Porphyra yezoensis Sauces Fermented With Lactic Acid Bacteria: Fermentation Properties, Flavor Profile, and Evaluation of Antioxidant Capacity in vitro

The demand for roasted seaweed sandwich (Porphyra yezoensis) product has risen in recent years. The product slicing process has created a huge number of scraps that are not utilized effectively. Three lactic acid bacteria (LAB) strains were used to ferment P. yezoensis sauces in this study, including Lactobacillus fermentum, Lactobacillus casei, Streptococcus thermophilus, and the mixed strains (1:1:1, v/v). The fermentation characteristics, antioxidant capacity in vitro, sensory properties, and flavoring substances of fermented P. yezoensis sauces were analyzed. After 21 days of fermentation, all LAB strains grew well in the P. yezoensis sauces, with protease activity increased to 6.6, 9.24, 5.06, and 5.5 U/mL, respectively. Also, the flavors of P. yezoensis sauces fermented with L. casei and L. fermentum were satisfactory. On this premise, gas chromatography-mass spectrometry (GC-MS) was used to investigate the changes in gustatory compounds in P. yezoensis sauces fermented with L. casei and L. fermentum. In general, 42 and 41 volatile flavor chemicals were identified after the fermentation of L. casei and L. fermentum. Furthermore, the fermented P. yezoensis sauce possessed greater DPPH scavenging activity and ferric-reducing ability power than the unfermented P. yezoensis. Overall, the flavor and taste of P. yezoensis sauce fermented by L. casei was superior.


INTRODUCTION
The East China Sea, South China Sea, Huanghai Sea, and other coastal regions produce Porphyra yezoensis. The protein level in dried P. yezoensis is 25-30%, while the carbohydrate amount is around 40% (1). It also contains vitamins like riboflavin and niacin (2). Moreover, P. yezoensis is rich in minerals that can assist youngsters and the elderly to absorb nutrition (3,4).
Dietary P. yezoensis products are popular in Japan and Korea while Chinese consumers eat less P. yezoensis products. The local market of P. yezoensis products in China is underdeveloped with few product varieties and influential manufactors (5). One of the main reasons is that P. yezoensis processing are essential, whereas the products have a distinct flavor that some domestic consumers cannot tolerate. Currently, the primary domestic seaweed processing methods are drying and roasting. Thus, more processing methods of P. yezoensis are needed to boosting its economic value and increase the market of P. yezoensis products.
The slicing of roasted seaweed sandwich into small pieces generates a large number of wastes, which are rich in protein and maltose. The processing of sandwich seaweed filet to Porphyra sauce helps save waste. In addition, microbal fermentation, expecially lactic acid fermentation can preserve the nutritional value of Porphyra sauce, give it a unique taste and flavor, enrich its product form, and creat new opportunities for the Porphyra market. To our knowledge, the fermentation of P. yezoensis sauces has been poorly studied. Zhang et al. investigated the basic nutrient content of dried Porphyra, and fermented it with LAB and Aspergillus oryzae (6). Fan et al. utilized A. oryzae and Rhodozyme to co-ferment Porphyra and optimized the fermentation based on the protease activity and sensory index of Porphyra sauces (7).
LAB is a probiotic that may generate high quantities of amino acids in its fermentation P. yezoensis supernatant. It coud be a suitable species to ferment Porphyra sauces (8). This study chose L. fermentum, L. casei, and S. thermophilus to independently and co-ferment waste materials of roasted seaweed sandwich. The changes of fermentation characteristics and flavor profile were evaluated. Furthermore, the DPPH scavenging activity and ferric-reducing ability power (FRAP) of fermented P. yezoensis sauces were also investigated. The findings may help to produce probiotic fermented P. yezoensis sauces with excellent nutritional value and flavor.

Bacterial Strains and Culture Conditions
S. thermophilus FJAT-46738, L. fermentum FJAT-46744, and L. casei FJAT-7928 were obtained from the Fujian Academy of Agricultural Sciences, China. The single colony of the three LAB strains were innoculted to 10 mL MRS broth, cultivated for 36 h at 40 • C except for L. fermentum (37 • C). These strains were cultured with the AnaeroPack System C-32 (Mitsubishi Gas Chemical Company, Inc. Tokyo).

Fermentation of P. yezoensis Sauce
The waste materials of roasted seaweed (P. yezoensis) sandwich was obtained from a local plant (Lianyungang Wende Food Co., Ltd. Lianyungang) in Lianyungang, Jiangsu, China. The seaweed sandwich scraps were crushed to a specific value (0.01 mm) by a beater. The aperture of the machine sieve hole should be kept at around 0.01 mm to promote the release of protein from the tissue (9). Ten grams of seaweed sandwich scrap powders and 15 mL water were mixed with a ratio (m/v) of 1:1.5 in 100 mL glass bottles with blue lid. The bottles were then sterilized at 121 • C for 30 min in a autoclave. This process both softened the tissue of P. yezoensis and inactivated the residue bacteria. After cooling to room tempreature, the activated L. fermentum, S. thermophilus, L. casei, and their mixtures (1:1:1 v/v) were, respectively, added into the bottles at a ratio of 3% (v/v). The blue lid bottles were then tightened and statically fermented at 40 • C. The viable bacteria level, pH, protease vitality, and sensory quality of the fermented products were measured at 0, 3, 7, 14, and 21 days.

Determination of Viable Cell and pH
One gram of fermented P. yezoensis sauces were weighed and serially diluted with sterile 0.9% (w/v) NaCl solution. One hundred microlilter of each dilutions were, respectively, spread on MRS agar plates with two replicates and incubated in AnaeroPack System C-32 at 40 • C for 36 h. The cell counts were expressed as colony numbers (Log 10 CFU per g). The P. yezoensis sauce without fermentation was used as control. An appropriate amount of fermented P. yezoensis sauces were sampled and the pH was measured by a digital pH meter (Instrument &amp; Electricity Scientific Instruments Company, Shanghai).

Sensory Evaluations
Ten grams of each fermented P. yezoensis sauce were placed in plastic cups with cup and individually tasted by the sensory panelists. The sensory panel consisted of 7 persons (2 males and 5 females, aged 19-24) from School of Food Science and Engineering, Jiangsu Ocean University. All of the members were well-tranined before sensory evaluation. The sensory descriptors agreed by the team members include intensities (seaweed flavor, sauce flavor and no fishy flavor), preferences (aroma, sour flavor, bitter flavor and aftertaste), and overall quality. The intensity, preference, and overall quality scales ranged from 0 (weak) to 10 (strong), 0 (bad or dislike) to 10 (good or like), and 0 (dislike) to 10 (like), respectively (10).
About 10 g fermented P. yezoensis sauces at 25 • C was randomly taken in the training and evaluation process. Water and white bread were provided to the assessors to wash the palate. The training and evaluation were organized in conformity to the International Organization for Standardization (ISO, 1993) and conducted in a sensory laboratory that complies with the American Society for Testing and Materials (ASTM) criteria.

Determination of Protease Activity
The protease produced during the fermentation could effectively enhanced biological activity of the products (11). The formaldehyde method was used to determine the protease activity of P. yezoensis sauce (12). Ten grams of fermented P. yezoensis sauce in a 250 mL tapered container was added with 80 mL 55 • C ddH 2 O, and incubated in water bath at 55 • C for 3 h. The samples were then boiled for 1 min to inactivate the inherent enzyme. The sample was cooled to room temperature and the volume was adjusted to 100 mL with ddH 2 O, and filtered through filter paper. Ten milliliter of the filtrate in a 150 mL tapered bottle was added with 50 mL ddH 2 O, 4-5 drops of phenolphthalein indicator, and titrated with 0.1 mol/L NaOH solution until the solution just turned red. The volume of consumed NaOH was recorded as V1. The sample in the tapered bottle was added with 10 mL formaldehyde, titrated with 0.1 mol/L NaOH to dark red as the endpoint. The volume of consumed NaOH was recorded as V2. The protease viability is then calculated as (Eq. 1): where V1 is the volume of NaOH consumed by titration before adding formaldehyde, mL; V2 is the volume of NaOH consumed by titration after adding formaldehyde, mL; C is the concentration of standard NaOH, mol/L; W is the moisture content of fermented P. yezoensis sauces, mL.

GC-MS Analysis
Agilent 5977A series GC/MSD system (Agilent Technologies, CA) and Agilent HP-INNOWAX (30 m × 0.25 mm × 0.25 µm) were used to analyze the volatile compounds in the fermented P. yezoensis sauces. Two grams of sauce sample in a 20 mL headspace bottle was added with 3 mL of saturated sodium chloride solution (refrigerated at −20 • C). The intake temperature was 250 • C and the initial column temperature was 40 • C. After incubation for 1 min, it was heated to 80 • C at a rate of 4 • C/min. This temperature was hold for 1 min, then heated to 160 • C at a rate of 2 • C/min, and finally heated to 220 • C at a rate of 10 • C/min. The temperature was then maintained for 10 min. Helium gas with high purity was used as the gas load and the flow rate was 1 mL/min. MS temperatures of the quaternary rod were 150 • C. The ion source (230 • C) was an electron-ion source with an electron energy of 70 eV and a scanning range of 35-350 m/z. The semi-qualitative analysis was done with mass spectrometry in the computer spectral library (NIST/WILEY). The compound was identified when matched to a known volatile component with a score ≥80. The internal standard in this study was 2-octanol (concentration of 10 mg/L) (13,14). The content of each volatile compound in the tested samples was calculated according to the following formula (Eq. 2).
where CX is the concentration of volatile compounds to be measured, MX is the peak area of the measured volatile compound, M intensor is the peak area of the internal standard, C intensor is the concentration of the internal standard. The data of samples were sorted and drawn with Origin 2021 to analyze the changes in volatile profile (15).

Assay of DPPH Radical Scavenging and Ferric-Reducing Activity
Active ingredients of each P. yezoensis sauce (5 g) was extracted with 50 mL ddH 2 O. Ultrasound (Wuxi Gangzheng Technology Co., Ltd. Wuxi) was utilized to disrupt the cell wall and assist the extraction. The ultrasound power was 880 W the ultrasonic duration was 30 min, and the ultrasonic temperature was 50 • C. The sample was then centrifuged at 8,000 g for 15 min, and the supernatant was collected (16,17). P. yezoensis sauce extract (1 mL) was mixed with 1 mL of DPPH solution (0.1 mM), and incubated at 37 • C in the dark for 30 min prior to measuring the absorbance at 517 nm (17). The scavenging activity of the DPPH radical was calculated as follows (Eq. 3).

− [(A sample − A control )/A blank ]×100
A control was made by using absolute ethanol instead of DPPH ethanolic solution, and A blank was prepared by replacing the polysaccharide sample solution with ddH 2 O. The Ferric-Reducing activity of fermented P. yezoensis sauces were determined by the FRAP assay with an commercial kits (Jiancheng Bioengineering Institute, Nanjing, China). The absorbance was recorded at 593 nm.

Statistical Analysis
All of the experiments were repeated triplicate. Data were expressed as the mean ± standard deviation. The data were analyzed by Excel, Origin 2021, and SPSS 20.0 software. Significance was defined at p < 0.05.

Growth of LAB and pH Changes
The changes of viable cell counts and pH value of P. yezoensis sauces fermented with S. thermophilus, L. casei, L. fermentum, and the mixed strains during 21 days of fermentation are shown in Figure 1. All the three LAB strains were able to grow in the rehydrated roasted seaweed sandwich scraps. The initial viable bacteria counts of L. casei, L. fermentum, S. thermophilus and the mixed fermentation group were 7.38 ± 0.02, 7.15 ± 0.01, 7.19 ± 0.01 and 7.47 ± 0.03 log CFU/mL, respectively. After 21 days of fermentation, the viable cell counts in P. yezoensis sauce fermented with L. casei, L. fermentum, S. thermophilus and the mixed strains were 7.1 ± 0.01, 7.4 ± 0.01, 6.9 ± 0.015 and 6.5 ± 0.01 CFU/mL, indicating that the L. casei and L. fermentum strains were able to adapt to the fermentation environment. The number of viable bacteria of P. yezoensis sauce fermented with the mixed strains was considerably low at the end of fermentation. This was possibly due to the low pH (2.7 ± 0.29), which was one of the important factors affecting the number of viable bacteria during lactic acid fermentation (18). After 3 days of fermentation, the viable cell count frist rose and then decreased in the fermented P. yezoensis sauces. In contrast, no viable bacteria were found in the unfermented P. yezoensis sauce that was previously sterilized.
The pH value of the P. yezoensis sauce decreased during fermentation with all the LAB strains ( Figure 1B). The pH values of the three strains have generally decreased. From 0 to Day 3, the pH value of fermented P. yezoensis sauces decreased the fastest. The pH value of each strain steadily dropped over a period of 3-21 days. When compared to the other two bacteria, L. casei has always had the lowest pH value. A low pH would efficiently inhibit the multiplication of pathogenic bacteria in samples and extend the shelf life (19).

Determination of Protease Activity
Microbial fermentation might enhance the protease activity of the product (20). Figure 1C revealed that the protease activity of P. yezoensis sauces was initially around 5.20 U/mL and increased during all the fermentation processes. The protease activity increased to the peak at Day 7 and then decreased in the following fermentation period. Among all the groups, the protease activity of P. yezoensis sauces fermented by L. casei were the greatest (11.88 U/mL at day 7) at the same stage, which was partly consistent with the low pH value. After 21 days of fermentation, the protease activities of P. yezoensis sauces fermented by L. fermentum, L. casei, S. thermophilus, and the mixed strains were increased to 6.60, 9.24, 5.06, and 5.50 U/mL, respectively. Furthermore, the protease activity of all fermented P. yezoensis sauces were significantly increased (p < 0.05) than the control.

Sensory Evaluation
The intensities of all the sensory attributes of fermented P. yezoensis sauces showed an increasing trend within the first week and then decreased gradually (Figure 2). The sensory quality of P. yezoensis sauces fermented by L. casei was the best among all the samples at the same fermentation period, followed by L. fermentum, S. thermophilus and the mixed strains. The fishy flavor, bitter flavor, and sour tastes in all samples got intensified with time, and the scent of seaweed and sauce flavor were more apparent in the L. casei fermented P. yezoensis sauce. Moreover, the sensory properties of the P. yezoensis sauce fermented by L. fermentum were relatively stable, and no significant changes in the flavors werwe observed. The aroma value increased the maximum at Day 7, and then declined significantly. Besides, the sensory quality of P. yezoensis sauces fermented by S. thermophilus were relatively low, among which the bitter and fishy smells were stonger than the aroma and sauce flavors. Taking all the sensory results into account, L. casei was more suitable for the P. yezoensis sauce fermentation.

Analysis of Volatile Composition
According to the above results, L. casei and L. fermentum strains were preferred for fermenting P. yezoensis sauce. Thus, GC-MS was employed to investigate flavor alterations in P. yezoensis sauce fermented by L. casei and L. fermentum. Figure 3 shows that during fermentation at 7 and 21 day, a total of 67 volatile compounds were detected from the P. yezoensis sauces. Specifically, 35, 41, and 42 volatile compounds were identified in the control group, L. fermentum group and L. casei group during fermentation, respectively. These volatile compounds mainly included aldehydes, alcohols, hydrocarbons, acids, phenols, ketones, esters, pyrazines, furans and benzodiazepines and others (Supplementary Table 1). At a given fermentation, the volatile profile changed with time due to the catabolic response of flavor precursor material (21). Alcohols presented the highest amount in all the 6 samples, accounting for 65.62%. Aldehydes and hydrocarbons accounted for 46.45 and 27.29% of the total volatile compounds, respectively. Alcohols and esters were mainly produced in the middle stage of fermentation, while hydrocarbons were produced in the later stage (22).

Assay of DPPH Radical Scavenging and Ferric-Reducing Activity
Polysaccharides, polyphenols, proteins, and porphyrins were the physiologically active compounds found in P. yezoensis, with anticancer, antioxidant, hypolipidemic, and anti-inflammatory properties (23,24). Fermentation with LAB can increase the biological activity of food by bio-transforming active molecules (25). The antioxidant capacities of P. yezoensis sauces extract before and after fermentation with L. casei and L. fermentum are shown in Figure 4. The free radical scavenging capability and ferric reducing antioxidant capacity of unfermented P. yezoensis sauce were only 20.4% and 0.23, respectively. The antioxidant activity of all the tested extract from fermentated P. yezoensis sauces steadly increased during the 21 days of fermentation. For the P. yezoensis sauce fermented by L. casei for 21 days, the free radical scavenging capability and ferric reducing antioxidant capacity increased to 78% and 0.73, respectively. Similarly, L. fermentum fermented samples increased to 67.8% and 0.58, respectively.

DISCUSSION
As more people become aware of the health benefits of laver, the laver industrial development has become more diverse and extended (26). The waste from the manufacturing of roasted seaweed sandwich were used to prepare the fermented P. yezoensis sauce. The obtained results revealed that L. casei was capable to grow in the the P. yezoensis sauce. The resulting seaweed sauce was featured by high enzyme vitality, and pronounced taste and flavor. In addition, compared with the control group and L. fermentum fermentation, L. casei fermentation improved the antioxidant activity of the sauce significantly, which was in line with the finding reported by Zubaidah et al., who fermented cabbage by L. casei (27).
The GC-MS analysis revealed that 42, 41, and 35 volatile flavor compounds were detected in the P. yezoensis sauces fermentated by L. casei, L. fermentum and the control group, respectively. These volatile compounds were responsible for P. yezoensis sauce flavor. Aldehydes are generated by fat oxidation, providing a fat scent (28). In the L. casei fermentation group one aldehyde was detected at Day 7, and 8 aldehydes were found at Day 21. The portion of aldehyde in the total volatile components increased from 2.71% at Day 7 to 46.45% at Day 21. However, in the L. fermentum group, the relative amount of aldehydes dropped from 25.53 to 6.64% and the number of compounds dropped from 6 to 3. In the control group, the ratio of aldehyde species increased from 2.49 to 8.47%, and the number of aldehyde species increased from 2 to 5. The unsaturated aldehydes have attractive aromas (29). For example, benzaldehyde possesses pleasant fruity flavor, and furfural refers to thew sweet and almond scent. These  components can contribute to the enhancement of the sauce flavor (30).
Hydrocarbon compounds were identified in P. yezoensis sauces, including β-ocimene, eicosane, heptadecane, 8heptadecene, tetradecane and others. Although alkyene compounds were regarded as odorless due to the cleavage of the alkoxygen radical, some olefins generated aldehydes and ketones under specific circumstances still can contribute to the overall flavor of P. yezoensis sauces (32). Several alkanes, when degraded under certain circumstances, generated a fishy odor (33). After 21 days of fermentation with L casei, the number of hydrocarbon species increased from 4 to 9, with the relative content of 8-heptadecene increased from 7.15 to 19.15%. During fermentation, the relative content of 8-heptadecene in unfermented group increased from 2.21 to 16.48%, but the relative content of 8-heptadecene during L. fermentum fermentation was no significant difference. It was widely established that 8-heptadecene tadecene was a distinctive flavor compound found in a wide variety of macroalgae (34).
P. yezoensis sauces contained a total of four acid compounds, namely acetic acid, butanoic acid, dodecane, 4-methyl, and 1-non-adecene. Among them, acetic acid is primarily an enzymatic response in the lipogenesis enzyme pathway, which has an oily fishy smell. Also, acetic acid may create ester bonds with sugar to enhance the flavor (35). The L. casei fermentation reduced the concentration of acetic acid, consequently atteunating the pungent smell of P. yezoensis sauces (36). At the same time, the release of acetic acid indicates that L. casei has the ability to metabolize lactic acid (37).
Esters are formed by esterifying carboxylic acid and alcoho, which are usually related with fruit or floral scents, and contributing to the flavor of food products (38,39). Ketones with a unique fruit smell, can be generated by heat oxidation or breakdown of amino acids (40). Pyrazines are nitrogencontaining heterocyclic compounds that are an intermediate in the Millard reaction (41). They often have a barbeque and nut flavor, an excellent dispersed fragrance, and an extreme deficient concentration. These volatile compounds were detected in P. yezoensis sauce fermented by L. casei. Therefore, it was determined that fermenting P. yezoensis sauce with L. casei produces the finest flavor.

CONCLUSION
In this study, three LAB strains fermented sandwich seaweed processing waste to prepare P. yezoensis sauce. The detection and analysis of the three LAB strains fermented P. yezoensis sauces revealed that the L. casei was able to adapt to the fermentation environment and had high protease vitality. Sensory evaluation results showed that fermentation of L. casei could enhanced the acceptability of P. yezoensis sauce. Following GC-MS detection, P. yezoensis sauce generated 42 volatile flavor compounds by fermentation of L. casei. These volatile chemicals are essential for P. yezoensis sauce taste. The antioxidant activity of P. yezoensis sauces fermented by L. casei was also higher. Overall, L. casei fermented P. yezoensis sauce had the best flavor and was appropriate for producing P. yezoensis sauce meals. The fermented P. yezoensis sauces has a distinctive taste and excellent nutritional value for the elderly, children and babies. This study can provide guidance for the development of LAB fermented P. yezoensis sauces in future studies.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
JY performed the experiments, analyzed the data, and wrote the manuscript. TG and FG analyzed the data and wrote the manuscript. HS, ZC, and ZW analyzed and discussed the data. SW, PS, and YT provided samples and discussed the data. WW designed the research content, analyzed the data, and modified the manuscript. All authors have read and agreed to the published version of the manuscript.