# Effects of Isochoric Freezing on the Quality Characteristics of Raw Bovine Milk
## 4 Conclusions  
This work describes the effects of isochoric freezing at \(-5\,^{\circ}\)C/77 MPa and \(10\,^{\circ}\)C/96 MPa on the physicochemical properties, indigenous enzyme activity, protein content, volatile organic compounds profile, lipid degradation, and microbiological activity of raw bovine milk. Overall, prolonged storage under isochoric freezing conditions, when compared to refrigeration (RF), resulted in superior quality across measured parameters. Prolonged isochoric freezing was also able to better preserve milk for longer storage periods as compared to HTST treated milk. Unlike conventional refrigeration and HTST treatment, IF-treated milk at \(-5\,^{\circ}\)C/77 MPa and \(-10\,^{\circ}\)C/96 MPa not only inhibited microbial growth, but also significantly reduced the microbial count, with IF (\(-10\,^{\circ}\)C) producing counts below the detectable limit. This anti-microbial effect, driven by the combination of enhanced pressure and reduced temperature, enabled the shelf-life extension of raw milk for up to 5 weeks (the longest storage period studied). At \(-10\,^{\circ}\)C/96 MPa, higher pressures resulted in faster and more effective microbial deactivation, but these conditions did impact the physicochemical properties when compared to unprocessed raw milk. In contrast, at \(-5\,^{\circ}\)C/77 MPa, the volatiles profile and lipid oxidation levels that were similar to fresh raw milk were retained at 5 weeks, and there were no physiochemical or microbiological indications that substantially longer storage would not be achievable. Compared to available data on room-temperature hyperbaric storage at 75 MPa and 100 MPa, the combined high-pressure low-temperature conditions offered by isochoric freezing appear to reduce unfavorable changes in lipid oxidation and the volatiles profile in raw milk; though direct experimental comparison would be needed to definitively conclude as much. Furthermore, hyperbaric storage requires active mechanical induction of pressure, whereas isochoric freezing produces passive self-pressurization driven only by temperature. As such, this preservation technique may prove more practical and adaptable to existing milk production and storage processes; low-temperature control infrastructure is already omnipresent within the dairy processing and distribution industry, and thus the only additional equipment necessary for this treatment would be isochoric storage containers. As a whole, this study indicates that isochoric freezing has the potential to significantly increase the shelf life of milk by reducing microbial activity, whilst maintaining its nutritional and physiochemical content. The application of isochoric freezing, with its potential to adjust milk's shelf life without a significant deviation in quality, could offer a new means of reducing milk waste driven by temporal market fluctuations, and addressing future milk capacitance uncertainties driven by climate change. Future work might include isochoric treatment of non-raw milk products, an examination of the absolute limits of milk shelf life under isochoric conditions, and the study of the evolution of milk bacterial profile following prolonged isochoric storage. Conceptualization, C.B.-S., M.J.P.-P. and B.R. ; Methodology, A.L.M., C.B.-S., A.K., G.T. and M.J.P.-P.; Formal analysis, A.L.M., C.B.-S., A.K. and G.T. ; Investigation, A.L.M., C.B.-S., A.K. and G.T. ; Writing--original draft, A.L.M. ; Writing--review & editing, A.L.M., C.B.-S., A.K., G.T., M.J.P.-P. and B.R. ; Visualization, A.L.M., A.K. and M.J.P.-P.; Supervision, C.B.-S., M.J.P.-P. and B.R. All authors have read and agreed to the published version of the manuscript. This work was supported by the USDA National Institute of Food and Agriculture, AFRI project Proposal #:2021-09570, Award #2022-67017-37098 \"Novel Isochoric Processing for Sustainable, Safe and High-Quality Preservation of Fluid Foods\". A.M. also recognizes support from the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE 2146752.