# Unraveling the Effects of Freezing and Frozen Storage Temperatures on Hop Secondary Metabolites and Antioxidants

## TL;DR

This study shows how different freezing and storage temperatures affect the chemical compounds and antioxidant properties of hops over time.

## Contribution

The study introduces a method to optimize hop freezing and storage temperatures based on metabolite and antioxidant retention.

## Key findings

- Freezing at −40 °C best preserves bitter acids, phenolic acids, and antioxidant capacity in hops.
- Xanthohumol and 8-prenylnaringenin levels peak at −30 and −20 °C, respectively.
- Over 360 days, storage time had a greater impact on metabolite changes than temperature.

## Abstract

This study evaluated the effect of freezing and frozen storage at three temperatures (−20, −30, −40 °C) on hop (Humulus lupulus L.) secondary metabolites and antioxidant capacity. These temperatures were selected based on the glass transition temperature (Tg’) of the maximally freeze-concentrated matrix. Cones were analyzed after freezing (t0) and up to 360 days (t360) by high-performance liquid chromatography with ultraviolet diode-array detection (HPLC-UV/DAD) for bitter acids, prenylflavonoids and phenolic acids, and by the Folin–Ciocalteu, ABTS the radical cation scavenging assay (ABTS) and the ferric-reducing antioxidant power assay (FRAP) assays for total phenolic content and antioxidant activity. Confocal laser scanning microscopy (CLSM) at t360 was used to relate microstructural damage to metabolite retention. Freezing at −40 °C ensured the highest retention of bitter acids, phenolic acids (gallic, syringic, vanillic, caffeic, chlorogenic), and antioxidant capacity, whereas xanthohumol and 8-prenylnaringenin reached their maximum levels at −30 and −20 °C, respectively. During frozen storage, changes in metabolite profiles were mainly driven by storage time rather than temperature; over 360 days, α-acids, colupulone, xanthohumol and selected phenolic acids increased, while most other compounds declined. Multivariate analysis and CLSM elucidated the relationships between process conditions, tissue structure and metabolite profiles, enabling the selection of freezing and storage temperatures to optimally preserve different targets of hop bioactives and overall indicating −40 °C as the most effective.