ponds, and the third segment of their shrimp shells was cut into 2 pieces along the back to measure the shrimp shell hardness. The texture analyzer test parameters were set as follows: the probe was selected as the P50 blade probe, the maximum pressure value of the induction element probe was 60 N, the mode was TPA, the trigger force was 0.6 N, the probe running speed was 60 mm / s, and the deformation amount was 100%. Three replicates were taken for each experimental treatment group, and the results were averaged, retaining two decimal places.

[0061] Table 3 Effects of different feeds on the shell quality of Procambarus clarkii

[0062]

[0063] As can be seen from Table 3, feeding Procambarus clarkii with different fermented feeds has an impact on the shell quality of the shrimp. From the data of the soft shell rate of Procambarus clarkii, it can be seen that compared with feeding Procambarus clarkii with unfermented crayfish feed, fermented feeds A - G can reduce the number of soft shell shrimps, which has a positive effect on reducing the soft shell rate of the shrimp. By comparing fermented feeds A - C, it can be seen that when the fermentation promoter is selected as linseed gum, the effect on improving the shrimp shell quality is the best. In addition, by comparing fermented feed A, fermented feed D and fermented feed F, it can be seen that Aspergillus oryzae and linseed gum have a synergistic effect on improving the shrimp shell quality. Finally, by comparing fermented feed A and fermented feed G, it can be seen that the addition of cutinase has a positive effect on improving the shrimp shell hardness.

[0064] 7. Effects on the meat quality of Procambarus clarkii

[0065] Randomly select 10 from each of the 9 experimental ponds. Referring to the methods described by Huang Chunhong et al. (2020) and Li Gaoshang et al. (2019), use a P / 36R probe to measure the hardness, elasticity, and chewiness of Procambarus clarkii meat in TPA mode. Each sample is measured 3 times and the average value is taken, retaining two decimal places.

[0066] The results are shown in Table 4.

[0067] Table 4 Effects of different feeds on the meat quality of Procambarus clarkii (unit: g)

[0068]

[0069] As can be seen from Table 4, feeding Procambarus clarkii with different fermented feeds has an impact on the meat quality of the shrimp. By comparing fermented feeds A - C, it can be seen that when the fermentation promoter is gum sesbania, it can reduce the hardness of the shrimp meat, improve the elasticity and chewiness of the shrimp meat, and comprehensively improve the meat quality and edible quality of the shrimp. In addition, by comparing fermented feed A, fermented feed D, and fermented feed F, it can be seen that there is a synergistic effect between Aspergillus oryzae and gum sesbania in improving the meat quality of the shrimp.

[0070] 8. Effects on the molting of Procambarus clarkii

[0071] Select 10 Procambarus clarkii with sound appendages and good vitality from each of the 9 experimental ponds. Isolate the breeding area in situ in each experimental pond to ensure that the size of the breeding area is suitable for the stocking density of the whole pond. After marking each Procambarus clarkii, carry out the breeding management as described above, and at the same time carry out daily water quality management. When the Procambarus clarkii in each experimental pond undergoes the second molt, observe the molting in sequence. The specific method is to take the time after the second molt is completed as the starting time, record the time required for the new shell of Procambarus clarkii to harden completely, and take the average value of the results, retaining two decimal places.

[0072] The results are shown in Table 5.

[0073] Table 5 Effects of different feeds on the molting of Procambarus clarkii (unit: h)

[0074]

[0075] As can be seen from Table 5, compared with feeding crayfish with unfermented feed, feeding Procambarus clarkii with fermented feed can improve the utilization effect of feed by Procambarus clarkii. The time required for the new shell to harden completely after molting is shortened, resulting in a shorter single molting cycle. To a certain extent, this will increase the number of molts during the breeding period, contribute to the weight gain of the shrimp, and improve the breeding efficiency. In addition, the shortening of the new shell hardening time after molting can, to a certain extent, reduce the risk of invasion by pathogens, toxins or other organisms during the long molting period, which also has a certain positive significance for improving the survival rate of the shrimp.

[0076] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention.

Claims

1. A special ecological fermented feed for Procambarus clarkii, characterized in that: The fermented feed is used to improve the shell quality and meat quality of Procambarus clarkii and shorten the time required for Procambarus clarkii to molt and harden the new shell. The fermented feed is prepared by fermenting the crayfish feed as a fermentation matrix through mixed bacterial liquid; The mixed bacterial liquid is prepared by adding cutinase, phytase, sodium butyrate, a fermentation accelerator and a mixed bacterial strain consisting of lactic acid bacteria, bacillus, yeast and Aspergillus oryzae into water; The fermentation promoter is sesame gum, and the addition amount of cutinase, phytase, sodium butyrate and fermentation promoter is 0.1-0.5% of the total mass of the mixed bacteria, and the number of live bacteria in the mixed bacteria is 2.5-3.0×10 8 cfu / g, and the live bacteria component ratio of the lactic acid bacteria, bacillus, yeast and Aspergillus oryzae is 1:2:1:0.

05.

2. A method for producing ecological fermented feed for Procambarus clarkii according to claim 1, characterized in that: The following steps are involved: (1) Select a mixed strain of lactic acid bacteria, Bacillus, yeast and Aspergillus oryzae, stir it evenly and let it stand, put the mixed strain after standing into water at 20-25°C, add cutinase, phytase, sodium butyrate and fermentation promoter, stir it evenly to form a mixed bacterial liquid; (2) Mixing the crayfish feed with the mixed bacterial solution obtained in step (1), adjusting the water content to 60-70%, and fermenting to obtain the ecological fermented feed for Procambarus clarkii.

3. The method for producing a special ecological fermented feed for Procambarus clarkii according to claim 2, characterized in that: In step (2), the fermentation process is divided into two stages. The fermentation conditions of the first stage are 25-30°C, pH 3.5-5.0, and fermentation for 72 hours. The fermentation conditions of the second stage are 28-32°C, pH 6.5-7.5, and fermentation for 48 hours.

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