Publication: Tuzlanmış derilerden zayıf halofil bakterilerin izole edilerek tanımlanması ve biyoteknolojik önemlerinin araştırılması
Abstract
Ham deri örnekleri, yüzüldükten sonra mamul deri hâline getirilmediğinde tuzlanarak depolanmaktadır. Derilerin korunmasında kullanılan tuzlar işlem görmemişse, tuzlardaki halofil bakteriler derileri kontamine ederek deri kalitesini düşürmekte ve ekonomik kayıplara sebep olmaktadır. Çalışmamızda tuzlanarak 6 ay 20°C’de depolanmış 10 adet koyun derisi örneğinde bulunan zayıf halofil bakterilerin izole edilerek moleküler olarak tanımlanması, biyoteknolojik açıdan önemli enzimlerinin tespit edilmesi, bakteriyosin üretim kapasitelerinin belirlenmesi ve deri sektöründe kullanılan kimyasal bir maddenin (sodyum dimetilditiyokarbomat) hangi konsantrasyon değerlerinde bu bakterileri inhibe edebildiği araştırılmıştır. Örneklerdeki toplam zayıf halofil, proteolitik ve lipolitik aktivite gösteren zayıf halofil bakteri sayıları sırasıyla 1x104 -4x105 kob/ g, 2x103 -4x104 kob/ g ve 1x102 -3x104 kob/ g olarak saptanmıştır. Örneklerden toplam 20 adet zayıf halofil bakteri izole edilmiştir. İzolatların optimum geliştiği tuz konsantrasyon, sıcaklık ve pH aralıkları sırasıyla %3-5, 37-45 °C ve 7-8 olarak tespit edilmiştir. İzolatların tamamı proteaz, 12 izolat lipaz (%60), 1 izolat üreaz (%5), 17 izolat DNaz (%85), 2 izolat pullulanaz (%10), 9 izolat ksilanaz (%45), 18 izolat selülaz (%90) aktivitesi göstermiştir. Hiçbir izolat amilaz ve β-galaktosidaz aktivitesi göstermemiştir. İzolatların tamamının Gram (+), çomak şekilli, katalaz ve oksidaz pozitif olduğu tespit edilmiştir. İzolatların farklı karbon kaynaklarından (D-ksiloz, D-mannoz, D-riboz, sukroz) asit üretebildiği saptanmıştır. İzolatların derilerin yapısında bulunan amino asitleri (L-alanin, L-sistein, L-glisin, L-treonin) parçalayabildiği tespit edilmiştir. İzolatlar, 16S rRNA dizi analiz sonuçlarına göre Bacillus aerius, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus haynesii, Bacillus kochii, Bacillus mojavensis, Bacillus pumilus, Bacillus rugosus ve Bacillus safensis olarak tanımlanmıştır. Tanımlanan izolatlardan Bacillus aerius bakteri suşu tarafından üretilen bakteriyosinin termostabil yapıda olduğu, yüksek pH ve tuz değerlerinde aktivitesini kaybetmediği, lipaz enzimiyle muamele edildiğinde aktivitesini koruduğu, proteinaz K enzimiyle muamele edildiğinde ise aktivitesini tamamen kaybettiği tespit edilmiştir ve deri endüstrisinde kullanılabilirliği öngörülmüştür. İzolatların deri endüstrisinde yaygın olarak kullanılan sodyum dimetilditiyokarbomat aktif içerikli kimyasal maddeye karşı duyarlılık durumları Kirby-Bauer disk difüzyon metoduyla araştırılmış ve izolatların bu kimyasala karşı duyarlı oldukları tespit edilmiştir. İzolatların belirtilen kimyasala karşı duyarlı olması sebebiyle bu maddenin ilgili bakterilerin inhibisyonu amacıyla deri endüstrisinde kullanılabilme potansiyelinin olduğu düşünülmektedir.
Rawhide samples are stored in salt curing in cases where they are not turned into finished leather immediately after skinning. If the salts used for the protection of the leather are not processed, the halophilic bacteria found in the salts contaminate the leather, reducing the leather quality and causing economic losses. In the present study, the isolation and molecular identification of slightly halophilic bacteria, determination of their biotechnologically important enzymes, and their bacteriocin production capacity, and their control with a chemical substance (sodium dimethyldithiocarbomate) were investigated in 10 salted sheepskin samples that were stored at 20 °C for 6 months. The total number of slightly halophilic bacteria, proteolytic slightly halophilic bacteria, and lipolytic slightly halophilic bacteria in the samples were determined as 1x104-4x105 CFU/ g, 2x103-4x104 CFU/ g, and 1x102-3x104 CFU/ g, respectively. Twenty slightly halophilic bacteria were isolated from the samples. The optimum salt concentration, temperature and pH ranges at which the isolates grew were 3-5%, 37-45°C, and 7-8, respectively. While all isolates showed protease activity, the number and percentage of isolates showing lipase, urease, DNase, pullulanase, xylanase, and cellulase activities were 12 (60%), 1 (5%), 17 (85%), 2 (10%), 9 (45%), and 18 (90%), respectively. None had amylase and β-galactosidase activities. All isolates were Gram (+), rod-shaped, catalase and oxidase positive. The isolates were found to be able to produce acid from different carbon sources (D-xylose, D-mannose, D-ribose, sucrose) and to break down amino acids (L-alanine, L-cysteine, L-glycine, L-threonine) found in the skin structure. According to the results of 16S rRNA sequence analysis, the isolates were identified as Bacillus aerius, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus haynesii, Bacillus kochii, Bacillus mojavensis, Bacillus pumilus, Bacillus rugosus and Bacillus safensis. It has been established that the bacteriocin produced by Bacillus aerius from the identified isolates is resistant to heat, and does not lose its activity at high levels of salt and pH. Conversely, when treated with proteinase K enzyme, it loses its activity entirely but retains its effectiveness when treated with lipase enzyme. Additionally, its potential use within the leather industry has been predicted. The Kirby-Bauer disc diffusion method was used to investigate the susceptibility of the isolates against the chemical substance containing sodium dimethyldithiocarbomate as an active ingredient, which is commonly used in the leather industry. As the isolates exhibited sensitivity to the chemical, it is thought that this substance could be utilized within the leather industry to combat associated bacterial growth.
Rawhide samples are stored in salt curing in cases where they are not turned into finished leather immediately after skinning. If the salts used for the protection of the leather are not processed, the halophilic bacteria found in the salts contaminate the leather, reducing the leather quality and causing economic losses. In the present study, the isolation and molecular identification of slightly halophilic bacteria, determination of their biotechnologically important enzymes, and their bacteriocin production capacity, and their control with a chemical substance (sodium dimethyldithiocarbomate) were investigated in 10 salted sheepskin samples that were stored at 20 °C for 6 months. The total number of slightly halophilic bacteria, proteolytic slightly halophilic bacteria, and lipolytic slightly halophilic bacteria in the samples were determined as 1x104-4x105 CFU/ g, 2x103-4x104 CFU/ g, and 1x102-3x104 CFU/ g, respectively. Twenty slightly halophilic bacteria were isolated from the samples. The optimum salt concentration, temperature and pH ranges at which the isolates grew were 3-5%, 37-45°C, and 7-8, respectively. While all isolates showed protease activity, the number and percentage of isolates showing lipase, urease, DNase, pullulanase, xylanase, and cellulase activities were 12 (60%), 1 (5%), 17 (85%), 2 (10%), 9 (45%), and 18 (90%), respectively. None had amylase and β-galactosidase activities. All isolates were Gram (+), rod-shaped, catalase and oxidase positive. The isolates were found to be able to produce acid from different carbon sources (D-xylose, D-mannose, D-ribose, sucrose) and to break down amino acids (L-alanine, L-cysteine, L-glycine, L-threonine) found in the skin structure. According to the results of 16S rRNA sequence analysis, the isolates were identified as Bacillus aerius, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus haynesii, Bacillus kochii, Bacillus mojavensis, Bacillus pumilus, Bacillus rugosus and Bacillus safensis. It has been established that the bacteriocin produced by Bacillus aerius from the identified isolates is resistant to heat, and does not lose its activity at high levels of salt and pH. Conversely, when treated with proteinase K enzyme, it loses its activity entirely but retains its effectiveness when treated with lipase enzyme. Additionally, its potential use within the leather industry has been predicted. The Kirby-Bauer disc diffusion method was used to investigate the susceptibility of the isolates against the chemical substance containing sodium dimethyldithiocarbomate as an active ingredient, which is commonly used in the leather industry. As the isolates exhibited sensitivity to the chemical, it is thought that this substance could be utilized within the leather industry to combat associated bacterial growth.