Anticancer Activity of Lactiplantibacillus plantarum subsp. plantarum IIA-1A5-Fermented Milk on Chemically Induced-Colorectal Cancer of BALB/c Mice

Anticancer Activity of Lactiplantibacillus plantarum subsp. plantarum IIA-1A5-Fermented Milk on Chemically Induced-Colorectal Cancer of BALB/c Mice | 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 Anticancer Activity of Lactiplantibacillus plantarum subsp. plantarum IIA-1A5-Fermented Milk on Chemically Induced-Colorectal Cancer of BALB/c Mice Iis Erlina, Irma Isnafia Arief, Cahyo Budiman, Anantha Sena, Kazuhito Fujiyama This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7681425/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 Colorectal cancer (CRC) remains a significant global health challenge, with current treatment options often limited by toxicity, resistance, and adverse effects. Probiotics, particularly lactic acid bacteria (LAB), offer promising alternatives through bioactive metabolites with anticancer and immunomodulatory properties. The indigenous Indonesian strain Lactiplantibacillus plantarum subsp. plantarum IIA-1A5 exhibits unique metabolic profiles and has demonstrated in vitro anticancer potential. This study evaluated the anticancer effects of L. plantarum IIA-1A5–fermented milk in an AOM and DSS-induced murine model, focusing on aberrant crypt foci (ACF), tumor cell density, malondialdehyde (MDA), cortisol levels, gut microbiota composition, and NF-κB expression. The curative intervention group (P5) exhibited the most pronounced effects, including significant reductions in ACF, tumor cell density, MDA levels, and serum cortisol, reflecting decreased oxidative and physiological stress. Importantly, NF-κB expression was markedly suppressed, indicating attenuation of pro-inflammatory signaling pathways pivotal to CRC progression. Favorable modulation of gut microbiota was also observed, particularly through suppression of Escherichia coli. Compared to 5-fluorouracil (5-FU), L. plantarum IIA-1A5 demonstrated comparable efficacy in mitigating oxidative stress, inflammation, and microbial dysbiosis. These findings highlight the potential of L. plantarum IIA-1A5 as a functional probiotic with both preventive and therapeutic applications in colorectal cancer management. Further mechanistic and clinical studies are warranted to validate these effects and assess their translational relevance in humans. azoxymethane (AOM) dextran sodium sulfate (DSS) Indonesian indigenous bacteria probiotics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Colorectal cancer (CRC) constitutes a significant global health issue, ranking third in incidence and second in cancer-related mortality worldwide, with 1.93 million new cases and over 903,000 fatalities recorded in 2022 (Bray et al., 2024 ). In Indonesia, colorectal cancer was the fourth most common cancer in 2020 (Sung et al., 2021 ). It primarily affects individuals over the age of 50, with men being at higher risk (Lewandowska et al., 2022 ). Risk factors for colorectal cancer encompass both non-modifiable elements (age, sex, genetics) and modifiable variables, including food, obesity, smoking, and alcohol use (Goodarzi et al., 2019 ). Current treatment modalities, including surgery, chemotherapy, and radiotherapy, demonstrate disparate success rates and frequently entail significant adverse effects. 5-Fluorouracil (5-FU), a commonly employed chemotherapeutic agent, may result in toxicity, drug resistance, and recurrence, whereas radiation might induce prolonged damage by the production of reactive oxygen species (Gmeiner and Okechukwu, 2023 ). Moreover, the financial burden associated with CRC treatment remains substantial (Bhimani et al., 2022 ). Attempts to provide alternative treatments for CRC have been widely studied by proposing lactic acid bacteria (LAB)-based probiotics as a promising avenue to be further explored due to their potent anticancer properties. This is particularly attributed to the ability of these probiotics to produce bioactive substances capable of interfering with the growth or development of cancer cells. Bioactive substances produced from probiotics, especially those from lactic acid bacteria (LAB), have surfaced as viable alternatives for cancer treatment (Zhao et al., 2023 ). LAB, designated as Generally Recognized as Safe (GRAS), generates antibacterial and anticarcinogenic metabolites, such as short-chain fatty acids (SCFAs), reuterin, and bacteriocins (Iacob et al., 2019 ). Probiotics may potentially inhibit colorectal cancer by modulating gut microbiota and immune function (Kahouli et al., 2013 ). One promising LAB-based probiotic is Lactiplantibacillus plantarum subsp. plantarum IIA-1A5, a indegenous Indonesian strain (GenBank accession no. OR473281.1) characterized by unique fatty acid, amino acid, and volatile profiles, including SCFAs, many of which are linked to anticancer properties. In vitro studies identified 83 volatile compounds with known anticancer activity, while its strain-specific surface layer protein (slp) gene. This gene is recognized as strain-specific and has a role in anticancer mechanisms, primarily via apoptosis induction and cell cycle inhibition (Zhang et al., 2020 ). The anticancer potential of L. plantarum IIA-1A5 has been reported; however, the primary challenge lies in its delivery, as patient acceptance may differ between consumption as a probiotic supplement and as a food product. Therefore, the development of functional foods incorporating this strain is essential to ensure both the stability and efficacy of its anticancer activity. Previous studies have demonstrated that L. plantarum IIA-1A5 can serve as a starter culture for fermented milk, which not only complies with the Indonesian National Standard (SNI) for food safety but also exhibits significant anticancer activity against WiDr colorectal cancer cells in vitro (Adiyoga et al., 2022 ). Nevertheless, these findings have not yet been validated in in vivo models. Considering that discrepancies often arise between in vitro and in vivo outcomes due to environmental variations affecting bioactive compounds, further in vivo investigations are warranted to substantiate the anticancer potential of L. plantarum IIA-1A5. This study is therefore aimed at comprehensively evaluating the anticancer potential of L. plantarum IIA-1A5–fermented milk in a murine model of colorectal cancer induced by azoxymethane (AOM) and dextran sodium sulfate (DSS). The current study describes the therapeutic efficacy of this indigenous Indonesian probiotic strain, particularly in the treatment group (P5), where its administration resulted in a marked reduction in aberrant crypt foci, tumor cell density, malondialdehyde levels, and cortisol concentrations. Moreover, it demonstrates favorable modulation of the gut microbiota, including a notable decline in Escherichia coli populations. Taken together, these findings position L. plantarum IIA-1A5 as a promising functional bioactive agent capable of mitigating oxidative stress, modulating physiological stress responses, and enhancing intestinal microbial homeostasis. Further mechanistic and clinical investigations are warranted to substantiate these outcomes and to explore their translational relevance for colorectal cancer prevention and therapy in humans. Materials and Methods 2.1 Production of Fermented Milk Containing L. plantarum subsp. plantarum IIA-1A5 The production of fermented milk was carried out following the method described by (Arief et al., 2015 ). The activation of L. plantarum IIA-1A5 (registered in GenBank, accession no. OR473281.1) began with retrieving the stock culture and inoculating it into 9 mL of de Man, Rogosa, and Sharpe broth (MRSB) (Oxoid, Basingstoke, UK, followed by incubation at 37°C for 24 h. Subculturing was performed repeatedly until the culture demonstrated stable growth and full adaptation to the medium. Sterile milk was then prepared and inoculated with 10% (v/v) of the activated culture. To increase biomass, the culture was further transferred into fresh sterile milk. Fermentation was conducted in an incubator (Memmert IN110, Memmert GnbH, Schwabach, Germany) at 37°C for 18 h. The initial bacterial population in the fermented milk was determined by plating on de Man, Rogosa, and Sharpe agar (MRSA) (Oxoid, Basingstoke, UK, according to standard procedures. Only fermented milk with a viable cell count exceeding 10⁹ CFU/mL was used for subsequent experiments. 2.2 In vivo Animal Experiments The in vivo study was conducted following ethical approval from the Directorate of Research and Innovation (DRI), IPB University (Ethical Clearance No. 287–2024 IPB), under the supervision of a licensed attending veterinarian. A total of 25 male BALB/c mice, aged 6–8 weeks and weighing between 18 and 30 grams, obtained from iRATco Laboratory Indonesia, were used as experimental subjects (Zhang et al., 2022 ). All animals underwent a 7-day acclimatization period under controlled environmental conditions prior to the start of the experiment. Following acclimatization, treatments were administered over a 70-day period. Mice were orally administered fermented milk containing L. plantarum IIA-1A5 twice daily once in the morning and once in the evening at a dosage of 0.3 mL per administration. At the end of the treatment period, the mice were humanely euthanized via cervical dislocation. The experimental animals were randomly assigned to five groups (n = 5 per group). The treatment scheme for each group is presented in Table 1 . Table 1 Experimental Animal Treatment Groups Groups Treatment P1 (Normal control) No induction with azoxymethane (AOM) and dextran sodium sulfate (DSS). P2 (Negative Control) Induction with AOM and DSS only P3 (Positive Control) Induction with AOM and DSS followed by administration of the chemotherapeutic agent 5-fluorouracil (5-FU). P4 (Preventive Group) Induction with AOM and DSS, with fermented milk containing Lactiplantibacillus plantarum subsp. plantarum IIA-1A5 administered both after induction and starting 7 days prior to induction. P5 (Curative Group) Induction with AOM and DSS, with fermented milk containing L. plantarum IIA-1A5 administered only after induction. 2.3 Induction of Colorectal Cancer Colorectal cancer was induced in mice following the protocol by (Martínez-Gregorio et al., 2022 ), utilizing azoxymethane (AOM) and dextran sodium sulfate (DSS). AOM (Sigma-Aldrich, St. Louis, MO, USA) was administered as a single intraperitoneal injection at a dose of 12.5 mg/kg body weight on the first day of treatment. Subsequently, DSS was provided in the drinking water at a concentration of 2% in three induction cycles: Phase I (days 8–14), Phase II (days 29–34), and Phase III (days 50–55). 2.4 Post-Treatment Animal Observations 2.4.1 Cancer Distribution (Aberrant Crypt Foci - ACF) Cancer distribution in the colon tissue of BALB/c mice was assessed macroscopically using ImageJ software by identifying surface nodules, which serve as key morphological indicators of colorectal cancer (Budijanto et al., 2023 ). This evaluation provided visual insight into the location and extent of cancer spread. 2.4.2 Histopathological Analysis Histopathological analysis was conducted to examine cellular abnormalities (Sun et al., 2023 ). Intestinal tissues were fixed in 10% neutral buffered formalin, sectioned (5–10 mm), and processed through dehydration and clearing with graded alcohols, toluene, and paraffin. Tissues were embedded, sectioned at 4–5 µm, and stained using the Harris Hematoxylin-Eosin method. Dehydration and clearing were completed with alcohol and xylene. 2.4.3 Cortisol Hormone Analysis Cortisol levels were measured using the ELISA method as described by (Gong et al., 2015 ). A 25 µL serum sample was processed with biotin and enzyme conjugates, incubated, washed, and reacted with TMB substrate. Absorbance was measured at 450 nm, and cortisol concentrations were determined using a standard curve. 2.4.4 Malondialdehyde (MDA) Analysis MDA levels were measured using the thiobarbituric acid (TBA) assay as described by (Gonzalez and Paulson, 2022 ). Samples were homogenized, treated with TCA, centrifuged, and reacted with TBA, followed by heating and cooling. Absorbance was measured at 530 nm using a spectrophotometer, with TEP as the standard. 2.4.5 Microbiological Analysis Microbiological analysis targeted lactic acid bacteria and Escherichia coli. Intestinal samples were serially diluted and cultured on MRSA (Oxoid, Basingstoke, UK), for LAB enumeration. E. coli was assessed using the BAM method on EMBA (Oxoid, Basingstoke, UK), characteristic colonies were identified by a metallic green sheen after 24 h incubation at 37°C (Arief et al., 2015 ). 2.4.6 Immunohistochemical of Nuclear Factor-kappa B (NF-κB) NF-κB expression was analyzed by immunohistochemistry using anti-NF-κB p65 antibody as described by Sivakumar et al., ( 2020 ). Tissue sections were incubated with primary antibody (1:50), followed by HRP-conjugated secondary antibody and DAB, then counterstained with Mayer’s hematoxylin, dehydrated, cleared, and mounted. 2.5 Statistical analysis Data are presented as the mean ± standard deviation from five independent replicates. Differences among means were evaluated using one-way ANOVA followed by Tukey’s post hoc test. Statistical analyses were performed with Minitab 20 software, with significance considered at the 5% confidence level. However, ACF data were presented descriptively and not subjected to statistical analysis. Results 3.1 Aberrant Crypt Foci (ACF) As shown in Fig. 1 , the highest number of aberrant crypt foci (ACF) was observed in the negative control group (P2), while the lowest was found in the curative treatment group (P5). No ACF formation was detected in the normal control (P1). The presence of ACF was confirmed microscopically (Fig. 2 ), where black arrows indicate clusters of enlarged, irregular crypts, clearly visible in P2, P3, P4, and P5 but absent in P1. 3.2 Histopathological Tumor cell density in colonic tissue varied significantly among groups (Fig. 3 ). Statistical analysis confirmed significant differences among all groups (p < 0.05), as indicated by the distinct superscript letters. The negative control showed the highest density, while the normal control remained unaffected. Treatment with 5-fluorouracil reduced tumor cell density but was less effective than fermented milk. Both preventive and curative administration of L. plantarum IIA-1A5 markedly suppressed tumor cell growth, with the curative group showing the greatest reduction, approaching normal levels. These findings highlight the potential of fermented milk as both a chemopreventive and therapeutic agent against colorectal cancer. Histopathological analysis (Fig. 4 ) revealed intact colonic architecture in the normal control, while the negative control showed extensive epithelial hyperplasia and tumor infiltration. The positive control displayed partial improvement, whereas preventive treatment attenuated tissue abnormalities. The curative group exhibited the most preserved structure, closely resembling normal tissue. 3.3 Cortisol Hormone Serum cortisol concentrations in colorectal cancer–induced mice are presented in Fig. 5 . The normal control group (P1) exhibited the lowest cortisol level, whereas the negative control group (P2) showed the highest concentration. The positive control (P3) and preventive treatment (P4) groups demonstrated intermediate values, while the curative treatment group (P5) displayed levels that were closer to the normal control. Statistical analysis revealed significant differences among the groups (p < 0.05). In particular, P2 was significantly higher than P1 and P5, whereas P3 and P4 did not differ significantly from either P2 or P5 but were both higher than P1. These findings suggest that the curative treatment (P5) more effectively modulated cortisol levels toward the normal profile compared with the preventive treatment (P4). 3.4 Malondialdehyde (MDA) The malondialdehyde (MDA) concentrations in colorectal cancer–induced mice across different treatment groups are shown in Fig. 6 . The normal control group (P1) exhibited the lowest MDA levels, whereas the negative control group (P2), which was exposed to carcinogens without treatment, displayed the highest concentration. The positive control group (P3, 5-FU treatment) presented reduced MDA levels compared to P2, and similar reductions were observed in the preventive (P4) and curative (P5) groups administered fermented milk with L. plantarum IIA-1A5. Statistical analysis confirmed significant differences among the groups (p < 0.05). In particular, P2 was significantly higher than P1, while P3, P4, and P5 did not differ significantly from either P1 or P2. These findings suggest that both preventive and curative treatments with L. plantarum IIA-1A5 effectively lowered MDA levels toward those observed in the positive control group. 3.5 Microbiologycal The microbiological profile of Escherichia coli is illustrated in the colorectal cancer–induced mice across the different treatment groups, as shown in Fig. 7 , whereas the total lactic acid bacteria (LAB) counts are presented in Fig. 8 . LAB levels were relatively consistent across all groups, with no statistically significant differences observed (p > 0.05). In contrast, E. coli counts varied significantly among treatments (p < 0.05). The highest E. coli levels were detected in the normal control (P1) and negative control (P2), while the positive control (P3) exhibited reduced counts compared with both controls. Notably, the preventive (P4) and curative (P5) groups administered fermented milk containing L. plantarum IIA-1A5 showed markedly lower E. coli counts, with P4 demonstrating the most pronounced reduction. These findings suggest that L. plantarum IIA-1A5 treatment effectively suppressed E. coli proliferation while maintaining LAB populations across groups. 3.6 Immunohistochemical of Nuclear Factor-kappa B (NF-κB) The NF-κB expression levels in colorectal cancer–induced mice across different treatment groups are presented in Fig. 9 . Statistical analysis confirmed significant differences among the groups (p < 0.05). Specifically, P2 was significantly higher than P1, P4, and P5, whereas P3 was significantly lower than P2 but remained higher than P1 and P5. These findings indicate that both preventive and curative treatments with L. plantarum IIA-1A5 effectively reduced NF-κB expression toward baseline levels, with the curative treatment (P5) showing modulation comparable to the normal control. Figure 10 provides immunohistochemical (IHC) visualization of NF-κB expression in colonic tissues. Positive NF-κB expression is indicated by brown staining (arrows), localized within the nucleus and cytoplasm of epithelial and inflammatory cells, thereby supporting the quantitative results observed in Fig. 9 . Discussion 4.1 Aberrant Crypt Foci (ACF) and Histopathological The distribution of aberrant crypt foci (ACF) and histopathological alterations provided complementary evidence for the role of L. plantarum IIA-1A5 in suppressing colorectal cancer (CRC) progression. As shown in Figs. 1 and 3 , the negative control group (P2) exhibited the highest number of ACF, consistent with the established role of azoxymethane (AOM) in inducing early epithelial alterations that initiate colorectal carcinogenesis (Kowalczyk et al., 2016 ). In contrast, the positive control group (P3, 5-FU treatment) demonstrated a marked reduction in ACF, corroborating previous findings that 5-fluorouracil effectively suppresses AOM-induced ACF formation (Stastna et al., 2019 ). Microscopic observations further supported these results, revealing clusters of enlarged and irregular crypts in groups P2–P5 but not in P1, thereby reinforcing the role of ACF as preneoplastic lesions and reliable biomarkers of CRC initiation (Genaro et al., 2019 ). Histopathological analysis provided further validation. The negative control (P2) displayed severe epithelial hyperplasia, disrupted crypt morphology, and tumor infiltration, reflecting successful induction of colorectal carcinogenesis by the AOM and DSS protocol, which promotes inflammation-driven malignant transformation (Liu et al., 2022 ; Modesto et al., 2022 ). By contrast, the normal control retained intact colonic architecture. Treatment with 5-FU partially ameliorated these pathological changes by reducing tumor cell density, consistent with its mechanism of action in inhibiting thymidylate synthase and disrupting nucleic acid synthesis (Longley et al., 2003 ). Notably, both the preventive (P4) and curative (P5) groups receiving L. plantarum IIA-1A5–fermented milk demonstrated improved tissue preservation compared with the positive control, with the curative group showing the closest resemblance to normal colon morphology. The reduction in ACF and tumor burden following probiotic intervention aligns with earlier evidence indicating that probiotics can decrease neoplastic lesions in experimental CRC models (Hajrezaie et al., 2014 ; Jacouton et al., 2017 ). L. plantarum is believed to exert anticancer effects through multiple mechanisms, including reshaping the gut microbiota, enhancing host immune responses, promoting apoptosis, and producing bioactive metabolites such as short-chain fatty acids (SCFAs), which regulate oxidative stress and epithelial proliferation (Chen and Li 2020 ; Ghanavati et al., 2020 ). In addition, bacteriocins and surface layer proteins synthesized by L. plantarum may contribute to mucosal protection and attenuation of proinflammatory signaling, thereby suppressing tumorigenesis. Collectively, these mechanisms explain the substantial improvement in colonic morphology observed in probiotic-treated groups. Although both preventive and curative interventions were beneficial, the curative group (P5) achieved a more pronounced reduction in ACF and tumor density, suggesting that probiotic efficacy may be greater during active disease progression. This observation supports the hypothesis that probiotics not only prevent tumor initiation but also exert therapeutic effects by modulating host immunity, counteracting microbial dysbiosis, and suppressing oncogenic pathways such as NF-κB and Wnt/β-catenin (Ghafouri-Fard et al., 2021 ; Modesto et al., 2022 ). These findings underscore the importance of treatment timing, indicating that post-induction administration may yield superior protective outcomes. Taken together, the integrated analysis of ACF distribution and histopathology highlights the dual preventive and therapeutic roles of L. plantarum IIA-1A5 in colorectal carcinogenesis. The reduction of preneoplastic lesions, restoration of colonic architecture, and group-dependent differences provide strong experimental evidence that fermented milk containing L. plantarum IIA-1A5 can suppress inflammation-driven colorectal cancer development. These results further support the growing perspective that probiotics may serve as adjuvants to conventional chemotherapy, offering tumor-suppressive benefits while potentially mitigating treatment-associated side effects. Continued investigations, particularly well-designed clinical studies, are necessary to validate the translational potential of L. plantarum IIA-1A5 in CRC management 4.2 Cortisol Hormone In the normal control group (P1), serum cortisol concentrations remained within the physiological range, whereas the negative control group (P2) exhibited marked elevations, consistent with previous reports that AOM and DSS-induced carcinogenesis disrupts glucocorticoid balance and promotes stress-driven epithelial dysplasia (Li et al., 2019 ). Chronic hypercortisolemia is known to exacerbate intestinal inflammation and immune dysregulation, thereby accelerating colorectal cancer (CRC) progression (La Vecchia and Sebastián, 2020 ). Prolonged activation of the hypothalamic pituitary adrenal (HPA) axis has also been associated with tumor growth and reduced therapeutic efficacy. Administration of 5-fluorouracil (P3) partially normalized cortisol levels compared with P2, reflecting its antiproliferative effects while simultaneously stimulating HPA axis activity (Lempesis et al., 2023 ). Fermented milk containing L. plantarum IIA-1A5 demonstrated treatment-dependent outcomes: preventive supplementation (P4) produced modest reductions, whereas curative administration (P5) restored cortisol concentrations to near-normal levels. These endocrine changes paralleled histopathological findings, with P5 showing greater preservation of colonic architecture and fewer tumor lesions than P4, suggesting enhanced therapeutic efficacy when administered post-induction (Agalakova, 2025 ). The cortisol profile was closely aligned with both ACF and histopathological data. Elevated cortisol in P2 coincided with higher ACF counts and severe epithelial dysplasia, while normalization in P5 corresponded with reduced ACF burden and improved tissue integrity. This concordance underscores the dual role of L. plantarum IIA-1A5 in modulating systemic stress responses and suppressing tumorigenesis. Supporting evidence indicates that probiotics regulate the HPA axis and enhance stress resilience, as demonstrated for L. rhamnosus , L. helveticus , and B. longum (Bercik et al., 2011 ; Bravo et al., 2011 ; Ohland et al., 2013 ). Collectively, these findings highlight L. plantarum IIA-1A5–fermented milk as a promising functional food with synergistic anticancer and anti-stress effects. 4.3 Malondialdehyde (MDA) The negative control group (P2), exposed to carcinogens without treatment, exhibited the highest malondialdehyde (MDA) levels, indicating severe oxidative stress, consistent with enhanced lipid peroxidation in colorectal cancer models (Reuter et al., 2010 ). Carcinogens such as AOM and DSS induce persistent inflammation, creating a pro-oxidative microenvironment that accelerates tumor progression (Longley et al., 2003 ). Administration of 5-fluorouracil (5-FU, P3) reduced MDA levels by suppressing tumor proliferation but also promoted reactive oxygen species (ROS) generation, with cancer cell survival mediated by Nrf2-driven antioxidant responses (Blondy et al., 2020 ). Both preventive (P4) and curative (P5) groups treated with L. plantarum IIA-1A5–fermented milk showed MDA reductions comparable to those observed in the 5-FU group. This probiotic scavenges ROS, stimulates enzymatic antioxidants (SOD, GSH-Px, CAT), enhances glutathione synthesis, and its extracellular polysaccharides further support free radical neutralization (Moslehishad et al., 2013 ; Silva et al., 2019 ; Liu et al., 2022 ; Jiang et al., 2024 ). L. plantarum has also been reported to neutralize dietary and endogenous carcinogens such as N-nitrosodimethylamine (Nowak et al., 2014 ). Elevated MDA in P2 corresponded with a high aberrant crypt foci (ACF) burden, severe epithelial dysplasia, and increased cortisol, reflecting the interplay of oxidative stress, tissue damage, and endocrine disruption. Conversely, normalization of MDA in P5 was associated with reduced ACF counts, improved colonic architecture, and restored cortisol levels. These findings suggest that L. plantarum IIA-1A5–fermented milk mitigates colorectal carcinogenesis by attenuating oxidative stress, modulating systemic stress responses, and preserving tissue integrity. 4.4 Microbiologycal Lactic acid bacteria (LAB), particularly Lactobacillus spp ., are key members of the Firmicutes phylum , playing a critical role in maintaining gut homeostasis through the production of short-chain fatty acids (SCFAs) and modulation of host immune responses (Li et al., 2017 ; Bader et al., 2018 ). In this study, LAB levels remained consistent across all experimental groups, indicating that neither carcinogen exposure nor therapeutic interventions significantly disrupted the beneficial microbial population. In contrast, Escherichia coli counts exhibited marked variability. The highest levels were observed in the normal control (P1) and CRC-induced negative control (P2), consistent with previous reports that AOM and DSS-induced colorectal carcinogenesis perturbs microbial homeostasis and promotes the proliferation of pathogenic taxa such as Escherichia shigella (Deng et al., 2022 ). The 5-FU treated group (P3) displayed reduced E. coli abundance, reflecting chemotherapeutic modulation of the gut microbiota, characterized by decreased Firmicutes and Proteobacteria and increased Bacteroidetes, which collectively suppress opportunistic pathogens (Stringer et al., 2009 ; Li et al., 2017 ; Deng et al., 2022 ). Although Proteobacteria constitute a minor fraction of the gut microbiome, they exert significant influence on host metabolism and inflammatory processes (Pérez-Cobas et al., 2013 ; Li et al., 2019 ). Notably, administration of L. plantarum IIA-1A5 in both preventive (P4) and therapeutic (P5) groups markedly reduced E. coli levels, with the most pronounced effect observed in P4, while preserving LAB populations. These findings support the probiotic’s capacity to alleviate CRC-associated dysbiosis (dos Reis et al., 2019 ). The antimicrobial activity of L. plantarum IIA-1A5 is attributed to plantaricin IIA-1A5, which inhibits enteric pathogens including Salmonella, Shigella , and enteropathogenic E. coli (Sihombing et al., 2015 ). Similarly, synbiotic formulations containing Lactobacillus spp. have demonstrated efficacy in suppressing E. coli and restoring microbial balance (Astawan et al., 2012 ). Importantly, the observed reduction in E. coli and the maintenance of LAB in P4 and P5 correlated with decreased aberrant crypt foci (ACF), improved histological architecture, lower malondialdehyde (MDA) levels, and reduced serum cortisol. These integrated findings indicate that L. plantarum IIA-1A5 exerts a multifaceted protective effect against CRC progression through modulation of the gut microbiota, mitigation of oxidative stress, and preservation of intestinal tissue integrity. 4.5 Immunohistochemical of Nuclear Factor-kappa B (NF-κB) Data Fig. 9 demonstrates markedly elevated NF-κB expression in the negative control group (P2) induced with AOM and DSS. This observation aligns with previous studies showing that AOM and DSS induces chronic inflammation through activation of pro-inflammatory pathways, including NF-κB, a critical mediator in the initiation and promotion of colorectal carcinogenesis (CRC) (Kanehara et al., 2019 ). NF-κB activation drives the production of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β, thereby creating a microenvironment conducive to tumor cell proliferation and survival (Sun et al., 2016 ). Probiotic intervention exhibited superior efficacy in mitigating inflammation compared to 5-FU chemotherapy. Preventive (P4) and curative (P5) administration of L. plantarum IIA-1A5 significantly suppressed NF-κB expression, achieving levels comparable to, or even lower than, those of the normal control group (P1), underscoring its potent anti-inflammatory capacity. The mechanisms underlying this effect likely involve multiple pathways, including modulation of gut microbiota composition, enhanced production of short-chain fatty acids (SCFAs) such as butyrate which inhibit NF-κB activation via histone deacetylase (HDAC) inhibition and reinforcement of intestinal epithelial tight junctions, thereby limiting bacterial and LPS translocation, which activates NF-κB via Toll-like receptors (Peng et al., 2020 ). Consistent with these findings, Reis et al., ( 2024 ) reported that probiotic supplementation effectively suppresses NF-κB, reducing chronic inflammation, a key driver of preneoplastic lesion formation and colorectal tumor progression. NF-κB inhibition by probiotics is further supported by mechanisms including EPS TLR4 interactions, upregulation of IκBα, and suppression of MAPK signaling, collectively mitigating pro-inflammatory cytokine overexpression (Morsli et al., 2025 ). In this study, NF-κB downregulation correlated with improved colonic histology and reduced inflammatory markers such as MDA and cortisol, confirming that L. plantarum IIA-1A5 effectively modulates inflammatory signaling and restores gut homeostasis. These results highlight the potential of L. plantarum as both a protective and therapeutic agent in managing colorectal cancer–associated chronic inflammation. Conclusion In conclusion, administration of this probiotic formulation, particularly in the curative group (P5), resulted in a marked reduction in aberrant crypt foci, tumor cell density, malondialdehyde levels, and serum cortisol, while concurrently promoting favorable modulation of gut microbiota, including a decrease in Escherichia coli populations. Notably, NF-κB expression was significantly suppressed, demonstrating the probiotic’s capacity to attenuate pro-inflammatory signaling pathways that are central to colorectal carcinogenesis. Collectively, these findings underscore the potential of L. plantarum IIA-1A5 as a multifunctional bioactive agent capable of mitigating oxidative stress, modulating systemic stress responses, suppressing inflammation, and restoring intestinal microbial balance. Further mechanistic studies and clinical trials are warranted to validate these effects and clarify the translational relevance of this probiotic in colorectal cancer prevention and therapy. Declarations Conflicts of Interest The authors declare no potential conflict of interest. Ethics Approval This article was conducted following ethical approval from the Directorate of Research and Innovation (DRI), IPB University (Ethical Clearance No. 287–2024 IPB). Funding This research was funded by the Indonesian Ministry of Higher Education, Science, and Technology. Author Contribution Conceptualization: Erlina I, Arief II, Budiman C. Data curation: Erlina I, Arief II. Formal analysis: Erlina I, Arief II, Budiman C. Methodology: Erlina I, Arief II, Budiman C. Software: Erlina I. Validation: Erlina I, Arief II, Budiman C, Sena A, Fujiyama K. Investigation: Erlina I. Writing - original draft: Erlina I. Writing - review & editing: Erlina I, Arief II, Budiman C, Sena A, Fujiyama K. Acknowledgement The authors gratefully acknowledge the financial support from the Ministry of Higher Education, Science, and Technology of the Republic of Indonesia (Kemdiktisaintek) through the PPS-PMDSU research grant scheme in 2025 (Contract No. 006/IT3.D10/PT.01.03/P/B/2025) and the PT research grant scheme in 2024 (Contract No. 22345/IT3.D10/PT.01.03/P/B/2024). References Adiyoga R, Arief II, Budiman C, Abidin Z. 2022. In vitro anticancer potentials of Lactobacillus plantarum IIA-1A5 and Lactobacillus acidophilus IIA-2B4 extracts against WiDr human colon cancer cell line. Food Sci Technol. 42:1–7.doi:10.1590/fst.87221. Agalakova NI. 2025. Modulation of Endoplasmic Reticulum Stress in Experimental Anti-Cancer Therapy. 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08:50:06","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":22737,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/22c18dcc4b98cffefa58455a.png"},{"id":93382293,"identity":"e331bd51-a836-4187-9292-786f304f666b","added_by":"auto","created_at":"2025-10-13 09:06:06","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":19340,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/22c48a8c4e11ba0f03544dbe.png"},{"id":93381913,"identity":"41e92e12-619d-47a2-9a2d-fc6be62fbf87","added_by":"auto","created_at":"2025-10-13 08:58:06","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":20965,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/1cd018cbccafa5a82e877c58.png"},{"id":93381008,"identity":"936a7935-f9a3-4331-a2fa-ac8b2f0715c2","added_by":"auto","created_at":"2025-10-13 08:50:06","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":27055,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/01aa62301af2405f7fb3cfe1.png"},{"id":93381024,"identity":"f50d3e37-5751-4164-a694-be1d00b1d822","added_by":"auto","created_at":"2025-10-13 08:50:08","extension":"xml","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":147670,"visible":true,"origin":"","legend":"","description":"","filename":"a03c69cb6eb044688172475fac7f6fb41structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/57dfdd1272ff125148f1c444.xml"},{"id":93381917,"identity":"632bdf63-717e-4c1e-9eaa-42970d0b45af","added_by":"auto","created_at":"2025-10-13 08:58:07","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":156175,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/a9844944fd3d45e62432b917.html"},{"id":93380989,"identity":"d0471c15-032a-451b-9f71-8d59b28bfa43","added_by":"auto","created_at":"2025-10-13 08:50:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":29178,"visible":true,"origin":"","legend":"\u003cp\u003eACF (Aberrant Crypt Foci) count in mice induced with colorectal cancer across different treatment groups. P1: Normal control; P2: Negative control (AOM and DSS); P3: Positive control (5-FU); P4: Preventive treatment (fermented milk); P5: Curative treatment (fermented milk).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/d3b685672360602695045af3.png"},{"id":93381902,"identity":"bdd56074-f49a-4313-9d3b-136b73a1cf4e","added_by":"auto","created_at":"2025-10-13 08:58:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6915975,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of ACF staining using methylene blue. P1: Normal control; P2: Negative control (AOM and DSS); P3: Positive control (5-FU); P4: Preventive treatment (fermented milk); P5: Curative treatment (fermented milk). The arrow indicates aberrant crypt foci (ACF), which represent early preneoplastic lesions in colorectal carcinogenesis.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/4bcf7351b084149e8f04413a.png"},{"id":93380990,"identity":"a2d3e322-b748-4f9e-bbbf-3be11f763ee4","added_by":"auto","created_at":"2025-10-13 08:50:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32434,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of tumor cell density in colorectal cancer-induced mice across different treatment groups. P1: Normal control; P2: Negative control (AOM and DSS); P3: Positive control (5-FU); P4: Preventive treatment (fermented milk); P5: Curative treatment (fermented milk).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/33d405ee81eae7c098c1423f.png"},{"id":93381022,"identity":"3a5425b9-9ec4-4aa2-a6d3-0a7cfc8a29a3","added_by":"auto","created_at":"2025-10-13 08:50:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":14895460,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathology with HE staining. P1: Normal control; P2: Negative control (AOM and DSS); P3: Positive control (5-FU); P4: Preventive treatment (fermented milk); P5: Curative treatment (fermented milk). The black arrow denotes foci of abnormal/neoplastic cells within the colonic tissue. Scale bar = 50 µm.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/f992a07ed6de5cc4281ebb6f.png"},{"id":93381001,"identity":"4a5e4664-7c52-4abb-89b5-5c5a6bf33216","added_by":"auto","created_at":"2025-10-13 08:50:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":45039,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of serum cortisol levels in colorectal cancer-induced mice across different treatment groups. P1: Normal control; P2: Negative control (AOM and DSS); P3: Positive control (5-FU); P4: Preventive treatment (fermented milk); P5: Curative treatment (fermented milk)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/1c60c0960380ab14b3de059a.png"},{"id":93380994,"identity":"81e795b8-0ea6-4b2a-bc6f-09c7171b6430","added_by":"auto","created_at":"2025-10-13 08:50:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":28386,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of MDA in colorectal cancer-induced mice across different treatment groups. P1: Normal control; P2: Negative control (AOM and DSS); P3: Positive control (5-FU); P4: Preventive treatment (fermented milk); P5: Curative treatment (fermented milk)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/0d764aa259bdfcee412f5bec.png"},{"id":93382289,"identity":"4daa34c5-b580-4d44-a7f4-2e24046caa9a","added_by":"auto","created_at":"2025-10-13 09:06:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":24978,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of BAL in colorectal cancer-induced mice across different treatment groups. Microbiologycal in Colorectal Cancer-Induced Mice (BAL). P1: Normal control; P2: Negative control (AOM and DSS); P3: Positive control (5-FU); P4: Preventive treatment (fermented milk); P5: Curative treatment (fermented milk)\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/97bb5dc4b28e8572c0b61633.png"},{"id":93381018,"identity":"45ee6774-45c7-4db9-8489-972feedc49e4","added_by":"auto","created_at":"2025-10-13 08:50:07","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":26595,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of \u003cem\u003eE. Coli\u003c/em\u003e in colorectal cancer-induced mice across different treatment groups. P1: Normal control; P2: Negative control (AOM and DSS); P3: Positive control (5-FU); P4: Preventive treatment (fermented milk); P5: Curative treatment (fermented milk)\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/7a0776fc57e67264b212042c.png"},{"id":93381020,"identity":"cda133f8-a894-47cf-bdd0-472be1f8b416","added_by":"auto","created_at":"2025-10-13 08:50:07","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":33214,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of NF-κB expression in colonic tissue of mice across different treatment groups. P1: Normal control; P2: Negative control (AOM and DSS); P3: Positive control (5-FU); P4: Preventive treatment (fermented milk); P5: Curative treatment (fermented milk).\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/05cfba6122e40a67bd5abe0e.png"},{"id":93381910,"identity":"40c6e974-b033-4dd6-8bcd-90a979e3d1fb","added_by":"auto","created_at":"2025-10-13 08:58:06","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":10726224,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical (IHC) visualization of NF-κB expression in colonic tissue across different treatment groups. P1: Normal control; P2: Negative control (AOM and DSS); P3: Positive control (5-FU); P4: Preventive treatment (fermented milk); P5: Curative treatment (fermented milk). Brown coloration (arrow) indicates positive NF-κB expression localized in the nucleus and cytoplasm of epithelial and inflammatory cells. Scale bar = 50 µm.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/41f62c960e2cea1facf1b2c6.png"},{"id":94235344,"identity":"236001b5-6ef1-40af-bee1-12bee538386b","added_by":"auto","created_at":"2025-10-24 01:46:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":30682831,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7681425/v1/d746797b-d7cb-413e-a72a-5b77440e1c7d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anticancer Activity of Lactiplantibacillus plantarum subsp. plantarum IIA-1A5-Fermented Milk on Chemically Induced-Colorectal Cancer of BALB/c Mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) constitutes a significant global health issue, ranking third in incidence and second in cancer-related mortality worldwide, with 1.93\u0026nbsp;million new cases and over 903,000 fatalities recorded in 2022 (Bray et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In Indonesia, colorectal cancer was the fourth most common cancer in 2020 (Sung et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It primarily affects individuals over the age of 50, with men being at higher risk (Lewandowska et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Risk factors for colorectal cancer encompass both non-modifiable elements (age, sex, genetics) and modifiable variables, including food, obesity, smoking, and alcohol use (Goodarzi et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Current treatment modalities, including surgery, chemotherapy, and radiotherapy, demonstrate disparate success rates and frequently entail significant adverse effects. 5-Fluorouracil (5-FU), a commonly employed chemotherapeutic agent, may result in toxicity, drug resistance, and recurrence, whereas radiation might induce prolonged damage by the production of reactive oxygen species (Gmeiner and Okechukwu, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, the financial burden associated with CRC treatment remains substantial (Bhimani et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAttempts to provide alternative treatments for CRC have been widely studied by proposing lactic acid bacteria (LAB)-based probiotics as a promising avenue to be further explored due to their potent anticancer properties. This is particularly attributed to the ability of these probiotics to produce bioactive substances capable of interfering with the growth or development of cancer cells. Bioactive substances produced from probiotics, especially those from lactic acid bacteria (LAB), have surfaced as viable alternatives for cancer treatment (Zhao et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). LAB, designated as Generally Recognized as Safe (GRAS), generates antibacterial and anticarcinogenic metabolites, such as short-chain fatty acids (SCFAs), reuterin, and bacteriocins (Iacob et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Probiotics may potentially inhibit colorectal cancer by modulating gut microbiota and immune function (Kahouli et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOne promising LAB-based probiotic is \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e subsp. \u003cem\u003eplantarum\u003c/em\u003e IIA-1A5, a indegenous Indonesian strain (GenBank accession no. OR473281.1) characterized by unique fatty acid, amino acid, and volatile profiles, including SCFAs, many of which are linked to anticancer properties. In vitro studies identified 83 volatile compounds with known anticancer activity, while its strain-specific surface layer protein (slp) gene. This gene is recognized as strain-specific and has a role in anticancer mechanisms, primarily via apoptosis induction and cell cycle inhibition (Zhang et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe anticancer potential of \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 has been reported; however, the primary challenge lies in its delivery, as patient acceptance may differ between consumption as a probiotic supplement and as a food product. Therefore, the development of functional foods incorporating this strain is essential to ensure both the stability and efficacy of its anticancer activity. Previous studies have demonstrated that \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 can serve as a starter culture for fermented milk, which not only complies with the Indonesian National Standard (SNI) for food safety but also exhibits significant anticancer activity against WiDr colorectal cancer cells \u003cem\u003ein vitro\u003c/em\u003e (Adiyoga et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Nevertheless, these findings have not yet been validated in \u003cem\u003ein vivo\u003c/em\u003e models. Considering that discrepancies often arise between \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e outcomes due to environmental variations affecting bioactive compounds, further \u003cem\u003ein vivo\u003c/em\u003e investigations are warranted to substantiate the anticancer potential of \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5.\u003c/p\u003e\u003cp\u003eThis study is therefore aimed at comprehensively evaluating the anticancer potential of \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5\u0026ndash;fermented milk in a murine model of colorectal cancer induced by azoxymethane (AOM) and dextran sodium sulfate (DSS). The current study describes the therapeutic efficacy of this indigenous Indonesian probiotic strain, particularly in the treatment group (P5), where its administration resulted in a marked reduction in aberrant crypt foci, tumor cell density, malondialdehyde levels, and cortisol concentrations. Moreover, it demonstrates favorable modulation of the gut microbiota, including a notable decline in \u003cem\u003eEscherichia coli\u003c/em\u003e populations. Taken together, these findings position \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 as a promising functional bioactive agent capable of mitigating oxidative stress, modulating physiological stress responses, and enhancing intestinal microbial homeostasis. Further mechanistic and clinical investigations are warranted to substantiate these outcomes and to explore their translational relevance for colorectal cancer prevention and therapy in humans.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Production of Fermented Milk Containing \u003cem\u003eL. plantarum subsp. plantarum\u003c/em\u003e IIA-1A5\u003c/h2\u003e\u003cp\u003eThe production of fermented milk was carried out following the method described by (Arief et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The activation of \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 (registered in GenBank, accession no. OR473281.1) began with retrieving the stock culture and inoculating it into 9 mL of de Man, Rogosa, and Sharpe broth (MRSB) (Oxoid, Basingstoke, UK, followed by incubation at 37\u0026deg;C for 24 h. Subculturing was performed repeatedly until the culture demonstrated stable growth and full adaptation to the medium. Sterile milk was then prepared and inoculated with 10% (v/v) of the activated culture. To increase biomass, the culture was further transferred into fresh sterile milk. Fermentation was conducted in an incubator (Memmert IN110, Memmert GnbH, Schwabach, Germany) at 37\u0026deg;C for 18 h. The initial bacterial population in the fermented milk was determined by plating on de Man, Rogosa, and Sharpe agar (MRSA) (Oxoid, Basingstoke, UK, according to standard procedures. Only fermented milk with a viable cell count exceeding 10⁹ CFU/mL was used for subsequent experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 In vivo Animal Experiments\u003c/h2\u003e\u003cp\u003eThe in vivo study was conducted following ethical approval from the Directorate of Research and Innovation (DRI), IPB University (Ethical Clearance No. 287\u0026ndash;2024 IPB), under the supervision of a licensed attending veterinarian. A total of 25 male BALB/c mice, aged 6\u0026ndash;8 weeks and weighing between 18 and 30 grams, obtained from iRATco Laboratory Indonesia, were used as experimental subjects (Zhang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). All animals underwent a 7-day acclimatization period under controlled environmental conditions prior to the start of the experiment. Following acclimatization, treatments were administered over a 70-day period. Mice were orally administered fermented milk containing \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 twice daily once in the morning and once in the evening at a dosage of 0.3 mL per administration. At the end of the treatment period, the mice were humanely euthanized via cervical dislocation. The experimental animals were randomly assigned to five groups (n\u0026thinsp;=\u0026thinsp;5 per group). The treatment scheme for each group is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\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\u003eExperimental Animal Treatment Groups\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP1\u003c/p\u003e\u003cp\u003e(Normal control)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNo induction with azoxymethane (AOM) and dextran sodium sulfate (DSS).\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP2\u003c/p\u003e\u003cp\u003e(Negative Control)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInduction with AOM and DSS only\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP3\u003c/p\u003e\u003cp\u003e(Positive Control)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInduction with AOM and DSS followed by administration of the chemotherapeutic agent 5-fluorouracil (5-FU).\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP4\u003c/p\u003e\u003cp\u003e(Preventive Group)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInduction with AOM and DSS, with fermented milk containing \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e subsp. \u003cem\u003eplantarum\u003c/em\u003e IIA-1A5 administered both after induction and starting 7 days prior to induction.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP5\u003c/p\u003e\u003cp\u003e(Curative Group)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInduction with AOM and DSS, with fermented milk containing \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 administered only after induction.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Induction of Colorectal Cancer\u003c/h2\u003e\u003cp\u003eColorectal cancer was induced in mice following the protocol by (Mart\u0026iacute;nez-Gregorio et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), utilizing azoxymethane (AOM) and dextran sodium sulfate (DSS). AOM (Sigma-Aldrich, St. Louis, MO, USA) was administered as a single intraperitoneal injection at a dose of 12.5 mg/kg body weight on the first day of treatment. Subsequently, DSS was provided in the drinking water at a concentration of 2% in three induction cycles: Phase I (days 8\u0026ndash;14), Phase II (days 29\u0026ndash;34), and Phase III (days 50\u0026ndash;55).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Post-Treatment Animal Observations\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1 Cancer Distribution (Aberrant Crypt Foci - ACF)\u003c/h2\u003e\u003cp\u003eCancer distribution in the colon tissue of BALB/c mice was assessed macroscopically using ImageJ software by identifying surface nodules, which serve as key morphological indicators of colorectal cancer (Budijanto et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This evaluation provided visual insight into the location and extent of cancer spread.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2 Histopathological Analysis\u003c/h2\u003e\u003cp\u003eHistopathological analysis was conducted to examine cellular abnormalities (Sun et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Intestinal tissues were fixed in 10% neutral buffered formalin, sectioned (5\u0026ndash;10 mm), and processed through dehydration and clearing with graded alcohols, toluene, and paraffin. Tissues were embedded, sectioned at 4\u0026ndash;5 \u0026micro;m, and stained using the Harris Hematoxylin-Eosin method. Dehydration and clearing were completed with alcohol and xylene.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.4.3 Cortisol Hormone Analysis\u003c/h2\u003e\u003cp\u003eCortisol levels were measured using the ELISA method as described by (Gong et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). A 25 \u0026micro;L serum sample was processed with biotin and enzyme conjugates, incubated, washed, and reacted with TMB substrate. Absorbance was measured at 450 nm, and cortisol concentrations were determined using a standard curve.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.4.4 Malondialdehyde (MDA) Analysis\u003c/h2\u003e\u003cp\u003eMDA levels were measured using the thiobarbituric acid (TBA) assay as described by (Gonzalez and Paulson, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Samples were homogenized, treated with TCA, centrifuged, and reacted with TBA, followed by heating and cooling. Absorbance was measured at 530 nm using a spectrophotometer, with TEP as the standard.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.4.5 Microbiological Analysis\u003c/h2\u003e\u003cp\u003eMicrobiological analysis targeted lactic acid bacteria and Escherichia coli. Intestinal samples were serially diluted and cultured on MRSA (Oxoid, Basingstoke, UK), for LAB enumeration. E. coli was assessed using the BAM method on EMBA (Oxoid, Basingstoke, UK), characteristic colonies were identified by a metallic green sheen after 24 h incubation at 37\u0026deg;C (Arief et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.4.6 Immunohistochemical of Nuclear Factor-kappa B (NF-κB)\u003c/h2\u003e\u003cp\u003eNF-κB expression was analyzed by immunohistochemistry using anti-NF-κB p65 antibody as described by Sivakumar et al., (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Tissue sections were incubated with primary antibody (1:50), followed by HRP-conjugated secondary antibody and DAB, then counterstained with Mayer\u0026rsquo;s hematoxylin, dehydrated, cleared, and mounted.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e\u003cp\u003eData are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation from five independent replicates. Differences among means were evaluated using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. Statistical analyses were performed with Minitab 20 software, with significance considered at the 5% confidence level. However, ACF data were presented descriptively and not subjected to statistical analysis.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Aberrant Crypt Foci (ACF)\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the highest number of aberrant crypt foci (ACF) was observed in the negative control group (P2), while the lowest was found in the curative treatment group (P5). No ACF formation was detected in the normal control (P1). The presence of ACF was confirmed microscopically (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), where black arrows indicate clusters of enlarged, irregular crypts, clearly visible in P2, P3, P4, and P5 but absent in P1.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Histopathological\u003c/h2\u003e\u003cp\u003eTumor cell density in colonic tissue varied significantly among groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Statistical analysis confirmed significant differences among all groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as indicated by the distinct superscript letters. The negative control showed the highest density, while the normal control remained unaffected. Treatment with 5-fluorouracil reduced tumor cell density but was less effective than fermented milk. Both preventive and curative administration of \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 markedly suppressed tumor cell growth, with the curative group showing the greatest reduction, approaching normal levels. These findings highlight the potential of fermented milk as both a chemopreventive and therapeutic agent against colorectal cancer. Histopathological analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) revealed intact colonic architecture in the normal control, while the negative control showed extensive epithelial hyperplasia and tumor infiltration. The positive control displayed partial improvement, whereas preventive treatment attenuated tissue abnormalities. The curative group exhibited the most preserved structure, closely resembling normal tissue.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Cortisol Hormone\u003c/h2\u003e\u003cp\u003eSerum cortisol concentrations in colorectal cancer\u0026ndash;induced mice are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The normal control group (P1) exhibited the lowest cortisol level, whereas the negative control group (P2) showed the highest concentration. The positive control (P3) and preventive treatment (P4) groups demonstrated intermediate values, while the curative treatment group (P5) displayed levels that were closer to the normal control. Statistical analysis revealed significant differences among the groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In particular, P2 was significantly higher than P1 and P5, whereas P3 and P4 did not differ significantly from either P2 or P5 but were both higher than P1. These findings suggest that the curative treatment (P5) more effectively modulated cortisol levels toward the normal profile compared with the preventive treatment (P4).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Malondialdehyde (MDA)\u003c/h2\u003e\u003cp\u003eThe malondialdehyde (MDA) concentrations in colorectal cancer\u0026ndash;induced mice across different treatment groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The normal control group (P1) exhibited the lowest MDA levels, whereas the negative control group (P2), which was exposed to carcinogens without treatment, displayed the highest concentration. The positive control group (P3, 5-FU treatment) presented reduced MDA levels compared to P2, and similar reductions were observed in the preventive (P4) and curative (P5) groups administered fermented milk with \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5. Statistical analysis confirmed significant differences among the groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In particular, P2 was significantly higher than P1, while P3, P4, and P5 did not differ significantly from either P1 or P2. These findings suggest that both preventive and curative treatments with \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 effectively lowered MDA levels toward those observed in the positive control group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Microbiologycal\u003c/h2\u003e\u003cp\u003eThe microbiological profile of \u003cem\u003eEscherichia coli\u003c/em\u003e is illustrated in the colorectal cancer\u0026ndash;induced mice across the different treatment groups, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, whereas the total lactic acid bacteria (LAB) counts are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. LAB levels were relatively consistent across all groups, with no statistically significant differences observed (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast, \u003cem\u003eE. coli\u003c/em\u003e counts varied significantly among treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The highest \u003cem\u003eE. coli\u003c/em\u003e levels were detected in the normal control (P1) and negative control (P2), while the positive control (P3) exhibited reduced counts compared with both controls. Notably, the preventive (P4) and curative (P5) groups administered fermented milk containing \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 showed markedly lower \u003cem\u003eE. coli\u003c/em\u003e counts, with P4 demonstrating the most pronounced reduction. These findings suggest that \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 treatment effectively suppressed \u003cem\u003eE. coli\u003c/em\u003e proliferation while maintaining LAB populations across groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Immunohistochemical of Nuclear Factor-kappa B (NF-κB)\u003c/h2\u003e\u003cp\u003eThe NF-κB expression levels in colorectal cancer\u0026ndash;induced mice across different treatment groups are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. Statistical analysis confirmed significant differences among the groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Specifically, P2 was significantly higher than P1, P4, and P5, whereas P3 was significantly lower than P2 but remained higher than P1 and P5. These findings indicate that both preventive and curative treatments with \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 effectively reduced NF-κB expression toward baseline levels, with the curative treatment (P5) showing modulation comparable to the normal control. Figure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e provides immunohistochemical (IHC) visualization of NF-κB expression in colonic tissues. Positive NF-κB expression is indicated by brown staining (arrows), localized within the nucleus and cytoplasm of epithelial and inflammatory cells, thereby supporting the quantitative results observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Aberrant Crypt Foci (ACF) and Histopathological\u003c/h2\u003e\u003cp\u003eThe distribution of aberrant crypt foci (ACF) and histopathological alterations provided complementary evidence for the role of \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 in suppressing colorectal cancer (CRC) progression. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the negative control group (P2) exhibited the highest number of ACF, consistent with the established role of azoxymethane (AOM) in inducing early epithelial alterations that initiate colorectal carcinogenesis (Kowalczyk et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In contrast, the positive control group (P3, 5-FU treatment) demonstrated a marked reduction in ACF, corroborating previous findings that 5-fluorouracil effectively suppresses AOM-induced ACF formation (Stastna et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Microscopic observations further supported these results, revealing clusters of enlarged and irregular crypts in groups P2\u0026ndash;P5 but not in P1, thereby reinforcing the role of ACF as preneoplastic lesions and reliable biomarkers of CRC initiation (Genaro et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHistopathological analysis provided further validation. The negative control (P2) displayed severe epithelial hyperplasia, disrupted crypt morphology, and tumor infiltration, reflecting successful induction of colorectal carcinogenesis by the AOM and DSS protocol, which promotes inflammation-driven malignant transformation (Liu et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Modesto et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). By contrast, the normal control retained intact colonic architecture. Treatment with 5-FU partially ameliorated these pathological changes by reducing tumor cell density, consistent with its mechanism of action in inhibiting thymidylate synthase and disrupting nucleic acid synthesis (Longley et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Notably, both the preventive (P4) and curative (P5) groups receiving \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5\u0026ndash;fermented milk demonstrated improved tissue preservation compared with the positive control, with the curative group showing the closest resemblance to normal colon morphology.\u003c/p\u003e\u003cp\u003eThe reduction in ACF and tumor burden following probiotic intervention aligns with earlier evidence indicating that probiotics can decrease neoplastic lesions in experimental CRC models (Hajrezaie et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Jacouton et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eL. plantarum\u003c/em\u003e is believed to exert anticancer effects through multiple mechanisms, including reshaping the gut microbiota, enhancing host immune responses, promoting apoptosis, and producing bioactive metabolites such as short-chain fatty acids (SCFAs), which regulate oxidative stress and epithelial proliferation (Chen and Li \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ghanavati et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, bacteriocins and surface layer proteins synthesized by \u003cem\u003eL. plantarum\u003c/em\u003e may contribute to mucosal protection and attenuation of proinflammatory signaling, thereby suppressing tumorigenesis. Collectively, these mechanisms explain the substantial improvement in colonic morphology observed in probiotic-treated groups.\u003c/p\u003e\u003cp\u003eAlthough both preventive and curative interventions were beneficial, the curative group (P5) achieved a more pronounced reduction in ACF and tumor density, suggesting that probiotic efficacy may be greater during active disease progression. This observation supports the hypothesis that probiotics not only prevent tumor initiation but also exert therapeutic effects by modulating host immunity, counteracting microbial dysbiosis, and suppressing oncogenic pathways such as NF-κB and Wnt/β-catenin (Ghafouri-Fard et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Modesto et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These findings underscore the importance of treatment timing, indicating that post-induction administration may yield superior protective outcomes.\u003c/p\u003e\u003cp\u003eTaken together, the integrated analysis of ACF distribution and histopathology highlights the dual preventive and therapeutic roles of \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 in colorectal carcinogenesis. The reduction of preneoplastic lesions, restoration of colonic architecture, and group-dependent differences provide strong experimental evidence that fermented milk containing \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 can suppress inflammation-driven colorectal cancer development. These results further support the growing perspective that probiotics may serve as adjuvants to conventional chemotherapy, offering tumor-suppressive benefits while potentially mitigating treatment-associated side effects. Continued investigations, particularly well-designed clinical studies, are necessary to validate the translational potential of \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 in CRC management\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Cortisol Hormone\u003c/h2\u003e\u003cp\u003eIn the normal control group (P1), serum cortisol concentrations remained within the physiological range, whereas the negative control group (P2) exhibited marked elevations, consistent with previous reports that AOM and DSS-induced carcinogenesis disrupts glucocorticoid balance and promotes stress-driven epithelial dysplasia (Li et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Chronic hypercortisolemia is known to exacerbate intestinal inflammation and immune dysregulation, thereby accelerating colorectal cancer (CRC) progression (La Vecchia and Sebasti\u0026aacute;n, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Prolonged activation of the hypothalamic pituitary adrenal (HPA) axis has also been associated with tumor growth and reduced therapeutic efficacy.\u003c/p\u003e\u003cp\u003eAdministration of 5-fluorouracil (P3) partially normalized cortisol levels compared with P2, reflecting its antiproliferative effects while simultaneously stimulating HPA axis activity (Lempesis et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Fermented milk containing \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 demonstrated treatment-dependent outcomes: preventive supplementation (P4) produced modest reductions, whereas curative administration (P5) restored cortisol concentrations to near-normal levels. These endocrine changes paralleled histopathological findings, with P5 showing greater preservation of colonic architecture and fewer tumor lesions than P4, suggesting enhanced therapeutic efficacy when administered post-induction (Agalakova, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe cortisol profile was closely aligned with both ACF and histopathological data. Elevated cortisol in P2 coincided with higher ACF counts and severe epithelial dysplasia, while normalization in P5 corresponded with reduced ACF burden and improved tissue integrity. This concordance underscores the dual role of \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 in modulating systemic stress responses and suppressing tumorigenesis. Supporting evidence indicates that probiotics regulate the HPA axis and enhance stress resilience, as demonstrated for \u003cem\u003eL. rhamnosus\u003c/em\u003e, \u003cem\u003eL. helveticus\u003c/em\u003e, and \u003cem\u003eB. longum\u003c/em\u003e (Bercik et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Bravo et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ohland et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Collectively, these findings highlight \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5\u0026ndash;fermented milk as a promising functional food with synergistic anticancer and anti-stress effects.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Malondialdehyde (MDA)\u003c/h2\u003e\u003cp\u003eThe negative control group (P2), exposed to carcinogens without treatment, exhibited the highest malondialdehyde (MDA) levels, indicating severe oxidative stress, consistent with enhanced lipid peroxidation in colorectal cancer models (Reuter et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Carcinogens such as AOM and DSS induce persistent inflammation, creating a pro-oxidative microenvironment that accelerates tumor progression (Longley et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Administration of 5-fluorouracil (5-FU, P3) reduced MDA levels by suppressing tumor proliferation but also promoted reactive oxygen species (ROS) generation, with cancer cell survival mediated by Nrf2-driven antioxidant responses (Blondy et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Both preventive (P4) and curative (P5) groups treated with \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5\u0026ndash;fermented milk showed MDA reductions comparable to those observed in the 5-FU group. This probiotic scavenges ROS, stimulates enzymatic antioxidants (SOD, GSH-Px, CAT), enhances glutathione synthesis, and its extracellular polysaccharides further support free radical neutralization (Moslehishad et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Silva et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Jiang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eL. plantarum\u003c/em\u003e has also been reported to neutralize dietary and endogenous carcinogens such as N-nitrosodimethylamine (Nowak et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eElevated MDA in P2 corresponded with a high aberrant crypt foci (ACF) burden, severe epithelial dysplasia, and increased cortisol, reflecting the interplay of oxidative stress, tissue damage, and endocrine disruption. Conversely, normalization of MDA in P5 was associated with reduced ACF counts, improved colonic architecture, and restored cortisol levels. These findings suggest that \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5\u0026ndash;fermented milk mitigates colorectal carcinogenesis by attenuating oxidative stress, modulating systemic stress responses, and preserving tissue integrity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e4.4 Microbiologycal\u003c/h2\u003e\u003cp\u003eLactic acid bacteria (LAB), particularly \u003cem\u003eLactobacillus spp\u003c/em\u003e., are key members of the \u003cem\u003eFirmicutes phylum\u003c/em\u003e, playing a critical role in maintaining gut homeostasis through the production of short-chain fatty acids (SCFAs) and modulation of host immune responses (Li et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bader et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this study, LAB levels remained consistent across all experimental groups, indicating that neither carcinogen exposure nor therapeutic interventions significantly disrupted the beneficial microbial population.\u003c/p\u003e\u003cp\u003eIn contrast, Escherichia coli counts exhibited marked variability. The highest levels were observed in the normal control (P1) and CRC-induced negative control (P2), consistent with previous reports that AOM and DSS-induced colorectal carcinogenesis perturbs microbial homeostasis and promotes the proliferation of pathogenic taxa such as \u003cem\u003eEscherichia shigella\u003c/em\u003e (Deng et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The 5-FU treated group (P3) displayed reduced \u003cem\u003eE. coli\u003c/em\u003e abundance, reflecting chemotherapeutic modulation of the gut microbiota, characterized by decreased \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eProteobacteria\u003c/em\u003e and increased Bacteroidetes, which collectively suppress opportunistic pathogens (Stringer et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Deng et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although \u003cem\u003eProteobacteria\u003c/em\u003e constitute a minor fraction of the gut microbiome, they exert significant influence on host metabolism and inflammatory processes (P\u0026eacute;rez-Cobas et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNotably, administration of \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 in both preventive (P4) and therapeutic (P5) groups markedly reduced E. coli levels, with the most pronounced effect observed in P4, while preserving LAB populations. These findings support the probiotic\u0026rsquo;s capacity to alleviate CRC-associated dysbiosis (dos Reis et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The antimicrobial activity of \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 is attributed to \u003cem\u003eplantaricin\u003c/em\u003e IIA-1A5, which inhibits enteric pathogens including \u003cem\u003eSalmonella, Shigella\u003c/em\u003e, and enteropathogenic \u003cem\u003eE. coli\u003c/em\u003e (Sihombing et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Similarly, synbiotic formulations containing \u003cem\u003eLactobacillus spp.\u003c/em\u003e have demonstrated efficacy in suppressing \u003cem\u003eE. coli\u003c/em\u003e and restoring microbial balance (Astawan et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eImportantly, the observed reduction in E. coli and the maintenance of LAB in P4 and P5 correlated with decreased aberrant crypt foci (ACF), improved histological architecture, lower malondialdehyde (MDA) levels, and reduced serum cortisol. These integrated findings indicate that \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 exerts a multifaceted protective effect against CRC progression through modulation of the gut microbiota, mitigation of oxidative stress, and preservation of intestinal tissue integrity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e4.5 Immunohistochemical of Nuclear Factor-kappa B (NF-κB)\u003c/h2\u003e\u003cp\u003eData Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e demonstrates markedly elevated NF-κB expression in the negative control group (P2) induced with AOM and DSS. This observation aligns with previous studies showing that AOM and DSS induces chronic inflammation through activation of pro-inflammatory pathways, including NF-κB, a critical mediator in the initiation and promotion of colorectal carcinogenesis (CRC) (Kanehara et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). NF-κB activation drives the production of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β, thereby creating a microenvironment conducive to tumor cell proliferation and survival (Sun et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eProbiotic intervention exhibited superior efficacy in mitigating inflammation compared to 5-FU chemotherapy. Preventive (P4) and curative (P5) administration of \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 significantly suppressed NF-κB expression, achieving levels comparable to, or even lower than, those of the normal control group (P1), underscoring its potent anti-inflammatory capacity. The mechanisms underlying this effect likely involve multiple pathways, including modulation of gut microbiota composition, enhanced production of short-chain fatty acids (SCFAs) such as butyrate which inhibit NF-κB activation via histone deacetylase (HDAC) inhibition and reinforcement of intestinal epithelial tight junctions, thereby limiting bacterial and LPS translocation, which activates NF-κB via Toll-like receptors (Peng et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eConsistent with these findings, Reis et al., (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported that probiotic supplementation effectively suppresses NF-κB, reducing chronic inflammation, a key driver of preneoplastic lesion formation and colorectal tumor progression. NF-κB inhibition by probiotics is further supported by mechanisms including EPS TLR4 interactions, upregulation of IκBα, and suppression of MAPK signaling, collectively mitigating pro-inflammatory cytokine overexpression (Morsli et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In this study, NF-κB downregulation correlated with improved colonic histology and reduced inflammatory markers such as MDA and cortisol, confirming that \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 effectively modulates inflammatory signaling and restores gut homeostasis. These results highlight the potential of \u003cem\u003eL. plantarum\u003c/em\u003e as both a protective and therapeutic agent in managing colorectal cancer\u0026ndash;associated chronic inflammation.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, administration of this probiotic formulation, particularly in the curative group (P5), resulted in a marked reduction in aberrant crypt foci, tumor cell density, malondialdehyde levels, and serum cortisol, while concurrently promoting favorable modulation of gut microbiota, including a decrease in \u003cem\u003eEscherichia coli\u003c/em\u003e populations. Notably, NF-κB expression was significantly suppressed, demonstrating the probiotic\u0026rsquo;s capacity to attenuate pro-inflammatory signaling pathways that are central to colorectal carcinogenesis. Collectively, these findings underscore the potential of \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 as a multifunctional bioactive agent capable of mitigating oxidative stress, modulating systemic stress responses, suppressing inflammation, and restoring intestinal microbial balance. Further mechanistic studies and clinical trials are warranted to validate these effects and clarify the translational relevance of this probiotic in colorectal cancer prevention and therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare no potential conflict of interest.\u003c/p\u003e\n\u003ch2\u003eEthics Approval\u003c/h2\u003e\n\u003cp\u003eThis article was conducted following ethical approval from the Directorate of Research and Innovation (DRI), IPB University (Ethical Clearance No. 287\u0026ndash;2024 IPB).\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis research was funded by the Indonesian Ministry of Higher Education, Science, and Technology.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eConceptualization: Erlina I, Arief II, Budiman C. Data curation: Erlina I, Arief II. Formal analysis: Erlina I, Arief II, Budiman C. Methodology: Erlina I, Arief II, Budiman C. Software: Erlina I. Validation: Erlina I, Arief II, Budiman C, Sena A, Fujiyama K. Investigation: Erlina I. Writing - original draft: Erlina I. Writing - review \u0026amp; editing: Erlina I, Arief II, Budiman C, Sena A, Fujiyama K.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors gratefully acknowledge the financial support from the Ministry of Higher Education, Science, and Technology of the Republic of Indonesia (Kemdiktisaintek) through the PPS-PMDSU research grant scheme in 2025 (Contract No. 006/IT3.D10/PT.01.03/P/B/2025) and the PT research grant scheme in 2024 (Contract No. 22345/IT3.D10/PT.01.03/P/B/2024).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdiyoga R, Arief II, Budiman C, Abidin Z. 2022. In vitro anticancer potentials of Lactobacillus plantarum IIA-1A5 and Lactobacillus acidophilus IIA-2B4 extracts against WiDr human colon cancer cell line. 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Clin Med Insights Oncol. 17.doi:10.1177/11795549231188225.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","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":"azoxymethane (AOM), dextran sodium sulfate (DSS), Indonesian indigenous bacteria, probiotics","lastPublishedDoi":"10.21203/rs.3.rs-7681425/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7681425/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eColorectal cancer (CRC) remains a significant global health challenge, with current treatment options often limited by toxicity, resistance, and adverse effects. Probiotics, particularly lactic acid bacteria (LAB), offer promising alternatives through bioactive metabolites with anticancer and immunomodulatory properties. The indigenous Indonesian strain \u003cem\u003eLactiplantibacillus plantarum subsp. plantarum\u003c/em\u003e IIA-1A5 exhibits unique metabolic profiles and has demonstrated in vitro anticancer potential. This study evaluated the anticancer effects of \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5\u0026ndash;fermented milk in an AOM and DSS-induced murine model, focusing on aberrant crypt foci (ACF), tumor cell density, malondialdehyde (MDA), cortisol levels, gut microbiota composition, and NF-κB expression. The curative intervention group (P5) exhibited the most pronounced effects, including significant reductions in ACF, tumor cell density, MDA levels, and serum cortisol, reflecting decreased oxidative and physiological stress. Importantly, NF-κB expression was markedly suppressed, indicating attenuation of pro-inflammatory signaling pathways pivotal to CRC progression. Favorable modulation of gut microbiota was also observed, particularly through suppression of Escherichia coli. Compared to 5-fluorouracil (5-FU), \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 demonstrated comparable efficacy in mitigating oxidative stress, inflammation, and microbial dysbiosis. These findings highlight the potential of \u003cem\u003eL. plantarum\u003c/em\u003e IIA-1A5 as a functional probiotic with both preventive and therapeutic applications in colorectal cancer management. Further mechanistic and clinical studies are warranted to validate these effects and assess their translational relevance in humans.\u003c/p\u003e","manuscriptTitle":"Anticancer Activity of Lactiplantibacillus plantarum subsp. plantarum IIA-1A5-Fermented Milk on Chemically Induced-Colorectal Cancer of BALB/c Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-13 08:50:00","doi":"10.21203/rs.3.rs-7681425/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":"d88bbd04-16d8-4740-8562-76f6e6ff4418","owner":[],"postedDate":"October 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-24T01:38:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-13 08:50:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7681425","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7681425","identity":"rs-7681425","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}