CN111592668A

CN111592668A - Crosslinking modification method of antibacterial gelatin

Info

Publication number: CN111592668A
Application number: CN202010577764.2A
Authority: CN
Inventors: 李俊英; 于宁; 马烽; 耿海滨; 杨鹏飞; 孙海霖
Original assignee: Qilu University of Technology
Current assignee: Qilu University of Technology
Priority date: 2020-06-23
Filing date: 2020-06-23
Publication date: 2020-08-28

Abstract

The invention relates to a cross-linking modification method of antibacterial gelatin, which comprises the following steps: using a natural biological material as a cross-linking agent, and carrying out cross-linking reaction with the antibacterial gelatin, wherein the pH of a solution in the cross-linking reaction is 9-11, the concentration of the cross-linking agent is 0.5-0.8 wt%, and the mass ratio of the cross-linking agent to the antibacterial gelatin is (0.03-0.08): 1, the crosslinking reaction temperature is 42-58 ℃, and the crosslinking reaction time is 2-10 h. The invention selects the natural biological cross-linking agent for cross-linking modification, improves the mechanical property of the antibacterial gelatin, does not generate toxic or side effect on human body, and improves the safety of the gelatin material. Meanwhile, the invention optimizes the crosslinking modification conditions to obtain the crosslinked gelatin with good biocompatibility and excellent mechanical property, thereby being more beneficial to the application in medical antibacterial materials.

Description

Crosslinking modification method of antibacterial gelatin

Technical Field

The invention relates to a cross-linking modification method of antibacterial gelatin, belonging to the technical field of antibacterial materials.

Background

Gelatin is a polypeptide compound obtained by hydrolyzing collagen in connective tissues such as animal skin and bone, and has been regarded as important because of its low immunological activity, biodegradability, and ability to promote cell proliferation and division. The gelatin is a natural renewable resource, has wide source, low price, good film forming property, biocompatibility and biodegradability, and degradation products of the gelatin are easy to absorb without generating inflammatory reaction, thereby being an excellent biomedical material. The pure gelatin has crisp texture, weak forming capability, low ductility and easy deterioration caused by bacterial erosion in a humid environment after being dried, so that different modification methods are needed to improve the mechanical property, the antibacterial property and the like of the gelatin material when in use. Common modification methods include chemical modification, physical blending and composite modification. The chemical modification mainly utilizes the reactivity of hydroxyl, carboxyl, amino and the like of a side chain of the gelatin, and combines a modified compound with the gelatin through grafting or crosslinking reaction to achieve the purpose of improving the performance. The physical blending achieves the effect of making up for the deficiencies by adding other materials with special properties.

In order to improve the mechanical properties of gelatin, chemical crosslinking and blending modification are currently common methods. The blending additives mainly comprise polylactic acid glycolic acid, polyurethane, carboxymethyl chitosan, sodium alginate and the like. Lulixin and the like (food science 31(2010)91-95) prepare the sodium alginate-sodium carboxymethylcellulose-gelatin blended film by a solution blending method, and the tensile strength of the film can reach 60.4MPa, and the breaking elongation of the film can reach 60.1%. The chemical cross-linking agents which are applied more frequently include glutaraldehyde, carbodiimide, iridoid compounds, vegetable tannin, saccharides and the like. The crosslinking modification of gelatin by glutaraldehyde and genipin was studied by Nguyen et al (J.biomedical Science and Engineering 3(2010)1117-1124) and Bigi et al (Biomaterials 23(2002) 4827-4832), respectively, and the Young's modulus of gelatin was significantly improved at an appropriate concentration of the crosslinking agent.

The gelatin has more active groups, chemical crosslinking is easy to carry out, the crosslinking degree is controllable, and the method is a good method for improving the performance of natural materials. There are also numerous patent documents reported on the chemical modification of gelatin, for example: chinese patent document CN105497970A discloses a medical dressing with adhesive function and rapid healing promotion, which is prepared by fully and organically combining carboxymethyl chitosan, sericin and gelatin through chemical crosslinking synthesis. Chinese patent document CN105400214A discloses a method for preparing hemostatic cotton by using hyaluronic acid cross-linked gelatin.

The existing crosslinking modification method has many defects and shortcomings, such as that the gelatin modified by glutaraldehyde can initiate Maillard reaction, and the reaction can generate carcinogenic substance acrylamide; enzymes are also a commonly used cross-linking agent, such as transglutaminase, but enzymes are not available on a large scale because of their high cost. Plant polyphenols such as tannic acid, chlorogenic acid, ferulic acid, rutin caffeic acid, etc. widely exist in plants, have wide sources, are safe and nontoxic, can react with some amino acids in protein such as tyrosine, lysine and cysteine, and can be used as natural protein crosslinking agent. Some scholars at home and abroad use polyphenol substances as protein cross-linking agents to improve the functional properties of proteins, so that the plant polyphenol has great research prospect and application value as the protein cross-linking agents.

Tannic acid is one of the most common phenolic compounds, has good oxidation resistance and strong binding capacity with protein and gelatin. A large number of hydroxyl groups in the tannin molecule may interact with carboxyl groups of gelatin through hydrogen bonds, and may also interact with amino groups in gelatin. Aromatic rings in tannic acid can also interact with the side chains of gelatin in a hydrophobic manner, thereby improving the performance of gelatin.

Usually, cross-linked raw gelatin has no antibacterial property, and most of the cross-linked gelatin with antibacterial property reported at present is mixed or co-cross-linked with chitosan, nano silver or other materials with antibacterial effect. After the antibacterial group is introduced into the gelatin, crosslinking modification is carried out, and the biologically safe crosslinked gelatin with good mechanical property can be obtained. The invention is therefore proposed.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a crosslinking modification method of antibacterial gelatin. As a medical antibacterial material, the invention selects a natural biological cross-linking agent with no toxicity and good biocompatibility to carry out cross-linking modification on the antibacterial gelatin. The mechanical property of the antibacterial gelatin is improved, and meanwhile, the antibacterial gelatin does not produce toxic or side effect on a human body, and the use safety of the gelatin material is improved.

The technical scheme of the invention is as follows:

a cross-linking modification method of antibacterial gelatin comprises the following steps:

the natural biological material is used as a cross-linking agent to carry out cross-linking reaction with the antibacterial gelatin, the pH value of the solution in the cross-linking reaction is 9-11, the concentration of the cross-linking agent is 0.5-0.8 wt%, the cross-linking reaction temperature is 42-58 ℃, and the cross-linking reaction time is 2-10 h.

According to the invention, the crosslinking reaction preferably has a pH of 10.

According to the invention, the crosslinker concentration is preferably from 0.6 to 0.78%.

According to the invention, it is preferred that the crosslinking reaction temperature is from 45 to 55 deg.C

According to the invention, the crosslinking reaction time is preferably from 5 to 6 hours.

According to the present invention, preferably, the natural biomaterial is tannic acid or anthocyanidin.

According to the invention, the mass ratio of the cross-linking agent to the antibacterial gelatin is preferably (0.03-0.08): 1, further preferably (0.06-0.07): 1.

according to the present invention, preferably, the antibacterial gelatin is quaternary ammonium salt cationic gelatin;

preferably, the quaternary ammonium salt cationic gelatin is prepared by the following method:

reacting epoxy quaternary ammonium salt with gelatin, inoculating into gelatin structure through ring-opening reaction of epoxy group, controlling reaction system pH at 6-12, reaction temperature at 45-55 deg.C, and reaction time for 2-6 h.

Preferably, the mass ratio of the epoxy quaternary ammonium salt to the gelatin is (0.1-1.2): 1, further preferably 0.78: 1.

preferably, the gelatin is type A gelatin with a molecular weight range of 50-200KD, and the mass concentration of the gelatin is 5% -15%.

Preferably, the epoxy quaternary ammonium salt is prepared by the following method:

the epoxy chloroalkane is used as a quaternizing agent to react with long-chain alkyl dimethyl tertiary amine in an organic solvent, the reaction temperature is 35-50 ℃, and the reaction time is 4-6 h.

Preferably, the alkyl dimethyl tertiary amine is an alkyl dimethyl tertiary amine with a carbon chain length of 12 to 18, more preferably 14 to 18, and even more preferably 16 or 18.

Preferably, the epichlorohydrin is epichlorohydrin.

The structural formula is as follows:

according to the present invention, preferably, the organic solvent is methanol or ethanol.

According to the invention, the reaction temperature is preferably between 35 and 45 ℃ and most preferably 40 ℃.

According to the invention, the reaction time is preferably 4 to 6h, most preferably 6 h.

The mechanical property of the gelatin depends on the property of the gelatin body, so that the antibacterial gelatin is used for preparing materials and needs to be cross-linked or blended for modification to improve the mechanical property. More active groups exist in the gelatin, chemical crosslinking is easy to carry out, and the crosslinking degree is controllable. As a medical antibacterial material, a cross-linking agent which is non-toxic and good in biocompatibility is selected as a key for modification, the plant tannin compound is preferably selected as the cross-linking agent, the proper pH value of the antibacterial gelatin and the cross-linking agent in the reaction is researched, and the influence of the concentration, the reaction temperature and the time of the cross-linking agent on the cross-linking degree is researched, so that the relationship between the key reaction condition and the cross-linking degree is established, and the controllable cross-linking is realized.

According to the present invention, a preferred embodiment comprises the steps of:

(1) dissolving gelatin solid particles in a buffer solution with the pH value of 10, preparing a gelatin solution with the mass fraction of 10 wt%, swelling for one hour at normal temperature, heating in a water bath at 50 ℃ in a water bath kettle, stirring with magnetic force, and dissolving for 1 hour;

(2) adding epoxy quaternary ammonium salt into 10 wt% gelatin solution, heating in water bath to 50 ℃, magnetically stirring, and reacting for 5h to obtain the antibacterial gelatin containing quaternary ammonium cations, wherein the mass ratio of the epoxy quaternary ammonium salt to the gelatin is 0.78: 1, the gelatin is type A gelatin with the molecular weight range of 50-200 KD;

(3) adding a tannic acid crosslinking agent into the antibacterial gelatin solution obtained in the step (2), heating in a water bath to 50 ℃, and reacting for 2h under magnetic stirring, wherein the mass ratio of the crosslinking agent to the antibacterial gelatin is (0.06-0.07): 1, the concentration of the cross-linking agent is 0.6-0.78%;

(4) and after the reaction is finished, taking out a part of the mixture to measure the content of the primary amino group, calculating the crosslinking degree, freeze-drying the rest of the mixture for 12 hours at the temperature of-59 ℃ by using a freeze dryer, continuously washing the dried mixture by using a methanol solution until no chloride ion is detected by using silver nitrate, and finally washing the dried mixture for 2 to 3 times by using distilled water to obtain the crosslinked antibacterial gelatin.

The modification route of the invention is as follows:

the invention has the following beneficial effects:

the invention selects the natural biological cross-linking agent for cross-linking modification, improves the mechanical property of the antibacterial gelatin, does not generate toxic or side effect on human body, and improves the safety of the gelatin material. Meanwhile, the invention optimizes the crosslinking modification conditions to obtain the crosslinked gelatin with good biocompatibility and excellent mechanical property, thereby being more beneficial to the application in medical antibacterial materials.

Drawings

FIG. 1 is an infrared spectrum of a cross-linked modified antibacterial gelatin prepared in example 1 of the present invention.

Fig. 2 is an XRD pattern of the cross-linked modified antibacterial gelatin prepared in example 1 of the present invention.

FIG. 3 is a diagram of the inhibition zone of the cross-linked modified antibacterial gelatin prepared in example 1 of the present invention on Escherichia coli.

FIG. 4 is a diagram of the inhibition zone of the cross-linked modified antibacterial gelatin prepared in example 4 of the present invention on Escherichia coli.

FIG. 5 is a cell micrograph of HeLa cells proliferating for 12 hours in the cytotoxicity test of the cross-linked modified antibacterial gelatin prepared in example 4 of the present invention.

Detailed Description

The present invention is further illustrated by, but is not limited to, the following specific examples.

The gelatin described in the examples is type A gelatin with a molecular weight range of 50-200 kD. In the examples, "%" is mass% unless otherwise specified.

The epoxy quaternary ammonium salt used in the examples was prepared as follows:

(1) weighing 8.37g of epoxy chloropropane, 5mL of methanol and adding the epoxy chloropropane into a constant pressure funnel, weighing 22.78g of N, N-dimethyl octadecyl amine, 5mL of methanol and adding the methanol into a three-neck flask, putting a stirring magneton, installing a thermometer, replacing 6-7 times with nitrogen, and ensuring that the whole reaction device is in an N state₂In the environment.

(2) Putting the whole set of reaction device into a water bath, starting magnetic stirring, slowly heating to 38 ℃, opening a constant-pressure funnel to dropwise add epichlorohydrin and methanol into a three-necked bottle, and keeping reaction for 6 hours after dropwise addition.

(3) The reaction product was poured into a 150ml single-neck flask, and the solvent methanol was removed by rotary evaporation for 2h, followed by washing with petroleum ether several times to remove unreacted raw materials, during which the washing solution was centrifuged.

(4) And (3) placing the washed sample in a vacuum drying oven, and drying at 40 ℃ for 24h to obtain 25g of epoxypropyl dimethyl octadecyl ammonium chloride.

The quaternary ammonium salt cationic gelatin described in the examples was prepared as follows:

reacting the epoxy quaternary ammonium salt with gelatin, controlling the pH value of a reaction system to be 8-12, controlling the reaction temperature to be 40-60 ℃, and controlling the reaction time to be 2-10 h.

Example 1

(1) dissolving 2.5g of gelatin in 22.5g of phosphate buffer solution with pH of 10 to prepare gelatin solution with the mass fraction of 10 wt%, swelling for one hour at normal temperature, heating in water bath at 50 ℃, and dissolving for 1 hour by magnetic stirring.

(2) To a prepared 10 wt% gelatin solution was added 0.486g N, N-dimethylepoxypropyloctadecyl ammonium chloride so that the molar ratio of primary amino groups in the gelatin to epoxy groups in the quaternary ammonium salt was 1: 0.5, the reaction was carried out for 5h while maintaining the temperature at 50 ℃ under magnetic stirring.

(3) After the quaternization reaction is finished, 1mL of prepared 200mg/mL crosslinking agent tannic acid aqueous solution is added into a reaction system, so that the mass ratio of the crosslinking agent to the antibacterial gelatin is 0.07: 1, the concentration of the cross-linking agent is 0.76 percent, the temperature is kept at 50 ℃, and the reaction is carried out for 2 hours under the magnetic stirring.

(4) And after the reaction is finished, putting the mixture into a freeze dryer for freeze drying for 12 hours at the temperature of-59 ℃, and circularly eluting the mixture by using methanol under the drive of a constant flow pump after freeze drying until no chloride ions are detected by silver nitrate. And finally washing with distilled water for more than 3 times.

(5) And freeze-drying the eluted modified gelatin again to obtain the cross-linked modified antibacterial gelatin. Preparing the product into 10% aqueous solution, adding 1g of glycerol into 20g of the aqueous solution, uniformly stirring, pouring the mixture into a polytetrafluoroethylene mold, drying the polytetrafluoroethylene mold for 24 hours at normal temperature to form a film, and performing mechanical property test.

The infrared spectrum of the cross-linked modified antibacterial gelatin obtained in this example was measured, as shown in fig. 1.

The XRD pattern of the cross-linked modified antibacterial gelatin obtained in this example was measured as shown in fig. 2.

Example 2

(1) dissolving 2.5g of gelatin in 22.5g of buffer solution with pH of 9 to prepare gelatin solution with the mass fraction of 10 wt%, swelling for one hour at normal temperature, heating in a water bath at 50 ℃ in a water bath kettle, and dissolving for 1 hour along with magnetic stirring.

(2) To a prepared 10 wt% gelatin solution was added 0.972g of N, N dimethyl glycidyl octadecyl ammonium chloride, so that the molar ratio of primary amino groups in the gelatin to epoxy groups in the quaternary ammonium salt was 1: 1, reacting for 5h under magnetic stirring and keeping the temperature at 45 ℃.

(3) After the quaternization reaction is finished, 1mL of prepared cross-linking agent tannic acid aqueous solution of 200mg/mL is added into a reaction system, so that the mass ratio of the cross-linking agent to the antibacterial gelatin is 0.06: 1, the concentration of the cross-linking agent is 0.74%, the temperature is kept at 45 ℃ and the reaction is carried out for 2h under magnetic stirring.

Example 3

(1) dissolving 2.5g of gelatin in 22.5g of phosphate buffer solution with pH of 11 to prepare gelatin solution with the mass fraction of 10 wt%, swelling for one hour at normal temperature, heating in water bath at 50 ℃, and dissolving for 1 hour by magnetic stirring.

(2) 1.458g of N, N-dimethylepoxypropyloctadecyl ammonium chloride was added to the prepared 10 wt% gelatin solution so that the molar ratio of primary amino groups in the gelatin to epoxy groups in the quaternary ammonium salt was 1: 1.5, and keeping the temperature of 55 ℃ for reaction for 5 hours under magnetic stirring.

(3) After the quaternization reaction is finished, 1mL of prepared 200mg/mL crosslinking agent tannic acid aqueous solution is added into a reaction system, so that the mass ratio of the crosslinking agent to the antibacterial gelatin is 0.05: 1, the concentration of the cross-linking agent is 0.73 percent, the temperature is kept at 55 ℃, and the reaction is carried out for 2 hours under the magnetic stirring.

Example 4

(2) 1.944g of N, N-dimethylepoxypropyloctadecyl ammonium chloride was added to the prepared 10 wt% gelatin solution so that the molar ratio of primary amino groups in the gelatin to epoxy groups in the quaternary ammonium salt was 1: 2, reacting for 5 hours under magnetic stirring and keeping the temperature at 50 ℃.

(3) After the quaternization reaction is finished, 1mL of prepared cross-linking agent tannic acid aqueous solution of 200mg/mL is added into a reaction system, so that the mass ratio of the cross-linking agent to the antibacterial gelatin is 0.045: 1, the concentration of the cross-linking agent is 0.72 percent, the temperature is continuously kept at 50 ℃, and the reaction is carried out for 2 hours under the magnetic stirring.

Example 5

(2) 2.916g of N, N-dimethylepoxypropyloctadecyl ammonium chloride was added to the prepared 10 wt% gelatin solution so that the molar ratio of primary amino groups in the gelatin to epoxy groups in the quaternary ammonium salt was 1: 3, reacting for 5 hours under the magnetic stirring and keeping the temperature at 50 ℃.

(3) After the quaternization reaction is finished, 1mL of prepared 200mg/mL crosslinking agent tannic acid aqueous solution is added into a reaction system, so that the mass ratio of the crosslinking agent to the antibacterial gelatin is 0.037: 1, the concentration of the cross-linking agent is 0.69%, the temperature is kept at 50 ℃, and the reaction is carried out for 2 hours under the magnetic stirring.

Comparative example 1

As described in example 4, except that:

the crosslinking reaction pH was controlled to 12.

The conversion rate of the reaction is reduced because the collagen polypeptide is hydrolyzed.

Comparative example 2

As described in example 4, except that:

the crosslinking reaction pH was controlled to 8.

The collagen polypeptide is near the isoelectric point at the moment, and certain influence is exerted on the aggregation of the collagen polypeptide, so that the conversion rate is not high.

Comparative example 3

As described in example 4, except that:

the crosslinking reaction temperature was controlled at 60 ℃.

Excessive temperatures cause excessive hydrolysis of the collagen polypeptide.

Comparative example 4

As described in example 4, except that:

the crosslinking reaction temperature was controlled at 40 ℃.

Lower temperatures are detrimental to the quaternization reaction.

Comparative example 5

As described in example 4, except that:

the mass ratio of the cross-linking agent to the antibacterial gelatin is 0.1: 1, controlling the concentration of the cross-linking agent to be 0.9 percent.

The concentration of the cross-linking agent is larger, and more active groups of the cross-linking agent remain, so that the cross-linking structure is not compact, and the tensile strength of a formed film is reduced.

Comparative example 6

As described in example 4, except that:

the mass ratio of the cross-linking agent to the antibacterial gelatin is 0.02: 1, controlling the concentration of the cross-linking agent to be 0.3 percent.

The concentration of the cross-linking agent is too low to allow complete reaction of the primary amine groups in the gelatin matrix and incomplete cross-linking.

Test example 1

The crosslinking degree and mechanical properties of the crosslinked and modified antibacterial gelatins prepared in examples 1 to 5 and comparative examples 1 to 6 were tested. And detecting the content of free amino groups by a derivative fluorescence method to obtain the crosslinking degree.

Degree of crosslinking ═ (amino content before crosslinking-amino content after crosslinking)/amino content before crosslinking.

The mechanical property test of the prepared film is completed on a WDW-02 type microcomputer control electronic universal tester. The test results are shown in table 1.

TABLE 1 degree of crosslinking and mechanical Properties

As is clear from Table 1, the crosslinking-modified antibacterial gelatins of examples 1 to 5 of the present invention have a significantly higher degree of crosslinking than those of comparative examples 1 to 6 and are superior in tensile strength.

Test example 2

The final products of examples 1-5 and comparative examples 1-6 were tested for antimicrobial properties. Staphylococci (gram positive bacteria) and escherichia coli (gram negative bacteria) are respectively selected as antibacterial experimental strains, the minimum inhibitory concentration of the cross-linked and modified antibacterial gelatin obtained in examples 1-5 and comparative examples 1-6 is determined by using a solid plate and a liquid culture method, and the antibacterial property of the cross-linked and modified product is evaluated. The minimum inhibitory concentrations are shown in table 2. The inhibition zone of the cross-linked modified antibacterial gelatin of example 1 is shown in fig. 3, and the inhibition zone of the cross-linked modified antibacterial gelatin of example 4 is shown in fig. 4.

TABLE 2 minimum inhibitory concentrations

As can be seen from table 2, fig. 3 and 4, the cross-linked modified antibacterial gelatin of the present invention has excellent antibacterial properties.

Test example 3

HeLa cells are selected as experimental objects, and the cytotoxicity of the product is evaluated through a cell proliferation experiment. The results are shown in Table 3.

The cross-linked modified antibacterial gelatin obtained in example 4 was tested for cytotoxicity, and a cell micrograph of HeLa cells proliferated for 12 hours, as shown in FIG. 5.

TABLE 3 OD value for cytotoxicity experiments

As can be seen from Table 3, the OD value of the present invention is very different from that of the blank sample, and even some sample values are larger than that of the blank sample, which indicates that the toxicity of the cross-linked modified antibacterial gelatin of the present invention is very low.

Claims

1. A cross-linking modification method of antibacterial gelatin comprises the following steps:

the natural biological material is used as a cross-linking agent to carry out cross-linking reaction with the antibacterial gelatin, the pH of the solution in the cross-linking reaction is 9-11, the concentration of the cross-linking agent is 0.5-0.8 wt%, the cross-linking reaction temperature is 42-58 ℃, and the cross-linking reaction time is 2-10 h.

2. The method for modifying crosslinking of antibacterial gelatin according to claim 1, wherein the pH of the crosslinking reaction is 10.

3. The method for crosslinking and modifying antibacterial gelatin according to claim 1, wherein the concentration of the crosslinking agent is 0.6 to 0.78%.

4. The method for cross-linking and modifying antibacterial gelatin according to claim 1, wherein the cross-linking reaction temperature is 45 to 55 ℃;

preferably, the crosslinking reaction time is 5 to 6 hours.

5. The method for cross-linking and modifying antibacterial gelatin according to claim 1, wherein the natural biomaterial is tannic acid or anthocyanidin.

6. The method for crosslinking and modifying antibacterial gelatin according to claim 1, wherein the mass ratio of the crosslinking agent to the antibacterial gelatin is (0.03-0.08): 1.

7. the method for cross-linking and modifying antibacterial gelatin according to claim 1, wherein the antibacterial gelatin is quaternary ammonium cationic gelatin;

8. The method for crosslinking and modifying antibacterial gelatin according to claim 7, wherein the mass ratio of the epoxy quaternary ammonium salt to the gelatin is (0.1-1.2): 1.

preferably, the gelatin is type A gelatin with a molecular weight range of 50-200KD, and the mass concentration of the gelatin is 5% -15%;

using epoxy chloroalkane as a quaternizing agent to react with long-chain alkyl dimethyl tertiary amine in an organic solvent, wherein the reaction temperature is 35-50 ℃, and the reaction time is 4-6 h; the alkyl dimethyl tertiary amine is alkyl dimethyl tertiary amine with the carbon chain length of 12-18, and the epoxy chloroalkane is epichlorohydrin.

9. The method for cross-linking and modifying antibacterial gelatin according to claim 8, wherein the organic solvent is methanol, ethanol;

preferably, the reaction temperature is 35-45 ℃.

10. The method for cross-linking and modifying antibacterial gelatin according to claim 1, comprising the steps of: