ردیابی باکتریوسین گروه IIa و IIb در سویه‌های بومی لاکتوباسیلوس جداسازی شده از محصولات لبنی سنتی
محورهای موضوعی :
علوم و صنایع غذایی
پریسا پورعبدی سرابی
1
,
علیرضا تاری نژاد
2
,
محمد امین حجازی
3
,
محمد مجیدی
4
1 - دانشجوی کارشناسی ارشد دانشگاه شهید مدنی آذربایجان، دانشکده کشاورزی، دانش‌آموخته کارشناسی ارشد گروه بیوتکنولوژی کشاورزی، تبریز، ایران
2 - دانشیار دانشگاه شهید مدنی آذربایجان، دانشکده کشاورزی، دانشیار گروه بیوتکنولوژی کشاورزی، تبریز، ایران
3 - دانشیار پژوهشکده بیوتکنولوژی صنایع غذایی، پژوهشگاه بیوتکنولوژی کشاورزی ایران، سازمان تحقیقات، آموزش و ترویج کشاورزی، تبریز، ایران
4 - استادیار دانشگاه شهید مدنی آذربایجان، دانشکده کشاورزی، استادیار گروه بیوتکنولوژی کشاورزی، تبریز، ایران
تاریخ دریافت : 1399/01/06
تاریخ پذیرش : 1399/09/06
تاریخ انتشار : 1399/07/01
کلید واژه:
پروبیوتیک,
باکتریوسین,
ردیابی,
زیرهم‌چینی,
چکیده مقاله :
باکتری های اسیدلاکتیک، مولکول های پروتئینی ضد باکتریایی باکتریوسین با منشأ ژنتیکی متنوع تولید می‌کنند. بنابراین، شناسایی رده باکتریوسینی این باکتری‌ها، می‌تواند منجر به درک درستی از عملکرد ضد باکتریایی این پپتیدها شود. هدف از این پژوهش غربالگری باکتری‌های اسیدلاکتیک بومی مولد باکتریوسین‌های زیرگروه IIa و IIb بود. ابتدا جدایه‌های با ویژگی ضد باکتریایی علیه دو باکتری شاخص لیستریا اﯾﻨﻮﮐﻮا (Listeria innocua)و اشریشیا کولای (Escherichia coli) با استفاده از آزمون انتشار از دیسک غربالگری شدند. وزن مولکولی پپتیدهای ترشح‌شده از باکتری های اسیدلاکتیک در محیط کشت با روش ترسیب سولفات آمونیوم تخمین زده شد. برای شناسایی جدایه های لاکتیکی مولد باکتریوسین از توالی های بالادستی و پایین‌دستی ژن ساختاری باکتریوسین در واکنش زنجیره ای پلیمراز استفاده گردید. پنج جدایه اسیدلاکتیک با بیشترین قطر هاله عدم رشدی علیه دو باکتری شاخص گزینش و وزن مولکولی پپتیدهای ترشح‌شده توسط این سویه‌ها کمتر از 10 کیلودالتون برآورد گردید. توالی‌یابی محصول واکنش زنجیره ای پلیمراز منجر به شناسایی مناطق ژنی plnN،plnJK وplnEF در جدایه N و مناطق ژنی plnJK وplnEF در جدایه M گردید. بنابراین، جدایه M و N دارای باکتریوسین گروه IIb بوده و از قابلیت خوبی برای استفاده در افزایش سطح کیفی فراورده های لبنی و غذای دام برخوردار می باشند. نتایج زیر هم‌چینی توالی های به‌دست‌آمده نشان داد که سویه‌های بومی موجود در ایران تشابه بسیار بالایی (99 درصد) با لاکتوباسیلوس پلانتاروم (Lactobacillus plantarum) سویه‌های FQ و ATCC BAA-793 دارد. در صورت انجام آزمایش‌های تکمیلی در ارتباط با جداسازی باکتریوسین‌های موجود در سویه‌های بومی M و N، می‌توان از اثر مهاری آن‌ها علیه باکتری‌های بیماری‌زای غذایی استفاده نمود.
چکیده انگلیسی:
Lactic acid bacteria produce bacteriocin as antibacterial protein molecules with diverse genetic origins. Therefore, identifying the bacteriocin class of native lactic acid bacteria could lead to an understanding of the antibacterial function of these peptides. In this study, presence of two subgroup of bacteriocins (IIa and IIb) in native strain of Lactic acid bacteria was investigated. After screening of this strains against two Index microorganisms including Listeria innocua (ATCC 33090), and Escherichia coli (ATCC 1399) by disk diffusion assay, the five strains with highest diameter of growth inhibitory against these index pathogens was selected. The molecular weight of peptides secreted by lactic acid bacteria was estimated by ammonium sulfate precipitation. Upstream and downstream sequences of the bacteriocin structural gene were used to identify bacteriocin-producing lactic isolates in the polymerase chain reaction. The results of SDS - PAGE showed bands with a molecular weight of less than 10 kDa for these strains. Sequencing of the polymerase chain reaction product led to the identification of plnN, plnJK and plnEF gene regions in N isolate and plnJK and plnEF gene regions in M isolate. Therefore, isolates M and N have good potential for use in increasing the quality level of dairy products and animal feed. The multalign results of sequences showed showed that the native strains in Iran are very similar (99%) with Lactobacillus plantarum FQ and ATCC BAA-793 strains. Inhibitory effects against foodborne pathogens can be used if additional experiments are performed to isolate present bacteriocins in M and N native strains.
منابع و مأخذ:
· Anderssen, EL., Diep, DB., Nes, IF., Eijsink, VG. and Nissen-Meyer, J. (1998). Antagonistic activity of Lactobacillus plantarum C11: two new two-peptide bacteriocins, plantaricins EF and JK, and the induction factor plantaricin A. Applied and Environmental Microbiology, 64(6): 2269-2272.
· Banerjee, SP., Dora, KC. and Chowdhury, S. (2013). Detection, partial purification and characterization of bacteriocin produced by Lactobacillus brevis FPTLB3 isolated from freshwater fish. Journal of Food Science and Technology, 50(1): 17-25.
· Bastos, MCF., Coutinho, BG. and Coelho, MLV. (2010). Lysostaphin: a staphylococcal bacteriolysin with potential clinical applications. Pharmaceuticals, 3(4): 1139-1161.
· Campos, CA., Rodríguez, Ó., Calo-Mata, P., Prado, M. and Barros-Velázquez, J. (2006). Preliminary characterization of bacteriocins from Lactococcus lactis, Enterococcus faecium and Enterococcus mundtii strains isolated from turbot (Psetta maxima). Food Research International, 39(3): 356-364.
· Cho, G-S., Huch, M., Hanak, A., Holzapfel, WH. and Franz, CM. (2010a). Genetic analysis of the plantaricin EFI locus of Lactobacillus plantarum PCS20 reveals an unusual plantaricin E gene sequence as a result of mutation. International Journal of Food Microbiology, 141: S117-S124.
· Cho, G-S., Huch, M., Hanak, A., Holzapfel, WH. and Franz, Charles M.A.P. (2010b). Genetic analysis of the plantaricin EFI locus of Lactobacillus plantarum PCS20 reveals an unusual plantaricin E gene sequence as a result of mutation. International Journal of Food Microbiology, 141: S117-S124.
· Deshmukh, PV. and Thorat, PR. (2014). Detection antimicrobial efficasy of novel bacteriocin produced from Lactobacillus similis RL7. Advanced Research, 2(1): 987-995.
· Diep, DB., Håvarstein, L., Nissen-Meyer, J. and Nes, IJA. (1994). The gene encoding plantaricin A, a bacteriocin from Lactobacillus plantarum C11, is located on the same transcription unit as an agr-like regulatory system. Applied and Environmental Microbiology, 60(1): 160-166.
· Eijsink, VG., Axelsson, L., Diep, DB., Håvarstein, LS., Holo, H. and Nes IF. (2002). Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication. Antonie Van Leeuwenhoek, 81(1-4): 639-654.
· Gholamzadeh, MA., Hejazi, MA. and Hosseinzadeh Gharaje, N. (2017). Determination of bacteriocin encoding gene in six native strains of Lactobacillus plantarum. Iranian Journal of Food Science and Technology, 14(66): 17-25. [In Persian]
· Haas, H., Budowle, B. and Weiler, G. (1994). Horizontal polyacrylamide gel electrophoresis for the separation of DNA fragments. Electrophoresis, 15(1):153-158.
· Jacobsen, CN., Nielsen, VR., Hayford, A., Møller, PL., Michaelsen, K., Paerregaard, A. et al., (1999). Screening of probiotic activities of forty-seven strains of Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans. Applied and Environmental Microbiology, 65(11): 4949-4956.
· Joerger, M. and Klaenhammer, T. (1990). Cloning, expression, and nucleotide sequence of the Lactobacillus helveticus 481 gene encoding the bacteriocin helveticin J. Journal of Bacteriology, 172(11): 6339-6347.
· Karami, S., Roayaei, M., Hamzavi, H., Bahmani, M., Hassanzad-Azar, H., Leila, M. and Rafieian-Kopaei, M. (2017). Isolation and identification of probiotic Lactobacillus from local dairy and evaluating their antagonistic effect on pathogens. International Journal of Pharmaceutical Investigation, 7(3):137-141.
· Kim, YS. and Cha, HJ. (2006). High-throughput and facile assay of antimicrobial peptides using pH-controlled fluorescence resonance energy transfer. Antimicrobial Agents and Chemotherapy, 50(10): 3330-3335.
· Klaenhammer T R. (1984). A general method for plasmid isolation in lactobacilli. Current Microbiology, 10(1): 23-28.
· Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259): 680-685.
· Liu, W., Zhang, L., Yi, H., Shi, J., Xue, C., Li, H. et al., (2014). Qualitative detection of class IIa bacteriocinogenic lactic acid bacteria from traditional Chinese fermented food using a YGNGV-motif-based assay. Journal of Microbiological Methods, 100: 121-127.
· Marie, KP., François, ZN., Abbasi, A., Anwar, F., Ali, SA., Victor, SD. et al., (2012). Characterization of a bacteriocin produced by Lactobacillus plantarum Lp6SH isolated from" Sha'a", a maize-based traditionally fermented beverage from cameroon. International Journal of Biology, 4(2):149-158.
· Nuding, S., Zabel, LT., Enders, C., Porter, E., Fellermann, K., Wehkamp, J. et al., (2009). Antibacterial activity of human defensins on anaerobic intestinal bacterial species: a major role of HBD-3. Microbes and Infection, 11(3): 384-393.
· Ogunbanwo, S.T., Sanni, A.I. and Onilude, A.A. (2003). Characterization of bacteriocin produced by Lactobacillus plantarum F1 and Lactobacillus brevis OGI. African Journal of Biotechnology, 2: 219-227.
· Papagianni, M. and Anastasiadou, S. (2009). Pediocins: The bacteriocins of Pediococci. sources, production, properties and applications. Microbial Cell Factories, 8(1): 1-16.
· Pingitore, EV., Salvucci, E., Sesma, F. and Nader-Macias, ME. (2007). Different strategies for purification of antimicrobial peptides from lactic acid bacteria (LAB). Communicating Current Research and Educational Topics and Trends in Applied Microbiology, 1:557-568.
· Tafkiki, S. and Hanifian, S. (2018). Inhibitory effect of native enterococci isolates on some of the foodborne bacterial pathogens. Journal of Food Hygiene, 9(33): 35-47. [In Persian]
· Tarinejad, A., Pourabdi Sarabi, P. and Hejazi, M.A. (2017). Antagonistic effect of native lactic acid bacteria against foodborne bacterial pathogens. Journal of Food Hygiene, 8(29): 25-38. [In Persian]
· Todoriki, K., Mukai, T., Sato, S. and Toba, T. (2001). Inhibition of adhesion of food‐borne pathogens to Caco‐2 cells by Lactobacillus strains. Journal of Applied Microbiology, 91:154-159.
· Van Belkum, MJ. and Stiles, ME. (2000). Nonlantibiotic antibacterial peptides from lactic acid bacteria. Natural Product Reports, 17(4): 323-335.
· Van Reenen, C., Chikindas, M., Van Zyl, W. and Dicks, L. (2003). Characterization and heterologous expression of a class IIa bacteriocin, plantaricin 423 from Lactobacillus plantarum 423, in Saccharomyces cerevisiae. International Journal of Food Microbiology, 81(1): 29-40.
· Vuyst, L. and Vandamme, E.J. (2012). Bacteriocins of lactic acid bacteria: Microbiology, genetics and applications. Springer press.
· Więckowicz, M., Schmidt, M., Sip, A. and Grajek, W. (2011). Development of a PCR‐based assay for rapid detection of class IIa bacteriocin genes. Letters in Applied Microbiology, 52:281-289.
· Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamura, K. (2018). MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35:1547-1549.
· Zacharof, M.P. and Lovitt, R.W. (2012). Bacteriocins produced by lactic acid bacteria a review article. APCBEE Procedia, 2: 50-56.
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· Anderssen, EL., Diep, DB., Nes, IF., Eijsink, VG. and Nissen-Meyer, J. (1998). Antagonistic activity of Lactobacillus plantarum C11: two new two-peptide bacteriocins, plantaricins EF and JK, and the induction factor plantaricin A. Applied and Environmental Microbiology, 64(6): 2269-2272.
· Banerjee, SP., Dora, KC. and Chowdhury, S. (2013). Detection, partial purification and characterization of bacteriocin produced by Lactobacillus brevis FPTLB3 isolated from freshwater fish. Journal of Food Science and Technology, 50(1): 17-25.
· Bastos, MCF., Coutinho, BG. and Coelho, MLV. (2010). Lysostaphin: a staphylococcal bacteriolysin with potential clinical applications. Pharmaceuticals, 3(4): 1139-1161.
· Campos, CA., Rodríguez, Ó., Calo-Mata, P., Prado, M. and Barros-Velázquez, J. (2006). Preliminary characterization of bacteriocins from Lactococcus lactis, Enterococcus faecium and Enterococcus mundtii strains isolated from turbot (Psetta maxima). Food Research International, 39(3): 356-364.
· Cho, G-S., Huch, M., Hanak, A., Holzapfel, WH. and Franz, CM. (2010a). Genetic analysis of the plantaricin EFI locus of Lactobacillus plantarum PCS20 reveals an unusual plantaricin E gene sequence as a result of mutation. International Journal of Food Microbiology, 141: S117-S124.
· Cho, G-S., Huch, M., Hanak, A., Holzapfel, WH. and Franz, Charles M.A.P. (2010b). Genetic analysis of the plantaricin EFI locus of Lactobacillus plantarum PCS20 reveals an unusual plantaricin E gene sequence as a result of mutation. International Journal of Food Microbiology, 141: S117-S124.
· Deshmukh, PV. and Thorat, PR. (2014). Detection antimicrobial efficasy of novel bacteriocin produced from Lactobacillus similis RL7. Advanced Research, 2(1): 987-995.
· Diep, DB., Håvarstein, L., Nissen-Meyer, J. and Nes, IJA. (1994). The gene encoding plantaricin A, a bacteriocin from Lactobacillus plantarum C11, is located on the same transcription unit as an agr-like regulatory system. Applied and Environmental Microbiology, 60(1): 160-166.
· Eijsink, VG., Axelsson, L., Diep, DB., Håvarstein, LS., Holo, H. and Nes IF. (2002). Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication. Antonie Van Leeuwenhoek, 81(1-4): 639-654.
· Gholamzadeh, MA., Hejazi, MA. and Hosseinzadeh Gharaje, N. (2017). Determination of bacteriocin encoding gene in six native strains of Lactobacillus plantarum. Iranian Journal of Food Science and Technology, 14(66): 17-25. [In Persian]
· Haas, H., Budowle, B. and Weiler, G. (1994). Horizontal polyacrylamide gel electrophoresis for the separation of DNA fragments. Electrophoresis, 15(1):153-158.
· Jacobsen, CN., Nielsen, VR., Hayford, A., Møller, PL., Michaelsen, K., Paerregaard, A. et al., (1999). Screening of probiotic activities of forty-seven strains of Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans. Applied and Environmental Microbiology, 65(11): 4949-4956.
· Joerger, M. and Klaenhammer, T. (1990). Cloning, expression, and nucleotide sequence of the Lactobacillus helveticus 481 gene encoding the bacteriocin helveticin J. Journal of Bacteriology, 172(11): 6339-6347.
· Karami, S., Roayaei, M., Hamzavi, H., Bahmani, M., Hassanzad-Azar, H., Leila, M. and Rafieian-Kopaei, M. (2017). Isolation and identification of probiotic Lactobacillus from local dairy and evaluating their antagonistic effect on pathogens. International Journal of Pharmaceutical Investigation, 7(3):137-141.
· Kim, YS. and Cha, HJ. (2006). High-throughput and facile assay of antimicrobial peptides using pH-controlled fluorescence resonance energy transfer. Antimicrobial Agents and Chemotherapy, 50(10): 3330-3335.
· Klaenhammer T R. (1984). A general method for plasmid isolation in lactobacilli. Current Microbiology, 10(1): 23-28.
· Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259): 680-685.
· Liu, W., Zhang, L., Yi, H., Shi, J., Xue, C., Li, H. et al., (2014). Qualitative detection of class IIa bacteriocinogenic lactic acid bacteria from traditional Chinese fermented food using a YGNGV-motif-based assay. Journal of Microbiological Methods, 100: 121-127.
· Marie, KP., François, ZN., Abbasi, A., Anwar, F., Ali, SA., Victor, SD. et al., (2012). Characterization of a bacteriocin produced by Lactobacillus plantarum Lp6SH isolated from" Sha'a", a maize-based traditionally fermented beverage from cameroon. International Journal of Biology, 4(2):149-158.
· Nuding, S., Zabel, LT., Enders, C., Porter, E., Fellermann, K., Wehkamp, J. et al., (2009). Antibacterial activity of human defensins on anaerobic intestinal bacterial species: a major role of HBD-3. Microbes and Infection, 11(3): 384-393.
· Ogunbanwo, S.T., Sanni, A.I. and Onilude, A.A. (2003). Characterization of bacteriocin produced by Lactobacillus plantarum F1 and Lactobacillus brevis OGI. African Journal of Biotechnology, 2: 219-227.
· Papagianni, M. and Anastasiadou, S. (2009). Pediocins: The bacteriocins of Pediococci. sources, production, properties and applications. Microbial Cell Factories, 8(1): 1-16.
· Pingitore, EV., Salvucci, E., Sesma, F. and Nader-Macias, ME. (2007). Different strategies for purification of antimicrobial peptides from lactic acid bacteria (LAB). Communicating Current Research and Educational Topics and Trends in Applied Microbiology, 1:557-568.
· Tafkiki, S. and Hanifian, S. (2018). Inhibitory effect of native enterococci isolates on some of the foodborne bacterial pathogens. Journal of Food Hygiene, 9(33): 35-47. [In Persian]
· Tarinejad, A., Pourabdi Sarabi, P. and Hejazi, M.A. (2017). Antagonistic effect of native lactic acid bacteria against foodborne bacterial pathogens. Journal of Food Hygiene, 8(29): 25-38. [In Persian]
· Todoriki, K., Mukai, T., Sato, S. and Toba, T. (2001). Inhibition of adhesion of food‐borne pathogens to Caco‐2 cells by Lactobacillus strains. Journal of Applied Microbiology, 91:154-159.
· Van Belkum, MJ. and Stiles, ME. (2000). Nonlantibiotic antibacterial peptides from lactic acid bacteria. Natural Product Reports, 17(4): 323-335.
· Van Reenen, C., Chikindas, M., Van Zyl, W. and Dicks, L. (2003). Characterization and heterologous expression of a class IIa bacteriocin, plantaricin 423 from Lactobacillus plantarum 423, in Saccharomyces cerevisiae. International Journal of Food Microbiology, 81(1): 29-40.
· Vuyst, L. and Vandamme, E.J. (2012). Bacteriocins of lactic acid bacteria: Microbiology, genetics and applications. Springer press.
· Więckowicz, M., Schmidt, M., Sip, A. and Grajek, W. (2011). Development of a PCR‐based assay for rapid detection of class IIa bacteriocin genes. Letters in Applied Microbiology, 52:281-289.
· Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamura, K. (2018). MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35:1547-1549.
· Zacharof, M.P. and Lovitt, R.W. (2012). Bacteriocins produced by lactic acid bacteria a review article. APCBEE Procedia, 2: 50-56.
