Exam 4 Food Analysis

Question 1

Definitions of lipids, total fats, fats, and oils

Answer 1

*LIPIDS: in general, soluble in ether, chlorform, and other organic solvents, but are sparingly soluble in water
*OILS: liquid triacylglycerols at room temp
*FATS: solid triacylglycerols at room temp
*TOTAL FATS: sum of fatty acids from C4 to C24, calculated as triacylglycerls; however doesn’t include C2 & C3 (acetate and propionate)

Question 2

FDA definitions for: total fat content, saturated fat, polyunsaturated fat, monounsaturated fat, trans fatty acids

Answer 2

*Total fat content: total lipid fatty acids expressed as triacylglycerides
*Saturated fat: sum of all fatty acids without double bonds
*Polyunsaturated fat: cis, cis-methylene-interrupted polyunsaturated fatty acids
*Monounsaturated fat: cis-monounsaturated fatty acids
*Trans fatty acids: trans isomers, not conjugated

Question 3

Preparation methods for fat extraction

Answer 3

*Predrying sample: makes sample easier to grind, breaks fat-water emulsions to more easily dissolve fats in organic solvents, and helps free fat from food tissue (ex. vacuum oven)
*Particle size reduction: inc extraction efficiency (ex. grinding in liquid nitrogen)
*Acid and/or alkaline hydrolysis: breaks btoh covalenty and ionically bound lipids into easily extractable lipid forms

Question 4

Soxhlet extractor (semi-continuous)

Answer 4

*dry sample if contains more than 10% H2O
*if comparing many food samples: dry them all
*weigh sample into thimble & place in Soxhlet
*extract with petroleum ether or hexane
*remove solvent (dry) & calc fat content
*solvent builds up in the extraction chamber for 5-10 min and completely surrounds the sample & then siphons back to boiling flask (ex. soxhlet method)

Question 5

Chloroform-methanol extraction

Answer 5

*chloroform: methanol is added to create a monophasic system
*the addition of aqueous salt solution helps to partition the sample into a byphasic system
*methanol layer (at top): contains salts, carbohydrates, some proteins
*in the middle: cellular debris
*chloroform layer (bottom): contains lipids

Question 6

Mojonnier extraction (alkaline hydrolysis method)

Answer 6

*use mojonnier flask
*treat sample, in series, with ammonium hydroxide, 95% ethanol, ethyl ether, and petroleum ether (i.e. use combination of solvents)
*repeat extration series; evaporate solvent; weight fat
*moisture removal from sample not necessary
*similar to roese-gottlieb method

Question 7

Babcock method

Answer 7

*sulfuric acid digests protein, generates heat, and releases fat
*after centrifugation of samples, fat content is measured volumetrically
*you don’t need to dry the sample

Question 8

In what form are fatty acids during FAME analysis

Answer 8

fatty acid methyl esters

Question 9

What is learned from GC of fatty acids

Answer 9

fat content

Question 10

GC analysis of fatty acids process

Answer 10

*after adding an internal standard and an antioxidant, sample is treated by acid and/or alkaline hydrolysis, then fat is extracted with ether
*fatty acids are converted to fatty acid methyl esters (FAMEs= palmitic acid/hexadecanoic acid) then separated by GC, and quanitfied with reference to the internal standard
*sum of fatty acids equals total fat

Question 11

Refractive index

Answer 11

*degree of unsaturation (RI decreases linearly as unsaturation dec)
*a measure of purity (contaminants change RI)
*also a means of identification (each lipid matrix will have a characteristic RI response)

Question 12

Iodine value

Answer 12

measure of degree of unsaturation
*defined as grams of iodine absorbed by 100g of sample
*measures: iodine required for absorption by double bonds
*application: used for fat characterization, to follow hydrogenation process, and as a measure of oxidation (a dec in unsaturation occurs during oxidation)

Question 13

Solid fat content

Answer 13

*determines amt of solids in fat (vs. liquid)
*SFC preffered over SFI bc: its an actual measurement, less subject to error, & faster
*measured by: percent solid fat, by continuous wave or pulsed NMR

Question 14

Saponification value

Answer 14

*an index for avg molecular wt of triacylglycerides in the sample
*defined as the mg of KOH required to saponify 1g of sample
*application: determines the avg fatty acid chain length of an oil or fat
*measures: alkali required to saponify fat/oil

Question 15

Why are free fatty acids analyzed

Answer 15

*indication of adequacy of refining
*breakdown after storage or use

Question 16

What are unique characteristics of proteins that are related to protein analysis

Answer 16

*nitrogen content
*peptide bond
*aromatic amino acids
*dye binding capacity
*ultraviolet absorptivity

Question 17

What is measured with the Kjeldahl & Dumas protein analysis methods

Answer 17

*Kjeldahl:
-measure the presence of N by method involving digestion, neutralization, distillation, and titration
-measures total organic N (including ammonia)
*Dumas:
-N is measured by gas chromatography using thermal conductivity detector
-measures total N, including inorganic

Question 18

Dye binding

Answer 18

*anionic dye binding: dye binds to protein (basic AA: His, Arg, Lys & free terminal amino group of protein) to percipitate protein
*bradford: dye changes color when bound to protein (due to amphoteric nature of proteins)

Question 19

Copper binding

Answer 19

*biuret: peptide bonds
*lowry: peptide bond + Tyr & Trp
*BCA: peptide bonds + four amino acids (Cys, Cystine, Trp, Tyr)
proteins reduce cupric ions to cuprous ions under alkaline conditions; cuprous ions react with BCA reagent to give purple color
*based on peptide bonds and the presence of four amino acids: Cys, Cystine, Trp, & Tyr

Question 20

UV absorbance

Answer 20

*UV absorption:
-280 nm (Tyr & Trp)
-220 nm (peptide bond)
*non-destructive
*rapid
*has to be a pure & clear solution

Question 21

Salting out

Answer 21

proteins are precipitated from solution as ionic strength is inc

Question 22

Isoelectric precipitation

Answer 22

proteins aggregate & precipitate at their pI bc there is no electrostatic repulsion

Question 23

Solvent fractionation

Answer 23

addition of ethanol or acetone dec dielectric constant of aqeous solution and dec solubility of most proteins

Question 24

Dialysis

Answer 24

*used to separate molecules in solution by the use of semipermeable membranes that permit passage of small molecules but not large molecules
*protein solution is placed into dialysis tubing that has been clamped at one end & the other end is sealed, the bag is placed in large volume of water or buffer (usually have to switch water out ~3 times)

Question 25

What is electrophoresis

Answer 25

defined as the migration of charged molecules in a solution through an electrical field

Question 26

what is PAGE & SDS-PAGE

Answer 26

*polyacrylamide gel electrophoresis
*PAGE with anionic detergent, sodium dodecyl sulfate (SDS), is used to separate protein subunits by size

Question 27

Isoelectric focusing

Answer 27

proteins separated by charge in an electric field on a gel matrix in which a pH gradient has been generated using ampholytes, under constant voltage, proteins migrate to the location on the gradient at which pH equals the pI of the protein, size is not a factor

Question 28

How are two systems combined for 2D electrophoresis of proteins

Answer 28

proteins separated in tube gels by isoelectric focusing, the tube gel containing the separated proteins is then placed on top of an SDS-PAGE slab gel, & proteins are separated

Question 29

What is a good method for AA analysis

Answer 29

*cation-exchange chromatography (elution by stepwise inc in pH & ionic strength), postcolumn derivatization with ninhydrin or o-phthalaldehyde, then quantify spectrophotometrically
*precolumn derivatization using phenylthiocarbamyl, separation by reversed-phase liquid chromatography, then quantify by UV spectroscopy
*with either chromatography method: use internal std (ex. norleucine) & std curves

Question 30

How are total carbohydrates calc for nutrition labeling

Answer 30

*total carbohydrate= 100% - [% Moisture + % Ash + %Lipid + %Protein]

Question 31

FDA definitions of carbohydrates for nutrition labeling purposes

Answer 31

a statement of the grams of total carbohydrate in a serving expressed to the nearest gram, except that if a serving contains less than 1 gram

Question 32

What information is determined from phenol-sulfuric assay

Answer 32

*often used for research purposes to determine “total carbohydrate”
*principle: carbohydrates are heated in the presence of acid to produce furan derivatives, that condence with phenol, to form compounds that can be quantified spectrophotometrically

Question 33

What information is determined from a reducing end assay

Answer 33

*principle: based on reduction of cupric ions in alkaline solution to cuprous ions by reducing sugars: amt of cuprous ions generated is measired to quanitfy reducing sugars

Question 34

What is the method for identification of mono & oligo saccharides

Answer 34

pulsed electrochemical detector (ECD)

Question 35

How must sugars be treated by GC analysis

Answer 35

*convert sugars to volatile derivatives
*use flame ionization detector

Question 36

What is gelatinized starch

Answer 36

*converted totally into D-glucose by enzymes specific for starch & D-glucose is quantified enzymatically

Question 37

Dietary fiber

Answer 37

*the non-digestible soluble & insoluble carbohydrates (w/ 3 + monomeric units) & lignin that are intrinsic & intact in plants; isolated or synthetic non-digestible carbohydrates (w/ 3+ monomeric units) determined by FDA to have physiological effects that are beneficial to human health

Question 38

What are the basic steps in dietary fiber determination

Answer 38

*defeat sample if >10% lipid
*treat mixture w/ alkaline protease
*treat hot mixture w/ thermostable a-amylase
*treat mixture w/ glucoamylase
*add 4 volumes of 95% ethanol
*collect residue + precipitate by filtration
*wash w/ ethanol & acetone
*air dry then oven dry & weigh
*substract weights of protein & ash determined on duplicate samples

Question 39

List several components of dietary fiber

Answer 39

*insoluble: cellulose, lignin, resistant starch
*soluble: native pectin, hydrocolloids, non-digestible oligosaccharides