Screening of a Novel Lactiplantibacillus plantarum MMB-05 and Lacticaseibacillus casei Fermented Sandwich Seaweed Scraps: Chemical Composition, In Vitro Antioxidant, and Volatile Compounds Analysis by GC-IMS

Lactic acid fermentation is a promising method for developing sandwich seaweed scraps. The objectives of this study were to investigate the effect of fermentation with Lactiplantibacillus plantarum MMB-05, Lactiplantibacillus casei FJAT-7928, mixed bacteria (1:1, v/v) and control on the physicochemical indexes, in vitro antioxidant activity, and volatile compounds of Porphyra yezoensis sauce. Sensory evaluation was also performed. The results indicated that all lactic acid bacteria strains grew well in P. yezoensis sauce after 72 h of fermentation, with the viable cell counts of L. plantarum MMB-05 exceeding 10.0 log CFU/mL, the total phenolic content increasing by 16.54%, and the lactic acid content increasing from 0 to 44.38 ± 0.11 mg/mL. Moreover, the metabolism of these strains significantly increased the content of umami, sweet and sour free amino acids in P. yezoensis sauce. The total antioxidant capacity of L. plantarum MMB-05, L. casei FJAT-7928, mix and control groups increased by 594.59%, 386.49%, 410.27%, and 287.62%, respectively. Gas chromatography-ion mobility spectrometry (GC-IMS) analysis suggested that aldehydes and ketones accounted for the largest proportion, and the relative contents of acids and alcohols in P. yezoensis sauce increased significantly after lactic acid bacteria fermentation. In addition, the analysis of dynamic principal component analysis (PCA) and fingerprinting showed that the volatile components of the four treatment methods could be significantly distinguished. Overall, the L. plantarum MMB-05 could be recommended as an appropriate starter for fermentation of sandwich seaweed scraps, which provides a fundamental knowledge for the utilization of sandwiched seaweed scraps.


Introduction
With the improvement of people's living standards, consumers are gradually inclined to choose convenient, nutritious, and functional foods, and pay more attention to the quality of food [1]. Sandwiched seaweed is one of the very popular seafood products with high commercial prospects. It is mainly made of Porphyra yezoensis, with crisp and fragrant product characteristics, and is a new product in recent years. Laver is a kind of important cultivated red seaweed with high nutritional value and biotechnology potential. It is rich in protein, carbohydrates, amino acids, and polyphenols, which mainly grown in China, Japan, and South Korea, and had an annual harvest of 0.222 million tons in 2020 [2][3][4]. Among them, P. yezoensis is one of the most economical seaweed species in China [5].

Determination of Growth Value and pH Value of Strains
The preserved L. plantarum MMB-05 was inoculated in 10 mL MRS liquid medium. It was incubated at 37 • C and inoculated in fresh MRS liquid medium with 1% inoculation for 72 h, and sampled regularly. The OD value was measured at 600 nm, and pH value of the strain was recorded.
The antibacterial activity was determined by well diffusion agar method. Escherichia coli CICC 10,003, Staphylococcus aureus CICC 23,656, and Bacillus cereus CICC 23,828 were used as indicator bacteria. The isolated L. plantarum MMB-05 was inoculated in MRS liquid medium, cultured at 37 • C for 48 h, centrifuged at 11,860× g for 15 min, then the supernatant was taken and stored at 4 • C for later use. The indicated bacteria were inoculated in the solid medium with 1% inoculation, drilled with a sterile punch, and 50 µL supernatant was added to each hole. After incubation at 37 • C for 12-16 h, the size of the inhibition zone was measured. The diameter (mm) of the inhibition zone was measured with a vernier caliper, indicating the antibacterial activity of the fermentation broth.

Preparation of P. yezoensis Sauces and Fermentation
The preserved L. plantarum MMB-05 and L. casei FJAT-7928 were inoculated into MRS liquid medium in glycerol tube, and after two subliminations the culture reached logarithmic stage. Meanwhile, the mixed bacteria (1:1, v/v) and the control group (without lactic acid bacteria) were set to perform the fermentation under the same conditions. We accurately weighed 100 g of dried seaweed sandwich and added 150 mL of distilled water to form a paste. The mixture was autoclaved at 121 • C for 30 min. After cooling to room temperature, 7.5 mL of bacterial solution was added to the P. yezoensis sauce for fermentation [22]. The fermentation temperature was set at 37 • C, and the static fermentation time was 0, 12, 24, 48, and 72 h, respectively.

Extraction of Supernatant
The supernatant of P. yezoensis sauce was extracted by the water extraction method after fermentation [25]. The sample was accurately weighed, diluted, and mixed with 50 mL deionized water. The magnetic stirrer was stirred for 30 min and centrifuged at 14,130× g for 15 min. This process was repeated twice under the same conditions, and the precipitation was discarded to obtain the supernatant.

Sensory Evaluation
Sensory evaluation was used to compare the differences in various fermented P. yezoensis sauces, which was performed in standard sensory laboratories. The four groups of P. yezoensis sauce fermented for 24, 48, and 72 h, were evaluated by 30 public officials (12 women and 18 men, aged [21][22][23][24][25]. None of them had a taste disturbance, and each participant had at least six months of sensory experiments. Nose clips were worn throughout the sensory process to prevent potential flavor and odor interactions. Each sensory assessor scored the samples based on their fishy taste, sour taste, bitter taste, aroma, salty taste, color, and overall quality of the samples, on a rating scale of 1-9 points where 1 represents the worst quality, and 9 the highest [26].

Determination of Basic Physicochemical Indexes
A digital viscometer (NDJ-5S, Shanghai Fangrui Instrument Co., LTD, Shanghai, China) was used for viscosity measurement. Approximately 100 g P. yezoensis sauce was taken and stir with a rotor at a speed of 60 r/min for 20 min. A water activity meter (HD-6, Wuxi Huako Instrument Co., LTD, Wuxi, China) was used to measure the water activity (a w ) of the P. yezoensis sauce. The color difference of the P. yezoensis sauce was determined by a color meter (Shanghai Yi Electrophysical Optical Instrument Co., LTD, Shanghai, China).
The total viable counts were tested according to a previous procedure described by Du et al. [23]. The cell counts were expressed as colony numbers (Log CFU/mL). The precision pH meter (PHS-3C, Lei Ci Instrument Factory, Shanghai, China) was used for measurement. Briefly, 5 g of P. yezoensis sauce was taken, diluted in 10 mL of distilled water, and then measured after mixing. The total sugar content was determined according to GB/T9695. , and the content was expressed as glucose equivalent.

Total Phenolic and Total Flavonoids Contents
The total phenolic content was determined by a modified Folin-Ciocalteu method, according to the procedure reported by Sowmya et al. [27]. Take 1 mL of the fermentation supernatant into a test tube, add 1 mL of folin phenol reagent and mix thoroughly, allow to stand for 5 min. Then, add 2 mL of 7.5% Na 2 CO 3 solution, let this stand for 5 min, take a water bath at 45 • C for 10 min, and the absorbance was then measured at a wavelength of 765 nm. The total phenol content was expressed as the equivalent (mg GEA/g DW) of gallic acid per mL of fermentation supernatant.
The content of total flavonoids was assessed spectrophotometrically according to the method proposed by Kim et al. with a slight modification [28]. Two mL of fermentation supernatant was taken in a test tube and neutralized by 0.6 mL of 10% AlCl 3 , then it was mixed and allowed to stand for 6 min. 4 mL of NaOH solution was absorbed and allowed to stand for 15 min. The absorbance values were measured at the wavelength of 510 nm with rutin as the standard. The total flavonoids content was expressed as equivalent (mg RE/g DW) of rutin per mL of fermentation supernatant.

Determination of Organic acid Content and Antioxidant Activities
The organic acid profile was analyzed on the Shimadzu LC-2010A system (Shimadzu, Tokyo, Japan) following a previously described method, with minor modifications [29]. The chromatographic column was Agilent TC-C18 (4.6 × 25 mm, 5 µm) with the detection wavelength of 210 nm. Isogradient elution was performed on 0.08 M KH 2 PO 4 solution (pH 2.5). The column temperature, flow rate, and injection volume, were 30 • C, 0.7 mL/min and 20 µL.

Determination of Free Amino Acid Content
Free amino acids were quantified according to the method by Adeyeye et al. [30] with minor modification. The P. yezoensis sauce (2.0 g) was homogenized for 5 min (3 × 1000 rpm) with 15 mL of hydrochloric acid (0.1 M) and centrifuged at 14,130× g for 10 min after standing for half an hour. This centrifugation process was repeated twice. Then, the supernatant was combined to a constant volume of 25 mL. Take 10 mL of the solution and add 10 mL of 10% (w/v) trichloroacetic acid (TCA), allow to stand for 1 h and then centrifuge it at 14,130× g for 10 min at 4 • C. The supernatant was adjusted to pH 2.0 with 6 M NaOH and 1 M NaOH. Finally, the content was determined using an amino acid analyzer after a 0.22 µm aqueous filter membrane. The analytical conditions were as follows: analytical column, 2622PH 4.6 mm I.D. × 60 mm; flow rate, 0.40 mL/min; column temperature, 57 • C; reaction temperature, 135 • C; detection wavelength, 570 nm; injection volume, 20 µL.

Determination of Volatile Substances by GC-IMS
The determination of volatile substances was done according to the method previously established, with minor modifications [31,32]. Briefly, the accurately weighed 2.0 ± 0.1 g of P. yezoensis sauce and placed in a 20 mL headspace bottle. The vial was then kept in a water bath for 20 min at 80 • C, after which samples were injected. The analytical conditions were as follows: column, wax, 30 m; ID, 0.53 mm; film thickness, 1 µm (RESTEK, USA); column temperature, 60 • C; carrier gas, N 2 ; IMS temperature, 45 • C; injection volume, 500 µL; injection needle temperature, 85 • C; incubation speed, 500 rpm; analysis time, 40 min; drift gas, 150 mL/min.

Statistical Analysis
All analyses were performed in triplicate and data is presented as means ± standard deviations. One-way analysis of variance (ANOVA) with Tukey method and significant difference expressed at p < 0.05 level. VOCal, a qualitative and quantitative analysis software, was used for the identification of compounds, and the NIST database and the IMS database were used for qualitative analysis of volatile substances. The spectral differences and fingerprints were compared by Gallery Plot and Reporter plug-in and volatile substances of four fermentation samples were analyzed by dynamic principal component analysis (PCA). Excel 2010, SPSS statistics 26 and Origin 2021 were used for data processing and drawing.

Identification of L. plantarum MMB-05
A total of ten presumptive LAB strains were isolated from pickle brine and used for the fermentation of sandwich seaweed scraps. The results showed that all selected strains were grow in P. yezoensis sauce, and the number of viable cells showed a trend of increasing first and then decreasing during fermentation (Table S1). However, after 96 h of fermentation, the number of colonies of MMB-05 remained at 11.43 ± 0.08 CFU/mL. Therefore, the strain of MMB-05 was selected for the further study. The physiological and biochemical identification was carried out according to the methods in the literature [33]. The colonies were milky white, opaque, with regular edges and smooth surfaces. Microscopic examination showed that a single strain was straight, or curved short rod, or chain, with positive gram stain ( Figure S1). According to Table 1, it could be concluded that the target strain accords with the characteristics of lactic acid bacteria. A comparison of the developmental trees established by BLAST procedure showed that the sequence similarity between this strain and L. plantarum was 100%, and confirmed that the screened strain was L. plantarum ( Figure S2). Therefore, the bacterium was named as L. plantarum MMB-05.

Growth Characteristics
The growth characteristics of L. plantarum MMB-05 was shown in Figure 1, L. plantarum MMB-05 entered the logarithmic phase from 2 to 8 h, and then entered the stable phase. At the same time, the pH value of L. plantarum MMB-05 decreased from 6.04 ± 0.10 to 3.84 ± 0.00 and entered a rapid decline stage from the 4th hour, which showed a good capacity for acid production. Obviously, the pH value was negatively correlated with the growth value. The decrease in pH is associated with the accumulation of metabolite lactic acid [34].  Figure 2A shows the growth of L. plantarum MMB-05 at different NaCl concentrations. There was certain growth under different salt concentrations, and the growth value decreased significantly with the increase of salt content (p < 0.05). It is worth noting that all concentrations showed a trend of rising first and then stabilizing. The strain still grew at 16% NaCl concentration. This is in line with the study conducted by Hiroshi et al. [35], which showed that strains could grow to a certain degree at 0-15% concentrations. In addition, L. plantarum MMB-05 had significant antibacterial effect against E. coli, S. aureus and B. cereus, with diameters of 11.00 ± 0.35, 7.60 ± 0.14 and 11.15 ± 0.21 cm, respectively,   16% NaCl concentration. This is in line with the study conducted by Hiroshi et al. [35], which showed that strains could grow to a certain degree at 0-15% concentrations. In addition, L. plantarum MMB-05 had significant antibacterial effect against E. coli, S. aureus and B. cereus, with diameters of 11.00 ± 0.35, 7.60 ± 0.14 and 11.15 ± 0.21 cm, respectively, which was generally consistent with the results of relevant studies ( Figure 2B). Cheong et al. [36] found that 12 strains of L. plantarum had fungicidal activity. Zangeneh et al. [37] concluded that the supernatant of L. plantarum had a significant inhibitory effect on pathogenic bacteria, especially S. aureus and E. coli.  Figure 2A shows the growth of L. plantarum MMB-05 at different NaCl concentrations. There was certain growth under different salt concentrations, and the growth value decreased significantly with the increase of salt content (p < 0.05). It is worth noting that all concentrations showed a trend of rising first and then stabilizing. The strain still grew at 16% NaCl concentration. This is in line with the study conducted by Hiroshi et al. [35], which showed that strains could grow to a certain degree at 0-15% concentrations. In addition, L. plantarum MMB-05 had significant antibacterial effect against E. coli, S. aureus and B. cereus, with diameters of 11.00 ± 0.35, 7.60 ± 0.14 and 11.15 ± 0.21 cm, respectively, which was generally consistent with the results of relevant studies ( Figure 2B). Cheong et al. [36] found that 12 strains of L. plantarum had fungicidal activity. Zangeneh et al. [37] concluded that the supernatant of L. plantarum had a significant inhibitory effect on pathogenic bacteria, especially S. aureus and E. coli.

Sensory Evaluation
The sensory evaluation results of fermented P. yezoensis sauce are presented in Figure  3. Regardless of what kind of LAB starter was used, the aroma, color, and overall quality of the final product were very high, indicating that the fermented P. yezoensis sauce was acceptable to consumers. However, the overall quality of the control group was much less than that of the lactic acid bacteria fermentation group. At 24 h of fermentation, the fish and sour taste of the lactic acid bacteria fermentation group were higher. At the end of the fermentation, the bitter flavor, fishy smell, and sour flavor decreased, with the fishy smell in L. plantarum MMB-05 fermentation group being the lowest, followed by L. casei FJAT-7928 and mix fermentation groups. Compared with the control group, the P. yezoensis sauce fermented by lactic acid bacteria had little difference in salty taste, but the aroma was better than that of the control group. In terms of the overall quality, L. plantarum MMB-05 fermentation group scored the highest, followed by L. casei FJAT-7928, mixed fermentation, and control fermentation groups. All the flavor and odor changes may be due to the production of different volatile compounds during lactic acid bacteria fermentation. Overall, the edible properties of P. yezoensis sauce was significantly improved, and the texture and sensory properties of P. yezoensis sauce were enhanced after the fermentation of lactic acid bacteria.

Sensory Evaluation
The sensory evaluation results of fermented P. yezoensis sauce are presented in Figure 3. Regardless of what kind of LAB starter was used, the aroma, color, and overall quality of the final product were very high, indicating that the fermented P. yezoensis sauce was acceptable to consumers. However, the overall quality of the control group was much less than that of the lactic acid bacteria fermentation group. At 24 h of fermentation, the fish and sour taste of the lactic acid bacteria fermentation group were higher. At the end of the fermentation, the bitter flavor, fishy smell, and sour flavor decreased, with the fishy smell in L. plantarum MMB-05 fermentation group being the lowest, followed by L. casei FJAT-7928 and mix fermentation groups. Compared with the control group, the P. yezoensis sauce fermented by lactic acid bacteria had little difference in salty taste, but the aroma was better than that of the control group. In terms of the overall quality, L. plantarum MMB-05 fermentation group scored the highest, followed by L. casei FJAT-7928, mixed fermentation, and control fermentation groups. All the flavor and odor changes may be due to the production of different volatile compounds during lactic acid bacteria fermentation. Overall, the edible properties of P. yezoensis sauce was significantly improved, and the texture and sensory properties of P. yezoensis sauce were enhanced after the fermentation of lactic acid bacteria. 7928 and mix fermentation groups. Compared with the control group, the P. yezoensis sauce fermented by lactic acid bacteria had little difference in salty taste, but the aroma was better than that of the control group. In terms of the overall quality, L. plantarum MMB-05 fermentation group scored the highest, followed by L. casei FJAT-7928, mixed fermentation, and control fermentation groups. All the flavor and odor changes may be due to the production of different volatile compounds during lactic acid bacteria fermentation. Overall, the edible properties of P. yezoensis sauce was significantly improved, and the texture and sensory properties of P. yezoensis sauce were enhanced after the fermentation of lactic acid bacteria.

Physicochemical Indexes
Aw is one of the most important factors affecting fungal growth. The aw values of P. yezoensis sauce fermented by lactic acid bacteria decreased significantly (p < 0.05) over the course of this study ( Table 2). The aw value of all groups reached about 0.75 after 72 h of fermentation. Among them, the aw value of P. yezoensis sauce fermented by L. plantarum MMB-05 was the lowest, while the aw value of control group was 0.81. After 72 h, the aw values of the four groups varied significantly (p < 0.05). All the above results confirmed that the aw value of the samples could be reduced to below 0.80 by the fermentation of the three lactic acids bacteria. The reduction of water activity could reduce the water available

Physicochemical Indexes
A w is one of the most important factors affecting fungal growth. The a w values of P. yezoensis sauce fermented by lactic acid bacteria decreased significantly (p < 0.05) over the course of this study ( Table 2). The a w value of all groups reached about 0.75 after 72 h of fermentation. Among them, the a w value of P. yezoensis sauce fermented by L. plantarum MMB-05 was the lowest, while the a w value of control group was 0.81. After 72 h, the a w values of the four groups varied significantly (p < 0.05). All the above results confirmed that the a w value of the samples could be reduced to below 0.80 by the fermentation of the three lactic acids bacteria. The reduction of water activity could reduce the water available to microorganisms, which could help improve the safety of products and inhibit the growth of miscellaneous bacteria [38]. Therefore, inoculation of lactic acid bacteria to ferment P. yezoensis sauce can significantly improve the preservation and safety of the product. As a visual quality characteristic, color is an influential parameter of food acceptability for consumers. At the end of fermentation, the L* values of the four groups were all in the range of 25-29 (Table 2), which was in accordance with the results for sandwich nori [39]. With the prolongation of fermentation time, the viscosity value of all groups decreased significantly (p < 0.05) ( Table 2). Meanwhile, the viscosity value of P. yezoensis sauce also showed significant differences between the different groups. The viscosity values of L. plantarum MMB-05, L. casei FJAT-7928, and the mix fermentation groups, decreased by 52.84%, 30.39%, and 57.69% after 72 h of fermentation, while the viscosity value of the control group was 494.69 cp, possibly due to the fermentation of sugars and proteins by lactic acid bacteria producing a large number of free amino acids and organic acids.

Viable Cell Counts, pH, and Total Sugar
Changes in viable cell counts, pH, and the total sugar content of the four groups of P. yezoensis sauce are shown in Figure 4. All strains could grow normally in the sauce after inoculation, and the count of viable cell increased first and then decreased ( Figure 4A). During the fermentation period, the count of viable cell in the L. plantarum MMB-05 fermentation group was consistently higher than that in the other groups, especially after 24 h (up to 13.24 ± 0.01 log CFU/mL). During the fermentation period of 0 to 24 h, the count of viable cell in the L. casei FJAT-7928 fermentation group was slightly higher than the mix fermentation group, and the opposite trend occurred after 24 h of fermentation. The count of viable cell after 72 h fermentation was always higher than the number after initial inoculation, indicating that all three strains could grow in the P. yezoensis sauce for a long time, and the growth status of L. plantarum MMB-05 was more prominent.
The pH value of the P. yezoensis sauce in each group showed a continuous downward trend, which decreased significantly during the 0-24 h, and was negatively correlated with the number of viable bacteria ( Figure 4B). In the early stage of fermentation, the rapid reduction of pH value could effectively inhibit the reproduction of other acid-intolerant bacteria, thereby reducing the accumulation of harmful metabolites. The initial pH values of L. plantarum, L. casei, mix and control groups, were 5.12 ± 0.01, 5.48 ± 0.01, 5.24 ± 0.01, and 5.82 ± 0.01, respectively, which dropped sharply to 3.6 ± 0.01, 3.91 ± 0.01, 3.73 ± 0.02, and 5.76 ± 0.01, at the end of fermentation. The pH of other fermenting bacteria has been reported to vary from 3.3 to 4.6 at the end of the fermentation, depending on the fermentation temperature, the amount of carbohydrates available, or the additives used during the fermentation [40]. P. yezoensis sauce are shown in Figure 4. All strains could grow normally in the sauce after inoculation, and the count of viable cell increased first and then decreased ( Figure 4A). During the fermentation period, the count of viable cell in the L. plantarum MMB-05 fermentation group was consistently higher than that in the other groups, especially after 24 h (up to 13.24 ± 0.01 log CFU/mL). During the fermentation period of 0 to 24 h, the count of viable cell in the L. casei FJAT-7928 fermentation group was slightly higher than the mix fermentation group, and the opposite trend occurred after 24 h of fermentation. The count of viable cell after 72 h fermentation was always higher than the number after initial inoculation, indicating that all three strains could grow in the P. yezoensis sauce for a long time, and the growth status of L. plantarum MMB-05 was more prominent.  The pH value of the P. yezoensis sauce in each group showed a continuous downward trend, which decreased significantly during the 0-24 h, and was negatively correlated with the number of viable bacteria ( Figure 4B). In the early stage of fermentation, the rapid reduction of pH value could effectively inhibit the reproduction of other acid-intolerant bacteria, thereby reducing the accumulation of harmful metabolites. The initial pH values of L. plantarum, L. casei, mix and control groups, were 5.12 ± 0.01, 5.48 ± 0.01, 5.24 ± 0.01, and 5.82 ± 0.01, respectively, which dropped sharply to 3.6 ± 0.01, 3.91 ± 0.01, 3.73 ± 0.02, and 5.76 ± 0.01, at the end of fermentation. The pH of other fermenting bacteria has been reported to vary from 3.3 to 4.6 at the end of the fermentation, depending on the fermentation temperature, the amount of carbohydrates available, or the additives used during Microorganisms consume carbohydrates for their metabolic activities during fermentation. Figure 4C shows the total sugar content in the fermented P. yezoensis sauce with different strains. At the end of the fermentation, the total sugar content of the control group was 193.16 ± 0.19 mg/L. Compared with the other two lactic acid bacteria fermentation groups, L. plantarum MMB-05 had the greatest effect on the total sugar content of P. yezoensis sauce, and its total sugar content decreased by 20.29% after fermentation. The results showed that the strain consumed more carbohydrate than other strains, and the reduction of carbohydrate during fermentation was associated with the conversion of lactic acid and the metabolism, growth, and reproduction of L. plantarum [41]. This also explains the higher number of viable bacteria in the P. yezoensis sauces inoculated with L. plantarum MMB-05.

Total Phenolic, Flavonoids Contents and Organic Acids
The total phenolic content of the three lactic acid bacteria fermentation groups continued to increase throughout the fermentation process ( Figure 5A). After 12 h of fermentation, the total phenolic content of P. yezoensis sauce fermented by three lactic acid bacteria was significantly higher than that in the control group. Moreover, the total phenolic content of L. plantarum MMB-05 fermentation group was significantly higher than the other groups (p < 0.05), a 16.54% increase than the initial content after 12 h of fermentation. These results indicate that L. plantarum fermentation could significantly increase the total phenol content of the fermentation supernatant of P. yezoensis sauce. This was in line with the increase in total phenolic content reported by Di Cagno et al. [42] and Kwaw et al. [43] during fermentation. These increases in the total phenol content could be related to the release of corresponding glycoside element by microbial enzymes, such as decarboxylase, reductase, and tannase [44,45].  With the increase of fermentation time, the total flavonoids content in the four groups showed a trend to increase first and then decrease ( Figure 5B). During the first 48 h of fermentation, the total flavonoids content in the supernatant of the L. plantarum MMB-05, L. casei FJAT-7928 and mix groups increased from 41.17, 41.33, and 39.50 mg/mL to 122.17, 99.50, and 105.50 mg/mL (p < 0.05), respectively. The content of total flavonoids in the control group was significantly lower than that in the other three groups. It was found that different kinds of lactobacillus species had different ability to release total flavonoids by fermentation. The increase in total flavonoids content is related to changes in the acid level in the fermentation environment, which release the combined flavonoid compounds. Another reason for the increase in flavonoid content is that microorganisms could secrete enzymes such as β-glucosidase during fermentation, thereby releasing more flavonoids [46].
Organic acids are important components that affect sensory and chemical properties of food products as well as microbial stability [47]. Changes in organic acid content during fermentation were examined in this study ( Figure 6). A total of six organic acids were detected, and lactic acid was produced in the P. yezoensis sauce fermented by lactic acid bacteria during fermentation, compared with the control group. At the same time, the content of succinic acid in P. yezoensis sauce fermented by L. plantarum MMB-05 increased by 74.69%, and the content of succinic acid in P. yezoensis sauce fermented by L. casei FJAT-7928 and the mix groups decreased. Other organic acids were low in content and therefore have little effect on the flavor of P. yezoensis sauce. The kind and content of organic acids in P. yezoensis sauce fermented by different lactic acid bacteria were different. After fermentation by lactic acid bacteria, the saccharide content in supernatant decreased obviously due to the conversion of some saccharide substances into organic acids. Overall, the total organic acid content of the three lactic acid bacteria fermentation groups gradually increased as the fermentation time increased.

In Vitro Antioxidant Capacity
Three systems (ABTS + radical scavenging capacity, FRAP, and total antioxidant capacity) were selected in our study to evaluate the antioxidant activity the supernatants of four P. yezoensis sauces. Figure 7A shows that the ABTS scavenging ability of different lactic acid bacteria strains increased with the increase of fermentation time. Among them, the scavenging ability of L. plantarum MMB-05 fermentation group was relatively high, which increased from 8.33 to 13.23%, whereas the ABTS scavenging ability did not change significantly in the control group.
The supernatants of the four P. yezoensis sauces showed varying degrees of reducing activity against the iron ions ( Figure 7B), which increased with the fermentation time. During fermentation, the reducing activity of L. plantarum MMB-05 was significantly higher than that of the other three samples. At the end of fermentation, the maximum FRAP value was achieved by the three lactic acid bacteria fermentation groups. Similarly, Figure 6. Changes in organic acid contents during LAB stains fermentation of P. yezoensis sauce.

In Vitro Antioxidant Capacity
Three systems (ABTS + radical scavenging capacity, FRAP, and total antioxidant capacity) were selected in our study to evaluate the antioxidant activity the supernatants of four P. yezoensis sauces. Figure 7A shows that the ABTS scavenging ability of different lactic acid bacteria strains increased with the increase of fermentation time. Among them, the scavenging ability of L. plantarum MMB-05 fermentation group was relatively high, which increased from 8.33 to 13.23%, whereas the ABTS scavenging ability did not change significantly in the control group.

Free Amino Acids
The accumulation of free amino acids during the fermentation process could not only improve the taste of the samples, but also the α-keto acids produced by microbial metab Figure 7. ABTS + radical scavenging (A), FRAP (B) and total antioxidant capacity (C), in P. yezoensis sauce fermented by LAB stains. Note: abcd represents the significant difference between groups (different fermentation time in the same group). ABC represents the significant difference between the same fermentation time of different groups (p < 0.05). The supernatants of the four P. yezoensis sauces showed varying degrees of reducing activity against the iron ions ( Figure 7B), which increased with the fermentation time. During fermentation, the reducing activity of L. plantarum MMB-05 was significantly higher than that of the other three samples. At the end of fermentation, the maximum FRAP value was achieved by the three lactic acid bacteria fermentation groups. Similarly, Chen et al. [48] also found that the scavenging activity of ABTS + and could be enhanced when Eurotium cristatum YL-1 was used for solid-state fermentation in soybeans.
In addition, the total antioxidant capacity of the three lactic acid bacteria fermentation groups increased significantly after 12 h of fermentation (p < 0.05) ( Figure 7C). During the fermentation process from 48 to 72 h, the total antioxidant capacity of L. plantarum MMB-05 group increased significantly from 2.22 ± 0.70 to 15.42 ± 0.52 U/mL. However, the total antioxidant capacity of L. casei FJAT-7928 and mix fermentation groups did not increase significantly. Meanwhile, the total antioxidant capacity of the control group remained unchanged. Antioxidant activity was associated with the presence of total phenols, which is why the results showed a similar increasing trend. It is undeniable that the improvement of antioxidant activity is related to the growth and metabolism of thallus, which may release some bioactive peptides [49].

Free Amino Acids
The accumulation of free amino acids during the fermentation process could not only improve the taste of the samples, but also the α-keto acids produced by microbial metabolism could enter the tricarboxylic acid cycle and promote the formation of flavor substances. According to the classification of amino acids taste by Kato et al. [50] and Schoenberger et al. [51], free amino acids were divided into umami amino acids (Asp and Glu), sweet amino acids (Asp, Thr, Ser, Gly, Ala, Val, Phe, and Pro), bitter amino acids (Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Pro, and Arg), and sour amino acids (Glu and His).
Changes in free amino acid contents during fermentation were compared and shown in Table 3. Among the 17 kinds of free amino acids, except the control group, the content of free amino acids increased significantly with the fermentation in the other three groups (p < 0.05). The contents of umami, sweet, sour, and bitter amino acids, in the other three fermentation groups, increased with time. The initial contents of sweet amino acids in L. plantarum MMB-05, L. casei FJAT-7928 and the mix fermentation groups were 4.5 ± 0.01, 3.37 ± 0.04, and 4.13 ± 0.01 mg/g, respectively, which increased to 6.14 ± 0.02, 5.81 ± 0.01, and 5.92 ± 0.02 mg/g at the end of fermentation. At the end of fermentation, the contents of umami amino acids, sour amino acids, bitter amino acids, and total free amino acids, in the L. plantarum MMB-05 fermentation group increased by 39.93%, 35.84%, 32.31%, and 36.33%, respectively. The bitter amino acids content of the L. casei FJAT-7928 fermentation group increased by 64.15%, and the bitter amino acids content of the mix fermentation group increased by 45.98%. It is worth noting that glutamic acid, alanine, and histidine, accounted for more than 50% of the total free amino acids content. During fermentation, the increase in free amino acids content is mainly due to the hydrolysis of proteases and peptidases [52]. However, the contents of umami amino acids and sour amino acids decreased gradually in the control group, while the opposite occurred with the bitter amino acids, and the content of sweet amino acids showed a trend of decreasing first and then rising, resulting in a slightly worse overall flavor of the P. yezoensis sauce. Table 3. Changes of free amino acid contents in P. yezoensis sauces during fermentation (mg/g).
The principal component analysis of free amino acids in P. yezoensis sauce was carried out ( Figure 8A), and three principal components were obtained, accounting for 41.5%, 30.7%, and 12.9%, respectively. The PC1 values of the three lactic acid bacteria fermentation groups increased with the fermentation, and the close distance between the L. plantarum MMB-05 and the mix fermentation groups indicated that the changes in free amino acid contents were similar in both groups. However, the values of PC1 and PC2 in the control group did not change significantly with the fermentation. As described in Figure 8B, lactic acid fermentation resulted in higher PC1 values for Pro, Ala, Thr, Gly, and His, especially during the later fermentation stages. From the overall flavor analysis, the flavor and nutrition of free amino acids were more prominent in the L. plantarum MMB-05 fermentation group.
The GC-IMS 3D topography overlook diagram was displayed in Figure 9A. The red vertical line at abscissa 1.0 is the RIP peak (reaction ion peak, normalized). The vertical axis represents the GC retention time (s) and the horizontal axis represents the ion migration time (normalized). Each point flanking the RIP peak represents a volatile organic compound, mainly occurring during a migration time of 1.0-1.75 and a retention period
The GC-IMS 3D topography overlook diagram was displayed in Figure 9A. The red vertical line at abscissa 1.0 is the RIP peak (reaction ion peak, normalized). The vertical axis represents the GC retention time (s) and the horizontal axis represents the ion migration time (normalized). Each point flanking the RIP peak represents a volatile organic com-pound, mainly occurring during a migration time of 1.0-1.75 and a retention period of 300-1500 s. The color represents the concentration of the substance, with white indicating a lower concentration and red indicating a higher concentration; the darker the color, the higher the concentration. The signal peaks in L. plantarum MMB-05 were deducted from L. plantarum MMB-05 as a reference to obtain the differential spectra ( Figure 9B). The blue area indicates that the substance is lower than L. plantarum MMB-05 in this sample, while the red area indicates higher than L. plantarum MMB-05. The blue area indicates that the substance was lower than L. plantarum MMB-05 in this sample, while the red area indicates higher than L. plantarum MMB-05. Similarly, the darker the color, the greater the difference. It is necessary to conduct in-depth statistical analysis (Table 4) to integrate the volatility profile information of the different samples.
Foods 2022, 11, x FOR PEER REVIEW of 300-1500 s. The color represents the concentration of the substance, with white ing a lower concentration and red indicating a higher concentration; the darker th the higher the concentration. The signal peaks in L. plantarum MMB-05 were d from L. plantarum MMB-05 as a reference to obtain the differential spectra (Figure blue area indicates that the substance is lower than L. plantarum MMB-05 in this while the red area indicates higher than L. plantarum MMB-05. The blue area i that the substance was lower than L. plantarum MMB-05 in this sample, while the indicates higher than L. plantarum MMB-05. Similarly, the darker the color, the gre difference. It is necessary to conduct in-depth statistical analysis (Table 4) to integ volatility profile information of the different samples.

Principal Component Analysis of Volatiles Compounds
Principal component analysis was used to identify various fermented P. yezoensis sauces ( Figure 9C). The total contribution rate of the first two principal components was 89%, with PC1 and PC2 accounted for 69% and 20%, respectively, which was sufficient to explain the similarity between the different samples [53]. From the degree of aggregation and dispersion of the samples, it could be seen that the fermentation group of L. plantarum MMB-05 was closer to the mix fermentation group, indicating that the flavor composition was similar in both groups, while the fermentation group of L. casei FJAT-7928 was far apart, indicating that there was a certain difference in the flavor components of the two groups. This phenomenon is largely consistent with the change of free amino acid content. Studies have shown that different strains of lactic acid bacteria metabolize nutrients depending on their specific transport modes. It is known that L. plantarum is facultative hetero-fermentative and L. casei is homogenous. Therefore, they produce different types and amounts of metabolites and have different effects on environmental characteristics, such as pH and oxygen availability, which in turn may affect the volatility of fermented samples [54].  Note: Compounds percentage of total area of identified volatile compounds. RI is the retention index, Rt is retention time, Dt is migration time. ABCD represented the significant difference between groups (same fermentation time in different groups) (p < 0.05). Values are expressed as averages ± standard deviation (n = 3).
The relative contents of aldehydes, ketones, acids, and alcohols, were higher after fermentation. Aldehydes and ketones contributed significantly to the early fermentation flavor of fermented P. yezoensis sauce. In our study, due to the short fermentation time, the flavor of P. yezoensis sauce is mainly composed of aldehydes and ketones. Compared with the control group, the contents of aldehydes in the three lactic acid bacteria fermentation groups decreased significantly, with little change in ketones, and significantly higher contents occurred for acids and alcohols compounds.
Aldehydes could be formed by oxidative degradation of polyunsaturated fatty acids under the action of microorganisms, with most volatile compounds having low thresholds values and pleasant smells, such as being grassy, malty, fruity, and cheesey [55]. Among them, phenylacetaldehyde has the fruit and sweet taste, and benzaldehyde has the taste of almond. Nonanal is confirmed to be the main body of fishy smell [56]. The contents of nonanal in the three lactic acid bacteria fermentation groups decreased after fermentation, indicating that the extension of fermentation time could reduce the fishy taste of P. yezoensis sauce.
Alcohols are mainly produced by lipid oxidation, amino acids, and carbohydrate metabolism [57]. The high threshold values of alcohols contribute little to the overall flavor of the sauce, but some low threshold value of unsaturated alcohols contribute to the overall flavor of the sauce. For example, the relative content of ethanol in the samples fermented by lactic acid bacteria increased to some extent, resulting in a soft alcohol odor. 1-Octen-3-ol is the key substance that produces fishy smell in laver. After fermentation by L. plantarum MMB-05, its relative content decreased significantly, which had a certain impact on the deodorization of P. yezoensis sauce.
The relative content of acids increased significantly after fermentation by lactic acid bacteria, which was associated with the acid production of lactic acid bacteria. Acids have a fruity, cheesy, fatty, and rancid odor, and three short-chain acids were identified from the samples in our study, namely acetic acid-M, acetic acid-D, and propionic acid. Acid is the precursor of esters, and the appropriate content of short-chain acid can enrich the flavor of seaweed sauce and make it refreshing and sweet [58,59].
The relative content of ketones did not change significantly after lactic acid bacteria fermentation. Ketones are stable in properties and have a pleasant aroma of fruit and milk. Among them, 3-hydroxy-2-buxy-3-butanone has a milky flavor, and acetone has sweet, fruity, and etherous aroma. The contents of 3-hydroxy-2-buxy-3-butanone and acetone in the three kinds of lactic acid bacteria fermentation groups were much higher than those in the control group, which had a certain effect on the flavor of P. yezoensis sauce. 2-heptanone may be associated with deterioration of raw materials and will bring unpleasant off-flavors to the consumer [60].
2-Pentylfuran has soy, fruity, earthy, green, and vegetable-like aromas, making an important contribution to the flavor. Furfural, contributing to a sweet and almond-like