Determination of Lactic Acid Bacterial Numbers of Lyophilized or Frozen Natural Lactic Acid Bacterial Liquids Prepared with Different Methods and Stored for Different Times

Year 2024, , 29 - 41, 31.03.2024

Sadık Serkan Aydın , Nihat Denek

https://doi.org/10.30607/kvj.1288653

Abstract

In this study, it was aimed to determine the lactic acid bacterium (LAB) counts and viability of fermented lactic acid bacteria liquids (pre-fermented juice (PFJ)) prepared with different levels of sucrose (3%, 5% and 10%) addition and incubation at different times (2, 5 and 10 days), using different cryoprotectants (trisodiumcitrate (TRIS) and dimethylsulfoxide (DMSO)), freezing and lyophilization, and at the end of different storage periods (one and three months). In frozen PFJs, the highest LAB counts were obtained in the TRIS and 5% sucrose supplemented group incubated for 5 days in a one-month storage period, and in the TRIS and 10% sucrose supplemented group incubated for 5 days at the end of the three-month storage period (p<0.01). At the end of one month of storage, the highest LAB numbers in lyophilized PFJs were obtained in groups incubated for 5 days and supplemented with 10% sucrose with TRIS and DMSO. In lyophilized PFJs subjected to 3-month storage, the highest LAB numbers were determined in the TRIS and 5%-10% sucrose-supplemented groups incubated for 5 days and in the DMSO and 5% sucrose-supplemented group incubated for 2 days (p<0.01). When the results obtained in the study were evaluated in general, it was observed that the viability ratios of LAB decreased due to the sucrose level and the prolongation of incubation and storage time. It can be said that cryoprotectant additive has a positive effect on the preservation of LAB and lyophilization process is advantageous compared to freezing in the deep freezer.

Keywords

Lactic acid bacteria, Freezing, Lyophilization

Project Number

18012

Farklı Şekillerde Hazırlanarak Değişik Sürelerde Depolanan Liyofilize Edilmiş ve Dondurulmuş Doğal Laktik Asit Bakteri Sıvılarının Laktik Asit Bakteri Sayılarının Belirlenmesi

Abstract

Bu çalışma kapsamında, farklı seviyelerde sükroz ilavesi (%3, %5 ve %10) ve farklı sürelerde inkübasyonla (2, 5 ve 10 gün) hazırlanmış fermente edilmiş laktik asit bakteri (LAB) sıvılarının (pre-fermented juice (PFJ)), farklı kryoprotektan maddeler (trisodyum sitrat (TRIS) ve dimetil sülfoksit (DMSO)) kullanılarak, dondurma ve liyofilizasyon ile farklı depolama süreleri (bir ve üç ay) sonunda LAB sayıları ve canlılığının belirlenmesi amaçlanmıştır. Dondurulan fermente edilmiş LAB sıvılarında en yüksek LAB sayıları bir aylık depolamada 5 gün inkübe edilen TRIS ve %5 sükroz katkılı grupta elde edilirken, üç aylık depolama süresi sonunda ise 5 gün inkübe edilen TRIS ve %10 sükroz katkılı grupta elde edilmiştir (p<0.01). Liyofilize edilmiş PFJ’lerde en yüksek LAB sayıları bir aylık depolama süresi sonunda TRIS ve DMSO katkılı %10 sükroz eklenerek 5 gün inkübe edilen gruplarda elde edilmiştir. Üç aylık depolama uygulanan liyofilize edilmiş PFJ'lerde ise en yüksek LAB sayıları 5 gün inkübe edilen TRIS ve %5-%10 sükroz katkılı gruplarda ve 2 gün inkübe edilen DMSO ve %5 sukroz katkılı grupta belirlenmiştir (p<0.01). Çalışmada elde edilen sonuçlar genel olarak değerlendirildiğinde; sükroz seviyesi ile inkübasyon ve depolama süresinin uzamasına bağlı olarak LAB’nin canlılık oranlarında azalmalar görülmüştür. Kryoprotektan katkısının LAB’nin korunmasında olumlu etki yaptığı, liyofilizasyon işleminin ise derin dondurucuda dondurulma işlemine göre avantajlı olduğu söylenebilir.

Keywords

Dondurulma, Laktik asit bakterisi, Liyofilizasyon

Supporting Institution

Harran Üniversitesi Bilimsel Araştırma Projeleri Koordinatörlüğü

Project Number

18012

References

• Avcıoğlu Y. (2013). Attenüe Brucella melitensis (REV-1) aşısının farklı stabilizatörlerle liyofilizasyonu. Kilis 7 Aralık Üniversitesi Fen Bilimleri Enstitüsü, Yüksek Lisans Tezi.
• Bâati, L., Fabre-Gea, C., Auriol, D., & Blanc, P. J. (2000). Study of the cryotolerance of Lactobacillus acidophilus: effect of culture and freezing conditions on the viability and cellular protein levels. International Journal of Food Microbiology, 59(3), 241-247. https://doi.org/10.1016/S0168-1605(00)00361-5
• Brock, T. Dale, Madigan, M. T, Martinko, J. M, & Parker, J. (2003). Brock biology of microorganisms. 10th ed. Upper Saddle River (N.J.): Prentice-Hall.
• Chang, T., & Zhao, G. (2021). Ice inhibition for cryopreservation: materials, strategies, and challenges. Advanced Science, 8(6), 2002425. https://doi.org/10.1002/advs.202002425
• Dumont, F., Marechal, P. A., & Gervais, P. (2004). Cell size and water permeability as determining factors for cell viability after freezing at different cooling rates. Applied and Environmental Microbiology, 70(1), 268-272. https://doi.org/10.1128/AEM.70.1.268-272.2004
• Ferry, R. M. (1995). The freeze-dring of bacteria and viruses. Methods in Molecuolar Biology, 38(2).
• Fonseca, F., Meneghel, J., Cenard, S., Passot, S., & Morris, G. J. (2016). Determination of intracellular vitrification temperatures for unicellular micro organisms under conditions relevant for cryopreservation. PLoS One, 11(4), e0152939. https://doi.org/10.1371/journal.pone.0152939
• Greaves, R. I. N. (1964). Fundamental aspects of freeze-drying bacteria and living cells. Aspects Theoriques et Industriels de la Lyophilisation, Rey L (ed).
• Giulio, B. D., Orlando, P., Barba G., Coppola, R., Rosa, M. D., Sada, A., De Prisco, P. P., & Nazzaro, F. (2005). Use of alginate and cryo-protective sugars to improve the viability of lactic acid bacteria after freezing and freeze-drying. World Journal of Microbiology and Biotechnology, 21, 739-746. https://doi.org/10.1007/s11274-004-4735-2
• Haigh, P. M., Appletont, M., & Clench, S. F. (1987). Effect of commercial inoculant and formic acid±formalin silage additives on silage fermentation and intake and on liveweight change of young cattle. Grass and Forage Science, 42(4), 405-410. https://doi.org/10.1111/j.1365-2494.1987.tb02131.x
• Halkman, A., & Doğan, H. B. (2000). Gıda Mikrobiyolojisi ve Uygulamaları. Genişletilmiş 2. Baskı. Sim Matbaası, Ankara.
• Masuko, T., Hariyama, Y., Takahashi, Y., Cao, L. M., Goto, M., & Ohshima, M. (2002). Effect of addition of fermented juice of epiphytic lactic acid bacteria prepared from timothy [Phleum pratense] and orchardgrass [Dactylis glomerata] on fermentation quality of silages. Journal of Japanese Society of Grassland Science, 48(2): 120-125.
• Meryman, H. T. (1960). Principles of freeze‐drying. Annals of the New York Academy of Sciences, 85(2), 630-640.
• Morichi, T., Irie, R., Yano, N., & Kembo, H. (1965). Protective effect of arginine and its related compounds on bacterial cells during freeze-drying. Agricultural and Biological Chemistry, 29(1), 61-65. https://doi.org/10.1080/00021369.1965.10858347
• Ohshima, M., Ohshima, Y., Kimura, E., & Yokota, H. O. (1997). Fermentation quality of alfalfa and Italian ryegrass silages treated with previously fermented juices prepared from both the herbages. Animal Science and Technology, 68, 41-44.
• Oluwatosin, S. O., Tai, S. L., & Fagan-Endres, M. A. (2022). Sucrose, maltodextrin and inulin efficacy as cryoprotectant, preservative and prebiotic–towards a freeze dried Lactobacillus plantarum topical probiotic. Biotechnology Reports, 33, e00696. https://doi.org/10.1016/j.btre.2021.e00696
• Öztürk, S., & Çakır, İ. 2015. Mikroorganizma kültürlerinin korunmasında kullanılan kurutma yöntemleri. Akademik Gıda, 13(1), 94-100.
• Peiren, J., Buyse, J., De Vos, P., Lang, E., Clermont, D., Hamon, S., Bégaud, E., Bizet, C., Pascual, J., Ruvira, M. A., Macián, M. C., Arahal, D. R. (2015). Improving survival and storage stability of bacteria recalcitrant to freeze-drying: a coordinated study by European culture collections. Applied Microbiology and Biotechnology, 99, 3559-3571. https://doi.org/10.1007/s00253-015-6476-6
• Pınarkara Y. (2008). Liyofilizasyon işlemi Esnasında Bazı Laktik Asit Bakterilerinin Canlılıkları Üzerine Kriyojenik Koruyucu Maddelerin Etkileri. Selçuk Üniversitesi, Fen Bilimleri Enstitüsü, Doktora Tezi.
• Rambhatla, S., & Pikal, M. J. (2003). Heat and mass transfer scale-up issues during freeze-drying, I: atypical radiation and the edge vial effect. Aaps Pharmscitech, 4, 22-31.
• Sparkes, J. D., & Fenje, P. (1972). The effect of residual moisture in lyophilized smallpox vaccine on its stability at different temperatures. Bulletin of the World Health Organization, 46(6), 729
• Shurman, S., Sutakwa, A., & Nadia, L. S. (2021, March). Effects Of Sucrose Addition To Lactic Acid Concentrations And Lactic Acid Bacteria Population Of Butterfly Pea (Clitoria Ternatea L.) Yogurt. In Journal of Physics: Conference Series, 1823(1), 012038. https://doi.org/10.1088/1742-6596/1823/1/012038
• Strasser, S., Neureiter, M., Geppl, M., Braun, R., & Danner, H. (2009). Influence of lyophilization, fluidized bed drying, addition of protectants, and storage on the viability of lactic acid bacteria. Journal of Applied Microbiology, 107(1), 167-177. https://doi.org/10.1111/j.1365-2672.2009.04192.x
• Tedeschi, R., & De Paoli, P. (2011). Collection and preservation of frozen microorganisms. In: Dillner, J. (eds) Methods in Biobanking. Methods in Molecular Biology, vol 675. Humana Press, Totowa, NJ, P. 313-326. https://doi.org/10.1007/9