Characterization and lipolytic activity of lactic acid bacteria isolated from Thai fermented meat

Article history: Received on: 24/12/2014 Revised on: 18/02/2015 Accepted on: 20/03/2015 Available online: 28/03/2015 Twenty six strains of lactic acid bacteria (LAB) were isolated from Thai fermented meat products, including 7 samples of Nham (fermented pork), 7 Sai-krog-prieo (fermented sausage) and one Mum (fermented beef). The isolates were identified as Lactobacillus pentosus (5 isolates), Lactobacillus sp. (11 isolates), Pediococcus pentosaceus (5 isolates) and each isolate of Pediococcus lolii, Leuconostoc fallax, Weissella thailandensis, W. cibaria, and W. paramesenteroides based on their phenotypic characteristics and 16S rRNA gene sequence similarities (99.8-100 %). The isolates were primary screened for their lipolytic activity on agar plates. The rodshaped isolates showed 0.031±0.030-0.938±0.127 U/ml of lipase activity in broth supplemented with Tween 20, Tween 40, Tween 60 or Tween 80. The isolate SS50-1 identified as Lactobacillus pentosus showed the highest activity in Tween 80 (0.938±0.127 U/ml). The coccal isolates showed lipase activity ranged from 0.029±0.0061.090±0.033 U/ml when Tween 20, Tween 40, Tween 60 or Tween 80 was used as a substrate. The isolate SS48-4 identified as Pediococcus lolii showed the highest activity in Tween 80 (1.090±0.033 U/ml).


INTRODUCTION
Lipase, the hydrolase of glycerol esters EC 3.1.1.3,has high catalytical potential to catalyse the hydrolysis and transesterification of triacylglycerols, enantioselective synthesis, and hydrolysis of a variety of esters.Lipase is extensively distributed in plants, animals and microorganisms.The strains of Bacillus, Pseudomonas, Burkholderia, Acinetobacter and Staphylococcus, and fungi, Aspergillus terreus and Fusarium heterosporum are reported to produce lipase (Walavalkar and Bapat, 2001;Mrozik et al., 2006;Gayathri et al., 2013).Lipase and esterase of lactic acid bacteria distributed in cheese and milk products and involved milk fat hydrolysis, alcoholysis and esterification including the control of bioflavor and safety in fermented sausages (Meyers et al., 1996;de Fátima Silva Lopes et al., 1999;2002;Demeyer et al., 2000;Hollanda et al., 2005).Microbial lipases are commercially significant in food industry because of low production cost, greater stability and wider availability than plant and animal lipases.Lipases are commonly used in the production of a variety of products, ranging from fruit juices, baked foods, vegetable fermentation and in flavour development (Hasan et al., 2005).Bacterial lipase is linked to their role as biocatalysts in many biochemical processes such (Joshi and Vinay, 2007).In Thailand, there are many kinds of fermented meat such as Nham (fermented pork), Sai-krog-prieo or Mum (fermented pork or beef sausage), Plara (fermented fish) and Kung-chom (fermented shrimp) that have been consumed daily by Thai people (Tanasupawat and Komagata, 2001).In Nham (fermented pork), the changes in lipid composition and fatty acid profile during fermentation have been reported (Visessanguan et al., 2006).However, a lipolysis of pork fat by bacterial cultures has not been studied in Thailand compared to the meat starter culture, Staphylococcus xylosus that was applied as starter in meat fermentation (Sørensen, 1997).The aim of the research was to isolate, identify, and screen the lipase activity of lactic acid bacteria (LAB) isolated from Thai fermented meat.

Sources and Isolation methods
Fifteen fermented meat including 7 Nham (fermented pork) samples collected in Utaradit and Bangkok provinces, 7 Saikrog-prieo (fermented sausage) samples collected in Mahasarakham and one Mum (fermented beef) collected in Phetchabun province, Thailand (Table 1) were used for the isolation.Bacterial strains were isolated by spread plate technique using one gram of the fermented food samples diluted in 99 ml of 0.1% peptone solution and then mixed by stomacher for 2 min.It was then 10-fold serially diluted with peptone solution, and 0.1 ml of each proper diluted sample was transferred to Tryptic soy agar (TSA) plate before being spread with a glass spreader and incubated at 37°C for 48 h.The bacterial cells were counted and the colonies which showed different appearance were picked up for purification and then were transferred to TSA slant.

Genotypic characterization
The 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the primers 20F (5'-AGTTTGATCCTGGCTC-3'), 1530R (5'-AAGGAGGTGAT CCAGCC-3'), 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3').The amplified 16S rRNA gene sequence was analyzed by Macrogen Inc., Korea.Sequence alignment was employed using the BLAST software from the Gen Bank.Multiple alignments of the sequences determined were performed with a program CLUSTAL_X (version 1.83; Thompson et al., 1997).Gaps and ambiguous bases were eliminated prior to construction of a phylogenetic tree.A phylogenetic tree was constructed by the neighbour-joining method (Saitou and Nei, 1987) with the program MEGA version 6 (Tamura et al., 2013).The confidence values of individual branches in the phylogenetic tree were determined by using the bootstrap analysis of Felsenstein (1985) based on 1000 replications.

Screening for lipolytic activity
All of the isolates were screened for lipolytic activity on agar plate.The medium consisted of peptone 1%, CaCl 2 .2H 2 O 0.01%, agar 2% and 1% of Tween 20, Tween 40, Tween 60 or Tween 80 (Barrow and Feltham, 1993) and were incubated at 30 °C for 48 h.The lipolytic activity of the isolates was detected by the appearance of an opaque zone around the colonies.
The selected isolates that showed their lipolytic activity to different substrates on agar medium were cultivated and 0.1% (v/v) seed cultures (0.5 McFarland standard) were inoculated in 50 ml Nutrient broth (NB) (250 ml) with 1% Tween as substrates and then incubated at 30 °C on a rotary shaker (200 rpm) for 24 h.The fermentation broth was collected and centrifuged at 10,000 rpm, 4 °C for 10 min and 50 µl of the supernatant was used as crude enzyme for the assay.
Lipase activity was determined by a spectrophotometric assay with ρ-nitrophenyl palmitate (ρ-NPP) as a substrate.The reaction mixture consisted of 135 µl of 0.4% Triton X, 0.1% gum arabic in 50 mM Tris-HCl buffer (pH 7) and 15 µl of 30 mg, p-NPP in 10 ml of isopropyl alcohol.The mixture was added with 50 µl of crude enzyme incubated at 37˚C for 1 h, the colour change of activity was measured at 405 nm (Arora, 2013).The enzyme activity was calculated as described by Lee et al., 2003.

Isolation of isolates
Eight rod-shaped lactic acid bacteria were isolated from Nham collected in Utaradit and Bangkok provinces and 8 isolates from Sai-krog-prieo collected in Mahasarakham province.One coccal isolate was isolated from Mum collected in Phetchabun province, 3 isolates were isolated from Nham collected in Utaradit and Bangkok provinces and 6 isolates were from Sai-krog-prieo collected in Mahasarakham province (Table 1).The total bacterial cell count in Nham, Sai-krog-prieo and Mum ranged from 2.63 x 10 8 -5.75 x 10 9 ; 2.05 x 10 8 -1.71 x 10 10 and 1.39 x 10 9 CFU/g, respectively.

Identification of isolates
Twenty-six isolates were Gram-positive none spore forming, catalase and oxidase negative lactic acid producing bacteria.They fermented glucose homofermentatively and some isolates that produced gas from glucose were heterofermentative.They did not grow at 50 °C.They showed negative reaction to nitrate reduction, hydrolysis of arginine and starch.All isolates were divided into seven groups based on the cell form, growth at different temperatures, pH and NaCl concentrations including the acid production from carbohydrates (Table 2) as described here.
Leuconostoc group (Group 5) contained an isolate SS49-1.Cells were cocci in chains and non spore forming.Colonies were smooth, circular, convex and white in colour.They fermented glucose heterofermentatively and produced gas from glucose.They grew at 20-40 °C, pH 4-7 and in 6% NaCl.Acid was produced from glucose, D-mannitol, methyl-α D-glucoside, and D-xylose but it did not produce acid from any carbohydrates as in Table 2.The isolate SS49-1 (1,327 bp) showed 99.9% 16S rRNA gene sequence similarity to Leuconostoc fallax DSM 20189 T .Therefore, this isolate was identified as Leu.fallax (Martinez-Murcia and Collins, 1991).
The enzyme potential of lactic acid bacteria is an important factor in the formation of characteristic taste of meat products.The ripening of the product are complex and are resulted from the interaction of the remaining enzymes of muscle and fat tissue and the action of bacterial enzymes.This study, LAB isolates showed low lipase activity the same as Lactobacillus plantarum, Lactobacillus sakei and Lactobacillus brevis strains isolated during different stages of fermented traditional Bulgarian meat product that reported by Stoyanovski et al. (2013).Production of naturally fermented sausages is a tradition in Southern Europe, Scandinavia and Latin America.
Specific flavor, odor, color and structure of the sausages are due to the characteristics of the raw meat and spices used, natural microflora including lactic acid bacteria.During the ripening of dry sausage biochemical and physicochemical processes occur, which alter the composition and the structure of meat to form end products that give specific flavor, taste, color and other features of the product.Enzymatic activity of microflora, including the lactic acid bacteria plays an essential role in these processes (Demeyer et al., 2000;Vestergaard et al., 2000;Papamanoli et al., 2003).Our results showed that LAB in fermented meat may play the formation of characteristic taste of the meat products.

Fig. 1 :LipaseFig. 2 :
Fig. 1: Neighbour-joining tree based on 16S rRNA gene sequences showing relationships among LAB isolates and related species.The numbers on the branches indicate the percentage bootstrap values of 1,000 replicates; only values >50% are indicated.Bar, 0.01 substitutions per nucleotide position.

Table 1 :
Sources, location, isolate number, identification and 16S rRNA gene sequence similarity (%) of the representative LAB isolates.

Table 2 :
Phenotypic characteristics of LAB isolates.