Optimization of deodorization process and flavor mechanism of raw and cooked salted kelp based on microbial fermentation and GC-IMS technology

Abstract

The fishy odor of salted kelp, mainly derived from halogenated hydrocarbons, sulfur-containing compounds, and free fatty acids, limits its sensory quality and market value. This study evaluated microbial deodorization using six food-grade strains—Pichia kluyveri, Saccharomyces cerevisiae, Wickerhamomyces anomalus, Lactiplantibacillus plantarum, Limosilactobacillus fermentum and Pediococcus pentosaceus—in single and co-fermentation systems. GC-IMS, relative odor activity values, and sensory evaluation identified 1-octen-3-one as the dominant off-odor compound. Fermentation significantly reduced 1-octen-3-one while increasing aroma-active volatiles such as isovaleraldehyde, isopentanol, and isopropyl propionate, likely through enzymatic degradation and microbial biosynthesis. Sensory analysis confirmed improvements in aroma and overall acceptability with minimal changes in color and texture. These findings clarify microbial modulation mechanisms of kelp flavor and support the development of value-added fermented kelp products.

Highlights

• •Comparison of the Deodorizing Effects of Single and Composite Strains on Salted Kelp.
• •Establish a microbial fermentation system suitable for whole salted kelp blocks.
• •Fermentation does not compromise the fundamental phenotypic traits of kelp.
• •This fermentation method enables the optimization of specific flavors.

1. Introduction

Kelp (Laminaria japonica), family Kelpaceae, is an endemic species in the North Pacific Ocean. Kelp has been heavily exploited as an important marine resource for high value-added products in food and pharmaceuticals. In recent years, researchers have discovered that kelp contains a large amount of fresh-flavored substances, which makes it a common ingredient in seasonings. Food preservation through the addition of salt is one of the ancient food preservation processes around the world, and the salting process not only extends the storage period of kelp, but also promotes the formation of its characteristic flavor.

The organoleptic qualities of salted kelp—especially its odor profile—are receiving increased attention as demand for “clean label,” minimally processed, and naturally flavored foods continues to grow. However, despite its nutritional value, the strong fishy odor poses significant challenges to the food processing industry. This odor originates from a complex mixture of volatile compounds such as halogenated hydrocarbons, aldehydes, ketones, sulfur-containing compounds, and free fatty acids (Wei et al., 2024). Even at very low levels, these compounds exert a strong impact on the flavor of food products, thus reducing consumer acceptance and limiting product diversification. Therefore, removing the fishy flavor of salted kelp has become a critical technical challenge.

In a wide range of aquatic product studies, fishy odor control strategies are diverse and include external environmental conditioning (clean water, microbial degradation) (Dai et al., 2024), masking and encapsulation (spices, β-cyclodextrins) (Zhou et al., 2024), adsorption (activated charcoal, molecular sieves), thermal treatments (steam, vacuum deodorization), acid-base reactions (washing, chemical conversion), and others. However, most of these methods suffer from limited masking effects or adverse effects on nutrition and flavor. For example, Dai et al. concluded that heat treatment and adsorption could remove some volatiles, but resulted in nutrient loss and flavor deterioration (Dai et al., 2024); Zhou et al. found that β-cyclodextrin encapsulation or spice masking could only improve the organoleptic qualities in the short term (Zhou et al., 2024). In contrast, biological methods have significant advantages. Among the existing studies, Wei et al. treated 1 × 1 cm small pieces of kelp with a combination of natural antioxidant impregnation and microbial fermentation, and confirmed that this strategy significantly reduced fishy substances and improved the overall aroma quality (Wei et al., 2024). However, such studies based on fragmentation treatment have somewhat weakened the natural structural integrity of kelp and still fall short of actual consumption and industrial processing patterns. To overcome this limitation, the present study directly used whole kelp (3 × 10 cm) as the fermentation substrate to achieve deodorization while maintaining natural morphology, making it more relevant for application and industrial popularization.

In summary, although physical and chemical methods can improve the quality of kelp to a certain extent, there are unavoidable limitations, while microbial fermentation provides a more promising way to achieve efficient deodorization and product reprocessing by virtue of its targeted metabolism and flavor optimization advantages. Microbial fermentation, as a representative of green food processing technology, has shown unique advantages in the field of flavor modulation (Allahgholi et al., 2023; Zhu et al., 2022). Food-grade microorganisms such as Lactiplantibacillus plantarum and Saccharomyces cerevisiae are widely used in food fermentation, which can significantly improve the organoleptic quality of products by degrading odor precursors, inhibiting the production of off-flavor substances, and promoting the synthesis of aroma-enhancing compounds such as alcohols and esters. For example, Zou et al. introduced L. plantarum as a starter bacterium in fermented beef–soybean paste, which significantly promoted the accumulation of alcohols and esters and reduced undesirable odor compounds (Zou et al., 2025); J. Zhou et al. used L. plantarum and Staphylococcus carnosus in co-fermentation of dried lamb to effectively degrade 4-branched-chain alkyl off-flavored fatty acids and improve color and texture (Zhou, Ying, et al., 2022); Zhu et al. introduced S. cerevisiae, L. plantarum, and Lactobacillus aceticus into mixed fermentation of prune pomace, which significantly enhanced the floral and fruity ester content while retaining polyphenols and antioxidant components (Zhu et al., 2022).

Lactic acid bacteria and yeast are probiotic microorganisms commonly used in food fermentation. Lactic acid bacteria are able to produce acid, inhibit spoilage bacteria, and synthesize flavor-enhancing substances such as alcohols and esters, whereas yeasts have advantages in alcohol and ester production and flavor richness (Zhu et al., 2022; Zou et al., 2025). Single-organism fermentation and complex fermentation with lactic acid bacteria and yeast each have their own advantages in food flavor regulation. Single-bacteria fermentation is easy to control, stable in flavor, and can highlight the metabolic characteristics of specific strains, while complex fermentation can achieve metabolic complementation and enhance flavor complexity. Acid production by Lactobacillus provides a suitable environment for yeast to grow, while yeast can produce esters and alcohols, which Lactobacillus lacks, thereby optimizing the aroma structure (Fang et al., 2023). In practice, the flavor of fermented foods can be further enhanced by modulating exogenous factors (e.g., oxygen, temperature, humidity) and strain combinations (Tian et al., 2023). For example, Previous studies have shown that co-inoculation of L. plantarum and S. cerevisiae can simultaneously improve product quality and functionality. For example, Fang et al. (Fang et al., 2023) reported phytic acid degradation and enhanced flavor compounds in sourdough; Chen et al. (Chen et al., 2023) observed improved antioxidant activity and flavor in cider; and Sun et al. (Sun et al., 2024) demonstrated that co-fermentation significantly modulated volatile dynamics in oat fermentation. Collectively, these findings indicate that L. plantarum and S. cerevisiae co-fermentation enhances both flavor quality and antioxidant properties in dairy, cereal, and fruit-based products. In addition, the application of this combination in fermented feeds also showed positive effects on microbial diversity and metabolite production (Wu et al., 2025). Overall, monobacterial fermentation is suitable for flavor stabilization and production of specific metabolites, whereas complex fermentation with lactic acid bacteria and yeast is more effective for complex flavor optimization, providing an important strategy for the food industry.

Previous studies have shown that these microorganisms can effectively degrade aldehydes, alcohols, and other off-flavor compounds in various matrices, and promote the positive transformation of flavor substances (Aguirre-Garcia et al., 2024; Zhou, Guan, et al., 2022; Zhu et al., 2022; Zou et al., 2025). Thus, it is reasonable to assume that they may play a similar role in the deodorization of salted seaweed. However, most existing work focuses on fresh seaweed or roughly processed products, and research on microbial deodorization of salted seaweed still faces challenges such as an imperfect strain screening system, lack of optimized process parameters, and unclear mechanisms of flavor change. Therefore, optimizing the deodorization process of whole salted kelp not only improves its quality but also provides a reference for its reprocessing in the food industry.

Based on this, in this study, under the premise of maintaining the tissue integrity of salted kelp, single and complex bacterial lineage fermentation were used to mildly deodorize salted kelp by regulating fermentation intensity and time. The fermented samples were analyzed using gas chromatography-ion mobility spectrometry (GC-IMS), relative odor activity value (ROAV) analysis, and principal component analysis (PCA) to detect key volatile flavor compounds, evaluate the deodorization effect, and explore the underlying mechanisms. The ultimate aim of this study is to propose a practical deodorization strategy for salted kelp and to provide technical support for its industrial reprocessing.

2. Materials and methods

2.1. Materials and chemicals

The salted kelp was provided by Dalian Xinlongshun Food Co. (Dalian, China). Analytically pure standard compounds including 2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, 2-octanone, and 2-nonanone were purchased from Aladdin Holding Group Limited (Shanghai, China). MRS agar, MRS broth, wort agar, and wort broth media were purchased from Biomicrobial Technology Co., Ltd. (Beijing, China).

The microbial strains used for fermentation were obtained from the China Center of Industrial Culture Collection (CICC, Beijing, China; https://www.china-cicc.org/). The strains included Pichia kluyveri (CICC 32844), Saccharomyces cerevisiae (CICC 32883), Wickerhamomyces anomalus (CICC 33313), Lactiplantibacillus plantarum (CICC 21809), Limosilactobacillus fermentum (CICC 21829), and Pediococcus pentosaceus (CICC 21862). All strains were stored and activated according to the CICC guidelines prior to use.

2.2. Sample preparation

The salted kelp stored at −20 °C was thawed, defective (diseased or yellowed) parts were removed, and flat pieces of uniform thickness were selected. After washing with water to remove excess salt, samples were divided into two groups: raw salted kelp (Yraw) and cooked salted kelp (Ycook, boiled in water). For fermentation experiments, both raw and cooked kelp were rinsed with sterile water and cut into uniform strips of 3 × 10 cm, which were used as the experimental substrate. “Yraw and Ycook served as blank control groups, in which kelp samples were treated with sterile water under identical fermentation conditions but without microbial inoculation.”

2.3. Strain screening

Based on the list of edible mushroom species and previous literature (Aguirre-Garcia et al., 2024; Allahgholi et al., 2023; Chen et al., 2023; Fang et al., 2023; Sun et al., 2024; Tian et al., 2023; Wu et al., 2025; Zhou, Ying, et al., 2022; Zhu et al., 2022; Zou et al., 2025), six candidate strains from six different genera were selected for microbial fermentation trials. These included lactic acid bacteria (Lactiplantibacillus plantarum CICC 21809, Limosilactobacillus fermentum CICC 21829, and Pediococcus pentosaceus CICC 21862) and yeasts (Pichia kluyveri CICC 32844, Saccharomyces cerevisiae CICC 32883, and Wickerhamomyces anomalus CICC 33313). Strain selection was based on two criteria: (i) safety for food-grade applications (Bourdichon et al., 2021); and (ii) reported efficacy in degrading fishy odor compounds such as aldehydes and ketones in seafood matrices (Liang et al., 2023; Ma et al., 2023). Lactic acid bacteria were cultivated in MRS broth at 37 °C under anaerobic conditions for 24–48 h, while yeasts were cultured in malt extract agar (MEA) medium at 28 °C with shaking for 72 h. All cultures were adjusted to a final cell density of 10⁸ CFU/mL before inoculation. Fermentation experiments were conducted separately on raw and cooked salted kelp substrates. Each sample was subjected to preliminary sensory screening to identify strains with the greatest deodorization potential.

2.4. Microbial fermentation of kelp

• (1)Single-strain fermentation

Three yeasts (Pichia kluyveri, Saccharomyces cerevisiae, and Wickerhamomyces anomalus) and three lactic acid bacteria (LAB) (Lactiplantibacillus plantarum, Limosilactobacillus fermentum, and Pediococcus pentosaceus) were selected for kelp fermentation. For yeast preparation, each strain was streaked onto MEA agar plates and incubated at 28 °C for 72 h (S. cerevisiae), 48 h (W. anomalus), or 72 h (P. kluyveri), according to their optimal colony-forming rates. A well-isolated colony was transferred into 5 mL MEA broth and cultured at 28 °C and 200 r/min for 18 h. The resulting culture was serially diluted with sterile saline, plated on MEA agar, and incubated at 28 °C for 24 h to enumerate colonies. Based on preliminary evaluations of yeast metabolic activity in kelp soaking solution, the final yeast inoculum was adjusted to 1 × 10⁷ CFU/mL. This concentration was sufficient to achieve stable deodorization performance while maintaining consistent fermentation kinetics.

LAB strains were streaked onto MRS agar and incubated at 37 °C for 24 h. Expanded cultures were prepared following the procedure described in Section 2.3, and the bacterial suspension was adjusted to 1 × 10⁸ CFU/mL before use as the LAB seed inoculum. Yeasts exhibit relatively slower growth and odor-related metabolic conversion in kelp soaking solutions, whereas LAB grow rapidly and produce acid efficiently. Therefore, different inoculum concentrations were applied: 1 × 10⁷ CFU/mL for yeasts to achieve stable deodorization, and 1 × 10⁸ CFU/mL for LAB to ensure rapid acidification and effective suppression of off-odor formation. These adjustments were validated by pH monitoring, VOC transformation profiles, and sensory evaluation.

• (2)Complex fermentation.

Based on the results of single-strain fermentations, a mixed yeast–LAB system was developed. Yeast and LAB suspensions prepared as described above were co-inoculated into sterile water-soaked kelp. Fermentation was conducted under controlled temperature and shaking conditions. To ensure balanced microbial growth and metabolic activity, the fermentation temperature was set according to the optimal range for LAB, which was also compatible with yeast viability and functionality. The specific operational parameters for the mixed fermentation, including inoculation ratios, fermentation temperature, and additional process settings, are provided in Table 1.

Table 1.

Co-fermentation conditions for different strain combinations.

Group ID	Strain combination (order = ratio order)	Inoculation ratio	Fermentation time (h)	Temperature (°C)	Inoculum level (%)
Yr2913	L. fermentum + W. anomalus	1:1	3	37	2
Yr6213	P. pentosaceus + W. anomalus	1:1	2	37	3
Yc0944	L. plantarum + P. kluyveri	1:1	1	37	3
Yc0913	L. plantarum + W. anomalus	1:1	2	37	2
Yc0988	L. plantarum + S. cerevisiae	1:1	3	37	3
Yc2944	L. fermentum + P. kluyveri	1:1	1	37	3
Yc2913	L. fermentum + W. anomalus	3:1	1	37	4
Yc2988	L. fermentum + S. cerevisiae	1:1	1	37	5

Note: The co-fermentation treatments were conducted in sterilized kelp-soaking solution under shaking conditions (150 r/min). The inoculation ratio refers to the relative volume ratio of the two seed cultures. All experiments were performed at 37 °C unless otherwise specified. Each treatment was conducted in triplicate.

Each fermentation group was labeled using an abbreviation combining the substrate type (Yc = cooked kelp; Yr = raw kelp) and the strain code. The specific mapping is as follows: Except for Saccharomyces cerevisiae, designated as strain 88, all other strains are named using the last two digits of their code.

2.5. Sensory evaluation

All sensory evaluations were approved by the Ethics Committee of Dalian Ocean University (approval no. DLOU-20240503, Liaoning, China) and conducted strictly following GB/T 39625–2020 standards. Six trained panelists (3 females, 3 males, aged 23–45) from the School of Food Science and Engineering participated after providing informed consent. Prior to the formal tests, the panel received one month of systematic training (6 h per week) and passed triangle tests to verify their discrimination ability. Personnel selection requirements are detailed in Table S1.

The sensory evaluation method was adapted from Dooley et al. (Dooley et al., 2010). All tests were conducted in a standard sensory evaluation room (25 ± 2 °C, white lighting). Samples were randomly coded and presented in lidded paper cups. Panelists evaluated odor acceptability using a 10-cm linear scale. The marked distance—reflecting both deodorization effectiveness and the absence of off-odors—was divided by 10 cm and multiplied by 100 to obtain a percentage score (0 = completely unacceptable; 100 = fully acceptable). The final score for each sample represents the mean of six trained panelists. The same evaluation procedure was applied to assess deodorization performance under different fermentation conditions, where higher scores indicate better sensory quality. With detailed criteria listed in Table 2.

Table 2.

Sensory Evaluation Table of Kelp.

Evaluation	Color	Fishiness	Fermented Odor	Taste
19–25	It has the dark green color that kelp should have, with uniform color and luster	Basically no kelp fishy flavor, easy to accept	Aroma from fermentation, no off-flavors, easy to accept	Inherent flavor of kelp, no bitterness or astringency, crunchy and chewy texture.
13–18	Too dark or too light in color, appearing green or brown, slightly shiny	Slightly fishy seaweed odor, acceptable	No fermented aroma or slightly fermented flavor, acceptable	Slightly bitter and astringent, with a crunchy, chewy texture
7–12	The color is light olive green in color and loses its luster.	It has a fishy kelp flavor, which is acceptable	Fermentation produces strong, overly sour, alcoholic or other off-flavors that are difficult to accept	Bitter and astringent flavor, soft texture, loss of chewiness
0–6	Completely lost the green color and luster that kelp should have, taking on a yellowish color	Fishy flavor too strong, not easy to accept	Excessive and unacceptable odor	Obviously bitter and astringent at the same time other miscellaneous flavors are heavy, the taste is very poor, not easy to accept

2.6. GC-IMS analysis

Following S. Jiang et al. with modifications, 1 g of sample was placed in a 20 mL headspace vial (Jiang et al., 2024), incubated at 60 °C for 20 min, and 500 μL of headspace gas was injected into the inlet at 85 °C (splitless mode). Three replicates were analyzed for each sample.

Chromatographic conditions: column temperature 60 °C; carrier gas high-purity nitrogen (≥ 99.999 %); programmed flow: 2.0 mL/min (2 min), ramped to 10.0 mL/min (8 min), then to 100.0 mL/min (10 min), held 20 min. Total runtime: 40 min; inlet temperature: 80 °C.

IMS conditions: tritium source (³H); migration tube 53 mm; field strength 500 V/cm; tube temperature 45 °C; drift gas nitrogen ≥99.999 %, 75 mL/min; positive ion mode.

2.7. Calculation of relative odor activity value (ROAV)

ROAV was calculated to identify key odor compounds (Wang, Chen, et al., 2025). The ROAV of the compound contributing most to kelp flavor was set as 100, and values of other volatiles were calculated as:

$$\text{ROAV}={\mathrm{C}}_i/C_{\mathit{max}}\times {\mathrm{T}}_{\max }/{\mathrm{T}}_{\mathrm{i}}\times 100$$ (1)

where Cᵢ = relative content of compound i (%); Tᵢ = threshold of compound i (μg/kg); Cₘₐₓ and Tₘₐₓ = relative content and threshold of the compound with the greatest contribution.Compounds with ROAV ≥1 were recognized as key odorants, while 0.1 ≤ ROAV <1 indicated important contributors.

2.8. Texture analysis

Texture was analyzed following Akomea-Frempong et al. with modifications. Kelp strips were cut into 3 × 3 cm blocks (1.5–2.0 mm thick) (Akomea-Frempong et al., 2022). Measurements were performed using a TA/50 probe, TPA mode, 1000 N range, 1.5 N trigger force, 30 % deformation, 5 mm rise, and 60 mm/min speed. Six replicates were averaged.

2.9. Color analysis

Following Yang et al., three kelp strips from each group were randomly selected. Three measurement points per strip were analyzed using a colorimeter for L*, a*, and b* values. Hue was calculated as arctan(b*/a*)(Yang et al., 2023). Color difference (ΔE) between deodorized and control samples was calculated as:

$$\mathrm{\Delta }{E}^{\ast }=\sqrt{{\left(\mathrm{\Delta }{L}^{\ast }\right)}^2+{\left(\mathrm{\Delta }{a}^{\ast }\right)}^2+{\left(\mathrm{\Delta }{b}^{\ast }\right)}^2}$$ (2)

2.10. Chlorophyll content

Chlorophyll was determined following Özkan & Bilek with modifications (Özkan & Bilek, 2015). Absorbance at 664 and 630 nm was measured using 90 % acetone as reference; absorbance at 750 nm was subtracted to correct turbidity. Concentrations of chlorophyll a and c were calculated using (Jeffrey & Humphrey (Jeffrey & Humphrey, 1975):

$${\rho }_a\left(\mathrm{\mu g}/\mathrm{mL}\right)=11.47E_{664}-0.4E_{630}$$ (3)

$${\rho }_c\left(\mathrm{\mu g}/\mathrm{mL}\right)=24.36E_{630}-3.73E_{664}$$ (4)

$$\rho \left(\mathrm{\mu g}/\mathrm{mL}\right)={\rho }_a+{\rho }_c$$ (5)

If expressed per fresh weight:

$$w\left(\mathrm{mg}/\mathrm{kg}\right)=\mathit{cv}/m$$ (6)

where c = chlorophyll concentration (μg/mL), v = extract volume (mL), m = fresh weight (g).

2.11. Data analysis

All measurements were performed in triplicate. Retention indices were calculated from retention times and compared with NIST 2020 and VOCal IMS libraries. VOCal software (Reporter, Gallery Plot, Dynamic PCA) was used to generate 2D/3D spectra, fingerprints, and PCA plots. Statistical differences were evaluated using SPSS, with significance set at p < 0.05.

3. Results and discussion

3.1. Sensory evaluation of raw and cooked salted kelp before and after microbial deodorization

The results of sensory evaluation (Fig. 1) showed significant differences in color, texture, and aroma between raw (Yraw) and cooked (Ycook) salted kelp before deodorization. These differences may be explained by the fact that heating disrupts the cellular structure of kelp, which not only alters texture but also facilitates the release of volatile compounds responsible for aroma changes. For example, Zhou et al. demonstrated that heating triggers cell wall disruption, further affecting texture and chewiness(Zhou et al., 2024).

Fig. 1.

Sensory evaluation of the deodorization effect of single and complex fermentation of different strains of bacteria on raw and cooked salted seaweeds (Yraw:raw salted kelp without microbial inoculation; Yr29 (Limosilactobacillus fermentum (CICC 21829), fermentation with raw kelp); Yr62 (Pediococcus pentosaceus (CICC 21862), fermented with raw kelp); Yr13 (Wickerhamomyces anomalus (CICC 33313), fermented with raw kelp); Yr2913 (Limosilactobacillus fermentum (CICC 21829) + Wickerhamomyces anomalus (CICC 33313) co-fermentation with raw kelp); Yr6213 (Pediococcus pentosaceus (CICC 21862) + Wickerhamomyces anomalus (CICC 33313) co-fermentation with raw kelp);

Ycook: cooked salted kelp without microbial inoculation; Yc09 (Lactiplantibacillus plantarum (CICC 21809), fermented with cooked kelp); Yc29 (Limosilactobacillus fermentum (CICC 21829), fermented with cooked kelp); Yc44 (Pichia kluyveri (CICC 32844), fermented with cooked kelp); Yc13 (Wickerhamomyces anomalus (CICC 33313), fermented with cooked kelp);Yc88 (Saccharomyces cerevisiae (CICC 32883), fermented with cooked kelp); Yc0944 (Lactiplantibacillus plantarum (CICC 21809) + Pichia kluyveri (CICC 32844) co-fermentation with cooked kelp); Yc0913 (Lactiplantibacillus plantarum (CICC 21809) + Wickerhamomyces anomalus (CICC 33313) co-fermentation with cooked kelp);Yc0988 (Lactiplantibacillus plantarum (CICC 21809) + Saccharomyces cerevisiae (CICC 32883) co-fermentation with cooked kelp); Yc2913(Limosilactobacillus fermentum (CICC 21829) + Wickerhamomyces anomalus (CICC 33313) co-fermentation with cooked kelp); Yc2944(Limosilactobacillus fermentum (CICC 21829) + Pichia kluyveri (CICC 32844) co-fermentation with cooked kelp); Yc2988(Limosilactobacillus fermentum (CICC 21829) + Saccharomyces cerevisiae (CICC 32883) co-fermentation with cooked kelp).

On this basis, the study further investigated the deodorization effects of six microbial strains (Fig. 1A), which showed significant strain-dependent and substrate-specific differences. In raw kelp, Pediococcus pentosaceus, Wickerhamomyces anomalus, and Limosilactobacillus fermentum performed best, while Pichia kluyveri was the least effective. In cooked kelp, P. pentosaceus was largely inactivated, likely due to heat sensitivity, whereas the other strains contributed to flavor improvement. Notably, W. anomalus maintained stable performance across both substrates (score: 65.00 ± 2.52), indicating good process adaptability and industrialization potential. Based on these results, L. fermentum, P. pentosaceus, and W. anomalus were selected for fermentation of raw kelp, while all strains except P. pentosaceus were selected for cooked kelp (Table 3).

Table 3.

Definitions of fermentation group abbreviations and Group Details.

groups	Group abbreviation	strains	Strain Code (CICC)	Description
Raw Salted Kelp	Yraw	Control	–	Unfermented
	Yr29	Limosilactobacillus fermentum	21829	Single-strain fermentation
	Yr62	Pediococcus pentosaceus	21862	Single-strain fermentation
	Yr13	Wickerhamomyces anomalus	33313	Single-strain fermentation
	Yr6213	Pediococcus pentosaceus + Wickerhamomyces anomalus	21862 + 33313	Co-fermentation
	Yr2913	Limosilactobacillus fermentum + Wickerhamomyces anomalus	21829 + 33313	Co-fermentation
Cooked Salted Kelp	Ycook	Control	–	Unfermented
	Yc09	Lactiplantibacillus plantarum	21809	Single-strain fermentation
	Yc29	Limosilactobacillus fermentum	21829	Single-strain fermentation
	Yc44	Pichia kluyveri	32844	Single-strain fermentation
	Yc13	Wickerhamomyces anomalus	33313	Single-strain fermentation
	Yc88	Saccharomyces cerevisiae	32883	Single-strain fermentation
	Yc0944	Lactiplantibacillus plantarum + Pichia kluyveri	21809 + 32844	Co-fermentation
	Yc0913	Lactiplantibacillus plantarum + Wickerhamomyces anomalus	21809 + 33313	Co-fermentation
	Yc0988	Lactiplantibacillus plantarum + Saccharomyces cerevisiae	21809 + 32883	Co-fermentation
	Yc2944	Limosilactobacillus fermentum + Pichia kluyveri	21829 + 32844	Co-fermentation
	Yc2913	Limosilactobacillus fermentum + Wickerhamomyces anomalus	21829 + 33313	Co-fermentation
	Yc2988	Limosilactobacillus fermentum + Saccharomyces cerevisiae	21829 + 32883	Co-fermentation

Note: the prefix “Y” indicates kelp, while “r” and “c” denote raw and cooked kelp substrates, respectively. The four-digit code (e.g., 2913, 6213) corresponds to the two strains used in co-fermentation, listed in the same order as their digits. (e.g., 29 = Limosilactobacillus fermentum, 13 = Wickerhamomyces anomalus). Except for Saccharomyces cerevisiae, designated as strain 88, all other strains are named using the last two digits of their code.

Post-fermentation sensory analysis (Fig. 1B–D) showed that microbial treatments significantly improved the sensory quality of both raw and cooked kelp. For instance, the Yr13 (Wickerhamomyces anomalus (CICC 33313), fermented with raw kelp)treatment in raw kelp reduced the fishy taste by 33.8 %, the Yr2913 (Limosilactobacillus fermentum (CICC 21829) + Wickerhamomyces anomalus (CICC 33313) co-fermentation with raw kelp) treatment achieved significant deodorization, and the Yr62 (Pediococcus pentosaceus (CICC 21862), fermented with raw kelp) treatment maintained higher color and texture scores due to less structural damage. These findings are consistent with Zeng et al., who reported that Lactobacillus fermentation improves the texture of black rice yogurt through extracellular polysaccharides (EPS)(Zeng et al., 2025). In cooked kelp, the Yc88 (Saccharomyces cerevisiae (CICC 32883), fermented with cooked kelp) treatment achieved the highest deodorization efficiency, reducing fishy flavor by 51.6 %, while the Yc0988 (Lactiplantibacillus plantarum (CICC 21809) + Saccharomyces cerevisiae (CICC 32883) co-fermentation with cooked kelp) group showed high flavor acceptability despite a lower composite score. The Yc2913(Limosilactobacillus fermentum (CICC 21829) + Wickerhamomyces anomalus (CICC 33313) co-fermentation with cooked kelp) combination treatment significantly enhanced fermentation aroma and preserved color, with a synergistic effect similar to the aroma-enhancing effects reported by Akomea-Frempong et al. in sugar kelp fermentation (Akomea-Frempong et al., 2021; Gao et al., 2010).

In conclusion, single strains such as Saccharomyces cerevisiae exhibit high deodorization efficiency, whereas composite strains provide greater advantages by synergistically improving both aroma and color, offering a feasible technological pathway for microbial deodorization and flavor optimization of salted kelp.

3.2. Analysis of volatile compounds and investigation of odor mechanisms

3.2.1. Characterization of VOCs and thermal effects on flavor profile

To clarify the mechanism of microbial deodorization in salted kelp, this study combined gas chromatography-ion mobility spectrometry (GC-IMS) with relative odor activity value (ROAV) analysis to characterize volatile organic compounds (VOCs). By referencing odor thresholds (Van Gemert, 2011) and integrating sensory data, the approach enabled accurate assessment of each VOC's contribution to overall aroma. Such an integrative method ensures that both chemical and sensory perspectives are considered, improving the reliability of aroma characterization. Similar strategies have been applied in marine algae research, as demonstrated by Che et al., who validated GC-IMS for distinguishing processing-induced volatile differences in Undaria pinnatifida (Che et al., 2025).

The GC-IMS spectra (Fig. 2A–2E) revealed that both Yraw and Ycook contained diverse VOCs existing as monomers and dimers. The difference plot highlighted that thermal treatment markedly remodeled VOC composition: red regions indicated increased VOCs after heating, whereas blue represented decreased VOCs. These observations indicate that thermal processing not only impacts pigment-based color formation, but also drives significant changes in flavor-related compounds. Strong evidence for this has been provided by study: J. Li et al. found that heat treatment significantly altered the physicochemical properties and volatile flavor substances of Argentine gun squid, with moderate heat treatments promoting the formation of ketones and alcohols, while excessive temperatures led to a reduction in aldehydes (Li, Li, et al., 2024). Together, these studies support the idea that thermal processing plays a pivotal role in shaping the flavor profile of foods by modulating VOC composition. However, such changes were not always favorable, and elevated levels of sulfur-containing compounds and aldehydes upon heating may have exacerbated fishy flavor perceptions, consistent with the results of the sensory evaluation.

Fig. 2.

GC-IMS and multivariate analysis of volatile compounds in raw and cooked salted kelp fermented by different microorganisms: (A)(B)(C) Three-dimensional GC-IMS spectra of volatile compounds. (D)(F)(H) Two-dimensional GC-IMS spectra. (E)(G)(I) Differential two-dimensional spectra. (J)(L) Principal component analysis. (K)(M) Flavor difference analysis based on Euclidean distance. (Yraw:raw salted kelp without microbial inoculation; Yr62 (Pediococcus pentosaceus (CICC 21862), fermented with raw kelp); Yr13 (Wickerhamomyces anomalus (CICC 33313), fermented with raw kelp); Yr2913 (Limosilactobacillus fermentum (CICC 21829) + Wickerhamomyces anomalus (CICC 33313) co-fermentation with raw kelp);

Ycook: cooked salted kelp without microbial inoculation; Yc88 (Saccharomyces cerevisiae (CICC 32883), fermented with cooked kelp); Yc2913(Limosilactobacillus fermentum (CICC 21829) + Wickerhamomyces anomalus (CICC 33313) co-fermentation with cooked kelp).

3.2.2. Microbial fermentation-induced remodeling of volatile aroma

To further investigate fermentation-mediated deodorization, the volatile profiles of raw and cooked fermented kelp were analyzed (Fig. 2). Five representative treatments were selected to ensure both coverage and mechanistic insight. In the raw group, Yr62 was used as a moderate microbial activity reference, while Yr13 and Yr2913 were included for their strong deodorization potential and distinctive volatile transformations. In the cooked group, Yc88 (Saccharomyces cerevisiae) and Yc2913 (Wickerhamomyces anomalus) were chosen due to their pronounced effects on volatiles and sensory traits. These selected treatments enabled comparisons across single-strain vs. co-fermentation and raw vs. cooked matrices, providing insights into how microbial metabolism interacts with processing conditions.

A total of 87 VOCs, including aldehydes, ketones, alcohols, esters, acids, and pyrazines, were identified by VOCal software, many of which were key odor-active compounds. Fig. 2 shows the raw samples (Yraw, Yr62, Yr13, Yr2913) and cooked samples (Ycook, Yc88, Yc2913). Three-dimensional and two-dimensional GC-IMS spectra showed that strain-specific fermentation significantly remodeled the volatile organic compound (VOC) composition of the salted kelp, altering its retention time and ion mobility. Difference plots showed that Yc88 and Yc2913 treatments significantly enhanced ester, alcohol, and ketone signals, suggesting that microbial metabolism may contribute to the formation of pleasurable aroma through esterification and reduction reactions. This change was highly consistent with the results of the sensory evaluation: samples treated with Wickerhamomyces anomalus (Yr13, Yc2913) or Saccharomyces cerevisiae (Yc88) showed a significant decrease in off-flavor compounds - especially 1-octen-3-one - and a significant increase in fruity and floral components (e.g. isopentanol, isopropyl propionate and isovaleric acid). octen-3-one - while the content of fruity and floral components (e.g. isoamyl alcohol, isopropyl propionate, and isovaleraldehyde) was significantly increased. This suggests that microorganisms may improve flavor through a dual mechanism: On the one hand, fishy VOCs are enzymatically degraded through pathways such as alcohol dehydrogenase and carbonyl reductase(Liang et al., 2023; Ma et al., 2023); on the other hand, they are generated through secondary metabolism to produce pleasurable aroma compounds, thus enhancing the overall flavor complexity. These results are in line with previous studies on flavor modulation by yeast in fermented foods. For example, Zou et al. reported that co-fermentation of lactic acid bacteria with yeast in fermented beef–soybean paste enhanced alcohol and ester aromas while suppressing undesirable odorants (Zou et al., 2025).

3.2.3. Mechanistic implications for strain selection and deodorization

The selected strains (Yr62, Yr13, Yr2913, Yc88, Yc2913) were chosen for their distinct deodorization performance in raw versus cooked kelp. Thermal processing alters substrate availability by releasing sugars, amino acids, and polyphenols, making the optimal strains for cooked kelp different from those for raw kelp. Single strains like S. cerevisiae or W. anomalus primarily produce esters and alcohols, enhancing fruity and floral notes, whereas mixed-strain fermentations leverage complementary enzymatic activities to more effectively degrade fishy VOCs and generate desirable aromas. Therefore, the rational selection of strains should consider both kelp processing state and microbial metabolic complementarity to achieve optimal deodorization and flavor enhancement.

In summary, microbial fermentation not only efficiently removes the fishy taste but also generates rich and pleasant aroma through metabolites, providing a solid scientific basis for flavor optimization in salted kelp. GC-IMS coupled with ROAV analyses not only verified the deodorization effect of the selected strains but also revealed their specific functions in modulating the aroma profile of salted kelp. Collectively, these findings support the feasibility of targeted microbial fermentation as an effective strategy for improving the flavor of marine-derived foods. In the future, studies combining metabolomics and enzymatic analyses will help to further elucidate the specific metabolic pathways of VOC transformation and their mechanisms of action.

3.3. Fingerprinting and cluster analysis of volatile compounds in raw and cooked salted kelp before and after fermentation

3.3.1. VOC profiling and characteristic changes in raw and cooked kelp

Volatile organic compounds (VOCs) of raw and cooked salted kelp samples from different fermentation treatments were qualitatively and visually characterized using gas chromatography–ion mobility spectrometry (GC-IMS), and the corresponding fingerprints are presented in Fig. 3A and Fig. 3B. The horizontal axis of the fingerprints indicates the identified compounds, the vertical axis indicates the sample number, and the color shades represent the relative abundance—with lighter colors corresponding to higher concentrations.

Fig. 3.

Effects of different microbial treatments on the composition of volatile flavor compounds (VOCs) in raw and cooked salted kelp. (A) (B) Fingerprints; (C) (D) Heat map of species abundance; (E) (F) Histograms of relative contents of various volatile substances in different treatment groups (Yraw:raw salted kelp without microbial inoculation; Yr62 (Pediococcus pentosaceus (CICC 21862), fermented with raw kelp); Yr13 (Wickerhamomyces anomalus (CICC 33313), fermented with raw kelp); Yr2913 (Limosilactobacillus fermentum (CICC 21829) + Wickerhamomyces anomalus (CICC 33313) co-fermentation with raw kelp);

Ycook: cooked salted kelp without microbial inoculation; Yc88 (Saccharomyces cerevisiae (CICC 32883), fermented with cooked kelp); Yc2913(Limosilactobacillus fermentum (CICC 21829) + Wickerhamomyces anomalus (CICC 33313) co-fermentation with cooked kelp).

In raw salted kelp (Fig. 3A), the focal compound classes include aldehydes (red boxes), ketones (orange/yellow boxes), olefins (green boxes), alcohols (purple boxes), and isovaleraldehyde (black boxes). Aldehydes are usually considered one of the main sources of fishy odor in aquatic products (e.g., hexanal, heptanal, nonanal). In this study, we observed a progressive and consistent decrease in the relative content of aldehydes across all fermentation treatments, suggesting that microbial fermentation may effectively mitigate odor by metabolically decomposing or transforming these compounds. In addition, Dai et al. also emphasized microbial fermentation as an important strategy for the reduction of off-flavors in aquatic products (Dai et al., 2024), supporting our inference that microbes are directly involved in aldehyde degradation. Ketones such as 1-octen-3-one and 1-penten-3-one—both associated with earthy and fishy flavors—decreased significantly in yeast-fermented samples, consistent with the sensory improvement observed. Olefins such as hexenal and acrolein were significantly increased in the Yr62 group, conferring grassy and spicy odors; however, ROAV analysis may underestimate the contribution of acrolein due to its very low odor threshold (Van Gemert, 2011). Alcohols such as n-hexanol and isoamyl alcohol increased markedly in yeast-fermented samples, enriching fruity and alcoholic aroma profiles. Notably, isovaleraldehyde accumulated mainly in Yr13 and Yr2913, adding both flavor complexity and a characteristic chocolate-like note.

A similar trend was observed in cooked kelp, where Yc88 and Yc2913 showed considerable decreases in fishy ketones and increases in alcohols and desirable aldehydes, indicating that microbial metabolism was effective in deodorization. Meanwhile, alcohols such as isoamyl alcohol, n-butanol, isobutanol, and n-hexanol increased significantly, contributing fruity and alcoholic notes to the samples. Aldehydes such as isobutyraldehyde, 2-methylbutyraldehyde, and isovaleraldehyde—associated with banana, malt, and chocolate aromas—also accumulated significantly, highlighting the central role of yeast strains (Saccharomyces cerevisiae and Wickerhamomyces anomalus) in aroma formation. This is consistent with, C. Li et al. who showed that co-fermentation of lactic acid bacteria with yeast improved flavor by enzymatically degrading fishy compounds while increasing alcohol and ester abundance in fermented snapper surimi (Li, Cui, et al., 2024).

3.3.2. Cluster analysis and classification of VOC transformations

Based on VOC fingerprinting combined with hierarchical clustering and chemical classification analyses, the results are illustrated in (Fig. 3C -3F), microbial fermentation significantly reshaped the volatile compound profiles of salted kelp. In raw kelp, the unfermented control group (Yraw) was clearly separated from all fermentation groups, with Yr13 clustering closely with Yr2913. This indicates that the combined action of lactic acid bacteria and yeast led to similar flavor modulation effects. This finding is consistent with the strain complementation reported by Viesser et al. in cocoa bean fermentation, where yeast primarily produces alcohols and esters while lactic acid bacteria produce organic acids, together forming a complex flavor matrix (Viesser et al., 2021). Quantitative analysis of VOC categories further showed a shift from aldehyde-dominated flavors to ketone-, alcohol-, ester-, and furan-rich profiles in post-fermentation samples, with the Yr2913 group showing the highest relative abundance of alcohols and esters, indicating the strongest flavor-enhancing capacity. These observations align well with Sun et al.,who demonstrated that co-inoculation of Saccharomyces cerevisiae and Lactobacillus significantly increased alcohol and ester levels in oat fermentation (Sun et al., 2024).

Hierarchical clustering of cooked kelp VOCs (Fig. 3D) further showed that fermentation groups were distinctly separated from the control, especially in the aldehyde and ester regions, suggesting that microbial fermentation had a strong modulating effect on VOC composition. The elevated contents of octanal and isovaleraldehyde in Yc2913 may result from synergistic interactions between heat-induced lipid oxidation and microbial metabolism. Fang et al. similarly observed that mixed fermentation of lactic acid bacteria and yeast in sourdough systems markedly altered the abundance of aldehydes and esters, which was attributed to the metabolic interactions and enzymatic activities between the strains (Fang et al., 2023). The VOC chemical classification analysis (Fig. 3F) revealed that esters and alcohols increased significantly while ketones and some aldehydes decreased in Yc88 and Yc2913, further confirming that microbial fermentation not only degraded fishy compounds but also generated pleasant aroma molecules. In particular, Yc2913 displayed a balanced aldehyde-to-ester ratio, which was consistent with ROAV analysis and correlated with superior aroma quality, emphasizing the critical role of LAB–yeast synergy in flavor enhancement.

3.4. Identification of key aroma active compounds based on ROAV analysis

In order to clarify the contribution of individual volatile compounds to the overall aroma profile of fermented salted kelp, relative odor activity values (ROAV) were analyzed in this study for raw samples (Yraw), heat-treated samples (Ycook), and their fermentation products (Yr62, Yr13, Yr2913, Yc88, and Yc2913). More than 80 volatile organic compounds (VOCs), including aldehydes, ketones, alcohols, esters, and furans, were identified as shown in Table 4, Table 5 (see Tables S2, S3 and S4 for the complete list of compounds).

Table 4.

Comparative Analysis of Characteristic Flavor Compounds in Unfermented Versus Fermented Fresh Salted Kelp.

Compound Name	Odor Description	Relative Content				Threshold(μg/kg)	ROAV
		Yraw	Yr62	Yr13	Yr2913		Yraw	Yr62	Yr13	Yr2913
Aldehydes
Decenal	Sweet orange-like aroma with fatty and waxy undertones	0.32 %	0.46 %	0.42 %	0.46 %	3.1	1.05	1.72	2.52	3.35
2,6-Nonadienal	Intense cucumber-like odor	0.51 %	0.38 %	0.28 %	0.29 %	3.9	1.32	1.13	1.33	1.65
(E)-2-Nonenal	Fatty, green, waxy, cucumber- and melon-like aroma	1.55 %	0.65 %	0.63 %	0.77 %	1.1	14.33	6.89	10.81	15.61
Isovaleraldehyde	Chocolate-like and fatty scent	0.12 %	0.16 %	1.31 %	1.45 %	0.35	3.37	5.34	70.33	92.63
Propionaldehyde	Pungent and grassy odor	6.09 %	6.32 %	5.78 %	6.14 %	36	1.72	2.05	3.02	3.81
Ketones
1-Octen-3-one (Monomer)	Strong earthy, mushroom-like, and vegetal aroma, with faint notes of fish and chicken	1.18 %	1.03 %	0.64 %	0.54 %	0.12	100.00	100.00	100.00	100.00
1-Octen-3-one (Dimer)	Strong earthy, mushroom-like, and vegetal aroma, with faint notes of fish and chicken	0.20 %	0.15 %	0.09 %	0.07 %	0.12	17.38	14.87	13.62	13.25
1-Penten-3-one (Monomer)	Strong pungent odor	1.50 %	1.54 %	1.12 %	0.91 %	1	15.24	18.04	21.12	20.29
1-Penten-3-one (Dimer)	Strong pungent odor	2.53 %	2.17 %	1.50 %	0.32 %	1	25.76	25.39	28.18	7.08
Alcohols
1-Octen-3-ol	Mushroom-like aroma with hints of lavender, rose, and hay	0.37 %	0.51 %	0.47 %	0.35 %	2.7	1.38	2.23	3.28	2.92
Acetals
Diethyl acetal (Ethylal)	Floral and fruity fragrance	0.55 %	0.56 %	0.36 %	0.43 %	4.9	1.15	1.33	1.38	1.94

Table 5.

Comparative analysis of common characteristic flavor compounds of unfermented and fermented cooked salted kelp.

Compound Name	Odor Description	Relative Content			Threshold(μg/kg)	ROAV
		Ycook	Yc88	Yc2913		Ycook	Yc88	Yc2913
Aldehydes
Decenal	Sweet orange-like aroma with fatty and waxy undertones	0.50 %	0.50 %	0.49 %	3.1	2.56	3.17	2.35
2,6-Nonadienal	Intense cucumber-like odor	0.29 %	0.36 %	0.35 %	3.9	1.16	1.80	1.34
(E)-2-Nonenal	Fatty, green, waxy, cucumber- and melon-like aroma	0.67 %	0.53 %	0.65 %	1.1	9.57	9.41	8.78
Isovaleraldehyde	Chocolate-like and fatty scent	0.10 %	1.78 %	1.14 %	0.35	4.56	100.00	48.28
Propionaldehyde	Pungent and grassy odor	6.94 %	6.09 %	6.53 %	36	3.04	3.32	2.70
Ketones
1-Octen-3-one (Monomer)	Strong earthy, mushroom-like, and vegetal aroma, with faint notes of fish and chicken	0.76 %	0.59 %	0.81 %	0.12	100.00	96.18	100.00
1-Octen-3-one (Dimer)	Strong earthy, mushroom-like, and vegetal aroma, with faint notes of fish and chicken	0.09 %	0.07 %	0.11 %	0.12	12.40	11.30	13.48
1-Penten-3-one (Monomer)	Strong pungent odor	1.51 %	1.19 %	1.57 %	1	23.82	23.43	23.37
1-Penten-3-one (Dimer)	Strong pungent odor	1.25 %	0.89 %	1.86 %	1	19.71	17.55	27.68
Alcohols
1-Octen-3-ol	Mushroom-like aroma with hints of lavender, rose, and hay	0.48 %	0.39 %	0.42 %	2.7	2.79	2.82	2.31
Acetals
Diethyl acetal (Ethylal)	Floral and fruity fragrance	0.76 %	0.51 %	0.52 %	4.9	2.46	2.04	1.57

In raw salted kelp and its fermentation products, 1-octen-3-one consistently dominated the ROAV analysis (ROAV = 100) due to its earthy and mushroomy flavor characteristics, suggesting a central role in the base aroma. However, fermentation treatment significantly reduced the actual concentration of the compound, indicating that yeast strains, particularly Saccharomyces cerevisiae, were capable of partially degrading or transforming 1-octen-3-one. This metabolic activity effectively reduced the fishy flavor and improved sensory quality. This is consistent with the study of Dai et al. (Dai et al., 2024), who pointed out that the modulation of key fishy VOCs through microbial metabolism is an effective strategy to reduce the off-flavors of aquatic products. Therefore, yeast fermentation not only suppresses dominant fishy compounds but also generates new aroma-active metabolites, thereby reshaping the overall flavor structure of salted kelp.

In the present study, a significant increase in 3-octanone together with hexanal was observed in fermentation groups Yr13 and Yr2913, highlighting the role of strain specificity in the remodeling of the aroma profile. Xie et al. also confirmed that 1-octen-3-one was the key aroma-active compound in button mushroom, highlighting that specific volatiles can play a dominant role in shaping overall flavor perception. This observation parallels our findings, where 1-octen-3-one was also identified as a crucial contributor to fishy odor (Xie et al., 2024). In contrast, the VOC composition of Yr62 closely resembled that of Yraw, indicating its limited metabolic transformation capacity. The additional accumulation of 3-octanone (musty/keto aroma) and hexanal (grassy/green aroma) in Yr13 and Yr2913 was highly consistent with sensory evaluation results, reinforcing the role of strain specificity in regulating aroma. Similarly, Sun et al. (Sun et al., 2024) also showed that inoculation with specific strains dynamically altered VOC composition, conferring unique aroma characteristics. Collectively, these studies, together with the present results, emphasize the pivotal role of microbial metabolism in the regulation of fermented food flavors.

Notably, the concentration and ROAV of isovaleraldehyde (chocolate/fat aroma) increased significantly after fermentation: 1.31 % and 1.45 % in Yr13 and Yr2913 compared to 0.12 % in Yraw, corresponding to ROAVs of 70.33 and 92.63. This finding suggests that isovaleraldehyde became a major positive contributor to aroma optimization during fermentation. Meanwhile, auxiliary volatiles such as 1-hexanol (green aroma), isopropyl propionate (fruity/pear aroma), and 2-pentylfuran (vegetable aroma) showed ROAVs >1, indicating their role in enriching the aroma hierarchy.

In heat-treated samples (Ycook), lipid oxidation products such as octanal and nonanal became the main features of the volatile profile, consistent with previous reports that cooking promotes thermal degradation of fatty acids, thereby generating aldehydes. For example, Hu et al. (Hu et al., 2022) confirmed lipid oxidation as the main source of these aldehydes during baking. Although 1-octen-3-one remained the dominant aroma contributor, the production of isovaleraldehyde (fruity aroma) and hexanal (grassy aroma) increased significantly in fermentation samples Yc88 and Yc2913. The synergistic accumulation of these compounds in Yc2913 indicated that microbial metabolism reinforced the remodeling of the aroma profile, particularly by balancing fishy and fruity notes.

In this study, isovaleraldehyde (3-methylbutanal) was significantly enriched in both raw and cooked fermented kelp, highlighting its central role in microbial aroma formation. This compound is mainly derived from the metabolism of branched-chain amino acids (e.g., valine, leucine) or the conversion of glycolytic intermediates (e.g., α-ketoglutarate). Yeast strains such as S. cerevisiae can generate isovaleraldehyde from these precursors and further esterify it to ethyl isovalerate, which imparts a sweet banana aroma. For example, Y. Huang et al. reported a high content of isovaleraldehyde in milk products, suggesting its important role in dairy flavor formation (Huang et al., 2024). These studies strongly support the present findings, confirming isovaleraldehyde as a key aroma-active compound in microbial fermentation. Its enrichment in kelp fermentation may be closely linked to the metabolic traits of specific yeast strains, providing new insights into the diversity and complexity of kelp flavors.

3.5. Multivariate flavor difference analysis: PCA with Euclidean distance clustering

In this study, multivariate analysis of volatile organic compounds (VOCs) of salted kelp under different fermentation conditions was performed using gas chromatography-ion mobility spectrometry (GC-IMS), including principal component analysis (PCA) and Euclidean distance clustering (Fig. 2 J-M). The results showed that the unfermented control group (Yraw) and the inefficient strain group (Yr62) clustered close to each other on the PC1 axis, suggesting a similar composition of VOCs and limited transformation. In contrast, samples fermented by Wickerhamomyces anomalus (Yr13) alone and co-fermented by Lactobacillus fermentum and W. anomalus (Yr2913) were significantly shifted in the PCA plot, reflecting strain-specific modulation of VOCs. This pattern was consistent with the sensory assessment (Fig. 1B), where Yr13 and Yr2913 together significantly improved the aroma profile.

Further analysis of the Euclidean distance thermogram (Fig. 2K) showed a clear separation of Yraw from Yr13 and Yr2913, whereas the separation between Yr13 and Yr2913 suggests that more extensive metabolic remodeling occurs during the co-fermentation process. Additionally, the proximity of Yr62 to Yraw confirms that it has a limited deodorizing effect. These results suggest that co-fermentation can increase the complexity of VOCs through microbial interactions, a finding that is consistent with previous studies. For example, Fang et al. showed that mixed fermentation improved the sourdough flavor profile, further supporting the conclusion that co-fermentation enhances VOC complexity and flavor(Fang et al., 2023).

In addition, Liang et al. found that co-fermentation of S. cerevisiae with L. paracasei significantly reduced off-flavors by altering the VOC composition in lobster (Gracilaria lemaneiformis) (Liang et al., 2023). This further supports the potential of co-fermentation for improving kelp flavor.

In summary, single-strain fermentation (Yr13) was suitable for generating specific metabolites and improving aroma, whereas co-fermentation of lactobacilli and yeast (Yr2913) optimized kelp flavor by remodeling VOC structure and increasing aroma complexity through inter-strain interactions. These observations align with existing fermented food studies, which indicate that mixed-strain fermentation can generate diverse active aroma compounds through metabolic complementation.

The limited deodorization effect of Yr62 in Yraw may stem from its inadequate regulation of metabolic pathways related to VOCs. These pathways are influenced by microbial characteristics, fermentation conditions, and substrate composition. S. Liu et al. reviewed the effects of microbial fermentation on the structure and bioactivity of polysaccharides in plant foods, indicating that microbial metabolic activities during fermentation may degrade polysaccharides by secreting sugar-active enzymes (e.g., glycoside hydrolases), which may decrease molecular weights and affect bioactivity (Liu et al., 2024). However, this review focused on polysaccharides and did not address microbial regulation of VOC metabolic pathways. Therefore, the limited deodorization effect of Yr62 and Yraw may be related to insufficient regulation of VOC metabolism.

In the heat-treated samples (Fig. 2L), PCA analysis showed that PC1 and PC2 together explained 84 % of the total variance, effectively separating kelp samples from different fermentation treatments. Among them, the S. cerevisiae alone fermentation (Yc88) and co-fermentation groups (Yc2913) formed distinctly separated clusters from the heated control group (Ycook), suggesting that fermentation substantially reshaped the VOC composition. Euclidean distance clustering (Fig. 2M) further confirmed the differences among Yc88, Yc2913, and Ycook.

Regarding key odor-active VOCs, the earthy/fishy odor markers 1-octen-3-one and hexanal were elevated in Yraw for the raw kelp samples, but were significantly reduced by fermentation. Notably, elevated levels of isovaleraldehyde (ROAV = 48.28) and hexanal in Yr2913 provided a base for sweet and fruity aromas, effectively masking undesirable odors, while Yr13 showed increased levels of isoamyl alcohol and n-butanol, enhancing fruity and wine-like aromas, consistent with sensory results. In cooked kelp samples, heat treatment resulted in lipid oxidation and elevated aldehydes (e.g., octanal), creating off-flavors. Fermentation, especially with S. cerevisiae, increased isovaleraldehyde content and decreased 1-octen-3-one, resulting in improved sensory quality.

These results are consistent with previous studies showing that yeast can convert lipid-derived intermediates into esters and aldehydes to enhance aroma complexity. For example, Tian et al. found that Saccharomyces cerevisiae regulates aroma component production through metabolic diversity in dairy fermentation (Tian et al., 2023).

In summary, multivariate and compound-level analyses (Fig. 2J-M) indicate that microbial strain selection and thermal pretreatment synergistically shape the VOC profile of salted kelp. Importantly, co-fermentation provides a targeted and sustainable strategy to reduce off-flavors and enhance flavor, highlighting its potential for application in value-added seaweed product development.

3.6. Effect of fermentation conditions on the texture of kelp

In order to assess the effect of fermentation on the textural and sensory properties of salted kelp, key textural parameters (hardness, elasticity, chewiness, etc.) of raw and cooked kelp samples were determined in this study (Table 6). The results showed that fermentation significantly reduced hardness and elasticity, while improvements in aroma partially compensated for the sensory discomfort caused by textural softening. In the raw kelp group, the Yr13 treatment showed the lowest hardness (55.25 N), indicating significant degradation of kelp tissues; nevertheless, it had the best deodorization effect, which was closely associated with the substantial reduction of 1-octen-3-one and enrichment of isovaleraldehyde. These results are consistent with previous studies. Liang et al. found that co-fermentation of S. cerevisiae and L. paracasei significantly improved the organoleptic qualities of Lungwort (Gracilaria lemaneiformis) by reducing off-flavors through altering VOC composition, indirectly affecting the product's taste (Liang et al., 2023). Furthermore, S. Liu et al. reviewed that microbial fermentation can alter food texture and optimize flavor profiles through degradation of phytopolysaccharides and modulation of metabolically active enzymes, providing mechanistic explanations for structural and flavor regulation in fermentation of plant-based foods (Liu et al., 2024).

Table 6.

Effects of fermentation conditions on the texture, color, and chlorophyll content of kelp.

Groups	Texture					Color						Chlorophyll Content
	Hardness (N)	cohesion	resilient (mm)	Adhesive (N)	chewability (mJ)	L*	a*	b*	Hue	ΔE*	Δa*	Chlorophyll a (mg/kg)	Chlorophyll c (mg/kg)	Chlorophyll c (mg/kg)	Elimination rate of Chlorophyll a (%)	Elimination rate of Chlorophyll c (%)	Elimination rate of Chlorophyll a + c (%)
Yraw	58.15 ±0.78a	0.95 ±0.04a	0.58 ±0.04a	55.12 ± 1.94a	32.01 ± 1.80a	33.51 ±0.96b	−2.49 ±0.24a	5.21 ±0.70a	115.71 ±3.10a	–	–	25.64 ±2.98a	8.78 ±1.36a	34.42 ±3.39a	–	–	–
Yr62	56.03 ±1.66ab	0.93 ±0.04a	0.43 ±0.05b	52.04 ±2.94ab	22.08 ± 1.62b	34.69 ±0.38b	−1.21 ±0.78b	4.51 ±0.93a	103.61 ±7.56b	2.15 ±0.74a	1.28 ±0.78b	19.14 ±0.26b	3.95 ±0.83d	23.09 ±1.03 b	24.44 ±8.89bc	54.50 ±9.66a	32.32 ±7.53ab
Yr13	55.25 ± 2.64b	0.88 ±0.06a	0.47 ±0.09b	48.78 ± 3.78b	22.88 ± 4.98b	37.65 ±1.67a	2.20 ±0.59c	6.66 ±0.75b	71.62± 5.25c	6.53 ±1.50c	4.68 ±0.59c	12.70 ±2.28cd	6.85 ±1.51bc	19.56 ±3.57b	50.14 ±9.37a	19.00 ±25.92a	42.73 ±12.25a
Yr2913	57.55 ±2.06ab	0.92 ±0.10a	0.47 ±0.06b	52.73 ±6.49ab	24.84 ± 5.16b	36.57 ±2.61a	−2.02 ±0.47a	6.97 ±1.10b	106.04 ±2.46b	3.83 ±2.51b	0.47 ±0.47a	14.93 ±2.39c	5.81 ±1.09c	20.73 ±1.74b	41.07 ±11.91ab	31.25 ±23.63a	39.27 ±7.51a
Ycook	65.23 ± 5.51a	0.91 ±0.10a	0.57 ±0.04ab	59.28 ± 6.75a	33.29 ± 2.41a	34.92 ±1.04ab	−1.47 ±0.20a	5.59 ±0.65b	104.88 ±2.77a	–	–	10.61 ±0.63de	2.61 ±0.94d	13.21 ±1.31c	–	–	–
Yc88	65.82 ± 5.14a	0.73 ±0.06b	0.58 ±0.05a	48.46 ± 7.23b	27.76 ± 4.21b	33.77 ±1.06b	0.40 ±0.29b	3.97 ±0.29a	84.21 ±4.21b	2.89 ±0.60a	1.87 ±0.29b	9.84 ±2.13e	3.18 ±1.24d	13.02 ±3.34c	7.42 ±18.81c	−26.82 ±45.62a	2.32 ±20.51b
Yc2913	63.08 ± 3.51a	0.86 ±0.05a	0.52 ±0.03b	54.15 ±2.69ab	28.32 ± 2.91b	36.47 ±2.63a	−1.53 ±0.97a	7.22 ±1.24c	101.12 ±8.03a	3.70 ±0.90b	−0.06 ±0.97a	24.78 ±44.49a	8.01 ±2.79ab	32.79 ±7.14a	−134.44 ±45.14d	−285.64 ±285.96b	−152.88 ±73.14c

Note: The same lowercase letter in the same column indicates no significant difference (p > 0.05), whereas different lowercase letters indicate significant differences (p < 0.05). As significance was evaluated across both raw and cooked kelp together, a unified lowercase letter system (a–c) was applied rather than separate uppercase/lowercase labeling.

In summary, single-organism fermentation (Yr13) was able to produce specific metabolites and improve aroma, whereas lactic acid bacteria co-fermented with yeast (Yr2913) remodeled VOC structure and increased aroma complexity through inter-strain interactions, accompanied by moderate textural softening. This balance of aroma enhancement and textural changes provides a viable strategy for flavor optimization of salted kelp and supports its application in value-added kelp product development.

In the cooked kelp group, the Yc88 samples maintained good elasticity and chewiness, although cohesion decreased to 0.73. Their significant accumulation of isovaleraldehyde (ROAV = 92.63) enhanced the sweet and fruity aroma, further improving sensory acceptability. It is worth noting that alcohols and aromatic aldehydes not only contribute to aroma, but may also affect textural properties through interactions with cell wall polysaccharides, proteins, and other components. For example, Y. Zhou et al. found that white ginseng extract improved the textural and flavor properties of roasted chicken through interactions with proteins and polysaccharides (Zhou, Guan, et al., 2022). These findings suggest a potential synergistic effect between flavor enhancement and textural modulation during fermentation, in which accumulation of key volatiles such as isovaleraldehyde can mitigate adverse sensory effects caused by textural changes.

3.7. Effect of fermentation conditions on the color of kelp

In this study, the effect of microbial fermentation on the color of salted kelp was evaluated based on the analysis of Δa value, which reflects the reduction in green color and reddening caused by chlorophyll degradation. In the raw kelp samples (Table 6), all fermentation treatments significantly increased the Δa value, with the largest increase observed in Yr13, suggesting that chlorophyll degradation was most pronounced and accompanied by noticeable reddening, which was consistent with the decrease in sensory color scores.

In cooked kelp, the Δa value of Yc88 changed from negative to positive, indicating a significant decrease in greenness. However, sensory evaluation did not show a corresponding deterioration, likely due to the inherent instability of the raw pigment or the small magnitude of color change, detectable only instrumentally. Xiao et al. also found that even with significant Δa value changes, human visual sensitivity to subtle color differences remains limited (Xiao et al., 2022).

At the mechanistic level, different microorganisms exhibited differential roles in pigment degradation and transformation. During yeast fermentation, large CO₂ production and a rapid pH decrease can accelerate magnesium loss from chlorophyll, resulting in the formation of olive-green demagnesium chlorophytin, which may further decompose under prolonged fermentation, leading to a rapid increase in Δa value and a distinct “green-to-red” phenomenon. This aligns with Stévant et al. (Stévant et al., 2025), who observed accelerated magnesium loss and rapid pH decrease during fermentation. In contrast, the Δa values in lactobacilli-treated samples showed smaller increases, possibly due to milder metabolism, and did not significantly induce chlorophyll conversion to pheophytin. Direct evidence for chlorophyll stabilization by Lactobacilli remains lacking, requiring further verification.

Overall, color changes induced by microbial fermentation were mainly influenced by strain type, fermentation conditions, and pH dynamics. Yeast was more likely to induce pronounced chlorophyll degradation and reddening, whereas lactic acid bacteria may delay color change. This suggests that precise regulation of kelp appearance can be achieved through strain selection and process optimization.

Although studies directly exploring fermentation-pigment interactions are limited, it has been reviewed that microbial metabolism and secreted enzymes (oxidoreductases, transferases, hydrolases) may influence pigment conversion pathways. F. Wang et al. emphasized that differential microbial metabolism not only shapes flavor but also affects cosmetic qualities of fermented foods(Wang, Wang, et al., 2025).

In summary, pigment stability and conversion during kelp fermentation depend not only on single-strain metabolism but also on the synergistic and enzymatic networks of multiple microorganisms, providing theoretical support and practical guidance for enhancing kelp appearance through targeted fermentation.

3.8. Effect of fermentation conditions on chlorophyll content

To assess the effect of microbial fermentation on the pigment composition of salted kelp, chlorophyll a and c contents and their removal rates were determined for raw and cooked kelp (Table 6). The results showed that fermentation significantly affected pigment stability (p < 0.05). In the raw kelp group, Yr13 had the lowest chlorophyll a content (0.013 mg/kg) and the highest overall pigment loss, likely due to prolonged W. anomalus fermentation. The highest chlorophyll c removal rate (54.5 %, p < 0.01) was observed in Yr62, suggesting that short-term fermentation can induce chlorophyll c degradation, consistent with (Stévant et al., 2025).

In the cooked kelp group, co-fermentation of lactic acid bacteria with W. anomalus (Yc2913) significantly increased chlorophyll a and c contents by 127.3 % and 166.7 %, respectively (p < 0.01), potentially due to the protective effects of microbial metabolites. The negative chlorophyll c removal rate of Yc88 (−26.8 %), although not statistically significant, reflects pigment instability. Akomea-Frempong et al. also observed significant differences in pigment stabilization among microbial treatments, consistent with our findings (Akomea-Frempong et al., 2021).

Correlation analysis indicated that chlorophyll a degradation was the main driver of total pigment loss (r = 0.93, p < 0.01). Xiong et al. confirmed that chlorophyll a degradation dominates total pigment loss, in agreement with our results (Xiong et al., 2024).

Overall, selecting suitable strains effectively slowed pigment degradation and improved seaweed color. However, increasing pigment retention alone (e.g., Yc2913) does not necessarily improve sensory perception. Therefore, simultaneous optimization of color and flavor is critical for product quality.

4. Conclusions

These findings identify 1-octen-3-one as the primary molecular marker of fishy odor in salted kelp, with microbial fermentation by Wickerhamomyces anomalus and Saccharomyces cerevisiae significantly reducing its concentration. Based on previous reports, it is inferred that the metabolic activity of these strains may convert unsaturated ketones and aldehydes into more acceptable volatiles (e.g., isovaleraldehyde, isopentanol, ethyl esters) through oxidative cleavage, reduction, or esterification. Based on this, a dual deodorization mechanism is inferred: (i) enzymatic degradation and masking of off-flavors, and (ii) secondary metabolism-driven formation of pleasant aroma compounds

Building on this mechanism, clear strain-specific differences emerged between single-strain and co-fermentation. While single strains such as S. cerevisiae or W. anomalus primarily enhanced fruity and floral attributes via alcohol and ester production, co-fermentation leveraged complementary enzymatic activities, leading to more efficient off-odor removal and a broader spectrum of desirable volatiles. This synergistic advantage underscores the potential of co-fermentation as a targeted strategy for flavor optimization in complex matrices like kelp. To establish the mechanistic basis of these effects, future research should integrate metabolomics, transcriptomics, and enzyme activity assays to identify catalytic nodes and regulatory pathways, thereby guiding the rational design of microbial deodorization strategies for kelp and other marine-derived foods.

CRediT authorship contribution statement

Fangjie Cao: Writing – original draft, Methodology, Conceptualization. Xinyi Che: Validation, Software. Xingyu Liu: Software. Tingmei Yan: Validation. Yutong Li: Writing – review & editing. Shu Liu: Supervision. Yichao Ma: Visualization, Investigation. Dandan Ren: Funding acquisition. Hui Zhou: Investigation. Qiukuan Wang: Validation. Yunhai He: Writing – review & editing, Methodology, Conceptualization. Han Zhang: Writing – review & editing, Methodology.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary material 1

Data availability

The data that has been used is confidential.