Qiao et al. AMB Expr (2018) 8:176 https://doi.org/10.1186/s13568-018-0706-0 ORIGINAL ARTICLE Open Access Egg yolk immunoglobulin interactions with Porphyromonas gingivalis to impact periodontal infammation and halitosis Wu Qiao1, Fang Wang2† , Xiaochen Xu3, Shujun Wang4, Joe Mac Regenstein5, Bin Bao1,6* and Ming Ma1,6* Abstract Porphyromonas gingivalis is a pathogenic Gram-negative anaerobic bacterium that colonizes the subgingival region of gums. These bacteria can invade periodontal tissues, form plaques, and produce volatile sulfur compounds (VSC) and volatile organic compounds (VOC). Egg yolk immunoglobulin (IgY) that was specifcally produced in egg yolks after chickens were challenged with P. gingivalis could control and prevent oral diseases caused by P. gingivalis. The releases of P. gingivalis ofensive metabolic odors in vitro and in vivo were determined using a Halimeter and GCMS. With IgY bacterial growth was inhibited, and the relative amounts of VOC and VSC were decreased. The scores for the oral health index and the levels of IL-6 and TNF-α are also decreased. All treatment groups showed signifcant anti- infammatory efects, which strongly suggests that specifcally IgY against P. gingivalis may be an efective treatment for the prevention and protection of periodontal infammation and halitosis. Keywords: Egg yolk immunoglobulin, Porphyromonas gingivalis, Periodontal infammation, Halitosis, Volatile sulfur compounds, Volatile organic compounds Introduction and Jenkinson 2000). Te immune response results in About 700 diferent microbial species cause a number tissue destruction and loss of alveolar bone and connec- of diferent infections and infammation in the oral cav- tive tissue, eventually leading to periodontal infamma- ity (Kuramitsu et al. 2007; Tsuzukibashi et al. 2014). Te tion (Larsson 2017). VSC and VOC cause the unpleasant most common microbial oral diseases are periodon- odors, which are referred to as halitosis (Lee et al. 2014). tal infammation (Ke et al. 2016) and halitosis (Tanda Some approaches have been reported and applied et al. 2015). Tese diseases may be caused by various to avoid oral diseases caused by P. gingivalis. Tey anaerobic bacteria such as Streptococcus mutans, Por- included inhibition of cell growth and initial cell adhe- phyromonas gingivalis, Prevotella intermedia, and Fuso- sion, and degradation of odors emitted using antibacte- bacterium nucleatum (Zhen et al. 2008; Patra et al. 2014). rial agents, and polyclonal and monoclonal antibodies. P. gingivalis is one of the major pathogen that colonizes Honey as a therapeutic option in periodontitis treat- the subgingival region of gums. Tese bacterial can ment acted as an antibacterial against P. gingivalis, but produce compounds that contribute to dental plaque, did not inhibited the formation of bioflms in an in vitro acquired immune response and release of unpleasant study (Eick et al. 2014). P. gingivalis was found at a fre- odor, such as protein adhesins, hemin binding proteins, quency of 35% in patients with chronic periodontitis proteinases, volatile sulfur compounds (VSC) and vola- and clinical isolates were highly sensitive to metroni- tile organic compounds (VOC) (Holt et al. 1999; Lamont dazole and tetracycline (Gamboa et al. 2014). However, antibiotic resistance attributed to the use of metronida- *Correspondence: [email protected]; [email protected] zole and tetracycline has become an increasing problem †Fang Wang—Co-frst author (Patil et al. 2013). Parenteral or intraoral administration 1 College of Food Science and Technology, Shanghai Ocean University, No. 999 Hucheng Ring Road, Pudong New Area, Shanghai 201306, China of KAS2-A1-specifc polyclonal antibodies protected Full list of author information is available at the end of the article against the development of P. gingivalis-induced bone © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Qiao et al. AMB Expr (2018) 8:176 Page 2 of 12 resorption and inhibited proteolytic activity, binding to Materials and methods host cells/proteins and co-aggregation with other peri- Materials odontal bacteria (O’Brien-Simpson et al. 2016). VSC Tis study used IgY against P. gingivalis (25,600 titer, were produced primarily by anaerobic bacteria that 370 mmol/l, according to the manufacturer) from Maxam produced large amounts of proteinases, many trypsin- Ltd. (Shanghai, China), and P. gingivalis (ATCC33277) like (van den Velde et al. 2007). And oral malodors were was purchased from American Type Culture Collection reduced by inhibition of P. gingivalis growth and neu- (Manassas, VA, USA). All other chemicals were analytical tralization of VSC (Lee and Baek 2014). Streptococcus reagent grade or better from Chinese suppliers. thermophilus, a probiotic bacterium, inhibited growth Te simple preparing process of P. gingivalis as an anti- of P. gingivalis, and reduced P. gingivalis-producing gen: P. gingivalis was maintained on Brain Heart Infusion VSC emissions. Nevertheless, S. thermophilus showed Broth (BHI) supplemented with 10% sheep blood. Stable- diferent antibacterial activity with diferent culture- phase P. gingivalis was dissolved in PBS (0.05 M, pH7.4) conditions. A possible reason may be that P. gingivalis and stored at 4 °C. Te solution mixed with Freund’s can inhibit the activity of antibacterial agents of S. ther- adjuvant in a ratio of 1:1 was used as the antigen. mophilus or has weak resistance for the antibacterial agent (Lee and Baek 2014). Growth inhibition Recently, chicken egg yolk immunoglobulin, referred Te artifcial saliva was prepared using a method to as immunoglobulin Y (IgY), has been shown to be a reported previously (Saunders et al. 2000; Bjorklund et al. specifc antibody that was produced in egg yolks after 2011). Briefy, the artifcial saliva was supplemented with chickens were challenged with bacteria, viruses, or mucin from porcine stomach (Sigma Chemical Co., St. parasites. Tus, IgY could specifcally control and pre- Louis, MO, USA) 2500 mg/l. Te saliva also contained vent diseases caused by their corresponding antigen (Li KCl 1160 mg/l, NaHCO3 375 mg/l, KH2PO4 355 mg/l, et al. 2012). Tese may be a convenient and economical NH4Cl 235 mg/l, KSCN 220 mg/l, CaCl2 210 mg/l, urea alternative to antibiotics to give passive immunization 175 mg/l, MgCl2 45 mg/l, thiamine 0.007 mg/l, ribofavin (Horie et al. 2004; Chalamaiah et al. 2017). IgY against 0.05 mg/l, folic acid 0.0001 mg/l, nicotinic acid 0.03 mg/l, Pseudomonas aeruginosa therapy facilitated rapid bac- pyridoxine 0.6 mg/l, pantothenic acid 0.08 mg/l, biotin terial clearance and moderated infammation in P. 0.0008 mg/l, B12 (cyanocobalamin) 0.003 mg/l, vitamin aeruginosa lung infections. It may serve as an adjunct K (menaphthone) 0.015 mg/l, bovine serum albumin to antibiotics in reducing early colonization (Tom- (Sinopharm Chemical Reagent Co., Shanghai, China) sen et al. 2016). IgY against Solobacterium moorei sig- 25 mg/l, choline 15 mg/l, uric acid 10 mg/l, alanine nifcantly inhibited growth and bioflm formation of S. 3.3 mg/l, arginine 1.9 mg/l, aspartic acid 1.6 mg/l, glu- moorei and decreased the bacterial level in the oral cav- tamic acid 3.9 mg/l, glycine 8.9 mg/l, histidine 1.0 mg/l, ity of mice after infection with S. moorei (Lee and Baek leucin 2.9 mg/l, iso-leucine 2.9 mg/l, lysine 2.7 mg/l, 2014). IgY against P. gingivalis inhibited dental plaque methionine 0.03 mg/l, phenylalanine 2.9 mg/l, pro- formation (Hamajima et al. 2007) and hemagglutinat- line 0.2 mg/l, serine 2.1 mg/l, threonine 2.9 mg/l, tyros- ing activity of P. gingivalis (Tezuka et al. 2006) in vitro, ine 2.1 mg/l, valine 1.8 mg/l, creatinine 0.1 mg/l, and 5 which may be useful in developing passive immuniza- α-amylase 3 × 10 U/l (Sigma). It was sterilized by passing tion against periodontal diseases caused by P. gingivalis through a 0.22 μm-flter (50 ml, Termo Fisher Scientifc, infection. Shanghai, China). P. gingivalis were cultured in brain Because of rats’ periodontal anatomy similar to heart infusion broth (BHI) (Shandong Haibo Technol- humans they are often used in the study of periodontal ogy Information System Co., Ltd., Qingdao, Shandong, diseases (Helieh and David 2011). In addition, rodent China). Colonies were counted using a hemocytom- models refect the state of the periodontal more sensi- eter (Shanghai Refned Biochemical Reagent Instrument tively. Terefore, periodontal diseases commonly have Co., Ltd., Shanghai, China). Bacterial liquid (0.5 ml) was been induced by placing a bacterial plaque retentive added to 4.5 ml artifcial saliva at a fnal concentration of ligature in the gingival sulcus around the molar teeth in 108 CFU/ml. Ten 10 μl of IgY at concentrations of 370, animal models (Breivik et al. 2000). Te purpose of this 7.4 and 0.0037 mmol/l (diluted using 0.9% NaCl) was put study was to evaluate the efect of IgY against P. gingivalis into 5 ml tubes and were identifed as the high-dose, mid- on the growth of P. gingivalis, emission of VSC and VOC dose, and low-dose group, respectively. Te mixtures in vitro and in vivo, the scores for oral health indexes and without IgY were used as a control. Tese tubes were the levels of IL-6 and TNF-α to determine if IgY might be placed into an anaerobic package (Anaero Pack, Mit- an efective treatment for the prevention of periodontal subishi Gas Chemical Co., Tokyo, Japan), and incubated infammation and halitosis caused by P.