Amaranth Meal and Environmental Carnobacterium maltaromaticum Probiotic Bacteria as Novel Stabilizers of the Microbiological Quality of Compound Fish Feeds for Aquaculture

: Fish feed should be characterized by microbiological stability to guarantee the optimal health of farmed ﬁsh. The aim of this study was to determine the e ﬃ cacy of amaranth meal ( Amaranthus cruentus ) and a highly active environmental strain of probiotic bacteria, Carnobacterium maltaromaticum , as novel supplements that stabilize the quantitative and qualitative composition of microbiota in compound ﬁsh feeds for aquaculture, regardless of storage temperature. The total viable counts of mesophilic bacteria at 28 ◦ C (TVC 28 ◦ C), hemolytic mesophilic bacteria (Hem 37 ◦ C), Staphylococcus sp. bacteria, aerobic spore-forming bacteria (ASFB), sulﬁte-reducing anaerobic spore-forming Clostridium sp. bacteria, yeasts, and molds were analyzed in control feed (CF), in feed supplemented with amaranth meal (AF), and in feed supplemented with amaranth meal and C. maltaromaticum (ACF), stored at a temperature of 4 ◦ C and 20 ◦ C for 98 days. Amaranthus cruentus and C. maltaromaticum signiﬁcantly reduced bacterial counts in ﬁsh feeds, regardless of the temperature and duration of storage. The antibacterial and antifungal e ﬀ ects of the tested additives were statistically signiﬁcant ( p ≤ 0.05). The studied novel supplements contribute to the microbiological safety of compound ﬁsh feeds. The tested additives could be recognized as the key ingredients of organic, environmentally friendly ﬁsh feeds, which guarantee the high quality of ﬁsh intended for human consumption. M.G.A.-A. and A.C.-R.; software, I.G.; validation, I.G., J.P., M.W. and P.N.; formal analysis, J.P. and P.N.; investigation, I.G., J.P., M.W. and P.N.; resources, I.G., M.W., M.G.A.-A. and A.C.-R.; data curation, I.G. and J.P.; writing—original draft preparation, I.G. and M.W.; writing—review and editing, M.W. and A.G.-P.; visualization, A.G.-P.; supervision, I.G.

other authors [40][41][42] demonstrated that amaranth meal increased the survival and growth rates of probiotic bacteria and improved the microbial stability of foods. The combined use of environmental probiotic bacteria and amaranth meal as stabilizers of the microbiological quality of fish feeds remains insufficiently researched. These facts have prompted the authors to evaluate the effectiveness of a highly active environmental isolate of C. maltaromaticum and amaranth meal in stabilizing the microbiological quality of fish feed. The aim of this study was to determine the efficacy of amaranth meal (Amaranthus cruentus) and a highly active environmental strain of probiotic bacteria, C. maltaromaticum, as novel supplements that stabilize the quantitative and qualitative composition of microbiota in compound fish feeds for aquaculture, regardless of storage temperature.

Isolation and Identification of C. Maltaromaticum Probiotic Bacteria
A probiotic strain of C. maltaromaticum was isolated from water samples collected from the benthic zone of Lake Legińskie (at a depth of 34 m) located in north-eastern Poland (N = 53 • 58 51" N and E = 21 • 8 4"). The strain had been isolated during a previous study conducted by the Department of Environmental Microbiology of the University of Warmia and Mazury in Olsztyn.
The isolate was identified to species level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF VITEK ® MS) at the Department of Microbiology, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, in Mexico City, Mexico. The identification was additionally verified by 16S rDNA (recombinant DNA) sequencing with the BigDye Terminator v3.1 kit in the ABI 3730xl genetic analyzer (Applied Biosystems, Foster City, USA). In addition, 16S rDNA genes were sequenced by PCR with the use of 27F (5 -AGAGTTTGATCATTGGCTCAG-3 ) and 1492R (5 -GGTACC-TTGTTACGACTT-3 ) primers according to the method described by Gillan et al. [43]. The BLAST program available on the website of the National Center of Biotechnology Information [44] was used to identify DNA sequences. The results of 16S rDNA sequencing are presented in Table S1 (Supplementary Materials).
After the identification process, C. maltaromaticum was considered as a probiotic strain based on the hemolysis assay, and its acid and bile tolerance properties, according to the guidelines developed by a joint FAO/WHO working group [45]. The hemolytic activity of C. maltaromaticum was determined on tryptone soya agar (TSA; Oxoid, Basingstoke, UK) with 5% addition of defibrinated sheep blood incubated at 37 • C for 48 h [46]. The bile salt tolerance test of the studied strain was performed in MRS broth culture medium (Sigma -Aldrich, Germany) containing 0.5%, 1.0%, or 2.0% bile salts (Oxoid LP0055) according to the procedure proposed by Succi et al. [47]. The C. maltaromaticum isolate was tested for acid tolerance based on its growth on medium with varying pH (1.5, 2.5, 3.5, and 4.5), as described by Vijayarama et al. [48].

Determination of the Metabolic Activity of Probiotic Bacteria Based on the Utilization of Different Carbon Sources
The applicability of the environmental C. maltaromaticum isolate for further analysis was determined by analyzing the bacteria's metabolism based on its utilization of various carbon sources. The biochemical activity of the C. maltaromaticum probiotic isolate and its potential to compete for nutrients with feed microbiota were estimated using the OmniLog ® System (Biolog, USA). A 96-well plate containing various carbon compounds was inoculated with the evaluated bacterial strain. The plate was incubated, and biochemical parameters were read in a microstation reader. The strain's utilization of different carbon compounds as sources of energy was determined based on the intensity of color reactions.

Compound Feed
The experiment was performed on three types of extruded compound feeds: Control feed (CF) without the addition of amaranth meal, experimental feed containing 20% of amaranth meal (AF), and experimental feed containing 20% of amaranth meal and C. maltaromaticum probiotic bacteria (ACF). The composition of each feed is presented in Table 1. All feeds were formulated based on the recommendations of Hart et al. [49] and NRC [50]. The feeds were extruded with a co-rotating twin screw extruder (Metalchem, Poland) equipped with a Ø 4.5 mm pellet stencil. The following extrusion processing parameters were applied: Screw speed-105-125 rpm, cutter speed-50 rpm, head temperature-120 • C, barrel temperature of 130-150 • C in 30 s, die diameter-2.0 mm. Compound feeds were enhanced with a mixture of fish oil and soybean oil (5% each). The tested strain of C. maltaromaticum was added to the oil mixture. Next, the probiotic oil suspension was added to two experimental feed samples. Tthe oil mixture was pumped into the feed at 0.9 Mpa for 5 min with the use of a vacuum pump. The feed contained 40.0% crude protein, 15.0% crude fat, 3.0% crude ash, 37.0 nitrogen-free extract (NFE), and 5% water.  4 1.00 1.00 1.00 Mineral premix 5 2.00 1.00 1.00 C. maltaromaticum (CFU·g −1 ) 0.00 0.00 1.5 × 10 9

Experimental Design
The prepared feeds (CF, AF, and ACF) were used in an experiment that lasted for 98 days. The control feed (CF) was divided into two equal parts, and the feed containing 20% amaranth meal (AF) was divided into four equal parts under sterile conditions. Every CF and AF sample was placed in a separate, sterile, and tightly closed vessel made of dark glass. Two samples (CF 4 • C, AF 4 • C) were chill-stored at a temperature of 4 • C, and two samples (CF 20 • C, AF 20 • C) were stored at a temperature of 20 • C throughout the experiment. Cultures of the environmental C. maltaromaticum strain were added to the remaining two samples (AF) at 1.5 × 10 9 CFU·g −1 ( Table 1). One of the samples containing probiotic bacteria (ACF 4 • C) was chill-stored at 4 • C, and the other sample (ACF 20 • C) was stored at 20 • C for 98 days.
All analyses were performed according to Polish Standard [51]. The potential pathogenicity of Hem 37 • C, Staphylococcus sp., and Clostridium sp. bacteria was determined based on their hemolytic activity on tryptone soya agar (TSA; Oxoid, Basingstoke, UK) with 5% addition of defibrinated sheep blood incubated at 37 • C for 48 h. Hemolysis was confirmed when a transparent zone was formed around the inoculated colonies [46]. Mean microbial counts were calculated based on the values determined in three replicates of the same sample of compound fish feed. Finally, the counts of all analyzed microorganisms were expressed in CFU·1 g −1 of compound feed.

Statistical Analysis
The mean values, standard deviations, standard errors, and confidence interval (CI = 95%, N = 3) of microbial counts in feeds (CF, AF, ACF) stored at a temperature 4 • C and 20 • C were calculated. The relationships between C. maltaromaticum bacterial counts and microbial (TVC 28 • C, Hem 37 • C, ASFB, Staphylococcus sp., Clostridium sp., yeasts, and molds) counts were determined by Spearman's non-parametric rank correlation test (p ≤ 0.05). The significance of differences in microbial counts between the analyzed types of fish feed (CF, AF, ACF) stored at different temperatures (4 and 20 • C) and for different periods of time (7,14,21,28,35,42,49,56,63,70,77, 84, 91, and 98 days) was determined by one-way analysis of variance (ANOVA). Leven's test was used to assess the homogeneity of variance. The verified hypothesis was rejected when Leven's test produced statistically significant results. The Kruskal-Wallis test, a non-parametric version of the classical one-way ANOVA, was then applied. Statistical analyses were performed in the Statistica 13.3 software package (TIBCO Software Inc., Palo Alto, USA) [52].

Probiotic Properties of Carnobacterium Maltaromaticum
The studied C. maltaromaticum isolate was not capable of causing hemolysis, which suggested that the strain was not pathogenic.
The strain tolerated the tested pH values. After 3 h acid exposure, the isolate's survival rate was higher at pH 2.5 (76.1%) than at pH 1.5 (65%), and it reached 82.3% at pH 3.5 and 87.8% at pH 4.5. The bile salt tolerance test revealed a small difference in the survival rates of C. maltaromaticum. The highest isolate viability (85.2%) was observed at a 2% concentration of bile salts, whereas the lowest viability (79.5%) was noted at a 0.5% concentration of bile salts; 83.2% of C. maltaromaticum bacteria survived at a 1.0% concentration of bile salts (data not shown).
The C. maltaromaticum isolate tested in our study could be classified as a probiotic strain based on the results of the above analyses and according to the guidelines developed by a joint FAO/WHO working group [45].

Metabolic Activity of C. maltaromaticum Probiotic Bacteria
The results of the analyses examining the utilization of various carbon sources by the environmental C. maltaromaticum isolate are presented in Figure 1. The analyses performed in the Omnilog Gen III system (Biolog, Hayward, CA, USA) revealed that the evaluated strain actively metabolized 70 carbon sources. The studied C. maltaromaticum strain was capable of growth at pH 5 and 6, and in the presence of 1%, 4%, and 8% NaCl. The tested isolate did not metabolize the following substrates: L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, histidine, D-gluconic acid, and mucic acid. The analyzed strain did not metabolize vancomycin, tetrazolium blue chloride, L-pyroglutamic acid, α-ketoglutaric acid, α-ketobutyric acid, and acetoacetic acid. These results confirmed the very high biochemical activity of the studied environmental probiotic isolate, and suggested its potential to compete for nutrients with feed microbiota.

Metabolic Activity of C. maltaromaticum Probiotic Bacteria
The results of the analyses examining the utilization of various carbon sources by the environmental C. maltaromaticum isolate are presented in Figure 1. The analyses performed in the Omnilog Gen III system (Biolog, Hayward, CA, USA) revealed that the evaluated strain actively metabolized 70 carbon sources. The studied C. maltaromaticum strain was capable of growth at pH 5 and 6, and in the presence of 1%, 4%, and 8% NaCl. The tested isolate did not metabolize the following substrates: L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, histidine, D-gluconic acid, and mucic acid. The analyzed strain did not metabolize vancomycin, tetrazolium blue chloride, L-pyroglutamic acid, -ketoglutaric acid, -ketobutyric acid, and acetoacetic acid. These results confirmed the very high biochemical activity of the studied environmental probiotic isolate, and suggested its potential to compete for nutrients with feed microbiota.  Figure 1. The results of a metabolic activity test analyzing the chemical sensitivity of an environmental Carnobacterium maltaromaticum probiotic isolate and its ability to utilize different carbon sources (GEN III MicroPlate™). Purple color-metabolic activity of the C. maltaromaticum isolate, white color-no metabolic activity of the C. maltaromaticum isolate.

The Quantitative and Qualitative Composition of Bacterial Microbiota in Compound Fish Feeds
The mean (of three replicates) counts of mesophilic bacteria (TVC 28 • C), hemolytic mesophilic bacteria (Hem 37 • C), Staphylococcus sp., Clostridium sp., aerobic spore-forming bacteria (ASFB), yeasts and molds in CF, AF, and ACF, and C. maltaromaticum bacteria stored at a temperature of 4 • C and Appl. Sci. 2020, 10, 5114 7 of 15 20 • C during the 98-day experiment are presented in Figure 2. The mean values, standard deviations, standard errors, and confidence interval of three replicates of microbial counts are shown in Table S2 (Supplementary Materials). In CF, microbial counts differed by several orders of magnitude, depending on the analyzed microbial group and the temperature and time of feed storage. In CF 4 • C samples, TVC 28 • C and Clostridium sp. counts increased several-fold after 14 and 28 days of storage, respectively, relative to initial values. The counts of other microbial groups (Hem 37 • C, Staphylococcus sp., yeasts, and molds) in CF 4 • C samples continued to decrease in successive weeks of the experiment. The noted decrease ranged from 10 1 to 10 5 CFU across the analyzed microbial groups, subject to storage time (Figure 2A).   Table S2 (Supplementary Materials).
The counts of nearly all microorganisms (excluding ASBF) increased by around 100% in CF 20 • C samples after 14, 28, and 42 days. In CF 20 • C samples, TVC 28 • C and yeast counts peaked on day 28 at 92 × 10 7 and 12 × 10 4 CFU·g −1 , respectively. The highest counts of potentially pathogenic bacteria (Hem 37 • C, Staphylococcus sp., Clostridium sp.) were noted after 42 days of feed storage. The maximum counts of Hem 37 • C, Staphylococcus sp., Clostridium sp., and molds were determined at 2.8 × 10 6 , 3.0 × 10 2 , 45, and 2.5 × 10 3 CFU·g −1 , respectively. A minor decrease in microbial counts was noted in successive weeks of the experiment. However, on day 98, the counts of all evaluated microorganisms in CF 20 • C samples were several-fold to several hundred-fold higher than those in CF 4 • C samples ( Figure 2B).
In feed samples supplemented with 20% amaranth meal stored at a temperature of 4 • C (AF 4 • C), the counts of all analyzed microbial groups decreased by several orders of magnitude after 14 days of the experiment. On day 28, Hem 37 • C (20 CFU·g −1 ) was the only potentially pathogenic microorganism in the studied samples. Toward the end of the experiment, AF 4 • C samples were colonized only by TVC 28 • C (500 CFU·g −1 ) and ASFB (5 CFU·g −1 ) ( Figure 2C).
In AF 20 • C samples, the decrease in the counts of potentially pathogenic Hem 37 • C bacteria was considerably lower than that in AF 4 • C samples. On day 28, Hem 37 • C counts in AF 20 • C samples were determined at 1.0 × 10 4 CFU·g −1 , and they were 500-fold higher than those in AF 4 • C samples on the same day. The counts of TVC 28 • C, ASBF, and yeasts were also several-fold to several dozen-fold higher in AF 20 • C samples than in AF 4 • C samples on the same days ( Figure 2D). Hem 37 • C, Staphylococcus sp., and Clostridium sp. survived for longer periods of time in AF 20 • C than in AF 4 • C. Hem 37 • C, Staphylococcus sp., and Clostridium sp. were eliminated from AF 20 • C samples only after 56 days, and from AF 4 • C-already after 14 or 28 days of the experiment ( Figure 2C,D).
Feed samples supplemented with 20% amaranth meal and a highly active environmental strain of C. maltaromaticum probiotic bacteria (ACF 4 • C, ACF 20 • C) were characterized by the lowest counts ( Figure 2E,F) and the lowest survival rate of all analyzed microbial groups, regardless of storage temperature (Table S3). Potentially pathogenic Staphylococcus sp., Clostridium sp., and Hem 37 • C bacteria were not detected in ACF 4 • C and ACF 20 • C samples already after 7 days. In the first two weeks of the experiment, TVC 28 • C counts decreased around 1000 fold, ASFB counts decreased more than 100-fold, and yeast counts decreased several fold in ACF 4 • C and ACF 20 • C samples relative to the initial values. On day 98, ACF 4 • C samples were colonized only by TVC 28 • C and ASBF at 10 and 5 CFU·g −1 , respectively ( Figure 2E). TVC 28 • C and ASFB counts were higher in ACF 20 • C at 120 and 20 CFU·g −1 , respectively ( Figure 2F). Additionally, Spearman's test revealed significant (p ≤ 0.05) negative correlations between C. maltaromaticum counts and almost all microbial populations (except for Clostridium sp. and molds) in ACF, regardless of storage temperature ( Table 2).
The differences in the quantitative and qualitative composition of bacterial and fungal microbiota in the analyzed types of fish feeds (CF, AF, and ACF) stored at different temperatures (4 • C and 20 • C) and for different periods of time were confirmed by the statistical analysis ( Table 3). The Kruskal−Wallis test revealed significant (p ≤ 0.05) differences in the counts of all analyzed microorganisms between the evaluated feeds (CF, AF, and ACF) and in ASFB and yeast counts in feed samples stored for different periods of time. Significant (p ≤ 0.05) differences were also observed in Staphylococcus sp., Clostridium sp., and mold counts in feed samples stored at different temperatures, and in TVC 28 • C, Hem 37 • C, Staphylococcus sp., and Clostridium sp. counts in feed samples stored for different periods of time. Table 2. The values of correlation coefficients between microbial counts in feed supplemented with 20% amaranth meal and C. maltaromaticum probiotic bacteria (ACF) stored at 4 and 20 • C. The correlations between microbial counts in ACF 4 • C (N = 14) and ACF 20 • C (N = 14) samples were analyzed with Spearman's test.

Discussion
The analyses of the quantitative and qualitative composition of microbiota in fish feed samples revealed significant differences (p ≤ 0.05) across the examined types of feed (CF, AF, ACF), feed storage temperatures, and feed storage times. Control feed (CF) was characterized by the highest counts, highest survival rates, and longest survival times of all analyzed microbial groups, which indicates that feed ingredients promote the growth of both specific feed microorganisms and potentially pathogenic microorganisms [37,53,54]. Similar results were reported by Petreska [4] and Gołaś et al. [55] who analyzed the counts of heterotrophic mesophilic bacteria and selected potentially pathogenic bacteria, yeasts, and molds in commercial feeds administered to intensively reared Silurus glanis L.
In our study, the counts of all specific feed microbiota and potentially pathogenic microorganisms (Hem 37 • C, Staphylococcus sp., Clostridium sp.) in feed supplemented with 20% amaranth meal (AF 4 • C, AF 20 • C) decreased by 1 to 4 orders of magnitude relative to those determined in CF 4 • C and CF 20 • C. The survival times of potentially pathogenic bacteria (Hem 37 • C, Staphylococcus sp., Clostridium sp.) were also significantly shorter in AF 4 • C and AF 20 • C than in CF 4 • C and CF 20 • C. The obtained results and the presence of significant differences (p ≤ 0.05) in the counts of all analyzed microbial groups between CF and AF samples indicate that feed supplementation with 20% amaranth meal inhibits the growth of bacterial and fungal microbiota regardless of storage temperature or duration ( Table 3). The antibacterial and antifungal properties of amaranth meal are also confirmed by the decrease in the counts of the remaining microbial groups (TVC 28 • C, ASFB, yeasts, molds) in AF 4 • C and AF 20 • C samples in successive weeks of the experiment. The above could be attributed to the fact that amaranth meal contains lignins whose antioxidant, antibacterial, antiviral, and fungistatic properties contribute to the maintaining of the adequate microbiological quality of feed [31,[56][57][58][59]. The addition of amaranth meal stabilizes natural microbiota in animal feeds, enhances the nutritional value of feeds, and improves performance.
Research studies have confirmed the beneficial influence of amaranth-supplemented feeds on the health status and body weight gains of rats [60], intensively farmed pigs [61,62], chickens [63], calves, lambs, sheep, and ruminants [56]. Studies investigating the effect of amaranth-supplemented feeds on fish in different farming systems also demonstrated that amaranth meal stimulated the immune system of fish [64], their growth performance, and the enzymatic activity of their gut microbiota [5,65]. The results of the present study indicate that amaranth meal can be effectively used to improve the quality and microbiological safety of fish feeds.
The counts of all studied microorganisms (TVC 28 • C, Hem 37 • C, ASFB, Staphylococcus sp., Clostridium sp., yeasts, and molds) were lowest in ACF 4 • and ACF 20 • relative to AF and CF stored at the corresponding temperatures. The counts, percentage viability, and survival times of the evaluated microbial groups were considerably lower in ACF 4 • and ACF 20 • than in AF 4 • and AF 20 • (Figure 2C-F; Table S3), which indicates that amaranth meal and C. maltaromaticum probiotic bacteria exert antibacterial and antifungal effects on natural microbiota and potentially pathogenic microorganisms in compound feed. The synergistic effects of the tested feed additives could be attributed to the symbiotic relationship between amaranth meal and the evaluated probiotic bacteria, and their ability to inhibit the growth and development of various microbial groups and genera. An in vitro study [57,66] revealed that amaranth is a source of bioactive compounds that suppress the proliferation of many microorganisms, including Staphylococcus aureus, Bacillus, Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Proteus mirabillis, Klebsiella pneumoniae, and Candida albicans. Amaranth meal also promotes the development of many species of probiotic bacteria, such as Lactobacillus plantarum, L. paralimentarius, L. helveticus, L. sakei, Pediococcus pentosaceus, L. paralimentarius, Enterococcus mundtii, E. hermanniensis, E. durans, Enterococcus sp., and Leuconostoc mesenteroides, whose metabolic activity enhances the nutritional value and health benefits of food products [67][68][69]. An in vitro study conducted by Gullón et al. [70] demonstrated that amaranth was characterized by a high prebiotic potential and promoted the growth of probiotic microflora isolated from the human digestive tract. By inhibiting the growth and development of naturally occurring microorganisms and pathogenic microbiota in foodstuffs and feedstuffs [5,66] probiotic bacteria and amaranth contribute to improving fish welfare and performance in various aquaculture systems [71][72][73].
The lowest counts of all evaluated microbial groups and genera and the shortest microbial survival times were noted in ACF samples regardless of storage temperature and storage time, which indicates that amaranth meal and C. maltaromaticum probiotic bacteria exert synergistic effects on the quantitative and qualitative composition of feed microbiota. Feed supplementation with 20% amaranth meal and C. maltaromaticum (ACF) bacteria completely inhibited the growth of most analyzed microorganisms (excluding ASFB and TVC 28 • C) in feeds stored at 4 • C and 20 • C for 7 days. The results of our in vitro study were validated statistically, which suggests that the novel tested additives contribute to the microbiological stability of fish feeds regardless of storage conditions and storage time.

Conclusions
The results of the present study, which investigated the supplementation of compound fish feeds with innovative additives, amaranth meal, and a highly active environmental strain of probiotic bacteria, C. maltaromaticum, indicate that the tested additives exert synergistic effects and contribute to the microbiological stability of fish feeds regardless of the temperature and time of storage. The evaluated components decreased the counts, percentage viability, and survival times of various groups and genera of microorganisms that occur naturally in feeds, which suggests that they can minimize feed losses resulting from the growth and metabolic activity of autochthonous and allochthonous microbiota in feeds that are stored for excessive periods of time and/or at inadequate temperature. Excessive microbial growth lowers the nutritional value of feed, and decreases nutrient digestibility and assimilability, which may negatively affect fish performance in aquaculture. The addition of 20% amaranth meal and a highly active environmental strain of probiotic bacteria, C. maltaromaticum, to fish feed inhibited the growth of potentially pathogenic microbiota (Hem 37 • , Staphylococcus sp., and Clostridium sp.) in vitro, which is important for the growth rate and welfare of fish. Due to their novel synergistic health-promoting properties, amaranth meal and environmental C. maltaromaticum bacteria could be recognized as the key ingredients of organic, environmentally friendly fish feeds, which guarantee the high quality of fish intended for human consumption.