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Comparison of pH stat and O-phthalaldehyde method for degree of hydrolysis

measurement of alcalase and flavourzyme digested casein.

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Indian J Dairy Sci (2018)71(1):107-109
SHORT COMMUNICATION
Comparison of OPA and pH stat methods for measurement of degree of
hydrolysis of alcalase and flavourzyme digested casein
Priyanka Singh Rao, Mayur A, Nichal Bodemala Harisha, Rajesh Bajaj and Bimlesh Mann
Received: 29 July 2017/Accepted: 11 September 2017
Abstract: Casein hydrolysates produced using commercial
proteases alcalase (endoproteases) and flavourzyme (endo- and
exoproteases) were analysed for degree of hydrolysis through
two methods; the pH stat and O-phthalaldehyde method. The pH
stat method is useful for measurement of DH for the casein
hydrolysed using enzyme with endoprotease activity up to 20%
degree of hydrolysis, with identical values compared to OPA
method while it is unsuitable for the measurement of sodium
caseinate digest with enzymatic preparation having exoproteases
activity.
Keywords: Degree of hydrolysis, Exoproteases,
Endoproteasess, O-phthalaldehyde, pH stat
Milk protein hydrolysates are drawing more interest of researcher
since last decade, due to their functional and health enhancing
properties (Udenigwe and Aluko, 2012). The composition of
hydrolysate relies on different factors such as degree of
hydrolysis, type of proteolytic enzymes and enzyme activity
(endo- or exo- activity) etc. Endoproteases break amide bonds
within the protein chain while exoproteases cleave terminal amino
acids. Alcalase is serine alkaline endoproteases produced by
Bacillus licheniformis with high specificity for sulfur-containing,
aliphatic, acidic, hydroxyl, aromatic and basic amino acids while
flavourzyme act as an endo- and exoproteases responsible for
release of free amino acids mainly from C-terminal (Uhlig et al.,
1998). The degree of hydrolysis (DH) is quantification of enzymatic
break down of protein which stands for the percentage of peptide
bonds cleaved. Over the period of protein hydrolysis, commonly
the DH is measured by pH stat and OPA method. During the
Priyanka Singh Rao, Mayur A , Nichal Bodemala Harisha
Dairy Chemistry Division
National Dairy Research Institute, Karnal 132001, Haryana, India
Rajesh Bajaj()
Dairy Chemistry Division
National Dairy Research Institute, Karnal 132001, Haryana, India
Email: rbajaj1375@gmail.com;Tel: 0184-2259161
Bimlesh Mann
Dairy Chemistry Division
ICAR-National Dairy Research Institute, Karnal 132001, Haryana, India
107
hydrolysis reaction at neutral or alkaline pH range, the liberation
of protons takes place, which leads to the lowering of pH of
reaction mixture. By calculating the volume of alkali used in
maintaining a constant pH of reaction mixture during the reaction,
the percentage of peptide bonds cleaved can be determined
(Adler-Nissen, 1986). The advantage of pH stat method is that it
gives a direct measure of the amount of alkali used during the
course of reaction. In OPA method, reaction of OPA reagent and
primary amino groups, in the presence of a thiol leads to the
formation of 1-alkylthio-2-alkyl-substituted isoindoles (Medina
Hernandez et al., 1990). Further, the iso-indoles formed can be
measured at 340 nm. Spellman et al. (2003) compared three
methods (OPA, TNBS and pH stat) for the quantification of DH in
whey protein hydrolysate. The TNBS method was found more
accurate for DH determination in whey protein hydrolysate
irrespective of type of enzyme used but it has long incubation
and cooling period and cannot be recommended for real-time
monitoring of DH. As the limited data is available on pH stat
method using endo and exoprotease for different proteins,
therefore, the objective of this study was to compare the pH stat
and OPA methods for the determination of degree of hydrolysis
in casein hydrolysates produced using commercial proteases
alcalase (endoproteases) and flavourzyme (endo- and
exoproteases).
The commercial enzyme Alcalase 2.4 L FG (P4860, endoproteases
from Bacillus licheniformis) and flavourzyme 500 L (P6110,
endoprotease and exoproteases from Aspergillus oryzae) were
purchased from sigma (St. Louis, MO, USA). All the enzymes
were stored at 4ºC. O-phthalaldehyde reagent was purchased
from Himedia (India). Commercial casein was obtained from
Modern dairy Limited, Karnal. Fresh solution of sodium caseinate
(5 % w/v) was prepared in distilled water and heated at 85°C for 15
min to inactivate the native proteases present in the solution
before the addition of enzymes and kept it for overnight for
hydration. The prepared sodium caseinate was hydrolyzed with
alcalase and flavourzyme using pH stat autotitrator (Titroline
7000, SI-Analytics), equipped with a reaction vessel (50 ml), stirrer,
thermometer and a pH electrode. The conditions used for
hydrolysis using alcalase included 3000 units/ g protein and
flavourzyme 2500 units/g protein at pH 7.5, 50 °C. The pH of
solution was kept constant throughout the hydrolysis using 1N

Fig 1: Time dependent degree of hydrolysis of casein
hydrolysate obtained using alcalase by pH stat method
(A) and OPA method (B)
Fig 2: Time dependent degree of hydrolysis of casein
hydrolysate obtained using flavourzyme by pH stat
method (A) and OPA method (B)
NaOH. The samples were drawn at a regular interval of 15 minute
up to 60 minute, followed by 30 minute interval up to 240 minute.
The enzymatic activity was terminated by heating the solution at
90°C for 15 min and the supernatant was used further to determine
the degree of hydrolysis using OPA method.
Quantification of DH by the pH stat method was conducted using
the following formula:
equation (1)
Where B is the alkali consumption in mL, Nb is normality of
alkali, α the Average degree of dissociation of the α -NH2 groups,
MP is mass of protein being hydrolysed (g) and htot is the total
number of peptide bonds in the protein substrate (meqv/ g
protein).
The degree of dissociation (α) for the α -NH2 groups was
calculated as follows
108
Indian J Dairy Sci (2018) 71(1):107-109
equation (2)
Where, pK represents the average dissociation value for the α-
amino groups liberated during hydrolysis. At 50°C (the
hydrolysis temperature used in the present study), the average
pK value for the α -amino groups of peptides and proteins is 7.1.
At pH 7.5 (the pH value for all hydrolysis reactions) α can be
calculated as 0.71, therefore the 1/ α value used was 1.40. The htot
for casein hydrolysates is 8.2 meqv per g protein (Adler-Nissen,
1986).
The degree of hydrolysis for the casein hydrolysates was
determined following the method given by Nielsen et al. (2001)
with a slight modification. The OPA reagent was prepared by
mixing 50 ml of sodium tetraborate (100mM), 5ml of SDS (20%), 2
ml of OPA reagent (80 mg OPA was dissolved in 2ml ethanol) and
200µl β-mercaptoethanol. The solution was made up to 100 mL
with deionized water. The assay was carried out by adding 400µl
of sample to 3ml of OPA reagent. After vortex, incubated for
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Indian J Dairy Sci (2018)71(1):107-109
exactly 2 min at room temperature and the absorbance was
measured at 340 nm using double beam UV-Vis spectrophotometer
(Model UV-2700, Shimadzu, Tokyo, Japan). L-serine (0.9516 meq/
L) was used as positive control and distilled water as negative
control. The calculation of DH was performed using equation (3).
𝑺𝒆𝒓𝒊𝒏𝒆 –𝑵𝑯𝟐 = (𝐴_(〖340 𝑆𝑎𝑚𝑝𝑙𝑒〗^.) − 𝐴_(〖340 𝐵𝑙𝑎𝑛𝑘〗^.))/((𝐴_(〖340 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑〗^.)
− 𝐴_(〖340 𝐵𝑙𝑎𝑛𝑘〗^.))) ∗ 0.9516 𝑚𝑒𝑞𝑣 /𝐿 ∗ 0.1 ∗ (100/𝑋) ∗ 𝑃
- equation (3)
Where, P is protein content solution, x is g sample, 0.1 is sample
volume in liter (L),
h = (serine-NH2 –β)/α meqv /g protein
% DH = h/htot * 100
The time dependent hydrolysis of sodium caseinate using
alcalase is presented in figure 1. Initially there was a rapid increase
in DH corresponding to 7.6% in 15 minutes, which increased to
12% in 30 minutes. Further increase in DH was slowed down and
reached plateau after 120 minute and kept relatively constant for
rest of hydrolysis reaction around 20%. The degree of hydrolysis
determined using OPA method for the casein hydrolysates
obtained using alcalase treatment showed relatively rapid increase
in DH for initial 60 minute, corresponding to 20.84%. Further
increase in DH was relatively slower, reaching maximum after 210
minute to 28%, unlike 20% observed with pH stat titration method,
wherein it became constant after 120 minute for alcalase
hydrolysed sodium caseinate with no change in pH in following
hydrolysis.
The results shows that the values of DH measured by pH-stat
method represented closer to OPA method up to 20% DH, while
further increase in DH up to 28%, resulted in underestimation
using pH stat method. It has been reported that when proteinase
such as alcalase is used for preparing hydrolysates with moderate
DH values (i.e. below 20%), the relative content of dipeptides and
free amino acids was likely to be low (Adler- Nissen, 1986). It
indicates that during hydrolysis the change in the value of 1/α
would be negligible and obtained DH values by the pH stat
method would be comparable.
The rate of hydrolysis of sodium caseinate using flavourzyme is
shown in figure 2. The initial slower rate of reaction was observed,
with DH corresponding to 5.71% and 6.81% using pH stat and
OPA method, respectively. Using OPA method, a continuous
increase in hydrolysis of sodium caseinate was observed up to
210 minute reaching 36.84% DH. However, using pH-stat method
no significant difference in DH was observed throughout the
hydrolysis reaction up to 240 minute with DH reaching maximum
9.5%. Thus the difference in DH observed might be due to
flavourzyme preparation which has both endo- and exoprotease
109
activity. Relatively low DH observed might be due to the fact
that pK value of α-amino group is considerably higher for
tripeptides, dipeptides and amino acids, than for polypeptides
(Adler-Nissen, 1986). This might lead to an under-estimation of
the value for 1/α used in the calculation of DH by the pH stat
method (see Eq. 1) and a consequent under-estimation of DH. In
case of exoprotease action, DH values calculated by the pH stat
method was lower than values obtained from OPA method, as
the hydrolysate would contain relatively high amounts of free
amino acids (Castro-Sato et al., 2015). Similar observations were
made by Spellman et al. (2003) using exoprotease rich preparation
debitrase HYW20 for hydrolysis of whey protein concentrate.
Conclusions
Accuracy of DH measurement method was found to be dependent
on the type of enzyme activity used in the hydrolysis reaction.
The accuracy of pH stat method was comparable to OPA method
for monitoring casein hydrolysis with endoprotease activity like
alcalase up to 20% while it was not suitable for the enzymatic
preparations containing exoproteases activity like flavourzyme.
Acknowledgements
This work was supported by the SERB-MOFPI Project (SERB/
MOFPI/0028/2013), Department of Science and Technology, Govt.
of India, New Delhi, India.
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