Fermentation of Kefir with Traditional Freeze-Dried Starter Cultures Successfully Recreates Fresh Culture Fermented Kefir

Maanasa Mudoor Sooresh¹Ashani Jayawickrama¹Amaya Silva¹Sally Nguyen¹Sheri Schmidt^2,3James J Harynuk^2,3Benjamin P Willing^1*Benjamin C.T. Bourrie^1,4*

• ¹University of Alberta Department of Agricultural, Food, and Nutritional Sciences, Edmonton, Canada
• ²University of Alberta Department of Chemistry, Edmonton, Canada
• ³The Metabolomics Innovation Centre, Edmonton, Canada
• ⁴MacEwan University, Edmonton, Canada

Interest in fermented foods and their purported health benefits has led to increased scientific research investigating the development of starter cultures which maintain the characteristics of traditional products while allowing for industrial scale production. One such fermented food that is gaining steady attention for industrial production is kefir. To improve the ease of use and maintenance of starter cultures without compromising desirable fermentation characteristics and potential health benefits, this study investigated the impact of freeze-drying a previously described reconstituted kefir consortia with two lyoprotectants trehalose and milk. The reconstituted kefir community consisted of 5 bacterial species (Lentilactobacillus kefiri, Lactobacillus kefiranofaciens, Lactococcus cremoris, Leuconostoc mesenteroides and Acetobacter pasteurianus) and 4 yeast species (Saccharomyces cerevisiae, Pichia fermentans, Monosporozyma unispora, and Kluyveromyces marxianus) previously developed by our group which has shown promise in reducing plasma cholesterol levels and cardiovascular disease risk factors in both mouse and human trials. We tested the effect of freeze drying on the viability of the microbes and conducted kefir fermentations comparing fresh and freeze-dried starter cultures. Completed kefir fermentations were compared for pH, microbial composition, volatile compounds, organic acids, and sugar consumption. We found that all starter cultures were able to ferment milk to a similar pH, however the freeze-dried cultures prepared with milk took a longer time to complete fermentation. The total bacterial and yeast counts were comparable across the fermentations performed as was the composition of bacteria and yeast present as determined by shotgun metagenomic sequencing. High performance liquid chromatography (HPLC) analysis showed no difference in the levels of lactic acid, acetic acid, ethanol, glucose, and galactose. Additionally, solid-phase microextraction followed by two-dimensional gas chromatography-time-of-flight mass spectrometry (SPME-GC×GC-TOFMS) showed that kefir fermented with freeze-dried starter cultures did not change the volatile profile compared to fresh cultures. Together these findings indicate that freeze-dried starter cultures consisting of traditional kefir microorganisms are able to recreate the fresh starter culture version of this product. This provides encouraging evidence for the development of commercially viable starter cultures that are capable of recreating traditional functional fermented foods.