Citation: Pinto, C.A.; Galante, D.; Espinoza-Suarez, E.; Gaspar, V.M.; Mano, J.F.; Barba, F.J.; Saraiva, J.A. Development Control and Inactivation of Byssochlamys nivea Ascospores by Hyperbaric Storage at Room Temperature. Foods 2023, 12, 978. https://doi.org/10.3390/ foods12050978 Academic Editor: Matthew D. Moore Received: 3 February 2023 Revised: 15 February 2023 Accepted: 21 February 2023 Published: 25 February 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). foods Article Development Control and Inactivation of Byssochlamys nivea Ascospores by Hyperbaric Storage at Room Temperature Carlos A. Pinto 1 , Diogo Galante 1 , Edelman Espinoza-Suarez 1 ,Vítor M. Gaspar 2 , João F. Mano 2 , Francisco J. Barba 3 and Jorge A. Saraiva 1, * 1 LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal 2 CICECO—Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal 3 Nutrition and Food Science Area, Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Faculty of Pharmacy, Universitat de València, Avda. Vicent Andrés Estellés, 46100 València, Spain * Correspondence: jorgesaraiva@ua.pt Abstract: This study tested hyperbaric storage (25–150 MPa, for 30 days) at room-temperature (HS/RT, 18–23 ◦ C) in order to control the development of Byssochlamys nivea ascospores in apple juice. In order to mimic commercially pasteurized juice contaminated with ascospores, thermal pasteurization (70 and 80 ◦ C for 30 s) and nonthermal high pressure pasteurization (600 MPa for 3 min at 17 ◦ C, HPP) took place, and the juice was afterwards placed under HS/RT conditions. Control samples were also placed in atmospheric pressure (AP) conditions at RT and were refrigerated (4 ◦ C). The results showed that HS/RT, in samples without a pasteurization step and those pasteurized at 70 ◦ C/30 s, was able to inhibit ascospore development, contrarily to samples at AP/RT and refrigera- tion. HS/RT for samples pasteurized at 80 ◦ C/30 s evidenced ascospore inactivation, especially at 150 MPa, wherein an overall reduction of at least 4.73 log units of ascospores was observed to below detection limits (1.00 Log CFU/mL); meanwhile, for HPP samples, especially at 75 and 150 MPa, an overall reduction of 3 log units (to below quantification limits, 2.00 Log CFU/mL) was observed. Phase-contrast microscopy revealed that the ascospores do not complete the germination process under HS/RT, hence avoiding hyphae formation, which is important for food safety since mycotoxin development occurs only after hyphae formation. These findings suggest that HS/RT is a safe food preservation methodology, as it prevents ascospore development and inactivates them following commercial-like thermal or nonthermal HPP pasteurization, preventing mycotoxin production and enhancing ascospore inactivation. Keywords: Byssochlamys nivea; mycotoxins; food safety; hyperbaric storage; thermal pasteurization; high pressure processing 1. Introduction Molds belong to the kingdom of fungi, which also includes yeasts and common mush- rooms. Molds may be classified into six filo: Ascomycota, Basidiomycota, Chytriomycota, Deuteromycota (an informal group of unrelated fungi that solely reproduce asexually), Glomeromycota, and Zygomycota. The Ascomycota produce extraordinarily resistant conidiospores (asexually produced spores) and ascospores (sexually produced spores) [1]. As the ascospores mature, they are released into the air and may travel great distances on the wind, contaminating a broad variety of fields, food-related settings, and eventually food items themselves, posing a hazard to food quality and safety [2]. The extreme resistance of ascospores to thermal and nonthermal processing, oxidative stress, UV-radiation, etc., is due to their exquisite characteristics, such as a protoplast very rich in trehalose and mannitol, which makes their interior very viscous, several heat-shock proteins, and a thick, Foods 2023, 12, 978. https://doi.org/10.3390/foods12050978 https://www.mdpi.com/journal/foods