KR102105829B1 - Zinc oxide nanocolloid dispersion produced by hot melt extrusion method and uses thereof - Google Patents

Abstract

The present invention is a method for producing a zinc oxide nanocolloidal dispersion comprising the step of heat-melting extruding a mixture of zinc oxide (ZnO) and a solubilizer using a hot melt extruder, zinc oxide nano produced by the above method The present invention relates to a feed additive composition for enhancing zinc oxide absorption, comprising a colloidal dispersion and the zinc oxide nanocolloidal dispersion as an active ingredient.

Description

Zinc oxide nanocolloid dispersion produced by hot melt extrusion method and uses thereof}

The present invention relates to a zinc oxide nanocolloidal dispersion prepared using a hot melt extrusion method and uses thereof.

A micronutrient is a generic term for vitamins and minerals that are considered to be ingested in a small amount compared to nutrients ingested in large quantities, such as carbohydrate, fat, and protein, among nutrients present in the living body. Vitamins and vitamin analogs known as physiologically meaningful nutrients include vitamins B1, B2, B6, B12, A, D, K, C, E, niacin, pantothenic acid, biotin, Folic acid, choline, inositol, ubiquinone, carotene, etc., and inorganic substances include calcium (Ca), phosphorus (P), magnesium (Mg), and sodium (Na), potassium (K), chlorine (Cl), iron (Fe), copper (Cu), zinc (Zn), manganese (Mn), iodine (I), cobalt (Co), molybdenum (Mo), selenium (Se), chromium (Cr), and the like. Micronutrients are often present in trace amounts, but are essential for improving animal survival, growth, health and fertility.

Zinc acts as an essential component of various enzymes in the body, promotes metabolism of nutrients, activates reproduction hormones, and acts as an essential element for the growth of new animals. However, when feeding is made by adding zinc to livestock feed, the absorption rate of zinc is low, and most of it is excreted through the manure, and zinc contained in the manure suppresses the growth of microorganisms for decomposition of the manure, thereby suppressing the growth of manure. It also increases soil pollution by delaying decomposition. Pig farms prefer zinc additives with a high absorption rate to prevent sub-linking phenomena in animals, and accordingly, studies on zinc additives that have high zinc utilization in livestock and can reduce environmental problems are needed.

On the other hand, Korean Registered Patent No.1753222 discloses 'Danggui-solid dispersion prepared by melt extrusion and its manufacturing method', and Korean Registered Patent No. 1390946 discloses limaprost, a pharmaceutically active ingredient, as tamsulo. Although the 'method for preparing a low-content pharmaceutical composition using high-temperature melt extrusion' of Shins (Tamsulosin) and Glimepiride has been disclosed, zinc oxide nanocolloid dispersions prepared using the heat-melt extrusion method of the present invention and uses thereof No description has been made.

The present invention was derived by the above-described demands, in the present invention, a feed treated with zinc oxide (ZnO), which has not been subjected to a zinc oxide-free basal diet and a heat-melting extrusion method, at a high concentration (2,500 ppm). Compared to the fed weaned pigs, the feed treated with zinc oxide nanocolloidal dispersion prepared using a hot melt extrusion method at a low concentration (500-1,000 ppm) was fed to the fed piglets, resulting in productivity (feed efficiency) and zinc oxide. It was confirmed that the absorption rate, the antibacterial activity, and the effect of promoting intestinal beneficial bacteria were excellent, and the present invention was completed by confirming that ingestion of the zinc oxide nanocolloid dispersion did not affect the digestibility of weaned pigs and the immunoglobulin content in serum.

In order to solve the above problems, the present invention provides a method for producing a zinc oxide nanocolloidal dispersion comprising extruding a mixture of zinc oxide (ZnO) and a solubilizer using a hot melt extruder. .

In addition, the present invention provides a zinc oxide nanocolloidal dispersion prepared by the above method.

In addition, the present invention provides a feed additive composition for improving the absorption rate of zinc oxide containing the zinc oxide nanocolloid dispersion as an active ingredient.

The zinc oxide nanocolloidal dispersion of the present invention has excellent efficiency as a feed additive even when used at low concentrations, thereby improving the productivity of animals, and the particle size is significantly reduced compared to zinc oxide that has not been subjected to heat melt extrusion. It is expected that the absorption rate will be increased to suppress the emission of environmental pollutants, thereby bringing about the effect of preventing environmental pollution.

1 is a schematic diagram of the production of zinc oxide nanocolloidal dispersion (HME-ZnO) using the hot melt extrusion (HME) of the present invention.
Figure 2 is a result showing the particle size distribution of the zinc oxide nano-colloidal dispersion prepared using a hot melt extrusion method.
3 is a result of measuring the antibacterial activity of HME-ZnO and ZnO against E.coli K88 strain. A; 5 mg / ml HME-ZnO treatment, B; 2.5 mg / ml HME-ZnO treatment, C; 1 mg / ml HME-ZnO treatment, D; 5 mg / ml ZnO treatment, E; 2.5 mg / ml ZnO treatment, F; 1 mg / ml ZnO treatment.

In order to achieve the object of the present invention, the present invention is a zinc oxide nanocolloidal dispersion comprising the step of hot melt extrusion using a hot melt extruder of a mixture of zinc oxide (ZnO) and a solubilizer. Hereinafter, a method for manufacturing HME-ZnO is provided.

In the method according to an embodiment of the present invention, the solubilizing agent is PEG (polyethylene glycol) 6000, PEG 400, PEG 1000, PEG 8000, PEG 20000 or Soluplus (Soluplus; polyvinyl capralactam-polyvinyl acetate-polyethylene glycol graft copolymer) ), Preferably PEG 6000, but is not limited thereto.

In the method according to an embodiment of the present invention, the zinc oxide and the solubilizer may be mixed in a weight ratio of 2 to 4: 6 to 8, and preferably in a weight ratio of 3: 7, but limited thereto Does not work.

In the method according to an embodiment of the present invention, the mixture may further include a surfactant and an ionizing agent, and the surfactant is preferably Span 80 (sorbitan monooleate) and Tween 80 (polyoxyethylene sorbitan monooleate). May be, the ionizing agent is preferably acetic acid or Omija, but is not limited thereto. The Omija means, but is not limited to, Omija powder, Omija juice or Omija extract.

In the method according to an embodiment of the present invention, in the heat-melting extrusion, the temperatures of the barrel and the die are maintained at 50-60 ° C and 40-50 ° C, respectively, and the screw speed is 110-190 rpm, and the extrusion speed is It may be carried out at 40 ~ 50 g / min, preferably the temperature of the barrel and the die is 55 ℃ and 45 ℃, respectively, the speed of the screw is 160 rpm, the extrusion speed may be performed at 45 g / min However, it is not limited thereto.

In the method according to an embodiment of the present invention, the heat-melting extruder includes a double screw, and preferably includes an extrusion die having a diameter of 0.8 to 1.2 mm. Compared to single screw extruders, double screw extruders offer the following advantages: high dispersibility, easy material supply, low risk of overheating, high process productivity and effective adjustment of process parameters. The strong force generated through the double screw system as described above can uniformly disperse zinc oxide in a PEG 6000 base, and at the same time, the nano-sized particles of zinc oxide are covered with an organic polymer (PEG 6000, etc.) and oxidize. The PEG 6000 coating on zinc particles reduces surface energy and aggregate generation.

The method for manufacturing HME-ZnO according to an embodiment of the present invention specifically includes a double screw in a mixture of zinc oxide and PEG 6000 in a weight ratio of 2 to 4: 6 to 8, and the extrusion die diameter Using a heat-melting extruder of 0.8 to 1.2 mm, the temperature of the barrel and die are 50 to 60 ° C and 40 to 50 ° C, respectively, the screw speed is 110 to 190 rpm, and the extrusion speed is 40 to 50 g / min. It may be prepared by melt extrusion, and more specifically, a mixture of zinc oxide and PEG 6000 in a weight ratio of 3: 7 includes a double screw, and a barrel and a hot melt extruder having an extrusion die diameter of 1 mm are used. The temperature of the die is 55 ° C and 45 ° C, the speed of the screw is 160 rpm, and the extrusion speed may be manufactured by thermal melting extrusion at 45 g / min, but is not limited thereto.

In addition, the present invention provides a zinc oxide nanocolloidal dispersion prepared by the above method.

In the zinc oxide nanocolloid dispersion according to an embodiment of the present invention, the size of the zinc oxide nanocolloid dispersion may be 50 to 500 nm, and preferably 317 nm, but is not limited thereto. It is possible to adjust the size of the dispersion as much as possible.

In addition, the present invention provides a feed additive composition for improving the absorption rate of zinc oxide containing the zinc oxide nanocolloid dispersion as an active ingredient. The feed additive composition of the present invention contains a zinc oxide nanocolloid dispersion prepared by heat-melting extrusion of a mixture of zinc oxide (ZnO) and a solubilizer as an active ingredient using a hot melt extruder as an active ingredient, When added to feed, it is possible to improve the zinc oxide absorption rate, productivity and antibacterial activity of the target animal.

The target livestock according to the present invention may be pigs, chickens, cows, sheep, and the like, preferably pigs, but is not limited thereto.

The feed additive composition of the present invention may be administered alone to livestock including pigs, cows, etc., or in combination with other feed additives in an edible carrier. In addition, the feed additive composition can be easily administered as a top dressing or by mixing them directly into livestock feed or separately from the feed, in a separate oral dosage form, or in combination with other ingredients. Typically, a single daily intake or a divided daily intake may be used, as is well known in the art.

The feed additive composition of the present invention corresponds to the supplementary feed under the feed management method.

The feed additive composition of the present invention may be added to the feed, and "feed" means any natural or artificial diet, one meal or the like, or one component of the meal, for the animal to eat, eat, and digest. can do. The type of the feed is not particularly limited, and a feed commonly used in the art may be used. Non-limiting examples of the feed, vegetable feed, such as grains, muscles, food processing by-products, algae, fiber, pharmaceutical by-products, fats and oils, starches, peels or grain by-products; And animal feeds such as proteins, inorganics, fats and oils, minerals, fats and oils, single cell proteins, animal planktons and food.

In one embodiment of the present invention, the feed additive composition may be 1.5 to 4.5% by weight, preferably 3% by weight, based on the feed composition, but is not limited now.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, the content of the present invention is not limited to the following examples.

Materials and methods

1. Characterization of zinc oxide particles

The particle properties of the zinc oxide nanocolloidal dispersion (HME-ZnO) prepared using the hot melt extrusion method were analyzed at a concentration of 10 mg / mL based on zinc oxide (ZnO) without the hot melt extrusion method. The average diameter, polydispersity index and zeta potential of HME-ZnO particles in distilled water are manufactured by the manufacturer using dynamic light scattering (DLS) and laser Doppler method (ELS-Z1000; Otsuka Electronics, Japan). Measured according to the instructions.

2. Antibacterial activity measurement

2-1. Measure absorbance and colony count

The zinc oxide particles (purity of 99.7% or more, average size of 30 nm) used for the measurement of antibacterial activity were purchased from Inframat Advanced Materials LLC (UK). Secondary distilled water was added to the zinc oxide stock, mixed by vortexing to a final concentration of 100 mg / ml, and the aliquot of the suspension was added to Mueller-Hinton medium (MH; Becton Dickinson Co. ., USA).

After mixing LB culture medium (Luria-Bertani) 5 ml, 1.41 × 10 ⁹ CFU / ml of E. coli K88 strain, zinc oxide and zinc oxide nanocolloid dispersion (HME-ZnO) 1 After adding the concentrations of mg / ml, 2.5 mg / ml and 5 mg / ml, respectively, and incubating for 16 hours at 37 ° C under aerobic conditions, absorbance was measured at 600 nm to confirm the bacterial population. The strains formed by inoculating and culturing LB solid medium after counting 1 ml of each of the tubes in which zinc oxide was treated by concentration and continuously diluting with PBS (phosphate buffer solution) solution were counted.

2-2. Statistical analysis

All experiments were repeated 3 times, and data were expressed as mean ± standard deviation (SD), and statistical analysis was performed by ANOVA and Student's t-test.

3. Animal Experiment

3-1. Animals, feed and feeding

Animal experiments were conducted at an experimental farm at Kangwon National University (Korea). A total of 360 weaned piglets (Landrace × Yorkshire × Duroc) were divided into 4 treatment groups based on the initial average weight (6.82 ± 0.02 kg) and sex (females, males). They were divided and reared. Each of the four treatment groups 1) weaned pigs fed a basic feed with no treatment, 2) weaned pigs fed a feed treated with zinc oxide (hereinafter, ZnO) at a concentration of 2,500 ppm, and 3) HME at a concentration of 500 ppm. -ZnO-treated feed was fed into weaned piglets and 4) 1,000 ppm concentration of HME-ZnO-treated feed was fed into weaned piglets. The experimental feed exceeded the nutrient requirements suggested by the National Research Council (NRC), and the feed prepared at different mixing ratios was divided into stages (1st stage; 0-14 days, 2nd stage; 15-28 days). (Table 1).

3-2. Specification Performance Survey

The weight of weaned piglets was measured individually at the start of the experiment and at the end of each step, and feed consumption was measured at the end of each step. Based on the measured body weight and feed consumption, the average daily gain (ADG), the average daily feed intake (ADFI), and the feed efficiency (gain to feed ratio, G: F) were analyzed.

3-3. Measurement of nutrient digestibility

To measure digestibility, chromium, an indigestible marker, was added to the feed at a concentration of 2.5 g / kg, feeding at 7 days after each step, and repeating each for the last 3 days of each step. 4 feces randomly selected from sugar are collected, dried in an air oven at 60 ° C. for 72 hours, crushed with a grinder (Thomas Model 4 Wiley Mill, Thomas Scientific, USA), and then dried (dry matter, DM), total The content of energy (gross energy, GE) and crude protein (CP) was measured.

3-4. Measurement of immunoglobulin content in serum

At the end of each step, blood was collected from a jugular vein of randomly selected weaned pigs at each repetition using a disposable vacuum tube that does not contain an anticoagulant. The collected blood samples were centrifuged for 15 minutes under conditions of 3,000 x g and 4 ° C to separate the serum, and then using a radical immune-diffusion kit (Tripple J Farms, USA), immunoglobulins in serum (IgG, IgA and IgM) ) Was measured.

3-5. Morphological changes in the small intestine and microbial content in the intestine

To analyze the intestinal microbial content of weaned piglets and morphological changes in the small intestine piglets, two randomly selected weaned piglets at each repetition were analyzed after sacrifice on the 28th day of the end of the experiment.

First, in order to analyze the strains present in the intestine, the contents of cecum were placed in a sterilized plastic bottle and then stored and used at 7 ° C. The contents of 1 g were diluted with 9 ml of Butterfield phosphate buffer, serially diluted with Butterfield phosphate buffer dilution solution, and inoculated and cultured in 0.1 ml of various solid media (2 replicates). The intestinal microbial group consists of total anaerobic microorganisms (medium: plate count agar, Difco Laboratories, USA), genus Lactobacillus (medium: MRS agar + 0.2 g / L NaN ₃ +0.5 g / L L-cystine hydrochloride monohydrate), Closted It was analyzed as a genus of lithium (medium: Tryptose sulphite cycloserine agar, Oxoid, UK), and when culturing total anaerobes and Clostridium spp., Gas-pak Anaerobic system (BBL, No. 260678, Difco, USA) ), And the microbial population was log-transformed before statistical analysis.

In addition, for the morphological analysis of the small intestine, the contents of the duodenum, jejunum and ileum are removed, washed with saline, glutaraldehyde 30 g / L, paraformaldehyde 20 It was immersed in a fixed solution (0.1 M collidine buffer, pH 7.3) containing g / L and 15 g / L of acrolein. Each sample was fabricated using paraffin embedding, and azur A and eosin staining were performed, followed by intact um-villi units present in the cross section of each sample. (crypt-villus unit) 10 were selected. The villus height is measured from the tip of the villus to the villus-crypt junction, and the crypt depth is the depth of penetration between adjacent villi. The ratio of villi height and villi and depth (VH / CD) was calculated by dividing villi height by villi and depth.

3-6. Defecation Index Measurement

The defecation index of weaned pigs is a score that identifies and scores the feces of weaned pigs at the same time every day. The scores for fecal conditions are as follows: 1) hard feces, 2) firm well formed, 3) soft and partially formed feces, 4) weak feces ( loose, semi-liquid feces, 5) watery feces.

3-7. Zinc oxide absorption rate measurement

Feed according to the method of AOAC (Official Methods of Analysis of the Association of Official Analytical Chemists International. 18th ed.Gaithersburg, MD, USA) using high-frequency inductively coupled plasma (ICP) emission spectroscopy The zinc oxide content in feces and plasma was measured to analyze the absorption rate of zinc oxide in the body.

First, the content of zinc oxide in the feed and feces is 1 g of feed and feces in a muffle furnace at 600 ° C for 1 hour to make dried ash, cool, and then add 10 ml of 50% HCl (v / v) And dissolved. The dissolved sample was left overnight with the lid closed, filtered 2-3 times with a Whatman filter in a 100 ml flask, diluted with deionized distilled water, and measured.

In addition, the content of zinc oxide in plasma is 1 ml of blood sample placed in a porcelain crucibles, dried in an oven at 105 ° C. for 4 hours, placed in a muffle furnace at 600 ° C. for 1 hour, cooled, and then It measured by the same method.

3-8. Statistical analysis

Statistical analysis of all data obtained in the experiment was performed through the General Linear Model (GLM) process of the SAS (Statistical Analysis System) program. Significant differences between treatment means were divided into Tukey's Honestly Significant Difference test. The repeat sugar per weaned pig was used as an experimental unit for the analysis of all variables, and was determined to be statistically significant when the p value was less than 0.05.

Preparation Example 1. Preparation of zinc oxide nanocolloid dispersion

Zinc oxide (ZnO) and polyethylene glycol (polyethylene glycol, PEG) 6000 are mixed immediately before extrusion in a 3: 7 ratio, and a double screw heat-melting extruder (STS-25HS, Hankook EM Ltd., Pyoungtaek) having a circular outlet (1.0 mm diameter) , Korea) and extruded at a rate of 45 g / min to prepare zinc oxide nanocolloidal dispersion (HME-ZnO). The temperature of the barrel and die were set at 55 ° C and 45 ° C, respectively, and the screw speed was set at 160 rpm.

Example 1. Particle characterization of zinc oxide nanocolloid dispersion

The average diameters of HME-ZnO particles and ZnO particles prepared in Preparation Example 1 are about 317 nm and several microns (µm), respectively, and it can be seen that the particle size of zinc oxide is significantly reduced by performing a hot melt extrusion process. There was (Table 2). In addition, considering the polydispersity index, it was confirmed that the particle size distribution of HME-ZnO is narrow (FIG. 1).

Example 2. Analysis of antibacterial activity

To analyze the antimicrobial activity of HME-ZnO, colonies formed after treating and culturing HME-ZnO and ZnO at a concentration of 1 mg / ml, 2.5 mg / ml, and 5 mg / ml, respectively, in a medium in which the E.coli K88 strain was cultured Was observed.

As a result, it was observed that the number of colonies formed in the HME-ZnO treatment group was significantly reduced compared to the ZnO treatment group, and in particular, the number of colonies was decreased depending on the treatment concentration of HME-ZnO (FIG. 2). Through this, it was thought that HME-ZnO of the present invention could be used as an antibacterial agent for treating diseases caused by E. coli.

Example 3. Animal test results

3-1. Specification performance (Growth performance)

HME-ZnO (500 ppm and 1,000 ppm) and ZnO (2,500 ppm) were tested for the performance characteristics of weaned pigs fed with feed.

As a result, the feed efficiency (G: F) calculated by dividing the daily gain (ADG) and the daily gain by the daily feed intake (ADFI) in the HME-ZnO and ZnO treatment groups compared to the control group that consumed nothing-treated basic feed It was confirmed that it was significantly increased (Table 3), and through this, it was found that treatment with HME-ZnO in the feed could increase the productivity of weaned pigs when feeding them.

In particular, it was found that the effect of improving daily weight gain and feed efficiency in the low concentration HME-ZnO treatment group and the high concentration ZnO treatment region was almost the same, and it was found that ingesting the low concentration of HME-ZnO has an excellent effect in improving animal productivity. .

3-2. Nutrient digestibility

HME-ZnO (500 ppm and 1,000 ppm) and ZnO (2,500 ppm) treated feedstock dry matter (DM) digestibility, gross energy (GE) content and crude protein (crude protein) Digestibility was analyzed.

As a result, in the case of building digestibility, there was no significant difference between the control group that consumed the basic feed that did not process anything and the HME-ZnO and ZnO treatment groups, and the total energy content and crude protein digestibility were also significantly different between the control group and the HME-ZnO. There was no (Table 4).

Through this, it was found that even if a solubilizer (PEG 6000) was added for the preparation of HME-ZnO, the digestibility of weaned piglets was not affected.

3-3. Intestinal microbial content analysis

Beneficial bacteria ( Lactobacillus spp.) And harmful bacteria (Clostridium) present in the ileum, cecum and large intestine of weaned piglets fed with feeds treated with HME-ZnO (500 ppm and 1,000 ppm) and ZnO (2,500 ppm) Contents of the genus; clostridium spp. And coliform bacteria were analyzed.

As a result, the beneficial bacteria in the intestine increased significantly in the HME-ZnO or ZnO treatment group compared to the control group consuming the basic feed that was not treated with anything, and the intestinal harmful bacteria were significantly decreased in the HME-ZnO or ZnO treatment group compared to the control group. It was confirmed (Table 5).

Particularly, even when HME-ZnO or ZnO is treated, the content of total anaerobic bacteria present in the intestine is not changed, and the content of beneficial and harmful bacteria in the intestine in the low concentration HME-ZnO treatment and the high concentration ZnO treatment is not changed. Through similar things, it was judged that the level of beneficial microorganisms in the intestine could be maintained well even if a low concentration of HME-ZnO was ingested.

3-4. Defecation Index Analysis

Feeds treated with HME-ZnO (500 ppm and 1,000 ppm) and ZnO (2,500 ppm) were checked and scored for fecal conditions of weaned piglets.

As a result, there was a significant difference between the control group consuming no-treated feed and the HME-ZnO or ZnO treatment group, and it was confirmed that the defecation index score was slightly increased in the HME-ZnO treatment group compared to the ZnO treatment group (Table 6). Through this, it was found that when feeding the feed supplemented with a low concentration of HME-ZnO, the defecation state of weaned pigs could be improved.

3-5. Zinc oxide absorption rate analysis

HME-ZnO (500 ppm and 1,000 ppm) and ZnO (2,500 ppm) were analyzed for zinc oxide absorption of feces, plasma, and feed of weaned pigs fed with feed. As a result, it was found that the zinc oxide content in the feces was significantly reduced in the HME-ZnO treatment group compared to the ZnO treatment group, and through this, it was found that the HME-ZnO ingested through the feed was well absorbed into the body of weaned piglets.

In addition, the content of zinc oxide in plasma increased in HME-ZnO at a concentration of 1,000 ppm and ZnO treatment at a concentration of 2,500 ppm, compared to the control group that consumed no-treated base feed, and through this, even when low concentrations of HME-ZnO were consumed, weaned piglets It can be seen that the zinc oxide absorption rate of can be improved (Table 7).

3-6. Analysis of morphological changes in the small intestine

HME-ZnO (500ppm and 1,000ppm) and ZnO (2,500ppm) were fed feed duodenum, villus height (VH) and crypt depth (CD) in the plant and ileum. It was measured, and as the value (VH / CD) divided by villi height divided by villi and depth increased, it means that the digestive absorption capacity was improved.

As a result, the VH / CD value increased in the HME-ZnO and ZnO treated groups compared to the control group consuming the untreated basic feed (Table 8).

3-7. Analysis of immunoglobulin content in serum

As a result of measuring the immunoglobulin content in the serum, the immunoglobulin G (IgA) and immunoglobulin M (IgM) and immunoglobulin M (IgM) content in the serum were compared to the control and HME- It was confirmed that there was no significant difference in the ZnO or ZnO treatment section (Table 9). Through this, it is thought that zinc oxide, regardless of the formulation, does not affect the immune response of weaned piglets.

Claims (11)

A mixture of zinc oxide (ZnO) and PEG (polyethylene glycol) 6000 in a weight ratio of 2 to 4: 6 to 8 includes a double screw, and a barrel using a hot melt extruder having an extrusion die diameter of 0.8 to 1.2 mm. Zinc oxide nanocolloidal dispersions comprising the steps of heat-melting extrusion at a temperature of die of 50-60 ° C and 40-50 ° C respectively, a screw speed of 110-190 rpm, and an extrusion rate of 40-50 g / min. How to manufacture. delete delete delete delete delete delete delete delete delete delete

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