## 4 Conclusions  
The main contribution of this work is the description of the fermentation of UHT whole milk with milk kefir grains in repeated fermentations of 24 or 48 h, comparing the microbiological, physicochemical, and volatile compositions, as well as the visual appearance of the different kefir beverages.  
Figure 10: Evolution of fermentation metabolites (LA: lactic acid, AA: acetic acid, EtOH: ethanol, and GOH: glycerol) in fermented beverages from the four subcultures of fermentation I during refrigerated storage at 4 \({}^{\circ}\)C.  
Figure 9: Evolution of pH and nutrients (L: lactose and Pr: proteins) in fermented beverages from the four subcultures of fermentation I during refrigerated storage at 4 \({}^{\circ}\)C.  
Fermenting milk for 48 h with kefir grains produced a beverage containing high counts of LAB (>10\({}^{6}\) UFC/mL in the four subcultures) and yeasts (>10\({}^{6}\) UFC/mL, except in the four subcultures). However, the concentration of volatile compounds decreased considerably compared to the kefir beverages obtained from the 24-h subcultures. Additionally, the increase in the fermentation time from 24 to 48 h negatively affected the visual appearance of the beverage due to the separation of whey and curd. In contrast, fermenting milk for 24 h was an appropriate strategy to produce kefir beverages with counts of probiotic cells (LAB and yeasts) higher than 10\({}^{6}\) UFC/mL, with relatively high concentrations of aromatic volatile compounds, and low concentrations of lactose, organic acids, and alcohols. Furthermore, a fermentation time of 24 h is sufficient to obtain a probiotic beverage with a good visual appearance compared to the beverages from the repeated 48-h subcultures.  
Storage at 4 \({}^{\circ}\)C for 28 days of fermented milks from the four 24-h subcultures resulted in a slight reduction in the concentrations of lactose and proteins, as well as in the counts of LAB, AAB, and yeasts. However, in all cases, the counts of these microbial groups remained higher than 10\({}^{6}\) UFC/mL. This indicates that these beverages can be appropriately preserved by refrigerated storage for at least four weeks.  
However, kinetic fermentation experiments should be conducted to clarify the relationships between the time course of different culture variables, such as culture pH, cell growth, nutrients, fermentation products, volatile compounds, and kefir grain weight. To achieve this objective, our research team is studying the kinetics of these culture variables in fermentations of different substrates with both milk and water kefir grains. Additionally, we are quantifying and characterizing the different bacteria and yeasts present in both the kefir grains and fermented samples using PCR or DNA sequencing techniques.  
Conceptualization, N.P.G.; methodology, D.L.B.T. and P.G.D.-R.; software, N.P.G.; validation, D.L.B.T., S.C.D. and P.G.D.-R.; formal analysis, N.P.G. and J.M.D.; investigation, N.P.G. and D.L.B.T.; resources, N.P.G. and J.M.D.; data curation, N.P.G., S.C.D. and D.L.B.T.; writing--original draft preparation, N.P.G. and D.L.B.T.; writing--review and editing, N.P.G. and J.M.D.; visualization, N.P.G. and D.L.B.T.; supervision, N.P.G.; project administration, N.P.G. and J.M.D.; funding acquisition, N.P.G. and J.M.D. All authors have read and agreed to the published version of the manuscript.  
[PERSON] thanks the National University of Jaen (Peru) and the National Fellowship Program (PRONABEC--Peru) for their research grant (President of the Republic 2017-I-PRONABEC--Peru). [PERSON] would like to express gratitude to the Conselleria de Cultura, Educacion, Formacion Profesional e Universidades of Xunta de Galicia (Spain) for their financial support (ED481B-2022-020).  
Data Availability Statement: Data are contained within the article.  
The authors declare no conflicts of interest.