Assessment and Enhancement of Methods for Exosome Isolation from Camel Milk

Assessment and Enhancement of Methods for Exosome Isolation from Camel Milk | 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 Assessment and Enhancement of Methods for Exosome Isolation from Camel Milk 回 杨, 塔娜 乌伦 This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4910547/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Camel milk is a source of exosomes with potential immunomodulatory and antioxidant benefits. However, the efficiency of exosome extraction from camel milk is crucial for maximizing their potential applications. Objective This study aims to optimize the exosome isolation process from camel milk to increase the yield and quality of the extracted exosomes. Methods We employed various pretreatment strategies prior to ultracentrifugation, including chymosin-assisted, isoelectric point (PI)-precipitation, and ethylenediaminetetraacetic acid (EDTA)-assisted methods. For the characterization of the isolated exosomes, we utilized nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blotting to evaluate size distribution, structural integrity, and specific exosomal protein markers, respectively. Results The chymosin-assisted technique yielded exosomes with a more intact and defined double-layered membrane structure, lower non-exosomal protein background, and a higher presence of the exosomal marker CD63 as evidenced by western blotting analysis ( P < 0.05), compared with the other methodologies tested. Conclusion Chymosin pretreatment combined with ultracentrifugation significantly enhances the isolation of high-quality exosomes from camel milk, indicating that this approach may be the most effective for purifying exosomes for downstream biomedical applications. Further studies are encouraged to validate and refine this method for large-scale extraction. Camel Milk Exosome Extraction method Chymosin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Camels, despite inhabiting harsh semi-arid and arid climatic zones, are capable of producing a substantial quantity of valuable milk( 1 – 3 ). This milk is characterized by a significant mineral content, including sodium, potassium, iron, copper, zinc, and magnesium, and serves as a plentiful source of unsaturated fatty acids( 1 , 3 – 5 ). Furthermore, camel milk surpasses cow milk in terms of vitamin C concentration( 6 – 8 ). Notably, it exhibits elevated protein levels that effectively impede bacterial contaminants, namely lactoperoxidase, lactoferrin, immunoglobulin, and lysozyme( 3 , 5 – 8 ). Consequently, camel milk possesses superior nutritional value, thereby promoting optimal health( 6 – 8 ). Recent studies have shown that exosomes derived from diverse animal milk sources possess an enhanced capacity for intestinal cell absorption and subsequent systemic distribution through the bloodstream, thereby enhancing their nutritional and health benefits( 9 – 12 ). Dairy exosomes, which are membranous nanoparticles with a size of 30–150 nm, facilitate intercellular communication by transmitting mRNAs, microRNAs, and proteins( 13 – 16 ). Previous studies have examined the properties of exosomes derived from camel milk( 17 – 20 ). Isolation and purification is the initial pivotal stage of the initial pivotal stage of exosome research( 21 , 22 ). Although multiple methodologies are available for their isolation, a comparative analysis of the techniques could aid in determining the optimal extraction and refinement protocols for specific exosomes( 17 , 20 – 22 ), advancing our understanding of the distinctive characteristics of exosomes obtained from various sources, and determining their potential applications in the food industry( 23 ). Overall, the efficacy and integrity of dairy exosomes are substantially influenced by the extraction method( 24 – 27 ). It is worth noting that contemporary research predominantly employs high-speed centrifugation for extracting exosomes from camel milk, without further enhancement of the methodology( 17 – 20 ). Ultracentrifugation emerged as an early and prominent technique for exosome extraction, predicated on the differential sedimentation coefficients of exosomes and other cellular components( 28 , 29 ), thereby facilitating the isolation of exosomes by segregating cells, cellular debris, and organelles( 29 ). However, this method's overreliance on centrifugal force can reduce the purity of the isolated exosomes-a key factor for their use in food nutrition studies. The primary complication in purifying milk-derived exosomes is the similarity in size and density of natural milk components like casein and lipid globules, which makes traditional separation methods insufficient( 28 , 29 ). Current techniques like differential centrifugation, density gradient centrifugation, and immunoaffinity separation are either inefficient or expensive( 28 ). There's a significant need for a new, cost-effective, and convenient purification method for camel milk exosomes to advance their application in scientific and industrial domains. This study aimed to determine the optimal method for isolating exosomes from camel’s milk using isoelectric point (PI)-assisted, EDTA-assisted, and chymosin-assisted techniques. The performance of each technique was subsequently determined by transmission electron microscopy (TEM) for shape analysis, nanoparticle tracking analysis (NTA), protein concentration determination, and western blot analysis of the isolated exosomes. We purposed that employing diverse pretreatment methods prior to high-speed centrifugation would enhance the extraction efficiency of camel-milk exosomes to varying degrees. The principal objective of this study was to assess and improve the extraction techniques for exosomes from camel milk while offering empirical evidence to inform future research in this field. 2 Materials and Methods 2.1 Experimental materials and Experimental design This research was approved by the Research Ethical Committee of the Qinghai University Medical College (SL202311-05). We randomly selected a cohort of 10 healthy camels with similar pregnancy histories of 4-5‐year‐old (first litters) and housing conditions (all grazing on pastures at the Delingha Camel Breeding Base, Qinghai Province, China). Milk (200 mL) was manually collected from each camel at least 50 d after parturition and pooled before being distributed into 50 mL centrifuge tubes. The samples were preserved at -80 °C until further experimentation. In short, our research was categorized into three groups of ultracentrifugation techniques, namely chymosin-assisted, isoelectric point-assisted, and ethylenediaminetetraacetic acid-assisted. Three groups of experiments were designed as follows ( Figure 1 ). To compare the morphology, size, and protein concentration of the extracted exosomes, we employed nanoparticle tracking analysis, transmission electron microscopy, and western blotting. 2.2 Extraction of exosomes All extraction processes were conducted at 4 °C and the resultant precipitate of exosomes was subsequently resuspended in a 200 μL PBS buffer solution and stored at -80 °C. 2.2.1 Chymosin-assisted technique Measure 50 mL of milk samples with a calibrated pipette and transfer into pre-labeled centrifuge tubes. Ensure all equipment is properly calibrated before use. Centrifuge the samples at 2000 rpm for 15 minutes at 4 °C using a refrigerated centrifuge. Ensure the centrifuge is balanced and calibrated with the appropriate rotor for the defined speed and duration. After centrifugation, carefully collect the intermediate layer, as shown in Supplemental Figure 1, avoiding disturbance of other layers, using a sterile pipette. Adjust the pH of the collected intermediate layer to 6.0 ± 0.05, by adding a precise volume of a 10% glacial acetic acid solution. Confirm pH adjustment with a calibrated pH meter. Prepare a chymosin solution by dissolving 0.035 mg of chymosin in 1 L of 1% NaCl solution (to achieve a final concentration of 0.035 mg/L). All reagents should be of analytical grade, and volumes should be measured with calibrated instruments. Activate the chymosin solution in a water bath maintained at 37 °C ± 0.5 °C for 30 minutes. Add the activated chymosin solution to the pH-adjusted intermediate layer and mix gently but thoroughly. Incubate the resulting mixture in a water bath at 37 °C ± 0.5 °C for 30-35 minutes, ensuring a stable temperature throughout the incubation period. Centrifuge the mixture at 12,000 rpm for 30 minutes at 4 °C. Discard the supernatant (whey), and gently retain the residual mixture to minimize losses. Take 25 mL of the residual mixture and centrifuge at 12,000 rpm for 60 minutes at 4 °C. Filter the resulting supernatant through a 0.45 μm filter using a vacuum filtration system or a suitable syringe filter setup. Centrifuge the filtrate at 75,000 rpm for 60 minutes at 4 °C. Pass the supernatant through a 0.22 μm filter to remove any remaining particulate matter. Subject the filtrate to a final ultracentrifugation step at 150,000 rpm for 2 hours at 4 °C in an ultracentrifuge. Ensure that the ultracentrifuge is capable of achieving the specified speed and that the temperature is maintained. Carefully collect the sediments at the bottom of the tube, avoiding any contamination or mixing with the discarded supernatant. 2.2.2 Isoelectric point (PI)-assisted technique As detailed in Section 2.2.1, a 25 mL intermediate layer was carefully extracted. Its pH was then fine-tuned to 4.6, equivalent to the isoelectric point of chymosin, using an ice-cold solution of 10% acetone. Following the pH adjustment, the sample was incubated for 10 minutes at a consistent 37°C. Afterwards, the sample was subjected to centrifugation at 12,000 rpm for a 30-minute duration, allowing the collection of the whey content. Finally, to complete the process, follow-up procedures including additional centrifugation and filtration measures were carried out as meticulously prescribed in Section 2.2.1. 2.2.3 EDTA-assisted technique Carefully obtain a 25 mL intermediate layer following the step-by-step guidance provided in Protocol 2.2.1. Next, add 1 mL of accurately prepared and measured 250 μmol/L EDTA to the sample, ensuring a thorough mix for a uniform distribution. Let the mixture rest for 10 minutes at a controlled temperature of 37 °C (± 0.5 °C) for consistent reaction conditions. Afterward, centrifuge the treated sample at 37,500 rpm for 60 minutes at 4 °C, strictly adhering to calibration parameters for reproducible outcomes. Finally, proceed with the centrifugation and filtration steps detailed in Protocol 2.2.1, applying the same meticulous standards throughout each step of the process. 2.3 TEM observation of exosomes The exosomes acquired through the three distinct methods were suspended and deposited onto separate 300-mesh copper grids (EMS, Hatfield, PA, USA) and dried in a fume hood. Following blotting and air-drying, the samples were stained with 2% uranyl acetate (10 µL) and visualized using an HT7700 transmission electron microscope (Hitachi, Tokyo, Japan). 2.4 Nanoparticle tracking analysis The acquired exosomes were suspended and their quantity and dimensions were examined by NTA with a ZetaView Particle Metrix (PMX-120, Particle Metrix, Ammersee, Germany). Prior to analysis, the instrument was calibrated with 100 nm polystyrene beads (Thermo Fisher Scientific, Fremont, CA, USA). The concentration of nanoparticles (particles/mL) was determined using NTA 3.2 software (Dev Build 3.2.16), and the in-built batch process was employed for each sample. 2.5 Western blot analysis The protein concentrations of the obtained exosomes were assessed using a BCA assay kit (Beyotime, Haimen, China) in accordance with the manufacturer’s instructions. The exosomes were suspended and subsequently lysed in ice-cold RIPA lysis buffer (Beyotime) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Lysates containing equal amounts of protein were separated by SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA) and subsequently transferred onto 0.22 µm PVDF membranes (Pall Corporation, Hercules, CA, USA). After multiple rinses with TBS (Signalway Antibody, Greenbelt, MD, USA) and subsequent blocking with 5% nonfat milk (Fisher Scientific, Pittsburgh, PA, USA) at room temperature for 1 h, the membranes were incubated overnight at 4 °C with anti-CD63 (1:3,000; ab216130, Abcam, Waltham, MA, USA) or anti-CD81 (1:3,000; ab109201, Abcam) primary antibodies. After thorough washing, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000; L3012, Signalway Antibody) in the dark at room temperature for 1 h. The membranes were treated with ECL reagent (Tanon, Shanghai, China) and immunoreactive protein bands were visualized using a ChemiDoc MP imaging system (Bio-Rad). 2.6 Statistical analysis All experiments were conducted in triplicate. Chromaticity analysis of the bands was performed using ImageJ software (version 2.0.0; National Institutes of Health, Bethesda, MD, USA) and plotting was performed using GraphPad Prism 8 software (GraphPad Software, Boston, MA, USA). The obtained data were analyzed using single-factor variance analysis with SPSS 20.0 (IBM SPSS, Armonk, NY, USA) and the results are presented as the mean ± standard deviation. P < 0.05 was considered significant. 3 Results 3.1 TEM analysis In the present study, TEM was used to compare the bilayer structures of exosomes extracted using the three different methods (Figure 2). The use of chymosin yielded exosomes with a distinct cup-like structure, whereas the PI-assisted method resulted in exosomes with rough structures and indistinct backgrounds. Meanwhile, the EDTA-assisted extraction method resulted in exosomes with no clearly discernable structures and significantly larger particle diameters than expected. 3.2 NTA observations Our findings indicated that the chymosin- and PI-assisted methods generated exosomes within the expected size range (30-150 nm). Given that the vesicles acquired using the EDTA-assisted technique did not align with the exosome profile, the subsequent analysis concentration and yield only pertained to the exosomes acquired using chymosin- and PI-assisted techniques. Although the concentration of exosomes obtained with lactase assistance was slightly higher than that obtained with PI assistance, the difference was not statistically significant (P > 0.05; Figure 3, Table 1). 3.3 Western blot analysis In our experiment, the protein concentration detection of different samples is shown in Supplemental table 1. SDS-PAGE analysis revealed significant aggregation between 20-25 kDa in extracts of the EDTA-assisted technique (Figure 4). The corresponding bands were observed for CD63 and CD81 proteins in exosomes obtained using all three methods (Figure 5). Notably, the chymosin-assisted method exhibited a significantly higher level of the surface marker protein CD63 than that for the other two methods (P < 0.01). 4 Discussion Successful isolation of camel milk exosomes is important for advancing the production of camel milk-based nutritious food products. The differences in the composition of fat globules and casein in camel milk complicate the extraction of exosomes(6, 31-33). The optimization of exosome extraction from bovine milk involves the incorporation of additional steps, namely chymosin-, PI-, and EDTA-assisted protein precipitation followed by ultracentrifugation(17-20). We followed the same procedures for camel milk and analyzed the obtained exosomes according to their morphological characteristics, particle size, and signature proteins, among other properties, using TEM, NTA, protein concentration determination, and western blot analysis in accordance with established standards. TEM enables visualization of the exosome structure, morphology, and particle size(13, 14). The quality of exosome extraction can be assessed based on the morphology of particles within the observed field of view(34). In the present study, the use of chymosin yielded exosomes with a distinct cup-like structure, whereas the PI-assisted method resulted in exosomes with rough structures and indistinct backgrounds. Meanwhile, the EDTA-assisted extraction method resulted in exosomes with no clearly discernable structures and significantly larger particle diameters than expected. Yamauchi et al. found that isoelectric precipitation produced exosomes with rough surfaces(35). Rahman et al. showed that acidification with PI-assisted isolation of exosomes from bovine milk resulted in a disrupted and rough surface structure(36). However, it is worth noting that the dehydration process involved in sample preparation may alter the morphology of exosomes(37). In summary, our TEM results were generally consistent with those of previous studies. NTA enables the expeditious quantitative measurement of exosome size and quantity while preserving their inherent structure(34). Our findings indicated that the chymosin- and PI-assisted methods generated exosomes within the expected size range (30-150 nm). Yamauchi et al. utilized hydrochloric acid for isoelectric precipitation to eliminate casein from cow’s milk, which resulted in a lower concentration of exosomes compared to those obtained through ultracentrifugation(35), consistent with the findings of the current study. Notably, El-Kattawy et al. reported a mean yield of 348.5 ± 41 mg/L for camel-milk exosomes obtained through conventional high-speed centrifugation(18). In contrast, our chymosin-assisted method resulted in a yield of 449.3 ± 38.6 mg/L. The utilization of the Chymosin-assisted technique yielded superior efficiency compared to the conventional extraction method. Due to the presence of carboxyl and amino groups, the chelating agent EDTA can form chelates with various substances, thereby aiding in titration(38). In our experiment, the use of EDTA had a minimal impact on the protein content of camel milk. Additionally, SDS-PAGE analysis revealed significant aggregation between 20-25 kDa in extracts of the EDTA-assisted technique. The diverse subtypes of casein molecules in camel milk have a molecular weight range of 20-25 kDa(39). Casein is a prominent protein found in the milk of various mammals, such as cows, sheep, and humans(40). It exhibits a rigid, compact structure and poses significant challenges in terms of digestion(40, 41). Casein is primarily utilized as a fortifier in the production of solid foods within the realm of food processing(41). Additionally, it may also be employed as a binder, filler, and carrier(41). While the consumption of casein aids in mineral absorption, it is more prone to elicit allergic reactions within the body, particularly among individuals with autism who are commonly advised to adhere to a diet devoid of casein(42, 43). Consequently, it is likely that the EDTA-assisted method was not entirely effective at eliminating casein in camel milk. Current markers utilized for exosomes include CD9, CD63, TSG101, and CD81(14, 19, 34). Camel milk exosome isolation was confirmed by western blotting of CD63 and CD81(18). The exosomes obtained through all three methods in this study exhibited the presence of corresponding bands for both proteins. Notably, the chymosin-assisted method exhibited a significantly higher level of the surface marker protein CD63 than that for the other two methods (P < 0.05). The research findings of Hanne et al. demonstrate that the unique proteomic signature of chymosin precipitated casein holds significant potential for the advancement of research and development in the dairy Nutrition product industry(44). And CD63 actively engages in diverse cellular processes and assumes a pivotal function in the sorting of cellular proteins within late endosomes and polyvesicles, thereby fostering the generation of exosomes(45). By specifically targeting CD63, the precise administration of extracellular drugs can be accomplished, thereby facilitating targeted therapy(46). Moreover, in the context of immune response, exosomes that express CD63 play a facilitative role in antigen presentation and the activation of CD4+T cells, thereby contributing to immune regulation(47). Based on these findings, we purposed that chymotrypsin-assisted extraction of camel-milk exosomes may better preserve their immune functionality to a certain extent. In this study, we focused on exploring the impact of different pre-treatments using centrifugation on the efficacy of exosome isolation. Although this work provided in-depth insights into a single technical approach, we have not yet comparatively assessed the comprehensive performance of various exosome isolation methods. Considering the widespread application of exosomes in the field of biomedicine, such as in disease diagnosis, therapeutic efficacy monitoring, and treatment, future studies must delve into how exosome isolation methods can be precisely tailored according to the specific requirements of different applications（20，23，28，29）. This process should strive not only to improve the purity of exosomes for more accurate biomarker analysis but also to maintain the integrity and functionality of the exosomes, thus avoiding interference caused by the quality of exosomes in clinical applications（20，23，34-37）. Furthermore, investigating how to reduce the generation of impurities and their associated interference will undeniably become a critical aspect of future exosome research（23，34，37）. Through such efforts, we can look forward to significant advancements in areas such as the development of drug carriers, the discovery of disease biomarkers, and the improvement of therapeutic methods. 5 Conclusions The objective of this study was to enhance the isolation of exosomes from camel milk using various pretreatments for ultracentrifugation, namely chymosin-, PI-, and EDTA-assisted methods. Based on NTA, TEM, and western blotting, we found that the chymosin-assisted method generated exosomes with distinct double-layer membrane structures, minimal background noise, and higher expression of the selective marker CD63 compared to those of the alternative techniques. These results suggest that the pretreatment of camel milk with chymosin prior to ultracentrifugation may promote the effective isolation of exosomes. This study exclusively employs the ultracentrifugation method for exosomes extraction, thereby imposing certain limitations and necessitating further research in the future. Declarations Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Ethics statement The animal study was approved by Qinghai University Animal Ethics Committee. The study was conducted in accordance with the local legislation and institutional requirements. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Author Contributions Hui YANG: Conceptualization, Methodology, Investigation, Formal analysis, writing the original draft, writing the review, and editing. Ri-Li GE: supervision, conceptualization, and methodology. Demtu ER: Supervision, Conceptualization, Methodology, and reviewing. Tana WUREN: Supervision, Conceptualization, Methodology, Writing, review, and editing. Funding This work was supported by the Qinghai Provincial Key Laboratory for Application of High-Altitude Medicine (Grant No. 2022-ZJ-Y15). Acknowledgments The authors are very grateful for the language polishing service provided by Editage. References Mtibaa I, Zouari A, Purcaro G, Attia H, Ayadi MA, Danthine S. (2021). Crystallization mechanisms in camel milk cream during physical ripening: Effect of temperature and ripening duration. Food and Bioproducts Processing . 127 :435-42. DOI: 10.1016/j.fbp.2021.03.016 Zouari A, Lajnaf R, Lopez C, Schuck P, Attia H, Ayadi MA. (2021). Physicochemical, techno-functional, and fat melting properties of spray-dried camel and bovine milk powders. Journal of Food Science . 86 ( 1 ):103-11. DOI: 10.1111/1750-3841.15550 Lajnaf R, Zouari A, Trigui I, Attia H, Ayadi MA. (2020). 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Chemical interactions among caseins during rennet coagulation of milk. Journal of Dairy Science . 105 ( 2 ):981-9. DOI: 10.3168/jds.2021-21071 Wąsik M, Nazimek K, Nowak B, Askenase PW, Bryniarski KJN. (2019). Delayed-type hypersensitivity underlying casein allergy is suppressed by extracellular vesicles carrying miRNA-150. Nutrients . 11 ( 4 ):907. DOI: 10.3390/nu11040907 González-Domenech PJ, Diaz-Atienza F, Gutiérrez-Rojas L, Fernández-Soto ML, González-Domenech CMJN. (2022). A Narrative Review about Autism Spectrum Disorders and Exclusion of Gluten and Casein from the Diet. Nutrients . 14 ( 9 ):1797. DOI: 10.3390/nu14091797 Jensen HB, Poulsen NA, Møller HS, Stensballe A, Larsen LBJJodr. (2012). Comparative proteomic analysis of casein and whey as prepared by chymosin-induced separation, isoelectric precipitation or ultracentrifugation. Journal of Dairy Research . 79 ( 4 ):451-8. DOI: 10.1017/S0022029912000404 Mathieu M, Névo N, Jouve M, Valenzuela JI, Maurin M, Verweij FJ, et al. (2021). Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9. Nature Communications . 12 ( 1 ):4389. DOI: 10.1038/s41467-021-24384-2 de Goeij BE, Vink T, Ten Napel H, Breij EC, Satijn D, Wubbolts R, et al. (2016). Efficient payload delivery by a bispecific antibody–drug conjugate targeting HER2 and CD63. Molecular Cancer . 15 ( 11 ):2688-97. DOI: 10.1158/1535-7163.MCT-16-0364 Torti SV, Torti FM. (2021). CD63 orchestrates ferritin export. Blood . 138 ( 16 ):1387-9. DOI: 10.1182/blood.2021013181 Table TABLE 1 Particle size and concentration of exosome analyzed by NTA. Concentration （ Particles / mL ） Dilution Factor Original Concentration （ Particles / mL ） Median （ × 50 ）（ nm ） Average yield （ mg/L ） Chymosin-Assisted 1.03 ± 0.30×10 8 256000 2.63 ± 0.80 ×10 13 109.70 ± 42.59 449.30 ± 38.60 PI-Assisted 7.70± 0.50×10 7 256000 2.06 ± 0.40×10 13 119.30 ± 37.80 324.60 ± 38.70 EDTA-Assisted 1.81± 0.30×10 8 256000 9.10 ± 0.49×10 13 183.80 ± 18.29 492.50 ± 35.35 Note: ANOVA followed by pairwise comparison was used to assess statistical significance. not significant for pairwise comparison ( P > 0.05). Additional Declarations No competing interests reported. Supplementary Files SUPPLEMENTALTablesFIGURE1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-4910547","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":351337116,"identity":"bfc028be-8937-4e99-bfaf-0185cbd36bd7","order_by":0,"name":"回 杨","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"回","middleName":"","lastName":"杨","suffix":""},{"id":351337117,"identity":"cb2178ee-9b1e-4768-bdcf-198138431c10","order_by":1,"name":"塔娜 乌伦","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYHAD5gPMYPoAAXU8CCZbAslaeAyI02LPfvbgB8a2w/n80j0fbxe2Mcjx3Uhg/FyAzxaevGQJoBbLmXPObrae2cZgLHkjgVl6Bl6H5RhIMG47bGBwI3ebNG8bQ+KGGwlszDz4tPC/Mf4B0mJ/I+cZSEs9YS0SOWYQWyRy2EBaEgwIarnxxswi8V+6gcSNNGNrnnMShjPPPGyWxqeFvT/H+MaHM9YG/DOSH97mKbOR5zuefPAzPi1gkAClJcCIgbGBkAYEkCBe6SgYBaNgFIwkAABgVUQyX0Zc7QAAAABJRU5ErkJggg==","orcid":"","institution":"Qinghai University","correspondingAuthor":true,"prefix":"","firstName":"塔娜","middleName":"","lastName":"乌伦","suffix":""}],"badges":[],"createdAt":"2024-08-14 04:01:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4910547/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4910547/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64229815,"identity":"64326ecf-1764-4657-abf9-23a2c7291c2a","added_by":"auto","created_at":"2024-09-10 14:40:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":131513,"visible":true,"origin":"","legend":"\u003cp\u003eDesign of the entire experimental protocol. The specific experimental methods are detailed in sections 2.1 and 2.2 of Materials and Methods.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4910547/v1/f659d8900bb0fa6bb903058e.png"},{"id":64230408,"identity":"acb30a7a-672f-4bbc-b87b-cff66522b13d","added_by":"auto","created_at":"2024-09-10 14:48:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":133886,"visible":true,"origin":"","legend":"\u003cp\u003eThe electron microscopic analysis of camel milk reveals the presence of exosomes. The exosome structures are indicated by arrowheads, with a scale bar of 100nm. \u003cstrong\u003e(A)\u003c/strong\u003eThe morphology of exosomes extracted using chymosin-assisted ultracentrifugation techniques;\u003cstrong\u003e (B)\u003c/strong\u003e The morphology of exosomes extracted using isoelectric point-assisted ultracentrifugation techniques;\u003cstrong\u003e (C) \u003c/strong\u003eThe morphology of exosomes extracted using ethylenediaminetetraacetic acid-assisted ultracentrifugation techniques.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4910547/v1/618a5298951e7604e3a03076.png"},{"id":64229816,"identity":"2f9323ee-e066-4763-a7b0-99e77315b8a7","added_by":"auto","created_at":"2024-09-10 14:40:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":112791,"visible":true,"origin":"","legend":"\u003cp\u003eExosome size distribution analysis was conducted using nanoparticle tracking analysis (NTA).\u003cstrong\u003e (A-C)\u003c/strong\u003eThe purpose was to observe the distribution of exosomes in a random field of view. \u003cstrong\u003e(A)\u003c/strong\u003eExtraction through chymosin-assisted ultracentrifugation methods; \u003cstrong\u003e(B)\u003c/strong\u003eExtraction through isoelectric point-assisted ultracentrifugation methods; \u003cstrong\u003e(C)\u003c/strong\u003eExtraction through ethylenediaminetetraacetic acid-assisted ultracentrifugation methods.\u003cstrong\u003e(D-F) \u003c/strong\u003eThe image shows the size (x axis: diameter in nanometers) and concentration (y axis: particles/ml) of exosomes. \u003cstrong\u003e(D)\u003c/strong\u003eExtraction through chymosin-assisted ultracentrifugation methods; \u003cstrong\u003e(E)\u003c/strong\u003eExtraction through isoelectric point-assisted ultracentrifugation methods; \u003cstrong\u003e(F)\u003c/strong\u003eExtraction through ethylenediaminetetraacetic acid-assisted ultracentrifugation methods.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4910547/v1/79ec40ebf8c47dedbbe2737b.png"},{"id":64229813,"identity":"84e1e431-250c-4b67-99de-789511a82438","added_by":"auto","created_at":"2024-09-10 14:40:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":264468,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE and Coomassie blue staining.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4910547/v1/40bb5aa52087bfc69c7a9605.png"},{"id":64229817,"identity":"c6f6471a-7fb6-439a-8b79-7ca7170edbc8","added_by":"auto","created_at":"2024-09-10 14:40:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":81554,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blots revealed the presence of exosomal markers, CD63 and CD81, in isolated exosomes. Western blots were quantified，the protein expression was quantified with β-actin as an internal reference. **: \u003cem\u003eP\u003c/em\u003e \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4910547/v1/33859e960bff925b7d616f61.png"},{"id":91147979,"identity":"80afe901-8dbc-458b-a7e6-91ccfa0ad251","added_by":"auto","created_at":"2025-09-12 06:39:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1534470,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4910547/v1/eb934da1-2b8d-4be1-b28b-1ab5c7cb970a.pdf"},{"id":64230409,"identity":"174d09bf-0dd0-4668-ace3-6c31484b3cc3","added_by":"auto","created_at":"2024-09-10 14:48:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":158484,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTALTablesFIGURE1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4910547/v1/a0d07fb8abef6372c434ae89.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment and Enhancement of Methods for Exosome Isolation from Camel Milk","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eCamels, despite inhabiting harsh semi-arid and arid climatic zones, are capable of producing a substantial quantity of valuable milk(\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). This milk is characterized by a significant mineral content, including sodium, potassium, iron, copper, zinc, and magnesium, and serves as a plentiful source of unsaturated fatty acids(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Furthermore, camel milk surpasses cow milk in terms of vitamin C concentration(\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Notably, it exhibits elevated protein levels that effectively impede bacterial contaminants, namely lactoperoxidase, lactoferrin, immunoglobulin, and lysozyme(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Consequently, camel milk possesses superior nutritional value, thereby promoting optimal health(\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent studies have shown that exosomes derived from diverse animal milk sources possess an enhanced capacity for intestinal cell absorption and subsequent systemic distribution through the bloodstream, thereby enhancing their nutritional and health benefits(\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Dairy exosomes, which are membranous nanoparticles with a size of 30\u0026ndash;150 nm, facilitate intercellular communication by transmitting mRNAs, microRNAs, and proteins(\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Previous studies have examined the properties of exosomes derived from camel milk(\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Isolation and purification is the initial pivotal stage of the initial pivotal stage of exosome research(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Although multiple methodologies are available for their isolation, a comparative analysis of the techniques could aid in determining the optimal extraction and refinement protocols for specific exosomes(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), advancing our understanding of the distinctive characteristics of exosomes obtained from various sources, and determining their potential applications in the food industry(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Overall, the efficacy and integrity of dairy exosomes are substantially influenced by the extraction method(\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is worth noting that contemporary research predominantly employs high-speed centrifugation for extracting exosomes from camel milk, without further enhancement of the methodology(\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Ultracentrifugation emerged as an early and prominent technique for exosome extraction, predicated on the differential sedimentation coefficients of exosomes and other cellular components(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), thereby facilitating the isolation of exosomes by segregating cells, cellular debris, and organelles(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). However, this method's overreliance on centrifugal force can reduce the purity of the isolated exosomes-a key factor for their use in food nutrition studies. The primary complication in purifying milk-derived exosomes is the similarity in size and density of natural milk components like casein and lipid globules, which makes traditional separation methods insufficient(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Current techniques like differential centrifugation, density gradient centrifugation, and immunoaffinity separation are either inefficient or expensive(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). There's a significant need for a new, cost-effective, and convenient purification method for camel milk exosomes to advance their application in scientific and industrial domains.\u003c/p\u003e \u003cp\u003eThis study aimed to determine the optimal method for isolating exosomes from camel\u0026rsquo;s milk using isoelectric point (PI)-assisted, EDTA-assisted, and chymosin-assisted techniques. The performance of each technique was subsequently determined by transmission electron microscopy (TEM) for shape analysis, nanoparticle tracking analysis (NTA), protein concentration determination, and western blot analysis of the isolated exosomes. We purposed that employing diverse pretreatment methods prior to high-speed centrifugation would enhance the extraction efficiency of camel-milk exosomes to varying degrees. The principal objective of this study was to assess and improve the extraction techniques for exosomes from camel milk while offering empirical evidence to inform future research in this field.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003ch2\u003e2.1 Experimental materials and Experimental design\u003c/h2\u003e\n\u003cp\u003eThis research was approved by the Research Ethical Committee of the Qinghai University Medical College (SL202311-05). We randomly selected a cohort of 10 healthy camels with similar pregnancy histories of 4-5‐year‐old\u0026nbsp;(first litters) and housing conditions (all grazing on pastures at the Delingha Camel Breeding Base, Qinghai Province, China). Milk (200 mL) was manually collected from each camel at least 50 d after parturition and pooled before being distributed into 50 mL centrifuge tubes. The samples were preserved at -80 \u0026deg;C until further experimentation. In short, our research was categorized into three groups of ultracentrifugation techniques, namely chymosin-assisted, isoelectric point-assisted, and ethylenediaminetetraacetic acid-assisted. Three groups\u0026nbsp;of\u0026nbsp;experiments were designed\u0026nbsp;as\u0026nbsp;follows (\u003cstrong\u003eFigure 1\u003c/strong\u003e). To compare the morphology, size, and protein concentration of the extracted exosomes, we employed nanoparticle tracking analysis, transmission electron microscopy, and western blotting.\u003c/p\u003e\n\u003ch2\u003e2.2 Extraction of exosomes\u003c/h2\u003e\n\u003cp\u003eAll extraction processes were conducted at 4 \u0026deg;C and the resultant precipitate of exosomes was subsequently resuspended in a 200 \u0026mu;L PBS buffer solution and stored at -80 \u0026deg;C.\u003c/p\u003e\n\u003ch3\u003e2.2.1 Chymosin-assisted technique\u003c/h3\u003e\n\u003cp\u003eMeasure 50 mL of milk samples with a calibrated pipette and transfer into pre-labeled centrifuge tubes. Ensure all equipment is properly calibrated before use. Centrifuge the samples at 2000 rpm for 15 minutes at 4 \u0026deg;C using a refrigerated centrifuge. Ensure the centrifuge is balanced and calibrated with the appropriate rotor for the defined speed and duration. After centrifugation, carefully collect the intermediate layer, as shown in Supplemental Figure 1, avoiding disturbance of other layers, using a sterile pipette. Adjust the pH of the collected intermediate layer to 6.0 \u0026plusmn; 0.05, by adding a precise volume of a 10% glacial acetic acid solution. Confirm pH adjustment with a calibrated pH meter. Prepare a chymosin solution by dissolving 0.035 mg of chymosin in 1 L of 1% NaCl solution (to achieve a final concentration of 0.035 mg/L). All reagents should be of analytical grade, and volumes should be measured with calibrated instruments. Activate the chymosin solution in a water bath maintained at 37 \u0026deg;C \u0026plusmn; 0.5 \u0026deg;C for 30 minutes. Add the activated chymosin solution to the pH-adjusted intermediate layer and mix gently but thoroughly. Incubate the resulting mixture in a water bath at 37 \u0026deg;C \u0026plusmn; 0.5 \u0026deg;C for 30-35 minutes, ensuring a stable temperature throughout the incubation period. Centrifuge the mixture at 12,000 rpm for 30 minutes at 4 \u0026deg;C. Discard the supernatant (whey), and gently retain the residual mixture to minimize losses. Take 25 mL of the residual mixture and centrifuge at 12,000 rpm for 60 minutes at 4 \u0026deg;C. Filter the resulting supernatant through a 0.45 \u0026mu;m filter using a vacuum filtration system or a suitable syringe filter setup. Centrifuge the filtrate at 75,000 rpm for 60 minutes at 4 \u0026deg;C. Pass the supernatant through a 0.22 \u0026mu;m filter to remove any remaining particulate matter. Subject the filtrate to a final ultracentrifugation step at 150,000 rpm for 2 hours at 4 \u0026deg;C in an ultracentrifuge. Ensure that the ultracentrifuge is capable of achieving the specified speed and that the temperature is maintained. Carefully collect the sediments at the bottom of the tube, avoiding any contamination or mixing with the discarded supernatant.\u003c/p\u003e\n\u003ch3\u003e2.2.2 Isoelectric point (PI)-assisted technique\u003c/h3\u003e\n\u003cp\u003eAs detailed in Section 2.2.1, a 25 mL intermediate layer was carefully extracted. Its pH was then fine-tuned to 4.6, equivalent to the isoelectric point of chymosin, using an ice-cold solution of 10% acetone. Following the pH adjustment, the sample was incubated for 10 minutes at a consistent 37\u0026deg;C. Afterwards, the sample was subjected to centrifugation at 12,000 rpm for a 30-minute duration, allowing the collection of the whey content. Finally, to complete the process, follow-up procedures including additional centrifugation and filtration measures were carried out as meticulously prescribed in Section 2.2.1.\u003c/p\u003e\n\u003ch3\u003e2.2.3 EDTA-assisted technique\u003c/h3\u003e\n\u003cp\u003eCarefully obtain a 25 mL intermediate layer following the step-by-step guidance provided in Protocol 2.2.1. Next, add 1 mL of accurately prepared and measured 250 \u0026mu;mol/L EDTA to the sample, ensuring a thorough mix for a uniform distribution. Let the mixture rest for 10 minutes at a controlled temperature of 37 \u0026deg;C (\u0026plusmn; 0.5 \u0026deg;C) for consistent reaction conditions. Afterward, centrifuge the treated sample at 37,500 rpm for 60 minutes at 4 \u0026deg;C, strictly adhering to calibration parameters for reproducible outcomes. Finally, proceed with the centrifugation and filtration steps detailed in Protocol 2.2.1, applying the same meticulous standards throughout each step of the process.\u003c/p\u003e\n\u003ch2\u003e2.3 TEM observation of exosomes\u003c/h2\u003e\n\u003cp\u003eThe exosomes acquired through the three distinct methods were suspended and deposited onto separate 300-mesh copper grids (EMS, Hatfield, PA, USA) and dried in a fume hood. Following blotting and air-drying, the samples were stained with 2% uranyl acetate (10 \u0026micro;L) and visualized using an HT7700 transmission electron microscope (Hitachi, Tokyo, Japan).\u003c/p\u003e\n\u003ch2\u003e2.4 Nanoparticle tracking analysis\u003c/h2\u003e\n\u003cp\u003eThe acquired exosomes were suspended and their quantity and dimensions were examined by NTA with a ZetaView Particle Metrix (PMX-120, Particle Metrix,\u0026nbsp;Ammersee, Germany). Prior to analysis, the instrument was calibrated with 100 nm polystyrene beads (Thermo Fisher Scientific, Fremont, CA, USA). The concentration of nanoparticles (particles/mL) was determined using NTA 3.2 software (Dev Build 3.2.16), and the in-built batch process was employed for each sample.\u003c/p\u003e\n\u003ch2\u003e2.5 Western blot analysis\u003c/h2\u003e\n\u003cp\u003eThe protein concentrations of the obtained exosomes were assessed using a BCA assay kit (Beyotime, Haimen, China) in accordance with the manufacturer\u0026rsquo;s instructions. The exosomes were suspended and subsequently lysed in ice-cold RIPA lysis buffer (Beyotime) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Lysates containing equal amounts of protein were separated by SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA) and subsequently transferred onto 0.22 \u0026micro;m PVDF membranes (Pall Corporation, Hercules, CA, USA). After multiple rinses with TBS (Signalway Antibody, Greenbelt, MD, USA) and subsequent blocking with 5% nonfat milk (Fisher Scientific, Pittsburgh, PA, USA)\u0026nbsp;at\u0026nbsp;room temperature for 1 h, the membranes were incubated overnight at 4 \u0026deg;C with anti-CD63 (1:3,000; ab216130, Abcam, Waltham, MA, USA) or anti-CD81 (1:3,000; ab109201, Abcam) primary antibodies. After thorough washing, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000; L3012, Signalway Antibody) in the dark at room temperature for 1 h. The membranes were treated with ECL reagent (Tanon, Shanghai, China) and immunoreactive protein bands were visualized using a ChemiDoc MP imaging system (Bio-Rad).\u003c/p\u003e\n\u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e\n\u003cp\u003eAll experiments were conducted in triplicate. Chromaticity analysis of the bands was performed using ImageJ software (version 2.0.0; National Institutes of Health, Bethesda, MD, USA) and plotting was performed using GraphPad Prism 8 software (GraphPad Software, Boston, MA, USA). The obtained data were analyzed using single-factor variance analysis with SPSS 20.0 (IBM SPSS, Armonk, NY, USA) and the results are presented as the mean \u0026plusmn; standard deviation. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered significant.\u003c/p\u003e"},{"header":"3 Results","content":"\u003ch2\u003e3.1 TEM analysis\u003c/h2\u003e\n\u003cp\u003eIn the present study, TEM was used to compare the bilayer structures of exosomes extracted using the three different methods (Figure 2). The use of chymosin yielded exosomes with a distinct cup-like structure, whereas the PI-assisted method resulted in exosomes with rough structures and indistinct backgrounds. Meanwhile, the EDTA-assisted extraction method resulted in exosomes with no clearly discernable structures and significantly larger particle diameters than expected.\u003c/p\u003e\n\u003ch2\u003e3.2 NTA observations\u003c/h2\u003e\n\u003cp\u003eOur findings indicated that the chymosin- and PI-assisted methods generated exosomes within the expected size range (30-150 nm). Given that the vesicles acquired using the EDTA-assisted technique did not align with the exosome profile, the subsequent analysis concentration and yield only pertained to the exosomes acquired using chymosin- and PI-assisted techniques. Although the concentration of exosomes obtained with lactase assistance was slightly higher than that obtained with PI assistance, the difference was not statistically significant (P \u0026gt; 0.05; Figure 3, Table 1).\u003c/p\u003e\n\u003ch2\u003e3.3 Western blot analysis\u003c/h2\u003e\n\u003cp\u003eIn our experiment, the protein concentration detection of different samples is shown in Supplemental table 1. SDS-PAGE analysis revealed significant aggregation between 20-25 kDa in extracts of the EDTA-assisted technique (Figure 4). The corresponding bands were observed for CD63 and CD81 proteins in exosomes obtained using all three methods (Figure 5). Notably, the chymosin-assisted method exhibited a significantly higher level of the surface marker protein CD63 than that for the other two methods (P \u0026lt; 0.01).\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eSuccessful isolation of camel milk exosomes is important for advancing the production of camel milk-based nutritious food products. The differences in the composition of fat globules and casein in camel milk complicate the extraction of exosomes(6, 31-33). The optimization of exosome extraction from bovine milk involves the incorporation of additional steps, namely chymosin-, PI-, and EDTA-assisted protein precipitation followed by ultracentrifugation(17-20). We followed the same procedures for camel milk and analyzed the obtained exosomes according to their morphological characteristics, particle size, and signature proteins, among other properties, using TEM, NTA, protein concentration determination, and western blot analysis in accordance with established standards.\u003c/p\u003e\n\u003cp\u003eTEM enables visualization of the exosome structure, morphology, and particle size(13, 14). The quality of exosome extraction can be assessed based on the morphology of particles within the observed field of view(34). In the present study, the use of chymosin yielded exosomes with a distinct cup-like structure, whereas the PI-assisted method resulted in exosomes with rough structures and indistinct backgrounds. Meanwhile, the EDTA-assisted extraction method resulted in exosomes with no clearly discernable structures and significantly larger particle diameters than expected. Yamauchi et al. found that isoelectric precipitation produced exosomes with rough surfaces(35). Rahman et al. showed that acidification with PI-assisted isolation of \u0026nbsp;exosomes from bovine milk resulted in a disrupted and rough surface structure(36). However, it is worth noting that the dehydration process involved in sample preparation may alter the morphology of exosomes(37). In summary, our TEM results were generally consistent with those of previous studies.\u003c/p\u003e\n\u003cp\u003eNTA enables the expeditious quantitative measurement of exosome size and quantity while preserving their inherent structure(34). Our findings indicated that the chymosin- and PI-assisted methods generated exosomes within the expected size range (30-150 nm). Yamauchi et al. utilized hydrochloric acid for isoelectric precipitation to eliminate casein from cow\u0026rsquo;s milk, which resulted in a lower concentration of exosomes compared to those obtained through ultracentrifugation(35), consistent with the findings of the current study. Notably,\u0026nbsp;El-Kattawy et al. reported a mean yield of 348.5 \u0026plusmn; 41 mg/L for camel-milk exosomes obtained through conventional high-speed centrifugation(18). In contrast, our chymosin-assisted method resulted in a yield of 449.3 \u0026plusmn; 38.6 mg/L. The utilization of the Chymosin-assisted technique yielded superior efficiency compared to the conventional extraction method.\u003c/p\u003e\n\u003cp\u003eDue to the presence of carboxyl and amino groups, the chelating agent EDTA can form chelates with various substances, thereby aiding in titration(38). In our experiment, the use of EDTA had a minimal impact on the protein content of camel milk. Additionally, SDS-PAGE analysis revealed significant aggregation between 20-25 kDa in extracts of the EDTA-assisted technique. The diverse subtypes of casein molecules in camel milk have a molecular weight range of 20-25 kDa(39). Casein is a prominent protein found in the milk of various mammals, such as cows, sheep, and humans(40). It exhibits a rigid, compact structure and poses significant challenges in terms of digestion(40, 41). Casein is primarily utilized as a fortifier in the production of solid foods within the realm of food processing(41). Additionally, it may also be employed as a binder, filler, and carrier(41). While the consumption of casein aids in mineral absorption, it is more prone to elicit allergic reactions within the body, particularly among individuals with autism who are commonly advised to adhere to a diet devoid of casein(42, 43). Consequently, it is likely that the EDTA-assisted method was not entirely effective at eliminating casein in camel milk.\u003c/p\u003e\n\u003cp\u003eCurrent markers utilized for exosomes include CD9, CD63, TSG101, and CD81(14, 19, 34). Camel milk exosome isolation was confirmed by western blotting of CD63 and CD81(18). The exosomes obtained through all three methods in this study exhibited the presence of corresponding bands for both proteins. Notably, the chymosin-assisted method exhibited a significantly higher level of the surface marker protein CD63 than that for the other two methods (P \u0026lt; 0.05). The research findings of Hanne et al. demonstrate that the unique proteomic signature of chymosin precipitated casein holds significant potential for the advancement of research and development in the dairy Nutrition product industry(44). And CD63 actively engages in diverse cellular processes and assumes a pivotal function in the sorting of cellular proteins within late endosomes and polyvesicles, thereby fostering the generation of \u0026nbsp;exosomes(45). By specifically targeting CD63, the precise administration of extracellular drugs can be accomplished, thereby facilitating targeted therapy(46). Moreover, in the context of immune response, \u0026nbsp;exosomes that express CD63 play a facilitative role in antigen presentation and the activation of CD4+T cells, thereby contributing to immune regulation(47). Based on these findings, we purposed that chymotrypsin-assisted extraction of camel-milk exosomes may better preserve their immune functionality to\u0026nbsp;a\u0026nbsp;certain\u0026nbsp;extent.\u003c/p\u003e\n\u003cp\u003eIn this study, we focused on exploring the impact of different pre-treatments using centrifugation on the efficacy of exosome isolation. Although this work provided in-depth insights into a single technical approach, we have not yet comparatively assessed the comprehensive performance of various exosome isolation methods. Considering the widespread application of exosomes in the field of biomedicine, such as in disease diagnosis, therapeutic efficacy monitoring, and treatment, future studies must delve into how exosome isolation methods can be precisely tailored according to the specific requirements of different applications（20，23，28，29）. This process should strive not only to improve the purity of exosomes for more accurate biomarker analysis but also to maintain the integrity and functionality of the exosomes, thus avoiding interference caused by the quality of exosomes in clinical applications（20，23，34-37）. Furthermore, investigating how to reduce the generation of impurities and their associated interference will undeniably become a critical aspect of future exosome research（23，34，37）. Through such efforts, we can look forward to significant advancements in areas such as the development of drug carriers, the discovery of disease biomarkers, and the improvement of therapeutic methods.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eThe objective of this study was to enhance the isolation of exosomes from camel milk using various pretreatments for ultracentrifugation, namely chymosin-, PI-, and EDTA-assisted methods. Based on NTA, TEM, and western blotting, we found that the chymosin-assisted method generated exosomes with distinct double-layer membrane structures, minimal background noise, and higher expression of the selective marker CD63 compared to those of the alternative techniques. These results suggest that the pretreatment of camel milk with chymosin prior to ultracentrifugation may promote the effective isolation of exosomes. This study exclusively employs the ultracentrifugation method for exosomes extraction, thereby imposing certain limitations and necessitating further research in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study was approved by Qinghai University Animal Ethics Committee. The study was conducted in accordance with the local legislation and institutional requirements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHui YANG:\u003c/strong\u003e Conceptualization, Methodology, Investigation, Formal analysis, writing the original draft, writing the review, and editing. \u003cstrong\u003eRi-Li GE:\u003c/strong\u003e supervision, conceptualization, and methodology. \u0026nbsp;\u003cstrong\u003eDemtu ER:\u003c/strong\u003e Supervision, Conceptualization, Methodology, and reviewing. \u003cstrong\u003eTana WUREN:\u003c/strong\u003e Supervision, Conceptualization, Methodology, Writing, review, and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Qinghai Provincial Key Laboratory for Application of High-Altitude Medicine (Grant No. 2022-ZJ-Y15).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are very grateful for the language polishing service provided by Editage.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMtibaa I, Zouari A, Purcaro G, Attia H, Ayadi MA, Danthine S. 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DOI: 10.1016/j.joen.2009.11.011\u003c/li\u003e\n\u003cli\u003eHamouda M, Sboui A, Salhi I, Hammadi M, Souchard JP, Bouajila J, et al. (2022). Effect of heat treatment on the antioxidant activities of camel milk alpha, beta and total caseins. \u003cem\u003eCellular and Molecular Biology\u003c/em\u003e. \u003cem\u003e68\u003c/em\u003e(\u003cem\u003e7\u003c/em\u003e):194-9. DOI: 10.14715/cmb/2022.68.7.32\u003c/li\u003e\n\u003cli\u003eKansu A, Urganci N, Bukulmez A, Kutluk G, Taskin DG, Keskin LS, et al. (2023). Growth, tolerance and safety outcomes with use of an extensively hydrolyzed casein-based formula in infants with cow\u0026rsquo;s milk protein allergy. \u003cem\u003eFrontiers in Pediatrics\u003c/em\u003e. \u003cem\u003e11\u003c/em\u003e:1230905. DOI: 10.3389/fped.2023.1230905\u003c/li\u003e\n\u003cli\u003eAmaro-Hern\u0026aacute;ndez J, Olivas G, Acosta-Mu\u0026ntilde;iz C, Guti\u0026eacute;rrez-M\u0026eacute;ndez N, Rios-Velasco C, Sepulveda DJJoDS. (2022). Chemical interactions among caseins during rennet coagulation of milk. \u003cem\u003eJournal of Dairy Science\u003c/em\u003e. \u003cem\u003e105\u003c/em\u003e(\u003cem\u003e2\u003c/em\u003e):981-9. DOI: 10.3168/jds.2021-21071\u003c/li\u003e\n\u003cli\u003eWąsik M, Nazimek K, Nowak B, Askenase PW, Bryniarski KJN. (2019). Delayed-type hypersensitivity underlying casein allergy is suppressed by extracellular vesicles carrying miRNA-150. \u003cem\u003eNutrients\u003c/em\u003e. \u003cem\u003e11\u003c/em\u003e(\u003cem\u003e4\u003c/em\u003e):907. DOI: 10.3390/nu11040907\u003c/li\u003e\n\u003cli\u003eGonz\u0026aacute;lez-Domenech PJ, Diaz-Atienza F, Guti\u0026eacute;rrez-Rojas L, Fern\u0026aacute;ndez-Soto ML, Gonz\u0026aacute;lez-Domenech CMJN. (2022). A Narrative Review about Autism Spectrum Disorders and Exclusion of Gluten and Casein from the Diet. \u003cem\u003eNutrients\u003c/em\u003e. \u003cem\u003e14\u003c/em\u003e(\u003cem\u003e9\u003c/em\u003e):1797. DOI: 10.3390/nu14091797\u003c/li\u003e\n\u003cli\u003eJensen HB, Poulsen NA, M\u0026oslash;ller HS, Stensballe A, Larsen LBJJodr. (2012). Comparative proteomic analysis of casein and whey as prepared by chymosin-induced separation, isoelectric precipitation or ultracentrifugation. \u003cem\u003eJournal of Dairy Research\u003c/em\u003e. \u003cem\u003e79\u003c/em\u003e(\u003cem\u003e4\u003c/em\u003e):451-8. DOI: 10.1017/S0022029912000404\u003c/li\u003e\n\u003cli\u003eMathieu M, N\u0026eacute;vo N, Jouve M, Valenzuela JI, Maurin M, Verweij FJ, et al. (2021). Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9. \u003cem\u003eNature Communications\u003c/em\u003e. \u003cem\u003e12\u003c/em\u003e(\u003cem\u003e1\u003c/em\u003e):4389. DOI: 10.1038/s41467-021-24384-2\u003c/li\u003e\n\u003cli\u003ede Goeij BE, Vink T, Ten Napel H, Breij EC, Satijn D, Wubbolts R, et al. (2016). Efficient payload delivery by a bispecific antibody\u0026ndash;drug conjugate targeting HER2 and CD63. \u003cem\u003eMolecular Cancer\u003c/em\u003e. \u003cem\u003e15\u003c/em\u003e(\u003cem\u003e11\u003c/em\u003e):2688-97. DOI: 10.1158/1535-7163.MCT-16-0364\u003c/li\u003e\n\u003cli\u003eTorti SV, Torti FM. (2021). CD63 orchestrates ferritin export. \u003cem\u003eBlood\u003c/em\u003e. \u003cem\u003e138\u003c/em\u003e(\u003cem\u003e16\u003c/em\u003e):1387-9. DOI: 10.1182/blood.2021013181\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTABLE 1\u0026nbsp;\u003c/strong\u003eParticle size and concentration of exosome analyzed by NTA.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"680\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.08823529411765%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.08823529411765%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentration\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eParticles / mL\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.61764705882353%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDilution Factor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.08823529411765%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eOriginal Concentration\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eParticles / mL\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.264705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003e\u0026times;\u003c/strong\u003e\u003cstrong\u003e50\u003c/strong\u003e\u003cstrong\u003e）（\u003c/strong\u003e\u003cstrong\u003enm\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.852941176470589%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverage yield\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003emg/L\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.08823529411765%\" valign=\"top\"\u003e\n \u003cp\u003eChymosin-Assisted\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.08823529411765%\" valign=\"top\"\u003e\n \u003cp\u003e1.03 \u0026plusmn; 0.30\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.61764705882353%\" valign=\"top\"\u003e\n \u003cp\u003e256000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.08823529411765%\" valign=\"top\"\u003e\n \u003cp\u003e2.63 \u0026plusmn; 0.80 \u0026times;10\u003csup\u003e13\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.264705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e109.70 \u0026plusmn; 42.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.852941176470589%\" valign=\"top\"\u003e\n \u003cp\u003e449.30 \u0026plusmn; 38.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.08823529411765%\" valign=\"top\"\u003e\n \u003cp\u003ePI-Assisted\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.08823529411765%\" valign=\"top\"\u003e\n \u003cp\u003e7.70\u0026plusmn; 0.50\u0026times;10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.61764705882353%\" valign=\"top\"\u003e\n \u003cp\u003e256000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.08823529411765%\" valign=\"top\"\u003e\n \u003cp\u003e2.06 \u0026plusmn; 0.40\u0026times;10\u003csup\u003e13\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.264705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e119.30 \u0026plusmn; 37.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.852941176470589%\" valign=\"top\"\u003e\n \u003cp\u003e324.60 \u0026plusmn; 38.70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.08823529411765%\" valign=\"top\"\u003e\n \u003cp\u003eEDTA-Assisted\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.08823529411765%\" valign=\"top\"\u003e\n \u003cp\u003e1.81\u0026plusmn; 0.30\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.61764705882353%\" valign=\"top\"\u003e\n \u003cp\u003e256000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.08823529411765%\" valign=\"top\"\u003e\n \u003cp\u003e9.10 \u0026plusmn; 0.49\u0026times;10\u003csup\u003e13\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.264705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e183.80 \u0026plusmn; 18.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.852941176470589%\" valign=\"top\"\u003e\n \u003cp\u003e492.50 \u0026plusmn; 35.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: ANOVA followed by pairwise comparison was used to assess statistical significance. not significant for pairwise comparison (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Camel, Milk, Exosome, Extraction method, Chymosin","lastPublishedDoi":"10.21203/rs.3.rs-4910547/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4910547/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCamel milk is a source of exosomes with potential immunomodulatory and antioxidant benefits. However, the efficiency of exosome extraction from camel milk is crucial for maximizing their potential applications.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThis study aims to optimize the exosome isolation process from camel milk to increase the yield and quality of the extracted exosomes.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe employed various pretreatment strategies prior to ultracentrifugation, including chymosin-assisted, isoelectric point (PI)-precipitation, and ethylenediaminetetraacetic acid (EDTA)-assisted methods. For the characterization of the isolated exosomes, we utilized nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blotting to evaluate size distribution, structural integrity, and specific exosomal protein markers, respectively.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe chymosin-assisted technique yielded exosomes with a more intact and defined double-layered membrane structure, lower non-exosomal protein background, and a higher presence of the exosomal marker CD63 as evidenced by western blotting analysis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), compared with the other methodologies tested.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eChymosin pretreatment combined with ultracentrifugation significantly enhances the isolation of high-quality exosomes from camel milk, indicating that this approach may be the most effective for purifying exosomes for downstream biomedical applications. Further studies are encouraged to validate and refine this method for large-scale extraction.\u003c/p\u003e","manuscriptTitle":"Assessment and Enhancement of Methods for Exosome Isolation from Camel Milk","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-10 14:40:24","doi":"10.21203/rs.3.rs-4910547/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"683d96d8-e7ad-4030-aa8f-9f45e26dcff0","owner":[],"postedDate":"September 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-12T06:38:29+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-10 14:40:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4910547","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4910547","identity":"rs-4910547","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}