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Part II. Techniques for protein quality evaluation: methodology

8. Some chemical and microbiological assay procedures for the evaluation of protein quality

A. Recommended Methods for Kjeldahl Techniques in Total Nitrogen Determination
B. Methods for Analysis of Amino Acid Composition of Pure Protein and Protein Foodstuffs
C. Microbiological Assays and Prediction of Protein Efficiency Ratio
References

Within this chapter more detail is given on some selected chemical and microbiological techniques that may be used for assay of protein quality. All are given in sufficient detail that they can be used without further reference to the original literature. References are given, however, to original reports because these generally give considerably more information on the procedures and precautions that must be taken to obtain accurate results. Where the original literature is available, users are recommended to consult it. It goes without saying that care and attention to detail should be followed throughout, and samples of known composition should be analysed periodically wherever possible so as to ensure accuracy and reproducibility,

Techniques discussed range from the direct determination of nitrogen in food materials by the Kjeldahl technique to the prediction of PER from digestibility and Tetrabymena growth data. The Kjeldahl procedure is a fundamental and basic determination that is needed as a part of every other assay discussed in this volume, whether it be a chemical predictive procedure, a rat bioassay, or a human multi-level balance technique. The in vitro predictive techniques for PER are, in contrast, new and relatively untried procedures for the more rapid prediction of PER. These were developed in the United States because PER, even though known to be severely limited as an assay procedure, remains the "official" procedure for regulatory purposes within North America, and rapid methods for predicting this value are urgently needed by industry. It is thus emphasized that inclusion of procedures in this publication does not constitute official' endorsement of these and only these procedures, but merely that detailed information on a wide range of assays should be made readily available to workers in the field.

It should be noted that the consideration of techniques for scoring of proteins and mixed dietaries from amino acid data has already been discussed and described in chapter 3, together with a fully worked example. Ordinary scoring techniques will thus not be considered further in this chapter except for the rather specialized use in the C-PER procedure.

Sampling and Sample Preparation

A representative sample is essential if analytical data are to be meaningful. The selection of representative samples is most important for the determination of nitrogen and amino acids because samples may be small, particularly in comparison with those used in animal and human evaluations of protein quality.

The sample must be not only representative but also homogeneous. The necessity for consideration of homogeneity increases as sample size for the analytical procedure decreases. To assure homogeneity, samples are usually dried and ground prior to analysis. Associated analyses of moisture and fat may be necessary before final results can be expressed in terms of the original material.

Detailed consideration of sampling procedures for all types of material is available in standard analytical textbooks (1 - 3).

A. Recommended Methods for Kjeldahl Techniques in Total Nitrogen Determination

The procedures are adapted from those of the Association of Official Analytical Chemists (AOAC) and are described for both macro (4) and semi-micro (5) scale. The macro Kjeldahl procedure is for samples difficult to homogenize, and the sample size may be between 1 and 3 9; the semi-micro determination is designed for small quantities (< 300 mg) of homogeneous material. The procedures as described are for food samples and assume that nitrogen is not present in significant quantities in the form of nitrate or N-N or N-O linkages.

Reagents (should be nitrogen-free)

• Sulphuric acid (H₂S0₄ ) S.G. 1.84.
• Mercuric oxide (HgO), reagent grade.
• Potassium sulphate ( K₂ SO. ), reagent grade.
• Sodium hydroxide-sodium thiosulphate solution: Dissolve 600 g NaOH and 50 g Na₂S₂0₃ 5H₂0 in distilled water and make to 1 litre.
• Indicator solution: Methyl red 0.2 9/100 ml ethanol; methylene blue 0.2 9/100 ml ethanol. For mixed indicator, 2 parts methyl red solution are added to 1 part methylene blue solution.
• Boiling chips and/or glass beads.

For macro Kjeldah/ procedure

• Zinc granules, reagent grade.
• Standard HCI or H₂SO₄ solution, 0.5 N or 0.1 N. - Standard NaOH solution, 0.1 N. Note: Standard acid and alkali should be checked against each other.

For semi-micro Kjeldahl procedure

• Boric acid, reagent grade, saturated solution. - Standard HCI or H₂SO₄, 0.02 N.

(i) Macro Kjeldahl Procedure

Apparatus

• Kjeldahl flasks, hard glass, 500-800 ml.
• Heating rack: Heat input adjusted so that 250 ml water at 25°C can be brought to boil in about five minutes.
• Distillation assembly: Mechanical carry-over of NaOH during distillation must be prevented and outlet of condenser must ensure the complete absorption of the ammonia in acid in receiver.

Method

A weighed sample (1-2 9) is placed in flask together with 20 g K₂SO₄, 1.0 g HgO, and 25 ml H₂SO₄. If sample is larger than 2 9, 10 ml of sulphuric acid should be added per extra gram of sample. The flask should be heated gently till frothing ceases (small quantities of paraffin wax can be added to reduce frothing) and then brought to a steady boil. Boiling is continued until the solution clears, and for an additional 30 minutes.

The flask is allowed to cool; 200 ml of distilled water is added and the material further cooled until below 25°C. Sodium hydroxide-sodium thiosulphate solution is added in a layer together with a few zinc granules to prevent bumping.

The quantity of alkali added must be sufficient for the mixture to be strongly alkaline after mixing. Approximate calculations of the quantity needed can be made as follows: A little more than 2 ml of alkalithiosulphate are needed to neutralize each millilitre of sulphuric acid remaining after digestion is complete. During the digestion, approximately 10 ml of acid are used by each gram of fat and 4 ml by each gram of carbohydrate.

The flask is connected to the distillation apparatus and the tip of the condenser is immersed in a selected volume of standard acid containing about 10 drops methyl red indicator. The flask is rotated to mix the contents, and the contents are then heated until all the ammonia has distilled (at least 150 ml distillate). The excess standard acid is titrated with standard NaOH solution. Determinations should be made on all reagents alone and a blank correction applied. The strength and volume of acid used for receiving the evolved ammonia should be based on the expected nitrogen content of the sample used.

Calculation

By normal acid-base titration techniques, with due allowance for reagent blank, the volume of standard 0.1 N acid that was equivalent to the ammonia evolved should be calculated. If this volume is v ml and w is the weight in grams that wasdigested, then

N g/kg = vx1.401/ w as 1 ml 0.1 N acid = 1.401 mg N.

Appropriate factors should be calculated for other acid normalities.

(ii) Semi-micro Kjeldahl Procedure

Apparatus

- Kjeldahl flasks: 30 ml hard glass flasks, 10 ml size for very small samples. - Digestion rack: Commercial heating apparatus, either electric or gas, should be adjusted so that a 30 ml flask containing 15 ml of water at 25°C should come to a boil in not less than two but not more than three minutes. - Distillation apparatus: A onepiece glass commercial-type distillation apparatus is recommended.

Method

Weigh out a sample containing between 1 and 3 mg nitrogen. Total dry weight should not exceed 100 mg; larger samples are discussed below. The sample is transferred to a 30 ml digestion flask and potassium suphate (1.9 + 0.1 9), mercuric oxide (80 + 10 mg), and 2.0 ml concentrated sulphuric acid are added. If sample size is greater than 20 mg dry weight, 0.1 ml H2SO4 should be added for each 10 mg dry material. Small boiling chips should be added and the sample heated gently until all water has been evolved and frothing has ceased; very small pieces of paraffin wax can be used to ease frothing. Digestion time should be between 1/2 hour and 1 hour.

The digest is cooled, diluted with a small quantity of distilled ammonia-free water, and transferred to the distillation apparatus. The flask should be rinsed with successive small quantities of water. An Erlenmeyer flask (100 ml) containing 5 ml saturated boric acid and a few drops of mixed indicator is prepared and placed under the apparatus with the condenser tip below the surface of the solution. Sodium hydroxidesodium thiosulphate solution (10 ml) is added to the apparatus and the ammonia steam distilled. Care should be taken that the water does not become warm, as this would invalidate the assay. At least 15-20 ml of distillate should be collected. After the condenser tip has been rinsed with distilled water, the solution should be titrated with the standard acid until the first appearance of violet end-point. A reagent blank should be determined and subtracted from the titration volume and the nitrogen content calculated:

N g/kg = [(ml HCI-ml blank) x normality x 14.01] / weight (9)

Provided that the proportions of reagents to sample are maintained, larger samples ( ~ 100 mg-300 mg) containing larger amounts of nitrogen can be digested. In these cases, after cooling, the digest should be quantitatively washed into a 25 ml or 50 ml volumetric flask, again cooled and made to volume with distilled water. A suitable aliquot should be transferred to the distillation apparatus and distilled as previously described. Care should be taken that the actual quantity of sulphuric acid so transferred does not exceed the capacity of the 10 ml of sodium hydroxide-sodium thiosulphate solution; the solution being distilled should always be strongly alkaline. The calculation is as described above, with adjustments made for the aliquot volume and dilution volume used:

N g/kg = [(ml HCl-ml blank) x normality x 14.01 x final volume] / weight (g) x aliquot volume

B. Methods for Analysis of Amino Acid Composition of Pure Protein and Protein Foodstuffs

(i) Protein Samples (Pure Proteins or High-Protein Concentration Homogeneous Samples)

1. Weigh out (or pipette, if in solution) 2 - 5 mg of protein into a standard 18 x 150 mm Pyrex test tube* previously washed with dilute NaOH, rinsed, and oven-dried.
2. If the sample is solid material, pipette into the tube either (a) 0.5 ml of reagent grade concentrate HCI followed by 0.5 ml of distilled water, or (b) 1.0 ml of glass-distilled 6N HCI per mg protein. If the sample is a liquid, pipette into the tube an equal volume of reagent-grade concentrate HCI.
3. With an oxygen-gas flame, work the neck of the tube at a point about 1.5 cm from the end in such a way as to draw it down to a capillary with a lumen of about 1 - 3 mm. Take care not to allow the glass wall of the tube to become unduly thin at any point. The "necking down" of the tube can be accomplished most conveniently if a length of glass rod about 3 mm in diameter is temporarily attached to the tube and then aligned so that it is in line with the centre of the tube. During the entire operation, care should be taken not to allow the contents of the tube to get up into the hot area of the neck.
4. Remove the glass rod and insert the tube into a dry ice/alcohol bath in a small Dewar flask.
5. After the acid-sample mixture has solidified in the tube, attach a line from a high vacuum mechanical pump and evacuate to 50 microns Hg pressure or less. A Tygon tube-glass adapter combination is convenient for attaching the sample tube. There should be a dry ice/alcohol trap in the line from the sample tube to the pump to freeze out all volatile and corrosive gases before they reach the pump.
6. After the sample tube has been fully evacuated, lift the tube from the dry ice/ alcohol bath and with a pinpoint oxygen-gas flame, seal off the tube at the neck while it is still under vacuum.
7. Place the sealed, evacuated tube in an upright position for 22 hours in an oven regulated at 110 + 1 C. A mechanical, forced-draft recirculating oven is recommended for this purpose.
8. Remove the tube from the oven, keeping it upright to prevent the contents from getting up into the top. Chill the tube and contents in a refrigerator or ice bath.
9. Remove the top of the tube by (a) making a deep scratch with a triangular file, and (b) touching the tip of a glass rod that has been heated white-hot to the edge of the scratch.
10. Any visible sediment or precipitate (humin) in the hydrolysate must be removed. Centrifugation is recommended, although filtration can be used if a previously washed and dried (to remove ammonia or other ninhydrin-positive compounds) filter is used.
11. Remove the HCI, evaporating the sample to dryness. If the total volume is 2 ml or less, the HCI can be most easily removed as follows: (a) place the cold tube, inclined at about 15 from horizontal, inside the lower portion of a 160 mm (i.e., ground flange) desiccator; (b) replace the desiccator plate; (c) set on top of the plate a small shallow dish containing NaOH pellets; (d) close the desiccator and evacuate continuously with an efficient water aspirator. The HCI will evaporate overnight. If the total volume in the tube before evaporation is more than 2 ml, or if the sample material is needed for immediate analysis, the sample should be taken to dryness in a rotary evaporator or similar device, water added, and the sample taken to dryness again.
12. Add a suitable volume of pH 2.2 citrate buffer (0.20N Na+) to the tube (or flask) containing the dried film of hydrolysed sample. After all soluble material has completely dissolved, the sample is ready for analysis. Any solid humin material that may have appeared during drying must also be removed by filtration (e.g., Whatman, no. 52). If the sample is not to be analysed immediately, transfer it to a glass-stoppered flask and store at 4 C.
13. Pipette suitable aliquots for analysis in each column.
14.  

(ii) Food Samples (Low-Protein Concentration Samples Containing Carbohydrate and/or Lipids)

1. Weigh out a food sample containing 12 + 1 mg N into a 1,000 ml round-bottomed flask fitted with a ground glass neck.
2. Add 300 ml of 6N HCI prepared with distilled water and reagent-grade, concentrated hydrochloric acid.
3. Connect a reflux condenser and arrange a thin glass tube to supply a slow stream of purified N2 gas bubbles below the surface of the acid.
4. Reflux for 24 hours in an electric glass mantle heater, maintaining N2 bubbles throughout the whole period.
5. Cool to room temperature and filter under suction through a medium-porosity, sintered glass filter to remove humin. Wash twice with small quantities of distilled water.
6. Transfer to the flask of a rotary evaporator and evaporate to dryness under vacuum, keeping the temperature of the sample below 40 C.
7. Wash twice with 5 - 10 ml samples of distilled water and re-evaporate to dryness again below 40 C.
8. Dissolve sample in pH 2.2 sodium citrate buffer, transfer to a 25 ml volumetric flask, and make to volume with combined washings and new buffer. If filtration is necessary, it should be done through a previously washed and dried sintered glass filter.
9. The sample can be analysed immediately or stored in a closed container in a refrigerator or, for longer storage, in a deep freeze.
10. Depending on the sensitivity of the amino acid analyser system,aliquots for analysis will be 0.10 - 0.25ml.

(iii} Performic Acid Pre-oxidation for Sulphur Amino Acid Analysis (Cysteine and Cystine as Cysteic Acid and Methionine as Methionine Sulphone) (6)

1. Prepare the performic acid reagent as follows: To 72 ml of 98 - 100 per cent formic acid add 1.5ml methanol (to prevent freezing) and 7.5 ml of 30 per cent H₂O₂. The mixture should be held at room temperature for two hours in a closed vessel for maximum performic acid formation.
2. Weigh out food sample containing about 7 mg N and transfer to a 500 ml ground-neck, round-bottomed flask containing a few drops of the freshly prepared performic acid so as just to dampen the sample. The mixture is cooled to about 10 - 15 C for 30 minutes in a deep freeze. Then 25 ml of performic acid mixture is added, and the mixture is swirled and refrigerated overnight at 4 C.
3. Add 25 ml of distilled water and 2 ml of 40 per cent HBr (to remove excess performic acid); swirl to mix.
4. Evaporate to dryness on the rotary evaporator. Some NaOH should be present in the receiver to absorb the HBr evolved.
5. Add 100 ml 6N HCI and proceed exactly as in method ii, above, steps 2 - 9.
6. For chromatography, 0.1 - 0.25 ml samples are used, but the runs are extended only to the complete emergence of methionine sulphone.

(iv) Determination of Tryptophan

Adapted from Hugli and Moore (7). This simplified procedure has been found satisfactory. For fullest accuracy, however, all the precautions described by the authors should be followed.

Required reagents - Partially hydrolysed potato starch (antioxidant during hydrolysis): Approxi mately 50 9 of potato starch are added to 99 ml of acetone to which 1 ml of concentrated HCI is added. The mixture is held at 50 C for two hours; 25 ml of 1 M sodium acetate are then added to neutralize the hydrolysate, and the slurry is poured into a chromatograph tube and washed with 2 litres of distilled water and then with acetone. The product is then dried in a desiccator using vacuum.

Commercial partially hydrolysed starch is equally suitable, but should be washed with acetone and dried. In each case, the starch is ground to a fine powder before using.

• Sodium citrate buffer (for loading): 0.20N pH 4.25 without Brij 35.
• Sodium citrate buffer (for chromatography): 0.21N pH 5.4 made from standard short-column buffer (0.35 N) for basic amino acids by diluting 3 parts buffer with 2 parts de-ionized water and adjusting pH.

Procedure

1. Weigh accurately a homogeneous sample containing about 1.5 mg N into a polypropylene centrifuge tube (cut with razor blade to 10.9 x 50 mm size).
2. Add partially hydrolysed potato starch to make total weight in tube approximately 60 mg.
3. Add 0.2 ml H20, 1.0 mi 5 N NaOH and 2 drops of 1 per cent octanol in toluene. Mix using a vortex test tube shaker.
4. Insert into standard soft glass test tubes; draw tube into a constriction and evacuate using an electric vacuum pump (high vacuum required) while the sample is cooled in dry ice/acetone. Seal.
5. Hydrolyse for 16 hours at 110 C.
6. Cool, open with care, add about 1 ml of sodium citrate buffer at pH 4.25 and mix with the now gelatinous hydrolysate.
7. Place 10 ml volumetric flask into an ice bath, add 0.42 ml 6N HCI and allow acid to cool. Transfer hydrolysate from tube to flask using 0.5 ml quantities of pH 4.25 buffer. Do not allow temperature to rise. Note: quantities and normalities of acid and alkali should be accurate, since sample is being exactly neutralized.
8. When total volume is about 8 - 9 ml, allow to rise to room temperature and make to volume.
9. Before chromatography (which should be on same day if possible), samples should be centrifuged to clear.
10. hromatography: A 1 ml sample of the neutralized hydrolysate at pH 4.26 (at pH 2.2, the normal pH used for sample loading, there is rapid tryptophan breakdown) is added to the short basic column (0.9 x 12 cm). Beckman PA-35 resin or equivalent is used. Buffer flow rate is 50 ml/hr at a temperature of 52 C. The buffer is sodium citrate (0.21 N for sodium at pH 5.4). Separation is dependent on the specific characteristics of the analyser used. An example of times of elusion obtained using the conditions indicated above is: neutral + acidic amino acids 20 minutes, tryptophan 38 minutes, and Iysine 68 minutes. Lysinoalanine is frequently formed in the alkaline hydrolysis of proteins, and the chromatographic technique used should be checked to be sure that tryptophan and Iysinoalanine can indeed be separated.

(v) General Notes on Chromatography

Operating methodology is dependent on the analyser system being used, and manufacturer's recommendations should be followed. Titanous chloride, however, as a replacement for stannous chloride in the ninhdrin solution (8) improves both the baseline and the ninhydrin stability and is recommended for routine use when available.

The use of internal standards, norleucine for acid and neutral amino acids and aamino4-guanidino-propionic acid for basic amino acids, has been recommended in chapter 2, and should always be used in analysis of protein hydrolysates. The use of these internal standards can serve as a check on ninhydrin deterioration and thus reduce the number of amino acid standard runs that are required. Temperature of column, buffer flow rate, pH, and ionic concentration of buffer are all critical operational criteria, and it is essential that they be kept constant when unknown samples are being compared to standards. The emergence of cystine is an extremely useful indicator of the correctness of operational conditions. It is moved rapidly forward or backward in relation to other peaks by quite small changes in pH. As cystine peaks in food samples can often be very small or even absent, care should be taken that the absence of cystine is a true phenomenon and not an artifact caused by a change in pH making cystine emerge undetected under either alanine or valine. Because the pH for optimal separation is somewhat relative, storage of samples of satisfactory buffers in a refrigerator is recommended so that new batches can be readily adjusted to pH values previously found satisfactory.

Results of amino acid analysis are usually expressed as milligrams of amino acid per gram of nitrogen, the nitrogen having been determined either on the orignal food material or on the hydrolysate, or both. In the latter case, when nitrogen was determined on both, comparison of the nitrogen values obtained can give useful indications of completeness of recovery throughout the entire hydrolytic and evaporation procedure.

Before amino acid data are reported, calculation should be made for total nitrogen recovery. This is determined by multiplication of the milligrams per gram nitrogen found for each amino acid (and for ammonia) by the nitrogen content of each amino acid (or ammonia) and summing these values. While 100 per cent recovery is rarely attainable, poor nitrogen recoveries are often indicative of poor technique.

(vi) Available Lysine

FDNB-reactive Iysine (9, 10)

Reagents

• 1 -fluoro-2, 4-dinitrobenzene (FDNB): For each sample use 0.4 ml FDNB dissolved in 15 ml ethanol.
• Methyl chlorocarbonate CICOOCH₃ (alternate names: methoxy-carbonyl chloride or methyl chloroformate).
• Sodium bicarbonate solution: Dissolve 80 g in 1 litre water.
• Diethyl ether: Important to use peroxide-free material.
• Phenolphthalein solution: Dissolve 40 mg in 100 ml 60 per cent ethanol.
• Sodium hydroxide solution: Dissolve 120 g in 1 litre water.
• Carbonate buffer: Dissolve 19.5 g NaHCOa and 1g Na₂CO₃, in 250 ml water and adjust to pH 8.5.
• Mono 6-N-dinitrophenyl-lysine hydrochloride monohydrate (DNP-lysine): Dissolve

314 mg in 250 ml 8 N HCI. Dilute 10 ml to 100 ml with water and use as a working standard containing the equivalent of 0.1 mg Iysine in a 2 ml aliquot.

Apparatus

- Round-bottomed flasks: 100 ml or 150 ml capacity with necks 8 cm long. - Test tubes: Use stoppered glass tubes graduated at 10 ml. -Shaker: Apparatus with gentle horizontal motion is preferred. - Heating equipment: An apparatus is needed for heating round-bottomedflasks under reflux. Heating mantles are recommended. - Spectrophotometer.

Determination

1. Weigh a quantity of finely ground sample containing about 12 mg reactive Iysine into a round-bottomed flask. For materials of low Iysine content, use a maximum sample weight of 1 9 and adjust subsequent dilutions.
2. Add four antibump glass balls and 10 ml sodium bicarbonate solution. Shake gently by hand until sample is fully wet, but avoid scattering.
3. Add 15 ml FDNB solution, stopper the flask, and shake gently on a mechanical shaker for two hours.
4. Remove ethanol (but not the water) by evaporation on a boiling water bath. This can be checked by weighing the flask until it has lost 12.5 9.
5. Cool the mixture, add 30 ml 8.1 N HCI, and reflux gently for 16 hours.
6. Filter the contents of the flask while still hot through a paper, such as Whatman 541, into a 250 ml volumetric flask.
7. Wash the digestion flask and residue thoroughly with water until the total filtrate is almost 250 ml. When filtrate is cool, make up to volume and mix. (A precipitate of dinitrophenol may form and should be allowed to settle).
8. Pipette 2 ml clear filtrate into each of two stoppered test tubes, A and B. (Should any dinitrophenol be in the aliquot it will be removed by ether during the next stage and not cause interference.} It is convenient to take six hydrolysates through the following stages in one batch.
9. Extract the contents of tube B with 5 ml diethyl ether. Remove and discard as much of the ether layer as possible using a dropping pipette on each occasion.
10. Place tube in hot water (about 80 C) until effervescence from residual ether has ceased, and then cool.
11. Add one drop phenolphthalein solution and then the NaOH solution from a dropping pipette until the first pink appears.
12. Add 2 ml pH 8.5 carbonate buffer and 5 drops (about 0.01 ml each) of methoxycarbonyl chloride. Stopper the tube, shake vigorously, and leave for about eight minutes.
13. Add 0.75 ml concentrated HCI cautiously, with agitation to prevent excess frothing. Remove the remaining gas by gentle shaking.
14. Extract three times with diethyl ether as described above and remove the residual ether as before by placing the tube in hot water.
15. Cool tube and make contents up to 10 ml with water.
16. During the pauses between the manipulation of tube B, tube A is extracted three times with diethyl ether and the residual ether is removed as before. Cool the tube and make the contents up to 10 ml with 1 N HCI.
17. Measure the absorbances of the contents of tubes A and B against water at 435 nm. Reading A minus reading B (blank) is the net absorbance attributable to DNPlysine.
18. Pipette 2 ml of working standard DNP-lysine solution into the tubes A and B and take each tube through steps 9 - 17. The working standard should have a net absorbance of about 0.4 at 435 nm in a 1 cm cuvette and the blank a value of 0.01.
19. The result may be calculated as follows:
    Ws x At x v x 100 x 100 x Cf FDNB-reactive Iysine (9/16 gN) = Wt x As x a x CP
    Ws = weight of standard,expressed as mg Iysine in 2 ml Wt = weight of test material in mg As = net absorbance of standard At = net absorbance of test sample v = volume of filtered hydrolysate (250 ml recommended) a = aliquot of filtrate taken for analysis (2 ml recommended) CP = % crude protein (%N x 6.25) in test sample
    Cf = correction factor for hydrolytic losses. 1.09 can be used for materials virtually free of carbohydrates and 1.2 for vegetable materials.

C. Microbiological Assays and Prediction of Protein Efficiency Ratio

(i) The Four-Enzyme In Vitro Assay for Protein Digestibility

This assay as reported by Satterlee et al. 111 ) is a modification of the procedure of Hsu et al. (12). All samples used for the in vitro digestion study were ground to a fine powder that was able to pass though an 80mesh screen. Glass distilled water was used in preparing all solutions.

1. Ten ml of glass distilled water is added to the powdered sample (amount of sample added to give 6.25 mg protein/ml).

2. The sample is allowed to hydrate for at least one hour, but no more than 24 hours, at 5 C.

3. A three-enzyme solution containing 1.6 mg trypsin,* 3.1 mg chymotrypsin,* and 1.3 mg of peptidase* per ml of glass distilled water is equilibrated to pH 8.0 at 37°C.

4. The sample is also equilibrated to pH 8.0 at 37 C.

5. Upon equilibrating the sample at pH 8.0, 37 C, 1 ml of the three-enzyme solution is added to the sample suspension, and the resulting mixture is stirred while being held at 37° C.

6. At exactly 10 minutes from the time the enzymes trypsin-chymotrypsiri-peptidase are added to the protein sample, stirring in a 37 C water bath, 1 ml of a bacterial protease** solution (7.95 mg enzyme/ml) is added to the sample.

7. Immediately, the solution is transferred to a 55 C water bath.

8. Nine minutes after adding the bacterial protease solution to the sample (19 minutes into the assay), the sample is removed from the 55 C water bath and transferred back to the 37 C water bath.

9. At exactly 10 minutes after the sample has received the bacterial protease (1 minute back in the 37 C water bath), the pH of the enzyme hydrolysate is recorded.

10. The pH measured in step 9 is recorded as the 20-minute pH.

11. In vitro protein digestibility of the sample is then calculated using the following equation: % digestibility = 234.84 - 22.56 x, where x is the pH after the 20-minute incubation (from step 9).

Note: With each sample or set of samples run, a control (ANRC sodium caseinate) must first be run and must have a 20-minute pH of 6.42 + 0.05. This control is needed to ensure the presence of proper enzyme activity prior to running any samples.

(ii) The Inoculation and Growth of Tetrahymena thermophila WH14 on a Food Sample (11)

1. Determine in vitro protein digestibility, and at the same time partially digest the sample. (See section i, above.)
2. Dilute hydrolysate with water to yield a nitrogen concentration of 0.83 mg/ml.(24-hour (66-hour growth ) , growth )
3. Withdraw 3 ml of hydrolysed sample and place in culture tube.
4. Add 3 ml nucleotide solution.
5. Autoclave at 121 C for ten minutes.
6. Aseptically add 5 ml dextrin-vitamin solution
7. Inoculate with 0.02 ml Tetrahymena.
8. Incubate 24 8. Incubate 66 hours at 25°C. 1hours at 25°C.
9. Stop growth by adding 5 ml isotonic formalin.
10. Filter culture through 150g nylon screen and wash screen.
11. Count cell numbers on the Coulter counter or haemocytometer. Record growth at 24 and 66 hours as_ x 104 organisms/ml.

(iii) Computation Procedure for Protein Efficiency Ratio from Digestibility and Tetrahymena Data (T-PER)

When using the haemocytometer, proceed as follows:

1. Determine the in vitro protein digestibility of the protein source using the procedures detailed in section i, above.
2. Obtain the Tetrahymena count of the casein control at 66 hours using the procedures detailed in section ii. If the Tetrahymena count is not within the range of 22.91 to 26.45 x 104 organisms/ml, the procedure should be discontinued at this point, as the casein control is not within the acceptable range for the model.
3. Obtain the Tetrahymena count of the sample at 66, hours using the procedures detailed in section ii.
4. Determine the T-PER by using the following equation: T-PER = 1.192 + 0.04608 (in vitro digestibility) 0.1612 (casein count) + 0.03968 (sample count).

When using the Coulter counter, proceed as follows:

1. Determine the in vitro digestibility of the protein source using the procedures detailed in section i.
2. Obtain the Tetrahymena count of the casein control at 66 hours using the procedures detailed in section ii. If the Tetrahymena count is not within the range of 25.78 to 30.60 x 104 organisms/ml, the procedure should be discontinued at this point as the casein control is not within the acceptable range for the model.
3. Obtain the Tetrahymena count of the sample at 24 and 66 hours using the procedure detailed in section ii.
4. Because the Coulter counter will count both particulate matter and Tetrahymena, an adjustment to the true 66-hour count is necessary. At 24 hours, little if any growth has occurred, as the Tetrahymena are adjusting to the environment. Thus, the 24-hour Coulter count is assumed to represent only particulate matter. This same amount of particulate matter will be present and counted by the Coulter counter at 66 hours. To obtain an accurate Tetrahymena count at 66 hours, subtract the 24-hour Coulter count from the 66-hour count. This is termed "adjusted 66-hour sample count."
5. Determine the T-PER by using the following equation: T-PER = 7.1116 + 0.01524 (in vitro digestibility) 0.25007 (casein count) + 0.03251 (adjusted 66-hour sample count).

Computation Procedure for Protein Efficiency Ratio from Digestibility and Amino Acid Composition Data {CPER)

The C-PER assay is an in vitro assay that has been developed to predict or estimate the PER of food proteins. The assay was developed utilizing more than 85 different foods and food ingredients. The PER for each protein was first determined and was then followed by the determination of in vitro protein apparent digestibility and the essential amino acid profile for each food or food ingredient.

By use of a computer, a model was developed that matched the in vitro digestibility and essential amino acid (EAA) profile of the samples to their respective rat-based PERs. This model is termed the C-PER.

The steps for the calculation of C-PER are listed below. First, the EAA profiles of the unknown sample and a casein standard are corrected for their digestibilities (steps 1 and 2). Each EAA (corrected for digestibility) is then compared to the ideal quantity of that EAA as given in the 1973 FAD/WHO report (steps 3 and 4). The model then rates each protein by examining each EAA and assigning a penalty weight to each EAA, with the weight being increased as the EAA is found to be a lower and lower per cent of what is required by the FAD/WHO pattern (13) for that EAA (step 5). The sample score is then compared to the score for the casein standard (the same type of comparisons that are done when actual rat PER runs are made), and corrected to a casein value equivalent to a PER of 2.5 (steps 7 and 8).

Procedure

1. Determine the in vitro digestibility of the protein and of the reference ANRC casein (see section i, above).
2. Determine the content of each essential amino acid in the sample and the reference ANRC casein, in terms of 9 of EAA/100 9 of protein (P).
3. Express each EAA as a percentage of the FAD/WHO 1973 standard, using the equation EAA% = [ EAA content (from step 2) / (FAO/WHO standard) ] x ( protein digestibility )(from step 1 )

The FAO/WHO standard for each EAA (9 of EAA/100 9 of the protein) is as follows:

4. If all EAA percentages are equal to or less than 100 per cent, proceed to step 5. If any EAA percentages are greater than 100 percent, reduce them to 100 percent and proceed to step 5.
5. Assign a weight to each EAA% (rounding the percentage to the nearest integer) as follows:

and compute the following:

X = ~ EAA% x associated weight Y = ~ weights

6. Divide the sum of the weights (Y) by the sum of the weighted reciprocals (X) for the protein sample and for the casein reference to obtain the "EAA score" for each.
7. Divide the EAA score of the sample by the EAA score of the casein reference to express the sample protein as a ratio of the casein standard (SPC).
8. Compute the C-PER as follows: C-PER = - 2.1074 + 7.1312 (SPC) - 2.5188 (SPCl2

References

1. AOAC, Official Methods of Ana/ysis of the Association of Official/ Analytical Chemists, 1 2th ed,, ed. William Horwitz (Washington, D.C., 1975).

2. M.A. Josly n, Methods in Food Analysis, 2nd ed. (Academic Press, New York and London, 1 970).

3 Y, Pomeranz. and C.E. Meloan, food Analysis: Theory and Practice /Avi Publishing Co., Westport, Conn., USA, 1971).

4. AOAC,Official Methods of Analysis, p.15.

5 Ibid, p. 858.

6. S. Moore, "On the Determination of Cystine as Cysteic Acid," J. Biol. Chem., 238: 235-237 (1963).

7, T.E. Hugh and S.J. Moore, "Determination of Tryptophan Content of Proteins by lon Exchange Chromatography of Alkaline Hydrolysates," J. Biol.. Chem., 247: 2828-2834 (1972).

8. L.P. James, "Amino Acid Analysis: The Reduction of Ninhydrin Reagent with Titanous Chloride,"J. Chromat., 59: 178-18011971).

9. K.J. Carpenter, "The Estimation of Available Lysine in Animal Protein Foods," Biochem. J., 77: 604-610 11960).

10. V.H. Booth, "Problems in the Determination of Lysine," J. Sci. Fd. Agric.., 22: 658-664 11971).

11. L.D. Satterlee, H.F. Marshall, and J.M. Tennyson, "Measuring Protein Quality," J. Amer. Oil Chem. Soc., 56: 103-109 (1979).

12. H.W. Hsu, N.E. Sutton, M.O. Banjo, L.D. Satterlee, and J.G. Kendrick, "The C-PER and T-PER Assays for Protein Quality," Fd. Techno/., 32 (12): 69-73 (1978).

13. Joint FAD/WHO Ad Hoc Expert Committee, Energy and Protein Requirements, WHO Technical Report Series, no. 522; FAO Nutrition Meetings Report Series, no, 52 (WHO, Geneva; FAO, Rome, 1973).

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