VIETNAM NATIONAL UNIVERSITY – HO CHI MINH CITY

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY

OFFICE FOR INTERNATIONAL STUDY PROGRAMS

REPORT

Analytical Methods For Protein In Food

Subject: Food Analysis

Lecturer: Nguyễn Thị Lan Phi

Class: CC02 – Semester: 202 – Group: 15

No. Full name Number ID Duties

1 Nguyễn Huỳnh Ngọc Thảo 1852747 Essay chapter 1, powerpoint

2 Hoàng Phương Quyên 1852705 Essay chapter 2

3 Nguyễn Thu Hà 1852340 Essay chapter 3, summary essay

CONTENTS

Abstract.........................................................................................................................1

CHAPTER1:INTRODUCTION..................................................................................1

1.1.Definitions............................................................................................................... 1

1.2. General classifications........................................................................................... 1

1.3. General composition in foods............................................................................... 2

1.4. Physical and chemical properties......................................................................... 3

1.5 General considerations........................................................................................... 6

CHAPTER 2. ANALYSIS METHODOLOGY.......................................................... 7

2.1.Introduction............................................................................................................ 7

2.1.1 Principles.......................................................................................................... 8

2.1.2 Applications..................................................................................................... 9

2.2 Sample preparation.............................................................................................. 10

2.3. Analysis procedures and calculations................................................................ 11

CHAPTER 3. DISCUSSION AND CONCLUSIONS............................................ 13

3.1. Discussion ........................................................................................................... 13

3.3. Conclusion............................................................................................................ 14

REFERENCES........................................................................................................... 15
TABLES

Table 1................................................................................................................................... 3

FIGURES

Figure 1.................................................................................................................................. 9
Abstract

The reported protein content of foods depends on the analytical method used for
determination, making a direct comparison between studies difficult. The aim of this study
was to examine and compare protein analytical methods, including Kjeldahl, Bradford, Lowry
methods. Some of these methods require extraction preceding analysis. The efficacy of
protein extraction differs depending on food matrices and thus extraction yield was
determined. Overall, most analytical methods overestimated the protein contents. The
inaccuracies were linked to indirect measurements, i.e., nitrogen determination and
subsequent conversion to protein, or interference from other chemical substances. Amino acid
analysis is the only protein analysis method where interfering substances do not affect the
results. Although there is potential for improvement in regards to the hydrolysis method, we
recommend that this method should be the preferred for food protein determination.

CHAPTER 1. INTRODUCTION
1.1. Definitions

A protein is an exceedingly complex natural molecule made up of amino acid residues

linked by peptide bonds. Proteins are found in all living species and contain a variety of
biologically important molecules such as enzymes, hormones, and antibodies. A protein
molecule is much larger than a sugar or salt molecule, and it is made up of several amino
acids linked together in long chains, much like beads on a string. Proteins contain around 20
distinct amino acids that exist naturally. The amino acid content and sequence of proteins with
similar functions are similar. Although it is not yet possible to deduce all of a protein's
functions from its amino acid sequence, the properties of the amino acids that make up
proteins have been shown to have established relationships between structure and function.

1.2. General classifications

Based on the chemical nature, structure, shape and solubility, protein are classified as:

- Simple proteins: They are composed of only amino acid residue. On hydrolysis these
proteins yield only constituent amino acids. It is further divided into:
+ Fibrous protein: Keratin, Elastin, Collagen.
+ Globular protein: Albumin, Globulin, Glutelin, Histones

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 - Conjugated proteins: They are combined with non-protein moiety. Eg. Nucleoprotein,
Phosphoprotein, Lipoprotein, Metalloprotein etc.
- Derived proteins: They are derivatives or degraded products of simple and conjugated
proteins. They may be:
+ Primary derived protein: Proteans, Metaproteins, Coagulated proteins.
+ Secondary derived proteins: Proteosesn or albunoses, peptones, peptides.

1.3. General composition in foods

Proteins are macromolecular polypeptides—i.e., very large molecules (macromolecules)

composed of many peptide-bonded amino acids. Most of the common ones contain more than
100 amino acids linked to each other in a long peptide chain.

The amount of protein in meals varies greatly. Animal-based foods and legumes are good
providers of protein. Table 1 shows the protein composition of a variety of foods.

Table 1: Protein content of selected foods.

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 1

1.4. Physical and chemical properties

1.4.1. Physical Properties of Proteins

Colour and Taste

Proteins are colourless and usually tasteless. These are homogeneous and crystalline.

Shape and Size

The proteins range in shape from simple crystalloid spherical structures to long fibrillar
structures. Two distinct patterns of shape have been recognized :
- Globular proteins- These are spherical in shape and occur mainly in plants, esp., in
seeds and in leaf cells. These are bundles formed by folding and crumpling of protein
chains. e.g., pepsin, edestin, insulin, ribonuclease etc.

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Nielsen, S. S. (2017). Food Analysis (Fifth Mæhre, H., Dalheim, L., Edvinsen, G., Elvevoll,
E. and Jensen, I., 2018. Springer Publishing. Chapter 18 page 318
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- Fibrillar proteins- These are thread-like or ellipsoidal in shape and occur generally in
animal muscles. Most of the studies regarding protein structure have been conducted
using these proteins. e.g., fibrinogen, myosin etc.

Molecular Weight
The proteins generally have large molecular weights ranging between 5 × 103 and 1 ×
106. It might be noted that the values of molecular weights of many proteins lie close to or
multiples of 35,000 and 70,000.

Colloidal Nature
Because of their giant size, the proteins exhibit many colloidal properties, such as; Their
diffusion rates are extremely slow and they may produce considerable light-scattering in
solution, thus resulting in visible turbidity (Tyndall effect).

Denaturation
Denaturation refers to the changes in the properties of a protein. In other words, it is the
loss of biologic activity. In many instances the process of denaturation is followed by
coagulation— a process where denatured protein molecules tend to form large aggregates
and to precipitate from solution.

Amphoteric Nature
Like amino acids, the proteins are amphoteric, i.e., they act as acids and alkalies both.
These migrate in an electric field and the direction of migration depends upon the net
charge possessed by the molecule. The net charge is influenced by the pH value. Each
protein has a fixed value of isoelectric point (pl) at which it will move in an electric field.

Ion Binding Capacity

The proteins can form salts with both cations and anions based on their net charge.

Solubility
The solubility of proteins is influenced by pH. Solubility is lowest at isoelectric point
and increases with increasing acidity or alkalinity. This is because when the protein
molecules exist as either cations or anions, repulsive forces between ions are high, since all
the molecules possess excess charges of the same sign. Thus, they will be more soluble
than in the isoelectric state.

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Optical Activity
All protein solutions rotate the plane of polarized light to the left, i.e., these are
levoratotory.

1.4.2. Chemical Properties of Proteins

Hydrolysis
Proteins are hydrolyzed by a variety of hydrolytic agents.
- By acidic agents: Proteins, upon hydrolysis with conc. HCl (6–12N) at 100–110°C for
6 to 20 hrs, yield amino acids in the form of their hydrochlorides.
- By alkaline agents: Proteins may also be hydrolyzed with 2N NaOH.

Reactions involving COOH Group

Reaction with alkalies (Salt formation)
Reaction with alcohols (Esterification)
Reaction with amines

Reactions involving NH2 Group

Reaction with mineral acids (Salt formation): When either free amino acids or proteins are
treated with mineral acids like HCl, the acid salts are formed.
Reaction with formaldehyde: With formaldehyde, the hydroxy-methyl derivatives are
formed.
Reaction with benzaldehyde: Schiff ‘s bases are formed
Reaction with nitrous acid (Van Slyke reaction): The amino acids react with HNO2 to
liberate N2 gas and to produce the corresponding α-hydroxy acids.
Reaction with acylating agents (Acylation)
Reaction with FDNB or Sanger’s reagent
Reaction with dansyl chloride

Reactions involving both COOH AND NH2 Group

Reaction with triketohydrindene hydrate (Ninhydrin reaction)
Reaction with phenyl isocyanate: With phenyl isocyanate, hydantoic acid is formed which
in turn can be converted to hydantoin.
Reaction with phenyl isothiocyanate or Edman reagent
Reaction with phosgene: With phosgene, N-carboxyanhydride is formed

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 Reaction with carbon disulfide: With carbon disulfide, 2-thio-5-thiazolidinone is produced

Reactions involving R Group or Side Chain

Biuret test, Xanthoproteic test, Millon’s test, Folin’s test, Sakaguchi test, Pauly test, Ehrlich
test

Reactions involving SH Group

Nitroprusside test: Red colour develops with sodium nitroprusside in dilute NH4OH. The
test is specific for cysteine.
Sullivan test: Cysteine develops red colour in the presence of sodium 1, 2-naphthoquinone-
4-sulfonate and sodium hydrosulfite.

1.5 General considerations

Divide the amount of protein evenly between meals. Most of us have a habit of eating a
lot of protein at the end of the day, which is absolutely not good for health. According to
nutritionists, you should evenly distribute protein in breakfast, lunch, dinner and snacks. For
women, you should consume about 45g of protein per day, and for men, 55g. Distributing
protein intake evenly helps control hunger hormones, so you'll stay fuller longer and won't
have to snack. After exercising, in about 30 minutes, you need to add about 20-30g of protein
to help your body recover and reduce pain caused by exercise.

Don't just add protein. Protein is one of three important nutrients in the diet. However,
you also note that too much of any one nutrient is not good, all nutrients should be maintained
in balance. Therefore, if you consume too much protein, you will be at risk of acute or chronic
health conditions. For example, a lack of carbohydrates will cause you to feel tired,
light-headed, and have low blood sugar.

Add just the right amount of protein your body needs. According to nutritionists, you
should add 0.8g of protein/kg of body weight. However, you should also note that the amount
of protein you add will depend on many factors such as age, activity level and muscle weight.
Therefore, you should calculate the amount of protein consumed daily, and pay attention to
the consumption of carbohydrates and fats to be able to adjust your diet.

Not all proteins are created equal. Almost everyone assumes that all proteins are the
same, but this is a misconception. In fact, there are many types of proteins that differ in both
quality and amino acid content. Meat and fish are the two best sources of protein. The protein
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content in meat has up to 9 essential amino acids for the body, but meat also contains a lot of
saturated fat. In contrast, fish is very high in protein and low in saturated fat. If you are a
vegetarian, you can also supplement protein by eating more lentils, quinoa, and hemp seeds.

Do not depend on protein supplements. Protein supplements may be the quickest and
easiest way to get amino acids. However, like protein foods, not all supplements are created
equal. The most popular supplement today is whey protein, which helps with muscle growth
and weight management. You should note that before using any functional food, you should
learn carefully about that product.

CHAPTER 2.ANALYSIS METHODOLOGY

2.1. Introduction

Proteins, together with carbohydrates and fats, play an important role in the formation and
maintenance of the human body and are the energy-giving components in the diet.
Furthermore, proteins have a variety of other roles in the body, including enzymatic activity
and the movement of nutrients and other biochemical molecules through cellular membranes.

Protein accessibility may be reduced due to food composition, food structure, or matrix,
and interactions between nutrients, resulting in an underestimate of protein quantity.
Furthermore, different approaches use different analytical principles to determine protein
content, which can be done directly or indirectly. When protein content is computed based on
amino acid residue analysis, it is known as direct protein determination. For example, after
determining the nitrogen content of a protein, or after chemical interactions with functional
groups within the protein, an indirect protein determination can be made.

2.2. Principles

The determination of nitrogen, peptide bonds, aromatic amino acids, dye-binding capacity,
ultraviolet absorptivity of proteins, and light scattering properties are among the core concepts
of these approaches. What is actually being measured must be considered in the selection of
an acceptable method for a given application, in addition to factors such as sensitivity,
accuracy, precision, speed, and cost of analysis.

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2.2.1. Kjeldahl Method

Proteins and other organic food components in a sample are digested with sulfuric acid
in the presence of catalysts in the Kjeldahl method. Ammonium sulfate is formed from the
total organic nitrogen. The digest is alkalized and distilled to produce a boric acid solution.
The generated borate anions are titrated with standardized acid, which in the sample is
transformed to nitrogen. Because nitrogen comes from non protein components, the result
of the analysis shows the food's crude protein content (note that the Kjeldahl method also
measures nitrogen in any ammonia and ammonium sulfate).Proteins and other organic food
components in a sample are digested with sulfuric acid in the presence of catalysts in the
Kjeldahl method. Ammonium sulfate is formed from the total organic nitrogen. The digest
is alkalized and distilled to produce a boric acid solution. The generated borate anions are
titrated with standardized acid, which in the sample is transformed to nitrogen. Because
nitrogen comes from non protein components, the result of the analysis shows the food's
crude protein content (note that the Kjeldahl method also measures nitrogen in any
ammonia and ammonium sulfate).

2.2.2. Bradford Dye-Binding Method

The hue of Coomassie Brilliant Blue G-250 changes from reddish to bluish as it binds to
protein, and the dye's absorption maximum shifts from 465 to 595 nm. The sample's
protein concentration is proportional to the change in absorbance at 595 nm. The Bradford
method, like other dye-binding methods, relies on the amphoteric property of proteins. The
dye added binds electrostatically when the protein-containing solution is acidified to a pH
less than the isoelectric point of the protein(s) of interest. The dye molecule's hydrophobic
interaction with the polypeptide backbone adjacent to positively charged sites in the
protein improves binding effectiveness. In the Bradford method, the dye bound to protein
has a different absorbance spectrum than the dye that isn't bound to protein.

2.2.3. Lowry Method

The Lowry method combines the biuret reaction with tyrosine and tryptophan residues
in the proteins reducing the Folin-Ciocalteu phenol reagent
(phosphomolybdic-phosphotungstic acid) by tyrosine and tryptophan residues in the
proteins in figure 1. The generated bluish color is measured at 750 nm (high sensitivity for
low protein concentration) or 500 nm (low sensitivity for high protein concentration) (low
sensitivity for high protein concentration). Miller and Hartree updated the original

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 approach to increase the linearity of the color response to protein concentration and to
eliminate the use of two unstable reagents in favor of one stable reagent.

Figure 1: slide chains of amino acids tyrosine (a) and tryptophan (b)

2.3. Applications

2.3.1. Kjeldahl Method

The Kjeldahl method is an AOAC-approved method for determining crude protein

content, and it has been used to evaluate a variety of other protein methods. The Kjeldahl
method is still used for some purposes, but due to the availability and benefits of
automated nitrogen combustion (Dumas) equipment, it is now only utilized in a few
nations.

2.3.2. Bradford Dye-Binding Method

Protein content in worts and beer products, as well as potato tubers, has been
determined using the Bradford method. This method has been improved to measure protein
levels in micrograms. The Bradford method has been widely utilized for the examination
of low quantities of proteins and enzymes in their purification and characterizations
because of its speed, sensitivity, and less interference than the Lowry approach. For better
compatibility with buffers and conditions typically employed in protein isolation,
improved Coomassie dye-based protein assays based on the Bradford method have been
developed.

2
Nielsen, S. S. (2017). Food Analysis (Fifth Mæhre, H., Dalheim, L., Edvinsen, G., Elvevoll,
E. and Jensen, I., 2018. Springer Publishing. Chapter 18 page 324
9
 2.3.3. Lowry Method

The Lowry method is widely used in protein biochemistry because of its simplicity and
sensitivity. However, without first removing the proteins from the food combination, it has
not been widely employed to determine proteins in food systems.

2.4. Sample preparation

Solid foods are pulverized fine enough to pass through a 20-mesh sieve. Analytical
samples should be homogenous. There are no additional preparations necessary.

2.4.1. Kjeldahl Method

The size of a sample required for chemical analysis is usually very small (10mg-1500mg)

2.4.2. Bradford Dye-Binding Method

Step 1. Turn on the Genova and allow it to warm up. See below for set up instructions.

Step 2. Gently mix the Bradford reagent and bring to room temperature.

Step 3. Prepare a series of protein standards ranging in concentration from 0.1 to 1.5mg/ml
such that the final volume for the assay is 0.1ml. Note that the Genova can accept up to 6
standards, not including the blank.

Step 4. Prepare the unknown samples in a similar way such that the final volume is 0.1ml.

Step 5. Add 3.0ml of Bradford reagent to each sample and standard, vortex and incubate at
room temperature for 5 to 45min. The protein-dye complex is stable for up to 60 minutes.

2.4.3. Lowry Method

Step 1. Dissolve 20 gm sodium carbonate in 260 ml water, 0.4 gm cupric sulfate (5x
hydrated) in 20 ml water, and 0.2 gm sodium potassium tartrate in 20 ml water. Mix all
three solutions to prepare the copper reagent.

Step 2. Prepare 100 ml of a 1% solution (1 gm/100 ml) of sodium dodecyl sulfate (SDS).

Step 3. Prepare a 1 M solution of NaOH (4 gm/100 ml).

Step 4. For the 2x Lowry concentrate mix 3 parts copper reagent with 1 part SDS and 1
part NaOH. Solution is stable for 2-3 weeks. Warm the solution to 37 degrees C if a white

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 precipitate forms, and discard if there is a black precipitate. Better, keep the three stock
solutions, and mix just before use.

Step 5. Prepare 0.2 N Folin reagent by mixing 10 ml 2 N Folin reagent with 90 ml water.
Kept in an amber bottle, the dilution is stable for several months.

2.5. Analysis procedures and calculations

2.5.1. Kjeldahl Method

Calculations:

Moles of HCl = moles NH3 = moles N in the sample

A reagent blank should be run to subtract reagent nitrogen from the sample nitrogen.

𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑎𝑐𝑖𝑑 𝑣𝑜𝑙𝑢𝑚𝑒 14𝑔 𝑁 100

%𝑁 = 𝑁𝐻𝐶𝑙 × 𝑔 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 ×1000
× 𝑚𝑜𝑙
× 1000

where:

N HCl = normality of HCl in moles/1,000 mL

Corrected acid vol. = (mL std. acid for sample) – (mL std. acid for blank)

14 = atomic weight of nitrogen

A factor is used to convert percent N to percent crude protein. Most proteins contain 16 %
N, so the conversion factor is 6.25 (100/16 = 6.25).

%𝑁 / 0. 16 = % 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑂𝑅 %𝑁 × 6. 25 = %𝑝𝑟𝑜𝑡𝑒𝑖𝑛

2.5.2. Bradford Dye-Binding Method

Step 1. Coomassie Brilliant Blue G-250 is dissolved in 95% ethanol and acidified with
85% phosphoric acid.

Step 2. Samples containing proteins (1–100 μg/mL) and standard BSA solutions are mixed
with the Bradford reagent.

Step 3. Absorbance at 595 nm is read against a reagent blank.

Step 4. Protein concentration in the sample is estimated from the BSA standard curve.

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2.5.3. Lowry Method

The following procedure is based on the modified procedure of Hartree:

Step 1. Proteins to be analyzed are diluted to an appropriate range (20–100 μg).

Step 2. K Na Tartrate-𝑁𝑎2𝑆𝑂3 solution is added after cooling then incubated at room

temperature for 10 min.

Step 3. 𝐶𝑢𝑆𝑂4 − 𝐾 Na Tartrate-NaOH solution is added after cooling then incubated at

room temperature for 10 min.

Step 4. Freshly prepared Folin’s reagent is added and then the reaction mixture is mixed
and incubated at 50 °C for 10 min.

Step 5. Absorbance is read at 650 nm.

Step 6. A standard curve of BSA is carefully constructed for estimating protein

concentration of the unknown.

Calculations:

∆ 𝐴750𝑛𝑚 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 = 𝐴750𝑛𝑚𝑆𝑡𝑑 − 𝐴750𝑛𝑚 𝐵𝑙𝑎𝑛𝑘

Plotting the ∆ 𝐴750𝑛𝑚 Standards vs Protein concentration.

Sample Determination:

∆ 𝐴750𝑛𝑚 𝑇𝑒𝑠𝑡 = 𝐴750𝑛𝑚𝑇𝑒𝑠𝑡 − 𝐴750𝑛𝑚 𝐵𝑙𝑎𝑛𝑘

Determine the mg protein from the Standard curve.

µg Protein = (µg of protein from the Standard curve) (df) df = Dilution factor

(µ𝑔 𝑃𝑟𝑜𝑡𝑒𝑖𝑛) (100)

% 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 = (µ𝑔 𝑠𝑜𝑙𝑖𝑑/𝑚𝑙 𝑅𝑒𝑎𝑔𝑒𝑛𝑡 𝐷)

100 = Conversion to percentage

For Products that are liquid:

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 (µ𝑔 𝑃𝑟𝑜𝑡𝑒𝑖𝑛)
µ𝑔 𝑃𝑟𝑜𝑡𝑒𝑖𝑛/𝑚𝑙 = (𝑚𝑙 𝑠𝑎𝑚𝑝𝑙𝑒/𝑚𝑙 𝑅𝑒𝑎𝑔𝑒𝑛𝑡 𝐷)

CHAPTER 3. DISCUSSION AND CONCLUSIONS

3.1 Discussion

The Kjeldahl method was selected as an example of this analytical principle in this study
since it is still acknowledged by the AOAC International as the standard method for
determining dietary protein. Following the nitrogen determination, a conversion factor is used
to compute crude protein concentration. The original, and still widely used, conversion factor
6.25 is based on the premise that dietary proteins contain 16 percent nitrogen and that all
nitrogen in meals is linked to proteins. However, because relative nitrogen concentration
changes across amino acids and amino acid composition varies between dietary proteins, they
are only preliminary estimates.

There are several disadvantages that should be considered.

Firstly, Kjeldahl Method not just protein AND, but total organic N is measured. It takes
a long time. Corrosive reagents are used. Precision is lower than with some other
approaches. In other words, this method does not measure actual protein, and
overestimations of protein can occur, because the normal nitrogen correction factor 6.25 is
used, and to calculate total protein content, species-specific conversion factors must be
used.

Secondly, the reagents in Bradford Dye-Binding method have a tendency to discolor the
test tubes. Because the stain would impact the absorbance result, the same test tubes could
not be used. This approach is also dependent on the passage of time. When more than one
solution is being tested, it is critical to ensure that each sample is incubated for the same
period of time in order to make valid comparisons. Additionally, this method works by
comparing the protein's absorbance to that of a reference protein. It is likely that the
concentration measured will be erroneous if the protein does not respond to the dye in the
same way as the reference protein.

Thirdly, Lowry method sensitive to low concentrations, the narrow pH range that it is
accurate in

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 In three methods, the Kjeldahl technique gives the highest protein levels in the majority of
samples, whereas the Lowry method showed greater results than the Bradford method.

3.2 Conclusion

In conclusion, the overestimation of protein content that results from estimating protein
from nitrogen poses a concern. One is the risk of food adulteration, as a high protein level
increases a product's economic worth. In other circumstances, the producer has added
non-protein nitrogen, such as melamine, to increase the apparent protein content and, as a
result, the commercial worth of the food product. This may threaten consumer food safety,
thus it's critical that such food adulteration is eliminated.

There are several methods for determining protein concentrations, each with its own set of
benefits and drawbacks. Because of the wide range of methodologies available, direct
comparisons between studies are challenging, and the analytical approach used should be
justified in light of the study's objectives. The results of this study revealed that protein
assessment based on nitrogen analysis overestimates protein amount in most dietary matrices
than that of amino acid analysis, whether or not species-specific conversion factors are
applied is debatable. To evaluate the protein content in foods, methods based on the specific
features of proteins and amino acids have been devised. The speed and sensitivity of the
various approaches differs.

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