401323v1 Full

bioRxiv preprint doi: https://doi.org/10.1101/401323; this version posted August 27, 2018.
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ANTIMICROBIAL, ANTIOXIDANT AND ANTI-INFLAMMATORY

ACTIVITIES OF PROTEINS OF Phaseoulus lunatus (FABACEAE)
BABY LIMA BEANS PRODUCED IN ECUADOR
J. TAMAYO1, T. POVEDA1, M. PAREDES1, G. VÁSQUEZ2, W. CALERO-CÁCERES3, 4*

1
Laboratory of Functional Foods, Faculty of Food Science and Engineering, Universidad Técnica de Ambato, Ambato,
Ecuador.
2
Escuela Superior Politécnica del Litoral, ESPOL, Facultad de Ingeniería Mecánica y Ciencias de la Producción, Campus
Gustavo Galindo Km. 30.5 Vía Perimetral, P.O. Box 09-01-5863.
3
UTA-RAM-OneHealth Group, Centro de Investigaciones Agropecuarias, Facultad de Ciencias Agropecuarias,
Universidad Técnica de Ambato, 180601 Cevallos, Tungurahua, Ecuador.
4
Dirección de Investigación y Desarrollo, Universidad Técnica de Ambato, Ambato, Ecuador.
*E-mail address: [email protected]
ABSTRACT
Phaseoulus lunatus L., a variety of baby lima bean, which is produced in the coastal region of
Ecuador, is a profitable crop of that country. Various cultivars of this common bean are considered
a sources for nutraceutical compounds, such as bioactive peptides. To assess the potential biologic
activities of protein isolates and hydrolysates of P. lunatus baby lima beans, this study evaluates the
proteins antimicrobial, antioxidant and anti-inflammatory activities. Antioxidant activity was
measured by the TBARS method. In-vitro anti-inflammatory activity was measured by the
inhibition of denatured protein as well as a diffusion method, according with CLSI guidelines by
antimicrobial activity. Both fractions (isolate and hydrolysates) showed anti-inflammatory and
antioxidant activity. However, protein hydrolysates (pH 5) had a better performance than protein
isolates. The same effect was observed in antimicrobial activity, when protein hydrolysates had a
broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria. These
preliminary studies suggest that P. lunatus baby lima beans could have a considerable biological
activity for nutraceutical applications.
Keywords: Phaseoulus lunatus, protein, baby lima bean, antimicrobial, antioxidant, anti-inflammatory.
bioRxiv preprint doi: https://doi.org/10.1101/401323; this version posted August 27, 2018. The copyright holder for this preprint (which was
1. INTRODUCTION
The genus Phaseolus (beans), included in the family Leguminosae, represent one of the most
ancient, common legumes consumed by humans around the world. In developing countries, this
legume specie is one of the principal sources of dietary proteins (BROUGHTON et al. 2003). Today,
in terms of morphological and genetic diversity, legumes are a complex genus with four genetic
groups (BITOCCHI AND NANNI 2012). Previous research has shown that the northwestern region of
South America (Colombia, Ecuador and northern Peru) represents the convergence of the two
principal genetic P. vulgaris varieties (Central American and Andean) (DEBOUCK et al. 1993). This
convergence shows a clear difference of morphology and molecular characteristics. Considering the
current correlation between population growth and bean consumption (BELLUCCI et al. 2014), a
detailed analysis of chemical and biological characteristics would help us better understand the
properties and differences between common bean varieties.
An increasing amount of literature demonstrates the biological properties of protein isolates of

legumes. These properties represent a considerable nutritional role and a source of essential amino
acids (BETANCUR-ANCONA et al., 2009; CARBONARO, MASELLI, & NUCARA, 2015). Varieties and
cultivars of common beans has been considered as sources of nutraceutical compounds, such as
bioactive peptides, polyphenols, and polysaccharides (NGOH and GAN 2016; CAMPOS-VEGA et al.
2010; BETANCUR-ANCONA et al. 2009; BITOCCHI and NANNI 2012).
The variety of baby lima bean that is produced in the coastal region of Ecuador is a profitable crop
for that country in terms of economic benefits (EL PRODUCTOR 2015). At the moment, no published
information about physicochemical and biological characteristics of this Ecuadoran variety of bean.
Considering the differences between Phaseolus germplasm according their precedence (BERNAL
and GRAHAM 2001), the differences are expected in chemical composition and biological activities.
This study evaluates the antimicrobial, antioxidant and anti-inflammatory activities of protein
extracts and protein hydrolisates of P. lunatus var. baby lima beans, in order to identify the beans
potential source of bioactive compounds for nutraceutical applications.
2. MATERIALS AND METHODS
2.1. Samples
P. lunatus var. baby lima beans were purchased in a local market in Machala, Ecuador. Seeds were
dehydrated in a Laboratory Incubator (ESCO, Singapore) at 50 °C for 48 hours. Subsequently,
dehydrated seeds were grinded into a fine flour powder with a mill MG 1511.
2.2. Protein isolates from baby lima beans

Baby lima bean protein concentrate was obtained according to the methodology of BARRIO &
AÑÓN, (2010) with minor modifications. Homogenized flour was diluted in a 1:10 solution using
distilled water. The pH level was set at 8 by using sodium hydroxide 2 M for 1 hour, shaken at 800-
900 rpm. Those samples were centrifuged at 4.400 rpm for 30 minutes. The supernatant was
recovered and treated at different pHs (3, 4, 5 and 6) using, if necessary, hydrochloric acid 2 N or
sodium hydroxide 2M. The mixture was vigorously homogenized and stored at 4 °C for 24 hours.
The supernatant was discarded and the precipitates were freezed at -80 °C and lyophilized using a
freeze dryer Bench Top Pro BTP-3ES0VW (SP Scientific, Stone Ridge, NY, USA), at -50 °C and 0.2 Pa.
The extraction performance was calculated with this formula: % performance = (Pf / Po) * 100.
Where Po: Initial weight of flour, Pf: Final weight of lyophilized sample. The protein quantification
was carried out by Biuret method (NIELSEN, 2017)
2.3. Protein hydrolysates

To obtain protein hydrolysates, physiological human gastric fluids were simulated according to the
methodology of MINEKUS et al. (2014). Prior to the ejecution, Simulated Gastric Fluid (SGF) and
Simulated Intestinal Fluid (SIF) were prepared. For SFG, 25 mg of pepsin (2000 U/mg; MP
Biomedicals, California, USA) was diluted in 50 mL of sodium chloride 0.35 M pH 2. For SIG, 61.6 mg
of sodium hydroxide (Merck Millipore, Darmstadt, Germany) and 680 mg of sodium phosphate
monobasic monohydrate (Sigma Aldrich, Missouri, USA) were dissolved in 100 mL of deionized
water and adjusted to pH 7. For the gastric digestion, E:S mixture was used: 100 mg of the protein
isolate was dissolved in 10 mL of NaCl 0.35 M pH 2.0. A relation 50:50 of diluted protein and
simulated gastric fluid (SGF) was made for each sample. Subsequently, the samples were incubated
during 2 hours at 37 °C and 500 rpm in a water bath (StableTemp, Cole Palmer, Illinois, USA).
Immediately, a duodenal digestion was done, using pancreatin (Merck Millipore, Darmstadt,
Germany) at a final concentration of 109 U/mL. A relation 1:1 (v/v) of the prior mixture and the
simulated intestinal fluid (SIF) was made for each sample. Finally, the samples were incubated for 2
hours at 37 °C at 500 rpm in a water bath. Afterwards, the enzymatic reaction was stopped using
200 µL of sodium bicarbonate 1 M (Merck Millipore, Darmstadt, Germany) at 80 °C for 10 minutes.
The samples were frozen at -80 °C and lyophilized. This protocol was carried out three times.
2.4. Antioxidant activities (TBARS assay)
TBARS assay (thiobarbituric acid reactive substances) is one of the most ancient and widely used
assays in order to quantify oxidative stress (DASGUPTA and KLEIN 2014), which is based in the
reaction between malondialdehyde and 2-thiobarbituric acid (TBA) that produces an adduct that
shows pink color species, who absorbs at 532-535 nm. For this study, with minor modifications, the
method of ROJANO, B. A., GAVIRIA, C. A. and SÁEZ (2008) was used. Aliquots of 0.2, 0.4, 1.0 and 2.0
mg of protein isolates or protein hydrolisates were mixed with 2 mL of distilled water (protein
solution). Then, 500 µL of olive oil (previously oxidized) was mixed with 500 µL of protein solution.
The samples were mixed at 450 rpm in a micro incubator Provocell Shaking (Esco, Singapore) for 13
hours, at 28 °C. Then, 1 mL TBA (1%) was placed and immediately incubated for 1 hour, at 95 °C.
Finally, the samples were chilled at 4 °C for 15 minutes in a freezer and then analyzed at 532 nm in
a UV Vis spectrophotometer (Thermo Scientific, USA). Butylated hydroxytoluene (BHT) ≥ 99%
(Merck, Darmstadt, Germany), as positive control, was diluted in ethanol 96% (Merck, Darmstadt,
Germany).
Oxidized olive oil was prepared with a heat treatment for 15 days. During the first eight days, the
oil was heated in a laboratory oven (VWR, Pennsylvania, USA) at 70 °C. During latter seven days,
the oil was heated at 40 °C. The results were compared with the positive control, quantifying the
antioxidant activity percentage with this formula: % AA = (M – C) / C * 100; Where AA: Antioxidant
activity, C: Oxidized oil absorbance (532 nm), M: Sample (protein sample + oxidized oil) absorbance
(532 nm).
2.5. Anti-inflammatory activity

The anti-inflammatory activity of the protein isolates and hydrolisates was analyzed by following
the methodology of PADMANABHAN P (2012), with minor modifications. Sodium diclofenac (25
mg/mL) was used as the positive control. Samples were homogenized, and aliquots of 0.2, 0.4, 1.0
and 2.0 mg were dissolved with 2 mL of deionized water. Then, the suspension was homogenized
with 0.2 mL of egg albumin and 2.8 mL of phosphate buffered saline (PBS) pH 6.4. This suspension
was mixed gently. The mixtures were incubated at 37°C for 15 minutes, then incubated at 70 °C for
10 minutes. The samples were finally chilled in cold water for 10 minutes, and the absorbance was
analyzed using a UV-Vis spectrophotometer at 660 nm, with distilled water as a blank. The obtained
data was compared with the results of the positive control (sodium diclofenac). The anti-
inflammatory activity percentage was determined with this formula: % anti-inflammatory activity =
(M-C)/C * 100, where C: Denatured egg albumin absorbance (without sample) and M: Sample
absorbance (protein suspension + egg albumin).
2.6. Antimicrobial activity

In order to evaluate the antimicrobial activity, these certified strains were used:
Staphylococcus aureus (ATCC® 25923), Escherichia coli (ATCC® 25922), Bacillus cereus (ATCC®
10876), Listeria monocytogenes (ATCC® 19115) and Pseudomonas aeruginosa (ATCC® 10145). The
guidelines of the Clinical and Laboratory Standards Institute (CLSI) were applied, with minor
modifications. A culture of each bacteria was inoculated in Tryptic Soy Broth TSB (Merck Millipore,
Darmstadt, Germany) until each culture´s optical density was 0.5 Mc Farland units. Then, in
Mueller-Hinton agar plates (Oxoid, Basingstoke, Hampshire, UK), the inoculum was struck with a
long cotton swab. The protein isolates and hydrolysates were evaluated at five concentrations: 500,
375, 250, 200 and 150 mg/mL, all diluted in distilled water. After the inoculum was struck in the
agar, 6mm wells were made with sterile plastic pipette tips (200 µL). 70 µL of protein solution or
controls were deposited into the 6mm wells. Gentamicin (500 µg/mL) was used as a positive control
and sterile deionized water was used as a negative control. After the plates stood for 30 minutes,
they were incubated for 48 hours, at 37 °C. The inhibition zones were measured after 24 hours and
48 hours. In order to determine the percentage of antimicrobial inhibition, this formula was used:
% Inhibition = (B – A)/(C – A)*100. Where A: Negative control diameter (mm), B: Sample inhibition
zone (mm), C: Positive control diameter (mm).
2.7. Statistical analysis

Statistical analysis was done by using Statgraphics Centurion 16.103 and Microsoft Excel® software
packages. Simple variance analysis (ANOVA) and Tukey’s comparative test at 95 % of confidence
were used to evaluate statistical differences between protein extraction conditions and biological
activities. Four (antioxidant activity) and three (anti-inflammatory and antimicrobial activities)
replicates of each experiment were done, and the results were summarized as a mean and standard
deviation.
3. RESULTS AND DISCUSSIONS
3.1. Preparation of protein isolates and hydrolizates
A proximate analysis of the baby lima bean flour was done at the National Institute of Agriculture
Research of Ecuador (INIAP Santa Catalina). The analyzed samples had a mean of 21.15% of protein.
This result is similar to the reported by FAO, 2005 (23.6%), TYLER, YOUNGS, & SOSULSKI, 1981
(22.8%) and SATHE, 2002 (17.5-28.7%).
Baby lima bean flour protein precipitation was based on isoelectric precipitation (CÁRDENAS et al.
2018). The pH was adjusted at four levels (3.0; 4.0, 5.0; 6.0) using HCl solution (2 N) and the
precipitates were recovered. The highest percentage of protein was obtained at pH 5.0 (19.56 ±
1,55 %). At pH 6, no precipitation was observed, however the solution was again frozen and
lyophilized. The precipitation effect was only present at pH <6. These precipitation effects could be
related to the fact that a majority of legumes proteins have their isoelectric points under pH 6 (EL-
SAYED M et al. 1986; KLUPŠAITĖ and JUODEIKIENĖ 2015).
The protein quantification of the isolates was determined by using the Biuret colorimetric method
(DOREY and DRAVES 1998). At pH 5, protein isolate represents 62.53 ± 0,02 % (dry weight base).
The protein isolates were characterized by Native-PAGE and SDS-PAGE (data pending for
publication) which showed a predominance of 2S albumins and 11S globulins (Basic and acid
subunities). Both gastric and intestinal digestibility were performed while simulating human
physiologic conditions. The SDS-PAGE shows five protein bands with sizes between 15 kDa and 37
kDa. These results suggest that the proteins from baby lime beans were not completely hydrolyzed
during human digestion. This effect was observed in related research, during which proteins from
different legumes had a high persistence during proteases digestion (M CARBONARO, GRANT, and
CAPPELLONI 2005; DESHPANDE & DAMODARAN 1989).
3.2. Antioxidant activity (TBARS method)
The antioxidant activity of protein isolates and hydrolysates were evaluated by the TBARS method.
The protein isolates obtained at pH 3, 4, 5 and 6; and concentrations of 100, 200, 500 and 1000
µg/mL were evaluated (Table 1). The differences between precipitation pHs, concentrations and
antioxidant activity were not statistically different (p < 0.05; simple variance analysis). However, at
pH 5, the highest inhibition of lipid peroxidation was observed in all four of the evaluated
concentrations (100, 200, 500, 1000 µg/mL), in relation to the obtained isolates at the pH 3, 4 and
6 levels.
Table 1. In vitro antioxidant activity of baby lima bean protein isolate using TBARS method.
Concentrations
Precipitation pH 100 µg/ml 200 µg/ml 500 µg/ml 1000 µg/ml
pH 3 10,82 ± 0,006a 15,69 ± 0,007a 22,30 ± 0,017a 29,99 ± 0,012a
pH 4 13,97 ± 0,012a 19,12 ± 0,011a 23,81 ± 0,022a 32,69 ± 0,008a
pH 5 15,66 ± 0,009a 19,65 ± 0,008a 24,45 ± 0,016a 33,19 ± 0,024a
pH 6 13,40 ± 0,006a 17,31 ± 0,009a 22,60 ± 0,010a 30,59 ± 0,027a
Data are expressed as the mean ± Standard deviation SD (n=4).
Values in the same column having different letters differ significantly (p<0.05). ANOVA and Tukey’s test
For this parameter, Butylated hydroxytoluene (BHT) ≥99% was used as a positive control at
concentrations of 100, 200, 500 and 1000 µg/mL. Between 70,82 ± 0,042 (100 µg/mL) and 91,73 ±
0,050 % (1000 µg/mL), BHT showed considerable values of antioxidant activity. Figure 1 shows the
relationship between protein isolates and hydrolysates at different pHs and the percentages of lipid
peroxidation inhibition. The maximum antioxidant activity in protein isolates was observed at pH 5
and 1000 µg/mL, with a value of 33,19 ± 0,024 %. The protein hydrolysate obtained at pH 5 and
1000 µg/mL was at a value of 77,17 ± 0,029 % of lipid peroxidation inhibition. As expected, in
comparison with the evaluated samples, the BHT showed considerable differences in their
antioxidant activity.
Isolated pH5 Protein Hydrolyzate BHT
91.73
84.13
77.17
77
70.82
63.43
54.87
44.03
35.84
29.37
26.71
20.91
100 ΜG/ML 200 ΜG/ML 500 ΜG/ML 1000 ΜG/ML
Figure. 1 Inhibition of lipid peroxidation (TBARS) of the protein isolate and hydrolysate from baby
lima bean at concentrations of 100, 200, 500 and 1000 μg/mL.
Figure 1 shows clear differences in antioxidant activity values in all of the evaluated concentrations
as well as between isolates and hydrolysates while displaying statistical differences (p<0.05; simple
variance analysis) between concentrations. In comparison with 100, 200 and 500 µg/mL, 1000
µg/mL was the highest observed peroxidation inhibition. This results supports studies which show
an inverse correlation between molecular weight and antioxidant activity (ZHAO et al. 2012). The
effect observed in different legume protein hydrolysates showed considerable antioxidant activity
(DO EVANGELHO et al. 2016; DURAK et al. 2013; ALASHI et al. 2014; CARRASCO-CASTILLA et al.
2012).
3.3. In-vitro anti-inflammatory activities
Table 2 shows the anti-inflammatory activities of protein isolates obtained at four pH levels (3.0,
4.0, 5.0 and 6.0) and hydrolysates (pH 5). Diclofenac sodium (25 mg/mL) was used as a positive
control at 100-1000 µg/mL. Statistical analysis do not show differences between anti-inflammatory
activities and the precipitation pH of the isolates (p<0.05; simple variance analysis). However, for
the protein isolates, the maximum activity was observed at pH 5 and 1000 µg/mL, with a value of
21,53 ± 0,39 % when inhibited by denatured protein.
Table 2. In vitro anti-inflammatory activity of baby lima bean protein isolate using albumin protein
denaturation method.
Concentrations
Precipitation pH 100 µg/ml 200 µg/ml 500 µg/ml 1000 µg/ml
pH 3 2,54 ± 0,018a 4,00 ± 0,038a 8,14 ± 0,046a 10,14 ± 0,038a
pH 4 4,24 ± 0,030a 7,36 ± 0,032a 12,55 ± 0,039a 17,52 ± 0,056a
pH 5 5,82 ± 0,048a 11,09 ± 0,054a 16,29 ± 0,037a 21,53 ± 0,039a
pH 6 2,34 ± 0,039a 5,42 ± 0,033a 10,72 ± 0,040a 15,80 ± 0,057a
Data are expressed as the mean ± Standard deviation SD (n=4).
Values in the same column having different letters differ significantly (p<0.05). ANOVA and Tukey’s test
Protein hydrolysates (pH 5) showed an improvement in the percentages of inhibition in relation with
protein isolates, with 30.62 ± 0.01 % of inhibition of denatured protein at 1000 µg/mL (Figure 2).
Positive controls of diclofenac (100 – 1000 µg/mL) showed anti-inflammatory activities in the range
of 32.14% -100.00 %, as reported in related research (CÁRDENAS et al. 2018). Related research
showed a clear inhibition of pro-inflammatory mediators of protein hydrolysates and unhydrolysed
proteins from legumes (NDIAYE et al. 2012; POWNALL, UDENIGWE, and ALUKO 2010). Therefore,
because baby lima bean proteins and hydrolysates have displayed an interesting anti-inflammatory
activity, further research could be considered.
Isolated pH5 Protein Hydrolyzate Diclofenac Sodium
100
75.85
% ANTI-INFLAMMATORY ACTIVITY
61.23
32.14
30.62
25.49
18.82
20.5
16.65
13.66
13.38
7.01
100 ΜG/ML 200 ΜG/ML 500 ΜG/ML 1000 ΜG/ML

CONCENTRATION
Figure 2. Anti-inflammatory Activity of protein isolate and hydrolysate from baby lima bean at
concentrations of 100; 200; 500 and 1000 μg/ml.
3.4. Antimicrobial activity
Several protein hydrolysates from plant and animal origin, and their peptides, have shown
antimicrobial activity against Gram-positive and Gram-negative bacteria. Other findings suggest
that certain peptides can alter the cytoplasmic membrane or inhibit nucleic acid synthesis, protein
synthesis or enzymatic reactions (BROGDEN 2005). However, the details of the peptides active
mechanics have not yet been completely determined due to the vast number of peptides with
potential biological activity (DASHPER, LIU, and REYNOLDS 2007).
a b c
d e f
Figure 3. Antimicrobial activity of the protein hydrolisate obtained at pH 5.

A) Protein hydrolysate without bacteria (negative control); B) L. monocytogenes; C) P. aeruginosa; in the D) E. coli; E)
S. aureus; F) B. cereus.
To evaluate the potential antimicrobial activity of P. lunatus baby lima bean protein isolate and
hydrolysates, the samples were screened against clinical bacterial strains (Figure 3). In the evaluated
bacteria, between 150-500 mg/mL, no inhibition zones were observed in non-hydrolyzed protein
samples. However, in four of five pathogenic bacteria evaluated (Table 3), their hydrolysates have
shown a considerable antimicrobial activity. Significant differences were found among hydrolysate
concentrations (p< 0.05; simple variance analysis).
Table 3. Bacterial inhibition areas from protein isolates and hydrolysates of P. lunatus baby lima
beans against certified bacterial strains.
Bacteria Concentration Isolate Hydrolysate Gentamycin
500 µg/ml (mm)
Inhibition Inhibition zone
zone (mm) (mm) Inhibition (%)
Escherichia coli 500 mg/ml <6 18,67 a ± 0,58 71,70 23,67 ± 0,58
(ATCC® 25922™)
375 mg/ml <6 15,67 b ± 0,58 54,72
c
250 mg/ml <6 12,33 ± 0,58 35,82
200 mg/ml <6 <6 0
Pseudomonas aeruginosa 500 mg/ml <6 12,33 a ± 0,58 33,31 25,00 ± 1,00
(ATCC® 10145) b
375 mg/ml <6 10,33 ± 0,58 22,78
250 mg/ml <6 8,33 c ± 0,58 12,26
200 mg/ml <6 <6 0
Listeria monocytogenes 500 mg/ml <6 18,33 a ± 0,58 51,37 30,00 ± 1,00
(ATCC® 19115™) 375 mg/ml <6 b
16,33 ± 0,58 43,04
c
250 mg/ml <6 13,67 ± 0,58 31,96
200 mg/ml <6 <6 0
Bacillus cereus 500 mg/ml <6 9,33 a ± 0,58 15,37 27,67 ± 0,58
(ATCC® 10876) b
375 mg/ml <6 7,33 ± 0,58 6,14
250 mg/ml <6 <6 0
200 mg/ml <6 <6 0
Staphylococcus. aureus 500 mg/ml <6 <6 0 35,00 ± 1,00
(ATCC® 25923™)
375 mg/ml <6 <6 0
250 mg/ml <6 <6 0
200 mg/ml <6 <6 0
The values represent the average of 3 measurements ± the standard deviation.

Different lowercase letters indicate, for each test, significant differences (p <0.05) between the distances of the different
concentrations of the protein hydrolyzed bean.
The distances are expressed in millimeters (mm); the positive control was gentamicin and sterile water was used as a
negative control.
The major percentage of inhibition was observed in 500 mg/mL of protein hydrolysate in Gram-
negative E. coli ATCC ® 25922, 71.7 % of which was related to the positive control (Gentamicin 500
µg/mL), followed by Gram-positive L. monocytogenes ATCC® 19115 with 51.37 %. Gram-negative P.
aeruginosa ATCC® 10145 shows a considerable inhibition of 33.31 %. A slight inhibition of 15.37%
was observed in Gram-positive B. cereus ATCC ® 10876. The results were negative with Gram-
positive S. aureus ATCC ® 25923TM. These results suggest that protein hydrolysates from P. lunatus
baby lima beans possess a broad-spectrum of antimicrobial activity against Gram-negative and
Gram-positive bacteria.
4. CONCLUSIONS
The P. lunatus baby lima bean protein isolates and hydrolysates displayed a considerable in vitro
biological effect, based on the observed antioxidant, anti-inflammatory and antimicrobial activities.
In comparison with protein isolates, protein hydrolysates performed better within the evaluated
parameters. However, more in depth research is needed in order to evaluate the effect of bioactive
peptides in baby lima bean proteins. Such peptides could be the source for notable biological activity
for nutraceutical applications.
ACKNOWLEDGMENTS
This work was supported by Dirección de Investigación y Desarrollo DIDE-Universidad Técnica de
Ambato (Project 2074-CU-P-2016 “Valorización de la Calidad Nutricional de Alimentos Tradicionales
de la Población Ecuatoriana VANFOOD”). This work have been reviewed in the English edition by
Max Long BA AAS CELTA (UNSIS EFL Instructor, tenured).
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bioRxiv preprint doi: https://doi.org/10.1101/401323; this version posted August 27, 2018.

The copyright holder for this preprint (which was

ANTIMICROBIAL, ANTIOXIDANT AND ANTI-INFLAMMATORY

J. TAMAYO1, T. POVEDA1, M. PAREDES1, G. VÁSQUEZ2, W. CALERO-CÁCERES3, 4*

*E-mail address: [email protected]

An increasing amount of literature demonstrates the biological properties of protein isolates of

2. MATERIALS AND METHODS

2.2. Protein isolates from baby lima beans

2.3. Protein hydrolysates

2.4. Antioxidant activities (TBARS assay)

2.5. Anti-inflammatory activity

2.6. Antimicrobial activity

2.7. Statistical analysis

3. RESULTS AND DISCUSSIONS

3.1. Preparation of protein isolates and hydrolizates

3.2. Antioxidant activity (TBARS method)

Isolated pH5 Protein Hydrolyzate BHT

100 ΜG/ML 200 ΜG/ML 500 ΜG/ML 1000 ΜG/ML

3.3. In-vitro anti-inflammatory activities

Isolated pH5 Protein Hydrolyzate Diclofenac Sodium

100 ΜG/ML 200 ΜG/ML 500 ΜG/ML 1000 ΜG/ML

3.4. Antimicrobial activity

Figure 3. Antimicrobial activity of the protein hydrolisate obtained at pH 5.

The values represent the average of 3 measurements ± the standard deviation.

Betancur-Ancona D, Martínez-Rosado R, Corona-Cruz A, Castellanos-Ruelas A, Jaramillo-Flores ME, Chel-Guerrero L,

Carrasco-Castilla J, Hernández-Álvarez AJ, Jiménez-Martínez C, Jacinto-Hernández C, Alaiz M, Girón-Calle J, Vioque J,

Nielsen SS, 2017. Food Analysis. Springer.

El Productor, 2015. Ecuador: La habichuela gana terreno. Available from: https://elproductor.com/noticias/ecuador-la-

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