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I know that papain is a proteolytic enzyme present in papaya. It is often used to tenderise meat. What is the function of papain in papaya? Is papain sweet? Does it function as an antibiotic?
Papain is a cysteine protease, for which Wikipedia should be sufficient:
Cysteine proteases play multi-faceted roles, virtually in every aspect of physiology and development in plants such as in growth and development, in senescence and apoptosis (programmed cell death), in accumulation and mobilization of storage proteins such as in seeds. In addition, they are involved in signalling pathways and in the response to biotic and abiotic stresses. In humans they are responsible for apoptosis, MHC class II immune responses, prohormone processing, and extracellular matrix remodeling important to bone development.
Papain, a Plant Enzyme of Biological Importance: A Review
Papain is a plant proteolytic enzyme for the cysteine proteinase family cysteine protease enzyme in which enormous progress has been made to understand its functions. Papain is found naturally in papaya (Carica papaya L.) manufactured from the latex of raw papaya fruits. The enzyme is able to break down organic molecules made of amino acids, known as polypeptides and thus plays a crucial role in diverse biological processes in physiological and pathological states, drug designs, industrial uses such as meat tenderizers and pharmaceutical preparations. The unique structure of papain gives it the functionality that helps elucidate how proteolytic enzymes work and also makes it valuable for a variety of purposes. In the present review, its biological importance, properties and structural features that are important to an understanding of their biological function are presented. Its potential for production and market opportunities are also discussed.
Volume 8 No. 2, 2012 , 99-104
Submitted On: 13 May 2012 Published On: 22 June 2012
How to Cite: Mamboya, F. & Amri, E. (2012). Papain, a Plant Enzyme of Biological Importance: A Review. American Journal of Biochemistry and Biotechnology, 8(2), 99-104. https://doi.org/10.3844/ajbbsp.2012.99.104
Copyright: © 2021 Florence Mamboya and Ezekiel Amri. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Arterivirus Papain-like Proteinase 1α
Danny D. Nedialkova , . Eric J. Snijder , in Handbook of Proteolytic Enzymes (Third Edition) , 2013
PLP 1α is a small papain -like cysteine proteinase domain, which is linked to an N-terminal zinc finger domain ( Figure 499.2 ). It cleaves the arterivirus pp1a and pp1ab replicase polyproteins in cis
35 residues downstream of its active-site His residue, and thus releases the N-terminal nsp1α subunit of the PRRSV and LDV replicase. The proteolytic activity of PLP 1α appears to have been lost during EAV evolution.
A polyclonal rabbit antiserum against EAV nsp1 was raised by immunization with a peptide representing the first 23 residues of pp1a  . This serum is available from the authors for research purposes on request. The production of a mouse monoclonal antibody (12A4) recognizing EAV nsp1 has also been documented  . Polyclonal antisera directed against nsp1α and nsp1β from a PRRSV-II have been described recently  .
Benefits Of Papain Enzyme
1. Stimulates Digestion
One of the key areas in which papain serves the body is in the realm of its protein-digestive properties. One case study found that when a male patient with gluten intolerance ate a gluten-free diet, he still experienced diarrhea, but when he additionally took 1800 mg of papain for one month, he had fewer loose stools and less malabsorption. This is just one study, and more research needs to be conducted.
2. Aids Skin and Wound Healing
Due to papain’s beneficial capacities, people have used it for many years as a topical application to burns, ulcers, irritations, bedsores and other wounds, and to assist recovery from sports injuries. Some practitioners have used it dental cavities. Papain’s enzymatic action is very specific, and it does not harm healthy skin. Traditional cultures in Hawaii and Tahiti made poultices out of the skins of papaya, as this part of the fruit has a particularly high concentration of papain. Traditional healers applied this substance to the skin to heal wounds, burns, rashes and insect stings.
3. Digests Mucus
Studies have found that papain digests sinus mucin, a glycoprotein found in mucus, and hence may have beneficial effects for people having sinus issues. Papain makes mucus less viscous, or runnier, and hence better able to be eliminated. Because of this feature, some researchers are studying how papain can help deliver nanoparticle medicines to the body so that they can get through the body’s natural mucosal barrier in the gut. Using papain with nanoparticles may not be the best for your health.
4. Supports Immune System Function
Studies have found that papain may have anti-cell proliferation properties. Some studies have shown papain delivers a strong effect while others found no difference between papain and controls. A review article found strong evidence for the overall immune function properties of papaya.
5. Resists Redness and Irritation
Studies confirm that the papain enzyme offers powerful resistance to redness and irritation. Papain helps aid the absorption of another beneficial substance, quercetin. One study found that when papain and bromelain were given along with quercetin, it helped swelling symptoms associated with prostate health.
6. Acts as an Antioxidant
Papain holds compounds that may aid in protecting the body from cellular damage caused by free radicals, which makes it an antioxidant. The compounds in papaya juice effectively scavenge, or counteract, highly reactive hydroxyl (OH-) free radicals, as well as super-oxides. Papain has an antioxidant level on par with Vitamins E and C. In one study, the Sunrise Solo cultivar (a type of papaya) was more effective as an antioxidant than two other cultivars.
7. Prevents Food Spoilage
Since research has shown papain has antifungal and antibacterial properties, and it is sometimes used to preserve foods naturally. It is a powerful agent commonly used in food preservation, reducing bacterial infestations and spoilage due to oxidation.
How Does Papain Tenderize Meat?
The most important characteristics of meat quality are tenderness, juiciness, and flavour. Consumers consider tenderness as the most important factor in determining eating satisfaction of beef (Issanchou, 1996, Boleman, Miller, Taylor, Cross, Wheeler, Koohmaraie, Johnson and Savell, 1997). Tenderness is defined as the ease of mastication, which involves the initial ease of penetration by the teeth, the ease with which the meat breaks into fragments and the amount of residue remaining after mastication (Lawrie, 1998).
Meat tenderness depends on spices, breed, age, sex and individual skeletal muscle tissue of the animal. Tenderness originates in structural and biochemical properties of scheletal muscle fibers, especially myofibrils and intermediate filaments, and of the intramuscular connective tissue, the endomysium and perimysium, which are composed of collagen fibrils and fibers (Takahashi, 1996). The mechanical stability of collagen fibrils increased markedly with chronological aging.
Meat produced from an old animal is tough and has a lower eating quality. Improvement of meat tenderness of aged cattle is necessary to increase functional properties and value of the meat (Shiba, 2004). Meat can be tenderized in different ways: mechanical methods (mincing or hitting to crush the conjunctive and muscle tissue), chemical methods by injecting in the muscle solutions with chemical substances (salt, sodium chloride, sodium polyphosphate, potassium lactate, sodium diacetate all dissolved into water, R.K., 2003), enzymatic methods used proteolytic enzymes like papain, bromelain or ficin.
Enzymatic tenderisation can be made in a humid way (by injecting in the muscle solutions with enzymatic preparations) or in a dry way (pressing tender mixtures on the meat surface). The end of the tenderisation process and the method employed for aging and thermal processing should be established to get an optimum sensorial quality of the meat.
Injection of beef cuts with papain cause an important improvement of functional properties of adult beef. Papain is a powerful proteases preparation, with great under-layer specificity, catalysing the breaking of the peptidic bonds in the protein molecules and their degradation products to amino acids. The increase of the papain level, from injection brine, as well as the increase of the time of ageing, determined a significant weakening of the meat structure. Papain tenderisation of the adult beef determined the improvement of tenderness, flavour, and juiciness. It is recommended to use papain doses as low as possible, in order to avoid advanced tenderisation and obtaining meats with soft structure, with a very low resistance to mastication and paste texture. Application of this technology could assist beef producers and processors to obtain meat products that can satisfy consumer’s expectation.
Papain’s ability to break down proteins has other uses besides meat tenderising. It is used in enzyme-cleaning solutions for contact lenses and in medicated ointments used to treat severe wounds.
Of course, there are other ways to tenderize meat without using chemicals. You can simply use a meat mallet to break down the large protein molecules by beating them into smaller pieces, but this method won’t work, as papain does, on both meat and contact lenses.
Function of papain in papaya - Biology
Papain, found naturally in papaya and ioften referred to as a “plant-based pepsin”, is an important industrial protein-degrading enzyme for the food and cosmetic industries. The cosmetic industry uses papain in exfoliating treatments to remove dead surface skin and there even are enzyme-based shampoos for house pets to clean the fur and make it easier to brush.
But lots of natural things can trigger allergic reactions.
Skin consists of several layers joined via cellular connections called “tight junctions”. In a new study, the authors showed that papain induces a breakdown of these cell-cell junctions. On the skin, papain results in a loss of the barrier function. That is why when humans or animals come in contact with papain, strong allergic reactions of the skin can be the result. The new work researched the effect of papain directly on the skin of mice as well as on skin cells in the petri dish.
“After just a short period of time, papain increased vascular permeability and inflammatory cells infiltrated the skin,” says Erika Jensen-Jarolim, Head of the Department of Comparative Medicine at the Messerli Research Institute.
Around two weeks after being exposed to papain, the researchers found antibodies to papain in the mice. These immunoglobulins are the cause of the allergic reaction toward the enzyme.
“Exposed mice not only experienced a loss of the barrier function of the skin, but also had a specific allergic sensitization toward papain. The animals developed an allergy,” says Jensen-Jarolim.
But the permeation of the skin barrier does not appear to be a prerequisite for sensitization toward papain.
“The enzyme remains allergenic even when its enzymatic function has been blocked,” explains Jensen-Jarolim. The disruption to the skin barrier, she says, is essential for the infiltration of other allergens and bacteria. In humans and in animals, diseases of the skin such as atopic dermatitis, commonly referred to as eczema, involve an increased permeability of the skin with a heightened risk for bacterial, fungal, or viral colonization. Besides genetic factors, allergenic enzymes from external sources may also contribute to the symptoms.
It is striking that papain has an enormous structural similarity with one of the most important house dust mite allergens. The authors conclude that sensitization toward these house dust mite allergens follows the same principle.
To assay the effect of latex on herbivorous lepidopteran larvae, we used the larvae of a line of Eri silkmoth (S. ricini, Saturniidae) maintained in our institute as an experimental insect. This species was originated and has been widely reared in northern India to produce silk. The larvae are oligophagous and their host plants include the castor oil plants, ailanthus, cassava, kesseru, and plumeria. However, it will eventually eat any kinds of plant leaves unless the leaves are too hard or hairy, and subsequently shows symptoms, such as death from poisoning and growth inhibition in response to the respective plant. The larvae are successfully used to perform bioassays to evaluate plant defense levels against herbivorous insects ( Fukui et al., 2002 ). M. brassicae and S. litura that had been kindly provided by Hokko Chemical Industry Co. Ltd., (Tokyo, Japan) and then maintained in our institute were also used for bioassays.
Whole young leaves that had reached approximately half their mature size and were still soft were collected from individuals grown in a greenhouse (C. papaya), grown outdoors (G. jasminoides), or from specimens naturally growing on Ishigaki Island, Okinawa, Japan (F. virgata), or in Tsukuba, Ibarki, Japan (M. bombycis, T. officinale, M. japonica, C. majus, and O. japonica). In the cases of F. virgata and all other plants except papaya, whole leaves collected from stems by cutting the petioles were directly used for bioassays. In the case of papaya, which has very large palmate leaves with five to seven lobes, whole palmate leaves were collected by cutting the petioles, then the lobes were collected by cutting each leaf at its base the lobes were used for bioassays. For each set of bioassays with various treatments for comparison, lobes that were collected from the same leaf and from adjacent positions were used. In these collecting processes, some amount of the plant latex was expected to have drained from the cuts. However, it was clear that a considerable amount of latex remained in collected leaves or petioles, based on the fact that we observed plenty of latex emerging after further cutting had been performed on the collected leaves or lobes. The drainage of latex in the collecting processes may have caused some underestimation of the biological effects of latex, but would never cause overestimation therefore, we concluded that it is not likely that the collecting procedures would have affected the most relevant result of this study (i.e. cysteine proteases in latex are the most important factors in plant defense systems, and can protect plants from even the most polyphagous pests).
In order to examine the biological effects of latex on insects, we attempted to wash latex from the leaves before feeding them to larvae. For this purpose, leaves (1–3 g) were cut into narrow leaf strips (2–5 mm in width) with scissors, then the leaf strips were washed two times in 500 ml of water. The leaves were patted dry with paper towels and then used for bioassays.
To examine the defensive roles of cysteine proteases, we performed bioassays using a cysteine protease-specific inhibitor, E-64, N-[N-( l -3-trans-carboxyoxirane-2-carbonyl)- l -leucyl]-agmatine, originally isolated from a culture extract of Aspergillus japonicus and its structure was determined ( Hanada et al., 1978a,b ). E-64 is a strict cysteine protease-specific inhibitor and shows inhibitory effects on cysteine proteases such as papain, ficin, bromelain, and cathepsin B in low concentrations, although it shows no inhibitory effects on proteases belonging to other protease groups such as trypsin, chymotrypsin, elastase, plasmin, and pepsin even at very high concentrations ( Hanada et al., 1978b ), and therefore, it is not likely that E-64 would affect the activities of digestive proteases in the midgut of lepidopteran insects, considering that major midgut proteases of lepidopteran insects are serine proteases, and cysteine proteases have not been found ( Terra et al., 1996 ). The structural basis of inhibition of cysteine proteases by E-64 has been studied recently, and those studies revealed that the epoxide ring of E-64 attacks and covalently binds to the active cysteine SH group in the catalytic site of a cysteine protease molecule, thereby irreversibly inhibiting cysteine protease ( Matsumoto et al., 1999 ). To examine the effect of the cysteine protease-specific inhibitor E-64, 5 m m solution of E-64 in 0.5% glycerol, then this solution was painted evenly on the surface of leaves at 200 µl g −1 fresh leaf (i.e. 0.36 mg E-64 g −1 fresh leaf). Care was taken to paint the leaves evenly so that the perimeter was well painted. Glycerol was added to make the E-64 adhesive to the leaf surface. In control leaves, shown in Figures 2 and 3 and Table 1, on which no E-64 was painted, 0.5% glycerol solution alone was painted. Artificial diets containing cysteine proteases were prepared based on the L4M diet (Nihon Nosan Kogyo Co., Japan), which mainly consists of soybean powder and is used for polyphagous insects. To 1 g of the L4M diet, 200 µl sodium phosphate buffer (50 m m , pH 7.0) that contained 4 mg of cysteine, and 0 or 20 mg of papain (P-3250 for papain diet A, P-4762 for papain diet B, Sigma, St Louis, MO, USA from C. papaya), ficin (F-6008, Sigma from F. carica), or bromelain (B-4882, Sigma from pineapple stem), whose proteolytic activities were determined as described below, were added, and these respective diets were used for the bioassays. The treated leaves or artificial diets were given to the 1st or 2nd instar larvae of Eri silkmoth, M. brassicae, and S. litura, and larval mass and mortalities were observed 2 and 4 days after the onset of the experiments. The leaves were exchanged with fresh ones every other day.
Two grams of treated or non-treated papaya leaves were homogenized in 30 ml of sodium phosphate buffer (50 m m , pH 7.0) cooled with ice. The homogenates were centrifuged two times (3000 g for 10 min, and 10 000 g for 10 min at 4°C) and the supernatants were collected. Two hundred microliters of sodium phosphate buffer (50 m m , pH 7.0) containing supernatants or enzymes was mixed with 1 ml of reaction solution containing 50 m m sodium phosphate (pH 7.0), 5 m m cysteine, 1 m m EDTA, and 1% casein as a substrate. Reactions were performed at 25°C for 30 min, then 1 ml of 20% trichloroacetate was added to terminate the reactions. After centrifugation (10 000 g, 10 min), supernatants were collected, then the absorptions at 280 nm (A280, 1 cm light path) were analyzed. One unit was defined as the enzyme activity that made a 0.001 increase in A280 per minute at the reaction condition described above.
How much papain is in a serving of papaya?
Papain is found in papaya but not in quantities considered to be therapeutic. The papain found in supplements is obtained from the sticky latex of the papaya fruit. Latex, by definition, is the milky emulsion that is exuded from certain plants and congeals when exposed to air.
After collection, the papaya latex is dried and purified. It then undergoes a process wherein the latex is liquified and the papain enzyme extracted in crystallized form. After powdering, the papain is incorporated into supplements and topical preparations.
In the present study we conducted two randomized controlled efficacy trials to evaluate the efficacy of papaya CPs against experimental T. suis infections in pigs, and we clearly demonstrated that a single application of CPs at the higher doses used for treatment is far more effective than the most widely used synthetic anthelmintic. Therefore, this study confirms that papaya CPs have anthelminthic properties that are extremely effective against Trichuris infections. Here a single-oral dose of 450 μmol of papaya CPs reduced the egg excretion and worm burden by more than 97%, regardless of the level of infection intensity. These efficacy results are superior not only to the efficacy results of a single-oral dose of 400 mg ALB in the current study, but also to most ERR efficacy results reported in human populations for ALB and MEB, either administered in a single oral dose on one day (1×1 ALB: 64.5% 1×1 MEB: 62.7%, Levecke et al., unpublished data), or even in several doses over consecutive days (2×1 ALB: 73.5%, 2×1 MEB: 87.1%, 3×1 ALB: 94.0%, 3×1 MEB: 97.3%, a double oral dose on 1 day (1×2 ALB: 94.8%, 1×2 MEB: 90.3%. Single dose CP treatment was also considerably more effective in removing T. suis than any of the other compounds trialed in recent years (1× levamisol: 0%, 1× ivermectin: 86.8%, 1× tribendimidine: 31.1%, 1× nitazoxanide: 13.4%) and drug combinations (1×1 ALB+MEB: 96.1% 2×1 ALB+MEB: 97.3%, 1×1 ALB+ivermectin: 91.1% and 97.5% ALB+diethylcarbamazine: 79.4% 2×1 ALB+nitazoxanide: 54.9% 1×1 MEB+levamisole: 85.0% 1×1 MEB+ivermectine: 96.7% pyrantel-oxantel: 86.9%.
Despite these promising results, there are some aspects that still need to be addressed before papaya CP can be considered as an acceptable alternative anthelminthic drug for MDA programs in the control of STH. These include formulation, dose and safety. At present, a dose 450 μmol papaya CPs represents a mass of
45 g, and this poses an important obstacle in treating both human patients and animals but particularly a pediatric population due to the quantity of material. Nevertheless, given that the CPs used in the current work were contained entirely in a solution (unlike in some earlier work where gel-like latex was used, Stepek et al., further concentration is possible, and a range of different vehicles/matrices can be exploited to generate a formulation and delivery system in a smaller volume that is more acceptable for oral delivery.
The dose–response trial indicated that the dose can be halved (225 μmol), without losing too much of its efficacy (
80%). Although this reduction in dose will also half the mass of CP, it still remains substantial (
22 g) and in terms of weight and bulk, more than alternative, albeit less effective, treatments.
‘Papain’ has found various applications in the daily life of people and in the food industry, and is therefore widely available for these purposes, with less regulation than conventional medicines. However, the amounts of papain used by the consumer for these tasks are clearly much lower than the minimal dose that showed anthelmintic efficacy against trichuriasis in our trials (115 μmol or
11 g). Some side effects of papaya CPs have been reported previously in animals and humans but these are relatively scarce. In the sheep trials reported by Buttle et al., some oral blistering was observed, but this was not serious at the doses used. Regurgitation of CP and their subsequent inhalation present a more serious threat, but again when administered to animals by well-trained staff oral delivery to mice did not result in losses. Novel formulations based on syrups would also most-likely eliminate this as a problem in treatment of people. CPs are proteins and there is also the risk of allergy developing to their frequent application. However, it should be emphasized here that the side effects have only been evaluated poorly to date and that this is one aspect of the use of papaya CPs that will have to be more thoroughly investigated before they can be marketed as anthelmintics.
The poor efficacy of ALB against T. suis (ERR: 59.0-64.4 WRR: 23.2-39.0) was not entirely unexpected, as the ERR results resemble those reported in a recent meta-analysis including five trials assessing the efficacy of a single oral-dose of 400 mg ALB in school children Cameroon, Ethiopia, Tanzania and Vietnam (ERR: 64.5% [95% CI: 44.4 84.7] Levecke et al., unpublished data). Although our results confirm the usefulness of pigs as a model for assessing drug efficacy against trichuriasis, we expected a more pronounced difference in efficacy across the two levels of infection intensity. The ERR for ALB against trichuriasis in the five aforementioned trials with school children ranged from 29.3% for a mean FEC at baseline of 1193 EPG, to 92.4% for a mean FEC at baseline of 420 EPG. In the current study, ALB provided comparable ERR results against low (mean FEC = 492 EPG, ERR = 64.4%) and heavy-intensity infections (mean FEC = 9267 EPG, ERR = 59.0%). Only for WRR was there a pronounced difference in efficacy (low: 23.2% vs. heavy: 39.0%).
Finally, our results indicate that egg excretion increased as a function of worm burden, suggesting that FEC are a valid proxy of worm burden for trichuriasis in pigs.
Enzyme activity [ править | править код ]
If papain is to be exploited commercially for an export market or local food industry use, it is important to be able to determine the enzyme activity. The method is known as assaying. The assaying could be carried out by, for example, the National Standards office.
Papain is used to hydrolyse (or breakdown) proteins. Therefore assays to measure papain activity are based on measuring a product of the hydrolysis. There are two main assay methods. The first relies on the ability of papain to clot milk. It is a low cost method but is time consuming. Also the lack of a standard method to find the clotting point and variations in the milk powder used can introduce errors.
In this method a known amount of papain sample (made by dissolving a known weight of papain in a known volume of a solution of acetic acid) is added to a fixed amount of milk (made by dissolving a known weight of milk powder in a known volume of water) which has been warmed to 30°C in a water bath.
The contents are thoroughly mixed and then observed until the first signs of clotting (formation of lumps) are detected. The time taken to reach this stage, from when the papain was added to the milk, is recorded. The experiment is then repeated using different known amounts of papain solutions. The different amounts of papain sample used should give a range of clotting times between 60 and 300 seconds for optimum results. The activity of the papain sample is then calculated by plotting a graph, finding the time taken to clot milk at an infinite concentration of papain and then using that value in a formula to calculate the activity.
To introduce a measure of standardisation the amount of milk can be fixed at a certain known concentration. This is done by reacting a known concentration of high grade papain with the milk. The concentration of milk powder solution can then be adjusted to obtain the desired clotting time under fixed reaction conditions. The 'activity of pure papain' at this known amount of milk can then be calculated. Testing the sample papain under the same reaction conditions and same (known) amount of milk will then give an activity relative to the pure papain.
The second method is based on the science of the absorption of light known as absorptiometry. This is the analytical technique for measuring the amount of radiation (or 'colour' of light) absorbed by a chemical solution.
It is known that, for example, a yellow-coloured solution will absorb blue light. (Blue is the 'complement colour' to yellow). The greater the concentration of yellow in the solution the more the absorption of blue light. This is a useful discovery because certain products of chemical reactions are coloured. The more intense the colour, the greater the concentration of product. Therefore by shining the relevant complement colour through the sample liquid the amount of light absorbed can be related to the concentration of product.
Not all 'colours' (or radiations of light) are visible to the human eye. The technique used when the 'colours' extend beyond the visible spectrum is known as spectrophotometry and the instrument used is called a spectrophotometer.
In the second method to determine the activity of a papain sample, a known amount of papain sample is mixed with a fixed amount of casein (the protein found in milk). The reaction is allowed to proceed for 60 minutes at 40°C. After this time, the reaction is stopped by the addition of a strong acid.
The product of the reaction is known as tyrosine which is known to absorb ultra-violet light (invisible to the human eye). The solutions containing the tyrosine are prepared for analysis using the spectrophotometer. The amount of ultra-violet light absorbed by the solution can be related to the number of tyrosine units produced by the papain sample. Hence the greater this number, the greater the activity of the papain sample.
Papain is a proteolytic enzyme derived from papaya used for its anti-inflammatory properties.
Generic Name Papain DrugBank Accession Number DB11193 Background
Papain, also known as papaya proteinase I, is a cysteine protease (EC 126.96.36.199) enzyme that is found in species of papaya, Carica papaya and Vasconcellea cundinamarcensis. The enzyme is found to be localized in the skin of papaya, and is collected from slashed unripe papayas as a crude latex. Papain is used in food, pharmaceutical, textile, and cosmetic industries. While it has been used for the treatment of inflammation and pain via topical administration, papain has also shown to have anthelmintic and tooth-whitening properties. Present in over-the-counter mixture products consisting of different digestive enzymes, its active site contains a catalytic diad that plays a role in breaking peptide bonds. Papain is also used as an ingredient in various enzymatic debriding preparations.