ISOLATION OF LACTIC ACID BACTERIA FROM TRADITIONAL PICKLE SAMPLES, THEIR IDENTIFICATION USING MOLECULAR METHODS, AND DETERMINATION OF SOME FUNCTIONAL PROPERTIES

Abstract

In this study, Lactic Acid Bacteria (LAB) were isolated and identified from 17 traditional pickled vegetable samples collected from different provinces and their functional roles were characterised. As a result of genotypic separation, 21 strains belonging to 9 different species were identified. These strains were further tested for their potential probiotic roles by testing their bile salts and low pH resistance and strains of Lactiplantibacillus plantarum and Levilactobacillus brevis demonstrated superior characteristics in terms of survival. The antibiotic resistance of isolates was found to be at very low levels. Importantly, while strain-specific effects were observed in terms of antifungal activity, they showed very strong activity in antibacterial activity. Finally, pickle isolates were tested for GABA production and Levilactobacillus brevis VB13 strain demonstrated 0.628±0.11 mg/mL GABA production. The results we obtained are important in terms of showing the diversity of LAB in pickles and the functional effect potential of these strains.

Keywords

• pickled vegetables
• Lactic Acid Bacteria (LAB)
• probiotic

Project Number

2021/69001-01-01

GELENEKSEL YOLLARLA ÜRETİLMİŞ TURŞU ÖRNEKLERİNDEN LAKTİK ASİT BAKTERİLERİNİN İZOLASYONU, MOLEKÜLER YÖNTEMLER KULLANILARAK TANIMLANMASI VE BAZI FONKSİYONEL ÖZELLİKLERİNİN BELİRLENMESİ

Abstract

Bu çalışmada ülkemizin farklı illerinden toplanmış 17 adet geleneksel turşu örneğinden Laktik Asit Bakterileri (LAB) izole edilip tanımlanmış ve izolatların fonksiyonel nitelikleri karakterize edilmiştir. Genotipik ayrıştırma sonucunda 9 farklı türe ait 21 suş tespit edilmiştir. Takiben bu 21 suşun potansiyel probiyotik değerlendirmeleri safra tuzlarına direnç ve düşük pH’da gelişim açısından test edilmiş ve Lactiplantibacillus plantarum ve Levilactobacillus brevis suşlarının yüksek canlılık gösterdiği tespit edilmiştir. İzolatlarının antibiyotik dirençlerinin ise oldukça düşük seviyede olduğu gözlenmiştir. Önemli olarak antifungal aktivite açısından suş spesifik etki gözlenirken, antibakteriyel aktivite noktasında oldukça güçlü aktivite sergilemişlerdir. Son olarak turşu izolatlarının GABA üretim potansiyelleri açığa çıkarılmış ve Levilactobacillus brevis VB13 suşunun 0.628±0.11 mg/mL GABA üretebildiği gösterilmiştir. Elde ettiğimiz sonuçlar turşuda bulunan LAB çeşitliliğini ve bu suşların fonksiyonel etki potansiyelini göstermesi bakımından önem arz etmektedir.

Keywords

• turşu
• Laktik Asit Bakterileri (LAB)
• probiyotik
• GABA üretimi

Supporting Institution

Bayburt Üniversitesi

Project Number

2021/69001-01-01

Thanks

Bu çalışma Bayburt Üniversitesi Bilimsel Araştırma Koordinatörlüğü (BAP) tarafından 2021/69001-01-01 nolu proje ile desteklenmiştir.

References

1. Abouloifa, H., Rokni, Y., Bellaouchi, R., Ghabbour, N., Karboune, S., Brasca, M., Salah, R. B., Chihib, N. E., Saalaoui, E., Asehraou, A. (2020). Characterization of Probiotic Properties of Antifungal Lactobacillus Strains Isolated from Traditional Fermenting Green Olives. Probiotics and Antimicrobial Proteins, 12(2): 683-696, doi:10.1007/s12602-019-09543-8.
2. Aljahani, A. H. (2020). Microbiological and physicochemical quality of vegetable pickles. Journal of the Saudi Society of Agricultural Sciences, 19(6): 415-421, doi:https://doi.org/10.1016/ j.jssas.2020.07.001.
3. Amini, E., Salimi, F., Imanparast, S., Mansour, F. N. (2022). Isolation and characterization of exopolysaccharide derived from Lacticaseibacillus paracasei AS20 (1) with probiotic potential and evaluation of its antibacterial activity. Letters in Applied Microbiology, 75(4): 967-981, doi.org/10.1111/lam.13771.
4. Bağder Elmacı, S., Tokatlı, M., Dursun, D., Özçelik, F., Şanlıbaba, P. (2015). Phenotypic and genotypic identification of lactic acid bacteria isolated from traditional pickles of the Çubuk region in Turkey. Folia Microbiologica, 60(3): 241-251, doi:10.1007/s12223-014-0363-x.
5. Behera, S. S., El Sheikha, A. F., Hammami, R., Kumar, A. (2020). Traditionally fermented pickles: How the microbial diversity associated with their nutritional and health benefits? Journal of Functional Foods, 70, 103971, https://doi.org/10.1016/j.jff.2020.103971.
6. Bhunia, A.K. (2018). Molds and Mycotoxins. In: Foodborne Microbial Pathogens, Springer, New York, NY, pp. 167-174.
7. Chakraborty, P., Dey, A., Gopalakrishnan, A. V., Swati, K., Ojha, S., Prakash, A., Kumar, D., Ambasta, R. K., Jha, S. K., Dewanjee, S. (2023). Glutamatergic Neurotransmission: A Potential Pharmacotherapeutic Target for the Treatment of Cognitive Disorders. Ageing Research Reviews, 101838, https://doi.org/10.1016/ j.arr.2022.101838.
8. Chen, O., Hong, Y., Ma, J., Deng, L., Yi, L., Zeng, K. (2021). Screening lactic acid bacteria from pickle and cured meat as biocontrol agents of Penicillium digitatum on citrus fruit. Biological Control, 158, 104606, https://doi.org/10.1016/ j.biocontrol.2021.104606.

9. Chen, Y.-s., Wu, H.-c., Pan, S.-f., Lin, B.-g., Lin, Y.-h., Tung, W.-c., Li, Y.-l., Chiang, C.-m., Yanagida, F. (2013). Isolation and characterization of lactic acid bacteria from yan-taozih (pickled peaches) in Taiwan. Annals of Microbiology, 63(2): 607-614, DOI 10.1007/ s13213-012-0510-z.
10. Cho, Y.-R., Chang, J.-Y., Chang, H.-C. (2007). Production of γ−Aminobutyric Acid (GABA) by Lactobacillus buchneri isolated from Kimchi and its neuroprotective effect on neuronal cells. Journal of Microbiology and Biotechnology, 17(1): 104-109.
11. Coda, R., Rizzello, C. G., Gobbetti, M. (2010). Use of sourdough fermentation and pseudo-cereals and leguminous flours for the making of a functional bread enriched of γ-aminobutyric acid (GABA). International journal of food microbiology, 137(2-3): 236-245, https://doi.org/10.1016/ j.ijfoodmicro.2009.12.010
12. Çetin, B. (2011). Production of probiotic mixed pickles (Tursu) and microbiological properties. African Journal of Biotechnology, 10(66): 14926-14931, DOI: 10.5897/AJB11.2621.
13. Demirbaş, F., İspirli, H., Kurnaz, A. A., Yilmaz, M. T., Dertli, E. (2017). Antimicrobial and functional properties of lactic acid bacteria isolated from sourdoughs. LWT-Food Science and Technology, 79: 361-366, https://doi.org/10.1016/ j.lwt.2017.01.067.
14. Dertli, E., Mercan, E., Arıcı, M., Yılmaz, M. T., Sağdıç, O. (2016). Characterisation of lactic acid bacteria from Turkish sourdough and determination of their exopolysaccharide (EPS) production characteristics. LWT - Food Science and Technology, 71:116-124, doi:doi.org/10.1016/ j.lwt.2016.03.030.
15. Di Biase, M., Le Marc, Y., Bavaro, A. R., De Bellis, P., Lonigro, S. L., Lavermicocca, P., Postollec, F., Valerio, F. (2022). A predictive growth model for pro-technological and probiotic Lacticaseibacillus paracasei strains fermenting white cabbage. Frontiers in Microbiology, 13, doi: 10.3389/fmicb.2022.907393.
16. Di Cagno, R., Coda, R., De Angelis, M., Gobbetti, M. (2013). Exploitation of vegetables and fruits through lactic acid fermentation. Food Microbiology, 33(1): 1-10, doi:https://doi.org/10.1016/ j.fm.2012.09.003.
17. Dušková, M., Morávková, M., Mrázek, J., Florianová, M., Vorlová, L., Karpíšková, R. (2021). Assessment of antibiotic resistance in starter and non-starter lactobacilli of food origin. Acta Veterinaria Brno, 89(4): 401-411, https://doi.org/10.2754/avb202089040401.
18. Encu, Ş. B., Soykut, E. A., Çakır, İ. (2022). geleneksel yoğurtlardan izole edilen laktik asit bakterilerinin MALDI TOF MS biotyper sistemi ile tanimlanmasi ve bazi starter kültür özelliklerinin belirlenmesi. Gıda, 47(6): 1059-1082, doi: 10.15237/ gida.GD22088.
19. Erten, H., Boyacı-Gündüz, C. P., Ağırman, B., Cabaroğlu, T. (2016). Fermentation, pickling and Turkish table olives. Handbook of vegetable preservation and processing, 209-230.
20. Hassanzadazar, H., Ehsani, A., Mardani, K. (2014). Antibacterial activity of Enterococcus faecium derived from Koopeh cheese against Listeria monocytogenes in probiotic ultra-filtrated cheese. Veterinary research forum: an international quarterly journal, 5 (3):169 – 175.
21. Huang, J., Mei, L. H., Wu, H., Lin, D. Q. (2007). Biosynthesis of γ-aminobutyric acid (GABA) using immobilized whole cells of Lactobacillus brevis. World Journal of Microbiology and Biotechnology, 23, 865-871, DOI 10.1007/s11274-006-9311-5.
22. Huang, Y., Luo, Y., Zhai, Z., Zhang, H., Yang, C., Tian, H., Li, Z., Feng, J., Liu, H., Hao, Y. (2009). Characterization and application of an anti-Listeria bacteriocin produced by Pediococcus pentosaceus 05-10 isolated from Sichuan Pickle, a traditionally fermented vegetable product from China. Food Control, 20(11): 1030-1035. https://doi.org/10.1016/j.foodcont.2008.12.008
23. Hwanhlem, N., Buradaleng, S., Wattanachant, S., Benjakul, S., Tani, A., Maneerat, S. (2011). Isolation and screening of lactic acid bacteria from Thai traditional fermented fish (Plasom) and production of Plasom from selected strains. Food Control, 22(3-4): 401-407, https://doi.org/ 10.1016/j.foodcont.2010.09.010.
24. Inoue, K., Shirai, T., Ochiai, H., Kasao, M., Hayakawa, K., Kimura, M., Sansawa, H. (2003). Blood-pressure-lowering effect of a novel fermented milk containing γ-aminobutyric acid (GABA) in mild hypertensives. European journal of clinical nutrition, 57(3): 490-495.
25. Ispirli, H., Dertli, E. (2021). Detection of fructophilic lactic acid bacteria (FLAB) in bee bread and bee pollen samples and determination of their functional roles. Journal of Food Processing and Preservation, 45(5), e15414, https://doi.org/10.1111/jfpp.15414.
26. İspirli, H., Demirbaş, F., Dertli, E. (2015). Characterization of functional properties of Enterococcus faecium strains isolated from human gut. Canadian journal of microbiology, 61(11): 861-870, https://doi.org/10.1139/cjm-2015-0446.
27. Jin, Y., Wu, J., Hu, D., Li, J., Zhu, W., Yuan, L., Chen, X., Yao, J. (2023). Gamma-Aminobutyric Acid-Producing Levilactobacillus brevis Strains as Probiotics in Litchi Juice Fermentation. Foods, 12(2):302, https://doi.org/10.3390/ foods12020302.
28. Jonganurakkun, B., Wang, Q., Xu, S. H., Tada, Y., Minamida, K., Yasokawa, D., Sugi, M., Hara, H., Asano, K. (2008). Pediococcus pentosaceus NB-17 for probiotic use. Journal of Bioscience and Bioengineering, 106(1): 69-73, https://doi.org/10.1263/ jbb.106.69.
29. Karasu, N. (2006). Turşu ve zeytinden antagonistik ve probiyotik özellikte laktik starter kültür eldesi. Pamukkale Üniversitesi Fen Bilimleri Enstitüsü Gıda Mühendisliği Anabilim Dalı Yüksek Lisans Tezi, Denizli, Türkiye, 78 s.
30. Kaya, Y., Erten, T., Vurmaz, M., İspirli, H., Şimşek, Ö., Dertli, E. (2022). Comparison of the probiotic characteristics of Lactic Acid Bacteria (LAB) isolated from sourdough and infant feces. Food Bioscience, 47, 101722, doi:https://doi.org/10.1016/j.fbio.2022.101722.
31. Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K. (2018). MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular biology and evolution, 35(6): 1547–1549, doi: 10.1093/molbev/msy096.
32. Lee, B.-H., Wu, S.-C., Shen, T.-L., Hsu, Y.-Y., Chen, C.-H., Hsu, W.-H. (2021). The applications of Lactobacillus plantarum-derived extracellular vesicles as a novel natural antibacterial agent for improving quality and safety in tuna fish. Food Chemistry, 340, 128104, https://doi.org/10.1016/ j.foodchem.2020.128104.
33. Liu, C., Xue, W.-j., Ding, H., An, C., Ma, S.-j., Liu, Y. (2022). Probiotic Potential of Lactobacillus Strains Isolated From Fermented Vegetables in Shaanxi, China. Frontiers in Microbiology, 12, 4168, https://doi.org/10.3389/fmicb.2021.774903.
34. Magnusson, J., Schnürer, J. (2001). Lactobacillus coryniformis subsp. coryniformis strain Si3 produces a broad-spectrum proteinaceous antifungal compound. Applied and environmental microbiology, 67(1):1-5, https://doi.org/10.1128/AEM.67.1.1-5.2001.
35. Mao, B., Yin, R., Li, X., Cui, S., Zhang, H., Zhao, J., Chen, W. (2021). Comparative genomic analysis of Lactiplantibacillus plantarum isolated from different niches. Genes, 12(2): 241, https://doi.org/10.3390/genes12020241.
36. Mathur, S., Singh, R. (2005). Antibiotic resistance in food lactic acid bacteria—a review. International journal of food microbiology, 105(3): 281-295, doi:10.1016/j.ijfoodmicro.2005.03.008.
37. Miura, D., Ito, Y., Mizukuchi, A., Kise, M., Aoto, H., Yagasaki, K. (2006). Hypocholesterolemic action of pre-germinated brown rice in hepatoma-bearing rats. Life sciences, 79(3): 259-264, https://doi.org/10.1016/j.lfs.2006.01.001.
38. Monteagudo-Mera, A., Rastall, R. A., Gibson, G. R., Charalampopoulos, D., Chatzifragkou, A. (2019). Adhesion mechanisms mediated by probiotics and prebiotics and their potential impact on human health. Applied microbiology and biotechnology, 103, 6463-6472, https://doi.org/10.1007/s00253-019-09978-7.
39. Moon, S. H., Kim, C. R., Chang, H. C. (2018). Heterofermentative lactic acid bacteria as a starter culture to control kimchi fermentation. LWT, 88, 181-188, doi:https://doi.org/10.1016/ j.lwt.2017.10.009.
40. Nakatani, Y., Fukaya, T., Kishino, S., Ogawa, J. (2022). Production of GABA-enriched tomato juice by Lactiplantibacillus plantarum KB1253. Journal of Bioscience and Bioengineering, 134(5): 424-431, https://doi.org/10.1016/ j.jbiosc.2022.08.008.
41. Nasiri Moslem, M., Faezi Ghasemi, M., Amirmozafari, N., Ranji, N. (2022). Antimicrobial activity of Pediococcus acidilactici PTCC 1954 and Leuconostoc mesenteroides PTCC 1953 isolated from organic meat sausages. Biological Journal of Microorganism, doi.10.22108/bjm.2022.134313.1473.
42. Park, K. B., Oh, S. H. (2007). Production of yogurt with enhanced levels of gamma-aminobutyric acid and valuable nutrients using lactic acid bacteria and germinated soybean extract. Bioresource technology, 98(8): 1675-1679, https://doi.org/10.1016/j.biortech.2006.06.006.
43. Plokhov, A. Y., Gusyatiner, M. M., Yampolskaya, T. A., Kaluzhsky, V. E., Sukhareva, B. S., Schulga, A. A. (2000). Preparation of γ-aminobutyric acid using E. coli cells with high activity of glutamate decarboxylase. Applied biochemistry and biotechnology, 88, 257-265.
44. Purutoğlu, K., İspirli, H., Yüzer, M. O., Serencam, H., Dertli, E. (2020). Diversity and functional characteristics of lactic acid bacteria from traditional kefir grains. International Journal of Dairy Technology, 73(1): 57-66, https://doi.org/10.1111/ 1471-0307.12633.
45. Sagdic, O., Ozturk, I., Yapar, N., Yetim, H. (2014). Diversity and probiotic potentials of lactic acid bacteria isolated from gilaburu, a traditional Turkish fermented European cranberrybush (Viburnum opulus L.) fruit drink. Food Research International, 64, 537-545, https://doi.org/ 10.1016/j.foodres.2014.07.045.
46. Saitou, N., Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular biology and evolution, 4(4): 406-425, https://doi.org/10.1093/ oxfordjournals.molbev.a040454.
47. Sathe, S., Nawani, N., Dhakephalkar, P., Kapadnis, B. (2007). Antifungal lactic acid bacteria with potential to prolong shelf‐life of fresh vegetables. Journal of Applied Microbiology, 103(6): 2622-2628, https://doi.org/10.1111/ j.1365-2672.2007.03525.x.
48. Schurr, B. C., Hahne, H., Kuster, B., Behr, J., Vogel, R. F. (2015). Molecular mechanisms behind the antimicrobial activity of hop iso-α-acids in Lactobacillus brevis. Food Microbiology, 46, 553-563, https://doi.org/10.1016/ j.fm.2014.09.017.
49. Štšepetova, J., Taelma, H., Smidt, I., Hütt, P., Lapp, E., Aotäht, E., Mändar, R. (2017). Assessment of phenotypic and genotypic antibiotic susceptibility of vaginal Lactobacillus sp. Journal of Applied Microbiology, 123(2): 524-534, doi:https://doi.org/10.1111/jam.13497.
50. Suzuki, S., Kimoto-Nira, H., Suganuma, H., Suzuki, C., Saito, T., Yajima, N. (2014). Cellular fatty acid composition and exopolysaccharide contribute to bile tolerance in Lactobacillus brevis strains isolated from fermented Japanese pickles. Canadian journal of microbiology, 60(4): 183-191, https://doi.org/10.1139/cjm-2014-0043.
51. Tamura, K., Nei, M., Kumar, S. (2004). Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences, 101(30): 11030-11035, https://doi.org/10.1073/pnas.0404206101.
52. Todorov, S., Onno, B., Sorokine, O., Chobert, J., Ivanova, I., Dousset, X. (1999). Detection and characterization of a novel antibacterial substance produced by Lactobacillus plantarum ST 31 isolated from sourdough. International journal of food microbiology, 48(3): 167-177, https://doi.org/ 10.1016/S0168-1605(99)00048-3.
53. Tokatlı, M. (2013). Ankara çubuk yöresi turşularından izole edilen laktik asit bakterilerinin tanımlanmaları, teknolojik ve fonksiyonel özelliklerinin belirlenmesi ve starter olarak kullanılma olanaklarının değerlendirilmesi. Ankara Üniversitesi Fen Bilimleri Enstitüsü Gıda Mühendisliği Anabilim Dalı, Doktora Tezi, Ankara, Türkiye, 183 s.
54. Tokatlı, M., Elmacı, S. B., İşleyen, N. A., Özçelik, F. (2019). Seçilmiş endojen laktik starter kültürler ile turşu üretimi. Gıda, 44(4): 742-757, https://doi.org/10.15237/gida.GD19081.
55. Tokatlı, M., Gülgör, G., Bağder Elmacı, S., Arslankoz İşleyen, N., Özçelik, F. (2015). In vitro properties of potential probiotic indigenous lactic acid bacteria originating from traditional pickles. BioMed Research International, https://doi.org/10.1155/2015/315819.
56. Villegas, J. M., Brown, L., de Giori, G. S., Hebert, E. M. (2016). Optimization of batch culture conditions for GABA production by Lactobacillus brevis CRL 1942, isolated from quinoa sourdough. LWT-Food Science and Technology, 67, 22-26, https://doi.org/10.1016/j.lwt.2015.11.027.
57. Wang, C.-Y., Lin, P.-R., Ng, C.-C., Shyu, Y.-T. (2010). Probiotic properties of Lactobacillus strains isolated from the feces of breast-fed infants and Taiwanese pickled cabbage. Anaerobe, 16(6): 578-585, https://doi.org/10.1016/ j.anaerobe.2010.10.003.
58. Wu, Q., Shah, N. P. (2017). High γ-aminobutyric acid production from lactic acid bacteria: emphasis on Lactobacillus brevis as a functional dairy starter. Critical Reviews in Food Science and Nutrition, 57(17): 3661-3672. https://doi.org/ 10.1080/10408398.2016.1147418.
59. Xiong, T., Guan, Q., Song, S., Hao, M., Xie, M. (2012). Dynamic changes of lactic acid bacteria flora during Chinese sauerkraut fermentation. Food control, 26(1): 178-181, https://doi.org/ 10.1016/j.foodcont.2012.01.027.
60. Yildirim, Z., Yildirim, M. (2001). Characterization of buchnericin LB produced by Lactobacillus buchneri LB. Turkish Journal of Biology, 25(1): 73-82.
61. Yuliana, N., Nurdjanah, S., Margareta, M. (2013). The Effect of a mixed-starter culture of lactic acid bacteria on the characteristics of pickled orange-fleshed sweet potato (Ipomoea batatas L.). Microbiology Indonesia, 7(1), 1-1, https://doi.org/10.5454/mi.7.1.1.
62. Zarzecka, U., Zadernowska, A., Chajęcka-Wierzchowska, W., Adamski, P. (2023). High-pressure processing effect on conjugal antibiotic resistance genes transfer in vitro and in the food matrix among strains from starter cultures. International Journal of Food Microbiology, 110104, https://doi.org/10.1016/j.ijfoodmicro.2023.110104.
63. Zeng, Y., Li, Y., Wu, Q., Zhang, J., Xie, X., Ding, Y., Cai, S. Z., Ye, Q. H., Chen, M. T., Xue, L., Wu, S., Zeng, H. Y., Yang, X. J., Wang J. (2020). Evaluation of the antibacterial activity and probiotic potential of Lactobacillus plantarum isolated from Chinese homemade pickles.Canadian Journal of Infectious Diseases and Medical Microbiology, 2020, 1-11, https://doi.org/10.1155/2020/8818989.
64. Zielińska, D., Rzepkowska, A., Radawska, A., Zieliński, K. (2015). In vitro screening of selected probiotic properties of Lactobacillus strains isolated from traditional fermented cabbage and cucumber. Current microbiology, 70, 183-194, DOI 10.1007/s00284-014-0699-0.