Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411411EnglishN2022June3Healthcare
Efficacy of Sublingual Therapeutic Bacterial Vaccine in Patients with Recurrent Sore Throat: A Randomized Controlled Trial
English0105Abdul ShakoorEnglish Abdul Malik MujahidEnglish Muhammad Junaid AshrafEnglish Izzah IslamEnglish Junaid Rasul AwanEnglish Waqas AslamEnglish
Introduction: Recurrent sore throat is a significant burden on the healthcare system. It is associated with significant morbidity and antimicrobial resistance due to frequent antibiotic use. The sublingual live-attenuated polyvalent bacterial vaccine has been used as an adjunct treatment in these patients for its immunomodulatory properties, increasing immune responses, and boosting the innate immune system. Objective: To determine the efficacy of the sublingual live-attenuated polyvalent bacterial vaccine in patients with recurrent sore throats. Materials and Methods: This randomized control trial was conducted at the Department of ENT, Services Institute of Medical Sciences, Lahore from July, 2019 to January 2020 A Total of 60 patients full filling the inclusion criteria were included in the study and were equally divided into two groups. Each group had 30 patients to compare the mean number of sore throat episodes in the treatment and control groups. After approval from the hospital ethical review committee (No. Estt/ 20980/S.H), informed consent was obtained from each patient. Group A patients received a sublingual live-attenuated polyvalent bacterial vaccine and whereas group B were given placebo (normal saline 0.9%). Treatment response was noted for a period of 3 months. Results: The mean number of sore throat episodes was 0.53 ± 0.63 in the sublingual polyvalent live-attenuated bacterial vaccine group compared to 1.67 ± 0.92 in the placebo group (p-value =0.0001). The mean duration of disease in Group A was 3.20 ± 1.56 years compared to 3.40 ± 1.63 years in Group B. Conclusion: Sublingual live-attenuated polyvalent bacterial vaccine is effective in reducing the number of recurrent sore throat episodes and need for frequent antibiotic use and/or tonsillectomies.
EnglishRecurrent sore throat, Sublingual polyvalent bacterial vaccine, Efficacy, Live attenuated, Outcome, Placebo group
Introduction: Recurrent sore throat is one of the common health problems that is associated with significant morbidity. It is a common manifestation of upper respiratory tract infection, affecting the quality of life and contributing to antimicrobial resistance due to the widespread use of antibiotics.1 Currently, there are no formal recommendations for the prophylaxis of recurrent episodes of throat infection. Several studies have shown that the oral administration of bacterial vaccines resulted in a reduction in recurrent episodes of sore throat in adults and children by decreasing the numbers of these episodes, duration and severity.2,3,4 Although this problem is relatively more common in children but still there is a significant burden of this disease in adults5. Group A beta-hemolytic streptococci are one of the most common organisms to cause this problem.6,7 The treatment is primarily based on anti-inflammatory medications and antibiotics and prophylaxis is rarely provided to these patients. A recurrent sore throat can also cause lifelong problems such as rheumatic fever and post-infective renal problems in children8
The sublingual route of bacterial preparations has been proposed as a safer and more effective immunotherapy.9 Sublingual vaccine is a polyvalent bacterial preparation. It contains strains of different inactivated bacteria which are frequently present in the UPRT. Such bacteria include Streptococcus pneumonia, Staphylococcus aureus and many other bacterias.10Macchiet al. evaluated the prophylactic role of an immunostimulating bacterial lysate in patients with recurrent URTI and showed promising results.11
As there is no local data available so result of this study will help to evaluate the role of the sublingual live-attenuated bacterial vaccine in reducing recurrent episodes of sore throat.
Objective: To determine the efficacy of sublingual live-attenuated polyvalent bacterial vaccine in patients with a recurrent sore throat.
Materials and methods: This RCT was conducted at the Department of ENT, Services Institute of Medical Sciences (SIMS), Lahore from July, 2019 to January, 2020. A Total of 60 patients full filling the inclusion criteria (All patients with sore throat > 4 episodes during last one year, ages between 10 to 40 years and both genders) were included. Patients having acute diseases, sensitive to polyvalent vaccine, who have already taken the polyvalent vaccine, Diabetic and immunocompromised patients were excluded from the study. Patients were randomly divided into two equal groups by using the ballot paper method. After the approval from the institutional review board, written informed consent was obtained from the patients or guardians. The parents/guardians of the patients were fully explained about the purpose, procedure, risks and benefits of the vaccine. Group-A received sublingual live attenuated polyvalent bacterial vaccines on daily basis for 3 months while group B received a placebo (0.9% Normal Saline) for the same duration once a day. All patients were followed up for three months at which outcome i.e., the number of episodes of sore throat was noted.SPSS version 23.0 was used to analyze the collected data. The mean and standard deviations were used for quantitative variables like age, duration of disease, BMI and number of episodes. The qualitative variables like gender, place of living (rural/urban) and occupation were presented as frequency and percentage. Independent ‘t’ test was used to compare the mean number of episodes of sore throat in both groups and p-value ≤ 0.05 was considered as significant.
Results: In this study, a total of sixty cases were included, out of that 19 (31.67%) were females and 41 (68.33%) were males with male to female ratio of 2.16:1. Group A patients were given the sublingual live attenuated polyvalent bacterial vaccine, while the Group B received placebo. Age range was from 10 to 40 years with mean age of 25.68 ± 7.20 years. The mean age in group A was 26.33 ± 7.70 years and in group B was 25.03 ± 6.73 years. The majority of the patients 31 (51.67%) were between twenty-six to forty years of age. The distribution of patients according to occupation and location is also shown(Table no: 1).
Mean duration of the disease was 3.30 ± 1.59 years. The mean duration of disease in group A was 3.20 ± 1.56 years and in group B was 3.40 ± 1.63 years. Majority of the patients 33 (55.0%) were of>3 years duration. Mean numbers of sore throat episodes were 0.53 ± 0.63 in group A and 1.67 ± 0.92 in the placebo group (p-value = 0.0001) (Table: 2) Stratification of mean episodes of sore throat with respect to age groups showed a significant difference in mean episodes of sore throat in all age groups among both groups. Similarly, a statistically significant difference was found in mean episodes of sore throat among both groups in both genders. Stratification of mean episodes with respect to the duration of disease also showed statistically significant differences among them. Stratification of episodes of sore throat with respect to the place of living, BMI and occupation is also shown (Table no: 3)
Discussion: Live vaccines have played a critical role from the beginning of vaccinology. Within the last two decades, the concept of live vaccines regained interest due to increased understanding and availability of molecular techniques for the preparation of safer live vaccines possible. It has led to the development of new bacterial vaccines that can avoid the downsides of intravenously administered vaccines.12,13 Furthermore, these vaccines can be designed to induce an immune response to itself or to a carried heterologous antigen. More than two thousand papers were published regarding the application of live vaccines; but few of those could be registered after the licensing process.14-17
We have conducted this study to compare the mean episodes of sore throat after 03 months of treatment with sublingual polyvalent live attenuated bacterial vaccine versus treatment with placebo for recurrent sore throat. In this study, the mean numbers of episodes were 0.53 ± 0.63 in the sublingual polyvalent live attenuated bacterial vaccine group and 1.67 ± 0.92 in the placebo group.
In a study by Macchi A et al.11, out of three groups, the group with sublingual bacterial vaccine had less numbers of respiratory tract infections and fewer requirements of antibiotics. Many questions regarding sublingual vaccinations still remain to be addressed. However, different studies have demonstrated promising aspects of sublingual immunization which are highly effective and safe in generating robust immune responses. Furthermore, it provides protective immunity by simultaneously eliciting systemic IgG and mucosal IgA antibodies as well as CTL responses. The result of these research studies suggests that against respiratory and genital organisms, sublingual vaccination could be a better choice than parental vaccines18,19,20. In another multicenter study, there was a fifty percent reduction in the number, severity, and duration of respiratory tract infections. 21
In another study, forty-seven patients were included and were divided into two groups randomly. In Group A, twenty-four patients received one sublingual tablet of MLBL per day for 10 consecutive days per month for 3 months and Group B patients (23) received daily one sublingual tablet of taste-masked placebo for 10 consecutive days per month for 3 months. During the treatment and after completion, the number of sore throats infection and their duration were statistically lower in the MLBL group than in the placebo group. The beneficial effects in vaccine treated group were maintained during the treatment and in 3 months follow-up after the completion of treatment.22
Conclusion: Sublingual live-attenuated polyvalent bacterial vaccine is effective in reducing the number of recurrent sore throat episodes and need for frequent antibiotic use and/or tonsillectomies. So, we recommend that routine use of sublingual polyvalent live attenuated bacterial vaccine should be encouraged for recurrent sore throats in order to reduce the morbidity of these patients and avoid unnecessary antibiotics use and tonsillectomies.
Acknowledgment
Authors acknowledge the immense help received from the scholars whose articles are cited and included in the references of this manuscript
Source of Funding
None
Conflict of interest
None
Authors Contribution
Dr. Abdul Shakoor
Principal contributor, conceptualization and design of research work
Dr. Abdul Malik Mujahid.
Co-contributor, plagiarism correction, final approval
Dr. Muhammad Junaid Ashraf
Co contributor, data analysis,
Dr. Izzah Fatima
Statistical analysis, literature search
Dr. Junaid Rasul Awan
Writing of manuscript, collection of data, drafting
Dr. Waqas Aslam
Literature search, result analysis, review of the manuscript
Englishhttp://ijcrr.com/abstract.php?article_id=4500http://ijcrr.com/article_html.php?did=4500
1. van de Pol AC, van der Gugten AC, van der Ent CK, Schilder AG, Benthem EM, Smit HA, et al. Referrals for recurrent respiratory tract infections including otitis media in young children. Int. J. Pediatr. Otorhinolaryngol. 2013;77(6):906-10.
2. Esposito S, Musio A. Immunostimulants and prevention of recurrent respiratory tract infections. J Biol Regul Homeost Agents. 2013;27(3):627-36.
3. Cazzola M, Anapurapu S, Page CP. Polyvalent mechanical bacterial lysate for the prevention of recurrent respiratory infections: a metaanalysis. Pulm Pharmacol Ther. 2012;25(1):62-8.
4. De Benedetto F, Sevieri G. Prevention of respiratory tract infections with bacterial lysate OM-85 bronchomunal in children and adults: a state of the art. Multidiscip Resp Med. 2013;8:33-10.
5. Lanzilli G, Traggiai E, Braido F, Garelli V, Folli C, Chiappori A, et al. Administration of a polyvalent mechanical bacterial lysate to elderly patients with COPD: effects on circulating T, B and NK cells. Immunol Lett. 2013;149(1–2):62-7.
6. Carlone S, Minenna M, Morlino P, Mosca L, Pasqua F, Pela R, et al. Clinical efficacy and tolerability of an immune-stimulant constituted by inactivated bacterial bodies in the prophylaxis of infectious episodes of airways: a double-blind, placebo-controlled, randomized, multicentre study. Multidiscipl Respirat Med. 2014;9:58.
7. Braido F, Melioli G, Candoli P, CavalotA, Di Gioacchino M, Ferrero V, et al. The bacterial lysate Lantigen B reduces the number of acute episodes in patients with recurrent infections of the respiratory tract: the results of a double-blind, placebo-controlled, multicenter clinical trial. Immunol Lett. 2014;162(2 Pt B):185-93.
8. Ambrose CS, Wu X, Knuf M, Wutzler P. The efficacy of intranasal live attenuated influenza vaccine in children 2 through 17 years of age: a meta-analysis of 8 randomized controlled studies. Vaccine. 2012; 30:886–92.
9. Balshem H, Helfand M, Schunemann HJ, Oxman AD, Kunz R, Brozek J, et al. GRADE guidelines: 3. Rating the quality of evidence. J Clin Epidemiol. 2011;64:401–6.
10. Caspard H, Heikkinen T, BelsheRB, Ambrose CS. A systematic review of the efficacy of live attenuated influenza vaccine upon revaccination of children. J Human Vaccines Immunotherap. 2016;12(7):1721-27.
11. Macchi A, Vecchia LD. Open comparative, randomized controlled clinical study of a new immunostimulating bacterial lysate in the prophylaxis of upper respiratory tract infections. Arzneimittelforschung. 2005;55(5):276-81.
12. Dietrich G, Griot-Wenk M, Metcalfe IC, Lang AB, Viret JF. Experience with registered mucosal vaccines. Vaccine. 2003; 21:678–683.
13. Lindberg AA. The history of live bacterial vaccines. Dev Biol Stand. 1995;84:211–219.
14. Kim S, Joo DH, Lee JB, Shim BS, Cheon IS, Jang JE, et al. Dual role of respiratory syncytial virus glycoprotein fragment as a mucosal immunogen and chemotactic adjuvant. PLoS One. 2012;7:e32226.
15. Park HJ, Ferko B, Byun YH, Song JH, Han GY, Roethl E, et al. Sublingual immunization with a live attenuated influenza a virus lacking the nonstructural protein 1 induces broad protective immunity in mice. PLoS One. 2012;7:e39921.
16. Shim BS, Choi JA, Song HH, Park SM, Cheon IS, Jang JE, et al. Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus. J Microbiol. 2013;51:130–135.
17. Shim BS, Choi YK, Yun CH, Lee EG, Jeon YS, Park SM, et al. Sublingual immunization with M2-based vaccine induces broad protective immunity against influenza. PLoS One. 2011;6:e27953.
18. Brandtzaeg P. Mucosal immunity: induction, dissemination, and effector functions. Scand J Immunol. 2009; 70:505–515.
19. Yuki Y, Kiyono H. New generation of mucosal adjuvants for the induction of protective immunity. Rev Med Virol. 2003; 13:293–310.
20. Hervouet C, Luci C, Cuburu N, Cremel M, Bekri S, Vimeux L, et al. Sublingual immunization with an HIV subunit vaccine induces antibodies and cytotoxic T cells in the mouse female genital tract. Vaccine. 2010; 28:5582–5590.
21. Grevers G, Palacios OA, Rodriguez B, Abel S, van Aubel A. Treatment of recurrent respiratory tract infections with a polyvalent bacterial lysate: results of an open, prospective, multinational study. Adv Ther. 2000 Mar-Apr;17(2):103-16.
22. Tricarico D, Varricchio A, D’Ambrosio S, Ascione E, Motta G. Prevention of recurrent upper respiratory tract infections in a community of cloistered nuns using a new immunostimulating bacterial lysate. A randomized, double-blind clinical trial. Arzneimittelforschung. 2004;54(1):57-63.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411411EnglishN2022June3Healthcare
Assess the Parameters of Teledentistry and its Acceptance Among Dental Clinicians
English0610Rehmatullah KandhroEnglish Khalida Naz MemonEnglish Jazib MemonEnglish Tahera AyubEnglish Naheed NajmiEnglish Yousuf Ahmed QureshiEnglish
Introduction: Teledentistry can be illustrated as the remote course of action of understanding, and treatment through the technique for information advancement as opposed to coordinating contact with any patient(s) included. Objective: This review was organized to assess the knowledge and parameters of teledentistry of the dentists in Pakistan concerning teledentistry as an emerging implement. Methodology: Descriptive Cross-sectional study was conducted among dentists working in Pakistan, by convenience sampling technique. And the data analysis was done on SPSS version 23. A Chi-Square test was used to control confounders and assess the relation of dentist response with respect to concerning age, gender, and other variables Results: Our participants have a high level of data with regard to teledentistry (85%) and they believe it’s a useful tool to be taken in pandemic situations (84.8%). Conclusion: In the present research, it was observed that most dental staff have adequate information in regard to teledentistry and they have highly obliged this gadget in curse conditions.
EnglishTeledentistry, Telemedicine, Dentists, Dental supervision, Modern gadget, COVID-19
INTRODUCTION:
Teledentistry can be marked as the remote arrangement of dental consideration, counsel, or treatment through the mode of information technology as opposed to direct contact with any patient.1
The expression "Telemedicine" truly signifies "recuperating from separation". As indicated by WHO, the term telemedicine can be characterized as: "The conveyance of social insurance administrations, where separation is a basic factor, by all medicinal services experts utilizing data and correspondence innovations for the trading of legitimate data for finding, treatment and avoidance of ailment and wounds, research and assessment and for the proceeding with the training of human services suppliers, all in light of a legitimate concern for propelling the strength of people and their communities.3,4
With the amalgamation of dentistry and telemedicine, another field of study has been presented which is known as Teledentistry. It was started as a service undertaking to permit correspondence between dental experts over huge separations just as to furnish continuing with dental preparation.5
Teledentistry is a site for oral professionals that uses electronic media to allow dental specialists to converse with patients from remote zones. It includes various applications not just in a city set up where a patient under unbelievable dental desolation or in a condition of dental crisis requires to see a dental power yet furthermore what's more, in common spaces where money is a remarkable worry for individuals living there as it can portray a critical obstruction to scan for a treatment.6,7
Teledentistry can address these issues by basically checking into the web page on the web and getting the opportunity to ease right away. Teledentistry approaches may hold the probability to address a colossal number of the issues identified with getting the chance to, cost, capacity, and the possibility of a dental idea.8-10
The web is the establishment of existing frameworks of teledentistry, being present-day and brisk, just as readied to transmit various measures of information.11
Correspondence and data advances used identified with the internet have become a central bit of insightful life in schools. Electronic teledentistry instruction engages understudies to pick the spot, time, and technique for learning.12
In continuing with capable dental training, present-day internet systems in like manner offer online videoconferencing, broadcasting exercises, and online instructional classes. Even though email remains a well-known system for store-and-forward conversation with limited data security, videoconferencing is so far a standard procedure for continuous counsel.13
With the quick development of cell phone innovation, the use of cell phones to accomplish and move pictures for the screening of dental conveys is incredibly expanded. The zoom and blaze highlights of cell phone cameras permit simpler catching of extra-oral just as intra-oral prints. The openness and compactness of cell phones give proficient intention to catching views in a brief timeframe.14,15
This article aims to assess the knowledge, parameters, and aspects of teledentistry among dental experts because deformities are increasing day by day in an era of pandemics (covid-19).
The primary work process of teledentistry includes the accompanying advances:
Capturing of pictures from cell phones.
Transmission of pictures from cell phone application legitimately to a protected framework.
Data recovery utilizing client ID and passwords.
Dental pictures are remotely inspected by dentists.
Bi-directional correspondence between a dental expert and a patient.
Improve findings, systematize, treat, and lessen the time and arrangement of patients.
Easy access to dental healthcare in remote areas, where there is less number of experts and specialists.
Objective:
Our research aims to evaluate parameters associated with teledentistry and its use and clinician acceptability of this modernized tool.
Methodology:
Study setting: Dental surgeons operating in Pakistan.
Study Design: Cross-sectional study
Duration of study: 2 weeks i.e. from 25th March 2022 to 8th April 2022.
Study population, sample size & sampling technique: Four hundred forty dental professionals were attended by convenience sampling
Data collection method, variables & analysis: The dental experts were drawn closer to complete the overview. The completed reviews were assembled and quantifiably researched with IBM SPSS variation 23 programs. Other than registering frequencies and rates, the collusion among factors was sought after by assuming the Pearson Chi-Square test to perceive differentiates appropriately for different variables with the level of significance set at p> 0.05. Separated from socio-segment factors, dental specialists' level of familiarity and expediency concerning teledentistry were recorded on a pre-validated questionnaire. Focus on people fuses subject matter experts, specialists, general dental specialists who work in Pakistan between some time of 24-50 years, and the individuals who didn't consent, residence officials, and undergraduate students were blocked from the review.
Results:
Five hundred dental experts were called to procure them outright comments of a respected figure of subjects i.e.440. The consistency scale was assumed as 88%.
Participants had a high phase of data (85%) considering teledentistry. We didn't decide on a solitary insignificant alliance between the period of dental specialists and their misgivings along gesture teledentistry (p ≤ 0.01)
Discussion:
The current cross-sectional review unfastened a subject that procured reflective concern conforming to potent enhancement in dentistry (teledentistry) among dental specialists in Pakistan.16
Considering the present circumstances of the world during the Covid-19 pandemic, when most of the world is in a state of lockdown/limited mobility, services like e-dentistry give speedy help to intense and painful conditions through consultations given over the phone.17
It is not a caprice strange to humankind and has been opening from the time of 1994, yet there have been enormous unique headways throughout the years in the field of dentistry. Conveying oral medical services to patients has drastically been affected during the pandemic, with dentistry considered as an exceptionally unsound calling in alive contexts.
In the flow of COVID-19 Teledentistry is the main solid method to dispense dialogue and cure, employing electronic bits.18
Teledentistry permits significant reserve correspondence by keeping away from the individual-to-person connection, noticing social removal, allowing the trade of clinical data and pictures, and functioning with distant dental consideration, patients' schooling, and direction which is suggested by medical care specialists across the globe.19
The existing composition spotlights the overtone of teledentistry. It holds subsets, for instance, “teleconsultation, telediagnosis, telemonitoring, and teletriage”, aside from holding significant capacities pertinent to dentistry. The perception emerging from our information is that dental specialists cherish teledentistry for its capacity to work on persistent consideration and improve oral well-being rehearses, which is consistent with a comparable review led in Canada, USA, where most of the dental specialists were on the side of the utilization of teledentistry in better medical care conveyance.20
In an ongoing exploration by “Yang et al. for an arrangement of well-being administrations during the Covid - 19 episode, it was seen that all the 48 clinics which were part of the research, suspended general non-critical treatment while giving critical dental administrations and 33 (68 %) medical clinics offered free online expert counsels for deciding if the condition required crisis treatment and gave home dental and oral consideration guidance”.21
A study about teledentistry in India saw that 58% of dental specialists concurred with the explanation that teledentistry is great for dental training furthermore, preparation over the web. From our perspective, 63.4% of dental specialists tackle that teledentistry assists in lessening tools for dental practices. 59.7% of respondents in a review completed in India concurred teledentistry abates charges for dental procedures. Teledentistry can profit Pakistan's human services framework as far as preventive consideration and illness treatment is concerned.
Although telemedicine is gradually picking up acknowledgment among the individuals of Pakistan just as the experts.
To appraise the current state and efficacy of teledentistry in Pakistan and its acceptance among patients and dental experts, the accompanying research is led.22-25
CONCLUSION
The present exploration shows that notwithstanding the vast majority of the members having satisfactory information about the expression "teledentistry", they communicated positive possibilities regarding its utilization.
It is needed to upgrade the information for and advance its use in this season of COVID-19, and, surprisingly, later. Planning new strategies and dental instruction intents is enforced. Teledentistry might keep on being utilized as a method for meeting and giving simple admittance to dental experts and patients.
In ongoing potential outcomes, it would be attainable that distant mechanical operations would be worked on due to teledentistry. Thusly, it might be said that teledentistry is a bracing and refined element with incessant potency.
Acknowledgment:
The authors acknowledge the immense help received from the scholars whose articles are cited and included in the references of this manuscript. The authors are also grateful to the authors/editors/publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.
Source of funding:
No financial support was taken.
Conflict of Interest:
There is no conflict of interest from any author.
Authors’ Contribution:
Rehmatullah Kandhro: Agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Substantial contribution to the designing, conceptualization, analysis, and interpretation of data for the article.
Khalida Naz Memon: Writing and editing of the article. Have contributions in conceptualizing the topic. Validation, Supervision, and providing the final approval for the publication of the content.
Jazib Memon: Substantial contribution to the design of the work.
Naheed Najmi and Yousuf Ahmed: Revised it critically for important intellectual content and final approval for publication.
Ethical Approval:
Ethical approval letter (NO.LUMHS/REC/-73) was taken by the Institutional Research Ethics Committee by Liaquat University Of Medical and Health Sciences Jamshoro on dated 24-03-2022
Englishhttp://ijcrr.com/abstract.php?article_id=4501http://ijcrr.com/article_html.php?did=4501Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411411EnglishN2022June3Healthcare
Chitinase Activity by Chitin Degrading Strain (Bacillus Salmalaya) in Shrimp Waste
English1117Abdulkhaliq J. AlsalmanEnglish Arshad FaridEnglish Mohammed Al MohainiEnglish Muhammad MuzammalEnglish Muhammad Hashim KhanEnglish Arezoo DadrasniaEnglish Yousef N. AlhashemEnglish Maitham A. Al HawajEnglish Shakira GhazanfarEnglish Eman M. AlmusalamiEnglish TEnglish
Introduction: Chitin degradation by chitinase enzyme can be used on a large scale for bioremediation of seafood waste and is environmentally friendly. Objective: The main aim of this study was to screen the potential of the strain as a chitin degrading agent. Methods: In this present study, the cell-free supernatant of Bacillus salmalaya was determined for its protein concentration. Bacillus salmalaya139SI was isolated from agricultural soil and it was identified by staining technique and colony morphology. The production of chitinase by Bacillus salmalaya139SI was optimized under different concentrations of substrate, pH and temperature. Results: Strain 139SI exhibited strong hemolytic activity and the crude protein concentration of Bacillus salmalaya was 84.09mg/ mL with OD value 0.462. Strain 139SI were also screened on colloidal chitin agar medium supplemented with mineral salt. Chikinase production was determined by clear zones of hydrolysis produced after 7 days of incubation at 37°C. The maximum chitinase production was observed in Brain Heart Infusion broth supplemented with 1.0% colloidal chitin at pH 7 and temperature 35°C after four days of incubation. Chitinase activity was observed when high concentrations of crude extract of 139SI able to degrade shrimp shell by showing the degradation zone at day 4. Conclusion: From the results, we concluded that the Bacillus salmalayahas potential to be a biofunctional chitinase that could degrade complex polysaccharides present in the organic wastes and applicable in cleaning the environment..
EnglishShrimp, Chitinase, Hemolytic activity, Bacillus salmalaya, Chitin, Degradation
Introduction
Shrimp has constituted a primary phase of crustacean consumption in the present years. An increase in shrimp waste is unavoidable owing to the increased amount of consumption. Shrimp waste is considered an imperative source of chitin. A critical sum of shrimp waste is delivered in Asia essentially in Thailand and India.1 Solid waste, consisting of the head, shell, and tail portions, accumulates owing to shrimp processing. The waste composed of the cephalothoraxes and exoskeleton 2 accounts for 50-70% of the weight of the raw material and is usually discarded. The recycling of chitinous waste is extremely important to keep the carbon-nitrogen balance in the ecosystem.2
Chitin is a sugar-like polymer and is available at a low cost. It appears to be safe for use in humans in the long term and has very low toxicity. Chitin plays a protective role in many lower eukaryotes similar to that of cellulose in plants. Metabolism of chitin in synthesis and degradation is essential for different morphogenesis.2 Chitin occurs as crystalline microfibrils in nature. It can be found as the main component in the construction processing of fungal cell walls, Mollusca radula, nematode eggshells, worm and arthropod exoskeletons, cephalopod beaks, and fish scales.3 The absence of chitin in vertebrates and plants makes the chitin metabolism potentially useful parasite-specific targets for chemotherapeutic attack.1
Marine bacteria for survival in aquatic ecosystems rapidly catabolize chitin. Some bacteria are producing chitinases probably to hydrolyze the diversity of chitins found in nature. As stated by Okonkoet al. (2006) several strains of microorganisms have been selected or genetically modified to increase the efficiency with which they produce enzymes.4 Chitinases are a large and diverse group of enzymes with different molecular structures, substrate specificity, and catalytic mechanisms. Many bacteria, including Serratia and Bacillus, produce four different chitinases, whereas filamentous fungi produce up to 20 different chitinases. As a result, there has been an increase in demand for chitin derivatives produced by the action of chitinases on chitin polymer for a variety of industrial, clinical, and pharmaceutical applications.4
Bacteria are thought to be the primary mediators of chitin degradation in nature. Their importance can be seen in both soil and water systems. The rate of chitin hydrolysis in soil systems is related to the bacterial population and abundance.5 In addition, the degradation process also depends on factors such as temperature and pH. Thus, the degradation of chitin by the chitinase enzyme can be used as bioremediation of seafood waste at a large scale and eco-friendly to the environment.6
The main aim of the current study was to analyze and screen the potential strain as a chitin-degrading agent. Therefore, the specific objectives were to screen the potential of Bacillus salmalaya in hydrolysis activity, to optimize cultural conditions for the production of chitinases like concentration, pH, and temperature, and to screen the potential of the strain as chitin degrading agent on shrimp shell.
Methodology.
Bacillus salmalaya139SI
Bacillus salmalaya139SI was originally isolated from soil obtained from a private farm in Selangor, Malaysia (2.99917°N 101.70778°E).7
Chitin from shrimp shells
Practical grade, chitin powder (Sigma, USA) was used and modified to form colloidal chitin as it is more homogenous distribution in agar media and act as a primary carbon source to bacteria in a medium for analysis of chitinase activity.
Growth condition and Chitinase production.
A single colony of 139SI, from the BHI agar plate, was cultured in 1 L of Difco ™, USA.Brain–Heart Infusion (BHI) medium containing 5 g/L KCl, 3 g/L dextroses, 2.5 g/L Na2HPO4, 14.5 g/L gelatin, 6 g/L BHI, and 6 g/L peptic digest of animal tissue with additional 1% (w/v) chitin powder from shrimp shell as an inducer in a shaking incubator at 150 rpm for 72 h at 35°C.The incubation continued until the OD600 equaled 1. The 7 days culture was centrifuged (8000 × g for 20 min) by using (SORVALL ST 16R) centrifuge and filtered through a sterile syringe filter with a pore size of 0.2µm (Minisart syringe filter) to remove all particles and dead microorganisms without any influence on their ingredients. The supernatant formed was a crude extract of the chitinase enzyme. The cell-free broth or supernatant was concentrated by freeze-drying and was stored at −20 °C.
Estimation of protein content
Purified and estimation enzymes were estimated by the following method of Lowry et al. (1951)8 using Bovine serum albumin as the standard.9
Preparation of colloidal chitin
Colloidal chitin was prepared from chitin powder (sigmaChemicals Company, USA) by the modified method of Hsu and Lockwood.10
Preparation of chitin agar medium
The chitin medium was prepared using moist form colloidal chitin based on the modified protocols combining elements from Hsu et al. and Ram?rez et al.10,11
Plate screening of Chitinase activity
Bacillus salmalaya139SI colonies from the plates were sub-cultured on Brain heart infusion broth (BHI; Difco) and incubated at 30°C for 24 hours. Later, a few drops of culture was placed onto the surface of BHI agar and spread using a sterile spreader or sterile cotton swab. This was incubated at 30°C for overnight. After incubation, the confluent growth of the culture was inoculated onto chitin agar using spot inoculation. Plates were incubated at 30°C for about a week (7 days). Following incubation, the bacterial cultures were observed for the production of chitinase. On the chitin agar, the clear zone of inhibition was noted around the colonies and was calculated using a Vernier caliper in (cm) and after 3 days of incubation, the chitinolytic index was calculated. The chitinolytic index was calculated using the below equation:
Optimization of cultural condition
Effect of substrate concentrations on chitinase production
Bacillus salmalaya strain 139SI was cultivated in different concentrations (0.3, 0.5, 0.8, and 1.0 %) of colloidal chitin enhance with minimal medium to determine the optimum concentration of colloidal chitin for chitinase production.
Effect of pH value on the chitinase production
Bacillus salmalaya strain 139SI was grown at a different pH ranges of the culture medium from 4 to 10. HCL was used for pH 4; phosphate buffer was used for pH 7 and NaOH for pH 10 in a minimal medium containing 1.0% colloidal chitin to determine the optimum pH for chitinase production.
Effect of temperature on the chitinase production
To determine the optimum temperature for chitinase production, Bacillus salmalaya strain 139SI was grown in the culture medium containing 1.0% colloidal chitin and incubated at different temperatures of 25°C, 30°C, 35°C, 40°C and 45°C up to 4 days.
The reaction of crude chitinase on shrimp shell
A number of prawn shells were prepared and treated with different concentrations of chitinase of strain139SI. Then, shell was observed in a month to identify the ability of chitinase in degradation action under a microscope (OLYMPUS SZ40).
Results:
Hemolytic activity
Bacillus salmalaya strain 139SIshowed a positive result as appears clear zone around the bacterial colony accompanied by lightened yellowish discoloration of the medium as in figure 1a. The complete lysis exhibits that strain 139SI has strong Beta (β) hemolysis because it could produce hemolysin substance, which is bacterial protein breakdown of the hemoglobin of the red blood cells and disrupting the structure of the membrane or punching a hole through the membrane.
Estimation of protein content
Bacillus salmalaya strain 139SI produced 0.32g of chitinase crust. The amount might be slightly not exactly as we used an old freeze-drying machine. Thus, there were some errors or mistakes that happened during freeze drying for instance incomplete drying since the process of removing moisture not working well. Based on the results from the BSA standard graph (figure 1b), Bacillus salmalaya strain 139SI has a high amount of crude protein concentration which is 84.09 mg/mL with an OD value of 0.462. Therefore, it proved chitinase crust of strain 139SI was pure. That might be due to the chitinase crust being influenced by other substances.
Plate screening of chitinase activity
In the primary screening of chitinase activity as shown in figure 2, Bacillus salmalaya139SI produced a prominent zone of hydrolysis on the colloidal chitin agar. The clear zone around the spot inoculation was increasing in diameter until day 7 with chitinolytic index 7. This indicated strain 139SI has a chitinolytic activity to break down the chitin compounds in the medium.
Effect of substrate concentrations on chitinase production.
Among the four different concentrations tested, the results showed Bacillus salmalaya strain 139SI produce chitinase maximally at the concentration of 1% of colloidal chitin with absorbance value (1.845±0.092), followed by colloidal chitin at 0.8% (1.541±0.103), 0.5% (1.418±0.068). Beyond 0.5%, the substrate concentration decreased enzyme activity (Table 1). But above 0.5% of colloidal chitin concentration, chitinase production was significantly increased.
Effect of pH value and temperature on the chitinase production.
In order to evaluate the effect of pH of the media on the chitinase production, Bacillus salmalaya139SI were grown at different pH (4, 7, and 10). The optimal pH for chitinase production was examined when kept at 37°C. Among the tested pH, neutral condition pH 7 (1.318±0.029) supported the maximum chitinase production. In pH 4, chitinase production was 0.235±0.040 followed by pH 10 was 0.297±0.135 (Table 2). Chitinase production was relatively stable at pH 7. However, for alkaline and acidic conditions, it rapidly lost its chitinase production. In addition, from the observation of the broth culture medium, the culture broth for acidic and alkaline are slightly clear compared to pH 7 medium that are cloudier which indicates cell growth. This test, it showed an excessively high or low pH led to poor cell growth and chitinase production.
Data presented in table 3 clearly indicated incubation temperature affects many biological processes, including the growth rate and enzyme production. The organism exhibited good growth as well as chitinase production at 35°C with an average of 1.732±0.072. The incubation temperature 30°C (1.460±0.036) also found to influence the chitinase production. It has been observed that in both lower and higher temperatures chitinase production decreased.
The reaction of crude chitinase on shrimp shells
Five different concentrations of crude chitinase were tested on the shrimp shell to check the chitin degradation ability. Among the five concentrations, 200 mg/ml until 300 mg/ml of crude chitinase was more effective in degrading ability compared to shrimp shell treated with lower concentration. This is because the degradation activity can be seen as early as day 4 and more clearly seen in the following week. At the end of the month, all concentrations showed the potential of degradation activity (Figure 3). In this study, a high concentration of crude chitinase was used to get the result within a month and also high concentration will produce more effective enzyme action mechanism in hydrolyzing the chitin. Thus, this study showed the degradation of chitin-composed material and action mechanisms of the chitinase enzyme were associated with the volume of the treatment given on the shrimp shell.
Discussion:
Hemolytic or also referred to as hemolysis is the breakdown of the membrane of red blood cells by a substance known as hemolysin. A hemolysin is a group of bacterial proteins, which causes the lysis of the red blood cell (RBC) membrane in the growth substrate 12. Many types of bacteria possess hemolytic proteins. These proteins act by desegregating into the membrane of RBC and disrupting the structure of the membrane.12 Bacteria are differentiated based on their hemolytic properties. In the study by Dadrasnia and Salmah (2015), Bacillus salmalaya was found to be a potential degrader of crude oil waste. In the study, haemolytic activity for Bacillus salmalaya139SI was detected as the presence of a definite clear zone around the colony.7 In the other finding by the same authors, Bacillus salmalaya139SI also identified as biosurfactant bacteria. As is known, biosurfactant bacteria were all positive in hemolytic activity as it is an initial test that has always been used to identify biosurfactant-producing bacteria. Therefore, hemolytic activity appears to be a good criterion in screening in the search for surfactant-producing strains. As reported by Abu et al., (2018), the bacterial colonies that were streaked on blood agar medium exhibited g β-hemolytic activities and were found to have the potential to produce bioflocculant to remove organic matter.13 Thus, isolated Bacillus salmalaya139SI were represented as members of a novel species of the genus Paenibacillus based on the hemolytic activities7 and also the ability to bind efficiently and degrade animal or human hemoglobin that could provide an effective heme source to ensure its successful growth and proliferation in vivo.13,14
In 2004, Stoykovet al.15 mentioned that the expression of the inducer can generate of a signal and increase in the production of chitinase. It also has been reported by Sato and Araki, (2008) showed the significance of medium composition for the production of chitinase from Bacillus cereus by supplementing the medium directly with chitin.16 The culture medium was incubated until reached OD value equaled 1 because it was the highest value culture density (cell number) of the strain with a constant growth rate, thereby exhibiting a maximum chitinase production. In the study by Salmah and Dadrasnia (2015), the maximum production of biosurfactant was also measured until the OD value equaled to 1 and was found to be a potential degrader of crude oil waste.7
In the study by Budi et al., (2000), Paenibacillus species have been reported to have a high activity of cell wall degrading enzymes and chitinase, making this species commonly applied as biocontrol agents.3 To give comprehensive proof, Kumar et al., (2012) mentioned that different species of Bacillus have been reported to produce chitinase and the result from their findings, Bacillus amyloliquefaciens SM3 produced the highest chitinolytic activity compared to other isolated chitinolytic bacteria.17 However, in the study by 18 he reported different strains will give different abilities to secrete extracellular degradative enzymes.
Results of this research found that Bacillus salmalaya strain 139SI has the potential to be a mediator of chitin degradation and may be useful for biotechnological applications and the production of transgenic microorganisms with superior biocontrol capabilities. Chitin degradation could therefore be explored as a general model for understanding microbial degradation of biopolymers in the biosphere.
In this study, a high concentration of crude chitinase was used to get the result within a month, and also high concentration will produce a more effective enzyme action mechanism in hydrolyzing the chitin. Thus, this study showed the degradation of chitin-composed material and action mechanisms of the chitinase enzyme were associated with the volume of the treatment given on the shrimp shell.
In the study by Sorokulova et al., (2009), they reported that the B. cereus strain performed better in shell waste decomposition and was used for large-scale fermentation in 12 L of 10% shrimp shell waste broth.19 The similar report supported by Abirami et al., 2016 observed that Bacillus licheniformisSSCL10 rapidly degrade the shrimp shell completely within 12 days while another isolate of Bacillus subtilis took more days for degradation activity.20 From the result obtained in this study, chitinase from Bacillus salmalaya139SI has efficiency in hydrolytic activity and it can also be used for the degradation of other chitin materials. Therefore, chitin degradation needs to be explored as a general model for understanding microbial degradation of biopolymers in the biosphere.
Conclusion
Chitinase is ubiquitous proteins that are widely distributed among all kingdoms of life. Chitinase as the name indicates is involved in the breakdown of chitin. The result concluded that Bacillus salmalaya are novel mesophilic bacterial strains that have strong hemolytic activity showing the best ability to produce a huge amount of chitinase in a short time. This enzyme may also be useful in the management of seafood waste industries. Colloidal chitin as the sole source of carbon can prove to be economical in terms of fermentation expenditure. Neutral pH along with a temperature around 35°C facilitates the highest yield. This work revealed that strain 139SI was also effective in hydrolyzing chitin medium and degrading shrimp shells at concentrations of 200, 250, and 300mg/ml of crude chitinase. Bacillus salmalaya139SI makes it a potential candidate for the bioremediation of seafood waste at a large scale. Strain 139SI performance was increased since it has the highest concentration of protein chitinase enzyme. However, much attention and research are needed for multiple potential applications in the future such as nanobiotechnology applications involving drug and gene delivery or in agriculture, food, and environmental protection.
Acknowledgments: The authors gratefully acknowledge Izzah Hazwani Binti Razali (Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia) for experimentation assistance and Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia, for providing financial support and lab facility for this research work.
Source of Funding: This research received no external funding or self-funding.
Conflict of Interest: The authors declare no conflict of interest.
Authors’ Contribution: All authors contributed equally.
Figure 1 (a) Red arrow showed a clear zone produced by pure colonies of 139SI after 24h incubated at 37°C under aerobic conditions. Bacillus salmalaya strain 139SI has strong hemolytic activity on the blood agar medium (b) figure showing the concentration of chitinase of strain 139SI plotted against absorbance at 700nm on BSA standard graph. The crude protein concentration of Bacillus salmalaya139SI was 84.09 mg/mL and its absorbance was 0.462 O.D.
Englishhttp://ijcrr.com/abstract.php?article_id=4502http://ijcrr.com/article_html.php?did=4502
1. Adrangi S, Faramarzi MA. From bacteria to human: a journey into the world of chitinases. Biotechnol. Adv. 2013 Dec 1;31(8):1786-95.
2. El-Sisi AS. Impact of replacement of gelatin with chitosan on the physicochemical properties of ice-milk. Int. J. Dairy Sci. 2015;10(1):36-43.
3. Budi SW, van Tuinen D, Arnould C, Dumas-Gaudot E, Gianinazzi-Pearson V, Gianinazzi S. Hydrolytic enzyme activity of Paenibacillus sp. strain B2 and effects of the antagonistic bacterium on cell integrity of two soil-borne pathogenic fungi. Appl. Soil Ecol. 2000 Oct 1;15(2):191-9.
4. Okonko IO, Olabode OP, Okeleji OS. The role of biotechnology in the socio-economic advancement and national development: An Overview. Afr. J. Biotechnol. 2006;5(23)..
5. Kielak AM, Cretoiu MS, Semenov AV, Sørensen SJ, van Elsas JD. Bacterial chitinolytic communities respond to chitin and pH alteration in soil. Appl. Environ. Microbiol. 2013 Jan 1;79(1):263-72.
6. Chen JK, Shen CR, Liu CL. N-acetylglucosamine: production and applications. Mar. Drugs. 2010 Sep;8(9):2493-516.
7. Ismail S, Dadrasnia A. Biotechnological potential of Bacillus salmalaya139SI: a novel strain for remediating water polluted with crude oil waste. PLoS One. 2015 Apr 13;10(4):e0120931.
8. Waterborg JH. The Lowry method for protein quantitation. The protein protocols handbook 2009 (pp. 7-10). Humana Press, Totowa, NJ.
9. Redmile-Gordon MA, Armenise E, White RP, Hirsch PR, Goulding KW. A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts. Soil Biol. Biochem.. 2013 Dec 1;67:166-73.
10. Hsu SC, Lockwood J. Powdered chitin agar as a selective medium for enumeration of actinomycetes in water and soil. Appl. Microbiol.. 1975 Mar;29(3):422-6.
11. Ram?rez MG, Avelizapa LR, Avelizapa NR, Camarillo RC. Colloidal chitin stained with Remazol Brilliant Blue R®, a useful substrate to select chitinolytic microorganisms and to evaluate chitinases. J. Microbiol. Methods. 2004 Feb 1;56(2):213-9.
12. World of Microbiology and Immunology. (2003). Blood Agar, Hemolysis and Hemolytic Reactions. Retrieved from https:// www.encyclopedia.com/science/encyclopedias-almanacs-transcripts-and-maps/blood-agar-hemolysis-and-hemolytic-reactions
13. Abu Tawila ZM, Ismail S, Dadrasnia A, Usman MM. (2018). Production and Characterization of a Bioflocculant Produced by Bacillus salmalaya 139SI-7 and Its Applications in Wastewater Treatment. Molecules, 23(10), 2689.
14. Guan SM, Nagata H, Shizukuishi S, Wu JZ. Degradation of human hemoglobin by Prevotella intermedia. Anaerobe. 2006 Oct 1;12(5-6):279-82.
15. Stoykov YM, Pavlov AI, Krastanov AI. Chitinase biotechnology: production, purification, and application. Eng. Life Sci. 2015 Jan;15(1):30-8.
16. Sato YU, Araki YO. Identification of inducers for chitinase B (ChiB) production in Bacillus cereus CH and estimation of its induction mechanism. J Environ Biotechnol. 2008 Dec;8:119- 21.
17. Kumar D. Identification and optimization of cultural conditions for chitinase production by Bacillus amyloliquefaciens SM3. J. Chem. Pharm. 2012;4(11):4816-21.
18. Waldeck J, Daum G, Bisping B, Meinhardt F. Isolation and molecular characterization of chitinase-deficient Bacillus lichenforms strains capable of deproteinization of shrimp shell waste to obtain highly viscous chitin. Appl. Environ. Microbiol. 2006 Dec;72(12):7879-85.
19. Sorokulova I, Krumnow A, Globa L, Vodyanoy V. Efficient decomposition of shrimp shell waste using Bacillus cereus and Exiguobacteriumacetylicum. J. Ind. Microbiol. Biotechnol.. 2009 Aug 1;36(8):1123-6.
20. Abirami S, Yogalsakshmi K, Pushpa AS, Kananan M. Screening and identification of chitin degrading bacteria from shrimp shell waste dumping soil environment and its media optimization for chitinase enzyme production. World J Pharm Pharm Sci.. 2016;5(11):743-57.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411411EnglishN2022June3Healthcare
A Study to Assess the Effect of Nursing Interventions on Selected Minor Ailments Among Antenatal Primigravida Women in Selected Areas
English1825Nikhila R. NairEnglish Martha Sunil RautEnglish Rupali Milind SalviEnglish Nisha NaikEnglish
Introduction: Minor ailments are common throughout pregnancy due to the physiological and psychological changes in women’s bodies. Constipation and foot edema are quite common ailments during pregnancy. As during pregnancy women’s increased weight pressures will also increase to the ankles, knees, and feet. To reduce selected minor ailments selected nursing interventions were provided in this study. Aim: To evaluate the effect of nursing interventions on selected minor ailments among antenatal primigravida women in selected areas. Methods and Material: Quasi-experimental pre-test post-test control group design was adapted. The study was conducted among antenatal primigravida women with selected minor ailments. 60 samples were selected using non-probability purposive sampling and data collection was done by using demographic variables, Pitting Edema Scale & Modified Constipation Assessment Scale. Nursing interventions provided to the experimental group. No intervention to control group. Results: The study findings showed that antenatal foot and leg exercises and consumption of fibrous fruits led to improvement in foot edema and constipation in the experimental group as compared to the control group who do not get any nursing interventions. Conclusion: Study concluded that selected nursing interventions were significantly effective in the improvement of foot edema and constipation among antenatal primigravida women.
EnglishAntenatal, Constipation, Edema, Minor ailments, Nursing interventions, Primigravida
INTRODUCTION:
Pregnancy means the time during which one or more offspring develop in the womb of the woman. The result of the pregnancy will be live birth or any minor or major complications that may cause.1 About 50 million women affect minor disorders of pregnancy in India.2 Minor ailments can be directly or indirectly related to increased levels of estrogen and progesterone present during pregnancy.3
Constipation and foot edema are quite common ailments during pregnancy. Constipation having Atonicity of the gut due to the effect of progesterone diminished physical activity and pressure of the gravid uterus on the pelvic colon which is unable to pass stool properly.4
Foot edema is caused by abnormal fluid retention in the tissues of the lower extremities. Exercising daily by pregnant women, throughout pregnancy will improve blood circulation, and reduce edema. Common intervention to reduce edema includes Foot exercises. To reduce or maintain constipation, fibrous and vitamin c fruits will be helpful.
If some minor ailment is left unattended or no care is provided then can become worsen and complicate pregnancy. Midwife nurses have a very important role in educating mothers and managing minor ailments.
The overall prevalence rate of foot edema during pregnancy was 8.5%.5 At some stage in pregnancy 8 out of 10 women have foot edema. Foot edema is found in about 80% of all pregnancies.6 It is necessary that pregnant women should be aware of how to take care of themselves during pregnancy so as to reduce the risk of minor disorders.
A study was conducted, to evaluate the knowledge of pregnant women regarding antenatal exercise and the effect of antenatal exercise on antenatal mothers. The study was conducted at Smt. Kashibai Navale Medical College and General Hospital, Pune. Data were collected from pregnant women by structured questionnaire and demonstrated antenatal exercise to pregnant women. Out of 30 samples, 11 were primigravida and 14 were the second gravida, and 5 were the third gravida this shows that multigravida women had more adequate knowledge regarding antenatal exercise than the primigravida women. The study conclude that most antenatal women were not having adequate knowledge and awareness of antenatal exercise.7
A study was conducted to check the intake of dietary fiber and the effect of fiber supplementation, 40 women have constipation were selected. Recorded their diet and bowel pattern for 4 weeks. After 2 weeks observation of the women were checked and divided into three groups and asked to take about 10 g of dietary fiber supplements per day. Results showed that changes were followed by an increase in the number of bowel movements and a stool became softer in Group A and B, and no changes in Group C who were not taking intervention. Hence study concluded that there was an effect of dietary supplements on constipation during pregnancy.8
If pregnant women consume a high-fiber diet, during pregnancy constipation can be reduced. Fiber-rich food will be helpful to keep the intestinal system running smoothly or soluble fiber is also helpful for water to remain in stool and make waste softer and easier to pass through the intestine.9 If minor ailments are neglected by pregnant women these ailments which minor leads to major complications to the mother and fetus.
That’s why want to demonstrate and teach them about foot and leg exercises and the importance of using fibrous fruit in their diet. The mother has to take care of herself and get a nursing intervention to prevent complications of selected minor ailments; therefore, this study aimed to determine the level of selected minor ailments & evaluate the effect of nursing interventions on selected minor ailments among antenatal primigravida women,
MATERIAL AND METHODS:
RESEARCH DESIGN
In this study Quasi-experimental, pre-test post-test control group design was used to assess the effect of nursing interventions on selected minor ailments among antenatal primigravida women in selected areas.
SETTING
The setting of the study was Dr. D. Y. Patil Hospital and Research Centre, Pimpri, Pune.
SAMPLE
The sample selected for the present study comprised antenatal primigravida women from 18 to 35 years of age.
INSTRUMENT
In this study, the tool consisted of the following:-
Demographic Variables: this includes 9 questions that obtain information regarding demographic data such as the age of the women, type of family, education, occupation, religion, gestational age, socio-economic status, and weight and bowel pattern of antenatal primigravida women.
Pitting Edema Scale: It consists of Pitting Edema Scale to assess foot edema among antenatal primigravida women. Measured the depth of the indention. Recorded how long it takes for skin to rebound back to its original position. Grading has been done on a scale from 1-4.
Modified Constipation Assessment Scale: It consists of the Modified Constipation Assessment Scale to assess constipation. In this 16 questions were asked to women which were present on the scale according to the answers soring was given to them.
INTERVENTION
Clinical Performa of foot exercises Assessment.
Nursing interventions-(Demonstrated antenatal foot and leg exercise). Exercises are done by antenatal primigravida women two times a day for 30 minutes each time (morning and evening) for 15 days. After 15 days again assessment was done by using Pitting Edema Scale to check the level of foot edema and to check the effect of the nursing intervention.
Diet recall of antenatal primigravida women consuming fruit (orange and banana)
(One Banana in the morning as well as one Orange in the evening for 15 days). After 15 days on the 16th-day assessment of constipation was done with the Modified Constipation Assessment Scale on the experimental and control group to check the effect of fruits on constipation.
ETHICAL CONSIDERATION
The research study was approved by Sub Ethical Committee (DYPV / CON/ 523/ 2020), and the Research & Recognition Committee (DPU / 656 / - 15 / 2020) of Dr. D. Y. Patil Vidyapeeth Pune.
DATA COLLECTION
After obtaining administrative permissions were procured from hospital authorities. The actual data was collected from 07/02/2021 to 22./02/2021
SCHEMATIC DIAGRAM
DATA ANALYSIS:
Descriptive and analytical statistics were done. The data is represented in mean and standard deviation. The association of the selected minor ailments with selected socio-demographic variables was analyzed by Fisher’s exact test. The paired sample t-test and two-sample t-test were used to check to mean differences. The level of significance was kept at pEnglishhttp://ijcrr.com/abstract.php?article_id=4503http://ijcrr.com/article_html.php?did=4503
1. Eunice Kennedy “Pregnancy: Condition Information” Shriver National Institute of Child Health and Human Development. 19 March 2015.
2. George M S, the effectiveness of planned teaching program on the management of selected minor ailments in terms of home remedies of primigravidae mothers during first trimester of pregnancy in selected hospitals, Bangalore, Karnataka, I-XIV: 1-175. 2005
3. Abdellah F G, Levine E. Better Patient Care through Nursing Research. New York: Macmillan Publishing Co. 1979
4. Konar H, Dutta DC, Comprehensive textbook of obstetrics, antenatal care, (6th ed.). Jaypee Brothers Medical Publishers. 2006
5. Davison J M, Edema in pregnancy, Kidney International Supplements, 59:S90 1997
6. Vasaiya M, Tiwari A, the effect of foot exercise and warm water .foot soak on foot edema among antenatal women - a literature review Int. J. Adv. Res. 1st, 7(3), 83-87. 2019
7. Bhanari A, Effectiveness of demonstration of antenatal exercise on the Knowledge of antenatal mothers attending antenatal OPD, Sinhgade J. Nurs, Vol. IV, Issue II/12,: 37-38,2016
8. Rachelle A. Pretorius, Debra J. Palmer, High-Fiber Diet during Pregnancy Characterized by More Fruit and Vegetable Consumption, Nutrients, vol-13(1): 35,2020
9. Anderson A S, Dietary factors in the etiology and treatment of constipation during pregnancy, BJOG BJOG-INT J OBSTET GY.93(3), 245-9, 1986
10. Vasaiya M, Tiwari A, Effect of foot exercise and warm water foot soak on foot edema among antenatal women- a literature review, Int. J. Adv. Res., 7(3), 83-87, 2019
11. Paonam D, A study to assess the effectiveness of fruit laxative on reduction of constipation among antenatal mothers in third trimester in selected areas, Bangalore,:1-13, 2008
12. Vennela KK, Knowledge on the management of minor ailments during pregnancy among antenatal mothers in NMCH, Int J Gynaecol Obstet 1(2): 20-22, 201
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411411EnglishN2022June3Healthcare
Relationship of Adiponectin Levels with Body Mass Index (BMI) in Pregnancy
English2630Akhtar YEnglish Fatima KEnglish Mehtab MEnglish Kashif SEnglish
Introduction: Low adiponectin level is an indication of insulin resistance during gestational weight gain, and could be used as a marker for diabetes or hypertension in pregnancy. Objective: To find the relationship between BMI and adiponectin levels in pregnant women. Methods: In this cross-sectional study, total 200 study participants were selected. Participants were 24 to 40 weeks pregnant, with or without gestational diabetes mellitus. Patients with pre-existing or pre-pregnancy diabetes were excluded from study. BMI was calculated at the time of the study. Adiponectin levels were measured on Metrolab ELISA, using Human Adiponectin ELISA kit by Biovendor, Germany. SPSS version 20 was used to analyze data. Results: Out of 200 participants, 100 had gestational diabetes. None of the participants was below normal weight, however, about 38% of participants were overweight, and about 58% of participants were obese, according to WHO-Asian criteria. Adiponectin level was below normal in more than 60% of patients. No difference was found between BMI within diabetes-based groups. But there was a significant difference between adiponectin levels in Group I and Group II. No relationship of BMI was found with diabetes-based groups, gestational age groups, or adiponectin categories. A significant relationship was found between the diabetic group and low serum adiponectin levels. Regression analysis showed that BMI was neither a predictor of diabetes nor adiponectin levels. However, adiponectin levels were a predictor of diabetes in pregnancy. Conclusion: Our study could not find a relationship of BMI with either adiponectin levels or gestational diabetes. Low adiponectin levels, however, could predict gestational diabetes.
EnglishGestational, Weight gain, Adiponectin, Pakistan, Pregnancy trimester, BMI
Introduction:
Weight gained by women during pregnancy is called gestational weight gain (GWG). This weight gain includes the fat and lean mass of the mother’s body, the weight of the baby, placenta, and amniotic fluid.1 Insufficient or excessive weight gained by the mother is of great concern for the healthcare team as this results in adverse health outcomes for both mother and the child. Excessive weight gain could result in the development of gestational diabetes mellitus in the mother and resultant macrosomia in the fetus, or it could lead to hypertension in the mother and resultant placental abruption causing fetal death. Conversely, insufficient weight gain could lead to preterm delivery.2 According to Institute of Medicine (IOM) guidelines 2009, recommended gestational weight gain is 12.7–18.2 kgs for underweight (Englishhttp://ijcrr.com/abstract.php?article_id=4504http://ijcrr.com/article_html.php?did=4504
1. Rogozinska, Zamora J, Marlin N,. Gestational weight gain outside the Institute of Medicine recommendations and adverse pregnancy outcomes: analysis using individual participant data from randomized trials. BMC Pregnancy Childbirth. 2019;19:322. doi:10.1186/s12884-019-2472-7
2. Ukah UV, Bayrampour H, Sabr Y,. Association between gestational weight gain and severe adverse birth outcomes in Washington State, US: A population-based retrospective cohort study, 2004–2013. Myers JE, ed. PLoS Med. 2019;16:e1003009. doi:10.1371/journal.pmed.1003009
3. Gariballa S, Alkaabi J, Yasin J, Al Essa A. Total adiponectin in overweight and obese subjects and its response to visceral fat loss. BMC Endocr Disord. 2019;19:55. doi:10.1186/s12902- 019-0386-z
4. Kumar R, Mi K, Badruddeen J A,. Solasodine with Coenzyme Q10 Supplementation Ameliorates High Fat Diet-Induced Metabolic Syndrome in Rats by Modulating Adipokines and Lipid Peroxidation. IJCRR. 2021;13(11):38-44. doi:10.31782/ IJCRR.2021.131109
5. Chaitali M, Cg R, Ac A. Adiponectin to Resistin Ratio concerning Insulin Resistance in Different Phenotypes of PCOS in Indian Population. IJCRR. 2021;13(19):09-13. doi:10.31782/ IJCRR.2021.131904
6. Mallardo M, Ferraro S, Daniele A, Nigro E. GDM-complicated pregnancies: focus on adipokines. Mol Biol Rep. 2021;48:8171- 80. doi:10.1007/s11033-021-06785-0
7. WHO. Appropriate body-mass index for Asian populations and its implications for policy and intervention strategies. Lancet. 2004;363:157-63. doi:10.1016/S0140-6736(03)15268-3
8. Most J, Marlatt KL, Altazan AD, Redman LM. Advances in assessing body composition during pregnancy. Eur J Clin Nutr. 2018;72:645-56. doi:10.1038/s41430-018-0152-8
9. Pheiffer C, Dias S, Jack B, Malaza N, Adam S. Adiponectin as a Potential Biomarker for Pregnancy Disorders. IJMS. 2021;22:1326. doi:10.3390/ijms22031326
10. Adu-Gyamfi EA, Fondjo LA, Owiredu WKBA. The role of adiponectin in placentation and preeclampsia. Cell Biochem Funct. 2020;38:106-17. doi:10.1002/cbf.3458
11. Liu H, Yang Y, Wang Y,. The ketogenic diet for treatment of intractable epilepsy in adults: A meta-analysis of observational studies. Epilepsia Open. 2018;3:9-17. doi:10.1002/epi4.12098
12. Raza, S., Ali, U., Zubairi, A.M., Salim, E. Mean Adiponectin Levels and Frequency of Hypoadiponectinemia in Gestational Diabetes Mellitus. PJMD. Published online February 28, 2021. doi:10.36283/PJMD10-1/007
13. Riaz M, Shaikh F, Fawwad A, Hakeem R, Shera AS, Hitman GA, et al. Maternal Nutrition during Early Pregnancy and Cardiometabolic Status of Neonates at Birth. J. Diabetes Res [Internet]. 2018 [cited 2022 Feb 4];1–8. Available from: https://www. hindawi.com/journals/jdr/2018/7382946/
14. UNICEF, Pakistan. National Nutrition Survey 2018. Published online 2018.
15. Khan GN, Ariff S, Kureishy S,. Effectiveness of wheat soya blend supplementation during pregnancy and lactation on pregnancy outcomes and nutritional status of their infants at 6 months of age in Thatta and Sujawal districts of Sindh, Pakistan: a cluster randomized controlled trial. Eur J Nutr. 2021;60:781-9. doi:10.1007/s00394-020-02276-3
16. Gul R, Iqbal S, Anwar Z, Ahdi SG, Ali SH, Pirzada S. Pre-pregnancy maternal BMI as a predictor of neonatal birth weight. Petry CJ, ed. PLoS ONE. 2020;15:e0240748. doi:10.1371/journal. pone.0240748
17. Asifa K, Aqsa AA, Sameena I. Socio-demographic determinants of BMI of Pakistani women: evidence from PDHS (2017-18) using quantile regression analysis. J Pak Med Assoc. 2021;71. doi:10.47391/JPMA.1459
18. Goudet S, Murira Z, Torlesse H, Hatchard J, Busch-Hallen J. Effectiveness of program approaches to improve the coverage of maternal nutrition interventions in South Asia. Matern Child Nutr. 2018;14. doi:10.1111/mcn.12699
19. Asim M, Ahmed ZH, Nichols AR. What stops us from eating: a qualitative investigation of dietary barriers during pregnancy in Punjab, Pakistan. Public Health Nutr. Published online April 19, 2021:1-10. doi:10.1017/S1368980021001737.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411411EnglishN2022June3Healthcare
Analysis and Characterization of Chitinase in Bacillus salmalaya Strain 139SI
English3136Abdulkhaliq J. AlsalmanEnglish Arshad FaridEnglish Mohammed Al MohainiEnglish Maitham A. Al HawajEnglish Muhammad MuzammalEnglish Muhammad Hashim KhanEnglish Arezoo DadrasniaEnglish Yousef N. AlhashemEnglish Shakira GhazanfarEnglish Eman M. AlmusalamiEnglish TEnglish
Introduction: Chitinases are enzymes that hydrolyze the internal ?-1,4 glycosidic linkages of chitin, a significant structural component of arthropod exoskeletons and fungus cell walls. Objective: Main objective of current study was to determine the protein and to check the antimicrobial, antifungal activity of chitinase in Bacillus salmalaya strain 139SI. Methodology: Purified and estimation enzymes were quantified by the method of Lowry method. Antimicrobial action of chitinase was done using standard Disc Diffusion method while the hyphal extension inhibition assay was used to test the antifungal activity of pure chitinase strains 139SI, 140SI, and 141SI. Results: In this study, Bacillus salmalaya 139SIexhibited strong hemolytic activity and their protein concentration was measured as 56.43 mg/mL. In addition, strain 139SI had strong antifungal activity against phytopathogenic fungus including Fusarium sp., R. solani and Phytophthora sp. Strain 139SI had the capability in degrading the peptidoglycan component of cell walls of gram-negative bacteria such as Escherichia coli but not against the gram-positive, Staphylococcus aureus. Chitinase activity was observed when 200?l crude extract of 139SI able to degrade 0.09 g chitin of shrimp shell by breaking down shrimp shell structure and bonds of chitin effectively as early as 2 days or up to 7 days. Conclusion: Hence, based on the results, B. salmalaya 139SI has potential to be a novel biofunctional chitinase that could use as a biological agent in degrading the chitin component of fungal cell walls and shells waste of many kind of insect and crustaceans for solving future problem in the agricultural sector and fishery industry.
EnglishChitinases, B. Salmalaya, Biofunctional, Peptidoglycan, Antimicrobial, Antifungal activity
Introduction
Many diseases, affect agricultural crops, resulting in considerable productivity losses including fungal infections.1 The cell wall of the fungus is the first structure that comes in contact with the host. Chitin is a homopolymer of N-acetylglucosamine (GlcNAc) units that makes up the major component of the fungal cell wall.2 As chitin is a major cell wall component of most pathogenic fungi, chitinase-producing bacteria can break down chitin, which can be employed as a biological control of fungal infection in crops. Chitinase also has been proven in as a biocontrol agent in degrading chitin in the exoskeleton of white fly.3
Chitin is a crucial part in the construction of fungal cell walls, as well as other vertebrates (fish scales) and invertebrates (mollusks, nematodes, worms, arthropods, cephalopods, etc.).4 Chitinase is a hydrolytic enzyme and belongs to the glycosyl-hydrolase family that hydrolyzes glycosidic links in chitin and converts polymeric chitin to chitooligosaccharides.5
Furthermore, seafood processing businesses, notably those in the processing foods industry, exploit marine resources to meet humanity's needs, resulting in massive amounts of chitin-containing waste in coastal areas.6 Despite its gigantic abundance, utilization of chitinous waste such as crab, shrimp, and lobster is still in its primitive stage due to the crystallinity and insolubility of chitin itself. Chitinase is capable in breakage of bonds typical to that of chitin in shrimp therefore, the degradation of chitin crustacean waste by chitinase enzyme is one of the industrial interest because it can be used as bioremediation of seafood waste at a large scale and has green characteristics, not harm the environments.6 The main objective of this study was to analyze and characterize chitinase activities in B. salmalaya strain 139SI. To analyze chitinase activities in B. salmalaya 139SI, 140SI and 141SI strains and their relationship with chitinase enzyme purification and observe the potential of the strains against various types of fungi and bacteria. To test the capability of the strains in degrading chitin-composed material such as shrimp shells.
Materials and Methodology
Isolation and screening of the strain
Bacillus salmalaya 139SI was first discovered in soil from a private farm in Selangor, Malaysia.7
Growth condition and Chitinase production
A single colony of 139SI, 140SI and 141SI from a Brain Heart infusion(BHI) agar plate was inoculated and grown in 1 litre of Difco™, USA.BHI medium containing potassium chloride (5 g/L), dextrose (3 g/L), disodium hydrogen phosphate (2.5 g/L), gelatin (14.5 g/L), BHI (6 g/L), and peptic digest of animal tissue (6 g/L) plus 1 percent (w/v) chitin powder from shrimp shell as inducer was shaken in a shaking incubator at 150 rpm for 72 hours at 35 °C. The 72-hour culture was centrifuged (8000×g for 15 minutes) and filtered through a Whatman no.1 filter using a SORVALL ST 16R centrifuge. The cell-free broth or supernatant was concentrated by freeze-drying (at the Microbiology laboratory, near PASUM) and was stored at −20 °C.
Protein determination
Purified and estimation enzymes were quantified by the method of Lowry method.8 Bovine serum albumin was used as standard. Firstly, Lowry reagent or alkaline copper sulfate solution was prepared by adding and mixing 50 ml solution A (2% sodium carbonate was added into 0.1 M NaOH) with 1 ml of solution B (0.5% copper sulfate was added into 1% sodium potassium tartrate solution). Then, Standard Protein Solution (BSA) was prepared by dissolving 200 mg of BSA into 100 ml of distilled water and diluting this stock BSA into 100 ml of distilled water. Finally, the solution was prepared and its absorbance was measured at 700 nm using the NV203 spectrophotometer.
In the test tubes, different dilutions of BSA solutions were prepared by mixing stock BSA solution (1 mg/ ml) and water as shown in table 1. The BSA concentration ranges from 0.05 to 1 mg/ml. 5 mL alkaline copper sulfate reagent was added to these various dilutions. After incubating for 10 minutes at room temperature, added 0.5 ml of reagent FolinCiocalteau solution (reagent solutions) to each tube and incubated again for 30 minutes or until the solution becomes blue. Each of the test tubes has a final volume of 6.5 ml. A standard calibration curve was created by plotting the absorbance against the protein concentration graph.
Assay of purified chitinase for antifungal
The hyphal extension inhibition assay was used to test the antifungal activity of pure chitinase strains 139SI, 140SI, and 141SI.9
Antimicrobial action of chitinase
The antimicrobial action of Chintinase was done using the standard Disc Diffusion method.10
100ug/ml original extract of chitinase crust 139SI, 140SI and 141SI were dropped onto disc (6.0/mm). Then, they placed 25mm perimeter from each other onto bacteria culture along with positive and negative control dick. The inoculated plates were cooled to allow the bioactive compounds to diffuse into the agar, then incubated at 37°C and checked for inhibitory zones around the wells after 24, 48, and 72 hours. A ruler was used to measure inhibition zones in millimeters under and around discs.
Effect of crude chitinase prawn shell
Two prawn shell were prepared and treated with different concentrations of purified chitinase of strain 139. Prawn shell 1 was treated with 200ug/ml of strain 139SI, while prawn shell 2 was treated with 400ug/ml of strain 139SI. Then, shell structure was observed in days 1, 3,7,14,21,28 and 41 days to identify the ability of chitinase in degradation action under a 1000x magnification compound microscope (NIKON 261871).
Results
Chitinase production
Chitinase crust (in powder form) was produced and extracted from 25ml of bacterial supernatant of each strain of Bacillussalmalaya after being freeze-dried. Strain 141SI produced a large amount which is 1.19g compared with strain 1140SI (1.06g) and 139SI (0.89g), respectively.
Enzyme purification
The concentration of chitinase of each strain plotted against absorbance at 700nm on the BSA standard graph is highlighted in figure 1. The concentration of purified protein of strain 139SI was 87.86 Mg ml -1 and its absorbance was 0.464 O.D. While, concentrations of purified protein of strain 140SI and 141SI were 82.70 Mg ml -1 and 56.43 Mg ml -1 and their absorbance were 0.435 O.D and 0.288 O.D, respectively. Based on the result, the regularity of purified enzyme chitinase in each sample can be simplified as shown; 139SI>141SI>140SI.
Antifungal
Based on Figure 2(a & b), the results showed that 100ug/ml chitinase could inhibit the growth of R. solani. The mean and standard error of the inhibition zone of strain 139SI within 2 days was 15 ± 9.90, while strain 140SI and 141 SI were 11.5 ± 4.95 and 13.5 ± 6.36, respectively. The inhibitory indices of all samples have a certain regularity against R. solani, which is 139SI> 141SI > 140SI.
Based on Figure 2(c & d), the results showed that 100μg/ml of all purified chitinase could inhibit the mycelial growth of Phytophthora sp. The mean and standard error of the inhibition zone of strain 139SI within 2 days was 19 ± 10.61, while strain 140SI and 141 SI were 16.5 ± 10.61 and 16.5 ± 7.78, respectively. The inhibitory indices of all samples have a certain regularity against Phytophthora sp. mycelial growth which is 139SI> 141SI > 140SI.
Based on Figure 2 (e &f), the results showed that 100μg/ml of each purified chitinase could inhibit the growth of Fusarium sp. The mean and standard error of the inhibition zone of strain 139SI within 2 days was 15±11.31 while strain 140SI and 141 SI were 11.5±7.78 and 13.5±9.19, respectively. The inhibitory indices of all samples have a certain regularity against Fusarium sp. mycelial growth which is 139SI> 141SI > 140SI.
Overview of the mean and standard error of purified chitinase in inhibiting the mycelial growth of three different types of phytopathogenic fungi; R.solani, Phytophthora sp., Fusarium sp. are shown in Table 2.
Antibacterial
100ug/ml of purified chitinase of each strain 139SI, 140SI and 141SI had been tested against S. aureus (gram +ve) and E. coli (gram -ve). Based on the observation after 24 hours, all purified chitinase were resistant towards S. aureus while, strain 139SI was more sensitive compared to strains 140SI and 141SI against E. coli (Figure 3 a & b).
Effect of crude chitinase on prawn waste
Two prawn shell were treated with different concentrations of chitinase 139SI. Prawn shell that was treated with 200ug/ml of chitinase was started to degrade at day 7 and their mass. While, prawn shell treated with 400ug/ml of chitinase was degraded as early as day 3 and their shell part obviously shrunken at day 7 (figure 4 a, b,c,d & e).
Discussion:
Enzyme purification or protein measurement Lowry assay was used to measure the amount of purified chitinase concentration in each strain. The quantity of protein was calculated using a BSA standard curve, and the advantages of this test include its sensitivity and, most significantly, accuracy.11 Crust chitinase of each sample was diluted twice before being measured at 700nm. Based on the result obtained from BSA standard graph (figure1), strain 139SI has the highest amount of purified chitinase enzyme concentration which is, 87.86 Mg ml-1 at 0.464 O.D. Strain 140SI contained 82.70 Mg ml-1 of chitinase enzyme concentration at 0.435 O.D. While strain 141SI only has 56.43 Mg ml-1 of purified chitinase at 0.288 O.D even though, it produced the highest amount of chitinase crust (powder form) in the previous assay (freeze-drying assay). Hence, it proved that the chitinase crust of 141SI was not pure compared to 139SI. The reason might be due to the chitinase crust being dominated by other substances. Increased concentration of crude extracts will be accompanied by the enhancement of cell viability.7 Meanwhile, the highest concentration of chitinase enzyme will lead to increases in performance, especially against various types of fungi, and bacteria and the ability in degrade chitin-composed material.
Pathogenic fungi can cause disease in humans and animals and contamination and severe damage to crops, which lead to huge economic losses.12 Significant growth retardation in the mycelial growth of R. solani, Fusarium sp. and phytophthora sp. by strain 139SI was observed. 139SI showed strong antifungal activity against all these types of fungi as strong as positive control inhibits fungal growth. While the zone of inhibition was less around the wells when applied with strain 141SI and followed with strain 140SI. The antifungal activity indices of all samples have a certain regularity, which is 139SI> 141SI > 140SI. Results above demonstrated inhibitory rates and antifungal activity increase with the rise of the concentration of purified chitinase. As the 139SI has the highest concentration of chitinase enzyme (87.86 mg/ml), hence it is more efficient in inhibiting all chitin-containing fungi. Antifungal activities must be along with hyphae distortion, heavy vacuolization, and swelling and lysing hyphae.13 It can be inferred that the over-expression of recombinant chitinase protein or enzyme has a strong and considerable antifungal effect.
Among all those three types of pathogenic fungi, 139SI was more sensitive towards phytophthora sp. and less effective against Fusarium sp. especially after 24 h incubation. Figure 2 depicts the inhibition zone of all pure chitinases, including 139SI, which ranges from 6 to 7 mm only after 24 hours. Therefore, the degree of inhibition is proportional to the amount of chitin in the cell wall of the target fungus. Chitinolytic enzymes, as well as the genes that code for them, could be used to create transgenic microorganisms with improved biocontrol capabilities and could be used to control fungal plant pathogens.13 The appearance of unambiguous zones of inhibition confirmed and identified antibacterial activity. Purified chitinase was used against two species of bacteria (S. aureus and E. coli), and microorganisms were considered positive for bioactive substances if an inhibitory zone of at least 8 mm wide was detected around the disc.14 Because B. salmalaya is a gram-positive bacterium, it showed no inhibition zone or antibacterial activity against S. aureus which is also a gram+ve bacteria and causes a wide range of infections ranging from skin infections to life-threatening diseases.14 Penicillin was employed as a positive control, with a 27mm inhibition zone.
While gram-negative bacteria such as E. coli were inhibited by 100ug/ml pure chitinase strain. Strains 139SI, 140SI, and 141SI had 12mm, 11mm, and 10mm inhibition zones against E. coli agar plate culture, respectively. As the concentration of pure protein in strain 139SI increased, the highest antibacterial activity was seen. 140SI, on the other hand, was less sensitive to E. coli due to a decrease in pure chitinase. Ampicillin was utilized as a positive control; however, it was shown to be resistant to E. coli. Resistance to β -lactam antimicrobial drugs in E. coli has been proven to be ineffective against pathogenic E. coli, which causes diarrhea, meningitis, and urinary tract infections, among other clinical syndromes.15
This study showed the degradation of chitin-composed material and action mechanisms of the chitinase enzyme was related to the volume of treatment given on prawn shells. The highest concentration of purified chitinase was given on prawn shell, hence the enzyme action mechanisms in hydrolyze chitin, a linear polymer of β-(1,4)-linked N-acetylglucosamine (NAG),16 was more effective. This fits the result obtained in this study where 400ug/ml of purified chitinase was more effective in degrading the chitin of the prawn shell as their cracked can be seen as early at day 3 and obviously shrunken at day 7 compared to the prawn shell that was treated with 200ug/ml chitinase, which their degrading part only appear at day 7 and shrunken, not obvious. The chitin found in the peritrophic matrix and the interior layers of exoskeletons of crustaceans such as prawns, shrimp, and crabs provide support for the muscle system as well as growth and development.17 Chitinase enzymes can directly degrade their chitin-containing structures by breaking down a typical bond that binds with chitin and as crustacean shells especially on prawn shell as a major carbon or nitrogen source for the production of chitinase.18 As a result, it is great for bioremediation and waste management, as well as releasing nutrients and keeping the carbon, nitrogen, and other biogeochemical cycles in check.19
Conclusion
Chitinase is an enzyme that aids in the breakdown of chitin-based materials. Among all the Bacillus salmalaya strains tested, 139SI, which has strong hemolytic activity, showed the best and strongest antifungal activity against three types of phytopathogenic fungi, including Fusarium sp., Phytophthora sp., and R. solani, suggesting that it could be used in the field to combat plant pathogenic fungi. This work revealed that strain 139SI also has capable in degrading peptidoglycan of gram-negative bacteria such as Escherichia coli and degrading prawn shell structure at a concentration of 400ug/ml of chitinase enzyme. Increased concentration of crude extracts will be accompanied by the enhancement of cell viability. As the 139SI has the highest concentration of purified chitinase enzyme (87.86 mg/ml), hence their performance was increased especially against various types of fungus, bacteria, and degrading chitin-composed material. However, a larger scale trial is needed in the future for the degradation of marine waste as chitinase enzymes have the potential to be used as a biocontrol agents in controlling plant diseases.
Acknowledgment
The authors gratefully acknowledge Nur Huza Aziera Binti Mohamad Huzairo (Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia) for experimentation assistance and Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia, for providing financial support and lab facility for this research work.
Source of funding: This research received no external funding.
Conflict of Interest: The authors declare no conflict of interest.
Authors’ Contribution: All authors contributed equally
Figure 2 (a) Effect of purified chitinase 139SI on mycelial growth of R. solani after 24h incubated. (b) Effect of purified chitinase on mycelial growth of R. solani after 48h incubated.(c) Purified chitinase against Phytophthora sp. After incubated 24 h. (d) Purified chitinase against Phytophthora sp. 48h. (e) Effect of purified chitinase on mycelial growth of Fusarium sp. after 24h incubated. (f) Effect of purified chitinase on mycelial growth of Fusarium sp. after 48h incubated.
Figure 3: (a) Agar plate is shown strain139SI,140SI and 141SI not sensitive to the tested gram-positive bacteria, S. aureus after incubated 24 h. (b) Strain 139SI has larger antibacterial zone inhibition (12mm) against gram-negative bacteria, E.coli, compared to both strains of 140SI(10mm) and 141SI (11mm) strain.
Figure 4. (a)Prawn shell 1 before treatment at 0 days. The red circle showed the original shape and structure before being treated and degrade by the chitinase enzyme. (b) The red circle showed the degradation area of prawn shell 1 after 7 days; reduction in shell prawn chitin material by 200ug/ml chitinase enzyme from the strain 139SI (c) Prawn shell 2, before treatment at day 0. Red circle showed the original shape and structure before treatment and degrade by chitinase enzyme strain139SI. (d) The red circle showed prawn shell 2 part was degraded by 400ug/ml purified chitinase strain139SI as early as 3 days after treatment. (e) Red circle showed prawn shell 2 part was obviously shrunken at day 7.
Englishhttp://ijcrr.com/abstract.php?article_id=4505http://ijcrr.com/article_html.php?did=4505
1. Toufiq N, Tabassum B, Bhatti MU, Khan A, Tariq M, Shahid N, et al. Improved antifungal activity of barley derived chitinase I gene that overexpress a 32 kDa recombinant chitinase in Escherichia coli. host. Braz. J. Microbiol. 2018 Apr;49:414-21.
2. Yan J, Yuan SS, Jiang LL, Ye XJ, Ng TB, Wu ZJ. Plant antifungal proteins and their applications in agriculture. Appl. Microbiol. Biotechnol. 2015 Jun;99(12):4961-81.
3. Seo DJ, Lee JH, Song YS, Park RD, Jung WJ. Expression patterns of chitinase and chitosanase produced from Bacillus cereus in suppression of phytopathogen. Microb. Pathog. 2014 Aug 1;73:31-6.
4. Moussian B, Schwarz H, Bartoszewski S, Nüsslein-Volhard C. Involvement of chitin in exoskeleton morphogenesis in Drosophila melanogaster. J. Morphol. 2005 Apr;264(1):117-30.
5. Pesch YY, Riedel D, Patil KR, Loch G, Behr M. Chitinases and Imaginal disc growth factors organize the extracellular matrix formation at barrier tissues in insects. Sci. Rep. 2016 Feb 3;6(1):1-4.
6. Chen JK, Shen CR, Liu CL. N-acetylglucosamine: production and applications. Mar drugs. 2010 Sep;8(9):2493-516.
7. Ismail S, Dadrasnia A. Biotechnological potential of Bacillus salmalaya 139SI: a novel strain for remediating water polluted with crude oil waste. PLoS One. 2015 Apr 13;10(4):e0120931..
8. Ramakrishnan N, Shanmugasundaram E. Short Communication Variations in argüíase and OTCase levels during growth in Aspergillus. J Indian Inst Sci. 1979:63.
9. Roberts WK, Selitrennikoff CP. Zeamatin, an antifungal protein from maize with membrane-permeabilizing activity. Microbio. 1990 Sep 1;136(9):1771-8.
10. Yaseen M, Kamran M, Farid A, Ismail S, Muzammal M, Amir KA, et al. Antibacterial, Hemagglutination, and Insecticidal Activity Studies on the Solvent Extracts of the Roots of Olea for guinea. Makara J. Sci.. 2022;26(1):8.
11. Olson BJ, Markwell J. Assays for determination of protein concentration. Curr Protoc Protein Sci. 2007 Sep;38(1): A-3A .
12. Rajaofera MJ, Jin PF, Fan YM, Sun QQ, Huang WK, Wang WB, et al. Antifungal activity of the bioactive substance from Bacillus atrophaeus strain HAB-5 and its toxicity assessment on Danio rerio. PesticBiochem Phys. 2018 May 1;147:153-61.
13. Lorito M, Harman GE, Hayes CK, Broadway RM, Tronsmo A, Woo SL, et al. Chitinolytic enzymes produced by Trichoderma harzianum: antifungal activity of purified endochitinase and chitobiosidase. Phytopathology. 1993 Mar 1;83(3):302-7.
14. Ghasemi S, Ahmadian G, Sadeghi M, Zeigler DR, Rahimian H, Ghandili S, et al. First report of a bifunctional chitinase/ lysozyme produced by Bacillus pumilus SG2. Enzyme Microb. Technol. 2011 Mar 7;48(3):225-31..
15. Livermore DM. beta-Lactamases in the laboratory and clinical resistance. Clin. Microbiol. 1995 Oct;8(4):557-84.
16. Park K, Nikapitiya C, Kim WS, Kwak TS, Kwak IS. Changes of exoskeleton surface roughness and expression of crucial participation genes for chitin formation and digestion in the mud crab (Macrophthalmus japonicus) following the antifouling biocide irgarol. Ecotoxicol. Environ. Saf. 2016 Oct 1;132:186-95.
17. Martin GG, Simcox R, Nguyen A, Chilingaryan A. Peritrophic membrane of the penaeid shrimp Sicyoniaingentis: structure, formation, and permeability. Biol. 2006 Dec;211(3):275-85.
18. Herrera-Estrella A, Chet I. Chitinases in biological control. EXS-BASEL-. 1999 Jan 1;87:171-84..
19. Waghmare SR, Ghosh JS. Chitobiose production by using a novel thermostable chitinase from Bacillus licheniformis strain JS isolated from a mushroom bed. Carbohydr. Res. 2010 Dec 10;345(18):2630-5.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411411EnglishN2022June3Healthcare
Factors Affecting the Production of Astaxanthin in the Microalgae Haematococcus pluvialis: A Review
English3746Muhsinin SoniEnglish Aligita WidhyaEnglish Rostinawati TinaEnglish Levita JuttiEnglish
Astaxanthin, a natural red pigment that belongs to the carotenoid group, has been known as a super antioxidant due to its very strong antioxidant activity (65 times higher than vitamin C, 54 times more potent than -carotene, and 14 times higher than vitamin E). Haematococcus pluvialis is known as microalgae with a high astaxanthin content. The benefit of astaxanthin in health issues is mainly its potential as the treatment for degenerative diseases caused by reactive oxygen or nitrogen species. Thus, it is important to develop Haematococcus pluvialis microalgae as a rich source of natural astaxanthin in the health and pharmaceutical industries.
EnglishAstaxanthin, Antioxidants, Haematococcus pluvialis, Carotenoids, Microalgae, Anticancer
INTRODUCTION
Astaxanthin (3,3'-dihydroxy-β-carotene-4,4'dione) is a secondary metabolite belonging to the carotenoid group.1–3 Astaxanthin has a high value in the pharmaceutical, nutraceutical, and cosmetic fields because of its potent antioxidant potential with an IC50 value of 39.1 ± 1.14 ppm.4 The antioxidant activity produced by astaxanthin is 65 times higher than that of vitamin C, 54 times more powerful than β-carotene, 14 times higher than vitamin E, and 20 times stronger than its synthetic form.5 Due to its potent antioxidant activity, astaxanthin can be used to treat several degenerative diseases caused by free radicals.6
Various sources of astaxanthin in nature can be obtained from several microorganisms such as the fungus Phaffia rhodozyma, microalgae Chlorella zofingiensis, and Haematococcus pluvialis.7–9 However, of these microorganisms, H. pluvialis is known to show the highest astaxanthin accumulation capacity of up to 4% dry weight under stress conditions.10,11
The market price of astaxanthin also varies, ranging from $2,500 to 7,000/kg. In 2014, the global market potential of astaxanthin was approximately 280 tonnes for $400 million. However, more than 95% of the market is synthetic astaxanthin types that are sourced from petrochemicals. This happens because the production cost of synthetic astaxanthin is relatively cheaper than natural astaxanthin obtained from microalgae.12 This synthetic type of astaxanthin has 20 times lower antioxidant power than the natural type.5 In addition, related to safety issues, synthetic astaxanthin types are still not allowed to be consumed by humans due to differences in stereochemical form with natural type. Therefore, its use is only permitted as feed and dye for aquaculture organisms.10
Astaxanthin production can be done by various methods, including culture, chemical synthesis, and genetic engineering. The culture method can be done by adding stress induction to microalgae because it is known that H. pluvialis is a microalgae that can accumulate astaxanthin under stress. These stress conditions can be caused by several factors, including light stress,13,14 nutritional deficiency,15 salinity stress,16,17 the addition of Fe2+,18,19 and so on. In addition, another method is chemical synthesis using asta-C15-triarylphosphonium salt and C10-dialdehyde with the Wittig reaction,20 which produces synthetic astaxanthin with antioxidant activity 20 times lower than natural astaxanthin. Then another method, genetic engineering, in several research journals has been widely reported overproduction of astaxanthin in several microorganisms such as fungi and bacteria.21 This review article contains biological and physiological conditions, biochemical content, and methods of producing astaxanthin from H. pluvialis by culture and genetic engineering.
Biology of H. pluvialis
Taxonomy
H. pluvialisis a biflagellate unicellular microalgae that lives in freshwater. According to Lorenz (1999),22 the classification of H. pluvialis microalgae is as follows:
Habitat
The habitat of H. pluvialisis spread evenly in the world, especially in temperate areas. This microalgae has been isolated in Europe, Africa, North America, and Himachal Pradeslv India.23,24 H. pluvialis is also found in various environmental conditions with extreme climates, which may be lethal to other types of microalgae. This is because H. pluvialis can defend itself by forming encysts (cells become closed with a thick membrane) quickly when under stress and extreme conditions.25
Morphology
The cell structure of H. pluvialis is similar to that of some groups of volvocalean green microalgae. The life cycle of H. pluvialis consists of four phases with different cellular morphology, namely macrozooid (zoospore), microzooid, palmella, and hematocyst (aplanospore).10,26 The following is the morphology of H. pluvialis microalgae with descriptions (A) Motile macrozoid cells (zoospores) with a size 50 m.
The macrozoid, microzoid, and palmella phases are also known as the green vegetative phase. The microzoid phase (zoospore) is when the cell has a spherical, elliptical or pear-shaped shape with two flagella of the same length and appears anteriorly and has cup-shaped chloroplasts (Figure 1A). In this phase, with the optimum environment, flagellated cells undergo rapid division and growth, producing 2-8 daughter cells.
Figure adapted from Shah et al. (2016),27 which is licensed under the Creative Commons Attribution License.
However, suppose the environmental conditions are unfavorable (stress). In that case, the cell will remove the flagella and begin to expand in size by forming an amorphous structure layered on the inside of the extracellular matrix and develops into non-motile cells called palmella (Figure 1B).28 In this phase, the H. pluvialis cell wall thickens and consists of three layers. The first layer is a trilaminar layer containing materials such as sporopollenin, an algaenan that is resistant to acetolysis.29 According to Kim et al. (2016),30 the content of algaenans in the cell walls of H. pluvialis microalgae will inhibit the extraction process using several solvents such as acetone, methanol, dichloromethane. The second and third layers contain mannose and cellulose.28,31,32
The hematocyst phase was also referred to as the non-motile phase with astaxanthin accumulation (Fig. 1C and 1D). This phase occurs when the state of stress continues. This stress state can be in the form of nutritional deficiency, light stress with a certain intensity, salinity stress, and the addition of certain chemicals that can induce stress. Under these conditions, the palmella will turn into an asexual form or hematocyst (aplanospore). Mature hematocysts accumulate large amounts of carotenoids, especially astaxanthin, stored in lipid droplets in the cytoplasm.28
Figure 2: Illustration of the life cycle of H. pluvialis.
Figure adapted from Wayama et al. (2013),33 which is licensed under the Creative Commons Attribution License.
After the environmental conditions return to normal and optimal, the hematocyst (aplanospore) will germinate again to form a microzoid (zoospore) which will re-initiate the start of a new vegetative growth cycle (Figure 3).33
Biochemical Content
The cellular content of the H. pluvialis microalgae varies between the green phase and the red phase due to its unique life cycle. The biochemical range of H. pluvialis in the green phase and red phase according to 34 is listed in Table 1.
Description (-): no data reported
According to Table 1, H. pluvialis produced 81.2% Astaxanthin (including ester) in the red phase. This amount is the highest compared to primary metabolites (Proteins, Lipids, Carbohydrates) and other carotenoid compounds. The green phase does not produce astaxanthin. H. pluvialis enters a logarithmic phase (growth phase) and produces more primary metabolites during this phase.
Astaxanthin
Sources of Astaxanthin
Natural sources of astaxanthin are found in several organisms, including algae, bacteria, fungi, salmon, shrimp, lobster.35 But for the mass production of astaxanthin, microorganisms such as fungi and microalgae are more widely used because of their rapid growth. Some of the natural astaxanthin-producing microorganisms are listed in Table 2.
According to Table 2, H. pluvialis is the microalgae that produce the most significant amount of astaxanthin (up to 3.8%) (excluding esters). HPLC and LC-MS methods for analyzing astaxanthin compounds. The biomass of H. pluvialis was homogenized and extracted with acetone several times. The extracts were combined, evaporated with a rotavopar, and then redissolved in acetone.
Astaxanthin Biosynthesis
Astaxanthin biosynthesis in H. pluvialis is a complex series of processes that occur under stress conditions along with triacylglycerol (TAG) accumulation. Both compounds are deposited in lipid droplets in the cytosol during the red phase. The formation of astaxanthin begins with the glycolysis process, which produces pyruvate and glyceraldehyde-3-phosphate (G3P). Furthermore, pyruvate, together with glyceraldehyde-3-phosphate (G3P), will form the compound Isopentenyl Pyrophosphate (IPP) as the primary precursor in the synthesis of carotenoids.
Astaxanthin belongs to the carotenoid group, is one of the C40 tetraterpenes synthesized from the isoprene unit Isopentenyl Pyrophosphate (IPP). In principle, IPP synthesis can originate from two different pathways: the mevalonate pathway (MVA) occurring in the cytosol and the non-mevalonate pathway (MEP) or the 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway occurring in chloroplasts.43–45
In H. pluvialis, IPP is synthesized from the non-mevalonate pathway. Furthermore, IPP undergoes isomerization to dimethylallyl diphosphate (DMAPP). Some research results indicate that the conversion is catalyzed by the enzyme isopentenyl pyrophosphate isomerase (IPI) encoded by the ipi1 and ipi2 genes during astaxanthin accumulation.2 However, the results of another study also stated that neither of the ipi1 and ipi2 genes was increased as long as H. pluvialis cells accumulated astaxanthin.46 Another study reported that another enzyme with similar activity, namely 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), was more likely to be responsible for catalyzing the intermediate conversion of IPP to DMAPP.46–48
Elongation of the isoprene chain begins with a DMAPP molecule, and the addition of three IPP molecules is catalyzed by the enzyme geranyl-geranyl pyrophosphate synthase (GGPS).49,50 The next step of this process is the formation of the compound C20 geranyl-geranyl pyrophosphate (GGPP). GGPP is converted to C40-phytoene as a precursor of astaxanthin and other carotenoids with the help of the phytoene synthase (PSY) enzyme encoded by the psy gene coupled with the head-to-tail condensation of two GGPP molecules.50
Figure 4. Biosynthesis of astaxanthin in H. pluvialis27
The formation of lycopene takes place through four desaturation steps catalyzed by two enzymes, namely the enzyme phytoene desaturase (PDS), which is encoded by the pds gene and z-carotene desaturase (ZDS), which is encoded by the zds gene.51,52 The desaturation reaction will increase the number of conjugated double bonds in the carbon chain to form chromophore groups in carotenoids, change the colorless molecule of phytoene to the lycopene, and produce a red color.50
Lycopene undergoes cyclization catalyzed by the enzyme lycopene cyclase (LCY-e and LCY b), which is encoded by the lcy gene. Cyclization of carotenoid biosynthesis in most organisms produces a-carotene (a precursor to lutein) and β-carotene (a precursor to carotenoids including astaxanthin). The last two oxygenation processes are catalyzed by the β -carotene ketolase (BKT) enzyme encoded by the bkt gene, and the β-carotene hydroxylase (CrtR-b or BKH) enzymes encoded by the bkh or crtR-b genes are the final stages of astaxanthin synthesis.53–55
Pharmacological Activity of Astaxanthin
Astaxanthin as a nutraceutical has a variety of pharmacological activities, including those in Table 3.
Description: ROS: Reactive Oxygen Species; KATO-III: Human gastric carcinoma cell line; SNU-1: Human gastric carcinoma cell line; GLUT4: Glucose transporter type 4; IRS-1: Insulin receptor substrate-1; INF-γ: Interferon -γ; IL-2: Interleukin-2; ALT: alanine aminotransferase; AST: aspartate aminotransferase.
Astaxanthin Production Method
Culture Method
Culture System
In general, microalgae culture systems are divided into three: photoautotrophic, heterotrophic, and mixotrophic systems. H. pluvialis allows cultivation using all three methods, either with open or closed systems.27 In the phototropic system, microalgae are very dependent on light as an energy source and CO2 as a carbon source, both light from lamps or the sun. In heterotrophic systems, microalgae growth requires organic carbon as an energy source. Commonly used organic carbon substrate sources include glucose, acetate, and glycerol.62 In this condition, the microalgae cell density achieved was higher than the phototropic condition, so that the cost required for harvesting was lower. The mixotrophic system is a combination of phototropic and heterotropic methods. The microalgae that grow in this system can assimilate sunlight and organic carbon as energy sources simultaneously or alternately.
Culture systems, especially those that require light (photoautotrophic), are divided into 2: closed and open culture systems. Advantages and disadvantages of culture with closed and open systems can be seen in Table 4.
Cultural Conditions
The success of microalgae culture is strongly influenced by several important factors, including nutritional and environmental factors. H. pluvialis culture conditions can be seen in Table 5.
Stress Induction
H. pluvialis can accumulate astaxanthin under stress. The accumulation of astaxanthin is a response of microalgae to protect themselves from oxidative stress conditions.70 Several studies of stress induction, either physically or chemically, are listed in Table 6.
Harvesting
An efficient harvesting technique is an important step that must be done to get a high concentration of harvested biomass. Several harvesting methods commonly used for H. pluvialis are flotation and centrifugation methods.27,73
Centrifugation Method
Centrifugation is a method of harvesting microalgae based on the application of rotary power to precipitate microalgae cells so that they are separated from the liquid growth medium. The separation was supported by the difference in density between the microalgae cells and the liquid medium in which the cells grew. The centrifugation method can produce microalgae in a paste with a solid content of up to 15%. Several studies also show that the faster the centrifugation cycle, the microalgae biomass obtained can reach up to 95%.
Flotation method
This method is a separation process based on gravity where the microalgae cells attach to air or gas bubbles so that the cells float on the surface. Under these conditions, microalgae cells can be harvested easily. In certain types of microalgae, cells can flow naturally if the lipid content in the cells increases. In the flotation method, the need for operational costs will be even greater if it involves flocculants.
Extraction
Extraction methods commonly used include maceration and percolation.74 Astaxanthin is a lipophilic compound and is soluble in organic solvents and oils. Organic solvents such as acetone, DMSO, methanol, n-hexane, and vegetable oils such as olive oil, soybean oil, and corn oil have been used for astaxanthin extraction.35,74
Genetic Engineering
The development of biotechnology today also supports the use of microalgae as a producer of bioactive compounds. Most of the bioactive compounds produced by microalgae are secondary metabolites, which have low cellular production. So that the mass production of bioactive compounds from microalgae culture (without modification and engineering) is still not efficient; on the other hand, the synthesis of bioactive compounds with chemicals, especially astaxanthin compounds, will produce products that are stereochemically different from the natural products so that they are not allowed to be consumed by humans.5 However, with the advancement of biotechnology, the "factory" of microalgae biomass can be made more optimal. The use of science and methods of mutagenesis and genetic engineering is a solution that must continue to be developed. Several studies on the production of carotenoid compounds such as astaxanthin by genetic engineering are listed in table 7.
CONCLUSION
This study provides valuable pieces of information on astaxanthin, particularly regarding its pharmacology activities, biosynthesis pathway, various methods of its production in microalgae, harvesting, and extracting techniques, that will add insight to uncover the critical area of astaxanthin from microalgae.
ACKNOWLEDGEMENT
The authors thank the Ministry of Education, Culture, Research, and Technology (grant No. 10/E1/KPT/2021). The funding source did not involve in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.
CONFLICT OF INTEREST
None declared.
FUNDING
Source of funding from Ministry of Education, Culture, Research, and Technology (grant No. 10/E1/KPT/2021).
AUTHOR’S CONTRIBUTIONS
Soni Muhsinin (SM) is responsible for the conception of the study. SM and Widhya Aligita (WA) collected the articles, drafted them, and wrote the manuscript. Tina Rostinawati (TR) and Jutti Levita (JL) supervised, reviewed, and finalized the manuscript. All authors have read and approved the final manuscript to be published.
Englishhttp://ijcrr.com/abstract.php?article_id=4506http://ijcrr.com/article_html.php?did=4506Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411411EnglishN2022June3Healthcare
Effect of an Indigenous Cleanser on the Microbial Biofilm on Acrylic Denture Base - A Pilot Study
English4752Vidya S. BhatEnglish Mrinali Maria ViegasEnglish Sanath Kumar ShettyEnglish Vipin CEnglish
Introduction: Edentulous patients wearing dentures may develop Candidiasis or denture sore mouth due to improper cleaning of the dentures. Denture cleansers remove not only the biofilm, but also stains and other forms of debris of food on dentures. Since people prefer natural substances for daily use, a natural indigenous cleanser was prepared to replace the chemical denture cleansing solution. Aim: The aim of this study was to check the effect of soapnut on Candida and Streptococcus mutans biofilm formed on dentures compared with control and commercially available Clanden tablet. Materials and Methods: 18 heat cure acrylic blocks were fabricated and (DPI heat cure) and divided into two groups for biofilm formation of C.albicans and S.mutans with 9 blocks in each group. Three solutions: distilled water(control), commercial cleanser (Clanden tablet- Global Dent Aids Pvt Ltd) and indigenous solution(soapnut) were used to check biofilm cleansing efficiency by crystal violet staining method and optical density (OD) analysis using spectrophotometry. 24 hours bacterial culture was carried out to check for zone of inhibition of the test solution. The biofilm cleansing ability tests was done thrice in triplicate. The statistical analysis was performed by Student T-test Results: The highest OD values were obtained in the S.mutans group tested with control (Water). Least values were obtained in C.albicansgroup tested with Clandon tablet. But, similar result was seen with the indigenous solution as well. No significant difference was seen between the effects of the two test specimens on either group of microorganisms compared with that of the control group. A zone of inhibition was seen around the indigenous solution. Conclusion: Indigenous denture cleanser is an effective solution for daily denture hygiene maintenance.
EnglishAcrylic denture base, Denture cleansers, Indigenous solution, Soapnut (Sapindusmukorossi), Candida, S.Mutansbiofilm
INTRODUCTION
Improper cleaning of dentures may lead to denture sore mouth or candidiasis. Denture stomatitis is an oral inflammation related to denture wearing and poor oral hygiene, which occurs in 70% of denture wearers. 1Associations of denture stomatitis have been reported with mucosal trauma due to poor denture fit, prolonged duration of denture use, bacterial and fungal (primarily Candida) infection, and poor denture hygiene.2 Candida cells in the oral cavity are associated with biofilms, which differ substantially from planktonic cells due to their higher antifungal resistance.3
In the case of removable partial dentures, poor oral and denture hygiene can lead to colonization of Streptococci and that may cause inflammation of tissues and caries in abutment teeth whereas in implant-supported removable it can lead to peri-implantitis and implant failure.
Microorganisms like Candida albicans are commensals of the oral cavity which get attached to the denture in the same way that dental plaque grows on natural teeth. As time progresses, the biofilm that develops on dentures may harden and become difficult to remove. Various methods like mechanical aids, chemical methods, and microwave irradiation have been used to maintain denture hygiene. The common mechanical methods used are brushing, sonic vibrators, and ultrasonic cleaners.4
Brushing dentures is a challenging task for old individuals.5Manyelderly patients suffering from dementia and poor dexterity cannot adequately brush their dentures because of disease. Various forms such as tablets, pastes and powders are available. Their relative efficacies have varied with different manufacturers, not only in removing the biofilm but also in staining food and other debris on dentures. The primary cleaning agent used is denture cleaners is sodium hypochlorite. Commercially available denture cleansers usually contain many chemical substances which could be harmful/allergic to the patients.6
Since nowadays people prefer natural products for day-to-day use, naturally available substances and traditionally used cleansers can be tested for cleaning ability. Soap nut (Sapindusmukorossi) also known as soapberry, ritha, aritha is an indigenous fruit found in upper Indo-Gangetic, Shivaliks and Sub Himalayan tracts. The fruit contains saponin which it is widely used as a cleanser for washing hair, and removing freckles and psoriasis.7
Powdered seeds had been used for arthritis, the common cold, nausea etc. The soapnut has anti-fungal and antibacterial properties.8 Saponin from the saopnuts has been extracted by using solvents such as ethanol and water.9 Traditionally it was used as a detergent for clothes and also to polish gold ornaments.10 However there has been no study carried out to check the efficiency of soapnut on denture biofilm. The aim of this study was to check the effect of soapnut on Candida and Streptococcus mutans biofilm formed on dentures compared with a control and commercially available tablet.
MATERIALS AND METHODS
The experimental invitro study was conducted after obtaining ethical clearance. The bacterial and fungal strains were procured from the Yenepoya Research Centre microbial repository. The sample size was calculated based on triplicate values i.e three samples for each group of microorganisms and cleanser solution.
METHODOLOGY
Fabrication of acrylic blocks
A 2mm thickness of modelling wax sheet was flasked and dewaxed. The mould space obtained was packed with heat-cure clear acrylic (DPI heat cure resin) and cured in a short curing cycle. The sheet of heat cure acrylic was polished to obtain smooth surfaces devoid of any roughness and irregularities and cut into blocks of 10mm x 10mm x 2mm each (Fig.1).
These were disinfected with surgical spirit and stored in distilled water. A total of 18 blocks were obtained and divided into group A (C. albicans) and group B (S.mutans) with 9 blocks in each group.
Preparation of cleansing solutions:
Ripe soap nuts collected from a tree in Mangalore were dried at room temperature for 10 days (fig 2). 10- 12 dry Soap nuts were boiled in 60 ml of water for 30 minutes at 270ºC. The nuts were strained from the liquid and the concentrated soap nut solution was stored in a sterilized container and refrigerated. (Fig 3) The solution was sterilized under germicidal Ultra Violet light prior to use.
One Clandon tablet (Global Dent Aids Pvt Ltd) was dissolved in 50 ml of water and this solution was used for testing.
Antimicrobial activity:
The antimicrobial assay was done by disc diffusion method. The 4hour microbial culture was uniformly spread on Muller-Hinton agar plates. The microorganism strains used were ATCC 90028 and ATCC 25175 for C. albicans and S.mutans respectively. A known concentration of test compounds were placed on the inoculated plates. The zone of inhibition was measured after incubating for 24hours at 37º C.A ruler was used to measure the diameter of the disk plus the surrounding clear area in millimetres (mm).
Biofilm assay by crystal violet stain method
Overnight bacterial culture was inoculated to sterile conical flasks containing potato dextrose AGAR growth media. After 8 hours of shaking incubation, the acrylic denture resin squares were immersed in the culture and allowed to form biofilm under static conditions in multiwall culture plates. (Fig 4)
After 48 hours the acrylic blocks were taken out and immersed in distilled water, soapnut and commercial denture cleanser for 8 hours. (Fig 5). They were washed and allowed to dry and the biofilm was fixed using 95 % methanol for ten minutes. The dried and fixed biofilms were stained with crystal violet (0.1%). Acrylic blocks were rinsed with PBS and air-dried again. The stained biofilm was solubilised in an acetic acid solution (33%) and the optical density (OD) of each sample was recorded at a wavelength of 580 nm using a spectrophotometer.
Analyses:
The experiments were performed thrice in triplicate. Statistical analysis was performed by student’s t test using SPSS Version 22 software (SPSS Inc., Chicago, USA). p values 0.05
DISCUSSION
Denture hygiene maintenance is the most important step to be followed by the patient. Denture cleaning methods used on daily basis may be mechanical or chemical. Studies show a positive effect of mechanical and chemical cleaning and of the combination of both on denture cleanliness.12
According to a study by Peracini et al. mechanical cleaning method was the most prevalent among various denture users.13 Various pastes used to clean dentures contain abrasive agents that may lead to surface roughness of the denture. This surface roughness would increase the adherence of plaque and microorganisms on the abraded denture surface. Also, mechanical cleaning with pastes requires patients to manually clean the dentures, which becomes cumbersome in old age due to loss of dexterity and increased dependence.
Commercial denture cleaning products are based on chemicals like sodium hypochlorite, peroxides, neutral peroxides with enzymes, or acids.
We used 3 different solutions to check the efficiency on microbial biofilm growing on the denture surface. Since in recent years people have adapted to naturally available substances for their daily use, a cleansing solution was extracted from an indigenous fruit and compared with that of a chemical cleansing tablet. The control solution used was water and it showed to have the least cleaning efficiency on biofilm formed on the denture surface. This proved that use of water alone would not lead to any cleaning of dentures.
The denture cleansing tablet used was Clandon which is an effervescent tablet. Effervescent denture cleansing tablets contain alkaline peroxides; the tablet used in this study contains sodium perborate monohydrate. The tablets have an effervescent action when they come in contact with water by producing an alkaline solution of hydrogen peroxide containing active oxygen. This effervescence has a mechanical action of removing debris, and oxygen has antimicrobial and stain-removing effects.14
The main disadvantage of conventional chemical cleansers, such as sodium hypochlorite, is their whitening properties on dentures when used for prolonged periods.15,16 The increased resistance of pathogenic organisms to synthetic antifungal and antibacterial agents is drawing attention to plant extracts having antifungal efficiency. For geriatric patients cost and availability may be a factor while selecting a denture cleanser.17
Nowadays various plant materials having antifungal properties are being tested in the research field. The main advantage of opting for naturally occurring substances over commercial chemical solutions is minimal chances of harmful effects as well as allergic reactions.
A study was conducted to check the efficiency of lemongrass extract on the C. Albicans denture biofilm and found that there was a reduction in cell counts. 18 Another study to check the effect of Triphala as a denture cleanser revealed that it had superior cleansing ability compared to chlorhexidine gluconate.19
In our study, an indigenous naturally occurring cleanser was tested for antimicrobial activity and cleaning efficiency on acrylic denture material. Sapindustrifoliatus Linn., a small deciduous tree belongs to the family Sapindaceae which is known as soapnut in English, Ritha in Bengali and Ponnangottai in Tamil.20 The major constituents of the fruits are saponins (10%-11.5%), sugars (10%) and mucilage.21 In a study conducted to evaluate the antimicrobial activity of soapnut against S.mutans and C.albicansfound that soapnut had higher antifungal activity against C.albicans.22
But in our study, the antibacterial action of soapnut was higher than antifungal activity according to the biofilm assay test. The action of soapnut is mainly by saponins. Plant-derived saponins are secondary metabolites and are traditionally used as detergent. It has amphiphilic nature due to the presence of lipid-soluble aglycone and water-soluble chains in its structure.23 Saponins are divided into two groups which are triterpenoid and steroid glycosides. Glycoside structure characterizations are varied by the number of sugar units attached at different positions.24
Various methods for extraction of saponins have been used, but according to a study by Merve et al., Ethanol water solution (50 % v/v) with 1:10 solid- liquid ratio was the best. Bioassay directed isolation and characterization of a new acetylated triterpene saponin, named hederagenin 3-O-(2,4-O-diacetyl-α-L--arabinopyranoside)-(1-3)-α-L-rhamnopyranosyl- (1-2)- α-L-arabinopyranoside (1), together with six known triterpenoidal hederagenin saponin (2-7) from S. Mukorossi.25
The indigenous soapnut solution had a significant effect against the denture biofilm but slightly lesser compared to the commercially available tablet cleanser. The antimicrobial activity test by disc diffusion method also showed an inhibitory zone formed around the well-containing soapnut solution, thus proving it has antimicrobial action.
Conclusion: The indigenous soapnut solution had a significant effect against the denture biofilm but slightly lesser compared to the commercially available tablet cleanser. The antimicrobial activity test by disc diffusion method also showed an inhibitory zone formed around the well-containing soapnut solution, thus proving it has antimicrobial action Thus naturally available sources could be an economic and chemical-free alternative.
Limitations:
Effects of the indigenous solution on the Physical and esthetic properties of the acrylic material were not evaluated in the present study.
Biofilm formation was not carried out in an environment simulating the oral cavity
Acknowledgement: We acknowledge Yenepoya Research Center for the facilities provided. We acknowledge the immense help received from the scholars whose articles are cited and included in the references of this manuscript. The authors are also grateful to authors/editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.
Conflict of Interest: Nil
Source of funding: Self-funded
Authors’ Contribution: Dr.Vidya Bhat –concept, guide and overall conduct of study, Dr.Mrinali- methodology and conduct of study. Mr.Vipin- Microbial assay
Dr. Sanath Shetty-approval of study design
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