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Marine halomonas for producing low-temperature beta-galactosidase and application thereof

A technology of galactosidase and Halomonas, applied in the preparation of low-lactose milk, in the field of production of low-temperature β-galactosidase, can solve the problem of low activity

Inactive Publication Date: 2011-09-07
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, only the β-galactosidase extracted from Kluyveromyces lactis (Kluyveromyces lactis) transformed by the Dutch DSM Group can be used in the food industry to meet the GRAS level (generally regard assafe) of the US FDA. The optimum temperature is around 40°C, but its activity is very low at low temperatures. Therefore, there is currently no real low-temperature β-galactosidase used in the food industry, and it is urgent to discover new low-temperature β-galactosidases.

Method used

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  • Marine halomonas for producing low-temperature beta-galactosidase and application thereof
  • Marine halomonas for producing low-temperature beta-galactosidase and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0017] Embodiment 1: the cultivation of bacterial strain P6009-1

[0018] The culture medium of bacterial strain P6009-1 is: peptone 5g, yeast extract 1g, iron phosphate 0.1g, artificial seawater (or old seawater) 1000ml, pH 7.6. Artificial seawater formula: NaCl 25.0g, NaCl 2 SO 4 4.0g, KCl 0.7g, NaHCO 3 0.20g, KBr 0.10g, H 3 BO 3 0.03g, NaF 0.003g, 53mL 1.0mol / LMgCl 2 solution, 10mL 1.0mol / l CaCl 2 Solution, 0.90ml 0.1mol / L SrCl 2 Solution, distilled water 1000ml, pH adjusted to 6.5. The strain P6009-1 was routinely inoculated in the culture medium and cultured at 28°C.

Embodiment 2

[0019] Embodiment 2: the crude enzyme liquid preparation of bacterial strain P6009-1

[0020] Lactose-induced culture was used to inoculate the P6009-1 strain in Zobell 2216E liquid medium (peptone 5g, yeast extract 1g, iron phosphate 0.1g, NaCl 60g, 1000ml distilled water, pH7.6), 28°C, 130r / min shaker culture After 2 days, the strain was activated, and then transferred to a fermentation liquid medium (lactose 20g, peptone 5g, yeast extract 1g, iron phosphate 0.1g) for culture, 28°C, 130r / min shaker culture for 3 days, and the fermentation broth was centrifuged at 5000r / min. min×15min×4°C, discard the supernatant. Resuspend the pellet with 0.05mol / L pH7.2 phosphate buffer, transfer to a centrifuge tube, wash the cells twice with phosphate buffer, and centrifuge at 5000r / min×15min×4°C, discard the supernatant. The bacteria were resuspended in 0.05mol / L pH7.2 phosphate buffer. Cells were disrupted with an ultrasonic pulverizer in an ice bath: sonicate for 1 s each time, and s...

Embodiment 3

[0021] Embodiment 3: the influence of metal ion on crude enzyme activity

[0022] Take 10 μL of crude enzyme solution, add 87 μL of 20g / L lactose, and then add 3 μL of 0.2M MnCl 2 , FeCl 2 , FeCl 3 , MgCl 2 , ZnCl 2 , LiCl, CoCl 2 , BaCl 2 , SrCl 2 , CuCl 2 , NaCl, CaCl 2 The solution was incubated at different temperatures, and whether the lactose was decomposed into galactose and glucose was detected by thin-layer chromatography. The specific operation was as follows: take 5 μL of the reaction solution and spot it on a silica gel thin-layer plate with a capillary tube, and use n-butanol: Pyridine: water (5:4:1) was used as the developing agent for development. When the front of the solvent reached about 1 cm away from the edge of the thin-layer plate, take out the thin-layer plate, evaporate the solvent, and spray 20% sulfuric acid-methanol solution, 115 ° C Bake color. With 0.2% lactose, 0.2% galactose, and 0.2% glucose as the standard reference substance, observe...

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Abstract

The invention relates to the technical field of marine microorganisms. Since milk is generally conveyed and preserved at a low temperature, the capability of decomposing lactose at the low temperature is one of conditions which are necessary for industrially applying the beta-galactosidase. A marine halomonas strain P6006-1 provided by the invention is separated from sea water of an East China Sea region through culturing and has been preserved at the China Center for Type Culture Collection (CCTCC for short) in Wuhan city on January 7th, 2011, with a collection number of CCTCC No. M2011001. The invention also provides a method for producing the low-temperature beta-galactosidase by using the marine halomonas. The low-temperature beta-galactosidase produced by using the marine halomonas can be used for decomposing the lactose in cow milk; and the most proper reaction temperature is 30 DEG C.

Description

technical field [0001] The invention relates to the technical field of marine microorganisms, in particular to a strain of Halomonas marine strain P6009-1, a method for producing low-temperature β-galactosidase using the Halomonas marine bacteria, and its use in preparing low-lactose milk Applications. Background technique [0002] The full name of β-galactosidase (E.C.3.2.1.23) is β-D-galactoside galactohydrolase, which is widely distributed in animals, plants and microorganisms. The β-galactosidase in animals mainly exists on the surface of the small intestine , whose role is to decompose lactose into glucose and galactose for use by organisms. At the same time, it can also transfer galactose to lactose through transglycosylation to generate oligosaccharides. [0003] Lactose is a disaccharide formed by connecting D-galactose and D-glucose through β-1,4 glycosidic bonds. It mainly exists in mammalian milk, with about 7% in human milk and about 5% in cow milk . Milk is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/38A23C9/12C12R1/01
Inventor 刘小宇卢小玲焦炳华王国祥高云胡波刘军华
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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