Proximate analysis

Department of Community Nutrition – Faculty of Human Ecology – IPB

Presented by AZG team

Content

1. Introduction
2. Basic technique
3. Moisture analysis
4. Ash analysis
5. Fat analysis
6. Protein analysis
7. Carbohydrate analysis
8. Fiber analysis

References

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Introduction

Source: Miller 1996, p. 626

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Importance of nutrient analysis

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Introduction

Trends and demands

• Food industry
• Governmental agencies
• Universities
• Consumers
• National and international regulations 🡪 official methods
• Challenge
• Monitor food composition
• Ensure quality and safety of food supply
• Analysis part of quality management (farm to fork)

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Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 4/42

Introduction

Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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Choice and validity of method

Validity of method

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 5/42

Definisi presisi dan akurasi secara grafik

Introduction

Official method

• AOAC International

🡪 an organization begun in 1884 to serve the analytical methods needs of government regulatory and research agencies. The goal of AOAC International is to provide methods that will be fit for their intended purpose (i.e., will perform with the necessary accuracy and precision under usual laboratory conditions).

• AACC International (American Association of Cereal Chemists)
• AOCS (American Oil Chemists’ Society)
• SNI

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Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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Table of content of 2007 official methods of analysis of AOAC International

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Basic technique

Proximate

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Refers to determining the major components of moisture, ash (total minerals), lipids , protein , and carbohydrates

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% total carbohydrate

= 100% - (% moisture + % total fat + % crude protein + % ash)

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🡺 Nutritional labelling

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Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 9/42

Basic technique

Titimetry

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Traditional technique

Stoichiometry based measurement

• Relationship between the relative quantities of substances taking part in a reaction or forming a compound, typically a ratio of whole integers
• Can be used to predict how much reactant is needed to create a certain amount of product or to predict how much of the product will be formed from a certain amount of reactant

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Source: Nielsen 2010, p. 231.

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 10/42

Basic technique

Spectrophotometry

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Basic principle

• Interaction of electromagnetic radiation with matter
• UV, Vis, IR, NMR

Sources: Nielsen 2010, p. 380

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 11/42

Basic technique

Spectrophotometry

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Sources: Nielsen 2010, p. 380

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 12/42

Basic technique

Chromatography

🡪a general term applied to a wide variety of separation techniques based on the partitioning or distribution of a sample (solute) between a moving or mobile phase and a fixed or stationary phase

Sources: Belitz et al. 2009; Nielsen 2010, p. 476.

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 13/42

Basic technique

Chromatography

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Sources: Nielsen 2010, p. 476

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Moisture analysis

Department of Community Nutrition – Faculty of Human Ecology – IPB

Content

1. Introduction: why? Moisture content? Water in food?
2. Oven drying method
3. Distilation method
4. Chemical method
5. Physical method

References

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 16/42

Why?

• Total solids: Dry matter remains after moisture removal
• Moisture is quality factor (preservation and stability): dried milk, powdered egg, jams, glucose syrup, processed meat, etc.
• Convenience for packaging and transportation
• Computation of nutritional value
• Determination of uniform basis

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Water in food?

• Free water: retains its physical properties, dispersing agent (colloid) /solvent (salt)
• Adsorbed water: held tightly, occluded in cell walls/protoplasma
• Water of hydration: bound chemically: lactose monohydrate, Na₂SO₄.10H₂O

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Introduction (1/2)

Sources: Fennema 1996, p. 323, Belitz et al. 2009, p. 8; Kusnandar 2010, p. 227; Nielsen 2010, p. 135.

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 17/42

Introduction (2/2)

Sample handling

• Short contact to atmosphere
• Minimal friction during grinding
• Minimal headspace during storage
• Control temperature fluctuation: moist go to cold point

🡪 RH 50%: lose 0.01% moisture in 5s

🡪 RH 70%: lose 0.01% moisture in 10s

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Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 18/42

2 OVEN DRYING METHOD

• Heated under specified condition, calculated the weight loss, simultaneous is allowed
• Internationally approved
• 1 mol in 1 L 🡪 increase the boliling point 0.512^oC
• Two stages is preferable
• Liguid product: steam bath and oven drying
• Dried product: air dried, ground, oven dried
• Decomposition of other food constituents:
• Carbohydrate 100^oC 🡪 6C + 6H₂O
• Hydrolysis 🡪 utilize the moisture, ex: sucrose
• Volatile compounds 🡪 acetic, propionic, butyric acids, alcohols, esters, etc.
• Oxidation of fatty acid 🡪 weight gain
• 365^oC: Critical temperature 🡪 CO₂, CH₄, H₂O, CO

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2 OVEN DRYING METHOD

• Temperature control:

Convection (atmospheric) (w/o fan; 10^oC), forced draft (with fan ;1^oC), vacuum

• Use! tongs, dessicator, prior dried the pans (3h, 100^oC)
• Standard pan: 5.5cm diameter with insert cover (glass fiber disc)
• Crust/lump:

20-30 g of sand/ 3 g of sample: sand pan technique for sticky fruits

• High carbohydrate sample:

🡪 vacuum oven (no more than 70^oC, reduced pressure 25-100 mm Hg)

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 21/42

2 OVEN DRYING METHOD

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 22/42

2 OVEN DRYING METHOD

• Microwave analyzer:
• First precise and rapid technique (10 min vs 1-16 h)
• In process adjustment of the moisture content

Examples:

• Tomato product (AOAC 985.26)
• Meat and poultry product (AOAC 985.14)

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• Infrared drying:
• Involved penetration of heat into the sample (10-25 min)
• High speed
• Suitable for qualitative in-process use
• No AOAC

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 23/42

3 DISTILLATION

• High boiling point solvent, immicible in water

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Process:

• Codistilling the moisture and the solvent 🡪 collecting the mixture that distills off 🡪 measuring the volume of the water

Classification:

• Direct and reflux distillations
• Toluene (110.6^oC), Xylene (137-140^oC), Benzene, Tetrachlorethylene (121^oC, no fire hazard)
• Widely used: reflux with toluene

Examples: Spices (AOAC 986.21), cheese (AOAC 969.19)

Potential error:

1. Unbreaking emulsion 🡪 need cooling
2. Clinging of water droplets to dirty apparatus
3. Water production from the decomposition of sample

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3 DISTILLATION

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 25/42

4 CHEMICAL METHOD

KARL FISCHER TITRATON

• Application:

Low moisture food high in sugar or protein:

Dried fruits and vegetables (AOAC 967.19 E-G), candies, chocolate (977.10), Roasted coffee, oils and fats (984.20)

• Rapid, accurate, no heat
• Involved the reduction of iodine by SO₂ in the presence of water

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• 1 mol water 🡪 1 mol SO₂, 3 mol pyridine (C₅H₅N), 1 mol methanol
• 3.5 mg water = 1 ml reagent
• End point: dark-red-brown

Sources: Fennema 1996, p. 323; Belitz et al. 2009, p. 9; Kusnandar 2010, p. 207; Barrett and

Elmore 1998, p. 1.

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Manual- (left) and automated- (right) KARL FISCHER TITRATON

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 27/42

5 PHYSICAL METHOD

1. Dielectric method:

electrical properties of the water, for sample with moisture no more than 30-35%, ex: cereal, grain

By measuring the change in capacitance or resistance electricity

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2. Hydrometry :

(specific gravity or density and moisture content).

Best for one solute in a medium of water

2.1 Hydrometer (archimedes principle)

ex: beverages, sugar/salt solution (saccharometer), milk (lactometer), .

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Sources: Fennema 1996, p. 323; Belitz et al. 2009, p. 9; Kusnandar 2010, p. 207; Barrett and

Elmore 1998, p. 1.

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 28/42

5 PHYSICAL METHOD

2.2 Pycnometer

Comparing the specific grafity between sample and water

Ex: Sugar syrups (AOAC 932.14B), milk (AOAC 925.22)

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 29/42

5 PHYSICAL METHOD

3. Refractometer

How water in a sample effect the refraction of the light

Ex: syrups (AOAC 9.32.14C), fruits and fruits products (AOAC 932.12, 967.20, 983.17), soft drink, orange juice, milk

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 30/42

5 PHYSICAL METHOD

4. Infrared analysis:

Absorption at wavelengths characteristic of the molecular vibration in water, possible at line

Ex: milk (fat, protein, lactose and total solid, AOAC 972.16)

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5. Freezing point:

Physical property of sample changed

by a change in a solute concentration

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 31/42

Ash analysis

Department of Community Nutrition – Faculty of Human Ecology – IPB

Presented by AZG team

Content

1. Introduction

a. Definition of ash

b. Importance of ash in food

c. Ash contents in food

2. Sample preparation

3. Analytical technique

a. Dry ashing

b. Wet ashing

c. Microwave ashing

d. Other ash measurements

References

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Introduction

Definition

• Ash 🡪 inorganic residue remaining after either ignition or complete oxidation of organic matter in a foodstuff.

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Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 34/42

Introduction

Importance of ash

• Ash content represents the total mineral content in foods.
• Part of proximate analysis for nutritional evaluation.
• Ashing is the first step in preparing a food sample for specific elemental analysis.
• Because certain foods are high in particular minerals, ash content becomes important.
• One can usually expect a constant elemental content from the ash of animal products, but that from plant sources is variable.

Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 35/42

Introduction

Ash contents in food

• The ash content of most fresh foods rarely is greater than 5%.
• Pure oils and fats generally contain little or no ash; products such as cured bacon may contain 6% ash, and dried beef may be as high as 11.6% (wet weight basis).
• Fats, oils, and shortenings vary from 0.0 to 4.1% ash, while dairy products vary from 0.5 to 5.1%.
• Fruits, fruit juice, and melons contain 0.2–0.6% ash, while dried fruits are higher (2.4–3.5%).
• Flours and meals vary from 0.3 to 1.4% ash.
• Pure starch contains 0.3% and wheat germ 4.3% ash.
• Grain and grain products with bran would tend to be higher in ash content than such products without bran.
• Nuts and nut products contain 0.8–3.4% ash, while meat, poultry, and seafoods contain 0.7–1.3% ash.

Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 36/42

Ash content of selected foods

Sample preparation

Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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• A 2–10-g sample generally is used for ash determination
• Milling, grinding, and the like probably will not alter the ash content much
• Preparatory step for specific mineral analyses, contamination by microelements is of potential concern
• Remember, most grinders and mincers are of steel construction
• Repeated use of glassware can be a source of contaminants as well
• The water source used in dilutions also may contain contaminants of some microelements
• Distilled-deionized water always should be used

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 38/42

Sample preparation

Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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Plant materials

• Generally dried by routine methods prior to grinding
• Drying temperature is little consequence for ashing
• The sample may be used for multiple determinations – protein, fiber, and so on – which require consideration of temperature for drying
• Fresh stem and leaf tissue probably should be dried in two stages (i.e., first at a lower temperature of 55◦C, then a higher temperature) especially to prevent artifact lignin
• Plant material with 15% or less moisture may be ashed without prior drying

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 39/42

Sample preparation

Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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Fat and sugar products

• Pre-treatments prior to ashing 🡪 high fat, moisture (spattering, swelling), or high sugar content (foaming) 🡪 loss of sample
• Meats, sugars, and syrups need to be evaporated to dryness on a steam bath or with an infrared (IR) lamp. One or two drops of olive oil (which contains no ash) are added to allow steam to escape as a crust is formed
• Smoking and burning may occur upon ashing for some products (e.g., cheese, seafood, spices). Allow this smoking and burning to finish slowly by keeping the muffle door open prior to the normal procedure
• Sample may be ashed after drying and fat extraction 🡪 In most cases, mineral loss is minimal during drying and fat extraction
• Fat-extracted samples should be heated until all the ether has been evaporated

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 40/42

Dry ashing

Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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• Refers to the use of a muffle furnace capable of maintaining temperatures of 500–600◦C.

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• Water and volatiles are vaporized, and organic substances are burned in the presence of oxygen in air to CO2 and oxides of N2. Most minerals are converted to oxides, sulfates, phosphates, chlorides, and silicates.

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• Elements such as Fe, Se, Pb, and Hg may partially volatilize with this procedure, so other methods must be used if ashing is a preliminary step for specific elemental analysis.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 41/42

Dry ashing

Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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Dry ashing

Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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Advantages of conventional dry ashing

• Safe
• Requires no added reagents or blank subtraction
• Little attention is needed once ignition begins
• Usually a large number of crucibles can be handled at once
• Resultant ash can be used additionally in other analyses for most individual elements, acid-insoluble ash, and water-soluble and insoluble ash

Disadvantages

• Length of time required (12–18 h or overnight)
• Expensive equipment
• Loss of the volatile elements and interactions between mineral components and crucibles
• Volatile elements at risk of being lost include As, B, Cd, Cr, Cu, Fe, Pb, Hg, Ni, P, V, and Zn.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 43/42

Wet ashing

• A procedure for oxidizing organic substances by using acids and oxidizing agents or their combinations.
• Minerals are solubilized without volatilization.
• Wet ashing often is preferable to dry ashing as a preparation for specific elemental analysis.
• Wet ashing often uses a combination of acids and requires a special perchloric acid hood if that acid is used.
• Primary for specific mineral analysis and metallic poisons. Often, analytical testing laboratories use only wet ashing in preparing samples for certain mineral analyses (e.g., Fe, Cu, Zn, P), because losses would occur by volatilization during dry ashing.

Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 44/42

Wet ashing

Advantages

• Minerals will usually stay in solution
• Little or no loss from volatilization because of the lower temperature
• Oxidation time is short and requires a hood, hot plate, and long tongs, plus safety equipment

Disadvantages

• Takes virtually constant operator attention
• Use corrosive reagents
• Only small numbers of samples can be handled at any one time
• If the wet digestion utilizes perchloric acid, all work needs to be carried out in an expensive special fume hood called a perchloric acid hood

Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 45/42

Wet ashing

• Single acid used does not give complete and rapid oxidation of organic material 🡪 a mixture of acids often is used
• Combinations of the following acid solutions are used most often:

(1) nitric acid

(2) sulfuric acid-hydrogen peroxide, and

(3) perchloric acid.

• Different combinations are recommended for different types of samples.
• The nitric–perchloric combination is generally faster than the sulfuric–nitric procedure (AOAC Method 975.03)
• Many analytical laboratories avoid if possible the use of perchloric acid in wet ashing and instead use a combination of nitric acid with either sulfuric acid, hydrogen peroxide, or hydrochloric acid
• Wet oxidation with perchloric acid is extremely dangerous 🡪explotion

Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 46/42

• Both wet ashing and dry ashing can be done using microwave instrumentation, rather than the conventional dry ashing in a muffle furnace and wet ashing in a flask or beaker on a hot plate
• CEM Corporation (Matthews, NC) has developed a series of instruments for dry and wet ashing, as well as other laboratory systems for microwave-assisted chemistry
• Ashing procedures by conventional means can take many hours, the use of microwave instrumentation can reduce sample preparation time to minutes
• Microwave wet ashing (acid digestion) may be performed safely in either an open- or closed-vessel microwave system

Sources: Fennema 1996, p. 322; Belitz et al. 2009, p. 8; Nielsen 2010, p. 135; Kusnandar 2010, p. 223; Nielsen 2010, p. 135; Barrett and Elmore 1998, p. 2.

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Microwave ashing

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 47/42

Microwave ashing

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Sources: Nielsen 2010, p. 380

Microwave closed-vessel digestion system

Microwave open-vessel system

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Fat analysis

Department of Community Nutrition – Faculty of Human Ecology – IPB

Total Lipid Concentration

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 50/42

Total Lipid Concentration

• It is important to be able to accurately determine the total fat content of foods for a number of reasons:
• Economic (not to give away expensive ingredients)
• Legal (to conform to standards of identity and nutritional labeling laws)
• Health (development of low fat foods)
• Quality (food properties depend on the total lipid content)
• Processing (processing conditions depend on the total lipid content)
• The analytical techniques can be conveniently categorized three different types: (i) solvent extraction; (ii) non-solvent extraction and (iii) instrumental methods.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 51/42

Total Lipid Concentration

• Solvent Extraction 
• Lipids are soluble in organic solvents, but insoluble in water
• Sample Preparation
• The preparation of a sample for solvent extraction usually involves a number of steps:
• Drying sample. 🡪 because many organic solvents cannot easily penetrate into foods containing water
• Particle size reduction 🡪 to produce a more homogeneous sample and to increase the surface area of lipid exposed to the solvent
• Acid hydrolysis 🡪 to break the bonds which hold the lipid and non-lipid components together
• Solvent Selection 🡪 The ideal solvent for lipid extraction would completely extract all the lipid components from a food, while leaving all the other components behind.
• The efficiency of solvent extraction depends on the polarity of the lipids present compared to the polarity of the solvent.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 52/42

Total Lipid Concentration

• Batch Solvent Extraction
• Mixing the sample and the solvent in a container, e.g., a separatory funnel 🡪 shaken vigorously 🡪 organic solvent and aqueous phase separate (either by gravity or centrifugation) 🡪 aqueous phase is decanted off 🡪 the concentration of lipid in the solvent is determined by evaporating the solvent and measuring the mass of lipid remaining: %Lipid = 100 � (M_lipid/M_sample).
• Have to be repeated a number of times to improve the efficiency of the extraction process.
• The efficiency of the extraction of a particular type of lipid by a particular type of solvent can be quantified by an equilibrium partition coefficient, K = c_solvent/c_aqueous, where c_solvent and c_aqueous are the concentration of lipid in the solvent and aqueous phase, respectively. 
• The higher the partition coefficient the more efficient the extraction process.

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 53/42

Total Lipid Concentration

• Semi-Continuous Solvent Extraction
• Commonly used to increase the efficiency of lipid extraction from foods.
• The Soxhlet method: sample is dried 🡪 ground into small particles 🡪 placed in a porous thimble 🡪 placed in an extraction chamber, which is suspended above a flask containing the solvent and below a condenser 🡪 flask is heated 🡪 the solvent evaporates and moves up into the condenser 🡪 converted into a liquid that trickles into the extraction chamber 🡪 the solvent builds up in the extraction chamber and completely surrounds the sample 🡪 the sample overflows and trickles back down into the boiling flask 🡪 the solvent passes through the sample 🡪 extracts the lipids and carries them into the flask 🡪 The lipids remain in the flask because of their low volatility 🡪 At the end of the extraction process, the flask containing the solvent and lipid is removed 🡪 the solvent is evaporated 🡪 the mass of lipid remaining is measured (M_lipid).
• The percentage of lipid (M_sample): %Lipid = 100 � (M_lipid/M_sample)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 54/42

Total Lipid Concentration

• Continuous Solvent Extraction
• The Goldfish method: the extraction chamber is designed so that the solvent just trickles through the sample rather than building up around it 🡪 reduces the amount of time required to carry out the extraction (disadvantage: channelling of the solvent, i.e., the solvent may preferentially take certain routes through the sample 🡪 the extraction is inefficient)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 56/42

Total Lipid Concentration

• Nonsolvent Liquid Extraction Methods.
• Babcock Method 
• A specified amount of milk is accurately pipetted into a specially designed flask (the Babcock bottle) 🡪 Sulfuric acid is mixed with the milk 🡪 digests the protein, generates heat, and breaks down the fat globule membrane that surrounds the droplets 🡪 releasing the fat. The sample is then centrifuged while it is hot (55-60^oC) 🡪 the liquid fat rise into the neck of the Babcock bottle 🡪 The neck is graduated to give the amount of milk fat present in wt%.
• The Babcock method takes about 45 minutes to carry out, and is precise to within 0.1%.
• It does not determine phospholipids in milk, because they are located in the aqueous phase or at the boundary between the lipid and aqueous phases.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 57/42

Total Lipid Concentration

• Nonsolvent Liquid Extraction Methods.
• Gerber Method   
• This method is similar to the Babcock method except that a mixture of sulfuric acid and isoamyl alcohol, and a slightly different shaped bottle, are used.
• It is faster and simpler to carry out than the Babcock method.
• The isoamyl alcohol is used to prevent charring of the sugars by heat and sulfuric acid which can be a problem in the Babcock method since it makes it difficult to read the fat content from the graduated flask.
• This method is used mainly in Europe, whilst the Babcock method is used mainly in the USA.
• As with the Babcock method, it does not determine phospholipids.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 58/42

Protein analysis

Department of Community Nutrition – Faculty of Human Ecology – IPB

Contents

1. Introduction

(aim, basic principles, considerations, constrains,

international standardized techniques)

2. Some rapid qualitative tests
3. Kjeldahl
4. Spectroscopy UV-VIS
5. Amino acid analyzer
6. Study case

​

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 60/42

1 Introduction (1/4)

To obtain:

• Protein content: nutrition labelling, pricing 🡪 melamine scandal?
• Content of a particular protein in a mixture: functional property investigation,
• Protein content during isolation and purification of a protein
• Amino acid composition
• Nutritive value of a protein
• Nonprotein nitrogen

​

Sources: Nielsen 2010, p. 135.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 61/42

1 Introduction (2/4)

Basic principles

Determinations of:

• Nitrogen
• Peptide bounds
• Aromatic amino acids
• Dye-binding capacity
• Ultraviolet absoptivity of proteins
• Light scattering properties

Sources: Nielsen 2010, p. 135.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 62/42

1 Introduction (3/4)

Factors for consideration

• Food components: lipids and charbohidrates
• Sensitivity, accuracy, precision, speed, cost of analysis
• Purpose
• Standard (international, regional, national)

​

Constraints

🡪 other components with similar physicochemical properties, e.g. : non coded amino acids, small peptides, nucleic acids, phospholipids, amino sugars, porphyrin, some vitamins alkaloids, uric acid, urea, ammonium ions.

​

​

​

​

Sources: Nielsen 2010, p. 135.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 63/42

1 Introduction (4/4)

International standardized techniques:

1. Kjeldahl method
2. Dumas method
3. Infrared spectroscopy
4. Biuret method
5. Anionic dye-binding method
6. Spectroscopy UV-VIS
7. Amino acid analyzer

​

Sources: Nielsen 2010, p. 136ff; AOAC International 1999

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 64/42

3 Kjeldahl (1/5)

Principle

🡪 Quantify the N content through 3 main steps:

​

1. Digestion

Sample + H₂SO₄ + heat + catalyst 🡪 (NH₄)₂SO₄ + CO₂ + H₂O

catalyst: HgO, Selenium dioxide:Copper sulfate = 3:1

​

2. Neutralization + destillation

(NH₄)₂SO₄ + 2 NaOH 🡪 Na₂SO₄ + 2 H₂O + 2 NH₃

2 NH₃ + 2 H₃BO₃ 🡪 2 NH₄H₃BO₃

indicators: methylene blue & methyl red (blue)

​

3. Titration

2 NH₄H₃BO₃ + 2 HCl 🡪 🡪 2 NH₄Cl + 2 H₃BO₃ (pink)

Sources: Nielsen 2010, p. 136ff; AOAC 1999, Ch. 33, p. 10ff; Andarwulan et al. 2011, p. 120

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 65/42

3 Kjeldahl (2/5)

Calculation

% N = (corrected HCl vol.) x N HCl x 14.007 x 100%

mg of sample

= ml x (mol/1000 ml) x (g N/mol) x (1/mg sampel) x 100%

= (g N/1000 mg sampel) x 100%

= (g N/g sampel) x 100%

= % w/w (w.b.)

% P = % N x F

​

w: weight

w.b.: wet basis

F: conversion factor (see Nielsen 2010, p. 137)

Sources: Nielsen 2010, p. 137; AOAC 1999, Ch. 33, p. 10ff; Andarwulan et al. 2011, p. 120

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 66/42

3 Kjeldahl (3/5)

Advantages

1. Applicable to all types of foods
2. Relative inexpensive (manual system)
3. Official method for crude protein content

​

Limitation

1. Calculate total N 🡪 crude protein

How to recognize protein nitrogen and nonprotein nitrogen of the sample? precipitation by tricloroacetic acid (TCA)

2. Time consuming (2 hours) 🡪 Dumas method (3 minutes)
3. Poorer precision 🡪 Spectroscopy method
4. Corrosive reagent 🡪 Dumas method

Sources: Nielsen 2010, p. 138; AOAC 1999, Ch. 33, p. 12ff.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 67/42

3 Kjeldahl (4/5)

Alternative: Dumas method

Principles (700-1000^oC)

​

Advantages

• No hazardous chemical
• Quick

​

Disadvantages

• Expensive
• Crude protein
• Sample with high content of fat 🡪 explosion

Sources: Nielsen 2010, p. 138; AOAC 1999

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 68/42

3 Kjeldahl (5/5)

Precipitation of protein by TCA

• 2,2,2-trichloroacetic acid
• The most efficient precipitating agent for native protein
• Reversible association reaction
• Protect the native conformation

of protein

• Without cleaving the protein
• A stable “molten globule-like“

🡪 parcially structured inter-

mediate state

• Stabilizing the β-strands at the

N- and C- terminal ends

​

​

​

Sources: Rajalingam et al. 2009, p. 980ff

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 69/42

Basic principles of spectroscopy

• Interaction of electromagnetic radiation with matter
• UV, Vis, IR, NMR

4 Spectroscopy UV-VIS (1/7)

Sources: Nielsen 2010, p. 380

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 70/42

​

​

4 Spectroscopy UV-VIS (3/7)

Sources: Nielsen 2010, p. 138ff; Upstone 2000, p. 13

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 71/42

Calculation

Sampel : 50 ml whole goat milk with total solid 13.57%

Specific gravity at 20^oC is 1.033 kg/L

Assay method: BCA (562 nm), dilution: 1 ml 🡪 100 ml

Average absorbance of 1 ml diluted sample was 0.4576

Calculate the protein content both wet basis and dry basis!

The absorbance analysis for BSA standard (Nielsen 2010, p. 145):

​

​

​

y = 1.11 x + 0.058

​

4 Spectroscopy UV-VIS (4/7)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 72/42

Result

Protein content of the sample:

3.48% w.b.

25.64% d. b.

4 Spectroscopy UV-VIS (5/7)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 73/42

Advantages

1. Simple and rapid
2. No corrosive agent
3. Does not measure nonprotein N
4. More precise than Kjeldahl method
5. Some methods are official
1. Biuret: cereal, meat, soybean
2. Dye-binding: wheat flour, soy products, meat, milk
3. IR: milk, grains, cereal, meat, dairy product

​

4 Spectroscopy UV-VIS (6/7)

Sources: Nielsen 2010, p. 138ff; AOAC International 1999

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 74/42

​

Limitation

1. Instruments are quite expensive
2. Some methods are not common for food mixture (need precipitation and extraction)
3. Protein standard and calibration are needed
4. Some coumpounds may interfere the analysis
5. The basic principles and limitations of each essay should be taking into consideration in selecting the method

4 Spectroscopy UV-VIS (7/7)

Sources: Nielsen 2010, p. 138ff; AOAC International 1999

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 75/42

1. Jelaskan mekanisme pengendapan protein pada saat titik isoelektrik (pI)!
2. Jelaskan kenapa urea terdeteksi sebagai protein pada analisis protein metode Biuret?

​

6 Study case

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 76/42

3. Which method is the most appropriate for:

1. Protein analysis for nutritional labelling?
2. Qualitative protein test for a novel food?
3. Protein adulteration test of imported food? (melamine scandal)

​

​

​

​

​

• Rapid analysis for protein content of milk and cereal?
• Analysing the content and the composition of essential amino acids of milk?

​

​

​

6 Study case

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 77/42

4. Metode analisis apa yang paling cepat dan tepat untuk tujuan berikut ini? Serta jelaskan prinsip kerja dari metode yang dipilih!

1. Analisis kadar protein dari biskuit bayi untuk tujuan Nutritional labelling
2. Analisis kadar protein dari kacang kedelai dan gandum
3. Analisis kadar air dari gula merah
4. Analisis kadar air dari bawah putih
5. Analisis kadar air dari jus apel
6. Analisis kandungan asam amino non-protein dengan menggunakan alat tradisional

​

6 Study case

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 78/42

Carbohydrates analysis

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 79/42

Contents

1. Introduction: aim, basic principles, considerations, constraints and some common techniques
2. Qualitative tests
3. Total carbohydrate
4. Sugars and oligosaccharides
5. Polysaccharides: starch

Study case

References

​

Note : Non-starch polysaccharides (Fibers 🡪 next class)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 80/42

1 Introduction (1/6)

To obtain:

• Qualitative analysis: compositional information
• Quantitative analysis: order of ingredient labels
• Amounts of spesific components , e.g.: … ?
• Adulteration of food ingredients and product, e.g.: …?
• Content of:

1. Total carbohydrate

2. Dietary fiber

3. Other carbohydrate ≈ Complex carbohydrate 🡪 … - …

4. Sugars 🡪 … + …

5. Sugar alcohols

​

Sources: BeMiller 2010, p. 149ff; Andarwulan et al. 2011, p. 144

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 81/42

1 Introduction (2/6)

Basic principles

• Remaining compounds 🡪 extraction
• Reducing group 🡪 reduction of Cu (II) to Cu (I)
• Polarization properties
• Stoichiometric reactions
• Interaction with enzymes
• Dye-binding capacity
• Colour formation
• Light scattering properties

Source: BeMiller 2010, p. 151ff

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 82/42

1 Introduction (3/6)

Factors for consideration

• Purpose and cost
• Standard (international or regional)

​

Constraints

• Interferences

🡪 Food components: lipids, proteins and other compounds may

interfere the spectrophotometric method

🡪 Maillard reaction: keto and aldehydo groups of sugars + amino

groups interferes the colour and destroys the sugars

• Carbohydrates has wide range of solubility
• Particular sugars are acid labile

🡺Often extraction is urgently required

​

​

Sources: BeMiller 2010, p. 135; Andarwulan et al. 2011, p. 158f;

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 83/42

1 Introduction (4/6)

Sources: BeMiller 2010, p. 151, AOAC International; Andarwulan et al. 2011, p. 158f

(chloroform-methanol)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 84/42

1 Introduction (5/6)

Some considerations in extraction

• AOAC: presweetened, ready-to-eat breakfast cereals

🡪 lipids: hexane, sugars: 50% ethanol

• Mono and oligo 🡪 neutral; contaminants 🡪 charged

🡺 weak anion-exchange is used: carbonate (CO₃^2-) or hydrogencarbonate (HCO₃^-)

• Acid susceptibility (sucrose) 🡪 anion-exchange resin first then cation-exchange resin
• Residue: polysaccharides, proteins

​

​

Sources: BeMiller 2010, p. 151; AOAC International; Andarwulan et al. 2011, p. 158f

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 85/42

1 Introduction (6/6)

Some common techniques:

1. By different
2. Specific qualitative test for carbohydrates
3. Polarimetric method
4. Refractometric method
5. Gravimetric method
6. Colorimetric method
7. Enzymatic method
8. Chromatography: paper, GC, thin layer, HPLC

​

Sources: BeMiller 2010; AOAC International; Andarwulan et al. 2011,

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 86/42

​

​

Sources: BeMiller 2010, p. 153; Andarwulan et al. 2011, p. 157f; Winarno 1997, p. 45ff

2 Qualitative tests (1/4)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 87/42

​

​

Sources: BeMiller 2010, p. 153; Andarwulan et al. 2011, p. 157f; Winarno 1997, p. 45ff

2 Qualitative tests (2/4)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 88/42

3 Total carbohydrate

​

3.1 By difference

3.2 Colorimetric method

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 89/42

3.1 By difference (1/2)

Principle

• Quantify the total carbohydrate as the remaining component

​

Calculation

1. % total carbohydrate

= 100% - (% moisture + % total fat + % crude protein + % ash)

2. % available carbohydrate

= % total carbohydrate – (% fiber)

3. % other carbohydrate / % complex carbohydrate

= % total carbohydrate – (% fiber + % sugars)

Sugars: sum of all free mono- and di- saccharides

​

🡪 What are the constituent components of total carbohydrate, available carbohydrate and complex carbohydrate?

Sources: BeMiller 2010, p. 151; AOAC Interntaional; Andarwulan et al. 2011, p. 155

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 90/42

3.1 By difference (2/2)

Advantages

1. Applicable to all types of foods
2. Simple and rapid
3. Official method for total carbohydrate in food labelling

​

Limitation

1. Not specific
2. Poorer precision
3. Can be quantified if only the content of other components are recognized
4. Not applicable for baby food (under 4 years old) 🡪 some regulations

Sources: BeMiller 2010, p. 151; AOAC Interntional

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 91/42

3.2 Colorimetric method (1/3)

🡪 phenol/phenol-sulfuric acid method

​

Principles

Carbohydrate + sulfuric acid + phenol + heat 🡪 yellow orange

• measured at 490 nm

​

• Pentoses + sulfuric acid 🡪 dehydrated to furfural
• Hexoses + sulfuric acid 🡪 dehydrated to hydroxymethyl furfural
• Furfural + hydroxymethyl furfural + phenol 🡪 yellowgold

Sources: BeMiller 2010, p. 151; AOAC Interntional; Nielsen 2010, p. 49

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 92/42

3.2 Colorimetric method (2/3)

• phenol/phenol-sulfuric acid method

​

High pentoses

• E.g.: wheat bran and corn bran containt high of xylose
• Standard: xylose
• Absorbance: 480 nm

​

High hexoses

• E.g.: glucose (common samples)
• Standard: glucose
• Absorbance: 490 nm

Sources: BeMiller 2010, p. 151; AOAC Interntional; Nielsen 2010, p. 49

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 93/42

3.2 Colorimetric method (3/3)

• phenol/phenol-sulfuric acid method

​

Advantages

• Applicable for all foodstuff
• Internationally standardized (AOAC Method 44.1.30)

​

Disadvantages

• Extraction is needed prior to the analysis
• The exact mixture of carbohydrate standard is not easy to determine

Sources: BeMiller 2010, p. 151; AOAC Interntional; Nielsen 2010, p. 49

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 94/42

4 Sugars and oligosaccharides

​

4.1 Total sugars

4.2 Reducing sugars

4.3 Oligosaccharides

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 95/42

4.1 Total sugars (1/2)

4.1.1 Polarimetric method

🡺 Polarization degree ≈ sugar content

​

[α] = 100.α/LC = 100.α/LPD

​

[α]: specific rotation

α: rotation angle of the solution

L: lenght of the flask (dm)

C: concentration (g/100ml)

P: weight of the compound per 100 g solution

D: speific grafity

​

​

Sources: Andarwulan et al. 2011, p. 161f

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 96/42

4.1 Total sugars (2/2)

Advantage: Rapid, non-destructive and quite precise

Limitation

• Other compounds which have polatization properties
• The sample should be transparent
• The sugar content of the sample should not out of the range of polarimater capacity

​

4.1.2 Colorimetric method

• Carbohydrates + strong acid (hydrolysis) 🡪 furfural 🡪 + anthrone 🡪 blue-green (630 nm)

​

​

​

Sources: Andarwulan et al. 2011, p. 161f

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 97/42

4.2 Reducing sugars

​

​

Sources: BeMiller 2010, p. 154; Andarwulan et al. 2011, p. 163

Main principles 🡪 Reduction of Cu(II) to Cu(I) by reducing sugars

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 98/42

4.2 Reducing sugars

​

​

Basic principle of Luff-Schoorl method

2 Cu²⁺ (excess) + reducing sugars 🡪 Cu₂O (precipitate)

2 Cu²⁺(remaining) + 4 I^- 🡪 2 CuI₂

2 CuI₂ 🡪 2 CuI (precipitate) + I₂

Titration

I₂ + 2 S₂O₃^2- 🡪 2 I^- + S₄O₆^2-

​

How to calculate the content of non-reducing sugars?

🡪 Total sugar – reducing sugars

​

How to calculate the content of sucrose?

🡪 Inversion then calculate using conversion factor = 0.95

Sources: BeMiller 2010, p. 154; Andarwulan et al. 2011, p. 163

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 99/42

4.3 Oligosaccharides

Alternative methods:

1. Paper chromatography
2. HPLC
3. GC
4. Enzymic method
5. Mass spectrometry
6. Thin-layer chromatography
7. Capillary electrophoresis

Source: BeMiller 2010, p. 155-160

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 100/42

5 Polysaccharides: starch (1/4)

To obtain:

• Total starch
• Content of amylose
• Content of amylopectin

​

Total starch

1. Hydrolyze starch with:

- acid:

- enzymatic: α-amylase and glucoamylase (amyloglucosidase)

🡺 D-Glucose

2. Analyse the content of total D-Glucose
3. Total starch = [D-Glucose] x 0.9

​

Sources: BeMiller 2010, p. 161; AOAC International; Andarwulan et al. 2011, p. 166ff

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 101/42

Sources: BeMiller 2010, p. 161; AOAC International

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 102/42

5 Polysaccharides: starch (3/4)

Constraints

• Other enzymic activity that would release D-glucose

e.g.: cellulases, invertase/sucrase, β-glucanase

• Other enzymes that would destroy the hydrogen peroxide (enzymic determination of D-glucose)
• The occurance of resistant starch 🡪 starch that escape digestion in the small intestine

- RS1 🡪 inaccessible since trapped whithin a food matrix

- RS2 🡪 nature of the starch granule (uncooked starch)

- RS3 🡪 retrograded starch (recristallized after gelatinization)

- RS4 🡪 has been modified structurally

🡺using dimethyl sulfoxide or included in analysis of fiber

Sources: BeMiller 2010, p. 161; AOAC International 1999

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 103/42

5 Polysaccharides: starch (4/4)

Content of amylose

• Colorimetric
• Dye binding capacity
• Amylose + iodine 🡪 blue (625 nm)

​

Content of amylopectin

🡺 Total of starch – total of amylose

Source: Andarwulan et al. 2011, p. 166ff

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 104/42

Which method is the most appropriate for:

1. Total carbohydrates for food labelling

2. Calculate the content of :

a. Complex carbohydrate

b. Sugars

c. Sugar alcohols

d. β-glucan

e. Inulin

f. Amylopectin

g. Sucrose

3. Total calories for food labelling

4. Total lactose in dairy products

​

​

​

​

Study case

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 105/42

Fiber analysis

​

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 108/42

Contents

1. Introduction:

1.1 What is fiber?

1.2 The role of fiber

1.3 Basic characteristics

1.4 Classification

2. Component of fiber
3. Technical analysis

3.1 Aim of analysis

3.2 Enzymatic-gravimetric technique

3.3 Other possible methods

Study case

References

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 109/42

1.1 What is fiber

​

American Association of Cereal Chemists (Anonymous, 2001a):

‘Dietary fibre is the edible parts of plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the large intestine. Dietary fibre includes polysaccharides, oligosaccharides, lignin and associated plant substances. Dietary fibres promote beneficial physiological effects including laxation, and/or blood cholesterol attenuation, and/or blood glucose attenuation.’

​

Sources: McCleary 2003, p. 4, BeMiller 2010, p. 166

1 Introduction (1/8)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 110/42

1.1 What is fiber

​

Food and Nutrition Board of the US Institute of Health (Anonymous, 2001b):

‘Dietary Fiber consists of nondigestible carbohydrates and lignin that are intrinsic and intact in plants. Added Fiber consists of isolated, nondigestible carbohydrates that have beneficial physiological effects in humans. Total Fiber is the sum of Dietary Fiber and Added Fiber.’

​

​

​

Sources: McCleary 2003, p. 4, BeMiller 2010, p. 167

1 Introduction (2/8)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 111/42

1.1 What is fiber

Dietary fiber

• Those food components which are not ‘available‘ for digestion and absorption in the small intestine to affect blood glucose levels
• The sum of the nondigestible components of a foodstuff or food product
• Identical physiological behaviour: non-digestible oligosaccharides (NDO) and resistant starch as dietary fiber components
• Examples: cellulose, hemicelluloses, lignin, pectin

​

Sources: McCleary 2003, p. 1f; BeMiller 2010, p. 165ff.

1 Introduction (3/8)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 112/42

1.1 What is fiber

Crude fiber

• The residue remaining after acid and basic hydrolysis
• Covers: 50-80% of cellulose, 10-50% of lignin, 20% of hemicelluloses

​

Q1: Is the fiber always carbohydrates?

Q2: Is all polysaccharides other than nonresistant starch are included in fiber?

Q3: What about oligosaccharides?

Sources: McCleary 2003, p. 1f; BeMiller 2010, p. 165ff.

1 Introduction (4/8)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 113/42

1 Introduction (5/8)

1.2 Basic characteristics

• Mostly is a huge polymer
• Complex structure
• Contains many hydroxyl groups
• High water capacity building

Sources: McCleary 2003, p. 1f; BeMiller 2010, p. 165ff.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 114/42

1 Introduction (6/8)

1.3 The role of fiber

• Inadequate fiber ∞ prevalence of heart disease and certain cancers
• Help protect against colon cancer
• Help to keep blood lipids within the normal range

🡪 reduce the risk of obesity, hypertension, cardiovascular

• Diabetics 🡪 slow D-glucose absorption and reduce insulin secretion

e.g.: pectin and hydrocolloids

• Prevent constipation and diverticular disease 🡪 e.g.: mixture of hemicellulose and cellulose
• DRI for dietary fiber 25 g per 2000kcal per day
• Pentosan fraction 🡪 prevent colon cancer and reducing cardiovascular disease

​

Sources: BeMiller 2010, p. 166

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 115/42

1 Introduction (7/8)

1.4 Classification

Soluble fiber

• Soluble in water
• + water 🡪 gel
• Includes:

1. Hemicelluloses not entrapped in a lignocellulosic matrix

2. Native pectin

3. Majority of hydrocolloids/food gums

• Some sources: beans, peas, prunes, jagung, wortel, cauliflower, nuts, pisang, apel, oat dan barley.
• Sesuai untuk makanan cair; sup, minuman, puding

​

​

Sources: McCleary 2003, p. 1f; BeMiller 2010, p. 166

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 116/42

1 Introduction (8/8)

1.4 Classification

Insoluble fibre

• Insoluble in water
• Includes:

1. Cellulose

2. Microcrystalline cellulose added as a food ingredient

3. Lignin

4. Hemicelluloses entrapped in a lignocellulosic matrix

5. Resistant starch

• Some sources : buah-buahan masak, brokoli, kacang buncis, sprout, kubis, radishes, green peppers, bran dan wholegrain bran
• Sesuai untuk makanan padat dan panggang

​

​

Sources: McCleary 2003, p. 1f; BeMiller 2010, p. 165ff

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 117/42

2 Component of fiber (1/5)

Cellulose

• Linear polimer of β-D-glucopyranosyl (PD ≥1000)
• H-bounding between parallel polymers 🡪 strong microfibrils 🡪 strong and rigid
• Abundant in primary and secondary plant cell walls

​

​

Sources: BeMiller 2010, p. 167

Cell-wall polysaccharides of land plants

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 118/42

Hemicellulose

• Heterogeneous group of polysaccharides
• Branched polymer
• Associate with cellulose
• Linear units: D-xylose, D-mannose, D-galactose
• Branch units: L-arabinose, D-galactose, and uronic acids

​

​

Sources: BeMiller 2010, p. 167

2 Component of fiber (2/5)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 119/42

Pectin

• Linear chain of α-D-galactopyranosyluronic acid
• Interspersed by neutral sugar
• Mostly water soluble
• Can be extracted by acid and chelators like EDTA, ammonium oxalat
• Present in the middle lamella of plant tissue

​

Sources: BeMiller 2010, p. 167

2 Component of fiber (3/5)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 120/42

Hydrocolloids/food gums as dietary fibers

• Marine algae 🡪 alginates and carrageenans
• Higher land plant 🡪 cellulose, hemicellulose, pectic polysaccharides
• Non structural components 🡪 guar gums, locust bean gum, inulin
• Bacterial polysaccharides 🡪 xanthan and gellan
• Applications: ice cream, salad dressing, yoghurt, etc.

Sources: BeMiller 2010, p. 163ff

2 Component of fiber (4/5)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 121/42

Lignin

• Non carbohydrate
• Three dimensional
• Water insoluble
• Major component of

cell walls of higher land

plants

• May be covalently linked

to hemicellulose

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Sources: BeMiller 2010, p. 167

2 Component of fiber (5/5)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 122/42

3 Technical analysis

3.1 Aim of analysis

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To obtain:

• Fiber content: nutrition labelling

🡪 Soluble-, insoluble-, and total- dietary fiber

• Analyse the purity
• Calculate the caloric content
• Analyse the specific important fiber

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Sources: BeMiller 2010, p. 166.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 123/42

3 Technical analysis

3.2 Enzymatic-gravimetric technique

Introduction

• Simulated digestion by incubating the food sample with enzymes
• Developed by Williams & Olmstedt (1935), Thomas (1972), Hellendoorn et al. (1975), Furda (1977, 1981), Schweizer & Würsch (1979, 1981), Asp & Johansson (1981)
• Official method for analysis of dietary fibre: AOAC 2000 method 985.29
• Specific for food labeling and control purposes
• AOAC method 991.43
• AACC International Method 32-07.01

Sources: McCleary 2003, p. 1; BeMiller 2010, p. 162.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 124/42

3 Technical analysis

Basic principles

1. Enzymic treatments for starch and protein removal
2. Precipitation of soluble dietary fiber components by aqueous ethanol
3. Isolation and weighing of the dietary fiber residue and correction for protein and ash in the residue

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​

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Sources: McCleary 2003, p. 1; BeMiller 2010, p. 162.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 125/42

3 Technical analysis

Stages

1. Sample preparation
2. Gelatinization
3. Digestion
4. Precipitation
5. Isolation and weighting

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Sources: McCleary 2003, p. 1; BeMiller 2010, p. 162.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 126/42

3 Technical analysis

1. Sample preparation
1. Sample should be low in fat (not more than 10% lipid), dry, and finely ground (0.3-0.5mm mesh screen)
2. Gelatinization
1. Aim 🡪 gelatinize starch granules and make them more susceptible to hydrolysis
2. Heating/boiling at 95-100^oC for 35 min

​

Sources: McCleary 2003, p. 1; BeMiller 2010, p. 162.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 127/42

3 Technical analysis

3. Digestion

• Digestible carbohydrates and proteins are selectively solubilized by enzyme-catalyzed hydrolysis

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Starch

🡪 most problematic component in fiber analysis

🡪 should be completely digested to be removed

Incomplete 🡪 overestimate result

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​

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Sources: McCleary 2003, p. 1; BeMiller 2010, p. 168

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 128/42

3 Technical analysis

Enzymes for digestion

• α-amylase 🡪 unbranched segments of 1,4-linked α-D-glucopyranosyl units 🡪 maltooligosaccharides (PD=3-6)

Termostable amylase is used 🡪 pH 8.2, T 95-100^oC for 35 min,

normally together with gelatinization stage

• Protease 🡪 to break down the protein (T 60^oC for 35 min)
• Debraching enzymes: pullulanase and isoamylase 🡪 1,6 -linked α-D-glucopyranosyl units 🡪 short linear molecules
• Glucoamylase (amyloglucosidase) 🡪 starts at the nonreducing ends of starch chains an relese D-glucose, one unit at a time 🡪 both 1,4 and 1,6 α-D-glucosyl (pH 4.1-4.8, T 60^oC for 30 min)

Sources: McCleary 2003, p. 1; BeMiller 2010, p. 168

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 129/42

3 Technical analysis

4. Precipitation
1. + 4 volumes of 95% ethanol
5. Isolation and weighting
1. Nonsolibilized and / or nondigested materials are collected by filtration
2. Fiber residue is recovered, dried, and weighted
3. Filtration then washed with 78% ethanol, 95% ethanol, and acetone 🡪 dried, weighted 🡪 TDF

Q4: Alternativelly how to calculate TDF?

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Sources: McCleary 2003, p. 1; BeMiller 2010, p. 162.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 130/42

Source: BeMiller 2010, p. 171

Flow diagram of

enzymatic-gravimetric technique

• IDF, SDF

(after digestion/stage 3)

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3 Technical analysis

Calculation

Source: BeMiller 2010, p. 172

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 132/42

3 Technical analysis

Advantages

1. Applicable to all types of foods
2. Quite good precision
3. Official method for fiber content
4. Quite inexpensive compare to other methods

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Limitation

1. Resistant starch is calculated as fiber
2. Quite time consuming

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 133/42

References

Nielsen SS. 2010. Food analysis. 4th Ed. Springer Science + Business Media, LLC: New York, Dordrecht, Heidelberg, London.

Nielsen SS. 2010. Food analysis: Laboratory manual. 2nd Ed. Springer Science + Business Media, LLC: New York, Dordrecht, Heidelberg, London.

Kusnandar F. 2010. Kimia pangan: Komponen makro. Dian Rakyat: Jakarta.

Andarwulan N, Kusnandar F, Herawati D. 2011. Analisis pangan. Dian Rakyat: Jakarta.

Winarno FG. 1997. Kimia pangan dan gizi. PT Gramedia Pustaka Utama: Jakarta.

Belitz H-D, Grosch W, Schieberle P. 2009. Food Chemistry. 4th revised and extended Edition. Springer-Verlag: Berlin, Heidelberg.

Fennema OR. 1996. Food Chemistry. 3rd Ed. Marcel Dekker, Inc. : New York, Basel, Hongkong.

AOAC International. http://www.aoac.org/

Huang T, Jander G, and de Vos M. 2011. Non-protein amino acids in plant defense against insect herbivores: Representative cases and opportunities for further functional analysis. Phytochemistry, Vol. 72 Issue 13, p1531-1537.

Barrett GC and Elmore DT. 1998. Amino acids and peptides. Cambridge University Press: New York.

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Thank you

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AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 135/42

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