Abstract
High proteolytic activity and several biological functions (antimicrobial, antioxidant, antihypertensive, among others) have been attributed to lactic-acid bacteria (LAB) isolated from fish and peptides obtained from proteolysis. Therefore, the objective of this research was isolating, characterizing, and identifying LAB with proteolytic activity by spontaneous fermentation from common carp (Cyprinus carpio) reared in ponds and wild ones obtained from Lago de Chapala, Jalisco, Mexico. Spontaneous fermentation from complete carp specimens was observed, considering two sampling points (skin and intestines) at 15 °C at 5 and 10 days. Isolated LAB—from both reared and wild specimens—were identified and morphologically characterized; identification was performed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Proteolytic activity was assessed by the presence of the proteolytic halo. A total of five genera and eight different LAB proteolytic species were isolated from all the carp samples. At 10 days, greater proteolytic LAB diversity was obtained from the intestine (Tukey’s, p < 0.05); the proteolytic halo with the greatest diameter was recorded in wild carp skin with Lactiplantibacillus plantarum S5P2 (2.8 cm) at 5 days of fermentation, followed by Leuconostoc mesenteroides S5I1 (2.73 cm) and Leuconostoc pseudomesenteroides S5P2 (2.66 cm) (p < 0.05). In conclusion, proteolytic capability of LAB isolated from carp (Cyprimus carpio)—both wild and reared—is influenced by the ecosystem where they develop. These proteolytic LAB may be used in biotechnological industries to obtain bioactive peptides by fermenting substrates rich in proteins.
Keywords: Wild and reared carp, Lactic-acid bacteria, MALDI-TOF MS, Proteolytic activity, Spontaneous fermentation
Introduction
Lactic-acid bacteria (LAB) are Gram-positive, do not produce spores, and may show coccus, coccobacillus, or rod morphology. They are not generally aerobic but different from the majority, grow in presence of O2 as aerotolerant anaerobe [1], and are oxidase (OX-), catalase (CAT-), and benzidine negative. LAB ferment glucose for the production of short-chain organic acids, such as lactic acid, CO2, and ethanol, besides proving they have an enzymatic system that provides them with the capacity of hydrolyzing proteins.
Protein hydrolysis by LAB is performed by a quite complex proteolytic system; they have a cell wall-bound extracellular proteinase, which is the first contact with the substrate protein in charge of hydrolyzing peptides of different sizes. They also have a transport system responsible for carrying peptides obtained from this first hydrolysis to the cell interior and continue hydrolyzing these peptides through a joint protease of different specificity—turning them into smaller peptides including amino acids [2]. The efficiency of this system is influenced by the available proteins in the culture medium.
The available proteins, amino acids, and carbohydrates in the culture medium favor the genetic expression of LAB proteolytic system [3]. Protein presence in the culture medium—as the only nutrient source where LAB develop—forces gene expression cell wall-bound extracellular proteinase development to use available proteins as source of energy [4]. The expression or overexpression of these genes is related to the amount of protein as major nutrient to which these native LAB are exposed to during their development, as well as their exposure time to these proteins [5]. LAB with proteolytic capacity have been identified from diverse sources as food, which include meat, vegetables, cereals, and fermented food, such as fish and dairy products. They may also be found in mouth and intestines of—besides humans—animals, such as poultry and fish as carp [6–9].
Carps are an important LAB source. They have been isolated from skin and intestine, and their presence is influenced by the ecosystem where they develop. The isolated LAB from the intestines of these fish vary according to the aquatic environment since they are related with fish food. Thus, isolated LAB from meat as fish have a high proteolytic capacity compared to those isolated from plant sources [10]. Different authors have attributed proteolytic activity to isolated LAB from carp. These properties can be exploited to obtain bioactive peptides, such as antimicrobials, antioxidants, and antihypertensives [11–13]. One way of native LAB recovery from carp is by means of spontaneous fermentation since this type promotes LAB development with the capacity of surviving in environments rich in proteins to obtain greater recovery with proteolytic capacity [14].
Some proteolytic LAB, such as Pedicoccus and Enterococcus isolated from fish, could be used in generating bioactive peptides starting from protein substrates [9]. Therefore, the objective of this research is isolating, characterizing, and identifying LAB with proteolytic activity by means of skin and intestine spontaneous fermentation from carps reared in ponds and wild ones.
Materials and methods
Raw matter
To isolate lactic-acid bacteria starting from a total of 12 carps—six reared in ponds from the sea market of Zapopan and six wild from Lago de Chapala, both in Jalisco, Mexico—the samples were transferred in cold chain to the Microbiological Control Laboratory at CIATEJ (Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco). Fish skin and intestines from each fish were considered experimental samples, making a total of 24.
Obtaining powder from carp (Cyprinus carpio) fillet
Carp fillet powder—from both wild and reared carps—was obtained from bits of fresh common carp (Cyprinus carpio) fillet 2 cm3 and dried in convection oven at 55 °C until a humidity percentage from 5–10% and water activity (aw) 0.3–0.4 were obtained. The bits of dried fillet were ground in a hammer mill (IKA MF 10.2, IKA Works, Inc., Wilmington, NC, USA); then, the obtained powder was sieved in mesh no. 60, vacuum-packed, and stored at 4 °C until use.
Culture medium and reagents
To isolate LAB, peptone water, de Man, Rogosa, and Sharpe (MRS) bacteriological agar (BD Difco, Franklin Lakes, NJ, USA) were used. Determination of the proteolytic activity was formulated in agar plate assay (APA) medium (magnesium sulfate heptahydrate 0.1 g/L; manganese sulfate tetrahydrate 0.05 g/L; dipotassium phosphate 2 g/L; glucose (Sigma-Aldrich, St. Louis, MO, USA) 10 g/L; agar 15 g/L (BD Difco, Franklin Lakes, NJ, USA) and supplemented with 1 mg/mL of carp muscle powder) besides staining solution (SS) Coomassie Brilliant Blue (CBB) R 250 0.25 g/L (Bio-Rad, Hercules, CA, USA); methanol (JT Baker, Avantor, Inc., Radnor, PA, USA) 450 mL/L; glacial acetic acid 100 mL/L (Fermont, Productos Químicos Monterrey, NL, MX); distilled water 450 mL/L and discoloration solution (DS) methanol 200 mL/L (Fermont, Productos Químicos Monterrey, NL, MX); acetic acid 150 mL/L (Fermont, Productos Químicos Monterrey, NL, MX) and distilled water 650 mL/L.
Isolation of native carp (Cyprinus carpio) lactic-acid bacteria
Native LAB isolation was performed by means of spontaneous fermentation of the carps—both reared and wild—at 15 °C to allow LAB development, and two incubation times (5 and 10 days) were considered. Each carp was sampled in two different points, skin (Sk) and intestines (In); these treatments are shown in Table 1. Serial decimal dilutions were performed in peptone water to each one of the samples; then, samples were sown in MRS agar and incubated at 32 ± 2 °C/48 h. Subsequently, isolates showing colonies with LAB characteristics were selected. After that, they were re-sown successively in MRS agar until pure cultures were obtained and subjected to CAT and Gram staining tests. The isolates were preserved at − 20 °C with glycerol at 80%, for further use.
Table 1.
Treatment design for isolating native lactic-acid bacteria (LAB) from carps; Wi wild; Re reared
| Treatments | Experimental conditions | ||
|---|---|---|---|
| Origin | Fermentation time | Sampling point | |
| Wi10In | Wild | 10 | Intestine |
| Wi10Sk | Wild | 10 | Skin |
| Wi5In | Wild | 5 | Intestine |
| Wi5Sk | Wild | 5 | Skin |
| Re10In | Reared | 10 | Intestine |
| Re10Sk | Reared | 10 | Intestine |
| Re5Sk | Reared | 5 | Intestine |
| Re5Sk | Reared | 5 | Skin |
Identification of lactic-acid bacteria by MALDI-TOF
The identification of LAB isolates was performed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) with MICROFLEX LT platform (Bruker, Daltonik, Bremen, DE), following De la Torre et al.’s [15] methodology with some modifications. The fresh colonies were transferred to a BC MSP 96 target-polished steel plate (Bruker, Daltonik, Bremen, DE) in duplicate. Each sample placed on the plate was covered with 1 µL matrix HCCA (10 mg/mL alpha-cyano-4-hydroxycinnamic acid (Sigma-Aldrich, St. Louis, MO, USA) solution in 50% acetonitrile and 2.5% trifluoroacetic acid), and subsequently left to dry for 5 min.
Isolate identification was performed by means of protein profile with MALDI biotyper complete solution flexible control (MBT FC) method; the equipment was calibrated with 1 µL of bacterial test standard (BTS) (Bruker Daltonics, MA, USA) calibration; 40 laser shots were performed in six randomized positions within each one of the samples (isolates), generating mass spectra automatically in intervals from 2000 to 20,000 Da.
Determination of lactic-acid bacteria proteolytic activity
Proteolytic activity of the isolates was measured by the APA technique (adapted from Drosinos et al. [16]); the isolates were sown on the Petri dish center (60 × 15 mm) with APA following the puncture technique. Subsequently, the Petri dishes were incubated at 32 °C/120 h, and after incubation time, they were flooded with SS at 25 ± 2 °C (room temperature) for 1 h. Then, SS was discarded and replaced by DS (discoloration solution) at 25 ± 2 °C (room temperature) for 30 min. The proteolytic activity was observed by the presence of a clear zone around the bacterial growth.
Experimental design and statistical analyses
A completely randomized experimental design was used for each treatment with three replicates. Data of the response variables were analyzed by a multifactorial analysis of variance (ANOVA) (factor 1 carp origin; factor 2 fermentation time; and factor 3 type of organ sampled). The LAB proteolytic activity variables were analyzed with a one-way ANOVA for the different LAB species isolated and characterized (31 treatments). The treatments and different factors were separated by Tukey’s multiple comparison of means. All the tests and statistical analyses were performed at a level of significance of 5% (p < 0.05) with the statistical program StatGraphics Centurion XV (StatPoint, 2005) (StatGraphics Centurion XV version 15.02.06. Warrenton, VA, USA. www.statgraphics.com).
Results
Lactic-acid bacterial growth in carp during spontaneous fermentation
The highest lactic-acid bacterial concentration induced by spontaneous fermentation was observed in the treatment Re10Sk (reared carps) (Fig. 1) with a value higher than 9.42 log of CFU/g different from the treatment Wi10In (wild carps) that showed the lowest LAB concentration with a value of 6.25 log UFC/g. The rest of the samples did not show statistically significant (p < 0.05) differences with values close to 8 log UFC/g. LAB concentration in wild carps in the samples taken from skin did not show statistically significant differences at 5 and 10 days. However, wild carp intestine samples showed higher LAB concentration at 5 than at 10 days of fermentation, whereas in the reared carp skin samples, significant differences were observed in those taken at 10 days showing higher LAB concentration than those at 5 days. LAB presence in carp has been reported previously in intestine concentrations from 5 to 7 log UFC/g [11, 12, 17].
Fig. 1.
Lactic-bacterial growth (LAB) in carp by spontaneous fermentation from different origin and organ at 5 and 10 days. Different letters indicate significant differences according to Tukey’s (p < 0.05). The bar in the rectangle represents standard error; Re = reared; Wi = wild; In = intestine; Sk = skin
Lactic-acid bacterium isolation and morphological characterization
A total of 162 LAB isolates were obtained, of which all showed their own morphological characteristics, such as circular colony morphology and flat colony elevation; all isolates were Gram-positive and CAT-positive [18]. Starting from the reared carps, a total of 86 isolates were obtained and 76 from the wild ones (Table 2).
Table 2.
Lactic-acid bacterium (LAB) isolates from wild and reared carps
| Carp origin | Treatment | Number of isolates |
|---|---|---|
| Wild | Wi5Sk | 21 |
| Wi5In | 19 | |
| Wi10Sk | 19 | |
| Wi10In | 17 | |
| Reared | Re5Sk | 26 |
| Re5Sk | 21 | |
| Re10Sk | 16 | |
| Re10In | 23 |
Sk skin; In intestine
*The results are the average of three replicates
Identification of isolated lactic-acid bacteria
From the total isolates (162), 85% (137) was identified, possibly because some of the isolated bacteria were not found in the equipment library, which was used for protein characterization—81 from reared carps and 56 from wild ones (Table 3).
Table 3.
Identification of lactic-acid bacteria (LAB) isolates of reared and wild carps
| Carp origin | Identified bacteria | Number of isolates | Abundance (%) |
|---|---|---|---|
| Wild | Lactiplantibacillus plantarum | 14 | 25 |
| Lactococcus lactis | 11 | 20 | |
| Leuconostoc mesenteroides | 9 | 16 | |
| Latilactobacillus sakei | 8 | 14 | |
| Leuconostoc pseudomesenteroides | 4 | 7 | |
| Weissella viridescens | 3 | 6 | |
| Pediococcus pentosaceus | 3 | 5 | |
| Leuconostoc citreum | 2 | 4 | |
| Latilactobacillus curvatus | 2 | 4 | |
| Reared | Latilactobacillus sakei | 47 | 58 |
| Leuconostoc mesenteroides | 15 | 19 | |
| Lactococcus lactis | 9 | 11 | |
| Lactiplantibacillus plantarum | 5 | 6 | |
| Leuconostoc pseudomesenteroides | 1 | 1 | |
| Latilactobacillus fuchuensis | 1 | 1 | |
| Lactococcus raffinolactis | 1 | 1 | |
| Leuconostoc citreum | 1 | 1 | |
| Limosilactobacillus reuteri | 1 | 1 |
*The results are the average of three replicates
In total, seven genera and 13 different species were obtained from all the samples both five proteolytic and two non-proteolytic (Table 3). A total of six LAB species were found both in reared and wild carps (Lactiplantibacillus plantarum, Lactococcus lactis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Latilactobacillus sakei, and Leuconostoc citreum); three LAB species were only found in reared carps (Latilactobacillus fuchuensis, Lactococcus raffinolactis, and Limosilactobacillus reuteri) and three only in wild ones (Latilactobacillus curvatus, Weissella viridescens, and Pediococcus pentosaceus). Some of the carps found in this study were also reported by Hagi et al. [11], such as L. lactis, L. raffinolactis, and L. curvatus in common carp, silver carp, and crucian carp intestines from Lake Kasumigaura in Japan. On the other hand, while Hagi and Hoshino [19] were searching for probiotic LAB in carp intestines, they reported some of those found in this research, such as L. raffinolactis, L. fuchuensis, and L. sakei. Giri et al. [20] also isolated L. reuteri from common carp intestine. Furthermore, LAB, such as L. mesenteroides and P. pentoseceus, were found in processed fish products [17], which were also identified in this study. Lactiplantibacillus plantarum and W. viridescens have been isolated from rainbow trout (Oncorhynchus mykiss) intestine [21, 22] and L. pseudomesenteroides from fish Mugil cephalis [23]. Additionally, some of the bacteria reported in this study have been also found in fermented feed based on fish, as Thai, where L. citreum have been isolated [24].
Of the isolated and identified bacteria from wild carps, L. plantarum followed by L. lactis represented 45% of abundance, different from L. sakei, which was the bacteria that showed greater abundance in reared carps with a percentage of 58%. Other research studies reported L. lactis and L. raffinolactis as predominant LAB in carp from Ibaraki Japan [19], which make evident that predominance of some genera and species is related to the ecosystem where these fish develop [13].
Genera and species of lactic-acid bacteria obtained
Table 4 shows LAB according to their origin, fermentation time, and sampling area. As it can be observed, time did not show a significant (p < 0.05) difference as to diversity. With this respect, two LAB (L. fuchuensis and L. raffinolactis) genera were isolated at 5 days of fermentation but not at 10 days of spontaneous fermentation, whereas three LAB (L. reuteri, P. pentosaceus, and L. curvatus) were isolated at 10 days of fermentation but not at 5 days. Spontaneous fermentation time could have affected LAB presence in carp because they do not survive in acid pH and others have some metabolic mechanisms that allow them to survive in this condition [25].
Table 4.
Obtaining lactic-acid bacteria (LAB) according to carp origin, fermentation time, and sampling area
| Carp origin | Fermentation time (days) | Sampling area | |
|---|---|---|---|
| Skin | Intestine | ||
| Reared | 5 | Latilactobacillus sakei | Latilactobacillus fuchuensis |
| Lactococcus lactis | Latilactobacillus sakei | ||
| Leuconostoc pseudomesenteroides | Lactococcus lactis | ||
| Lactococcus raffinolactis | |||
| Leuconostoc citreum | |||
| Leuconostoc mesenteroides | |||
| 10 | Lactiplantibacillus plantarum | Lactiplantibacillus plantarum | |
| Latilactobacillus sakei | Limosilactobacillus reuteri | ||
| Lactococcus lactis | Latilactobacillus sakei | ||
| Leuconostoc mesenteroides | Leuconostoc mesenteroides | ||
| Wild | 5 | Lactiplantibacillus plantarum | Lactiplantibacillus plantarum |
| Lactococcus lactis | Lactococcus lactis | ||
| Leuconostoc mesenteroides | Leuconostoc citreum | ||
| Leuconostoc pseudomesenteroides | Leuconostoc mesenteroides | ||
| Weissella viridescens | |||
| 10 | Latilactobacillus sakei | Latilactobacillus curvatus | |
| Lactococcus lactis | Lactiplantibacillus plantarum | ||
| Leuconostoc citreum | Latilactobacillus sakei | ||
| Pediococcus pentosaceus | Lactococcus lactis | ||
| Weissella viridescens | Leuconostoc citreum | ||
| Leuconostoc mesenteroides | |||
| Leuconostoc pseudomesenteroides | |||
| Weissella viridescens | |||
With respect to organs, intestines have shown a greater diversity in LAB genera and species compared with those found in skin. In 1990, Cahill [13] reported a greater diversity in LAB compared to those in water related to the ones found in skin. This difference can be explained because the intestine provides favorable ecological niches for these microorganisms. On the other hand, LAB found in skin are a reflex of their environment [13].
Table 4 shows eight LAB carp skin samples isolated, whereas 10 were isolated in intestines; P. pentosaceus was only recovered in skin and L. fuchuensis, L. raffinolactis, and L. reuteri only in intestines. These LAB have been previously reported in the intestine in different research; as mentioned previously, Hagi and Hoshino [19] reported the presence of L. fuchuensis and L. raffinolactis in carp intestine. On the other hand, L. reuteri was also recovered in carp intestine as reported by Giri et al. [20] attributing its presence because it is a bacterium that protects fish from metal toxicity.
The adaptability of the isolated LAB in carp may be explained because these bacteria can develop a proteolytic enzymatic system to fraction proteins. The isolated LAB in this research, such as L. plantarum, L. curvatus, and L. sakei, have been used as starters in fermentations to obtain bioactive peptide with different functions (antimicrobial, antioxidant, anti-inflammatory, antihypertensive, among others) starting from substrates, such as milk, pig meat, fish, and shellfish, among other sources [26, 27]. Thus, obtaining bioactive peptides is feasible starting from protein substrate fermentation, using these bacteria as culture starters.
Proteolytic capacity determination of isolated lactic-acid bacteria
A total of 43 LAB showed proteolytic activity with a total of 137, which represents 31.4% using the APA medium supplemented with 1 mg/mL of carp muscle powder as protein source. Figure 2 shows the relationship of the total isolated and identified with respect to those that showed proteolytic capacity. In all the treatments, the identified proteolytic LAB are shown with less incidence than the total. The isolated LAB from wild carps showed the greatest number of proteolytic LAB than those reared.
Fig. 2.
Comparison of total native lactic-acid bacteria (LAB) and total proteolytic native LAB. Different letters for each LAB type indicate significant differences according to Tukey’s (p < 0.05). The meaning of the treatment abbreviation can be seen in Table 1. The bar in the rectangle represents standard error; Wi = wild; Re = reared; In = intestine; Sk = skin
According to fermentation time, 46.5% was obtained in treatment Wi10In and 64% in Wi10Sk at 10 days; at 5 days of fermentation, 50% was obtained in treatment Wi5In and 35% in Wi5Sk. In reared carp, 17% was obtained in treatment Re10In and 15% in Re10Sk at 10 days of fermentation; at 5 days of fermentation, 29% was obtained in treatment Re5In and 20% in Re5Sk. The presence of isolated LAB from fresh water and marine fish with proteolytic activity has been reported by different authors [9, 28, 29]. While searching for proteolytic microorganisms from fish as carp, Sudeepa et al. [29] found that the genera previously called Lactobacillus (including Lactiplantibacillus, Latilactobacillus, and Limosilactobacillus, found in this research) [30] and Lactococcus are two of the LAB genera that have shown greater proteolytic activity jointly with Bacillus, Pseudomonas, and Sarcina, among others.
Semi-quantification of proteolytic capacity of isolated lactic-acid bacteria
Figure 3 shows treatment comparison where evidently a greater proteolytic activity was observed in the isolated LAB from the treatments Wi5In and Wi5Sk, which have no statistical difference. As observed, these treatments are from wild carps with the same spontaneous fermentation time at day 5. With this respect, authors as Torino et al. [31] have reported obtaining antihypertensive peptides fermented with L. plantarum at day 4, which is related with spontaneous fermentation time when a greater proteolytic activity was observed in this research. Thus, these bacteria could be used to obtain peptides since they have shown a favorable proteolytic activity at 5 days of fermentation. The isolated LAB from these wild carps showed a greater proteolytic activity on day 5 of spontaneous fermentation in both organs (skin and intestine). These results indicate that no spontaneous fermentation greater than 5 days are required to isolate proteolytic LAB. In accordance, the proteolytic LAB from wild carps showed greater activity with respect to those reared, which can be explained because their activity may be due to the proteolytic enzyme system development caused by environmental conditions, such as the presence of protein as food source for LAB [5].
Fig. 3.
Proteolytic activity of the treatments in carp skin and intestines. Letters indicate significant differences according to Tukey’s (p < 0.05). The meaning of the treatment abbreviation can be seen in Table 1. The bar in the rectangle represents standard error; Wi = wild; Re = reared; In = intestine; Sk = skin
Average proteolytic activity by genera and species
The average proteolytic activity of the isolated genera and species is shown in Table 5. As observed, the genus and species with a greater proteolytic halo was L. plantarum (1.54 cm in diameter), which in the majority were wild carp isolates. The sampling point was the intestine because LAB have an environment composed of proteins in the intestine, so a more efficient proteolytic system had to be developed. The second genera and species with greater proteolytic capacity was L. pseudomesenteroides with a proteolytic halo of 1.32 cm; and in the third place, L. sakei with a proteolytic halo average of 1.11 cm. Other research works have demonstrated that L. sakei strains have greater adaptation to substrates with high protein concentration, such as meat, thus, the reason for being the third LAB with greater proteolytic activity and having shown the greatest number of isolates than the rest of the proteolytic LAB genera and species [32].
Table 5.
Proteolytic activity of lactic-acid bacteria (LAB) by genera and species
| Native LAB genera and species | Number or isolates | Proteolytic activity (cm)* |
|---|---|---|
| Latilactobacillus curvatus | 1 | 0.27 ± 0.03 |
| Lactiplantibacillus plantarum | 7 | 1.54 ± 0.19 |
| Latilactobacillus sakei | 12 | 1.11 ± 0.13 |
| Lactococcus lactis | 8 | 0.86 ± 0.19 |
| Leuceuconostoc citreum | 2 | 1.08 ± 0.40 |
| Leuconostoc mesenteroides | 9 | 0.94 ± 0.18 |
| Leuconostoc pseudomesenteroides | 3 | 1.32 ± 0.38 |
| Weisella viridescens | 2 | 0.78 ± 0.27 |
*Average plus/minus standard error
According to the results, four different genera and eight proteolytic species were found. Table 6 shows the identification of all LAB isolates that showed proteolytic activity in all the treatments. As observed, the genus previously called Lactobacillus was the one that had the greatest incidence with 46.5%, Leuconostoc with 30%, Lactococcus with 18.6%, and Weissella with 4.6%.
Table 6.
Identification of proteolytic lactic-acid bacteria (LAB) with proteolytic halo
| LAB with proteolytic halo | Treatment | Halo (cm) |
|---|---|---|
| Latilactobacillus curvatus S10I1 | Wi10In | 0.27 |
| Lactiplantibacillus plantarum C10I1 | Re10In | 0.43 |
| Lactiplantibacillus plantarum S10I1 | Wi10In | 0.93 |
| Lactiplantibacillus plantarum S5I1 | Wi5In | 2.40 |
| Lactiplantibacillus plantarum S5I2 | Wi5In | 1.42 |
| Lactiplantibacillus plantarum S5P1 | Wi5Sk | 1.00 |
| Lactiplantibacillus plantarum S5P2 | Wi5Sk | 2.80 |
| Lactiplantibacillus plantarum S5P3 | Wi5Sk | 1.66 |
| Latilactobacillus sakei C10I1 | Re10In | 1.07 |
| Latilactobacillus sakei C10P1 | Re10Sk | 0.93 |
| Latilactobacillus sakei C10P2 | Re10Sk | 0.23 |
| Latilactobacillus sakei C5I1 | Re5In | 1.17 |
| Latilactobacillus sakei C5I2 | Re5In | 0.40 |
| Latilactobacillus sakei C5I3 | Re5In | 0.30 |
| Latilactobacillus sakei C5P1 | Re5Sk | 0.17 |
| Latilactobacillus sakei S10P1 | Wi10Sk | 1.13 |
| Latilactobacillus sakei S10P2 | Wi10Sk | 1.97 |
| Latilactobacillus sakei S10P3 | Wi10Sk | 1.57 |
| Latilactobacillus sakei S10P4 | Wi10Sk | 2.27 |
| Latilactobacillus sakei S10P5 | Wi10Sk | 2.13 |
| Lactococcus lactis C5I1 | Re5In | 1.10 |
| Lactococcus lactis C5I2 | Re5In | 1.03 |
| Lactococcus lactis C5I3 | Re5In | 0.90 |
| Lactococcus lactis C5P1 | Re5Sk | 0.43 |
| Lactococcus lactis C5P2 | Re5Sk | 0.26 |
| Lactococcus lactis S10P1 | Wi10Sk | 0.53 |
| Lactococcus lactis S10P2 | Wi10Sk | 2.20 |
| Lactococcus lactis S5I1 | Wi5In | 0.47 |
| Leuconostoc citreum S10I1 | Wi10In | 1.90 |
| Leuconostoc citreum S10P1 | Wi10Sk | 0.26 |
| Leuconostoc mesenteroides C10I1 | Re10In | 0.20 |
| Leuconostoc mesenteroides C10P1 | Re10Sk | 0.27 |
| Leuconostoc mesenteroides C5I1 | Re5In | 0.27 |
| Leuconostoc mesenteroides C5I2 | Re5In | 0.23 |
| Leuconostoc mesenteroides S10I1 | Wi10In | 0.87 |
| Leuconostoc mesenteroides S10I2 | Wi10In | 0.93 |
| Leuconostoc mesenteroides S5I1 | Wi5In | 2.73 |
| Leuconostoc mesenteroides S5I2 | Wi5In | 0.77 |
| Leuconostoc mesenteroides S5I3 | Wi5In | 2.20 |
| Leuconostoc pseudomesenteroides S5P1 | Wi5Sk | 0.23 |
| Leuconostoc pseudomesenteroides S5P2 | Wi5Sk | 2.66 |
| Leuconostoc pseudomesenteroides S10I1 | Wi10In | 1.07 |
| Weissella viridescens S10P1 | Wi10Sk | 0.83 |
| Weissella viridescens S5P1 | Wi5Sk | 0.73 |
The LAB genera of Lactobacillus (which now includes Lactiplantibacillus, Latilactobacillus, and Limosilactobacillus), Lactococcus, and Weissella have been reported in different research works with proteolytic capacity, which have been isolated strains from viscera and intestines of fresh water fish and shellfish waste [9, 33].
The genera and species that showed the greatest incidence of all the isolated proteolytic LAB was L. sakei with 28%, followed by L. mesenteroides (21%), L. lactis (18.6%), L. plantarum (16.3%), and L. pseudomesenteroides (7%); L. citreum and W. viridescens showed the same incidence percentage (4.6%).
According to the results found in fermentation time, apparently it has an influence on genera and species besides its proteolytic activity. Proteolytic LAB that were isolated at two fermentation times (5 and 10 days) were L. plantarum, L. pseudomenseteroides, W. viridescens, L. lactis, L. sakei, and L. mesenteroides; at 10 days of fermentation, L. citreum and L. curvatus also showed proteolytic activity. LAB with proteolytic bacteria were found in two sampling areas (skin and intestine) (L. plantarum, L. pseudomesenteroides, L. lactis, L. sakei, and L. mesenteroides). Furthermore, W. viridescens was recovered in skin and L. curvatus and L. citreum in the intestines.
The LAB with the greatest average proteolytic halo (2.73 cm) were isolated from wild carps at 5 days of fermentation (L. plantarum S5P2, L. mesenteroides S5I1, and L. pseudomesenteroides S5P2). Moreover, these three LAB did not show significant differences as to proteolytic halo diameter.
However, three L. plantarum bacteria did not show the same proteolytic halo, of which L. plantarum S5P2 (from wild carp at 5 days of skin fermentation) was the strain that showed the greatest proteolytic activity of all the isolated strains with a halo of 2.8 cm. In contrast, L. plantarum S5P1 and L. plantarum S5P3 showed halos of 1.66 cm and 1.0 cm, respectively, despite the three of them were isolated in the same conditions. The same behavior was observed in L. mesenteroides S5I1 (from wild carp at 5 days of intestine fermentation) showing a halo of 2.73 cm, which was the second isolate with greater proteolytic activity compared with L. mesenteroides S5I3 and L. mesenteroides S5I2 with halos of 2.2 cm and 0.77 cm, respectively. Another example of this behavior was L. pseudomesenteroides S5P2 (from wild carp at 5 days of skin fermentation) with a proteolytic halo of 2.66 cm, which was the third LAB with the greatest proteolytic halo; L. pseudomesenteroides S10I1 and L. pseudomesenteroides S5P1 (isolated from wild carp at 10 days of intestine fermentation) showed halos of 1.07 cm and 0.23 cm, respectively. As it can be observed, the proteolytic activity of the first two bacteria—isolated in the same conditions—showed different proteolytic capability. In this case, despite each LAB was isolated in the same condition, regardless of being the same genus and species, they may express their proteolytic genes differently. In contrast, Cahill et al. [11] mentioned that this proteolytic capacity is very much related to the ecosystem where the bacteria develop.
Conclusions
This study isolated 162 lactic-acid bacteria (LAB) from both wild and reared carps, of which 137 LAB isolates were identified by means of MALDI-TOF—81 from reared carps and 56 from wild ones. From the identified isolates, 31.4% showed proteolytic activity that corresponded to five different genera and eight species (L. curvatus, L. plantarum, L. sakei, L. lactis, L. citreum, L. mesenteroides, L. pseudomesenteroides, and W. viridescens). The proteolytic LAB obtained from wild carps corresponded to the treatments Si5In and Si5Pi, which were the ones that showed greater proteolytic halo: L. plantarum S5P2 (2.8 cm), L. mesenteroides S5I1 (2.73 cm), and L. pseudomesenteroides S5P2 (2.66 cm). These three proteolytic LAB were isolated at 5 days of spontaneous fermentation, which shows that an efficient proteolytic system from wild carps does not need more time of spontaneous fermentation for LAB recovery. Future research should evaluate the use of the strains that showed greater proteolytic halo for the production of bioactive peptides with different biological functions, such as antioxidant, antimicrobial, and antihypertensive, among others, starting from fermentation of protein-rich substrates.
Acknowledgements
The authors are grateful to CIATEJ (Centro de Investigación y Asistencia y Tecnología de Jalisco) where all the project was developed and Diana Fischer for the English translation and edition.
Funding
This research was financed by FODECIJAL (“Fondo 2019 de Desarrollo Científico de Jalisco”) to address state problems from the project “Desarrollo de un alimento nutracéutico inhibidor de la enzima convertidora de la Angiotensina, principal responsable de la hipertensión arterial, a partir de músculo de carpa (Cyprinus carpio)”. The authors also received project funding through the FODECIJAL fund from COECYTJAL (Consejo Estatal de Ciencia y Tecnología de Jalisco), and María Luisa Sahagún-Aguilar received doctoral scholarship from CONACYT (Consejo Nacional de Ciencia y Tecnología) Mexico.
Declarations
Conflict of interest
The authors declare no competing interests.
Footnotes
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