Hydrolysis of Food and Feed Samples

3.1 INTRODUCTION

Foods and feeds are made up of chemical compounds that contain essential amino acids for growth and nutrition. Analyzing the amino acid content is important to ensure proper nutrition. However, these products are produced in bulk processes. As with pharmaceutical production, bulk processes may vary in their output during a production run. Consideration must be given to what constitutes a representative sample. A subsampling strategy is usually required. Amino acid analysis of these products requires multiple approaches to properly analyze the samples for total protein composition. Because foods and feeds are produced in bulk and contain nonprotein constituents, liquid hydrolysis is recommended.

Note: The sulfur-containing amino acids cysteine and methionine and the amino acid tryptophan are not stable in standard acid hydrolysis. Alternative methodologies can be employed to effectively analyze these amino acids.

In this section, we present three different hydrolysis procedures:

• Acid hydrolysis – to determine the total protein content and composition
• Performic acid oxidation – to measure the sulfur-containing amino acids cysteine and methionine
• Alkaline hydrolysis – to assess tryptophan recovery

3.2 ACID HYDROLYSIS OF FOODS AND FEEDS

When analyzing amino acids bound in proteins, the peptide bonds must be broken to free the amino acids for analysis (Figure 1). For feed protein samples to be hydrolyzed, the pH of the sample material and the presence of solids should be considered. As mentioned in the previous sections, the rate or extent of hydrolysis varies across the amino acids present in proteins. This is especially true for proteins bound in food materials. As in any hydrolysis method, the parameters selected must be the result of careful experimentation.

In the analysis of feeds, three sample preparation factors must be kept in mind:

1. Sample processing
2. Sample amount hydrolyzed
3. Volume of acid used for hydrolysis

3.2.1 Sample processing

Feed grains and similar samples are not normally uniform. To make them as uniform as possible, the samples should be ground to a fine powder. High-fat samples can be defatted by standard procedures prior to final grinding. A fine powder allows effective hydrolysis of the feed proteins. The AOAC method (4.1.11, 994.12b, J.AOAC Int. 88, 2005, Amino Acid Analysis of Feeds) calls for the test sample to be ground until it passes through a 0.25 mm, or 60 mesh, sieve (250 µm particle size).

3.2.2  Sample amount

The AOAC method for feed analysis of amino acids recommends the following calculation to be used to determine the sample amount used in a feed analysis.

Calculate the approximate amount of test portion to use as follows:

Ws = 1000/Ns

where Ns = nitrogen content of test portion (%), and Ws = weight of test material equivalent to 10 mg nitrogen content (mg).

In general, this will fall in the range of 100–1000 mg of the material for each sample analyzed.

3.2.3 Acid volume

As noted previously, a large weight excess of acid is necessary for effective hydrolysis of protein samples. Feeds are no different. However, unlike the 100-fold excess discussed in Section 2.1.2, the ratio of acid weight added to sample weight ranges from 50- to 500-fold in the AOAC Method 994.12. This range and the presence of bulk nonprotein particulate materials in feeds point to the need for a careful range-finding study prior to routine use for feed analysis.

3.2.4 Internal standards

The use of an internal standard (IS) best compensates for variable hydrolysis of the individual amino acids of the sample. Waters recommends using Norvaline (Nva) as IS for AccQ•Tag Ultra on UPLC, and Alpha-Aminobutyric Acid (AABA) or Norleucine for AccQ•Tag on HPLC. The internal standard recommended in the AOAC method 994.12 for feed analysis is Norleucine. Caution should be taken when choosing an IS to ensure the desired resolutions can be achieved between the amino acid peaks in the chromatography.

3.2.5  AOAC 994.12 Amino Acids in Feeds

The literature contains a variety of methods that can be adapted to the analysis of amino acids in feeds. The method presented here is adapted from AOAC Method 994.12 Amino Acids in Feeds.

3.2.5.1  Apparatus and glassware:

• Analytical balance, readability ±0.1 mg
• Balance, top loading
• Bottle, 50 mL; polyethylene
• Digestion tubes, boiling flasks are suitable
• Digestion block, heating mantle or water bath is suitable
• Filter units, 0.22 µm (Millex GS, Millipore are suitable)
• pH meter, calibrated with buffers of pH 2.0, 4.0, and 7.0
• Reflux condenser
• Rotary evaporator
• Glass beakers, 250 and 1000 mL
• Erlenmeyer flask, 150 mL
• Round-bottom evaporating flask, 1000 mL
• Graduated cylinders, 100, 500, and 1000 mL
• Volumetric flask, 1000 mL
• Volumetric pipets, 10 and 20 mL
• Sintered glass filter, porosity 10–15 µm
• Syringes

3.2.5.2 Reagents

• DL-norleucine, crystals
• Hydrochloric acid, concentrated
• Sodium hydroxide, 30% solution (30 g/100 mL)
• Phenol, crystals
• Thiodiglycol, 98% solution
• Tri-sodium citrate dihydrate
• pH buffers, pH 2.0, 4.0, and 7.0

3.2.5.3 Preparation of solutions

Sodium citrate buffer—pH 2.20

1. Weigh 19.60 g tri-sodium citrate, dihydrate in 1000 mL beaker.
2. Dissolve in approximately 800 mL H₂O.
3. While stirring, add 10 mL 98% thiodiglycol solution, and 15 mL HCl.
4. Transfer solution quantitatively into a 1000 mL volumetric flask and dilute to mark with H₂O.
5. Filter buffer solution through sintered glass filter.
6. Adjust pH to 2.20 with HCl or 2 M NaOH.

6 M HCl–phenol solution

1. Weigh 1 g phenol crystals into tared 1000 mL beaker.
2. Dissolve crystals in 500 mL H2O. While stirring, slowly add 500 mL HCl.

Hydrochloric acid solution—1 M

1. Pour approximately 800 mL H₂O into 1000 mL volumetric flask.
2. Add 83.3 mL HCl, using pipet.
3. Dilute to the mark with H₂O and mix thoroughly.

Hydrochloric acid solution—0.1 M

1. Pour approximately 800 mL H₂O into 1000 mL volumetric flask.
2. Add 100 mL 1 M HCl, using pipet.
3. Dilute to the mark with H₂O and mix thoroughly.

Sodium hydroxide solution (2 M NaOH)

1. Weigh 80.0 g NaOH in tared 1000 mL beaker.
2. Slowly dissolve pellets in beaker in ca 600 mL H₂O.
3. Cool solution and transfer quantitatively to a 1000 mL volumetric flask.
4. Dilute to mark with H₂O and mix thoroughly.

Internal standard solution

1. Accurately weigh 195–200 mg DL-norleucine crystals into a tared 150 mL Erlenmeyer flask.
2. Dissolve with 100 mL 1 M HCl.
3. Transfer solution quantitatively into a 1000 mL volumetric flask and dilute to mark with H₂O.

3.2.5.4 Hydrolysis procedure

1. Accurately weigh 100–1000 mg finely ground test sample to the nearest 0.1 mg (equivalent to ca. 10 mg nitrogen content) into labeled digestion tubes. Use calculation described in above section to determine actual amount of sample to use.
2. Add 50 mL 6 M HCl-phenol solution to weighed portion and briefly stir. Add 2–3 pieces of boiling chips to solution.
3. In a fume hood, hydrolyze under reflux for 24 h at 110–120 °C using heating block or water bath equilibrated to temperature.
4. Remove digestion tubes from heat and cool to room temperature.
5. Add 20 mL norleucine internal standard solution to each test solution using a volumetric pipet. Mix solutions by swirling flasks.
6. Filter hydrolysates through sintered glass filter into labeled 1000 mL round-bottom evaporating flasks.
7. Connect flasks to rotary evaporators and evaporate at 60 °C to dryness.
8. Wash by adding approximately 20 mL H2O and repeat evaporation. Repeat washing and evaporating steps twice.
9. Remove flask from evaporator.
10. Add 50 mL sodium citrate buffer to evaporated hydrolysate, mix well, and transfer into labeled 50 mL polyethylene bottle.
11. The sample is ready to be prepared for derivatization.

3.3 PERFORMIC ACID OXIDATION FOR THE DETERMINATION OF CYSTEINE, CYSTINE, AND METHIONINE

Cysteine and methionine are critical amino acids in the analysis of feed materials; both are growth-limiting for the animals consuming the feed. Because standard acid hydrolysis conditions do not work for these specific amino acids, an alternative oxidation using performic acid is commonly performed. This approach converts cysteine and cystine to cyanuric acid, and methionine to methionine sulfone (Figure 3). The samples can then be acid-hydrolyzed and derivatized effectively.

In literature, there are multiple versions of performic acid oxidation. Although the overall processes are similar, their specifics differ. This guide presents two procedures to use as possible starting points. The first method is from the AOAC 994.12. The second is an alternative method based on MacDonald et al, 1985. Careful evaluation and optimization is necessary before you commit to a specific approach.

3.3.1  Performic acid oxidation, method 1, AOAC 994.12

3.3.1.1  Apparatus and glassware

• Analytical balance, readability ±0.1 mg
• Balance, top loading
• Bottle, 50 mL; polyethylene
• Digestion tubes, boiling flasks are suitable
• Digestion block, heating mantle or water bath is suitable
• Filter units, 0.22 µm (Millex GS, Millipore are suitable)
• pH meter, calibrated with buffers of pH 2.0, 4.0, and 7.0
• Reflux condenser
• Rotary evaporator
• Glass beakers, 250 and 1000 mL
• Erlenmeyer flask, 150 mL
• Round-bottom evaporating flask, 1000 mL
• Graduated cylinders, 100, 500, and 1000 mL
• Volumetric flask, 1000 mL
• Volumetric pipets, 10 and 20 mL
• Sintered glass filter—porosity 10–15 µm
• Ice bath
• Syringes

3.3.1.2 Reagents

• Formic acid, 88%
• Hydrogen peroxide, 30%
• Sodium metabisulfite
• DL-norleucine, crystals
• Hydrochloric acid, concentrated
• Sodium hydroxide, 30% solution (30 g/100 mL)
• Phenol, crystals
• Thiodiglycol, 98% solution
• Tri-sodium citrate dihydrate
• pH buffer, pH 2.0, 4.0, and 7.0

3.3.1.3  Preparation of solutions

Sodium citrate buffer—pH 2.20

1. Weigh 19.60 g tri-sodium citrate; dihydrate in 1000 mL beaker.
2. Dissolve in approximately 800 mL H₂O.
3. While stirring, add 10 mL 98% thiodiglycol solution and 15 mL HCl.
4. Transfer solution quantitatively into a 1000 mL volumetric flask and dilute to mark with H₂O.
5. Filter buffer solution through a sintered glass filter.
6. Adjust pH to 2.20 with HCl or 2 M NaOH.

6 M HCl–phenol solution

1. Weigh 1 g phenol crystals into tared 1000 mL beaker.
2. Dissolve crystals in 500 mL H₂O. While stirring, slowly add 500 mL HCl.

Hydrochloric acid solution—1 M

1. Pour ca. 800 mL H2O into 1000 mL volumetric flask.
2. Add 83.3 mL HCl, using pipet.
3. Dilute to the mark with H₂O and mix thoroughly.

Hydrochloric acid solution—0.1 M

1. Pour ca. 800 mL H₂O into 1000 mL volumetric flask.
2. Add 100 mL 1 M HCl, using pipet.
3. Dilute to the mark with H₂O and mix thoroughly.

Sodium hydroxide solution (2 M NaOH)

1. Weigh 80.0 g NaOH in tared 1000-mL beaker.
2. Slowly dissolve pellets in beaker in about 600 mL H₂O.
3. Cool solution and transfer quantitatively to a 1000-mL volumetric flask.
4. Dilute to mark with H₂O and mix thoroughly.

Internal standard solution

1. Accurately weigh 195–200 mg of DL-norleucine crystals into a tared 150 mL Erlenmeyer flask.
2. Dissolve with 100 mL 1 M HCl. Transfer solution quantitatively into a 1000 mL volumetric flask and dilute to mark with H₂O.

Performic acid reagent

1. Prepare in hood. Weigh 25 mg phenol crystals in a 25-mL test tube.
2. Add 0.5 mL of 30% H₂O₂, using a micropipette.
3. Add 4.5 mL of 88% formic acid solution.
4. Cover test tube with stopper and let mixture stand 30 minutes at room temperature.
5. After 30 minutes, place the test tubes in an ice bath and cool the performic acid mixture for 15 minutes.
6. Prepare reagent just before use.

 3.3.1.4 Performic acid oxidation

1. Accurately weigh 100–1000 mg of finely ground test sample to the nearest 0.1 mg (equivalent to about 10 mg nitrogen content) into labeled hydrolysis tubes. Use the calculation described earlier to determine the actual amount of sample to use.
2. Put a magnetic stirrer into each tube and place digestion tubes in an ice bath (0 °C).
3. After both the performic acid and the test portion have cooled at least 15 minutes, add 5 mL of performic acid reagent into each digestion tube; stopper or cap all tubes and stir for 15 minutes.
4. Return digestion tubes to the ice bath and let samples oxidize 16 hours.
5. Remove the glass stoppers and add about 0.84 g sodium metabisulfite to decompose the performic acid. Stir for 15 minutes to liberate SO₂.
6. The sample is now ready for the acid hydrolysis stage.

3.3.1.5 Hydrolysis procedure

1. Add 50 mL of 6 N HCl–phenol solution to weighed portion and briefly stir. Add 2–3 pieces of boiling chips to the solution.
2. In a fume hood, hydrolyze under reflux for 24 hours at 110–120 °C using a heating block or water bath equilibrated to temperature.
3. Remove the digestion tubes from heat and cool to room temperature.
4. Add 20 mL of norlecucine internal standard solution to each test solution using a volumetric pipet. Mix the solutions by swirling flasks.
5. Proceed with steps (a–d) or (e–g) below.
1. Filter hydrolysates through a sintered glass filter into labeled 1000 mL, round-bottom, evaporating flasks.
2. Connect the flasks to rotary evaporators and evaporate under vacuum at 40 °C to approximately 0.5 mL Note: Do not let solution evaporate to dryness.
3. Remove flask from evaporator.
4. Add 50 mL sodium citrate buffer to evaporated hydrolysate, mix well, and transfer into labeled 50 mL polyethylene bottle.
5. Filter hydrolysates into 250 mL vacuum flask through sintered glass filter, then transfer filtrate to 250 mL beaker.
6. Place beaker in ice bath.
7. Partly neutralize hydrolysates with ca 40 mL 7.5 M NaOH, while stirring. (Note: Temperature cannot exceed 40 °C.) Adjust pH to 2.20 using 2 M NaOH.

     6. The sample is ready to be prepared for derivatization.

3.3.2 Performic acid oxidation and acid hydrolysis for cystine and methionine (method 2) based on MacDonald et al, 1985

Note: Literature contains many different references for this assay. No two agree on the amounts of performic acid and hydrogen bromide or the evaporation temperature. Changing the amounts and/or temperature will affect the time of evaporation in Step 7.

3.3.2.1  Materials

• 25 x 150 mm tubes with caps
• Ice and ice bath
• Volumetric pipets
• Vacuum evaporator
• Clean nitrogen source
• Oven or heat block
• Volumetric glassware
• Performic acid
• Hydrogen peroxide
• Formic acid
• Ultrapure water
• HBr solution, 48% by weight

3.3.2.2  Performic acid reagent

1. Add one volume of 30% hydrogen peroxide to nine volumes of 88% formic acid.
2. Let the mixture stand for one hour, swirling it frequently.
3. Place mixture in an ice bath for 30 minutes, and then use it immediately.

 3.3.2.3  Procedure

1. Weigh samples corresponding to approximately 20 mg protein (to the nearest mg) into 25 x 150 mm tubes.
2. Place the samples into an ice bath for 30 minutes.
3. Add 10 mL of cold performic acid to the samples, swirl them gently, and cap with a Teflon-lined cap.
4. Store the samples (still in plenty of ice) at 0 °C overnight (16 hours) in a refrigerator.
5. After 16 hours, with the samples still at 0 °C, add 3 drops of octanol and then 3 mL of chilled 48% HBr, swirling the samples slowly. Do this step in a hood. Allow the samples to stand at 0 °C for 30 minutes.
6. Evaporate to dryness with a vacuum evaporator at 37 °C. This will take 1–2 hours.
7. Add 5 mL of 6 N HCI to the tubes. Purge with nitrogen for 30 seconds, and then cap immediately.
8. Place the samples in a 110 °C oven for 24 hours.
9. Remove them from the oven and allow to cool.
10. Add 10 mL of working internal standard, 5.0 µmol/mL, and mix thoroughly. Note: The actual concentration of the internal standard used should be calculated to give the same amount on injection into the instrument.
11. Quantitatively transfer the samples into 250 mL volumetric flasks, rinsing the sample tubes with HPLC-grade water, and filling the flasks to mark with the washings.
12. Filter approximately 1 mL of samples from Step 12, through a 0.45-µm sample filter. If derivatization does not immediately follow, store the capped samples in a freezer. Note: Centrifugation can replace this filtration step. Centrifuge to produce a clear supernatant.

3.4  ALKALINE HYDROLYSIS FOR THE ANALYSIS OF TRYPTOPHAN IN FEEDS

Under the standard conditions of acid hydrolysis, tryptophan (Trp) is unstable and cannot be analyzed effectively. Base hydrolysis is proposed as an alternative method for the release and analysis of this amino acid in feeds. This method uses 4.2 M NaOH to hydrolyze the protein. An advantage of this method is that upon completion there is no need for a derivatization step. Instrumental analysis using UV detection at 280 nm is all that is required.

The procedure presented here is adapted from AOAC Method 988.15 Tryptophan in Foods and Food and Feed Ingredients.

3.4.1  Apparatus and materials

• Modified micro-Kjeldahl flasks (available from Ace Glass, Inc.) – 25 mL micro-Kjeldahl flasks with 12 mm I.D., 15 cm long neck. Neck constricts to about 6 mm I.D., 5 cm above the bulb of flask.
• Vacuum pump
• Membrane filter apparatus and filters of 0.45 µm
• Dry ice
• Water bath
• Ethanol for water bath
• Volumetric pipets and glassware
• pH meter

3.4.2  Reagents

• Water, purified by Milli-Q system (Millipore Corp.), or equivalent
• 4.2 M NaOH
• 1-octanol
• pH 4.25 sodium citrate buffer solution
• HCl
• Tryptophan standard solution
• Stock Solution—1 mg/mL. Dissolve 250 mg L-tryptophan in 100 mL of H2O with six drops HCl. Dilute to 250 mL with H₂O.
• Working Solution I—0.1 mg/mL. Dilute 10 mL stock solution to 100 mL with H₂O.
• Working Solution II—0.04 mg/mL. Dilute 4 mL stock solution to 100 mL with H₂O.
• Refrigerate standard solutions when not in use. Prepare fresh solutions monthly.

3.4.3  Preparation of test sample

1. Grind laboratory sample in a centrifugal mill fitted with a 1 mm screen; mix thoroughly.
2. Weigh a test portion containing 100 mg protein into a modified micro-Kjeldahl flask.
3. For test samples with >5% lipids:
   1. Add 10 mL of petroleum ether to the weighed test portion in flask.
   2. Swirl gently to mix and sonicate for 20 minutes. Let settle. Centrifuge if necessary.
   3. Siphon off as much petroleum ether as possible, taking care not to remove any solids.
   4. Evaporate the remaining petroleum ether under a gentle stream of N₂. Move on to hydrolysis.
4. For test samples with <5% lipids: move on to hydrolysis.

3.4.4  Base hydrolysis procedure

1. De-aerate 4.2 M NaOH by bubbling with N₂ for 10 minutes.
2. Add 10 mL of de-aerated 4.2 M NaOH to each flask.
3. Add three drops of 1-octanol.
4. Immediately freeze the treated solution in a dry ice/ethyl alcohol bath. Then remove the flask from the bath and evacuate to 10 mm.
5. Turn off the vacuum and seal the flasks.
6. Place the sealed flask in a beaker containing H₂O at room temperature until the test solution melts.
7. Place the flask in a 110 °C oven for 20 hours.
8. Let flasks cool to room temperature.
9. Rinse the neck of flask with 1 mL of pH 4.25 sodium citrate buffer solution, collecting rinse in a beaker.
10. Quantitatively transfer hydrolysate to same 50 mL beaker, rinsing the flask with two portions of pH 4.25 sodium citrate buffer solution.
11. Neutralize the solution with 3.5 mL of HCl and stir vigorously. Adjust pH to 4.25 ± 0.05.
12. Quantitatively transfer the solution to a 25 mL volumetric flask and dilute to volume with H₂O.
13. Pour the solution into a 40 mL centrifuge tube and centrifuge for 20 minutes at 1150 G.
14. Filter the supernatant through glass filter paper (Whatman GF/A).
15. Transfer the filtrate to a centrifuge tube and centrifuge for 10 minutes at 23,000 G.

Note: At this point you can take an aliquot of the supernatant to UV (289 nm) analysis, without derivatization.