Evaluation of the Effectiveness of Cell-Protective Agents on the Survival of Lactic Acid Bacteria after Freeze-Drying

Premsuda Saman

ผู้แต่ง

Premsuda Saman Biodiversity Research Center, Thailand Institute of Scientific and Technological Research, Thailand

คำสำคัญ:

สารปกป้องเซลล์, การทำแห้งแบบแช่เยือกแข็ง, แบคทีเรียกรดแลกติก

บทคัดย่อ

การศึกษานี้มีวัตถุประสงค์เพื่อประเมินผลของสารปกป้องเซลล์ 4 ชนิด ได้แก่ Isomalto-oligosaccharide (IMO) Trehalose (TRE) Fructo-oligosaccharide (FOS) และ Skim milk (SM) ต่อความมีชีวิตของ L. farciminis L. acidophilus และ P. pentosaceus ภายหลังการทำแห้งแบบแช่เยือกแข็งและเก็บรักษาที่อุณหภูมิ 4 องศาเซลเซียส เป็นเวลา 24 สัปดาห์ ผลการทดลองพบว่าปริมาณเซลล์ตั้งต้นมีค่าใกล้เคียงกัน (13 log CFU/g) แต่เมื่อเวลาผ่านไปมีความแตกต่างอย่างมีนัยสำคัญทางสถิติ การวิเคราะห์ด้วย Two-way Repeated Measures ANOVA แสดงให้เห็นว่าทั้งชนิดสารปกป้องเซลล์และระยะเวลาเก็บรักษามีผลต่อความอยู่รอดของเซลล์ (p < 0.05) โดย IMO สามารถรักษาจำนวนเซลล์ได้สูงที่สุด โดยที่สัปดาห์ที่ 24 มีค่าเฉลี่ยสูงกว่ากลุ่มทดลองอื่น ๆ (L. farciminis = 11.23 log CFU/g L. acidophilus = 10.66 log CFU/g และ P. pentosaceus = 11.17 log CFU/g) TRE มีผลการปกป้องในระดับปานกลาง ขณะที่ FOS มีประสิทธิภาพต่ำที่สุด โดย L. acidophilus ลดลงเหลือเพียง 9.40 log CFU/g ที่สัปดาห์ที่ 24 ส่วน SM มีผลแตกต่างตามสายพันธุ์แบคทีเรียกรดแลคติก ผลการศึกษานี้แสดงให้เห็นว่า IMO มีประสิทธิภาพสูงสุดในการเก็บรักษาเซลล์โพรไบโอติกในระยะยาว

เอกสารอ้างอิง

Bharti, S. K., Krishnan, S., Kumar, A., Gupta, A. K., Ghosh, A. K., & Kumar, D. (2015). Mechanism-based antidiabetic activity of fructo- and isomalto-oligosaccharides: Validation by in vivo, in silico and in vitro interaction potential. Process Biochemistry, 50(3), 317–327. https://doi.org/10.1016/j.procbio.2014.10.014

Chapot-Chartier, M.P., & Kulakauskas, S. (2014). Cell wall structure and function in lactic acid bacteria. Microbial Cell Factories, 13(Suppl 1), S9. https://doi.org/10.1186/1475-2859-13-S1-S9

Chen, H., Chen, S. W., Li, C. N., & Shu, G. (2015). Response surface optimization of lyoprotectant for Lactobacillus bulgaricus during vacuum freeze-drying. Preparative Biochemistry & Biotechnology, 45(5), 463–475. https://doi.org/10.1080/10826068.2014.923451

Cruz, M. G. D., Silva, A. M. S. D., Prada-Mejia, K. D., Koolen, H. H. F., Tavares, G. C., & Valladão, G. M. R. (2025). Skim milk as a multifunctional cryoprotectant for fish probiotic Enterococcus spp.: Impact on viability during lyophilization and long-term storage. Microorganisms, 13(11), 2486. https://doi.org/10.3390/microorganisms13112486

Cui, S., Hang, F., Liu, X., Xu, Z., Liu, Z., Zhao, J., Zhang, H., & Chen, W. (2018). Effect of acids produced from carbohydrate metabolism in cryoprotectants on the viability of freeze-dried Lactobacillus and prediction of optimal initial cell concentration. Journal of Bioscience and Bioengineering, 125(5), 513–518. https://doi.org/10.1016/j.jbiosc.2017.12.009

El Ahmadi, K., Haboubi, K., El Allaoui, H., El Hammoudani, Y., Bouhrim, M., Eto, B., Shahat, A. A., & Herqash, R. N. (2025). Isolation and preliminary screening of lactic acid bacteria for antimicrobial potential from raw milk. Frontiers in Microbiology, 16, 1565016. https://doi.org/10.3389/fmicb.2025.1565016

Florowska, A., Krygier, K., Florowski, T., & Dłużewska, E. (2016). Prebiotics as functional food ingredients preventing diet-related diseases. Food & Function, 7(5), 2147–2155. https://doi.org/10.1039/c5fo01459j

Goffin, D., Delzenne, N., Blecker, C., Hanon, E., Deroanne, C., & Paquot, M. (2011). Will isomalto-oligosaccharides, a well-established functional food in Asia, break through the European and American market? The status of knowledge on these prebiotics. Critical Reviews in Food Science and Nutrition, 51(4), 394–409. https://doi.org/10.1080/10408391003628955

Haindl, R., Neumayr, A., Frey, A., & Kulozik, U. (2020). Impact of cultivation strategy, freeze-drying process, and storage conditions on survival, membrane integrity, and inactivation kinetics of Bifidobacterium longum. Folia Microbiologica, 65, 1039–1050. https://doi.org/10.1007/s12223-020-00815-3

Han, D., Bao, X., Wang, Y., Liao, X., Wang, K., Chen, J., Li, X., Yang, Z., & Wang, Y. (2024). The impact of lactic acid bacteria inoculation on the fermentation and metabolomic dynamics of indigenous Beijing douzhi microbial communities. Frontiers in Microbiology, 15, 1435834 https://doi.org/10.3389/fmicb.2024.1435834

Ibrahim, I., Ayariga, J. A., Xu, J., Boakai, R. K., Ajayi, O. S., & Owusu-Kwarteng, J. (2023). A comparative study of skimmed milk and cassava flour on the viability of freeze-dried lactic acid bacteria as starter cultures for yogurt fermentation. Foods, 12(6), 1207. https://doi.org/10.3390/foods12061207

Karan, D., Sharma, D., Kanwal, A., & Kanwar, N. (2025). Freeze-drying: Increasing probiotic stability. In D. Gugulothu, S. Sharma, & M. K. Chauhan (Eds.), Freeze-drying technology in pharmaceutical and biomedical product development: Impact, progress and challenges of lyophilization (pp. 349–368). Springer Nature Singapore. https://doi.org/10.1007/978-981-95-0221-9

Ketabi, A., Dieleman, L. A., & Gänzle, M. G. (2011). Influence of isomalto-oligosaccharides on intestinal microbiota in rats. Journal of Applied Microbiology, 110(5), 1297–1306. https://doi.org/10.1111/j.1365-2672.2011.04984.x

Kothari, D., Patel, S., & Goyal, A. (2014). Therapeutic spectrum of nondigestible oligosaccharides: Overview of current state and prospect. Journal of Food Science, 79(8), R1491–R1498. https://doi.org/10.1111/1750-3841.12536

Miao, Z., Zhao, Y., & Huo, X. (2016). Assessment and determination of lyoprotectant for survival of freeze-dried Lactobacillus rhamnosus. Acta Universitatis Cibiniensis, Series E: Food Technology, 20(1), 105–113. https://doi.org/10.1515/aucft-2016-0009

Musi, V., Aiello, E., Arena, M. P., Grieco, S., Sangiorgi, F., Russo, P., Fiocco, D., Capozzi, V., & Spano, G. (2026). Freeze-drying effects on viability and cellular stability in a subset of sourdough lactic acid bacteria strains. Current Microbiology, 83, 76. https://doi.org/10.1007/s00284-025-04673-5

Naghili, H., Tajik, H., Mardani, K., Razavi Rouhani, S. M., Ehsani, A., & Zare, P. (2013). Validation of drop plate technique for bacterial enumeration by parametric and nonparametric tests. Veterinary research forum: an international quarterly journal, 4(3), 179–183. https://pmc.ncbi.nlm.nih.gov/articles/PMC4312378/

Niu, D., Qiao, J., Li, P., Tian, K., Liu, X., Singh, S., & Lu, F. (2017). Highly efficient enzymatic preparation of isomalto-oligosaccharides from starch using an enzyme cocktail. Electronic Journal of Biotechnology, 26, 46–51. https://doi.org/10.1016/j.ejbt.2016.12.002

Ren, H., Zentek, J., & Vahjen, W. (2019). Optimization of production parameters for probiotic Lactobacillus strains as feed additive. Molecules, 24(18), 3286. https://doi.org/10.3390/molecules24183286

Rini, D. M., Xu, W., & Suzuki, T. (2024). Current research on the role of isomaltooligosaccharides in gastrointestinal health and metabolic diseases. Preventive Nutrition and Food Science, 29(2), 93–105. https://doi.org/10.3746/pnf.2024.29.2.93

Saman, P., Chaiongkarn, A., Moonmangmee, S., & Artjariyasripong, S. (2012). Prebiotic isomalto-oligosaccharide production from economic crops of Thailand. Asia-Pacific Journal of Science and Technology, 17(5), 794–799. https://so01.tci-thaijo.org/index.php/APST/article/view/83290

Senaratne, W. M. T. N., & Jayaweera, J. A. A. S. (2024). Comparison of microbial preservation methods: A narrative review. Germs, 14(4), 375–386. https://doi.org/10.18683/germs.2024.1447

Shin, D., Elbegbayar, E., Baek, Y., Jeong, E., Lee, K., Kang, K., Lee, K., & Lee, H. G. (2023). Effects of different cryoprotectants on the viability of microencapsulated Lactobacillus plantarum CJLP133 during long-term storage. Journal of Food Measurement and Characterization, 17(1), 1–8. https://doi.org/10.1007/s11694-023-01863-2

Shu, G., Xin, Y., Chen, L., Dan, H., Zhangteng, L., & Chen, H. (2019). Comprehensive optimization of composite cryoprotectant for Saccharomyces boulardii during freeze-drying and evaluation of its storage stability. Preparative Biochemistry & Biotechnology, 49(9), 846–857. https://doi.org/10.1080/10826068.2019.1630649

Siaterlis, A., Deepika, G., & Charalampopoulos, D. (2009). Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying. Letters in Applied Microbiology, 48(3), 295–301. https://doi.org/10.1111/j.1472-765X.2008.02529.x

Tymczyszyn, E. E., Sosa, N., Gerbino, E., Hugo, A., Gómez-Zavaglia, A., & Schebor, C. (2012). Effect of physical properties on the stability of Lactobacillus bulgaricus in a freeze-dried galacto-oligosaccharides matrix. International Journal of Food Microbiology, 155(3), 217–221. https://doi.org/10.1016/j.ijfoodmicro.2012.02.008

Van Engeland, C., Haut, B., & Debaste, F. (2025). A closer look at the potential mechanisms of action of protective agents used in the drying of microorganisms: A review. Microorganisms, 13(2), 435. https://doi.org/10.3390/microorganisms13020435

Wang, G., Luo, L., Dong, C., Zheng, X., Guo, B., Xia, Y., Tao, L., & Ai, L. (2021). Polysaccharides can improve the survival of Lactiplantibacillus plantarum subjected to freeze-drying. Journal of Dairy Science, 104(3), 2606–2614. https://doi.org/10.3168/jds.2020-19110

Wang, J., Wu, P., Dhital, S., Yu, A., & Chen, X. D. (2025). Impact of freezing and freeze-drying on Lactobacillus rhamnosus GG survival: Mechanisms of cell damage and the role of pre-freezing conditions and cryoprotectants. Foods, 14(10), 1817. https://doi.org/10.3390/foods14101817

Yoha, K. S., Moses, J. A., & Anandharamakrishnan, C. (2023). Effect of different drying methods on the functional properties of probiotics encapsulated using prebiotic substances. Applied Microbiology and Biotechnology, 107(4), 1575–1588. https://doi.org/10.1007/s00253-023-12398-3

Zhang, Z., Sun, N., Chen, J., & Li, X. (2022). Determination of glass transition temperature of IMO/D-mannitol aqueous solution and analysis of collapse phenomenon during freeze-drying process of solution. Science and Technology of Food Industry, 43(4), 33–40. https://doi.org/10.1111/ijfs.16111