Construction of Dose-Response Curve

● Toxicology = study the adverse effect (harm) of hazards and poisons to living organisms.
● Dose = amount of exposure to an agent.
● Response = reaction to the dose (how a chemical affects a living thing)
- For example, drinking one glass of milk may be fine, but drinking a gallon of milk will produce a
very undesirable response (bloating and gastric discomfort).
● Increased dose -> produce more effects
● The effect of a chemical depends on the dose (amount of chemical that enters the body) but also on the
resulting concentration (amount of chemical compared with body size/volume), the length of exposure to the
chemical, and the route of exposure.
● Response Dose The classic “S-shaped” dose response curve. (response = x axis, dose = y axis)
● Examining the risk-benefit ratio in respect to the side effects of pharmaceutical treatments is important
in understanding another aspect of toxicology since chemical toxicity of a substance varies between persons
Results
The mung beans were able to grow in sugar, ethanol, coca cola, coffee, milk, and apple juice substances. However,
the mung beans cannot grow in soy sauce, this might be due to the fact that soy sauce contains complex molecules
that can not be digested by the plant. Soy sauce contains high salinity that can cause plants to dehydrate as a result
of osmosis where the water (altogether with nutrients) inside the plants move across to a higher concentration.
Consequently, the plants will not be able to absorb and utilize water, leading to death.

Treatments
1. Sugar (sucrose): fructose and glucose
● Human: provide energy
- downside: weight gain, dental caries, fatty liver, type 2 diabetes
- Limitation: 25 grams for women, 37.5 grams for man
● Plant: stimulate the growth of plant by providing the necessary energy for growth and even
increasing total phenolic compounds as well as antioxidant activitiy
- excessive sucrose uptake: disrupt the metabolism of the plants
- Harmful for mungbean growth bcs sugar solution is source of energy for pathogenic
microorganisms
2. Cocacola: high in caffeine, fructose corn syrup
● Human
- Benefit: energizing,
- Downside: increase weight gain as the fructose produces less leptin, a hormone to control
appetite and gives less satiety
● Plant
- Benefit: enhance the growth and development as soda water is filled with the required
macronutrients for plants (carbon, hydrogen, phosphorus, oxygen, sodium, sulfur, and
potassium)
 - Downside: inhibit the growth of plants due to the high amounts of sugar as they could not
absorb the complex structure of sugar and dehydration from osmosis where water exits
the plant caused by different concentrations
3. Ethanol
● Human
- Benefit: could reduce the risk of coronary heart disease
- Downside: cardiovascular disease, nervous system damage, cancer, liver damage
● Plant
- Benefit: stimulate seed germination (With ethanol, dehusking could occur due to the
hydrolysis, hence water could penetrate into the seed and grow)
- Downside: stunted growth
4. Apple juice
● Human
- Benefit: reducing the risk of chronic disease, diabetes, heart disease, and cancer.
- Downside: diarrhea and poor blood sugar regulation
● plant:
- Downside: remove minerals, vitamins and other nutrients bcs of the acidic pH
5. Milk
● Human
- Downside: plagues in the artery, and heart diseases, lactose intolerance
● Plant
- Benefit: support plant’s growth due to high calcium content which could prevent stunting
and underdeveloped fruit. Milk act as antifungal agent
-
6. Soy sauce: high sodium
● Human
- Downside: escalate blood pressure levels which eventually leads to kidney damage,
stroke and heart diseases, allergy
● Plant
- Downside: dehydrate plant due to high salinity -> death
7. Coffee: high in caffeine
● Human
- Benefit: increased metabolism and maintaining liver
- Downside: anxiety, insomnia, restlessness
● plant
- Benefit: provide nitrogen, calcium, and magnesium for plants which promotes strong stem
growth
- Downside: inhibit the absorption of vitamins and minerals (for sprouting)
Ochratoxin A in Herbs and Spices
● Toxic mold contamination -> happens bcs of lack adequate quality control, weather conditions during the
growing season, improper harvesting and storage practices
● Herbs & spices that found the presence of mycotoxins: chamomile, black and white tea leaves, ginkgo
leaves, paprika, and cumin
● Mycotoxins that are present in herbs and spices = aflatoxins and ochratoxin A
❖ Aflatoxins = produced by Aspergillus flavus and Aspergillus parasiticus
- Source: growing soil, decaying vegetation, hay, and grains
- Most toxic type: aflatoxin B1
❖ Ochratoxin A = produced by different Aspergillus and Penicillium species
- Ochratoxin A = secondary metabolite (not protein)
- Sifatnya Carcinogenic
- Ochratoxin A is Nephrotoxic -> toxic to kidneys and can lead tumors in the upper urinary
tract
- Source: cereal grains, dried fruits, wine and coffee
● SDS-PAGE or Sodium Dodecyl-Sulfate PolyAcrylamide Gel Electrophoresis = used to separate protein
molecules with molecular masses between 5 and 250 kDa ( can detect fungi-produced Ochratoxin A)
- Principle: a charged molecule migrates to the electrode with the opposite sign when placed in an
electric field. The tiny molecules tend to move faster due to their less resistance at the time of
electrophoresis.
- Developed by: Ulrich K. Laemmli
- to get the visualization of protein degradation and the existence of contaminating protein
- Mechanism: The tertiary and quaternary structure will be denatured to ensure their capability of
flowing through the gel in the electric field
- smaller proteins = positively charged | larger proteins being = negatively charged
- Smaller protein molecules -> located nearer the anode
● TAE buffer: for the separation of nucleic acids (DNA & RNA)
● Nucleic acids have an overall negative charge due to the presence of phosphodiester bonds in their
phosphate backbone and will move towards the positive electrode (anode). Nucleic acid molecules will move
through the agarose pores in different speeds in inverse correlation with their size – smaller sized fragments
will move faster through the gels and can be found in the lower part of the agarose gel.
● The smaller molecules migrate faster due to less resistance during electrophoresis. The structure and the
charge of the proteins also influence the rate of migration. Sodium dodecyl sulfate and polyacrylamide
eliminate the influence of structure and charge of the proteins, and the proteins are separated based on the
length of the polypeptide chain.
● Electrophoresis is usually done in two different matrix types with different pore sizes: acrylamide and
agarose. Acrylamide is commonly used for very small DNA and most protein electrophoresis. Agarose is the
matrix of choice for most nucleic acid electrophoresis as the larger pore enables larger DNA (or RNA)
fragments to separate clearly. In fact, agarose gel electrophoresis is the most common method for analysis
of DNA samples.
● SDS is a detergent present in the SDS-PAGE sample buffer
 - Function: break the disulphide bonds of proteins disrupting the tertiary structure of proteins
● 4 main step of SDS PAGE:
1. Agarose concentration
- Agarose = composed of polymers of uncharged repeating disaccharides
❖ The agarose % is inversely proportional to the pores within the gel and so has
an inverse relationship with the size of DNA fragments that may be properly
resolved (Low percentage agarose gel will have large pores (~300 nm) which
enables large DNA fragments to be clearly resolved)
❖ Commonly used: agarose gel 0.8% (w/v) or 1.0 % (w/v)
2. Running buffer
- Two types of buffers commonly used: Tris-Acetate-EDTA (TAE) and Tris-Borate-EDTA
(TBE). But TAE is preferred better separation of larger fragments, which is greater
than 3 kb and better for cloning
- Downside of TBE: TBE may precipitates during storage and TBE reduces the amount of
DNA that can be recovered from the gel.
3. Running voltage
- Most agarose gels are run at a constant voltage and usually run at 10 V / cm
- Lower voltage size (1-5 V/cm) used if large DNA fragments (> 5 kb) is desired
4. Staining method
- gel staining: to visualize the nucleic acid fragments
- Most nucleic-acid binding dyes work by intercalating in between the base pair stacks of
nucleic acids
- Nucleic acid stain: Ethidium Bromide (EtBr) -> commonly used
● Hexane → to extract edible oils from seeds and vegetables and as a cleaning agent
● NaCl → It keeps proteins soluble and increases the ionic strength of the buffer (facilitates the disruption of
molecular interactions)
● Methanol → prevent the gel-swelling, precipitate the proteins in the gel in acidic condition and solubilize the
dye in coomassie solution
● Laemmli SDS sample buffer → protein sample preparation, which is used in the Laemmli SDS-PAGE
system. It is a widely used system for protein electrophoresis. Further, it is employed in the polyacrylamide
gel analysis of proteins.
● A running buffer contains ions that conduct current through the gel. When proteins are loaded into wells
at the top edge and current is applied, the proteins are drawn by the current through the matrix slab and
separated by the sieving properties of the gel.
● Coomassie Blue stain = to stain the protein bands in polyacrylamide gels
- to dissolve the dye in a mixture of methanol, acetic acid, and water. This stain will permeate the gel,
stain the protein, and also fix the protein in place.
- The dye binds more tightly to the proteins than to the gel matrix
- The stain can be removed by omitted the dye is omitted or destain
Results

There was no band present in the SDS page. Failure to acquire bands from the SDS page was mainly caused by the
poor practices during the insertion of samples to the gel. Supposedly, after the second stop of the micropipette was
used, the micropipette should be lifted before releasing from the second stop. However, with respect to the common
practices where the tip of the pipette has no contact with liquid, experimenters habitually release the second stop
right after ejecting the samples. Consequently, bubbles were present and the samples were not inserted properly as
the samples were diluted in the tris-glycine-SDS buffer, leading to no band formation. Another possible mistake was
the samples failed to reach the well’s bottom which caused difficulty in the identification. It is nonetheless unusual for
Ochratoxin A to be detected through the SDS-page method as Ochratoxin A is not a protein, but a secondary
metabolite.

Proper method to detect the presence of Ochratoxin A

1. Liquid Chromatography Mass Spectrometry (LC-MS)

- Benefit: has been used to identify the Ochratoxin A and produce more accurate data
- The method can identify as few as 5-20 pg of inserted OT A, which corresponds to a detection limit
of 0.2 ppb in paprika powder.
2. ELISA (Enzyme-Linked Immunosorbent Assay)
- applied to assess ochratoxin in feed, animal serum, and barley.
- Benefit: simple, can process massive samples in a day, and specificity of the antibody
3. HPLC-FD (High-Performance Liquid Chromatography with Fluorescence Detection)
- commonly used to detect and quantify the mycotoxin in foods
- Benefit: higher sensitivity and specificity than ELISA method
- Limit detection: 0.004-0.100 mg/kg
- Quantification limit; 0.01-0.30 mg/kg

Detection of Cow’s DNA Presence in Buffalo Mozzarella Cheese

Cheese

● Cheddar cheese = hard cheese from pasteurized cow milk

● Feta cheese = use sheep milk (either 100% or combined with 30% goat milk)
- Characteristics: crumbly texture
● Dali in Horbo cheese = from batak indonesia
- Characteristics: soft like tofu, chewy and umami
● Mozzarrella cheese = use buffalo milk
- Characteristics: soft, chewy, meltability

PCR (Polymerase Chain Reaction) = used DNA enzymatic replication principle. Using a thermal cycler and a
reagent mixture, the DNA is cycled through a series of different temperatures (2 – 3 temperature points) and
amplified due to the presence of a heat stable DNA polymerase

● Mechanism: short segment of DNA is amplified using primer mediated enzymes. DNA Polymerase
synthesizes new strands of DNA complementary to the template DNA. The DNA polymerase can add a
 nucleotide to the pre-existing 3’-OH group only. Therefore, a primer is required. Thus, more nucleotides are
added to the 3’ prime end of the DNA polymerase.
● 3 Steps in PCR:
1. Denaturation = template DNA is denatured producing a single strand by heating the sample at
temperature > 90˚C
2. Annealing = primers are annealed to the original strand to synthesize new strands. These primers
are usually 20-25 nucleotides in length (Shorter primers may result in an increased chance of
mismatch).
3. Extension = Heat-stable DNA polymerase extends the primers by adding the complementary
dNTPs from the 3’ (-OH) end of the primer. This step is usually done at 72 ˚C, with 1 minute
allocated for each 1000 bp of products
- Enzyme: Taq polymerase (synthesized and duplicated the DNA consisting of one old
DNA strand and one new DNA strand)
● Food lysis buffer → isolating genomic DNA from a variety of food samples
● Proteinase K solution → destruction of proteins in cell lysates (tissue, cell culture cells) and for the release
of nucleic acids
● Chloroform → protects genomic DNA during a catastrophe. Chloroform increases the efficiency of phenol to
denature the protein. Here, chloroform allows proper separation of the organic phase and aqueous phase
and keeps DNA protected into the aqueous phase
● Buffer PB → used in DNA cleanup procedures and enables efficient binding of single- or double-stranded
PCR products to the spin-column membrane.
● Buffer AW2 → wash away contaminants from the DNA.
● Buffer EB → elution buffer
● Gotaq Green Mastermix → gives robust amplification equal
● Reverse and forward primer → The forward primer attaches to the start codon of the template DNA (the
antisense strand), while the reverse primer attaches to the stop codon of the complementary strand of DNA
(the sense strand).
● Nanodrop = determine the DNA purity
- 2 indications of the ratio used: 260/280 and 260/230

NOTES: numbers describe the absorbance at wavelengths of 230, 260, and 280 nm

- Absorbance at 230 nm = how pure a sample is from salts or other contaminants

- 260 nm = whether the compound was DNA or RNA
- 280 nm = the presence of a high amount of protein which correlates to nucleic acids.
● 3 general step in DNA extraction:
○ Lysis = the nucleus and the cell are broken open, thus releasing DNA. This process involves
mechanical disruption and uses enzymes and detergents like Proteinase K to dissolve the cellular
proteins and free DNA
○ Precipitation = separates the freed DNA from the cellular debris. It involves use of sodium (Na+)
ions to neutralize any negative charge in DNA molecules, making them less water soluble and
more stable. Alcohol is then added -> causing precipitation of DNA from the aqueous solution since
it does not dissolve in alcohol.
 ○ Purification = removes all the remaining cellular debris and unwanted material. Once the DNA is
completely purified, it is usually dissolved in water again for convenient storage and handling.

Results

The PCR method were paired with gel electrophoresis. PCR is to only determine the presence of bovine’s
DNA, gel electrophoresis with agarose gel was done to determine the molecular weight of said DNA. The experiment
used a 100 bp DNA ladder with a range of 100-3,000 bp for the standard. The results showed the samples that
contain cow’s DNA: mozzarella, cheddar, and feta cheese which all of them was observable at bp Plus DNA Ladder
at the range between 200-300. Furthermore, there was no cow’s DNA detected in local cheese samples (Dali in
Horbo). Additionally, there was DNA detected in a feta cheese sample which might be caused by the similarities
between the DNA between cow and goat because both of them have the same diploid chromosome number which is
2n=60.

The optimal amount for the 260/280 ratio is 1.80 for DNA, while for RNA is 2.00 which means if the amount
is below 1.70, it should be avoided because the sample is already contaminated and it is not pure already. On the
other hand, the optimal amount for the ratio 260/230 is 2.0 or above 2.0. Based on the result of the experiment, there
was only one sample which had 260/280 value above 2.0 which means that there were other proteins present in
almost all of the samples that lead to not accurate detection and no sample had 260/230 above 2.0. Thus, it could be
determined that all of the samples were contaminated by salt and other impurities and the samples were not
completely pure. Other factors showed that there was inconsistency by the results since the duplicate mozzarella and
cheddar cheese samples had a band in one gel but not the other.

Improvements:

● pedestal used need to be cleaned frequently

● no air bubbles present in the pedestal -> create a bad graph during measuring
● Carefully handling the samples with low concentration
● Use negative and positive control to decrease the possibility of false results
- Positive control = presence of target sequence
- Negative control = all of the chemicals used but no nucleic acids.

Effect of different heat treatments on bacterial contents

● Aim: determine the Bacillus subtilis thermal resistance in milk as well as observing the effect of the heat
treatments on the bacterial count.
● Plate Count Agar = used for the enumeration of bacteria in food, water and other materials of sanitary
importance
● Bacillus subtilis = gram positive bacteria that are able to form spores and able to withstand toxic chemicals,
desiccation, radiation, and heat.
- Can caused food spoilage
- Best temperature: 37oC.
● D- value: the time required to achieve 90% reduction or 10% remaining in the population of specific
microorganism
 ● F- value: time that a product receives a lethal amount of sterilization
● Z-value: temperature change required to produce one log reduction in D-value
● Cfu/mL = total number of colonies x dilution factor)/volume of cultured plate

Effect of heat treatments to the bacteria

● Bacillus subtilis are killed by heat treatment at 140oC for 15 minutes
● Wen et al. (2022) stated applying heat treatments to Bacillus subtilis at 40o-70o C actually promotes the
spore germination.
Results
All of the milk samples had Too Numerous To Count (TNTC) values which inferred that heat treatment was
ineffective in eradicating the Bacillus species. This is due to the fact that Bacillus spores were extremely resistant to a
wide range of heating. Furthemore, the results indicated that it was not possible to calculate the D-value, F-value, and
Z-value since the samples depicted TNTC results. For future studies, it is recommended to conduct the experiment
more aseptically and heat the samples for a longer period of time. Moreover, it also recommended using a higher
dilution factor so most of the samples could be counted and it is important to make sure that the heat already
achieved the desired temperature before heating the sample so the bacteria could die.

Note:
● the bacterial content can also be more easily observed if the milk sample heated at 80°C and 90°C is treated
to a greater dilution factor as the highest dilution employed in this experiment is the lower dilution treatment
(10-2).

How does the SDS run from + to - current?