Optimizing Lipid Removal and Protein Digestion in Human Milk for Microplastic Analysis using Candida rugosa Lipase, KOH Digestion, and Hydrogen Peroxide Oxidation

Optimizing Lipid Removal and Protein Digestion in Human Milk for Microplastic Analysis using Candida rugosa Lipase, KOH Digestion, and Hydrogen Peroxide Oxidation | 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 Optimizing Lipid Removal and Protein Digestion in Human Milk for Microplastic Analysis using Candida rugosa Lipase, KOH Digestion, and Hydrogen Peroxide Oxidation Dhea Maisarah Ahmad Nasri, Nur Rasyiqah Syamsul, Fikriah Faudzi, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8153566/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract This study successfully developed and evaluated a multi-step digestion protocol for the effective extraction and identification of microplastics (MPs) from human breast milk (HBM). The method combined hexane-mediated lipid removal, enzymatic digestion using Candida rugosa lipase at a milk-to-enzyme ratio of 20:1, protein digestion with potassium hydroxide (KOH) at 1:3 ratio, and oxidative treatment with hydrogen peroxide (H₂O₂) at 10% concentration. Ethanol was found essential for reducing sample viscosity, particularly post-saponification, thereby facilitating efficient filtration. Results showed that extended incubation times during enzymatic digestion notably enhanced lipid removal and improved clarity of the digestate, contributing to higher extraction efficiency. This protocol establishes a reproducible framework for reliable isolation and downstream spectroscopic identification of MPs in breast milk. Despite these advances, improvements in mechanical handling and contamination control are necessary to further enhance accuracy and consistency. The present findings contribute important methodological advancements to the field and support the urgent need for standardised, scalable protocols in monitoring neonatal exposure to environmental microplastic contaminants. Future work should expand on validation across diverse sample sets and explore correlations between MP presence and neonatal health outcomes. Microplastics human milk lipid extraction enzymatic digestion KOH digestion Introduction Microplastics (MPs), defined as plastic particles smaller than 5 mm, have emerged as pervasive environmental contaminants with global ecological and human health implications. They were widely detected in diverse ecosystems and increasingly within the human body (Lakshmayya et al. 2023). MPs originate either from the degradation of larger plastic debris, synthetic textile fibers, or from intentionally manufactured microbeads and resins used in consumer products. Their small size facilitates the bioavailability and accumulation in multiple organs including the lungs, liver, kidneys, gastrointestinal tract, and placenta, thus raising concerns about potential health risks (Roslan et al. 2024). Although long-term effects and chronic toxicity associated with MPs are not yet fully understood, recent evidence suggests that their presence in tissues and fluids may contribute to inflammation, oxidative stress, and endocrine disruption through both physical effects from particle (shape and size) and/or chemical leaching (Nawab et al. 2024). Particularly, a recent report confirming the occurrence of MPs in human breast milk (HBM) has raise pressing concerns regarding early-life (neonatal) exposure, since HBM is not only a critical source of nutrition and bioactive compounds for the immune system and growth of infants (Ragusa et al. 2022). Moreover, MPs may adsorb and transport hazardous chemicals such as perfluoroalkyl substances (PFAS), phthalates, and other organic pollutants, which raising potential consequence for infant health (Dzierżyński et al. 2024; Mišľanová et al. 2024). Therefore, the presence of MPs in HBM highlights an urgent need for more robust and standardized analytical protocol that capable of detecting and quantifying MPs in complex biological matrices. However, accurate detection of MPs in HBM is practically challenging and hindered due to its complex composition. Content of HBM is majorly well known with the composition of around 87%–88% water and 124 g/L of solid macronutrients, which include approximately 7% (60–70 g/L) carbohydrates, 1% (8–10 g/L) protein, and 3.8% (35–40 g/L) fat (Kim & Yi 2020). Protein and carbohydrates contents can interfere with the recovery, isolation, identification, and quantification of MPs (Kutralam-Muniasamy 2022). Standard digestion methods for biological samples, including alkaline, acid, or enzymatic treatment followed by oxidative degradation have been individually employed with varying level of success in the biota sample recovery (Rani et al. 2023; Roslan et al. 2024). However, their combination is insufficiently evaluated and its effectiveness in the extraction of MPs in HBM remains limited, and incomplete removal of fats and proteins will result in matrix interferences during further microscopic and spectroscopic identification (Rani et al. 2023). Therefore, the optimization of a multi-step digestion protocol that ensures efficient lipid removal, protein degradation and minimal polymer damage is essential for reliable microplastic quantification in HBM. This study proposes an integrated workflow combining hexane-based lipid extraction, enzymatic digestion using Candida rugosa lipase, alkaline digestion with potassium hydroxide (KOH) and oxidative digestion with hydrogen peroxide (H₂O₂) treatments of HBM prior to physical analysis of MPs. The objective is to evaluate and optimize the synergistic application of these aims to optimize these treatments to enhance substrate clarity (i.e. by enhancing lipid removal and organic matter degradation), improve MPs recovery rates and minimize artefacts during identification. This is critical toward developing a reproducible and validated protocol for microplastics in complex human fluids. Experimental Materials Samples This study was ethically approved on 22 nd March 2025 by the IIUM Research Ethical Committee (IREC), Malaysia in accordance with the principles outlined in the Declaration of Helsinki, the International Conference on Harmonisation - Good Clinical Practice (ICH-GCP), the Malaysian Guidelines for Good Clinical Practice (Malaysia GCP), and the Council for International Organizations of Medical Sciences (CIOMS) International Ethical Guidelines (Approval ID: IREC 2025-058). Donated human breast milk (HBM) samples were obtained from the HMMC, Malaysia, with collected HBM sample were anonymised to ensure confidentiality. Chemicals The key chemicals and reagents used in this study included hexane (HMBG Chemicals, Germany), 30% H₂O₂ solution (HMBG Chemicals, Germany), 70% ethanol (HMBG Chemicals, Germany), lipase Type VII from Candida rugosa with an activity of 700 units/mg solid (Sigma-Aldrich, United States), pH 7.2 buffer tablets (Sigma-Aldrich, United States), KOH pellets (Sigma-Aldrich, United States), and ultra-pure distilled water (milli-Q). All chemical and reagents used are analytical grade. The Candida rugosa lipase was stored at 2–4 °C prior to use, as recommended by the manufacturer, while other chemicals such as hexane, H₂O₂, and KOH were stored at room temperature in designated chemical safety cabinets. Milk Processing Donated HBM samples were handled according to the standard procedures employed by the milk bank and stored at −20°C to preserve their quality. Upon thawing, samples displayed distinct physical differences were noted and were categorized into two groups: (1) Milk Type A (high-fat content) characterized by a thicker, creamier consistency with visible lipid clumps; and (2) Milk Type B (low-fat content) due to its diluted consistency. This classification was necessary for assessing digestion performance across different fat levels. A structured naming convention was applied for sample tracking. For lipase digestion, tubes were labelled L1 to L10, with one sample (“+”) was intentionally spiked with microplastics (MPs) to evaluate whether enzymatic digestion affected MP integrity. Meanwhile, OL1 and OL2 referred to samples treated with lipase stored for two weeks under refrigeration. Additional sample groups included S1 – S3 (test samples), N1– N3 (negative controls using Milli-Q water), and P1– P3 (positive controls spiked with known MPs: Nylon-66, PET, and PP). This labelling system was applied consistently and systematically throughout the digestion and oxidation to maintain clarity, minimize procedural errors, and improve reproducibility in data interpretation. Lipase Solution Preparation The buffer solution was prepared by dissolving one tablet of phosphate buffer (pH 7.2) in 1 L of distilled water, mixing with a glass rod and kept at 4oC in the refrigerator until use. A 5% lipase solution was prepared by weighing 0.5 g of lipase powder and gently mixing it with 10 mL of cold phosphate buffer solution. The prepared lipase solution was then refrigerated until use. Lipid Digestion using Lipase from Candida rugosa For each sample, 10 mL of HBM was used. The HBM sample was preheated to 37 °C in a water bath. Once the milk reached the optimum temperature, the lipase solution was added at various milk-to-enzyme ratios (1:1, 2:1, 5:1, 10:1, and 20:1). The resulting mixtures were then incubated at 37 °C – 37.4 °C for a minimum of 2 days, with extended incubation period up to 7 days for selected samples to assess the procedural variation. The pH of each sample was measured before and after digestion procedure, with a post-digestion pH value of approximately 4 indicated successful lipid hydrolysis and digestion. Protein Digestion using KOH Following the enzymatic lipid digestion step, protein digestion was conducted using a KOH solution to further degrade the protein and organic matrices of HBM. A 10% (w/v) KOH solution was prepared by dissolving 50 g of KOH pellets in distilled water and diluting to a final volume of 500 mL. For each 10 mL milk sample, the HBM-to-KOH ratio was adjusted stepwise at 1:1 or 1:1.5, 1:3 and 1:7. Samples were then mixed gently and incubated at 40 – 45°C for 2 – 4 hours to facilitate protein denaturation and solubilization. The reaction was carried out in a beaker closed with aluminium foil to allow pressure release while minimizing airborne contamination. A color change from a cloudy white suspension to dark brown solution indicated progressive protein degradation. The appearance of turbidity or curd formation was monitored visually throughout the progress. Samples that remained highly viscous or slimy were further treated with an additional 70% ethanol in a 1:1 ratio (sample: ethanol) to aid in the disruption of emulsions and reduce filtering resistance. Following digestion, all samples were allowed to cool to room temperature before proceeding to the oxidation step or filtration. Digestion of Residual Lipids and Remaining Organic Matters using H₂O₂ After protein digestion, residual lipids and organic matter were treated with 10% H₂O₂. In brief, treated milk samples were combined with 3%, 10% or 30% H2O2 solution at ratio 1:1, 1:2, 1:3 (milk-to-H2O2). The mixture was left at room temperature for ~12 hours. In cases where lipid or protein digestion was incomplete following enzymatic or KOH treatments, a secondary oxidation step involving incubation at 40 °C – 50°C for 20 minutes was introduced (Shang et al. 2021; Enders et al. 2016). During this process, the mixture was stirred gently and heated until the solution became clear or faintly colored, indicating successful digestion of residual organic matter. Bubble formation occurred minimally, and cessation of bubbling indicated that the oxidation process has stopped. Positive and Negative Controls Negative controls consisted of three samples prepared using only ultra-pure distilled water (Milli-Q) without HBM. Positive controls were prepared by spiking HBM with known MPs; three nylon 66 (NY66) fragments (blue), three polyethylene terephthalate (PET) fragments (green), and three polypropylene (PP) fragments (pink) with the size of approximately around 1mm each. These polymers were selected based on their common presence in consumer packaging and have been previously detected in breast milk and food-contact plastics (Liu et al. 2023; Ragusa et al. 2022). Results and Discussion Lipid Digestion using Lipase from Candida rugosa The observations across different experimental conditions varied significantly. Among all tested milk-to-enzyme ratios, the most effective digestion was achieved at a 20:1 ratio, yielding the clearest sample with efficient lipid breakdown. In contrast, Sample L1 (1:1 ratio) exhibited poor lipid separation and remained turbid, while Sample L2 (2:1 ratio) showed slight improvement with reduced surface fat. Samples L3 to L5 (5:1 to 10:1 ratios) demonstrated increased clarity and lower viscosity, with Sample L3 presenting the least viscous appearance among them. Sample L6, processed at room temperature without incubation, showed no digestion, underscoring the importance of optimal enzymatic temperature. Interestingly, Sample L7, incubated for an extended period of seven days, developed a distinct oil layer, suggesting prolonged incubation continues lipid hydrolysis, consistent with known applications of Candida rugosa lipase in fat and oil modification (Anuar et al., 2015), albeit with diminishing returns. Lipase stability was confirmed as refrigerated enzymes stored for two weeks in Tests OL1 and OL2 still facilitated effective digestion. The spiked Sample “+” containing microplastics exhibited increased turbidity and sliminess following KOH treatment, indicating that undigested or partially digested organic matter can interfere with subsequent steps and complicate microplastic recovery. This observation further suggests that the enzymatic digestion procedure is more suitable for samples with relatively low microplastic burdens, such as human biological matrices, while it may be less effective in more complex environmental matrices with higher microplastic loads, such as river water or sediments. Overall, these results systematically document visual and physical changes in milk samples subjected to varying enzyme concentrations, incubation times, and enzyme storage conditions. Enzymatic digestion using Candida rugosa lipase was employed as an initial step to hydrolyze the abundant lipids in HBM and to evaluate its efficacy in breaking down other milk components. A significant challenge encountered was the lack of clear methodological guidance in the literature regarding optimal buffer composition, lipase concentration, and enzyme-to-milk volume ratios suitable for complex HBM matrices. Consequently, a trial-and-error approach was undertaken to optimize these parameters. Initial attempts using a one percent (w/v) lipase solution directly added to breast milk yielded no significant digestion, even after prolonged incubation, suggesting that the lipase requires a stabilizing medium to maintain catalytic activity (Anuar et al., 2015). Accordingly, phosphate buffer (pH 7.2) was selected for lipase solubilization due to its compatibility with enzyme stability and its capacity to maintain an optimal pH for Candida rugosa lipase activity. This finding aligns with prior research by Hansen et al. (2020), which demonstrated superior performance of liquid buffer formulations of Candida antarctica lipase B (CALB) compared to powdered or free enzyme forms. Liquid lipase formulations, particularly those combined with glycerol as a carrier in biphasic systems, have been noted to be more effective, economical, and practical. Therefore, preparation of Candida rugosa lipase in a buffered aqueous solution was essential for efficient lipid hydrolysis in human breast milk samples. A 5% (w/v) lipase solution was prepared by dissolving 5 g of lipase powder in 100 mL of distilled water. Given the lack of standardized protocols for lipid removal in HBM, the use of a 5% solution provided a feasible and effective starting point for preliminary trials and protocol optimization. The preparation process requires careful handling to avoid foaming, as the small volume made the solution susceptible to bubble formation that could affect the enzyme distribution. The working hypothesis initially assumed that a 1:1 ratio would yield optimal lipid degradation, however the improved clarity observed in Sample L2 compared to L1 revealed that both enzyme concentration and incubation time significantly influence lipid digestion efficiency. The absence of surface oil in Sample L1 suggested incomplete lipid hydrolysis or retention of emulsified lipids. This counterintuitive outcome may be explained by enzyme saturation or steric hindrance effects, where excessive enzyme concentrations lead to overcrowding of enzyme molecules at the oil-water interface, physically limiting substrate access to the catalytic sites (Hansen et al., 2020; Mhadmhan et al., 2024). Such steric hindrance has been reported to influence both the selectivity and reaction rates of lipase-catalyzed processes by modulating substrate accessibility to the enzyme’s active site (Mhadmhan et al., 2024). This supports the hypothesis that enzyme-substrate interactions depend not only on enzyme concentration but also on structural factors, highlighting the delicate balance required for optimal catalytic efficiency. In this study, the success of lipid digestion using Candida rugosa lipase was primarily assessed through direct visual observation and qualitative assessment of sample clarity (Table 1). A noticeable reduction in turbidity was one of the clearest indicators of effective lipid hydrolysis, occurring as emulsified triglycerides were cleaved into smaller, more water-soluble components such as free fatty acids and glycerol. These observations are consistent with previous reports demonstrating lipase activity predominantly at the oil–water interface (Salihu et al. 2011, das Neves et al. 2024). Similar findings in studies using immobilized lipase for dairy effluent treatment also demonstrated the successful degradation of lipids and further support the notion that interfacial enzyme activity effectively reduces turbidity and lipid content (das Neves et al. 2024). Table 1 Observation Under Different Ratios and Conditions Using Lipase Milk type Sample Milk vol. (mL) Lipase vol. (mL) Milk-to-lipase ratio KOH vol. (mL) KOH ratio Ethanol vol. (mL) Ethanol ratio H₂O₂ vol. (mL) H₂O₂ ratio H₂O₂ percentage Observation B L1 10 5 2:1 30 1: 3 0 - 20 1: 2 30% Greyish colour and high turbidity. Saponification stayed for 6 hours. B L2 10 2 5:1 30 1: 3 0 - 30 1: 3 30% Greyish colour and high turbidity. Slimy solution. A + 10 2 5:1 70 1: 7 100 1: 1 20 1: 2 3% Soft mucus texture was present. Cloudy solution turned less cloudy after ethanol was added. No changes in cloudiness after being left overnight at room temperature. A L3 10 1 10:1 30 1: 3 41 1 : 1 20 1: 2 3% Clear fat and liquid separation after overnight. Liquid appeared clear, lipid stayed at the bottom of the beaker but will mix with liquid if stirred. B L4 10 2 5:1 30 1: 3 42 1: 1 20 1: 2 3% Clear lipid and liquid separation after overnight. Liquid appeared clear, lipid stayed at the bottom of the beaker but would mix with liquid if stirred. Clearer than L3 and L4. A L5 10 5 2:1 30 1: 3 90 1: 2 20 1: 2 3% Clear lipid and liquid separation after overnight. Liquid appeared clear, lipid stayed at the bottom of the beaker but would mix with liquid if stirred. A L6 10 10 1: 1 60 1: 6 100 1: 1 20 1: 2 30% Thick fatty layer on the bottom of the beaker. B OL1 10 0.5 20:1 10 1: 1 20.5 1: 1 20 1: 2 3% No clear separation between lipids and liquid, lipids are solidified. B OL2 10 1 10:1 15 1: 3/2 26 1: 1 20 1: 2 3% Clear lipid and liquid separation after overnight. Liquid appeared clear, lipids stayed at the bottom of the beaker but would mix with liquid if stirred. B L7 10 0.5 20:1 30 1: 3 40.5 1: 1 20 1: 2 10% Clear lipid and liquid separation after overnight. Liquid appeared clear, lipid stayed at the bottom of the beaker but would mix with liquid if stirred. Showed the smallest amount of fat layer compared to L8, L9, and L10. B L8 10 1 10:1 30 1: 3 41 1: 1 20 1: 2 10% Clear lipid and liquid separation after overnight. Liquid appeared clear, lipid stayed at the bottom of the beaker but would mix with liquid if stirred. B L9 10 1.5 6:1 30 1: 3 41.5 1: 1 20 1: 2 10% Clear lipid and liquid separation after overnight. Liquid appeared clear, lipid stayed at the bottom of the beaker but would mix with liquid if stirred. B L10 10 2 5:1 30 1: 3 42 1: 1 20 1: 2 10% Clear lipid and liquid separation after overnight. Liquid appeared clear, lipid stayed at the bottom of the beaker but would mix with liquid if stirred. The spiked sample “+” which contained microplastics, showed incomplete digestion despite observable pH changes, suggesting that an increased lipase concentration might contribute additional organic load due to protein content from the enzyme itself. This excess organic matter may require more KOH and H₂O₂ during subsequent chemical digestion to achieve complete digestion. The slimy consistency observed after KOH treatment at 1:7 ratio supported this assumption, as soap-like gel resulting from saponification can likely hinder filtration (Dawson et al. 2020). The application of ethanol helped reduce this sliminess, supporting its use in dissolving saponified lipids and improving sample fluidity and filterability. Samples L3-L5 showed progressively improved results with optimized enzyme ration, with Sample L3 exhibiting the most consistent clarity and lower viscosity. The effective performance of old (stored) lipase samples (Samples OL1 and OL2) confirmed the stability of Candida rugosa lipase over 1 to 2 weeks of cold storage, aligning with the manufacturer’s data and previous findings by das Neves et al. (2024). Sample L7, which was incubated for seven days (one week), produced a distinct oil layer, thus demonstrated that sufficient time is vital for enzyme-substrate interaction in achieving complete lipid hydrolysis. Among all tested ratios, the 20:1 milk-to-enzyme ratio produced the most effective for lipid digestion, yielding the clearest and least viscous filtrate (Table 1). Comparative testing between milk types revealed that Milk Type A (high-fat content) was more difficult to filter due to higher lipid residue, which was resolved by mild heating and stirring to promote saponification and improve separation efficiency. This result highlights that matrix composition (milk consistency) and physical properties (viscosity) of the sample play a critical role in digestion success. In our case, controlled modifications significantly improved clarity and filtration efficiency. Collectively, these findings demonstrated that enzyme stability, preparation method, buffer environment, and incubation time are critical to successful digestion outcomes in complex biological matrices like HBM. However, as a limitation of current study, the optimization was conducted under a limited range of buffer conditions and enzyme concentrations. Therefore, future research should also focus on refining buffer composition, maintaining pH control, and optimizing enzyme-to-substrate ratios to further enhance lipid breakdown while preserving microplastic integrity. Role of KOH in Digestion of Organic Matters KOH was particularly useful in samples that exhibit poor lipid separation or persistent cloudiness following lipase digestion, especially in high-fat milk samples such as Milk Type A (Table 1). Its strong alkaline properties allow it to hydrolyze lipids and proteins by breaking ester and peptide bonds, which helps reduce turbidity and facilitate clarification of solutions. This effect was especially evident in samples where enzymatic digestion alone was insufficient to disintegrate the dense organic matrix or remove emulsified residues in the sample solution. However, the results revealed that increasing the KOH concentration beyond an optimal point did not necessarily improve digestion efficiency. In fact, higher KOH ratios such as 1:7, as seen in Sample “+”, led to increased turbidity and a slimy consistency, which interfered with subsequent processing and filtration. This observation is relatable with the findings by Salimon et al. (2011) who reported that ethanolic KOH at 1.75 M and 65 °C yielded optimal FFA release and clarity during Jatropha seed oil processing, while excessive concentration reduced efficiency. Similarly, in the current study of dairy digestion it showed that intermediate KOH ratios (1:3) provided the best balance between organic matter removal and reliable solution clarification. Whereas both lower and higher ratios, led to excessive turbidity and slimy residue due to incomplete or unstable digestion (Salimon et al. 2011). Although KOH effectively digested proteins, it was less efficient in removing residual lipids that had remained undigested from earlier enzymatic steps. The use of color changes as an indicator (from milky white to light or dark drown) was consistent with Maillard reaction, which served as a visual cue for successful protein breakdown (Tamanna et al. 2015; Kathuria et al. 2023; Lund & Ray 2017). This well-known chemical reaction between amino acids and reducing sugars produces brown pigmentation and aromatic compounds (Rani et al. 2023) and is widely recognized in food chemistry for giving distinct color and flavor (Tamanna et al. 2015). However, in the context of this study, the Maillard reaction served as a secondary confirmation of protein degradation, demonstrating that alkaline hydrolysis successfully altered the organic composition of the samples. Incubation at 45 °C for 4-5 hours appeared to be a critical parameter of digestion performance. The appearance of mucus-like residue and turbid suspension during stirring was likely due to incomplete digestion of complex organic matrices. However, once the sample settled, the appearance of a clear brown supernatant and white sediment suggested near-complete decomposition of soluble organic material, while insoluble particulates likely represented inorganic matter or residual microplastics. Moreover, the addition of ethanol played an important supporting role in reducing viscosity and enabling successful filtration particularly in samples with dense organic residue such as P1 to P3. Ethanol is known as co-solvent to reduce viscosity and coagulate proteins thus lowering the surface tension of fatty mixture and enhances the breakdown of the lipid residue, thus providing positive effect on filtration performance (Ferreira et al. 2019). Conversely, the use of KOH without heating, as in Sample L6 (1:6 ratio at room temperature), failed to achieve sufficient lipid digestion. This result reinforces the importance of both temperature and incubation time as essential variables for the success of chemical digestion via alkaline hydrolysis. Sample OL2’s treatment with a 1:1.5 ratio and the resulting Maillard reaction suggested that intermediate ratios may also be effective under optimal conditions. However, as this condition was not replicated, further testing is required before recommending it as a standardized parameter. Noteworthy, the observation that samples with shorter lipase incubation times correlated with poorer KOH digestion outcomes supports the idea that each step of the protocol is interdependent. Inadequate lipid removal during the early enzymatic phase likely will limit KOH access to the remaining proteins and lipids, resulting in incomplete digestion. H₂O₂ in the Oxidation Process of Remaining Organic Matter Although lipid and protein digestion are important for removing biological residues, the oxidation of remaining organic matter using peroxide such as H₂O₂ represents a critical final step for sample clarification. The oxidative strength of 30% H₂O₂ facilitates the breakdown of residual organic substances that persist after enzymatic or solvent-based digestion (Roslan et al. 2024; Fiore et al. 2024). Its widespread use in microplastic sample preparation has been attributed to its strong oxidizing potential and minimal impact on polymer integrity when used under controlled conditions (Rani et al. 2023). Studies such as Phofl et al. (2021) have also demonstrated that H 2 O 2 effectively degrades organic debris without significantly altering the polymeric structure of common plastics with only slight change of particle size distribution when using in combination of iron (II) catalyst (Fenton reagent), if exposure time and temperature are optimized (Pfohl et al. 2021). However, in samples with poor pre-treatment, particularly those with residual lipids or traces of hexane, the performance of H₂O₂ was notably reduced. This aligned with Sheriff et al. (2024), that emphasized that incomplete pre-treatment quality directly affects the efficacy of H₂O₂ in complex biological matrices. Moreover, Zhou et al. (2022) also noted that while H₂O₂ is effective, it requires optimal conditions such as temperature control, absence of interfering solvents, and adequate pre-digestion to achieve complete oxidation. For instance, in the present study, a secondary oxidation step involving incubation at 40°C for 20 mins proved beneficial, as it enhanced reaction kinetics and improved the visual clarity of the digestate. Although heating was initially approached with caution due to the presence of ethanol, mild heating up to 40°C was both safe and effective. Nevertheless, literature on the combined used of heated H₂O₂ and ethanol remains scarce, thus presents a potential safety concern particularly relating to peroxide-alcohol reactivity, and should be evaluated further before scaling up. To established cost-effective and safer oxidation protocol, trials with lower H₂O₂ concentrations (3% and 10%) were performed. Identifying the minimum effective concentration is crucial for enhancing cost-efficiency, minimizing safety risks and completing oxidation process. In Sample L1, passive oxidation using low-concentration peroxide achieved partial degradation but left a greyish tint and surface foam indicated incomplete oxidation. In contrast, Sample L2 showed that increasing H₂O₂ concentration alone did not guarantee improvement, since the presence of ethanol dilution or temperature variations also played significant contribution. Meanwhile, the use of 30% H₂O₂ at 50°C in L2 proved hazardous, as excessive foaming occurred within minutes, suggesting instability when heating high-concentration H₂O₂ in samples with organic matter. This supported the need to avoid aggressive heating of strong peroxide solutions (Pfohl et al. 2021). The introduction of 3% H₂O₂ in Sample ‘+’ demonstrated that lower concentrations can still achieve oxidation, though at a slower rate, indicating that peroxide strength directly influences the rate of organic matter degradation. As such, at lower concentrations, oxidation proceeds through gradual generation of hydroxyl radicals, resulting in a gentler but prolonged digestion process that preserves the surface morphology of microplastic particles (Tagg et al. 2017). The role of ethanol was further verified through comparison between Samples OL1 and OL2, where ethanol addition improved clarity even under identical oxidation protocols. Ethanol likely enhances miscibility between aqueous peroxide and hydrophobic residues, thus helps in reducing viscosity of organic suspensions for subsequent filtration. Centrifugation, however, did not significantly improve clarity in undigested samples, confirming that incomplete oxidation will limit phased separation regardless of applied centrifugal forces, and thus was not effective in improving clarity or filterability. This important observation highlights that chemical oxidation, rather than mechanical separation, remains the determining steps for achieving digestate transparency that are suitable for microplastic recover efficiency and accurate quantification. The shift to 10% H₂O₂ in later samples (L7–P3) established a practical balanced between oxidative strength and polymer integrity. The 10% H₂O₂ concentration accelerated oxidation more efficiently than 3% yet avoided the hazard risk and excessive foaming observed with 30% solution. This allowed integration with ethanol and mild heating, reducing oxidation time safely and effectively. Thus, a combination of a 1:2 sample-to-10% H₂O₂ ratio and controlled heating (<50 °C) provides a reliable, reproducible, and safe protocol for removing residual organic matter in HBM samples. Methodological Limitations Methodological limitations inherent to exploratory research with biological samples were encountered and should be considered for future studies. The variability in breast milk composition, including differences in lipid and protein content, influenced digestion efficiency and sample consistency. Mechanical handling presented occasional challenges, such as sample loss and risks of layer disturbance during lipid separation. The use of hexane for lipid extraction, while effective, sometimes interfered with subsequent reactions due to incomplete phase separation. Sample size was limited, reflecting both availability constraints and ethical considerations common in human milk research. Controls included reagent blanks but lacked procedural blanks to fully account for environmental contamination, which may explain detection of unexpected polymers in controls. Additionally, the absence of standardized protocols required empirical optimization of digestion conditions, potentially contributing to variability. Despite these factors, the study provides valuable preliminary insights and a flexible methodological foundation for future investigations into microplastic extraction from complex biological matrices like human breast milk. Conclusions As a conclusion, this study managed to explore a multi-step protocol for extracting and identifying MPs from human breast milk using a combination of hexane-based lipid extraction, enzymatic digestion with Candida rugosa lipase, KOH protein digestion, and H₂O₂ oxidation. Among the various conditions tested, the most effective combination was found to be a milk-to-enzyme ratio of 20:1, followed by KOH digestion at a 1:3 ratio and H₂O₂ oxidation at 10% concentration. Ethanol played a crucial role in reducing sample viscosity, especially after saponification, and facilitated effective filtration. The findings demonstrated that longer incubation periods, particularly for enzymatic digestion, significantly improved lipid removal, and downstream clarity. Nevertheless, this study provides a valuable foundation for future work in standardizing MP extraction from human samples. Going forward, improvements in mechanical handling and contamination control are recommended to enhance accuracy and reproducibility. Thus, this research contributes to the growing body of evidence on human exposure to MPs and highlights the need for reliable, scalable methods in environmental health monitoring. Declarations Participant Consent Statement: All participants, or their legal guardians, provided informed consent for the collection and use of human breast milk samples in this study. The study was conducted following approval by the IIUM Research Ethical Committee (IREC) (Approval ID: IREC 2025-058). The ethics committee confirmed that the procedures complied with the Declaration of Helsinki, ICH-GCP, Malaysian GCP, and CIOMS guidelines. All collected samples were anonymized to ensure donor confidentiality. Where applicable, the requirement for individual consent was waived by the approving ethics committee. Acknowledgements The authors expressed their gratitude to the Sultan Ahmad Shah Medical Centre @IIUM (SASMEC), Malaysia for granting the SASMEC Research Grant (ID: SRG25-167-0167). The open access funding was provided by the International Islamic University Malaysia. Data Availability All data supporting the findings of this study are available in the paper. Author Contributions Conceptualization: Norafiza Zainuddin, Hamizah Ismail; Methodology: Norafiza Zainuddin, Dhea Maisarah Ahmad Nasri, Fikriah Faudzi, Nur Rasyiqah Shamsul; Formal analysis and investigation: Dhea Maisarah Ahmad Nasri; Writing - original draft preparation: Dhea Maisarah Ahmad Nasri; Writing - review and editing: Norafiza Zainuddin, Dhea Maisarah Ahmad Nasri, Sabiqah Tuan Anuar, Muhammad Syafiq Musa, Mufti Petala Patria; Funding acquisition: Norafiza Zainuddin; Resources: Hamizah Ismail; Supervision: Norafiza Zainuddin, Fikriah Faudzi Conflict of interest Dhea Maisarah Ahmad Nasri, Nur Rasyiqah Syamsul, Fikriah Faudzi, Hamizah Ismail, Sabiqah Tuan Anuar, Muhammad Syafiq Musa, Mufti Petala Patria, Norafiza Zainuddin declare that they have no conflicts of interest. References Anuar ST, Mugo SM, Curtis JM (2015) A flow-through enzymatic microreactor for the rapid conversion of triacylglycerols into fatty acid ethyl ester and fatty acid methyl ester derivatives for GC analysis. Analytical Methods 7:5898–5906. doi:10.1039/c5ay00800j das Neves AM, Visioli LJ, Enzweiler H, Paulino AT (2024) Lipase from Candida rugosa incorporated in pectin hydrogel via immobilization for hydrolysis of lipids in dairy effluents and production of fatty acids. Journal of Water Process Engineering 58:104821. doi:10.1016/j.jwpe.2024.104821 Dawson AL, Motti CA, Kroon FJ (2020) Solving a sticky situation: Microplastic analysis of lipid-rich tissue. Frontiers in Environmental Science 8:563565. doi:10.3389/fenvs.2020.563565 Di Fiore C, Ishikawa Y, Wright S (2024) A review on methods for extracting and quantifying microplastic in biological tissues. Journal of Hazardous Materials 464:132991. doi:10.1016/j.jhazmat.2023.132991 Dzierżyński E, Gawlik PJ, Puźniak D, Flieger W, Jóźwik K, Teresiński G, Forma A, Wdowiak P, Baj J, Flieger J (2024) Microplastics in the human body: Exposure, detection, and risk of carcinogenesis: A state-of-the-art review. Cancers 16:3703. doi:10.3390/cancers16213703 Enders K, Lenz R, Beer S, Stedmon CA (2016) Extraction of microplastic from biota: Recommended acidic digestion destroys common plastic polymers. ICES Journal of Marine Science 74:326–331. doi:10.1093/icesjms/fsw173 Ferreira AC, Sullo A, Winston S, Norton IT, Norton-Welch AB (2019) Influence of ethanol on emulsions stabilized by low molecular weight surfactants. Journal of Food Science 85:28–35. doi:10.1111/1750-3841.14947 Hansen RB, Agerbaek MA, Nielsen PM, Rancke-Madsen A, Woodley JM (2020) Esterification using a liquid lipase to remove residual free fatty acids in biodiesel. Process Biochemistry 97:213–221. doi:10.1016/j.procbio.2020.06.005 Kathuria D, Hamid, Sunakshi G, Thakur A (2023) Maillard reaction in different food products: Effect on product quality, human health and mitigation strategies. Food Control 153:109911. doi:10.1016/j.foodcont.2023.109911 Kim SY, Yi DY (2020) Components of human breast milk: From macronutrient to microbiome and microRNA. Clinical and Experimental Pediatrics 63:301–309. doi:10.3345/cep.2020.00059 Kutralam-Muniasamy G, Shruti VC, Pérez-Guevara F, Roy PD (2023) Microplastic diagnostics in humans: "The 3Ps" progress, problems, and prospects. Science of The Total Environment 856:159164. doi:10.1016/J.SCITOTENV.2022.159164 Lakshmayya NSV, Panday A, Yadavalli R, Reddy CN, Mandal SK, Agrawal DC, Mishra B (2023) Food Contamination with Micro-plastics: Occurrences, Bioavailability, Human Vulnerability, and Prevention. Current Nutrition & Food Science 20:797–810. doi:10.2174/1573401319666230915164116 Liu L, Zhang X, Jia P, He S, Dai H, Deng S, Han J (2023) Release of microplastics from breastmilk storage bags and assessment of intake by infants: A preliminary study. Environmental Pollution 323:121197. doi:10.1016/j.envpol.2023.121197 Lund MN, Ray CA (2017) Control of Maillard reactions in foods: Strategies and chemical mechanisms. Journal of Agricultural and Food Chemistry 65:4537–4552. doi:10.1021/acs.jafc.7b00882 Mhadmhan S, Yoosuk B, Henpraserttae S (2024) Selective lipase-catalyzed hydrolysis for removal of diglyceride in palm oil. Separation and Purification Technology 349:127897. doi:10.1016/j.seppur.2024.127897 Mišľanová C, Valachovičová M, Slezáková Z (2024) An overview of the possible exposure of infants to microplastics. Life (Basel, Switzerland) 14:371. doi:10.3390/life14030371 Nawab A, Ahmad M, Khan MT, Nafees M, Khan I, Ihsanullah I (2024) Human exposure to microplastics: A review on exposure routes and public health impacts. Journal of Hazardous Materials Advances 16:100487. doi:10.1016/j.hazadv.2024.100487 Pfohl P, Roth C, Meyer L, et al (2021) Microplastic extraction protocols can impact the polymer structure. Microplastics and Nanoplastics 1:8. doi:10.1186/s43591-021-00009-9 Ragusa A, Notarstefano V, Svelato A, Belloni A, Gioacchini G, Blondeel C, Zucchelli E, De Luca C, D’Avino S, Gulotta A, Carnevali O, Giorgini E (2022) Raman microspectroscopy detection and characterisation of microplastics in human breastmilk. Polymers 14:2700. doi:10.3390/polym14132700 Rani M, Ducoli S, Depero LE, Prica M, Tubić A, Ademovic Z, Morrison L, Federici S (2023) A complete guide to extraction methods of microplastics from complex environmental matrices. Molecules 28:5710. doi:10.3390/molecules28155710 Roslan NS, Lee YY, Ibrahim YS, Tuan Anuar S, Yusof KMKK, Lai LA, Brentnall T (2024) Detection of microplastics in human tissues and organs: A scoping review. Journal of Global Health 14:04179. doi:10.7189/jogh.14.04179 Salihu A, Alam MZ, Abdulkarim MI, Salleh HM (2011) Optimization of lipase production by Candida cylindracea in palm oil mill effluent based medium using statistical experimental design. Journal of Molecular Catalysis B: Enzymatic 69:66–73. doi:10.1016/j.molcatb.2010.12.012 Salimon J, Abdullah BM, Salih N (2011) Hydrolysis optimization and characterization study of preparing fatty acids from Jatropha curcas seed oil. Chemistry Central Journal 5:67. doi:10.1186/1752-153X-5-67 Shang Y, Wang X, Chang X, Sokolova IM, Wei S, Liu W, Fang JC, Hu M, Huang W, Wang Y (2021) The effect of microplastics on the bioenergetics of the mussel Mytilus coruscus assessed by cellular energy allocation approach. Frontiers in Marine Science 8:754789. doi:10.3389/fmars.2021.754789 Sheriff I, Awang NA, Halim H, Ikechukwu OS, Jusoh AF (2024) Extraction and analytical methods of microplastics in wastewater treatment plants: Isolation patterns, quantification, and size characterization techniques. Desalination and Water Treatment 100399–100399. doi:10.1016/j.dwt.2024.100399 Tagg AS, Harrison JP, Ju-Nam Y, Sapp M, Bradley EL, Sinclair CJ, Ojeda JJ (2017) Fenton’s reagent for the rapid and efficient isolation of microplastics from wastewater. Chemical Communications 53:372–375. doi:10.1039/c6cc08798a Tamanna N, Mahmood N (2015) Food processing and Maillard reaction products: Effect on human health and nutrition. International Journal of Food Science 2015:1–6. doi:10.1155/2015/526762 Zhou Q, Chen J, Zhang D, Pan X (2022) Evaluation of organic matter removal by H2O2 from microplastic surface by nano-physicochemical methods. Green Analytical Chemistry 3:100035. doi:10.1016/j.greeac.2022.100035 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 02 Mar, 2026 Reviewers invited by journal 23 Feb, 2026 Editor assigned by journal 20 Nov, 2025 Submission checks completed at journal 20 Nov, 2025 First submitted to journal 19 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8153566","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":548081021,"identity":"21caefef-0eb8-401d-b1d6-8d9395838ca5","order_by":0,"name":"Dhea Maisarah Ahmad Nasri","email":"","orcid":"","institution":"International Islamic University Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Dhea","middleName":"Maisarah Ahmad","lastName":"Nasri","suffix":""},{"id":548081022,"identity":"d023de25-9a2f-438d-9e92-ba25cf8368f1","order_by":1,"name":"Nur Rasyiqah Syamsul","email":"","orcid":"","institution":"International Islamic University Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Nur","middleName":"Rasyiqah","lastName":"Syamsul","suffix":""},{"id":548081023,"identity":"4cdc682c-82b6-44bc-b031-bb3fd73818f9","order_by":2,"name":"Fikriah Faudzi","email":"","orcid":"","institution":"International Islamic University Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Fikriah","middleName":"","lastName":"Faudzi","suffix":""},{"id":548081024,"identity":"de614e5a-5d99-4521-a900-004ebd5627c6","order_by":3,"name":"Hamizah Ismail","email":"","orcid":"","institution":"Sultan Ahmad Shah Medical Centre @IIUM (SASMEC)","correspondingAuthor":false,"prefix":"","firstName":"Hamizah","middleName":"","lastName":"Ismail","suffix":""},{"id":548081025,"identity":"696d027b-26e9-4594-8cfe-c4e4d86edf85","order_by":4,"name":"Sabiqah Tuan Anuar","email":"","orcid":"","institution":"Universiti Malaysia Terengganu","correspondingAuthor":false,"prefix":"","firstName":"Sabiqah","middleName":"Tuan","lastName":"Anuar","suffix":""},{"id":548081026,"identity":"345623eb-7844-4a91-9703-8dd1c559e6a6","order_by":5,"name":"Muhammad Syafiq Musa","email":"","orcid":"","institution":"National University of Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Syafiq","lastName":"Musa","suffix":""},{"id":548081027,"identity":"66ee3303-b9f4-4a72-9d7f-5a34e484946b","order_by":6,"name":"Mufti Petala Patria","email":"","orcid":"","institution":"Universitas Indonesia","correspondingAuthor":false,"prefix":"","firstName":"Mufti","middleName":"Petala","lastName":"Patria","suffix":""},{"id":548081028,"identity":"a6c22785-4ebb-4d82-b420-bde91ba143f5","order_by":7,"name":"Norafiza Zainuddin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIie3OsQqCUBSA4XuTbLnWeoLIV1BaGgRf5YjQ5DOUETT1AEn1DkHgnAi6+ABKU7QWODo0dJG24lpbxP3hDGf4OIcQmexns4CQztw/1svxIzLhhEXfET6Az6WJ6JtFUlY4tnvBZR5rHhl2c2wfmIDQbeIGKwQFTo4fayEZ9XNUTyKigDciWgUqeRJn30RUTugdgelFVJNZI2GcKAwBjJzWBI0mAjBxlQGCYWaOH+1CMIPsvCy2AqKv3ZhecWoP0/RS3kJL76Zukl8F5OUqH+q3RI+9j1ZfE5lMJvvjHpERTPeMz7vYAAAAAElFTkSuQmCC","orcid":"","institution":"International Islamic University Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Norafiza","middleName":"","lastName":"Zainuddin","suffix":""}],"badges":[],"createdAt":"2025-11-19 09:38:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8153566/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8153566/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97315185,"identity":"e0223544-0901-4810-9cd8-28d85959576b","added_by":"auto","created_at":"2025-12-03 06:33:27","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":49210,"visible":true,"origin":"","legend":"","description":"","filename":"AhmadNasrietal.2025ChemicalPapers.docx","url":"https://assets-eu.researchsquare.com/files/rs-8153566/v1/5d8e7d0cfa607229b2cddfe3.docx"},{"id":97315181,"identity":"a9840977-0de0-433f-9b8b-604e3748ac92","added_by":"auto","created_at":"2025-12-03 06:33:25","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9309,"visible":true,"origin":"","legend":"","description":"","filename":"43af0822efac49048b9851892e65160d.json","url":"https://assets-eu.researchsquare.com/files/rs-8153566/v1/184005a1fb8e15f6c07569f2.json"},{"id":97369476,"identity":"778382d9-b79f-40d6-aa6f-3e92c8cd88fa","added_by":"auto","created_at":"2025-12-03 16:25:01","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109360,"visible":true,"origin":"","legend":"","description":"","filename":"43af0822efac49048b9851892e65160d1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8153566/v1/81a4250998483c1ba555f7d0.xml"},{"id":97315182,"identity":"bbd9a877-4974-4770-b258-97c9ecf6e479","added_by":"auto","created_at":"2025-12-03 06:33:25","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":106211,"visible":true,"origin":"","legend":"","description":"","filename":"43af0822efac49048b9851892e65160d1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8153566/v1/286e5b801b818ecd26306aff.xml"},{"id":97315183,"identity":"c4b6c455-3645-4bf2-a6e6-4ee11d9581ef","added_by":"auto","created_at":"2025-12-03 06:33:25","extension":"html","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114620,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8153566/v1/d19d4ef85ba358526b17314e.html"},{"id":97664586,"identity":"a57c46dc-2910-493e-92da-d5607a7f79c1","added_by":"auto","created_at":"2025-12-08 09:10:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":891216,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8153566/v1/15f2bc17-ec40-4888-b9d4-bc38886816ba.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":" Optimizing Lipid Removal and Protein Digestion in Human Milk for Microplastic Analysis using Candida rugosa Lipase, KOH Digestion, and Hydrogen Peroxide Oxidation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicroplastics (MPs), defined as plastic particles smaller than 5 mm, have emerged as pervasive environmental contaminants with global ecological and human health implications. They were widely detected in diverse ecosystems and increasingly within the human body (Lakshmayya et al. 2023). MPs originate either from the degradation of larger plastic debris, synthetic textile fibers, or from intentionally manufactured microbeads and resins used in consumer products. Their small size facilitates the bioavailability and accumulation in multiple organs including the lungs, liver, kidneys, gastrointestinal tract, and placenta, thus raising concerns about potential health risks (Roslan et al. 2024). Although long-term effects and chronic toxicity associated with MPs are not yet fully understood, recent evidence suggests that their presence in tissues and fluids may contribute to inflammation, oxidative stress, and endocrine disruption through both physical effects from particle (shape and size) and/or chemical leaching (Nawab et al. 2024). Particularly, a recent report confirming the occurrence of MPs in human breast milk (HBM) has raise pressing concerns regarding early-life (neonatal) exposure, since HBM is not only a critical source of nutrition and bioactive compounds for the immune system and growth of infants (Ragusa et al. 2022). Moreover, MPs may adsorb and transport hazardous chemicals such as perfluoroalkyl substances (PFAS), phthalates, and other organic pollutants, which raising potential consequence for infant health (Dzierżyński et al. 2024; Mi\u0026scaron;ľanov\u0026aacute; et al. 2024). Therefore, the presence of MPs in HBM highlights an urgent need for more robust and standardized analytical protocol that capable of detecting and quantifying MPs in complex biological matrices.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, accurate detection of MPs in HBM is practically challenging and hindered due to its complex composition. Content of HBM is majorly well known with the composition of around 87%\u0026ndash;88% water and 124 g/L of solid macronutrients, which include approximately 7% (60\u0026ndash;70 g/L) carbohydrates, 1% (8\u0026ndash;10 g/L) protein, and 3.8% (35\u0026ndash;40 g/L) fat (Kim \u0026amp; Yi 2020). Protein and carbohydrates contents can interfere with the recovery, isolation, identification, and quantification of MPs (Kutralam-Muniasamy 2022). Standard digestion methods for biological samples, including alkaline, acid, or enzymatic treatment followed by oxidative degradation have been individually employed with varying level of success in the biota sample recovery (Rani et al. 2023; Roslan et al. 2024). However, their combination is insufficiently evaluated and its effectiveness in the extraction of MPs in HBM remains limited, and incomplete removal of fats and proteins will result in matrix interferences during further microscopic and spectroscopic identification (Rani et al. 2023). Therefore, the optimization of a multi-step digestion protocol that ensures efficient lipid removal, protein degradation and minimal polymer damage is essential for reliable microplastic quantification in HBM. This study proposes an integrated workflow combining hexane-based lipid extraction, enzymatic digestion using \u003cem\u003eCandida rugosa\u003c/em\u003e lipase, alkaline digestion with potassium hydroxide (KOH) and oxidative digestion with hydrogen peroxide (H₂O₂) treatments of HBM prior to physical analysis of MPs. The objective is to evaluate and optimize the synergistic application of these aims to optimize these treatments to enhance substrate clarity (i.e. by enhancing lipid removal and organic matter degradation), improve MPs recovery rates and minimize artefacts during identification. This is critical toward developing a reproducible and validated protocol for microplastics in complex human fluids.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cp\u003e\u003cem\u003eMaterials\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSamples\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was ethically approved on 22\u003csup\u003end\u003c/sup\u003e March 2025 by the IIUM Research Ethical Committee (IREC), Malaysia in accordance with the principles outlined in the Declaration of Helsinki, the International Conference on Harmonisation - Good Clinical Practice (ICH-GCP), the Malaysian Guidelines for Good Clinical Practice (Malaysia GCP), and the Council for International Organizations of Medical Sciences (CIOMS) International Ethical Guidelines (Approval ID: IREC 2025-058). Donated human breast milk (HBM) samples were obtained from the HMMC, Malaysia, with collected HBM sample were anonymised to ensure confidentiality.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChemicals\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe key chemicals and reagents used in this study included hexane (HMBG Chemicals, Germany), 30% H₂O₂ solution (HMBG Chemicals, Germany), 70% ethanol (HMBG Chemicals, Germany), lipase Type VII from \u003cem\u003eCandida rugosa\u003c/em\u003e with an activity of 700 units/mg solid (Sigma-Aldrich, United States), pH 7.2 buffer tablets (Sigma-Aldrich, United States), KOH pellets (Sigma-Aldrich, United States), and ultra-pure distilled water (milli-Q). All chemical and reagents used are analytical grade. The \u003cem\u003eCandida rugosa\u003c/em\u003e lipase was stored at 2\u0026ndash;4 \u0026deg;C prior to use, as recommended by the manufacturer, while other chemicals such as hexane, H₂O₂, and KOH were stored at room temperature in designated chemical safety cabinets.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMilk Processing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDonated HBM samples were handled according to the standard procedures employed by the milk bank and stored at \u0026minus;20\u0026deg;C to preserve their quality. Upon thawing, samples displayed distinct physical differences were noted and were categorized into two groups: (1) Milk Type A (high-fat content) characterized by a thicker, creamier consistency with visible lipid clumps; and (2) Milk Type B (low-fat content) due to its diluted consistency. This classification was necessary for assessing digestion performance across different fat levels.\u003c/p\u003e\n\u003cp\u003eA structured naming convention was applied for sample tracking. For lipase digestion, tubes were labelled L1 to L10, with one sample (\u0026ldquo;+\u0026rdquo;) was intentionally spiked with microplastics (MPs) to evaluate whether enzymatic digestion affected MP integrity. Meanwhile, OL1 and OL2 referred to samples treated with lipase stored for two weeks under refrigeration. Additional sample groups included S1 \u0026ndash; S3 (test samples), N1\u0026ndash; N3 (negative controls using Milli-Q water), and P1\u0026ndash; P3 (positive controls spiked with known MPs: Nylon-66, PET, and PP). This labelling system was applied consistently and systematically throughout the digestion and oxidation to maintain clarity, minimize procedural errors, and improve reproducibility in data interpretation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLipase Solution Preparation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe buffer solution was prepared by dissolving one tablet of phosphate buffer (pH 7.2) in 1 L of distilled water, mixing with a glass rod and kept at 4oC in the refrigerator until use. A 5% lipase solution was prepared by weighing 0.5 g of lipase powder and gently mixing it with 10 mL of cold phosphate buffer solution. The prepared lipase solution was then refrigerated until use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLipid Digestion using Lipase from Candida rugosa\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor each sample, 10 mL of HBM was used. The HBM sample was preheated to 37 \u0026deg;C in a water bath. Once the milk reached the optimum temperature, the lipase solution was added at various milk-to-enzyme ratios (1:1, 2:1, 5:1, 10:1, and 20:1). The resulting mixtures were then incubated at 37 \u0026deg;C \u0026ndash; 37.4 \u0026deg;C for a minimum of 2 days, with extended incubation period up to 7 days for selected samples to assess the procedural variation. The pH of each sample was measured before and after digestion procedure, with a post-digestion pH value of approximately 4 indicated successful lipid hydrolysis and digestion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eProtein Digestion using KOH\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the enzymatic lipid digestion step, protein digestion was conducted using a KOH solution to further degrade the protein and organic matrices of HBM. A 10% (w/v) KOH solution was prepared by dissolving 50 g of KOH pellets in distilled water and diluting to a final volume of 500 mL. For each 10 mL milk sample, the HBM-to-KOH ratio was adjusted stepwise at 1:1 or 1:1.5, 1:3 and 1:7. Samples were then mixed gently and incubated at 40 \u0026ndash; 45\u0026deg;C for 2 \u0026ndash; 4 hours to facilitate protein denaturation and solubilization. The reaction was carried out in a beaker closed with aluminium foil to allow pressure release while minimizing airborne contamination. A color change from a cloudy white suspension to dark brown solution indicated progressive protein degradation. The appearance of turbidity or curd formation was monitored visually throughout the progress. Samples that remained highly viscous or slimy were further treated with an additional 70% ethanol in a 1:1 ratio (sample: ethanol) to aid in the disruption of emulsions and reduce filtering resistance. Following digestion, all samples were allowed to cool to room temperature before proceeding to the oxidation step or filtration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDigestion of Residual Lipids and Remaining Organic Matters using H₂O₂\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter protein digestion, residual lipids and organic matter were treated with 10% H₂O₂. In brief, treated milk samples were combined with 3%, 10% or 30% H2O2 solution at ratio 1:1, 1:2, 1:3 (milk-to-H2O2). The mixture was left at room temperature for ~12 hours. In cases where lipid or protein digestion was incomplete following enzymatic or KOH treatments, a secondary oxidation step involving incubation at 40 \u0026deg;C \u0026ndash; 50\u0026deg;C for 20 minutes was introduced (Shang et al. 2021; Enders et al. 2016). During this process, the mixture was stirred gently and heated until the solution became clear or faintly colored, indicating successful digestion of residual organic matter. Bubble formation occurred minimally, and cessation of bubbling indicated that the oxidation process has stopped.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePositive and Negative Controls\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNegative controls consisted of three samples prepared using only ultra-pure distilled water (Milli-Q) without HBM. Positive controls were prepared by spiking HBM with known MPs; three nylon 66 (NY66) fragments (blue), three polyethylene terephthalate (PET) fragments (green), and three polypropylene (PP) fragments (pink) with the size of approximately around 1mm each. These polymers were selected based on their common presence in consumer packaging and have been previously detected in breast milk and food-contact plastics (Liu et al. 2023; Ragusa et al. 2022).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLipid Digestion using Lipase from Candida rugosa\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe observations across different experimental conditions varied significantly. Among all tested milk-to-enzyme ratios, the most effective digestion was achieved at a 20:1 ratio, yielding the clearest sample with efficient lipid breakdown. In contrast, Sample L1 (1:1 ratio) exhibited poor lipid separation and remained turbid, while Sample L2 (2:1 ratio) showed slight improvement with reduced surface fat. Samples L3 to L5 (5:1 to 10:1 ratios) demonstrated increased clarity and lower viscosity, with Sample L3 presenting the least viscous appearance among them. Sample L6, processed at room temperature without incubation, showed no digestion, underscoring the importance of optimal enzymatic temperature. Interestingly, Sample L7, incubated for an extended period of seven days, developed a distinct oil layer, suggesting prolonged incubation continues lipid hydrolysis, consistent with known applications of \u003cem\u003eCandida rugosa\u003c/em\u003e lipase in fat and oil modification (Anuar et al., 2015), albeit with diminishing returns. Lipase stability was confirmed as refrigerated enzymes stored for two weeks in Tests OL1 and OL2 still facilitated effective digestion. The spiked Sample \u0026ldquo;+\u0026rdquo; containing microplastics exhibited increased turbidity and sliminess following KOH treatment, indicating that undigested or partially digested organic matter can interfere with subsequent steps and complicate microplastic recovery. This observation further suggests that the enzymatic digestion procedure is more suitable for samples with relatively low microplastic burdens, such as human biological matrices, while it may be less effective in more complex environmental matrices with higher microplastic loads, such as river water or sediments. Overall, these results systematically document visual and physical changes in milk samples subjected to varying enzyme concentrations, incubation times, and enzyme storage conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEnzymatic digestion using \u003cem\u003eCandida rugosa\u003c/em\u003e lipase was employed as an initial step to hydrolyze the abundant lipids in HBM and to evaluate its efficacy in breaking down other milk components. A significant challenge encountered was the lack of clear methodological guidance in the literature regarding optimal buffer composition, lipase concentration, and enzyme-to-milk volume ratios suitable for complex HBM matrices. Consequently, a trial-and-error approach was undertaken to optimize these parameters. Initial attempts using a one percent (w/v) lipase solution directly added to breast milk yielded no significant digestion, even after prolonged incubation, suggesting that the lipase requires a stabilizing medium to maintain catalytic activity (Anuar et al., 2015). Accordingly, phosphate buffer (pH 7.2) was selected for lipase solubilization due to its compatibility with enzyme stability and its capacity to maintain an optimal pH for \u003cem\u003eCandida rugosa\u003c/em\u003e lipase activity. This finding aligns with prior research by Hansen et al. (2020), which demonstrated superior performance of liquid buffer formulations of \u003cem\u003eCandida antarctica\u003c/em\u003e lipase B (CALB) compared to powdered or free enzyme forms. Liquid lipase formulations, particularly those combined with glycerol as a carrier in biphasic systems, have been noted to be more effective, economical, and practical. Therefore, preparation of \u003cem\u003eCandida rugosa\u003c/em\u003e lipase in a buffered aqueous solution was essential for efficient lipid hydrolysis in human breast milk samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA 5% (w/v) lipase solution was prepared by dissolving 5 g of lipase powder in 100 mL of distilled water. Given the lack of standardized protocols for lipid removal in HBM, the use of a 5% solution provided a feasible and effective starting point for preliminary trials and protocol optimization. The preparation process requires careful handling to avoid foaming, as the small volume made the solution susceptible to bubble formation that could affect the enzyme distribution. The working hypothesis initially assumed that a 1:1 ratio would yield optimal lipid degradation, however the improved clarity observed in Sample L2 compared to L1 revealed that both enzyme concentration and incubation time significantly influence lipid digestion efficiency. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe absence of surface oil in Sample L1 suggested incomplete lipid hydrolysis or retention of emulsified lipids. This counterintuitive outcome may be explained by enzyme saturation or steric hindrance effects, where excessive enzyme concentrations lead to overcrowding of enzyme molecules at the oil-water interface, physically limiting substrate access to the catalytic sites (Hansen et al., 2020; Mhadmhan et al., 2024). Such steric hindrance has been reported to influence both the selectivity and reaction rates of lipase-catalyzed processes by modulating substrate accessibility to the enzyme\u0026rsquo;s active site (Mhadmhan et al., 2024). This supports the hypothesis that enzyme-substrate interactions depend not only on enzyme concentration but also on structural factors, highlighting the delicate balance required for optimal catalytic efficiency.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, the success of lipid digestion using \u003cem\u003eCandida rugosa\u003c/em\u003e lipase was primarily assessed through direct visual observation and qualitative assessment of sample clarity (Table 1). A noticeable reduction in turbidity was one of the clearest indicators of effective lipid hydrolysis, occurring as emulsified triglycerides were cleaved into smaller, more water-soluble components such as free fatty acids and glycerol. These observations are consistent with previous reports demonstrating lipase activity predominantly at the oil\u0026ndash;water interface (Salihu et al. 2011, das Neves et al. 2024). Similar findings in studies using immobilized lipase for dairy effluent treatment also demonstrated the successful degradation of lipids and further support the notion that interfacial enzyme activity effectively reduces turbidity and lipid content (das Neves et al. 2024). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Observation Under Different Ratios and Conditions Using Lipase\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMilk type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMilk vol. (mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLipase vol. (mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMilk-to-lipase ratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKOH vol. (mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKOH ratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEthanol vol. (mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEthanol ratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eH₂O₂ vol. (mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eH₂O₂ ratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eH₂O₂ percentage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eObservation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eL1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGreyish colour and high turbidity. Saponification stayed for 6 hours.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eL2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5:1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGreyish colour and high turbidity. Slimy solution.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5:1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e70\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 7\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSoft mucus texture was present. Cloudy solution turned less cloudy after ethanol was added. No changes in cloudiness after being left overnight at room temperature.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eL3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10:1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1 : 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eClear fat and liquid separation after overnight. Liquid appeared clear, lipid stayed at the bottom of the beaker but will mix with liquid if stirred.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eL4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5:1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eClear lipid and liquid separation after overnight. Liquid appeared clear, lipid stayed at the bottom of the beaker but would mix with liquid if stirred. Clearer than L3 and L4.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eL5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2:1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eClear lipid and liquid separation after overnight. Liquid appeared clear, lipid stayed at the bottom of the beaker but would mix with liquid if stirred.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eL6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eThick fatty layer on the bottom of the beaker.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOL1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNo clear separation between lipids and liquid, lipids are solidified.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10:1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e15\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 3/2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e26\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eClear lipid and liquid separation after overnight. Liquid appeared clear, lipids stayed at the bottom of the beaker but would mix with liquid if stirred.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eL7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20:1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e40.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eClear lipid and liquid separation after overnight. Liquid appeared clear, lipid stayed at the bottom of the beaker but would mix with liquid if stirred. Showed the smallest amount of fat layer compared to L8, L9, and L10.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eL8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10:1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eClear lipid and liquid separation after overnight. Liquid appeared clear, lipid stayed at the bottom of the beaker but would mix with liquid if stirred.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eL9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e41.5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eClear lipid and liquid separation after overnight. Liquid appeared clear, lipid stayed at the bottom of the beaker but would mix with liquid if stirred.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eL10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5:1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e42\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1: 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eClear lipid and liquid separation after overnight. Liquid appeared clear, lipid stayed at the bottom of the beaker but would mix with liquid if stirred.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe spiked sample \u0026ldquo;+\u0026rdquo; which contained microplastics, showed incomplete digestion despite observable pH changes, suggesting that an increased lipase concentration might contribute additional organic load due to protein content from the enzyme itself. This excess organic matter may require more KOH and H₂O₂ during subsequent chemical digestion to achieve complete digestion. The slimy consistency observed after KOH treatment at 1:7 ratio supported this assumption, as soap-like gel resulting from saponification can likely hinder filtration (Dawson et al. 2020). The application of ethanol helped reduce this sliminess, supporting its use in dissolving saponified lipids and improving sample fluidity and filterability.\u003c/p\u003e\n\u003cp\u003eSamples L3-L5 showed progressively improved results with optimized enzyme ration, with Sample L3 exhibiting the most consistent clarity and lower viscosity. The effective performance of old (stored) lipase samples (Samples OL1 and OL2) confirmed the stability of \u003cem\u003eCandida rugosa\u003c/em\u003e lipase over 1 to 2 weeks of cold storage, aligning with the manufacturer\u0026rsquo;s data and previous findings by das Neves et al. (2024). Sample L7, which was incubated for seven days (one week), produced a distinct oil layer, thus demonstrated that sufficient time is vital for enzyme-substrate interaction in achieving complete lipid hydrolysis. Among all tested ratios, the 20:1 milk-to-enzyme ratio produced the most effective for lipid digestion, yielding the clearest and least viscous filtrate (Table 1).\u003c/p\u003e\n\u003cp\u003eComparative testing between milk types revealed that Milk Type A (high-fat content) was more difficult to filter due to higher lipid residue, which was resolved by mild heating and stirring to promote saponification and improve separation efficiency. This result highlights that matrix composition (milk consistency) and physical properties (viscosity) of the sample play a critical role in digestion success. In our case, controlled modifications significantly improved clarity and filtration efficiency. Collectively, these findings demonstrated that enzyme stability, preparation method, buffer environment, and incubation time are critical to successful digestion outcomes in complex biological matrices like HBM. However, as a limitation of current study, the optimization was conducted under a limited range of buffer conditions and enzyme concentrations. Therefore, future research should also focus on refining buffer composition, maintaining pH control, and optimizing enzyme-to-substrate ratios to further enhance lipid breakdown while preserving microplastic integrity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRole of KOH in Digestion of Organic Matters\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKOH was particularly useful in samples that exhibit poor lipid separation or persistent cloudiness following lipase digestion, especially in high-fat milk samples such as Milk Type A (Table 1). Its strong alkaline properties allow it to hydrolyze lipids and proteins by breaking ester and peptide bonds, which helps reduce turbidity and facilitate clarification of solutions. This effect was especially evident in samples where enzymatic digestion alone was insufficient to disintegrate the dense organic matrix or remove emulsified residues in the sample solution.\u003c/p\u003e\n\u003cp\u003eHowever, the results revealed that increasing the KOH concentration beyond an optimal point did not necessarily improve digestion efficiency. In fact, higher KOH ratios such as 1:7, as seen in Sample \u0026ldquo;+\u0026rdquo;, led to increased turbidity and a slimy consistency, which interfered with subsequent processing and filtration. This observation is relatable with the findings by Salimon et al. (2011) who reported that ethanolic KOH at 1.75 M and 65 \u0026deg;C yielded optimal FFA release and clarity during Jatropha seed oil processing, while excessive concentration reduced efficiency. Similarly, in the current study of dairy digestion it showed that intermediate KOH ratios (1:3) provided the best balance between organic matter removal and reliable solution clarification. Whereas both lower and higher ratios, led to excessive turbidity and slimy residue due to incomplete or unstable digestion (Salimon et al. 2011).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough KOH effectively digested proteins, it was less efficient in removing residual lipids that had remained undigested from earlier enzymatic steps. The use of color changes as an indicator (from milky white to light or dark drown) was consistent with Maillard reaction, which served as a visual cue for successful protein breakdown (Tamanna et al. 2015; Kathuria et al. 2023; Lund \u0026amp; Ray 2017). This well-known chemical reaction between amino acids and reducing sugars produces brown pigmentation and aromatic compounds (Rani et al. 2023) and is widely recognized in food chemistry for giving distinct color and flavor (Tamanna et al. 2015). However, in the context of this study, the Maillard reaction served as a secondary confirmation of protein degradation, demonstrating that alkaline hydrolysis successfully altered the organic composition of the samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIncubation at 45 \u0026deg;C for 4-5 hours appeared to be a critical parameter of digestion performance. The appearance of mucus-like residue and turbid suspension during stirring was likely due to incomplete digestion of complex organic matrices. However, once the sample settled, the appearance of a clear brown supernatant and white sediment suggested near-complete decomposition of soluble organic material, while insoluble particulates likely represented inorganic matter or residual microplastics. Moreover, the addition of ethanol played an important supporting role in reducing viscosity and enabling successful filtration particularly in samples with dense organic residue such as P1 to P3. Ethanol is known as co-solvent to reduce viscosity and coagulate proteins thus lowering the surface tension of fatty mixture and enhances the breakdown of the lipid residue, thus providing positive effect on filtration performance (Ferreira et al. 2019).\u003c/p\u003e\n\u003cp\u003eConversely, the use of KOH without heating, as in Sample L6 (1:6 ratio at room temperature), failed to achieve sufficient lipid digestion. This result reinforces the importance of both temperature and incubation time as essential variables for the success of chemical digestion via alkaline hydrolysis. Sample OL2\u0026rsquo;s treatment with a 1:1.5 ratio and the resulting Maillard reaction suggested that intermediate ratios may also be effective under optimal conditions. However, as this condition was not replicated, further testing is required before recommending it as a standardized parameter. Noteworthy, the observation that samples with shorter lipase incubation times correlated with poorer KOH digestion outcomes supports the idea that each step of the protocol is interdependent. Inadequate lipid removal during the early enzymatic phase likely will limit KOH access to the remaining proteins and lipids, resulting in incomplete digestion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eH₂O₂ in the Oxidation Process of Remaining Organic Matter\u003c/em\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough lipid and protein digestion are important for removing biological residues, the oxidation of remaining organic matter using peroxide such as H₂O₂ represents a critical final step for sample clarification. The oxidative strength of 30% H₂O₂ facilitates the breakdown of residual organic substances that persist after enzymatic or solvent-based digestion (Roslan et al. 2024; Fiore et al. 2024). Its widespread use in microplastic sample preparation has been attributed to its strong oxidizing potential and minimal impact on polymer integrity when used under controlled conditions (Rani et al. 2023). Studies such as Phofl et al. (2021) have also demonstrated that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e effectively degrades organic debris without significantly altering the polymeric structure of common plastics with only slight change of particle size distribution when using in combination of iron (II) catalyst (Fenton reagent), if exposure time and temperature are optimized (Pfohl et al. 2021).\u003c/p\u003e\n\u003cp\u003eHowever, in samples with poor pre-treatment, particularly those with residual lipids or traces of hexane, the performance of H₂O₂ was notably reduced. This aligned with Sheriff et al. (2024), that emphasized that incomplete pre-treatment quality directly affects the efficacy of H₂O₂ in complex biological matrices. Moreover, Zhou et al. (2022) also noted that while H₂O₂ is effective, it requires optimal conditions such as temperature control, absence of interfering solvents, and adequate pre-digestion to achieve complete oxidation. For instance, in the present study, a secondary oxidation step involving incubation at 40\u0026deg;C for 20 mins proved beneficial, as it enhanced reaction kinetics and improved the visual clarity of the digestate. Although heating was initially approached with caution due to the presence of ethanol, mild heating up to 40\u0026deg;C was both safe and effective. Nevertheless, literature on the combined used of heated H₂O₂ and ethanol remains scarce, thus presents a potential safety concern particularly relating to peroxide-alcohol reactivity, and should be evaluated further before scaling up.\u003c/p\u003e\n\u003cp\u003eTo established cost-effective and safer oxidation protocol, trials with lower H₂O₂ concentrations (3% and 10%) were performed. Identifying the minimum effective concentration is crucial for enhancing cost-efficiency, minimizing safety risks and completing oxidation process. In Sample L1, passive oxidation using low-concentration peroxide achieved partial degradation but left a greyish tint and surface foam indicated incomplete oxidation. In contrast, Sample L2 showed that increasing H₂O₂ concentration alone did not guarantee improvement, since the presence of ethanol dilution or temperature variations also played significant contribution. Meanwhile, the use of 30% H₂O₂ at 50\u0026deg;C in L2 proved hazardous, as excessive foaming occurred within minutes, suggesting instability when heating high-concentration H₂O₂ in samples with organic matter. This supported the need to avoid aggressive heating of strong peroxide solutions (Pfohl et al. 2021).\u003c/p\u003e\n\u003cp\u003eThe introduction of 3% H₂O₂ in Sample \u0026lsquo;+\u0026rsquo; demonstrated that lower concentrations can still achieve oxidation, though at a slower rate, indicating that peroxide strength directly influences the rate of organic matter degradation. As such, at lower concentrations, oxidation proceeds through gradual generation of hydroxyl radicals, resulting in a gentler but prolonged digestion process that preserves the surface morphology of microplastic particles (Tagg et al. 2017). The role of ethanol was further verified through comparison between Samples OL1 and OL2, where ethanol addition improved clarity even under identical oxidation protocols. Ethanol likely enhances miscibility between aqueous peroxide and hydrophobic residues, thus helps in reducing viscosity of organic suspensions for subsequent filtration. Centrifugation, however, did not significantly improve clarity in undigested samples, confirming that incomplete oxidation will limit phased separation regardless of applied centrifugal forces, and thus was not effective in improving clarity or filterability. This important observation highlights that chemical oxidation, rather than mechanical separation, remains the determining steps for achieving digestate transparency that are suitable for microplastic recover efficiency and accurate quantification. The shift to 10% H₂O₂ in later samples (L7\u0026ndash;P3) established a practical balanced between oxidative strength and polymer integrity. The 10% H₂O₂ concentration accelerated oxidation more efficiently than 3% yet avoided the hazard risk and excessive foaming observed with 30% solution. This allowed integration with ethanol and mild heating, reducing oxidation time safely and effectively. Thus, a combination of a 1:2 sample-to-10% H₂O₂ ratio and controlled heating (\u0026lt;50 \u0026deg;C) provides a reliable, reproducible, and safe protocol for removing residual organic matter in HBM samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMethodological Limitations\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMethodological limitations inherent to exploratory research with biological samples were encountered and should be considered for future studies. The variability in breast milk composition, including differences in lipid and protein content, influenced digestion efficiency and sample consistency. Mechanical handling presented occasional challenges, such as sample loss and risks of layer disturbance during lipid separation. The use of hexane for lipid extraction, while effective, sometimes interfered with subsequent reactions due to incomplete phase separation. Sample size was limited, reflecting both availability constraints and ethical considerations common in human milk research. Controls included reagent blanks but lacked procedural blanks to fully account for environmental contamination, which may explain detection of unexpected polymers in controls. Additionally, the absence of standardized protocols required empirical optimization of digestion conditions, potentially contributing to variability. Despite these factors, the study provides valuable preliminary insights and a flexible methodological foundation for future investigations into microplastic extraction from complex biological matrices like human breast milk.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eAs a conclusion, this study managed to explore a multi-step protocol for extracting and identifying MPs from human breast milk using a combination of hexane-based lipid extraction, enzymatic digestion with \u003cem\u003eCandida rugosa\u003c/em\u003e lipase, KOH protein digestion, and H₂O₂ oxidation. Among the various conditions tested, the most effective combination was found to be a milk-to-enzyme ratio of 20:1, followed by KOH digestion at a 1:3 ratio and H₂O₂ oxidation at 10% concentration. Ethanol played a crucial role in reducing sample viscosity, especially after saponification, and facilitated effective filtration. The findings demonstrated that longer incubation periods, particularly for enzymatic digestion, significantly improved lipid removal, and downstream clarity. Nevertheless, this study provides a valuable foundation for future work in standardizing MP extraction from human samples. Going forward, improvements in mechanical handling and contamination control are recommended to enhance accuracy and reproducibility. Thus, this research contributes to the growing body of evidence on human exposure to MPs and highlights the need for reliable, scalable methods in environmental health monitoring.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eParticipant Consent Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants, or their legal guardians, provided informed consent for the collection and use of human breast milk samples in this study. The study was conducted following approval by the IIUM Research Ethical Committee (IREC) (Approval ID: IREC 2025-058). The ethics committee confirmed that the procedures complied with the Declaration of Helsinki, ICH-GCP, Malaysian GCP, and CIOMS guidelines. All collected samples were anonymized to ensure donor confidentiality. Where applicable, the requirement for individual consent was waived by the approving ethics committee.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors expressed their gratitude to the Sultan Ahmad Shah Medical Centre @IIUM (SASMEC), Malaysia for granting the SASMEC Research Grant (ID: SRG25-167-0167). The open access funding was provided by the International Islamic University Malaysia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available in the paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Norafiza Zainuddin, Hamizah Ismail; Methodology: Norafiza Zainuddin, Dhea Maisarah Ahmad Nasri, Fikriah Faudzi, Nur Rasyiqah Shamsul; Formal analysis and investigation: Dhea Maisarah Ahmad Nasri; Writing - original draft preparation: Dhea Maisarah Ahmad Nasri; Writing - review and editing: Norafiza Zainuddin, Dhea Maisarah Ahmad Nasri, Sabiqah Tuan Anuar, Muhammad Syafiq Musa, Mufti Petala Patria; Funding acquisition: Norafiza Zainuddin; Resources: Hamizah Ismail; Supervision: Norafiza Zainuddin, Fikriah Faudzi\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDhea Maisarah Ahmad Nasri, Nur Rasyiqah Syamsul, Fikriah Faudzi, Hamizah Ismail, Sabiqah Tuan Anuar, Muhammad Syafiq Musa, Mufti Petala Patria, Norafiza Zainuddin declare that they have no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAnuar ST, Mugo SM, Curtis JM (2015) A flow-through enzymatic microreactor for the rapid conversion of triacylglycerols into fatty acid ethyl ester and fatty acid methyl ester derivatives for GC analysis. Analytical Methods 7:5898\u0026ndash;5906. doi:10.1039/c5ay00800j\u003c/li\u003e\n \u003cli\u003edas Neves AM, Visioli LJ, Enzweiler H, Paulino AT (2024) Lipase from Candida rugosa incorporated in pectin hydrogel via immobilization for hydrolysis of lipids in dairy effluents and production of fatty acids. Journal of Water Process Engineering 58:104821. doi:10.1016/j.jwpe.2024.104821\u003c/li\u003e\n \u003cli\u003eDawson AL, Motti CA, Kroon FJ (2020) Solving a sticky situation: Microplastic analysis of lipid-rich tissue. Frontiers in Environmental Science 8:563565. doi:10.3389/fenvs.2020.563565\u003c/li\u003e\n \u003cli\u003eDi Fiore C, Ishikawa Y, Wright S (2024) A review on methods for extracting and quantifying microplastic in biological tissues. Journal of Hazardous Materials 464:132991. doi:10.1016/j.jhazmat.2023.132991\u003c/li\u003e\n \u003cli\u003eDzierżyński E, Gawlik PJ, Puźniak D, Flieger W, J\u0026oacute;źwik K, Teresiński G, Forma A, Wdowiak P, Baj J, Flieger J (2024) Microplastics in the human body: Exposure, detection, and risk of carcinogenesis: A state-of-the-art review. Cancers 16:3703. doi:10.3390/cancers16213703\u003c/li\u003e\n \u003cli\u003eEnders K, Lenz R, Beer S, Stedmon CA (2016) Extraction of microplastic from biota: Recommended acidic digestion destroys common plastic polymers. ICES Journal of Marine Science 74:326\u0026ndash;331. doi:10.1093/icesjms/fsw173\u003c/li\u003e\n \u003cli\u003eFerreira AC, Sullo A, Winston S, Norton IT, Norton-Welch AB (2019) Influence of ethanol on emulsions stabilized by low molecular weight surfactants. Journal of Food Science 85:28\u0026ndash;35. doi:10.1111/1750-3841.14947\u003c/li\u003e\n \u003cli\u003eHansen RB, Agerbaek MA, Nielsen PM, Rancke-Madsen A, Woodley JM (2020) Esterification using a liquid lipase to remove residual free fatty acids in biodiesel. Process Biochemistry 97:213\u0026ndash;221. doi:10.1016/j.procbio.2020.06.005\u003c/li\u003e\n \u003cli\u003eKathuria D, Hamid, Sunakshi G, Thakur A (2023) Maillard reaction in different food products: Effect on product quality, human health and mitigation strategies. Food Control 153:109911. doi:10.1016/j.foodcont.2023.109911\u003c/li\u003e\n \u003cli\u003eKim SY, Yi DY (2020) Components of human breast milk: From macronutrient to microbiome and microRNA. Clinical and Experimental Pediatrics 63:301\u0026ndash;309. doi:10.3345/cep.2020.00059\u003c/li\u003e\n \u003cli\u003eKutralam-Muniasamy G, Shruti VC, P\u0026eacute;rez-Guevara F, Roy PD (2023) Microplastic diagnostics in humans: \u0026quot;The 3Ps\u0026quot; progress, problems, and prospects. Science of The Total Environment 856:159164. doi:10.1016/J.SCITOTENV.2022.159164\u003c/li\u003e\n \u003cli\u003eLakshmayya NSV, Panday A, Yadavalli R, Reddy CN, Mandal SK, Agrawal DC, Mishra B (2023) Food Contamination with Micro-plastics: Occurrences, Bioavailability, Human Vulnerability, and Prevention. Current Nutrition \u0026amp; Food Science 20:797\u0026ndash;810. doi:10.2174/1573401319666230915164116\u003c/li\u003e\n \u003cli\u003eLiu L, Zhang X, Jia P, He S, Dai H, Deng S, Han J (2023) Release of microplastics from breastmilk storage bags and assessment of intake by infants: A preliminary study. Environmental Pollution 323:121197. doi:10.1016/j.envpol.2023.121197\u003c/li\u003e\n \u003cli\u003eLund MN, Ray CA (2017) Control of Maillard reactions in foods: Strategies and chemical mechanisms. Journal of Agricultural and Food Chemistry 65:4537\u0026ndash;4552. doi:10.1021/acs.jafc.7b00882\u003c/li\u003e\n \u003cli\u003eMhadmhan S, Yoosuk B, Henpraserttae S (2024) Selective lipase-catalyzed hydrolysis for removal of diglyceride in palm oil. Separation and Purification Technology 349:127897. doi:10.1016/j.seppur.2024.127897\u003c/li\u003e\n \u003cli\u003eMi\u0026scaron;ľanov\u0026aacute; C, Valachovičov\u0026aacute; M, Slez\u0026aacute;kov\u0026aacute; Z (2024) An overview of the possible exposure of infants to microplastics. Life (Basel, Switzerland) 14:371. doi:10.3390/life14030371\u003c/li\u003e\n \u003cli\u003eNawab A, Ahmad M, Khan MT, Nafees M, Khan I, Ihsanullah I (2024) Human exposure to microplastics: A review on exposure routes and public health impacts. Journal of Hazardous Materials Advances 16:100487. doi:10.1016/j.hazadv.2024.100487\u003c/li\u003e\n \u003cli\u003ePfohl P, Roth C, Meyer L, et al (2021) Microplastic extraction protocols can impact the polymer structure. Microplastics and Nanoplastics 1:8. doi:10.1186/s43591-021-00009-9\u003c/li\u003e\n \u003cli\u003eRagusa A, Notarstefano V, Svelato A, Belloni A, Gioacchini G, Blondeel C, Zucchelli E, De Luca C, D\u0026rsquo;Avino S, Gulotta A, Carnevali O, Giorgini E (2022) Raman microspectroscopy detection and characterisation of microplastics in human breastmilk. Polymers 14:2700. doi:10.3390/polym14132700\u003c/li\u003e\n \u003cli\u003eRani M, Ducoli S, Depero LE, Prica M, Tubić A, Ademovic Z, Morrison L, Federici S (2023) A complete guide to extraction methods of microplastics from complex environmental matrices. Molecules 28:5710. doi:10.3390/molecules28155710\u003c/li\u003e\n \u003cli\u003eRoslan NS, Lee YY, Ibrahim YS, Tuan Anuar S, Yusof KMKK, Lai LA, Brentnall T (2024) Detection of microplastics in human tissues and organs: A scoping review. Journal of Global Health 14:04179. doi:10.7189/jogh.14.04179\u003c/li\u003e\n \u003cli\u003eSalihu A, Alam MZ, Abdulkarim MI, Salleh HM (2011) Optimization of lipase production by Candida cylindracea in palm oil mill effluent based medium using statistical experimental design. Journal of Molecular Catalysis B: Enzymatic 69:66\u0026ndash;73. doi:10.1016/j.molcatb.2010.12.012\u003c/li\u003e\n \u003cli\u003eSalimon J, Abdullah BM, Salih N (2011) Hydrolysis optimization and characterization study of preparing fatty acids from Jatropha curcas seed oil. Chemistry Central Journal 5:67. doi:10.1186/1752-153X-5-67\u003c/li\u003e\n \u003cli\u003eShang Y, Wang X, Chang X, Sokolova IM, Wei S, Liu W, Fang JC, Hu M, Huang W, Wang Y (2021) The effect of microplastics on the bioenergetics of the mussel Mytilus coruscus assessed by cellular energy allocation approach. Frontiers in Marine Science 8:754789. doi:10.3389/fmars.2021.754789\u003c/li\u003e\n \u003cli\u003eSheriff I, Awang NA, Halim H, Ikechukwu OS, Jusoh AF (2024) Extraction and analytical methods of microplastics in wastewater treatment plants: Isolation patterns, quantification, and size characterization techniques. Desalination and Water Treatment 100399\u0026ndash;100399. doi:10.1016/j.dwt.2024.100399\u003c/li\u003e\n \u003cli\u003eTagg AS, Harrison JP, Ju-Nam Y, Sapp M, Bradley EL, Sinclair CJ, Ojeda JJ (2017) Fenton\u0026rsquo;s reagent for the rapid and efficient isolation of microplastics from wastewater. Chemical Communications 53:372\u0026ndash;375. doi:10.1039/c6cc08798a\u003c/li\u003e\n \u003cli\u003eTamanna N, Mahmood N (2015) Food processing and Maillard reaction products: Effect on human health and nutrition. International Journal of Food Science 2015:1\u0026ndash;6. doi:10.1155/2015/526762\u003c/li\u003e\n \u003cli\u003eZhou Q, Chen J, Zhang D, Pan X (2022) Evaluation of organic matter removal by H2O2 from microplastic surface by nano-physicochemical methods. Green Analytical Chemistry 3:100035. doi:10.1016/j.greeac.2022.100035\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"chemical-papers","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"chpa","sideBox":"Learn more about [Chemical Papers](http://link.springer.com/journal/11696)","snPcode":"11696","submissionUrl":"https://www.editorialmanager.com/CHPA/default.aspx","title":"Chemical Papers","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Microplastics, human milk, lipid extraction, enzymatic digestion, KOH digestion","lastPublishedDoi":"10.21203/rs.3.rs-8153566/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8153566/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study successfully developed and evaluated a multi-step digestion protocol for the effective extraction and identification of microplastics (MPs) from human breast milk (HBM). The method combined hexane-mediated lipid removal, enzymatic digestion using \u003cem\u003eCandida rugosa\u003c/em\u003e lipase at a milk-to-enzyme ratio of 20:1, protein digestion with potassium hydroxide (KOH) at 1:3 ratio, and oxidative treatment with hydrogen peroxide (H₂O₂) at 10% concentration. Ethanol was found essential for reducing sample viscosity, particularly post-saponification, thereby facilitating efficient filtration. Results showed that extended incubation times during enzymatic digestion notably enhanced lipid removal and improved clarity of the digestate, contributing to higher extraction efficiency. This protocol establishes a reproducible framework for reliable isolation and downstream spectroscopic identification of MPs in breast milk. Despite these advances, improvements in mechanical handling and contamination control are necessary to further enhance accuracy and consistency. The present findings contribute important methodological advancements to the field and support the urgent need for standardised, scalable protocols in monitoring neonatal exposure to environmental microplastic contaminants. Future work should expand on validation across diverse sample sets and explore correlations between MP presence and neonatal health outcomes.\u003c/p\u003e","manuscriptTitle":" Optimizing Lipid Removal and Protein Digestion in Human Milk for Microplastic Analysis using Candida rugosa Lipase, KOH Digestion, and Hydrogen Peroxide Oxidation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-03 06:33:21","doi":"10.21203/rs.3.rs-8153566/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"125804821113148903524417501095612721017","date":"2026-03-02T05:40:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-23T10:16:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-20T10:54:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-20T08:50:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chemical Papers","date":"2025-11-19T09:36:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chemical-papers","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"chpa","sideBox":"Learn more about [Chemical Papers](http://link.springer.com/journal/11696)","snPcode":"11696","submissionUrl":"https://www.editorialmanager.com/CHPA/default.aspx","title":"Chemical Papers","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a729146c-c455-45cd-b4a1-1aed9fe6c0ff","owner":[],"postedDate":"December 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T10:27:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-03 06:33:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8153566","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8153566","identity":"rs-8153566","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}