A Review of Sterilization Methods and Their Commercial Impacts on Polysaccharide-Based Biomaterials

Abstract

The most significant barrier against biopolymers’ commercialization is their sensitivity to external factors and poor material properties. In recent years, significant progress has been made to enhance these materials so that they are able to provide their unique physiological benefits while maintaining acceptable material performance. As these materials have developed, so too has their application in the food and medical industry, which often requires them to undergo sterilization. Sterilization is a process in which all microbial life and spores are removed from the surface and within materials and is a regulatory requirement for some food packaging products and all medical applications. Sterilization is carried out primarily using radiation, chemical, and heat treatment, which are all effective in disrupting cell regulation and causing cell death. These processes are known to induce structural and/or chemical changes in materials as well as potential migratory or leaching effects. This review aims to provide a comprehensive evaluation of these sterilization processes and the effects they have on polysaccharides, while established data is discussed that provides insight into their market viability post-sterilization and the importance of further characterization using sterilization.

1. Introduction

Over the past 50 years, synthetic polymers and their usage, applications, and presence in industry have grown exponentially. The plastic industry is still seeing rapid growth and it is estimated that plastic production will exceed 600 mt (Million Tons) in the next 10 years [1]. This rapid growth can be credited to polymer materials’ excellent versatility in terms of properties and characteristics [2], which allow for a broad range of applications in food, medical and manufacturing settings. Synthetic polymers provide an almost limitless range of properties that can be tailored to fit general purpose applications, as seen with plastic bags, containers and utensils, to extremely niche applications in drug delivery and medical devices [3,4].

Polymers exhibit these desirable characteristics because of their polymeric structure, in which long chains of repeating units called monomers are covalently bonded and can consist of several different arrangements. These are generally accepted to be “linear,” “branched,” “cross-linked,” and “networked,” [5] with each arrangement becoming more complex and harder to separate. Each different arrangement contributes to the characterization of these materials and, crucially, their ability to be reused and recycled [6]. While the versatility these molecular structures provide is advantageous, it has also become a critical environmental issue, particularly in recent years [7]. The synthetic nature of these elements provides them with excellent properties, such as being hydrophobic and inert, which are desirable for many applications in industry; however, it also creates materials that are inherently non-degradable by environmental factors and impervious to enzymatic degradation through microbial action [8].

The inability of these materials to degrade naturally along with consistent mismanagement of plastic waste has led to significant negative environmental impacts. Plastic pollution is a global crisis, with only 7% of plastic waste actually being recycled annually and over 50% ending up in landfill [9]. Annual plastic waste production is forecast by the OECTD to rapidly increase to over 1000 mt by 2060 [10]. While countries around the world struggle to develop effective waste management systems, increased pressure has been put on companies to employ greener processing methods and materials to help alleviate the issue of plastic pollution and bolster long-term and environmentally sustainable practices. This has resulted in a push for sustainable and biodegradable materials, particularly in the food and medical industries, which will see the global production capacity of bioplastics rise from 2.18 mt in 2023 to a predicted 7.4 mt in 2028 [11].

The increased demand for sustainable materials has led to the development and implementation of a range of biomaterials, with PLA seeing the most widespread use because of its notable mechanical properties and relatively cheap production costs compared to other biomaterials [12,13]. Biomaterials consist of organic compounds and are easily degraded in the presence of water through hydrolysis or enzymes specific to the material, such as cellulase for cellulose and amylase for starches [14]. The relative abundance of organic materials for use in biopolymers has enhanced their market viability with extensive preliminary available data on proteins, polysaccharides, starches, and polyphenols and their applications in food and medical settings [15,16,17,18]. Biopolymers provide an extensive range of benefits when compared to traditional synthetic polymers. Data has shown that the use of organic compounds in packaging films has proven physiological benefits and antimicrobial properties [19]. These materials have also become significant in the medical industry in the form of packaging, medical devices and more recently as drug delivery excipients providing safe and non-toxic byproducts when they degrade within the body [20].

The push for sustainable materials has, however, been fraught with challenges. Biomaterials are produced through entirely separate mechanisms to conventional synthetic polymers. Materials such as PLA and PHB are created through bacterial fermentation as opposed to petrochemical processing; this has resulted in notable material costs which can exceed 50% of the cost of production in some cases [21]. The consistency of the term “biodegradable” has also been heavily scrutinized in recent years. This has come from a broad range of biomaterials, including PLA’s inability to degrade unless under specific circumstances, which are typically uncommon in nature [22]. As a result, this has led to apprehension about these materials’ introduction to the market. Recent developments into marine polysaccharides have addressed some common issues faced by traditional biomaterials. Seaweed-based polysaccharides are extremely abundant materials and are considered third-generation feedstocks which require no land to cultivate and grow at rates up to ten times faster than terrestrial materials [23]. Algal polysaccharides have become a popular choice for biodegradable packaging [24], drug delivery excipients [25], and medical devices [26] because of their excellent biocompatibility and physiological benefits [27].

While the outlook for biomaterials is still extremely positive because of their sustainability, physiological benefits, and biocompatibility, there are still significant barriers preventing them from entering the commercial market as food packaging and medical devices. The most significant issue with biomaterials is their poor material performance compared to traditional synthetics [28]. The mechanical performance and barrier properties of biomaterials have been consistent hindrances to these materials’ commercialization and are often further reduced due to sterilization processes related to stringent regulatory measures for food packaging and medical applications.

Sterilization is not essential for all food packaging but is often a prerequisite for packaging of sterile food, some ready-to-eat products, and high-risk products such as baby formula, and is carried out on all medical devices, packaging, and drugs. It is the process of removing all microbial life and endospores from the surface and penetrating a material. Sterilization is an important process that ensures that food packaging, medical devices, drugs, and excipients are free from harmful bacteria and spores that could cause potential outbreaks or cause serious harm to members of the population considered high-risk or vulnerable.

Methods of sterilization include dry and wet heat, irradiation, chemicals, solvents, and gas, and the selection of a method of sterilization for a given material is dependent on material characteristics such as thermal, UV, and moisture resistance. Sterilization, while often a requirement for use in food packaging and medical applications, has been known to degrade the materials being sterilized and, in many cases, causes notable reductions in mechanical and optical properties through discoloration, chemical or heat-based degradation, and crosslinking and chain scission through free radical interactions caused by irradiation processes [29]. This review focuses on the studied effects that a range of sterilization techniques have had on conventional polysaccharides and how these effects can impact the physiochemical and migratory properties of common polysaccharides and their market viability thereafter.

2. Sterilization Mechanisms and Their Effects on Polysaccharide Biomaterials

Polysaccharides’ ability to produce biodegradable and biocompatible materials has resulted in their usage in the food and medical device industry increasing dramatically in recent years. The growing fears related to microplastic toxicity and the long-standing issue with plastic waste have resulted in the promotion of sustainable, circular and, most importantly, biocompatible approaches to new products. While these new products are rigorously tested to fall in line with ISO and product regulations, the necessity for sterilization, in the medical industry, can modify the materials’ properties in undesirable ways. Data relating to the sterilization of polysaccharide materials is crucial for understanding changes in material properties and how these may impact the performances of medical devices or food packaging.

2.1. Ionizing Radiation

Many methods of sterilization employ a combination of temperature, pressure, humidity, and chemical treatments. To keep chemical and physical interactions to a minimum, particularly in complex and sensitive medical devices, ionizing radiation is commonly used. The benefit of radiation methods is that they are non-invasive and highly effective sterilizing agents; however, the formation of free radicals and the potential for radiolysis at higher dosages can lead to unwanted changes in material properties and leaching from device to bloodstream. While these methods produce no byproducts, they can still alter the material through crosslinking and chain scission and they require specialized training, particularly in the case of gamma radiation which uses a live radioactive element to sterilize materials.

2.2. Electron Beam

Electron (E) beam sterilization has become a popular method of sterilization for a range of materials due to its non-invasive nature. Materials that are susceptible to thermal degradation at elevated temperatures or undesirable interactions with chemicals and solvents are typically sterilized by means of radiation. The use of e-beam sterilization has been beneficial, particularly in the field of medicine, for its excellent material penetration at higher voltages, allowing it to effectively sterilize complex and sensitive devices at high speed and with no physical interaction. E-beam sterilization has also found success in the packaging industry because of this property and the added benefit of being able to produce ionizing radiation with no radioactive elements [30].

The system of irradiation used for E-beam is an electron accelerator. Electric fields are generated in the accelerator wherein electromagnetic (micro)waves are used to accelerate electrons emitted thermionically to ~99.6% of the speed of light. The beam can be focused using lenses or small plates to direct the electrons and effectively sterilize the target product. E-beam and other irradiation techniques are some of the most effective sterilizing methods because of ionizing radiation’s effects on microbial life and endospores. Electrons which encounter organisms cleave the backbone structure of DNA, which destroys all potential contaminants [31]. These systems are typically energy-intensive, ranging from 1 MeV (Mega electron Volt) to 10 MeV for denser materials. The penetrative effects of E-beam sterilization increase with increasing MeV; however, regulations such as ISO I11137-1:2006 generally require materials to be further evaluated for induced radioactivity over 10 MeV [32].

While the ionizing effects of e-beam radiation are a considerable advantage for sterilizing materials, this same property can induce unwanted negative side-effects which directly impact material performance. Biopolymers have often been criticized for their poor material performance compared to traditional synthetics and the process of irradiation is known to cause the formation of free radicals in polymeric materials, which can cause chain scission or crosslinking [33]. The data relating to the sterilization of biopolymers through e-beam is extensive compared to data on other sterilization techniques.

Wei Liang et al. performed detailed characterizations of potato starch using EBI (electron beam irradiation), where they found that the process of EBI induced significant changes in the isolated starch samples. The samples experienced degradation, yellowing, and reductions in molecular weight while still retaining their original morphological properties [30]. A similar study by Xing Zhou et al. investigated the effects of EB on waxy maize starch films where the results were consistent with established data. They found reductions in MW, a notable reduction in tensile properties at higher dosages, and an increase in short-chain production which significantly increased the solubility. It was also found to have increased modulus properties at lower dosages due to increased relative crystallinity from crosslinking [31].

Common reinforcing materials such as cellulose lack persistent data, with studies referring primarily to pulp degradation and effects as part of an organic–non-organic mix or composite. Driscoll et al. investigated the effects of EBI that had doses varying from 0 to 1000 kGy with values in excess of 50–100 kGy, far above a common dosage [32]. These results did emphasize the degrading effects of EBI dosages with a reduction in MW of 2%, 93%, and a 97% reduction at 10 kGy, 100 kGy and 1000 kGy, respectively. The relative crystallinity of the cellulose reduced from 87% to 45% at 1000 kGy [33]

Data for non-terrestrial polysaccharides are sparse, with alginate, agar, and chitosan being the most frequently characterized through means of EBI. Yuan et al. characterized carboxymethyl chitosan hydrogels, in which it was found that although the irradiation inhibited the materials’ ability to flow and caused greater degrees of yellowing at each dose, there were no significant alterations to the group structure and molecular weight of the chitosan material post-irradiation [34]. While these results are promising, a paper by Urszula Gryczka investigated and emphasized the necessity of long-term analysis of chitosan undergoing ionizing radiation. It was found to cause elevated degrees of deacetylation and oxidative degradation, dependent on dosage, over a five-year period [35]. Data for agar is extremely limited with minor references made to sterilization through EBI; however, a singular study by Luliano et al. completed a range of sterilization techniques including EBI on TPS (thermoplastic starch) agar–agar blends. The results were consistent with the established literature, with the material becoming more rigid and exhibiting observable increases in WVP (water vapor permeability) of 10% and 14% [36].

Apart from starches, alginates appear to have the most extensive range of data available although it is still exceptionally limited and primarily related to medical applications. Puspitasari et al. studied the degradation of a high- and low-viscosity sodium alginate using a low-energy electron beam. Color changes and reductions in molecular weight were observed, and it was concluded that for LEEB applications, low-viscosity alginates were preferrable. Research by Farno et al. studied LEEB irradiation of 3D scaffolds of alginate/chitosan and their polyelectrolyte complexes. Depolymerization was observed at dosages of 25 kGy with reductions in molecular weight and overall material cohesion. The results also found an optimal dose of 280 keV sustained sterility and maintained scaffold properties [37].

2.3. Gamma Radiation

Like E-beam sterilization, gamma produces ionizing radiation which effectively removes all endospores and microbial life from surfaces and within devices and materials. While the phenomena of crosslinking and scission are persistent through each irradiation method, the process has several distinct differences that separate it from other forms of radiation treatment, including its emission of radioactive elements. This is due to the difference in ionizing particles in each method. E-beam radiation uses high- or low-energy electrons to sterilize materials, whereas gamma radiation uses photons (gamma rays) produced from the radioactive decay of cobalt-60 (60 CO) that can penetrate much deeper into the material that is being sterilized [38]. Gamma sterilization is an inherently more dangerous and time-consuming process because of the radioactivity, the slow rate of decay of cobalt-60, and the greater penetrating abilities of photons. Due to their limited interactions with outer shell electrons resulting from their neutral charge, photons can travel deeper into irradiated materials [39]. As such, compared to other forms of sterilization, gamma radiation requires a setup that is often subject to more stringent requirements, including the necessity of thick radiation shielding. Gamma radiation has become a preferred method of sterilization, as it allows large batch processing of in-package devices and materials. The diffuse nature of photons means they do not need to be directed or reflected to effectively target an object for sterilization [40]. Due to its excellent penetrability, this type of radiation can sterilize complex and dense medical devices without causing chemical or heat degradation.

Polysaccharides have a variable range of data relating to gamma radiation, as their use in medical and packaging applications has seen sharp increases in recent years. While terrestrial polysaccharides have been well characterized, non-terrestrial or marine polysaccharides remain largely unexplored. Starch is one of the most common polysaccharides that is employed in the biopolymer industry and radiation studies have been carried out on a range of starches from varying sources. Chung et al. studied the effects of gamma radiation on corn starches with different ratios of amylose to amylopectin. The study found increased IR absorbance at each dosage (1, 5, 10, 25, 50 kGy) as well as significant reductions in the pasting abilities and crystallinity of the starch materials. While the degrading effects were significant for a range of starches tested, one specimen, Hylon VII, exhibited significant resistance to gamma radiation at 5 kGy and showed signs of increased viscosity at this range [41].

A study by Atrous et al. characterized the effects of gamma radiation on starch/clay composites at 1–4% wt. The study observed that the tensile strength and gelling fraction of starch blends steadily increased from dosages of 10 kGy, 20 kGy, and 30 kGy but sharply declined by ≥50% at 40 kGy for all samples. The elongation at break of each sample steadily declined in all cases, while the increase in clay content enhanced the thermal stability of the material [42]. Similar results found by Atrous et al. showed that high doses of gamma irradiation of >20 kGy induced significant deterioration of starch’s cellular structure and swelling power. The study also found that apparent amylose content in wheat samples was higher than in potato samples at each dosage, which aligns with results presented by Chung et al. which posit that stronger sources of amylopectin will reduce degradation via gamma irradiation [43].

Marine polysaccharides have seen little characterization in terms of gamma sterilization, with chitosan and alginate having the most established data while carrageenan fucoidan and agar have extremely sparse data pertaining to sterilization. While sterilization through gamma radiation is not readily available for carrageenan, some of the effects of ionizing radiation have been cataloged by V. Abad in a review of radiation-modified carrageenan. The effects of gamma radiation induce chain scission and thus reduce the molecular weight of the material. This reduction in molecular weight has been linked an increase in reducing sugars which have anti-oxidant effects [44]. Similarly, fucoidan has seen little characterization in terms of gamma radiation; however, a study by Choi et al. investigated the effects it had on the structure of fucoidan itself. The study observed the formation of additional carboxyl groups in the material through gamma irradiation and also concluded that radiation up to a dosage of 100 kGy had no significant effect on the sulfate functional groups of the material [45]. A similar study carried out by Jong-il Choi and Hyun-Joo Kim used gamma radiation to prepare fucoidan at dosages of 30, 50, and 100 kGy. The study showed a sharp initial decrease in molecular weight that normalized over time. The study also investigated the anti-cancer properties of fucoidan and found that at dosages of 100 kGy the cytotoxicity significantly increased to 47% from 35% in non-irradiated specimens [46].

Alginates have undergone considerably more analysis compared to other marine polysaccharides, likely due to their broad usage in the medical field. A study by Lee et al. investigated the effects of gamma radiation degradation sodium alginate. They found that over the range of 0.1 kGy to 200 kGy the rate of degradation increased exponentially but that it sharply declined/ceased at dosages higher than 200 kGy. The viscosity of the alginate was also significantly reduced at dosages as low as 10 kGy and the materials exhibited a color change to brown as the dosage increased [47]. Huq et al. studied the effects of gamma radiation on alginate films and beads. They found that minor dosages in the range of 0.1–0.5 kGy resulted in an increase in mechanical properties and gelling power in the films and beads. The study also observed that dosages of 5 kGy and above completely removed the film-forming capabilities of the alginate material.

Like alginate, the data obtained for chitosan in recent years are well established compared to data on other polysaccharides. Jarry et al. investigated the effects of gamma irradiation on chitosan/polyol and found that the viscosity of chitosan was significantly reduced, which severely inhibited the thermogelling properties of the mixture. Another study by Lim et al. characterized the mechanical, thermal, and molecular properties of chitosan films at dosages of 3.7, 11, 18.3, and 25 kGy. The study showed a considerable increase in mechanical performance at 3.7 kGy, with a notable increase in tensile strength of 60%. The mechanical properties continued to increase until dosages reached 25 kGy. The material also showed a steady decrease in molecular weight, as expected from the chain scission attributed to ionizing radiation, with reductions in viscosity also observed [38]. Further studies on chitosan films were conducted by Li et al. Their study focused on konjac glucomannan/chitosan films and characterized the mechanical and structural properties of the films. IR spectra showed increased absorbance at each dose with little interaction/cleavage observed at dosages of 25 kGy. The results showed that dosages of 0 kGy–80 kGy had negligible impacts on mechanical performance; however, the maximum increase in crystallization occurred at dosages of 25 kGy. The results of the study suggest that konjac glucomannan can inhibit the degradation of chitosan by gamma radiation [39].

2.4. X-Ray

X-ray sterilization is a less conventional method of sterilization that has seen some use in recent times due to its superior ability to penetrate materials compared to gamma and e-beam methods. X-ray, while it is a much more costly and complex process, offers distinct advantages over gamma sterilization, with the ability to sterilize denser and more complex devices at a much faster rate. The principle of sterilization remains the same with X-ray sterilization: ionizing radiation penetrates the molecular structure of microbial life and endospores present on the material, damaging their DNA and effectively sterilizing the material [40]. X-ray sterilization is carried out using many of the same principles as e-beam sterilization. Electrons are thermionically released from a cathode inside an electron gun, where they are sped up to 99% of the speed of light using an electric field. Instead of being fired directly at the materials, as is the case for e-beams, these electrons are directed at an anode which commonly consists of tungsten. The sudden arrest of velocity causes a phenomenon known as “Bremsstrahlung,” which leads to the emission of X-ray [48]. This illustrates that the ionizing electrons produced during this process can interact with the two outermost shells of the anodes’ molecules, creating photons as a result [49].

Data relating to X-ray sterilization on biomaterials are extremely sparse. This is likely due to the complexity of operation and large costs associated with this process. While there is little data available, there are still several studies that have worked to catalog the effects of X-ray radiation on a range of starches, wool keratin, collagen, and alginate. Kerf et al. characterized corn, potato, and drum-dried corn starches and their disintegration properties following X-ray treatment. The disintegration rates of tablets containing α-lactose monohydrate, magnesium stearate, and corn, potato, or ddc-irradiated starches at dosages of 0, 10, 50, and 100 kGy were examined. The tablets showed decreased disintegration time per increased radiation dosage. The solubility of each sample also notably increased with increasing dosages, with the solubility of corn starch increasing from 24% at 0 kGy to 75% at 100 kGy [50]. Another study by Leccia et al. investigated the effects of a high-flux X-ray synchrotron micro beam on alpha keratin fibers. The study observed disulfide bond cleavage as well as mass loss associated with chain scission.

2.5. Ultraviolet Radiation

Ultraviolet (UV) radiation is a less common form of sterilization that also sterilizes via photochemical processes. Typical UV rays are considered non-ionizing and are categorized as “UVA and UVB”. In a sterilization environment, UVC rays are used; these are shorter-wavelength rays composed of photons that interact with electrons in the outer orbit of irradiated molecules to alter their structure and cause irreversible damage to the DNA of living organisms [51]. The most common apparatus used for UVC sterilization are lamps or rods which can effectively sterilize surfaces and thin devices. The penetrating power of UV rays is much weaker than that of other radiation methods. This is likely the primary reason for the lack of data relating to the sterilization of biomaterials used in complex medical devices and dense packaging [52].

Various sources of starches have been characterized through UV radiation in studies by Bajer et al. They observed that out of the tested starches from corn, waxy corn, potato, and wheat, potato starch was the most vulnerable to UV radiation. UV radiation induced chain scission in each of the samples, except for potato starch due to its higher water content and lower crystallinity [53]. Kurdziel et al. also investigated the effects of UVB radiation on potato and corn starch and amylopectin. The results showed that potato starches exhibited much more degradation than corn starches and amylopectin samples [54]. This result is consistent with previous discussions describing amylopectin’s resistance to degradation by radiation via gamma rays, which suggests that, based on available data, amylopectin is effective at reducing the degrading effects of radiation in materials. A study on wheat and spelt starches by Nowak et al. investigated their physiochemical and molecular properties after UV radiation. They found that the molecular weight of the irradiated spelt starches fluctuated through different periods of exposure with an initial decrease followed by an increase in Mw post 5 h of radiation. The study observed that the highest-molecular-weight chains appeared after 50 h of radiation, which suggests that UV radiation can be used to alter the modifiable properties of the starches for further applications [55].

The data for marine polysaccharides are not as extensively available, though some evaluation is possible. Sedayu et al. investigated the effects of surface crosslinking on carrageenan film using sodium benzoate as a crosslinking agent. The results of the study showed a 35–55% increase in tensile strength and 144% increase in modulus with 52% reduction in elongation at break. The study also observed a decrease in water transmission (WVP) of 21% but increased solubility and water uptake of 23%/22% [56]. The effects of photo crosslinking are positive, but the necessity of a crosslinking agent here suggests a certain resistance to UV in the carrageenan alone. A study by Prasetyaningrum et al. studied the effects that UV/O₃ had on k-carrageenan. They observed that UV radiation or O₃ alone did not significantly alter the material except for minor reductions in viscosity [57]. This result does give merit to the assumption that carrageenan has some inherent UV resistance. Fucoidan also suffers from a distinct lack of data relating to UV radiation exposure. Some studies have shown that fucoidan may exhibit anti-photoaging effects in vivo [58] and anti-bacterial activity post UV sterilization [59], but the studied effects of UV exposure are not well covered.

Both chitosan and alginate remain the most widely characterized marine polysaccharides, with a larger number of apparent studies carried out with UV radiation. Meynaud et al. studied the effects of UV radiation on chitosan bioactivity. Their study found results that suggest that UV exposure has no significant performance-inhibiting effects on chitosan solutions. The results showed little to no structural modifications in the material through IR testing and the material retained its anti-fungal properties [60]. A study by Sionkowska et al. studied the effects of UV radiation on chitosan/tannic acid films. The study revealed that UV radiation caused significant degradation in the films. The tensile strength and elongation % of the chitosan films reduced from 70 to 41 MPa and 3.2–0.9% over six hours of exposure. The Young’s modulus of the chitosan film increased from 1.75 to 3.01 MPa over the same period. A study by Zhu et al. used riboflavin as a crosslinking agent under UV light to improve the properties of chitosan films. The results of photo-crosslinking showed a 50% reduction in WVP at 2% wtRf; however, the mechanical properties suffered sharp reductions at 0% wt and 2% wt with a minor increase in TS at 4% wt–6% wt [61]. Alginate has less evident characterization with UV sterilization; however, several studies on hydrogels have been conducted. Al-Sabah et al. studied the effects of UV sterilization on sodium alginate hydrogels. The UV exposure caused significant reductions in internal pore size in the hydrogel as well as minor reductions in swelling capacity. The Young’s modulus and the porosity of the hydrogel saw slight increases over the same period, which is evidence of crosslinking taking place [62]. Harriz Iskandar investigated the crosslinking capabilities of UV exposure on alginate-based hydrogels. He observed an enhancement in the gelation matrix of the hydrogel over a 1120 min exposure time. The study also revealed that the stability of the hydrogels was also enhanced, allowing them to last for up to 10 weeks in comparison to previous data that indicated a 4-week lifespan. The mechanical properties of the hydrogels were also enhanced because of UV exposure [63].

2.6. Ethylene Oxide

Ethylene oxide (EtO) is commonly used for sterilization, particularly for medical devices. This stems from its ability to penetrate difficult-to-reach areas in medical devices and its ability to effectively sterilize products without using heat or humidity. EtO is a well-established and extremely well-controlled sterilization process that enables the sterilization of sensitive medical devices without the risk of heat- or radiation-based degradation [64]. EtO is a direct-acting alkylating agent, which does not require metabolic activation, and exhibits antimicrobial action via inactivation of DNA, RNA, and proteins found within bacteria, viruses, fungi, and spores [65,66,67]. The addition of alkyl groups which are bound to the sulfhydryl, hydroxyl, amino and carboxyl groups prevents regular cellular activity and the ability to reproduce, thus rendering microbes nonviable [67]. Although EtO sterilization is a carefully controlled process, there is an innate danger stemming from the reactive nature of the substance itself. EtO is an effective sterilizing agent due to its reactivity with DNA pairs. The reactive nature of EtO is a result of its strained epoxide structure [68], which, when in contact with DNA pairs, will covalently bond, causing alkylation which disrupts cell functions. These characteristics also make EtO extremely toxic to human life. It is considered a mutagen and carcinogen and requires strict regulatory control in industrial settings [69]. The process of EtO sterilization is typically carried out in a vacuum chamber over four distinct phases: vacuum, exposure, sterilizing, and aerating. Once a vacuum has been created, the chamber’s humidity and temperature are stabilized so that the material can be exposed to the gas. The process of exposure, sterilizing, and aerating can take up to 60 h, making EtO the most time-intensive sterilization process. EtO is one of the most widely used processes for materials that have low thermal resistance or are susceptible to water-based degradation. As such, the established data on this method should be extensive compared to that of many of the other discussed methods.

Starch has seen very little characterization with EtO sterilization, as is the case with many of the other biopolymers discussed. This could be because starch is a common additive to biopolymer blends and is often not a strictly singular processing material. Many materials such as collagen and cellulose also fall into this category. The lack of data relating to EtO sterilization of these materials, despite it being one of the most common methods for these sensitive materials, supports this claim.

Chitosan and alginate remain the most studied materials in sterilization studies, with a small number of studies available for EtO. Marreco et al. studied the effects of EtO sterilization on the mechanical and morphological properties of chitosan membranes. Their study evaluated several compositions of chitosan fibers and found that EtO sterilization slightly increased the membrane thickness for all but the first few test samples. The tensile strength of the samples was reduced by an average of 20%, with slight inconsistencies shown in the % strain at break over the four tests [70].

2.7. Autoclaving

Autoclaving is a common form of sterilization. It uses a relatively simple method of pressurized steam to kill bacteria and fungal spores through denaturation of proteins, inactivation of DNA, and cell membrane damage. Typically, autoclaving is carried out at 115 °C for 20 min. Autoclaving is considered a sterilization method with low penetrability. Because of this, the items that are most compatible with autoclave sterilization are glassware, pipettes, cultures, bags, and liquids [71]. Autoclaving is considered an extremely safe method of sterilization with little to no risk of contamination of the sterilized material or danger to the operators. The process uses a chamber where the airflow can be regulated and removed to create a vacuum. After air is removed, the chamber is filled with steam, causing the pressure and temperature to increase to a baseline level at which sterilization will take place. The simplicity of autoclaving makes it a preferable option for lab equipment and it can be used to sterilize large batches per cycle [72].

Within industrial settings, the use of autoclaving for biopolymer sterilization is negligible. This incompatibility stems from the low C-C bond strength in molecular structures of biopolymers [73] and the heat and humidity produced via autoclaving. Biopolymers are considered biodegradable and biocompatible largely because of hydrolysis, which is the cleavage and degradation of the backbone structure when it comes into contact with water. The occurrence of this phenomenon significantly increases the risk of degradation in the material during a process such as autoclaving, resulting in an expected lack of data.

Hoffman et al. investigated the effects of autoclaving on the properties of xyloglucan scaffolds, where they were sterilized at 121 °C and 1 bar for 15 min. They found that samples sterilized using autoclave showed significant reductions in maximum stress and equilibrium modulus. The results of FTIR showed no significant alterations to the peaks of the materials using autoclave; this indicates that no major structural or chemical reactions occurred because of the autoclave process [74]. Another study by Hashimoto et al. studied the effects of thermal treatments on the secondary structure of silk fibroin. They found that at temperatures above 60 °C, the structure of fibroin changed from silk I to silk II; however, the structural changes were not significant enough to cause adverse effects [75]. These results suggest that silk fibroin is a potential candidate for further research on heat treatment and heat-based sterilization methods.

Starch has seen some characterization through heat treatments, although the data does not provide a solid foundation for a meta-analysis. Zheng et al. investigated the effects of heat-based sterilization on the physiochemical properties of proso millet starch. They found that autoclave treatment increases the water-holding capacity of starch by 91% while also causing a loss of crystallinity in the samples. The viscosity of the samples was also heavily impacted by autoclave treatment, although DSC analysis showed enhanced thermal performance post-autoclave. The established data for starch is minimal, but this study suggests that starch undergoes significant structural degradation in the presence of heat and humidity which has a considerable effect on the crystallinity of the sample [76].

Marine Polysaccharides show a similar lack of data to many other biopolymers. While some data exists for alginate, fucoidan, and chitosan, studies relating to the effects of autoclave on agar and carrageenan are extremely sparse. Zhu et al. investigated the potential of fucoidan as a marine prebiotic using autoclave as the sterilization method for their work. The FTIR analysis of fucoidan showed no significant alterations to the functional groups post-sterilization; however, the molecular weight analysis showed a considerable reduction. Such a reduction in molecular weight indicates that the fucoidan experiences depolymerization in the presence of high-temperature sterilization, which may affect the integrity of the materials it is contained within [77]. Alginate remains one of the most well documented marine polysaccharides used for autoclave sterilization; there are several relevant studies on the topic. Stoppel et al. investigated the effects of autoclave sterilization on the mechanical properties of alginate hydrogels. They found that autoclave significantly affected the swelling capacity of the gels and there was a steady increase in stiffness across the measured frequency range [69]. Another study on alginate hydrogels by Ofori-Kwakye & Martin investigated the effects of autoclave sterilization on the mechanical and morphological properties of calcium alginate gels. The study found that by increasing calcium content, the modulus steadily increases, with a similar effect occurring as the alginate grade increases. The study observed a significant reduction in the shear modulus with gels formed from calcium alginate solutions that were autoclaved at 121 °C for 15 min [78]. It is evident from these two studies that autoclaving can negatively impact the mechanical properties of alginate hydrogels. The application of heat results in a stiffer gel with heavily impacted swelling properties. Further investigation of the chemical and thermal profiles of these gels under these circumstances would be necessary to fully understand how autoclaving can impact materials’ performance. The effects of autoclave sterilization on chitosan have also been documented in several studies. Juan et al. investigated the effects of autoclave sterilization of different chitosan materials, flakes, solutions, and hydrogels. The results suggest that chitosan flakes dispersed in water and autoclaved at 121 °C for 20 min did not induce any significant degradation in the samples, while autoclaving in standard conditions caused significant depolymerization of the materials [79].

A study by Gossla et al. investigated the effects of autoclave sterilization on chitosan fibers. The results of the study showed that autoclave sterilization induced a minor change in UTS from 177 Mpa to 153 Mpa and no significant changes to the tensile strength were noted. Conventional autoclaving also caused a minor reduction in Young’s modulus from 14 GPa to 12 GPa, although autoclaving while submerged in water caused a significant reduction to 6 GPa. This suggests some discrepancies between previous studies in which chitosan flakes did not suffer any degradation [80]. While the results do not match, the difference between flakes and fibers could be significant, and the lack of mechanical testing in the study conducted by Juan et al. may have contributed to this discrepancy.

2.8. Dry Heat

Dry heat is another relatively simple method of sterilization. Dry heat methods use convective heat transfer to circulate hot air in a chamber which sterilizes the target material. The air temperature ranges from 105 to 190 °C, with typical cycle times of 180 min to 11 min, respectively. This method of sterilization is desirable for materials that may be susceptible to degradation in the presence of water and/or humidity. The penetrative ability provided by dry heat is also beneficial for products of complex geometry, provided that airflow is permitted over its surface [81]. Dry heat has the potential to inactivate all microbes, including pyrogens; however, it is less effective against prions than moist heat. Primarily, the microbial inactivation occurs as a result of protein denaturation, dehydration, and oxidative free radical damage [82,83,84]

In the context of biopolymers, the use of dry heat is relatively uncommon. Unlike autoclaving, which will induce a hydrolytic reaction, dry heat induces more significant heat degradation in materials. The higher operating temperatures and exposure times found in dry heat sterilization heavily impact its viability in the biopolymer industry.

Starch has seen a great deal of characterization with various heat treatment methods. Much like previously discussed biopolymers, sterilization studies have not frequently been carried out, but heat treatment studies can be used as a proxy to determine the potential effects and viability of dry heat sterilization. Noranizan et al. investigated the changes in physiochemical properties of several types of starch from varying botanical sources. The studies were carried out using wheat, tapioca, sago, and potato starches, and they were tested for one hour at 100 °C and 110 °C for one hour and 120 °C for one and two hours. The results showed that none of the samples exhibited any swelling power after two hours, with wheat being the only sample to exhibit swelling capacity after one hour at 110 °C. The swelling capacity of all the starch samples was severely impacted (>50%) after 1 h at 110 °C, with the exception of wheat, which showed a 20% increase [85]. A study by Liu et al. studied the effects heat treatment had on waxy potato starch. The potato starches were tested at 110 °C for 0.5 h, 1.5 h, and 2.5 h, and a steady reduction in crystallization temperature was observed at each time interval. The solubility and swelling power of the samples also increased over time [86]. These results show some consistency with established data from autoclaving of starches where the overall solubility was enhanced; however, starch samples in the previously discussed data exhibited a total lack of crystallinity post-sterilization, which is not the case for the study by Liu et al. While there is a lack of data to complete a comprehensive analysis, it does suggest that humidity has a significant impact on the reductions in the crystallinity of the samples.

Marine polysaccharides also exhibit temperature sensitivity; however, there have been some relevant studies conducted to characterize several polysaccharides. Eha et al. investigated the effects of short-term heat treatment on commercial carrageenan. The study was carried out by exposing the carrageenan to temperatures of 75–115 °C for 15 min and no significant difference was found in this range in the carrageenan gels. The study found that above this range, the gelling and melting temperature of carrageenan gels steadily decreased, and it was concluded that to avoid significant degradation in processing, high temperatures should not be used [87]. While characterization of agars’ thermal degradation and some heat treatment studies are available, the context in which they have been completed is not entirely applicable to the context of sterilization. The data does suggest that heat treatments results in a more coarse microstructure and that agar thermally degrades in a single-step fashion, which may be beneficial for lower-temperature and more short-term heat-based sterilization [88,89]. A comprehensive study by Saozo et al. investigated the properties of calcium alginate planar films after they underwent heat treatment at 180 °C for 0, 4, 8, 12, 20, and 24 min. The results of the study found overall reductions in physiochemical properties in the materials, with the planar films becoming thinner and more brittle over time, suggesting some degree of thermal degradation in the alginate films at this temperature [90]. The effects of dry heat on chitosan have been investigated by Lim et al., who found that dry heat at a temperature of 80 °C or less produced chitosan with lower glass transition temperatures and improved solubility. Higher temperatures produced chromophores in the chitosan and caused reduced solubility and a significant color change: white > yellow > brown [91].

2.9. Ozone

Ozone has seen some use as a sterilizing agent in industry. Ozone’s effectiveness comes from a process known as “oxidative bursts” in which it perforates the cell wall of microorganisms and disrupts cell activity. This is due to the reactive nature of ozone (03) in which it will always freely give up an oxygen atom. As cell proliferation occurs, more ozone atoms enter the cell membrane and react internally to further eliminate microbial elements, leaving only oxygen as a by-product. Ozone has some distinct advantages, including its ability to penetrate and sterilize complex geometries and heat-sensitive materials [92]. While ozone boasts some advantages, it is still a relatively new process with some pertinent issues. Ozone has been shown adverse effects when exposed to human respiratory systems, with long-term exposure suspected to be fatal [93]. With regard to the literature relating to ozone sterilization, the process may be unsuitable for a large number of polymers due to materials’ ability to adsorb ozone, causing unwanted chemical or structural degradation [94].

Not many studies on ozone sterilization are available; however, Rediguieri et al. investigated the effects of ozone on PLGA (Poly (lactic-co-glycolic acid)) scaffolds. The scaffolds were sterilized in a vacuum chamber and exposed to “pulses” or 20 min intervals of ozone gas for 2, 4, or 8 cycles. Results from FTIR and the morphological profile of the samples showed no significant changes due to ozone exposure, with the infrared analysis showing no additional or altered peaks. The tensile properties of the scaffolds remained unchanged at two and four pulses but did suffer considerable reductions at eight pulses for tensile strength (3.41–2.91 Mpa) and modulus (118–88 MPa). The study showed that ozone was an effective sterilization method for PLGA scaffolds, removing all microbial life without negatively impacting cell proliferation [95].

Another study by Tyubaeva et al. focused on the effects of ozone sterilization on PHB fibers. The fibers were subjected to 1–600 min of ozone exposure in a flow-through reactor at 0.1 L/min. The study found that the mechanical properties of the fibers were significantly enhanced at 7 min of exposure, with max strength increasing from 1.7 to 3.5 N and elongation % increasing from 3.4 to 7.6%. The crystallinity of the fibers saw a large initial increase at 4 min then a steady decrease until the 20 min mark, where it began to steadily increase again. DSC results showed broader melting peaks as exposure time increased, as the material’s crystalline structure was degraded by the ozone, while the peak also shifted to the right over time [96].

2.10. Supercritical Carbon Dioxide

Supercritical Carbon Dioxide (ScCO₂) is a promising alternative to many forms of sterilization that induce degradation in base materials. The inert nature of carbon dioxide and its low surface tension allow it to penetrate deep into materials and completely sterilize objects with complex geometry without the risk of chemical reaction and toxic residues [97]. CO₂ reaches its critical state at a low temperature of ~30 °C and pressure of ~38 MPa, which is well within the acceptable range for even the most sensitive biopolymers. The supercritical nature of carbon dioxide allows it to possess the penetrative properties of a gas while maintaining the dissolution properties of its liquid form, which gives it excellent biocidal properties [98]. The mechanism of sterilization for ScCO₂ involves the denaturation of proteins through the formation of carbonic acid from chemical reactions with H₂O within the cell wall [99]. ScCO₂ has also been observed to extract lipids from the cell membranes which maintain cell function [100].

Similarly to other gas-based processes, ScCO₂ is carried out in a vacuum chamber where pressure and temperature are controlled to the supercritical point of the gas. Unlike ETO sterilization, no toxic residue is present after the initial sterilization phase, making long aeration phases unnecessary, which means that ScCO₂ is a considerably faster process. While the biocidal effects of ScCO₂ are considered extremely effective, the nature or mechanism of sterilization by carbon dioxide renders it ineffective against some forms of spores and endospores. Eliminating microbes primarily through dissolution and reaction with H₂O is an effective method for bacterial strains consisting of high water content. Spores generally consist of a highly dehydrated core with a low-permeability outer spore cortex, which makes carbon dioxide an ineffective sterilizing agent [101]. The effectiveness of ScCO₂ against spores has been enhanced through the use of additives and slight increases in operation temperatures; however, many of these developments fail to produce a 9-log (99.99%) reduction in spores in the material [102,103].

The characterization of biopolymers using ScCO₂ is likely limited because the process is ineffective against endospores, which is a significant barrier for the medical device industry. While the data are limited in this area, there have been a small number of studies carried out. Bento et al. studied the effects of sequential ScCO₂ drying and sterilization on alginate–gelatin aerogels. The sterilization took place over a two-hour period using a 7.5 min static period followed by a 4–6 min depressurization cycle. FTIR analysis of the aerogel sterilized at 100 and 250 bar showed a broadened band at 1600 cm⁻¹ with no additional peaks forming, indicating that the process did not induce any chemical reactions in the material. DSC results showed no significant changes from the control sample, while the samples sterilized at 250 bar showed a notable increase in the Young’s modulus. The process was concluded to be effective with no detectable microbes on the samples post-sterilization [104].

2.11. Cold Plasma

An effective and environmentally safe method for sterilization exists in the form of cold plasma cleaning. The majority of surface contaminants, many of which are of a high molecular weight, contain organic bonds (C-H, C=O) which are readily broken by the vacuum ultraviolet energy generated by plasma [105]. A secondary cleaning action is performed by the various oxygen species generated within the plasma field, which subsequently react with organic contaminants for CO, CO₂, and H₂O, resulting in an ultraclean surface [106]. Numerous authors have displayed the effectiveness of cold plasma treatment in the inactivation of a wide variety of microorganisms, including E. coli [107], L. monocytogenes [71], S. aureus [72], as well as fungal contaminants [73]. In addition to its antimicrobial activity, cold plasma allows for surface modifications, molecular interactions, and the ability to tailor the functional properties of polysaccharide-based materials. Wan et al. showed that cold plasma treatment of whey protein isolate–carboxymethyl chitosan films significantly improved both the mechanical performance and preservative properties of the films when applied as a pork preservative. It was demonstrated that the application of the cold plasma increased the aggregation of the two polymers, thus enhancing viscosity and viscoelasticity. This was due to the intermolecular interaction caused by the cold plasma leading to an increase in disulfide bond formation. As polysaccharide-based packaging materials are known to be susceptible to moisture uptake, cold plasma treatment has been utilized to tailor the solubility of starch-based materials. Guo et al. utilized dielectric blocking discharge plasma treatment for the modification of potato starch films. Their results demonstrated that the surface of the produced films had been flattened post-treatment and exhibited the highest tensile strength and lowest water vapor permeability of tested materials [108]. Sifuentes-Nieves et al. utilized plasma treatments to modify starch with varying amylose content ranging from 30 to 70%, with higher amylose content displaying an increased degree of hydrophobicity. The application of plasma promotes the oxidation of OH-groups to C=O groups, forming new hydrogen bonds and thus increasing the hydrophobicity of the materials [109].

3. Safety and Performance Considerations for Biomaterial Sterilization

3.1. Migratory Effects Caused by Sterilization

One of the most notable dangers associated with the sterilization of polysaccharide materials is the potential for the creation and subsequent migration of toxic byproducts. When considering the nature of synthetic polymers, and their inherent biotoxicity, it is likely safe to assume that the plasticizers, stabilizers, dyes, and other processing compounds used are not prioritized as being biocompatible when compared to biomaterials. The necessity of sterilization in the food and pharmaceutical industries can heavily impact these materials and the way they behave and interact with external stimuli (Table 1), which has resulted in regulatory compliance and standards to dominate the industry

For polysaccharide-based materials, the use of non-toxic and biodegradable additives is paramount in the formation of safe medical devices, food packaging, and utility items. The necessity of biodegradation often requires the action of hydrolysis, making these material and their constituent parts highly hydrophilic. This affinity for water can lead water-soluble gases such as EtO and carbon dioxide to absorb and become trapped in these materials, leading to leaching of cytotoxic compounds [110], which is a serious regulatory concern.

While EtO can be absorbed as a consequence of polysaccharides’ ability to degrade via hydrolysis, autoclaving can induce hydrolysis in biomaterials [101], causing a breakdown of the materials’ structure and leaching of polymer components. The cleavage of glycosidic bonds in polysaccharides causes the polymer to reduce into simple sugars, acids and water [102]. While typically non-toxic in nature, simple sugars can leach into food products and react with proteins during food preparation, causing a Maillard reaction which has been known to produce mutagenic compounds with free amines in food products [103]. Similarly, dry heat treatments can cause thermal degradation in heat sensitive biopolymers and at significant temperatures >180 °C glycerol, a common plasticizer, can thermally dehydrate to form toxic acrolein [104].

Methods that can produce significant oxidation in biomaterials can be considered the highest-risk carriers in a regulatory context. The formation of free radicals that induce chain scission or crosslinking in materials can have a considerable impact on the material properties post-sterilization when compared to other methods. Radiolysis of polysaccharide materials has been shown to be associated with the formation of formyl free radicals, which react with hydrogen to form formaldehyde [105], a compound known for its extreme toxicity [106]. Minor secondary reactions of radiolysis can also produce acetic acid, particularly in the degradation of hemicellulose materials [107]. In low concentrations, acetic acid is generally safe as a food additive; however, higher concentrations, particularly when inhaled, have adverse effects [71], and the potential for continuous free radical action and interaction with food content post-sterilization is not well covered in the literature. Ozone sterilization is also a highly oxidative technique which can form carboxylic acids as well as aldehydes in polysaccharide materials [72]

Table 1. Summary of migratory effects including (i) the method of sterilization employed, (ii) the mechanism employed in the sterilization, (iii) the specific effect on the polysaccharide, and (iv) the potential byproducts caused by the sterilization process.

Method	Mechanism	Effects on Polysaccharides	Potential Byproducts	Reference
Ethylene Oxide (EtO)	Alkylation, gaseous diffusion	Absorption into hydrophilic matrix; slow desorption	Residual EtO; cytotoxicity; potential DNA alkylation	[111]
Autoclaving	Moist heat, hydrolysis	Glycosidic bond cleavage; structural degradation	Release of simple sugars; Maillard reaction forms mutagens	[112]
Dry Heat	High-temperature thermal degradation	Dehydration of plasticizers like glycerol	Acrolein/thermal degradation products	[113]
Gamma/E-Beam Radiation	Radiolysis via free radicals	Oxidative chain scission; sugar ring opening	Formaldehyde (from formyl radicals); acetic acid	[114]
Ozone Sterilization	Strong oxidizer surface oxidation	Oxidation of sugar residues and additives	Aldehydes; carboxylic acid degradation of structural groups	[115]
UV-C Radiation	Surface photolysis, oxidation	Limited penetration; surface oxidation	Surface-level carbonyls; aldehydes (low concentration)	[116]
Supercritical CO2	Penetrating, inert under typical conditions	Physically absorbed; minimal chemical reactivity	Minimal unless additives are used (H2O2/peracetic acid)	[81]

Table 1. Summary of migratory effects including (i) the method of sterilization employed, (ii) the mechanism employed in the sterilization, (iii) the specific effect on the polysaccharide, and (iv) the potential byproducts caused by the sterilization process.

Method	Mechanism	Effects on Polysaccharides	Potential Byproducts	Reference
Ethylene Oxide (EtO)	Alkylation, gaseous diffusion	Absorption into hydrophilic matrix; slow desorption	Residual EtO; cytotoxicity; potential DNA alkylation	[111]
Autoclaving	Moist heat, hydrolysis	Glycosidic bond cleavage; structural degradation	Release of simple sugars; Maillard reaction forms mutagens	[112]
Dry Heat	High-temperature thermal degradation	Dehydration of plasticizers like glycerol	Acrolein/thermal degradation products	[113]
Gamma/E-Beam Radiation	Radiolysis via free radicals	Oxidative chain scission; sugar ring opening	Formaldehyde (from formyl radicals); acetic acid	[114]
Ozone Sterilization	Strong oxidizer surface oxidation	Oxidation of sugar residues and additives	Aldehydes; carboxylic acid degradation of structural groups	[115]
UV-C Radiation	Surface photolysis, oxidation	Limited penetration; surface oxidation	Surface-level carbonyls; aldehydes (low concentration)	[116]
Supercritical CO2	Penetrating, inert under typical conditions	Physically absorbed; minimal chemical reactivity	Minimal unless additives are used (H2O2/peracetic acid)	[81]

3.2. Alteration of Physiochemical Properties

E-beam technologies have been shown to compromise the molecular structure of biopolymer materials in sterilization applications of both medical devices and food packaging applications [117,118]. The consistency of a minor—or in some cases significant—boost to a material’s elastic modulus at mid–high-range doses is a direct result of free radical interactions within the material [119,120]. Chain scission was seen to reduce the relative crystallinity of materials, which induced notable degradations in material performance, while crosslinking’s’ modulus-boosting effect resulted in a consistently more rigid and less flexible material [118]. It is important to note that while these interactions persistently produced a reduction in mechanical, rheological, and optical properties, they have also produced increased WVP, density, and solubility in organic compounds including starches, cellulose and collagen, as previously discussed, which resulted in them exhibiting increased modifiability due to alterations of their molecular structures [36].

Gamma radiation of materials induces phenomena that are characteristic of ionizing radiation. The processes of chain scission and crosslinking are present in much of the data discussed. A notable effect that gamma appears to have with biopolymers is a sharp increase in their gelling properties, with some studies suggesting gamma radiation as a means of gel preparation [121]. The effects of gamma radiation on biopolymers from the established literature show promising results. In some cases, considerable increases in mechanical performance have been observed without the expected reduction in elongation and increased rigidity of the material. While gamma sterilization still interacts with and cleaves the backbone structures of irradiated materials, the reduction in chain scission is likely due to the reduced dosages required for effective sterilization. The data also suggests that some potential degradation inhibitors—namely konjac glucomannan and, to a lesser extent, amylopectin—in starches have been observed to reduce the overall degradation and short-chain reductions, respectively.

While the effects of gamma radiation are discussed in the context of biopolymers, the data that is most readily available exists within the area of material modification. Gamma sterilization of materials is generally complete at a specific and generally low dosage of 0.1–25 kGy [122], but much of the data available for material modification through irradiation greatly exceeds these parameters. The effects of gamma sterilization on many biopolymers is still relatively unexplored and, according to the data discussed, can produce desirable properties in the irradiated materials. Further characterization of biopolymers using gamma radiation may allow for effective dosing for material enhancements.

The effects of X-ray sterilization on biomaterials are evidently not well studied [123]. The lack of data is indicative that conventional methods of ionizing radiation are preferable to X-ray for several reasons; however, the importance of material characterization in this context cannot be understated. Further evaluating the potential of X-ray sterilization could provide some unique benefits, such as the previously discussed ability of gamma radiation to significantly increase the gelling properties of materials. While the data are sparse, it is logical to conclude that the common effects of ionizing radiation would be present in irradiated biopolymers, although to what extent is unknown. However, the reduced exposure time required for X-ray, comparable to that of e-beam, suggests that the degradation effects may not be as prevalent as the long-term exposure seen in gamma sterilization methods.

According to established data, UV radiation is not a widely adopted method for sterilization of biopolymers. It is evident that many naturally occurring elements exhibit some range of UV absorbance or blocking capabilities [124]. This inherent resistance to UV has led to these materials’ use in sunscreen [125,126], UV-blocking windows, paints, and topical products. There is little data on direct sterilization, and many of the cited studies report UV exposure rates that ranged from one hour to several days of exposure. Typical UV sterilization procedures are not carried out over extended periods, so it is difficult to identify prevalent trends; however, in many studies, it appears that immediate exposure to UV radiation, i.e., the first hour, induces the most significant changes to materials’ structure if they do not exhibit UV resistance. Moreover, this resistance to UV radiation was beneficial in several studies in the context of photo crosslinking using a surfactant crosslinking agent, which produced notable mechanical improvements in the material. The ability for amylopectin to resist several types of radiation is also significant, with several studies across multiple sterilization methods citing similar results. The presence of amylopectin appears to inhibit degradation both physically and morphologically in the materials it is present in. While UV sterilization methods may not be applicable for a range of naturally occurring polymers due to their resistant nature, the photodegradation of these polymers is still an essential characterization for their environmental performance.

While the effects of autoclave sterilization are not well documented for a significant number of biopolymers, from the established literature, it can be concluded that the effects are primarily negative. Xyloglucan appears to exhibit a higher degree of resistance to heat treatment than other biopolymers, which is evident from the lack of degradation resulting from exposure to both heat and humidity. A common phenomenon observed in these studies, particularly with starch, is a reduction in crystallinity in the samples [64,127]. Overall, the outlook for autoclave treatment on these materials is poor. The materials’ innate vulnerability to water through hydrolytic reactions and the compounding effect humidity has on this phenomenon makes autoclaving a mostly ineffective method of sterilization for biomaterials [68,69,128].

The established data for EtO sterilization is exceptionally limited. With EtO being an ideal sterilizing agent for many sensitive materials, this is somewhat unexpected. The data that is available seems to indicate that EtO sterilization can affect the mechanical properties of the material as well as the processability, permeability, and viscosity [62]. The limited data may also be due to the duration of EtO processes. To eliminate any degree of trace chemicals from sterilized materials a long period of aeration is necessary, which is typically 8–12 h in length. This, paired with the 6–12 h EtO exposure times, makes EtO sterilization one of the longest-duration methods available [129].

The data for ozone-based sterilization methods for biopolymers is quite limited; however, from observation of the available literature, ozone appears to be an effective sterilizing agent. Ozone exposure induces chain scission in PHB fibers even at relatively short durations, although it produces a significant increase in fibers’ mechanical performance [96]. The reduction in crystallinity also effects the thermal profile of the fibers; this contrasts with the PLGA, which showed no notable structural or morphological differences post-sterilization [95]. The lack of data relating to ozone makes it difficult to suggest any significant trends in the context of its effect on a range of biopolymers. More relevant data would be required in this area to fully understand ozone’s potential as a biopolymer sterilizing agent, although the relative ease of synthesis and the fact that it does not require the application of heat and humidity point towards it being a suitable process for biopolymer sterilization.

Despite the lack of data, ScCO₂ is a promising sterilization technique that offers a safer alternative to intensive processes such as EtO and thermal sterilization. The low operating temperatures make this process particularly suitable for biopolymer sterilization [130]; however, the consistent use of additives such as hydrogen peroxide to eliminate endospores is a significant disadvantage. According to the available data ScCO₂, has negligible effects on the materials it is used with, while its cycle times are far shorter than those of processes such as EtO and Gamma radiation [131]. More data is required for ScCO₂ to fully understand its potential in both the food and medical industries, but the further development of this process, using an effective method that results in a frequent 9-log reduction in spores, will present a sterilization process that may be suitable for a large portion of biopolymers in industry.

Table 2. A summary of sterilization methods and their physicochemical impacts on polysaccharide-based materials on the basis of mechanical, morphological, thermal, and molecular changes.

Sterilization Method	Mechanical	Morphological	Thermal	Molecular	Reference
E-Beam	Increased elastic modulus at moderate–high doses; reduced flexibility	Chain scission reduces crystallinity; crosslinking increases rigidity	Potential increase in solubility and density	Increased modifiability; oxidative degradation; WVP increases	[132]
Gamma	Sometimes improves mechanical performance without sacrificing elongation	Backbone cleavage; enhanced gelling behavior in some polymers	Effects vary by dose and polymer	Reduced chain scission at sterilization doses crosslinking observed	[132]
X-ray	Undocumented but likely less severe than gamma due to shorter exposure	Effects inferred to be similar to those of gamma radiation	Not well characterized	Expected ionizing radiation effects; more research required	[40]
UV	Mechanical improvement with photo-crosslinking agents	Surface degradation in sensitive materials	Not widely observed due to surface-limited effects	Limited degradation in UV-resistant polymers like amylopectin	[53]
Autoclaving	Generally negative; reduced mechanical integrity	Complete loss of crystallinity in starches; hydrolytic breakdown	Thermally induced degradation in hydrophilic polymers	Glycosidic bond cleavage; sugar leaching; Maillard reactions	[76]
Dry Heat	Not well documented; likely poor due to thermal sensitivity	Loss of crystallinity varies without humidity	Distinct from autoclave; less hydrolysis	Thermal dehydration of plasticizers; acrolein formation possible	[86]
EtO	Slight increase in mechanical strength; increased brittleness	Unclear due to limited data	Minimal thermal impact	Trace residues require long aeration and slow desorption	[70]
Ozone	Mechanical improvement in some fibers like PHB	Reduction in crystallinity; minimal impact on PLGA	Alters thermal behavior in degraded fibers	Chain scission; aldehyde and carboxylic acid formation	[96]
ScCO2	Maintains mechanical performance	Minimal structural degradation	Operates at low temperature; thermally gentle	Negligible chemical changes unless oxidative additives are used	[104]

presents a summarized version of the physiochemical effects of sterilization methods on polysaccharaide-based materials.

4. Commercial Advancements in Polysaccharides

With mounting pressure on businesses to adopt sustainable and circular practices, the biopolymer market has seen countless innovations over the past few years. A focus on renewable and non-toxic materials has pushed the continued development of biodegradable polymers in the food and medical industries. Recent controversies involving microplastics and the growing understanding of their effects on the environment and the physical dangers they pose have pushed consumers and businesses to pursue user-friendly alternatives.

The development of marine polysaccharides has greatly benefited from these circumstances, with a considerable range of applications as food packaging alternatives and physical benefits being explored [18]. These materials represent a cheap and extremely abundant source of material but have yet to be fully harnessed due to the difficult nature of their extraction; however, the growing understanding of these materials and their potential in industry [133,134,135,136] has inspired greater focus on an effective and scalable solution [137,138,139,140]. As the terms “renewable” and “sustainable” become more commonplace in the materials industry, the development of higher-yield extraction methods for many biopolymers have seen similar advancements [141,142,143,144]. Advancements in the scalability and effectiveness of extraction methods are a major component of these materials’ desirability for commercial use, although the most significant factor is their performance under diverse and strenuous conditions.

The enhancement of biopolymers and their resulting resistance to external processes such as sterilization are paramount if these materials are to be considered as replacements for synthetic polymers. The development of biopolymer composites and blends has greatly increased the functional characteristics of biopolymers, particularly with additives such as microcrystalline cellulose [145,146], gelatin [147], and chitosan [148,149]. The significance of the continued development and characterization of biopolymers in this regard cannot be understated, as materials’ performance under varying conditions and the ease of extraction and synthesis contribute significantly to their implementation in industry as a whole.

5. Conclusions and Future Directions

The interaction between the sterilization method and biopolymer structure varies significantly between methods and between materials. The inherent sensitivity of biomaterials and their susceptibility to undergo structural modifications with the application of different sterilization methods is relatively unexplored. Considering the structural properties of biopolymers and their viability for commercialization post-sterilization requires significantly more data than are available. The broad range of available methods and the data relating to them exhibit high variance in the degree of exposure times, dosages, and processing parameters.

The opportunity to fully understand these processes and their effects on material properties requires a commitment to the comprehensive study of sterilization processes and their optimizations in the context of sterilizing biopolymers. The scope of characterization by sterilization is clear from the established literature, with each method of sterilization presenting a notably different interaction between process and material that can only be generalized due to the lack of consistent data for each material.

The further development and application of biopolymers in the food and medical industry as active packaging, medical devices, and drug delivery agents will see greater exposure to various sterilization processes. To ensure these materials’ success in industry, the effects of sterilization must be known, and we must consider the development and enhancement of new, less intensive methods. Sterilization presents an opportunity to enhance materials’ properties through chain modification. In many cases, the data suggests that the materials’ amorphous properties were preserved while providing notable increases in modulus and tensile strength; however, without consistent data or optimizations for the processes, these enhancements can be negated or may lead to degradation. The necessity for adequate regulation and compliance to be applied to these materials post-sterilization is evident from the migratory effects that are possible to achieve via sterilization. Further examination of these migratory effects, particularly when exposed to external stimuli such as foods, and in vitro, will be essential for providing safe, sterile, and sustainable packaging and medical devices.

The importance of sterilization and its effects on biopolymers is significant. Relative to these materials’ development and usage in industry, it is clear that the development and generation of data for sterilization methods should be considered an essential characterization parameter for all biopolymers. This review provides a comprehensive evaluation of much of the available data relating to the sterilization of common polysaccharides, detailing the structural effects and impact on material performance and migratory effects thereafter. This review attempts to highlight the significant gap in the literature on the sterilization of biopolymers and both the necessity and opportunity for further research in this area.