Do softgel manufacturing or gastric conditions break down carrageenan into poligeenan?

Abstract

Carrageenan (CGN) is a high molecular weight (typically 200–800 kDa) polysaccharide derived from red seaweed. It is used as a food additive and as a functional excipient in pharmaceutical and nutraceutical products. In softgels, CGN can offer advantages over gelatin, including pH tolerance up to 12, thermal stability during processing (70–90 °C), and resistance to cross-linking. However, under extreme conditions, such as prolonged exposure to strong acid and temperatures exceeding 100 °C, CGN may degrade into low-molecular-weight fragments (<20 kDa), known as poligeenan, which has been linked to toxicological effects. This study investigates whether conditions used in softgel manufacturing or simulated gastrointestinal digestion could cause CGN to degrade into poligeenan or similar low-molecular-weight species. Iota carrageenan from three different batches used in food, nutraceutical, and pharmaceutical products was subjected to controlled stress conditions, including high temperature and low pH. Molecular weight distributions were measured using gel permeation chromatography (GPC). Carrageenan exhibited excellent thermal stability at 95 °C and 105 °C without significant molecular weight reduction when not exposed to acid. Under gastric-simulating conditions (pH 1.2 at 37 °C), moderate hydrolysis occurred, but fragments remained above the 20 kDa threshold. In contrast, combined acid and high-temperature treatment induced substantial degradation, yielding fragments under 20 kDa. No low-molecular-weight fragments consistent with poligeenan (<20 kDa) were observed under relevant conditions, reinforcing carrageenan's safety profile for use as a functional excipient in controlled-release dosage forms and dietary supplements.

Graphical abstract

Highlights

• •Carrageenan stability tested under softgel and GI-relevant stress conditions
• •No poligeenan formed under gastric pH and softgel processing temperatures
• •Degradation only observed under extreme acid and heat combinations
• •Gel permeation chromatography used to track molecular weight shifts
• •Supports carrageenan's safety as a pharmaceutical excipient

1. Introduction

Carrageenan (CGN) is a high-molecular-weight, sulfated polysaccharide derived from red seaweed and commonly used as a multifunctional food additive and excipient in pharmaceutical and nutraceutical products [1]. It serves as a gelling, thickening, and stabilizing agent, with broad applicability in food, pharmaceutical and other industries. One of the applications of carrageenan is in softgel capsules and controlled-release systems. Carrageenans have also been reported to exhibit a range of biological activities, including antiviral, immunomodulatory, and antimicrobial effects, and have been investigated for biomedical applications such as tissue engineering and biomaterials development [2].

CGN exists in three primary forms: kappa (κ), iota (ι), and lambda (λ), which differ in sulfate content and gelling properties (Fig. 1). The functionality and safety of CGN are closely linked to its molecular weight distribution, which typically ranges from 200 to 800 kDa. Analytical studies, including gel permeation chromatography (GPC) coupled with inductively coupled plasma (ICP) detection, have confirmed that commercial food-grade or pharmaceutical-grade CGN lacks significant quantities of low-molecular-weight fragments associated with toxicity [1].

Fig. 1.

Types of carrageenan. Carrageenan is a sulfated galactan extracted from red seaweed. The three primary types [kappa (κ), iota (ι), and lambda (λ)] differ in sulfate content and presence of 3,6-anhydro-d-galactose. κ-Carrageenan contains one sulfate group and forms strong, rigid gels with potassium ions. ι-Carrageenan has two sulfate groups and forms soft, elastic gels with calcium ions. λ-Carrageenan, with three sulfate groups and no 3,6-anhydro bridge, does not gel but imparts viscosity. These structural features define their solubility and functional behavior in pharmaceutical and food formulations.

Despite its broad functionality, the safety of CGN has been debated due to its potential to degrade into low-molecular-weight components under the combined conditions of strong acidity and high temperature. Of particular concern is poligeenan (PGN), a chemically distinct degradation product with molecular weights below 20 kDa, formed through extended exposure of CGN to strong acid hydrolysis at elevated temperatures [3]. Poligeenan is not approved for food or pharmaceutical use due to its demonstrated pro-inflammatory and toxic effects [3,4].

Poligeenan (PGN) is not produced biologically and is not present in native carrageenan. Rather, it is generated through extensive acid hydrolysis of carrageenan under harsh, non-physiological conditions, typically at very low pH (about 0.9-1.3) and elevated temperatures above 80 °C for several hours. These conditions are substantially more severe than those encountered in the gastrointestinal tract or during standard softgel manufacturing. In humans, gastric pH under fasting conditions is generally about 1.3-3 and rises transiently after food intake before gradually returning toward baseline as gastric emptying proceeds. In addition, gastric residence time is limited, typically on the order of 3-4 h. Taken together, these conditions are not generally sufficient to produce the extensive hydrolysis required to convert carrageenan into poligeenan.

Toxicological concern around poligeenan arises from its well-documented pro-inflammatory activity in experimental systems. Unlike high-molecular-weight carrageenan, poligeenan has been associated with gastrointestinal injury, activation of innate immune signaling, disruption of epithelial barrier integrity, oxidative stress, and alterations in gut microbial composition [5]. Proposed mechanisms include activation of Toll-like receptor-mediated pathways and downstream NF-kB signaling, resulting in increased expression of pro-inflammatory mediators such as IL-8, TNF-a, and IL-6. Poligeenan has also been reported to reduce tight junction protein expression, which may increase intestinal permeability, and to promote oxidative and microbial changes that can further amplify inflammatory responses.

Confusion surrounding CGN's safety has been exacerbated by the historical use of the term “degraded carrageenan,” which led to toxicological findings derived from poligeenan being misattributed to CGN [3]. Although regulatory authorities such as the U.S. Food and Drug Administration (FDA), the Joint FAO/WHO Expert Committee on Food Additives (JECFA), and the European Commission have concluded that CGN is safe under intended use conditions [3,5,6], concerns persist regarding its stability during digestion or processing. Specifically, questions remain as to whether human gastrointestinal conditions or standard softgel manufacturing processes could induce hydrolytic degradation of CGN to poligeenan-like fragments.

Numerous pharmacokinetic studies have demonstrated that CGN is not systemically absorbed and is excreted unchanged in feces following oral administration in rats, guinea pigs, and primates [3,[7], [8], [9], [10], [11], [12], [13]]. Even in models colonized with human gut microbiota, no significant biotransformation or molecular weight reduction has been demonstrated [3,10]. Nonetheless, in vitro studies using highly sulfated λ-carrageenan (which contains three sulfate groups, compared to two in ι-carrageenan and one in κ-carrageenan) have reported inflammatory responses in cultured epithelial cells. However, these effects have not been consistently replicated in in vivo models or human studies [6,11,12].

A review of the literature revealed no studies directly examining whether carrageenan undergoes molecular weight degradation under actual softgel manufacturing or gastrointestinal physiological conditions, or whether such conditions could lead to the formation of poligeenan.

To address this gap, the present study aimed to assess whether iota-carrageenan degrades into low-molecular-weight fragments, specifically those consistent with poligeenan (<20 kDa), under physiologically relevant or softgel processing conditions. For this reason, gel permeation chromatography (GPC) was selected as the primary analytical method because it directly measures molecular weight distribution and enables detection of low-molecular-weight degradation products. Three commercial lots of iota-carrageenan were subjected to controlled stress conditions involving elevated temperature, acidic pH, and their combinations. Molecular weight distributions were measured using gel permeation chromatography (GPC), a sensitive technique for detecting polymer fragmentation. This study focused on qualitative comparisons of molecular weight distribution profiles, reporting observed shifts in peak molecular weight (Mp) and distribution patterns as indicators of degradation trends across treatment conditions and time points.

2. Materials and methods

2.1. Design of experiment

Three batches of commercially available iota-carrageenan (GENUTINE® Carrageenan, supplied by CP Kelco) were utilized to assess the variability of this naturally derived material. These samples were not standardized, meaning no dextrose was added to alter their gel strength and viscosity. Dextrose can appear as low molecular weight fractions in tests, possibly being mistaken for poligeenan. To evaluate its stability, carrageenan samples from three different lots underwent three distinct treatment conditions: 1) elevated temperature alone; 2) acid treatment at physiological temperature; and 3) acid treatment at elevated temperature. The experimental design is summarized in Table 1 below.

Table 1.

Design of experiment for carrageenan stability study.

Summary of test conditions across three variables: temperature (37 °C, 95 °C, 105 °C), pH (neutral, 1.2, 3.0), and duration (15 to 120 min). All three carrageenan lots were subjected to each condition. A total of 42 samples were prepared; each was run in triplicates using GPC method.

Temperature (°C)	pH Condition	Exposure Time (min)
37	1.2 (HCl)	15, 30, 60, 120
95	Neutral	60, 120
95	1.2 (HCl, H2SO4), 3.0 (CH3COOH)	60, 120
105	Neutral	60, 120

2.2. Analytical procedure for molecular weight distribution

Gel permeation chromatography (GPC) was used to assess the molecular weight distribution of carrageenan samples following various stress conditions. A refractive index (RI) detector measured the concentration of eluting polymer species, generating chromatograms that plot RI response (y-axis) against logarithmic molecular weight (log Mw, x-axis). In these chromatograms, larger molecular weight species elute earlier (left side of the x-axis), while smaller degraded fragments elute later (right side). Thus, a left-shift in peak position indicates intact, high-molecular-weight carrageenan, whereas the appearance or intensification of right-shifted peaks signals polymer degradation. The RI response at a given molecular weight reflects the relative abundance of molecules within that range; it is not a direct measure of absolute concentration or molecular weight. This analytical approach is particularly suited for identifying poligeenan, a degradation product of carrageenan defined by its molecular weight <20 kDa. In this study, any emergence of signal in the Log Mw ∼4.3–4.5 region (corresponding to Mw ≤ 20 kDa) was used as an indicator of potential poligeenan formation.

2.2.1. Sample preparation

Carrageenan is used widely in food and pharmaceutical and products as a gelling or viscosity modifying agent. The typical usage level is less than one percentage (1.0%). Thus, one percent (1.0%) iota-carrageenan solution was selected for the experiments based on the usage levels in most commercial food and pharmaceutical products. A bulk solution of 0.1 M NaNO₃ was prepared and filtered through a 0.22 μm membrane prior to use. Carrageenan powder was dissolved in this mobile phase by heating to 90 °C with continuous stirring until a clear solution was obtained. To reduce the sample viscosity, the solution was then diluted to 0.2% (w/w) using the same mobile phase and aliquoted into glass vials (∼20 g per vial) for analytical testing. The study evaluated the effect of four distinct stress scenarios on the molecular weight distribution of iota-carrageenan:

• iHigh temperature only (neutral pH)

To simulate thermal exposure during softgel manufacturing, samples were incubated at 95 °C and 105 °C under neutral pH for 60 and 120 min. No acid was added in this condition.

• iiAcid treatment at ambient temperature (∼25 °C)

To isolate the effect of acidic pH without thermal stress, samples were treated with.

• oHydrochloric acid (HCl) at pH 1.2
• oSulfuric acid (H₂SO₄) at pH 1.2
• oAcetic acid (CH₃COOH) at pH 3.0

These acidified samples were maintained at ambient temperature and sampled after 60 and 120 min. The pH was subsequently adjusted to 4–5 to stop the hydrolysis process.

• iiiCombined acid and high temperature stress

To simulate harsh degradation conditions, acidified samples (pH 1.2 with HCl or H₂SO₄, and pH 3.0 with CH₃COOH) were incubated at 95 °C. Samples were collected after 60 and 120 min and neutralized to halt further degradation.

• ivSimulated physiological conditions (gastric pH at body temperature)

To mimic conditions in the human stomach, samples were adjusted to pH 1.2 using HCl and incubated at 37 °C. Aliquots were withdrawn at 15, 30, 60, and 120 min. Each was neutralized to pH 4–5 immediately upon removal. This in vitro model represents a simplified and conservative approximation of gastric conditions, using a low pH to reflect a worst-case acidic environment. It does not account for the dynamic and heterogeneous nature of the human gastrointestinal system, including fluctuations in pH, enzymatic activity, gastric motility and transit processes.

2.2.2. Gel permeation chromatography method

A Gel Permeation Chromatography system (Model 1260 Infinity, Agilent Technologies) was utilized to run the GPC experiments. A TSKgel® GMPWxl column (30 cm × 7.8 mm) with a particle size of 13 μm was utilized to determine the molecular weight distribution of the samples. A calibration curve was created using various molecular weights of polyethylene oxide as standards. The mobile phase consisted of 0.1 M NaNO₃ and elution was performed with the filtered mobile phase at a flow rate of 0.45 mL/min. An injection volume of 80 μL was used for the samples and all measurements were conducted in triplicates to ensure reproducibility.

3. Results

3.1. Baseline molecular weight distributions of untreated carrageenan

Fig. 2 illustrates the molecular weight distributions of control carrageenan samples that were neither heat-treated nor acid-treated. The average molecular weight distributions for these control samples were as follows: 1000 ± 250 kDa for Lot 1405995 (n = 2), 529 ± 31 kDa for Lot 2011234 (n = 6), and 580 ± 109 kDa for Lot 16278878 (n = 6). Lot 1405995 was run in duplicates due to equipment issues with the GPC instrument. The control carrageenan samples also exhibited low molecular weight fractions, commonly referred to as the “low molecular weight tail,” which constituted typically less than 10% of the total carrageenan fraction, with molecular weight distributions below 50 kDa. Such low-level fractions are commonly reported in carrageenan and may arise from natural variability in the raw material, partial depolymerization during extraction, or minor analytical contributions. Importantly, these fractions remained well above the poligeenan threshold (<20 kDa), indicating that poligeenan was absent in the control samples.

Fig. 2.

Molecular weight distributions of untreated carrageenan lots (control samples).

Gel permeation chromatography (GPC) profiles of untreated iota-carrageenan from three commercial lots. Each lot shows a high M_p with minor low-MW tails (<50 kDa), attributed to natural biosynthetic variability. No poligeenan (<20 kDa) fragments were detected.

3.2. Changes in molecular weight under high temperature (neutral pH)

Carrageenan is typically exposed to elevated temperatures (75–95 °C) during softgel manufacturing to facilitate dissolution. This section evaluates whether such thermal exposure, in the absence of acidic conditions, results in degradation. Fig. 3 shows GPC chromatograms for samples incubated at 95 °C and 105 °C for 60 and 120 min under neutral pH. No new peaks indicative of low molecular weight fragments were observed, suggesting that the carrageenan remained stable.

Fig. 3.

Molecular weight stability of carrageenan under thermal processing conditions (95–105 °C, neutral pH). Carrageenan samples were subjected to elevated temperatures (95 °C and 105 °C) at neutral pH for 60 and 120 min. No significant degradation or formation of low-molecular-weight fragments observed across all lots. Legend:

Table 2 summarizes the corresponding peak molecular weights. Minor reductions in Mp were observed, but these remained well above the 20 kDa threshold.

Table 2.

Peak molecular weight after heat treatment (neutral pH)

Molecular weight data from samples incubated at 95 °C and 105 °C. Changes in Mp are minimal and consistent with thermal stability of iota-carrageenan.

Treatment Condition (Temperature/Time)	Lot 1405995	Lot 2011234	Lot 16278878
	Peak 1 Mp (kDa)	Peak 1 Mp (kDa)	Peak 1 Mp (kDa)
Control	1019.4	529 ± 31	583 ± 109
95 °C/60 min	862.2	513 ± 29	546 ± 82
95 °C/120 min	829.0	463 ± 50	569 ± 101
105 °C/60 min	983.1	500 ± 50	536 ± 72
105 °C/120 min	912.9	443 ± 138	561 ± 33

Fig. 4 demonstrates the changes for the corresponding peak molecular weights when subject to temperature alone under neutral pH conditions. Minimum reductions in Mp were observed for both 95 °C and 105 °C, but these remained well above the 20 kDa threshold. Also, the reduction in molecular weights at 105 °C was slightly higher than that at 95 °C.

Fig. 4.

Changes in peak molecular weight under high temperature and neutral pH.

3.3. Changes in molecular weight under acidic conditions at ambient temperature

To isolate the impact of acid treatment alone, CGN samples were treated with HCl (pH 1.2), H₂SO₄ (pH 1.2), and CH₃COOH (pH 3.0) at ambient temperature (standard laboratory conditions). Fig. 5 shows slight broadening and shifting of the main peak, but no significant formation of low molecular weight fragments. These results indicate minimal degradation under acidic conditions at ambient temperature.

Fig. 5.

Molecular weight distributions of carrageenan samples after subjecting them to acidic treatment at ambient temperature. Molecular weight profiles of carrageenan lots treated at room temperature with HCl (pH 1.2), H₂SO₄ (pH 1.2), or CH₃COOH (pH 3.0). Moderate reductions in peak molecular weight observed, but no poligeenan-range fragments detected. Legend:

Table 3 presents Mp values post-treatment. No sample showed molecular weights in the poligeenan range (<20 kDa).

Table 3.

Peak molecular weight of carrageenan after acidic treatment at ambient temperature Comparison of Mp values for each lot after 1 and 2 h of acid exposure at room temperature. Mild reduction observed, but no significant low-MW fragment formation.

Lot 1405995 (Duration of hydrolysis)	pH 1.2. HCl (Peak MW, kDa)	pH 1.2, H2SO4 (Peak MW, kDa)	pH 3.0, CH3COOH (Peak MW, kDa)
Control	1019	1019	1019
1 h	993	828	718
2 h	1034	689	1092

Lot 2011234 (Duration of hydrolysis)	pH 1.2. HCl (Peak MW, kDa)	pH 1.2, H2SO4 (Peak MW, kDa)	pH 3.0, CH3COOH (Peak MW, kDa)
Control	529	529	529
1 h	318	378	490
2 h	318	435	351

Lot 16278878 (Duration of hydrolysis)	pH 1.2. HCl (Peak MW, kDa)	pH 1.2, H2SO4 (Peak MW, kDa)	pH 3.0, CH3COOH (Peak MW, kDa)
Control	583	583	583
1 h	433	430	470
2 h	350	352	327

Fig. 6 shows the changes of molecular weight after treatment at various pH levels at ambient temperatures.

Fig. 6.

Changes of molecular weight after treatment at various pH levels at ambient temperatures.

3.4. Changes in molecular weight under combined acid and high temperature stress

Samples were treated with acid (HCl, H₂SO₄, CH₃COOH) and heated to 95 °C to evaluate degradation under harsh conditions. Fig. 7 shows dramatic shifts in molecular weight distributions. Main peaks collapsed and new peaks appeared below 20 kDa, indicating formation of low-molecular-weight fragments consistent with poligeenan or further breakdown into even smaller fragments.

Fig. 7.

Molecular weight degradation under combined acid and high-temperature stress (95 °C). GPC curves for carrageenan samples exposed to pH 1.2 or 3.0 at 95 °C. Severe fragmentation was observed, with main peaks collapsing and Mp values dropping below 20 kDa, indicating formation of low-molecular-weight fragments consistent with poligeenan. Legend:

Table 4 confirms that treatment under these conditions caused extensive fragmentation across all three lots, with many Mp values below 20 kDa.

Table 4.

Peak molecular weight of carrageenan after combined acid and heat exposure (95 °C) Mp values from samples exposed to pH 1.2 (HCl or H₂SO₄) and pH 3.0 (CH₃COOH) at 95 °C. Fragmentation into poligeenan (<20 kDa) occurs under all acid types, especially after 2 h.

Lot 1405995 (Duration of hydrolysis)	pH 1.2. HCl (Peak MW, kDa)	pH 1.2, H2SO4 (Peak MW, kDa)	pH 3.0, CH3COOH (Peak MW, kDa)
Control	1019	1019	1019
1 h	1	1	17
2 h	1	1	8

Lot 2011234 (Duration of hydrolysis)	pH 1.2. HCl (Peak MW, kDa)	pH 1.2, H2SO4 (Peak MW, kDa)	pH 3.0, CH3COOH (Peak MW, kDa)
Control	529	529	529
1 h	1	1	28
2 h	1	1	12

Lot 16278878 (Duration of hydrolysis)	pH 1.2. HCl (Peak MW, kDa)	pH 1.2, H2SO4 (Peak MW, kDa)	pH 3.0, CH3COOH (Peak MW, kDa)
Control	583	583	583
1 h	1	1	26
2 h	1	1	13

3.5. Changes in molecular weight under simulated human physiological conditions

Simulated gastric conditions were applied (pH 1.2 at 37 °C) to test carrageenan's stability during digestion. Samples were withdrawn at multiple time points (15–120 min). Fig. 8 displays the molecular weight distributions over time.

Fig. 8.

Molecular weight distribution following acid exposure at 37 °C (simulated gastric conditions). GPC traces showing molecular weight shifts in carrageenan samples treated at pH 1.2 and 37 °C for up to 120 min. A second peak emerges over time, indicating partial hydrolysis. All fragments remain above the 20 kDa poligeenan threshold. Legend:

Table 5 quantifies the peak molecular weight values across lots. Even the lowest observed value (81 kDa) remained well above the 20 kDa threshold, suggesting no risk of poligeenan formation during digestion.

Table 5.

Peak molecular weight (M_p) of carrageenan samples after acid Treatment at 37 °C Peak 1 and Peak 2 molecular weight values (kDa) after treatment with pH 1.2 at 37 °C. Results show gradual MW reduction but no formation of poligeenan-level fragments.

Time (Min)	Lot 1405995		Lot 2011234		Lot 16278878
	Mp at Peak 1 (kDa)	Mp at Peak 2 (kDa)	Mp at Peak 1 (kDa)	Mp at Peak 2 (kDa)	Mp at Peak 1 (kDa)	Mp at Peak 2 (kDa)
Control	1019 ± 250	NA	529	NA	583	NA
15	622 ± 7	124 ± 31	543 ± 28	NA	530 ± 15	111 ± 63
30	471 ± 11	85 ± 0	432 ± 35	124 ± 5	392 ± 24	152 ± 13
60	401 ± 57	80 ± 45	365 ± 25	172 ± 16	210 ± 20	NA
120	373 ± 9	81 ± 14	217 ± 15	NA	185 ± 5	NA

Fig. 9 shows the changes of molecular weight at (pH 1.2 at 37 °C) conditions.

Fig. 9.

Changes of molecular weight after treatment at pH 1.2 and 37 °C.

4. Discussion

This study evaluated the molecular weight stability of iota-carrageenan under conditions simulating food and pharmaceutical manufacturing conditions and human gastrointestinal physiology. Gel permeation chromatography (GPC) was used to characterize the degradation profiles of three commercial carrageenan lots following exposure to (1) acidic conditions representative of the human gastrointestinal physiology, (2) elevated temperatures mimicking softgel processing, and (3) combined acid and heat stress to assess worst-case degradation scenarios.

Across all conditions of simulating human gastric exposure (pH 1.2 at 37 °C), carrageenan exhibited partial hydrolysis, reflected by the emergence of a second molecular weight peak. However, the resulting fragments remained well above the 20 kDa threshold used to define poligeenan (by more than 4-fold). These findings are consistent with in vivo pharmacokinetic data showing minimal gastrointestinal degradation and near-complete fecal recovery of intact carrageenan [3,8,10]. Our results provide in vitro confirmation that physiologically relevant acidity and exposure time do not result in meaningful molecular weight fragmentation.

Under thermal processing conditions representative of carrageenan gel preparation and softgel encapsulation (typically 80–95 °C), carrageenan remained chemically stable, with molecular weights consistently well above the threshold for poligeenan formation. Observed shifts in molecular weight were minimal and did not yield secondary peaks or low-molecular-weight tails. These results support previous literature asserting carrageenan's thermal resilience in neutral pH environments [3,5].

By contrast, combined exposure to acidic pH (1.2 and 3.0) and elevated temperatures (95 °C) induced significant degradation across all three carrageenan lots. Molecular weight distributions collapsed into low-kDa fragments, with Mp values falling well below the 20 kDa poligeenan threshold after just 1 h. These conditions represent a known pathway for generating poligeenan in laboratory settings and are not representative of either gastrointestinal or softgel manufacturing environments [1,3]. Therefore, poligeenan formation is not associated with carrageenan used in softgel encapsulations.

Due to the scope and design of this study, we employed a qualitative evaluation of molecular weight trends rather than inferential statistical comparisons. While data were collected in triplicate to confirm reproducibility, variability in sample complexity and inherent limitations of GPC, including resolution constraints in the low-molecular-weight range and dependence on calibration standards, limited statistical power. Interpretation of chromatographic peak shifts was therefore performed qualitatively. Nevertheless, the clear and consistent separation of degradation profiles, particularly under combined acid and heat stress, supports the robustness of our conclusions. This study also did not include viscosity or rheological characterization as a complementary measure of polymer chain integrity. While rheological properties can be sensitive to changes in polymer size, conformation, and intermolecular interactions, such measurements provide indirect evidence of degradation and cannot directly confirm whether low-molecular-weight fragments fall within the poligeenan range. Because the primary research question centered on the presence or absence of fragments below the 20 kDa threshold, GPC was considered the most appropriate method for this work. Another limitation is the absence of confirmatory structural techniques (e.g., FTIR, NMR) to chemically validate poligeenan formation. Although confirmatory structural analyses were not performed, the substantial molecular weight reductions observed, consistent fragmentation across acid types, and alignment with established degradation mechanisms strongly support the conclusion that significant hydrolysis, potentially yielding poligeenan, occurs only under extreme combined acid and heat conditions, not under physiological gastric conditions or during softgel manufacturing.

5. Conclusions

This study demonstrated that iota-carrageenan maintains its molecular integrity under conditions that closely simulate both human gastric physiology and standard industrial thermal processing. When exposed to simulated gastric pH (1.2) at 37 °C for up to 120 min, carrageenan exhibited limited hydrolysis with no evidence of fragmentation into poligeenan-range molecular weights. Similarly, exposure to elevated temperatures (95–105 °C) under neutral pH conditions produced only minor reductions in molecular weight without formation of low-molecular-weight species.

In contrast, carrageenan subjected to the combination of low pH and high temperature underwent extensive degradation. The resulting molecular weight distributions shifted significantly, with major peaks collapsing into fragments well below the 20 kDa threshold. These conditions are consistent with known poligeenan-generating processes and are not representative of human physiology or routine softgel manufacturing process conditions.

Together, these findings support the chemical stability of iota-carrageenan under realistic use scenarios and provide further evidence that poligeenan is not formed during digestion or typical pharmaceutical manufacturing processes. This reinforces carrageenan's continued use as a safe and functional excipient in oral and thermally processed dosage forms where its unique benefits (outlined in the introduction) can be fully leveraged. Future studies may explore degradation kinetics and expand to other carrageenan types to further refine our understanding of excipient behavior under controlled stress conditions as well as actual manufacturing and use conditions.

Research data availability

The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request, subject to company confidentiality restrictions.

Funding statement

Funding was provided by Catalent Inc.

CRediT authorship contribution statement

Soo Ah Jin: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft. Qi Fang: Conceptualization, Data curation, Formal analysis, Methodology, Validation, Visualization, Writing – original draft. Karunakar Sukuru: Conceptualization, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – review & editing.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:• All authors are full-time employees of Catalent Pharma Solutions, a contract development and manufacturing organization (CDMO) that provides softgel and drug delivery technologies relevant to the study.• This research was conducted as part of internal scientific investigations and does not reflect any contractual research for a specific client.

Data availability

The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request, subject to company confidentiality restrictions.