Extraction and identification of endophytes for skin health

Mahlangu, Siphiwe Godfrey

Extraction and identification of endophytes for skin health

Thesis / Dissertation

2026

Publisher

University of Cape Town

Department

Department of Chemical Engineering

Faculty

Faculty of Engineering and the Built Environment

Abstract

The health crisis of multidrug microbial resistance is responsible for approximately 700 000 deaths annually, worldwide. Interest in exploring ancient treatment methods to develop novel therapeutic strategies against multidrug-resistant (MDR) pathogens has increased in recent years. The ongoing increase in the number of infectious diseases exacerbated by antimicrobial resistance has intensified the need for novel, organic, and potent antimicrobial agents. In addition, antioxidant compounds are valuable in drug discovery owing to their defensive effects against oxidative stress, potentially treating various ailments including skin diseases and infections. Medicinal plants produce a plethora of bioactive compounds with antimicrobial and antioxidant properties, which play critical roles in drug development. Centella asiatica (L) Urban and Gunnera perpensa L are multipurpose medicinal plants indigenous to the Western Cape Province of South Africa. These medicinal plants treat inflammation, burns, ulcers, wounds, postoperative hypertrophic scars, eczema, and cough. However, the extensive use of medicinal plants for drug development can result in deforestation, potentially leading to the extinction of plant species. This can be prevented by using endophytes, as only a small portion of the plant sample is required for extraction. Thus, endophytes (endo-symbiotic bacteria and fungi) reside in host plants. Endophytes stimulate the host plant defence system and contribute to their medicinal properties. Endophytes sometimes produce compounds similar to those produced by the host plants, making them promising candidates for drug development. Hence, these compounds have proven valuable resources as they may have significant untapped potential to eradicate multidrug-resistant infections and oxidative stress in humans. In this study, fresh healthy plant samples were collected and 31 endophytic bacteria were isolated from surface-sterilized Centella asiatica leaves and Gunnera perpensa rhizomes, with 20 bacterial endophytes isolated from C. asiatica leaves and 11 endophytic bacteria isolated from G. perpensa rhizomes. C. asiatica isolates were identified as Pseudomonas sp. strain (SGM1, SGM2, SGM3, SGM4, SGM5, SGM6, SGM7), Novosphingobium sp. strain SGM8, Pseudomonas sp. strain (SGM9, SGM10), Chryseobacterium sp. strain SGM11, Enterobacter sp. strain SGM12, Enterobacter sp. strain SGM13, Pseudomonas sp. strain SGM14, Enterobacter sp. strain SGM15, Enterobacter sp. strain SGM16, Agrobacterium sp. strain SGM17, Pantoea sp. strain SGM18, Paraburkholderia sp. strain SGM19, and Pseudomonas sp. strain SGM20. Herbaspirillum sp. (GP-SGM1, GP-SGM2, GP-SGM3, GP-SGM11), Pseudomonas sp. (GP-SGM4 and GP-SGM5), and Novosphingobium sp. GP-SGM6, Chryseobacterium sp. GP-SGM7, Labedella sp. GP-SGM8, Brevibacterium sp. GP-SGM9, and Pseudomonas sp. GP-SGM10 were isolated from G. perpensa rhizomes and characterized by scanning electron microscopy (SEM). The growth kinetics of the 31 endophytic bacteria were evaluated using the specific maximum growth rate (µmax) and optical density (OD) values on 12-well microtiter plates. Standard bacterial growth trends were observed for C. asiatica strains, with the exception of Enterobacter sp. strain SGM15 (0.03 ± 0.02 hr-1) which portrayed slower cell growth, while Pseudomonas sp. strain SGM6 (0.27 ± 0.16 hr-1) displaying the highest µmax, followed by Enterobacter sp. strain SGM16 (0.25 ± 0.32 hr-1). Additionally, Pseudomonas sp. strain SGM3 exhibited the highest maximum OD (6.74 ± 0.14), while the lowest maximum OD was recorded for Novosphingobium sp. strain SGM8 (0.14 ± 0.06). G. perpensa strains growth trends revealed that Chryseobacterium sp. GP-SGM7 portrayed the highest µmax and OD values of 0.33 ± 0.01 hr−1 and 4.20 ± 0.04 while Herbaspirillum sp. GP-SGM11 portrayed noticeably slower growth (0.12 ± 0.05 hr−1) and least microbial growth (1.50 ± 0.00). The antibacterial activity was evaluated using the disc diffusion method against five pathogenic bacteria: Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. It was observed that B. cereus, S. aureus, K. pneumoniae, and P. aeruginosa were more susceptible to the antibacterial metabolites from most endophytic isolates from C. asiatica and G. perpensa isolates. All 31 endophytic crude metabolites were ineffective against E. coli. Pseudomonas sp. strain SGM2 from C. asiatica, and Chryseobacterium sp. GP-SGM7 from G. perpensa exhibited significant antibacterial activity. The DPPH and FRAP assays demonstrated that all C. asiatica and G. perpensa endophytic crude metabolites portrayed strong antioxidant activity in a dose-dependent manner with Pseudomonas sp. strain SGM2 displaying the greatest DPPH (88.09 ± 0.73 %) and Chryseobacterium sp. strain SGM11 showing the highest antioxidant activity in the FRAP assay (0.72 ± 0.01) of the C. asiatica endophytic strains and Chryseobacterium sp. strain GP-SGM7 and Pseudomonas sp. strain GP-SGM10 showed the highest scavenging activity (73.86 ± 1.01 % and 73.66 ± 1.68 %, respectively); greater reducing power of 0.54 ± 0.01 was also exhibited by Chryseobacterium sp. strain GP-SGM7 demonstrating the greatest antioxidant properties for G. perpensa strains. Pseudomonas sp. strain SGM2 of C. asiatica and Chryseobacterium sp. GP-SGM7 of G. perpensa were chosen as the isolates of interest for further alteration of cultivation parameters to maximize growth yields and metabolite production. The effects of different temperatures (23, 30, and 37°C) and glucose feed (0, 2, 4, and 8 g/L) on the growth patterns and production of antibacterial and antioxidant metabolites in Pseudomonas sp. strain SGM2 and Chryseobacterium sp. GP-SGM7 were grown in 2 L shake flasks. Cultivation of Pseudomonas sp. strain SGM2 and Chryseobacterium sp. GP-SGM7 at 30°C with 0 g/L glucose appeared to increase the microbial growth, as the highest maximum OD was observed under these conditions. However, increasing the temperature to 37°C with or without glucose supplementation resulted in a decrease in the microbial yield. However, at 37°C with 0 g/L glucose, µmax increased in Pseudomonas sp. strain SGM2 and Chryseobacterium sp. GPSGM7 was observed at 23 and 30°C in the presence of 0 g/L glucose. The antibacterial and antioxidant properties under these conditions revealed significant inhibition against S. aureus at 30°C (2, 4, and 8 g/L) and at 30°C with 0 g/L glucose, significant inhibition was observed against four pathogenic strains B. cereus, S. aureus, K. pneumoniae, and P. aeruginosa. No inhibition was observed for E. coli, and endophytic crude metabolites cultivated at 23 and 37°C (0, 2, 4, and 8 g/L) were not effective against all test strains. In addition, 30°C (0 g/L glucose) resulted in greater DPPH scavenging free radical activity for Pseudomonas sp. strain SGM2 (88.09 ± 0.73 %) at 20 mg/L concentration and highest DPPH for Chryseobacterium sp. GPSGM7 was observed at 30°C (0 and 4 g/L), with scavenging activity of 73.86 ± 1.01 % and 80.97 ± 0.43 %, respectively. Furthermore, Pseudomonas sp. strain SGM2 and Chryseobacterium sp. GP-SGM7 showed a significantly higher FRAP in the presence of 4 g/L glucose at 30°C. The absence of glucose and suboptimal and high temperatures resulted in decreased FRAP values. Optimum conditions for improved microbial yields and antibacterial and antioxidant production by Pseudomonas sp. strain SGM2 and Chryseobacterium sp. GP-SGM7 was grown at 30°C with 0 g/L glucose and applied to a 0.5 L Sixfor bioreactor runs. The effect of altering fermentation conditions on endophytes of interest for increased growth yields and metabolite production in a controlled environment was then investigated in a 0.5 L Sixfor bioreactor. This included testing at various pH levels (5, 6, and 7), agitation speeds (190, 250, and 500 rpm) and aeration rates (1 and 3 vvm). To validate the shake flask cultivation, an additional run was performed with 3 g/L glucose to assess the growth yields and efficacy of the metabolites produced under the selected parameters. When all changes were incorporated into the bioreactor fermentation conditions, a slight improvement was observed in biomass and antioxidant production, owing to the better agitation and oxygen availability experienced in the bioreactor. However, no antibacterial or antibiotic metabolites were produced under the tested conditions owing to the lack of antibacterial activity. The bioreactor samples were analyzed for glucose consumption trends using high-performance liquid chromatography (HPLC), and glucose utilization was detected under fermentation conditions of pH 7 – 500 rpm – 3 vvm (3 g/L glucose feed) with glucose depletion at 24 h. No residual glucose was detected under the other tested cultivation conditions. Moreover, comprehensive metabolite profiling of the crude secondary metabolites of Pseudomonas sp. strain SGM2 and Chryseobacterium sp. GP-SGM7 was analyzed using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Metabolite profiling of Pseudomonas sp. strain SGM2 and Chryseobacterium sp. GP-SGM7 under various reactor fermentation conditions yielded 86 and 93 metabolites, respectively, with distinct metabolites detected during fermentation at pH 7 – 500 rpm and 3 vvm (3 g/L glucose feed). Agitation speed, aeration rate, and pH are key parameters in endophyte biomass production. Although neutral pH, high agitation and aeration, and the addition of the carbon source glucose were conducive to endophytic growth and yielded the highest biomass, they did not necessarily facilitate antibiotic/antimicrobial production.

Keywords

multidrug microbial resistance

skin diseases

Reference:

Collections

PhD / Doctoral

Full item page