Slide content - Labs Flashcards
What is the mechanism of the BCA assay?
- Peptide bonds in protein reduce Cu2+ to Cu1+ in the presence of a base
- BCA reagent is alkaline (~pH 11)
- Temperature-dependent assay!!
- Every assay needs a new standard curve
- The amount of Cu2+ reduced is proportional to the amount of protein in the sample
- BCA reagent chelates the Cu1+ from the protein to form a complex
- 2 moles of BCA to 1 mole of Cu1+
- Not a reaction, no bonds are formed
- Creates a colored product that can be quantified at λ = 562 nm
The BCA assay involves a reaction between the BCA reagent and copper.
True or False?
False.
No bonds formed.
BCA reagent chelates the Cu+1 (reduced from Cu2+ by peptide bonds in protein) to form a complex
The BCA assay is temperature-dependent.
True or False?
True.
Every assay needs a new standard curve.
The BCA agent is acidic.
True or False?
False.
It is alkaline.
The BCA reagent is alkaline.
True or False?
True.
Why dry foods? [3]
-
Microbial activity
- Not enough water available for bacterial, yeast, or mould growth
- Does not necessarily kill microbes unless temp >60°C used
-
Enzymatic activity
- Produces conformational changes in the enzyme, affecting its catalytic activity
-
Chemical reactions
- Reactants cannot move as freely in the food and thus reaction rates are lower; however, oxidation rates may increase
What are advantages of air-drying?
Relatively inexpensive equipment
Can modify drying conditions (temp, air flow, humidity, time)
What are disadvantages of air drying?
- case hardening
- poor rehydration
- loss of bioactive compounds
- slow - especially in later stages
What are the 3 stages of freeze drying?
- Freezing phase – Freeze product to below it’s triple point so that sublimation rather than melting will occur
- Sublimation phase – Pressure is lowered and “heat” (-20°C) is added to shelves, causing water to sublimate; 95% of water is removed
- Adsorption phase – Ionically-bound water molecules are removed by raising the temperature (~ 0 - 30°C or above) to break the bonds between water and the materials; 1-5% residual moisture
Describe the drying phases in air drying.
- Heating phase (Product is brought up to drying temperature
- Constant rate zone (heat is added at the same rate as heat is loss due to evaporation of water; most water is removed
- Falling rate zone (Last 10% of water is removed; Rate of removal is slow due to bound water and case hardening)
What are advantages of freeze-drying? [4]
- no/little shrinkage
- no case hardening
- excellent rehydration
- good retention of bioactive compounds
What are disadvantages of freeze drying? [4]
- expensive process
- Slow
- Cannot achieve partial drying - all or nothing
- Porous structure; susceptible to oxidation and moisture uptake
What type of drying takes the longest?
Freeze drying
What type of drying produces the least dense product?
Freeze drying
What type of drying has the best rehydration?
Freeze-drying
What does the phenol sulfuric acid method measure?
- Total carbohydrate (using a standard curve)
- Other food components can interfere with absorbance if they absorb light at the same wavelength (e.g., flavonoids)
What does the enzymatic method measure?
Free glucose + fructose content (via NADPH)
Less subject to interference by food components due to specificity of enzyme active site.
Describe the phenol sulfuric assay mechanism.
- Sulfuric acid breaks down polysaccharides to monomers and dehydrates them to furfural derivatives
- Furfural derivatives react with phenol to produce yellow-gold colour (food dilution should be clear initially)
- Absorptivity of solution is measured at 490 nm; colour is stable for several hours
- Standard curve of representative sugar is used for quantification; the reaction is not stoichiometric
What wavelength is measured in the phenol sulfuric acid assay?
490 nm
What are advantages of the phenol-sulfuric acid assay? [5]
- Accuracy is within ±2% under proper conditions
- Simple
- Rapid
- Inexpensive
- Reagents are stable
What are disadvantages of the phenol sulfuric acid assay? [3]
- Harsh chemicals
- Subject to interference by colour and other food components
- Accuracy depends on how well standard solution represents sugar composition in food
What is the principle of the enzymatic method for glucose determination?
- Glucose is converted to glucose-6-phosphate (G6P) by hexokinase (HK) + ATP
- Glucose-6-phosphate dehydrogenase (G6P-DH) + NADP+ oxidizes G6P to gluconate-6-phosphate and forms NADPH which is then measured at 340 nm and = [glucose]
What is the principle of the enzymatic method for fructose determination?
- Fructose is converted to fructose-6-phosphate (F6P) by hexokinase (HK) + ATP
- F6P converted to G6P by phosphoglucose isomerase (PGI)
- G6P then again converted to gluconate-6-phosphate and NADP+ to NADPH
- NADPH measured at 340 nm and = [glucose + fructose]
What wavelength is measured in the enzymatic method?
340 nm
What are advantages of the enzymatic method? [4]
- Simple
- Rapid
- Allows for quantification of individual sugars
- Less subject to interference
What are disadvantages of the enzymatic assay? [3]
- Expensive
- Reagents not stable
- Total carbohydrates cannot be quantified
Yeasts and moulds commonly spoil foods that: [3]
Have low pH and low water activity and are stored at refrigerated temperatures
Why are yeasts and moulds undesirable?
- Some moulds can produce dangerous toxins (aflatoxins)
- Yeasts can cause sugar to ferment (ethanol and carbon dioxide production) or degrade in other ways
Describe the HGMF method.
- Can examine large sample volumes
- Used to concentrate microbes onto a grid membrane
- Membrane is placed on agar surface and colonies grow
- Hydrophobicity of grid confines microbial growth to within squares, allowing for up to 1600 “colonies” to be counted on one plate
What is potato dextrose agar?
- Most widely used medium for growing fungi and bacteria
- Contains a potato infusion (potatoes + water, then filtered) to promote the growth of yeasts and moulds
- Relatively low pH (~5.5) partially inhibits the growth of background microflora (bacteria)
- Trypan blue makes yeast colonies visible
- Chloramphenicol suppresses bacterial growth
- Colonies will appear:
- Yeast = blue
- Mould = blue-grey & fuzzy
What is the D-value?
How is it obtained?
- The D-value is the time is takes for a 1 log reduction in microbial cells (or 90% of a population) at a specific temperature
- Obtained by plotting log(surviving population) vs. time, followed by calculating -1/slope
What is the z-value?
How is it obtained?
- Z-value is the temperature change required for a 1 log reduction in the D-value (described as the thermal resistance of a microorganism)
- Obtained by plotting log(D-values) vs. temperature, followed by calculating -1/slope
How does milk turn into yogurt?
- Milk is inoculated with lactic acid bacteria starter cultures (Usually Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus
Describe the effect of temperature on pH.
- pH decreases with an increase in temperature. As the temperature increases, molecular vibrations increase, which causes water to ionise and form more H+
What is the role of phenolpththalein in titration?
Secondary indicator that changes from colourless to pink at the equivalence point.
How is equivalent weight determined?
- First determine molecular weight of the acid
- Then divide by the number of carboxylic acid groups on the molecule – this equals the number of titratable hydrogens
How is texture analysed (not viscosity)?
Puncture force (N)
Desribe what L, a, and b values correspond to in colourimetry.
-a = green
+a = red
-b = blue
+b = yellow
L = 0 = black
L = 100 = white
L = 50 = grey
What are the megazyme K-FRUGL assay kit components?
- Buffer (pH 7.6)
- NADP+ and ATP
- Hexokinase + glucose-6-phosphate dehydrogenase
- Phosphoglucose isomerase
- D-glucose and D-fructose standards (0.2 mg/mL)
