Enriching antimicrobial peptides from milk hydrolysates using pectin/ alginate food-gels

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Food Chemistry 352 (2021) 129220

Available online 9 February 2021

Enriching antimicrobial peptides from milk hydrolysates using pectin/

alginate food-gels

Jounghyun Um

, Jean Manguy

, Jo˜

ao Anes

, Jean-Christophe Jacquier

, Daniel Hurley

Eugene T. Dillon

, Kieran Wynne

, S´

eamus Fanning

, Michael O’Sullivan

, Denis C. Shields

Conway Institute of Biomolecular and Biomedical Research, School of Medicine, University College Dublin, Dublin, Ireland

UCD-Centre for Food Safety, School of Public Health, Physiotherapy and Population Science, University College Dublin, Dublin, Ireland

UCD Institute of Food and Health, School of Agriculture and Food Science, University College Dublin, Dublin 4, Ireland

UCD-Centre for Food Safety, School of Agriculture and Food Science, University College Dublin, Dublin 4, Ireland

Mass Spectrometry Resource, Conway Institute of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland

ARTICLE INFO

Keywords:

Antimicrobial peptide

Gel enrichment

Pectin

Alginate

Casein

Hydrolysate

Bioinformatics

Peptidomics

ABSTRACT

Cationic antimicrobial peptides have raised interest as attractive alternatives to classical antibiotics, and also

have utility in preventing food spoilage. We set out to enrich cationic antimicrobial peptides from milk hydro-

lysates using gels containing various ratios of anionic pectin/alginate. All processes were carried out with food-

grade materials in order to suggest food-safe methods suited for producing food ingredients or supplements.

Hydrolysed caseinate peptides retained in the gel fraction, identied by mass spectrometry, were enriched for

potential antimicrobial peptides, as judged by a computational predictor of antimicrobial activity. Peptides

retained in a 60:40 pectin:alginate gel fraction had a strong antimicrobial effect against 8 tested bacterial strains

with a minimal inhibitory concentration of 1.5–5 mg/mL, while the unfractionated hydrolysate only had a

detectable effect in one of the eight strains. Among 110 predicted antimicrobial peptides in the gel fraction, four

are known antimicrobial peptides, HKEMPFPK, TTMPLW, YYQQKPVA and AVPYPQR. These results highlight the

potential of pectin/alginate food-gels based processes as safe, fast, cost-effective methods to separate and enrich

for antimicrobial peptides from complex food protein hydrolysates.

1. Introduction

Milk proteins are a rich source of bioactive peptides and enzymatic

hydrolysis is the most common way to release these peptides which are

normally inactive within the mother protein (Meisel & FitzGerald, 2003;

Phelan, Aherne-Bruce, O’Sullivan, FitzGerald, & O’Brien, 2009). These

peptides perform a wide range of activities, including microbial inhi-

bition, angiotensin converting enzyme (ACE) inhibition, immunomo-

dulation, mineral binding, opioid activities, antithrombotic and

antioxidative functions (Sibel Akalın, 2014; Miguel, Recio, Ramos,

Delgado, & Aleixandre, 2006; Nongonierma & FitzGerald, 2016; Quir´

et al., 2007; Rival, Boeriu, & Wichers, 2001; Robert, Razaname, Mutter,

& Juillerat, 2004; Saito, Nakamura, Kitazawa, Kawai, & Itoh, 2000).

Functional milk peptides are suited for applications as added food in-

gredients, supplements, or drugs, as they likely have few negative side

effects due to the evolution of milk as food for the mammalian neonate

(Nielsen, Beverly, Qu, & Dallas, 2017). In particular, there is an

increasing interest in milk-derived antimicrobial peptides as they offer

advantages over traditional small molecule antibiotics due to their low

toxicity, structural diversity and absence or low levels of accumulation

in body tissues (Agyei & Danquah, 2011). Antimicrobial peptides

(AMPs) can be also used in the food industry as a natural tool to control

undesirable bacteria in food grade products designed for extended

storage stability, reducing the risk of food-borne illness and spoilage

without the use of chemical additives (Benkerroum, 2010; Sedaghati,

Ezzatpanah, Boojar, Ebrahimi, & Kobarfard, 2016). However, AMPs are

present in milk protein hydrolysates as a small component of complex

mixtures and further concentration or purication steps might be

required to increase their potency. Many AMPs are cationic amphipathic

peptides, containing a high number of cationic Arg, Lys or His residues

(Dziuba & Dziuba, 2014; Mohanty, Mohapatra, Misra, & Sahu, 2016).

The cationic residues interact with the anionic head groups of the mi-

crobial membrane lipids, sometimes generating pores which lead to cell

leakage (Chang, McLandsborough, & McClements, 2011; Peschel &

* Corresponding authors.

E-mail addresses: Michael.osullivan@ucd.ie (M. O’Sullivan), denis.shields@ucd.ie (D.C. Shields).

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier.com/locate/foodchem

https://doi.org/10.1016/j.foodchem.2021.129220

Received 18 September 2020; Received in revised form 22 January 2021; Accepted 22 January 2021

Food Chemistry 352 (2021) 129220

Sahl, 2006). Since AMPs are often cationic in nature, it is possible that

food grade, anionic biopolymers - such as the gel-forming poly-

saccharides alginate, pectin or carrageenan - may be exploited for their

enrichment from complex hydrolysates. Alginate is a naturally occurring

anionic polysaccharide comprised of guluronic and manuronic acids

that are typically obtained from brown seaweed, and has been used for

many applications due to its low toxicity, relatively low cost, and mild

gelation conditions, i.e. by addition of divalent cations such as Ca

(Lee

& Mooney, 2012). Pectin is an anionic polymer that occurs as a struc-

tural material in all land-growing plants (Harris, 2012). Pectin is a linear

chain of

-(1,4)-linked galacturonic acid residues with varying degrees

of methyl esterication (DM), which forms a gel in acidic media in the

presence of sugars (high DM pectin) or by interacting with Ca

ions

(low DM pectin) (Fraeye et al., 2010). Both are potentially useful

because they are anionic and widely used within the food industry.

There are two potential benets to using food gels for binding antibac-

terial peptides. Firstly, the alternatives, such as chemical synthesis of

antibacterial peptides or purication by preparative chromatography,

are expensive and inefcient (Elbarbary, Abdou, Nakamura, Park,

Mohamed, & Sato, 2012; Jaskiewicz, Orlowska, Olizarowicz, Migon,

Grzywacz, & Kamysz, 2016). Secondly, the cationic nature of antibac-

terial peptides may cause problems during storage in some food and

beverage systems, such as cloudiness arising from precipitation with

other food components (Chang et al., 2011), so that formulating the

peptides with a gel is likely to stabilise the peptides within the food.

In this study, we hypothesised that it would be possible to develop

food-grade gels to enrich cationic peptides from food hydrolysates, that

would display enhanced antimicrobial activity.

2. Materials and methods

The protein substrate used in the enzymatic hydrolysis was Sodium

caseinate (90% w/w protein). It was obtained from Armor Proteins (19

bis rue de la Liberation, 35,460 Saint-Brice-en-Cogles, France). The

enzymes used in the enzymatic hydrolysis of the sodium caseinate were

acid stable protease (EC # 3.4.23.18), alkaline protease (EC #

3.4.21.62), bromelain (EC # 3.4.22.32), cin (EC # 3.4.22.3), fungal

protease (EC # 3.4.23.18), neutral protease (EC # 3.4.24.28) and papain

(EC # 3.4.22.2). Ficin was obtained from Sigma-Aldrich (Ireland). The

other enzymes were obtained from BIO-CAT (9117 Three Notch Road

Troy, VA 22974, USA). The enzymes were supplied as dry powders and

were stored at 4 ◦C. Alginates (RF6650, GP5450, and LF10/60) were

obtained from FMC, Walnut Street, Philadelphia, USA. Apple pectin

powder was obtained from Solgar (500 Willow Tree Road, Leonia, NJ

07605, USA). Müeller-Hinton (MH) broth, Brain heart infusion (BHI)

broth, and phosphate buffered saline (PBS) were obtained from Sigma-

Aldrich (Ireland). The chemicals used in this research project were of

general purpose reagent grade or of higher quality. These were all

supplied by Sigma-Aldrich, Vale Road, Arklow, Wicklow, Ireland.

2.1. Preparation of Casein hydrolysates

A 10% [w/w] (protein basis) sodium caseinate (NaCas) substrate

solution was prepared in a beaker by weighing 22.2 g NaCas and dis-

solving it in deionised water bringing the nal solution weight to 200 g.

The NaCas was added gradually to the water and was stirred for 2 h until

the powder had fully dissolved. The solution was then stored in a fridge

overnight at 4 ◦C. The following day, the solution was equilibrated to

50 ◦C and the pH adjusted to 7 by adding 1 M NaOH. The ratio of enzyme

to substrate was 0.25 g enzyme per 100 g NaCas. Dry enzyme powder

(50 mg) was accurately weighed and was then dissolved in 1 mL of

deionised water, and immediately added to the substrate. The enzyme/

substrate mix was incubated at 50 ◦C for 5 h in a shaking water bath

under gentle agitation (100 RPM). After incubation, the solution was

heated to 85 ◦C for 10 min to inactivate the enzyme. Then the solution

was freeze-dried using a Super Modulyo freeze dryer (Edwards, UK) and

subsequently stored at −20 ◦C prior to use.

2.2. Preparation of food gels

Mixed solutions (2% [w/v]) of pectin (P) and alginate (A) in distilled

water, having P:A ratios of 0:100, 20:80, 40:60, and 60:40, were pre-

pared by stirring at room temperature for ~ 4 h to ensure full dispersion.

The resulting solution stood overnight at 4 ◦C to facilitate the release of

the air incorporated during mixing. Beads were manufactured by

extruding the solution using a peristaltic pump, drop-wise from 10 cm

high through a 200

L pipette tip into 300 mL of a 0.1 M CaCl

solution.

The beads were cured for 30 min in the CaCl

solution, washed 3 times

with distilled water and immersed for 2 h in 300 mL of distilled water.

The resulting gel beads were kept at 4 ◦C in 100 mL of distilled water for

further experiments.

2.3. Fractionation of the hydrolysate using a food-gel

Samples (2 g) of freeze-dried hydrolysates were dissolved in 100 mL

of deionized water. The pH was adjusted to 7 or 9 with 1 M NaOH. Gel

beads (50 g) were added to the hydrolysate solution and stirred at 300

rpm for 1.5 h to allow peptide binding to take place. The solution was

then ltered through a 2 mm sieve in order to separate the gel fraction

(GF) from the solution. After manually removing gel beads, the super-

natant fraction (SF) was kept to compare properties with both the GF

and the unfractionated hydrolysate. An aliquot (50 mL) of EDTA solu-

tion (0.2 M, pH 8) was poured into the recovered gels and mixed until

the gels were completely dissolved. The dissolved gel / EDTA solution

was then mixed with an equal volume of 99.8% [v/v] EtOH to precip-

itate the alginate and ltered through a lter paper (Grade 1, Whatman)

to separate the GF peptides from the polysaccharides.

2.4. Purication

The GF ltered through a lter paper was evaporated at 40 ◦C using

rotary evaporator (Rotavapor RII, Buchi, Switzerland) in order to

remove EtOH until the solution volume was reduced by half. An aliquot

(40 mL) of the solution was loaded onto C18 Solid Phase Extraction

columns (phenomenex Strata

-XL 100

m Polymeric Reversed Phase

10 g/60 mL Giga tubes) which had previously been washed rstly with

solution B (65% [v/v] acetonitrile, 35% [v/v] milliQ water, 0.1% [v/v]

TFA) and subsequently with solution A (2% [v/v] Acetonitrile, 98% [v/

v] milliQ water, 0.1% [v/v] TFA). Following sample loading, the column

was washed with 80 mL of solution A and adsorbed peptides subse-

quently eluted by 40 mL of solution B. The eluate was divided into 10 mL

aliquots in tubes. And these were subsequently evaporated to dryness

using a vacuum evaporator (miVac Duo concentrator, Genevac, UK).

The dried peptides were kept at −20 ◦C prior to further analysis. The

supernatant fraction was puried, dried and stored using the same

method as described above.

2.5. Peptide identication

Aliquots (2

g) of puried samples were re-suspended in 4

L of 2%

[v/v] acetonitrile (ACN), 0.05% Triuoroacetic acid (TFA) solution and

analysed on quadrupole Orbitrap (Q-Exactive, Thermo Scientic) mass

spectrometers equipped with a reversed-phase NanoLC UltiMate 3000

HPLC system (Dionex LC Packings, now Thermo Scientic). Peptide

samples were loaded onto C18 reversed phase columns (50 cm length,

75 µm inner diameter, 2 µm particles) and eluted with a linear gradient

from 10 to 40% B gradient (A: 0.1% [v/v] formic acid (FA), 3% [v/v]

acetonitrile; B: 0.1% [w/v] FA, 80% [v/v] acetonitrile) in 30 min at a

ow rate of 0.3

L/min. The injection volume was 1.5

L. The mass

spectrometer was operated in data dependent mode, automatically

switching between MS and MS2 acquisition. Survey full scan MS spectra

(m/z 300 – 1600) were acquired in the Orbitrap with a resolution of

J. Um et al.

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