Bifidobacterium longum, one of the main microorganisms in the human gut, is used as an adjunct to lactic acid starter cultures or sold as a probiotic product. Therefore, Bifidobacterium longum cell suspensions get freeze-dried with protective additives to prevent activity losses. To date, investigations covering growth and inactivation kinetics of Bifidobacterium longum during the whole process (cultivation, drying, and storage) have been lacking. In this study, the effect of cultivation conditions and shelf temperature as well as the influence of protectants (maltodextrin, glucitol, trehalose) at various concentrations on cell survival during freeze-drying was assessed. Drying was followed by a storage at + 4 °C and + 20 °C for 70 days to evaluate inactivation kinetics. The impact of the different factors was assessed by measuring surival rate and residual moisture content at various points of time over the whole process. In parallel cell membrane integrity and glass transition were determined to reveal inactivation effects. Cultivation strategy had a strong influence on survival with a huge potential for process improvement. A pH of 6.0 at the growth optimum of the strain provides better conditions regarding cell survival after drying than free acidification (non-regulated pH conditions). During the drying step, membrane leakage due to the removal of water is the main reason for the inactivation in this process step. In this study, the highest survival of 49% was obtained with cells dried at + 35 °C shelf temperature with an addition of maltodextrin (75% bacterial dry matter, w/w). The results show that Bifidobacterium longum cells are mostly inactivated during drying, whereas storage conditions at + 4 °C with an addition of 75% BDM maltodextrin relative to bacterial dry mass prevent cell loss completely.

Chair of Food and Bioprocess Engineering Technical University of Munich Weihenstephaner Berg 1 Freising Weihenstephan Germany

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Abe F, Miyauchi H, Uchijima A, Yaeshima T, Iwatsuki K. Effects of storage temperature and water activity on the survival of bifidobacteria in powder form. Int J Dairy Technol. 2009;62:234–239. doi: 10.1111/j.1471-0307.2009.00464.x. DOI

Ambros S, Hofer F, Kulozik U. Protective effect of sugars on storage stability of microwave freeze-dried and freeze-dried Lactobacillus paracasei F19. J Appl Microbiol. 2018;125:1128–1136. doi: 10.1111/jam.13935. PubMed DOI

Ambros S, Vollmer AH, Youssef NN, Kulozik U. Structural basis of the impact of microwave drying on survival and shelf life of Lactobacillus paracasei. LWT Food Sci Technol. 2018;98:291–298. doi: 10.1016/j.lwt.2018.08.051. DOI

Bauer SAW, Schneider S, Behr J, Kulozik U, Foerst P. Combined influence of fermentation and drying conditions on survival and metabolic activity of starter and probiotic cultures after low-temperature vacuum drying. J Biotechnol. 2012;159:351–357. doi: 10.1016/j.jbiotec.2011.06.010. PubMed DOI

Bruno FA, Shah NP. Viability of two freeze-dried strains of bifidobacterium and of commercial preparations at various temperatures during prolonged storage. J Food Sci. 2003;68:2336–2339. doi: 10.1111/j.1365-2621.2003.tb05769.x. DOI

Carvalho AS, Silva J, Ho P, Teixeira P, Malcata FX, Gibbs P. Protective effect of sorbitol and monosodium glutamate during storage of freeze-dried lactic acid bacteria. Lait. 2003;83:203–210. doi: 10.1051/lait:2003010. DOI

Champagne CP, Gardner N, Brochu E, Beaulieu Y. The freeze-drying of lactic acid bacteria. A review. Can Inst Food Sci Technol J. 1991;24:118–128. doi: 10.1016/S0315-5463(91)70034-5. DOI

Corcoran BM, Ross RP, Fitzgerald GF, Stanton C. Comparative survival of probiotic lactobacilli spray-dried in the presence of prebiotic substances. J Appl Microbiol. 2004;96:1024–1039. doi: 10.1111/j.1365-2672.2004.02219.x. PubMed DOI

Dianawati D, Vijay M, Nagendra PS. Survival of Bifidobacterium longum 1941 microencapsulated with proteins and sugars after freezing and freeze drying. Food Res Int. 2013;51:503–509. doi: 10.1016/j.foodres.2013.01.022. DOI

Dokic L, Jakovljevic J, Dokic P. Relation between viscous characteristics and dextrose equivalent of maltodextrins. Starch. 2004;56:520–525. doi: 10.1002/star.200400294. DOI

Drucker DB, Megson G, Harty DW, Riba I, Gaskell SJ. Phospholipids of Lactobacillus spp. J Bacteriol. 1995;177:6304–6308. doi: 10.1128/jb.177.21.6304-6308.1995. PubMed DOI PMC

Exterkate FA, Otten BJ, Wassenberg HW, Veerkamp JH. Comparison of the phospholipid composition of bifidobacterium and lactobacillus strains. J Bacteriol. 1971;106:824–829. doi: 10.1128/JB.106.3.824-829.1971. PubMed DOI PMC

Gibson GR, Wang X. Regulatory effects of bifidobacteria on the growth of other colonic bacteria. J Appl Bacteriol. 1994;77:412–420. doi: 10.1111/j.1365-2672.1994.tb03443.x. PubMed DOI

Higl B, Santivarangkna MSC, Först P. Bewertung und Optimierung von Gefrier- und Vakuumtrocknungsverfahren in der Herstellung von mikrobiellen Starterkulturen. Chemie Ingenieur Technik. 2008;80:1157–1164. doi: 10.1002/cite.200800069. DOI

Hsiao H-C, Lian W-C, Chou C-C. Effect of packaging conditions and temperature on viability of microencapsulated bifidobacteria during storage. J Sci Food Agric. 2004;84:134–139. doi: 10.1002/jsfa.1616. DOI

Kailasapathy K, Chin J. Survival and therapeutic potential of probiotic organisms with reference to Lactobacillus acidophilus and Bifidobacterium spp. Immunol Cell Biol. 2000;78:80–88. doi: 10.1046/j.1440-1711.2000.00886.x. PubMed DOI

Kiviharju K, Leisola M, Eerikainen T. Optimization of a Bifidobacterium longum production process. J Biotechnol. 2005;117:299–308. doi: 10.1016/j.jbiotec.2005.02.007. PubMed DOI

Knorr D. Technology aspects related to microorganisms in functional foods. Trends Food Sci Technol. 1998;9:295–306. doi: 10.1016/S0924-2244(98)00051-X. DOI

Koster KL, Maddocks KJ, Bryant G. Exclusion of maltodextrins from phosphatidylcholine multilayers during dehydration: effects on membrane phase behaviour. Eur Biophys J. 2003;32:96–105. doi: 10.1007/s00249-003-0277-z. PubMed DOI

Lian W. Survival of bifidobacteria after spray-drying. Int J Food Microbiol. 2002;74:79–86. doi: 10.1016/S0168-1605(01)00733-4. PubMed DOI

Matsuki T, Watanabe K, Fujimoto J, Kado Y, Takada T, Matsumoto K, Tanaka R. Quantitative PCR with 16S rRNA-gene-targeted species-specific primers for analysis of human intestinal bifidobacteria. Appl Environ Microbiol. 2004;70:167–173. doi: 10.1128/AEM.70.1.167-173.2004. PubMed DOI PMC

Min M, Bunt CR, Mason SL, Bennett GN, Hussain MA (2017) Effect of non-dairy food matrices on the survival of probiotic bacteria during storage. Microorganisms 5. 10.3390/microorganisms5030043 PubMed PMC

Novik GI, Astapovich NI, Grzegorzewicz A, Gamian A. Analysis of phospholipids in bifidobacteria. Microbiology. 2006;75:29–34. doi: 10.1134/S0026261706010061. PubMed DOI

Oldenhof H, Wolkers WF, Fonseca F, Passot S, Marin M. Effect of sucrose and maltodextrin on the physical properties and survival of air-dried Lactobacillus bulgaricus: an in situ Fourier transform infrared spectroscopy study. Biotechnol Prog. 2005;21:885–892. doi: 10.1021/bp049559j. PubMed DOI

Quigley EMM. Bifidobacterium longum. In: Floch MH, Walker WA, Ringel Y, editors. The microbiota in gastrointestinal pathophysiology: implications for human health, prebiotics, probiotics, and dysbiosis. Amsterdam: Elsevier Academic Press; 2017. pp. 139–141.

Reilly SS, Gilliland SE. Bifidobacterium longum survival during frozen and refrigerated storage as related to pH during growth. J Food Science. 1999;64:714–718. doi: 10.1111/j.1365-2621.1999.tb15117.x. DOI

Rong Y, Sillick M, Gregson CM. Determination of dextrose equivalent value and number average molecular weight of maltodextrin by osmometry. J Food Sci. 2009;74:C33–C40. doi: 10.1111/j.1750-3841.2008.00993.x. PubMed DOI

Saarela M, Virkajärvi I, Alakomi H-L, Mattila-Sandholm T, Vaari A, Suomalainen T, Mättö J. Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of Bifidobacterium animalis ssp. lactis cells produced without milk-based ingredients. J Appl Microbiol. 2005;99:1330–1339. doi: 10.1111/j.1365-2672.2005.02742.x. PubMed DOI

Saarela M, Virkajärvi I, Alakomi H-L, Sigvart-Mattila P, Mättö J. Stability and functionality of freeze-dried probiotic Bifidobacterium cells during storage in juice and milk. Int Dairy J. 2006;16:1477–1482. doi: 10.1016/j.idairyj.2005.12.007. DOI

Santivarangkna C, Higl B, Foerst P. Protection mechanisms of sugars during different stages of preparation process of dried lactic acid starter cultures. Food Microbiol. 2008;25:429–441. doi: 10.1016/j.fm.2007.12.004. PubMed DOI

Saxelin M. Lactobacillus GG—a human probiotic strain with thorough clinical documentation. Food Rev Int. 1997;13:293–313. doi: 10.1080/87559129709541107. DOI

Shamekhi F, Shuhaimi M, Ariff A, Manap YA. Cell viability of microencapsulated Bifidobacterium animalis subsp. lactis under freeze-drying, storage and gastrointestinal tract simulation conditions. Folia Microbiol (Praha) 2013;58:91–101. doi: 10.1007/s12223-012-0183-9. PubMed DOI

Sohail A, Turner MS, Coombes A, Bhandari B. The viability of Lactobacillus rhamnosus GG and Lactobacillus acidophilus NCFM following double encapsulation in alginate and maltodextrin. Food Bioprocess Technol. 2013;6:2763–2769. doi: 10.1007/s11947-012-0938-y. DOI

Sugahara H, Odamaki T, Fukuda S, Kato T, Xiao J, Abe F, Kikuchi J, Ohno H. Probiotic Bifidobacterium longum alters gut luminal metabolism through modification of the gut microbial community. Sci Rep. 2015;5:13548. doi: 10.1038/srep13548. PubMed DOI PMC

Villarreal MA, Díaz SB, Disalvo EA, Montich GG. Molecular dynamics simulation study of the interaction of trehalose with lipid membranes. Langmuir. 2004;20:7844–7851. doi: 10.1021/la049485l. PubMed DOI

Wang Y-C, Yu R-C, Chou C-C. Viability of lactic acid bacteria and bifidobacteria in fermented soymilk after drying, subsequent rehydration and storage. Int J Food Microbiol. 2004;93:209–217. doi: 10.1016/j.ijfoodmicro.2003.12.001. PubMed DOI

Yeung TW, Üçok EF, Tiani KA, McClements DJ, Sela DA. Microencapsulation in alginate and chitosan microgels to enhance viability of Bifidobacterium longum for oral delivery. Front Microbiol. 2016;7:494. doi: 10.3389/fmicb.2016.00494. PubMed DOI PMC