International Journal of Peptide Research and Therapeutics

https://doi.org/10.1007/s10989-019-09863-x

Enzymatic Hydrolysis of Phaseolus vulgaris Protein Isolate:

Characterization of Hydrolysates and Effect on the Quality of Minced
Beef During Cold Storage
Ahmed Mohamed Saad1 · Ali Osman Mohamed Osman1 · Alaa Samy Mohamed2 · Mohamed Fawzy Ramadan1

Accepted: 16 April 2019

© Springer Nature B.V. 2019

Abstract
The aim of this work was to hydrolyze kidney bean (Phaseolus vulgaris L.) protein isolate (KBPI) using pepsin (P7000)
to obtain kidney bean protein hydrolysates (KBPH) with different degrees of hydrolysis (DH). Antioxidant and antibacte-
rial activities of KBPH against selected pathogenic and spoilage bacteria were evaluated. In addition, applying KBPH as
a potential preservative in minced beef during cold storage was studied. The extent of protein degradation by pepsin was
determined by measuring DH and SDS-PAGE. KBPH obtained after 6 h of hydrolysis had the highest DH (33.7%), while
KBPH obtained after 1, 2, 3, 4 and 5 h had DH values of 11.6, 15, 20.7, 25.6, and 30, respectively. The highest antiradical
activity of KBPH (85% for DPPH· and 80% for A ­ BTS+ assay) was recorded after 6 h of hydrolysis. The hydrolyzate with
the highest antibacterial activity (DH = 33.7%) was selected for the antibacterial evaluation in situ and in vitro. The anti-
bacterial activity of KBPH (100 mg/mL) with a different degree of hydrolysis on bacterial growth was evaluated using the
disc-diffusion method. KBPH reduced the growth of gram-positive bacteria by 70–75% and the bacterial growth of gram-
negative bacteria by about 78–80%. Total viable, psychrotrophic bacterial and coliform bacterial counts were determined
in beef samples enriched with KBPH (100 and 200 µg/g) during storage at 4 °C for 15 days. Samples supplemented with
200 µg/g KBPH kept the level of the total viable count at 5.7 and 6.55 log after 12 and 15 days of storage, which increased
the secured storage time to be 14 or 15 days instead of 7 or 8 days in the case of the control sample. The results could be
useful to formulate functional peptides from KBPI that could be applied in novel foods and nutraceuticals.

Keywords Kidney bean · Proteolysis · Protein isolate · Antibacterial · SDS-PAGE · Degree of hydrolysis · Antioxidant
activity

Introduction

Red kidney bean (Phaseolus vulgaris L.) is an excellent

source of proteins (20–40 g/100 g), antioxidants, carbo-
hydrates, fibers, vitamins and minerals. Kidney bean is
* Mohamed Fawzy Ramadan consumed as a cheap protein source in many developing
hassanienmohamed@yahoo.com; mframadan@zu.edu.eg countries (Sathe et al. 1981; Boye et al. 2010; Dave Oomah
Ahmed Mohamed Saad et al. 2011; Ahmed et al. 2018). Studies revealed that kidney
ahmedm4187@gmail.com beans consumption has health-promoting impacts including
Ali Osman Mohamed Osman the reduction of the risk of type 2 diabetes, obesity, cardio-
ali_khalil2006@yahoo.com vascular diseases, and types of cancer (Dueñas et al. 2015).
Alaa Samy Mohamed Phaseolin (vicilin or 7–8S globulins), is the main stor-
dr_alla88@yahoo.com age protein in the kidney bean, and constituted from 75
1
Agricultural Biochemistry Department, Faculty to 82 g/100 g of total protein (Yin et al. 2008; Ahmed
of Agriculture, Zagazig University, Zagazig 44519, Egypt et al. 2018). Phaseolin is an oligomeric protein contain-
2
Food Science Department, Faculty of Agriculture, Zagazig ing 3 polypeptide subunits (α-, β-, and γ-phaseolin), while
University, Zagazig 44519, Egypt the molecular weight (MW) ranged from 43 to 53 kDa

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 International Journal of Peptide Research and Therapeutics

(Romero et al. 1975). Vicilin in kidney beans exhibited a bio-preservative in minced beef during refrigeration condi-
unique structural peculiarity by high subunit homogene- tions was studied.
ity and low susceptibility to trypsin digestion (Yin et al.
2008). Therefore, kidney bean protein isolate (KBPI)
exhibited a good emulsifying and gelation traits and have
Materials and Methods
potential as a functional ingredient in food formulations
such as meat, baking, and extruded products (Kimura et al.
Materials and Chemicals
2008; Tang 2008; Ahmed et al. 2018).
Natural bioactive peptides (BP) usually contain 2-20
Kidney bean (Phaseolus vulgaris) seeds were purchased
amino acids, which could be enzymatically hydrolyzed
from the local market (Zagazig, Egypt). Pepsin (P7000, a
and releasing BP with antioxidative potential (Chalamaiah
peptidase from porcine gastric mucosa with specific activ-
et al. 2017; Evangelho et al. 2017; Al-Ruwaih et al. 2019).
ity ≥ 250 U/mg) was purchased from Sigma (St. Louis, MO,
BP play an important role as a food preservative. Most
USA). Reagents for electrophoresis were from Bio-Rad
of the natural BP exhibited broad-spectrum antimicrobial
Laboratories (Richmond, CA, USA). Bacillus licheniformis,
activities against several spoilage bacteria (Sitohy et al.
Bacillus thuringiensis, Escherichia coli (O157:H7), and
2012; Li et al. 2014). BP could be produced from proteins
Escherichia coli (E32511) strains were obtained from the
during: (i) food fermentation using proteolytic starter cul-
Microbiology Department, Faculty of Agriculture, Zagazig
tures; (ii) in vivo digestion of proteins; (iii) manufactur-
University (Egypt).
ing of protein hydrolysates; or (iv) in vitro digestion by
proteolytic enzymes such as trypsin, alcalase, pepsin, and
pancreatin (Korhonen and Pihlanto 2006). These BP with Extraction and Characterization of Kidney Bean
low MW are more bioavailable and digestible to the human Protein Isolate (KBPI)
body and could be incorporated into nutraceuticals and
functional foods (Al-Ruwaih et al. 2019). Kidney bean Kidney bean seeds were ground at maximum speed using a
bioactive phytochemicals act as antioxidant, antifungal, Moulinex mixer (Type 716, France) to pass through a 1 mm
antihypertensive, and anticancer agents (Bernardini et al. sieve. The obtained powder was defatted with chloroform:
2011; Luna-Vital et al. 2015; Al-Ruwaih et al. 2019). methanol (3:1, v/v) in a Soxhlet apparatus for 8 h. The sol-
Pepsin, a peptidase from porcine gastric mucosa, vent was evaporated and the dried-defatted flour was stored
showed proteolytic activity against numerous proteins at 4 °C till further analyses. Protein isolate was extracted as
(Polanco-Lugo et al. 2014). It has been used to hydro- described by Fan and Sosulski (1974) with few modifica-
lyze kidney bean and pea proteins to improve the protein tions. The defatted meal was dispersed in deionized water
functional traits (Wani et al. 2015; Barac et al. 2011). (1: 20, w/v), while the dispersion was adjusted to pH 8.0
Al-Ruwaih et al. (2019) explored the influence of high- with NaOH (2N). The dispersion was stirred for 2 h at room
pressure (600 MPa) and the addition of alcalase (E/S, temperature, then centrifuged at 8000×g for 30 min at 20 °C.
0.5–1%) on the hydrolysis of kidney bean protein isolate The pellet was discarded, and the supernatant was adjusted
(KBPI) followed by evaluation of functional properties of to pH 4.5 with HCl (2N) to precipitate the protein. The pre-
the obtained hydrolysates. cipitate obtained by centrifugation at 5000×g for 20 min was
One of the main problems affecting food quality and re-dispersed in deionized water. The final dispersion was
safety is microbial contamination. Previous studies have homogenized and adjusted to pH 7.0 using NaOH (2N).
focused on the generation of BP, which demonstrated anti-
microbial potential in some foodstuffs (Osman et al. 2013,
Total Protein Content
2016). Different protein hydrolysates were applied as anti-
oxidant and antimicrobial agents in meat products (Mein-
The total protein content of KBPI was calculated by multiply
ert et al. 2015; Meshginfar et al. 2017). To the best of our
the total nitrogen by 6.25. Total nitrogen was measured by
knowledge, kidney bean protein hydrolysates (KBPH) was
micro Kjeldahl method (AOAC 1996).
not applied as an antioxidant and antibacterial agent in meat
products.
In the present study, kidney bean (Phaseolus vulgaris L.) Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
protein isolates (KBPI) were prepared, characterized and (SDS‑PAGE)
hydrolyzed using pepsin (P7000) to obtain KBPH. Antioxi-
dant and antibacterial activities of KBPH against selected A method of discontinuous SDS-Polyacrylamide slab gel
pathogenic and spoilage bacteria were evaluated. Further- electrophoresis based on Laemmli (1970) technique was
more, the effect of enrichment with KBPH as a potential used in the fractionation of KBPI.

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International Journal of Peptide Research and Therapeutics

Iso‑Electric Point (Protein pH‑Solubility Curves) (1970) in 3% and 12% acrylamide for resolving and stack-
ing gels.
The isoelectric point (pI) was recorded from the protein
pH-solubility curves as pH at which the protein is less Antibacterial Activity of KBPH
soluble. pH-solubility curves of proteins were measured
in the pH range of 2–10 (Chobert et al. 1991) with few Bacterial Strains
modifications. Each sample (125 mg) was dispersed in
25 mL distilled water and pH of solutions were adjusted Two Gram-positive bacteria (Bacillus licheniformis, and
to 2–10 using either 0.5 mol/L HCl or 0.5 mol/L NaOH. Bacillus thuringiensis) and two Gram-negative bacteria
The slurries were mixed for 60 min at 30 °C then centri- [Escherichia coli (O157:H7), and Escherichia ci (E32511)]
fuged at 1200×g at 4 °C for 20 min. The supernatant was were used to evaluate the antibacterial activity of different
filtered and total protein content in the supernatant was KBPH using a diffusion method to select the highest active
determined using the Kjeldahl technique (AOAC 1996). hydrolysate.
The solubility profile was obtained from triplicate deter-
minations by plotting averages of protein solubility (%) Agar Well‑Diffusion Assay
against pH:
Solubility(%) = Protein amount in the supernatant∕ Antibacterial activity of KBPH obtained after hydrolysis
for 1, 2, 3, 4, 5 and 6 h (at the concentration dose 100 µg/
Protein amount in the sample × 100
mL) was tested against Bacillus licheniformis, Bacillus
thuringiensis, Escherichia coli (O157:H7) and Escherichia
Preparation and Characterization of Kidney Bean coli (E32511) by well-diffusion test (Nanda and Saravanan
Protein Hydrolysate (KBPH) 2009). Pure cultures of bacterial strains were sub-cultured
on nutrient broth at 37 °C on a shaker at 200 rpm. The expo-
Lyo p h i l i z e d K B P I wa s d i s s o lve d i n 0 . 1 M nential phase cultures of these strains were adjusted to a con-
­Na2HPO4–NaH2PO4 buffer pH 6 (100 g/L) and hydrolyzed centration of 1.05 × 109 CFU/mL. Each strain was spread on
by pepsin treatment (E:S ratio 1:200, w/w) at 37 °C. The the plates using sterile cotton swabs. Wells (6 mm diameter)
hydrolysis was allowed to proceed for 4 h. The enzyme were made on Müller Hinton Agar (MHA) plates using a gel
was inactivated after the end of the hydrolysis by heat- puncturing tool. Aliquots (30 μL) of the KBPH solutions (0,
ing for 15 min in a boiling water bath. The hydrolysate 25, 50, 100, 200, 400 and 800 µg/mL) were added to each
was centrifuged at 4000×g at 4 °C for 30 min to remove well. After incubation at 37 °C for 24 h, the diameter (mm)
insoluble fragments. The supernatant was lyophilized at of the inhibition zone was recorded.
− 20 °C to obtain KBPH. Pepsin hydrolysis was performed
in triplicate.
Minimum Inhibitory Concentration (MIC)

The MIC of selected samples was determined according to

Dree of Hydrolysis (DH)
Yamamoto et al. (2003). Aliquots (30 μL) of the KBPH (0,
25, 50, 100, 200, 400 and 800 µg/mL) were transferred into
The DH was determined according to Hoyle and Merritt
each well. The lowest concentration of the samples which
(1994) after 1, 2, 3, 4, 5, and 6 h. One volume of 20%
exhibited a visible clear zone on MHA plates was considered
trichloroacetic acid (TCA) was added to the supernatant,
as the MIC.
centrifuged at 10,000 rpm for 10 min (4 °C), and the TCA-
soluble materials were collected. Total nitrogen in TCA
soluble materials and the substrate was measured. Bacterial Growth Curve (Turbidity Test)

Degree of hydrolysis(%) Turbidity ­(A600) assay was used to determine CFU/mL in

(10%TCA − Soluble nitrogen in the sample) nutrient broth media suspensions. KBPH with the highest
= × 100 degree of hydrolysis (at their MIC values = 100 µg/mL) was
(Total nitrogen in the sample)
added to the medium (10 mL) containing 100 μL Gram-
SDS–Polyacrylamide Gel Electrophoresis (SDS‑PAGE) positive or Gram-negative bacteria ­(109 CFU/mL) and tested
for their growth compared to control. Each treatment was
SDS-PAGE of KBPH obtained after different times (1, 2, incubated at 37 °C for different periods (0, 6, 12, 18 and
3, 4, 5, and 6 h) was estimated as described by Laemmli 24 h) before recording the turbidity.

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 International Journal of Peptide Research and Therapeutics

Antioxidant Activity of KBPH bio-preservative material for minced beef. The fresh beef
was obtained from the local market (Zagazig, Egypt) and
KBPH, obtained after hydrolysis for 1, 2, 3, 4, 5, and 6 h at minced finely in a sanitized meat mincer.
200 µg/mL, were evaluated as an antioxidant agent to select
the highest active KBPH using the following methods. Storage of Beef Supplemented with KBPH Under Cold
Conditions
DPPH· Radical Scavenging Activity Assay
Each minced beef sample (100 g) was placed in a stomacher
Radical scavenging activity against DPPH· was measured bag and homogenized in a stomacher at room temperature
according to Göçer and Gülçin (2011). One milliliter of each for 2 min. Treatments of the minced beef samples included
KBPH at different concentrations (0–400 µg/mL) was mixed no addition (negative control), and the addition of KBPH
with 3 mL of DPPH· (0.15 mM in 95% ethanol). The mixture at 100 µg/g and 200 µg/g. Stomacher bag with the sample
was shaken and incubated in the darkness at room tempera- from each treatment was wrapped and stored under aerobic
ture for 30 min. The absorbance at 517 nm was measured conditions for 15 days at 4 °C. Physicochemical and micro-
using UV/visible spectrophotometer (Jenway 6405, UK). biological analyses were carried out at intervals of preserva-
Ethanol was used as a control. The radical scavenging activ- tion (0–15 days) at 4 °C.
ity was calculated as a decrease in the absorbance of DPPH·
using the following equation.
Physicochemical Analysis of Minced Beef
Abs.control − Abs.sample
Radical scavenging activity(%) = × 100
Abs.control Analysis of moisture, protein, and fat content in beef sam-
ples was carried out according to AOAC (2002). pH was
The ­SC50 value (concentration of sample required to scav-
determined according to Özyurt et al. (2012). Five g of beef
enge 50% DPPH·) was evaluated (Bursal and Gülçin 2011).
sample was homogenized with 50 mL of distilled water
(pH 7) then the mixture was filtered. pH of the filtrate was
ABTS+ Radical Scavenging Activity Assay
recorded using pH meter (pH 211 HANNA instruments,
Woonsocket, USA).
TS+ antiradical activity of KBPH was measured according
to Tironi and Añón (2010). ­ABTS+ solution was prepared
by the reacting 7 mM of ­ABTS+ solution in 2.45 mM potas- Microbial Analysis
sium persulfate. The mixture was kept at room temperature
for 16 h in the dark before use. The solution was diluted with Microbial analysis of minced beef supplemented with KBPH
distilled water and equilibrated to give an absorbance of 0.70 at both concentrations (100 and 200 µg/g) was performed
at 734 nm at room temperature. 200 μL of sample (0–400 µg/ during intervals of preservation (0–15 days) at 4 °C accord-
mL) was added to 2 mL A ­ BTS+ solution and the absorbance ing to APHA (1992). The sample (10 g) was transferred to
nm was measured at 734. Solvent blank (negative control a stomacher bag containing 90 mL of peptone saline diluent
NC) was run for each assay. The scavenging percentage was (1 g peptone and 8.5 g NaCl in one L distilled water) and
calculated as follows: homogenized for 60 s at room temperature. A tenfold dilu-
tion series were prepared. Determinations were performed
Scavengening activity(%) for different bacterial counts using specific selective media
(Lee 2009). Coliform bacteria were determined by Mac-
[ ]
= (Abs0 − Abst) − (Abc0 − Abct)∕(Abc0) × 100 .
Conkey agar (Mast Group, Merseyside, UK) with a double
layer of the same medium incubated at 37 °C for 24 h. Total
where Abst and Abs0 = absorbance of the sample at viable count (TVC) was enumerated on Plate Count Agar
t = 20 min and t = 0. Abct and Abc0 = absorbance of the (PCA, Merck, Darmstadt, Germany) at 25 °C after 72 h.
negative control at t = 20 min and t = 0 (Gülçin 2011). Psychrotrophs were counted on PCA (Merck, Darmstadt,
Germany) at 7 °C after 10 days. Microbiological results were
Effect of KBPH on the Quality and Safety of Minced transformed to logarithms of the number of colonies forming
Beef Stored Under Cold Conditions units (CFU/g).

KBPH generated by pepsin with the highest antibacte-

rial activity (E/S ratio of 1:200, hydrolysis time = 6 h,
DH = 33.7%, and MIC = 400 µg/mL) was tested as a

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International Journal of Peptide Research and Therapeutics

Results and Discussion reduced when the pH increased from 2 to 5, which corre-
sponding to its pI, after which subsequent increments in pH
Characterization of KBPI increased progressively the protein solubility. KBPI mini-
mum solubility (11%) was at pH 4.6 which corresponds to its
SDS-PAGE was employed to elucidate the protein frac- pI. The highest protein solubility (65%) was observed at pH
tions and subunits (Boztaş et al. 2015; Kucuk and Gulcin 10. Solubility is one of the most important functional char-
2016; Caglayan and Gulcin 2018). Total protein content in acteristics of proteins. Good protein solubility is needed in
the KBPI was 92%. Figure 1 shows the pattern of the SDS- functional foods, especially for foams, emulsions, and gels,
PAGE electropherogram of KBPI. Eleven protein bands with because soluble proteins enhance the interfacial properties
MW ranging from 15 to 140 kDa are present in KBPI. The and give a homogenous dispersibility of the molecules in
49 and 55 kDa bands, corresponding to phaseolin subunits colloidal systems (Zayas 1979).
(between 40 and 55 kDa) were the most abundant proteins
(Montoya et al. 2006). In our study, the electrophoretic pat- Characterization of KBPH
tern was similar to those reported by Montoya et al. (2006)
and Carrasco-Castilla et al. (2012) who indicated that the The extent of protein degradation by pepsin was tested
phaseolin is the main protein of P. vulgaris. The polypeptide by assessing DH and SDS-PAGE. The KBPH recovered
with MW of 43 kDa which might correspond to 7S vicilin after 6 h of degradation recorded the highest DH (33.7%).
was detected in the KBPI (Rui et al. 2011; Mundi and Aluko As shown in Fig. 3, hydrolysates obtained after 1, 2, 3, 4
2012). Other subunits were also found in KBPI ranging from and 5 h have DH (%) values of 11.6, 15, 20.7, 25.6 and
93 to 17 kDa, with major subunits of 55, 46–40, 32, and 30, respectively. Betancur-Ancona et al. (2014) observed
21 kDa (Tang 2008; Rui et al. 2011; Shevkani et al. 2015).
The presence of polypeptides with similar MW in different
kidney bean lines was previously reported (Shevkani et al. 70
2015). 60
The pH-solubility curve of KBPI is shown in Fig. 2. The
solubility profile of KBPI indicates that protein solubility 50
Protein solubilty (%)

40

30

20

10

0
0 2 4 6 8 10

pH

Fig. 2  pH-solubility curve of KBPI

40

35

30

25
DH (%)

20

15

10

0
1 2 3 4 5 6

Hydrolysis me (h)

Fig. 1  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Fig. 3  Progression of hydrolysis with time for KBPI at 37 °C and pH
(SDS-PAGE) profiles of KBPI 2

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 International Journal of Peptide Research and Therapeutics

Fig. 4  SDS-PAGE of KBPH produced at different times (0, 1, 2, 3, 4,

5, and 6 h)

Table 1  Antibacterial activity of KBPH (100 mg/mL) with different ­ ram+ and G
Fig. 5  Inhibition zones in G ­ ram− bacteria as affected by
DH on bacterial growth of B. licheniformis and E. coli treatment with KBPH

Hydrolysis time (h) DH (%) B. licheniformis E. coli

90 min required less energy and time to gain the maximum
1 11.6 − −
proteolysis (Gómez-Ruiz et al. 2006).
2 15.0 − −
Eleven protein bands having MW between 15 and
3 20.7 − −
140 kDa were obtained in the KBPH (first lane, hydroly-
4 25.6 − −
sis time = 0 h) as presented in Fig. 4. The electrophoretic
5 30.0 − −
pattern of KBPH after 360 min of hydrolysis by pepsin
6 33.7 + +
showed that between 20 and 140 kDa wherein kidney bean
(−) no activity protein fractions were nearly disappeared (lane 7; hydrolysis
(+) high activity time = 6 h), which agrees with the highest DH. Abeyrathne
et al. (2015) showed that ovomucoid was completely hydro-
lyzed with pepsin and alcalase but α-chymotrypsin was not
the highest DH values of hard-to-cook bean (P. vulgaris) effective. Trypsin was not used as the primary enzyme due
protein concentrates (E/S 1:50, v/v) were 33.29% for alca- to its inhibitory activity.
lase–flavourzyme and 28.47% for pepsin–pancreatin. The
levels of hydrolysates produced after 90 min and 120 min
were equal. It was noted that extending the reaction time did
not improve the DH. However, hydrolysates produced after

Table 2  Inhibition zones Microorganism Concentration (µg/mL)

diameter (mm) of KBPH
(DH = 29%) 0 25 50 100 200 400 800
+
Gram
B. licheniformis – – – – – 24 ± 0.2 35 ± 0.1
B. theriogensis – – – – – 31 ± 0.3 39 ± 0.2
Gram−
E. coli (O157:H7) – – – – – 25 ± 0.1 36 ± 0.2
E. coli (E32511) – – – – – 30 ± 0.1 35 ± 0.3

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International Journal of Peptide Research and Therapeutics

Gram + Gram -
1.8
B. lichniforms E.coli E32511
1.5

1.2

0.9

0.6

0.3
O.D 600

0.0
1.8
B. theriogensis E.coli O157:H7
1.5

1.2

0.9

0.6

0.3

0.0
0 6 12 18 24 0 6 12 18 24

Incubation time (hr)

Fig. 6  Growth curves of ­G+ and ­G− bacteria in the presence of MIC (400 µg/mL KBPH)

Antibacterial Activity of KBPH treatments, but the protein was replaced by distilled water. It
was noted that native KBPI or control samples did not have
The antibacterial effect of KBPH (100 mg/mL) with dif- antibacterial effects.
ferent DH on bacterial growth for B. licheniformis and E. The diameter (mm) of the resulting inhibition zones were
coli (O157:H7) was evaluated by the disc-diffusion method. recorded in Table 2. As shown in Table 2 and Fig. 5, KBPH
KBPH with the highest antibacterial activity (DH = 33.7%) gave rise to dose-dependent inhibition zones. There was
was selected for the antibacterial evaluation. KBPH was no significant difference between Gram-negative bacteria
applied at different concentrations (0–800 µg/mL) to Petri and Gram-positive in their susceptibility to KBPH at dif-
dishes containing Müller Hinton Agar (MHA) infected with ferent doses. These results could be due to the cleavage of
pathogenic Gram-positive bacteria (Bacillus licheniformis antimicrobial peptides by pepsin action. Results agree with
and Bacillus theriogensis) and Gram-negative bacteria [E. those reported by Salami et al. (2010) and Abdel-Hamid
coli (O157:H7) and E. coli (E32511)], incubated at 37 °C et al. (2016).
for 24 h (Table 1). Native KBPI was applied similarly at the The growth curves of the control bacteria reached the
same concentrations. Control was prepared exactly like the highest turbidity after about 18 h at 37 °C. At this time,

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 International Journal of Peptide Research and Therapeutics

140 bacterial cell wall, leading to the destruction of the cell wall
120
DPPH (Hancock and Rozek 2002; Gobbetti et al. 2004; Jenssen
ABTS et al. 2006). In addition, peptide hydrophobicity plays an
100 important role in the disturbance of the bacterial cell mem-
80 brane and cell wall. The MIC of KBPH was determined
%RSA

against all experimental bacterial strains, wherein MIC

60
against the four studied bacteria was 400 µg/mL (Table 2).
40

20 Antioxidant Activity of KBPH

0
The hydrolysates were tested for radical scavenging activ-
1 2 3 4 5 6
Time (h)
ity using DPPH· and A ­ BTS+ tests (Köse et al. 2015; Aksu
et al. 2016; Köksal et al. 2017). DPPH· antiradical assay is
an in vitro method to measure the ability of antioxidants to
Fig. 7  Radical scavenging activity of KBPH against DPPH· and
­ABTS+ quench free radicals (Xie et al. 2008). ­ABTS+ is a stable free
radical but readily quenched by antioxidant. The antiradical
­ BTS+ indicates the ability of the peptide to
activity against A
Table 3  Changes in the pH values of minced beef as affected by act as a hydrogen donor or electron donor in the free radical
KBPH supplementation during cold storage (0–15 days) reactions (Prior et al. 2005).
Storage time Control KBPH (µg/g) The respective antioxidant activity S ­ C50 (µg/mL) of the
(day) KBPH was accomplished with the highest DH was 95 μg/mL
100 200
for DPPH· and 115 μg/mL for ­ABTS+. The results suggested
0 5.30 ± 0.12 5.30 ± 0.12 5.30 ± 0.12 that there was a relationship between the hydrolysis time (h),
3 5.83 ± 0.15 5.66 ± 0.11 5.55 ± 0.22 DH and the antioxidant potential. The highest activity (85%
6 6.23 ± 0.12 5.98 ± 0.25 5.70 ± 0.24 for DPPH· and 80% for A ­ BTS+ assay) obtained after 6 h of
9 6.65 ± 0.16 6.16 ± 0.16 5.80 ± 0.15 hydrolysis (Fig. 7).
12 6.78 ± 0.20 6.19 ± 0.11 6.05 ± 0.14
15 6.98 ± 0.21 6.25 ± 0.10 6.13 ± 0.27 Preservative Impact of KBPH on Minced Beef Stored
Under Refrigeration Conditions

KBPH reduced the growth of gram-positive bacteria by Physicochemical Properties

70–75%. Similarly, KBPH inhibited the bacterial growth of
gram-negative bacteria by about 78–80% as shown in Fig. 6. The initial pH of the minced beef sample was 5.30 (Table 3).
The antibacterial activity of KBPH with the highest degree The pH values of the minced beef samples were increased
of hydrolysis (DH = 33.7%) might be attributed to its net during cold storage especially for the control sample (non-
charge or hydrophobic traits. The majority of antibacterial supplemented), while supplementation with KBPH remark-
peptides are positively charged; therefore they bind electro- ably limited this increase. This might be evidently due to
statically to the negatively charged compounds found on the the inhibition of microbial growth and consequently the

Table 4  Total viable count, psychrotrophic bacterial count, and coliform bacterial counts in minced beef as affected by KBPH supplementation
during storage
Storage Total viable count (Log CFU/g) Psychrotrophic bacterial count (Log Coliform bacterial counts (Log CFU/g)
time (day) CFU/g)
Control KBPH (µg/g) Control KBPH (µg/g) Control KBPH (µg/g)
100 200 100 200 100 200

0 3.00 ± 0.11 3.00 ± 0.11 3.00 ± 0.11 2.00 ± 0.10 2.00 ± 0.10 2.00 ± 0.10 1.20 ± 0.11 1.20 ± 0.11 1.20 ± 0.11
3 3.80 ± 0.14 3.60 ± 0.13 3.10 ± 0.16 2.75 ± 0.17 2.56 ± 0.15 2.11 ± 0.12 1.62 ± 0.16 1.50 ± 0.10 1.40 ± 0.10
6 5.30 ± 0.15 4.30 ± 0.18 3.40 ± 0.17 4.59 ± 0.15 3.67 ± 0.16 3.10 ± 0.19 2.16 ± 0.11 1.80 ± 0.16 1.50 ± 0.15
9 6.58 ± 0.17 5.20 ± 0.15 4.55 ± 0.11 5.55 ± 0.15 5.20 ± 0.12 4.33 ± 0.18 2.44 ± 0.14 1.90 ± 0.14 1.60 ± 0.16
12 8.45 ± 0.11 6.80 ± 0.15 5.70 ± 0.18 6.46 ± 0.13 6.31 ± 0.14 5.11 ± 0.21 2.84 ± 0.14 2.30 ± 0.10 1.87 ± 0.11
15 8.98 ± 0.16 7.30 ± 0.19 6.55 ± 0.17 7.88 ± 0.15 6.50 ± 0.21 6.15 ± 0.23 3.53 ± 0.13 2.75 ± 0.17 2.10 ± 0.13

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International Journal of Peptide Research and Therapeutics

reduction in the hydrolysis of protein and lipids. The dec- Informed Consent All authors have agreed to submit the manuscript
rement in pH values reflects the degree of beef spoilage in its current form for publication.
through protein breakdown which causes free amino acids Research Involving Human Participants No tests, measurements or
production leading to the induction of amines, N ­ H3, and experiments were performed on humans as part of this work.
other alkaline reaction compounds (Karabagias et al. 2011).

Microbial Changes in Beef During Storage at 4 °C

References
Abdel-Hamid M, Goda HA, De Gobba C, Jenssen H, Osman A (2016)
Total viable, psychrotrophic bacterial and coliform bacterial Antibacterial activity of papain hydrolysed camel whey and its
counts were determined in beef samples during storage at fractions. Int Dairy J 61:91–98
4 °C for 15 days (Table 4) either in the control or KBPH-sup- Abeyrathne EDNS, Lee HY, Jo C, Suh JW, Ahn DU (2015) Enzy-
matic hydrolysis of ovomucoid and the functional properties of
plemented (100 and 200 µg/g) samples. It was noted that all its hydrolysates. Poultry Sci 94(9):2280–2287
bacterial counts were increased during storage either in the Ahmed J, Al-Ruwaih N, Mulla M, Rahman MH (2018) Effect of
control or KBPH-enriched samples. However, lower values high-pressure treatment on functional, rheological and structural
of these counts were observed in KBPH-supplemented sam- properties of kidney bean protein isolate. LWT-Food Sci Technol
91:191–197
ples. This reduction effect of KBPH was most conspicuous in Aksu K, Özgeriş B, Taslimi P, Naderi A, Gülçin İ, Göksu S (2016)
the case of total viable bacteria and was dose-dependent at all Antioxidant activity, acetylcholinesterase and carbonic anhy-
the time intervals of storage (3–15 days), confirming that this drase inhibitory properties of novel ureas derived from phenethy-
action might be due to the chemical composition of KBPH lamines. Arch Pharm 349(12):944–954. https​://doi.org/10.1002/
ardp.20160​0183
and not to any other interfering factors. It has been observed Al-Ruwaih N, Ahmed J, Mulla MF, Arfat YA (2019) High-pressure
that the control minced beef reached levels of 5.3 and 6.5 assisted enzymatic proteolysis of kidney beans protein isolates
log after 6 and 9 days of storage at 4 °C, indicating that the and characterization of hydrolysates by functional, structural,
secured storage period could be 7 or 8 days according to the rheological and antioxidant properties. LWT-Food Sci Technol
100:231–236
acceptable range of total bacterial count (2.8–4.3 CFU/cm2) AOAC (1996) Official methods of analysis. Association of Official
(Todd 2004). Beef samples enriched with 200 µg/g KBPH Analytical Chemistry, Arlington
kept the level of the total viable count at 5.7 and 6.55 log AOAC (2002) Official methods of analysis, 17th edn. Association of
after 12 and 15 days of storage, which increased the secured Official Analytical Chemists, International Inc., Arlington
APHA (1992) Compendium of methods for the microbiological exami-
time of storage to be 14 or 15 days instead of 7 or 8 days in nation of foods, 3rd edn. American Public Health Association,
the case of control beef sample. Washington, D.C
Barac M, Cabrilo S, Pesic M, Stanojevic S, Pavlicevic M, Macej O
(2011) Functional properties of pea (Pisum sativum L.) protein
isolates modified with chymosin. Int J Mol Sci 12(12):8372–8387
Conclusions Bernardini R, Harnedy P, Bolton D, Kerry J, O’Neill E, Mullen AM,
Hayes M (2011) Antioxidant and antimicrobial peptidic hydro-
KBPH were successfully prepared using pepsin, wherein lysates from muscle protein sources and byproducts. Food Chem
the hydrolysates exhibited strong antioxidant and antimi- 124:1296–1307
Betancur-Ancona D, Sosa-Espinoza T, Ruiz-Ruiz J, Segura-Campos
crobial activities. The functionality of KBPH improved M, Chel-Guerrero L (2014) Enzymatic hydrolysis of hard-to-cook
significantly with respect to the native protein. Supple- bean (Phaseolus vulgaris L.) protein concentrates and its effects
mentation of minced beef with KBPH increased the safe on biological and functional properties. Int J Food Sci Technol
storage period by about 7 days when compared to control. 49(1):2–8
Boye J, Zare F, Pletch A (2010) Pulse proteins: processing, characteri-
This delay in the spoilage emergence is apparently attrib- zation, functional properties and applications in food and feed.
uted to the antimicrobial activity of KBPH. The obtained Food Res Int 43(2):414–431
results may be useful to produce functional peptides from Boztaş M, Çetinkaya Y, Topal M, Gülçin İ, Menzek A, Şahin E, Tanc
KBPI, which might be incorporated into the formulation M, Supuran CT (2015) Synthesis and carbonic anhydrase isoen-
zymes I, II, IX, and XII inhibitory effects of dimethoxy-bromo-
of novel foods and nutraceuticals. Additional research on phenol derivatives incorporating cyclopropane moieties. J Med
KBPH in vitro digestion is needed to understand their sta- Chem 58(2):640–650. https​://doi.org/10.1021/jm501​573b
bility in the gastrointestinal tract. Bursal E, Gülçin İ (2011) Polyphenol contents and in vitro antioxidant
activities of lyophilised aqueous extract of kiwifruit (Actinidia
deliciosa). Food Res Int 44:1482–1489
Caglayan C, Gulcin İ (2018) The toxicological effects of some aver-
Compliance With Ethical Standards mectins on goat liver carbonic anhydrase enzyme. J Biochem Mol
Toxicol 32(1):e22010. https​://doi.org/10.1002/jbt.22010​
Conflict of interest The authors declare that there are no conflicts of Carrasco-Castilla J, Hernández-Álvarez AJ, Jiménez-Martínez C,
interest. Jacinto-Hernández C, Alaiz M, Girón-Calle J, Dávila-Ortiz G

13
 International Journal of Peptide Research and Therapeutics

(2012) Antioxidant and metal chelating activities of Phaseolus on the enzyme activity. Environ Toxicol Pharmacol 44:134–139.
vulgaris L. var. Jamapa protein isolates, phaseolin and lectin https​://doi.org/10.1016/j.etap.2016.04.011
hydrolysates. Food Chem 131(4):1157–1164 Laemmli UK (1970) Cleavage of structural proteins during the assem-
Chalamaiah M, Yu W, Wu J (2017) Immunomodulatory and antican- bly of head of bacteriophage T4. Nature 227:680–685
cer protein hydrolysates (peptides) from food proteins: a review. Lee PS (2009) Quantitation of microorganisms. In: Goldman E, Green
Food Chem 245:205–222 LH (eds) Practical handbook of microbiology, 2nd edn. CRC
Chobert JM, Touati A, Bertrand-Harb C, Dalgalarrondo M, Nicolas Press, Boca Raton
MG, Haertlé T (1991) In vitro proteolysis and functional prop- Li YQ, Han Q, Feng JL, Tian WL, Mo HZ (2014) Antibacterial char-
erties of reductively alkylated [3-casein derivatives]. J Dairy acteristics and mechanisms of ε-poly-lysine against Escherichia
Res 58:285–298 coli and Staphylococcus aureus. Food Cont 43:22–27
Dave Oomah B, Patras A, Rawson A, Singh N, Compos-Vega R Luna-Vital DA, Mojica L, de Mejía EG, Mendoza S, Loarca-Piña G
(2011) Chemistry of pulses. In: Tiwari BK, Gowen A, McKenna (2015) Biological potential of protein hydrolysates and peptides
B (eds) Pulse foods processing, quality and nutraceutical appli- from common bean (Phaseolus vulgaris L.): a review. Food Res
cations. Academic Press, Elsevier, London, pp 9–55 Int 76:39–50
Dueñas M, Martínez-Villaluenga C, Limón RI, Peñas E, Frias J Meinert L, Broge EH, Bejerholm C, Jensen K (2015) Application of
(2015) Effect of germination and elicitation on phenolic com- hydrolyzed proteins of animal origin in processed meat. Food Sci
position and bioactivity of kidney beans. Food Res Int 70:55–63 Nutr 4(2):290–297
Evangelho JAD, Vanier NL, Pinto VZ, De Berrios JJ, Dias ARG, da Meshginfar N, Sadeghi Mahoonak A, Ghorbani M, Aalami M (2017)
Rosa Zavareze E (2017) Black bean (Phaseolus vulgaris L.) pro- Effects of protein hydrolysate from sheep visceral on oxidative
tein hydrolysates: physicochemical and functional properties. stability of soybean oil and chicken sausage. J Food Process Pre-
Food Chem 214:460–467 serv 41:e12875. https​://doi.org/10.1111/jfpp.12875​
Fan TY, Sosulski FW (1974) Dispersion and isolation of proteins from Montoya CA, Lalles JP, Beebe S, Montagne L, Souffrant WB, Leterme
legume flours. Can Inst Food Sci Technol J 7:256–259 P (2006) Influence of the Phaseolus vulgaris phaseolin level of
Gobbetti M, Minervini F, Rizzello CG (2004) Angiotensin I-convert- incorporation, type and thermal treatment on gut characteristics
ing enzyme-inhibitory and antimicrobial bioactive peptides. Int J in rats. Br J Nutr 95(01):116–123
Dairy Technol 57:173–188 Mundi S, Aluko RE (2012) Physicochemical and functional properties
Göçer H, Gülçin İ (2011) Caffeic acid phenethyl ester (CAPE): cor- of kidney bean albumin and globulin protein fractions. Food Res
relation of structure and antioxidant properties. Int J Food Sci Int 48:299–306
Nutr 62(8):821–825 Nanda A, Saravanan M (2009) Biosynthesis of silver nanoparticles
Gómez-Ruiz JÁ, Taborda G, Amigo L, Recio I, Ramos M (2006) from Staphylococcus aureus and its antimicrobial activity against
Identification of ACE-inhibitory peptides in different Spanish MRSA and MRSE. Nanomed 5:452–456
cheeses by tandem mass spectrometry. Eur Food Res Technol Osman AO, Mahgoub S, Sitohy M (2013) Preservative action of 11S
223(5):595–601 (glycinin) and 7S (β-conglycinin) soyglobulin on bovine raw milk
Gülçin İ (2011) Antioxidant activity of eugenol: a structure-activity stored either at 4 or 25 °C. J Dairy Res 80:174–183
relationship study. J Med Food 14(9):975–985 Osman A, Goda H, Abdel-Hamid M, Badran S, Otte J (2016) Anti-
Hancock REW, Rozek A (2002) Role of membranes in the activi- bacterial peptides generated by Alcalase hydrolysis of goat whey.
ties of antimicrobial cationic peptides. FEMS Microbiol Lett LWT-Food Sci Technol 65:480–486
206:143–149 Özyurt G, Kuley K, Balikçi E, Kaçar Ç, Gökdogan S, Etyemez M
Hoyle NT, Merritt JH (1994) Quality of fish protein hydrolysate from (2012) Effect of the icing with rosemary extract on the oxida-
Herring (Clupea harengus). J Food Sci 69:615–619 tive stability and biogenic amine formation in sardine (Sar-
Jenssen H, Hamill P, Hancock REW (2006) Peptide antimicrobial dinella aurita) during chilled storage. Food Bioprocess Technol
agents. Clin Microbiol Rev 19:491–511 5:2777–2786
Karabagias I, Badeka A, Kontominas MG (2011) Shelf life extension Polanco-Lugo E, Dávila-Ortiz G, Betancur-Ancona DA, Chel-Guerrero
of lamb meat using thyme or oregano essential oils and modified LA (2014) Effects of sequential enzymatic hydrolysis on struc-
atmosphere packaging. Meat Sci 88:109–116 tural, bioactive and functional properties of Phaseolus lunatus
Kimura A, Fukuda T, Zhang M, Motoyama S, Maruyama N, Utsumi protein isolate. Food Sci Technol 34(3):441–448
S (2008) Comparison of physicochemical properties of 7S and Prior RL, Wu X, Schaich K (2005) Standardized methods for the
11S globulins from pea, fava bean, cowpea, and French bean with determination of antioxidant capacity and phenolics in foods and
those of soybean French bean 7S globulin exhibits excellent prop- dietary supplements. J Agric Food Chem 53:4290–4302
erties. J Agric Food Chem 56:10273–10279 Romero J, Sun SMM, McLeester RC, Bliss FA, Hall TC (1975) Herit-
Köksal E, Bursal E, Gülçin İ, Korkmaz M, Çağlayan C, Gören AC, able variation in a polypeptide subunit of the major storage protein
Alwasel SH (2017) Antioxidant activity and polyphenol content of the bean, Phaseolus vulgaris L. Plant Physiol 56:776–779
of Turkish thyme (Thymus vulgaris) monitored by LC–MS/MS. Rui X, Boye JI, Ribereau S, Simpson BK, Prasher SO (2011) Compara-
Int J Food Prop 20(3):514–525. https​://doi.org/10.1080/10942​ tive study of the composition and thermal properties of protein
912.2016.11684​38 isolates prepared from nine Phaseolus vulgaris legume varieties.
Korhonen H, Pihlanto A (2006) Bioactive peptides: production and Food Res Int 44:2497–2504
functionality. Int Dairy J 16(9):945–960 Salami M, Moosavi-Movahedi AA, Ehsani MR, Yousefi R, Haertle T,
Köse LP, Gülçin İ, Gören AC, Namiesnik J, Martinez-Ayala AL, Gor- Chobert JM (2010) Improvement of the antimicrobial and anti-
instein S (2015) LC–MS/MS analysis, antioxidant and anticho- oxidant activities of camel and bovine whey proteins by limited
linergic properties of galanga (Alpinia officinarum Hance) rhi- proteolysis. J Agric Food Chem 58:3297–3302
zomes. Ind Crops Prod 74:712–721. https​://doi.org/10.1016/j. Sathe SK, Iyer V, Salunkhe DK (1981) Functional properties of the
indcr​op.2015.05.034 Great Northern Bean (Phaseolus vulgaris L.) proteins, amino acid
Kucuk M, Gulcin İ (2016) Purification and characterization of car- composition, in vitro digestibility and application to cookies. J
bonic anhydrase enzyme from black sea trout (Salmo trutta Lab- Food Sci 47:8–11
rax Coruhensis) kidney and inhibition effects of some metal ions

13
International Journal of Peptide Research and Therapeutics

Shevkani K, Singh N, Kaur A, Rana JC (2015) Structural and func- Yamamoto Y, Togawa Y, Shimosaka M, Okazaki M (2003) Purifi-
tional characterization of kidney bean and field pea protein iso- cation and characterization of a novel bacteriocin produced by
lates: a comparative study. Food Hydrocoll 43:679–689 Enterococcus faecalis strain RJ-11. Appl Environ Microbiol
Sitohy MZ, Mahgoub SA, Osman AO (2012) In vitro and in situ anti- 69:105746–105753
microbial action and mechanism of glycinin and its basic subunit. Yin SW, Tang CH, Wen QB, Yang XQ, Li L (2008) Functional proper-
Inter J Food Microbiol 154(1):19–29 ties and in vitro trypsin digestibility of red kidney bean (Phaseo-
Tang CH (2008) Thermal denaturation and gelation of vicilin-rich pro- lus vulgaris L.) protein isolate: effect of high-pressure treatment.
tein isolates from three Phaseolus legumes: a comparative study. Food Chem 110:938–945
LWT-Food Sci Technol 41(8):1380–1388 Zayas JF (1979) Solubility of proteins. functionality of proteins in food.
Tironi VA, Añón MC (2010) Amaranth proteins as a source of antioxi- Springer, Berlin, pp 6–22
dant peptides: effect of proteolysis. Food Res Inter 43:315–322
Todd EC (2004) Microbiological safety standards and public health Publisher’s Note Springer Nature remains neutral with regard to
goals to reduce foodborne disease. Meat Sci 66(1):33–43 jurisdictional claims in published maps and institutional affiliations.
Wani IA, Sogi DS, Shivhare US, Gill BS (2015) Physico-chemical
and functional properties of native and hydrolyzed kidney bean
(Phaseolus vulgaris L.) protein isolates. Food Res Int 76:11–18
Xie Z, Huang J, Xu X, Jin Z (2008) Antioxidant activity of pep-
tides isolated from alfalfa leaf protein hydrolysate. Food Chem
111:370–376

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