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رویکرد ابن سینا به غربالگری و ارزیابی پیش از فعالیت ورزشی
No full textBackground and Aim: Since one of the primary goals of physical activity is to maintain and improve health and enhance the quality of life, achieving this goal requires determining a suitable and safe physical activity for each individual. The American College of Sports Medicine and the American Heart Association have established criteria for screening and assessing individuals' health status and physical fitness levels before prescribing physical activity. Given the valuable status of exercise in maintaining and promoting health in Iranian medicine, this study is the first to investigate Avicenna's approach on assessment and screening before physical activity.
Methods: This research was conducted by reviewing Avicenna's book "The Canon of Medicine" and performing an electronic search using relevant keywords.
Ethical Considerations: In compiling this study, the principles of trustworthiness, citation integrity, authenticity of sources and avoidance of bias in referring to texts or analyses were observed.
Results: Avicenna considers the examination of four factors essential before recommending physical activity:
Medical History Review: In chronic diseases, pressure should not be applied to the weak organ.
Assessment of Physical Weakness and Strength: Strenuous exercises should not be prescribed for individuals with a weak constitution.
Determination of Temperament: Individuals with a warm and dry temperament may be harmed by performing certain exercises.
Examination of Signs of Bodily Fullness (Plethora): In individuals whose bodies are filled with waste materials due to an improper lifestyle, exercising before cleansing the body of these materials can be harmful.
Conclusion: According to Avicenna's approach, prescribing physical activity should be done after careful assessment. Specific groups such as children, the elderly and patients require special considerations, which aligns with modern research. The two topics of temperament and bodily fullness can provide insightful research ideas for pre-activity screening and assessment, especially since, based on the principles of Iranian medicine, intense physical activity in a state of fullness may lead to sudden cardiac death.زمینه و هدف: با توجه به اینکه یکی از اهداف اصلی فعالیت ورزشی، حفظ و بهبود سلامت و ارتقای کیفیت زندگی است، دستیابی به این هدف مستلزم تعیین فعالیت ورزشی مناسب و ایمن برای هر فرد میباشد. کالج پزشکی ورزشی آمریکا و انجمن قلب آمریکا، معیارهایی برای غربالگری و ارزیابی سطح سلامت و آمادگی جسمانی افراد پیش از تجویز فعالیت ورزشی تعیین کردهاند. با عنایت به جایگاه ارزشمند ورزش در حفظ و ارتقای سلامت در طب ایرانی، این مطالعه برای نخستین بار به بررسی دیدگاه ابن سینا در مورد ارزیابی و غربالگری پیش از فعالیت ورزشی میپردازد.
روش: این پژوهش با مرور کتاب «قانون» ابن سینا و جستجوی الکترونیکی با استفاده از کلیدواژههای مرتبط انجام شده است.
ملاحظات اخلاقی: در تدوین این مطالعه، اصل امانتداری و صداقت استناد به متون مورد استفاده، اصالت منابع و پرهیز از جانبداری در مراجعه به متون یا تحلیلها، رعایت شده است.
یافتهها: ابن سینا پیش از توصیه به فعالیت ورزشی، بررسی چهار مورد را ضروری میداند:
1- بررسی سوابق بیماری: در بیماریهای مزمن، نباید به عضو ضعیف فشار وارد کرد.
2- ارزیابی میزان ضعف و قوت (بُنیه) بدنی: برای افراد دارای بنیه ضعیف نباید ورزشهای سنگین تجویز شود.
3- تعیین مزاج: ممکن است افراد با مزاج گرم و خشک از انجام برخی از ورزشها آسیب ببینند.
4- بررسی علایم امتلاء بدنی: در اشخاصی که به دلیل سبک زندگی نادرست، بدنشان انباشته از مواد زاید است، ورزش کردن قبل از پاکسازی بدن میتواند مضر باشد.
نتیجهگیری: بر اساس رویکرد ابن سینا تجویز فعالیت ورزشی باید بعد از ارزیابی دقیق انجام شود. گروههای خاص مانند کودکان، سالمندان و بیماران، نیازمند توجهات خاص هستند که همسو با پژوهشهای نوین است. دو مبحث مزاج و امتلاء بدنی میتوانند در غربالگری و ارزیابی پیش از فعالیت ورزشی ایدههای پژوهشی راهگشایی باشند؛ به ویژه با توجه به این که بر اساس مبانی طب ایرانی، فعالیت شدید بدنی در حالت امتلاء ممکن است به مرگ ناگهانی قلبی منجر شود
مقایسه ساختار و عملکرد قلب پسران ورزشکار نخبه کشتی با غیرورزشکاران شهر اردبیل: ساختار و عملکرد قلب کشتی گیران
No full textمقدمه: فعالیت ورزشی شدید و مداوم موجب تغییرات سازگاری در ساختار و عملکرد قلب میگردد که به پدیده «قلب ورزشکار» شناخته میشود. هدف از این پژوهش، مقایسه شاخصهای ساختاری، سیستولیک و دیاستولیک قلب پسران ورزشکار نخبه کشتی با همسالان غیرورزشکار بود.
روش کار: در این مطالعه مقطعی، ۴۰ پسران جوان سالم (۲۰ ورزشکار نخبه کشتی و ۲۰ غیرورزشکار) شرکت کردند. ارزیابی قلب با استفاده از اکوکاردیوگرافی داپلر دوبعدی در حالت استراحت انجام شد. برای مقایسه گروهها، آزمون t مستقل و تحلیل کوواریانس (ANCOVA) با کنترل سن و شاخص توده بدنی (BMI) بهکار رفت.
یافته ها: نتایج نشان داد ورزشکاران نخبه دارای توده بطن چپ (195 ± 18 g در برابر 158 ± 15 g ؛ p<0.001)، کسر تزریقی بالاتر (64.5 ± 3.2% در برابر 60.1 ± 3.5%؛ p<0.001)، و حجم ضربهای بیشتر بودند. علاوه بر آن، شاخصهای دیاستولیک شامل نسبت E/A بالاتر (1.7 ± 0.2)، نسبت ′E/e پایینتر (7.1 ± 1.1) و IVRT کوتاهتر (70 ± 8 ms) بیانگر شلشدگی مؤثرتر میوکارد بودند. نتایج پس از تعدیل برای سن و BMI نیز معنادار باقی ماندند.
نتیجه گیری: این یافتهها نشان میدهد تمرینات منظم و شدید کشتی موجب هیپرتروفی فیزیولوژیک، بهبود عملکرد انقباضی و پرشدگی قلبی میشود؛ بهطوری که قلب ورزشکاران نخبه از کارایی بالاتر و سازگاری مطلوبتری نسبت به افراد غیرورزشکار برخوردار است
تعیین ارتباط بین دانشآفرینی سازمانی با تابآوری سازمانی از طریق شادکامی در محیط کار در بین معلمان تربیت بدنی شهرستان ممسنی: تعیین ارتباط بین دانشآفرینی سازمانی با تابآوری سازمانی از طریق شادکامی در محیط کار در بین معلمان تربیت بدنی شهرستان ممسنی
No full textهدف اصلی این پژوهش تعیین ارتباط بین دانشآفرینی سازمانی با تابآوری سازمانی از طریق شادکامی در محیط کار در بین معلمان تربیت بدنی شهرستان ممسنی بود. پژوهش حاضر، از لحاظ هدف، کاربردی و از نظر روش، توصیفی و از نوع همبستگی بوده است. جامعه آماری این پژوهش کلیه معلمان مدارس متوسطه اول در شهرستان ممسنی در سال تحصیلی 1404-1403 به تعداد 500 نفر بود. برای تعیین حجم نمونه از جدول مورگان استفاده و تعداد 220 نفر بعنوان حجم نمونه انتخاب شد. شیوه نمونه گیری به صورت طبقه ای تصادفی بود. برای گردآوری اطلاعات از پرسشنامه پسشنامه شادکامی در محل کار کرولف (2007)، پرسشنامه تابآوری سازمانی پرایاگ و همکاران (2018) و پرسشنامه دانش آفرینی رحیمی و همکاران (2011) استفاده شد. برای تجزیه و تحلیل دادهها از روش همبستگی، رگرسیون و معادلات ساختاری بر پایه نرم افزارهای SPSS27 و PLS3 استفاده شد. نتایج نشان داد که بین دانشآفرینی سازمانی با تابآوری سازمانی از طریق شادکامی در محیط کار در معلمان رابطه مثبت و معناداری وجود دارد. بین دانشآفرینی سازمانی و مولفههای آن با تابآوری سازمانی و شادکامی در محیط کار معلمان رابطه مثبت و معناداری وجود دارد. بین شادکامی در محیط کار و مولفههای آن با تابآوری سازمانی معلمان رابطه مثبت و معناداری وجود دارد. دانشآفرینی سازمانی قابلیت پیشبینی تابآوری سازمانی معلمان را دارد. همچنین دانشآفرینی سازمانی قابلیت پیشبینی شادکامی در محیط کار معلمان را دارد، و در نهایت مشخص گردید که شادکامی در محیط کار قابلیت پیشبینی تابآوری سازمانی معلمان را دارد
Biological Mechanisms and Therapeutic Potential of PRP-Fibrin Glue in Managing Refractory Pleural Effusion: Insights from a Pilot Study
No full textBackground: Refractory pleural effusion (RPE) is a serious post-cardiac surgery complication, often unresponsive to conventional chest tube drainage. This pilot clinical trial investigates platelet-rich plasma fibrin glue (PRP-FG), a non-invasive treatment previously effective for postoperative chylothorax and pneumothorax, to assess its efficacy in managing RPE resistant to standard thoracostomy.
Methods: The study enrolled 19 patients with unilateral or bilateral RPE unresponsive to conventional treatment. The primary outcome was successful treatment, defined as pleural effusion volume <50 mL/day for two consecutive days within one-week post-treatment, accompanied by symptomatic improvement.
Results: Participants had a mean age of 43.58 ± 19.58 years, with 52.6% having undergone coronary artery bypass grafting (CABG) and 36.8% congenital cardiac surgery. All patients showed reduced effusion volumes post-PRP-FG treatment. However, five patients (26.3%) required a second PRP-FG application, and three (15.8%) needed a third to achieve optimal outcomes. Pleural effusion volume significantly decreased from 624.21 ± 275.02 mL to 25.00 ± 20.07 mL (mean difference: 599.21 mL, p < 0.001, effect size d = 2.29). No recurrence of effusion was observed during follow-up. No severe adverse effects were reported; minor side effects, such as pleuritic pain and transient dyspnea, were managed with symptomatic treatment. One patient with poorly controlled diabetes died due to an unrelated sternal wound infection.
Conclusion: PRP-FG offers a safe, cost-effective, and minimally invasive treatment for RPE resistant to conventional methods. Larger, multicenter studies with extended follow-up are warranted to confirm its efficacy in managing persistent pleural effusion
An Exploratory Study Using an Artificial Neural Network to Predict Fatigue from Mobile Phone Use: A Population-Based Survey in Algeria
Full text linkBackground: Mobile phones are widely used, raising questions about the possible health effects of the radiofrequency electromagnetic fields they emit. Fatigue is frequently reported in this context, but it is influenced by multiple, interacting factors that are difficult to model with classical statistical methods. Artificial neural networks (ANNs) may help explore these complex relationships.
Methods: An ANN model was developed to estimate fatigue associated with cell phone use. Data were collected via a semi-structured questionnaire completed by 478 Algerian participants. The survey recorded sociodemographic data and patterns of mobile phone use. The network had a 5-10-1 architecture, and its performance was evaluated by mean squared error (MSE) and the coefficient of determination (R²). A simple MATLAB interface was created to allow user input and display model outputs with a colour-coded indicator.
Results: The ANN achieved an MSE of 0.5993, indicating that it reproduced some general patterns in the data. However, the coefficient of determination was low (R² = 0.0338), showing that only a small proportion of the variability in fatigue scores was explained and that individual predictions were imprecise.
Conclusion: This exploratory study suggests that ANN-based models are feasible for analysing fatigue associated with mobile phone use, but the findings should be regarded as preliminary and are subject to some limitations. Larger, more diverse samples and richer, preferably objective, exposure and health measures will be required before such tools can be used for reliable risk assessment or public health guidance
Exploring how core beliefs shape risky self-injury in teens: the bridge of emotional control challenges
Full text linkBackground: Teen years represent a pivotal phase of growth, fraught with distinct risks like tendencies toward self-injurious actions. Our research explored the straightforward connection between core life principles and such behaviors among teens, while probing how challenges in managing feelings act as an intermediary in this dynamic.
Methods: Employing a correlative design with structural equation modeling, we targeted Tehran high schoolers aged 14–18 from the 2023–2024 school term. Through multi-phase clustered random selection, 356 youths joined, filling out tools like the Self-Harm Inventory, abbreviated Portrait Values Questionnaire, and compact Difficulties in Emotion Regulation Scale. We crunched numbers via SPSS 27.0 and AMOS.
Results: The proposed framework aligned well with real-world findings. As predicted, stronger life principles linked inversely to self-injury (β= 0.11, P=0.044). Emotional control issues tied positively to these behaviors (β=0.37, P=0.001). Notably, feeling management woes bridged part of the gap between principles and self-injury (β=-0.06, P=0.007).
Conclusion: This study highlights that personal values, emphasizing meaning, purpose, and prosocial behaviors (e.g., benevolence, universalism), protect against adolescent self-harm by enhancing emotion regulation. Integrating values-based interventions and emotion regulation training into prevention programs is recommended for clinicians and educators to reduce self-harm risks effectively
بررسی وضعیت مخاطرات زیستی و بهداشتی استقرار صنایع آلاینده در استانهای ایران و ارائه راهکارهایی اخلاق مدار برای رفع آن
No full textBackground and Aim: The particles and gases that constitute air pollution cause harmful health effects in humans. Evidence from various studies indicates that reducing air pollution levels leads to improved health outcomes. One reason countries resist reducing environmental pollutants is the decline in production levels due to reduced use of fossil fuels. Meanwhile, the concentration of polluting industries in some areas has resulted in environmental costs being imposed on polluted regions due to the deterioration of health indicators.
Methods: This article, with a broad perspective and irrespective of political issues, seeks to propose a model for the placement of industries aimed at enhancing health indicators to create the most favorable conditions. To achieve the research objectives, two models were utilized: a regional data input-output model and econometric analysis.
Ethical Considerations: In this research, ethical considerations in a library study including the authenticity of texts, honesty and trustworthiness, have been considered.
Findings: Based on the findings of the research, it can be stated that the way of setting up industries in the country is not in an optimal situation from the moral and economic point of view, because the most polluting areas are not likely to cause the most environmental damage and some areas have a much higher share of pollution per capita. They allocate themselves.
Conclusion: Based on the research findings, policymakers can adopt two strategies to reduce environmental costs and improve health indicators: the first strategy involves establishing restrictions on industrial placement in each region according to the distance of per capita emissions per unit area. In the second step, if any firm violates these regulations in any region, an environmental tax should be imposed in areas with higher density, and these taxes should be distributed among the citizens of that region to compensate for these costs or be allocated to infrastructure aimed at offsetting environmental costs.زمینه و هدف: ذرات و گازهایی كه آلودگی هوا را تشكیل میدهند، باعث اثرات مضر بهداشتی در انسان میشوند. شواهدی از مطالعات مختلف نشان میدهد كه كاهش سطح آلودگی هوا منجر به بهبود نتایج سلامتی میشود. یكی از دلایلی كه كشورها در مقابل كاهش آلایندههای زیستمحیطی از خود مقاومت نشان میدهند، كاهش سطح تولیدات به واسطه كاهش استفاده از سوختهای فسیلی است این در حالی است كه تجمع صنایع آلاینده در برخی از مناطق باعث شده هزینههای زیستمحیطی به واسطه نامطلوبشدن شاخصهای سلامت بر مناطق آلاینده تحمیل شود.
روش: این مقاله با نگاه كلی، فارغ از موضوعات سیاسی به دنبال الگویی است كه با هدف ارتقای شاخصهای سلامت، جایابی صنایع را به نحوی پیشنهاد میدهد كه وضعیتی بهینه ایجاد شود. جهت دستیابی به اهداف تحقیق از دو الگوی داده ستانده منطقهای و اقتصادسنجی استفاده شده است.
ملاحظات اخلاقی: در این تحقیق جنبههای اخلاقی در مطالعه كتابخانهای شامل اصالت متون، صداقت و امانتداری رعایت شده است.
یافتهها: براساس یافتههای تحقیق میتوان بیان نمود كه نحوه استقرار صنایع در كشور از نظر اخلاقی و اقتصادی در وضعیت بهینه قرار ندارد، زیرا آلایندهترین مناطق بیشترین خسارت زیستمحیطی را متحمل نمیشوند و برخی از مناطق به صورت سرانه سهم بسیار بالاتری از انتشار آلودگی را به خود اختصاص میدهند.
نتیجهگیری: براساس نتایج تحقیق سیاستگذاران جهت كاهش هزینههای زیستمحیطی، بهبود شاخصهای سلامت و ارتقای اخلاقمداری زیستی دو راهكار میتوانند استفاده نمایند: راهكار اول در هر منطقه با توجه به فاصله میزان سرانه انتشار آلایندگی به ازای واحدهای سطح محدودیتهای استقرار صنایع وضع گردد و در گام دوم در صورت تخطی هر بنگاه در هر منطقه مالیات آلایندگی در مناطق با تراكم بالاتر وضع و این مالیاتها جهت جبران این هزینهها بین شهروندان آن منطقه توزیع گردد و یا اینكه صرف زیرساختهای جبران هزینههای زیستمحیطی گردد
نقش مشارکت ورزشی بر نشاط اجتماعی و بهره¬وری سازمانی کارکنان شرکت آذین الکترونیک پیشرو
No full textمقدمه: هدف از پژوهش حاضر بررسی نقش مشارکت ورزشی بر نشاط اجتماعی و بهرهوری سازمانی کارکنان شرکت آذین الکترونیک پیشرو بود.
روش کار: روش پژوهش حاضر توصیفی از نوع همبستگی، بر اساس زمان حالنگر، از نظر هدف کاربردی و به لحاظ جمعآوری اطلاعات میدانی بود. جامعة آماري پژوهش شامل همه کارکنانی هستند که در سال 1404 در شرکت آذین الکترونیک پیشرو تهران فعالیت داشتند که تعداد کل آنها بنا بر آمار اعلام شده از سوی شرکت الکترونیک پیشرو 500 نفر بودند. نمونه آماری طبق جدول مورگان تعداد 217 نفر از بين جامعه آماري بودند که به صورت در دسترس انتخاب شدند. از پرسشنامههای مشارکت ورزشی گیل، گروس و هادلستون (1983)، نشاط اجتماعی تمیزی فر و عزیزی مهر (1396) و بهرهوری سازمانی شریفی (1391) برای اندازهگیری متغیرهای تحقیق استفاده شد.
یافتهها: مشارکت ورزشی بر نشاط اجتماعی و بهرهوری سازمانی کارکنان شرکت آذین الکترونیک پیشرو نقش داشت.
نتیجهگیری: بنابراین با کمک مشارکت ورزشی میتوان منجر به افزایش نشاط اجتماعی و بهرهوری سازمانی کارکنان شرکت آذین الکترونیک پیشرو شد. لذا پیشنهاد میشود که مدیران به وضعیت نشاط اجتماعی و بهرهوری سازمانی در کارکنان توجه بیشتری نمایند تا از طریق مشارکت ورزشی به نشاط اجتماعی و بهرهوری سازمانی کمک نمایند
Metabolomic Profiling of Industrially Associated Lactococcus Strains for Consortia Design
Full text linkBackground and Objective: The metabolomic profile represents the totality of all low-molecular-weight metabolites. It reflects the physiological state of micro-organisms and their potential for biotechnological uses. The aim of this study was to analyze the metabolomic profile of Lactococcus strains, which allowed the researchers to study the characteristics of these microorganisms. As a result, principles for designing lactic acid bacteria consortia with pronounced production-significant chara-cteristics for the creation of next-generation fermented milk products can be developed.
Material and Methods: The study microbes were Lactococcus strains. The content of organic and amino acids in the experimental samples was assessed by capillary electrophoresis. The contents of vitamins, mono- and disaccharides were assessed using high-performance liquid chromatography.
Results and Conclusion: The metabolomic profiles of 16 industrially associated Lactococcus strains were analyzed. All strains produced lactic acid, with the highest content observed in Strain Ll1, at 8677.8 mg·kg-1. The experimental samples showed a significant increase in methionine content, with the highest value of 311.2 mg·(100 g)-1 in Strain Ll3 and cystine content, with the highest value of 45.6 mg·(100 g)-1 in Strain Ld1. Strain Lc8 showed the ability to synthesize methionine and cystine, which might be promising in cheese production, since these sulfur-containing amino acids contribute to the aroma and flavor of fermented milk products. This strain produced formic, succinic and acetic acids and could catabolize citric acid and galactose. The Strains Ld1, Ld2 and Ld3 showed galactose accumulation, indicating a lack of enzymatic activity for the oxidation of this monosaccharide. Literature data on variability in vitamin synthesis levels between the strains have been verified. Thus, only Strain Ld2 synthesized vitamin B2, the content of which significantly exceeded the control and reached 103.18 ±2.06 μg·(100 g)-1. Five strains were capable of synthesizing vitamin B6 up to 247.69 ±5.45 μg·(100 g)-1. The vitamin B9 content increased, compared to the control, in eight samples, with maximum values of 69.59 ±1.46 μg·(100 g)-1 for Strains Ll3. The dataset on lactococci metabolomic profiles can serve as a basis for the further development of methodological systems, mathematical models and algorithms for selecting strains for the creation of consortia and the production of products, including those with functional characteristics. The results can serve as a basis for the design of consortia of lactic acid bacteria in biotechnological approaches to produce novel fermented milk products with specific characteristics, including functional ones, which include positive effects on human organs when consumed regularly.
Keywords: Lactic acid bacteria, Lactococcus, Functional characteristics, Metabolomic profiles, Metabolites
Introduction
Fermented dairy products contain various metabolites produced by starter cultures that offer health benefits to the human body. These include organic acids, exopolysacchar-ides, vitamins and nitrogenous compounds such as peptides and amino acids. Metabolites are formed as a result of the vital activity of lactic acid bacteria (LAB), which ferment lactose into lactic acid, hydrolyze proteins and lipids to peptides, amino acids and free fatty acids. Some metabolites can regulate human metabolism, decrease inflammation or include antimicrobial effects [1]. Lactococcus bacteria (Streptococcaceae family) are among industrially asso-ciated bacteria in the dairy industry. They are Gram-positive, facultative anaerobic catalase-negative cocci [2]. Their primary role in fermentation processes includes efficient conversion of lactose into lactic acid, thereby increasing medium acidity, which enhances antimicrobial characteristics and extends the shelf life of the final product [3]. Lactose metabolism occurs via the pentose phosphate pathway and/or the Embden-Meyerhof-Parnas pathway (glycolysis) through the action of the enzyme β-galactosidase, which hydrolyzes lactose into glucose and galactose. Glucose enters glycolysis, forming pyruvate, which is subsequently reduced to L-lactic acid by lactate dehydrogenase. Another major route of lactose catabolism is the pentose phosphate pathway, in which lactose is phosphorylated to lactose-6-phosphate during transport and then cleaved into glucose and galactose-6-phosphate by phospho-β-galactosidase. Then, galactose-6-phosphate is converted into tagetose-6-phosphate by isomerase, which turns into glyceraldehyde-3-phosphate and dihydroxyace-tone phosphate. Glyceraldehyde-3-phosphate enters glycol-ysis and leads to pyruvate and L-lactic acid formation [4]. The accumulation of L-lactic acid leads to a decrease in the pH of the medium, which determines the intensity and pathway of lactic acid fermentation. Other key factors affecting this process are the acidification rate and the presence of oxygen and carbon dioxide in the medium. Within the Genus Lactococcus, L. lactis subsp. lactis stands out for its higher rate of lactose utilization and the ability to ferment maltose and trehalose [2].
Moreover, acid production is indirectly linked to exopolysaccharide (EPS) production. The EPS are high-molecular-weight carbohydrate polymers (either homo or heteropolysaccharides) secreted by microorganisms into the extracellular environment. Their precursors are intermediate metabolites of glycolysis and the pentose phosphate pathway glucose-6-phosphate, fructose-6-phosphate, uridine diphosphate glucose and other nucleotide sugars. Since these intermediates serve as precursors for lactic acid, a competitive relationship for carbohydrate substrates occurs [5]. The highest activity of the enzymatic complex of EPS synthesis is achieved at lower acidity levels during lactate biosynthesis (pH 5.0–5.5) [6, 7]. The production of EPS leads to the formation of a slimy layer surrounding the cell, which forms a colloidal aggregate in the form of an amorphous substance with little or no cell adhesion or a cohesive capsule [8]. Acid formation and then EPS production by Lactococcus spp. contribute to the texture of dairy fermented products, resulting in a smooth, creamy, homogeneous consistency with possibly slight ropiness.
Cow milk contains 0.4–0.5 mg·(100 g)-1 of potassium and sodium citrates. Among Lactococcus species, only L. lactis subsp. lactis var. Diacetylactis is capable of citrate metabolism. End products of citrate metabolism include diacetyl, acetoin, 2,3-butanediol, acetaldehyde, ethanol and lactic acid. The induction of the citrate permease catalytic enzyme is caused by the presence of lactic acid rather than citrate, which is synthesized from glucose and citrate under acidic pH conditions [9]. Thus, acid formation affects citrate metabolism. The most valuable metabolic products in this biosynthesis are diacetyl and carbon dioxide, as they improve the texture and flavor of fermented dairy products.
Lactococcus species include a limited ability to synthesize amino acids de novo from inorganic carbon sources and are therefore auxotrophic. However, fermented dairy products can be enriched with amino acids through the proteolysis of milk caseins [10]. Extracellular proteinases of Lactococcus hydrolyze caseins into oligopeptides, which are then transported into the cell and cleaved by intracellular peptidases into amino acids [11]. The activity of proteolytic enzymes and transport systems depends on the availability of nitrogen, acidity and the content of free amino acids and is controlled by regulatory systems. Particularly, L. lactis subsp. cremoris cleaves β-casein with the similar protease specificity as L. lactis subsp. lactis, but has less active proteolysis [12]. Amino acids are used for cellular biosynthesis and as precursors of biologically active compounds. Several amino acids are degraded to volatile compounds (aldehydes, ketones and alcohols), which can affect the sensory characteristics of fermented dairy products [12, 13]. Partial hydrolysis of milk proteins by lactococci, with lactose reduction, enhances digestibility in the human gastrointestinal tract (GIT) and may decrease the risk of intolerance, compared to raw milk.
Amino acids play a critical role in the synthesis of the bacteriocin of nisin (a polycyclic peptide composed of 34 amino acid residues, including the non-proteinogenic amino acid lanthionine) which is active against a broad spectrum of Gram-positive bacteria [14]. Studies have shown that the presence of amino acids such as alanine, isoleucine, serine, glutamic acid, tyrosine and tryptophan in the culture medium enhances bactericidal activity against strains of Escherichia coli and Staphylococcus aureus [14, 15]. Regarding vitamin biosynthesis, Lactococcus species are largely auxotrophic due to their adaptation to nutrient-rich environments such as milk, where several vitamins are readily available. Nevertheless, certain strains have the ability to synthesize B-group vitamins, such as folates (B9) and riboflavin (B2) [16]. Folate production levels vary significantly between the strains. Efforts to enhance B9 biosynthesis in LAB have involved optimized cultivation conditions and genetic modification through strain selection and metabolic engineering [17]. The ability of Lactococcus strains to produce riboflavin is limited and highly strain-dependent. Some strains function as “riboflavin producers” and are used to enrich dairy products with improved nutritional value, as well as imparting a yellowish color to whey and cheeses [18, 19].
The metabolomic profile reflects the key characteristics of the microorganism that determine its technological potential in dairy production. Industrially associated characteristics of strains include technological significance in the production of dairy products (e.g., fermentation time and viscosity) Understanding the genetic and regulatory mechanisms underlying metabolite biosynthesis pathways in Lactococcus species enables the targeted selection of starter cultures and development of microbial consortia, which is critical for creating novel biotechnological products. The aim of this study was to analyze the metabolomic profile of Lactococcus strains, which allowed the researchers to study the characteristics of these microorganisms. As a result, principles for designing LAB consortia with pronounced production-significant charact-eristics for the creation of next-generation fermented milk products can be developed.
Materials and Methods
2.1. Experimental samples
The microorganisms of this study were Lactococcus strains from the collection of the All-Russian Research Institute of the Dairy Industry (“VNIMI”), selected based on an evaluation of the most important characteristics for dairy processing, including the viability after long-term storage, acidification rate, titratable acidity and pH, apparent viscosity and sensory characteristics. The selected strains included four strains of L. lactis subsp. lactis biovar diacetylactis (Ld1, Ld2, Ld3 and Ld4), eight strains of L. lactis subsp. cremoris (Lc1, Lc2, Lc3, Lc4, Lc5, Lc6, Lc7 and Lc8) and four strains of L. lactis subsp. lactis (Ll1, Ll2, Ll3 and Ll4). Before use, the strain was preserved at -80 °C under glycerol. The strain was then cultured in milk at 30 °C. A second-generation culture was used. The initial cell concentration was 107 CFU·ml-1. To obtain experimental samples, sterile skimmed milk (the "Standard" brand Complimilk, Belarus) was fermented with the test strain at a concentration of 5% and incubated at (30 °C ±2) until a clot formed (рН 4.5 ±0.2). The control was sterile skim milk. The milk was prepared by dissolving 90 g of dry skim milk in 1000 ml of tap water and sterilized by autoclaving using N-Bioteck sterilizer, Korea, at 121°C for 3 min. The initial pH was 6.8 and was set in a thermostat with the experimental samples at 30 °C. A limitation of this study was the number of strains (40). The study was limited to assessing the characteristics of representatives of a genus, lactococci, the most common type of LAB used in food production. The studied strains were isolated from homemade fermented products and natural sources in a temperate climate zone. Moreover, the study did not include genetically modified strains.
2.2. Assessment of organic and amino acids
The content of organic acids in the experimental samples was assessed using capillary electrophoresis and "Kapel 205" system, equipped with a spectrophotometric detector and a quartz capillary (75-µm inner diameter, 60-cm total length). Samples were pre-diluted with distilled water. The buffer electrolyte was prepared using benzoic acid, diethanolamine, cetyltrimethylammonium bromide and Trilon B. Separation was carried out at 20 kV with UV detection at 254 nm. Electropherograms were processed using "Elforan" software.
For the assessment of amino acid composition, the samples underwent acid hydrolysis and, alkaline hydrolysis for tryptophan, to convert protein-bound amino acids into free forms. All amino acids, except tryptophan, were derivatized into phenylisothiocarbamyl derivatives and quantified via capillary electrophoresis. Tryptophan was assessed directly without derivatization using borate buffer, +25 kV voltage and UV detection at 219 nm. Glutamic acid, aspartic acid and cystine were analyzed in phosphate buffer with β-cyclodextrin under +25 kV voltage, 50 mbar pressure and UV detection at 254 nm. The rest of amino acids (arginine, lysine, tyrosine, phenylalanine, histidine, leucine and isoleucine, methionine, valine, hydroxyproline, proline, threonine, serine, alanine and glycine) were assessed using similar method without pressure. All electropherograms were processed using "Elforan" software. For such amino acids as tryptophan, glutamic acid and glutamine, and aspartic acid and asparagine, the associated error was ±20%; for arginine, tyrosine, phenylalanine, histidine and methionine, the associated error was ±23%; for lysine, leucine and isoleucine, valine, proline, threonine, serine, alanine and glycine, the associated error was ±18%; for cystine and cysteine, the associated error was ±24%; and for organic acids, the associated error was ±20%.
2.3. Assessment of mono and disaccharide contents
The contents of mono and disaccharides in the experimental samples were assessed using high-perfor-mance liquid chromatography (HPLC) in accordance with GOST 54760-2011, “Component milk products and infant milk products. Assessment of mono and di-sugars mass concentration using HPLC method.” The analysis was carried out using MAESTRO liquid chromatograph (INTER-LAB, Russia), with a Zorbax carbohydrate analysis column (5 μm, 4.6 × 250 mm; Agilent Technologies, USA), CAU-X-320 electronic analytical balance (CAS, Korea), variable-volume single-channel pipette (100–1000 μl; BIOHIT, Finland; Sartorius Biohit Liquid Handling, Germany) and Sigma 1-14 microcentrifuge (Sigma Laborz-entrifugen, Germany). The associated measurement error was ±12%.
2.4 Assessment of vitamins
Vitamin C was assessed using Agilent 1260 chromato-graphy system equipped with a gradient 4-channel pump, a diode array detector, a column thermostat and an automatic sample delivery system. The associated measurement error was ±34%. The analyte was extracted by diluting the sample aliquot 1:2 with an extracting solution (3% metaphosphoric acid). It was vortexed and centrifuged at 10.850× g. The supernatant was filtered through a 0.22-μm filter. The resulting filtrate was used for analysis. Separation of components was carried out by reversed-phase chromate-graphy on an Agilent Extend-C18 4.6*250 mm column. Vitamin C was eluted under isocratic conditions using mobile phase of 30 mM phosphate buffer, pH 2.6, at a flow rate of 1 ml·min-1. Detection was carried out using diode array detector at 254 nm.
B-group vitamins were assessed using HPLC with an Agilent 1260 Infinity II HPLC device combined with an Ultivo Triple Quad LC/MS mod. 6465, Singapore, Agilent Technologies and an Agilent 1260 Infinity II HPLC device with a diode array detector G7115A. Chromatographic separation was carried out using Agilent InfinityLab 120 Poroshell 120 Phenyl-Hexyl column (3.0 × 100 mm, 2.7 μm). For sample preparation, 1 g of the sample was mixed with 4 cm³ of deionized water, vortexed and then 5 cm³ of acetonitrile and 0.1 g of ascorbic acid were added to the mixture. The mixture was re-vortexed, ultrasonicated for 30 min and centrifuged at 3.650× g for 10 min. The sample was stored at -4 to -6 °C and filtered through a 0.22-μm filter.
2.5. Statistics
The reliability of data was verified through the execution of experiments in at least three independent replicates. Furthermore, MS Office Excel 2016 was used for data analysis and graph creation. Experimental data were presented in“mean value ± measurement error (relative)”. The data on vitamins were present as mean ± standard deviation. To calculate the significance of differences, ANOVA statistical method was used, with the accepted limitation of statistical significance of differences between options at p ≤ 0,05. The study was carried out using equipment of the Collaborative Center of the All-Russian Dairy Research Institute (CKP “VNIMI”).
Results and Discussion
3.1. Assessment of amino acids
Amino acids are promising metabolites for biotech-nological uses. These compounds are used as artificial sweeteners, flavoring agents and feed additives and for pharmaceutical purposes [19, 20]. During the production of fermented dairy products, specific amino acids generated via casein proteolysis are responsible for the formation of thiols, alcohols, esters and aldehydes, contributing to a broad range of flavors [20]. Sulfur-containing amino acids play a critical role in the development of sensory charac-teristics, as well as in the antioxidant activity of fermented dairy products [21]. In the experimental samples containing the industrially associated Lactococcus strains, the amino acids were assessed. Compared with the control (milk), a significant increase in the content of methionine and cystine was detected in the experimental samples. The results are presented in Figure 1.
A significant increase in amino acid content relative to the control was observed for methionine (Figure 1A) in Strains Lc2, Lc6, Lc8, Lc4, Ll2, Ll3, Ll4, with the highest content in Strain Ll3 at 311.2 mg·(100 g)-1. The sulfur-containing amino acid methionine plays a role in the initiation of transcription and is critical in various methyltransferase reactions. Aromatic compounds such as 3-methylbutanal, methanethiol, dimethyl sulfide, 2-methylpropanol and dimethyl trisulfide are synthesized from methionine [22]. Among these, methanethiol is particularly important for its contribution to the desirable flavor of cheddar cheese, especially in combination with diacetyl and butyric acid [23]. Increase in cystine content (Figure 1B) was observed in Strains Ld1, Ld2 and Lc8, with the highest content in Strain Ld1 [45.6 mg·(100 g)-1], compared to the control. Cystine is the oxidized form of cysteine, which serves as a key metabolite for synthesizing most sulfur-containing cellular compounds. Cysteine is involved in protein folding, assembly and stability through the formation of disulfide bonds. Additionally, cysteine-containing proteins such as thioredoxin and glutathione play essential roles in protecting cells against oxidative stress [24]. The strain with the conditional designation (Lc8) is capable of synthesizing methionine and cystine. The levels of other amino acids were similar to or less than those of the control, suggesting their active utilization by microorganisms during growth.
3.2. Assessment of organic acid content
One of the fundamental criteria for selecting Lactococcus strains for starter culture consortia is their ability to produce acid. Organic acids such as acetic, lactic, citric, succinic and propionic acids are produced by LAB during carbohydrate metabolism. As intermediates in metabolic pathways, these acids enhance the taste of products and extend shelf life through their antimicrobial and antioxidant characteristics; thereby, increasing consumer value [25,26]. The results of assessment of the content of organic acids are presented in Figure 2.
All the samples showed a significant increase in lactic acid content, compared to the control (Figure 2). The highest levels were observed in Strains Ld1 and Ll1, reaching 8116.5 and 8677.8 mg·kg-1, respectively. Lactic acid synthesis indicates the β-galactosidase enzymatic activity of these microorganisms. Lactic acid in fermented milk products acts as an acidity regulator and preservative. When ingested, lactic acid accelerates metabolism, decreases the number of pathogenic bacteria and also has antioxidant characteristics.
Only trace quantities of citric acid were detected in Samples Lc1, Lc3 and Lc5, which indicated the presence of a citrate pathway for the breakdown of this substrate. The consumption of citric acid depends on the presence of genes encoding the enzymes citrate permease and citrate lyase, which ensure the transport of citrate into the cell and its catabolism into oxaloacetate [27]. As a result of breakdown by lactococci, gas formation occurs in dairy products. In the production of certain products such as cheeses, this is addressed as a positive phenomenon, leading to the formation of "eyes." However, if its presence is assessed, this can lead to product defects. The breakdown of citric acid by these microorganisms leads to the formation of acetate, lactic acid, diacetyl and acetoin. Diacetyl and acetoin create flavor and aroma in food products, which is promising for the production of st
Purification and Antimicrobial Use of Egg-white Lysozyme against Staphylococcus aureus
Full text linkBackground and Objective: Staphylococcus aureus is a significant foodborne and zoonotic pathogen. This study aimed to enhance the anti-Staphylococcus aureus activity of egg-white lysozyme through heat treatment and synergistic combinations with natural antimicrobials.
Material and Methods: The lysozyme was purified from egg white via ammonium sulfate precipitation and cation-exchange chromatography, yielding a homogeneous protein. Anti-Staphylococcus aureus activity of native lysozyme, heat-treated lysozyme and its combination with ferulic acid or Mycobacterium smegmatis acyltransferase was assessed, respectively. All experiments were carried out in triplicate and statistical analysis was carried out using SPSS software.
Results and Conclusion: The specific activity of lysozyme to Micrococcus lysodeikticus was 27,407.4 U.mg-1. The lysozyme IC50 against Staphylococcus aureus was 300.8 µg.ml-1, with transmission electron microscopy verifying bacteriolytic action. Heat treatment under optimized conditions (90°C, 15 min, pH 6.2) significantly enhanced the lysozyme antibacterial activity by 35.1%, which was correlated with structural changes evidenced by circular dichroism spectroscopy. Furthermore, synergistic effects were observed when heat-treated lysozyme was combined with ferulic acid or Mycobacterium smegmatis acyltransferase (MsAcT), leading to prolonged inhibition and decreased viable bacterial counts. The findings of this research demonstrated that structural modifications and combinatorial strategies could effectively improve the efficacy and application potential of lysozyme as a natural antimicrobial agent in food safety.
Keywords: Egg-white lysozyme, Ferulic acid, Heat-induced fibrillar aggregates, Mycobacterium smegmatis acyltransferase, Staphylococcus aureus
Introduction
Staphylococcus aureus is a Gram-positive bacterium widely spread in nature and a common foodborne pathogen. The enterotoxins (SEs) of the bacteria show significant heat resistance, rendering them difficult to completely eliminate through conventional cooking methods and posing a significant risk of food poisoning. Furthermore, S. aureus facilitates cross-infection between humans and animals via the food chain through contamination of animal feed, subsequent infection of animals and transmission to humans, establishing it as an important zoonotic pathogen. The S. aureus has been detected in various animal species and a wide range of food products, with its prevalence continuously increasing on a global scale. Therefore, S. aureus is still a high-priority target in food safety monitoring [1, 2].
To combat S. aureus contamination in food processing, diverse biological control strategies have been investigated. These include inhibition using lactic acid bacteria (LAB) probiotics [3], Lysostaphin [4], bacteriophages [5,6], essential oils (EO) [5] and lysozyme [7,8]. From these, egg-white lysozyme has widely been used due to its high catalytic activity, simple preparation and cost-effectiveness [7,9]. It inhibits S. aureus through two primary mechanisms of (1) lytic mechanism as lysozyme hydrolyzses β-1, 4-glycosidic bonds to peptidoglycan, which causes cell wall damage, induces cell lysis and results in bactericidal activity; and (2) the non-lytic mechanism, where under denaturing conditions, it suppresses growth through inherent protein characteristics such as hydrophobicity and cationic effects [8,10]. While the antimicrobial characteristics of native egg-white lysozyme are well-documented, its efficacy under common food processing conditions, particularly those involving heat, needs further investigation. Moreover, strategies to enhance its activity, especially in a heat-treated state, through combination with other natural antimicrobial agents are still under-investigation. Under various reaction conditions, the interactions between other natural antibacterial agents and egg-white lysozyme can lead to various effects on its antimicrobial activity. For example, in an alkaline solution, theaflavin covalently binds to egg-white lysozyme, resulting in significant decrease of its antibacterial activity [11]. However, in an amyloid fibril hydrogel, the interaction between epigallocatechin gallate (EGCG) and egg-white lysozyme significantly broadens the antibacterial spectrum of egg-white lysozyme [12]. Therefore, an in-depth investigation into the interaction conditions between natural antibacterial factors and egg-white lysozyme is greatly important for enhancing the antibacterial efficiency of egg-white lysozyme.
This research aimed to isolate and purify egg-white lysozyme using chromatography and to assess the effect of heat treatment on its anti-S. aureus activity. Furthermore, the study prepared a synergistic combination of heat-treated lysozyme with ferulic acid and acyltransferase to develop an enhanced strategy for suppressing S. aureus growth. The experimental results demonstrated that heat-induced structural modification (fibrillar aggregation) enhanced lysozyme antimicrobial mechanism beyond native peptidoglycan hydrolysis. Moreover, it has first been reported that a synergistic combination of heat-treated lysozyme with ferulic acid and MsAcT against S. aureus significantly improves the efficacy and time of inhibition.
Materials and Methods
2.1. Strains, biochemical reagents and chemical reagents
Micrococcus lysodeikticus CGMCC 1.4547 and S. aureus CGMCC 1.282 were purchased from China General Microbiological Culture Collection Center, China. Bovine serum albumin (BSA) and standard protein molecular weight marker were purchased from Takara Biomedical Technology, China. Moreover, CM-Sepharose fast flow chromatography column was purchased from GE Healthcare, China. Ferulic acid, caffeic acid, gallic acid and N-acetylglucosamine (NAG) included analytical grade unless otherwise specified and purchased from Shanghai Macklin Biochemical Technology, China.
2.2. Purification of egg-white lysozyme
Egg white was initially diluted by 50-fold using 50 mmol.l-1 pH8.0 tris-HCl buffer and the resulting protein solution was ultrasonicated for 10 min. The supernatant was collected by centrifugation at 12,000 rpm for 5 min at 4 °C. Solid ammonium sulfate was added to the supernatant to achieve 40% (w/v) saturation and the mixture was set to precipitate for 2 h at 4 °C. After centrifugation, the precipitate was removed and the collected supernatant was further loaded onto a CM-Sepharose fast flow column (5 × 20 cm) that was pre-equilibrated with 50 mmol.l-1 tris-HCl buffer (pH 8.5). The lysozyme was eluted with a 5-fold column volume of 50 mmol.l-1 tris-HCl buffer (pH 8.5) with increasing concentrations of NaCl (0.1 and 0.5 mol.l-1) at a flow rate of 20 ml.h-1, respectively. The active fractions were pooled. Protein concentration was assessed using Bradford method with BSA as a standard. The homogeneity of the purified egg-white lysozyme was assessed using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
2.3. Activity assay of egg-white lysozyme
The cell lysis activity of lysozyme from egg white to M. lysodeikticus was quantitatively assessed using spectrophotometeric assay method, which was described by the National Standard of the People's Republic of China (GB/T 30990-2014, Determination of Lysozyme Acyivity) as well as Naveed et al. [13]. Briefly, after incubation overnight, M. lysodeikticus was transferred into the fresh LB liquid media at 1% (v/v) and incubated at 37 °C for 2 h at 220 rpm. The cells were harvested by centrifugation and resuspended in 50 mmol.l-1 Na2HPO4-Na2HPO4 buffer (pH 6.2) to a final concentration of 5 × 105 CFU.ml-1. The reaction system contained 2.5 ml of cell suspension and 0.5 ml of lysozyme solution, while the control system received 0.5 ml of inactivated lysozyme solution. The reaction was carried out at 50 mmol.l-1 Na2HPO4-Na2HPO4 buffer (pH 6.2) for 5 min at 25 °C and the absorbance of the reaction system was continuously monitored at 450 nm. One unit of lysozyme activity (U) was defined as the quantity of enzyme necessary to decrease OD450 by 0.001 per minute under the standard assay condition.
2.4. Anti-Staphylococcus aureus activity of egg-white lysozyme
Briefly, S. aureus in the logarithmic growth phase was used as an indicator microorganism to assess antibacterial activity of the purified lysozyme. To set the growth curve, overnight-cultured S. aureus was subcultured into the fresh LB liquid media at a 1% (v/v) inoculum and incubated at 37 °C for 14 h at 220 rpm. Samples were collected every 30 min to measure the optical density (OD) of the culture broth at 600 nm. The growth curve was plotted with OD600 on the y-axis and incubation time on the x-axis.
The antibacterial assessments were carried out using a method described by Carrillo et al. [14] with slight modification. Following 2 h of subculture, S. aureus suspensions were adjusted to 5 × 105 CFU.ml-1 through serial dilution in LB liquid media. Aliquots of S. aureus suspensions were mixed with equal volumes of the purified lysozyme at various concentrations and incubated at 37 °C for 6 h at 220 rpm. The OD600 of the mixture was recorded after a 6-h subculture. The control group used 50 mmol.l-1 Na2HPO4-Na2HPO4 filter-sterilized buffer (pH 6.2) instead of lysozyme. Antibacterial activity of the purified lysozyme to S. aureus was assessed using the antibacterial ratio. Antibacterial ratio was calculated using the following equation of R = (A - B) ÷ A × 100%; in which, R was the antibacterial ratio (%); A was the OD600 value of the control group; and B was the OD600 value of the experimental group. The antibacterial effect curve was plotted with the antibacterial ratio on the y-axis and the logarithmic of the purified lysozyme concentration on the x-axis. The IC50 was defined as the purified lysozyme concentration, which resulted in a 50% decrease in the level of the antibacterial ratio, compared with untreated groups after a 6-h treatment. The IC50 value was calculated using GraphPad Prism software. The cell morphology of S. aureus after a 6-h lysozyme treatment (IC50 concentration) was reported using transmission electron microscopy (TEM).
2.5. Effects of heat treatment on anti-Staphylococcus aureus activity of the purified egg-white lysozyme
To assess the anti-S. aureus effects of the heat-treated egg-white lysozyme, a specific concentration of the purified lysozyme solution was incubated at various temperatures for a set duration before assessing its anti-S. aureus activity using water bath. In this study, three key parameters were primarily investigated, including heat treatment temperature, time and initial lysozyme concentration. The temperatures were set at 37, 50, 60, 70, 80, 90 and 100 °C, respectively. The treatment times were set at 5, 10, 15, 60, 120 and 240 min, respectively. The initial lysozyme concentration were set at 601.6, 1000, 2000, 4000 and 6000 μg.ml-1, respectively. The three highlighted factors were optimized using one-factor-at-a-time method. After heat treatment, equal volumes of the lysozyme solution and S. aureus suspension were thoroughly mixed and co-cultured at 37 °C for 6 h at 220 rpm. The antibacterial ratio was then quantified. Untreated lysozyme was used as control group.
To assess the effect of heat treatment on lysozyme molecular structure, circular dichroism (CD) spectroscopy was used to analyze changes in its secondary structural components before and after thermal exposure. The CD spectra were assessed using Jasco J-1500 spectropolarimeter (Jasco, Japan) with a 1-mm cell in the far-UV region from 190 to 300 nm. The concentration of lysozyme was 0.1 mg.ml-1 in 5 mmol.l-1 phosphate buffer (pH 6.2).
2.6. Inhibitory effects of phenolic acids combined with heat-treated egg-white lysozyme on Staphylococcus aureus
2.6.1. Compatibility screening of various phenolic acids with egg-white lysozyme
In this experiment, the inhibitory effects of egg-white lysozyme combined with three phenolic acids (ferulic acid, caffeic acid and gallic acid) at various concentrations on S. aureus growth were assessed, respectively. The final concentrations of each phenolic acid were 200, 400, 800 and 1000 μg.ml-1, while the final concentration of egg-white lysozyme was set at 300.8 μg.ml-1. The anti-S. aureus activity analysis and antibacterial ratio calculation methods were based on those described in Section 1.4. For the control group, phenolic acids or lysozyme were replaced with 50 mmol.l-1 Na2HPO4-Na2HPO4 buffer solution (pH 6.2).
2.6.2. Effects of the heat-treated lysozyme and ferulic acid combination on Staphylococcus aureus growth
The method for assessing the growth curves of S. aureus was based on Section 1.4. The heat treatment procedure for egg-white lysozyme was carried out according to Section 1.5, with the final concentration of heat-treated lysozyme adjusted to 300.8 μg.ml-1. For the control group, ferulic acid or lysozyme were substituted with 50 mmol.l-1 Na2HPO4-Na2HPO4 buffer solution (pH 6.2).
2.7. Numberized subsection inhibitory effects of Mycobacterium smegmatis acyltransferase combined with heat-treated egg-white lysozyme on Staphylococcus aureus growth
The mechanism by which, M. smegmatis acyltransferase (MsAcT) combined with heat-treated egg white lysozyme inhibited the growth of S. aureus is illustrated in Fig. 6.1. The preparation of the purified MsAcT was based on the methods described by Jia et al. [15]. Briefly, recombinant Escherichia coli BL21(DE3) strain was inoculated to Luria-Bertani (LB) broth and grown at 30 °C. The IPTG was added to culture broth to the final concentration of 1 mmol.l-1, when the OD600 reached 0.6–0.8. After 14 h interval, cell pellet was collected, resuspended using loading buffer (20 mmol.l-1 pH 7.4 Na2HPO4-NaH2PO4, 20 mmol.l-1 imidazole and 500 mmol.l-1 NaCl) and then lysed using sonication. The supernatant from the cell lysate was collected and directly loaded on the HisTrap HP affinity chromatography column pre-equilibrated with loading buffer, respectively. Recombinant protein was eluted with a linear gradient of 20 ml of 20–500 mmol.l-1 imidazole in the buffer with a flow rate of 0.8 ml.min-1. The fractions with pure MsAcT were pooled and dialyzed against 20 mmol.l-1 Na2HPO4-NaH2PO4 (pH 6.2) buffer overnight at 4 ◦C. The hydrolysis activity of MsAcT to NAG was assessed according to Jiang et al. [16] and Muzzarelli and Rocchetti [17]. In brief, the reaction mixture contained 20 μg.ml-1 NAG in 20 mmol.l-1 Na2HPO4-Na2HPO4 buffer (pH 6.2). Appropriately diluted MsAcT was added into the reaction mixture to create a linear dependence of the reaction rate to protein concentration. The reaction was carried out at 37 ◦C and the kinetics was detected for 3 h at 202 nm.
The specific procedure for inhibiting S. aureus growth using MsAcT combined with heat-treated egg-white lysozyme was carried out as follows: MsAcT, heat-treated egg white lysozyme and buffer solutions were sterilized using 0.22-μm filters. Then, 200 μl of appropriately diluted log-phase S. aureus cells were mixed with 200 μl of 1 mg.ml-1 MsAcT solution, while the control group received 200 μl of 50 mmol.l-1 NaH2PO4-Na2HPO4 solution (pH 6.2). After incubation at 37 °C for 3 h, 200 μl of the heat-treated egg-white lysozyme (final concentration of 300.8 μg.ml-1) were added to the experimental and control groups and mixed thoroughly, followed by incubation at 37 °C for 3 h. The samples were then centrifuged at 12,000 rpm for 3 min to separate the supernatant and the bacterial cell pellet. The protein concentration in the supernatant was measured to calculate the increase in protein, while the number of viable bacteria in the pellet was assessed according to the National Food Safety Standard of China (GB4789.2-2022, Food Microbiological Examination: Determination of Total Bacterial Count).
Results and Discussion
3.1. Purification and characterization of egg-white lysozyme
The purification of egg-white lysozyme was achieved through ammonium sulfate precipitation and CM-Sepharose fast flow column chromatography, resulting in 8.7-fold purification and yield of 41.0% (Table 1). Cation exchange chromatography verified particularly effective, accounting for 5.8-fold increase in specific activity. The purified enzyme was verified as homogenous using SDS-PAGE, showing a single band at 14.3 kDa (Fig. 1, Lane 6), which was similar to the molecular mass of egg-white lysozyme [18]. Its specific activity was assessed as 27,407.4 U.mg-1 using M. lysodeikticus cells as substrate. This value was less than that reported by Chen et al. [18], a discrepancy likely attributable to the use of whole cells in this assay instead of the isolated cell walls used in the highlighted study.
A gradual loss of total activity was observed throughout purification. Particularly, lysozyme was detected in pellets at a relatively low ammonium sulfate saturation (40%), suggesting the potential formation of insoluble aggregates under these conditions, as previously documented [19, 20]. For practical uses, it is critical to state that the growth state of M. lysodeikticus, affected by culture conditions and equipment, significantly affects the assessed specific activity [11, 13, 21]. Therefore, standardizing the substrate by setting the growth curve and harvesting log-phase cells under local laboratory conditions is essential for accurate activity assessment. In this study, the buffer solution for the assessment of the egg-white lysozyme activity according to the National Standard of the People's Republic of China (GB/T 30990-2014) requirements included 50 mmol.l-1 Na2HPO4-NaH2PO4 buffer (pH 6.2). In practical fields, particularly in the food processing industry, buffer solutions with pH 6.2 are rarely used.
Therefore, it is essential to assess optimal pH and temperature under the specific conditions necessary for the target uses, when assessing using effectiveness of egg-white lysozyme.
3.2. Anti-Staphylococcus aureus activity and mechanism of the purified egg-white lysozyme
The growth curve of S. aureus was characterized, identifying a log phase from 1 to 8 h (Fig. 2A), similar to the previous reports [22]. Cells from the exponential phase (2 h) were used for the assays. The IC50 of native lysozyme against S. aureus was 300.8 μg.ml-1 (Fig. 2B). The antibacterial efficacy of lysozyme is affected by the composition and sequence of bacterial cell wall peptidoglycan, as well as the physiological state of the enzyme [23, 24]. Lysozyme fights microbes through bacteriostatic, bactericidal and bacteriolytic mechanisms [25]. The transmission electron microscopy (TEM) images provided direct evidence of the bacteriolytic action, showing damaged S. aureus cell walls with distinct light/dark contrast and the collapse of cells, leading to leakage of intracellular contents (Fig. 2C, arrows). Despite this significant effect, the inhibitory activity of the native lysozyme is difficult to sustain over extended times (Figs. 2A, 5A), highlighting a limitation for its use as a standalone antimicrobial agent.
3.3. Enhancement of anti-Staphylococcus aureus activity using heat treatment and structural changes
Heat treatment under optimized conditions (600 μg.ml-1, 90°C, 15 min, pH 6.2) enhanced the anti-S. aureus activity of lysozyme by 35.1%, achieving 82.5% inhibition compared to the native enzyme (Fig. 3A–C). This increase in activity after thermal denaturation was similar to that against other microbes such as SARS-CoV-2 and Bacillus subtilis [26, 27, 28]. Structural analysis revealed the reason behind this enhancement as a significant rearrangement of secondary structure occurred, with α-helix content decreasing from 35.59 to 23.60% and β-sheet, β-turn and random coil structures increasing (Fig. 3D, Table 2). This unfolding and proliferation of β-sheets drived the formation of fibrillar aggregates [30,29], which were postulated to perforate microbial membranes, a mechanism distinct from the native enzyme peptidoglycan hydrolysis [27]. This suggested that structural modification wa a viable strategy to improve the efficacy of lysozyme.
3.4. Synergistic anti-Staphylococcus aureus effects of heat-treated egg-white lysozyme and phenolic acids
From the phenolic acids (ferulic, caffeic and gallic acids), ferulic acid showed the highest inhibition (52.2%) at 200 μg.ml-1, though differences diminished at higher concentrations (1000 μg.ml-1), where all acids reached ~99% inhibition (Fig. 4A–C). Ferulic acid is reported to inhibit S. aureus by suppressing tetK and MsrA efflux pumps on the bacterial membrane [31,32].
A combination of 400 μg.ml-1 ferulic acid with native lysozyme (300.8 μg.ml-1) showed a synergistic effect, increasing inhibition by respectively 12.3 and 29%, compared to either compound alone (Fig. 4A). This synergy was further increased, when ferulic acid was combined with heat-treated lysozyme, reducing bacterial biomass (OD600) by additional 18.7%, compared to the combination with native enzyme (Fig. 5A). The optimal protocol involved sequential addition of heat-treated lysozyme added at time zero, followed by ferulic acid after 6 h. This not only delayed the entry into the log phase by an additional hour but also decreased final biomass by 48.8% (Fig. 5B). This demonstrated that combining lysozyme with other antibacterial compounds, particularly after structural modification, could significantly enhance and prolong its inhibitory effect, potentially broadening its antibacterial spectrum [12,33]. However, when designing such combinations, concentration and addition sequence of egg-white lysozyme and phenolic acid, biocompatibility, reaction condition and differences in the mechanisms of action must carefully be addressed. Otherwise, adverse effects may occur. For e