Characterization of antimicrobial peptides produced by Lactobacillus acidophilus LA-5 and Bifidobacterium lactis BB-12 and their inhibitory effect against foodborne pathogens

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LWT - Food Science and Technology 153 (2022) 112449

Available online 11 September 2021

Characterization of antimicrobial peptides produced by Lactobacillus

acidophilus LA-5 and Bidobacterium lactis BB-12 and their inhibitory effect

against foodborne pathogens

Saber Amiri

, Reza Rezaei Mokarram

, Mahmoud Sowti Khiabani

Mahmoud Rezazadeh Bari

, Mohammad Alizadeh Khaledabad

Department of Food Science and Technology, Faculty of Agriculture, Urmia University, Urmia, Iran

Department of Food Science and Technology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

ARTICLE INFO

Keywords:

Antimicrobial peptides

Commercial probiotics

Dairy by-product

Optimization

Characterization

ABSTRACT

This study aimed to optimize the production of antimicrobial peptides (AMPs) by pure and mixed cultures of

Lactobacillus acidophilus LA-5 and Bidobacterium lactis BB-12 in milk model media and then characterize the

properties of produced AMPs. The optimal conditions for AMPs bio-production by L. acidophilus LA-5, B. lactis BB-

12, and their mixed culture were temperatures of 38.71, 42.00, and 39.00 ◦C, fermentation times of 26.15 h,

60.00 h, and 36.00 h, and yeast extract concentrations of 4.45%, 6.00%, and 5.00% in cheese whey. At optimal

conditions, the inhibition activities of AMPs against L. monocytogenes were 14.03 ±0.68 mm, 13.33 ±0.81 mm,

and 11.02 ±1.04 mm by L. acidophilus LA-5, B. lactis BB-12, and their mixed culture, respectively. The SDS-page

analysis indicated that L. acidophilus LA-5 and B. lactis BB-12 produced AMPs with two and three fractions,

respectively. The Fourier-transform infrared spectroscopy and nuclear magnetic resonance tests showed the

typical characteristics of a peptide for produced AMPs. The DSC test indicated that produced AMPs had high-

temperature stability. These AMPs were bioactive in a wide range of pH (3–9) and temperature (40–100 ◦C

for 30 min). The results of minimum inhibitory concentration and minimum bactericidal concentration showed

that the AMPs had antimicrobial effects against important food-borne pathogens.

1. Introduction

In the recent decade, the application of bio-preservatives in the food

industry is progressively increasing due to the consumers’ interests.

Probiotic bacteria and their pharmabiotic metabolite, bacteriocins

(BACs), are the most commonly-used bio-preservatives in different

foodstuffs (Amiri, Mokarram, Khiabani, Bari, & Alizadeh, 2020). BACs

are ribosomally bio-synthesized extracellular antimicrobial peptides.

These metabolites have antagonistic activity against bacteria with ge-

netic similarity to the producing bacteria, although the producer bac-

teria are immune to their own BACs (Zou, Jiang, Cheng, Fang, & Huang,

2018). These antimicrobial peptides (AMPs) are synthesized by

Gram-positive (G

) or Gram-negative (G

−

) bacteria, as well as by certain

probiotics. BACs have an extensive spectrum of antibacterial activities

and are safe replacements of antibiotics. In the last two decades, BACs

have received increasing interests as natural food bio-preservatives and

are used in food products for biological control of spoilage and patho-

genic bacteria (Wannun, Piwat, & Teanpaisan, 2016; Zhou, Zeng, Han,

& Liu, 2015). Furthermore, BACs have been considered as bioactive

molecules with potential activities on human health, such as antiviral

agents and anticancer agents, in current years (Amiri, Moghanjougi,

Bari, & Khaneghah, 2021; Chikindas, Weeks, Drider, Chistyakov, &

Dicks, 2018; Kaur & Kaur, 2015).

Lactobacilli and Bidobacteria are two common commercial probiotic

genera with widespread usage in functional foods and are the main

microbiota of the humans’ intestine. Lactobacillus acidophilus is a G

short rod-shaped, homo-fermentative, and microaerophilic bacterium.

Bidobacterium lactis is a G

, rod-shaped, non-gas-producing, non-

motile, catalase-negative, and anaerobic bacterium (Amiri et al., 2020,

pp. 53–79).

Different agro-industrial residuals, especially dairy efuents, are

used as a microbial culture medium rather than expensive cultivation

* Corresponding author.

** Corresponding author.

E-mail addresses: sa.amiri@urmia.ac.ir (S. Amiri), rmokarram@tabrizu.ac.ir (R. Rezaei Mokarram).

Contents lists available at ScienceDirect

LWT

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

https://doi.org/10.1016/j.lwt.2021.112449

Received 21 October 2020; Received in revised form 12 June 2021; Accepted 9 September 2021

LWT 153 (2022) 112449

media for microbial fermentation (Pescuma, de Valdez, & Mozzi, 2015).

Whey and permeate are the main dairy efuents that remain in tradi-

tional cheeses and ultra-ltered white cheese producing processes,

respectively. These low-cost wastes are available in large quantities and

can be used as a carbon source due to their high concentrations of

lactose (Amado, V´

azquez, Pastrana, & Teixeira, 2016).

Production of BACs has been investigated by different probiotics

(Engelhardt, Szakm´

ar, Kisko, Mohacsi-Farkas, & Reichart, 2018; de

Lima, de Moura; Fernandes; Cardarelli, 2017; Salman et al., 2020;

Schirru et al., 2014; Ünlü, Nielsen, & Ionita, 2015). However, there is no

report about the bio-production of AMPs by L. acidophilus LA-5, B. lactis

BB-12, and their mixed culture. Therefore, the present study aims to

show that 1) new microbial interactions can result in interesting product

characteristics, and 2) novel substrates can be selected for fermentative

processing with the use of microorganisms known from traditional

fermentation processes. The latter concept is called ‘cross-over fermen-

tation’, in which a microorganism is taken from a traditional fermen-

tation process and is introduced to a new substrate and/or to a new

microbial partner in a mixed culture (Dank et al., 2021). This study

demonstrates that the culture of L. acidophilus LA-5 in a new medium

with B. lactis BB-12 can lead to the production of a new fermentation

product with new and strong antimicrobial properties.

2. Materials and methods

2.1. Probiotic bacteria

The probiotics (LA-5 and BB-12) were obtained from Chr. Hansen,

DK-2970 Hørsholm, Denmark and weighted according to the manufac-

turer’s recommendation. LA-5 was grown in de Man Rogosa and Sharpe

(MRS) broth (Merck, Darmstadt, Germany) with 0.1% tween 80

(AppliChem, Darmstadt, Germany) at 37 ◦C for 24 h. BB-12 was grown

in MRS broth with 0.1% tween 80, 0.05% L-cysteine (AppliChem,

Darmstadt, Germany), and 0.1% lithium chloride (Sigma-Aldrich, St.

Louise, Missouri, USA) in the same condition. After that, the cell cultures

were centrifuged at 5000×g for 15 min and washed twice in 0.85% w/v

NaCl solution. The pellet was resuspended in a normal saline solution to

obtain a suspension containing approximately 10

CFU/mL of pro-

biotics (Amiri, Rezazadeh-Bari, Alizadeh-Khaledabad,

Rezaei-Mokarram, & Sowti-Khiabani, 2021; Amiri et al., 2020, pp.

53–79).

2.2. Preparation of cultivation media

Fresh cheese whey and milk permeate were purchased from Sahar

and Aynaz dairy industries (local dairy plants in Urmia City, Iran),

respectively. First, the antibacterial activity of cheese whey and milk

permeate was determined by indicator strains to conrm the absence of

antibacterial metabolites in the growth matrix. Then, the pH of cheese

whey and milk permeate was adjusted to 4.5 by 5N HCl, both were

heated at 121 ◦C for 15 min, and then a centrifuge separated the pre-

cipitates at 2360×g for 5 min. After that, the pH was adjusted based on

the experimental design and autoclaved (121 ◦C, 15 min) afterward.

Next, yeast extract (Sigma-Aldrich, St. Louise, USA) and linoleic acid

with 99% purity (Sigma-Aldrich, St. Louise, USA) were added depending

on the statistical design using a syringe lter (pore size =0.45

m). The

fermentation bioprocess was done in 100 mL asks inoculated by pro-

biotics (10

CFU/mL). Finally, they were incubated at different tem-

peratures and time conditions (Amiri et al., 2020) according to the

experimental design. Table 1 shows the composition and characteristics

of cheese whey and milk permeate after preparation as cultivation

media.

2.3. Partial purication and inhibitory activity of AMPs

For this purpose, cell-free supernatant was achieved by the centri-

fugation of the cultivation medium at 7000×g at 4 ◦C for 30 min. Then,

the cell-free supernatant was partially puried using gradient salt pre-

cipitation by 20, 40, 60, and 80% ammonium sulfate at 4 ◦C. After that,

the precipitates were collected by a centrifuge at 10,000×g at 4 ◦C for

30 min. The pellets (AMPs) were liqueed in potassium phosphate

buffer (50 mM, pH 7.0) and the AMPs’ suspension was dialyzed against

distilled water at 4 ◦C using a dialysis bag with 14 kDa molecular weight

cut off (Sigma Aldrich, St. Louise, USA) for 48 h. Then, the dialyzed

AMPs were freeze-dried and used for further characterization (Sar-

ikhani, Kermanshahi, Ghadam, & Gharavi, 2018).

The inhibitory activity of AMPs was estimated using the agar well

diffusion method as described below. First, Trypticase soy agar (Merck,

Darmstadt, Germany) was cooled to 47 ◦C and inoculated with Listeria

monocytogenes ATCC 19115, as an indicator strain at a nal concentra-

tion of 10

CFU/mL, cultured for 24 h at 37 ◦C in Trypticase soy broth

(TSB) (Merck, Darmstadt, Germany). Then, it was poured into a sterile

plate at room temperature. After solidication, wells with 6 mm in

diameter were cut and lled with 50

L of AMPs neutralized to pH 7 with

1N NaOH solution. The plates were kept in a refrigerator (4 ◦C) for 2 h to

diffuse supernatant and then incubated at 37 ◦C for 24 h. Finally, the

inhibition zone diameters were determined (Ünlü et al., 2015).

2.4. Characteristics of AMPs

2.4.1. Determination of molecular weight

Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(Mini-PROTEAN® Tetra Cell, Bio-Rad Laboratories, USA) was used to

estimate the molecular weight of partially puried AMPs at 120 V for 3

h. Then, the gel was stained by the Coomassie brilliant blue R-250

(Sigma Aldrich, St. Louise, USA). The molecular weight of the AMPs was

determined by evaluation with the size marker 11–180 kDa (Cina Clon,

Tehran, Iran) using a gel documentation system BIOMATE (Sarikhani

et al., 2018).

2.5. Functional group analysis

The functional groups of puried AMPs were investigated by a

Bruker TENSOR 27, Fourier Transform Infra-red (FTIR) spectrometer

(Bruker Optics, Ettlingen, Germany). For this determination, 5 mg of the

freeze-dried AMPs was mixed with KBr powder (200 mg) and pushed

into a tablet. The tablet was scanned in the 400-4000 cm

−1

(Perumal &

Venkatesan, 2017).

2.6. Nuclear magnetic resonance (NMR) spectroscopy

An NMR spectrometer (Bruker DRX500, Bruker Spectrospin Ltd,

Coventry, UK) was used to record the NMR spectra of AMPs. For this test,

50 mg of the freeze-dried AMPs was dissolved in 1 mL of D

O. The so-

lutions were analyzed at 70 ◦C at 400 MHz and 100 MHz for

H and

NMR, respectively (Lin & Pan, 2017).

2.7. Differential scanning calorimetry (DSC) of AMPs

Thermal properties were determined using a Jade DSC, PerkinElmer,

Table 1

The composition and characteristics of cheese whey and milk permeate.

Characteristics Cheese whey Milk permeate

pH 6.54 ±0.02 6.23 ±0.02

Dry matter (%) 6.03 ±0.02 6.47 ±0.03

Total sugars content (%) 4.44 ±0.10 5.49 ±0.05

Total proteins content (%) 0.3 ±0.005 0.1 ±0.002

Fat content (%) 0.1 ±0.00 0 ±0.00

Total phosphorus content (%) 1.29 ±0.14 0.88 ±0.01

Potassium content (ppm) 36.51 ±1.25 34.41 ±1.41

Sodium content (ppm) 10.99 ±1.04 13.04 ±1.76

S. Amiri et al.

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Characterization of antimicrobial peptides produced by Lactobacillus acidophilus LA-5 and Bifidobacterium lactis BB-12 and their inhibitory effect against foodborne pathogens

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