Lactobacillus spp. Strains Isolation, Identification, Preservation and Quantitative Determinations from Gut Content of 45-Day-Old Chickens Broilers

24/August/2020 Approved: 15/October/2020 ABSTRACT The objective of this study was to isolate, identify, preserve and determine the quantitative level of the Lactobacillus strains from the gut content of 45-day-old chickens broilers; to test the viability of these strains preserved at 4 ºC and room temperature (20 ± 2 ºC). Lactobacillus strains were isolated, phenotypically identified and preserved from the gut content of 17 chickens broilers. Identification was performed by morphological, cultural and biochemical characters examination, using apiwebTM and ABIS online software. The quantitative level of Lactobacillus strains in intestinal content (10 5 – 10 9 CFU/g) and the viability of strains preserved at 4 ºC and at room temperature (from 8 days to 9 months) was also determined. Twenty-three strains of L. acidophilus , L. brevis , L. plantarum , L. fermentum and L. salivarius from the gut content of chickens broilers were isolated, phenotypically identified, and preserved. Of these, L. plantarum , L. fermentum and L. acidophilus biotype 1 strains were technologically and ecologically suitable to continue the testing


INTRODUCTION
Recent research on the structure of the normal intestinal microbiota of chickens revealed the presence of Lactobacillus spp. (Lu et al., 2003;Wei et al., 2013;Waite & Tailor, 2014;Duar et al., 2017), known for its beneficial effects on the host's health. Lactobacilli have a symbiotic role in host fitness, their metabolites contributing to the digestive process and counteracting pathogens (Duar et al., 2017). Zou et al. (2018) showed that Lactobacillus induce a polarizing effect on the chicken cecal microbiome, suggesting a major influential role of this genus in local microbiome, with negative (with Ruminococcaceae, Lachnospiraceae) or positive (with other lactobacilli, Bacteroides, Clostridiales and Christensenellaceae) correlations. At hatching, the poultry microbiota from cecum consists, predominantly, as Enterococcus, coliforms and clostridia (Coates & Fuller, 1977) but, from the 4 th day of age, Lactobacillus becomes a significant component of the intestinal microbiota (Zhu et al., 2002). At the 7 th day of age, the ileal mucosal microbiota is dominated by Lactobacillus, followed by Lachnospiraceae and Enterococcus (Cressman et al., 2010). After the 14 th day of age, cecum and small intestine of broilers chicks develop various communities (Pedroso & Lee, 2015) but, from day 21 to 42 of age, Lactobacillus became the most abundant organism in the small intestine. Of this genus, L. salivarius, L. johnsoni, L. reuteri, L. oris and L. crispatus were detected (Nakphaichit et al., 2011). This diversity raises the issue of selecting the best strain for developing bacterial-based feed additives in poultry nutrition.
The objective of our work was to isolate, identify, preserve and assess the quantitative level of the Lactobacillus strains from the gut content Sorescu I, Dumitru M, Ciurescu G

Lactobacillus spp. Strains Isolation, Identification, Preservation and Quantitative Determinations from Gut Content of 45-Day-Old Chickens Broilers
of 45-day-old chicken broilers, in order to further test their probiotic traits and to select the best strains as intestinal flora stabilizers in chicken nutrition.

MATERIALS AND METHODS
Birds were treated in accordance with Romanian legislation (law no. 305/2006) for handling and protection of animals used for experimental purposes. The birds' care and use protocol were approved by the Animal Care and Use Committee at the National Research-Development Institute for Biology and Animal Nutrition (INCDBNA-IBNA) Balotești, Romania, following the principles of EU Directive 2010/63/EU and Romanian Law on Animal Protection.

Lactic acid bacteria isolation and determination of total bacterial count
The Mountzouris method (2007) completed by Sorescu et al., (2019) was applied. Sample preparation: 1 g intestinal content (ileum and cecum, respectively) per capita from seventeen chicks (Cobb 500, 45-day-old) was homogenized with 7 ml Oxoid BHI (Brain Heart Infusion) broth and 2 ml glycerol, and immediately frozen at -20 ºC until testing (no more than three months). After defrost, decimal dilutions from every sample were inoculated on Oxoid Man, Rogosa, Sharpe (MRS) agar. Further instead, the procedure presented in other paper (Sorescu et al., 2019) for the isolation and counting of Lactobacillus CFU has been applied.

Identification of bacterial strains
Phenotypic identification of isolated bacterial strains was performed by morphological, cultural and biochemical characters examination, according to Bergey's Manual of Systematic Bacteriology (Garrity et al., 2009), ABIS on line software (Stoica & Sorescu, 2018) and apiweb TM API50CHL software BioMerieux (France), following the protocol described in (Sorescu et al., 2019). The results obtained by Pelinescu et al. (2009) were also considered.

Preservation of bacterial strains
The medium-term preservation (months) was done by culture in MRS broth, the viability of bacterial strains being evaluated after 45 days, 3, 7 and 9 months.
Long-time preservation (years) was done at -80 ºC, with addition of glycerol 20%, and bacteria viability is to be assessed every 2 years.
The morphological, cultural and biochemical characteristics of the identified strains are presented in Table 1.

Lactobacillus spp. Strains Isolation, Identification, Preservation and Quantitative Determinations from Gut Content of 45-Day-Old Chickens Broilers
Details on the meaning and mode of calculation of %SIM for ABIS and API %ID were presented in a previous article (Sorescu et al., 2019). Table 4 presents the results of viability test for Lactobacillus strains which are preserved at 4 ºC and at room temperature (20 ± 2 ºC). L. salivarius and L. acidophilus are included in the vertebrate adapted lifestyle lactobacilli group and were isolated from human, pigs, hamsters, horses, chickens (Duar et al., 2017) and turkeys (Sorescu et al., 2019). L. fermentum and L. plantarum belongs to the nomadic species group of lactobacilli (Duar et al., 2017). L. fermentum was isolated from fermenting plant material, sewage, milk products, mouth and faeces of humans, and intestines of pig, rat, cattle, mouse and birds (Garrity et al., 2009), including turkeys and 26-day-old chicks (Sorescu et al., 2019). L. plantarum was isolated from fruit flies, vertebrate digestive tract, plants, dairy products, environments, silage (Duar et al., 2017). L. brevis has a free-living lifestyle (Duar et al., 2017) and was isolated from silage, beer, milk, cheese, sauerkraut, sourdough, cow manure, mouth, feces and intestinal tract of human, cattle, rats, pigs and birds (Garrity et al., 2009), including 26-day-old chicks .
The strains described in this paper, isolated from the intestinal content, can be important for developing probiotic compounds for the same bird species because they are host-adapted and have a high ecological fitness. Moreover, this higher fitness is relevant in the process of outcompeting the pathogens.

Lactobacillus spp. Strains Isolation, Identification, Preservation and Quantitative Determinations from Gut Content of 45-Day-Old Chickens Broilers
with the absorption and metabolism of these carbohydrates in the host gut, if these strains are used in poultry nutrition. L. delbrueckii subsp. delbrueckii was identified only from 26-day-old chickens and L. plantarum only from 45-day-old chickens.
As in turkeys (Sorescu et al., 2019) and 26-day-old chickens  cases, in the intestinal cecum content of 45-day-old chickens, the numbers of CFU lactobacilli/g were higher (10 8 -10 9 ) than in the ileum area (10 5 -10 8 ), obviously especially in the case of isolation of the same species from both intestinal segments (L. acidophilus biotype 3, L. plantarum, L. fermentum, L. salivarius). Unlike the results on turkeys (probably due to the age difference -73d versus 26d, and 45d and the species) and similar to the results on 26-day-old chickens, L. fermentum and L. plantarum strains had relative higher presence (up to 10 9 CFU/g) than other lactobacilli (up to 10 8 CFU/g) in the intestinal content of the 45-dayold chickens, which suggests a possible ecologic and, therefore, probiotic advantage for them. This fact is interesting, because the L. fermentum and L. plantarum are considered to be nomadic species, while the L. acidophilus and L. salivarius strains are adapted to vertebrate species.
As identification systems, both software (apiwebTM and ABIS) proved to be appropriate, especially for L. plantarum, L. salivarius and L. brevis biotype 2, where the same taxonomic classification was obtained, but with different percentage results, the way of calculating them being totally different. Instead, for L. acidophilus biotype 1, L. acidophilus biotype 3, which Table 3 -The results of parallel identification of strains by apiweb TM soft, API50CHL V.5.1, BioMerieux (France) and ABIS online software.