Valorization of chitosan for sustainable soap production: Development,performance evaluation,and hygiene applications

Abstract

This study investigates a new method for making soap by using chitosan (CH) obtained from discarded cephalopod waste. This strategy aims to tackle the environmental issues related to trash disposal. Low molecular weight chitosan (LMWCH) was developed by extracting chitin, converting into chitosan and subjecting it to gamma irradiation for reducing the molecular weight. The chitosan has a molecular weight of 982 Da, making it well-suited for being included in soap compositions and providing strong anti-microbial activity. The soap that was produced demonstrated desirable attributes, such as a moisture content of 17.13%, a pH level of 9, and a stable fatty acid content, indicating its potential for successful commercial use. The soap had significant antibacterial activity against common pathogens such as Methicillin Resistant Staphylococcus aureus (MRSA) and Candida albicans (C.albicans). After 36 h, it produced zones of inhibition measuring 7.7 ± 1.05 mm and 8.4 ± 0.5 mm respectively. The research’s reliability is based on its meticulous experimental technique, which included comprehensive characterization of both the chitosan derivative and the final soap product. Moreover, conducting antimicrobial testing on widely recognized pathogens enhances the credibility of the soap’s possible practical uses in hygiene goods. Overall, this study underscores the feasibility of utilizing waste materials for sustainable soap production while offering potential antimicrobial benefits, thus contributing to both environmental conservation and public health.

Keywords

Chitosan soap gladius anti-microbial activity free fatty acid Fourier transform infrared spectroscopy matrix-assisted laser desorption ionization—time of flight

Introduction

The human body’s largest organ, the skin, acts as a critical barrier protecting us from the external environment. It shields us from physical, chemical, and biological threats, functioning as our first line of defense. This complex structure, composed of the epidermis, dermis, and hypodermis, plays a vital role in maintaining our health.¹ For centuries, soap has been a cornerstone of hygiene, effectively removing dirt, grease, and microorganisms from surfaces.² Traditionally soaps are produced through saponification, a process where fats (animal or vegetable) react with strong bases, soap offers a versatile cleaning agent.^3,4 However, with the emergence of antibiotic-resistant bacteria and fungal overgrowth concerns, there is a growing need for hygiene solutions with enhanced functionality. This is particularly relevant when considering antibiotic-resistant bacterial species which can cause a variety of infections, and Candida fungal species that can lead to candidiasis when overgrown. Recent studies displayed that the bacterial species has developed resistance even against higher concentration of soap leading to the ineffectiveness of the soap as a disinfectant agent.⁵ Further, to eradicate Candida fungal strains from the skin, the skin should be treated with soap followed by applying ethanolic gel. This validated the ineffectiveness of using soap alone to disinfect Candida fungal strains from the skin.⁶

Population expansion has historically been associated with waste generation. However, the problems associated with the seafood business are frequently disregarded. Over time, trash and by-products from the sector have grown dramatically. Often, these wastes are dumped in the ocean, burned, or dumped in landfills, which causes bacterial contamination, foul odors, and coastal pollution. It is less practical to completely eradicate these wastes. Reusing these wastes to make useful products should be the primary objective; this change could have positive effects on the environment and the economy^7,8 Chitosan (CH), a natural biopolymer derived from chitin (extracted from marine waste), presents exciting possibilities in various fields, including biomedical and cosmetic applications.⁹ CH was approved by United States Food and Drug Administration as safe and therefore, it opens a wide range of opportunities in the bio related industries.¹⁰

CH is found to possess antimicrobial property against pathogens isolated from skin. Costa et al.¹¹ studied the activity of CH against clinical isolates of drug resistant bacterial strains. The study revealed that the CH exhibited anti-quorum sensing activity leading to the inhibition of biofilm formation of the isolated strains. Similar study by Costa et al.¹² validated that the CH inhibits the methicillin resistant bacterial cell adhesion which declines the strain’s capability to form biofilm. Another study headed by Alburquenque et al. ¹³ studied the effectiveness of CH against 105 clinal isolates of Candida species and found that the CH had activity against ~89.9% of the isolates where 68.6% were completely mitigated. Likewise, the data reported in the literature is summarized in Table 1 for better understanding.

Table 1.

Anti-microbial activity of CH.

Sl. No.	Agent	Microorganisms	Description	Reference
1	CH	Pseudomonas aeruginosa (SS29), Escherichia coli (SS2, SS9), Proteus mirabilis (SS77), and Staphylococcus aureus (LM15)	The team compared the effect of antibacterial activity of CH against bacterial strains and found that the low molecular CH displayed better results	Bano et al.¹⁴
2	N-(2-hydroxypropyl)-3-trimethylammonium chitosan chlorides	methicillin-resistant Staphylococcus aureus	CH derivatives as antimicrobial topical agents	Hoque et al.¹⁵
3	CH	Acinetobacter baumannii	CH was prepared using green synthesis route and displayed activity against drug resistant strain	El-Naggar et al.¹⁶
4	CH	Candida albicans	Low molecular weight CH display antifungal activity	Peña et al.¹⁷
5	CH	Candida albicans	Water soluble CH display better activity than 1% acetic acid soluble CH	Tayel et al.¹⁸
6	CH	Candida albicans	CH inhibits the biofilm formation over medical devices	Pu et al.¹⁹
7	CH	C. tropicalis, C. krusei, C. guilliermondii and C. parapsilosis	Medium molecular weight CH displayed activity against pathogens and display no cytotoxicity against huma fibroblast cells	Palmeira-de-Oliveira et al.²⁰

From these literature data, it is evident that CH derived from the marine sources has the ability to fight against drug resistant bacterial strains and Candida species. In this regard, CH-enriched soaps hold promise by combining the cleaning efficacy of traditional soaps with the potential benefits of CH. Studies suggest that reducing CH’s molecular weight can improve its incorporation into soap, potentially enhancing lathering and stability while offering additional antimicrobial activity.²¹ The inherent antimicrobial properties of low molecular weight chitosan (LMWCH) may provide broader protection against bacteria, fungi, and other microbes compared to conventional soaps.²¹ As the soap is in direct contact with the skin, the toxicity related studies should be also considered for CH. The study conducted by Peng et al.²² concluded that the LMWCH displayed lesser toxicity toward human fibroblast HS27 cells than CH. While traditional soaps offer effective cleaning, LMWCH-enriched soaps hold the potential for enhanced functionality and improved skin health through their increased antimicrobial activity with more biocompatibility.

By harnessing the antimicrobial properties of LMWCH and exploring its incorporation into soap formulations, this study aims to address both environmental and public health concerns through a novel approach. Specifically, we focus on extracting LMWCH from underutilized marine waste, Sepia lessoniana gladius, to develop sustainable and potentially enhanced antimicrobial soaps. This study focuses on extraction, conversion, and characterization of LMWCH derived from gladius of Sepia lessoniana (S. lessoniana), a source of underutilized waste. The LMWCH obtained will then be incorporated into soap formulations to evaluate their physiochemical properties and antimicrobial efficacy was evaluated against Methicillin Resistant Staphylococcus aureus (MRSA) and Candida albicans (C.albicans).

Materials and methods

Sample collection

In Kasimedu, a city located on the south eastern coast of Tamil Nadu, India, gladii were collected from seafood processing facilities. The collected gladii were first subjected to a cleaning process using regular tap water to remove any adhering impurities. Following this, the gladii were spread out and allowed to dry in a shaded area. Finally, the dried gladii were ground into a powder using a blender and stored for subsequent purification steps.

Extraction of chitosan and GIR of chitosan

The CH extraction process employed a modified method based on previously reported work.²³ The extraction of CH from squid gladius involves three steps. First, deprotenization using 1N NaOH for 24 h breaking peptide bonds to remove proteins. Then, demineralization with 1% HCl for 36 h breaks minerals such as CaCO₃. The final deacetylation using 50% NaOH overnight removes acetyl groups from chitin, thereby resulting in CH.²⁴ (Figure 1). Irradiation of the CH samples occurred within a Gamma Chamber 5000, a self-shielded Co-60 Gamma Irradiator variant complying with American National Standards ANSI-N 4~3. L. The CH, housed in glass ampoules, received a dose rate of 4 kGy h⁻¹. The irradiation time varied, resulting in a range of doses delivered to the samples, starting from 75 Gy. A Fricke dosimeter served to determine the precise dosage rate employed.⁹

Fourier transform infrared spectroscopy

The analysis of Fourier Transform Infrared Spectroscopy (FT-IR) involved preparing a KBr pellet containing the sample.²³ Here, 10 mg of the LMWCH was mixed with dry potassium bromide (KBr). This mixture was then pressed into a pellet for analysis using a Bruker Alpha FT-IR spectrometer (USA). The spectrometer collected infrared radiation across a broad range of frequencies, spanning 4000–500 cm⁻¹.

Matrix-assisted laser desorption ionization—Time of flight

To determine the molecular weight of the LMWCH, a Bruker Daltonics UltrafleXtreme Matrix-Assisted Laser Desorption Ionization—Time of Flight (MALDI-TOF/MS) instrument was configured for positive ion mode with a reflectron detector and specific voltage settings. These settings included a 26 kV accelerating voltage, a 16 kV focus lens voltage, and a 19.95 kV extraction lens voltage. The system utilized a pulsed nitrogen laser operating at a frequency of 50 Hz and a wavelength of 337 nm. For improved accuracy, an external calibration was performed using standard CH. Sample preparation involved mixing the LMWCH analyte with a saturated solution of a matrix (cyano-4-hydroxycinnamic acid) in a 1:1 ratio. A small droplet (0.25 µl) of this mixture was then deposited onto a stainless-steel plate. Finally, the laser was fired, and the instrument measured the time-of-flight (TOF) to calculate the molecular weight of the LMWCH.²³

Preparation of soap

LMWCH soap was prepared with coconut oil with slight modification.²¹ The LMWCH of 2 g was dissolved in 200 ml of 0.1 m acetic acid. The dissolved LMWCH was then washed with water by centrifugation at 2000 rpm (Remi, India) for five times to remove the unbound acetic acid. The obtained precipitate was then dried for 2 h at a comfortable temperature of 40ºC. In another container, 3.5 g of NaOH (lye) were dissolved in 10 ml of distilled water to prepare a lye solution. Next, 25 ml of coconut oil were continuously stirred and heated to a gentle 40ºC using a magnetic stirrer for 15 min. The dried LMWCH 5% (w/v) was then carefully added to the warm coconut oil while constantly stirring to ensure everything blended well. Once the coconut oil and LMWCH were thoroughly mixed, the lye solution was slowly added while continuing to stir. The mixture was stirred until it became thick and creamy, indicating the soap had formed. Finally, the resulting LMWCH soap mixture was poured into a mold and left untouched for 24 h to solidify at room temperature (Figure 2).

Properties of soap

PH

A 1% LMWCH soap solution was formulated. First, a precisely weighed 1 g sample of the LMWCH soap was added to a known volume of 100 ml of distilled water in a beaker. To ensure homogenous distribution of the LMWCH soap particles throughout the solution, the mixture was gently stirred. Finally, the pH of the resulting solution was measured.

Moisture content

The moisture content of the LMWCH soap was determined following the method outlined in Sherazi.²⁵ First, the weight of an empty petri dish (W1) was carefully recorded. Then, a precisely weighed LMWCH soap sample (0.493 g) was placed within the petri dish. The combined weight of the dish and sample (W2) was then meticulously documented. To remove any moisture, the petri dish containing the soap sample was placed in a drying oven at 100–105°C for 2 h. After drying, the dish was retrieved from the oven and allowed to cool in a desiccator for 30 min. Finally, the weight of the petri dish with the completely dry LMWCH soap sample (W3) was meticulously recorded. This value, along with the previously obtained weights, was used in the following equation (1) to calculate the exact moisture content of the soap sample.

M o i s t u r e c o n t e n t (%) = {\frac{(W 2 - W 3)}{(W 2 - W 1)}} * 100

(1)

Stability test

The stability of the LMWCH soap was investigated to assess the potential influence of environmental factors on its shelf life. To achieve this, the soap was exposed to various temperatures. Visual inspection was employed to evaluate the organoleptic properties of the LMWCH soap, focusing on color, odor, consistency, texture, and signs of sterility. Additionally, a battery of physicochemical tests was conducted. It is important to note that all measurements were performed in triplicate to ensure data reproducibility.²⁶

Free fatty acid

To check the Free Fatty Acid (FFA) content of the LMWCH soap. A 10 g sample of the LMWCH soap was precisely weighed and added to a 250 ml conical flask containing 100 ml of C₂H₆O. To dissolve the soap, the mixture was stirred continuously on a hot plate set at 60°C using a magnetic stirrer. A few drops of phenolphthalein indicator were then added to the solution. The mixture was titrated with NaOH solution until a pink endpoint was reached. The initial and final burette readings were recorded, and the FFA content was calculated using the following formula (2),²⁷

P e r c e n t a g e o f f r e e f a t t y a c i d = \frac{V \times N \times E q u i v a l e n t w e i g h t o f F F A}{W e i g h t o f t h e s o a p s a m p l e}

(2)

$V$ —Volume of NaOH, $N$ —Normality of NaOH.

Total fatty matter

Total Fatty Matter (TFM) refers to the total quantity of fatty substances, primarily fatty acids, which can be isolated from a soap sample using mineral acids. Soaps with a TFM content of at least 75% are classified as Grade 1, while those with a minimum of 65% TFM are considered Grade 2. Soaps with higher TFM content generally produce more lather, have a longer shelf life, and offer more effective yet gentle skin cleansing. The minimum TFM requirement for the lowest quality soap (Grade 3) is at least 60%. To determine the TFM of the LMWCH soap, 5 g of the sample was dissolved in 100 ml of hot water. To achieve an acidic environment, approximately 40 ml of 0.5 N HNO₃ was added. The mixture was then heated until a layer of fatty acids formed and floated on the surface. This layer was solidified by cooling the mixture in ice water. After separation, the remaining aqueous solution was treated with 50 ml of CHCl₃ to extract any residual fatty acids. The combined isolated fatty matter was then obtained by evaporating the solvent, and the final yield was recorded. The following equation (3) was used to calculate the total fatty matter content of the soap sample,²⁸

\begin{array}{l} P e r c e n t a g e o f \\ f a t t y m a t t e r = \frac{(y - x) \times 100}{W e i g h t o f s o a p s a m p l e} \end{array}

(3)

$x$ —Weight of the petri plate, $y$ —Weight of the petri plate with soap sample after drying, Weight of LMWCH soap sample—5 g

Anti-bacterial activity

The antibacterial activity of the LMWCH soap was assessed using a modified well diffusion method adapted from Valgas et al.²⁹ Methicillin-resistant Staphylococcus aureus (MRSA) ATCC strain was obtained from the Department of Microbiology at Chettinad Hospital and Research Institute for this experiment. Bacteria were evenly spread on a sterile nutrient agar plate using a sterile cotton swab. Then, 20 µl of three different solutions (CH, LMWCH, and LMWCH soap solution (1 mg/ml)) were added to five small wells on each plate. The plates were incubated for a day at 36°C. After incubation, bacterial growth was observed across the entire plate, except in areas where it was inhibited by the test solutions. The size of these inhibition zones was measured. The experiment was repeated twice, and a commercial antibiotic Chloramphenicol (HiMedia) was used as a control.

Anti-fungal activity

The antifungal activity of the LMWCH soap against C. albicans was evaluated using a modified Kirby-Bauer diffusion method.^30,31 A culture of C. albicans was grown in Nutrient Broth medium for 24 h at 37°C. The agar plate surface was then inoculated by spreading a volume of the C. albicans culture over the entire surface. Disks containing the LMWCH soap solution, along with controls, were placed on the inoculated agar plates. All plates were incubated for 24 h at 37°C. The diameter of the clear zone of inhibition surrounding each sample was measured at 12, 24, and 36 h using Vernier calipers (Lab world, India). Chloramphenicol was included as a positive control to assess fungal growth inhibition. Following incubation, the data on the diameter of the inhibition zones were statistically analyzed using GraphPad Prism 9.0.0 software.

Results and discussion

Fourier transform infrared spectroscopy

The FT-IR analysis presented in (Figure 3) reveals the presence of various functional groups within the LMWCH sample. The broad peak observed in the range of 3269.32 cm⁻¹ corresponds to the stretching vibrations of O-H bonds in phenols and alcohols, suggesting these moieties are present within the LMWCH structure. The peaks at 2906.05 and 1454.30 cm⁻¹ correspond to the characteristic stretching vibrations of alkanes and carboxylic acids, respectively.³² This indicates the presence of both aliphatic and acidic functionalities in the LMWCH. Furthermore, the peak observed at 1273.88 cm⁻¹ can be assigned to aromatic amines, suggesting their incorporation into the LMWCH structure.³³ The C-H stretching vibrations confirmed at 1153.27 cm⁻¹ further support the presence of organic components in the LMWCH. The peak positions are tabulated (Table 2). The similarity between the FT-IR spectrum of the LMWCH and that of reported CH polysaccharides suggests a potential shared structural motif. Chitin is a well-known polysaccharide and the deacetylated derivative of which is CH, is known to possess hydroxyl, amine, and saccharide functionalities.³⁴ The presence of these same functional groups in the LMWCH, as evidenced by FT-IR analysis, strengthens the possibility of a similar structural makeup between LMWCH and CH. The FT-IR analysis shows that the LMWCH sample has functional groups, such as hydroxyl, amine, and saccharide groups, that are similar to those in chitosan (CH). This resemblance raises the possibility that LMWCH and chitosan have comparable chemical makeup and functions. The FT-IR study indicates that these functional groups are present in LMWCH, which suggests that LMWCH may also be applicable in related situations. For example, the hydroxyl and amine groups may facilitate the creation of hydrogels for controlled drug release or allow LMWCH to interact with biological systems.

Table 2.

Functional groups and assignments of FT-IR peaks.

S.No	Wave number (cm⁻¹)	Functional group	Assignment
1	3296.31	O-H bonded	Alcohols, Phenols
2	2981.34	O-H Stretch	Carboxylic acids
3	2906.05	O-H Stretch	Carboxylic acids
4	1641.17	–C=C–Stretch	Alkenes
5	1454.30	C–H	Alkanes
6	1383.35	C–H	Alkanes
7	1324.47	C–N Stretch	Aromatic amines
8	1273.88	C–N Stretch	Aromatic amines
9	1153.27	C–H	Alkyl halides
10	1084.39	C–N Stretch	Aromatic amines
11	1043.71	C–N Stretch	Aromatic amines
12	876.76	C–H	Aromatics

Matrix-assisted laser desorption ionization—Time of flight

The MALDI-TOF spectrum of the LMWCH, presented in (Figure 4), reveals a symmetrical distribution of glucosamine (Glc) and N-acetyl glucosamine (GlcNAc) units, each with a mass of 200 Da. This suggests controlled disruption during the production process. The presence of both homo- and hetero-polymerization indicates a heterogeneous population of LMWCH molecules. The spectrum shows a simple Glc homopolymer (base peak at 538 m/z) and a possible hexamer (982 Da), which could represent the upper limit for the LMWCH molecular weight. Further investigation is necessary to compare this data with the desired structure and confirm the precise molecular weight distribution. While Sherazi²⁵ reports fragmentation of a 12-mer CH oligosaccharide, the non-irradiated CH in this study exhibits a molecular weight of 1732 Da. LMWCH may have various physical and chemical properties due to a reduction in molecular weight and the presence of discrete polymerization patterns. For instance, improved solubility, increased reactivity, and a greater capacity for interaction with biological systems are frequently linked to polysaccharides with a reduced molecular weight. Because of these characteristics, LMWCH may be especially helpful in situations where quick dissolution or increased biological activity are required, including in medication delivery systems or as nutritional supplements. Furthermore, the discovery of both homo- and hetero-polymerization emphasizes the LMWCH structure’s potential complexity, which could have an impact on the way it functions and behaves in different applications. The material’s performance in practical applications as well as it interacts with other molecules may be impacted by the heterogeneity seen.

Preparation of soap

The solidification process within a square mold for 48 h yields a final soap bar (Figure 5). The soap weighs 35 g and measures 5.3 cm on each side with a thickness of 1.20 cm. Notably, the soap exhibits a pale-yellow color without the addition of any coloring agents.

PH

The prepared soap exhibits a pH of 9, which falls within the typical range observed for handmade soaps (pH 8–10).³⁵ Commercially produced soaps can have a wider pH range, but many also fall within this category. This slightly alkaline nature (pH 9) is a consequence of the saponification reaction. During this process, fats and oils (triglycerides) react with a strong base (lye) to form soap (fatty acid salts) and glycerol.³⁶ As lye is a strong base, the resulting soap solution has a slightly higher pH than neutral (pH 7). While a slightly alkaline pH is characteristic of soap, it’s important to consider the ideal range for skin health. Human skin naturally maintains a slightly acidic pH, typically between 4.5 and 5.5.³⁷ Soaps with a very high pH can disrupt this natural balance, potentially leading to dryness, irritation, or even increased susceptibility to infection.³⁸ However, a pH of 9 is generally considered safe for most skin types. In fact, some studies suggest that mildly alkaline soaps may even offer benefits for certain skin conditions.³⁹

Moisture content

The empty petri dish weighed 46.284 g (W1), while the petri dish containing the soap before drying weighed 46.786 g (W2) (Figure 6). Following drying, the weight of the petri dish with soap (W3) was recorded as 46.700 g. Using the equation provided in section 2.6.2, the moisture content of the prepared soap was calculated to be 17.13%. This value falls within the desirable range (12%–20%) for bar soaps.⁴⁰ Soaps with a higher moisture content (typically within the 10%–25% range)⁴¹ tend to be softer due to less interaction between soap molecules. However, this can also make the soap more susceptible to cracking or breaking.⁴² Conversely, soap with lower moisture content may be harder and more durable but potentially more drying to the skin.⁴¹ The type of fats and oils used, along with the presence of any additives, can all affect how much water the soap can retain and thus influence the ideal moisture content.⁴¹

Stability test

The prepared soap exhibited remarkable stability across all tested temperatures (4°C, room temperature, 40°C) in terms of odor, texture, appearance, sterility and pH (Table 3 and Figure 7). This indicates a well-formulated soap with minimal susceptibility to temperature-induced degradation. Fats and oils with different melting points affect soap hardness and lather. Soaps with high-melting-point fats, like cocoa butter, are more stable at warmer temperatures compared to those with lower-melting-point oils, like coconut oil.⁴³ The observed stability across a range typically encountered during storage and use (4–40°C) is particularly promising. Studies on soap stability have shown that some soap formulations can be susceptible to degradation at extreme temperatures.⁴⁴ This study shows the soap’s resilience within practical temperatures, but further tests at extreme temperatures and long-term storage studies are needed. These would assess its full stability range and performance over time, providing a clearer understanding of its durability and suitability.

Table 3.

Stability assessment of the soap.

S. No	Parameters/tests	4°C ± 2°C	RT (32°C ± 2°C)	40°C ± 2°C
1.	Color	No color difference was observed	No color difference was observed	No color difference was observed
2.	Odor	No change	No change	No change
3.	Strength	Hard	Hard	Hard
4.	pH	9	9	9
5.	Microbial load (bacteria)	Sterility was maintained for 72 h	Sterility was maintained for 72 h	Sterility was maintained for 72 h
6.	Sterility	No microbial contamination	No microbial contamination	No microbial contamination

Free fatty acid

The analysis of FFA revealed a content of 0.21% in the LMWCH soap sample, which falls well within the acceptable range for high-quality bar soaps (typically below 1% FFA).⁴⁵ This value signifies the proportion of unbound fatty acids present in the soap. A low FFA content of 0.21% indicates a well-made soap with minimal unreacted free fatty acids. This translates to several potential benefits for the soap, including good lathering properties, a mild feel on the skin, and a longer shelf life. Prior research suggests a positive correlation between lower FFA content and superior lathering ability. Studies have shown that soaps with lower FFA content generate richer and more voluminous foam.⁴⁶ This enhanced lathering is likely due to the improved interaction between the fatty acid molecules and water molecules, facilitating the formation and stabilization of micelles, the microscopic structures responsible for soap lather.⁴⁷ Additionally, a study suggests that a reduction in FFA chain length disrupts the normal arrangement of lipids, potentially contributing to a milder feel on the skin.⁴⁸ Finally, soaps with a lower FFA content are generally considered to have a longer shelf life.⁴⁵

Total fatty matter

Analysis of the TFM content (Figure 8) revealed a value of 79.56% in the LMWCH soap, placing it well within the desirable range for high-quality bar soaps (typically 70%–85% TFM).⁴⁰ This value signifies the overall quantity of fatty acids present in the soap. Soaps with a high TFM content, like the LMWCH soap, offer several potential benefits. Due to the higher concentration of fatty acids, which act as the primary cleansing agents in soap, the LMWCH soap is likely to be very effective at removing dirt and grime during use.⁴⁷ Additionally, a high TFM content often correlates with soap’s ability to generate a rich and voluminous lather. This lather plays a role in the perception of cleanliness and can enhance the user experience.⁴⁹ Therefore, the LMWCH soap, with its high TFM content, is likely to produce a satisfying lather during use.

Anti-bacterial activity

LMWCH soap solution demonstrated significant antibacterial activity against the clinically important pathogen, Methicillin-Resistant Staphylococcus aureus (MRSA). Tested at a concentration of 1 mg/ml, the soap solution inhibited MRSA growth, as evidenced by a clear inhibition zone measuring 6.3 ± 0.8 mm in diameter (Figure 9). These results indicate that LMWCH soap solution effectively suppresses MRSA growth, outperforming both CH solution and the LMWCH group (p < 0.05; Figure 10). The observed significant inhibition of MRSA by LMWCH soap solution at a concentration of 1 mg/ml. This finding underscores the potential of LMWCH as a potent antibacterial agent.⁵⁰ The mechanism of antibacterial activity for LMWCH is not fully elucidated, but it appears to be heavily influenced by the molecular weight. When the molecular weight of CH (a key component of LMWCH) is reduced, it may more readily penetrate the bacterial cell wall, potentially leading to increased activity against human pathogens and dermal pathogen.⁵¹ This aligns with recent studies demonstrating the promising antibacterial properties of low-molecular-weight chitosan products in various applications, including scaffolds,⁵² food packaging material,⁵³ and wound care patches.⁵⁴

Anti-fungal activity

The antifungal activity of the LMWCH soap solution was evaluated against C. albicans, using chloramphenicol as a positive control. As expected, chloramphenicol effectively inhibited fungal growth. The diameter of the clear zone of inhibition surrounding the LMWCH-treated C. albicans cultures was measured at 12, 24, and 48h of incubation (data summarized in Figure 11). Compare to the CH the LMWCH soap inhibits (8.5 ± 1.5) the fungal growth significantly (p< 0.05). inhibition disappeared after 48h of incubation. This observation aligns with the findings of Alburquenque et al.,¹³ who reported a similar phenomenon with Syzygium Aromaticum essential oil in natural bath soap. The potential application of LMWCH as an antifungal agent against clinical Candida strains is promising due to its low toxicity to humans. This characteristic opens new avenues for exploring LMWCH in various therapeutic and environmental contexts.⁵⁴

Conclusion and recommendation

This study examined the process in developing sustainable soaps that contain LMWCH obtained from discarded mollusk shells. The LMWCH, produced by converting chitin to CH and subjecting it to gamma irradiation, reached a desired molecular weight of 982 Da and was effectively incorporated into soap compositions. The soaps that were produced exhibited desirable characteristics, such as a moisture content of 17.13%, a pH level of 9, a low FFA content of 0.21%, and a high TFM content of 79.56%. These qualities indicate that the soaps are stable and of excellent quality. The results emphasize the potential of using discarded mollusk shells as a renewable source of CH for soap manufacturing, demonstrating its suitability for hygiene goods. Future study shall be done on fine-tuning the concentration of LMWCH to achieve a balance between performance and sustainability, conducting extensive stability tests to evaluate the durability of the product over an extended period, and carrying out consumer acceptability testing to examine the market feasibility. Furthermore, it is imperative to conduct a thorough economic analysis to assess the cost-effectiveness of utilizing waste mollusk shells in comparison to conventional sources of CH. Environmental impact evaluations, which include life cycle analyses, will be essential for quantifying the ecological advantages and sustainability of the process. Exploring these study topics will improve our comprehension and maximize the potential of waste mollusk shells as a useful resource in the production of sustainable products.

Footnotes

Glossary

CH—Chitosan, LMWCH—Low molecular weight chitosan, FFA—Free Fatty Acid, TFM—Total Fatty Matter, FT-IR—Fourier Transform Infrared Spectroscopy, MALDI-TOF—Matrix-Assisted Laser Desorption Ionization—Time of Flight, MRSA—Methicillin Resistant Staphylococcus aureus, C. albicans—Candida albicans.

Authors’ contributions

The LMWCH soap preparation, moisture content, pH, manuscript draft was done by AS. The anti-bacterial and anti-fungal activity was carried out by versus The free fatty acid, total fatty matter test was done by AJ. The stability test and manuscript draft were done by AR. SR offered comments on the data and analyses as well as critically examined the final manuscript draft. The final manuscript was read and approved by all writers.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The corresponding author (SR) is grateful to the Ministry of Earth Sciences [Ref No. MoES/PAMC/DOM/69/2023(E-13080)], Government of India, for providing funds with related facilities.

ORCID iD

Saravanan Ramachandran

Data availability

All data generated or analyzed during this study are included in this published article.

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