# US 11,999,769 B2 · Method for isolating and purifying bacteriocin against drug-resistant Staphylococcus aureus

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- Number: US 11,999,769 B2
- Document ID: US11999769B2
- Status: Granted
- Kind: B2
- Application number: 18/054,546
- Filed: 2022-11-11
- Published: 2023-11-02
- Granted: 2024-06-04
- Earliest claimed: 2022-04-30
- Assignees: Zhejiang Gongshang University
- Applicants: Zhejiang Gongshang University
- Inventors: Qing Gu; Mengdi Dai; Qingqing Zhou; Ping Li; Danli Wu
- Web page: https://patentgrep.com/patent/US11999769B2

## CPC

- C07K 14/335: from Lactobacillus (G)
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- Y02A 40/90: in food processing or handling, e.g. food conservation
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present disclosure discloses a method for isolating and purifying Lactobacillus rhamnosus ZFM216 bacteriocin, the method including the following steps: preparing L. rhamnosus ZFM216 fermentation supernatant; treating the fermentation supernatant with macroporous adsorption resin method to obtain a crude protein extract; isolating the crude protein extract by gel chromatography to obtain a preliminary purified protein solution; and then performing purification by reversed-phase high performance liquid chromatography (HPLC) to obtain an L. rhamnosus ZFM216 bacteriocin solution. The present disclosure further provides use of the L. rhamnosus bacteriocin in anti-Staphylococcus aureus.

## Claims

### Claim 1

A method for isolating and purifying Lactobacillus rhamnosus ZFM216 bacteriocin, comprising the following steps:
1) preparing L. rhamnosus ZFM216 fermentation supernatant:
inoculating MRS liquid medium with L. rhamnosus ZFM216 for fermentation, and
centrifuging the fermentation broth to obtain the fermentation supernatant;
the preservation number of L. rhamnosus ZFM216 being CCTCC M 2020325;
2) treating the fermentation supernatant with macroporous adsorption resin to obtain a crude protein extract;
3) isolating the crude protein extract by gel chromatography to obtain a preliminary purified protein solution; and then performing purification by reversed-phase high performance liquid chromatography (RP-HPLC) to obtain an L. rhamnosus ZFM216 bacteriocin solution;
wherein in step 1), MRS liquid medium, pH 6.4, is inoculated with L. rhamnosus ZFM216 cultured to the logarithmic growth phase at an inoculum concentration of 2% v/v for performing stationary culture at 37° C. for 21 h; and the obtained fermentation broth is centrifuged at 8000 rpm, 4° C., for 30 min to obtain the fermentation supernatant;
in step 2), 1 L of the fermentation supernatant is allowed to flow through the macroporous adsorption resin, rinsed with 2 L of ultrapure water at a flow rate of 2 mL/min, and then eluted with 500 mL of 20% ethanol at a flow rate of 2 mL/min, and the eluate is a crude protein extract; and
during the isolation by gel chromatography in step 3), the crude protein extract is subjected to gel chromatography and eluted with ultrapure water as the eluent at a flow rate of 1 mL/min; and the eluate corresponding to the first absorption peak under the absorbance of 215 nm/280 nm is collected to obtain a preliminary purified protein solution; and then the preliminary purified protein solution is filtered through a 0.22 m membrane, and precisely purified by preparative C18 reversed-phase high performance liquid chromatography (RP-HPLC) with a chromatographic column at an injection volume of 5 mL to obtain an L. rhamnosus ZFM216 bacteriocin solution.

### Claim 2 (depends on 1)

The method according to claim 1, wherein 0.05% trifluoroacetic acid (TFA) by volume of ultrapure water is added to the ultrapure water as mobile phase A, and 0.05% TFA by volume of acetonitrile is added to the acetonitrile as mobile phase B; the gradient elution procedure is as follows:
Time
Flow rate
Mobile
Mobile
(min)
(mL/min)
phase A
phase B
0
4
95
5
15
4
95
5
16
4
50
50
26
4
5
95
30
4
5
95
31
4
95
5
45
4
95
5
the eluate corresponding to the 3rd absorption peak under the absorbance of 280 nm is collected, and named as peak 3 eluate, the peak 3 eluate contains L. rhamnosus ZFM216 bacteriocin.

### Claim 3 (depends on 2)

The method according to claim 2, wherein 5 mL of peak 3 eluate is freeze-dried to obtain L. rhamnosus ZFM216 bacteriocin, which is reconstituted with 1 ml of ultrapure water to obtain a peak 3 reconstituted solution, that is, the peak 3 reconstituted solution is an L. rhamnosus ZFM216 bacteriocin solution.