fdsci2100 prelim 1

Trends and demands that dictate analysis

Consumers - expect quality, safety, nutrition, value
Food industry - manage product quality; uses “select suppliers,” specifications are delivered as “certificate of analysis”
Government regulations -
→ nutrition labeling
→ mandatory and voluntary standards
→ GMPs (good manufacturing practices
→ HACCP (hazard analysis and critical control points) → adopted by FDA, other US federal agencies, and codex alimentarius commission

Types of samples analyzed in quality control program

Ingredients (raw materials) → processing (process control sample) → finished product

Competitor samples - samples from similar products of other businesses (competitors)
Complaint samples - samples submitted for analysis due to consumer complaint

Reasons to measure food components

Check quality of raw ingredients
Formulate and develop new products
Check composition changes during processing
Evaluating new processes for making food products
Identifying source of problem for unacceptable products
Develop/check nutritional label + other gov regulations

Published official methods

AOAC International (association of official analytical chemists) - methods for most foods
AACC International (American association of cereal chemists, international) - methods mostly for cereal products
AOCS (American oil chemist’ society) - methods for mostly fats and oils
American public health association - Standard methods for the examination of milk and dairy products + water and waste water
US Pharmacopeia - food chemicals codex for food additives

Sampling

Population: group of items or individuals under discussion
Sample: subject of the population that has been selected to study or measure

Population → Sampling to produce sample → Analysis to produce Results → conclusion goes back to population

Types of sampling (4)

Simple random sample (SRS) - each member of population has equal and independent chance of being selected at each stage in the selection

Stratified random sampling - separating population elements into overlapping groups (Strata) and selecting a SRS from each strata

Systematic sampling - select a sample unit from a list of units, like with production-line sampling

Judgment sampling - drawing samples based on the judgment and experience of the investigator

Statistical considerations

Sample size: determined based on either precision or power analysis
→ precision analysis based on confidence internval
→ calculate maximum error acceptable of desired level of confidence
→ Use estimated mean and standard deviation to calculate sample size

How to choose sampling method?

Consider:
→ nutrition labeling
→ Quality control
→ acceptance of raw materials/ingredients/products (ex: accepting or rejecting the lot - whole milk, minimal amount of fat %)
→ Investigation of food poisoning
→ Contamination and accident contamination

Sampling Approaches and errors

Approaches
Continuous sampling: Usually done during manufacturing. Samplers mechanically take a fraction of material for sampling while it is being processed
Manual sampling: truckloads, boxes, sacks → physically collecting a small representative portion of a food product, ingredient for analysis or quality control

Errors
Lack of randomness: due to instrumental limitation, human bias, heterogeneous mixture, particle shape/size, surface adhesiveness
Changes in composition during or after sample: gain or loss of water, volatile loss, oxidation (lipids!), contamination

Preparation of samples (8)

1.General lot size reduction:
- ex: sack of rice - sample from 4 corners and centers at top, bottom, and center

2. Liquid sample
- Small sample: mix well
- Large sample: representative sampling → from top, center, bottom

3. Grinding dry materials: mills, blenders, pestle and mortar

4. Grinding moist materials: Blenders, homogenizer, ultra low temp (-70C?… just really cold)

5. Enzyme inactivation: store at low temp, freeze dry, shift pH, high temperatures

6. Minimize lipid changes: colder temps = slower oxidation (ex: rate of oxidation at -20C is ~1/16 that at RT), add antioxidants to reduce free radicals (Vit E, Vit C, BHA, BHT), avoid lighting, remove oxygen/store under nitrogen

7. Controlling microbial growth and contamination: freezing, drying, preservatives (sodium benzoate, sodium azide), and acid/base

8. control moisture loss or gain

Reliability of an analytical method depends on..

Specificity (selectivity), accuracy, precision, sensitivity and detection limit, linearity and linear range

Specificity (selectivity)

The ability to detect the component of interest specifically in the presence of other components → affected mostly by presence of interfering substances

Broad spectrum vs very specific
Ex: Crude fat analysis: broad specificity → analyzes compounds soluble in an organic solvent such as triglycerides, phospholipids, cholesterol, fat-soluble vitamins (ADEK) so basically fats
However, determining just one of these categories, like phospholipids alone, is very specific

Ex: GC-TEA (Gas chromatograph-Thermal Energy Analyzer) - analysis of N-nitrosamines → selectively analyzes compounds with NO structure, but ig since its a a group of structures its pretty broad

Accuracy

The nearness of an experimental value to the true value

Ex: body weight → he measure his weight with two diff scales and got diff answers → you gotta calibrate your instruments to ensure accuracy!
dart board → avg position of darts is at the bullseye

Methodology to evaluate:
→Calibration
→Standard reference material - purity
→% recovery - how much you recover from your analysis → ex: if you add in a known amount, after analysis can you get 100% recovery back? depends on what you’re looking for, but generally want >60%
→Comparison to a well-characterized method → Golden assay! if you have a new assay, you must compare to the golden child

Precision

The reproducibility of replicate measurements → agreement between values in a set of data

If all replicate measurements agree well, they may not necessarily be accurate or close to the true value

ex: dart board → all darts land around the same area of the board

Evaluating precision:
→Mean - average set of data → add all data, divide by sample size
→Standard deviation (SD) - measure spread of experiment values and show how close the values are to each other → sqrt of (value - mean)²/(sample size - 1)
→ Coefficient of variation (CV) = SD/mean x 100% → CV = <5% is acceptable → lower CV = higher precision/reproducibility

Sensitivity and detection limit

Sensitivity: ratio b/w the magnitude of the instrumental response and the amount of the compound analyzed

Detection limit: the lowest concentration of an analyte that can be detected with a statistical significance
→ In instrumental analysis, the limit of detection can be researched when the signal-to noise ratio (S/N) is 2:1
→ S/B: response to analyte divided by electronic noise of detection system → you want signal to be stronger than noise, higher ratio = good!
→ The detection limit is basically the spot where the signal begins to be larger than the electronic noise → when the signal is lower than the electronic noise it will not show
→ as we dev new technologies, we can detect lower and lower concentrations of things

Linearity and linear range

the interval between the upper and lower levels of analyte in a linear dose-response → directly proportional to analyte concentration within a given range

this is where you wanna do your analysis

Standard curves

Used to determine unknown concentrations
→ concentration of substance must be proportional to measurement
→ linear regression most often used to construct curve
→ specificity of ferulic and gallic acid (flavonoids ex) were very good… the other guys were not as much i guess?

Types of errors

Systematic/determinate errors: ones that can be determined and eliminated
→Ex: due to methods, equipment and materials (material purity!), personal judgments (follow procedure!), mistakes

Random/indeterminate errors: cannot be determined and controlled
→ the effect of many small, uncontrollable variables and personal judgments that lead to uncertainty in a measured value

Nutrition labeling

Nutrition labels have lots of info → calories, % daily value for all macronutrients, breakdown of fat and carbs, sodium, vitamins and minerals
Daily value - used to describe RDI and DRV

RDI - reference daily intake → used for essential vitamins and minerals
DRV - Daily reference value → used for food components: like total fat, sat fat, cholesterol, total carbs, dietary fat, sodium, potassium, protein → based on 2000 or 2500 reference calories

moisture and total solids

moisture: measure of water content of a material
Moisture content affects product shelf life → more water limits shelf life, drying a product can significantly increase shelf life

total solids: dry matter after moisture removal

- water solids = 100%? not always because there may be volatile compounds that were lost

Structure of H2O

A dipolar molecule where the H has a partial + charge and the O has a partial - charge

One water can bind 4 other molecules through hydrogen bonds!

Free water

retains physical properties and acts as a solvent → can easily form ice, which causes damage to tissue structure after freezing

Immobilized water: blocked by cell membrane or subcellular structures, does not flow freely
Capillary water: held by capillary force, or intercellular water
Fluid water: freely moves, like blood or coconut water

Bound water

AKA adsorbed water, is held tightly in cell walls, bound to proteins and carbohydrates with hydrogen bonds

50g/100g protein; 30-40g water/100g starch

Properties:
Not easy to form ice: freezing point -40C
Can not be used as a solvent

ex: salt on ice to lower freezing point and melt ice by becoming bound to the water?

Water of crystallization

is chemically bound

Ex: lactose monohydrate

Drying agents
sodium sulfate Na2SO4 × 10H2O → good at removing water from organic solvents
Calcium sulfate is good for a desiccator (a machine that protects moisture sensitive things by keeping them dry) because it cannot remove water from organic solvents

creating important temperatures?

0C: ice water bath
-20C: Ice bath with salt
+40 and +70C: hot water bath (controlled)
-70C: dry ice + ethanol → at this point theres no more cellular response so you can preserve things for a long time
-186C: liquid nitrogen

Importance of analysis of moisture

quality control and stability
economic value
reduced moisture lowers cost of transport → like concentrated orange juice
Calculation of nutritional value of foods requires moisture content
→ dry weight vs wet weight, must make a fair comparison so you cannot compare the two
→ use dry weight to determine the effect of processing on nutrition content

Methods for determination of moisture content

Drying methods: oven, vacuum oven, microwave oven

Distillation methods: Toluene (110.6C), xylene (137-140C), good for low water content food (like 2-3%)

Chemical methods: Karl fischer titration, also good for low water content food

Physical methods: infrared determination, hydrometry (alcoholometer, lactometer to measure specific gravities), freezing point

Instrumental methods: gas chromatography - methanol extraction

Oven drying methods

sample is heated under carefully specified conditions:
Time and temperature, ex: 102C for 5 hours to extract water because 102C is just above boiling point of water

Then, loss of weight is taken as a measure of the moisture content of the sample

advantages: simple, relatively rapid, analyzes larger numbers of samples at the same time

Drying curve

Exponential/linear area: free water is lost first and fastest

Curve where slope begins to approach 0: bound water is lost at a slower rate

Near plateau: crystallization water is lost

Plateau: constant weight area, weight is unchanging and water is not lost?

After plateau: water loss may increase due to decomposition, especially in high sugar foods

Run drying to before decomposition to get a nice drying curve

Basic considerations when drying

Decomposition:
Fructose in foods decomposes at temperatures above 70C, so fructose solutions should dry at 60C for 7-9hr or at 70C for 4hr in a vacuum oven
C6H12O6 → 6C + 6H2O

Loss of volatiles: acetic acid, propionic acid, butyric acid, alcohols, esters, and aldehydes

Reabsorption of moisture after drying: cool and store dried samples in a desiccator

Handling and preparation of pans:
Pans for moisture analysis must be pre-dried
Handle and prepare pans with tongs to avoid fingerprints or water from hands contaminating

Drying temperatures and time

Temperature: consider the boing point of water (100C) → average temperature used is 102C (70C-150C)

Time: average time is 1-6 hours

Constant weight: we usually dry to constant weight → when two successive weighings show a negligible loss in weight at 1-hr intervals (<2mg for a 5g sample)

Calculations: %moisture (wt/wt), total solids (wt/wt)

% moisture (wt/wt) = [(wet weight - dry weight of sample)/ wet weight of sample] x 100

Total solids (wt/wt) = (dry weight of sample %/wet weight of sample) x 100

Ovens and things

Forced draft oven = hot air oven

Vacuum oven:
→Dries samples under reduced pressure (25-100mmHg) for 3-6 hour
→Temperature depends on samples - high-sugar products and fruits use 70C
→For samples with high concentrations of volatiles, we need a correction factor to compensate for the loss
→Heat is not conducted well in a vacuum so the pan must directly contact oven shelves

Microwave drying oven:
→very quick, must be calibrated otherwise not as accurate
→must ensure even heat distribution
→not great for low moisture things

Distillation

Used with liquid in liquid samples to separate them by taking advantage of differences in boiling points

Sample is suspended in immiscible solvent (one that does not mix like xylene or toluene) in distillation flask and distilled at the boiling temperature of the solvent (130C for xylene) for 1 hour
→ So the stuff that gets separated is the stuff at the lower boiling point i think and the stuff left behind will have a higher boiling point

the mixture of water and an immiscible solvent is condensed and collected → then the volume of water is measured

Cold water goes in through the bottom and hot water flows out through the top to maintain the temperature of the condenser tube so that whatever youre collecting actually condenses

Ash?? Like the Ketchum?

inorganic residue from the incineration or complete oxidation of organic matter

Determined by weighing the dry mineral residue of organic materials after heated at high temperatures (around 55C) - represents total mineral content in foods

Ash = total minerals = organic materials - (C, H, O, N)

The amount and composition of ash in a food product depend on the nature of food and on the methods of ashing

Ash content in foods

In non-fat dry milk has a lot more ash % than regular milk because it has less water?

Processed meats have higher ash % than fresh meats because of preservatives like nitrates an nitrites added in

Minerals in our body

Major minerals:
Calcium and phosphorus - 75%
Sodium, potassium, Chlolride, Sulfure, Mg - 25%

Trace minerals:
Fe, Zn, Se, Cu, F. I
→ Anemia - Fe deficiency
→ Se deficiency - Keshan disease… but High amounts linked to diabetes?
→Iodine important for thyroid hormones

• Deficiency leads to goiter (enlargement of thyroid gland), miscarriages, mental retardation

• Fortify the salt with iodine!!

• Dairy also has a lot of iodine
  
  Toxic minerals:

  • Hg, Pb, Cd, As

    • Fish contaminated wth Hg

    • Some rice contaminated with cadmium (Cd)

Body composition

• Non-minerals: 96%

  • Oxygen 65%

  • Carbon 18%

  • Hydrogen 10%

  • Nitrogen 3%

• Minerals: 4%

  • Calcium and phosphorus: ~3%

  • K,S,Cl,Na,Mg: ~1%

  • Trace elements: so little

Why analyze ash and minerals? How to sample? Methods of analysis?

For nutrition labeling calculation
Quality control - Ex: making sure you reached the required nutrient amount for fortification

sampling:
Remove impurities; especially for fruits and vegetables → don’t want dust or dirt contamination!
Lipid content: Lipids are free of minerals, but lipid content in animal foods vary widely → must mix sample well and use representative sample
Contamination: container, reagents → use high purity water to wash and prevent mineral contamination??

Methods: Dry ashing vs wet ashing

Dry ashing

Refers to use of muffle furnace at 500-600C

Principle: When sample is heated to high temperatures, all organic matter is incinerated, leaving inorganic material to be quantified gravimetrically

Crucible materials: you need something to withstand the high temperatures
Ex: Quartz dishes (1100C), porcelain (1200C), Platinum (1773C)

Procedures:
1. Pre-dry crucible or pan to constant weight and keep it in the desiccator
2. Weigh a 5-10g sample (generally use the sample for moisture)
3. Put into a muffle furnace - start at 250C and raise temp to 550C (range 500-600) within 1 hour, and hold until sample turns to white gray ash (12-18 hrs or overnight) or to constant weight (<2mg chg)
4. Cool down the crucible in desiccator prior to weighing

Wet ashing

To digest or oxidize organic samples using acids, or mixed acids. AKA Wet digestion or wet oxidation

Principle: Organic matter is oxidized using acids and oxidizing agents, leaving inorganic matter

Commonly uses single acids: Nitric acids (HNO3) or sulfuric acid (H2SO4)
But industry often uses mixed acids because its faster
→Sulfuric acid-nitric acid (1:1) mix
→Perchloric acid-nitric acid (1:2) mix
Perchloric acid is very strong oxidant, but can explode.
SUPER SPEED → 10 min compared to 8hr with Sulfuric acid-nitric acid mix to digest 5g wheat

Dry ashing advantages and disadvantages

advantages:
→ Relatively safe
→ Simple; requires little technician time
→ Large muffle furnaces are available so large numbers of samples can be ashed at one time

disadvantages:
→ Long time (12-18 hrs)
→ Things with lots of volatile elements may not be read accurately
→ Requires special procedures for high fat and high sugar foods
ex: must slow down raising temperature otherwise its hard to digest high sugar? Must get low temps in high fats and must use representative sample

Wet ashing advantages disadvantages

advantages:
→ Good for volatile-containing samples
→ Good as first step in mineral analysis
→ shorter time than dry ashing

disadvantages
→ chemicals are dangerous! Corrosive! May explode!
→ requires special perchloric acid hood
→ requires constant attention while ashing

types of digestion

wet (acid), chemical (acid, base?), high temperature (dry), enzymatic, mechanical?

What are lipids?

they are defined by solubility, not structure

They are a group of chemicals that are insoluble in water, and soluble in non-polar organic solvents (like hexane, ether, chloroform, acetone)

ex of lipids:
→ triglycerides (fats and oils): made of fatty acids and glycerol
→ Phospholipids: glycerol + fatty acid + phosphate
→ sterols: cholesterols, like LDL, HDL
→ Fat soluble vitamins: A, D, E, K

The FDA’s regulatory definition of total fat for nutrition labeling purposes: the sum of Fatty acids from C4 to C24, calculated as triglycerides
Oils are liquid triglycerides at RT
Fats are solid triglycerides at RT

On a scale of polar to non polar, which solvent are you

Polar → non polar

Water → CH3OH → Acetone → Chloroform (CHCl3) → CH2Cl2 → Ether → Hexane

Ex: to extract vitamin A, you should select a nonpolar organic solvent like CHCl3, Ch2Cl2, ether, or hexane

Sources of lipids:

vegetable oils:
→ saturated: cocoa fat, palm oil
→ Oleics (Omega 9!): olive oil, canola, peanut oil, avocado → usually more healthy in terms of heart diseases
→ Linoleics (omega 6): soybean, sunflower, safflower, corn, cotton seet

Animal fats: butter fat, lard, tallow, fish oil

Major classes of lipids

In order of Simple and nonpolar → complex and polar

Simple: cholesterol esters, carotenoids, triglycerides (majority of fats), free fatty acids, cholesterol, diglycerides, monoglycerides

Complex: phospholipids

Structure of lipids and how to name them!

Triglycerides: glycerol esterified with 3 fatty acids → triglyceride = glycerol + 3 fatty acids bound together by ester bond
Glycerol: 3 C chain, each C has OH group attached
Fatty acid: R-COOH

Fatty acids have a methyl (omega) end and a carboxyl (alpha) end

Fatty acids are distinguished by their
→chain length (how many Cs),
→saturation (how many double bonds, saturated = 0),
→cis and trans isomers (For double bonds only, same side Hs vs opposite side Hs)
→number of double bonds (mono or poly)
→and position of double bond (omega 3 vs 6 vs 9)

All natural fatty acids have even number of Cs because Cs are added during fatty acid synthesis 2 at a time
→ we can synthesize odd # C FA though, which are useful for analysis to check recovery % → assay quality accuracy

Fatty acid nomenclature and specifically omega fatty acids

Fatty acids are named by number Cs:number double bonds

Ex:
Omega 3 fatty acids are linolenic → 18:3, EPA → 20:5, or DHA → 22:6

Omega fatty acid number is given by the first double bond from the methyl (omega) end
Ex: Omega 6 has six carbons b/w the terminal methyl group and the double bond

Linoleic acid (omega 6) fatty acid can also be conjugated since it has 2 double bonds → conjugated linoleic acid will have the double bonds separated by 1 single bond
Ex: unconjugated (9, 12) vs conjugated (9, 11 or 10,12)

Major families of unsaturated fatty acids

Omega 3:
linolenic - veg oil, nuts
EPA and DHA - fish

Omega 6:
Linoleic: veg oil
Arachidonic acid: animal tissue

Omega 9:
Oleic: veg oil

cis vs trans fatty acids

Double bonds are introduced when oils and fats are heated in the presence of metal catalysts and hydrogens

Cis and trans isomers are chemically different because trans fatty acids are not very kinky (they are linear). This means trans fatty acids have higher melting points bc they pack more easily and are thus solid at room temp, whereas cis isomers are bent and liquid at room temp

Saponification

Hydrolysis of a triglyceride by alkali into glycerol and alkali salts of fatty acids, which are called soaps

Carotenoids and tocopherols are non-saopnifiable

Triglyceride + 3KOH and heat → glycerol + fatty acid potassium salt (soap)
Soap is water soluble → important to take into consideration for lipid analysis?

Methylation

Acid-catalyzed esterification: free fatty acids are esterified by heating with excess of anhydrous methanol in the presence of an acidic catalyst

Free fatty acid (R-COOH) + methanol + H⁺ → replaces H on the carboxylic acid with a methyl group
This process makes the fatty acid way more reactive, stable, and volatile for analysis

Functions of lipids in foods

Processing:
Shortening → tenderizes/shortens baked products
Cooking medium (ex: deep frying) → is a good medium for heat transfer, flavor (majority of flavor compounds are lipid soluble), texture, extraction

Sensory:
Pleasant mouth-feel → creamy, smooth feel
Flavor carrier
Texture and color: crisp fried foods, emulsion, etc.
Off-flavors: oxidative and hydrolytic rancidity

Nutrition:
E source → 9kcal/g
Essential fatty acids: linoleic (Omega-6, 18:2) and alpha-linolenic (omega 3, 18:3)
Carrier of nutrients: fat soluble vitamins (ADEK); beta carotene
Omega-3 fatty acids

Importance of lipid analysis

→ To understand the effects of lipids on the food functional properties in the food processing and shelf life of the product
→ consumer concerns about effect of dietary fat on health (lipids related to chronic diseases, heart disease, cancer, obesity) → health concerns require to measure cholesterol content, amount of sat and unsat fats, trans fats, individual fatty acids
→ nutrition labeling → total fat, sat fat, trans fat, cholesterol
→ food regulation and food safety

Gas chromatography GC-FID

Gas chromatography flame ionization detector

Very good for determining fatty acid lipid profiles → measures sat and unsat FA, cholesterol, omega 3, trans fatty acids, conjugated linoleic acid
(soxhlet and mojonnier aint got nothing on tis one)

Adding synthesized odd # C fatty acid before analysis and remeasure after analysis to check % recovery and see if your analysis is correct → check against internal standards

High performance liquid chromatography HPLC-UV, Supercritical fluid extraction SFE, and mid infrared (IR) analysis

HPLC-UV
Can measure fat soluble vitamins (ADEK), cholesterol, carotenoids (beta carotene, beta cryptoxanthin, lutein, zeaxanthin, lycopene), and conjugated linoleic acid isomers

SFE
Uses a CO2 fluid under specific pressure and temperature → when CO2 behaves differently as a solvent… like changes in polarity??
Removes cholesterol from milk and leaves fatty acid and vitamins alone → but also we need some dietary cholesterol
Very expensive, not possible for commercial use

IR
Used for milk composition → fat, protein, lactose
Needs calibration for accuracy

Soxhlet method

For lipid analysis in solid foods

Principal: fat is extracted with petroleum ether, and extracted fat is dried to a constant weight and expressed as percent fat by weight

Sample Preparation:
Use solid food → dry sample to constant weight before extraction → grind to reduce particle size

Procedure:
Dry thimble and boiling flask, weigh
Weigh sample into dried thimble, dry sample
Place thimble in extraction flask
Add petroleum ether into boiling flask → assemble the whole thing together
Heat apparatus

How it works:
Solvent heats up, evaporates → Solvent vapors are condensed over the sample → rinses out vaporized fat → and condensed again for another rinse
Petroleum ether takes fat with it into bottom of flask and then the ether is evaporated away and then you just have fat

Calculation:
Fat content % = fat in sample (g)/sample weight(g) x 100%

Babcock method

Used for lipid analysis in raw milk → MILK CANT BE PASTEURIZED!

Add sulfuric acid to milk → digests protein, generates heat, release fat → isolate fat by centrifugation and adding hot water → fat is quantitated in the graduated portion of the babcock bottle volumetrically

The babcock bottle looks like an upside down wine glass but with volumetric lines on the neck

the fat gets stuck in the neck of the bottle and thats how you measure it

Mojonnier ether extraction

used for lipid analysis of liquid samples

Liquid:liquid extraction (aqueous:ether)
Ether is pretty polar

Add ammonium hydroxide NH4OH to denature protein and release fat from milk fat globules → extract into ether → collect ether layer → evaporate ether → weigh ether extractable material

Calc:
% fat (w/w) = g ether extractable material/g milk x 100

More accurate and precise than babcock

Amino acid structure

amino acids are the basic structural unit of protein

They contain an amino NH2 group, a carboxyl COOH group, an H atom, an alpha carbon in the middle, and an R side chain

In solution they show acid base properties and act as good buffers
→ Neutral → NH3 and COO- group
→ Acidic → NH3 and COOH group
→ Alkaline → NH2 and COO- group

Can be aromatic (have benzene ring structure) or aliphatic (no ring)
→ ring can be used for protein analysis

Peptides and proteins

Peptides: amino acids chained together via peptide between alpha amino group of one aa and alpha carboxyl group of a second aa, loses a water molecule

Proteins: high molecular weight polypeptides
Many are single polypeptide chains, like myoglobin
Some contain two or more chains, like hemoglobin (4 chains)

protein structure
Primary: amino acid sequence from covalent linkage of L-alpha-amino acids by peptide bonds
Secondary: coiling and folding of a chain (alpha helix, beta sheets)
Tertiary structure: folding loops and domains (folded 3D protein)
Quaternary: multiple subunits (protein teamwork)

Elemental composition of protein

Generally 50% C, 7% H, 23% O, 16% N

The N content acts as a conversion factor to determine protein content from nitrogen content

Different foods have different conversion factors depending on the %N of their proteins
→ Generally 6.25 is the conversion factor (100/16)
→ plant based proteins have higher N content and have a smaller conversion factor

Melamine

A protein that has a high nitrogen content
China put this in their infant formula → adulteration! bad for babies

Put into question whether we should be using N content to determine protein

Isoelectric point

the pH at which a protein is neutrally charged (equal ± charge)

At low pH, protein is +
At high pH, protein is -

Each protein has their own characteristic pI → can use this to separate proteins?

Unique protein characteristics used for analysis

Nitrogen content
Peptide bonds → can be determined by copper and sulfate reaction with peptide bond to get a color reaction
Aromatic amino acids → reduction potential (Lowry assay + peptide bond assay = super good analysis)
Dye binding capacity → proton specificity, color reaction
Ultraviolet absorptivity → UV light is absorbed by amino acids with aromatic rings

Importance of protein analysis

Nutrition labeling: total protein content
→ Essential amino acids (8):  isoleucine, leucine, valine, lysine, threonine, tryptophan, methionine, phenylalanine + children need histidine
→ limiting amino acid: what the food is lacking in
Ex: grain low in lysine, corn in tryptophan, legumes low in sulfur amino acids like methionine
Protein complementing → combine 2 protein sources to get the full shebang of essential aas in sufficient amonuts (ex: grains x beans)

Functions in foods: foaming (egg albumen), gelling, emulsification, coagulation, water binding and solubility

Sensory: flavor (MSG), texture, taste, and color

Biological activity determination: must determine protein content to determine enzyme activity per mg protein, expression of specific protein per mg protein, western blot analysis (gel electrophoresis - separate proteins by molecular weight)

Methods for protein analysis

Element (Nitrogen) →
Kjeldahl (GOLDEN!),
Nitrogen analyzer: Pyrolysis - high temps to release N

Colorimetric:
Biuret (peptide bondes)
Lowry (side chains, peptide bonds)
Bradford: Dye binding assay. Dye (Coomassie brilliant blue G-250) binds to protein to cause dye color change from 465 to 595 nm
Common

Spectroscopy
Ultraviolet 280nm absorption: targets specific side chains
Infrared spectroscopy (IR) → Peptide bonds in protein causes a specific wavelength absorption

Molecular size and charge: Electrophoresis

HPLC: for separating proteins, peptides, amino acids

Kjeldahl method principles protein analysis

Proteins in food sample are digested with sulfuric acid to release nitrogen and then trapped by being converted into ammonium sulfate (NH4)2SO4

The ammonium sulfate is then neutralized with NaOH to release NH3

NH3 is then distilled into a boric acid solution to form ammonium borate (NH4)3BO3, which can be titrated with acid to convert to total nitrogen in the sample to calculate amount of protein
Steam distillation! ammonia goes straight into the flask that contains the boric acid

The sample must pass 20-mesh screen and be homogenous

Kjeldahl calculation and applications

Mole HCl used = Mole NH3 produced = Mole N sample
% protein = [(mL std. acid for sample - mL std. acid for blank) x N_HCl]/w(g) x 14g N/mol x 6.25 × 100

Applications
Suitable for all types of foods
Accurate, but poorer precision than Biuret and Lowry → many steps involved
Official method for crude protein
Relatively simple
Inexpensive but slow >2hrs, can do many samples at the same time
Disadvantages: measures total nitrogen, not just protein nitrogen → can have problems with urea contamination or people adulterating with melamine to increase % protein

Biuret method protein analysis

Principle:
Biuret rxn = protein mixed with solution of NaOH and copper sulfate → peptide bonds in protein and peptides form a stable complex with Cu2+ (cupric ions) → violet color → absorbance read at 540nm against a reagent blank
Requires more than 2 peptide bonds → dipeptides and free amino acids won’t work

Applications:
Less expensive
Rapid and simple → less than 30 min
More specific, less interference → does not detect non-protein nitrogen (NPN)
Sensitivity: 2mg

Lowry method protein analysis

Principle
Based on reaction with Folin-Ciocalteau phenol reagent after alkaline copper treatment (Biuret reaction)
Reaction with Folin-Ciocalteau will reduce aromatic amino acids (tryosine and tryptophan) → blue color! read at 750nm → FC will also react with sugars, phenolics and stuff
Phenol reagent = phospho-18-molybdictungstic acid

Applications
Most widely used
Most sensitive method → 10-20ug → 100x more sensitive than Biuret, 10-20x more sensitive than UV
Less affected by sample turbidity bc of color → can do direct analysis, no need to purify interfering compounds out
More specific (first peptide bonds → then aromatic amino acids)
Simple and quick: 1 hr

UV 280nm absorption method protein analysis

Principle:
→ Tryptophan, tyrosine, and phenylalanine are the only amino acids that absorb UV light with absorption maxima at 200-230nm and 250-290nm
→ We use readings at 280nm for determination of peptides and proteins bc thats where we see tyrosine and tryptophan residues → then use Beer’s law to estimate concentration of protein
→ Tyrosine and tryptophan content in proteins from each food source is fairly constant

Applications:
Rapid
Sensitive: detect 100uL, 20x more sens thn Biuret, 10x lower than Lowry
NO interference from buffer salt
Nucleic acid interference: nucleic acids also have absorbance at 280nm → use 280nm/260nm ratio for purity analysis → ratio for pure protein and nucleic acids are 1.75 and 0.5, respectively → used to correct for nucleic acids