Optimizing germination conditions for Alicyclobacillus acidoterrestris spores, and achieving efficient inactivation via high hydrostatic pressure using a germination-inactivation strategy

Abstract

The 'germination-inactivation' strategy can potentially eliminate Alicyclobacillus acidoterrestris spores by exploiting the loss of their resistance that occurs during germination. However, the germination behavior of A. acidoterrestris spores remains incompletely characterized. In this study, the germination responses of A. acidoterrestris spores to various stimuli, including L-alanine, AGFK (L-asparagine, D-glucose, D-fructose, and KCl), and high hydrostatic pressure (HHP) were comprehensively examined. Surprisingly, it was observed that A. acidoterrestris spores germinate in acidic buffer, even in the absence of L-alanine or AGFK. Optimal acidic-induced germination efficiency was achieved at pH 4.0 following heat shock (70  °C, 15 min), and incubation at 37  °C. While heat treatment (65-75 °C) accelerated germination, acidic conditions (pH 3.0-4.0) were strictly necessary to initiate the process. Meanwhile, HHP-induced germination can also occur under acidic conditions (pH 3.0-5.0), demonstrating the acidophilic nature of A. acidoterrestris spores. Notably, spores subjected to HHP (200 or 500 MPa, 3 min, 30  °C) underwent spontaneous germination during subsequent incubation at 37  °C, irrespective of the medium's pH. Building on these findings, a combined 'HHP-incubation-pasteurization' approach was developed that achieved up to a 5.3 ± 0.51 log reduction in A. acidoterrestris spores within acidic systems. The results of this study provide a valuable foundation for the development of efficient sterilization techniques in acidic juice processing.

Keywords

Alicyclobacillus acidoterrestris spores / High hydrostatic pressure / Germination behavior / 'Germination-inactivation' strategy

Author contributions

The authors confirm their contributions to this paper as follows: study conception and design, funding acquisition: Rao L; writing − review and editing: Ding Y, Rao L; data curation, investigation: Ding Y, Lyu F, Gong Z; formal analysis: Yang D, Wang Y; data collection: Ding Y; project administration and supervision: Rao L, Yang D, Wang Y. All authors reviewed the results and approved the final version of the manuscript.

Data availability

All statistics generated or analyzed during this study are included in this published article.

Acknowledgements

This work was supported by National Key R&D Program of China (Grant No. 2023YFD2100303), Key Research and Development Program of Ningxia Hui Autonomous Region (Grant No. 2025BEE02003), National Natural Science Foundation of China (NSFC) (Grant Nos 32522084 and 32372470), Agricultural Research Outstanding Talents of China (Grant No. 13210317). And 2115 Talent Development Program of China Agricultural University.

Conflict of interest

The authors declare that they have no conflict of interest.

References

[1] Brito IPC, Silva EK. 2024. Pulsed electric field technology in vegetable and fruit juice processing: a review. Food Research International 184:114207 doi: 10.1016/j.foodres.2024.114207

[2] Sorrenti V, Burò I, Consoli V, Vanella L. 2023. Recent advances in health benefits of bioactive compounds from food wastes and by-products: biochemical aspects. International Journal of Molecular Sciences 24:2019 doi: 10.3390/ijms24032019

[3] Alves RMV, Sarantópoulos CIGL, Saron ES, Bordin MR. 2001. Stability of fruit juice drinks in aseptic packages. Packaging Technology and Science 14:79-86 doi: 10.1002/pts.538

[4] Raybaudi-Massilia RM, Mosqueda-Melgar J, Soliva-Fortuny R, Martín-Belloso O. 2009. Control of pathogenic and spoilage microorganisms in fresh-cut fruits and fruit juices by traditional and alternative natural antimicrobials. Comprehensive Reviews in Food Science and Food Safety 8:157-180 doi: 10.1111/j.1541-4337.2009.00076.x

[5] Snyder AB, Worobo RW. 2018. The incidence and impact of microbial spoilage in the production of fruit and vegetable juices as reported by juice manufacturers. Food Control 85:144-150 doi: 10.1016/j.foodcont.2017.09.025

[6] Lima Tribst AA, de Souza Sant’Ana A, de Massaguer PR. 2009. Review: microbiological quality and safety of fruit juices - past, present and future perspectives. Critical Reviews in Microbiology 35:310-339 doi: 10.3109/10408410903241428

[7] Walker M, Phillips CA. 2008. Alicyclobacillus acidoterrestris: an increasing threat to the fruit juice industry? International Journal of Food Science & Technology 43:250-260 doi: 10.1111/j.1365-2621.2006.01427.x

[8] Snyder AB, Martin N, Wiedmann M. 2024. Microbial food spoilage: impact, causative agents and control strategies. Nature Reviews Microbiology 22:528-542 doi: 10.1038/s41579-024-01037-x

[9] Chang SS, Kang DH. 2004. Alicyclobacillus spp. in the fruit juice industry: history, characteristics, and current isolation/detection procedures. Critical Reviews in Microbiology 30:55-74 doi: 10.1080/10408410490435089

[10] Danyluk MD, Friedrich LM, Jouquand C, Goodrich-Schneider R, Parish ME, et al. 2011. Prevalence, concentration, spoilage, and mitigation of Alicyclobacillus spp. inropical and subtropical fruit juice concentrates. Food Microbiology 28:472-477 doi: 10.1016/j.fm.2010.10.008

[11] Kakagianni M, Kalantzi K, Beletsiotis E, Ghikas D, Lianou A, et al. 2018. Development and validation of predictive models for the effect of storage temperature and pH on the growth boundaries and kinetics of Alicyclobacillus acidoterrestris ATCC 49025 in fruit drinks. Food Microbiology 74:40-49 doi: 10.1016/j.fm.2018.02.019

[12] Yokota A, Fujii T, Goto K. 2008. Alicyclobacillus: Thermophilic Acidophilic Bacilli. Tokyo: Springer. doi: 10.1007/978-4-431-69850-0

[13] Goto K, Tanaka T, Yamamoto R, Suzuki T, Tokuda H. 2008. Characteristics of Alicyclobacillus. In Alicyclobacillus:Thermophilic Acidophilic Bacilli, eds. Yokota A, Fujii T, Goto K. Tokyo: Springer. pp. 9-48 doi: 10.1007/978-4-431-69850-0_3

[14] Matsubara H, Goto K, Matsumura T, Mochida K, Iwaki M, et al. 2002. Alicyclobacillus acidiphilus sp. nov., a novel thermo-acidophilic, omega-alicyclic fatty acid-containing bacterium isolated from acidic beverages. International Journal of Systematic and Evolutionary Microbiology 52:1681-1685 doi: 10.1099/00207713-52-5-1681

[15] Goto K, Mochida K, Kato Y, Asahara M, Fujita R, et al. 2007. Proposal of six species of moderately thermophilic, acidophilic, endospore-forming bacteria: Alicyclobacillus contaminans sp. nov., Alicyclobacillus fastidiosus sp. nov., Alicyclobacillus kakegawensis sp. nov., Alicyclobacillus macrosporangiidus sp. nov., Alicyclobacillus sacchari sp. nov. and Alicyclobacillus shizuokensis sp. nov. International Journal of Systematic and Evolutionary Microbiology 57:1276-1285 doi: 10.1099/ijs.0.64692-0

[16] Wahia H, Fakayode OA, Mustapha AT, Zhou C, Dabbour M. 2024. Application and potential of multifrequency ultrasound in juice industry: comprehensive analysis of inactivation and germination of Alicyclobacillus acidoterrestris spores. Critical Reviews in Food Science and Nutrition 64:4561-4586 doi: 10.1080/10408398.2022.2143475

[17] McKnight IC, Eiroa MNU, Sant'Ana AS, Massaguer PR. 2010. Alicyclobacillus acidoterrestris in pasteurized exotic Brazilian fruit juices: isolation, genotypic characterization and heat resistance. Food Microbiology 27:1016-1022 doi: 10.1016/j.fm.2010.06.010

[18] Yamazaki K, Teduka H, Shinano H. 1996. Isolation and identification of Alicyclobacillus acidoterrestris from acidic beverages. Bioscience, Biotechnology, and Biochemistry 60:543-545 doi: 10.1271/bbb.60.543

[19] Vieira MC, Teixeira AA, Silva FM, Gaspar N, Silva CLM. 2002. Alicyclobacillus acidoterrestris spores as a target for Cupua-u (Theobroma grandiflorum) nectar thermal processing: kinetic parameters and experimental methods. International Journal of Food Microbiology 77:71-81 doi: 10.1016/S0168-1605(02)00043-0

[20] Silva FVM, Gibbs P. 2001. Alicyclobacillus acidoterrestris spores in fruit products and design of pasteurization processes. Trends in Food Science & Technology 12:68-74 doi: 10.1016/S0924-2244(01)00070-X

[21] Fan L, Zhang Y, Ismail BB, Muhammad AI, Li G, et al. 2024. Bacillus spore germination: mechanisms, identification, and antibacterial strategies. Critical Reviews in Food Science and Nutrition 64:11146-11160 doi: 10.1080/10408398.2023.2233184

[22] Setlow P, Christie G.2021. What's new and notable in bacterial spore killing! World Journal of Microbiology & Biotechnology 37: 144 doi: 10.1007/s11274-021-03108-0

[23] Lyu F, Zhang T, Gui M, Wang Y, Zhao L, et al. 2023. The underlying mechanism of bacterial spore germination: an update review. Comprehensive Reviews in Food Science and Food Safety 22:2728-2746 doi: 10.1111/1541-4337.13160

[24] Zhang T, Gong Z, Zhou B, Rao L, Liao X. 2025. Recent progress in proteins regulating the germination of Bacillus subtilis spores. Journal of Bacteriology 207:e00285-24 doi: 10.1128/jb.00285-24

[25] Yamamoto K. 2017. Food processing by high hydrostatic pressure. Bioscience, Biotechnology, and Biochemistry 81:672-679 doi: 10.1080/09168451.2017.1281723

[26] Sarker MR, Akhtar S, Torres JA, Paredes-Sabja D. 2015. High hydrostatic pressure-induced inactivation of bacterial spores. Critical Reviews in Microbiology 41:18-26 doi: 10.3109/1040841X.2013.788475

[27] Black EP, Setlow P, Hocking AD, Stewart CM, Kelly AL, et al. 2007. Response of spores to high-pressure processing. Comprehensive Reviews in Food Science and Food Safety 6:103-119 doi: 10.1111/j.1541-4337.2007.00021.x

[28] Porębska I, Rutkowska M, Sokołowska B. 2015. Decrease in optical density as a results of germination of Alicyclobacillus acidoterrestris spores under high hydrostatic pressure. High Pressure Research 35:89-97 doi: 10.1080/08957959.2015.1006630

[29] Sokołowska B, Skąpska S, Fonberg-Broczek M, Niezgoda J, Por-bska I, et al. 2015. Germination and inactivation of Alicyclobacillus acidoterrestris spores induced by moderate hydrostatic pressure. Polish Journal of Microbiology 64:351-359 doi: 10.5604/17331331.1170291

[30] Vercammen A, Vivijs B, Lurquin I, Michiels CW. 2012. Germination and inactivation of Bacillus coagulans and Alicyclobacillus acidoterrestris spores by high hydrostatic pressure treatment in buffer and tomato sauce. International Journal of Food Microbiology 152:162-167 doi: 10.1016/j.ijfoodmicro.2011.02.019

[31] Delbrück AI, Tritten Y, Nanni P, Heydenreich R, Mathys A. 2022. Moderate high-pressure superdormancy in Bacillus spores: properties of superdormant spores and proteins potentially influencing moderate high-pressure germination. Applied and Environmental Microbiology 88:e02406-21 doi: 10.1128/aem.02406-21

[32] Zhang J, Kang S, Zhang T, Wang J, Rao L, et al. 2025. Spontaneous germination of superdormant Bacillus subtilis spores under high hydrostatic pressure: influencing factors and underlying mechanism. Innovative Food Science & Emerging Technologies 100: 103937 doi: 10.1016/j.ifset.2025.103937

[33] Van Luong TS, Moir C, Chandry PS, Pinfold T, Olivier S, et al. 2020. Combined high pressure and heat treatment effectively disintegrates spore membranes and inactivates Alicyclobacillus acidoterrestris spores in acidic fruit juice beverage. Innovative Food Science & Emerging Technologies 66: 102523 doi: 10.1016/j.ifset.2020.102523

[34] Paidhungat M, Setlow P. 2000. Role of ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis. Journal of Bacteriology 182:2513-2519 doi: 10.1128/jb.182.9.2513-2519.2000

[35] Khan S, Wicander J, Korza G, Caldbeck R, Cowan AE, et al. 2025. Resistance and germination of spores of Bacillus species lacking members of a spore integral inner membrane protein family and locations of these proteins in spores. Journal of Bacteriology 207:e00217-25 doi: 10.1128/jb.00217-25

[36] Rueckert A, Ronimus RS, Morgan HW. 2005. Rapid differentiation and enumeration of the total, viable vegetative cell and spore content of thermophilic bacilli in milk powders with reference to Anoxybacillus flavithermus. Journal of Applied Microbiology 99:1246-1255 doi: 10.1111/j.1365-2672.2005.02728.x

[37] Sinigaglia M, Corbo MR, Altieri C, Campaniello D, D'amato D, et al. 2003. Combined effects of temperature, water activity, and pH on Alicyclobacillus acidoterrestris spores. Journal of Food Protection 66:2216-2221 doi: 10.4315/0362-028X-66.12.2216

[38] Wen J, Smelt JPPM, Vischer NOE, de Vos AL, Setlow P, et al. 2022. Heat activation and inactivation of bacterial spores: is there an overlap? Applied and Environmental Microbiology 88:e02324-21 doi: 10.1128/aem.02324-21

[39] Wuytack EY, Soons J, Poschet F, Michiels CW. 2000. Comparative study of pressure- and nutrient-induced germination of Bacillus subtilis spores. Applied and Environmental Microbiology 66:257-261 doi: 10.1128/AEM.66.1.257-261.2000

[40] Arnosti DN, Singer VL, Chamberlin MJ. 1986. Characterization of heat-shock in Bacillus subtilis. Journal of Bacteriology 168:1243-1249 doi: 10.1128/jb.168.3.1243-1249.1986

[41] Pinto CA, Moreira SA, Fidalgo LG, Inácio RS, Barba FJ, et al. 2020. Effects of high‐pressure processing on fungi spores: factors affecting spore germination and inactivation and impact on ultrastructure. Comprehensive Reviews in Food Science and Food Safety 19:553-573 doi: 10.1111/1541-4337.12534

[42] Pontius AJ, Rushing JE, Foegeding PM. 1998. Heat resistance of Alicyclobacillus acidoterrestris spores as affected by various pH values and organic acids. Journal of Food Protection 61:41-46 doi: 10.4315/0362-028X-61.1.41

[43] Freire V, Condón S, Gayán E. 2024. Impact of sporulation temperature on germination of Bacillus subtilis spores under optimal and adverse environmental conditions. Food Research International 182:114064 doi: 10.1016/j.foodres.2024.114064

[44] Paredes-Sabja D, Setlow P, Sarker MR. 2011. Germination of spores of Bacillales and Clostridiales species: mechanisms and proteins involved. Trends in Microbiology 19:85-94 doi: 10.1016/j.tim.2010.10.004

[45] Baker-Austin C, Dopson M. 2007. Life in acid: pH homeostasis in acidophiles. Trends in Microbiology 15:165-171 doi: 10.1016/j.tim.2007.02.005

[46] Blocher JC, Busta FF. 1985. Inhibition of germinant binding by bacterial spores in acidic environments. Applied and Environmental Microbiology 50:274-279 doi: 10.1128/aem.50.2.274-279.1985

[47] Setlow P. 2003. Spore germination. Current Opinion in Microbiology 6:550-556 doi: 10.1016/j.mib.2003.10.001

[48] Wuytack EY, Michiels CW. 2001. A study on the effects of high pressure and heat on Bacillus subtilis spores at low pH. International Journal of Food Microbiology 64:333-341 doi: 10.1016/S0168-1605(00)00478-5

[49] Zhang J, Ding Y, Kang S, Yang D, Wang Y, et al. 2024. Isolation and characterization of superdormant Bacillus subtilis spores under high hydrostatic pressure at 200 MPa and 500 MPa. Innovative Food Science & Emerging Technologies 96: 103769 doi: 10.1016/j.ifset.2024.103769

[50] Delbrück AI, Zhang Y, Hug V, Trunet C, Mathys A. 2021. Isolation, stability, and characteristics of high-pressure superdormant Bacillus subtilis spores. International Journal of Food Microbiology 343:109088 doi: 10.1016/j.ijfoodmicro.2021.109088

[51] Heydenreich R, Delbrück AI, Peternell C, Trunet C, Mathys A. 2024. Characterization of high-pressure-treated Bacillus subtilis spore populations using flow cytometry - shedding light on spore superdormancy at 550 MPa. International Journal of Food Microbiology 422:110812 doi: 10.1016/j.ijfoodmicro.2024.110812

[52] Lyu F, Jiang Z, Meng Q, Zhang T, Yang D, et al. 2025. Cortex degradation plays a critical role in modulating the pressure resistance of Bacillus spores and their inactivation under high-pressure treatments. Innovative Food Science & Emerging Technologies 105: 104177 doi: 10.1016/j.ifset.2025.104177