Effects of heat stress on the resistance of Lactic Acid Bacteria: Viability, morphological integrity, and acidification capacity in fermentation processe

Effects of heat stress on the resistance of Lactic Acid Bacteria: Viability, morphological integrity, and acidification capacity in fermentation processe | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of heat stress on the resistance of Lactic Acid Bacteria: Viability, morphological integrity, and acidification capacity in fermentation processe IBOURAHEMA COULIBALY This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7053884/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Lactic acid bacteria (LAB) are essential in food fermentation, contributing to preservation and flavor development. However, their survival under heat stress during food processing remains a challenge. This study investigates the effects of heat treatment (45°C–65°C) on the viability, morphology, and acidification capacity of Lactobacillus plantarum , Lactobacillus acidophilus , and Lactococcus lactis . Results showed that L. plantarum exhibited the greatest thermal resistance, with minimal viability loss at 55°C. SEM analysis revealed cellular damage, including membrane rupture, particularly at higher temperatures (60°C and 65°C). Acidification capacity declined with increasing heat, indicating reduced metabolic activity. These findings emphasize the need to understand strain-specific heat tolerance for optimizing fermentation processes and improving the quality of heat-sensitive products like yogurt and probiotics. Applied Biochemistry Lactic acid bacteria thermal resistance viability fermentation acidification morphology food processing Figures Figure 1 Figure 2 Figure 3 1. Introduction Lactic acid bacteria (LAB) play a vital role in the fermentation industry, contributing to food preservation, flavor development, and the promotion of health benefits, such as enhancing the gut microbiota [ 1 , 2 ]. These microorganisms are involved in the fermentation of various food products, including dairy, meat, and vegetables, where they exert significant influence on the final product's quality and safety [ 3 , 27 ]. LAB are widely valued for their ability to produce lactic acid during fermentation, which helps preserve food and imparts desirable sensory characteristics, including sourness and texture [ 4 ]. However, during thermal processing, such as pasteurization, LAB are subjected to heat stress, which can negatively impact their viability and functional properties [ 5 ]. This is a significant challenge in the food industry, as heat treatment is often necessary to ensure food safety, particularly in dairy and other fermented products. Understanding the heat resistance mechanisms of LAB is crucial for optimizing fermentation processes, improving product quality, and developing heat-resistant strains with enhanced survival rates during thermal treatment [ 6 , 7 ]. Previous studies have demonstrated that LAB possess mechanisms to respond to environmental stresses, including high temperature, osmotic pressure, and pH fluctuations. Among these, heat stress is particularly critical because it can damage cellular structures, leading to a loss of metabolic activity and overall viability [ 8 , 9 ]. One key adaptation to heat stress is the upregulation of heat shock proteins (HSPs), which play an essential role in protecting cellular components from thermal damage by facilitating protein folding and stabilizing cellular structures [ 10 , 11 ]. These stress-response mechanisms vary between different LAB strains, and several factors influence their thermotolerance, such as membrane composition, the presence of protective compounds like trehalose, and genetic adaptations to high temperatures [ 12 , 13 ]. This study aims to evaluate the effects of heat stress on the viability, morphological integrity, and acidification capacity of three LAB strains— Lactobacillus plantarum , Lactobacillus acidophilus , and Lactococcus lactis —which are commonly used in the food industry. By investigating these factors, we hope to gain a better understanding of how heat exposure impacts LAB, which can lead to improved industrial applications, such as the development of heat-resistant strains for pasteurization and fermentation processes [ 14 , 15 ]. 2. Materials and Methods 2.1. Bacterial strains and culture conditions Three LAB strains were selected for this study: Lactobacillus plantarum , Lactobacillus acidophilus , and Lactococcus lactis , each representing different genera and species commonly used in industrial fermentation processes. These strains were cultured in de Man, Rogosa, and Sharpe (MRS) broth, which is a standard medium for the growth of LAB due to its high nutrient content. Cultures were incubated at 37°C for 24 hours under anaerobic conditions to promote optimal growth, as LAB are facultative or obligate anaerobes. The choice of 37°C is representative of the temperature commonly encountered in the fermentation of dairy products and other foods where LAB are used. The strains were then harvested at their mid-log phase to ensure the viability of the cells during subsequent heat treatments [ 14 ]. 2.2. Heat treatment After the initial growth phase, bacterial cultures were subjected to heat stress at 55°C, 60°C, and 65°C for 30 minutes, which is a typical duration for assessing heat resistance in bacterial strains. These temperatures were chosen to simulate the thermal conditions that may be encountered during pasteurization and other food processing steps. The cultures were exposed to these temperatures in a water bath, which ensured even and controlled heat distribution. Immediately following the heat treatment, samples were cooled rapidly on ice to halt any further thermal effects, preserving the condition of the cells for post-treatment analysis. This cooling step is crucial to prevent any additional damage that could occur if the cells were allowed to return to room temperature at a slower pace, which could confound results [ 15 , 25 ]. 2.3. Viability assessment To assess bacterial viability after heat treatment, the plate count method was employed, which is a widely used technique for determining the number of viable microorganisms in a sample. The samples were serially diluted and plated on MRS agar, which provides the necessary nutrients for LAB growth. The plates were then incubated at 37°C for 48 hours, allowing the colonies to form. The colony-forming units per milliliter (CFU/mL) were counted, and the results were used to calculate the reduction in bacterial viability due to heat exposure. This method is reliable for distinguishing between viable and non-viable cells, as only live cells can form colonies under these conditions [ 16 , 28 ]. To assess the morphological alterations caused by heat stress, scanning electron microscopy (SEM) was employed, as it enables high-resolution visualization of bacterial surface structures [ 17 ]. Samples were first fixed with glutaraldehyde to preserve cellular integrity, then dehydrated through a graded ethanol series and coated with gold to facilitate conductivity. This preparation ensured optimal conditions for SEM imaging. The resulting micrographs revealed strain-specific responses to thermal stress, particularly regarding membrane integrity and cellular deformation, offering valuable insight into the structural resilience of LAB strains under heat exposure [ 29 ]. 2.5. Acidification capacity The acidification capacity of the heat-treated LAB strains was assessed by monitoring the pH of MRS broth inoculated with both treated and untreated cultures. Following inoculation, the samples were incubated at 37°C for 24 hours, consistent with standard protocols for evaluating LAB metabolic performance. After incubation, pH measurements were conducted using a calibrated pH meter. This approach allows for a reliable evaluation of bacterial metabolic activity, as lactic acid production during fermentation leads to a measurable decrease in pH. A significant reduction in acidification following heat exposure is indicative of diminished metabolic function or compromised fermentative ability. As described by [ 18 ], this method facilitates comparative analysis of the lactic acid production efficiency among LAB strains under thermal stress. 3. Results and Discussion 3.1. Viability Under Heat Stress The results of heat exposure on the viability of the three LAB strains were consistent with previous findings on the impact of thermal stress on bacterial survival [ 1 , 24 ]. All strains exhibited a reduction in viability as temperature and exposure time increased, with Lactobacillus plantarum showing the greatest thermotolerance. At 45°C, all strains maintained high viability (log CFU/mL > 8), which aligns with the natural environmental temperatures they may encounter in fermented food products. However, at 55°C and 65°C, a significant reduction in viable cell counts was observed, especially for Lactococcus lactis , which exhibited the most dramatic loss in viability. Table 1 Viability (log CFU/mL) after heat treatment Temperature °C Time (min) L. plantarum L. acidophilus Lc. lactic 45 10 8,7 8,5 8,3 55 20 7,2 6,5 6,9 65 30 4,8 3,6 2,9 These findings suggest that L. plantarum has an inherent capacity to withstand heat stress, which is particularly relevant for industrial applications involving pasteurization or fermentation at elevated temperatures [ 2 , 3 ]. L. acidophilus demonstrated moderate heat tolerance, but L. lactis was more sensitive to heat, consistent with other studies that have reported strain-specific variations in heat resistance [ 4 ]. These differences can be attributed to various factors, including the robustness of the cell wall structure and the presence of heat-shock proteins, which help the bacteria recover from thermal damage [ 5 ]. 3.2. Morphological changes The morphological changes observed under heat stress provide further insight into the mechanisms behind the observed reductions in viability. SEM images revealed substantial damage to the cell membranes of all three LAB strains, particularly at 65°C. The membrane shrinkage and surface ruptures observed in Lactobacillus acidophilus (Fig. 2 ) are indicative of thermal lysis, which disrupts cellular integrity and likely contributes to the observed reduction in viability [ 6 ]. Similar membrane damage has been reported in other LAB strains exposed to heat [ 7 ], suggesting that a disruption in membrane integrity is a common response to thermal stress. Interestingly, the degree of morphological damage was more pronounced in Lactococcus lactis , which exhibited severe membrane rupture, while L. plantarum appeared to retain its shape and structure better under the same conditions [ 8 ]. This suggests that L. plantarum might have more robust cellular mechanisms to withstand thermal stress, including stronger or more stable cell membranes, or higher levels of protective compounds like trehalose, which has been linked to heat resistance in bacteria [ 9 , 26 ]. This observation also has practical implications for industrial applications. Strains that maintain structural integrity under heat stress may be more suitable for products that undergo higher-temperature processes, as they are more likely to survive and retain functionality [ 10 , 22 ]. 3.3. Acidification capacity The decline in acidification capacity post-heat treatment is another critical finding, reflecting the functional impairment of LAB after heat exposure. The acidification ability of LAB is essential in fermentation processes, as it helps preserve food and imparts desirable sensory qualities [ 11 , 30 ]. In this study, the final pH of MRS broth was significantly higher after heat treatment, indicating that the heat-exposed LAB strains had a reduced ability to produce lactic acid. This aligns with previous studies showing that heat stress can affect metabolic activity, including the ability to ferment sugars [ 1 , 12 ]. At 65°C, the pH values for all strains were notably higher, especially for Lactococcus lactis , which showed the least acidification, reaching a final pH of 5.5 (Table 2 ). Table 2 Final pH value after 24 h incubation Strain Control 45°C 55°C 65°C L. plantarum 3,6 3,8 4,3 4,9 L. acidophilus 3,5 3,9 4,4 5,1 Lc. lactic 3,7 4,0 4,7 5,5 This suggests that L. lactis may have a reduced metabolic capacity under heat stress, possibly due to more extensive cellular damage or an inability to adapt to high temperatures. On the other hand, Lactobacillus plantarum and L. acidophilus showed relatively better acidification performance after heat exposure, with final pH values of 4.9 and 5.1, respectively [ 13 , 14 ]. These results highlight the importance of selecting strains with good thermal resilience for applications where maintaining metabolic activity under heat stress is critical [ 15 , 21 ]. Additionally, this finding underscores the complexity of bacterial responses to heat, where both viability and functionality (e.g., acid production) are influenced by a combination of factors such as cell wall integrity and stress response mechanisms [ 3 , 16 ]. 3.4.Implications for industrial applications The findings of this study provide valuable insights into the selection of LAB strains for industrial fermentation processes that involve heat exposure. Strains like Lactobacillus plantarum that exhibit both higher thermal resistance and better acidification capacity are more suitable for processes that involve moderate to high temperatures, such as pasteurization, yogurt production, and probiotic delivery [ 17 , 18 ]. Conversely, strains like Lactococcus lactis , which show significant loss in viability and functionality under heat stress, may not be as suitable for these applications unless subjected to protective treatments, such as pre-adaptation to heat or the use of cryoprotectants in fermentation media [ 19 , 23 ]. Furthermore, the study highlights the potential for improving heat tolerance in LAB strains through genetic and metabolic engineering. Understanding the molecular mechanisms that confer heat resistance such as the role of heat-shock proteins, trehalose synthesis, and cell membrane composition can help in developing more heat-tolerant strains for the food and pharmaceutical industries [ 5 , 20 ]. 4. Conclusion In conclusion, this study highlights the differential heat tolerance among lactic acid bacteria, with L. plantarum exhibiting superior resistance compared to L. acidophilus and Lc. lactis. Moderate thermal exposure up to 55°C had limited effects on viability, particularly for L. plantarum, while higher temperatures (60–65°C) caused significant morphological damage and reduced acidification activity. Scanning electron microscopy confirmed structural compromise of cell membranes, especially in thermosensitive strains. These findings underscore the necessity of selecting thermotolerant strains for industrial applications involving thermal processes. Understanding the heat response profiles of LAB is essential for optimizing fermentation performance and ensuring the functional integrity of probiotic products. This knowledge can inform starter culture formulation and enhance the quality of heat-processed fermented foods. Furthermore, strain-specific resilience offers opportunities to improve process robustness without compromising product efficacy. Future research should explore molecular mechanisms of thermal resistance and investigate potential protective strategies. Ultimately, targeted strain selection and process control can advance industrial fermentation and probiotic manufacturing. Declarations Acknowledgements The authors would like to express their sincere gratitude to the laboratories of Université Jean Lorougnon Guédé and Gembloux Agro-Bio Tech for providing the technical facilities required for this research. Special thanks go to Dr. Ibrahim Konaté for his scientific guidance, and to Kra Kouassi Athanase, Voko-Bi Rodrigue, Beugré Maxwell, and Mohamed Baghui for their valuable contributions throughout the study. The support from the PNR1 project “CoTrans-Alim” is gratefully acknowledged. We also thank all staff members and students who assisted in data collection and laboratory analyses. This work would not have been possible without their collective efforts References Papadimitriou K, Alegría Á, Bron PA et al (2016) Stress physiology of lactic acid bacteria. Microbiol Mol Biol Rev 80(3):837–890. 10.1128/MMBR.00076-15 Wouters JA, Rombouts FM, de Vos WM et al (1999) Heat shock proteins in lactic acid bacteria. Antonie Van Leeuwenhoek 76(1–4):235–242. 10.1023/A:1002067908930 Corcoran BM, Stanton C, Fitzgerald GF, Ross RP (2005) Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars. Appl Environ Microbiol 71(6):3060–3067. 10.1128/AEM.71.6.3060-3067.2005 Fiocco D, Capozzi V, Collins M et al (2007) Characterization of stress-induced genes of Lactobacillus plantarum and their role in stress tolerance. Appl Microbiol Biotechnol 74(5):1040–1050. 10.1007/s00253-006-0744-2 Chen C, Wang Y, Zhang Y et al (2024) Screening and genome analysis of heat-resistant and antioxidant Lactobacillus plantarum L19. Front Microbiol 15:1455849. 10.3389/fmicb.2024.1455849 Zhao X, Zhang M, Bai W (2013) Effects of thermal processing on viability, membrane integrity and protein expression of probiotic bacteria. J Food Eng 119(2):287–293. 10.1016/j.jfoodeng.2013.05.040 Karam L, Jama C, Mamet T et al (2013) Effect of thermal and osmotic treatments on the viability and physiology of lactic acid bacteria used in starter cultures. Food Control 34(2):494–500. 10.1016/j.foodcont.2013.05.036 Gänzle MG, Schwab C (2009) Ecology of exopolysaccharide formation by lactic acid bacteria: regulation, genetic determinants, and impact on technological properties. Int Dairy J 19(6–7):341–346. 10.1016/j.idairyj.2008.12.008 Shobharani P, Halami PM (2015) Heat shock response and cross protection against acid and bile stress in Lactobacillus plantarum . J Basic Microbiol 55(6):729–739. 10.1002/jobm.201400842 Zárate G, Chaia AP (2009) Effect of heat treatment on the cell surface hydrophobicity of Lactobacillus acidophilus and Lactobacillus casei . J Appl Microbiol 106(5):1612–1619. 10.1111/j.1365-2672.2008.04029.x Vinderola G, Reinheimer JA, Salminen S (2006) Probiotic bacteria: stress response mechanisms and stress factors relevant to probiotic viability and functionality. Food Microbiol 23(6):663–669. 10.1016/j.fm.2005.10.006 Rault A, Bouix M, Béal C (2009) Fermentation pH influences the physiological-state dynamics of Lactobacillus bulgaricus CFL1 during pH-controlled culture. Appl Environ Microbiol 75(13):4374–4381. 10.1128/AEM.00130-09 Zhang Y, Li X, Wang Y et al (2019) Mild heat stress limited the post-acidification caused by Lactobacillus rhamnosus hsryfm 1301 in fermented milk. J Dairy Sci Font de Valdez G, de Giori GS, de Ruiz Holgado AP, Oliver G (1983) Temperature effect on the growth, protein synthesis and ribonucleic acid of Lactobacillus casei . Appl Environ Microbiol 46(5):1231–1234 Burgain J, Gaiani C, Linder M, Scher J (2011) Encapsulation of probiotic living cells: From laboratory scale to industrial applications. J Food Eng 104(4):467–483. 10.1016/j.jfoodeng.2010.12.031 Ananta E, Heinz V, Knorr D (2004) Assessment of high pressure and thermal processing on bacteria using porosity and gas exchange characteristics of the cell envelope. Innov Food Sci Emerg Technol 5(1):1–9. 10.1016/j.ifset.2003.11.002 Wang W, Zhou W, Wang Y et al (2020) Microencapsulation of probiotic bacteria with alginate–chitosan microgels for yogurt production. LWT - Food Sci Technol 124:109155. 10.1016/j.lwt.2020.109155 Broadbent JR, Larsen RL, Deibel V, Steele JL (2010) Physiological and transcriptional response of Lactobacillus casei ATCC 334 to acid stress. J Bacteriol 192(8):2445–2458. 10.1128/JB.01618-09 Desmond C, Ross RP, O’Callaghan E et al (2002) Improved survival of Lactobacillus paracasei NFBC 338 in spray-dried powders containing gum acacia. J Appl Microbiol 93(6):1003–1011. 10.1046/j.1365-2672.2002.01776.x Barbosa J, Borges S, Teixeira P (2015) Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests. Food Control 51:323–330. 10.1016/j.foodcont.2014.11.036 Higashikawa F, Noda M, Awaya T et al (2010) Improvement of constipation and liver function by plant-derived Lactobacillus plantarum SN13T in humans. Microorganisms 58(1):37–44 Ahn J, Grun IU, Mustapha A (2002) Effects of lactobacilli on the inhibition of Helicobacter pylori and the production of lactic acid. J Food Sci 67(7):2765–2769. 10.1111/j.1365-2621.2002.tb08787.x Malmo C, LaStella D, Ryan K, Huppertz T (2022) Role of calcium ions in stress resistance of lactic acid bacteria. J Dairy Sci 105(2):1234–1242. 10.3168/jds.2021-21015 Wu T, Wang X, Li Y et al (2015) Influence of prebiotics and heat shock pretreatment on the survival of Lactobacillus rhamnosus under acidic and bile stress conditions. Food Res Int 74:68–73. 10.1016/j.foodres.2015.04.008 Sanders ME, Guarner F, Guerrant R et al (2013) An update on the use and investigation of probiotics in health and disease. Gut 62(5):787–796. 10.1136/gutjnl-2012-302504 Bourdichon F, Casaregola S, Farrokh C et al (2012) Food fermentations: Microorganisms with technological beneficial use. Int J Food Microbiol 154(3):87–97. 10.1016/j.ijfoodmicro.2011.12.030 Monteagudo-Mera A, Rastall RA, Gibson GR et al (2019) In vitro evaluation of the viability of commercial probiotics and their protective effect against enteropathogenic bacteria. J Funct Foods 57:392–401. 10.1016/j.jff.2019.04.030 De Angelis M, Gobbetti M (2017) Stress responses of lactic acid bacteria. Curr Opin Food Sci 13:68–73. 10.1016/j.cofs.2017.02.002 Slattery C, Cotter PD, O'Toole PW (2009) Analysis of bacterial stress responses in the selection of lactic acid bacteria for functional foods. J Appl Microbiol 107(6):1903–1914. 10.1111/j.1365-2672.2009.04376.x Tripathi MK, Giri SK (2014) Probiotic functional foods: Survival of probiotics during processing and storage. J Funct Foods 9:225–241. 10.1016/j.jff.2014.04.030 Additional Declarations The authors declare potential competing interests as follows: Yes Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7053884","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":481099093,"identity":"8c703001-7d9b-4dbd-b0fa-ce15ead1c24e","order_by":0,"name":"IBOURAHEMA COULIBALY","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIiWNgGAWjYBACNgkwdQDCkyhgYOAHMRIKiNZiwMAg2QDSYoDHGhQtDECVBgegDFyAT7r5mHRFzR053fYDbBIWBjZ2m8+vTvzwwIBBnl/sAFYtbDLH0iTPHHtmbHYmgU1CwiAteduNt5slgA4znDk7AYdfcswkG9gOJ247ANZyONnsxtkNIC0JBrfxafl3uH7b+QcQLcYzzm7+QVBLY9vhBLMbEFvsDPh7t+G3ReZYsmVj32HDbTceNlsA/ZIgcYN3m0WCgQROv8jPbj54s+HbYXmz88kHb0tU2Njz95/dfPNHhY08vzR2LUiAsYEZGEmJDRJglRKElEM1fWBgsGfgP0Cc6lEwCkbBKBgxAABETV8BkktJpwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-6744-2012","institution":"University Jean Lorougnon GUEDE","correspondingAuthor":true,"prefix":"","firstName":"IBOURAHEMA","middleName":"","lastName":"COULIBALY","suffix":""}],"badges":[],"createdAt":"2025-07-05 15:18:29","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":true,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-7053884/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7053884/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86220622,"identity":"9ac296e4-7ab7-424b-8221-3476df725984","added_by":"auto","created_at":"2025-07-08 07:00:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":34468,"visible":true,"origin":"","legend":"\u003cp\u003e(Log reduction in LAB viability after exposure to increasing temperatures) visually demonstrates these differences, with \u003cem\u003eL. plantarum\u003c/em\u003e showing the least reduction at higher temperatures, supporting its use in processes that require higher heat tolerance.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7053884/v1/5d0bbeaff465565035995c86.jpg"},{"id":86219747,"identity":"1a82bc28-a11f-4b36-b91b-1476eef906b3","added_by":"auto","created_at":"2025-07-08 06:52:31","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":48469,"visible":true,"origin":"","legend":"\u003cp\u003eDamage scores based on SEM analysis across temperatures.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7053884/v1/f1b5db2a2167b444de5ca5c0.jpg"},{"id":86219748,"identity":"c0de31fb-e095-4356-82a5-58fa230dd261","added_by":"auto","created_at":"2025-07-08 06:52:31","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39260,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of heat on pH reduction capacity of LAB strains.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7053884/v1/fca3c7cde79d464a3993877f.jpg"},{"id":86221325,"identity":"0b949caf-f6eb-46f8-a2f2-e0d4799a12dd","added_by":"auto","created_at":"2025-07-08 07:08:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":652084,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7053884/v1/3da766c6-1615-4ee9-88d4-31559ff2b2da.pdf"}],"financialInterests":"The authors declare potential competing interests as follows: Yes","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEffects of heat stress on the resistance of Lactic Acid Bacteria: Viability, morphological integrity, and acidification capacity in fermentation processe\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLactic acid bacteria (LAB) play a vital role in the fermentation industry, contributing to food preservation, flavor development, and the promotion of health benefits, such as enhancing the gut microbiota [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These microorganisms are involved in the fermentation of various food products, including dairy, meat, and vegetables, where they exert significant influence on the final product's quality and safety [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. LAB are widely valued for their ability to produce lactic acid during fermentation, which helps preserve food and imparts desirable sensory characteristics, including sourness and texture [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, during thermal processing, such as pasteurization, LAB are subjected to heat stress, which can negatively impact their viability and functional properties [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This is a significant challenge in the food industry, as heat treatment is often necessary to ensure food safety, particularly in dairy and other fermented products.\u003c/p\u003e\u003cp\u003eUnderstanding the heat resistance mechanisms of LAB is crucial for optimizing fermentation processes, improving product quality, and developing heat-resistant strains with enhanced survival rates during thermal treatment [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Previous studies have demonstrated that LAB possess mechanisms to respond to environmental stresses, including high temperature, osmotic pressure, and pH fluctuations. Among these, heat stress is particularly critical because it can damage cellular structures, leading to a loss of metabolic activity and overall viability [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. One key adaptation to heat stress is the upregulation of heat shock proteins (HSPs), which play an essential role in protecting cellular components from thermal damage by facilitating protein folding and stabilizing cellular structures [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These stress-response mechanisms vary between different LAB strains, and several factors influence their thermotolerance, such as membrane composition, the presence of protective compounds like trehalose, and genetic adaptations to high temperatures [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study aims to evaluate the effects of heat stress on the viability, morphological integrity, and acidification capacity of three LAB strains\u0026mdash;\u003cem\u003eLactobacillus plantarum\u003c/em\u003e, \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e, and \u003cem\u003eLactococcus lactis\u003c/em\u003e\u0026mdash;which are commonly used in the food industry. By investigating these factors, we hope to gain a better understanding of how heat exposure impacts LAB, which can lead to improved industrial applications, such as the development of heat-resistant strains for pasteurization and fermentation processes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Bacterial strains and culture conditions\u003c/h2\u003e\u003cp\u003eThree LAB strains were selected for this study: \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e, and \u003cem\u003eLactococcus lactis\u003c/em\u003e, each representing different genera and species commonly used in industrial fermentation processes. These strains were cultured in de Man, Rogosa, and Sharpe (MRS) broth, which is a standard medium for the growth of LAB due to its high nutrient content. Cultures were incubated at 37\u0026deg;C for 24 hours under anaerobic conditions to promote optimal growth, as LAB are facultative or obligate anaerobes. The choice of 37\u0026deg;C is representative of the temperature commonly encountered in the fermentation of dairy products and other foods where LAB are used. The strains were then harvested at their mid-log phase to ensure the viability of the cells during subsequent heat treatments [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Heat treatment\u003c/h2\u003e\u003cp\u003eAfter the initial growth phase, bacterial cultures were subjected to heat stress at 55\u0026deg;C, 60\u0026deg;C, and 65\u0026deg;C for 30 minutes, which is a typical duration for assessing heat resistance in bacterial strains. These temperatures were chosen to simulate the thermal conditions that may be encountered during pasteurization and other food processing steps. The cultures were exposed to these temperatures in a water bath, which ensured even and controlled heat distribution. Immediately following the heat treatment, samples were cooled rapidly on ice to halt any further thermal effects, preserving the condition of the cells for post-treatment analysis. This cooling step is crucial to prevent any additional damage that could occur if the cells were allowed to return to room temperature at a slower pace, which could confound results [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Viability assessment\u003c/h2\u003e\u003cp\u003eTo assess bacterial viability after heat treatment, the plate count method was employed, which is a widely used technique for determining the number of viable microorganisms in a sample. The samples were serially diluted and plated on MRS agar, which provides the necessary nutrients for LAB growth. The plates were then incubated at 37\u0026deg;C for 48 hours, allowing the colonies to form. The colony-forming units per milliliter (CFU/mL) were counted, and the results were used to calculate the reduction in bacterial viability due to heat exposure. This method is reliable for distinguishing between viable and non-viable cells, as only live cells can form colonies under these conditions [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo assess the morphological alterations caused by heat stress, scanning electron microscopy (SEM) was employed, as it enables high-resolution visualization of bacterial surface structures [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Samples were first fixed with glutaraldehyde to preserve cellular integrity, then dehydrated through a graded ethanol series and coated with gold to facilitate conductivity. This preparation ensured optimal conditions for SEM imaging. The resulting micrographs revealed strain-specific responses to thermal stress, particularly regarding membrane integrity and cellular deformation, offering valuable insight into the structural resilience of LAB strains under heat exposure [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Acidification capacity\u003c/h2\u003e\u003cp\u003eThe acidification capacity of the heat-treated LAB strains was assessed by monitoring the pH of MRS broth inoculated with both treated and untreated cultures. Following inoculation, the samples were incubated at 37\u0026deg;C for 24 hours, consistent with standard protocols for evaluating LAB metabolic performance. After incubation, pH measurements were conducted using a calibrated pH meter. This approach allows for a reliable evaluation of bacterial metabolic activity, as lactic acid production during fermentation leads to a measurable decrease in pH. A significant reduction in acidification following heat exposure is indicative of diminished metabolic function or compromised fermentative ability. As described by [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], this method facilitates comparative analysis of the lactic acid production efficiency among LAB strains under thermal stress.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Viability Under Heat Stress\u003c/h2\u003e\u003cp\u003eThe results of heat exposure on the viability of the three LAB strains were consistent with previous findings on the impact of thermal stress on bacterial survival [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. All strains exhibited a reduction in viability as temperature and exposure time increased, with \u003cem\u003eLactobacillus plantarum\u003c/em\u003e showing the greatest thermotolerance. At 45\u0026deg;C, all strains maintained high viability (log CFU/mL\u0026thinsp;\u0026gt;\u0026thinsp;8), which aligns with the natural environmental temperatures they may encounter in fermented food products. However, at 55\u0026deg;C and 65\u0026deg;C, a significant reduction in viable cell counts was observed, especially for \u003cem\u003eLactococcus lactis\u003c/em\u003e, which exhibited the most dramatic loss in viability.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eViability (log CFU/mL) after heat treatment\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTemperature \u0026deg;C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTime (min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eL. acidophilus\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eLc. lactic\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e45\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8,7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8,5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8,3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e55\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7,2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6,5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6,9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e65\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4,8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3,6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2,9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThese findings suggest that \u003cem\u003eL. plantarum\u003c/em\u003e has an inherent capacity to withstand heat stress, which is particularly relevant for industrial applications involving pasteurization or fermentation at elevated temperatures [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. \u003cem\u003eL. acidophilus\u003c/em\u003e demonstrated moderate heat tolerance, but \u003cem\u003eL. lactis\u003c/em\u003e was more sensitive to heat, consistent with other studies that have reported strain-specific variations in heat resistance [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These differences can be attributed to various factors, including the robustness of the cell wall structure and the presence of heat-shock proteins, which help the bacteria recover from thermal damage [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Morphological changes\u003c/h2\u003e\u003cp\u003eThe morphological changes observed under heat stress provide further insight into the mechanisms behind the observed reductions in viability. SEM images revealed substantial damage to the cell membranes of all three LAB strains, particularly at 65\u0026deg;C. The membrane shrinkage and surface ruptures observed in \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) are indicative of thermal lysis, which disrupts cellular integrity and likely contributes to the observed reduction in viability [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Similar membrane damage has been reported in other LAB strains exposed to heat [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], suggesting that a disruption in membrane integrity is a common response to thermal stress.\u003c/p\u003e\u003cp\u003eInterestingly, the degree of morphological damage was more pronounced in \u003cem\u003eLactococcus lactis\u003c/em\u003e, which exhibited severe membrane rupture, while \u003cem\u003eL. plantarum\u003c/em\u003e appeared to retain its shape and structure better under the same conditions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This suggests that \u003cem\u003eL. plantarum\u003c/em\u003e might have more robust cellular mechanisms to withstand thermal stress, including stronger or more stable cell membranes, or higher levels of protective compounds like trehalose, which has been linked to heat resistance in bacteria [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis observation also has practical implications for industrial applications. Strains that maintain structural integrity under heat stress may be more suitable for products that undergo higher-temperature processes, as they are more likely to survive and retain functionality [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Acidification capacity\u003c/h2\u003e\u003cp\u003eThe decline in acidification capacity post-heat treatment is another critical finding, reflecting the functional impairment of LAB after heat exposure. The acidification ability of LAB is essential in fermentation processes, as it helps preserve food and imparts desirable sensory qualities [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In this study, the final pH of MRS broth was significantly higher after heat treatment, indicating that the heat-exposed LAB strains had a reduced ability to produce lactic acid. This aligns with previous studies showing that heat stress can affect metabolic activity, including the ability to ferment sugars [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAt 65\u0026deg;C, the pH values for all strains were notably higher, especially for \u003cem\u003eLactococcus lactis\u003c/em\u003e, which showed the least acidification, reaching a final pH of 5.5 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFinal pH value after 24 h incubation\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e45\u0026deg;C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55\u0026deg;C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003e65\u0026deg;C\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eL. plantarum\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3,6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3,8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4,9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eL. acidophilus\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3,5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3,9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4,4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5,1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLc. lactic\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3,7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4,0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4,7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5,5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThis suggests that \u003cem\u003eL. lactis\u003c/em\u003e may have a reduced metabolic capacity under heat stress, possibly due to more extensive cellular damage or an inability to adapt to high temperatures. On the other hand, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e and \u003cem\u003eL. acidophilus\u003c/em\u003e showed relatively better acidification performance after heat exposure, with final pH values of 4.9 and 5.1, respectively [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese results highlight the importance of selecting strains with good thermal resilience for applications where maintaining metabolic activity under heat stress is critical [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Additionally, this finding underscores the complexity of bacterial responses to heat, where both viability and functionality (e.g., acid production) are influenced by a combination of factors such as cell wall integrity and stress response mechanisms [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.4.Implications for industrial applications\u003c/h2\u003e\u003cp\u003eThe findings of this study provide valuable insights into the selection of LAB strains for industrial fermentation processes that involve heat exposure. Strains like \u003cem\u003eLactobacillus plantarum\u003c/em\u003e that exhibit both higher thermal resistance and better acidification capacity are more suitable for processes that involve moderate to high temperatures, such as pasteurization, yogurt production, and probiotic delivery [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Conversely, strains like \u003cem\u003eLactococcus lactis\u003c/em\u003e, which show significant loss in viability and functionality under heat stress, may not be as suitable for these applications unless subjected to protective treatments, such as pre-adaptation to heat or the use of cryoprotectants in fermentation media [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFurthermore, the study highlights the potential for improving heat tolerance in LAB strains through genetic and metabolic engineering. Understanding the molecular mechanisms that confer heat resistance such as the role of heat-shock proteins, trehalose synthesis, and cell membrane composition can help in developing more heat-tolerant strains for the food and pharmaceutical industries [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn conclusion, this study highlights the differential heat tolerance among lactic acid bacteria, with L. plantarum exhibiting superior resistance compared to L. acidophilus and Lc. lactis. Moderate thermal exposure up to 55\u0026deg;C had limited effects on viability, particularly for L. plantarum, while higher temperatures (60\u0026ndash;65\u0026deg;C) caused significant morphological damage and reduced acidification activity. Scanning electron microscopy confirmed structural compromise of cell membranes, especially in thermosensitive strains. These findings underscore the necessity of selecting thermotolerant strains for industrial applications involving thermal processes. Understanding the heat response profiles of LAB is essential for optimizing fermentation performance and ensuring the functional integrity of probiotic products. This knowledge can inform starter culture formulation and enhance the quality of heat-processed fermented foods. Furthermore, strain-specific resilience offers opportunities to improve process robustness without compromising product efficacy. Future research should explore molecular mechanisms of thermal resistance and investigate potential protective strategies. Ultimately, targeted strain selection and process control can advance industrial fermentation and probiotic manufacturing.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThe authors would like to express their sincere gratitude to the laboratories of Universit\u0026eacute; Jean Lorougnon Gu\u0026eacute;d\u0026eacute; and Gembloux Agro-Bio Tech for providing the technical facilities required for this research. Special thanks go to Dr. Ibrahim Konat\u0026eacute; for his scientific guidance, and to Kra Kouassi Athanase, Voko-Bi Rodrigue, Beugr\u0026eacute; Maxwell, and Mohamed Baghui for their valuable contributions throughout the study. The support from the PNR1 project \u0026ldquo;CoTrans-Alim\u0026rdquo; is gratefully acknowledged. We also thank all staff members and students who assisted in data collection and laboratory analyses. This work would not have been possible without their collective efforts\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePapadimitriou K, Alegr\u0026iacute;a \u0026Aacute;, Bron PA et al (2016) Stress physiology of lactic acid bacteria. Microbiol Mol Biol Rev 80(3):837\u0026ndash;890. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/MMBR.00076-15\u003c/span\u003e\u003cspan address=\"10.1128/MMBR.00076-15\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWouters JA, Rombouts FM, de Vos WM et al (1999) Heat shock proteins in lactic acid bacteria. Antonie Van Leeuwenhoek 76(1\u0026ndash;4):235\u0026ndash;242. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1023/A:1002067908930\u003c/span\u003e\u003cspan address=\"10.1023/A:1002067908930\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCorcoran BM, Stanton C, Fitzgerald GF, Ross RP (2005) Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars. Appl Environ Microbiol 71(6):3060\u0026ndash;3067. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/AEM.71.6.3060-3067.2005\u003c/span\u003e\u003cspan address=\"10.1128/AEM.71.6.3060-3067.2005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFiocco D, Capozzi V, Collins M et al (2007) Characterization of stress-induced genes of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e and their role in stress tolerance. Appl Microbiol Biotechnol 74(5):1040\u0026ndash;1050. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00253-006-0744-2\u003c/span\u003e\u003cspan address=\"10.1007/s00253-006-0744-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen C, Wang Y, Zhang Y et al (2024) Screening and genome analysis of heat-resistant and antioxidant \u003cem\u003eLactobacillus plantarum\u003c/em\u003e L19. Front Microbiol 15:1455849. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2024.1455849\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2024.1455849\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao X, Zhang M, Bai W (2013) Effects of thermal processing on viability, membrane integrity and protein expression of probiotic bacteria. J Food Eng 119(2):287\u0026ndash;293. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jfoodeng.2013.05.040\u003c/span\u003e\u003cspan address=\"10.1016/j.jfoodeng.2013.05.040\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaram L, Jama C, Mamet T et al (2013) Effect of thermal and osmotic treatments on the viability and physiology of lactic acid bacteria used in starter cultures. Food Control 34(2):494\u0026ndash;500. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.foodcont.2013.05.036\u003c/span\u003e\u003cspan address=\"10.1016/j.foodcont.2013.05.036\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eG\u0026auml;nzle MG, Schwab C (2009) Ecology of exopolysaccharide formation by lactic acid bacteria: regulation, genetic determinants, and impact on technological properties. Int Dairy J 19(6\u0026ndash;7):341\u0026ndash;346. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.idairyj.2008.12.008\u003c/span\u003e\u003cspan address=\"10.1016/j.idairyj.2008.12.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShobharani P, Halami PM (2015) Heat shock response and cross protection against acid and bile stress in \u003cem\u003eLactobacillus plantarum\u003c/em\u003e. J Basic Microbiol 55(6):729\u0026ndash;739. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/jobm.201400842\u003c/span\u003e\u003cspan address=\"10.1002/jobm.201400842\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZ\u0026aacute;rate G, Chaia AP (2009) Effect of heat treatment on the cell surface hydrophobicity of \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e and \u003cem\u003eLactobacillus casei\u003c/em\u003e. J Appl Microbiol 106(5):1612\u0026ndash;1619. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1365-2672.2008.04029.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2672.2008.04029.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVinderola G, Reinheimer JA, Salminen S (2006) Probiotic bacteria: stress response mechanisms and stress factors relevant to probiotic viability and functionality. Food Microbiol 23(6):663\u0026ndash;669. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.fm.2005.10.006\u003c/span\u003e\u003cspan address=\"10.1016/j.fm.2005.10.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRault A, Bouix M, B\u0026eacute;al C (2009) Fermentation pH influences the physiological-state dynamics of \u003cem\u003eLactobacillus bulgaricus\u003c/em\u003e CFL1 during pH-controlled culture. Appl Environ Microbiol 75(13):4374\u0026ndash;4381. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/AEM.00130-09\u003c/span\u003e\u003cspan address=\"10.1128/AEM.00130-09\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Y, Li X, Wang Y et al (2019) Mild heat stress limited the post-acidification caused by \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e hsryfm 1301 in fermented milk. J Dairy Sci\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFont de Valdez G, de Giori GS, de Ruiz Holgado AP, Oliver G (1983) Temperature effect on the growth, protein synthesis and ribonucleic acid of \u003cem\u003eLactobacillus casei\u003c/em\u003e. Appl Environ Microbiol 46(5):1231\u0026ndash;1234\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBurgain J, Gaiani C, Linder M, Scher J (2011) Encapsulation of probiotic living cells: From laboratory scale to industrial applications. J Food Eng 104(4):467\u0026ndash;483. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jfoodeng.2010.12.031\u003c/span\u003e\u003cspan address=\"10.1016/j.jfoodeng.2010.12.031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnanta E, Heinz V, Knorr D (2004) Assessment of high pressure and thermal processing on bacteria using porosity and gas exchange characteristics of the cell envelope. Innov Food Sci Emerg Technol 5(1):1\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ifset.2003.11.002\u003c/span\u003e\u003cspan address=\"10.1016/j.ifset.2003.11.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang W, Zhou W, Wang Y et al (2020) Microencapsulation of probiotic bacteria with alginate\u0026ndash;chitosan microgels for yogurt production. LWT - Food Sci Technol 124:109155. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.lwt.2020.109155\u003c/span\u003e\u003cspan address=\"10.1016/j.lwt.2020.109155\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBroadbent JR, Larsen RL, Deibel V, Steele JL (2010) Physiological and transcriptional response of \u003cem\u003eLactobacillus casei\u003c/em\u003e ATCC 334 to acid stress. J Bacteriol 192(8):2445\u0026ndash;2458. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/JB.01618-09\u003c/span\u003e\u003cspan address=\"10.1128/JB.01618-09\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDesmond C, Ross RP, O\u0026rsquo;Callaghan E et al (2002) Improved survival of \u003cem\u003eLactobacillus paracasei\u003c/em\u003e NFBC 338 in spray-dried powders containing gum acacia. J Appl Microbiol 93(6):1003\u0026ndash;1011. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1046/j.1365-2672.2002.01776.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-2672.2002.01776.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarbosa J, Borges S, Teixeira P (2015) Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests. Food Control 51:323\u0026ndash;330. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.foodcont.2014.11.036\u003c/span\u003e\u003cspan address=\"10.1016/j.foodcont.2014.11.036\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHigashikawa F, Noda M, Awaya T et al (2010) Improvement of constipation and liver function by plant-derived \u003cem\u003eLactobacillus plantarum\u003c/em\u003e SN13T in humans. Microorganisms 58(1):37\u0026ndash;44\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhn J, Grun IU, Mustapha A (2002) Effects of lactobacilli on the inhibition of \u003cem\u003eHelicobacter pylori\u003c/em\u003e and the production of lactic acid. J Food Sci 67(7):2765\u0026ndash;2769. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1365-2621.2002.tb08787.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2621.2002.tb08787.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMalmo C, LaStella D, Ryan K, Huppertz T (2022) Role of calcium ions in stress resistance of lactic acid bacteria. J Dairy Sci 105(2):1234\u0026ndash;1242. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3168/jds.2021-21015\u003c/span\u003e\u003cspan address=\"10.3168/jds.2021-21015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu T, Wang X, Li Y et al (2015) Influence of prebiotics and heat shock pretreatment on the survival of \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e under acidic and bile stress conditions. Food Res Int 74:68\u0026ndash;73. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.foodres.2015.04.008\u003c/span\u003e\u003cspan address=\"10.1016/j.foodres.2015.04.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSanders ME, Guarner F, Guerrant R et al (2013) An update on the use and investigation of probiotics in health and disease. Gut 62(5):787\u0026ndash;796. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1136/gutjnl-2012-302504\u003c/span\u003e\u003cspan address=\"10.1136/gutjnl-2012-302504\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBourdichon F, Casaregola S, Farrokh C et al (2012) Food fermentations: Microorganisms with technological beneficial use. Int J Food Microbiol 154(3):87\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ijfoodmicro.2011.12.030\u003c/span\u003e\u003cspan address=\"10.1016/j.ijfoodmicro.2011.12.030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMonteagudo-Mera A, Rastall RA, Gibson GR et al (2019) In vitro evaluation of the viability of commercial probiotics and their protective effect against enteropathogenic bacteria. J Funct Foods 57:392\u0026ndash;401. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jff.2019.04.030\u003c/span\u003e\u003cspan address=\"10.1016/j.jff.2019.04.030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDe Angelis M, Gobbetti M (2017) Stress responses of lactic acid bacteria. Curr Opin Food Sci 13:68\u0026ndash;73. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cofs.2017.02.002\u003c/span\u003e\u003cspan address=\"10.1016/j.cofs.2017.02.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSlattery C, Cotter PD, O'Toole PW (2009) Analysis of bacterial stress responses in the selection of lactic acid bacteria for functional foods. J Appl Microbiol 107(6):1903\u0026ndash;1914. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1365-2672.2009.04376.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2672.2009.04376.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTripathi MK, Giri SK (2014) Probiotic functional foods: Survival of probiotics during processing and storage. J Funct Foods 9:225\u0026ndash;241. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jff.2014.04.030\u003c/span\u003e\u003cspan address=\"10.1016/j.jff.2014.04.030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Université Jean Lorougnon Guédé","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lactic acid bacteria, thermal resistance, viability, fermentation, acidification, morphology, food processing","lastPublishedDoi":"10.21203/rs.3.rs-7053884/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7053884/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLactic acid bacteria (LAB) are essential in food fermentation, contributing to preservation and flavor development. However, their survival under heat stress during food processing remains a challenge. This study investigates the effects of heat treatment (45\u0026deg;C\u0026ndash;65\u0026deg;C) on the viability, morphology, and acidification capacity of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e, and \u003cem\u003eLactococcus lactis\u003c/em\u003e. Results showed that \u003cem\u003eL. plantarum\u003c/em\u003e exhibited the greatest thermal resistance, with minimal viability loss at 55\u0026deg;C. SEM analysis revealed cellular damage, including membrane rupture, particularly at higher temperatures (60\u0026deg;C and 65\u0026deg;C). Acidification capacity declined with increasing heat, indicating reduced metabolic activity. These findings emphasize the need to understand strain-specific heat tolerance for optimizing fermentation processes and improving the quality of heat-sensitive products like yogurt and probiotics.\u003c/p\u003e","manuscriptTitle":"Effects of heat stress on the resistance of Lactic Acid Bacteria: Viability, morphological integrity, and acidification capacity in fermentation processe","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-08 06:52:26","doi":"10.21203/rs.3.rs-7053884/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3da7bab5-7c37-4252-a16e-32877dd7a26b","owner":[],"postedDate":"July 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51088501,"name":"Applied Biochemistry"}],"tags":[],"updatedAt":"2025-07-08T06:52:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-08 06:52:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7053884","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7053884","identity":"rs-7053884","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}