JOURNAL OF MEDICINAL FOOD

J Med Food 00 (0) 2019, 1–6

# Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2019.0039

Bioactivity of Peptides Released During Lactic Fermentation of Amaranth Proteins

with Potential Cardiovascular Protective Effect:
An In Vitro Study
Alexis Ayala-Niño,1 Gabriela M. Rodrı́guez-Serrano,2
Ruben Jiménez-Alvarado,3 Mirandeli Bautista-Avila,4 José A. Sánchez-Franco,3
Luis G. González-Olivares,1 and Alberto Cepeda-Saez5
1
Chemical Research Center, Autonomous University of Hidalgo State, Mineral de la Reforma, Hidalgo, Mexico.
2
Biotechnology Department, Autonomous Metropolitan University, Iztapalapa Unit, Mexico City, Mexico.
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3
Post Harvest Laboratory, Autonomous University of Hidalgo State, Tulancingo, Hidalgo, Mexico.
4
Natural Products Pharmacology and Synthesis Laboratory, Autonomous University of Hidalgo State,
Pachuca-Actopan, Hidalgo, Mexico.
5
Food Hygiene, Inspection and Control Laboratory, University of Santiago de Compostela, Campus Lugo,
University Campus, Lugo, Spain.

ABSTRACT Fermentation has shown to be an effective technique in bioactive peptides release. That is why in this study
antihypertensive, antithrombotic, and antioxidant activity was evaluated during amaranth proteins fermentation with Lacto-
bacillus casei Shirota and Streptococcus thermophilus 54102 in mono and combined culture. During fermentation an increase
of free amine groups was observed, and no statistical differences among monocultures were shown, getting higher concen-
tration in combined culture. This was related to antihypertensive and antioxidant activities, where the highest values were also
found in the combined process (45% of angiotensin-converting enzyme inhibition, and 168 lmol Trolox equivalents per liter
[TE/L] for 2,2-diphenyl-1-picrylhydrazyl, 268 lmol TE/L for 2,20 -azino-bis 3-ethylbenzothiazoline-6-sulfonic acid, and 381 lmol
Fe2E/L for ferric reducing ability of plasma). On the contrary, antithrombotic activity was not related to free amine groups
during fermentation, having the highest bioactivity in different moments in each experiment. L. casei Shirota and S. thermophilus
54102 are strains that are able to release bioactive peptides from amaranth protein, although amaranth is not a common matrix
for the development of lactic acid bacteria. In addition, in this study it was observed for the first time that lactic acid strains
are able to release bioactive peptides from amaranth protein. In addition, this methodology could be part for the devel-
opment of fermented beverages, different from fermented milk, to diversify matrix to obtain a novel functional food.

KEYWORDS: acid lactic bacteria  amaranth protein  bioactive peptides  fermentation

INTRODUCTION drolysis has been reported2–5; however, the fermentation of

amaranth proteins or its bioactivity has never been reported.

A maranth is a valuable source of protein for human

nutrition with a good balanced profile of amino acids
and acceptable levels of lysine, tryptophan, and sulfur amino
Fermentation is a worldwide technique used in food
processing, which can contribute to microbial safety and
offer one or more organoleptic, technological, nutritional, or
acids and it could be a precursor for the release of bioactive health advantages; milk being one of the main sources for
peptides encrypted in native proteins, which reach values developing fermented foods.6 During fermentation, en-
between 15% and 18%.1 In this way, the release of antith- crypted biological active peptides can be released from their
rombotic, antihypertensive, and antioxidant peptides from inactive forms that are present in the structure of its pre-
amaranth proteins by in vitro digestion and enzymatic hy- cursor protein.7 The length and specific sequences of these
peptides depend on the precursor protein and the starter
bacteria, because the proteolytic system is inherent to each
bacterial strain.8 The most common starter bacteria are
Lactobacillus casei and Streptococcus thermophilus that
Manuscript received 25 February 2019. Revision accepted 9 April 2019.
have the capacity to release bioactive peptides from milk.9
Address correspondence to: Dr. Luis G. González-Olivares, PhD, Centro de Investigaciones This because they possess a proteolytic system composed of
Quı´micas, Universidad Autónoma del Estado de Hidalgo, Carretera Pachuca-Tulancingo
km 4.5, Mineral de la Reforma Hidalgo, C.P. 46067, México, E-mail: lgonzales@uaeh
proteinases, peptidases, and peptide transport systems that is
.edu.mx vital to bacterial nutrition.10

1
 2 AYALA-NIÑO ET AL.

Some of the activities that peptides have shown are: (i) phosphate solution (0.21 M; pH 8.2) in a 1% (v/v) concen-
antithrombotic, inhibiting thrombin from its interaction with tration. The reaction was carried out mixing 2 mL of TNBS
fibrin thereby avoiding the formation of blood clots11; (ii) solution, 2 mL of phosphate buffer, and 0.250 mL of sample
antihypertensive, inhibiting angiotensin-converting at 50C. After 60 min of reaction, 2 mL of HCl 0.1 N were
enzyme-I (ACE-I), leading to a decrease in angiotensin II added to stop it, and absorbance was read at 340 nm. Results
and a concomitant increase in the bradykinin level, yielding were obtained using glycine calibration line constructed
an overall reduction in blood pressure12; and (iii) antioxidant at different concentrations (0, 0.05. 0.1, 0.15, 0.2, and
activity, preventing the formation or scavaging free radicals 0.25 mg/mL). Results were expressed as milligrams of
and reactive oxygen species.13 free amine groups per liter (mg/L NH:-).
Therefore in this study, fermentation was investigated as
a means of producing ACE-I and thrombin inhibitors and In vitro bioactivity studies
antioxidant capability provided by peptides from amaranth Antihypertensive activity. Inhibitory effect of ACE-I
proteins. (EC 3.4.15.1; Sigma-Aldrich) was evaluated spectrophoto-
metrically according to Cushman et al.,16 with some modi-
MATERIALS AND METHODS fications. The substratum, hippuril–histidyl–leucine (HHL;
Sigma-Aldrich), was dissolved in sodium borate buffer
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Amaranth protein extraction (0.1 M, pH 8.3, with 0.3 M sodium chloride) at a concen-
Raw amaranth seeds were obtained from Xochimilco, tration of 5 mM substrate. Then 10 lL of ACE (EC 3.4.15.1,
Mexico City, in July 2016. Seeds were milled in a Chopin 5.1 U/mg; Sigma-Aldrich) was added to 100 lL substrate
mill, using semolina fraction (molecule size between 200 and 40 lL sample and the reaction took place for 75 min at
and 800 lm). Protein extraction was realized following the 37C. The formed hipuric acid was extracted with ethyl
methodology of Martı́nez and Añón.14 Defatted amaranth acetate, resuspended in deionized water, and measured at
flour was obtained with 10 g of flour/100 mL of n-hexane for 220 nm, in a Power Wave XS UV-Biotek (software KC
24 h. The hexane was retired by filtration. The obtained solid Junior, Kansas, MO, USA) spectrometer. The inhibitory
was resuspended in 10g/100mL deionized water and sub- activity of ACE was calculated using the formula:
mitted to an alkaline pH (9) with 2 M NaOH for 30 min to
% Inhibitory activity ¼ (AbsC  AbsM)=(AbsC  AbsB) · 100
extract the protein. Then the protein was precipitated
changing the pH to 5 with 2 M HCl. Finally, the obtained where AbsC is the formed hippuric acid during the action of
protein was taken to a neutral pH using NaOH solution. It ACE without inhibitor; AbsB, not reacted HHL that has
was lyophilized and stored at 4C until use. been extracted with ethyl acetate; and AbsM, hippuric acid
formed after the action of ACE in presence of inhibitory
Amaranth protein fermentation substance.
For the fermentation, protein and glucose solution were Antithrombotic capacity. Microplate methodology by
mixed in a ratio of 8:2, pasteurized separately, and mixed Zhang et al.17 was used to evaluate antithrombotic activity.
under sterile conditions before their inoculation. The protein Thrombin (150 IU/L; EC 3.4.15.1; Sigma Chemical Co.)
solution was prepared at 40 g/L and glucose solution at and fibrinogen (Sigma Chemical Co.) were prepared with
20 g/L. Deionized water was used to prepare them. Fer- Tris-HCl buffer (0.05 M, pH 7.2 with NaCl 0.15 M), in
mentation with L. casei Shirota and S. thermophilus 54102 concentrations of 12 IU/L and 0.10%, respectively. Eighty
occurred in monoculture and in combination for 36 h. The microliters of sample was mixed with 280 lL of fibrinogen
selected microorganisms were added to fermentation me- measuring its absorbance at 405 nm (sample bank [SB]).
dium under sterile conditions at a concentration of 6.5 log10 Then 20 lL of thrombin was added, and absorbance was
colony-forming unit (CFU)/mL for L. casei Shirota and 6.7 measured again after 10 min off reaction at 37C (S). The
log10 CFU/mL for S. thermophilus 54102 for monocultures percentage of inhibition (% inhibition) was calculated with
and 3.2 log10 CFU/mL and 3.3 log10 CFU/mL, respec- the following equation:
tively, in combined culture. Then the inoculated fermen-
tation medium was incubated at 37C for 36 h. During the %Inhibition ¼ [(C  CB)  (S  SB)]=(C  CB) · 100
process 1 mL of sample was taken every 2 h, was centri-
fuged and the supernatant was stored for hydrolysis grade where CB (control blank) is the initial absorbance of the
and bioactivity analysis. negative control of inhibition; C (control), the absorbance of
the negative control at 10 minutes of incubation with thrombin;
Hydrolysis grade measured SB (sample blank), the initial absorbance of the sample;
by 2,4,6-trinitrobenzenesulfonic acid test and S (sample), the absorbance of the sample at 10 min of
incubation with thrombin.
Sashidhar et al.15 method of 2,4,6-trinitrobenzenesulfonic
acid (TNBS, 5%; Sigma-Aldrich, St. Louis, MO, USA) was Antioxidant activity. Antioxidant activity was evaluated
used to measure hydrolysis grade as soluble peptide con- by three different methods using the corresponding cali-
centration during fermentation. TNBS was prepared in bration curves.
 AMARANTH PEPTIDES RELEASED DURING LACTIC FERMENTATION 3

ABTS test. Antiradical capacity was measured with solution was prepared using acetate buffer (0.3 M, pH 3.6),
the radical cation 2,20 -azino-bis 3-ethylbenzothiazoline-6- FeCl3 (20 mM in HCl 40 mM), and 2,4,6-tris(2pyridyl)-s-
sulfonic acid (ABTS+; Sigma-Aldrich). Seven millimo- triazine (10 mM in HCl 40 mM) in a ratio of 10:1:1. Thirty
lars per liter of ABTS was prepared with 2.45 mM/L of microliters of sample, 900 lL of FRAP solution, and 90 lL
persulfate of potassium (Sigma-Aldrich), was incubated of distilled water were mixed. At the same time, a standard
for 16 h in the dark at room temperature, and finally diluted curve was created by adding the FRAP reagent to a range of
until reaching an absorbance of 0.7 – 0.01. Nine hundred Fe2+ (FeSO4) solutions of known concentrations. Samples
eighty microliters of the diluted ABTS+ solution was absorbance was measured at 593 nm and the antioxidant ac-
incubated with 20 lL of sample at room temperature for tivity was expressed as micromole Fe(II) equivalents per
7 min, and absorbance was measured at 754 nm. The results 100 g [lmolFe(II)E/100 g].
were compared with a Trolox calibrating curve, and antioxi-
dant capacity was expressed as milligram Trolox equiva- Statistical analysis
lents per liter (mg TE/L).18 All values were obtained by triplicate and expressed as
DPPH test. Antiradical activity was measured using standard deviation of the mean. The data were analyzed by
2,2-diphenyl-1-picrylhydrazyl (DPPH; Sigma-Aldrich) one-way analysis of variance test, and differences among
mean values were compared using Tukey test with a level of
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radical, which was prepared at 0.07% with ethanol. One

hundred microliters of the sample was mixed with 500 lL of significance at P < .05, using the SPSS System for WINTM
version 15.0. All values are represented in Supplementary
DPPH solution and was left at room temperature for 1 h
Tables S1 and S2.
and their absorbance was measured at 520 nm in a micro-
plate reader. Trolox calibrating curve was built and results
were expressed as mg TE/L.18 RESULTS AND DISCUSSION
Proteolytic capacity
Ferric reducing ability of plasma test. Ferric reducing
ability of plasma (FRAP) antioxidant activity was evaluated Free amine groups concentration was evaluated to know
according to the methodology of Benzie and Strain.19 FRAP the hydrolytic degree during fermentation. The concentration

FIG. 1. Free amine group concentration.

 4 AYALA-NIÑO ET AL.

Table 1. Inhibition of Angiotensin-Converting In vitro bioactivity studies

Enzyme and Thrombin
Bioactivities of all the samples during fermentation were
ACE Thrombin measured. However, because of the amount of data obtained
Not fermented protein 8.17 – 1.40 4.87 – 2.43
only the highest bioactivity time reported are given in
Strain Time % of Time % of Tables 1 and 2, basing the discussion on these results.
(h) inhibition (h) inhibition
Lactobacillus 34 38.38 – 0.98ab 16 85.46 – 3.01b ACE and thrombin inhibition
casei Shirota
Streptococcus 32 36.14 – 1.32a 8 75.60 – 4.87a ACE inhibition has been tested in several studies where
thermophilus 54102 the main source of protein has been milk, finding inhibitory
Combined 36 45.22 – 0.28b 32 93.49 – 3.75c activity >90% in fermentations with L. casei Shirota YIT
9029.24 Our results showed (Table 1) that the highest in-
Different superscript letters indicate statistically significant differences
between processes. hibitory activity was observed when fermentation was car-
ACE, angiotensin-converting enzyme. ried out in combining both microorganisms at 36 h of
process (45.22 – 0.28). At the same time, these results are
related to the concentration of amine groups obtained at the
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of free amine groups during fermentation realized by L. casei same time, where a higher concentration of amino groups is
Shirota and S. thermophilus 54102 in monoculture and hav- combined with a higher antihypertensive activity. Different
ing a slightly higher concentration in L. casei process than authors have reported the relation between hydrolysis de-
that observed in S. thermophilus fermentation (1945 – 77.78 gree and higher antihypertensive activity, where smaller
and 1645 – 63.63 mg/L NH:-, respectively) is given in peptides show higher inhibition of ACE.25,26 In addition,
Figure 1. On the contrary, when fermentation was carried out fermented milk produced by mixing different strains of
in a combined culture, free amine groups reached the highest lactic acid bacteria might contain a wider variety of func-
concentration (4245 – 35.35 mg/L NH:-). tional substances than milk inoculated with a single strain27;
The free amine group concentration differences between those substances could be peptides released during fer-
both microorganisms could be related to particularities in mentation process, explaining the higher inhibition in
each proteolytic system. Cell wall-bound proteinase is dif- combined culture. The size, the amino acidic chain, and the
ferent in each bacteria. PrtP is inserted in Lactobacillus function of peptides produced are influenced by the fer-
system and PrtS is found in streptococci,20,21 each one re- mentation conditions and the protein source.28 These could
alize different cuttings determinate by their affinity. In ad- explain the low ACE-I activity in this study compared with
dition, the members of the PepE/PepG (endopeptidases) activity of milk-derived peptides. Also, another character-
and PepI/PepR/PepL (proline peptidases) superfamilies and istic of ACE-I peptides of milk is that they usually present a
enzymes related to bioactive peptides released are absent proline residue at terminal-C. In contrast, amaranth proteins
in streptococci.20–22 This explains the higher concentration present a low concentration of proline (4%) in their primary
of free amine groups in L. casei fermentation against those structure.29
observed in streptococci fermentation. In addition, it can be observed that the higher antith-
As has been mentioned, combined culture had the highest rombotic activity in each fermentation system was reached
concentration of free amine groups. In other studies it has at different moments (Table 1); whereas the higher antith-
been reported that S. thermophilus and different strains of rombotic activity was reached at hour 8 for S. thermophilus,
lactobacillus stimulate each other’s growth by the ex- it was reached at hour 16 for L. casei, and in the combined
change of metabolites such as peptides, folic acid, and system the higher bioactivity was shown at hour 32, show-
carbon dioxide,23 which may result in a higher proteolytic ing different behavior to that observed by antihyperten-
activity. In addition, during the enumeration of viable sive activity. In addition, during all fermentations increases
bacteria, combined culture showed the highest viability followed by diminishments of inhibition percentages were
(data not shown). observed, diversifying the percentages of inhibition at

Table 2. Antioxidant Activity

DPPH ABTS FRAP

Not fermented protein 35.43 – 3.71 34.61 – 1.46 132.05 – 2.45
Strain Time lmol TE/mL Time lmol TE/mL Time lmol Fe2/mL
L. casei Shirota 20 109.9 – 7.3a 20 194.5 – 5.9b 20 316.3 – 35.1b
S. thermophilus 54102 20 104.1 – 9.7a 34 103.9 – 10a 10 225.6 – 19.6a
Combined culture 26 168.1 – 5.7b 26 268.4 – 11.8c 26 381.3 – 0.6c

Different superscript letters indicate statistically significant differences between processes.

DPPH, 2,2-diphenyl-1-picrylhydrazyl; FRAP, ferric reducing ability of plasma; ABTS, 2,20 -azino-bis 3-ethylbenzothiazoline-6-sulfonic acid; TE/mL, Trolox equivalents
per milliliter.
 AMARANTH PEPTIDES RELEASED DURING LACTIC FERMENTATION 5

different times. Pérez-Escalante et al.30 showed similar be- also a source of bioactive peptides, making it a new option
havior of antithrombotic activity during milk fermentation for the release of bioactive peptides that may prevent or treat
with L. casei. This can be explained by the specificity of cardiovascular diseases.
proteolytic system of each strain, which leads to the release
of different peptide structure.20–22 The proteolytic system AUTHOR DISCLOSURE STATEMENT
has particularities between lactic acid bacteria, which could
result in appearance and disappearance of some peptides No competing financial interests exist.
with inhibitory thrombin activity.31 This behavior has been
explained by Gasson and de Vos31 and it was called cascade SUPPLEMENTARY MATERIAL
pattern. The main differences are included in the transport Supplementary Table S1
systems of di- and tripeptide (Dpp and DtpT in Lactobacillus Supplementary Table S2
and Dpp in Streptococcus), and the oligopeptide system
(Opp system in Lactobacillus and Ami system in Strepto-
REFERENCES
coccus).32–34 Also, antithrombotic activity is related to its
structure rather than to its size, which might be related to 1. Barba de la Rosa AP, Gueguen J, Paredes-López O, Viroben G:
its affinity with thrombin active site.30 Fractionation procedures, electrophoretic characterization and
Downloaded by Mary Ann Liebert, Inc., publishers from www.liebertpub.com at 07/03/19. For personal use only.

amino acid composition of amaranth seed proteins. J Agric Food

Antioxidant activity Chem 1992;40:931–936.
2. Tovar-Pérez EG, Guerrero-Legarreta I, Farrés-González A,
In Table 2 antioxidant activity by DPPH, ABTS, and Soriano-Santos J: Angiotensin I converting enzyme inhibitory
FRAP is observed. Combined culture showed the highest peptide fractions from albumin 1 and globulin as obtained of
antioxidant activity in the three assays. In addition, it can be amaranth gran. Food Chem 2009;116:437–444.
observed that L. casei Shirota and combined fermentation 3. Orsini-Delgado MC, Tironi VA, Añón MC: Antioxidant activity
showed the highest antioxidant activity at the same time in of amaranth protein or their hydrolysates under simulated gas-
the three activities (hour 20 and 26, respectively), whereas S. trointestinal digestion. J Food Sci Technol 2011;44:1752–1760.
thermophilus 54102 fermentation had different maximum 4. Orsini-Delgado MC, Nardo A, Pavlovic M, Rogniaux H, Añón
antioxidant activity at different times. As in the antith- MC, Torini VA: Identification and characterization of antioxidant
rombotic activity, an increase of antioxidant activity fol- peptides obtained by gastrointestinal digestion of amaranth pro-
lowed by a diminishment was observed during fermentation. teins. Food Chem 2016;197:1160–1167.
These results correspond with those reported by Ha et al.34 5. Sabbione AC, Scilingo A, Añón MC: Potential antithrombotic
and Zou et al.35 They described that antioxidant activity is activity detected in amaranth proteins and its hydrolysates.
dependent on factors like molecular weight, amino acidic J Food Sci Technol 2015;60:171–177.
composition and sequence, having the higher activity in 6. Leroy F, De Vuyst L: Lactic acid bacteria as functional starter
peptides with >20 amino acid residues (molecular weight cultures for the food fermentation industry. Trends Food Sci
Technol 2004;15:67–78.
<3000 Da). They obtained the higher antioxidant activity in
7. Agyei D, Danquah MK: Industrial-scale manufacturing of
samples with greater amount of free amine groups. How-
pharmaceutical-grade bioactive peptides. Biotechnol Adv 2011;29:
ever, Ren et al.36 reported higher scavenging activity by 272–277.
peptides with molecular weight >4000 Da. The presence of 8. Li Z, Jiang A, Yue T, Wang J, Wang Y, Su J: Purification and
different peptides that act by different mechanisms and the identification of five novel antioxidant peptides from goat milk
influence of the proteolytic system (cutting by aminopepti- casein hydrolysates. J Dairy Sci 2013;96:4242–4251.
dases localized inside cells) explain the differences ob- 9. Inoue K, Shirai T, Ochiai H, Kasao M, Hayakawa K, Kimura M,
served in the antioxidant activities evaluated.37,38 In Sansawa H: Blood-pressure-lowering effect of a novel fermented
addition, it has been found that free amine acids may en- milk containing c-aminobutyric acid (GABA) in mild hyperten-
hance antioxidant activity, such as nucleophilic amine acids sives. Eur J Clin Nutr 2003;57:490–495.
with sulfur (Cys and Met), and those with aromatic rings 10. Hebert EM, Raya RR, Savoy de Giori G: Nutritional require-
(Trp, Tyr, and Phe) or imidazole (His).38 ments and nitrogen-dependent regulation of proteinase activity of
Lactobacillus helveticus CRL 1062. Appl Environ Microbiol
Conclusions 2000;66:5316–5321.
11. Scheraga HA: The thrombin-fibrinogen interaction. Biophys
Amaranth proteins are potential source to release peptides Chem 2004;112:117–130.
with the use of acid lactic bacteria with antihypertensive, 12. Donkor ON, Henriksson A, Singh TK, Vasiljevic T, Shah NP:
antithrombotic, and antioxidant activity. A fermentation ACE inhibitory probiotic yoghurt. Int Dairy J 2007;17:1321–1331.
with combined culture in specific with L. casei Shirota and 13. Chang OK, Seol KH, Jeong SG, Oh MH, Park BY, Perrin C,
S. thermophilus 54102 reach higher bioactivities (antioxi- Ham JS: Casein hydrolysis by Bifidobacterium longum
dant, antihypertensive, and antithrombotic) than the same KACC91563 and antioxidant activities of peptides derived
strains in monocultures, concluding that there exists a re- therefrom. J Dairy Sci 2013;96:5544–5555.
lation between the symbiotic cooperation during fermenta- 14. Martı́nez NE, Añón CM: Composition and structural character-
tion and the protein source. This shows amaranth protein as ization of amaranth protein isolates: An electrophoretic and ca-
a new matrix for lactic acid bacteria proliferation, which is lorimetric study. J Agric Food Chem 1996;44:2523–2530.
 6 AYALA-NIÑO ET AL.

15. Sashidhar RB, Capoor AK, Ramana D: Quantification of amino 27. Elkhtab E, El-Alfy M, Shennana M, Mohamed A, Yousef A:
group using amino acids as reference standards by trini- New potentially antihypertensive peptides liberated in milk
trobenzene sulfonic acid: A simple spectrophotometric method during fermentation with selected lactic acid bacteria and ko-
for the estimation of hapten to carrier protein ratio. J Immunol bucha cultures. J Dairy Sci 2017;100:9508–9520.
Methods 1995;167:121–127. 28. Toldrá F, Reig M, Aristoy MC, Mora L: Generation of bioactive
16. Cushman DW, Cheung HB, Sabo EF, Ondetti MA: Design of peptides during food processing. Food Chem 2018;267:395–404.
potent competitive inhibitors of angiotensin converting enzyme. 29. Pı́sařı́ková B, Krácmar S, Herzig I: Amino acids contents and
Carboxylalkanoyl and mercaptoalkanoyl aminoacids. Biochem- biological value of protein in various amaranth species. Czech J
istry 1977;16:5484–5491. Anim Sci 2005;50:169–174.
17. Zhang BS, Zhang W, Xu SY: Antioxidant and antithrombotic ac- 30. Pérez-Escalante E, Jaimez-Ordaz J, Castañeda-Ovando A,
tivity of raoeseed peptides. J Am Oil Chem Soc 2008;85:521–527. Contreras-López E, Añorve-Morga J, Gónzalez-Livares LG:
18. Delgado-Andrade C, Rufian-Henares JA, Morales FJ: Assessing Antithrombotic activity of milk protein hydrolysates by lactic
the antioxidant activity of melanoidins from coffee brews by acid bacteria isolated from commercial fermented milks. Braz
different antioxidant methods. J Agric Food Chem 2005;53: Arch Biol Technol 2018;61:1–10.
7832–7836. 31. Gasson MJ, de Vos WM: The proteolytic system of lactic
19. Benzie I, Strain J: The ferric reducing ability of plasma (FRAP) acid bacteria. In: Genetics and Biotechnology of Lactic Acid
as a measure if ‘‘Antioxidant power.’’ The FRAP assay. Anal Bacteria, Chapter 4 (Gasson MJ, de Vos WM, eds.). London,
Downloaded by Mary Ann Liebert, Inc., publishers from www.liebertpub.com at 07/03/19. For personal use only.

Biochem 1996;239:70–76. United Kingdom: Blackie Academic and Professional/Chapman

20. Siezen RJ, Renckens B, van Swam I, Peters S, van Kranenburg and Hall, 1994, pp. 169–210.
R, Kleerebezem M, de Vos WM: Complete sequences of four 32. Kunji ER, Mierau I, Hagting A, Poolman B, Konings WN: The
plasmids of Lactococcus lactis subsp. cremoris SK11 reveal proteolytic systems of lactic acid bacteria. Antonie Van Leeu-
extensive adaptation to the dairy environment. Appl Environ wenhoek 1996;70:187–221.
Microbiol 2005;71:8371–8382. 33. Hols P, Hancy F, Fontaine L, Grossiord B, Prozzi D, Leblond-
21. Rodrı́guez-Serrano GM, Garcı́a-Garibay M, Cruz-Guerrero AE, Bourget N, Decaris B, Bolotin A, Delorme C, Guédon SDEE,
Gómez-Ruiz L, Ayala-Niño A, Castañeda-Ovando A, González- Monnet V, Renault P, Kleerebezem M: New insight in the mo-
Olivares LG: Proteolytic system of Streptococcus thermophilus. lecular biology and physiology of Streptococcus thermophilus
J Microbiol Biotechnol 2018;28:1581–1588. revealed by comparative genomics. FEMS Microbiol Rev 2005;
22. Liu M, Bayjanov RJ, Renckens B, Nauta A, Siezen RJ: The 29:435–463.
proteolytic system of lactic acid bacteria revisited: A genomic 34. Ha GE, Chang OK, Han GS, Ham JS, Partk BY, Jeong SG:
comparison. BMC Med Genomics 2010;11:36–41. Comparison of antioxidant activities of hydrolysates of domestic
23. Sander-Sieuwerts S, Molenaar D, van Hijum SAFT, Beerthuyzen and imported skim Milk powder treated with papain. J Food Sci
M, Stevens MJA, Janssen PWM, Ingham CJ, de Bok FAM, de Anim Resour 2015;35:360–369.
Vos WM, van Hylckama-Vlieg JET: Mixed-culture tran- 35. Zou TB, He TP, Li HB, Tang HW, Xia WQ: The structure-
scriptome analysis reveals the molecular basis of mixed-culture activity relationship of the antioxidant peptides from natural
growth in Streptococcus thermophilus and Lactobacillus bul- proteins. Molecules 2016;12:1–14.
garicus. Appl Environ Microbiol 2010;76:7775–7784. 36. Ren J, Zhao M, Shi J, Wang J, Jiang Y, Cui C, Kakuda Y, Xue
24. González-González CR, Tuohy KM, Jauregi P: Production of SJ: Purification and identification of antioxidant peptides from
angiotensin-I-converting enzyme (ACE) inhibitory activity in grass carp muscle hydrolysates by consecutive chromatography
milk fermented with probiotic strains: Effects of calcium, pH and electrospray ionization-mass spectrometry. Food Chem
and peptides on the ACE-inhibitory activity. Int Dairy J 2011; 2008;108:727–736.
21:615–622. 37. Virtanen T, Pihlanto A, Akkanen S, Korhonen H: Development
25. Korhonen H, Pihlanto A: Bioactive peptides: Production and of antioxidant activity in milk whey during fermentation with
functionality. Int Dairy J 2006;16:945–960. lactic acid bacteria. J Appl Microbiol 2007;102:106–115.
26. Lee SY, Hur SJ: Antihypertensive peptides from animal products, 38. Sarmadi BH, Ismail A: Antioxidant peptides from food proteins:
marine organisms, and plants. Food Chem 2017;228:506–517. A review. Peptides 2010;31:1949–1956.