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. 2025 Oct 10:16:1655390.
doi: 10.3389/fmicb.2025.1655390. eCollection 2025.

Fermentation of kefir with traditional freeze-dried starter cultures successfully recreates fresh culture fermented kefir

Maanasa Mudoor Sooresh #  1 , Ashani Jayawickrama #  1 , Amaya Silva  1 , Sally Nguyen  1 , Sheri Schmidt  2   3 , Joseph Sebastian  1 , Shane Carey  1 , James Harynuk  2   3 , Benjamin P Willing  1 , Benjamin C T Bourrie  1   4
Affiliations

Fermentation of kefir with traditional freeze-dried starter cultures successfully recreates fresh culture fermented kefir

Maanasa Mudoor Sooresh et al. Front Microbiol. .

Abstract

Introduction: 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.

Methods: 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) underwent freeze-drying prior to viability testing and use as starter cultures in kefir fermentations. Completed kefir fermentations were analyzed for pH, microbial composition, volatile compounds, organic acids, and sugar consumption. Freeze-dried starter culture fermentations were compared to kefir made with fresh starter cultures of the same species and pitching rate.

Results: 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.

Conclusion: 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.

Keywords: fermented foods and beverages; freeze-drying; kefir; metabolomics; starter culture fermentation.

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Conflict of interest statement

BCTB and BPW hold a patent for the method used to produce the fresh culture pitched kefir used in this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
PCA of volatile metabolites in kefir after fermentation prepared with fresh (n = 3) or freeze-dried starter cultures (n = 6); PERMDISP p = 0.8463; PERMANOVA p = 0.5657.
Figure 2
Figure 2
Boxplots showing total useful peak area (TUPA) of butanoic acid (A), hexanoic acid (B), octanoic acid (C), butanoic acid ethyl ester (D), and butanal-3-methyl (E) between kefir fermentations performed with fresh culture, freeze-dried cultures with milk and freeze-dried cultures with trehalose as measured by GCxGC-TOFMS. Data are expressed as mean values with their standard errors (n = 3–6).
Figure 3
Figure 3
Boxplots showing concentration (mM) levels of glucose (A), galactose (B), acetic acid (C), lactic acid (D), and ethanol (E) between kefir fermentations performed with fresh culture, freeze-dried cultures with milk and freeze-dried cultures with trehalose as measured by HPLC. Data are expressed as mean values with their standard errors (n = 3–6).
Figure 4
Figure 4
Stacked bar charts representing relative abundance at the species level for each kefir sample at the completion of fermentation.
See this image and copyright information in PMC

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