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Enzyme Supplement Formulation
Townsend Letter for Doctors & Patients July 2001

I have spent the last decade working with various enzymes ranging from exotic one such as thermal stable DNA polymerases to the more common such as bromelain.1-3 As one with many years of experience with enzymes, I am often asked which enzymes and how much should go into a digestive product. I can understand this because frequently, it appears as if there is really no rhyme or reason as to what is in the blend. I am going to walk us all through how I would go about designing a general enzyme.

A wide variety of enzyme supplements are currently on the market. They are designed to address an equally wide range of health issue. Such health concerns include conditions such as lactose maldigestion, general gastrointestinal distress, flatulence, autism, cancer, weight management, etc. Each one of these areas is often addressed by a particular enzyme supplement. For example, the use of lactase (beta-galactosidase) for the digestion of lactose in lactose maldigesters. Many times, the particular enzyme that is required, such as lactase, is presented by itself. The choice then becomes very easy as to which enzyme one wants. However, often times, there are five, ten, and even more enzymes in a particular product. How does one decide which is right for them? The answer lies in a more important question. How does one's favorite supplement company decide what to offer in an enzyme product? Further, how do they determine how MUCH of each enzyme goes into that blend? Those are the two deciding questions in enzyme supplementation and will be address below.

Please keep in mind that this is just a general digestive aid. It is not meant to completely replace the body's own ability to carry out some digestion. Rather, it is designed to compliment and assist.

There are several issues that need addressing when developing an enzyme digestive aid. Most notably are the targeted ages (i.e. adults would more likely require lactase than juveniles) and geographical location (i.e. the U.S. consumes more beef and dairy than Asia on a per capita basis and might require a more aggressive protease formula). Without further definition, I have made some assumptions; 1) The consumer is an adult; 2) that adult's diet is represented, on average, by the examples I have provided.

The examples cover the range of lacto-ovo (consume egg and milk products) vegetarians to meat eaters. Additionally, each of the meals would appeal to different levels of health-consciousness. These examples seem reasonable but I have included the URL, for the USDA web site incase you prefer to find other representatives for the diet. I am basing my calculations on the ones I selected.

That being said, I have decided the best approach is to take the obvious facts first and that is, that there should be a Protease, a Amylase, and a Lipase.

The enzymes should be as stable as possible over the broadest pH range so they will be functional in both the gastric and intestinal environments. The fungal enzymes fit these requirements better than the bacterial and animal enzymes. Today, fungal enzymes are produced and sold after much

First, the simplest way to do this is to look at a typical nutritional box. From the Nutrition Facts Box on foods, 2000 Cal/day is used as the "average diet". We can make an assumption that the average diet is 30% calories from fat (67 grams), 30% protein (150g) and 40% CHO (200g). If 5.2% of the protein is tyrosine that would give you 7.8 grams or 43,000 micromoles of tyrosine (mw=181).

For an Acid Stable Protease with an activity of 2,000 SAPU / g,
one SAPU is the amount of enzyme, which will liberate one micromole of tyrosine per minute. How this relates to conditions in the stomach and to digesting insoluble protein (e.g. a beef steak) in the diet versus soluble protein (casein) has not been correlated.

In a biomedical text book (White, Handler and Smith) there were six animal proteins with an average tyrosine content of 5.2% (range from 0.8 to 12.6%. How this average relates to the average protein of someone's diet can be highly variable but it should be in the right order of magnitude.

Assuming you have 30 minutes (more on that later) to digest the food, you would liberate 30 micromoles of tryosine per SAPU during 30 minutes of digestion (assuming the enzyme is completely stable for that 30 minutes). For 43,000 micromoles (see above) of tryosine you would need 1,433 SAPUs or 717 mg of the Acid Stable Protease. If you assume that the protein intake is divided among 3 meals per day that would be 239 mg of Protease per meal.

Next, what about carbohydrates (CHO)? It is given that one Sandstedt Kneen Blish Unit (SKBU) will liberate 1.0 grams of limit-dextrin substrate per hour. Based on the activity, it is reasonable to say one SKBU will digest on gram of starch per hour. With 40 g of CHO in the diet, and given an activity of 100,000 SKBU / g, 0.4 mg would be required to digest all the CHO present in an hour.

Finally, it is known from our work that 1.3 g of a lipase at 50,000LU / g ( or a total of 65,000 U) will digest 300 grams of Olive Oil in 1 hour at 37C. Again, if one assumes all the fat is from the oil, then the 67 g would take 14,517U (65,000U /300g = xU/ 67g) to digest to completion. This is the amount in 181.5 mg of Lipase Concentrate at the activity mentioned.

That all being said, we have completely neglected any contribution by endogenous enzymes to the digestive process. Again, an assumption was made that we needed to completely supplement the diet. As a starting point, this then allows one to easily calculate different, moreindividualized, diet plans.

Next, the more difficult way to do this is to find some actual examples of meals, which seem typical. I have taken these off the USDA website and your more than welcome to find other variations, which may more closely suit your requirements. Three meals seem reasonable:

Meal I
- A Glass (cup) of Milk, reduced fat, 2%, protein fortified.

Total protein: (29.083g + 9.717g) 38.8 g
Total fat: (40.045g + 4.871g) 44.916 g
Total carbohydrate: (33.567g + 13.505g) 47.072g

Based on the activity of acid stable protease, the amount of enzyme required to digest the protein in this meal would be:
0.052 x 38.8g = tyrosine present = 10,970?M requiring 365.52 SAPUs for digestion or 182.76mg of the AFP at 2,000 SAPU/g would be required.

Certain assumptions are required here as well. From our assay for lipase activity, the simplest way to do this is to assume all the fat comes from Olive Oil. Without this assumption, the problem becomes unwieldy in complexity. Given due consideration, I believe you will find this a reasonable assumption for the sake of the calculations. Based on the activity of our fungal lipase, the amount of enzyme required to digest the fat, in this meal would be:

If 67g of oil requires 181.5 mg of Lipase Concentrate, then 44.916g requires 135.22 mg.

Next, assuming all the carbohydrate needs to be digested by an enzyme comparable to the human amylase, I would suggest using fungal alpha amylase. Based on the activity of this enzyme, the amount of enzyme required to digest the carbohydrate in this meal would be: Again, one Sandstedt Kneen Blish Unit (SKBU) will liberate 1.0 grams of limit-dextrin substrate per hour. Based on the activity, it is reasonable to say one SKBU will digest on gram of starch per hour. With 100,000 SKBU / g, and assuming all 47.072g of carbohydrate in the meal is starch, one would need 47.072 SKBU to complete the digestion in an hour or 0.4007 mg per meal / capsule.

Meal II
- HEALTHY CHOICE Chicken Teriyaki with Rice Medley, Mixed Vegetables in Butter Sauce and Apple Cherry Compote, frozen meal.
- Milk, reduced fat, fluid, 2% milkfat, protein fortified, with added vitamin A

Total protein: (17.006g + 9.717g) 26.723g
Total fat: (5.616g + 4.871g) 10.487g
Total carbohydrates: (37.097g + 13.505g) 50.602g

Protease required:
AFP required = 26.723g x 0.052 = 1.390g tyrosine = 7,555uM of tyrosine requiring 125.87 mg of AFP per meal.

Lipase required:
Again, if 67 g requires 181.5mg then 10.487g requires 28.4mg ofLipase @ 80,000 LU /g.

Amylase required:
With 100,000 SKBU / g, and assuming all 50.602g of carbohydrate in the meal is starch, one would need 50.602 SKBU to complete the digestion in an hour or 0.506 mg per meal / capsule.

Meal III
-GREEN GIANT, HARVEST BURGER, Original Flavor, All Vegetable Protein Patties, frozen.
- Milk, reduced fat, fluid, 2% milkfat, protein fortified, with added vitamin A
- 1 container Dannon Sprinkl'ins yogurts, fruit, low fat.

Total protein: (18.00g + 9.717g + 5.069g) 32.786g
Total fat: (4.140g + 4.871g + 1.253g) 10.264g
Total carbohydrates: (7.020g + 13.505g + 22.098g) 42.623g

Protease required:
32.786g x 0.052 = 1.704 g tyrosine = 9,393 ?M tyrosine = 156.48 mg of AFP is required per meal.

Lipase required:
If 67g required 181 mg of Lipase, then 10.264 g would require 27.8 mg

Amylase required:
With a starting material of say 100,000 SKBU / g, and assuming all 42.623g of carbohydrate in the meal is starch, one would need 42.623 SKBU to complete the digestion in an hour or 0.4262 mg per meal.

As a general rule-of-thumb, one glass (12oz) of milk requires 3.3 mg of Lactase (100,000 SKBU/g) to digest 80% of the lactose under conditions of assay. That translates to 330 LACU / glass of milk or per dosage. Again, that would be the amount required for digestion over a very short period of time so the number can be adjusted downward if a more extended digestion period is probable.

Finally, I suggest an inclusion of Phytase. Briefly, phytase digests phytic acid. Phytic acid (Inositol hexakisphosphate) is present in plant fiber and due to all the phosphate groups, has a net negative charge. The negative charge affords good binding to cations of nutrients such as calcium, magnesium, iron, manganese, etc. Additionally, digestion of phytic acid allows greater bio-availability of the freed phosphorus groups. Typically, 500 U are used per Kg of feed. Humans typically do not eat a Kg at a time (on average), so I considerably less. One tenth that amount seems reasonable for the average meal so 50 Units should be sufficient. Phytase is a fantastic enzyme and anyone concerned about mineral absorption / loss should look for it in their enzyme products.

What about Papain and Bromelain? Both are good digestive enzymes. They fall into the same family of cysteine proteases with similar substrate specificities and functions. There should be no problem substituting one for the other. However, I suggest "hedging your bet" and splitting the activity between them. Bromelain has been demonstrated to be an effective anti-inflammatory as well as digestive enzyme, so if you are forced to pick one over the other, I would recommend Bromelain. If one grinds through the calculations to find some sort of equivalency for Bromelain and papain a ratio that has worked well in our laboratory is 230 BTU for bromelain are equal to 4,500,000 PU for papain. Typically, we suggest around 20-25 U of bromelain and 250,000 PU of papain. There is a little more bromelain because, again, I tend to slightly favor it in the formulations.

In addition, I would recommend alpha galactosidase (AG) to take care of the oligosaccharides such as raffinose, stachyose, and melibiose, which are often responsible for complains of flatulence or indigestion. A 1:3 ratio of alpha amylase : alpha galactosidase seems prudent given the high prevalence of gas forming foods. As they are both naturally occurring carbohydrases in many of the organisms used to produce commercial enzymes, they are both amenable to digestive blends.

Finally, I said I would talk more about digestion time. This directly relates to the final formula. The actual dosage used in the formula can be scaled back somewhat from the numbers I used above. That is because in the examples I gave, I used very short digestion times. Also, to keep the calculations from becoming unwieldy, I assume all the digestion had to come from the supplemented enzymes. Those are valid assumptions and will help the formulator, but now we need to apply it to real life. Therefore, one can decrease the levels of those enzymes several fold and still have an efficacious and affordable product. A typical final formula might look something like this:


Acid Stable Protease 240 SAPU
Alpha Amylase 15 SKBU
Lipase 6111 LU
Lactase 139 LacU
Bromelain 20 BTU
Papain 210500 PU
Phytase 52.6 U
Alpha Galactosidase 53 U
Please note that I did not throw in "everything but the kitchen sink," as some blends seem to. For instance, cellulase is omitted. The jury is still out on this but for a general digestive blend, for people without any major problem, I don't see any reason to add it. However, it some instances it could greatly assist in the release of nutrients and should be considered based upon the application.

Enzyme supplementation can be a wonderful tool as a digestive aid.3 With a little time and attention, formulas can be created to address a wide range of specific health. As for what units to look for, I have used some of my favorites based on assays I am comfortable with. The units that are used really do not matter so long as one is able to calculate how much to use based on the enzyme assay. All units are defined by their assay and which ones are used usually is only a reflection of what any particular laboratory is doing at any given time. Some people look for FCC units but the problem is that some of the FCC assays are antiquated and based on old techniques and equipment. Again, the units are just labels of activity based on an assay. So long as you have some label of activity ( and not just the mass) then you can always do a conversion.

While the enzyme unit issue may appear incomprehensible, it easily understood with a little background. Armed with this, you will be able to determine when someone is trying to take advantage of your innocence by trying to confuse you with enzyme units. Let's start with an example of a protease, an enzyme that digests protein. A protease is not a protease is not a protease. What I mean by that is while yes, A,B, and C protease can cut protein X, they will probably all cut at different sites. Additionally, they might use different reactive clefts in the enzyme to do so. Also, they will all function different even in the same assay. That functionality is itself a function of several parameters such as time, temperature, and pH, not to mention, the structure of the enzyme itself. Similar proteases can have different structures. Those nuances are why there are different assays for seemingly similar enzymes. A distillation down to the least common denominator (i.e., saying they are collectively proteases and therefore the same) causes a loss of meaning. That is what makes it look incomprehensible and that is where many enzyme companies try to cash in by confusing you. Each of those assays, is designed to be able to determine the maximal amount of activity that enzyme is capable of, under those conditions. Those conditions, usually are dictated by food applications, because the lion's share of enzymes sold in the world is for food processing. Though, detergents may be close but that we are really not interested in that. It is not a good idea to base enzyme assays on how much blood detergent-X will get out. That would *really* flip consumers out. As an aside, what makes colors bright, is cellulase. It digests the textile frayed strands and makes them look sharper. Anyway, that issue is being addressed in that way.

You see, in enzymology, for all the reasons listed above, each lab has to determine how the enzymes perform for them. The units listed on a specification sheet only give an idea of where to start. When one gets enzymes "in" from a company, what is usual is to take that enzyme, read the data sheet and set up a standard curve of your own. Slight variations in methods (and that is common from lab to lab) can have dramatic differences in how the enzymes perform. Your lab may be at 23C and mine at 25C. That *will* make a notable difference in some cases. Those can be minimized but not eliminated by the scientific method. Also, the application is vital. That standard curve just gives the lab an idea of the activity that enzyme will have in their lab. Then, the lab/company needs to determine how well the enzyme performs in their application. Again, they then, usually, do serial dilutions and assay the enzyme in the application. Often times, they make up their own units during these assay procedures so that they mean more to them. For instance, who cares how many tyrosines a protease will liberate in one minute if you are trying to debitter a powdered drink mix, for instance. Answer: no one. It might give an approximation on where to start with your activity, but it really means very little. What will usually be done, is that lab will create a unit definition in their books based on debittering of protein. In a theoretical case, the assay is really how well the blend works in the application of being a digestive aid in humans. That being said, one suggests starting with a low concentration of the blend (half a cap or a quarter cap...whatever) and then working up until the desired effect is observed. That is how to establish a 'standard curve' if you will. That is the way good enzymology is done. Period.

Recognizing this, the Enzyme Technical Association (ETA), the decided governing body on enzymes in the United States states on their website, under Industry Guidelines for the Use of Enzymes in Dietary Supplements:


In order for consumers and health professionals to be able to compare enzymes, both in the commercial market place and in clinical and other published studies, it is necessary that enzyme potencies be expressed in scientifically sound units of activity.

The ETA guidelines for enzyme activities are:
1. Enzymes in dietary supplements should be measured and labeled on an activity basis, not a weight basis.
2. An authorized, compendial method of measuring and expressing enzyme activity such as Food Chemicals Codex (FCC), United States Pharmacopoeia (USP), Federation Internationale Pharmaceutique (FIP) or Japan Pharmacopoeia (JP) should be adopted whenever possible.
3. For enzymes not adequately covered in compendial sources, it is recommended that activity methods that are used have undergone scientifically sound development and validation procedures.
4. When developing new assay methods, widely available equipment and reagents should be used.
5. The assay temperature should be body temperature, 37oC, unless characteristics of the enzyme preclude this temperature.
6. An assay pH range of pH 4 to pH 5 is recommended whenever possible if the enzyme is to be ingested.
7. A well-defined substrate with adequate lot-to-lot uniformity should be used.
8. At a minimum, validation of enzyme assays should document assay specificity, assay variability, assay linearity and assay sensitivity.

Paying special attention to numbers 2 and 3, one can see that there are some recommended federations from which to use enzyme assays and there is also recognition that not all areas are covered by those. For enzyme units that are not included in the compendial sources mentioned in number two, then number three should apply. The qualification being that those assays are properly validated, which is exactly what any good enzyme laboratory will do anyway.

I have sketched some broad strokes and covered a lot of ground for you. Both patients and doctors should be asking the questions I raised to make sure their enzyme product is really formulated in their best interest. I have provided enzymes with units of activity that I prefer because they are common in the food industry. Others may, of course, be used but we have found in our laboratory that those presented work best.

1. Miller, K.S. and Brudnak, M. (1994) Expression Cloning: PCR Versus Episomal Vectors for Rescue of Transfected Genes. In PCR in Neuroscience (Methods in Neuroscience Vol. 26) Volume Editor G. Sarkar, Academic Press, Orlando, FL.
2. Mark Brudnak 2000. Enzyme Therapy - Part I Townsend Letter for Doctors & Patients. December 209:88-92.
3. Mark Brudnak 2001 Enzyme Therapy - Part II Townsend Letter for Doctors & Patients. January 210:94-98.


Please feel free to contact me.

Mark A. Brudnak PhD, ND
957 Lake Shore Road
Grafton, WI 53024

E-mail Mark

(Wisconsin is in the Central Time Zone)



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