WO2021236024A1

WO2021236024A1 - Preparation of biocellulose wound dressing comprising honey as an antimicrobial active ingredient

Info

Publication number: WO2021236024A1
Application number: PCT/TH2021/000012
Authority: WO
Inventors: Narin KAABBUATHONG; Chaiyapruk KATEPETCH; Supachai REAKASAME; Wantanee CHANVANITCHAGOOL
Original assignee: Ptt Public Company Limited
Priority date: 2020-05-21
Filing date: 2021-04-19
Publication date: 2021-11-25

Abstract

Preparation of Biocellulose wound dressing comprising honey as an antimicrobial active ingredient consists of steps of purifying Biocellulose wound dressing by boiling Biocellulose in sodium hydroxide solution in an autoclave and adjusting for a neutral pH by re-boiling the obtained Biocellulose in distilled water or deionized water in an autoclave, and then soaking the obtained Biocellulose in organic acid. Subsequently, the Biocellulose is soaked in honey for a required period. Subsequent, excess honey is wiped off, and the Biocellulose is left at room temperature and then sterilized by gamma radiation.

Description

PREPARATION OF BIOCELLULOSE WOUND DRESSING COMPRISING HONEY AS AN ANTIMICROBIAL ACTIVE INGREDIENT

Field of the Invention

Chemical science, materials science, and biology related to the preparation of Biocellulose wound dressing comprising honey as an antimicrobial active ingredient.

Background of the Invention

Biocellulose (or bacterial cellulose) is a pure cellulose nanofiber produced by bacterial cultured in appropriate culture media. Biocellulose has high purity, chemical resistance, large contact surface area, and high water-absorption, which shows that Biocellulose possesses a “hydrophilic” property, thereby enhancing Biocellulose water holding capacity 100 times than its dry weight and having its structural arrangement organized. In other words, fibers within Biocellulose have a tightly packed weaving quality, contributing to its strength and high elasticity.

Biocellulose is applied in food industry, paper industry as well as medical industry because these bacterial cellulose fibers can expedite the wound healing process of the body with no adherence to wounds, which thereby reduces pain while applying or changing wound dressings.

Besides, many small pores of Biocellulose help adsorb liquid from wounds (source: Academic Service Office, Burapha University, www.uniserv.buu.ac.th/fomm2/topic.asp?TOPIC_ID₌ 6043), help stimulate wound healing mechanism of the body without disturbance to re-epithelialization and reduce the chance of scar formation by keeping the skin moisturized. Biocellulose, therefore, possesses appropriate properties for wound dressing as shown in description of the following inventions.

The Thai patent application number 1201002468 discloses the preparation of wound dressing that comprises Biocellulose fibers and silver nanoparticles at the silver nanoparticle content of 1,000 micrograms per 100 square centimeters of wound dressing area. Biocellulose fibers prepared by this invention are derived from bacteria chosen from Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa or methicillin- resistant Staphylococcus aureus (MRSA). The Thai patent application number 1401005361 discloses composite Biocellulose fibers produced from natural ingredients together with at least one ingredient chosen from a group of monomolecular or bimolecular carbohydrate source, nitrogen source, humidifying agent and Acetobacter xylinum bacteria, wherein the said Biocellulose product is formed into a thin sheet or molded into shape corresponding with body organs, namely foot, hand, leg or arm.

The Thai patent application number 1601001745 discloses a production process for a composite material, consisting of cellulose fibers from microbes and textile materials, that comprises steps (a) preparation of microbial concentrate, (b) textile material preparation, and (c) production of the composite material that consists of cellulose from microbes and textile materials, wherein the said microbes are chosen from those in the groups of Acetobacter, Achromobacter, Aerobacter, Agrobacterium, Alcaligenes, Pseudomonas, Rhizobium, Sarcina, Zoogloea, Enterobacter amnigenus, Gluconacetobacter hansenii, Gluconacetobacter xylinum and Rhodococcus sp.

The Thai patent application number 1603002296 discloses a preparation method for Biocellulose fibers that contain pomegranate peel extract. Biocellulose fibers according to this invention are produced by Acetobacter xylinum bacteria in TISTR 109 strain cultured on the culture medium that comprises glucose yeast extract at a temperature of 30 degrees Celsius for 3-

5 days, and then Biocellulose fibers floating on the culture medium surface is soaked in sodium hydroxide. Subsequently, the obtained Biocellulose sheet is dried using the freeze-dried method and soaked again in pomegranate peel extract, and then re-dried using hot air oven method to yield a dry light-yellow Biocellulose sheet with tough and durable properties.

The Chinese patent number CN105169464B discloses wound dressing that comprises natural honey and nystatin antifungal (C47H75NO17) at concentration of 10-50 milligrams per liter.

The international patent application, publication number W02020060165A1 discloses a culture medium that comprises cellulase, magnesium sulfate, calcium chloride, sodium acetate, acetic acid, and honey or glucose or fructose for Komagataeibacter rhaeticus bacteria in the KOSS 15 strain for the purpose of Biocellulose fibers production.

The United States patent application number US 20170260297 A 1 discloses Biocellulose film prepared by Gluconacetobacter xylinus microorganisms to use as implant soft tissue. The film obtained from this invention contains Biocellulose in a range of 2-15 milligrams per square centimeter.

The United States patent number US10314306B2 discloses Biocellulose fibers produced from culturing bacteria which is chosen from Acetobacter xylinum, Gluconacetobacter sp., Agrobacterium sp., Rhizobium sp., Pseudomonas sp. or Sarcina sp.

The United States patent number US9987170B2 discloses Biocellulose wound dressing with thickness in a rage of 0.8-1.5 millimeters and water vapor transmission rate greater than 300 grams per square meter in the duration of 24 hours at a temperature of 38 degrees Celsius.

The European patent application number EP3181153A1 discloses a wound care product that comprises collagen protein and Biocellulose fibers produced from culturing bacteria which is chosen from the following microorganisms or microorganism groups: Acetobacter xylinum,

Sphaerotilus spp., Pseudomonas spp., Alcaligenes spp., Salmonella spp., Agrobacterium spp. or Rhizobium spp.

The United States patent application number US5766907A discloses a process of fixing microbes onto fibers produced from polytetrafluoroethylene (PTFE) polymer. The bacterial fixation in the said process relies on the fiber surface roughness. The obtained product can be applied to various work fields, such as use as a catalyst for synthesizing chemicals and use in eliminating various residual chemicals, and so on.

Based on the above-mentioned patents and patent applications, it is seen that research on preparation and application of Biocellulose fibers has gained much interest, particularly in the adoption of Biocellulose fibers as wound dressings that additionally comprise antimicrobial active ingredients. The disclosed inventions above are of rather complicated process and high costs, and antimicrobial active ingredients in the form of chemicals may cause allergies or irritation in those with the sensitive skin. This invention chooses to incorporate a natural substance that can exert antimicrobial activity, namely honey, into Biocellulose. This is because carbohydrate forms in honey are monosaccharides being certain glucose and fructose, and disaccharides being sucrose, maltose, isomaltose, maltulose and turanose. Elements of these various sugar types contribute to the sweetness of honey. In addition, honey proteins, which mostly are enzymes, have various functions. For example, diastase digests glycogen or starch to sugar; invertase digests sucrose to fructose and glucose; and glucose oxidase transforms glucose in honey to hydrogen peroxide (H2O2), which has a microbial inhibitory activity. Also, honey consists of amino acids, vitamins, particularly vitamin B3 (niacin), zinc mineral and antioxidants, and natural honey has high acidity; that is, having a pH between 3.24.5 that is adequate for inhibiting microbial growth as disclosed in the European patent application number EP3442605A1, thereby having high antimicrobial efficiency and high safety, and helping reduce allergic reactions or irritation from wound dressings with chemical components, and being fit for users of all genders and ages.

Summary of the Invention

Preparation of Biocellulose wound dressing comprising honey as an antimicrobial active ingredient comprises steps of purifying Biocellulose wound dressing by boiling Biocellulose in sodium hydroxide solution in an autoclave and adjusting for a neutral pH by re-boiling the obtained

Biocellulose in distilled water or deionized water in an autoclave, and then soaking the obtained Biocellulose in organic acid. Subsequently, the Biocellulose is soaked in honey. Then the excess honey is wiped off, and the Biocellulose is left at room temperature. In the final step, the Biocellulose, which comprises honey, is sterilized by gamma radiation.

The objective of this invention is to provide a preparation process for a wound dressing product from Biocellulose fibers that comprise honey-type natural antimicrobial active ingredient with high efficiency and safety as well as to study antimicrobial abilities of the wound dressing product from Biocellulose fibers and honey prepared by this invention.

Brief Description of the Drawing

Figure 1 shows a comparison of regular Biocellulose wound dressing (left) and Biocellulose wound dressing soaked in honey for 24 hours (right).

Detailed Description of the Invention

Unless specified otherwise, terms and definitions appearing in this invention correspond with general use and are understandable by those with expertise in the said field.

The preparation of wound dressing from Biocellulose comprising honey in order to exert antimicrobial activity according to this invention comprises the following steps: A. Purifying Biocellulose wound dressing by boiling Biocellulose in sodium hydroxide (NaOH) solution in an autoclave and adjusting for a neutral pH by re-boiling the obtained

Biocellulose in distilled water or deionized water in an autoclave, and then soaking the obtained Biocellulose in organic acid. Subsequently, the obtained Biocellulose is soaked and stored in distilled water, ready for the production in the next step.

B. Soaking the Biocellulose wound dressing obtained from A in honey for an appropriate duration at room temperature. Subsequently, excess honey is wiped off, and the Biocellulose is left at room temperature for one week.

C. Sterilization of the Biocellulose wound dressing comprising honey from B by gamma radiation.

In one preferred embodiment, Biocellulose wound dressing is chosen from Biocellulose wound dressing and/or composite materials consisting of cellulose fibers from microbes and textile materials.

In one preferred embodiment, appropriate concentration of sodium hydroxide solution to use in the step of purifying the Biocellulose wound dressing is 2-8% w/v.

In one preferred embodiment, appropriate autoclave temperature to use in the step of purifying the Biocellulose wound dressing is 100 150 degrees Celsius.

In one preferred embodiment, the organic acid to use in the step of purifying the Biocellulose wound dressing is acetic acid with concentration of 1-5 percent by volume.

In one preferred embodiment, appropriate time in soaking Biocellulose wound dressing in honey is 5-24 hours, yielding honey content in the Biocellulose wound dressing at 50-80 percent by weight. It has been found that the said Biocellulose wound dressing soaked in honey that is left at room temperature for one week remains the same without deformation.

In one preferred embodiment, the soaking of Biocellulose wound dressing in honey according to this invention will be carried out inside a laminar air flow cabinet.

In one preferred embodiment, gamma radiation dose for the sterilization of Biocellulose wound dressing that comprises honey is at 1-20 kilograys (kGy). Subsequently, study antimicrobial abilities of Biocellulose that comprises honey according to this invention at a temperature of 37 degrees Celsius by agar well diffusion, which is an initial measure in testing antimicrobial substances that can conveniently be performed in a laboratory and can be applied to test of activities of substances derived from animals, plants, and microbes against test microorganisms. Test results can be obtained within a short time.

The agar well diffusion is performed by diffusion of substances to be tested for their actions against test microorganisms in pores of agar with test microorganisms spreading across the agar surface. Should substances to be tested have activities to kill or inhibit the growth of test microorganisms, clear zones will appear as inhibition zones.

Examples provided hereafter will demonstrate clearer details of this invention and are not deemed as limitation of the scope of this invention.

Example 1 Purifying Biocellulose wound dressing can be performed by boiling Biocellulose in 4% w/v sodium hydroxide (NaOH) solution in an autoclave at a temperature of 115 degrees Celsius for

60 minutes and adjusting for a neutral pH by boiling the Biocellulose in distilled water in an autoclave at a temperature of 115 degrees Celsius for 60 minutes for 3 rounds and soaking the Biocellulose in acetic acid solution at a concentration of 2 percent by volume at room temperature for 12 hours. Subsequently, the obtained Biocellulose is soaked and stored in distilled water, ready for the production in the next step.

Example 2 Study of the effect of time on honey retention amount of Biocellulose material

A study of honey retention of Biocellulose material was conducted by soaking Biocellulose material sized l x l centimeter in honey for 1-24 hours and then measuring the increased weight of the Biocellulose material. Following the soaking in honey for 1, 2, 3, 4, 5, 6, 12 and 24 hours, test results were obtained as in Table 1.

Table 1 shows the increased weight of the Biocellulose material upon soaking in honey at different time points.

Based on the test results in Table 1, it was found that the Biocellulose material could absorb honey well. Appearance observation found that the honey had permeated into the Biocellulose material since the first hour of the test. As the duration used in soaking the Biocellulose material in honey was lengthened, the Biocellulose material gained more weight and was enlarged as demonstrated in Figure 1.

Because soaking Biocellulose wound dressing in honey for 24 hours can add as much as 80 percent by weight of the latter into the former, honey retaining Biocellulose wound dressing prepared under this condition is chosen for further studies in example 3 and example 4. Example 3 Study of gamma radiation effect on properties of Biocellulose wound dressing comprising honey according to this invention

The honey retaining Biocellulose wound dressing obtained from example 2 was taken to undergo sterilization with gamma radiation at dose of 0-20 kilograys (KGy) with Gammacell 220

(NORDION) and then placed on culture media in the controlled condition at a temperature of 37 degrees Celsius for 24-48 hours, using three types of culture media in order to cover those microbes - fungi, yeasts, general bacteria and bacteria that can produce lactic acid namely:

• Culture medium for both fungi and bacteria (Plate Count Agar: PCA)

• Culture medium for bacteria that can produce lactic acid (MRS Agar)

• Culture medium for yeasts (YM Agar) The experiment found that the gamma ray could help reduce the microbial contents in the honey retaining Biocellulose material prepared by the mode of this invention as shown in Tables 2 and 3. Table 2 Microbial contents in the honey retaining Biocellulose material at a test time point of 24 hours

Table 3 Microbial contents in the honey retaining Biocellulose material at a test time point of 48 hours.

Remark: - denotes no microbes found on the culture medium.

In addition, based on naked eye observation, it is found that the honey retaining Biocellulose wound dressing according to this invention was not denatured and neither dissolved nor transformed following the gamma radiation. Example 4 Study of test microorganism-inhibitory abilities of honey retaining Biocellulose material prepared by the mode of this invention

This example tested microorganism-inhibitory abilities of the honey retaining Biocellulose material prepared by this invention by a method of agar well diffusion. Test microorganisms chosen for the study were: • Staphylococcus aureus DMST 8013

• Staphylococcus epidermidis DMST 5868

• Methicillin-resistant Staphylococcus aureus (MRS A) DMST 20652

• Bacillus subtilis DMST 15896 · Klebsiella pneumoniae DMST 8216

• Escherichia coli DMST 703

• Salmonella typhi DMST 22842

• Propionibacterium acnes DMST 14916

• Clostridium perfringens DMST 16637 · Candida albicans DMST 5815

The test adjusted quantities of the above microbes against McFarland standard concentrations; the required concentration was McFarland No. 0.5. Sterile swabs were dipped into various culture tubes and then smeared on a standard culture medium for testing antimicrobials (Mueller Hinton agar: MHA). Subsequently, wells were made on the culture medium surface for adding the honey retaining Biocellulose material samples according to this invention that had exposed to the gamma ray at different doses from 0-20 kilograys to test in comparison to phenol solution at a concentration of 5% (w/v) in the condition of a temperature of 37 degrees Celsius for 24 hours. Subsequently, results were recorded by measuring inhibition zone diameters on the culture medium surface that represented inhibitory abilities of the tested samples against various microbial types as demonstrated in Table 4.

Table 4 Inhibition zone diameters in the unit of millimeters (mean ± SD)

Based on Table 4, the ability of the honey retaining Biocellulose according to this invention in inhibiting the growth of the test microorganisms chosen to study was higher than that of the 5_% w/v phenol solution, which is a commonly used oxidant with microbe-killing activity, as observed from the greater inhibition zone diameters, except for Bacillus subtilis and Klebsiella pneumoniae. In addition, for Staphylococcus aureus, the ability of the honey retaining Biocellulose according to this invention in inhibiting the microbial growth was increased upon the exposure to the gamma ray at increased doses, as observed from the greater inhibition zone diameters.

Best Mode of the Invention

As disclosed in the detail description of the invention.

Claims

1. Preparation of Biocellulose wound dressing comprising honey as an antimicrobial active ingredient comprises steps of:

A. Purifying Biocellulose wound dressing by boiling Biocellulose in sodium hydroxide solution in an autoclave and adjusting for a neutral pH by re-boiling the obtained Biocellulose in distilled water or deionized water in an autoclave, and then soaking the obtained Biocellulose in organic acid. Subsequently, the Biocellulose is soaked and stored in distilled water or deionized water, ready for the next step.

B. Soaking the Biocellulose wound dressing obtained from A in honey, and subsequently wiping off excess honey and leaving at room temperature.

The characteristic is that Biocellulose wound dressing consists of cellulose fibers from microbials and textile materials and contains honey at 50-80 percent by weight.

2. Preparation of Biocellulose wound dressing comprising honey as an antimicrobial active ingredient according to claim 1, wherein the Biocellulose wound dressing is chosen from Biocellulose wound dressing or composite material consisting of any one or more types of cellulose fibers from microbials and textile materials.

3. Preparation of Biocellulose wound dressing comprising honey as an antimicrobial active ingredient according to claims 1-2, wherein appropriate time in soaking the Biocellulose wound dressing in honey is 5-24 hours.

4. Preparation of Biocellulose wound dressing comprising honey as an antimicrobial active ingredient according to claims 1-3, wherein the appropriate soaking of the Biocellulose wound dressing in honey is performed inside a laminar air flow cabinet.

5. Preparation of Biocellulose wound dressing comprising honey as an antimicrobial active ingredient according to claims 1-4, wherein, in sterilization of the Biocellulose wound dressing comprising honey, appropriate dose levels of gamma radiation are 1-20 kilogray (kGy).

6. Preparation of Biocellulose wound dressing comprising honey as an antimicrobial active ingredient according to claims 1-5, wherein appropriate concentration of sodium hydroxide solution for use in the step of purifying the Biocellulose wound dressing is 2-8_% w/v.

7. Preparation of Biocellulose wound dressing comprising honey as an antimicrobial active ingredient according to claims 1-6, wherein appropriate organic acid to use in the step of purifying the Biocellulose wound dressing is acetic acid.

8. Preparation of Biocellulose wound dressing comprising honey as an antimicrobial active ingredient according to claims 1-7, wherein appropriate acetic acid concentration for use in the step of purifying the Biocellulose wound dressing is 1-5 percent by volume.

9. Preparation of Biocellulose wound dressing comprising honey as an antimicrobial active ingredient according to claims 1-8, wherein appropriate autoclave temperature for use in the step of purifying the Biocellulose wound dressing is 100 150 degrees Celsius.

10. Preparation of Biocellulose wound dressing comprising honey as an antimicrobial active ingredient according to claims 1-9, wherein appropriate honey content in the Biocellulose wound dressing is 50-80 percent by weight.