Razi Journal of Medical Sciences

fa اثر سویه‌های پروبیوتیکی معده زنبور عسل در کاهش گلوکز سرم نمونه های دیابتی در شرایط آزمایشگاهی Effect of honey bee stomach probiotic strains on in vitro glucose lowering in diabetic serum samples میکروبیولوژی Microbiology پژوهشي Research زمینه و هدف : دیابت نوع 2 یک بیماری چند عاملی است که عوامل ژنتیکی و محیطی می توانند در ایجاد آن نقش داشته باشند. با شیوع دیابت در سراسر جهان لزوم کاربرد درمان های نوین مورد توجه قرار گرفته است. هدف این مطالعه، بررسی توانایی سویه‌های پروبیوتیک جداشده از معده زنبور عسل در کاهش غلظت گلوکز در سرم بیماران دیابتی نوع 2 در شرایط آزمایشگاهی  بود. روش کار : باکتری های پروبیوتیک از معده 20 زنبور عسل جداسازی شدند و با استفاده از آزمون های بیوشیمیایی، تست مقاومت آنتی بیوتیکی، ارزیابی توانایی تخمیر دکستروز بعنوان منبع کربن، مورد بررسی قرار گرفتند. جهت شناسایی و تایید سویههای جداسازی شده، استخراج DNA و بررسی مولکولی بر اساس 16srRNA انجام شد. اثر پروبیوتیک های مورد مطالعه، بر کاهش گلوکز سرم جدا شده از 50 بیمار دیابت نوع 2، در شرایط ازمایشگاهی بررسی شد. داده ها آنالیز آماری شدند.  یافته ها : از معده 20 زنبور عسل، 30 ایزوله پروبیوتیک جداسازی شد. بر اساس تست های بیوشیمیایی و روش مولکولی، ایزوله ها شامل لاکتوباسیلوس اسیدوفیلوس، لاکتوباسیلوس پلانتاروم، لوکونوستوک مزنتروئیدس و باسیلوس سوبتیلیس بودند که اثر معنی داری را درکاهش گلوکز در بازه زمانی 6 ساعت پس از تلقیح باکتری به سرم خون بیماران دیابتی نوع 2، در شرایط ازمایشگاهی نشان دادند (0.05>P). نتیجه گیری : نتایج نشان داد که پروبیوتیک های معده زنبور عسل، می توانند در شرایط ازمایشگاهی پتانسل کاهش گلوکز سرم بیماران دیابتی را داشته باشند. برای تایید اثرات درمانی در بیماران دیابتی، مطالعات بالینی گسترده با حجم نمونه بیشتر و تنوع سویه های فراوان تر، توصیه می گردد.   Background and Aim: Type 2 diabetes mellitus is a multifactorial disease with a genetic susceptibility that causes high mortality and illness tolls. Type 2 diabetes mellitus is affected by different factors, consist of genetic damage and environmental disorders. Yet, lifestyle risk factors extremely show a significant function in causing this disease (1-6). Other funders to the pathogenesis of this disease contain entrance to healthy food and changes in the gut microbiota. Alterations in gut microbiota have been observed in diabetic patients. These variations can damage to glycemic control and show significantly roles in glucose metabolism. Development of Type 2 diabetes mellitus and its problems containing those marking the gut microbiota with novel antioxidant causes have arisen new treatment programs. One of the programs consist of using the probiotics. Probiotics may improve blood glucose regulation and reduce oxidative stress. Both glycemic control and oxidative stress remain to the pathogenesis of Type 2 diabetes mellitus by the gut microbiota regulation (11-21). Conversely, current researches demonstrated that probiotics, mainly the lactobacillus species, hold antioxidant effects and may be control and treatment Diabetes. Probiotics are living microbes that can offer health profits after used suitably and can gain them over some food as honey. Approving new dietary plans can permit to regulation normal blood sugar levels by controlling the gut microbiota in Diabetes patients (15-30). General studies have presented that probiotics as, Lactobacillus acidophilus, Lactobacillus casei, and plantarum incorporate major antidiabetic properties (33-40). Concurrently, oxidative stress is a critical contributor to the development and progression of Type 2 diabetes and its complications. This has spurred interest in novel therapeutic strategies that target these pathways. The use of probiotics represents a promising intervention. Specific probiotic strains, particularly from the Lactobacillus genus (e.g., L. acidophilus, L. casei, L. plantarum), have demonstrated significant potential in improving blood glucose regulation and reducing oxidative stress through their interaction with the gut microbiome. Honey, a natural substance, has been identified as a potential vector for these beneficial microbes, as it can contain viable probiotic bacteria, including those originating from the bee gut. This research aims were isolation and identification probiotic bacteria from the stomach of honey bees that exhibit antibacterial properties. So, Investigatation and validation the in vitro glucose-lowering efficacy of these isolated probiotic strains when introduced to diabetic serum samples. This work focuses on evaluating these novel probiotics in direct response to current scientific suggestions regarding their antidiabetic potential. methods: This study employed a comprehensive multi-step process, from classical microbiology to molecular biology, to ensure the isolated strains were safe and had strong probiotic potential, with a specific focus on applications for managing diabetes. For this matter breakdown of the Methodology contain: 1. Isolation and Preliminary Identification: Source: Stomachs of 20 honey bees. Culture Medium: De Man, Rogosa and Sharpe (MRS) agar, selective for Lactic Acid Bacteria (common probiotics like Lactobacillus). Biochemical and Morphological Tests: A standard battery of tests was used for initial classification reside: Gram staining: Isolates were likely Gram-positive rods (typical for Lactobacillus and Bifidobacterium). Catalase test: Isolates were likely catalase-negative (a key trait of many Lactic Acid Bacteria; they do not produce the catalase enzyme). Oxidase test: Isolates were likely oxidase-negative. Carbohydrate fermentation: Used to create a metabolic profile for identification. Growth at different temperatures, motility, acid and bile salt resistance: These tests screened for essential probiotic properties—survival through the stomach's acid and the small intestine's bile. 2. Assessment of Probiotic Properties: Antibacterial Activity: The isolated probiotics showed a statistically significant (P < 0.05) ability to inhibit the growth of three major pathogens (Proteus mirabilis, E. coli, S. aureus). The triple-repeat (triplicate) measurements ensure the results are reliable and reproducible. Acid and Bile Tolerance: Isolates were confirmed to survive the harsh conditions of the human gastrointestinal tract, a prerequisite for any effective probiotic. Antibiotic Resistance (Antibiogram): The profile was determined using the CLSI standard method. This is crucial for safety, ensuring the probiotics do not harbor transmissible resistance to common antibiotics. 3. Specialized Functional Assessment (Diabetes Focus): Glucose Tolerance: The ability of the bacteria to consume ("tolerate") glucose at different concentrations was tested in vitro. Clinical Application: This property was directly linked to a clinical study involving 50 diabetic patients. The results, presented as Mean ± SEM, showed that the probiotic application led to a statistically significant (P < 0.05) reduction in blood sugar levels compared to a control group. 4. Molecular Identification: DNA Extraction and PCR: The 16S rRNA gene, the gold standard for bacterial identification, was amplified. Sequencing and Phylogenetics: By sequencing the PCR product and comparing it to the NCBI database via BLAST, the researchers conclusively identified the isolated strain(s) to the species level (e.g., Lactobacillus acidophilus or a similar species common in bees). 5. Statistical Analysis: Software: SPSS. Test: One-way ANOVA followed by Tukey's post-hoc test. This is appropriate for comparing the means of more than two groups (e.g., control vs. different probiotic dosage groups). Significance: A P-value < 0.05 confirms that the observed differences (e.g., in pathogen inhibition or blood sugar reduction) were very unlikely to be due to random chance. Results: In the current research, 30 insulated of probiotic bacteria which had lactobacillus species characters were identified from Stomachs of 20 honey bees’ samples. Summary of the research Findings include: 1.Isolation and Identification:30 bacterial isolates with characteristics of Lactobacillus species were obtained from 20 honey bee samples. They were initially identified using standard morphology and biochemical tests. 2.Probiotic Properties: Antimicrobial Activity: All 30 isolates demonstrated the ability to inhibit the growth of the pathogenic bacteria Proteus mirabilis. Antibiotic Sensitivity: The isolates showed high sensitivity (50%) to penicillin and ampicillin but complete resistance (100%) to ceftazidime. Tolerance to Gut Conditions: All isolates survived simulated gut conditions, specifically acidic environments and the presence of bile salts, which is a key characteristic for potential probiotics. 3.Glucose-Lowering Effect (In Vitro): Out of the 30 isolates, 9 showed a significant ability to reduce glucose levels in a solution (dextrose powder) at various concentrations over a 6-hour period. Crucially, these 9 strains also significantly lowered blood glucose levels in vitro in serum samples taken from 50 diabetic patients. This effect was observed over 6 hours and was statistically significant (p < 0.05). 4.Molecular Confirmation: Molecular analysis (16S rRNA gene sequencing) was used to precisely identify the 9 most effective strains. They were found to be: Lactobacillus acidophilus, Lactobacillus plantarum, Leuconostoc mesenteroides, Bacillus subtilis. This molecular confirmation validated the initial biochemical identification methods. Conclusion: This study's in vitro results demonstrate that the isolated and identified several strains of bacteria from honey bees that exhibit strong probiotic potential. These probiotic strains effectively reduced blood sugar levels in samples from diabetic patients. They pronounced ability to lower glucose levels in vitro, including in diabetic serum samples. This suggests these specific strains hold potential for antidiabetic effects, warranting further investigation through in vivo studies in animal models and human clinical trials. Thus, these bee-derived probiotics could be promising candidates for further research into managing blood sugar levels, potentially leading to applications for diabetes management.  Current preclinical and clinical research is increasingly focused on the ability of various probiotic strains, particularly from the Lactobacillus genus, to regulate blood glucose levels and enhance antioxidant capabilities. Probiotics are emerging as a promising complementary therapy for managing Type 2 Diabetes Mellitus and its complications, primarily by modulating oxidative stress and reducing systemic inflammation (15-20). Recent studies corroborate that probiotics can help ameliorate oxidative stress and inflammatory markers in diabetic patients. However, translating these promising preclinical findings into standard clinical practice presents several challenges that must be addressed (29-36). Key limitations include: Strain-Specific Effects: Different probiotic strains possess unique biological and metabolic properties and interact with the host's immune system in distinct ways. Consequently, their effects can vary significantly across different populations. Delivery Method: The vehicle of administration (e.g., dairy products, capsules, fermented foods, honey) can influence the efficacy and integration into standardized treatment regimens. Dosage and Duration: Determining the optimal dosage, treatment duration, and the ideal timing for intervention remains unclear and appears to vary between strains. Despite these limitations, clinical evidence supporting the use of specific probiotics in diabetes management continues to grow (24-28). In conclusion, while this study indicates that probiotics may assist in diabetes treatment, their application requires rigorous validation. Future research must include long-term human studies to confirm efficacy, establish safety profiles, and rule out any potential toxicity associated with prolonged use. There is a critical need for large-scale, placebo-controlled clinical trials on diabetic patients from diverse populations, utilizing various well-defined probiotic strains under standardized protocols to fully realize their therapeutic potential.   باکتری پروبیوتیک, زنبورعسل, دیابت نوع 2, کاهش گلوکز. probiotic bacteria, honey bee, Type 2 diabetes, glucose lowering. 0 0 http://rjms.iums.ac.ir/browse.php?a_code=A-10-8659-1&slc_lang=fa&sid=1 elham siasi الهام سیاسی emi_biotech2006@yahoo.ca 3900319475328460090926 3900319475328460090926 Yes Department of Microbiology, School of science, NT.C., Islamic Azad University, Tehran, Iran. گروه میکروبیولوژی، دانشکده علوم زیستی، واحد تهران شمال، دانشگاه آزاد اسلامی ، تهران، ایران. mohaddeseh karimi nori محدثه کریمی نوری mohaddesehkarimi13@yahoo.com 3900319475328460090927 3900319475328460090927 No Department of Microbiology, School of science, NT.C., Islamic Azad University, Tehran, Iran. گروه میکروبیولوژی، دانشکده علوم زیستی، واحد تهران شمال، دانشگاه آزاد اسلامی ، تهران، ایران. laya takbiri لعیا تکبیری Laya6076@yahoo.com 3900319475328460090928 3900319475328460090928 No Department of Microbiology, School of science, NT.C., Islamic Azad University, Tehran, Iran. گروه میکروبیولوژی، دانشکده علوم زیستی، واحد تهران شمال، دانشگاه آزاد اسلامی ، تهران، ایران.