Archana Venkateshan - Analytical services Chemist

 
 
Archana Venkateshan - Analytical services Chemist
Proximates Testing – What & Why?
Fat, Protein, Moisture, Ash, Dietary Fibre

Archana Venkateshan - Analytical services Chemist




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                                                    © Silliker Australia Pty Ltd 2017
Archana Venkateshan - Analytical services Chemist
Speaker


Archana Venkateshan
Analytical services Chemist
Mérieux NutriSciences




         © Silliker Australia Pty Ltd 2017
Archana Venkateshan - Analytical services Chemist
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makes no claims, promises or guarantees about the
accuracy, completeness or adequacy of the information
presented in this         INFORM webinar.




                 © Silliker Australia Pty Ltd 2017
Archana Venkateshan - Analytical services Chemist
Who is Mérieux
NutriSciences?

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Archana Venkateshan - Analytical services Chemist
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                                              5
Archana Venkateshan - Analytical services Chemist
CONTENTS

 Why Multiple Methods for Each Test?

 Moisture
 Ash
 Protein
 Fat
 Dietary Fiber
 Calculation of Carbohydrates and
  Energy
 Conclusions

             © Silliker Australia Pty Ltd 2017
Archana Venkateshan - Analytical services Chemist
What Are Proximates?

Five categories:
      Ash
            1 main test method with 2 approaches
      Moisture
            3 main test methods
      Protein
            2 main test methods
      Fat
          3 main test methods
          Some specific test methods

      Fiber
          crude fiber
          Insoluble dietary fiber, Soluble dietary fiber
          More accurate methods – Integrated DF



      These results are used to calculate Carbohydrates & Calories



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Archana Venkateshan - Analytical services Chemist
What Do the Moisture Methods
           Actually Measure?




 Forced Air Oven    Vacuum Oven        Karl Fischer Titration
Evaporates Water   Evaporates Water   Measures water directly

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Archana Venkateshan - Analytical services Chemist
Moisture Method
Advantages/Disadvantages
Method                                          Advantages                                     Disadvantages

Forced Air- measures weight loss during         Applicable to most foods & ingredients         Not suitable for high sugar, volatile or
heating ( use of sand to increase surface are                                                  high fat foods due to > 100°C
and facilitate drying)
                                                Fast (2-3 hrs), inexpensive, easy to           Some foods require several hours
                                                perform
                                                Ovens = large capacity
Vacuum Oven- measures weight loss               Inexpensive                                    Longer test vs. Forced Air (3-5 hrs,
during heating                                                                                 sometimes overnight)
                                                Vaccum allows lower temps to be used           Vacuum maintenance system required
                                                Some foods & ingredients misbehave at
                                                higher temps
                                                Example: high sugar foods carmelize
                                                affecting results


Karl Fischer- measures water directly           Good sensitivity, no heat required             Longer test vs. Forced Air (3-5 hrs,
                                                                                               sometimes overnight)
                                                Can distinguish moisture from non-             Requires more sample preparation and
                                                moisture volatiles (i.e. alcohol in a flavor   titration skills. More expensive
                                                concentrate)                                   instrumentation and maintainence.

                                                Method of choice for many low-moisture         Reagents- safety, disposal concerns
                                                foods or any low moisture food high in
                                                sugar or protein.



                                                                                                                                          9
Archana Venkateshan - Analytical services Chemist
What Does the Ash Method
Actually Measure?

     The analysis of ash content in foods is simply the burning away
     of organic content, leaving inorganic minerals. This helps
     determine the amount and type of minerals in
     food; important because the amount of minerals can determine
     physiochemical properties of foods, as well as retard the growth of
     microorganisms.

Gentle Heating to Remove Water                Combustion to remove
     - Prevents Splattering                    all organic material




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The Ash Method


  Measures  the amount of non-organic material in the food by burning
  away all the organic materials.
       The remaining as are minerals as salts and oxides


  As   a hint: The %ash value is roughly 2X the amount of total metals




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What Do the Protein Methods
Actually Measure?

               Kjeldahl Protein Method

    ACID
      &
  Chemicals


                                  Ammonia – a strong base




         Total Nitrogen value     Total Nitrogen measured
        converted to %protein       by titration with acid
                                                             12
What Do the Protein Methods
             Actually Measure?
        Combustion (aka Dumas) Protein Method




 Total Nitrogen value
converted to %protein



                                  Total Nitrogen measured
                                           directly




                                                        13
The Different Protein Methods


  Kjeldahl   Protein
      Converts all nitrogen to ammonia.
      %Protein calculated using Protein Factors
      Acceptable for nutritional labels

  Combustion    (Dumas) Protein
      Converts all nitrogen to nitrogen molecules.
      %Protein calculated using Protein Factors
      Acceptable for nutritional labels




                                                      14
The Different Protein Methods


  Protein   Factors
      Converts %Total Nitrogen to %Protein.
      KNOWING WHAT THE PROTEIN SOURCE IN THE FOOD IS
       REQUIRED!
          Default for mixed foods = 6.25. Dairy = 6.38.   Wheat = 5.70.
          There are other factors

         Almonds 5.18
         Barley, Oats, Wholemeal 5.83
         Bran 6.31
         Brazil nuts & Peanuts 5.46
         Coconut & other tree nuts 5.30
         Gelatin and Collagen 5.55
         Rice 5.95
         Whole eggs 6.68




                                                                           15
Protein Method Advantages /
Disadvantages
Kjeldahl Advantages                               Kjeldahl Disadvantages
Applicable to all foods                           Measures all organic nitrogen not just protein nitrogen

Most recognized method (around since 1883)        Does not exclude economic adulteration agents (e.g.,
                                                  melamine)
Rapid methods routinely calibrated against this   Corrosive reagents- some present disposal and safety
method                                            concerns
Relatively simple, inexpensive                    Time consuming

Official crude protein method

Combustion Advantages                                Disadvantages
Applicable to all foods                              Measures all organic nitrogen not just protein
                                                     nitrogen
No toxic chemicals                                   Does not exclude economic adulteration agents
                                                     (e.g., melamine)
Automation  Increased speed and capacity,
Faster TAT*
Faster and easier to run

100% Nitrogen recovery

                                                                                                            16
What Do the Fat Methods
               Actually Measure?

  A
  C        triglycerides
  I
  D     waxes
 or                  esters
Base   other ether-soluble stuff


Ether Extracted %Fat = most fat + some other stuff
 Acid/Ether %Fat = all of the fat + more other stuff

                                   Measure the amounts of all fatty acids
                                   Mathematically express as “Fat”




        %Fat = only the triglycerides (FDA “fat”)                           17
The Different Fat Methods (Core Methods?)


  Soxhlet    Fat
         Measures the amount of surface fat and other ether-soluble stuff


  Acid    &/or Alkaline Digestion Fat Methods (Mojonnier Fat Methods)
       Measure all the fat and more other ether-soluble stuff
       This is the old FDA definition of fat
       Can overestimate the %fat. Sometimes used on food labels since over-
        declaring is allowed and this test is cheaper. Can also be used to justify
        zero fat claims.


    Ankom Fat analyser
         It is automated Soxhlet analyser applicable for raw meats , minced
          meats, pet foods , oil seeds and nuts.




                                                                                     18
The Different Fat Methods (Core Methods?)


  Fatty   Acid Profile Fat Method
      Measures only the triglycerides (current FDA definition of fat)
      Required method for nutritional labels
      Also measures each fatty acid separately
              Saturated, Monounsaturated, Polyunsaturated and trans- Fatty Acids
  We measure 37 fatty acid components using Gas chromatography
  technique.




                                                                                    19
What Do the Fiber Methods Actually Measure?




     Dietary Fiber is what
      arrives unchanged
    into the large intestine




                                              20
Dietary Fiber Methods Advantages /
Disadvantages
               Crude Fiber                           Dietary Fiber (AOAC 991.43)

   Advantages            Disadvantages              Advantages            Disadvantages
•Used as a quick        •Does not simulate       •Better simulates      **Some highly-
test and for pet        the human digestion      human digestion        soluble dietary fiber
foods                   system                   •Enzymes remove        is missed by this
•Gives a good           •**Not applicable to     other carbs. Fat,      method
estimate of %dietary    nutritional labels       ash, and protein are   Fructooligosaccharides
                                                                        (inulin), polydextrose,
fiber                                            removed                some resistant starches,
                                                                        etc.


     Codex Fiber (AOAC 2009.01 &
               2011.25)
   Advantages              Disadvantages
•Much better            **The method may
simulation of human     miss some hydrolyzed
digestion               FOS (aka liquid inulin
e.g.: includes 16 hr    or liquid FOS).
enzyme digestion step
•Captures all dietary
fiber including
highly-soluble forms


                                                                                                   21
Important to Consider the Matrix
Critical for proper method selection- This is Why It is Important to Ask
Fat Methods
   FAT_NH3
        Fluid milk, dry milk, ice-cream, cream, cottage cheese, butter, water soluble samples

   FAT_GRAVI
        Cheese, butter, salad dressing, pet food, cereals, mixed entrees

   FAT_ANKOM
        Meat (raw and processed)

        Oil seeds

        Feeds

        Nuts and nut butters




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Important to Consider the Matrix

 Moisture Methods
    MOIST-VO – 100
        Total moisture in cheese, fluid dairy, dry dairy powders, syrups (DE < 58), grains

    Moist-VO – 70 or 65
         Free moisture in powders, sugars, syrups (DE > 58)

    Karl Fischer
         Samples with non-moisture volatiles (flavors with alcohol etc.)




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Carbohydrates


  The amount of carbohydrates
  declared on a label is not measured
  directly.
  ThePrinciple of Measuring
  Carbohydrates
     Measure all the non-carbohydrate
      components
     Subtract those values from 100%
     The left-over amount is declared as
      the amount of carbohydrates in the
      food.


%Carbohydrates = 100% - %fat - %protein -%DF - %ash - %moisture




                                                                  24
Carbohydrates & Calories

    Results from the Proximate tests are also used to calculate Energy
           Energy (kJ /100g )= (Protein% x 17) + (Fat% x 37) + (Carbs% x 17) +
                                (DF% x 8 ) + (Alcohol% x 29)


     Energy Value ( kcal/100 g ) = Energy Value ( kilojoules /100 g ) / 4.18
    Note: 1 Cal is equivalent to 1 kcal
    Yes, the calculated %carbohydrates does include vitamins, caffeine, and other
     non-carbohydrates. The assumption is these levels are too small to matter.




                                                                                     25
Non-Carbohydrates


  What
      are the non-carbohydrate
  components?
     Protein
     Fat
     Ash (minerals and salts)
     Moisture




 %Carbohydrates = 100% - %fat - %protein - %ash - %moisture




                                                              26
Carbohydrates


  When     does the “By Difference” approach not work?
         When the product contains non-carbs that are not protein, moisture, fat,
          or ash.
  Examples
       Vitamins (in dietary supplements?)
       Non-protein nitrogen gets counted as protein

  The    error of the 4 analytical results can be cumulative
    Another approach for carbohydrate estimation is
         Carbohydrate = Total starch +Total sugars




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