ACTA VETERINARIA BRNO JCR分区-刊鹿论文编译

Properties of potentially probiotic Lactobacillus isolates from poultry intestines

Abstract:

The most commonly used probiotic bacteria belong to the genus Lactobacillus, being regarded as beneficial for poultry health and production. However, commercial probiotics do not always ensure both expected effects. In order to improve the utility properties of new preparations, the selection of new probiotic candidates should be made on the basis of the performance of the species within the poultry digestive tract. The aim of this study was to isolate and identify lactobacilli from poultry intestines, and to select probiotic candidates for subsequent in vivo trials. Digesta from 18 poultry specimens were collected, serially diluted, plated onto Wilkins-Chalgren anaerobe agar supplemented with 30% of rumen fluid and onto De Man, Rogosa and Sharpe agar plates, and incubated at 37 °C for 48 h under anaerobic conditions. Isolated colonies were subjected to Gram staining and catalase reaction. They were then pre-identified using matrixassisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS). Forty-four Lactobacillus strains belonging to 16 species were identified and subjected to evaluations of survival under simulated gastrointestinal conditions, autoaggregation and hydrophobicity. Most of the screened Lactobacillus reuteri strains as well as individual strains of L. acidophilus, L. amylovorus, L. gallinarum, L. ingluviei, L. johnsonii, L. oris, L. salivarius, L. saerimneri, and L. vaginalis showed high survival rates under gastrointestinal tract conditions and good surface properties. The results suggest their potential for further testing as probiotic candidates in in vivo trials. Lactobacillus, MALDI TOF MS, autoaggregation, hydrophobicity, probiotics Lactobacillus populations are autochthonous residents in the gastrointestinal tracts of humans and animals, including poultry (Stephenson et al. 2010). The presence of lactobacilli in the poultry intestinal ecosystem has previously been considered as beneficial to poultry health and production (Tannock 2004). Recent studies of Lactobacillus species ecology and their beneficial effects have brought new data (Wang et al. 2014; Adhikari and Kwon 2017; Duar et al. 2017b), and new probiotic candidates from this group of bacteria are being sought based on current knowledge. This renewed interest is stimulated by the urgent need to reduce the consumption of antimicrobials in the poultry industry while at the same time promoting health and improving performance indicators such as mean egg weight, body weight, and the feed conversion ratio (Angelakis and Rault 2010; Olnood et al. 2015; Shokryazdan et al. 2017). Commercial probiotic preparations containing lactobacilli do not always ensure these expected effects which, among other things, may be influenced by an inappropriate process of selecting Lactobacillus isolates (KizerwetterŚwida and Binek 2016). Moreover, commercial probiotic products commonly do not contain the probiotic strains stated on their labels or are even inappropriate for the stated target animal species (Lata et al. 2006; Šmídková and Čížek 2017). Selection criteria for new probiotic candidates should be based on actual knowledge as to the importance of particular Lactobacillus species in poultry intestines (Adhikari and Kwon 2017; Duar et al. 2017a,b) and their probiotic functional properties. In vitro selection procedures usually include survival of probiotic candidates under gastrointestinal ACTA VET. BRNO 2019, 88: 73–84; https://doi.org/10.2754/avb20198801073 Address for correspondence: Prof. MVDr. Alois Cizek, CSc. Department of Infectious Diseases and Microbiology Faculty of Veterinary Medicine University of Veterinary and Pharmaceutical Sciences Brno Palackeho tr. 1946/1, 61242 Brno, Czech Republic Phone: +420 607 652 460 E-mail: cizeka@vfu.cz http://actavet.vfu.cz/ conditions, their surface properties, capability for competitive exclusion and antibiotic susceptibility (Bujnakova et al. 2014; Kizerwetter-Świda and Binek 2016; Rajokaa et al. 2018). The objectives of the present study were to isolate and identify lactobacilli from poultry intestines and then to select probiotic candidates for subsequent in vivo trials. Materials and Methods Lactobacilli culture and growth conditions Whole caeca or jejuna from 18 randomly selected healthy chickens at 4 to 40 weeks of age were processed in an anaerobic chamber (10% CO2, 5% H2, and 85% N2 atmosphere; Concept 400, Baker Ruskinn, Sanford, Maine, USA) within 1 h after they were ethically sacrificed. Approximately 0.5 ml of caecal or jejunal digesta were collected and serially diluted in 4.5 ml of pre-reduced anaerobically sterilized dilution blanks and plated onto Wilkins-Chalgren anaerobe agar (Oxoid, Basingstoke, UK) supplemented with 30% of rumen fluid (Medvecky et al. 2018) and onto De Man, Rogosa and Sharpe (MRS) agar plates (Oxoid, Basingstoke, UK). All inoculated media were incubated at 37 °C for 48 h in an anaerobic chamber. Isolated colonies on the plates with the highest dilution were subjected to Gram staining and catalase reaction. They were then pre-identified using matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrometry (MS). Colonies pre-identified as lactobacilli were subcultured on Wilkins-Chalgren anaerobe agar or MRS agar plates and the pure cultures thus obtained were used for further studies. Six strains (L. amylovorus CCM4380, L. fermentum CCM7192, L. parabuchneri DSM5987, L. pentosus CCM4619, L. plantarum CCM7039, and L. salivarius subsp. salivarius CCM7274) were included as control strains in all procedures. All cultures of lactobacilli were stored in MRS broth (Oxoid, Basingstoke, UK) with 20% glycerol at −80 °C. Species identification using MALDI-TOF MS Pure cultures of Lactobacillus spp. isolates and control strains on MRS agar plates were subjected to MALDITOF MS on a Microflex LT instrument (Bruker Daltonik, Germany) as described by several authors (Duskova et al. 2012; Bujnakova et al. 2014; Dec et al. 2016). Briefly, the bacterial culture on MALDI plates was overlaid with 1 μl of matrix solution containing 10 mg/ml HCCA (a-cyano-4-hydroxycinnamic acid, SigmaAldrich, Prague, Czech Republic) dissolved in 50% acetonitrile (Sigma-Aldrich, Prague, Czech Republic) and 2.5% trifluoroacetic acid, and then air-dried. The mass spectra were processed using the MALDI Biotyper 3.0 software package (Bruker, Leipzig, Germany) containing 6903 reference spectra, including 243 for lactobacilli. Identification was performed according to the criteria recommended by the manufacturer (ID score: 1.700–1.999 probable genus identification; 2.000–2.299 secure genus identification, probable species identification; 2.300– 3.000 highly probable species identification). Tolerance to gastrointestinal conditions Tolerance of isolates to simulated gastric juice and bile salts was assayed as described by Jena et al. (2013). Briefly, for the bile tolerance test, MRS broth (Oxoid, Basingstoke, UK) was supplemented with 0.3% (w/v) bile salts (Sigma-Aldrich, Prague, Czech Republic). Simulated gastric juice contained 6.4 g NaHCO3, 0.239 g KCl, 1.28 g NaCl, and 0.1% (w/v) pepsin (Sigma-Aldrich, Prague, Czech Republic) per litre of MRS broth (pH 2.5). For both tests, 5 ml of lactobacilli culture grown overnight in MRS broth at 37 °C were pelleted and washed twice with 4 ml of phosphate buffered saline (PBS; pH 7.2). Subsequently, culture density was adjusted to McFarland turbidity standard 1.0 (Densi-La-Meter®, Erba Lachema, Brno, Czech Republic) (ca 3 × 108 cfu/ml) and inoculated into 10 ml of modified MRS broth containing bile salts or simulated gastric juice. Samples were then incubated at 37 °C for 30, 60, 90, 120, or 180 min. The number of viable cells was assessed by a serial dilution and plate count method. The results were expressed as growth rate (%) of Lactobacillus strains. Autoaggregation assay Autoaggregation capabilities were assessed according to the procedure described by Collado et al. (2007) with minor modifications. The lactobacilli culture grown overnight in MRS broth at 37 °C was pelleted, washed twice with PBS, and then resuspended in PBS. The optical density (OD) of the bacterial suspension was adjusted to McFarland turbidity standard 2.0 (Densi-La-Meter®, Erba Lachema, Brno, Czech Republic). Bacterial cell suspensions were incubated without agitation in closed measuring cuvettes (2 ml) at room temperature (22 ± 2 °C) for different time periods (0, 2, 4, 6, 24, and 48 h). At the determined time the absorbance was measured at 600 nm using a Biowave Cell Density Meter (WPA, Cambridge, UK). Autoaggregation assay was accomplished in one experiment with each isolate and was expressed as autoaggregation percentage (A% = 1 − (At/A0)/100), where At represents absorbance at the determined time and A0 the absorbance at t0. Cell surface hydrophobicity assay The degree of hydrophobicity of the cultures was specified according to procedures described previously (Rosenberg 1984; Jena et al. 2013) and which are based on affinity of bacterial cells for toluene 74

Author Listing： Alexandra Pokorna;Tereza Maňáková;Alois Čížek

Volume： 88

Pages： 73-84

DOI： 10.2754/AVB201988010073

Language： English

Journal： Acta Veterinaria Brno

ACTA VETERINARIA BRNO

ACTA VET BRNO

影响因子：0.6

是否综述期刊：否

是否OA：是

是否预警：不在预警名单内

发行时间：1969

ISSN：0001-7213

发刊频率：Quarterly

收录数据库：SCIE/Scopus收录

出版国家/地区：CZECH REPUBLIC

出版社：University of Veterinary and Pharmaceutical Sciences

期刊介绍

ACTA VETERINARIA BRNO is a scientific journal of the University of Veterinary and Pharmaceutical Sciences in Brno, Czech Republic.The scientific journal Acta Veterinaria Brno is dedicated to the publication of original research findings and clinical observations in veterinary and biomedical sciences. Original scientific research articles reporting new and substantial contribution to veterinary science and original methods that have not been submitted for publication elsewhere are considered for publication. A written statement to this effect should accompany the manuscript, along with approval for publication by the author´s head of department. The authors bear full responsibility for the contents of their contribution. Book reviews are published, too.

《布尔诺兽医学报》（Acta Veterinaria Brno）是捷克共和国布尔诺兽医和制药科学大学的科学期刊，专门发表兽医和生物医学科学的原创研究成果和临床观察。报告对兽医科学有新的和实质性贡献的原始科学研究文章以及尚未在其他地方提交发表的原始方法将被考虑发表。手稿应附有一份书面声明，沿着附有作者所在部门负责人的出版批准。作者对其稿件内容承担全部责任。书评也出版了。

年发文量	53
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平均审稿周期	较慢,18-36周
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