Next Article in Journal
Potential Prebiotic Effect of Caatinga Bee Honeys from the Pajeú Hinterland (Pernambuco, Brazil) on Synbiotic Alcoholic Beverages Fermented by Saccharomyces boulardii CNCM I-745
Previous Article in Journal
In Vitro Preliminary Characterization of Lactiplantibacillus plantarum BG112 for Use as a Starter Culture for Industrial Dry-Fermented Meats
Previous Article in Special Issue
Smart Fermentation Technologies: Microbial Process Control in Traditional Fermented Foods
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Fermentation
Volume 11
Issue 7
10.3390/fermentation11070404
fermentation-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Mixed Inoculation with Lacticaseibacillus casei and Staphylococcus carnosus Improves Safety, Gel Properties and Flavor of Giant Squid Surimi Without Added Seasonings

by
Hongliang Mu
,
Peifang Weng
and
Zufang Wu
*
College of Food Science and Engineering, Ningbo University, Ningbo 315832, China
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(7), 404; https://doi.org/10.3390/fermentation11070404
Submission received: 23 May 2025 / Revised: 4 July 2025 / Accepted: 6 July 2025 / Published: 14 July 2025
(This article belongs to the Special Issue Functional Properties of Microorganisms in Fermented Foods, 2nd Edition)
Browse Figures
Versions Notes

Abstract

The gel performance of giant squid is weak. Researchers have confirmed that adding some substances could improve the texture. However, the flavor has not been taken into account. In a previous study, we proved that mixed inoculation with Lacticaseibacillus casei and Staphylococcus carnosus with several seasonings adding could improve the texture of squid. Whether the addition of seasonings could affect the quality of samples or not and how fermentation affects the texture and flavor were not clear. In present study, we prepared fermented squid without seasonings. The results showed that compared with fermented samples with added seasonings, samples without seasonings might be safer, with fewer types and lower concentrations of biogenic amines. In samples without seasonings, non-inoculation had a higher pH and higher levels of biogenic amines. Meanwhile, mixed inoculation with L. casei and S. carnosus could ensure safety, improve texture and rheological properties. The water state of the fermented sample was also changed. The microstructure indicated that good network was formed in the fermented sample. After fermentation, the contents of several organic acids, free amino acids and volatile flavor compounds increased, and the results of the electronic nose test were also changed. In addition, starters were dominant during fermentation. These results indicated that mixed inoculation without seasonings might be a safer method than that with seasonings. In addition, mixed inoculation without seasonings could improve the texture and flavor of the squid. These results lay the foundation for improving fermented squid quality in further studies.

1. Introduction

Fermentation is an effective way to improve food quality [1,2] and preserve food [2]. In meat processing, fermentation can improve texture and flavor of samples [3,4,5]. Nowadays, the use of starter is necessary during meat fermentation [2], as spontaneously fermented meat might have safety risks, such as biogenic amines [6,7]. During meat fermentation, lactic acid bacteria (LAB) and coagulase-negative staphylococci (CNS) are among the mostly commonly used starters [8,9,10,11]. LAB are crucial for the fermentation due to the inhibition of other bacteria [8]; in addition, LAB are also important for improving food quality [12]. CNS work through their various enzymes, and are beneficial for the quality improvement [13].
Giant squid (Dosidicus gigas) might be an alternative source for surimi; however, its gelling ability is poor [14]. There have been studies reporting that adding some ingredients could influence the gel performance of giant squid, such as laver powder [15], organic salts [16] and egg white protein [17]. Flavor is an important feature of food. However, in the abovementioned studies, how the flavor of the squid gel changed was not clear. It is of great significance to study the change of flavor while improving the gel performance of squid. As we discussed above, fermentation could improve the flavor of meat; however, whether fermentation could improve the flavor of squid while enhancing the gel properties was not clear.
In a previous study, we confirmed that co-inoculation with L. casei and S. carnosus to squid could improve the texture of the product while ensuring safety [18]. In this study, whether the seasonings affected the safety of samples was not clear, as seasonings have been reported to influence the biogenic amine accumulation in fermented food [19]. Meanwhile, how fermentation improved texture was not addressed. The impact of fermentation on flavor was also not studied. Producing fermented squid without seasonings might benefit the research of inoculation rather than seasonings on the effect of fermented squid. In addition, the added seasonings might limit the acceptability of the samples. So, it might be necessary and important to produce fermented squid without added seasonings.
In present study, firstly, we evaluated the effect of inoculation on the safety of squid without seasonings to select the best starters. Then, the effect of mixed inoculation on the rheology, texture and color, microstructure, water state and bacterial community change in squid were evaluated. Lastly, the effect of mixed inoculation on the flavor of fermented squid was researched. Present study might contribute to a deeper understanding of the quality formation mechanism of fermented squid, laying the foundation for the production of a high-quality related product.

2. Materials and Methods

2.1. Materials

The squid, obtained from Ningbo Feirun Co., Ltd. (Ningbo, China), had the internal organs and skin removed and was cut into slices. Upon arrival, the samples were stored below −20 °C.

2.2. Preparation of Fermented Squid Surimi Sausage

L. casei ATCC 393 and S. carnosus ATCC 51365, obtained from Guangdong Microbial Culture Collection Center (GDMCC), were prepared as previously described [18]. In brief, L. casei ATCC 393 and S. carnosus ATCC 51365 were cultivated three times at 37 °C for 24 h. Then, they were harvested through centrifuging at 5000× g, 10 min, at 4 °C (TGL-18M, Bioridge, Shanghai, China) and diluted to about 7.3 × 109/mL and 3.7 × 109/mL cfu with sterile 0.85% NaCl.
The samples were prepared as previously with minor modifications [18]. In brief, after squid was thawed using tap water and cut into chunks, they were soaked in 2% sodium citrate for 15 h [20], washed and minced. Then, several substances were added, including glucose (2%), NaCl (3%), corn starch (8%), soy protein isolates (3%) and mixed phosphates (0.3%). Afterwards, three groups’ samples were prepared, as follows: (1). Control (CK, no starter added); (2). LC (only L. casei added) and (3) LS (L. casei and S. carnosus added). The starters in the samples were about 7.3 × 107/mL and 7.4 × 107/mL cfu for L. casei and S. carnosus, respectively. Then, the mixture was stuffed into a 30 mm (diameter) collagen casing, holes were punched with sterile toothpicks and fermented for 48 h at 30 °C, 85% RH with an incubator (RLD-450E-4, Ningbo Ledian, Ningbo, China).

2.3. Measurement of pH

pH was measured as previously described [18]. Briefly, the pH was measured with a pH meter (PB-10, Sartorius, Shanghai, China) after the samples were homogenized with 9 times of water (boiled and cooled). pH was measured in triplicate for every group.

2.4. Determination of Biogenic Amines

Biogenic amines (BAs) were extracted using a method described in China National Standard GB 5009.208—2016 with minor modifications [21]. Briefly, the samples were accurately weighed, then trichloroacetic acid (TCA) (the concentration was 5%) was added and homogenized. After shaking for 30 min and centrifuging (5000 rpm, 10 min), the supernatant was transferred to a 25 mL volumetric flask. The extraction process was repeated as described above. Finally, the total volume was adjusted to 25 mL with 5% TCA. The BAs were derivatized and detected as previously performed [18]. The standards (including tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine) were derivatized and detected as the same method. BAs were determined in triplicate for every group.

2.5. Texture Profile Analyses (TPA) and Color Test

The samples for TPA and color test were prepared as previously described [18]. Briefly, the samples were boiled with two-stage heated method (40 °C, 60 min; 90 °C, 30 min) and immersed into precooled boiled water for 30 min at 4 °C. Then samples were stored at 4 °C for further analysis.
TPA was performed with a TA. XT Plus texture analyzer (Stable Micro System, Godalming, UK) with a P/50 probe. The samples were cut into 20 mm length. The parameters were as follows: pre-test speed, 1 mm/s; test speed, 1 mm/s; post-speed, 5 mm/s; target model, strain; strain, 30%; time,5 s; trigger force, 5 g. TPA was performed in 4 replicates for every group.
The color was measured with a color meter (NH310, 3NH, Shenzhen, China) in 6 replicates for every group using D65 as the illuminant source.

2.6. Rheology Measurements

Rheology measurements were performed with a rheometer (Discovery HR-2, TA instruments, New Castle, DE, USA) using a 20 mm plate as the method described previously with modifications [22]. The gap was set as 1000 μm. The parameters were as follows: temperature 25 °C; strain, 0.1%; frequency: 0.1–10 Hz. Before the test, the plate was sealed with paraffin liquid to inhibit water evaporation. For every group, the rheology was measured in triplicate.

2.7. Observation of Microstructure

The microstructure of samples was observed as previously described with slight modifications [23]. The samples were fixed with 2.5% glutaraldehyde solution for 24 h in 4 °C after the samples were cut into small pieces. Afterwards, rinse three times with 0.1 M phosphate buffer (pH 7.2) for 15 min each time. Then, the samples were subjected to gradient dehydration using 30%, 40%, 50%, 60%, 70%, 80% and 90% ethanol for 15 min each time. After that, ethanol was removed using a mixture of ethanol and tert-butanol in ratio of 3:1, 1:1, 1:3 and 0:1 for 15 min each time. Finally, cover the sample with a small amount of tert-butanol and dry in freeze-dryer (Scientz-10N, Scientz, Ningbo, China). After sputter-coating with gold (E-1010, Hitachi Ltd., Tokyo, Japan), the sample was observed using a scanning electron microscope (S–3400N, Hitachi Ltd., Tokyo, Japan).

2.8. Water-Holding Capacity (WHC) Analysis

WHC was determined after the samples were stored overnight. WHC of the samples was analyzed as previously described with modifications [24]. After cutting off the edge of samples, then the samples were accurately weighed (W1) and wrapped with filter paper. After centrifugation at 10,000× g, 4 °C, for 10 min (TGL-18M, Bioridge, Shanghai, China), weigh the sample after removing the filter paper (W2). WHC was calculated as the following formula:
W H C % = W 2 W 1 × 100 %
WHC was measured in triplicate for every group.

2.9. Low-Filed Nuclear Magnetic Resonance (LF-NMR)

After weighing, the sample was placed into a 25 mm tube, then the LF-NMR spin-spin relaxation time (T2) was measured using the Carr–Purcell–Meiboom–Gill (CPMG) sequence with the nuclear magnetic resonance analyzer (NMI20-060H-1, Niumag, Suzhou, China). For every group, T2 was tested for four replicates, and every replicate was measured three times. The parameters were as follows: sampling frequency, 200 kHz; digital gain, 3; 90° pulse width, 7.52 μs; analog gain, 20 dB; waiting time, 10,000 ms; accumulative time, 2; 180° pulse width, 12 μs; echo time, 0.2 μs; number of echoes, 8000; peak offset, 0.01 ms.

2.10. Bacteria Community Structure Analysis

After extracting the DNA of the sample with a DNA extraction kit (D6356-F-96-SH, Guangzhou Magen, Guangzhou, China), use NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis to detect the concentration and quality of DNA. Afterwards, primers (343F:TACGGRAGGCAGCAG and 798R:AGGGTATCTAATCCT) with barcodes were used to amplify the V3-V4 region of 16S DNA. PCR products were checked by agarose gel electrophoresis. Then, the product was purified using Agencourt AMPure XP beads (Beckman Coulter Co., Brea, CA, USA), followed by a second round of PCR amplification and further purification. Then, the product was quantified with Qubit dsDNA Assay detection kit (Q32854, Invitrogen, Eugene, OR, USA). The concentrations were adjusted to sequence. Sequencing was performed on Illumina NovaSeq 6000 with 250 bp paired-end reads (Illumina Inc., San Diego, CA, USA). The adapter of the raw sequencing data was cut off using cutadapt [25]. Afterwards, DADA2 [26] was used to filter low quality sequences, denoise, merge, detect and cut off the chimer reads using the default parameters of QIIME 2 [27], and representative reads and ASV abundance tables were obtained. After selecting the representative read of each ASV using the QIIME 2 software, annotate and blast all representative reads with the Silva database (Version 138). Bacteria community structure analysis was performed in 6 replicates for every group.

2.11. Determination of Organic Acids

Extraction of organic acids was performed as previously described with minor modifications [28]. Briefly, add 9 times volume of water of the sample’s weight, then the mixture was homogenized, centrifuged at 10,000× g, 10 min, at 4 °C and filtered through 0.22 μm filter prior to analysis. The determination of organic acids was performed using liquid chromatography system (1260, Agilent, Santa Clara, CA, USA) as previously described with modifications [29,30]. The detection parameters were as follows: chromatographic column, C18 (250 mm × 4.6 mm × 5 μm); detection wavelength, 210 nm; mobile phase, 0.01 M potassium dihydrogen phosphate: methanol (98:2) (pH of 2.5); flow rate: 0.8 mL/min; injection volume, 20 μL; column temperature, 30 °C. The different concentrations of standards (including malic acid, lactic acid, acetic acid and citric acid) were injected into the liquid chromatography system as the same method. For every group, organic acid detection was performed in triplicate.

2.12. Analysis of Free Amino Acids

The extraction of free amino acids was performed as previously described with modifications [31]. Briefly, after weighing the sample, add 9 times of 5% TCA of the sample’s weight. The mixture was homogenized and stored at 4 °C for 1 h. After centrifuging at 10,000× g, 10 min, at 4 °C, the supernatant was filter through 0.22 μm filter to further analysis. The free amino acids were detected as previously described [32]. The mobile phase A included 0.5% tetrahydrofuran (v/v), 0.8% sodium acetate (m/v) and 0.0225% triethylamine (pH of 7.2), while mobile phase B was a mixture of 2% sodium acetate acetic acid solution (pH of 7.2) with acetonitrile and methanol in a ratio of 1:2:2 (v/v/v). The mobile phase was set as follows: 0 min, mobile phase A: 92%, mobile phase B: 8%; 27.5 min, mobile phase A: 40%, mobile phase B: 60%; 31.5 min, mobile phase A: 0%, mobile phase B: 100%; 34 min, mobile phase A: 0%, mobile phase B: 100%; 35.5 min, mobile phase A: 92%, mobile phase B: 8%. The peaks of free amino acids were identified through comparing the retention time with the standards, and the contents of free amino acids were calculated through linear equation of standards. The free amino acids were measured in triplicate for every group.

2.13. Analysis of Volatile Flavor Compounds Through Gas Chromatography–Mass Spectrometry (GC-MS)

The detection of volatile flavor compounds was performed as previously described with modifications [33]. The analysis was performed using a GC-MS system (8890 GC System+5977B/MSD, Agilent, Santa Clara, CA, USA). After weighing about 3 g of the sample, place it in a 20 mL headspace bottle, insert a solid-phase microextraction (SPME) fiber, extract at 60 °C for 40 min. Then, the SPME fiber was inserted into GC-MS injector port at 250 °C for 3 min. The analysis was performed using DB-624 capillary column (30 m × 0.25 mm × 1.4 μm, Agilent, Santa Clara, CA, USA).The parameters were as follows: temperature program, held at 40 °C for 3 min, then increased to 150 °C at 4 °C/min, held for 1 min, then increased to 200 °C at 5 °C/min, and then increased to 230 °C at 20 °C and held for 5 min; carrier gas was helium gas and the flow rate was of 1 mL/min; the operation was performed in splitless mode; ion source temperature, 250 °C; mass range, 30–400 m/z; solvent delay, 1 min. For both unfermented and fermented samples, the GC-MS was performed for no less than four replicates. The compounds were identified with the Agilent MassHunter Qualitative Analysis software (Version B.07.00, Agilent, Santa Clara, CA, USA) by comparing with the National Institute of Standards and Technology (NIST) reference database (NIST 14), and compounds with a score higher than 80 were reserved [34]. Compounds only detected in one sample of unfermented or fermented samples were ignored.

2.14. Electronic Nose Analysis

The aroma pattern of samples were detected using an electronic nose (PEN3, Airsense, Schwerin, Germany) with a method described previously with modifications [35]. Take about 5 g of the sample, equilibrate at room temperature for 60 min to further analyze. The parameters were as follows: flush time, 100 s; measurement time,180 s; chamber flow, 200 mL/min; initial injection flow, 200 mL/min. Principal component analysis (PCA) was performed with the software (WinMuster, Version 1.6.2.22) attached with the instrument. Electronic nose analysis was performed in triplicate for every group.

2.15. Statistical Analysis

The tests were performed in no less than triplicate (the replicates are presented in the corresponding section of Section 2). Analysis of variance (ANOVA) and the Student’s t-test were performed using Statistical Package for the Social Sciences 19.0 (SPSS Inc., Chicago, IL, USA).

3. Results and Discussion

3.1. Inoculation on Safety of Squid Surimi Sausage

In a previous study, we confirmed that inoculation can improve the texture of squid and ensure safety [18]. As mentioned above, how texture improved and how flavor was influenced were not clear. Several seasonings were added during sample preparation, which may affect its acceptance. Additionally, seasonings might affect the biogenic amines accumulation in fermented food [19]. In order to facilitate the study of the impact of fermentation on quality and exclude the influence of seasonings, in present study, no seasonings were added when preparing the samples. So, firstly, it is necessary to evaluate the safety of fermented squid surimi without added seasonings. Traditionally, fermented meats were processed without starters inoculated [2]. Nowadays, the use of starters is necessary to assure the safety and quality of fermented food [18]. Firstly, we compared the effect of inoculation on the safety of fermented giant squid.
The pH of different groups is shown in Figure 1a. It can be seen that before fermentation, the pH of the unfermented sample was around 7.4. After 48 h of fermentation, the pH of all three groups (including non-inoculated, LC and LS groups) decreased. The pH of the non-inoculated group was around 5.68, which was significantly higher (p < 0.05) than that of LC and LS, while the pH of LC and LS were 4.78 and 4.54, respectively. Although the pH of the non-inoculated group had decreased, consistent with the pH change in the no starter-added sausage between day 0 and day 7 in the previous study [10], it was still higher than 5.3, which does not meet the requirements (when the sausage is fermented at 30 °C, the maximum hour to reach a pH of 5.3 should be 48 h [36]), indicating a high safety risk. The pH levels of both LC and LS groups were under 5.3, suggesting that inoculation with L. casei and mixed inoculation can lead to acidification of the samples. The decrease in pH may be caused by organic acids produced by lactic acid bacteria [8]. The reason for the higher pH in the CK group may be due to the lower quantity of lactic acid bacteria or organic acids in the sample.
Among the eight biogenic amines, only four biogenic amines (including phenethylamine (PHE), putrescine (PUT), tyramine (TYR) and spermine (SPM)) were detected in the samples (Figure 1b), while only SPM was detected in the unfermented samples, consistent with previous findings [18]. For PUT and TYR, the concentration of CK (non-inoculated group) was significantly higher (p < 0.05) than those of LC and LS, and the concentration of LC was also higher (p < 0.05) than those of LS. In raw pork meat and lard, SPM was found to be one of the two BAs identified [37]. Previous study reported that the content of SPM decreased during the ripening of sausages [37]. Meanwhile, SPM can be consumed by microorganisms as nitrogen source [37]. Although the statistical results indicate that the SPM of the LS group was higher than that of the UF group (p < 0.05), combined considering the concentrations of UF, CK, LC and LS were low, between about 3 and 11 mg/kg, significantly lower than the cytotoxic concentration of SPM (653.56 mg/kg) [38], and there is no significant difference between these four groups (p > 0.05), SPM may not be the main BA during squid fermentation. The higher content in the LS group in the present study might be explained as or at least partially explained as water loss [37]. Whether the microorganisms contribute to the increase in SPM in this study need further investigation. In addition, a low concentration of PHE was detected in one sample of the LS group, suggesting PHE might not be the main BA. Both PUT and TYR were believed to be toxic or to cause adverse effects to humans [39,40,41]; however, the present results confirmed mixed inoculation could improve the safety of the fermented giant squid. Several studies have confirmed that inoculation to fermented meat could inhibit several kinds of BA accumulation [42,43]. In summary, these results implied that inoculation with L. casei and mixed inoculation can lower the pH and inhibit the increase in PUT and TYR. In addition, mixed inoculation was a better choice than single inoculation with L. casei, considering the pH and biogenic amine concentration.
Combined with the previous study [18], the results of BA in the present study showed that the concentration of BA detected in fermented sample without seasonings were lower than those with seasonings added (except for the LC group, where the concentration of PUT without seasonings was higher than that with seasonings added), indicating that the addition of seasonings had impact on the safety of fermented squid samples. For samples without added seasonings, although only four kinds of BAs were detected in the non-inoculated samples, its pH was relatively high. Based on the requirement [36], if not inoculated, it may be more likely to cause excessive growth of unwanted bacteria. Therefore, considering the pH and BA concentration, for squid surimi sausages, non-inoculation may have a higher safety risk, and mixed inoculation may be a safer choice in samples without added seasonings.

3.2. Mixed Inoculation on the Rheology of Squid Surimi Sausage

The complex viscosity of the samples is shown in Figure 1c. It can be seen that as the angular frequency increased, the viscosity of the unfermented samples gradually decreased from approximately 2306 to 58, while the viscosity of fermented samples decreased from 203,244 to 5063. It can be seen that the complex viscosity of fermented samples is significantly higher than that of unfermented samples at low frequency. As the frequency increased, the viscosity of both groups of samples decreased, but the viscosity of fermented samples was still much higher than that of unfermented samples. The increase in viscosity after fermentation was similar to the results of fermented milk–cereal-based composite substrate: before fermentation, the samples were fluid-like, and fermentation increased the viscosity [44]. Viscosity represents flow resistance and is mainly related to the interactions between components [45]. Increased viscosity might be due to biochemical and physicochemical changes [44].
Figure 1d and e show the storage modulus (G′) and loss modulus (G″) of the samples, respectively. G′ and G″ describe the elastic and viscous behavior of the internal structure of the sample [22]. In both unfermented and fermented samples, G′ was always greater than G″, suggesting that elasticity was the main property of the samples [22].The G′ of myofibrillar proteins (MPs) from different species is different, which may be related to differences in the molecular composition of different MPs, and MPs with higher G′ may have ordered macromolecular structures [46]. In the present study, the G′ of the fermented sample significantly increased, which may indicate that in fermented sample, large molecular structures were formed, resulting in the formation of a denser structure after sample cooking [46].
The rheological results showed that fermentation helps interact among various components in the product, thus helping to improve the formation of an orderly network structure and improve the gel quality of the product.

3.3. Mixed Inoculation on Texture and Color of Squid Surimi Sausage

The texture of the product before and after fermentation was compared (Table 1). After fermentation, the hardness and chewiness of the product increased (p < 0.05), while the cohesiveness decreased (p < 0.05). However, there was no significant change in the springiness (p > 0.05). Several studies have confirmed that compared with non-fermented samples, fermented meat had a higher hardness, including sour meat [47], silver carp sausage [48] and Suan yu [49]. The increase in hardness might be due to protein coagulation induced by pH decrease and water loss [47]. The color test results were consistent with findings in silver carp sausages [9]; that is, after mixed inoculation and fermentation, the L*, a* and b* values of the product increased (Table 1). In meat, changes in protein are believed to affect the color of product [50]. In summary, fermentation has a positive effect on improving the texture characteristics and color of products, which might benefit improvements in acceptability.

3.4. Mixed Inoculation on the Microstructure of Squid Surimi Sausage

The microstructure of samples is shown in Figure 2a,b. Unfermented samples had more porous structures, while fermented samples had more dense structures. The loose structure of unfermented samples may be direct evidence of weak interactions between unfermented samples [51]. As fermentation progressed, the intermolecular interactions of the samples increased, resulting in denser structure and increase in product hardness [51]. These results were consistent with the improvement in texture and rheological properties of the fermented samples mentioned above. In the fermentation of freshwater fish, inoculation with lactic acid bacteria has been reported to cause similar changes in the microstructure of the product [51].

3.5. Mixed Inoculation on Water-Holding Capacity and Water State of Squid Surimi Sausage

It can be seen from Figure 2c that the water-holding capacity of the fermented sample was significantly lower than that of the unfermented sample (p < 0.05). Generally, with the progress of fermentation, the moisture will decrease, such as fermented silver carp sausages [3]. We speculate that most of the water lost by the fermented sample during centrifugation may come from two-stage heating and cold-water immersion. As shown in the microstructure above, unfermented samples may be difficult to retain water during cooking due to poor network structure, resulting in less water loss after centrifugation and lower measured water-holding capacity. This inconsistency will be discussed in detail below.
LF-NMR is a useful method for studying the water state in samples, including distribution, movement and interaction with other macromolecules [52]. As shown in Figure 2d, there are a total of 2 peaks in the unfermented sample and 3 peaks in the fermented sample. The T21 (0–10 ms), T22 (10–100 ms), and T23 (100–1000 ms) relaxation time ranges correspond to bound water, immobilized water, and free water, respectively [52].The relaxation time represents the degree of freedom of water; the shorter the relaxation time is, the tighter the water and other molecules form a structure [53,54]. It can be seen from the figure that compared with the unfermented sample, the relaxation time of bound water and immobilized water is shifted to the left (Figure 2d–f), and the areas are also reduced (Figure 2h–i). These changes may indicate that these parts of water in the fermented sample may be more difficult to flow and formed more tightly packed structures with other molecules.
The fermented samples had a peak of 100–1000 ms (T23), while in the non-fermented samples, there was no T23 peak (Figure 2d,g,j), indicating that the cooked fermented samples had more free water. Therefore, after centrifugation, the fermented sample lost more water, corresponding to a lower water-holding capacity. The higher free-water content of fermented samples may be related to the sample cooking method in this study. As discussed above, generally, water will be lost during fermentation, such as with silver carp sausages [3], and the area of the peak corresponding free water should be reduced. After two-stage boiling and cold-water immersion of the sample, the fermented sample may absorb more water due to its good network as mentioned above, and the absorbed water might correspond to the possible free water, which might be easy to lose after centrifugation and might correspond to a lower water-holding capacity. Meanwhile, unfermented samples could absorb less water during cooking and soaking, and may lose less water during centrifugation, which corresponded to higher water-holding capacity. In summary, combined with the results of the microstructure and water-holding capacity, the results of LF-NMR confirmed that the bound water and immobilized water of fermented samples exhibited restricted mobility, while the increase in free water likely originated from water released during sample cooking and soaking, which also suggested that the unfermented samples had a poor ability to absorb free water during cooking and soaking.

3.6. Bacterial Community Structure Changes During Fermentation of Mixed Inoculation

The results of the rarefaction curve indicate that the sequencing strategy is sufficient for analyzing the bacterial community structure (not presented in the figure). Figure 3 shows the changes in the bacterial community during the fermentation process.
Although several phyla were detected in the samples at the beginning of fermentation, the proportion of Firmicutes and Proteobacteria exceeded 97% and 0.6%, respectively (Figure 3a). The combined proportion of these two phyla exceeded 98%. As fermentation proceeded, at 16 h and 32 h, the proportion of Firmicutes increased to over 99%, while the proportion of Proteobacteria decreased (Figure 3a). At 48 h, the combined proportion of these two phyla, especially Firmicutes, was still high, while the proportion of bacteria of other phyla continued at extremely low level (Figure 3a).
Among all the samples, more than 100 genera were detected. Figure 3b shows the top 15 genera during the fermentation. In the initial stage of fermentation, the dominant genera were Lactobacillus and Staphylococcus, exceeding 95% totally. As the fermentation reached 16 h, the proportion of Lactobacillus decreased, the least proportion was only about 17.7%, while the proportion of Staphylococcus increased significantly (p < 0.05) during the same period, with the highest proportion about 81.1%. This phenomenon may be explained by the fact that the L. casei in the initial stage of fermentation took some time to adapt to the environment and did not produce enough organic acids and other metabolites. Therefore, Staphylococcus was able to grow rapidly and exceeded Lactobacillus in quantity. The decrease in proportion of Lactobacillus between 0 h and 16 h was consistent with the results of 0 d and 9 d in the Chinese dry-fermented sausages inoculated with Lactobacillus plantarum and Staphylococcus xylosus [5]. At 32 h, the proportion of Lactobacillus increased significantly (p < 0.05), the highest proportion was about 95.5%, while the proportion of Staphylococcus decreased significantly (p < 0.05), with the least proportion only about 3.6%. At 48 h, the proportion of Lactobacillus remained was still high, over 90% in all samples, while the proportion of Staphylococcus was below 6.4%. The increase in proportion of Lactobacillus at later stage of fermentation suggested that Lactobacillus might grow faster than the other genus bacteria. The decrease in Staphylococcus might be caused by low pH and other anti-microbial substances due to LAB [8]. These results were in accordance with results of 9 d and 18 d in Chinese dry fermented sausages inoculated with Lactobacillus plantarum and Staphylococcus xylosus [5]. During the fermentation period, Lactobacillus and Staphylococcus were dominant, which contributed significantly to ensure product quality and safety. It was confirmed that Lactobacillus and Staphylococcus were believed to be related to lipolysis and proteolysis, and Staphylococcus could reduce the off-flavors and rancidity of samples [5]. In addition, LAB could inhibit the growth of other microorganisms [8]. The bacterial structure analysis confirmed that during squid fermentation, the safety of the sample could be ensured, while the flavor might be improved.

3.7. Mixed Inoculation on Flavor of Squid Surimi Sausage

As discussed above, several studies had confirmed that adding some substances to squid could improve texture [15,16]; however, how the flavor was influenced in those studies remained unclear. Fermentation has been proved to be an effective way of to affect the flavor of fermented meat products [55,56,57]. Therefore, it is worth to elucidate the flavor change during squid fermentation in present study.

3.7.1. Organic Acids

The change in organic acids during fermentation of squid surimi sausage are presented in Figure 4a. The content of lactic acid and acetic acid gradually increased. The content of citric acid in the initial stage of fermentation was relatively high. Afterwards, it gradually decreased during the fermentation. The citric acid in the initial stage may come from the sodium citrate used in the sample processing (in organic acid detection, the citric acid ion was transformed to citric acid). As the pH decreased, the citric acid ions might be transformed to citric acid, and then might be further utilized by microorganisms, as it is well known that citric acid is one of the components of the tricarboxylic acid cycle. In addition, the content of malic acid gradually decreased firstly, then increased at later stage.
In fermented meat products, many studies have confirmed that the organic acid contents increase during the fermentation [58,59,60]. The gradually increased content of organic acid will lead to the decrease in the pH, which will inhibit the growth of unwanted microorganisms [8], and may also benefit the flavor of meat [61]. The change in content of organic acids might be related to microbial activity [8], and the loss of water during the fermentation may also be involved.

3.7.2. Free Amino Acids

Free amino acids are not only compounds contributing to taste, but also important precursors of volatile compounds [61]. Several free amino acids have a special taste [61]. For example, alanine, threonine and proline are associated with sweetness [61] and aspartic acid and glutamic acid are believed to have umami and sour tastes [61]. In the fermentation of other meat products, there have been reports of increase in free amino acids, such as Suanyu [62], dry fermented sausage [63] and silver carp sausages [48].
Figure 4b and Table 2 show the change in free amino acid content during fermentation. It can be easily concluded that the content of many free amino acids such as aspartic acid and glutamic acid gradually increased. The trends of serine, glycine and arginine were different from the other amino acids. At the initial stage, the serine began to decrease and reached its lowest level at 32 h. At 48 h, the concentration increased slightly but remained below the initial concentration. Glycine showed a fluctuating increase throughout the fermentation process, and at 48 h, the concentration was higher than the at beginning of fermentation. As fermentation progressed, the concentration of arginine rapidly decreased, and at the later stage of fermentation, its content increased but still remained lower than at the initial stage. The mechanism of decrease in arginine after fermentation needs further investigation. The concentration of total free amino acids first decreased and then increased during the fermentation process. Analysis showed that at the end of fermentation, the total concentration of free amino acids was significantly higher (p < 0.05) than non-fermentation.
Microorganisms might contribute to the change in free amino acids. On one hand, some lactic acid bacteria, such as L. plantarum 120 [64], may have the ability to degrade proteins. On the other hand, CNS, including S. carnosus, have also been reported to possess various enzyme activities including protease [65]. These microorganisms may contribute to the degradation of proteins. Meanwhile, studies in silver carp have confirmed that decrease in pH could activate endogenous proteases, thereby participating in protein degradation [66]. In addition, as is well known, microorganisms can also use free amino acid to grow, thus affecting the contents of free amino acids. During the fermentation, water loss might also contribute to the change in the concentration of free amino acids.
In summary, present results showed that after fermentation, the free amino acids concentration was changed, with some amino acids with sweetness, umami and sour taste increasing, which might have positive effect on the flavor of the squid. We have discussed a possible mechanism of change in free amnio acids above; however, the specific mechanism needs further investigation.

3.7.3. Electronic Nose Test

The results of electronic nose tests are shown in Figure 4c,d. Statistical analysis showed significant differences in the values of multiple sensors (p < 0.05), and W5S, W1S, W1W, W2S and W2W exhibited distinct patterns on the radar chart (Figure 4c). Principal component analysis (PCA) was performed on the electronic nose results of two groups of samples (Figure 4d). The variance explained by the first principal component (PC1) was 99.78%, and that by PC2 was 0.20%, cumulatively accounting for 99.98% of the total variance. This indicates that these two components captured the primary variation in the samples. Figure 4d shows clear separation between the two sample groups, with data points from the same group clustering together and those from different groups being widely spaced. PCA results suggested that fermentation significantly altered the composition of volatile flavor compounds.

3.7.4. Volatile Flavor Compounds

As shown in Table 3, among the detected compounds, eight metabolites were only detected in the fermented samples (including 1,4-butanediol, 1-methylene-1H-indene,2,3-butanedione, 2-nonanone, acetic acid, acetoin, dimethyl phthalate, formamide). Among these substances, several compounds have unique flavors. For example, acetoin is considered to have a yogurt flavor and a pleasant fat cream flavor [67]. 2,3-butanedione is believed to impart a buttery or cheesy flavor to various foods [68]. 2-Nonanone is considered to contribute to the taste and aroma of some foods [69]. Acetic acid not only has antibacterial effects [8], but also serves as an important flavor compound [70]. The peak areas of four substances (including 4,4-dimethyl-1-pentene,2-heptanone, butyrolactone, 2-(2-ethoxyethoxy)-ethanol) increased after fermentation. Among these compounds, 2-heptanone also has a special flavor [69]. There were two substances (including 5-hydroxy-4-octanone, 3-methyl-butanal) that were only detected in unfermented samples, 3-methyl-butanal, produced by lactic acid bacteria in some fermented foods, was a flavor compound with different flavor attributes in different fermented meat [71]. In the present experiment, the substance was detected in the unfermented sample, which may come from starter. In the fermented sample, it was not detected, possibly due to differences in the growth conditions of the starter. In addition, the peak area of tetrahydrofuran in the fermented sample decreased. In a previous study, researchers have also detected tetrahydrofuran in squid products [72], and the underlying mechanisms for this change during fermentation needs further investigation.
The results of GC-MS indicated that fermentation may help improve the composition of volatile flavor compounds in squid surimi sausages, which may contribute to the improvement of product quality. However, it must be noted that although fermentation can increase various volatile flavor compounds compared to unfermented samples, the types of substances detected are still limited. In future, it might be necessary to add appropriate seasonings or other ingredients to improve flavor.

4. Conclusions

In present study, the results suggested that in mixed inoculation with L. casei and S. carnosus to squid, without added seasonings might be a better choice than that with seasonings added. In samples without the addition of seasonings, compared with unfermented samples, mixed inoculation could improve texture, color and the rheological properties of samples. In mixed inoculated samples, the network was denser, and water distribution was changed, with water less easy to flow. In addition, in the mixed inoculated group, the starters were dominant during fermentation. Compared with the unfermented samples, mixed inoculation could improve the flavor of the samples, with an increase in organic acids, several free amino acids and volatile flavor compounds, and the electronic nose test also showed differences between fermented and unfermented samples. It might be necessary to optimize fermentation conditions to obtain fermented squid with improved flavor and texture in future study.

Author Contributions

Conceptualization, H.M., P.W. and Z.W.; methodology, H.M.; formal analysis, H.M., P.W. and Z.W.; investigation, H.M.; resources, P.W. and Z.W.; data curation, H.M., P.W. and Z.W.; writing—original draft preparation, H.M.; writing—review and editing, H.M., P.W. and Z.W.; supervision, P.W. and Z.W. All authors have read and agreed to the published version of the manuscript.

Funding

We thank Ningbo University for providing a scholarship and financial support to H.M.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Sharma, R.; Garg, P.; Kumar, P.; Bhatia, S.K.; Kulshrestha, S. Microbial Fermentation and Its Role in Quality Improvement of Fermented Foods. Fermentation 2020, 6, 106. [Google Scholar] [CrossRef]
  2. Munekata, P.E.S.; Pateiro, M.; Tomasevic, I.; Domínguez, R.; da Silva Barretto, A.C.; Santos, E.M.; Lorenzo, J.M. Functional Fermented Meat Products with Probiotics—A Review. J. Appl. Microbiol. 2022, 133, 91–103. [Google Scholar] [CrossRef] [PubMed]
  3. Xu, Y.; Xia, W.; Yang, F.; Kim, J.M.; Nie, X. Effect of Fermentation Temperature on the Microbial and Physicochemical Properties of Silver Carp Sausages Inoculated with Pediococcus Pentosaceus. Food Chem. 2010, 118, 512–518. [Google Scholar] [CrossRef]
  4. Essid, I.; Hassouna, M. Effect of Inoculation of Selected Staphylococcus xylosus and Lactobacillus plantarum Strains on Biochemical, Microbiological and Textural Characteristics of a Tunisian Dry Fermented Sausage. Food Control 2013, 32, 707–714. [Google Scholar] [CrossRef]
  5. Xiao, Y.; Liu, Y.; Chen, C.; Xie, T.; Li, P. Effect of Lactobacillus plantarum and Staphylococcus xylosus on Flavour Development and Bacterial Communities in Chinese Dry Fermented Sausages. Food Res. Int. 2020, 135, 109247. [Google Scholar] [CrossRef]
  6. Sang, X.; Ma, X.; Hao, H.; Bi, J.; Zhang, G. Evaluation of Biogenic Amines and Microbial Composition in the Chinese Traditional Fermented Food Grasshopper Sub Shrimp Paste. LWT 2020, 134, 109979. [Google Scholar] [CrossRef]
  7. Rabie, M.; Simon-Sarkadi, L.; Siliha, H.; El-seedy, S.; El Badawy, A.A. Changes in Free Amino Acids and Biogenic Amines of Egyptian Salted-Fermented Fish (Feseekh) during Ripening and Storage. Food Chem. 2009, 115, 635–638. [Google Scholar] [CrossRef]
  8. Laranjo, M.; Potes, M.E.; Elias, M. Role of Starter Cultures on the Safety of Fermented Meat Products. Front. Microbiol. 2019, 10, 853. [Google Scholar] [CrossRef]
  9. Hu, Y.; Xia, W.; Ge, C. Characterization of Fermented Silver Carp Sausages Inoculated with Mixed Starter Culture. LWT 2008, 41, 730–738. [Google Scholar] [CrossRef]
  10. Du, S.; Cheng, H.; Ma, J.K.; Li, Z.J.; Wang, C.H.; Wang, Y.L. Effect of Starter Culture on Microbiological, Physiochemical and Nutrition Quality of Xiangxi Sausage. J. Food Sci. Technol. 2019, 56, 811–823. [Google Scholar] [CrossRef]
  11. Ren, H.; Deng, Y.; Wang, X. Effect of a Compound Starter Cultures Inoculation on Bacterial Profile and Biogenic Amine Accumulation in Chinese Sichuan Sausages. Food Sci. Hum. Wellness 2022, 11, 341–348. [Google Scholar] [CrossRef]
  12. Wang, Y.; Han, J.; Wang, D.; Gao, F.; Zhang, K.; Tian, J.; Jin, Y. Research Update on the Impact of Lactic Acid Bacteria on the Substance Metabolism, Flavor, and Quality Characteristics of Fermented Meat Products. Foods 2022, 11, 2090. [Google Scholar] [CrossRef]
  13. dos Santos Cruxen, C.E.; Funck, G.D.; Haubert, L.; da Silva Dannenberg, G.; de Lima Marques, J.; Chaves, F.C.; da Silva, W.P.; Fiorentini, Â.M. Selection of Native Bacterial Starter Culture in the Production of Fermented Meat Sausages: Application Potential, Safety Aspects, and Emerging Technologies. Food Res. Int. 2019, 122, 371–382. [Google Scholar] [CrossRef]
  14. Tolano-Villaverde, I.J.; Santos-Sauceda, I.; Santacruz-Ortega, H.; Ramírez-Wong, B.; Brown-Bojórquez, F.; Suárez-Jiménez, G.M.; Márquez-Ríos, E. Evaluation of the Gelling Ability of Actomyosin-Paramyosin from Giant Squid Mantle (Dosidicus gigas). Int. Food Res. J. 2020, 27, 712–719. [Google Scholar]
  15. Yang, R.; Xu, A.; Chen, Y.; Sun, N.; Zhang, J.; Jia, R.; Huang, T.; Yang, W. Effect of Laver Powder on Textual, Rheological Properties and Water Distribution of Squid (Dosidicus gigas) Surimi Gel. J. Texture Stud. 2020, 51, 968–978. [Google Scholar] [CrossRef]
  16. Chu, Y.; Deng, S.; Lv, G.; Li, M.; Bao, H.; Gao, Y.; Jia, R. Improvement of Gel Quality of Squid (Dosidicus gigas) Meat by Using Sodium Gluconate, Sodium Citrate, and Sodium Tartrate. Foods 2022, 11, 173. [Google Scholar] [CrossRef]
  17. Niu, F.; Li, X.; Lin, C.; Hu, X.; Zhang, B.; Pan, W. The Mechanism of Egg White Protein to Enhance the Thermal Gel Properties of Giant Squid (Dosidicus gigas) Surimi. Food Chem. 2025, 469, 142601. [Google Scholar] [CrossRef]
  18. Mu, H.; Weng, P.; Wu, Z. Effect of Inoculation with Lacticaseibacillus casei and Staphylococcus carnosus on the Quality of Squid (Dosidicus gigas) Surimi Sausage. Fermentation 2023, 9, 794. [Google Scholar] [CrossRef]
  19. Kim, S.Y.; Dang, Y.M.; Ha, J.H. Effect of Various Seasoning Ingredients on the Accumulation of Biogenic Amines in Kimchi during Fermentation. Food Chem. 2022, 380, 132214. [Google Scholar] [CrossRef]
  20. Yang, W.; Chen, S.; Yu, S.; Zhang, Y. Effects of Different Soaking Processes and Additives on the Acid Removal Efficiency of Squid. Jiangxi Fish. Sci. Technol. 2019, 5, 45–47. [Google Scholar]
  21. GB 5009.208—2016; National Food Safety Standard—Determination of Biogenic Amines in Food, 1st ed. Standards Press of China: Beijing, China, 2017; ISBN 155066·1-53568.
  22. Xu, X.; Cui, H.; Yuan, Z.; Xu, J.; Li, J.; Liu, J.; Liu, H.; Zhu, D. Effects of Different Combinations of Probiotics on Rheology, Microstructure, and Moisture Distribution of Soy Materials-Based Yogurt. J. Food Sci. 2022, 87, 2820–2830. [Google Scholar] [CrossRef] [PubMed]
  23. Mi, J.; Zhao, X.; Huang, P.; Hong, J.; Jia, R.; Deng, S.; Yu, X.; Wei, H.; Yang, W. Effect of Hydroxypropyl Distarch Phosphate on the Physicochemical Characteristics and Structure of Shrimp Myofibrillar Protein. Food Hydrocoll. 2022, 125, 107417. [Google Scholar] [CrossRef]
  24. Yu, X.; Wang, Y.; Xie, Y.; Wei, S.; Ding, H.; Yu, C.; Dong, X. Gelation Properties and Protein Conformation of Grass Carp Fish Ball as Influenced by Egg White Protein. J. Texture Stud. 2022, 53, 277–286. [Google Scholar] [CrossRef] [PubMed]
  25. Martin, M. Cutadapt Removes Adapter Sequences from High-Throughput Sequencing Reads. EMBnet.journal 2011, 17, 10–12. [Google Scholar] [CrossRef]
  26. Callahan, B.J.; McMurdie, P.J.; Rosen, M.J.; Han, A.W.; Johnson, A.J.A.; Holmes, S.P. DADA2: High-Resolution Sample Inference from Illumina Amplicon Data. Nat. Methods 2016, 13, 581–583. [Google Scholar] [CrossRef]
  27. Bolyen, E.; Rideout, J.R.; Dillon, M.R.; Bokulich, N.A.; Abnet, C.C.; Al-Ghalith, G.A.; Alexander, H.; Alm, E.J.; Arumugam, M.; Asnicar, F.; et al. Reproducible, Interactive, Scalable and Extensible Microbiome Data Science Using QIIME 2. Nat. Biotechnol. 2019, 37, 852–857. [Google Scholar] [CrossRef]
  28. Chen, D.W.; Zhang, M. Non-Volatile Taste Active Compounds in the Meat of Chinese Mitten Crab (Eriocheir sinensis). Food Chem. 2007, 104, 1200–1205. [Google Scholar] [CrossRef]
  29. Cawthray, G.R. An Improved Reversed-Phase Liquid Chromatographic Method for the Analysis of Low-Molecular Mass Organic Acids in Plant Root Exudates. J. Chromatogr. A 2003, 1011, 233–240. [Google Scholar] [CrossRef]
  30. Gao, H.; Wang, S.; Liao, S.; Hu, X. Study on Determination and Correlation of Soluble Sugars and Organic Acids in Peer Juice from Different Cultivars. Acta Agric. Boreali-Sin. 2004, 19, 104–107. [Google Scholar]
  31. Nie, X.; Zhang, Q.; Lin, S. Biogenic Amine Accumulation in Silver Carp Sausage Inoculated with Lactobacillus plantarum plus Saccharomyces Cerevisiae. Food Chem. 2014, 153, 432–436. [Google Scholar] [CrossRef]
  32. Hua, Q.; Gao, P.; Xu, Y.; Xia, W.; Sun, Y.; Jiang, Q. Effect of Commercial Starter Cultures on the Quality Characteristics of Fermented Fish-Chili Paste. LWT 2020, 122, 109016. [Google Scholar] [CrossRef]
  33. Mandha, J.; Shumoy, H.; Devaere, J.; Schouteten, J.J.; Gellynck, X.; De Winne, A.; Matemu, A.O.; Raes, K. Effect of Lactic Acid Fermentation on Volatile Compounds and Sensory Characteristics of Mango (Mangifera indica) Juices. Foods 2022, 11, 383. [Google Scholar] [CrossRef] [PubMed]
  34. Yu, H.; Li, P.; Yin, P.; Cai, J.; Jin, B.; Zhang, H.; Lu, S. Bacterial Community Succession and Volatile Compound Changes in Xinjiang Smoked Horsemeat Sausage during Fermentation. Food Res. Int. 2023, 174, 113656. [Google Scholar] [CrossRef]
  35. Gu, X.; Sun, Y.; Tu, K.; Pan, L. Evaluation of Lipid Oxidation of Chinese-Style Sausage during Processing and Storage Based on Electronic Nose. Meat Sci. 2017, 133, 1–9. [Google Scholar] [CrossRef]
  36. Fermentation Standards for Making Fermented Sausages. Available online: https://www.meatsandsausages.com/sausage-types/fermented-sausage/standards (accessed on 3 August 2024).
  37. Hernández-Jover, T.; Izquierdo-Pulido, M.; Veciana-Nogués, M.T.; Mariné-Font, A.; Vidal-Carou, M.C. Effect of Starter Cultures on Biogenic Amine Formation during Fermented Sausage Production. J. Food Prot. 1997, 60, 825–830. [Google Scholar] [CrossRef]
  38. del Rio, B.; Redruello, B.; Linares, D.M.; Ladero, V.; Ruas-Madiedo, P.; Fernandez, M.; Martin, M.C.; Alvarez, M.A. Spermine and Spermidine Are Cytotoxic towards Intestinal Cell Cultures, but Are They a Health Hazard at Concentrations Found in Foods? Food Chem. 2018, 269, 321–326. [Google Scholar] [CrossRef]
  39. Wang, H.; Zhang, H.; Liu, S.; Qin, L.; Chen, Q.; Kong, B. Analysis of Biogenic Amine in Dry Sausages Collected from Northeast China: From the Perspective of Free Amino Acid Profile and Bacterial Community Composition. Food Res. Int. 2022, 162, 112084. [Google Scholar] [CrossRef]
  40. Fan, H.; Luo, Y.; Yin, X.; Bao, Y.; Feng, L. Biogenic Amine and Quality Changes in Lightly Salt- and Sugar-Salted Black Carp (Mylopharyngodon piceus) Fillets Stored at 4 °C. Food Chem. 2014, 159, 20–28. [Google Scholar] [CrossRef]
  41. Zarei, M.; Najafzadeh, H.; Eskandari, M.H.; Pashmforoush, M.; Enayati, A.; Gharibi, D.; Fazlara, A. Chemical and Microbial Properties of Mahyaveh, a Traditional Iranian Fish Sauce. Food Control 2012, 23, 511–514. [Google Scholar] [CrossRef]
  42. Lu, S.; Xu, X.; Zhou, G.; Zhu, Z.; Meng, Y.; Sun, Y. Effect of Starter Cultures on Microbial Ecosystem and Biogenic Amines in Fermented Sausage. Food Control 2010, 21, 444–449. [Google Scholar] [CrossRef]
  43. Dias, I.; Laranjo, M.; Potes, M.E.; Agulheiro-Santos, A.C.; Ricardo-Rodrigues, S.; Fialho, A.R.; Véstia, J.; Fraqueza, M.J.; Oliveira, M.; Elias, M. Co-Inoculation with Staphylococcus equorum and Lactobacillus sakei Reduces Vasoactive Biogenic Amines in Traditional Dry-Cured Sausages. Int. J. Environ. Res. Public Health 2021, 18, 7100. [Google Scholar] [CrossRef] [PubMed]
  44. Ganguly, S.; Sabikhi, L.; Singh, A.K. Effect of Probiotic Fermentation on Physico-Chemical and Nutritional Parameters of Milk-Cereal Based Composite Substrate. J. Food Sci. Technol. 2022, 59, 3073–3085. [Google Scholar] [CrossRef] [PubMed]
  45. Amiri, A.; Sharifian, P.; Soltanizadeh, N. Application of Ultrasound Treatment for Improving the Physicochemical, Functional and Rheological Properties of Myofibrillar Proteins. Int. J. Biol. Macromol. 2018, 111, 139–147. [Google Scholar] [CrossRef]
  46. Wang, H.; Yang, Z.; Yang, H.; Xue, J.; Li, Y.; Wang, S.; Ge, L.; Shen, Q.; Zhang, M. Comparative Study on the Rheological Properties of Myofibrillar Proteins from Different Kinds of Meat. LWT 2022, 153, 112458. [Google Scholar] [CrossRef]
  47. Lv, J.; Li, C.; Li, S.; Liang, H.; Ji, C.; Zhu, B.; Lin, X. Effects of Temperature on Microbial Succession and Quality of Sour Meat during Fermentation. LWT 2019, 114, 108391. [Google Scholar] [CrossRef]
  48. Hu, Y.; Xia, W.; Ge, C. Effect of Mixed Starter Cultures Fermentation on the Characteristics of Silver Carp Sausages. World J. Microbiol. Biotechnol. 2007, 23, 1021–1031. [Google Scholar] [CrossRef]
  49. Zeng, X.; Xia, W.; Jiang, Q.; Yang, F. Effect of Autochthonous Starter Cultures on Microbiological and Physico-Chemical Characteristics of Suan Yu, a Traditional Chinese Low Salt Fermented Fish. Food Control 2013, 33, 344–351. [Google Scholar] [CrossRef]
  50. Wang, D.; Cheng, F.; Wang, Y.; Han, J.; Gao, F.; Tian, J.; Zhang, K.; Jin, Y. The Changes Occurring in Proteins during Processing and Storage of Fermented Meat Products and Their Regulation by Lactic Acid Bacteria. Foods 2022, 11, 2427. [Google Scholar] [CrossRef]
  51. Xu, Y.; Xia, W.; Yang, F.; Nie, X. Protein Molecular Interactions Involved in the Gel Network Formation of Fermented Silver Carp Mince Inoculated with Pediococcus pentosaceus. Food Chem. 2010, 120, 717–723. [Google Scholar] [CrossRef]
  52. Liu, Y.; Sun, Q.; Pan, Y.; Wei, S.; Xia, Q.; Liu, S.; Ji, H.; Deng, C.; Hao, J. Investigation on the Correlation between Changes in Water and Texture Properties during the Processing of Surimi from Golden Pompano (Trachinotus ovatus). J. Food Sci. 2021, 86, 376–384. [Google Scholar] [CrossRef]
  53. Li, D.Y.; Tan, Z.F.; Liu, Z.Q.; Wu, C.; Liu, H.L.; Guo, C.; Zhou, D.Y. Effect of Hydroxyl Radical Induced Oxidation on the Physicochemical and Gelling Properties of Shrimp Myofibrillar Protein and Its Mechanism. Food Chem. 2021, 351, 129344. [Google Scholar] [CrossRef] [PubMed]
  54. Pan, J.; Jia, H.; Shang, M.; Xu, C.; Lian, H.; Li, H.; Dong, X. Physiochemical Properties and Tastes of Gels from Japanese Spanish Mackerel (Scomberomorus niphonius) Surimi by Different Washing Processes. J. Texture Stud. 2018, 49, 578–585. [Google Scholar] [CrossRef] [PubMed]
  55. Shen, Y.; Wu, Y.; Wang, Y.; Li, L.; Li, C.; Zhao, Y.; Yang, S. Contribution of Autochthonous Microbiota Succession to Flavor Formation during Chinese Fermented Mandarin Fish (Siniperca chuatsi). Food Chem. 2021, 348, 129107. [Google Scholar] [CrossRef] [PubMed]
  56. Yue, Y.; Zhao, N.; Yin, S.; Zhang, S.; Ji, C.; Chen, Y.; Dai, Y.; Zhu, B.; Lin, X. Flavour Enhancement of Dry Fermented Sausages by Nitrite-Degrading Levilactobacillus brevis CHOL1: Combining Flavouromics and Lipidomics to Elucidate the Mechanism of Aroma Formation. Food Chem. 2025, 473, 143119. [Google Scholar] [CrossRef]
  57. Chen, L.; Wei, C.; Liu, R.; Wu, M.; Ge, Q.; Yu, H. Enhancement of the Flavor Compound 3-Methylbutanal and Sensory Quality in Sausages Fermented by Lactococcus lactis CGMCC 31087: Leucine and α-Ketoglutaric Acid as Supplements. Food Chem. 2025, 473, 143091. [Google Scholar] [CrossRef]
  58. Milićević, B.; Danilović, B.; Džinić, N.; Savić, D. Changes in the Organic Acids Content during the Production of the Petrovac Sausage. Adv. Technol. 2018, 7, 41–45. [Google Scholar] [CrossRef]
  59. Yoo, S.A.; Park, S.E.; Seo, S.H.; Son, H.S. Quality Characteristics of Fermented Sausage Prepared with Soy Sauce. Food Sci. Biotechnol. 2016, 25, 533–539. [Google Scholar] [CrossRef]
  60. Sidira, M.; Kandylis, P.; Kanellaki, M.; Kourkoutas, Y. Effect of Curing Salts and Probiotic Cultures on the Evolution of Flavor Compounds in Dry-Fermented Sausages during Ripening. Food Chem. 2016, 201, 334–338. [Google Scholar] [CrossRef]
  61. Dashdorj, D.; Amna, T.; Hwang, I. Influence of Specific Taste-Active Components on Meat Flavor as Affected by Intrinsic and Extrinsic Factors: An Overview. Eur. Food Res. Technol. 2015, 241, 157–171. [Google Scholar] [CrossRef]
  62. Wang, W.; Xia, W.; Gao, P.; Xu, Y.; Jiang, Q. Proteolysis during Fermentation of Suanyu as a Traditional Fermented Fish Product of China. Int. J. Food Prop. 2017, 20, S166–S176. [Google Scholar] [CrossRef]
  63. Liu, Y.; Wan, Z.; Yohannes, K.W.; Yu, Q.; Yang, Z.; Li, H.; Liu, J.; Wang, J. Functional Characteristics of Lactobacillus and Yeast Single Starter Cultures in the Ripening Process of Dry Fermented Sausage. Front. Microbiol. 2021, 11, 611260. [Google Scholar] [CrossRef] [PubMed]
  64. Wang, W.; Xia, W.; Gao, P.; Xu, Y. Sarcoplasmic Protein Hydrolysis Activity of Lactobacillus plantarum 120 Isolated from Suanyu: A Traditional Chinese Low Salt Fermented Fish. J. Food Process. Preserv. 2017, 41, e12821. [Google Scholar] [CrossRef]
  65. Heo, S.; Lee, J.H.; Jeong, D.W. Food-Derived Coagulase-Negative Staphylococcus as Starter Cultures for Fermented Foods. Food Sci. Biotechnol. 2020, 29, 1023–1035. [Google Scholar] [CrossRef] [PubMed]
  66. Yang, F.; Xia, W.; Zhang, X.; Xu, Y.; Jiang, Q. A Comparison of Endogenous and Microbial Proteolytic Activities during Fast Fermentation of Silver Carp Inoculated with Lactobacillus plantarum. Food Chem. 2016, 207, 86–92. [Google Scholar] [CrossRef]
  67. Xiao, Z.; Lu, J.R. Strategies for Enhancing Fermentative Production of Acetoin: A Review. Biotechnol. Adv. 2014, 32, 492–503. [Google Scholar] [CrossRef]
  68. Gao, R.; Chen, L.; Li, Y.; Wang, Y.; Yang, T.; Li, X.; Geng, J.; Guo, Z. Polyoxometalate Cluster-Guided Dynamic Nucleation and Hierarchical Growth of Branched WO3 Nanofibers with Ultrafine Pt Nanoparticles for Advanced Gas Sensing. Inorg. Chem. 2024, 63, 18285–18295. [Google Scholar] [CrossRef]
  69. Yan, Q.; Simmons, T.R.; Cordell, W.T.; Hernández Lozada, N.J.; Breckner, C.J.; Chen, X.; Jindra, M.A.; Pfleger, B.F. Metabolic Engineering of β-Oxidation to Leverage Thioesterases for Production of 2-Heptanone, 2-Nonanone and 2-Undecanone. Metab. Eng. 2020, 61, 335–343. [Google Scholar] [CrossRef]
  70. Hu, Y.; Zhang, L.; Wen, R.; Chen, Q.; Kong, B. Role of Lactic Acid Bacteria in Flavor Development in Traditional Chinese Fermented Foods: A Review. Crit. Rev. Food Sci. Nutr. 2022, 62, 2741–2755. [Google Scholar] [CrossRef]
  71. Chen, L.; Liu, R.; He, C.; Wu, M.; Ge, Q.; Yu, H. Biosynthesis Pathway of Flavor Compound 3-Methylbutanal Derived from Leucine by Lactococcus lactis 517: From a Transcriptomics Perspective. Food Biosci. 2024, 61, 104740. [Google Scholar] [CrossRef]
  72. Chen, T. Research on Thermal Processing Characteristics and Flavor of Dosidicus gigas Carcass Meat. Master’s Thesis, Shanghai Ocean University, Shanghai, China, 2023. [Google Scholar]
Fermentation 11 00404 g001
Figure 1. pH (a) and biogenic amines (b) of different groups of fermented giant squid; complex viscosity (c), storage modulus (d) and loss modulus (e) of unfermented and fermented giant squid. UF, CK, LC and LS refer to unfermented samples, non-inoculated samples, samples inoculated with L. casei and samples mixed inoculated with L. casei and S. carnosus, respectively.
Figure 1. pH (a) and biogenic amines (b) of different groups of fermented giant squid; complex viscosity (c), storage modulus (d) and loss modulus (e) of unfermented and fermented giant squid. UF, CK, LC and LS refer to unfermented samples, non-inoculated samples, samples inoculated with L. casei and samples mixed inoculated with L. casei and S. carnosus, respectively.
Fermentation 11 00404 g001
Fermentation 11 00404 g002
Figure 2. Microstructure of unfermented (a) and fermented (b) giant squid; WHC (c) and T2 relaxation time distribution (d) of unfermented and fermented giant squid; T2 relaxation time (e–g) and peak area (h–j) of unfermented and fermented giant squid. UF and LS represent unfermented samples and fermented samples, respectively.
Figure 2. Microstructure of unfermented (a) and fermented (b) giant squid; WHC (c) and T2 relaxation time distribution (d) of unfermented and fermented giant squid; T2 relaxation time (e–g) and peak area (h–j) of unfermented and fermented giant squid. UF and LS represent unfermented samples and fermented samples, respectively.
Fermentation 11 00404 g002
Fermentation 11 00404 g003
Figure 3. Changes in bacterial community structure at phylum (a) and genus (b) level during different stages of fermentation (Top 15). A, B, C and D refer to the samples at 0 h, 16 h, 32 h and 48 h, respectively. Some legend colors are not well displayed in the bar chart because their proportions are small.
Figure 3. Changes in bacterial community structure at phylum (a) and genus (b) level during different stages of fermentation (Top 15). A, B, C and D refer to the samples at 0 h, 16 h, 32 h and 48 h, respectively. Some legend colors are not well displayed in the bar chart because their proportions are small.
Fermentation 11 00404 g003
Fermentation 11 00404 g004
Figure 4. Changes in organic acids (a) and free amino acids (b) during different stages of giant squid fermentation; radar chart (c) and PCA (d) of the results of electronic nose test of unfermented and fermented giant squid. In (b), FAA is short for free amino acid. In (c), UF represent unfermented sample, while LS represent fermented sample mixed inoculated with L. casei and S. carnosus. In (d), red and bule represent unfermented samples and fermented samples, respectively.
Figure 4. Changes in organic acids (a) and free amino acids (b) during different stages of giant squid fermentation; radar chart (c) and PCA (d) of the results of electronic nose test of unfermented and fermented giant squid. In (b), FAA is short for free amino acid. In (c), UF represent unfermented sample, while LS represent fermented sample mixed inoculated with L. casei and S. carnosus. In (d), red and bule represent unfermented samples and fermented samples, respectively.
Fermentation 11 00404 g004
Table 1. The texture and color of unfermented samples and mixed inoculated samples. UF and LS indicate unfermented samples and samples mixed inoculated with L. casei and S. carnosus, respectively.
Table 1. The texture and color of unfermented samples and mixed inoculated samples. UF and LS indicate unfermented samples and samples mixed inoculated with L. casei and S. carnosus, respectively.
UFLS
TPA
Hardness (g)1545.88 ± 269.41 a5011.78 ± 263.37 b
Springiness0.95 ± 0.02 a0.94 ± 0.04 a
Cohesiveness0.86 ± 0.01 a0.77 ± 0.02 b
Chewiness (g)1262.38 ± 232.37 a3620.55 ± 162.65 b
Color
L*84.68 ± 0.74 a90.68 ± 0.46 b
a*−0.92 ± 0.40 a0.02 ± 0.32 b
b*2.95 ± 0.40 a4.76 ± 0.48 b
Different lowercase letters indicate significant difference between different samples (p < 0.05).
Table 2. Free amino acids concentration of squid surimi sausage during different stages of fermentation (mg/100 g).
Table 2. Free amino acids concentration of squid surimi sausage during different stages of fermentation (mg/100 g).
0 h16 h32 h48 h
asp6.82 ± 0.06 a9.60 ± 0.13 b11.55 ± 0.92 b17.58 ± 0.12 c
glu9.14 ± 0.14 a23.39 ± 0.29 b33.78 ± 4.27 bc49.69 ± 0.69 cd
ser1.85 ± 0.02 a1.64 ± 0.09 b1.33 ± 0.02 c1.64 ± 0.12 b
his15.43 ± 0.32 a18.64 ± 0.12 b21.58 ± 2.28 abc23.96 ± 0.21 c
gly5.74 ± 0.10 a6.39 ± 0.32 ab4.99 ± 0.92 ab7.25 ± 0.09 bc
thr6.19 ± 0.12 a6.99 ± 0.24 a7.12 ± 1.14 ac10.55 ± 0.03 bc
arg52.95 ± 0.96 a1.52 ± 0.05 b1.86 ± 0.27 b5.50 ± 0.14 c
ala14.76 ± 0.25 a16.76 ± 0.36 b18.48 ± 2.18 abc21.91 ± 0.31 c
tyr4.23 ± 0.60 a6.17 ± 0.15 a6.36 ± 1.15 ab7.38 ± 0.33 c
cys-s0.59 ± 0.08 a1.02 ± 0.11 b1.16 ± 0.25 bc1.32 ± 0.06 c
val4.86 ± 0.12 a8.14 ± 0.36 b8.71 ± 1.12 abc12.76 ± 0.23 c
met6.05 ± 0.33 a8.42 ± 0.21 b10.44 ± 1.26 abc13.03 ± 0.20 c
trp2.28 ± 0.10 a2.8 ± 0.10 a3.73 ± 0.76 b4.37 ± 0.26 b
phe4.17 ± 0.60 a6.07 ± 0.50 a7.42 ± 1.10 ac9.90 ± 0.08 bc
ile2.83 ± 0.23 a3.63 ± 0.40 a3.30 ± 0.55 ac5.25 ± 0.02 bc
leu5.49 ± 0.14 a7.92 ± 0.38 bc8.79 ± 1.26 acd13.94 ± 0.11 d
lys6.34 ± 0.12 a9.62 ± 0.38 bc10.57 ± 1.36 ac16.02 ± 0.45 d
pro11.63 ± 0.29 a15.23 ± 0.18 bc17.98 ± 1.46 cd18.59 ± 0.18 d
Total FAA161.32 ± 4.36 a153.96 ± 3.37 a179.14 ± 22.01 ab240.65 ± 1.22 b
Different lowercase letters indicate significant difference among different samples (p < 0.05).
Table 3. Volatile flavor compounds of unfermented and fermented samples.
Table 3. Volatile flavor compounds of unfermented and fermented samples.
No.Molecular FormulaMetaboliteAverage Peak AreaNumber of Samples Identifying the Compound/Number of Samples
unfermented sample
1C7H144,4-dimethyl-1-pentene 17,140.52/4
2C7H14O2-Heptanone55,164.52/4
3C8H16O25-Hydroxy-4-octanone45,685.52/4
4C5H10O3-methyl-butanal48,651.673/4
5C4H6O2Butyrolactone79,0393/4
6C6H14O32-(2-ethoxyethoxy)-ethanol81,0582/4
7C4H8OTetrahydrofuran394,964.54/4
fermented samples
1C4H10O21,4-Butanediol154,926.73/5
2C10H81-methylene-1H-indene42,636.52/5
3C7H144,4-dimethyl-1-pentene 18,7711/5
4C4H6O22,3-Butanedione223,684.25/5
5C7H14O2-Heptanone232,499.45/5
6C9H18O2-Nonanone258,866.65/5
7C2H4O2Acetic acid627,612.25/5
8C4H8O2Acetoin46,4444/5
9C4H6O2Butyrolactone283,026.25/5
10C10H10O4Dimethyl phthalate201,618.65/5
11C6H14O32-(2-ethoxyethoxy)-ethanol260,846.33/5
12CH3NOFormamide32,999.52/5
13C4H8OTetrahydrofuran243,857.73/5
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Mu, H.; Weng, P.; Wu, Z. Mixed Inoculation with Lacticaseibacillus casei and Staphylococcus carnosus Improves Safety, Gel Properties and Flavor of Giant Squid Surimi Without Added Seasonings. Fermentation 2025, 11, 404. https://doi.org/10.3390/fermentation11070404

AMA Style

Mu H, Weng P, Wu Z. Mixed Inoculation with Lacticaseibacillus casei and Staphylococcus carnosus Improves Safety, Gel Properties and Flavor of Giant Squid Surimi Without Added Seasonings. Fermentation. 2025; 11(7):404. https://doi.org/10.3390/fermentation11070404

Chicago/Turabian Style

Mu, Hongliang, Peifang Weng, and Zufang Wu. 2025. "Mixed Inoculation with Lacticaseibacillus casei and Staphylococcus carnosus Improves Safety, Gel Properties and Flavor of Giant Squid Surimi Without Added Seasonings" Fermentation 11, no. 7: 404. https://doi.org/10.3390/fermentation11070404

APA Style

Mu, H., Weng, P., & Wu, Z. (2025). Mixed Inoculation with Lacticaseibacillus casei and Staphylococcus carnosus Improves Safety, Gel Properties and Flavor of Giant Squid Surimi Without Added Seasonings. Fermentation, 11(7), 404. https://doi.org/10.3390/fermentation11070404

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop