1.

INTRODUCTION

1.1. Background

Protein is an essential nutrient that used for body metabolism, maintaining body weight,
body composition, and overall health. Estimated Average Requirement (EAR) for daily
protein intake is 0.7 g/kg body weight/day. The protein requirement increases with the
increase of age. Adolescence and adult people require 0.75 g/kg body weight/day; older
people requires 1.0 to 1.3 g/kg body weight/day dietary protein to optimize physical
function (Deutz et al., 2014; WHO, FAO, 2007). Protein in its native form has an
inactive amino acid composition, but its inactivity can be altered through the action of
digestive proteases (Silva & Malcata, 2005). In the body, protein continuously degrades
in a regulated process by nonspecific proteases (pepsin, trypsin, and chymotrypsin). It
will generate energy and release amino acids that are absorbed by the body and used for
metabolism (Reinstein & Chiechanover, 2006).

The amino acid compositions and its digestibility determine the quality of protein.
Protein digestibility is the ability of a protein to hydrolyze by digestive enzymes into a
smaller form. High digestibility of protein results in a higher amount of amino acids that
can be absorbed and readily used by the body; vice versa. If only small amounts of
protein are digested by the body so the rest will be discarded through feces (Anggraini
& Yunianta, 2015). Food processing has a role in the level of protein digestibility.
Minimal processing resulted in longer digestibility of protein. Enzymatic hydrolysis is a
method that used to increase protein digestibility and works by transforming complex
molecules into a simpler one. In food processing, the enzyme has been widely used and
provided various advantages. It can decrease biodegradability of the products, decrease
energy consumption levels, catalyze process under very mild reaction, and specific in
action. It has fewer waste products, fewer side reactions, does not destroy food
components and valuable attributes, neither results in a higher quality of a product
(Whitehurst & Oort, 2010).

1
 2

Milk protein is considering as an excellent source of essential amino acids for human
nutrition, growth, and development (Kanwar et al., 2009). Milk protein fragments
varying in size from 2 to 20 amino acids (Pessato & Tavano, 2015). Its properties relate
to their composition, structure, hydrophobicity, the position of amino acid residue, and
molecular weight (Bayarjargal et al., 2014). It has various functionalities and may play
important for biological functions such as antioxidant activity (Kumar et al., 2016).
Antioxidant activity can prevent free radicals. Synthetic antioxidant in food product
used under strict regulation. Therefore, the search for natural and safe antioxidant from
various natural sources has been gaining much interest (Mirzaei et al., 2016).

Through enzymatic hydrolysis, milk protein enters into the body already in simpler
forms. Protein is ready to use for body metabolism. It will be faster and optimizer the
digestion of the protein. It also has a capability as an antioxidant. The selected milk for
this research was pasteurized skim milk. It has a high quality of protein with a good
balance of all essential amino acids needed by humans. In the previous study “The
Effect of Enzymatic Hydrolysis Condition on Physical of Cow Milk”, milk was
hydrolyzed by alcalase, protamex, and flavourzyme. All the enzymes were expansive.
Therefore in this study, milk will be hydrolyzed by two kinds of commercial enzymes,
i.e. papain and bromelain enzymes. Papain and bromelain enzymes are endopeptidase
enzyme that has proteolytic activity. Both of the enzymes are rare to hydrolyze protein
in milk, so there limited information about it. It also becomes an alternative enzyme for
hydrolyze milk protein in addition alcalase enzyme, protamex enzyme or flavourzyme
which has been very often used. Papain enzyme and bromelain enzyme also cheaper and
easier to get compare with alcalase, protamex, and flavourzyme.
 3

1.2. Literature Review

1.2.1. Milk

Billions of people around the world consume milk every day due to it is a nutritious
food ingredient. It recognized as a source of protein for humans in all ages. It is not only
as a source of protein but also as a source of numerous nutrients such as magnesium,
selenium, calcium, riboflavin, vitamin B5, vitamin B12 and the other components that
give a significant contribution to human body and health (FAO, 2013). It contains high-
quality protein with a good balance of all essential amino acids that needed by humans
for growth, development and human nutrition (Kanwar et al., 2009). Milk contains 3%
of protein (80% is casein (αs1-, αs2, β-, and κ-casein) and 20% is whey). After casein
precipitation, whey in the supernatant and it contains amino acid and peptides (Tavares
& Malcata, 2013). The pH of milk ranges from 6.6 to 6.8 (Gemechu et al., 2015).
Skimmed milk is milk which most of the fat has been removed, its compositions (per
100 g product) are 90.8% water, 3.4% protein, 0.1% total fat, 5.1% lactose, and 34 kcal.
Pasteurization is a heat treatment process to reduce the number of pathogenic
microorganisms until in a level that does not contribute a significant health hazard, such
as Mycobacterium tuberculosis and Coxiella burnetii. Time and temperature for
pasteurization are 720C for 15 seconds (continues pasteurization) or 630C for 130
minutes (batch pasteurization) (FAO, 2013).

The bioactive peptide that is derived from milk protein has various functionalities such
as antioxidant activity. Antioxidant activity can be measured through DPPH radical-
scavenging activity (DPPH) method. The method based on the peptide activity to donate
hydrogen that can scavenge free radicals (Wu et al., 2015). DPPH is a method that
based on the ability to donate hydrogen atoms from peptides to DPPH as free radicals
that have one unpaired electron so that there is a stability (Nadia et al., 2017). The
antioxidant peptide donates a hydrogen atom to the oxidized DPPH as free radical. The
activity of free radical reduces and releases peptide radical. It reacts with the other
oxidized product and forms a stable product (Wu et al., 2015). The reaction between the
peptide and DPPH can be seen in Figure 1.
 4

Figure 1. Mechanism of Antioxidant Activity (Wu et al., 2015)

In the human body, antioxidant plays a role to remove free radicals during metabolism,
to prevent excessive accumulation of free radicals, to prevent the damage of cells or
tissues. The balance between antioxidant and oxidation need to maintain, so
physiological and biological function achieve normally. Protein hydrolysates are
precursors of bioactive peptide that can act as natural antioxidants. Due to health
reasons, people are likely to choose natural antioxidants. Natural antioxidants are
necessary because of their nature, small side effects or absolutely no side effects
(Kumar et al., 2016).

1.2.2. Enzymatic Hydrolysis

Enzymes have different catalytic activity and specific works. It can modify the
functional characteristics of the protein, improve the functional qualities, affect the
location, and the number of the peptide bonds (Baharuddin et al., 2016; Sbroggio et al.,
2016). Enzymatic protein hydrolysis is the cleavage of protein into amino acids and
smaller peptide sizes by the presence activity of protease enzyme. After protein
hydrolyzed, the reaction continued by hydrolyzing small peptide fractions. The result is
a varying lengths mixture of amino acid and polypeptide that has ability as bioactive
peptide and called as hydrolysate protein (Saidi et al., 2013; Whitehurst & Oort, 2010).
 5

Hydrolysate protein is the result of the enzymatic hydrolysis process. The molecular
weight is lower than the whole protein. It also has a simpler bond of peptides and free
amino acids. It is easy to be absorbed by the body, has a high water solubility, and good
emulsion. It utilizes in various fields such as food industry or pharmacy (Wijayanti et
al., 2016). In the food industry, it is for fortification into non-allergenic food
formulations for infants, diet food supplements, and also an emulsifier. It can also
improve the characteristics of various food products and act as flavoring. In the
pharmaceutical, it uses in the manufacture of dermatological products, such as facial
cleanser and skin moisturizing cream (Nurhayati et al., 2007).

The amino acid contains acidic and basic groups that call amphoteric properties. In
acidic solution, the amino acid has a positive charge or more hydrogen ion
concentration. In basic solution, the amino acid has a negative charge or lower hydrogen
ion concentration. H+ ion will bind with COO- group to form COOH. The remainder
(acid) will bind to NH2 to form NH3+ (Hughes, 2011).

Figure 2. Decrease-Increase Reaction of The pH (Hughes, 2011)

1.2.3. Papain and Bromelain Enzymes

Carica papaya L. commonly known as papaya belongs to the family of Caricaceae. It

has a proteolytic activity of papain enzyme. Its molecular weight about of 23,000
daltons (Ming et al., 2002). The activity decreases 20% when the pH 7.0 and heats at
700C during 30 minutes (Anggraini & Yunianta, 2015). It also decreases about 50%
when the pH increases to 7.5 (Pessato & Tavano, 2015).

Bromelain enzyme is isolated from pineapple (Ananas comosus L.) and it has an ability
to protein digestibility (Wuryanti, 2004). The mayor endopeptidases are stem bromelain
and fruit bromelain. The minor endopeptidases are ananain and comosain. Stem
 6

bromelain has a molecular weight range from 6-37 kDa, the optimum pH range from 6-
7, and the optimum temperature range from 50-60ºC. Fruit bromelain has a molecular
weight range from 24.5-32.5 kDa, the optimum pH range from 3-8, and the optimum
temperature range from 37-70ºC (Bala et al., 2012). Summarize activities of bromelain
enzyme are molecular weight ranged from 24.5-37 kDa, the optimum pH range from 3-
9, and the optimum temperature range from 37-70ºC.

Papain enzyme uses in the meat processing and as a common ingredient in the brewery.
It can also act as a clarifying agent and uses in bakery industry to produce high-quality
bread and pastry. It has for a long time uses in pharmaceuticals such as combating
dyspepsia, other digestive disorders, and disturbances of the gastrointestinal tract (Amri
& Mamboya, 2012). In food processing, bromelain enzyme uses in numerous
applications, mainly in tenderization, baking industry, fish protein hydrolysate, anti-
browning agent, alcohol production, animal feed, and etc. (Arshad et al., 2014).

Papain and bromelain enzymes as protease enzyme have an ability to cleave the peptide
bonds in polypeptide chains so that the amino acids and peptides are released (Bala et
al., 2012; Matijević et al., 2011). It makes a protein more easily to digest and absorbs
by the body (Anggraini & Yunianta, 2015). Based on their site of action, papain and
bromelain enzymes belongs to endopeptidase which contains an active site of cysteine.
Endopeptidase cleaves peptide bonds in the inside of peptide molecules distant from N
and C termini. The activity of all cysteine proteases depends on a catalytic dyad
consisting of cysteine and histidine. The order of Cys and His (Cys-His or His-Cys)
residues differs from the families. Generally, cysteine proteases are active only in the
presence of reducing agents such as HCN or cysteine (Freire, 1970). The specificity of
papain enzyme is wider than bromelain enzyme that has ability for breaking the lysine,
arginine, phenylalanine and tyrosine chains (Whitehurst & Oort, 2010).

1.3. Aim of The Study

The aim of this study was to know the difference activity of papain and bromelain
enzymes in hydrolyzing milk protein and to know the effects of hydrolysis to milk
 7

hydrolysate (pH measurement, determination of the degree of hydrolysis (DH), DPPH

radical-scavenging activity, and sensory analysis).