PERACETIC ACID-BASED FORMULATION ASSOCIATED WITH A GRINDING PROCESS, THE COMBINATION OF WHICH TRANSFORMS CULTURES AND STRAINS OF BIOHAZARDOUS INFECTIOUS WASTE GENERATED IN THE PRODUCTION OF VACCINES IN OVO INTO RAW MATERIAL FOR THE PREPARATION OF HIGH-PROTEIN COMPOSTS

Abstract

Disclosed is a formulation which, in association with a grinding system, transforms biohazardous wastes unto raw material for the preparation of composts, enabling biohazardous infectious waste to be inactivated for use and industrial biohazardous infectious waste to be inactivated for reuse as a protein source for producing protein products. The formulation can be used to eliminate microorganisms in waste resulting from the production of drugs and vaccines in ovo, with applications in biohazardous infectious waste and animal waste with a high protein content and decomposition potential for the preparation of composts.

Claims

1-3. (canceled)

4. A peracetic acid-based solution comprising: hydrogen peroxide in an amount from about 40-80%; acetic acid in an amount from about 10-40%; and sulfuric acid in an amount from about 1-10%, wherein the acetic acid (CH.sub.3COOH) and the hydrogen peroxide (H.sub.2O.sub.2) react to form peracetic acid, with the presence of the sulfuric acid (H2SO4) stabilizing a molecule.

5. The solution of claim 4, wherein the solution is diluted in 1% hydrogen peroxide, 0.75% acetic acid, and 0.5% sulfuric acid volume/volume with potable water.

6. The solution of claim 4, wherein the solution is at a concentration of about 200 to 400 ppm or the equivalent thereof of peracetic acid.

7. The solution of claim 4, wherein the solution enables at least one of a bactericidal, a fungicidal, and a viricidal action.

8. The solution of claim 4, wherein the solution enables destruction of biohazardous waste for reuse as raw material in the preparation of a protein product.

9. The solution of claim 8, wherein the biohazardous waste results from the production of vaccines “in ovo.”.

10. The solution of claim 8, wherein the raw material is a protein source.

11. The solution of claim 8, wherein the protein product is a high-protein compost.

12. The solution of claim 8, wherein the solution reduces the volume of the biohazardous waste and inactivates pathogens in the biohazardous waste.

13. The solution of claim 4, wherein the solution enables destruction of waste of animal origin with a high protein content.

14. A method of biohazardous waste destruction using waste destruction equipment, the method comprising: introducing the biohazardous waste into the waste destruction equipment; performing a first rinse on the biohazardous waste; destroying the biohazardous waste, wherein destroying the biohazardous waste includes: grinding and crushing the biohazardous waste by the waste destruction equipment; exposing the biohazardous waste to a chemical wash, wherein the chemical wash incudes a peracetic acid-based solution; and transforming the biohazardous waste into special waste, wherein the special waste includes leachate and solid waste; and collecting the special waste from the destroyed biohazardous waste, wherein the leachate is released through a drain valve of the waste destruction equipment and the solid waste is collected in a container.

15. The method of claim 14, wherein the biohazardous waste results from the production of vaccines “in ovo.”.

16. The method of claim 14, wherein performing the first rinse on the biohazardous waste includes rinsing the biohazardous waste in an upper chamber of the waste destruction equipment.

17. The method of claim 14, further comprising removing the solid waste from the waste destruction equipment with a screw conveyor.

18. The method of claim 14, wherein exposing the biohazardous waste to a chemical wash includes at least one of a bactericidal, a fungicidal, and a viricidal action.

19. The method of claim 14, wherein destroying the biohazardous waste includes reducing the volume of the biohazardous waste and inactivating pathogens in the biohazardous waste.

20. The method of claim 14, further comprising preparing the special waste for reuse as a raw material in the preparation of a protein product.

21. The method of claim 20, wherein the raw material is a protein source, and wherein the protein product is a high-protein compost.

22. The method of claim 14, further comprising destroying waste of animal origin with a high protein content.

23. A method of waste destruction using waste destruction equipment, the method comprising: introducing the waste to the waste destruction equipment; destroying the waste, wherein destroying the waste includes: grinding and crushing the waste by the waste destruction equipment; exposing the waste to a chemical wash, wherein the chemical wash incudes a peracetic acid-based solution; and transforming the waste into special waste, wherein the special waste includes leachate and solid waste; and collecting the special waste from the destroyed biohazardous waste.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0022] FIG. 1 shows the waste destruction process with the parameters used: 65 kg of waste from the production of vaccines in ovo in yellow bags, 700 ml of peracetic acid, in 10 liters of water for a 20-minute cycle. As a result, 50 liters of leachate is obtained which passes through the drain, as well as 15 kg of pathogen-free solid waste that is ready to be reused.

[0023] FIG. 2 shows the monitoring of cell cultures exposed to supernatants treated with peracetic acid/hydrogen peroxide, negative control (not exposed to supernatant) and positive control (exposed to untreated supernatant). The columns indicate the culture time, and the rows indicate the type of exposure.

[0024] FIG. 3 shows the monitoring of MCF7 (Michigan Cancer Foundation 7; this is a breast cancer cell line isolated in 1970 from a 69-year-old Caucasian woman), negative control (A-D), positive control exposed to the supernatant obtained from the biohazardous waste bags prior to processing (E-H), and post-process leaching (I-H). Arrows indicate cytopathic damage (cell death).

[0025] FIG. 4 shows the monitoring of HEPG2 (hepatocellular carcinoma), negative control (A-D), positive control exposed to the supernatant obtained from the biohazardous waste bags prior to processing (E-H), post-process leaching (I-H), and the cells exposed to the solids obtained after the integral grinding and crushing treatment that simultaneously disinfects the biohazardous waste, reducing its volume by up to 90% and rendering it harmless for disposal as ordinary waste. Arrows indicate cytopathic damage (cell death).

[0026] FIG. 5 shows the histogram of cell viability based on the result of the MTT assay, which is a method for determining the possible cytotoxic effect of an agent on tumor cell lines or primary cultures of normal cells; the Y axis represents the percentage of viability of each well, and the X axis represents the type of treatment and control. The bars have their standard deviation.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The characteristic details of the peracetic acid-based formulation associated with a grinding process, the combination of which transforms the crops and strains of biohazardous waste generated in the production of vaccines “in ovo” into raw material for the preparation of high-protein composts are unambiguously demonstrated in the following description and in the accompanying illustrative drawings.

[0028] For the application of the formulation, a grinding process was developed for the treatment of waste which includes the combination of the process and the peracetic acid-based formulation that transforms biohazardous waste into special waste. This process consists of grinding and chemical disinfection (reaction mixture) under controlled conditions for time and the concentration of the chemical sterilization solution, whereupon the volume of the waste is reduced by destroying the waste until it is unrecognizable and pathogens are rendered inactive. As a result of the process, leachate is obtained having characteristics that enable it to be disposed of in general drainage, as well as transformed solid waste that is free of pathogens and can be treated as special waste.

[0029] The waste destruction process consists of the following stages: loading, grinding, chemical washing, drainage, and solids output. In the loading stage, the biohazardous waste generated in the production of vaccines in ovo is introduced, and sprinklers carry out a first rinse in the upper chamber of the equipment. During grinding, the waste is destroyed and then exposed to a chemical wash through the action of a mixture of water with peracetic acid. Finally, the leachate comes out through a drain valve, and the sterilized solids are removed by means of a screw conveyor. This solid waste is collected in containers for subsequent reuse.

[0030] FIG. 1 shows the waste destruction process with the parameters used: 65 kg of waste from the production of vaccines in ovo in yellow bags, 700 ml of peracetic acid, in 10 liters of water for a 20-minute cycle. After the aforementioned stages, 50 liters of leachate are obtained, which pass through the drain, as well as 15 kg of pathogen-free solid waste that is ready to be reused.

[0031] In order to reuse biohazardous waste as raw material for composts and/or products that require an organic source, the complete inactivation of the waste must be verified, meaning that it must not have any pathogenic microorganism that continues to be considered a biohazardous waste. Therefore, between 72,000 to 88,000 parts per million (PPM) or 72 to 88 grams per liter of peracetic acid are obtained upon producing the mixture of hydrogen peroxide, acetic acid, and sulfuric acid. These components are found in the proportions of 40-80% (hydrogen peroxide), 10-40% (acetic acid), and 1-10% (sulfuric acid), with acetic acid (CH.sub.3COOH) and hydrogen peroxide (H.sub.2O.sub.2) reacting to form peracetic acid, and with the presence of sulfuric acid (H.sub.2SO.sub.4) stabilizing this molecule. Its stable formula is as follows:

##STR00001##

[0032] This solution was diluted to 1 and 0.5% volume/volume with potable water. This yielded solutions of 200 to 400 ppm or 200 to 400 mg per liter of peracetic acid. These solutions were used to validate their virucidal capacity in industrial biohazardous wastes contaminated with New Castle-type viruses from manufacturing vaccines in ovo.

Cell Culture Monitoring

[0033] A cell culture of HEK 293 cells (human embryonic kidney 293 cells) distributed in a 24-well plate was monitored for 96 hours. During the first 24 hours after the exposure of the supernatant liquid to the biohazardous waste, untreated and treated with the sterilizing solution, no cytopathic evidence was found, as can be seen in FIGS. 1A, 1G, 1K, 1O, 1S. The culture in the different wells of the controls and test samples continued to grow and proliferate as normal; however, at 72 hours the positive control wells that were exposed to the supernatant liquid of the waste without treatment with the sterilizing liquid began to manifest signs of cytopathy, as is shown in FIG. 1D. Finally, at 96 hours, severe cytopathic effects were observed, which are shown in FIG. 1E, whereas the other cultures exhibited normal growth, similar to that of the negative control (non-exposed cells).

[0034] On the other hand, HEPG2 and MCF7 cells exposed to leachate and solids only for HEPG2 exhibit a behavior similar to that of HEK293, with the exposed cultures not showing any signs of cytopathy, just like the negative control; this is shown in FIGS. 2 and 3. However, the cytopathic evidence is clear in the positive controls from 72 and 48 hours, respectively, which can be seen in FIG. 2F and FIG. 3C.

MTT Assay

[0035] After MTT processing, FIG. 2 shows that the cells that were exposed to the viral supernatant have a viability of 80-95% compared to the negative control. They do not mimic the behavior of the negative control, since the neutralization process and the possible salts that are formed might affect the cell culture, inducing cell death due to an osmotic imbalance.

[0036] However, the positive control has a viability of 45%, showing cell death due to the presence of the virus and the replication and proliferation thereof. In order to avoid having a low cell viability in the negative control that might affect the reading, the culture was not continued for another 24 hours. For HEPG2 and MCF7, MTT was not necessary, since cell death was evident at 96 hours, which can be seen in FIGS. 2D, 2M and FIGS. 3D, 3L, 3G.

Process Validation and Formulation With Waste With Other Microorganisms

[0037] The bactericidal and fungicidal action was verified using five different species: Escherichia coli, Pseudomona aureginosa representing the bacteria in the Gram-negative group, Staphylococcus aureus, Bacillus subtilius representing the bacteria in the Gram-positive group, and Candida afficans as a fungus. These microorganisms were allowed to grow on nutrient media to proliferation. After 24 hours, 3 batches of waste prepared with sterile residue from needles, syringes, sheets, compresses, and cotton pads were contaminated. Samples were taken from the various proliferating suspensions, with dilutions up to 10×.sup.-6 being prepared in triplicate. Each batch was then eliminated in the system with concentrations of 0.5 and 1% volume/volume for 5 minutes, respectively (washing). At the end of each process, 3 ml of the liquid sample were neutralized with sodium hydroxide. Subsequently, each sample was taken for subsequent culturing in specific agar solutions for each microorganism; these were allowed to incubate for 24 hours. After incubation, the colony-forming units (CFUs) were counted for purposes of quantification.

[0038] A clear elimination of pathogens from 6Log.sub.10 to 7Log.sub.10 was observed among all species, leaving the cultures exposed to the practical acid solution with no evidence of any colony formation. Formed colonies were observed in the positive controls, indicating the proliferation of the microorganism.

[0039] FIG. 5 shows the cell viability based on the result of the MTT assay.

[0040] The sterilizing liquid and the grinding process show efficacy in the elimination of the New Castle virus from the biohazardous waste that was discarded in the in ovo vaccine production process, and the absence of other microorganisms that might cause these wastes to be categorized as biohazards was likewise confirmed. The cultures that were inoculated with the treated and neutralized supernatant behaved like a healthy culture, whereas the control that was inoculated with the untreated supernatant fluid exhibited signs of mild (at 72 hours) to severe (94 hours) cytopathy. Likewise, the presence of other microorganisms such as bacteria and fungi was verified, demonstrating that the combination of peracetic acid and a grinding system is a binomial for the transformation of biohazardous waste to special waste; transformation of biohazardous waste to special waste by means of chemical washing to inactivate the viral proliferation of biohazardous wastes from the production of vaccines in ovo and enable this product to be labeled as raw material for the preparation of composts and/or products that require organic sources.

Resulting Formulation

[0041] The formulation that was obtained as a result of all the analyses described and yields the best results is:

[0042] A formulation for the transformation of biohazardous waste to raw material for the preparation of composts, comprising: [0043] a. peracetic acid for the elimination of microorganisms in biohazardous wastes from the production of drugs and vaccines in ovo, comprising: [0044] i. peracetic acid at 23% weight/weight in water, which is obtained by mixing hydrogen peroxide, acetic acid, and sulfuric acid; [0045] ii. the proportions of the above components are: [0046] 1. hydrogen peroxide 40-80%, [0047] 2. acetic acid 10-40%, and [0048] 3. sulfuric acid 1-10%; [0049] iii. wherein acetic acid (CH.sub.3COOH) and hydrogen peroxide (H.sub.2O.sub.2) react to form peracetic acid, with the presence of sulfuric acid (H.sub.2SO.sub.4) stabilizing this molecule; [0050] iv. the solution is diluted in 1% hydrogen peroxide, 0.75% acetic acid, and 0.5% sulfuric acid volume/volume with potable water; [0051] v. all of the above yields solutions of 200 to 400 ppm or the equivalent thereof of 200 to 400 mg per liter of peracetic acid.

[0052] In addition to the above, it is combined with a grinding process for biohazardous waste that produces a reaction mixture in order to increase the biocidal action of the PAA formulation.

[0053] The preceding description of the disclosed definitions is provided in order to enable any person skilled in the art to implement or use the present invention. Various modifications to the generic definitions and/or implementations defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not intended to be limited to the embodiments shown herein, but should be granted the broadest scope consistent with the following claims and the principles and novel features disclosed herein.