ارزیابی ویژگی‌های پروبیوتیکی و ضدقارچی مخمر جدا شده از خمیرترش باکویت

نوع مقاله : مقاله پژوهشی

نویسندگان

• سارا شهریاری ¹
• علیرضا صادقی ¹
• مریم ابراهیمی ²
• علیرضا صادقی ماهونک ¹
• علی مویدی ³

¹ گروه علوم و صنایع غذایی، دانشگاه علوم کشاورزی و منابع طبیعی گرگان، گرگان، ایران.

² مرکز تحقیقات سلامت فراورده‌های غذایی، دارویی و طبیعی، دانشگاه علوم پزشکی گلستان، گرگان، ایران.

³ دانشگاه علوم کشاورزی و منابع طبیعی گرگان

https://doi.org/10.22067/ifstrj.2022.74006.1119

چکیده

ارزیابی خصوصیات ضدمیکروبی و پروبیوتیکی فلورمیکروبی جدا شده از شبه غلات تخمیرشده جهت تامین کشت‌های میکروبی همراه و آغازگر از اهمیت به‌سزایی برخوردار است. در این پژوهش، مخمر غالب از خمیرترش باکویت، جداسازی و با استفاده از PCR شناسایی گردید. سپس ویژگی‌های پروبیوتیکی و همچنین اثر ضدقارچی این جدایه مخمری بر روی Aspergillus niger وAspergillus flavus مورد مطالعه قرارگرفت. توالی‌یابی محصولات PCR منجر به شناسایی مخمر Pichia kudriavzevii به‌عنوان جدایه مخمری خمیرترش باکویت شد. نرخ زنده‌مانی جدایه مذکور پس از تیمار متوالی اسید و صفرا در مقایسه با نمونه شاهد 26/79 درصد بود. همچنین اثر ضدباکتریایی آن در برابر Escherichia coli نسبت به سایر عوامل غذازاد مورد مطالعه به شکل معنی‌داری (05/0p<) بیشتر بود. با این حال، رشد Listeria monocytogenes در حضور جدایه مخمری 50/19 درصد کاهش یافت اما این جدایه، اثر بازدارنده‌ای بر Salmonella entrica نداشت. قابلیت‌های آبگریزی و خوداتصالی جدایه مذکور نیز به‌ترتیب، 07/64 و 40/67 درصد تعیین گردید. علاوه بر این، جدایه مخمری نسبت به تمامی آنتی‌بیوتیک‌های مورد بررسی، مقاومت نشان داد اما در برابر آنتی‌مایکوتیک‌های کتوکونازول و فلوکونازول، مقاوم و همچنین نسبت به ایتراکونازول دارای حساسیت نسبی بود. این جدایه مخمری، فاقد فعالیت همولیتیکی بود و اثر ضدقارچی آن در برابر A. niger و A. flavus نیز مورد تایید قرار گرفت. بر این اساس، جدایه P. kudriavzevii از قابلیت مناسبی برای استفاده به‌عنوان کشت پروبیوتیک و یا محافظت کننده در صنایع تخمیری برخوردار می‌باشد.

کلیدواژه‌ها

• جدایه مخمری
• خمیرترش باکویت
• ویژگی‌های پروبیوتیکی
• اثر ضدقارچی

موضوعات

• زیست فناوری مواد غذایی

عنوان مقاله [English]

Evaluation of probiotic and antifungal properties of the yeast isolated from buckwheat sourdough

نویسندگان [English]

• Sara Shahryari ¹
• Alireza Sadeghi ¹
• Maryam Ebrahimi ²
• Alireza Sadeghi Mahoonak ¹
• Ali Mayedi ³

¹ Department of Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gonrgan, Iran

² Health Research Center of Food, Drug and Natural Products, Golestan University of Medical Science, Gonrgan, Iran.

³ Gorgan

چکیده [English]

[1]Introduction: Evaluation of antimicrobial and probiotic properties of the microbiota isolated from fermented pseudo-cereals is important in order to prepare adjunct and starter cultures. Probiotics are live and active microorganisms that, if used in sufficient numbers, help the microbial balance of the gastrointestinal tract and improve its function. Probiotics are also used as a substitute for antibiotics and synthetic preservatives in the prevention and treatment of complications of many infectious and pathogenic gastrointestinal pathogens. Probiotic microorganisms include lactic acid bacteria (LAB) and yeasts, and despite numerous reports on the probiotic properties of LAB, the probiotic properties of yeasts are less reported. Probiotic yeasts are organisms resistant to antibacterial compounds and effective against pathogens, which can rapidly increase their population in the gastrointestinal tract. These yeasts have several properties such as antimicrobial effects, resistance to acid and bile, binding to mucosal surfaces, inhibitory activity against pathogens and also the inability to transmit antibiotic resistance genes. In the present study, after isolation of the predominant yeast from buckwheat sourdough, the probiotic and antifungal properties of the isolate were investigated. Based on the literature review, no study has been presented to evaluate the probiotic and antifungal capabilities of yeasts isolated from buckwheat sourdough.
 
Materials and Methods: In the present study, predominant yeast was isolated from buckwheat sourdough, and then it was identified using PCR. Subsequently, probiotic properties of the isolate including survival in present of low pH and bile salt, antibacterial effect, antibiotic susceptibility assay, aggregation, hydrophobicity, haemolytic activity as well as its antifungal activity against Aspergillus niger and Aspergillus flavus were studied. After spontaneous fermentation of buckwheat, predominant yeast was isolated using ten-fold dilution of the sourdough sample and its spread plating. The predominant isolate was identified through PCR amplification of a 650 bp target sequence from its ribosomal gene and sequencing of the PCR product. Then, survival of the yeast was determined at pH=2 and 0.3% bile salt as simulated gastrointestinal conditions. Subsequently, simultaneous culture of the yeast with some food-borne indicator bacteria in chromogenic media was used to investigate the inhibitory activity of the isolate against the studied bacteria. After that, resistance of the yeast isolate against the common antibiotics and some antimycotic agents was evaluated using disc method. Co-aggregation ability and hydrophobicity capability of the isolate were also determined based on the absorbance tests. In vitro safety of the yeast isolate was checked through its cultivation on blood agar containing sheep blood. Next stage, overlay bioassay was conducted to investigate antifungal effect of the yeast on the selected fungi. Finally, one way analysis of variance (ANOVA) with the least significant differences (LSD) post hock at p<0.05 was used for statistical analysis of the data. 
Results and Discussions: Sequencing results of the PCR products led to the identification of Pichia kudriavzevii as predominant yeast isolated from buckwheat sourdough. Survival rate of the isolate after treatment in simulated gastrointestinal conditions was 79.26% in comparison with the control. Antibacterial effect of the isolate on Escherichia coli was also significantly (P<0.05) higher than the other studied food-borne agents. Meanwhile, the growth of Listeria monocytogenes was decreased 19.50% in the present of the isolate. Whereas, the yeast isolate had no inhibitory effect on Salmonella enterica. Hydrophobicity and auto-aggregation capabilities of the isolate were also 64.07 and 67.40%, respectively. Furthermore, the isolate showed resistance towards all of the antibiotics tested, while it was resistant against ketoconazole and fluconazole, and the yeast was semi-sensitive towards itraconazole as antimycotic agents. The yeast isolate had no hemolytic activity, and its antifungal activity against A. niger and A. flavus was also verified. Accordingly, it is concluded that P. kudriavzevii isolate exhibits proper potential to be used as probiotic or protective culture in fermentation industries.
In accordance with the results, probiotic characteristics of P. kudriavzevii (as the predominant yeast isolated from buckwheat sourdough) were approved. Accordingly, the isolate can be used as a potential probiotic culture in food industries.

کلیدواژه‌ها [English]

• Yeast isolate
• Buck wheat sourdough
• Probiotic properties
• Antifungal effect

مراجع

1. AACC, International. (2010). Approved methods of the American association of cereal chemists. 11th Ed. The St. Paul.
2. Ahmed, A., Khalid, N., Ahmad, A., Abbasi, N. A., Latif, M. S. Z., Randhava, M. A. (2014). Phytochemicals and biofunctional properties of buckwheat. a review. The Journal of Agricultural Science, 152 (3), 349–369. https://doi.org/10.1017/S0021859613000166
3. Andrabi, S. T., Bhat, B., Gupta, M., Bajaj, B. K. (2016). Phytase-producing potential and other functional attributes of lactic acid bacteria isolates for prospective probiotic applications. Probiotics and Antimicrobial Proteins, 8(3), 121-129. https://doi.org/10.1007/s12602-016-9220-3
4. Angelov, A., Gotcheva, V., Hristozova, T., Gargova, S. (2005). Application of pure and mixed probiotic lactic acid bacteria and yeast cultures for oat fermentation. Journal of the Science of Food and Agriculture, 85 (12), 2134–2141. https://doi.org/10.1002/jsfa.2223
5. Angmo, K., Kumari, A., Bhalla, T. C. (2016). Probiotic characterization of lactic acid bacteria isolated from fermented foods and beverage of Ladakh. LWT-Food Science and Technology, 66, 428-435. https://doi.org/10.1016/j.lwt.2015.10.057
6. Arroyo López, F. N., Romero Gil, V., Bautista Gallego, J., Rodriguez Gomez, F., Jimenez Diaz, R., García García, P., Garrido Fernandez, A. (2012). Potential benefits of the application of yeast starters in table olive processing. Frontiers in Microbiology, 3, 161. https://doi.org/10.3389/fmicb.2012.00161
7. Bajaj, B. K., Claes, I. J., Lebeer, S. (2021). Functional mechanisms of probiotics. Journal of Microbiology, Biotechnology and Food Sciences, 2021, 321-327.‏
8. Banik, A., Mondal, J., Rakshit, S., Ghosh, K., Sha, S. P., Kumar, Halder, S., Ghosh, C., Mondal, K. C. (2019). Amelioration of cold-induced gastric injury by a yeast probiotic isolated from traditional fermented foods. Journal of Functional Foods, 59, 164–173. https://doi.org/10.1016/j.jff.2019.05.039
9. Banu,, Vasilean, I., Aprodu, I. (2011). Effect of select parameters of the sourdough rye fermentation on the activity of some mixed starter cultures. Food Biotechnology, 25(4), 275-291. https://doi.org/10.1080/08905436.2011.617251
10. Bevilacqua,, Corbo, M. R., Sinigaglia, M. (2012). Selection of yeasts as starter cultures for table olives: a step-by-step procedure. Frontiers in Microbiology, 3, 1-9. https://doi.org/10.3389/fmicb.2012.00194
11. Chelliah, R., Ramakrishnan, S. R., Prabhu, P. R., Antony, U. (2016). Evaluation of antimicrobial activity and probiotic properties of wild‐strain Pichia kudriavzevii isolated from frozen idli batter. Yeast, 33(8), 385-401.‏ https://doi.org/10.1002/yea.3181
12. Czerucka, D., Piche, T., Rampal, P. (2007). Review article: Yeast as probiotics: Saccharomyces boulardii. Alimentary Pharmacology & Therapeutics, 26(6), 767-778. https://doi.org/10.1111/j.1365-2036.2007.03442.x
13. De Vuyst, L., Neysens, P. (2005). The sourdough microflora: biodiversity and metabolic interactions. Trends in Food Science & Technology, 16(1-3), 43-56. https://doi.org/10.1016/j.tifs.2004.02.012
14. Fadda, M. E., Mossa, V., Deplano, M., Pisano, M. B., Cosentino, S. (2017). In vitro screening of Kluyveromyces strains isolated from Fiore Sardo cheese for potential use as probiotics. LWT-Food Science and Technology, 75, 100-106. https://doi.org/10.1016/j.lwt.2016.08.020
15. Fakruddin, M. D., Nur Hossain, M. D., Ahmed, M. M. (2017). Antimicrobial and antioxidant activities of Saccharomyces cerevisiae IFST062013, a potential probiotic, BMC Complementary and Alternative Medicine, 17(1), 1-11. https://doi.org/10.1186/s12906-017-1591-9
16. Fekri, A., Torbati, M. A., Yari Khosrowshahi, A., Bagherpour Shamlood, H., Azadmard- Damirchi, S. (2020). Functional effects of phytate-degrading, probiotic lactic acid bacteria and yeast strains isolated from Iranian traditional sourdough on the technological and nutritional properties of whole wheat bread. Food Chemistry, 306, 125620. https://doi.org/10.1016/j.foodchem.2019.125620
17. Fernandez-Pacheco, P., Arevalo-Villena, M., Bevilacqua, A., Corbo, M., Beriones Perez, A. (2018). Probiotic characterization in Saccharomyces cerevisiae strains: application in food industries. LWT-Food Science and Technology, 97, 332-340. https://doi.org/10.1016/j.lwt.2018.07.007
18. García-Hernández, Y., Rodríguez, Z., Brandão, L. R., Rosa, C. A., Nicoli, J. R., Iglesias, A. E., Pérez-Sanchez, T., Salabarría, R. B., Halaihel, N. (2012). Identification and in vitro screening of avian yeasts for use as probiotic. Research in Veterinary Science, 93(2), 798-802. https://doi.org/10.1016/j.rvsc.2011.09.005
19. Gil-Rodriguez, A. M., Carrascosa, A. V., Requena, T. (2015). Yeasts in foods and beverages: In vitro characterisation of probiotic traits. LWT-Food Science and Technology, 64(2), 1156-1162. https://doi.org/10.1016/j.lwt.2015.07.042
20. Goretti, M., Turchetti, B., Buratta, M., Branda, E., Corazzi, l., Vaughan-Martini, A., Buzzini, P. (2009). In vitro antimycotic activity of a Williopsis saturnus killer protein against food spoilage yeasts. International Journal of Food Microbiology, 131(2-3), 178–182. https://doi.org/10.1016/j.ijfoodmicro.2009.02.013
21. Greppi, A., Krych, L., Costantini, A., Rantsiou, K., Hounhouigan, D. J., Arneborg, N. (2015). Phytase-producing capacity of yeasts isolated from traditional African fermented food products and PHYPk gene expression of Pichia kudriavzevii International Journal of Food Microbiology, 205, 81–89. https://doi.org/10.1016/j.ijfoodmicro.2015.04.011
22. Greppi, A., Saubade, F., Botta, C., Humblot, C., Guyot, J.P., Cocolin, L. (2017). Potential probiotic Pichia kudriavzevii strains and their ability to enhance folate content of traditional cereal-based African fermented food. Food Microbiology, 62, 169-177.‏ https://doi.org/10.1016/j.fm.2016.09.016
23. Hammes, W. P., Brandt, M. J., Francis, K. L., Rosenheim, J., Seitter M. F., Vogelmann, S. A. (2005). Microbial ecology of cereal fermentations. Trends in Food Science & Technology, 16(1-3), 4-11. https://doi.org/10.1016/j.tifs.2004.02.010
24. Ilavenil, S., Vijayakumar, M., Kim, D. H., Valan Arasu, M., Park, H. S., Ravikumar, S., Choi, K. C. (2016). Assessment of probiotic, antifungal and cholesterol lowering properties of Pediococcus pentosaceus KCC‐23 isolated from Italian ryegrass. Journal of the Science of Food and Agriculture, 96(2), 593-601.‏ https://doi.org/10.1002/jsfa.7128
25. Janković, T., Frece, J., Abram, M., Gobin, I. (2012). Aggregation ability of potential probiotic Lactobacillus plantarum International Journal of Sanitary Engineering Research, 6(1), 19-24.
26. Kanafani, Z. A., Perfect, j. R. (2014). Resistance to Antifungal Agents: Mechanisms and Clinical Impact. Clinical Infectious Diseases, 46(1), 120-128.‏ https://doi.org/10.1086/524071
27. Kapetanakou, A. E., Kollias, J. N., Drosinos, E. H., Skandamis, P. N. (2012). Inhibition of carbonarius growth and reduction of ochratoxin A by bacteria and yeast composites of technological importance in culture media and beverages. International Journal of Food Microbiology, 152(3), 91–99. https://doi.org/10.1016/j.ijfoodmicro.2011.09.010
28. La Penna, M., Nesci, A., Etcheverry, (2004). In vitro studies on the potential for biological control on Aspergillus section Flavi by Kluyveromyces spp. Letters in Applied Microbiology, 38(4), 257–264. https://doi.org/10.1111/j.1472-765X.2003.01467.x
29. Magliani, W., Conti, S., Frazzi, R., Ravanetti, L., Maffei, D. L., Polonelli, L. (2005). Protective antifungal yeast killer toxin-like antibodies. Current Molecular Medicine, 5(4), 443-452.‏ https://doi.org/10.2174/1566524054022558
30. Magnusson, J., Ström, K., Roos, S., Sjögren, J., Schnürer, J. (2003). Broad and complex antifungal activity among environmental isolates of lactic acid bacteria. FEMS Microbiology Letters, 219(1), 129-135. https://doi.org/10.1016/S0378-1097(02)01207-7
31. Maia Danielski, G., Didimo Imazaki, P. H., Daube, G., Ernlund Freitas de Macedo, R., Clinquart, A. (2017). In vitro evaluation of the competing effect of Carnobacterium maltaromaticum isolated from vacuum packed meat against food pathogens. BAMST Symposium: Meet the Belgian Meat Researchers, Melle 7th.
32. Marquina, D., Santos, A., Peinado, J. M. (2002). Biology of killer yeasts. International Microbiology, 5(2), 65-71. https://doi.org/10.1007/s10123-002-0066-z
33. Montville, T. J., Matthews, K. R. (2012). Physiology, growth, and inhibition of microbes in foods. Food Microbiology, Fundamentals and Frontiers, 1-18.‏ https://doi.org/10.1128/9781555818463.ch1
34. Moroni, A., Arendt, E., Dal Bello, F. (2011). Biodiversity of lactic acid bacteria and yeast in spontaneously fermented buckwheat and teff sourdough. Food Microbiology, 28(3), 497-502. https://doi.org/10.1016/j.fm.2010.10.016
35. Moroni, A.V., Zannini, E., Sensidoni, G., Arendt, E. K. (2012). Exploitation of buckwheat sourdough for the production of wheat bread. European Food Research and Technology, 235(4), 659-668. https://doi.org/10.1007/s00217-012-1790-z
36. Moslehi Jenabian, S., Lindegaard, L., Jespersen, L. (2010). Beneficial effects of probiotic and food borne yeasts on human health. Nutrients, 2(4), 449-473. https://doi.org/10.3390/nu2040449
37. Ng, K. R., Lyu, X., Mark, R., Chen, W. N. (2019). Antimicrobial and antioxidant activities of phenolic metabolites from flavonoid-producing yeast: Potential as natural food preservatives. Food Chemistry, 270, 123-129.‏ https://doi.org/10.1016/j.foodchem.2018.07.077
38. Palla, M., Agnolucci, M., Calzone, A., Giovannetti, M., Di Cagno, R., Gobbetti, M., Rizzello, C. G., Pontonio, E. (2019). Exploitation of autochthonous Tuscan sourdough yeasts as potential starters. International Journal of Food Microbiology, 302, 59-68. https://doi.org/10.1016/j.ijfoodmicro.2018.08.004
39. Pedersen, L., Owusu-Kwarteng, J., Thorsen, L., Jespersen, L. (2012). Biodiversity and probiotic potential of yeasts isolated from Fura, a West African spontaneously fermented cereal. International Journal of Food Microbiology, 159(2), 144-151. https://doi.org/10.1016/j.ijfoodmicro.2012.08.016
40. Perricone, M., Bevilacqua, A., Corbo, M., Sinigaglia, M. (2014). Technological characterization and probiotic traits of yeasts isolated from Altamura sourdough to select promising microorganisms as functional starter cultures for cereal-based products. Food Microbiology, 38, 26-35. https://doi.org/10.1016/j.fm.2013.08.006
41. Rima, H., Steve, L., Ismail, F. (2012). Antimicrobial and probiotic properties of yeasts: from fundamental to novel applications. Frontiers in Microbiology, 3, 421.‏ https://doi.org/10.3389/fmicb.2012.00421
42. Rojo-Bezares, B., Sáenz, Y., Poeta, P., Zarazaga, M., Ruiz-Larrea, F., Torres, C. (2006). Assessment of antibiotic susceptibility within lactic acid bacteria strains isolated from wine. International Journal of Food Microbiology, 111(3), 234-240. https://doi.org/10.1016/j.ijfoodmicro.2006.06.007
43. Rolim, F. R. L., dos Santos, K. M. O., de Barcelos, S. C., do Egito, A. S., Ribeiro, T. S., da Conceição, M. L. (2015). Survival of Lactobacillus rhamnosus EM1107 in simulated gastrointestinal conditions and its inhibitory effect against pathogenic bacteria in semi-hard goat cheese. LWT-Food Science and Technology, 63(2), 807-813. https://doi.org/10.1016/j.lwt.2015.05.004
44. Ruggirello, M., Nucera, D., Cannoni, M., Peraino, A., Rosso, F., Fontana, M., Cocolin, L., Dolc, P. (2019). Antifungal activity of yeasts and lactic acid bacteria isolated from cocoa bean fermentations. Food Research International, 115, 519-525. https://doi.org/10.1016/j.foodres.2018.10.002
45. Saad, N., Delattre, C., Urdaci, M., Schmitter, J. M., Bressollier, P. (2013). An overview of the last advances in probiotic and prebiotic field. LWT-Food Science and Technology, 50(1), 1-16. https://doi.org/10.1016/j.lwt.2012.05.014
46. Saarela, M., Mogensen, G., Fonden, R., Mättö, J., Mattila-Sandholm, T. (2000). Probiotic bacteria: safety, functional and technological properties. Journal of Biotechnology, 84(3), 197-215. https://doi.org/10.1016/S0168-1656(00)00375-8
47. Sakandar, H. A., Usman, K., Imran, M. (2018). Isolation and characterization of gluten-degrading Enterococcus mundtii and Wickerhamomyces anomalus, potential probiotic strains from indigenously fermented sourdough (Khamir). LWT-Food Science and Technology, 91, 271-277. https://doi.org/10.1016/j.lwt.2018.01.023
48. Suvarna, S., Dsouza, J., Ragavan, M. L., Das, N. (2018). Potential probiotic characterization and effect of encapsulation of probiotic yeast strains on survival in simulated gastrointestinal tract condition. Food Science and Biotechnology, 27(3), 745-753.‏ https://doi.org/10.1007/s10068-018-0310-8
49. Viljoen, B. C. (2006). Yeast ecological interactions. Yeast'Yeast, Yeast'Bacteria, Yeast'Fungi interactions and yeasts as biocontrol agents. In Yeasts in food and beverages(pp. 83-110). Springer, Berlin, Heidelberg.‏ https://doi.org/10.1007/978-3-540-28398-0_4
50. White, T. J., Bruns, T., Lee, S. J. W. T., Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications. 18(1): 315-322.
51. Zhu, F., (2016). Chemical composition and health effects of Tartary buckwheat. Food Chemistry, 20, 231-245. https://doi.org/10.1016/j.foodchem.2016.02.050
52. Zielinski, H., Szawara-Nowak, D., Baczek, N., Wronkowska, M. (2019). Effect of liquid-state fermentation on the antioxidant and functional properties of raw and roasted buckwheat flours. Food Chemistry, 271, 291-297. https://doi.org/10.1016/j.foodchem.2018.07.182
53. Zullo, B.A., Ciafardini, G. (2019). Evaluation of physiological properties of yeasts strains isolated from olive oil and their in vitro probiotic trait. Food Microbiology, 78, 179-187. https://doi.org/10.1016/j.fm.2018.10.016