Enhancement of Antioxidative and Intestinal Anti-inflammatory

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Enhancement of antioxidative and intestinal anti-inflammatory activities of glycated milk casein after fermentation with Lactobacillus casei 4B15 Nam Su Oh, Jae Yeon Joung, Ji Young Lee, Younghoon Kim, and Sae Hun Kim J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 17, 2017

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Journal of Agricultural and Food Chemistry

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Enhancement of antioxidative and intestinal anti-inflammatory activities of

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glycated milk casein after fermentation with Lactobacillus casei 4B15

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Nam Su Oh†, Jae Yeon Joung† , Ji Young Lee† , Younghoon Kim*#, Sae Hun Kim*

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§

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†

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§

R & D Center, Seoul Dairy Cooperative, Ansan, Kyunggi 15407, South Korea Department of Biotechnology, College of Life Sciences and Biotechnology, Korea

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University, Seoul 02841, South Korea

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#

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University, Jeonju 54896, South Korea

Department of Animal Science and Institute of Milk Genomics, Chonbuk National

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N. S. Oh and J. Y. Joung contributed equally to this study.

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Corresponding author: S. H. Kim, and Y. Kim

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Tel:

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[email protected]

+82-63-270-2606;

Fax:

+82-63-270-2612;

E-mail:

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ACS Paragon Plus Environment

[email protected]

or


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Acknowledgements

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This research was supported by the High Value-Added Food Technology Development

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Program of the Korea Institute of Planning and Evaluation for Technology in Food,

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Agriculture, Forestry and Fisheries (iPET), and the Ministry for Food, Agriculture, Forestry

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and Fisheries of Republic of Korea (115006-03-2-SB010) and a grant from the Next-

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Generation BioGreen 21 Program (Project No. PJ0118142016), Rural Development

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Administration, Republic of Korea.

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Notes

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The authors declare no competing financial interests.

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ABSTRACT

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In this study, we investigated the glycoproteomics of glycated milk casein (GMC) and

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GMC fermented by Lactobacillus casei 4B15 (FGMC), and determined their biological

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implications. There was a significant increase in the antioxidative and anti-inflammatory

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activities of GMC with galactose, which was higher than that of GMC with glucose (GMC-

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glc). Further, the fermentation of GMC by Lactobacillus casei 4B15 synergistically enhanced

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the above activities compared to those of unfermented GMC. Especially, fermented GMC-glc

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(FGMC-glc) possessed remarkably improved reducing power and radical scavenging

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activities. Moreover, FGMC-glc ameliorated the inflammatory response and tight junction–

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related intestinal epithelial dysfunction. Additionally, hexose-derived glycation and

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modification sites in protein sequences of GMC were identified. In particular, glycosylation

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and sulfation of serine and threonine residues were observed, and distinct modification sites

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were detected after fermentation. Therefore, these results indicated that glycation-induced

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modification of casein and fermentation correlated strongly with the enhanced functional

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properties.

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Keywords: glycation, casein, Lactobacillus casei 4B15, antioxidant, pro-inflammatory

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cytokine, protein modification

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INTRODUCTION

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Milk proteins contain valuable components and biologically active substances 1, and

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have been used widely as functional ingredients in food applications. Glycation of milk

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proteins via the Maillard reaction improves their functional and nutritional properties 2.

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Glyco-conjugates, derived from glycation of proteins, have received substantial attention in

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recent years. The glycation reaction proceeds under mild and simple conditions, requiring no

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extraneous chemicals, and is thus superior to other types of chemical modifications of

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proteins. Therefore, glycation is used widely as a promising technique for protein

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modification in the food industry 3.

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Microbial fermentation with probiotics, which generates bioactive peptides, is an

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alternative for improving protein function. Certain probiotics are beneficial for human health

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in selected but varied clinical conditions such as in atopic dermatitis, colon cancer, irritable

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bowel syndrome, diabetes, and intestinal disorders

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effects of milk casein and its microbially hydrolyzed peptides are well-documented

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However, the effects of fermentation of casein glyco-conjugates on antioxidative activities

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and inflammation-associated intestinal disorders have not been explored. Oxidative stress

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induced by reactive oxygen species is a leading cause of numerous human chronic disorders

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such as cancer, inflammatory bowel disease (IBD), neurodegenerative and cardiovascular

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diseases, and chronic intestinal inflammatory disorders 8. Moreover, defective tight junction

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(TJ)-related intestinal barriers are a central pathogenic factor of inflammation, and the levels

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of pro-inflammatory cytokines are elevated in IBD, which increases the permeability of

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intestinal epithelial TJ 9.

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4-5

. For instance, the anti-inflammatory 6-7

.

Structural modification of proteins by glycation and improvement in protein function are highly correlated

2, 10

. However, most studies focus on either the structural changes of the 4


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glyco-conjugates or on the improvement of their functional properties, with minimal attention

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to the relationship between their structural and functional properties. Moreover, peptides

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derived from the fermentation of glyco-conjugates have rarely been studied using proteomic

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techniques such as peptide profiling and post-translational modification analysis. Recently,

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matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) and electrospray

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ionization-mass spectrometry have acquired a leading role in the structural characterization of

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proteins

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numerous structures independent of their physicochemical properties and conformation 11.

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. Mass spectrometry is a particularly powerful analytical tool for analyzing

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In this study, we glycated milk casein with monosaccharides such as glucose and

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galactose, followed by fermentation with Lactobacillus casei 4B15 (4B15), potential

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probiotics that demonstrated preliminary probiotic properties based on acid and bile tolerance,

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intestinal adhesion, antioxidation, and antimicrobial and casein-proteolytic activities.

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Thereafter, we investigated the glycation and fermentation properties of glycated milk casein

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(GMC) and GMC fermented by 4B15 (FGMC), respectively. Furthermore, we determined the

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effects of GMC and FGMC (throughout the fermentation of GMC) on antioxidative and anti-

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inflammatory activities and intestinal barrier function in Caco-2 cells. Additionally, we

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analyzed the glycation- and fermentation-induced properties of modified casein, and peptides

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derived from GMC after fermentation with 4B15 were analyzed using MALDI-TOF-MS/MS.

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MATERIALS AND METHODS Manufacture of Glycated Milk Protein and Fermentation

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Manufacture of Glycated Milk Protein. Casein (Erie Foods International, Inc., IL, USA)

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was dissolved together with either glucose or galactose in deionized water at a 2:1 (w/w) ratio

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of protein (50 mg/g) and monosaccharides (25 mg/g). The reaction was allowed to proceed

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with shaking at 60 rpm in a pilot scale instrument controlled by a pilot-scale pasteurization

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unit (Powerpoint International, Tokyo, Japan) at 75°C, and sample aliquots were collected

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every 3 h during the 24 h reaction period. Then, the reaction products were dialyzed twice in

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a 10,000 molecular weight cut-off ultrafiltration system (Sam Yeon Engineering, Seoul,

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Korea) to remove the monosaccharide remaining after reaction with proteins, and the

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dialyzed products were lyophilized. All experiments were performed in triplicates.

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Fermentation of Glycated Milk Protein. A probiotic Lactobacillus isolate of human

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origin (infant feces), Lactobacillus casei 4B15 (4B15, R & D center, Seoul Dairy

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Cooperative, Gyeonggi-do, Korea), was used in this study. Preliminarily, the probiotic

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potential of this isolate was evaluated using various tests, such as acid and bile tolerance,

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bacterial adhesion capacity, and antioxidative and antimicrobial activities. Additionally, the

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proteolytic activity and viability of the strain in milk casein were determined for fermentation

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of glycated milk casein (GMC). Strain identity was confirmed prior to use 16S rDNA

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sequencing. The probiotic Lactobacillus strain, 4B15 was deposited in Korean culture Center

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of Microorganisms (KCCM) and is accessible through accession number of KCCM11983P.

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The strain was activated three times in de Man, Rogosa, and Sharpe (MRS) (Difco

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Laboratories, MI, USA) broth at 37°C for 18 h before use. Fermentation of GMC with 4B15

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(FGMC) was performed in a GMC solution that contained 5% GMC and 2.5% 6


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monosaccharide. A total of 106 CFU/mL bacterial cells were inoculated into GMC solution

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and incubated at 37°C for 48 h. After centrifugation of the fermented GMC at 28,000 × g for

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10 min at 4°C, the supernatant was freeze-dried, and the lyophilized powder was used for this

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study.

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Characterization of Glycated Milk Protein

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Fluorescence Intensity. Fluorescence intensity at 370 nm excitation and 440 nm

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emission was estimated to determine the extent of glycation using a Synergy H1 plate reader

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(Bio-Tek Instruments Inc., Winooski, VT). The advanced Maillard product (AMP), which is

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produced in the early stage of the glycation reaction, was quantified using fluorescence

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intensity at an excitation of 330 nm and an emission of 420 nm.

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Chemical Composition. The furosine (ε-N-2-furoylmethyl-L-lysine) concentration was

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analyzed according to a previous method 12. The sugar content in samples was analyzed using

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high pressure liquid chromatography (HPLC) (Agilent Technologies, Waldbronn, Germany)

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based on AOAC Official Methods 980.13 and 972.16

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according to the AOAC Official Methods 990.19

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endotoxin quantification kit (Thermo Fisher Scientific Inc., Waltham, MA USA). Organic

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acids in samples were determined following the procedure reported by Donkor et al.

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an Aminex HPX-87C column (Bio-Rad, Hercules, CA, USA) on an Agilent 1260 HPLC

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system (Agilent Technologies, Santa Clara, CA, USA).

13

. Total solid (TS) was measured

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. Endotoxin was measured using the

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using

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Determination of Antioxidative Activity. The antioxidative activity of natural

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compounds depends on the experimental procedures used owing to the differences in their

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reaction mechanism. The antioxidative activities were determined by estimating the reducing 7



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power and radical scavenging ability using the ferric-reducing antioxidant power (FRAP)

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assay,

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sulphonic acid) (ABTS), and the hydroxyl radical scavenging activity assays, respectively.

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FRAP, DPPH, and ABTS assays were performed using the method of Oh et al.

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hydroxyl radical scavenging activity was measured using the deoxyribose method

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Deoxyribose is degraded upon exposure to hydroxyl radicals generated by Fenton reaction.

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The pH of the samples was adjusted to 7.5 with 0.1N NaOH and the supernatants were

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filtered using a 0.45 µm membrane filter prior to the experiments. The protein contents were

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estimated using the bicinchoninic acid (BCA) protein assay kit, and equal amounts of protein

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were used in all experiments (Thermo Fisher Scientific Inc., Waltham, MA, USA). All

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experiments were performed in triplicates.

2,2-diphenyl-1-picrylhydrazyl

(DPPH),

2,2'-azino-bis(3-ethylbenzothiazoline-6-

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, and

16

. 2-

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Measurement of mRNA Expression

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Cell Culture and Treatment. The human intestinal epithelial Caco-2 cells were obtained

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from the Korea Cell Line Bank (Seoul, Korea). Caco-2 cells were cultured in Dulbecco’s

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modified Eagle medium (DMEM) high glucose (HyClone, Logan, UT, USA) supplemented

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with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 µg/mL streptomycin at

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37°C in a humidified atmosphere containing 5% CO2. To determine the pro-inflammatory

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effect and tight junction permeability of lipopolysaccharide (LPS)-induced Caco-2 cells, the

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cells (5 × 105 cells/well) were treated with 2.5 mg/mL samples for 24 h before exposure to 1

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µg/mL LPS for 18 h. Samples were dissolved in media, filtered with a 0.22 µM filter, and

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added directly to the culture media. Each treatment was performed in triplicates in a single

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experiment.

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Quantitative Real-time PCR. The total cellular RNA was isolated from Caco-2 cells

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using the TRIZOL™ reagent (Invitrogen, Carlsbad, CA, USA) according to the

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manufacturer’s instructions and further purified using the acidic phenol/chloroform extraction.

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Then, cDNA was generated using the High-Capacity cDNA reverse transcription kit (Applied

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Biosystems, Foster City, CA, USA). All the cDNA samples were stored at -20°C until further

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use. Quantitative real-time PCR (CFX Connect™ Real-Time PCR Detection System, Bio-

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Rad) was used to detect the mRNA expression in the samples using KAPA™ SYBR® FAST

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qPCR kit universal master mix (2X)

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protocol was as follows: denaturation at 95°C for 10 min, annealing at 95°C for 15 s, and

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extension at the temperature of Tm for 30 s. The relative expression of the genes examined

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was calculated using the comparative threshold cycle method, and all the values were

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normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All the

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samples were run in triplicates. The sequences of the PCR primers for IL-8, MCP-1, TLR-4,

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ZO-1, OCLN, CLDN1, and the housekeeping gene GAPDH are listed in Supplementary Table

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1.

(Kapa Biosystems, Boston, MA, USA). The reaction

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Peptide Analysis by MALDI-TOF MS/MS

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Enzymatic Protein Hydrolysis. Samples were dried under vacuum and reconstituted in

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lysis buffer (6 mol/L urea, 100 mmol/L Tris-HCl, pH 7.8). Sample aliquots containing 1 mg

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proteins were reduced with dithiothreitol (DTT), alkylated with iodoacetamide (IAA),

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reduced again with DTT, and digested with trypsin at 37ºC overnight. The digestion was

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stopped with acetic acid, and the peptides were desalted using zip-tip C18 (Millipore,

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Darmstadt, Germany) with aqueous acetonitrile (50%, v/v) and trifluoro acetic acid (0.1%,

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v/v). 9



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MALDI-TOF/MS and MS/MS Analysis. The trypsin-digested GMC and fermented

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GMC were mixed with an equal volume of matrix solution (α-cyano-4-hydroxycinnamic acid,

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HCCA), and 1.5 µL mixture was spotted onto MALDI Anchorchip target or MALDI ground

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steel target plate. The MALDI-TOF/MS experiments were performed using a Bruker

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Autoflex (Bruker Daltonics, Bremen, Germany) equipped with a nitrogen laser (337 nm).

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Laser-desorbed positive ions were analyzed after acceleration by 19 kV in the reflector mode

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for peptide digest. External calibration was performed using a mix of angiotensins I and II,

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substance P, bombesin, adrenocorticotropic hormone (ACTH) clips 1–17 and 18–39, and

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somatostatin 28. For each displayed mass spectrum, at least 2,000 laser shots from several

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positions on the spots were collected. The mass spectra were searched against the

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nonredundant Swiss-Prot database using Mascot (Matrix Science, London, UK). The search

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was

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(www.matrixscience.com) using carbamidomethylation as a fixed modification and glycation,

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oxidation, deamidation, and phosphorylation as variable modifications.

performed

against

the

“Other

Mammalia”

(or

Bos

taurus)

database

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Statistical Analysis

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All data were expressed as means ± standard deviation (S.D.). Statistical significance for

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the differences between the groups was assessed using independent sample t-test. The SPSS

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software (version 22.0, IBM, Chicago, IL, USA) was used to perform all statistical tests.

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RESULTS Characterization of Glycated Milk Casein

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The properties of milk casein glycated with different monosaccharides during the 24 h

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glycation reaction are shown in Figure 1. The fluorescence intensity of GMC increased

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gradually as the reaction proceeded. In contrast, casein, which was used as a control, did not

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show any change in fluorescence intensity. Glycation of milk casein with galactose (GMC-

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gal) resulted in a significantly higher fluorescence intensity compared to that of milk casein

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glycated with glucose (GMC-glc). The curves for fluorescent AMP and furosine

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concentration produced similar patterns of fluorescence intensity changes in all samples.

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Thus, all three indicators of glycation supported the formation of glyco-conjugates derived

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from the heat treatment of milk casein with reducing sugars. Moreover, glycation of casein

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increased the concentration of organic acids such as acetic acid, formic acid, and propionic

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acid, which was more evident with galactose than with glucose. The chemical compositions

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of GMCs manufactured in a pilot-scale system, followed by ultrafiltration and freeze drying,

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are presented in Table 1. The TS content was > 95% (w/w) in both samples, and the

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percentages of protein and sugar residues in the TS were > 92% (w/w) and

Enhancement of Antioxidative and Intestinal Anti-inflammatory

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