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Article

Transforming Tilapia into Indoleacetic Acid-Containing Biostimulants: Synergistic Effect of Enzymolysis and Multi-Strain Fermentation

by
Hanyi Xie
1,
Bin Zhong
1,2,
Qimin Zhang
1,3,
Xi Hu
1,2,
Xuesen Xia
1,2,
Hong Xie
1,3 and
Zhenqiang Wu
1,*
1
School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China
2
Pan Asia (Jiangmen) Institute of Biological Engineering and Health, Jiangmen 529080, China
3
Yiwyi Biofabrication (Jiangmen) Co., Ltd., Jiangmen 529080, China
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(9), 525; https://doi.org/10.3390/fermentation11090525
Submission received: 11 July 2025 / Revised: 27 August 2025 / Accepted: 4 September 2025 / Published: 8 September 2025
(This article belongs to the Special Issue Fermentation of Organic Waste for High-Value-Added Product Production—2nd Edition)
Browse Figures
Versions Notes

Abstract

Following new trends in green development, many studies have focused on the high-value utilization of fish resources through green biological processes. This study innovatively introduced a one-step process of mixed strain–enzyme synergy (MES) with which to prepare tilapia hydrolysates and explored the synergistic effects of strains and enzymes on both the protein hydrolysis process and its products’ characteristics via comparative experiments. Further, soybean was used as a model crop to verify the agronomic effects of the hydrolysates. The addition of exogenous papain increased hydrolysis by 31.94% compared to the fermentation-only group. Peptides and amino acids contents in the mixed strains were higher than those in the single fermentation process (p < 0.05), while 8.46 mg/L of indoleacetic acid was produced through fermentation. Hydrolysates promoted the growth of lateral roots in soybean seedlings (p < 0.05) via the use of a 2500-fold dilution of the biostimulant, increasing the root area and stem length and reducing the sugar content of soybean seedlings by 1.59-, 1.44- and 1.69-fold compared to those in Hoagland’s nutrient solution. These results lay a foundation for the biological preparation of biostimulants for hydroponic vegetables through the utilization of fish waste resources, aligning with green development goals.

1. Introduction

The world’s population is continuing to increase, causing the area of arable land available for agriculture to decrease and thereby highlighting the utilization of the limited resources to meet high crop yield demands as a major issue. Chemical fertilizers and pesticides have been used in response to this problem, but their persistent misuse usually causes irreversible damage to the environment on which we depend [1]. In recent years, China has urged for the sustainable development of environmentally friendly strategies, leading to increased research on the use of biostimulants [2]. Seven main categories of plant biostimulants have been reported, including protein hydrolysates and other nitrogen (N)-containing compounds, chitosan and other biopolymers, seaweed extracts and botanicals, humic and fulvic acids, beneficial fungi and bacteria and inorganic compounds [3].
The metabolic effects of protein hydrolysates on plants include promoting plant root robustness and plant-associated bacteria growth, increasing the rate of photosynthesis and crop yields, and enhancing plant resilience [4,5]. In recent years, the demand for high-quality and convenient processed fishery products (such as fish fillets and fish balls) has increased, as has the accompanying fishery waste. The main types of such waste include minced meat, which is difficult to separate from fish bones during processing, and discarded fish meat, which fails to meet standards for shape or freshness but has qualified protein content. Additionally, Guangdong, a major tilapia-producing region, has experienced large-scale tilapia mortality in its aquaculture industry due to water pollution or improper management; these protein-rich dead tilapia (including their skin and meat) have become a major source of waste, with their recycling becoming an urgent local issue. Converting these high-protein wastes into protein hydrolysate-based biostimulants with positive regulatory effects on plants will reduce pollution and enhance the economic value of the waste.
Animal protein peptides and amino acids are mainly produced via chemical methods, and enzymatic and microbial fermentation [6,7]. The chemical method usually requires strong acids or bases to break the peptide bonds of proteins to obtain amino acids and small peptides, a highly efficient process involving a high degree of hydrolysis. However, the use of strong acids and bases places a high demand on the equipment and may destroy some unstable amino acids, thus altering the nutritional value and quality of the product. On the other hand, the use of enzymes is harmless to the environment and retains the nutritional value of the product, though their specificity may lead to low substrate hydrolysis levels and limited utilization of the raw material [8]. Additionally, microbial fermentation hydrolyzes proteins and produces biologically active metabolites, providing the products with more functional properties and nutritional value, though via a long production cycle with low efficiency. Most of the reported methods for producing peptides and amino acids combine animal endogenous and commercial proteases to reduce the number of commercial enzymes needed [9,10]. To increase protease yields, other researchers use genetic modification or a complex process of initial hydrolysis of protein feedstocks followed by fermentation with commercial proteases [11]. The structure and function of protein hydrolysis products are affected by the differences in the specificity of proteases and the degree of hydrolysis of proteins. Therefore, mixing bacteria that produce different types of proteases may also improve the utilization of proteins and obtain a richer variety of amino acids due to the different sites of action. The combination of enzyme digestion and fermentation technology can improve the efficiency of fermentation, enhance product quality and process stability, and reduce production costs. Moreover, its mild reaction conditions, product safety, and low pollution levels are in line with current concepts of green environmental protection. The use of mixed strains in fermentation can also give full play to the synergistic effect of the complex enzyme system secreted by each strain, thereby achieving efficient substrate utilization, among other effects [12].
Therefore, this study used skin-on tilapia meat from large-scale mortality events in Guangdong (caused by improper management) as its raw material, aiming to simultaneously improve the raw material utilization rate and the plant-regulating ability of the product. The study adopted a mixed strain–enzyme synergy strategy (MES) and successfully prepared fish-derived protein hydrolysate-based plant biostimulants containing indoleacetic acid (IAA). Additionally, by determining the contents of soluble protein, amino acid nitrogen (AAN), trichloroacetic acid (TCA)-soluble peptides, and IAA, we further clarified the interaction mechanism between papain and bacterial strains during the preparation process.

2. Materials and Methods

2.1. Materials

Tilapia weighing about 600–700 g were purchased from the local market (Guangdong, China), while papain (>200 U/mg) and other analytically pure reagents were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Salkowskis reagent and Hoagland nutrient solution were purchased from Fuzhou Phygene Biotechnology Co., Ltd. (Fuzhou, China). The bacterial strains used for fermentation included Bacillus velezensis KA2 and B. amyloliquefaciens BF2 from the School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China and Enterococcus faecalis GDMCC 62,869 (F) from Guangzhou Institute of Microbiology Group Co., Ltd., Guangzhou, China. The media for strain cultivation and isolation are the Luria–Bertani (LB) medium and Enterococcus faecalis Agar.
The LB medium g/L was characterized as follows: peptone 10.0, NaCl 5.0, glucose 1.0, yeast paste powder 5.0, agar 15.0, pH 7.0.
Enterococcus faecalis Agar g/L was characterized as follows: peptone 10.0, maltose 20.0, yeast infusion 10.0, sodium glycerophosphate 10.0, NaCl 5.0, lactose 1.0, bromocresol violet 0.015, NaN3 0.4, TTC 0.1, agar 13.0, pH 7.4.

2.2. Pretreatment of Tilapia

The obtained tilapia were slaughtered, and their heads, viscera, bones, scales, fins and tails were removed. The remaining fish meat with skin (FMS) was rinsed with water and cut into small pieces measuring about 3 cm × 3 cm, followed by crushing at 30,000 rpm for 2 min with a crusher (high-speed pulverizer, Xinxiang, China) to obtain FMS for fermentation. The composition of FMS is shown in Table 1. The contents of protein, K2O, and P2O5 in FMS were determined using the Kjeldahl method, ammonium molybdate spectrophotometry, and flame photometry, respectively [13]. The direct drying, Soxhlet, and ignition methods were applied to measure the moisture, ash, and fat contents, respectively [14].

2.3. Treatment of Fish Meat with Skin (FMS) with Bacteria Strains and Papain

The FMS was mixed in a ratio of 1:1 (v:v) with distilled water, followed by the addition of 5% (w/w) glucose as a carbon source for strain growth. The mixture was then sterilized at 121 °C for 15 min with a GI100TR model autoclave (Zealway Instrument Inc., Xiamen, China) and mixed with 0.1% papain (M1), 5% mixed-strain culture (MSC) (M2), or a combination of 0.1% papain and 5% MSC (M3). The MSC was composed of BF2, KA2 and F in a ratio of 1:2:2 (v:v:v); this ratio was selected based on the data presented in Supplementary Material S1. All substances were added based on the wet weight of FMS. The initial cell density of each strain during inoculation was uniformly controlled at OD600 = 0.8. The omitted components were substituted with an equivalent volume of sterile water, using no enzyme or bacteria as the control (CK). All the mixtures were incubated at 37 °C with shaking at 180 rpm. Daily sampling was conducted to determine the degree of amino acid nitrogen hydrolysis; on the 6th day of fermentation, additional samples were taken to measure the contents of soluble protein, AAN, and TCA-soluble peptides. Thereafter, the fermented mixture was centrifuged at 8000 rpm for 15 min at 4 °C to obtain detection samples.

2.4. Single or Mixed Microbial Fermentation of Fish Meat with Skin (FMS) in the Presence of Papain Synergism

The FMS was mixed with distilled water at a ratio of 1:0.8 (w:v). The medium contained 7% (w/w) and 0.7% (w/w) glucose and magnesium sulphate (MgSO4), respectively, calculated based on the wet weight of FMS. Four control groups (J1–J4) were set up and supplemented with 0.1% (w/w) papain. Each group was inoculated with 5% (v/v) of different strains: J1, J2, J3, and J4 with strains KA2, BF2, F, and MSC, respectively. All groups underwent bacterial-enzyme synergistic fermentation at 37 °C for 6 days to explore how single- vs. mixed-strain fermentation affects product differences under papain synergism. Samples were collected every 24 h, and the physicochemical indices of Group J4 were dynamically monitored to analyze the dynamic process of preparing the fish-derived protein hydrolysate-based biostimulants (FPBs) from FMS. All the fermented mixtures were centrifuged at 8000 rpm for 15 min at 4 °C to obtain detection samples. The Group J4 supernatant (mixed-strain fermentation) was defined as an FPB on the 6th day of fermentation.

2.5. Analytical Method

2.5.1. Determination of Soluble Protein and Trichloroacetic Acid (TCA)-Soluble Peptide

All the fermented samples were mixed with an equal amount of 15% TCA and allowed to stand at room temperature for 30 min to precipitate the proteins. The supernatant was then used to determine the TCA-soluble peptide and soluble protein contents according to the Lowry [15] and Bradford methods, respectively.

2.5.2. Analysis of Amino Acids and Hydrolysis Degree

The AAN content was tested using the colorimetric method. Exactly 2 mL of FPB was mixed with 4 mL of sodium acetate-acetic acid buffer solution (pH 4.8) and 4 mL of color developer (containing 37% methanol and acetylacetone), homogenized, and heated at 100 °C for 15 min. The solution was then cooled to room temperature, and its absorbance was measured at 400 nm, with the results (Ns) being calculated from the standard curve. The AAN content of the non-fermented supernatant (N0) treatment was also assayed using the same method. The total nitrogen content of the fermented broth (Nt) was determined using the Kjeldahl method [16], while the composition and content of free amino acids in single and mixed FPBs were determined using an automatic amino acid analyzer (membraPure A300 Advanced, membraPure GmbH, Hennigsdorf, Germany) with detection wavelengths of 440 nm and 570 nm. The degree of hydrolysis of AAN (DH) was calculated according to Equation (1).
DH ( % ) = N s − N 0 N t − N 0 × 100

2.5.3. Indoleacetic Acid Analysis

Indoleacetic acid content was analyzed using the Salkowskis colorimetric method [17]. An appropriately diluted FPB was mixed with Salkowskis reagent at a ratio of 1:2 (v:v) and kept in the dark for 30 min. Afterwards, the absorbance was measured at 530 nm and the IAA content was calculated by comparing it with the IAA standard curve.

2.6. Protease Activity Analysis

The crude enzyme solution was obtained by centrifugation at 8000 rpm for 10 min at 4 °C every 24 h during fermentation. The activities of acidic protease (pH 3.0) and neutral protease (pH 7.5) were each determined using 10 mg/mL casein solution at the corresponding pH as the substrate. The crude enzyme solution was mixed with the casein solution in equal parts and then heated at 37 °C for 10 min. The enzyme reaction was terminated by adding twice the volume of 0.4 M TCA solution for 10 min, followed by measuring the absorbance of tyrosine at 280 nm against its plotted standard curve [18].

2.7. Viable Bacteria Count Analysis

During the fermentation process, 100 μL of fermented broth was collected every 24 h and diluted with sterile water. Appropriate dilutions were then selected and coated onto LB and E. faecalis agar plates, incubated at 37 °C for 24 h, and counted. The log CFU/mL of viable bacteria was calculated as the ordinates and used to draw the curve of the change in the total number of viable bacteria.

2.8. Determination of Growth Soybean Seedlings

In this study, commercially available Hoagland nutrient solution, diluted 80 times, served as the commercial control, while ultrapure water was used as a blank control for soybean seedling cultivation. The FPB was diluted 100, 1000, and 2500 times with ultrapure water. All solutions (the diluted Hoagland solution, diluted FPB, and ultrapure water) were adjusted to pH 6.0 ± 0.1 before the experiment. Soybean seeds were first soaked in a 2% calcium hypochlorite solution for 10 min, rinsed 2–3 times in sterile water, soaked in sterile water for 12 h, and then germinated at 25 °C for 3 days. Soybean sprouts with consistent bud lengths were selected and immersed in the above-mentioned solutions for 48 h in the dark. Afterwards, the root area and stem length were calculated using Image J software, followed by counting the number of lateral roots. For reducing sugar content in dried soybean sprouts (40 °C drying), 3,5-dinitrosalicylic acid colorimetry was used as follows: the dried sprouts were crushed (50-mesh sieve), 0.4 g of the sample was weighed and mixed with 6 mL distilled water, and ultrasonic extraction was carried out at 50 °C for 30 min to obtain the reducing sugar solution for the assay.

2.9. Statistical Analysis

All statistical analyses were performed using Origin 2021 software (OriginLab Corporation, Northampton, MA, USA). Data were analyzed by analysis of variance using SPSS 27.0 software (IBM Corporation, Chicago, IL, USA), with p < 0.05 indicating a significant difference between samples.

3. Results and Discussion

3.1. Synergistic Effect of Papain and Mixed Bacterial Strains on Fish Meat with Skin (FMS) Hydrolysis

Soluble protein, TCA-soluble peptide, and AAN contents are important indicators of the hydrolysis effect of protein [19,20]. On the sixth day of fermentation, the soluble protein content in the M1, M2, and M3 experimental groups was reduced by 88.68, 91.47, and 97.41%, respectively, compared with the CK group. The contents of soluble proteins in Group M3 decreased by 77.16% and 69.67% compared with that in the M1 and M2 Groups, respectively (Figure 1a). On the other hand, the TCA-soluble peptide content in the M1, M2 and M3 groups was significantly increased after treatment (Figure 1b). The AAN contents in the M1, M2, and M3 groups were also increased 13.17-, 37.83- and 49.92-fold, respectively, compared to the CK group. Notably, the AAN content in the M3 group was significantly higher than that in the M1 and M2 groups, with increases of approximately 3.59- and 1.31-fold, respectively (Figure 1c). The DH was significantly promoted by the synergy of exogenous papain from the fourth day of fermentation. By the sixth day of fermentation, the DH in the M3 group (42.55%) was 31.94% higher than that in the M2 group (32.25%), with a significant reduction in the time required to produce equivalent amino acids (Figure 1d).
Myofibrillar proteins and collagens can be degraded by papain and their proportion in FMS is relatively high, followed closely by sarcoplasmic proteins, matrix proteins, keratin, and elastin [21]. The neutral protease produced by B. velezensis exhibited a hydrolysis activity of approximately 42.66% towards fish peptone [22], while those produced by B. amyloliquefaciens preferentially cleaved peptide bonds involving leucine and phenylalanine [23]. Peptidases on the cell surface of E. faecalis, such as leucine aminopeptidase, hydrolyzed alkaline amino acids at the terminals of polypeptides, generating smaller oligopeptides or free amino acids. Therefore, these peptidases were combined with the endonucleases produced by B. velezensis and B. amyloliquefaciens to promote the hydrolysis of proteins into peptides and amino acids of smaller molecular weight. Thus, the combination of microbial-secreted protease and papain was more effective in degrading soluble proteins and generating TCA-soluble peptides and AAN than when used separately, suggesting a synergistic effect between the hybrid strain and papain.

3.2. Effects of Single and Mixed Strains on Fish Meat with Skin (FMS) Degradation

The two core modes of microbial fermentation—single- and mixed-strain fermentation—produce products with very different characteristics due to the differences in the strains’ mechanisms of action [24]. Within the mixed-strain fermentation system, due to the differences in the site of action and the metabolic mechanisms of papain and proteases secreted by each strain, a mutually beneficial symbiotic relationship is usually established between strains [25]. When the three bacterial strains were mixed and fermented in specific ratios, the TCA-soluble peptide and AAN contents were significantly higher than those of single-strain fermentations (p < 0.05) (Figure 2a). For example, the contents of AAN reached 9.73 g/L on the sixth day of fermentation; however, in a study by Li et al. [26], the AAN content of fermented fish sauce reached only 0.795 g/100 mL and 1.039 g/100 mL on the 15th and 45th day of fermentation, respectively. The use of mixed-strain fermentation in the present study substantially increased the contents of threonine, glycine, alanine, lysine, and proline to 713.50, 1551.50, 1595.70, 8217.75, and 39.6 mg/L, respectively, compared to each single-strain fermentation (Figure 2b).
Collagen contains a large number of glycine-proline-hydroxyproline sequences that can be hydrolyzed by proteases and peptidases. Proline-specific peptidases and two nonspecific enzymes, namely leucine aminopeptidase and cytoplasmic nonspecific dipeptidyl peptidase, broke down N-terminal Pro, allowing proline to freely exist [27]. Proteases or aminopeptidases that specifically cleave the sites in threonine are unknown, though its release may be a function of some broad-spectrum aminopeptidases, while alanine may be hydrolyzed by leucine aminopeptidase [28]. Papain has a high affinity for the carboxyl-side peptide bond of basic amino acid residues, such as lysine, and usually induces detachment of lysine from the peptide chain [29]. Lysyl endonuclease specifically shears the peptide bond of the C-terminal lysine and the S-aminoethyl cysteine residues. On the other hand, microorganisms such as B. amyloliquefaciens, B. velezensis, and E. faecalis secrete a variety of endopeptidases, such as neutral protease, gelatinase, keratinase and metalloprotease, and aminopeptidase and carboxypeptidase [30,31,32,33], which form a complex enzyme system with exogenous papain during mixed-strain fermentation. With the increased enzyme cleavage sites, proteins were more efficiently and thoroughly hydrolyzed, leading to a substantial increase in the polypeptide, amino acid, and nitrogen contents in the products. Therefore, in the system with an addition of 0.1% papain hydrolysis, mixed-strain fermentation was more effective than the single-bacteria fermentation, significantly increasing the TCA-soluble peptide and AAN contents in the product, as well as balancing the free amino acid content.

3.3. Indoleacetic Acid (IAA) Synthesis in Fish Meat with Skin (FMS) Fermentation with Single and Mixed Strains

Microbial fermentation produces IAA, a crucial active ingredient in biostimulants. The synthesis process is jointly regulated by the unique physiological characteristics and environmental factors of the strain. In the present study, the IAA content produced by the fermentation of mixed strains significantly exceeded that of the single fermentation of B. velezensis KA2 (J1) and E. faecalis F (J3) (Figure 3). Although B. velezensis and B. amyloliquefaciens can secrete IAA [34,35], during mixed-strain fermentation, synergistic interactions between complex enzyme systems secreted by these microorganisms accelerate tryptophan release and activate the expression of tryptophan aminotransferase and indole acetaldehyde dehydrogenase genes involved in IAA synthesis through signalling mechanisms, such as community sensing, resulting in an accumulation of IAA [36].

3.4. Changes in Physicochemical Properties During the Transformation of Fish Meat with Skin (FMS)

There is a close and complex dynamic correlation between the growth of bacterial strains and various metabolites, implying the key role of the MES approach. In the initial stage of the synergistic fermentation, the content of reducing sugars sharply dropped on the first day, while the total number of viable strains in the system increased (Figure 4a), indicating that bacteria in the logarithmic growth phase absorb reducing sugars to promote their proliferation in the early growth stage. The microbial–enzyme synergy can efficiently utilize carbon sources to provide sufficient energy for the growth of strains, thus laying a solid foundation for smooth subsequent fermentation processes. However, with the continuous accumulation of fermentation products, the high concentration of metabolites, such as small-molecule peptides, free amino acids, antimicrobial peptides (bacteriocins), and sugar metabolic by-products, inhibited the growth of bacteria, leading to a decrease in the number of viable bacteria in the system, as previously suggested [37,38].
The neutral protease activity initially and gradually increased to a peak value of about 209 U/mL on the third day, then gradually decreasing and finally stabilizing at about 30 U/mL, while the acidic protease activity hardly fluctuated significantly in this process (Figure 4b). The pH value also gradually increased from weakly acidic (pH 5.95) to nearly neutral (pH 7.46) (Figure 4c), which is conducive to the production of enzymes by microorganisms [31]. On the other hand, the synergistic effect between bacteria and enzymes changed the environmental pH, thereby precisely regulating the activities of proteases to meet the needs of different fermentation stages. This greatly promoted the efficient hydrolysis and full utilization of proteins, as previously has been suggested [39]. However, the decline in neutral protease activity in the later stages could be due to the exhaustion of nutrients and comprehensive changes in factors, such as the osmotic pressure in the environment and the accumulation of products, resulting in the obstruction of its synthesis and activity maintenance.
The analysis of the trends of protein-related hydrolysates (Figure 4d,e) revealed the increased total viable cell count and content of peptides alongside a rapid decrease in the soluble proteins on the first day of fermentation, indicating that the bacterial strains secrete a large number of proteases to meet the needs of their rapid growth and reproduction. The microbial–enzyme synergy plays a crucial role in this process by providing a basis for subsequent transformation and energy metabolism. Thus, the increase in the AAN content (Figure 4e) at this stage connotes that during fermentation, the microbial-enzyme synergy could convert complex nitrogen-containing compounds into amino acid nitrogen, which can be directly absorbed and utilized by microorganisms [40]. Accompanied by protein hydrolysis and lowered reducing sugar contents, the strain fermentation produced alkaline amino acids and peptides, which increased the pH and thereby directly affected the activities of proteases, thus impacting the permeability of the microbial cell membrane and other metabolic pathways. As a result, strain fermentation created suitable conditions for other metabolic pathways and optimized the entire fermentation environment, which could further improve the fermentation efficiency and product quality [41]. Furthermore, these results indicate that within the “mixed strain–papain” system established in this study, the microbial–enzyme synergistic effect can effectively regulate the transformation dynamics of proteins into peptides and amino acids, significantly improving protein hydrolysis efficiency and thus highlighting the application potential and value of this synergistic mode in industrial production and scientific research.
The IAA content increased as the number of viable bacteria increased in the first two days of fermentation, while the enzyme activity remained almost unchanged (Figure 4f). The contents of tryptophan declined, indicating that the growth and reproduction of the bacterial strains and the synthesis of IAA occurred simultaneously during this stage. Within the subsequent 24 h, the proteases secreted by the enzyme-active strains and papain synergistically converted a large number of proteins in the substrate, resulting in a significant increase in the tryptophan content while hindering the synthesis of IAA. However, polypeptides accumulated as the substrates in the system were depleted while the activity of neutral protease and the viability of the strains decreased. Thereafter, the synthesis pathway of IAA was restarted, increasing the phytohormone. However, the content of tryptophan, which participates in the regulation of the synthesis of IAA and other metabolic pathways, frequently fluctuated content [42]. Thus, these dynamic changes in key indicators reflect the complex metabolic pathways and strain regulation mechanisms during the fermentation process [43].

3.5. Manurial Effect of Fish Meat with Skin (FMS) Hydrolysate on Soybean Seedlings Growth

Soybean seedlings were treated with different dilutions of FPBs for 3 days under darkness to validate the growth-promoting effect of the biostimulants prepared under the MES strategy in this study. The highest number of lateral roots was observed in soybean seedlings treated with an FPB diluted 100 times, followed by those treated with an FPB diluted 2500 times. The root area, stem length, and reducing sugar content of soybean seedlings treated with the FPB diluted 2500 times significantly increased 1.59-, 1.44-, and 1.69- fold compared to those treated with Hoagland nutrient solution (Figure 5). This could largely be due to the rich content of bionutrients in FPBs, such as peptides, amino acids, IAA, phosphorus, potassium, and other substances. The hydrolysate of animal-derived collagen has also been shown to increase root length growth and the number of lateral roots of tomato seedlings [44]. On the other hand, amino acids directly or indirectly promote the growth of plant roots through different pathways. For example, proline, glutamic acid, and lysine maintain cell homeostasis and stimulate cell division in adverse conditions, supply energy to plants by participating in nitrogen metabolism, and affect the growth and development of plants by participating in the synthesis of plant hormones and chlorophyll, respectively [45,46].
Indoleacetic acid increases the biomass and the growth of lateral roots by promoting cell elongation and division, regulating the differentiation of pericycle cells, and enhancing the absorption and transportation of nutrients [47]. Indoleacetic acid is regarded as a signalling molecule for phosphorus and potassium that binds to the receptors on the cell surface, thereby promoting root development and significant elongation of stems in soybean seedlings [48]. All the bacteria used in the study, including B. amyloliquefaciens, B. licheniformis, and B. subtilis, synthesized IAA. Hence, there was a positive correlation between the root length of the seedlings and the IAA produced by the strains (R2 = 0.6965) after the hydroponic tomato seedlings were exposed to said IAA [49]. Glucose, IAA, amino acids, and polypeptides in FPBs are considered signalling molecules that regulate the metabolism and maintain the metabolic balance within cells [50], while the reducing sugar content in plants is effectively increased by combining multiple molecular mechanisms [51]. Therefore, the FPB prepared in this study can promote the growth of root systems and nutrient accumulation in soybean seedlings.

4. Conclusions

The MES method outperforms single-strain-papain or mixed-strain-only fermentation by integrating the broad-spectrum hydrolytic properties of papain and the metabolic function of mixed strains, improving protein hydrolysis efficiency, enriching peptides and amino acids, and promoting the biosynthesis of IAA. During the transformation of fishery processing by-products (FMS), the reducing sugar content is positively correlated with the number of viable bacteria, forming a feedback network that can enhance product quality and yield. When prepared as a biostimulant with optimized dilution, it not only promotes the growth of soybean seedlings (e.g., increased root area and stem length) but also increases the reducing sugar content of seedlings (compared with commercial soilless culture nutrient solution), demonstrating the agricultural application potential of the MES method. However, this study has limitations. The transformation of FMS via the MES method is still only achievable at the laboratory scale; for successful large-scale application, the hurdles of papain cost, microbial community stability, and reactor efficiency must first be overcome. In addition, the efficacy of the biostimulant has only been verified in soybeans. Future research can further explore low-cost papain substitutes, test the biostimulant in multiple crops and different environments, and investigate the metabolic mechanism of the MES method. In conclusion, the MES method can optimize the transformation process of fishery waste, providing a path for its high-value utilization in agriculture.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11090525/s1, Supplementary Material S1. Effect of inoculation ratio of mixed strains on FMS protein hydrolysates (a) TCA soluble peptide content, (b) Amino acid nitrogen content.

Author Contributions

Conceptualization, Z.W.; data curation, X.H. and X.X.; formal analysis, H.X. (Hanyi Xie) and B.Z.; investigation, H.X. (Hanyi Xie), B.Z. and Q.Z.; methodology, H.X. (Hanyi Xie); resources, Z.W.; supervision, Z.W.; validation, Q.Z., X.X. and H.X. (Hong Xie); writing—original draft, H.X. (Hanyi Xie); writing—review & editing, B.Z., X.H., H.X. (Hong Xie) and Z.W. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Construction Funds for the Science and Technology Innovation Platform of Jiangmen, China (2021JK02402003) and Development Fund of Yiwyi Biofabrication (Jiangmen) Co., Ltd., China (2023Y1103011).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that support this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

Authors Qimin Zhang and Hong Xie were employed by the company Yiwyi Biofabrication (Jiangmen) Co., Ltd., Jiangmen, 529080, China. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Lykogianni, M.; Bempelou, E.; Karamaouna, F.; Aliferis, K.A. Do pesticides promote or hinder sustainability in agriculture? The challenge of sustainable use of pesticides in modern agriculture. Sci. Total Environ. 2021, 795, 148625. [Google Scholar] [CrossRef]
  2. Elkhlifi, Z.; Iftikhar, J.; Sarraf, M.; Ali, B.; Saleem, M.H.; Ibranshahib, I.; Bispo, M.D.; Meili, L.; Ercisli, S.; Kayabasi, E.T.; et al. Potential role of biochar on capturing soil nutrients, carbon sequestration and managing environmental challenges: A review. Sustainability 2023, 15, 2527. [Google Scholar] [CrossRef]
  3. Du Jardin, P. Plant biostimulants: Definition, concept, main categories and regulation. Sci. Hortic. 2015, 196, 3–14. [Google Scholar] [CrossRef]
  4. Burbano-Cuasapud, J.M.; Solarte-Toro, J.C.; Restrepo-Serna, D.L.; Alzate, C.A.C. Process Sustainability Analysis of Biorefineries to Produce Biofertilizers and Bioenergy from Biodegradable Residues. Fermentation 2023, 9, 788. [Google Scholar] [CrossRef]
  5. Colla, G.; Nardi, S.; Cardarelli, M.; Ertani, A.; Lucini, L.; Canaguier, R.; Rouphael, Y. Protein hydrolysates as biostimulants in horticulture. Sci. Hortic. 2015, 196, 28–38. [Google Scholar] [CrossRef]
  6. Nasri, R.; Abdelhedi, O.; Nasri, M.; Jridi, M. Fermented protein hydrolysates: Biological activities and applications. Curr. Opin. Food Sci. 2022, 43, 120–127. [Google Scholar] [CrossRef]
  7. Babu, N.S.; Suresh, P.V.; Kudre, T.G. Preparation and identification of a novel antioxidative peptide from fermented protein hydrolysate of chicken (Gallus gallus domesticus) meat. Process Biochem. 2025, 151, 167–176. [Google Scholar] [CrossRef]
  8. Zhou, J.; Li, D.; Duan, X.; Zhang, X.; Chen, C.; Chen, Y. Biological VFAs production from proteinaceous wastewater varied with protein type: The role of protein exposed enzyme cleavage sites and hydrolysates biotransformation capacity. Water Res. 2025, 275, 123201. [Google Scholar] [CrossRef]
  9. Nikoo, M.; Benjakul, S.; Gavlighi, H.A. Protein hydrolysates derived from aquaculture and marine byproducts through autolytic hydrolysis. Compr. Rev. Food Sci. Food Saf. 2022, 21, 4872–4899. [Google Scholar] [CrossRef]
  10. Chiodza, K.; Goosen, N.J. Emulsion formation during enzymatic protein hydrolysis and its effect on protein recovery and molecular weight distribution of protein hydrolysates from sardine (Sardina pilchardus) by-products. Biomass Convers. Biorefin. 2024, 14, 24069–24080. [Google Scholar] [CrossRef]
  11. Sun, T.; Xiao, W.; Jiang, C.; Wang, J.; Liu, Z. Producing amino acid fertilizer by hydrolysis of the fermented mash of food waste with the synergy of three proteases expressed by engineered Candida utilis. Bioresour. Technol. Rep. 2019, 7, 100268. [Google Scholar] [CrossRef]
  12. Yang, L.; Zhang, J.; Chen, Z.; Chen, Y.; Wang, C.; Yu, H.; Zuo, F.; Huang, W. Probiotic–enzyme synergy regulates fermentation of distiller’s grains by modifying microbiome structures and symbiotic relationships. J. Agric. Food Chem. 2025, 73, 5363–5375. [Google Scholar] [CrossRef]
  13. Zhong, B.; Xie, H.; Pan, T.; Su, B.; Xu, W.; Wu, Z. High acidity organic waste degradation and the potential to bioremediation of heavy metals in soil by an acid-tolerant Serratia sp. Environ. Geochem. Health 2024, 46, 321. [Google Scholar] [CrossRef]
  14. Neylon, E.; Nyhan, L.; Zannini, E.; Monin, T.; Münch, S.; Sahin, A.W.; Arendt, E.K. Food Ingredients for the Future: In-Depth Analysis of the Effects of Lactic Acid Bacteria Fermentation on Spent Barley Rootlets. Fermentation 2023, 9, 78. [Google Scholar] [CrossRef]
  15. Kononiuk, D.A.; Karwowska, M.; Bejarano, R.G.; Cacciola, F. Bioactive compounds in fermented sausages prepared from beef and fallow deer meat with acid whey addition. Molecules 2020, 25, 2429. [Google Scholar] [CrossRef] [PubMed]
  16. Yanohara, T.; Taoka, Y.; Yamamoto, M. Rapid Production of Fish Sauce from the Internal Organs of White Sturgeon, Acipenser transmontanus Richardson, 1836. Fermentation 2022, 8, 238. [Google Scholar] [CrossRef]
  17. Moropana, T.J.; Rensburg, E.L.J.V.; Makulana, L.; Phasha, N.N. Screening Aspergillus flavus, Talaromyces purpureogenus, and Trichoderma koningiopsis for plant-growth-promoting traits: A study on phosphate solubilization, IAA production, and siderophore synthesis. J. Fungi 2024, 10, 811. [Google Scholar] [CrossRef]
  18. Kumar, R.; Banerjee, T.K. Changes in the level of proteins, free amino acids and protease activities of clarias batrachus in response to sodium meta-arsenite intoxication. CLEAN—Soil Air Water 2013, 41, 1196–1200. [Google Scholar] [CrossRef]
  19. Sun, Y.; Gao, P.; Xu, Y.; Xia, W.; Hua, Q.; Jiang, Q. Effect of Storage Conditions on Microbiological Characteristics, Biogenic Amines, and Physicochemical Quality of Low-Salt Fermented Fish. J. Food Prot. 2020, 83, 1057–1065. [Google Scholar] [CrossRef] [PubMed]
  20. Gao, X.; Zhao, X.; Hu, F.; Fu, J.; Zhang, Z.; Liu, Z.; Wang, B.; He, R.; Ma, H.; Ho, C.-T. The latest advances on soy sauce research in the past decade: Emphasis on the advances in China. Food Res. Int. 2023, 173, 113407. [Google Scholar] [CrossRef]
  21. Xu, J.; Cao, H.; Zhang, B.; Yao, H. The mechanistic effect of bromelain and papain on tenderization in jumbo squid (Dosidicus gigas) muscle. Food Res. Int. 2020, 131, 108991. [Google Scholar] [CrossRef]
  22. Li, N.; Shen, B.; Liu, Y.; Weng, P.; Wu, Z. Heterologous expression and characterization of Bacillus velezensis SW5 serine protease involved in the hydrolysis of anchovy protein. J. Sci. Food Agric. 2023, 103, 3468–3478. [Google Scholar] [CrossRef]
  23. Hathwar, S.C.; Bijinu, B.; Rai, A.K.; Narayan, B. Simultaneous recovery of lipids and proteins by enzymatic hydrolysis of fish industry waste using different commercial proteases. Appl. Biochem. Biotechnol. 2011, 164, 115–124. [Google Scholar] [CrossRef] [PubMed]
  24. Lambo, M.T.; Chang, X.; Liu, D. The recent trend in the use of multistrain probiotics in livestock production: An overview. Animals 2021, 11, 2805. [Google Scholar] [CrossRef]
  25. Smid, E.J.; Lacroix, C. Microbe–microbe interactions in mixed culture food fermentations. Curr. Opin. Biotechnol. 2013, 24, 148–154. [Google Scholar] [CrossRef]
  26. Li, C.; Zhao, Y.; Wang, Y.; Wu, Y.; Chen, S. Improvement of the quality and safety of low-salt fish sauce by reconstruction of microbial community through cooperative fermentation of starters. Food Res. Int. 2025, 205, 115972. [Google Scholar] [CrossRef]
  27. Alkin, N.; Dunaevsky, Y.; Elpidina, E.; Beljakova, G.; Tereshchenkova, V.; Filippova, I.; Belozersky, M. Proline-specific fungal peptidases: Genomic analysis and identification of secreted DPP4 in alkaliphilic and alkalitolerant fungi. J. Fungi 2021, 7, 744. [Google Scholar] [CrossRef]
  28. Ren, Q.; Wang, Y.; Sun, S.; Zhang, L.; Sun, L.; Weng, L.; Liu, G.; Cao, M. Aminopeptidase Play a Critical Role in the Accumulation of Free Amino Acids in Abalone (Haliotis discus hannai) During Cold Storage. J. Ocean Univ. China 2023, 22, 1049–1058. [Google Scholar] [CrossRef]
  29. Rivero-Pino, F.; Leon, M.J.; Millan-Linares, M.C.; la Paz, S.M. Antimicrobial plant-derived peptides obtained by enzymatic hydrolysis and fermentation as components to improve current food systems. Trends Food Sci. Technol. 2023, 135, 32–42. [Google Scholar] [CrossRef]
  30. Jeon, S.H.; Seong, H.-J.; Kim, H.; Kim, D.; Yang, K.-Y.; Nam, S.-H. Improvement of branched-chain amino acid production by isolated high-producing protease from Bacillus amyloliquefaciens NY130 on isolated soy/whey proteins and their muscle cell protection. Food Chem. 2024, 450, 139327. [Google Scholar] [CrossRef] [PubMed]
  31. Lu, J.; Zhao, Y.; Hu, R.; Cheng, Y.; Qin, J.; Yang, J.; Fang, Y.; Lyu, M.; Wang, S. Screening and characteristics of marine Bacillus velezensis Z-1 protease and its application of enzymatic hydrolysis of mussels to prepare antioxidant active substances. Molecules 2022, 27, 6570. [Google Scholar] [CrossRef]
  32. Willett, J.L.E.; Robertson, E.B.; Dunny, G.M. The phosphatase Bph and peptidyl-prolyl psomerase PrsA are required for gelatinase expression and activity in Enterococcus faecalis. J. Bacteriol. 2022, 204, e00129-22. [Google Scholar] [CrossRef]
  33. Zeng, J.; Huang, W.; Tian, X.; Hu, X.; Wu, Z. Brewer’s spent grain fermentation improves its soluble sugar and protein as well as enzymatic activities using Bacillus velezensis. Process Biochem. 2021, 111, 12–20. [Google Scholar] [CrossRef]
  34. Yang, R.; Liu, P.; Ye, W.; Chen, Y.; Wei, D.; Qiao, C.; Zhou, B.; Xiao, J. Biological control of root rot of strawberry by Bacillus amyloliquefaciens strains CMS5 and CMR12. J. Fungi 2024, 10, 410. [Google Scholar] [CrossRef] [PubMed]
  35. Wei, P.; Gao, M.; Zhou, S.; Liu, G.; Wang, P.; Liu, C.; Yang, F.; Fu, H. Antifungal activity of Bacillus velezensis X3-2 against plant pathogens and biocontrol effect on potato late blight. Agriculture 2024, 14, 2224. [Google Scholar] [CrossRef]
  36. Ma, M.-Y.; Hu, L.-L.; Xu, W.-Y.; Zhang, W. L—Tryptophan anaerobic fermentation for indole acetic acid production: Bacterial enrichment and effects of zero valent iron. Bioresour. Technol. 2024, 400, 130691. [Google Scholar] [CrossRef] [PubMed]
  37. Li, X.; Rao, W.; Hu, S.; Zhu, S.; Ouyang, L.; Zhou, J. Study on the relationship between microbial community succession and physicochemical factors in the fermentation of rice-flavor Baijiu based on high-throughput and redundancy analysis techniques. LWT 2024, 213, 117031. [Google Scholar] [CrossRef]
  38. Caulier, S.; Nannan, C.; Gillis, A.; Licciardi, F.; Bragard, C.; Mahillon, J. Overview of the antimicrobial compounds produced by members of the Bacillus subtilis Group. Front. Microbiol. 2019, 10, 302. [Google Scholar] [CrossRef] [PubMed]
  39. Ren, H.; Wang, T.; Liu, R. Correlation analyses of amylase and protease activities and physicochemical properties of wheat bran during solid-state fermentation. Foods 2024, 13, 3998. [Google Scholar] [CrossRef]
  40. Wei, R.; Sun, X.; Chen, X.; Zhang, Y.; Li, Q.; Zhang, X.; Xu, N. Unraveling the microbial community and succession during the maturation of Chinese cereal vinegar Daqu and their relationships with flavor formation. Food Res. Int. 2025, 203, 115851. [Google Scholar] [CrossRef]
  41. Treece, T.R.; Tessman, M.; Pomeroy, R.S.; Mayfield, S.P.; Simkovsky, R.; Atsumi, S. Fluctuating pH for efficient photomixotrophic succinate production. Metab. Eng. 2023, 79, 118–129. [Google Scholar] [CrossRef] [PubMed]
  42. Zhang, P.; Jin, T.; Sahu, S.K.; Xu, J.; Shi, Q.; Liu, H.; Wang, Y. The distribution of tryptophan-dependent indole-3-acetic acid synthesis pathways in bacteria unraveled by large-scale genomic analysis. Molecules 2019, 24, 1411. [Google Scholar] [CrossRef]
  43. Liu, W.-H.; Chen, F.-F.; Wang, C.-E.; Fu, H.-H.; Fang, X.-Q.; Ye, J.-R.; Shi, J.-Y. Indole-3-acetic acid in Burkholderia pyrrocinia JK-SH007: Enzymatic identification of the indole-3-acetamide synthesis pathway. Front. Microbiol. 2019, 10, 2559. [Google Scholar] [CrossRef]
  44. Ambrosini, S.; Prinsi, B.; Zamboni, A.; Espen, L.; Zanzoni, S.; Santi, C.; Varanini, Z.; Pandolfini, T. Chemical characterization of a collagen-derived protein hydrolysate and biostimulant activity assessment of its peptidic components. J. Agric. Food Chem. 2022, 70, 11201–11211. [Google Scholar] [CrossRef]
  45. Sun, W.; Shahrajabian, M.H.; Kuang, Y.; Wang, N. Amino acids biostimulants and protein hydrolysates in agricultural sciences. Plants 2024, 13, 210. [Google Scholar] [CrossRef]
  46. Vaitkeviciene, R.; Burbulis, N.; Masiene, R.; Zvirdauskiene, R.; Jakstas, V.; Damasius, J.; Zadeike, D. Fermentation as a Promising Tool to Valorize Rice-Milling Waste into Bio-Products Active against Root-Rot-Associated Pathogens for Improved Horticultural Plant Growth. Fermentation 2022, 8, 716. [Google Scholar] [CrossRef]
  47. Ali, M.; Shi, L.; Khan, M.A.; Ali, A.; Hu, S.; Shen, J. Auxin biodynamics and its integral role in enhancing plant resilience to environmental cues. Physiol. Plant. 2025, 177, e70165. [Google Scholar] [CrossRef]
  48. Peng, Y.; Chen, K.; Wang, G.; Tan, J.; Wei, F.; Ma, Y. Nitrogen addition regulates the growth of Pinus tabuliformis by changing distribution patterns of endogenous hormones in different organs. New For. 2023, 54, 853–865. [Google Scholar] [CrossRef]
  49. Pappalettere, L.; Bartolini, S.; Toffanin, A. Auxin-producing bacteria used as microbial biostimulants improve the growth of tomato (Solanum lycopersicum L.) seedlings in hydroponic systems. BioTech 2024, 13, 32. [Google Scholar] [CrossRef]
  50. Heidarzadeh, A. Role of amino acids in plant growth, development, and stress responses: A comprehensive review. Discov. Plants 2025, 2, 237. [Google Scholar] [CrossRef]
  51. Li, R.; Qin, M.; Yan, J.; Jia, T.; Sun, X.; Pan, J.; Li, W.; Liu, Z.; El-Sheikh, M.A.; Ahmad, P.; et al. Hormesis effect of cadmium on pakchoi growth: Unraveling the ROS-mediated IAA-sugar metabolism from multi-omics perspective. J. Hazard. Mater. 2025, 487, 137265. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Differences in (a) soluble protein content, (b) TCA-soluble peptide content, (c) amino acid nitrogen (AAN) content, and (d) DH of AAN among treatment groups. CK: without papain and strains; M1: only 0.1% papain; M2: only 5% MSC (BF2: KA2: F = 1:2:2 (v:v:v)); and M3: both 0.1% papain and 5% MSC. The columns (data points on the curve) and error bars represent mean ± standard deviation (n = 3). Different letters indicate significant difference (p < 0.05) among treatments.
Figure 1. Differences in (a) soluble protein content, (b) TCA-soluble peptide content, (c) amino acid nitrogen (AAN) content, and (d) DH of AAN among treatment groups. CK: without papain and strains; M1: only 0.1% papain; M2: only 5% MSC (BF2: KA2: F = 1:2:2 (v:v:v)); and M3: both 0.1% papain and 5% MSC. The columns (data points on the curve) and error bars represent mean ± standard deviation (n = 3). Different letters indicate significant difference (p < 0.05) among treatments.
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Figure 2. Differences in protein hydrolysates of the synergistic fermentation products of single- and mixed-strain with papain. (a) TCA-soluble peptide content and amino acid nitrogen (AAN) content. (b) Composition and content of 17 free amino acids. J1: B. velezensis KA2, J2: B. amyloliquefaciens BF2, J3: E. faecalis F and J4: BF2: KA2: F = 1:2:2 (v:v:v). The columns and error bars in (a) represent mean ± standard deviation (n = 3). Different letters indicate significant difference (p < 0.05) among treatments.
Figure 2. Differences in protein hydrolysates of the synergistic fermentation products of single- and mixed-strain with papain. (a) TCA-soluble peptide content and amino acid nitrogen (AAN) content. (b) Composition and content of 17 free amino acids. J1: B. velezensis KA2, J2: B. amyloliquefaciens BF2, J3: E. faecalis F and J4: BF2: KA2: F = 1:2:2 (v:v:v). The columns and error bars in (a) represent mean ± standard deviation (n = 3). Different letters indicate significant difference (p < 0.05) among treatments.
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Figure 3. Differences in the indoleacetic acid (IAA) content of the synergistic fermentation products of single- and mixed-strain with papain. J1: B. velezensis KA2, J2: B. amyloliquefaciens BF2, J3: E. faecalis F and J4: BF2: KA2: F = 1:2:2 (v:v:v). The columns and error bars represent mean ± standard deviation (n = 3). Different letters indicate significant difference (p < 0.05) among treatments.
Figure 3. Differences in the indoleacetic acid (IAA) content of the synergistic fermentation products of single- and mixed-strain with papain. J1: B. velezensis KA2, J2: B. amyloliquefaciens BF2, J3: E. faecalis F and J4: BF2: KA2: F = 1:2:2 (v:v:v). The columns and error bars represent mean ± standard deviation (n = 3). Different letters indicate significant difference (p < 0.05) among treatments.
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Figure 4. Dynamics of (a) log CFU of viable bacteria and reducing sugar content, (b) protease activity, (c) pH, (d) soluble protein and TCA-soluble peptide content, (e) amino acid nitrogen content (AAN), (f) indoleacetic acid and tryptophan content of the process of MES conversion. The data for the curves represent mean ± standard deviation (n = 3).
Figure 4. Dynamics of (a) log CFU of viable bacteria and reducing sugar content, (b) protease activity, (c) pH, (d) soluble protein and TCA-soluble peptide content, (e) amino acid nitrogen content (AAN), (f) indoleacetic acid and tryptophan content of the process of MES conversion. The data for the curves represent mean ± standard deviation (n = 3).
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Figure 5. Indicators for evaluating the growth of soybean seedlings. (a) The number of lateral roots, (b) root area, (c) stem length, and (d) reducing sugar content (dry basis) of soybean seedlings treated with clear water (CK), Hoagland’s nutrient solution (H), and different dilutions of FPB (100, 1000, 2500). Boxplot (a–c) with n = 32. The columns and error bars in (d) represent mean ± standard deviation (n = 3). Different letters indicate significant difference (p < 0.05) among treatments.
Figure 5. Indicators for evaluating the growth of soybean seedlings. (a) The number of lateral roots, (b) root area, (c) stem length, and (d) reducing sugar content (dry basis) of soybean seedlings treated with clear water (CK), Hoagland’s nutrient solution (H), and different dilutions of FPB (100, 1000, 2500). Boxplot (a–c) with n = 32. The columns and error bars in (d) represent mean ± standard deviation (n = 3). Different letters indicate significant difference (p < 0.05) among treatments.
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Table 1. Composition of fish meat with skin (FMS).
Table 1. Composition of fish meat with skin (FMS).
ParametersValue
Moisture (%)77.99 ± 1.29
Protein content (%)17.93 ± 0.13
Ash content (%)0.88 ± 0.02
Fat content (%)4.12 ± 0.27
K2O (g/kg)18.27 ± 0.36
P2O5 (g/kg)3.42 ± 0.23
The indicators given include moisture (%), protein content (%), ash content (%), and fat content (%) for FMS wet basis and P2O5 and K2O for dry basis. Data are shown as mean ± standard deviation (n = 3).
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MDPI and ACS Style

Xie, H.; Zhong, B.; Zhang, Q.; Hu, X.; Xia, X.; Xie, H.; Wu, Z. Transforming Tilapia into Indoleacetic Acid-Containing Biostimulants: Synergistic Effect of Enzymolysis and Multi-Strain Fermentation. Fermentation 2025, 11, 525. https://doi.org/10.3390/fermentation11090525

AMA Style

Xie H, Zhong B, Zhang Q, Hu X, Xia X, Xie H, Wu Z. Transforming Tilapia into Indoleacetic Acid-Containing Biostimulants: Synergistic Effect of Enzymolysis and Multi-Strain Fermentation. Fermentation. 2025; 11(9):525. https://doi.org/10.3390/fermentation11090525

Chicago/Turabian Style

Xie, Hanyi, Bin Zhong, Qimin Zhang, Xi Hu, Xuesen Xia, Hong Xie, and Zhenqiang Wu. 2025. "Transforming Tilapia into Indoleacetic Acid-Containing Biostimulants: Synergistic Effect of Enzymolysis and Multi-Strain Fermentation" Fermentation 11, no. 9: 525. https://doi.org/10.3390/fermentation11090525

APA Style

Xie, H., Zhong, B., Zhang, Q., Hu, X., Xia, X., Xie, H., & Wu, Z. (2025). Transforming Tilapia into Indoleacetic Acid-Containing Biostimulants: Synergistic Effect of Enzymolysis and Multi-Strain Fermentation. Fermentation, 11(9), 525. https://doi.org/10.3390/fermentation11090525

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