A kind of Lactococcus lactis hks2 with lactic acid activity and its isolation and screening method and application

A technology of Lactococcus lactis and activity, applied in the field of strains, can solve the problems of no geographical population patent of Lactococcus lactis in Hainan Island, unstable purification effect, and few Lactococcus lactis, so as to improve the body's immunity and inhibit the effect significantly , the effect of strong acid production capacity

Active Publication Date: 2020-11-06
HAINAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The strains of Bacillus licheniformis in different growth environments have a good effect on the purification of aquaculture water in the local environment, but they often show poor purification effect and unstable purification effect on the remote environment.
At present, domestic patents on microecological preparations of lactic acid bacteria mainly focus on fermented strains for fertilizer production and food, and there are few patents on Lactococcus lactis added to breeding feed, let alone the geographical population of Lactococcus lactis in Hainan Island. patent

Method used

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  • A kind of Lactococcus lactis hks2 with lactic acid activity and its isolation and screening method and application
  • A kind of Lactococcus lactis hks2 with lactic acid activity and its isolation and screening method and application
  • A kind of Lactococcus lactis hks2 with lactic acid activity and its isolation and screening method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Example 1 Isolation and screening of Lactococcus lactis HKS2.

[0054] The above-mentioned isolation and screening method of Lactococcus lactis HKS2 of the present invention mainly includes the following steps:

[0055] (1) Sample collection

[0056] Seawater and live shrimp intestinal contents were collected from the waters of Wenchang shrimp farm in Hainan and Hainan tropical live Litopenaeus vannamei. A small amount of the collected seawater samples were taken for later use; the live shrimp intestines were put into sterilized physiological saline and cut Crush the shrimp intestines, shake and mix into a shrimp intestinal mixture; take a small amount of the mixture for use, and the entire operation process is performed on an ultra-clean bench;

[0057] (2) Expanded cultivation of sample strains

[0058] Inoculate the seawater sample and shrimp intestinal mixture described in step (1) into MRS liquid medium, respectively, and shake culture in a shaker at 30°C and 180r / min for 2...

Embodiment 2

[0065] Example 2 Molecular identification of Lactococcus lactis HKS2.

[0066] In order to identify the strains, first use scanning electron microscope to observe the cell morphology and some physiological and biochemical reactions of the tested strains. The scanning electron microscope images are as follows figure 2 As shown, the strain can be preliminarily judged to be Lactococcus lactis.

[0067] Use bacterial universal primers for PCR identification of the selected strains: extract template DNA according to the operating instructions of the bacterial DNA extraction kit. Use the upstream primer 5'-AGAGTTTGATCCTGGCTCA-3' of 16S rRNA conservative sequence, see SEQ ID No. 2 and the downstream primer 5'-GGTTACCTTGTTACGACTT-3, see SEQ ID No. 3, to perform PCR amplification on the 16srRNA gene fragment of Lactococcus lactis HKS2 It was amplified, cloned and sequenced. After sequencing, 1409bp fragments were obtained. The accession number in GenBank was MF037867. After sequencing, the...

Embodiment 3

[0068] Example 3 Test of acid production ability of Lactococcus lactis HKS2.

[0069] Acid production capacity test: first use an inoculating loop to pick out the target strains initially screened, inoculate the strains in MRS liquid medium aseptically, and then incubate them in a 30°C incubator with shaking at 180 rpm for 24 hours for use; prepare MRS solid medium ( Add 1% light CaCO 3 ), pour 20 mL of culture medium into each petri dish, and use 10 8 Spread 100μL of cfu / mL bacterial solution on the solid culture medium in the petri dish, incubate it in a 30℃ incubator for 48h, measure the diameter of the calcareous circle with a vernier caliper, set 3 replicates for each sample, and take the average value. It is finally determined that the diameter of the calcium-dissolving circle of Lactococcus lactis HKS2 is 5.00~7.00mm, such as figure 1 Shown by figure 1 The diameter of the calcium-dissolving circle shows that Lactococcus lactis HKS2 has a strong ability to produce acid.

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Abstract

The invention relates to a Lactococcus lactis HKS2 with lactic acid activity and its separation and screening method and application. The bacterial strain of the present invention is classified and named: (Lactococus lactis) HKS2, and has been preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M2017296, and the preservation date is May 31, 2017. The strain was isolated from a mixture of tropical cultured seawater in Hainan and the gastrointestinal tract of Litopenaeus vannamei. The Lactococcus lactis HKS2 of the present invention has no hemolytic activity, has better hydrophobic activity, higher self-aggregation rate, stronger artificial gastrointestinal fluid tolerance, stronger bile salt tolerance, better antibacterial ability, The Lactococcus lactis HKS2 of the present invention is used as a feed additive to feed Litopenaeus vannamei. It is verified by breeding experiments that it can better improve the immunity of the body and promote the growth of prawns, and the effect is stable. It is a strain with good application prospects.

Description

Technical field [0001] The invention belongs to the field of strains, and relates to a Lactococcus lactis. More specifically, the present invention relates to a Hainan native Lactococcus lactis HKS2 with lactic acid activity and a separation and screening method and application thereof. Background technique [0002] In order to pursue high-efficiency and rapid breeding benefits, continuously increase breeding density and expand breeding scale, resulting in a decline in the growth rate of aquatic animals, an increase in feed coefficient, frequent occurrence of common diseases such as vibriosis, and the abuse of antibiotics and other chemical fishery drugs. This not only increases the cost of breeding, but also poses a huge threat to the quality and safety of aquatic products and human health. Therefore, looking for an efficient, healthy and safe breeding method has become a current research hotspot in aquaculture technology. [0003] The application of microecological preparations ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N1/02A23K10/18A23K50/80C12R1/46
CPCA23K10/18A23K50/80C12N1/02C12N1/20C12N1/205C12R2001/46
Inventor 谢珍玉文金顺龙昊章翔
Owner HAINAN UNIV
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