Food Analysis Exam 4

qualitative reasons to test carbs

detect adulterants

• most carbs made up of CHO, carbs with a different content than that can contain adulterants

quantitative reasons to measure carbs

nutrition labeling, ingredient label declaration (which order ingredients are in)

which carbs are required on nutrition label

total carbs, sugars, dietary fibers

how are total carbs usually measured

calculated by difference from proximate analysis

what makes up total sugars

glucose+ fructose+ sucrose+ lactose + maltose

degree of polymerization

how many CHO units the carb chain is

general formula for carbs

CnH2nOn

difference between sugar and sugar alcohol

sugar alcohols are sugars with aldehyde replaced by an alcohol group

• sugar alcohols provide limited energy for the body but still add sweetness

what makes a sugar a reducing sugar

must have an aldehyde group or convertible ketone accessible

all ____ are reducing sugars

monosaccharides

which disacharides are reducing sugars

lactose and maltose

can sugar alcohols or polysaccharides be reducing sugars?

no

polysaccharides

long chains of monosaccharides (DP>20)

types and characteristics of starch

amylose- linear

amylopectin- branched

break down into glucose and absorbed in small intestine

what do gums and hydrocolloids do in food

thickening/gelling agents

examples of dietary fiber

cellulose, pectin, lignin

two methods of total carb analysis

• anthrone test

• phenol-sulfuric acid method

principle of anthrone test

sulfuric acid degrades CHO to furans which react with anthrone to make chromophore (can be measured using spectrophotometer)

what kind of sugars is the anthrone test the best with

hexose (6-carbon sugars)

anthrone test general procedure

1. mix unknown solution with sulfuric acid

   1. this hydrolyzes polysaccharides into monosaccharides (furan derivatives)

2. add the anthrone, solution turns blue/green

3. quantify w spectrophotometer at 620

principle of phenol sulfuric method

sulfuric acid and heat degrade CHO to furans which condense with phenol to make chromophore

what does the phenol-sulfuric method measure

reducing sugars, mono and disaccharides, starches, and non starch polysaccharides

phenol sulfuric acid method steps

1. mix unknown solutions with sulfuric acid to produce furan derivatives

2. add phenol to form orange solution

3. quantify at 490nm

sample prep of sugars

oven dry,

defat,

extract with 80% ethanol to solubilize sugars,

remove ethanol,

analyze

reducing sugar

sugar that gives up electron to reduce other chemical species

in reduction reaction what does an aldehyde turn into

carboxylic acid

conditions to analyze reducing sugars

done in basic buffer solution

principle of measuring reducing sugars

reduce Cu2+ to Cu in alkaline solution

salmongyi-nelson test

1. reducing sugar reduces Cu2+ to Cu+

2. Cu+ reduces AsMo complex (turns blue)

3. measure absorbance

most common method for total reducing sugars

somongyi-nelson test

Benedicts test

semi quantitative measurement of reducing sugars

1. mix sample with Benedicts reagent (copper II citrate)

2. heat+ reducing sugar+ reagent —> colored product

3. blue is no reducing sugar —> red is highest amt of reducing sugars

fehlings test

qualitative test for presence of reducing sugars

1. mix Fehlings A ad B to make unstable blue solution

2. heat + reducing sugar + Cu2+ form a red solution

lane eynon titration

similar to felhings test, allows quantitative analysis of reducing sugars

1. boil solution with Fehlings a and B

2. titrate unknown until turns red

instrumental techniques to measure carbohydrates

Anion exchange HPLC

Normal phase HPLC

GC

mobile and stationary phase of AE HPLC

stationary: cation with anion counter ion (negative charges stick)

mobile phase: acidic → basic

principle of AE HPLC

carbs have OH groups with high pKa (net negative charge)

AE column retains carbs at acidic pH and elutes with basic pH

elution order of AE HPLC

sugar alcohols, monosaccharides, di + oligosaccharides

mobile and stationary phase of NP HPLC

stationary: polar

mobile: nonpolar

NP HPLC carbohydrate principle

carbs are highly polar so they retained by the polar stationary phase and then separate based on size and polarity

elution order of NP HPLC for carbs

monosaccharides and sugar alcs → di and oligosaccharides

how to analyze mono and oligosaccharides on GC

1. reduce aldehyde to hydroxyl group

2. convert all hydroxyls to volatile derivatives

3. detect with flame ionizer detector or mass spectrometry

principle of enzymatic methods of carb analysis

specific enzyme reacts with specific carb of interest

GOPOD

measures glucose in a sample

principle of GOPOD

Glucose Oxidase reduces glucose and forms peroxide which reacts with dye and PerOxiDase to form colored compound

benefits of total reducing sugar analysis, instrumental techniques, and enzymatic methods

total reducing sugars: rapid, easy, cheap

instrumental methods: best for accuracy and precision, identification, and quantification

enzymatic methods: rapid, specific for analyte of interest

sample prep and isolation of polysaccharides

1. oven dry

2. defat

3. extract with hot ethanol (polysaccharides will be insoluble)

4. remove liquid layer

5. analyze insoluble layer

types of starch (5)

amylose, amylopectin, gelatinized starch, retrograded starch, resistant starch

two methods to measure starch

total starch assay and degree of gelatinization

types of dietary fiber (6)

cellulose, hemicellulose, lignin, resistant starch, pectin, beta glucan

three methods to analyze fiber

toral dietary fiber, soluble dietary fiber, insoluble dietary fiber

what is starch

polymers of D glucose with alpha 1,4 linkages

starch gelatinization

method to expose the starch which otherwise is unable to be accessed by enzymes

1. heat starch in water

2. crystalline structure is lost and swelling occurs

3. amylase leaches and starch becomes accessible to enzymes

total starch assay procedure

1. gelatinize starch in DMSO

2. digest with amylase (make starch fragments)

3. digest with glucoamylase (break into glucose monomers)

4. analyze glucose using GOPOD

degree of gelatinization assay principle

debranching followed by depolymerization of non gelatinized starch

• enzymes only degrade non gelatinized starch

what is the degree of gelatinization assay used for

used to determine extent of retrogradation

procedure of degree of gelatinization assay

1. suspend water in sample

2. debranch sample

3. convert amylose to maltose with beta amylase

4. measure reducing sugars

why is it important to remove sugars before analyzing starch

you have to break down the sugars into glucose to analyze with glucose specific assay— if other sugars are present they will be quantified as starch (overestimate)

dietary fiber definition

sum of non digestible components of a food ingredient or product

soluble fiber

fibers that dissolve in water

insoluble fibers

fibers that do not dissolve in water

fiber contribution to NFP

insoluble fiber can be subtracted from total CHO prior to calorie calculation

soluble fiber can not be subtracted from total CHO prior to calorie calculation

how to analyze total dietary fiber procedure

1. dry and defat

2. remove sugar and hot ethanol

3. remove starch with amylase and glucoamylase

4. deproteinate with protease

5. precipitate all dietary fiber with ethanol and remove stay

how to analyze insoluble and soluble fiber procedure

procedure

1. dry and defat

2. remove sugar with hot ethanol

3. remove starch with amylase and glucoamylase

4. deproteinate with protease

5. add water to dissolve soluble fiber

6. filter to separate soluble and insoluble fibers

7. insoluble portion is the insoluble fiber which can be removed and weighed

8. precipitate the soluble fiber with ethanol from the remaining solution and weigh

why is it a challenge to analyze dietary fiber and starch

they are difficult to differentiate because they are both polysaccharides

what must you do in sample prep stage when analyzing fiber

remove starch

• starch gelatinization

• enzymatic degradation of starches

physical methods for measuring carbs

specific gravity, refractometer, mass spec (MALDI-TOF)

protein structure

primary: amino acids

secondary: amino acids held together with peptide bonds (sheets + helices)

tertiary: secondary structures coming together

quaternary: multiple subunits

how much nitrogen is generally in protein

16%

organic nitrogen examples

amino acids, proteins, nucleic acids, urea

inorganic nitrogen examples

N2 gas, ammonium NH4, nitrate

protein factor based on nitrogen

6.25 (100/16)

challenges with 16% protein assumption

%N varies depending on amino acids present

three useful traits in proteins to distinguish them from other macromolecules when analyzing

nitrogen content, aromatic rings (tryptophan and tyrosine), and peptide bonds

kjeldahl test procedure

1. digest sample with sulfuric acid and heat

2. neutralize sample with base

3. distill ammonia with boric acid to become borate

4. titrate borate with HCl

what does kjeldahl test measure

organic nitrogen, then multiply by 6.25 to get total protein content in sample (assume 16%)

dumas method procedure

1. combust sample in O2

2. reduce sample via copper catalyst (converts N to N2)

3. Analyze via GC

what does Dumas test measure

total nitrogen (organic and inorganic)

how to calculate total protein content using Dumas

analyze sample using Gc and multiply nitrogen value by 6.25 to get crude protein content

what is the official AOAC method to analyze crude protein

dumas

synthetic NPN examples

melamine, ammonia

naturally occurring NPN examples

nucleic acids, vitamins, alkaloids,

can Dumas and kjeldahl differentiate between NPN and nitrogen from protein

no, they can only determine protein content not its source

TCA precipitation procedure

1. TCA is mixed with sample and filtered

2. TCA precipitates protein and NPN stays dissolved in solution

3. analyze NPN via kjeldahl

what does TCA precipitation measure

measures NPN— converts to protein using conversion factor

propose of TCA precipitation

detect adulteration

can melamine in milk and horse meat in beef be detected using TCA precipitation

Milk- yes because it would pick up on melamine

Beef- no because the horse meat

anionic binding assay procedure

1. mix protein and excess dye

2. centrifuge and remove precipitated protein

3. measure remaining dye via spectroscopy

what does anionic dye binding assay measure

measures remaining unbound dye which is inversely proportional to protein content

applications for anionic dye binding assay

milk, wheat flour, soy, meats

Bradford assay procedure

1. mix Bradford reagent and protein

2. measure absorbance at 595 (measures bound dye)

Bradford reagent composition

coomassie dye, acid, ethanol

what is used for standard curve in Bradford assay

BSA

applications of Bradford assay

beer, potatoes

what are the colors in the Bradford assay

turns from brown to blue (unbound to bound)

Biuret method procedure

1. mix buret reagent with proteins

2. measure absorbance at 540nm

biuret method measures

peptide bonds

biuret method principle

cu2+ ions complex with peptide bonds in alkaline solution and form a violet color

what is biuret reagent

NaOH, Cu2SO4, and Na-K tartrate

what does biuret method use for standard curve

BSA