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The aim of this study was to investigate the effect of altering the fatty acid profile of the lipid membrane on storage survival of freeze-dried probiotic, Lactobacillus acidophilus La-5, as well as study the membrane integrity and lipid oxidation. The fatty acid composition of the lipid membrane of L. acidophilus La-5 was significantly different upon growth in MRS (containing Tween 80, an oleic acid source), or in MRS with Tween 20 (containing C12:0 and C14:0), linoleic, or linolenic acid supplemented. Bacteria grown in MRS showed the highest storage survival rates. No indications of loss of membrane integrity could be found, and membrane integrity could therefore not be connected with loss of viability. Survival of bacteria grown with linoleic or linolenic acid was more negatively affected by the presence of oxygen, than bacteria grown in MRS or with Tween 20 supplemented. A small, but significant, loss of linolenic acid during storage could be identified, and an increase of volatile secondary oxidation products during storage was found for bacteria grown in MRS, or with linoleic, or linolenic acid supplemented, but not for bacteria grown with Tween 20. Overall, the results indicate that lipid oxidation and loss of membrane integrity are not the only or most important detrimental reactions which can occur during storage. By altering the fatty acid composition, it was also found that properties of oleic acid gave rise to more robust bacteria than more saturated or unsaturated fatty acids did., (© 2015 American Institute of Chemical Engineers.)