A method for artificially cultivating water earthworms

Abstract

The present invention provides a method for artificially cultivating water earthworms, wherein quercetin flavonoids are used to regulate biological flocs, and then water earthworms are fed, and unregulated common biological flocs are used as controls. Dyella sp. and Pantoea sp. The abundance of the two probiotics was 1.4 and 5.0 times that of the control. Legionella sp. and Mycobacterium sp. The abundance of the two pathogens was only 40.2% and 51.9% of that of the control; the weight gain and body length growth of a single water earthworm were 3.8 times and 5.0 times of that of the control, respectively, and the crude protein and crude fat contents of the water earthworm were 1.2 times and 1.1 times of that of the control, respectively. The present invention optimizes the microbial abundance of the biological flocs by using quercetin flavonoids, thereby significantly improving the growth rate and nutritional value of the water earthworms, obtaining high-quality water earthworms, and having obvious application value.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to a method for artificially cultivating water earthworms. Background Art

[0002] Water earthworms, also known as silkworms and red nematodes, belong to the phylum Annelida, class Aquatic Oligochaeta, order Parapora, family Trembling, and genus Lumbricus. They are small benthic animals that mostly live on the surface of riverbed sludge or in the silt on the banks of rivers. Water earthworms usually feed on bacteria, algae or humus-rich sediments in water bodies, as well as organic debris particles and micro-animals. They are not only consumers but also decomposers in ecosystems, promoting energy flow and material circulation in nature. Water earthworms have high nutritional value and contain a complete range of amino acids. They are the starting bait for many aquatic animals and the main bait for benthic fish. Li Yanhua et al. (2021) used water earthworms as the starting bait for sturgeon, which can well promote its growth and survival. Therefore, it is of great practical significance to study the growth and reproduction of water earthworms and cultivate high-quality water earthworms.

[0003] Common earthworm baits include lettuce, a mixture of flour and lettuce, cooked mashed potatoes, yeast, Elodea, Cladophora, Daphnia, and bioflocs. Kosiorek (1974) used lettuce as bait for earthworms and found that their growth rate was 0.069-0.078 mg·(piece·d)-1; while Finogenova et al. (1987) found that when bioflocs were used as substrates, the growth rate of earthworms could reach 0.25-0.28 mg·(piece·d)-1. It can be seen that using bioflocs to cultivate earthworms can increase their growth rate.

[0004] Biofloc is an irregular flocculent suspension composed of organic matter, protozoa, algae and filamentous fungi. The composition and structure of biofloc from different sources are different, especially in terms of the differences in dominant bacterial communities. Studies have shown that water earthworms have predation selectivity, which is mainly reflected in their preference for substrate particle size, organic matter content and microbial species (Zhu Hui et al., 2008), and the influence of microbial species on water earthworm predation selectivity is more obvious (McMurtry et al., 1983). Therefore, optimizing the microbial species of biofloc can promote water earthworm feeding and obtain better quality water earthworms. Summary of the invention

[0005] Purpose of the invention: In view of the needs of the prior art, the present invention provides a method for artificially cultivating water earthworms. The method uses quercetin flavonoids to adjust the types and abundance of microorganisms in bioflocs, and then feeds water earthworms. After adjustment by quercetin flavonoids, the bioflocs contain more probiotics such as Dyella sp. and Pantoea sp. (the abundance increases from 35.96% and 0.47% of ordinary bioflocs to 50.34% and 2.35% at this time, respectively), and contain fewer pathogens such as Legionella sp. and Mycobacterium sp. (the abundance decreases from 1.26% and 0.17% of ordinary bioflocs to 0.51% and 0.09% at this time, respectively); using the bioflocs to feed water earthworms, the weight gain and body length growth of a single water earthworm are 3.8 times and 5.0 times of those without adjustment, respectively, and the crude protein and crude fat contents of water earthworms are 1.2 times and 1.1 times of those without adjustment, respectively. The invention can improve the growth speed and nutritional value of water earthworms and has obvious application value.

[0006] Technical solution: In order to solve the above technical problems, the present invention provides a method for artificially cultivating water earthworms, comprising the following steps:

[0007] (1) Using simulated aquaculture wastewater, adding inoculum, quercetin flavonoids and carbon source to cultivate quercetin bioflocs;

[0008] (2) Domesticating earthworms;

[0009] (3) Artificial cultivation of water earthworms in breeding boxes.

[0010] Among them, the step (1) of cultivating quercetin bioflocs includes: preparing 80L of simulated aquaculture wastewater, adding inoculation solution at a volume ratio of 1%-10%, adding 0.15g-0.30g of quercetin flavonoids, and adding once every three to ten days. During the cultivation period, the water is not changed, aeration is continued, and a carbon source is regularly added to adjust the C / N to >10:1; after 3-5 days, bioflocs are formed, the mixed solution is taken and centrifuged at 5000r / min for 15min, the supernatant is removed, and the precipitate is the quercetin bioflocs.

[0011] Wherein, the inoculation solution is any fish pond water.

[0012] Wherein, the carbon source is glucose, brown sugar or molasses.

[0013] Among them, the step (2) of domesticating water earthworms includes: placing water earthworm seedlings in clean water, feeding 8g-12g of quercetin biofloc every day, and domesticating for 5 days.

[0014] Among them, the step (3) of artificially cultivating water earthworms includes: adding paddy field soil with a thickness of 2cm-4cm that has been air-dried, sieved and crushed into a breeding box, and then adding 1.5L-2L of water, and standing for 2 days; picking 5g-10g of water earthworms with bright red color and good activity after being domesticated in step (2), putting them into the breeding box, and feeding 2g-4g of quercetin biofloc every day.

[0015] Among them, the cultivation mode of step (3) is micro-flow water type, the cultivation time is 30 days, and the cultivation temperature is natural room temperature.

[0016] Wherein, the specification of the breeding box is 24.5cm×17cm×14cm.

[0017] Among them, the abundance of two probiotics, Dyella sp. and Pantoea sp., in the quercetin biofloc reached 50.34% and 2.35%, and the abundance of two pathogens, Legionella sp. and Mycobacterium sp., was as low as 0.51% and 0.09%; when the quercetin biofloc was used to feed water earthworms, the weight and body length of a single water earthworm increased by 0.69 mg and 5.06 mm, respectively, and the crude protein and crude fat contents of the water earthworms reached 58.80% and 12.98%, respectively.

[0018] Beneficial effects:

[0019] 1) The present invention uses quercetin flavonoids to regulate bioflocs, and then feeds water earthworms, so that the weight and body length of a single water earthworm increase by 0.69 mg (3.8 times that of ordinary bioflocs) and 5.06 mm (5.0 times that of ordinary bioflocs), respectively, and the crude protein and crude fat contents reach 58.80% (1.2 times that of ordinary bioflocs) and 12.98% (1.1 times that of ordinary bioflocs), respectively, which are significantly higher than ordinary bioflocs;

[0020] 2) The present invention develops a better quality bait for water earthworms, which is beneficial to improving the growth rate and nutritional value of water earthworms; it is worth pointing out that the present invention only changes and optimizes the microbial abundance of bioflocs, but does not change the microbial species of bioflocs, but achieves unexpected results. DETAILED DESCRIPTION

[0021] The materials used in the embodiments of the present invention are quercetin flavonoids purchased from Hunan Hill Natural Pharmaceutical Co., Ltd., water earthworms purchased from a flower and bird market in Haizhu District, Guangzhou, paddy field soil collected from Shuangjie Town, Jiangcheng District, Yangjiang City (N 21°55'45", E 111°49'34"), and the inoculation liquid is the fish pond water of Yangjiang Smart Agriculture Technology Development Co., Ltd. (N 21°54'11.01", E 111°55'50.33"). The formula of aquaculture simulated wastewater is: 70mg / L (NH4)2SO4, 78mg / L K2HPO4, 31mg / L KH2PO4, 95mg / LMgSO4·7H2O, 37mg / L KCl, 557mg / L C6H 12 O6, 0.67mL / L trace elements. The trace element formula is: 640mg / LEDTA, 550mg / LFeSO4·7H2O, 230mg / L ZnSO4·7H2O, 340mg / L MnSO4·H2O, 75mg / LCuSO4·5H2O, 47mg / L Co(NO3)2·6H2O, 25mg / L(NH4)5Mo7O 24 ·4H2O.

[0022] Example:

[0023] Cultivating water earthworms using quercetin biofloc includes the following steps:

[0024] S1) The steps of cultivating quercetin bioflocs are as follows: 80L of simulated aquaculture wastewater is prepared in a 250L bucket, and an inoculation solution is added at a volume ratio of 5%, and the amount of quercetin flavonoids added is 0.16g, and the frequency of addition is once every five days. During the cultivation period, the water is not changed, aeration is continued, and glucose is regularly added to adjust C / N=15:1. After 3 days, bioflocs are formed, and the mixed solution is centrifuged at 5000r / min for 15min, and the supernatant is removed. The precipitate is the quercetin biofloc.

[0025] S2) The steps of acclimatizing water earthworms are as follows: water earthworm seedlings purchased from the market are placed in clean water, and 8 g of the quercetin biofloc prepared in S1) of this example is fed daily for 5 days.

[0026] S3) artificial cultivation of water earthworms, comprising the following steps:

[0027] A. Add 2 cm thick air-dried, sieved and crushed paddy soil to a culture box with a size of 24.5 cm × 17 cm × 14 cm, then add 1.7 L of water and let it stand for 2 days;

[0028] B. Selecting water earthworms with bright red color and good activity from the water earthworms tamed in S2) of this embodiment;

[0029] C. Add 5 g (wet weight) of water earthworms to the culture medium after standing for 2 days, and cultivate them in micro-flow water at natural room temperature to reduce disturbance to the water earthworms while keeping the dissolved oxygen above 2 mg / L;

[0030] D. Feed 2 g of the quercetin biofloc prepared as bait in Example S1) regularly every day and cultivate for 30 days at natural room temperature.

[0031] S4) Determination indicators: after 30 days of the experiment, the average weight gain and average body length increase of a single water earthworm, crude protein and crude fat of the water earthworm.

[0032] Average weight gain of a single earthworm (M): At the beginning and end of the formal experiment, 300 earthworms were randomly selected, washed repeatedly with distilled water, and placed on a 100-mesh sieve. Absorbent paper was placed under the sieve to absorb the moisture on the surface of the earthworms. When the absorbent paper was no longer wet, it was transferred to an electronic balance to weigh its wet weight. The wet weight of the earthworms at the beginning of the formal experiment was recorded as M0, and the wet weight of the earthworms at the end of the formal experiment was recorded as M1, both in milligrams (mg).

[0033]

[0034] Average body length growth of a single water earthworm (L): At the beginning and end of the formal experiment, 100 water earthworms were randomly selected and placed on a glass dish, and a few drops of 1:1 ethanol solution were added. When the water earthworms stopped swinging and died, their body length was measured with a vernier caliper. The body length of the water earthworms at the beginning of the formal experiment was recorded as L0, and the body length of the water earthworms at the end of the formal experiment was recorded as L1, both in millimeters (mm).

[0035]

[0036] The crude protein and crude fat contents of water earthworms were determined by Kjeldahl method and Soxhlet extraction method, respectively.

[0037] After continuous cultivation of water earthworms with quercetin biofloc for 30 days, the average weight gain of a single water earthworm was 0.69 mg, the average body length increase was 5.06 mm, and the crude protein and crude fat of the water earthworms reached 58.80% and 12.98%.

[0038] Comparative Example:

[0039] Cultivating water earthworms using common bioflocs includes the following steps:

[0040] S1) The steps of cultivating common bioflocs are as follows: 80L of simulated aquaculture wastewater is prepared in a 250L bucket, and 5% volume of inoculation solution is added. During the cultivation period, the water is not changed, aeration is continued, and a carbon source is regularly added to adjust C / N=15:1. After 3 days, bioflocs are formed, and the mixed solution is centrifuged at 5000r / min for 15min, the supernatant is removed, and the precipitate is the common biofloc.

[0041] S2) The steps of domesticating water earthworms are as follows: water earthworm seedlings purchased from the market are placed in clean water, and 8 g of the common biofloc prepared in this comparative example S1) are fed every day for 5 days.

[0042] S3) artificial cultivation of water earthworms, comprising the following steps:

[0043] A. Add 2 cm thick air-dried, sieved and crushed paddy soil to a culture box with a size of 24.5 cm × 17 cm × 14 cm, then add 1.7 L of water and let it stand for 2 days;

[0044] B. Select the water earthworms with bright red color and good activity from the water earthworms tamed in this comparative example S2);

[0045] C. Place 5 g (wet weight) of water earthworms in the culture box after standing for 2 days, and cultivate them in micro-flow water at natural room temperature to reduce disturbance to the water earthworms while keeping the dissolved oxygen above 2 mg / L;

[0046] D. Feed 2 g of the common biofloc prepared in Comparative Example S1) regularly every day and cultivate for 30 days at room temperature.

[0047] S4) Determination indicators: after 30 days of the experiment, the average weight gain and average body length increase of a single water earthworm, crude protein and crude fat of the water earthworm.

[0048] After continuous cultivation of water earthworms with ordinary biofloc for 30 days, the average weight gain of a single water earthworm was 0.18 mg, the average body length increase was 1.01 mm, and the crude protein and crude fat of the water earthworms reached 48.11% and 11.97%.

[0049] In summary, after continuous cultivation of water earthworms with ordinary biofloc and quercetin biofloc for 30 days, there were significant differences in the growth performance and nutritional components of water earthworms in each group, as shown in Table 1:

[0050] Table 1 Growth performance and nutritional components of water earthworms fed with different baits

[0051]

[0052] Note: Three replicates were set for each treatment, and the reported experimental results are average values; the same lowercase letters after the data in the same column indicate no significant difference at p>0.05, and different lowercase letters indicate significant difference at p<0.05.

[0053] As shown in Table 1, compared with ordinary bioflocs, quercetin bioflocs can better promote the growth of water earthworms and improve the nutritional content of water earthworms. The weight and length of a single water earthworm increased by 0.69 mg and 5.06 mm, respectively, and the crude protein and crude fat contents reached 58.80% and 12.98%, respectively, and were significantly higher (p < 0.05) than ordinary bioflocs, indicating that feeding quercetin bioflocs better improved the nutritional value of water earthworm growth rate. It can be seen that quercetin bioflocs is a better quality bait.

[0054] The present invention has many specific application paths, and the above is only a preferred embodiment of the present invention. It should be pointed out that the above embodiments are only used to illustrate the present invention, rather than to limit the scope of protection of the present invention. For those of ordinary skill in the art, without departing from the principle of the present invention, several improvements can also be made, and these improvements should also be regarded as the scope of protection of the present invention.

Claims

1. A method for artificially cultivating water earthworms, characterized in that: The following steps are involved: (1) Using simulated aquaculture wastewater, adding inoculum, quercetin flavonoids and carbon source to cultivate quercetin bioflocs; (2) Domestication of water earthworms: Place water earthworm seedlings in clean water and feed them 8g-12g of quercetin biofloc every day for 5 days; (3) Artificially cultivating water earthworms in a breeding box: Add paddy field soil that has been air-dried, sieved and crushed to a thickness of 2 cm-4 cm into the breeding box, then add 1.5 L-2 L of water and let it stand for 2 days; pick 5 g-10 g of water earthworms with bright red color and good activity after being domesticated in step (2), put them into the breeding box, and feed 2 g-4 g of quercetin biofloc every day.

2. The method for artificially cultivating water earthworms according to claim 1, characterized in that: Step (1) of cultivating quercetin bioflocs comprises: preparing 80L of simulated aquaculture wastewater, adding inoculum at a volume ratio of 1%-10%, adding 0.15g-0.30g of quercetin flavonoids, adding once every three to ten days, not changing water during the cultivation period, continuously aerating, and regularly adding carbon source to adjust C / N>10:1; after 3-5 days, bioflocs are formed, taking the mixed solution and centrifuging it at 5000r / min for 15min, removing the supernatant, and the precipitate is the quercetin bioflocs.

3. The method for artificially cultivating water earthworms according to claim 1, characterized in that: The inoculating solution is any fish pond water.

4. The method for artificially cultivating water earthworms according to claim 1, characterized in that: The carbon source is glucose, brown sugar or molasses.

5. The method for artificially cultivating water earthworms according to claim 1, characterized in that: The cultivation mode of step (3) is micro-flow water, the cultivation time is 30 days, and the cultivation temperature is natural room temperature.

6. The method for artificially cultivating water earthworms according to claim 1, characterized in that: The specifications of the breeding box are 24.5cm◊17cm◊14cm.

7. The method for artificially cultivating water earthworms according to claim 1, characterized in that: The quercetin biofloc Dyella sp. and Pantoea sp. The abundance of the two probiotics reached 50.34% and 2.35%, and Legionella sp. and Mycobacterium sp. The abundance of the two pathogens was as low as 0.51% and 0.09%; when the quercetin biofloc was used to feed water earthworms, the weight and body length of a single water earthworm increased by 0.69 mg and 5.06 mm, respectively, and the crude protein and crude fat contents of the water earthworms reached 58.80% and 12.98%, respectively.

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