Culture-Based No-Detectable-Growth Assessment And Antioxidant Profiling of Fresh Aloe vera Gel Extract

Summary

This protocol describes the aseptic preparation of fresh Aloe vera inner-gel extract, potentiometric pH measurement, total antioxidant capacity assessment by an automated ABTS-based colorimetric assay, culture-based no-detectable-growth assessment under defined incubation conditions, and preliminary antimicrobial screening of a 70:30 (v/v) Aloe vera./N-acetyl-L-cysteine test mixture by disk diffusion and qualitative two-fold tube dilution.

Abstract

Aloe vera (Aloe barbadensis Miller) is widely used in topical and complementary medicine products. However, standardized laboratory workflows for simultaneous evaluation of total antioxidant capacity (TAC), pH behavior, culture-based no-detectable-growth status under defined conditions, and preliminary antimicrobial screening remain limited. Fresh inner-gel Aloe vera extract was prepared aseptically and formulated at 5%, 10%, 15%, and 20% (w/v). pH was measured potentiometrically, and TAC was determined by an automated ABTS radical cation-based colorimetric assay. Culture-based no-detectable-growth assessment was performed by inoculating the extract onto blood agar, eosin methylene blue agar, and Sabouraud dextrose agar, followed by aerobic incubation and visual inspection at 24 h, 48 h, and 72 h. Antimicrobial screening of a 70:30 (v/v) Aloe vera/N-acetyl-L-cysteine test mixture was performed against standard reference strains by disk diffusion and qualitative two-fold tube dilution assays. TAC differed significantly among groups (Kruskal-Wallis, p = 0.014), with the highest values observed in the 20% formulation. No visible bacterial or fungal growth was observed under the tested culture conditions during the 72 h observation period. The Aloe vera/N-acetyl-L-cysteine test mixture produced inhibition zones ranging from 11 mm to 17 mm, with the largest zone observed against Pseudomonas aeruginosa. This workflow provides a practical laboratory approach for preparing and preliminarily characterizing fresh Aloe vera.-based formulations for early-stage complementary medicine research.

Introduction

Aloe vera has continued to attract considerable scientific attention because of its long-standing use in traditional medicine and its expanding relevance in pharmaceutical, biomedical, cosmetic, and food research^1,2,3. This medicinal plant contains a diverse range of biologically active constituents, including polysaccharides, phenolic compounds, anthraquinones, vitamins, minerals, enzymes, and other low molecular weight metabolites that may contribute to its therapeutic and functional properties^1,2,3. Among these components, acemannan has been emphasized due to its biomaterial potential and reported roles in tissue repair, immunomodulation, and regenerative applications^4,5. In parallel, phenolic and other antioxidant-active compounds have been associated with the free-radical scavenging, protective, and biologically supportive effects of Aloe vera. preparations^2,3.

Recent years have witnessed increasing interest in the incorporation of Aloe vera into advanced formulation systems and bioactive materials. Aloe vera-based hydrogels, biomaterial inks, nanocomposites, and three-dimensional-printed scaffolds have been investigated for wound-healing and tissue-support applications because of their high hydration capacity, biocompatibility, and multifunctional biological properties^{4,6,8,9,10,11}. Experimental studies have shown that Aloe vera-containing materials may support wound management by contributing to a moist environment, improving physicochemical properties of the formulation, and providing antioxidant or antimicrobial support under selected in vitro, in vivo, and formulation-dependent conditions^{4,7,8,9,10,11}. In addition, Aloe vera has been explored in food preservation and formulation applications, where gel-based coatings and related systems have been associated with improved oxidative stability, better sensory quality, and enhanced microbiological performance^12,13. These developments indicate that Aloe vera. is increasingly regarded not only as a traditional herbal product but also as a multifunctional natural material with broad formulation-oriented potential^2,3,12,13.

The biological relevance of Aloe vera also extends to antimicrobial and microbiologically oriented applications. Recent studies have described inhibitory effects of Aloe vera or Aloe vera-containing preparations against clinically important microorganisms, including Propionibacterium acnes, Enterococcus faecalis, Candida species, and clinical isolates isolated from wound infections^14,15,16,17. Earlier in vitro evidence has also shown activity of inner-gel Aloe vera against Helicobacter pylori reference strains and clinical isolates¹⁸. Moreover, hybrid Aloe vera-containing systems have been reported to exhibit combined antimicrobial, antioxidant, and wound-healing capabilities, further supporting the multifunctional nature of this plant-derived material¹⁹. However, the biological performance of Aloe vera. is influenced by several methodological variables, including the plant fraction used, extraction procedure, concentration, handling conditions, freshness of the gel, and whether the tested preparation contains only inner gel or includes latex-associated components^2,20. These factors are especially important because inner gel and outer leaf fractions differ in both composition and toxicological profile²⁰.

Despite the growing body of literature, important methodological gaps remain. In many published studies, antioxidant behavior, physicochemical properties, microbiological assessment, and antimicrobial performance are often evaluated separately rather than within a single integrated workflow. For formulation-focused research, however, these parameters are closely interrelated. A preparation may display favorable antioxidant activity yet still require careful microbiological assessment and physicochemical characterization before it can be considered suitable for further experimental development. Therefore, laboratory workflows that combine redox-related assessment, pH evaluation, culture-based no-detectable-growth testing under defined conditions, and preliminary antimicrobial screening may provide a more practical and informative basis for the early-stage characterization of fresh Aloe vera. formulations.

Accordingly, this study aimed to evaluate fresh inner-gel Aloe vera extract formulations prepared at different concentrations in terms of pH characteristics, total antioxidant capacity, culture-based no-detectable-growth status under defined laboratory conditions, and preliminary antimicrobial screening by disk diffusion and qualitative two-fold tube dilution. By integrating these assessments within a single experimental workflow, the study was designed to provide a practical baseline approach for the preliminary characterization of Aloe vera.-based formulations intended for complementary medicine research.

Protocol

1. Preparation of Aloe vera. extract and dilution series

1. Harvest the mature leaves of an aloe vera plant grown at room temperature and rinse them thoroughly under running water to remove any residue.
2. In a biosafety level 2 (BSL-2) cabinet, disinfect the leaf surface with 70% ethanol using sterile gloves. After applying 70% ethanol, allow the leaf surface to air-dry completely for at least 2 min before aseptic incision.
3. Using a sterile scalpel, remove the distal tip and margins of the leaf, allow the yellow latex exudate to drain completely for approximately 30 min, and then fillet the leaf longitudinally. Carefully separate and collect only the transparent inner gel, avoiding the outer rind and residual latex layer.
4. Carefully transfer the Aloe vera inner gel into a sterile container and homogenize by intermittent vortexing for approximately 5-10 min at approximately 2,500 rpm (or equivalent medium-high vortex setting); perform the procedure at room temperature unless temperature control is required for the specific instrument.
5. Under aseptic conditions, prepare fresh Aloe vera. inner-gel extract formulations at 5%, 10%, 15%, and 20% (w/v) using sterile distilled water; for example, prepare 5% (w/v) as 5 g gel brought to a final volume of 100 mL, and adjust the remaining concentrations analogously.

2. pH measurement

1. Perform three-point pH meter calibration using standard pH 4.01, 7.00, and 10.01 buffer solutions with NIST traceability before each measurement series.
2. Measure pH for each concentration at room temperature (20–25 °C); record at least three technical replicates per concentration.

3. Total antioxidant capacity (TAC) measurement

1. Use the Aloe vera. extract formulations prepared in Step 1 (5%, 10%, 15%, and 20%) for TAC measurement.
2. Bring all reagents and samples to room temperature (20–25 °C) prior to analysis to ensure analytical consistency.
3. Calibrate the automated biochemical analyzer (Rel Assay, Turkey) according to the manufacturer’s standard operating procedures before initiating the assay.
4. Load the total antioxidant capacity (TAC) reagents based on the Erel method into the analyzer reagent compartments.
5. Pipette the Aloe vera. extract samples into the appropriate sample cups or reaction wells using calibrated micropipettes. (Sample volume: 5 µL)
6. Initiate the TAC assay run using the analyzer’s programmed protocol for the ABTS radical cation-based method, following the reagent volumes and incubation settings specified by the manufacturer.
7. Allow the reaction to proceed automatically, during which antioxidants present in the sample reduce the colored ABTS radical cation, resulting in a decrease in absorbance.
8. Measure the change in absorbance spectrophotometrically at the specified wavelength defined by the TAC method. (Wavelength: 660 nm)
9. Calculate total antioxidant capacity values using the analyzer’s software by comparing sample absorbance changes to the Trolox-equivalent calibration curve.
10. Express TAC results as mmol Trolox equivalents / L per unit volume of extract.
11. Perform all measurements in duplicate to ensure analytical reliability.

4. Culture-based no-detectable-growth assessment of Aloe vera. inner gel

1. Inoculate 10 µL of each extract onto blood agar, eosin methylene blue agar, and Sabouraud dextrose agar using a calibrated loop (10 µL) (colony counting method).
2. For culture-based no-detectable-growth assessment, aseptically dispense a standardized 50 µL aliquot using a calibrated micropipette onto blood agar, eosin methylene blue agar, and Sabouraud dextrose agar plates and gently spread it using a sterile disposable loop (10 µL) (Culture-based reproduction assessment using the direct drip method).
3. Incubate blood agar and eosin methylene blue agar plates aerobically at 37 °C and Sabouraud dextrose agar plates aerobically at 25–28 °C, then inspect the plates visually for growth at 24, 48, and 72 h. Incubate one uninoculated plate of each medium in parallel as a negative control.

5. Antimicrobial activity assessment of Aloe vera test mixture

1. Under sterile conditions, prepare a homogeneous test solution by mixing 70% sterile Aloe vera inner-gel extract with 30% N-acetyl-L-cysteine solution (10% w/v in sterile water).
   NOTE: This mixture reduces the high viscosity of the gel scraped from the leaf and obtains a more fluid consistency. Consider this composite formulation as a preliminary screening mixture. Therefore, interpret the antimicrobial findings to reflect the combined activity of both components.
2. Vortex the homogenized mixture thoroughly until a uniform solution is obtained.
3. Subculture the previously cryopreserved standard bacterial stocks of Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli. ATCC 25922, stored at -80 °C, onto standard blood agar (for Gram-positive bacteria) and standard eosin methylene blue agar (for Gram-negative bacteria), and incubate at 37 °C for 18-24 h.
4. At the end of incubation, prepare bacterial suspensions adjusted to 0.5 McFarland turbidity using sterile saline from the grown colonies, following standard inoculum preparation recommendations for antimicrobial susceptibility testing³⁸.
5. Inoculate Mueller-Hinton agar plates uniformly with each standardized bacterial suspension using a sterile swab.
6. Apply a standardized volume of 15 µL of the sterile Aloe vera/NAC test mixture onto each sterile 6 mm antimicrobial disc using a calibrated micropipette. The same loading volume, absorption time (at least 2 min), and air-drying time (approximately 5 min) were applied to all discs across replicates to ensure procedural consistency. This volume was selected as a practical loading amount for 6 mm discs in plant extract-based disk diffusion screening and was kept constant for all experiments.
7. Place the extract-impregnated discs onto the surface of the inoculated agar plates using sterile forceps.
8. Incubate the plates at 37 °C for 18–24 h under aerobic conditions.
9. After incubation, manually measure the diameter of each inhibition zone, including the disk diameter, to the nearest millimeter using a ruler. Perform the experiment as a single descriptive measurement, reporting the recorded zone diameters as individually observed values. Perform measurements by visually inspecting the plates according to standard disk diffusion reading recommendations³⁸.
10. Prepare two-fold serial dilutions (1/2, 1/4, 1/8, and 1/16) of the 70:30 (v/v) Aloe vera/NAC test mixture in Mueller-Hinton broth.
11. Inoculate each tube with 500 µL of the corresponding 0.5 McFarland standardized bacterial suspension prepared in sterile saline. Prepare a separate dilution series for each reference strain and incubate the inoculated tubes under the conditions specified below.
12. Incubate the inoculated tubes at 37 °C for 18–24 h.
13. Evaluate microbial growth visually by turbidity relative to the broth control; interpret the assay qualitatively rather than as a formal MIC determination.
14. Use a ciprofloxacin disc (5 µg) as a positive control in parallel with aloe vera/NAC-impregnated discs. Apply the same positive control disc for Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922 to confirm susceptibility under disc diffusion conditions.
    NOTE: Perform all microbiological procedures under BSL-2 conditions and record all zone diameter and turbidity observations immediately after incubation for subsequent descriptive analysis. This protocol combines aseptic preparation of A. vera.extract with complementary biochemical and microbiological assays to support the development of plant-based formulations for topical and complementary medicine use. The workflow is designed to be practical for routine laboratories: pH and redox screening (TAC) can be performed alongside sterility culture and two commonly used antimicrobial assays (disk diffusion and tube dilution).

Results

pH values of Aloe Vera. extract at different concentrations

The pH profile showed a non-monotonic pattern across concentrations. Mean pH values were 6.47, 6.60, 6.26, and 6.28 for the 5%, 10%, 15%, and 20% formulations, respectively. Overall group differences were significant (p = 0.024). The 5% and 10% formulations showed relatively higher pH values, whereas the 15% and 20% formulations clustered at lower pH values (p < 0.05) (Table 1 and ...

Discussion

The present study provides an integrated laboratory workflow for the preliminary characterization of fresh inner-gel Aloe vera extract formulations by combining pH measurement, total antioxidant capacity (TAC) analysis, culture-based no-detectable-growth assessment, and descriptive antimicrobial screening. Increasing concentrations of fresh inner-gel Aloe vera. were associated with higher TAC values, while no visible bacterial or fungal growth was observed during short-term culture monitoring under the ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
(-)80 C Deep Freezer	Thermofisher/USA	Sample storage	TSX40086A
Vortex	Thermofisher/USA	Sample preperation	88882010
Blood Agar	RTA/Turkey	Culture inoculation	2001
Eosine Methilene Blue Agar	RTA/Turkey	Culture inoculation	2010
SDA Agar	RTA/Turkey	Culture inoculation	2036
Müller Hinton Agar	RTA/Turkey	Culture inoculation	2022
Müller Hinton Broth	RTA/Turkey	Culture dilution	1018
Pipettes	Thermofisher/USA	Sample preperation	4642090N
Hanna pH Meters	Hitachi/JAPAN	Sample analyses	HI2211-02
Rel Assay Plate Reader	Rel Assay(Turkey)	Sample TAC analyses	M201 (Smart Model)
TAC Kits	Rel Assay(Turkey)	Kit	RL0017