Combined Thermomechanical–Biological Treatment for Corn By-Product Valorization into Added-Value Food (Feed) Material

The aim of this study was to apply the combined thermomechanical–biological treatment for corn processing by-product (CPBP) valorization to added-value food and feed material. The mechanical–thermal pre-treatment was performed by applying the extrusion technique. Extruded CPBPs (14, 16, and 18% moisture) were further biodegraded with Lactiplantibacillus plantarum-LUHS122 (Lpl), Liquorilactobacillus uvarum-LUHS245 (Lu), Lacticaseibacillus casei-LUHS210 (Lc), and Lacticaseibacillus paracasei-LUHS244 (Lpa). Acidity parameters, microbial characteristics, sugars concentration, amino and fatty acids profile, biogenic amines (BA), and antibacterial and antifungal properties of CPBP were analyzed. Fermented CPBP had a reduced count of mould/yeast. A significantly lower (p ≤ 0.05) count of total enterobacteria was found in most of the extruded–fermented CPBP. Fermentation of extruded CPBP (moisture of 16 and 18%) increased valine and methionine content. Cadaverine and spermidine were not found after treatment of CPBP, and the lowest content of BA was found in the extruded–fermented (Lpa, moisture 18%) CPBP. Applied treatment had a significant effect on most of the fatty acids. CPBP fermented with Lpl, Lu, and Lpa displayed inhibition properties against 3 of the 10 tested pathogenic/opportunistic bacterial strains. Extruded–fermented (Lu, Lc, and Lpa moisture of 14 and 18%) CPBP showed antifungal activity against Rhizopus. Extruded–fermented (14% moisture, Lpl) CPBP inhibited Rhizopus and Aspergillus fumigatus. In conclusion, combined treatment can improve certain parameters and properties of CPBP in order to produce safer and more nutritious ingredients for food and feed industries.


Introduction
Cereal grains are staple crops and provide food and energy for the population yearround because they are easy to store, and maintain essential nutrients for humans and animals [1,2]. The most important part of the cereal is starchy endosperm; however, most of the functional compounds are generally located in the outer part of the grain. Despite that, the utilization of cereal grain outer part in the food industry is very low (on average, 7.5%) due to the negative effects on overall acceptability of the product [1]. In addition to wheat, rye, and rice, corn (Zea mays L.) is cultivated globally in many regions [3,4]. Corn grain outer layer matrix is very complex, and contains hemicellulose, cellulose, protein, starch, crude The LAB strains Lactiplantibacillus plantarum-LUHS122, Liquorilactobacillus uvarum-LUHS245, Lacticaseibacillus casei-LUHS210, and Lacticaseibacillus paracasei-LUHS244 were used for the fermentation of CCon, Cex18, Cex16, Cex14. Characteristics, including carbohydrates metabolism, survival at low pH, gas production capacities, and antimicrobial and antifungal properties, of the LAB strains, used for corn by-product fermentation, are reported by Bartkiene et al. [15]. Prior to the experiments, LAB strains were multiplied in MRS broth (de Man-Rogosa-Sharpe, CM 0359, Oxoid Ltd., Hampshire, UK) at 30 ± 2 °C for 48 h. The corn by-products, water, and a suspension of LAB strain (3% of dry matter relative to the corn by-product mass) containing 8.9 log10 CFU/mL were incubated at 30 ± 2 °C for 24 h. For 100 g of corn by-product, 60 mL of water was used. Three parallel replicates of the fermentation were performed, and three parallel samples were analyzed. Corn by-products, non-processed, and extruded in a Twin Screw extruder (Jinan Shengrun Machinery Co., Ltd., Jinan, China), were obtained from SME "Ustukiu malunas" (Pasvalys, Lithuania). The temperatures in the different extrusion zones were I-60-61 • C, II-100-101 • C, and III-130-131 • C. Different moisture contents of the corn by-products substrate during the extrusion were tested (18, 16, and 14%). Extruder feed rate (F) was 8.2 ± 0.3 kg/h, and the nozzle diameter was 6 mm. The moisture content of the final corn by-product samples (after extrusion) was 11%. The samples were extruded at 130 • C and 14.6 rpm extruder screw speed. Three extruded corn by-product sample groups were prepared (C ex18 , C ex16 , C ex14 ) and non-extruded corn by-product samples were used as a control (C Con ).

Analysis of the Acidity Parameters and Microbiological Characteristics
The LAB strains Lactiplantibacillus plantarum-LUHS122, Liquorilactobacillus uvarum-LUHS245, Lacticaseibacillus casei-LUHS210, and Lacticaseibacillus paracasei-LUHS244 were used for the fermentation of C Con , C ex18 , C ex16 , C ex14 . Characteristics, including carbohydrates metabolism, survival at low pH, gas production capacities, and antimicrobial and antifungal properties, of the LAB strains, used for corn by-product fermentation, are reported by Bartkiene et al. [15]. Prior to the experiments, LAB strains were multiplied in MRS broth (de Man-Rogosa-Sharpe, CM 0359, Oxoid Ltd., Hampshire, UK) at 30 ± 2 • C for 48 h. The corn by-products, water, and a suspension of LAB strain (3% of dry matter relative to the corn by-product mass) containing 8.9 log10 CFU/mL were incubated at 30 ± 2 • C for 24 h. For 100 g of corn by-product, 60 mL of water was used. Three parallel replicates of the fermentation were performed, and three parallel samples were analyzed.

Analysis of the Acidity Parameters and Microbiological Characteristics
The pH was measured using a pH electrode (PP-15; Sartorius, Goettingen, Germany). The total titratable acidity (TTA) was evaluated for a 10 g portion of sample mixed with 90 mL of water; the results were expressed as mL of 0.1 mol/L NaOH solution required to achieve a pH value of 8.2. The concentration of L-(+) and D-(−)-lactic acid isomers was evaluated using a specific Megazyme assay Kit (Megazyme, Bray, Ireland). LAB, total Plants 2022, 11, 3080 4 of 18 bacteria (TBC), enterobacteria (TEC), and mould/yeast (M/Y) counts in the samples were determined according to Bartkiene [16].

Analysis of the Amino Acids Profile and Biogenic Amines Concentration
For amino acid analysis, analytes were extracted from homogenized sample with aqueous 0.1 M HCl solution and dansylation were performed according to the method of Hua-Lin Cai et al. [17], with some modifications. The concentrations of analytes were determined using The Varian ProStar HPLC system (Varian Corp., Palo Alto, CA, USA) and Thermo Scientific LCQ Fleet Ion trap mass detector. The detailed description of the method is given in Supplementary Data S1.
Biogenic amines (BA) were analyzed according to the method of Ben-Gigirey et al. [18] with some modifications by Bartkiene et al. [19]. Following BAs were analyzed: tryptamine, phenylethylamine, cadaverine, putrescine, histamine, tyramine, spermine (SPER), and spermidine. The extraction of BA was performed by using 0.4 M perchloric acid. The derivatization was carried out with a dansyl chloride solution in acetonitrile (10 mg/mL). The content of each BA was analyzed with the Varian ProStar HPLC system (Varian Corp., Palo Alto, CA, USA). The detailed description of the method is given in Supplementary Data S1.

Determination of Sugars Concentration in Corn By-Product Samples
Sugars concentration analysis of the non-treated and treated corn by-products was carried out with an Ultra Performance Liquid Chromatography system (Shimadzu Corp., Kyoto, Japan). A 2 mg/mL standard solution of a sugar mixture (fructose, glucose, sucrose, and maltose) was used for sugar detection. The detailed description of the method is given in Supplementary Data S1.

Evaluation of Fatty Acids Profile
The fatty acid (FA) composition of the corn by-product samples was determined using GCMS-QP2010 (Shimadzu, Japan) gas chromatograph with a mass spectrometer. The FA methyl esters (FAME) concentration was determined using 3-point calibration curve method and results were expressed as the percentage of total FAME concentration in the sample. The detailed sample preparation and chromatographic conditions is given in Supplementary Datas S1 and S3.

Evaluation of Antimicrobial Properties
The antibacterial activity of the non-treated and treated corn by-products against a variety of pathogenic and opportunistic bacterial strains was assessed by measuring the diameter of inhibition zones (DIZ, mm) in agar well diffusion assays. The list of pathogenic and opportunistic bacterial strains and detailed description of the method is given in Supplementary Data S1.
The antifungal activities of the non-treated and treated corn by-products against 10 different mould species were determined by the agar well diffusion assay [20]. The list of mould species and detailed description of the method is given in Supplementary Data S1.

Statistical Analysis
The physico-chemical data were expressed as the mean values (n = 3) of each sample ± standard error (SE), and the microbiological data were expressed as the mean values (n = 5) of each sample ± standard error (SE). The effects of the different treatments were analyzed by multivariate analysis of variance (ANOVA) and Tukey's honestly significant difference test (HSD) procedure, as post-hoc tests. A linear Pearson's correlation was used to quantify the strength of relationships between variables. The correlation coefficients were calculated using the statistical package SPSS for Windows (v15.0, SPSS, Chicago, IL, USA). Correlation strength interpretation was performed in accordance with Evans et al. [21]. The results were recognized as statistically significant at p ≤ 0.05.

Acidity Parameters and Microbiological Characteristics of the Corn By-Products
The changes in acidity parameters of corn by-product after fermentation are shown in Table 1. After 24 h of fermentation, the significant reduction in pH values and increase in TTA values of all samples were observed, compared to non-fermented samples. The lowest pH after 24 h of fermentation was found in the C conLpl , C conLu , and C conLc samples (3.34, 3.35, and 3.30, respectively) and the highest TTA was found for C ex18Lpl (5.5 • N). The content of L(+)-and D (−)-lactic acid isomers after 24 h of fermentation varied from 0.274 to 0.360 and 0.321 to 0.6 g/100 g, respectively. The highest content of L (+) isomer was produced in C conLpa and C ex14Lpa (0.360 and 0.362 g/100 g, respectively). The lowest content of D (−) isomer was found in C ex18Lpa (0.056 g/100 g).
The results also showed that increased moisture content of extruded corn by-products increased the pH and decreased TTA of samples, as well as the concentration of lactic acid isomers. At 16 and 18% corn by-products moisture, the pH of the fermented samples was the highest, and the TTA was the lowest compared to corn extrudates with lower moisture content ( Table 1). The concentration of lactic acid isomers was lower at higher moisture (18%) content of corn extrudates and varied from 0.095 to 0.221 g/100 g and 0.056 to 0.251 g/100 g, L (+) and D (−) content, respectively, compared to corn extrudates with moisture of 14% (Table 1).
Grain seeds fermentation is related with increased nutritional value, a high number of viable LAB, reduced pH, and a high concentration of organic acids [22][23][24]. In a previous study, the increase in TTA values with increasing fermentation time supported the decrease in pH performance, which was one of the most important changes during LAB fermentation [25,26]. Fast acidification by starter cultures, resulting in pH reduction, is considered critical from a food safety standpoint and plays a vital role in eliminating food pathogens and extending product quality [27,28].
Microbiological parameters of non-treated and treated corn by-products are shown in Table 2. Lactic acid bacteria (LAB) counts were significantly higher in the fermented (nonextruded) and extruded-fermented corn by-products, compared to the control samples. The highest LAB count was found in C conLc (9.39 log10 CFU/g). LAB count in the extruded (nonfermented) samples was lower or similar to that of the control samples. In corn by-products fermented with Lpl, Lu, and Lc, mould/yeast (M/Y) counts (3.72-3.80 log10 CFU/g) were significantly lower compared to the control. M/Y was significantly higher in most of the other extruded and extruded-fermented samples, compared to control. Significantly lower total bacteria count (TBC) was found in most of the fermented (C conLpl , C conLu, and C conLpa ) and extruded-fermented (C ex14Lpl , C ex14Lu , C ex16Lpa , C ex18Lu , C ex18Lc , and C ex18Lpa ) samples, compared to the control group, with the lowest (7.94-8.12 log10 CFU/g) being in C conLu , C ex18Lu , and C ex18Lpa . Total enterobacteria count (TEC) was significantly lower in all the fermented (non-extruded) samples and most of the extruded-fermented samples (C ex14Lc , C ex14Lu , C ex16Lpl , C ex16Lu , C ex16Lc , C ex18Lpl , C ex18Lu , C ex18Lc , and C ex18Lpa ), with the lowest being in C ex16Lc and C ex18Lpl (5.45 and 5.32 log10 CFU/g). The extruded (non-fermented) samples had significantly higher TEC than control. 8.13 ± 0.11 e 3.99 ± 0.05 b 7.94 ± 0.05 a 6.01 ± 0.11 c CFU-colony forming units; C-corn by-product samples; con-control samples (non-extruded, non-fermented); Lpl, Lu, Lc, Lpa-fermented with L. plantarum-LUHS122, L. uvarum-LUHS245, L. casei-LUHS210, and L. paracasei-LUHS244 strains, respectively; ex-extruded samples; 14, 16, 18-moisture content of the corn by-product samples. Data are represented as means (n = 5) ± SE. a-k-mean values within a column denoted with different letters are significantly different (p ≤ 0.05).
The Lactobacillus group produces many antimicrobial compounds, including lactic and acetic acids, that reduce environmental pH and are antagonistic to a wide range of pathogenic and opportunistic microorganisms [29]. Organic acids, produced by LAB, lower environment pH, and limit the growth of bacterial pathogens [30]. Our results are similar with Ayyash et al. [31], who found that compared with day 0, all Lactobacillus spp. populations increased (p < 0.05) in all grain ferments. In general, Lactobacillus spp. increased by~1.5 log (~7.0 logs to 9.0 logs) during 48 days of storage. The similar results were observed by Ferrero et al. [32], who reported that the yeast count decreased with Plants 2022, 11, 3080 7 of 18 fermentation and was below the detection limit at 250 days, while the mould count was under the detection limit after 30 days of fermentation, regardless of the treatment.

Amino Acids Profile and Biogenic Amines Formation in Corn By-Products
Essential amino acids (EAA) mass concentrations in corn by-products are presented in Table 3. The predominant EAA in control group were isoleucine (Ile), valine (Val), tryptophan (Trp), and threonine (Thr) and their content ranged from 0.15 to 0.36 g/100 g. Lysine was not found in all samples, while the presence of leucine (<0.02 g/100 g) was observed in all fermented (not extruded) samples, as well as extruded-fermented C ex14Lpl and C ex14Lu samples. Significant changes (p ≤ 0.05) were found in the contents of all EAA between the tested samples. Extrusion increased the content of Phe and Val in C ex14 and C ex18 , respectively, as well as Met in C ex16 and C ex18 , compared to the control samples. Fermentation of the control samples increased the contents of Phe and histamine (His). Fermentation of the extruded samples increased the contents of Phe, His, and Thr in C ex14Lpl ; Val in C ex14Lpl and all extruded samples with moisture content of 16 and 18%; methionine in all extruded samples with moisture content of 16 and 18%, compared to control. The contents of Trp and Ile were reduced or similar in fermented, extruded, or extruded-fermented samples, compared to control.  Non-essential amino acid (NEAA) concentrations in corn by-products are given in Table 4. The presence of arginine (<0.08 g/100 g) was observed in all fermented (not extruded) samples, as well as extruded-fermented C ex14Lpl , C ex14Lu , and C ex16Lc samples. The contents of alanine and proline were reduced in the fermented, extruded, or extrudedfermented samples, compared to the control. Tyrosine (Tyr) and glutamine (Glu) were reduced in all the extruded and extruded-fermented samples, compared to the control. However, fermented (non-extruded) corn by-products shared similar contents of Tyr and Glu with the control group. After treatment of corn by-products, arginine appeared in all fermented (non-extruded) samples, as well as in some extruded-fermented samples (C ex14Lpl , C ex14Lu , and C ex16Lc ). Aspartic acid and glycine were significantly higher in fermented (non-extruded) samples and C ex14Lpl , compared to the control group. Serine was significantly higher in C conLu and C ex14Lpl , compared to the control group. Cysteine (Cys) was significantly higher in all extruded and extruded-fermented samples with moisture content of 16 and 18%, compared to the control group. The highest content (1.40-1.69 g/100 g) of Cys was found in the extruded-fermented corn by-products with a moisture content of 18%. Asp-aspartic acid; Glu-glutamine; Asn-asparagine; Ser-serine; Gly-glycine; Arg-arginine; Ala-alanine; Tyr-tyrosine; Cys-cysteine; Pro-proline. C-corn by-product samples; con-control samples (non-extruded, non-fermented); Lpl, Lu, Lc, Lpa-fermented with L. plantarum-LUHS122, L. uvarum-LUHS245, L. casei-LUHS210, and L. paracasei-LUHS244 strains, respectively; ex-extruded samples; 14, 16, 18-moisture content of the corn by-product samples; <LOD-lower than the limit of detection (6.78 µmol/L for Arg). Data are represented as means (n = 3) ± SE. a-f-mean values within a column denoted with different letters are significantly different (p ≤ 0.05).
The increased content of some amino acid in fermented corn by-products could be explained by the proteolytic activity of LAB and endogenous proteases which are activated under the acidic conditions [33]. Moreover, certain amino acids and peptides are also used by LAB for their metabolism. Our results partly agree with Onyango et al. [34], that aspartic acid, glycine, cystine, and methionine increased after fermentation, while contents of all other amino acids showed no significant changes. Another study showed that fermentation of corn milling by-products improved the content of free amino acids and polypeptides [35]. The increased content of free amino acids after wheat bran fermentation with LAB was also observed [36]. Protein structure could be destroyed due to the conditions of the extrusion process as high temperature and pressure [37]. Thermal degradation of lysine, valine, leucine, threonine, and isoleucine was reported [14]. It was found that the extrusion of wheat bran led to a quantitative decrease in amino acids and the protein and moisture content of raw material, while the barrel temperature had no significant influence on cysteine and methionine content in rice-based snacks [38,39]. Moisture levels also influence lysine retention, but conflicting results have been observed [40]. However, the increase in the concentration of amino acids (for arginine, histidine, proline, and alanine by 80, 3, 13, and 11%, respectively) in corn after extrusion, relative to the native form, was observed by Kholodilina et al. [38]. It was also reported that extrusion increased all amino acids, except Lys and Pro in corn [41].
The concentrations of BAs in corn by-products that were extruded and fermented by LAB, are presented in Figure 2. Histamine, tryptamine, putrescine, tyramine, and spermine were not found in all the tested samples. Phenylethylamine (PHE) was found in all corn by-products, while cadaverine and spermidine were found only in non-treated samples. Results showed that treatment had a significant effect (p ≤ 0.05) on the concentration of PHE in corn by-products. Fermentation with Lpl and Lc strains, as well as extrusion (moisture content of 14 and 16%) of control samples significantly increased PHE content by on average 18.4%. However, after extrusion, PHE was significantly lower in the C ex18 sample, compared to the control group. In most of cases, fermentation of the extruded samples led to the increased content of PHE, compared to only extruded corn by-products or control group. Fermentation with L. paracasei-LUHS244 significantly reduced the content of PHE in C conLpa and C ex18Lpa samples, compared to non-treated corn by-products and that was the lowest concentration of BA found between all samples.

Sugars Concentration in Corn By-Product Samples
The contents of fructose, glucose, sucrose, and maltose in non-treated and treated corn by-products were analyzed and the results are given in Figure 3. There was a significant effect (p ≤ 0.05) of treatment on the fructose, glucose, and sucrose concentration in corn by-product samples. Significantly lower (p ≤ 0.05) contents of fructose and glucose (on average by 42.2 and 47%, respectively) were found in all fermented (non-extruded) corn by-products, compared to non-treated samples. The same tendency was also observed with fructose content in Cex14, Cex14Lpl, and Cex16Lpl samples (lower, on average, by 53.1%), while fructose was not determined in the rest of samples. Glucose was not found in all extruded and extruded-fermented samples. The presence of sucrose was observed after extrusion in Cex14, Cex16, and Cex18. After fermentation of the extruded samples, the content of this sugar was found to be significantly lower (p ≤ 0.05) in Cex14Lu and Cex16Lpl by 66.1 and 48.6%, respectively, compared to only extruded samples. Maltose was not found in any of the tested samples.
The variations of fructose, glucose, and sucrose concentrations in fermented corn byproducts are related with the LAB activity. During fermentation, the conversion of available carbohydrates by LAB, which could also possess polysaccharide-degrading activity, yields such compounds as lactic, and acetic acid [49,50]. As carbohydrates undergo a series of changes under the extrusion conditions, the lower concentrations of fructose, the Biogenic amines (BA) are low-molecular-weight nitrogenous organic bases, which can accumulate in high concentration in food due to microbial activity and cause toxic effects in consumers. Some food microorganisms are able to degrade BA once they have been synthesized in the food matrix [42,43]. Corn can also be a source of biogenic amines. Some biogenic amines can be naturally present in corn whereas others can be introduced during production, processing, and storage. They can be formed by thermal or microbial decarboxylation of amino acids and may be used as an index of quality or hygienic conditions of products [44]. Cadaverine and spermidine can be naturally found in raw plant foods, while PHE can be produced by microorganisms from phenylalanine [45,46].
The reports on the effect of extrusion processing or LAB fermentation on biogenic amine changes in corn products are scarce. Previous research reported that no significant changes in the formation of amines occurred during brewing fermentation, except for tryptamine and tyramine [47]. In the another study, the content of PHE in hemp (Cannabis sativa L.) seed paste fermented with L. uvarum-LUHS245 and L. casei-LUHS210 increased by 5% and decreased by 52%, respectively, compared with untreated samples [48].

Sugars Concentration in Corn By-Product Samples
The contents of fructose, glucose, sucrose, and maltose in non-treated and treated corn by-products were analyzed and the results are given in Figure 3. There was a significant effect (p ≤ 0.05) of treatment on the fructose, glucose, and sucrose concentration in corn by-product samples. Significantly lower (p ≤ 0.05) contents of fructose and glucose (on average by 42.2 and 47%, respectively) were found in all fermented (non-extruded) corn by-products, compared to non-treated samples. The same tendency was also observed with fructose content in C ex14 , C ex14Lpl , and C ex16Lpl samples (lower, on average, by 53.1%), while fructose was not determined in the rest of samples. Glucose was not found in all extruded and extruded-fermented samples. The presence of sucrose was observed after extrusion in C ex14 , C ex16 , and C ex18 . After fermentation of the extruded samples, the content of this sugar was found to be significantly lower (p ≤ 0.05) in C ex14Lu and C ex16Lpl by 66.1 and 48.6%, respectively, compared to only extruded samples. Maltose was not found in any of the tested samples.
The FA profile of all corn by-products was dominated by polyunsaturated FA (PFA, 45-54%), followed by monounsaturated FA (MFA, 33-35%), and then saturated FA (SFA, 13-21%) ( Table 5). Significantly higher content of SFA was found in several extrudedfermented Cex14Lc, Cex18Lpl Cex18Lc, compared to the rest of the samples (p ≤ 0.05), while MFA content was similar in all tested corn by-products. PFA content was significantly lower (p ≤ 0.05) only in Cex16Lu, compared to other samples. The higher content of PFA is favourable The variations of fructose, glucose, and sucrose concentrations in fermented corn byproducts are related with the LAB activity. During fermentation, the conversion of available carbohydrates by LAB, which could also possess polysaccharide-degrading activity, yields such compounds as lactic, and acetic acid [49,50]. As carbohydrates undergo a series of changes under the extrusion conditions, the lower concentrations of fructose, the absence of glucose and a higher content of sucrose in extruded corn by-products could be explained by the influence of extrusion processes. Due to the impact of shear forces, as well as feed moisture content and temperature, the insoluble fibres in corn by-products could be broken down into soluble compounds with reduced molecular weight [51]. Lower moisture content in feed causes a more intensive decomposition of insoluble dietary fibres [11]. Moreover, the formation of Maillard browning products during extrusion lowers the content of reducing sugars in corn by-products.

Corn By-Products Fatty Acids Profile
The fatty acids (FA) profile of non-treated and treated corn by-products is given in Table 5 and Supplementary Data S2. The FA profile of samples revealed that linoleic acid (C18:2 n6, 41-53%) was the highest followed by oleic (C18:1 n9, 32-33%), palmitic (C16:0, 9-13%), stearic (C18:0, 2-4%), and α-linolenic (C18:3 n3, 1-3%) acids (Supplementary Data S2). Similar results have been reported for predominant FA by other authors [52,53]. The rest of FA were observed at low levels (less than 1%). It was found that treatment had a significant effect (p ≤ 0.05) on most of the FA (C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, C15:0, C15:1, C16:0, C16:1, C17:0, C18:0, C18:3, C20:1, C21:0, C20:2, C22:0, C20:3, C24:0, and C22:6) in corn by-products. Palmitic and stearic acids were significantly higher in C ex18Lpl and C ex18Lc , compared to the rest of the samples. Compared to non-treated corn-by products, α-linolenic was significantly higher in extruded and extruded-fermented samples, with C ex18Lu , C ex18Lc , and C ex18Lpa being the highest. In the case of other FA, clearer tendencies cannot be drawn. However, significant differences in the content of oleic and linoleic acid between samples were not found. The FA profile of all corn by-products was dominated by polyunsaturated FA (PFA, 45-54%), followed by monounsaturated FA (MFA, 33-35%), and then saturated FA (SFA, 13-21%) ( Table 5). Significantly higher content of SFA was found in several extrudedfermented C ex14Lc , C ex18Lpl C ex18Lc , compared to the rest of the samples (p ≤ 0.05), while MFA content was similar in all tested corn by-products. PFA content was significantly lower (p ≤ 0.05) only in C ex16Lu , compared to other samples. The higher content of PFA is favourable in food because they improve blood sugar level, possess blood cholesterol and pressure lowering abilities, and fight against inflammatory reactions and various cancers [54]. The PFA/SFA ratio ranged from 2.2 to 4.2 with the highest being for C conLpl , C conLpa , and C ex14Lpl and the lowest being for C ex18Lpl and C ex18Lc . Ratios of all tested samples were higher than 0.4 as recommended by the World Health Organization [55]. The group of omega 6 FA was the highest (41-53%) followed by omega 9 (32-34%), and omega 3 (1-4%) in all tested samples (Table 5). Significant changes in the content of omega 6 and omega 9 were not observed in all tested samples. However, compared to non-treated samples, the content of omega 3 was significantly higher in all extruded and extruded-fermented corn by-products, except C ex14Lu . The omega 6/omega 3 FA ratio ranged from 11.4 to 48.2 with the highest being for C conLpa , and the lowest being for C ex18Lc . The decrease in omega 6/omega 3 ratio is desirable for the prevention of cardiovascular diseases, diabetes, obesity, and cancer but there are no recommended specific values [56]. Some fermented samples (C conLpl , C conLu , C conLc , C conLpa , and C ex18Lpl ) contained a very small amount (lower than 0.08%) of trans FA.
Availability of data is limited to being on the effect of fermentation and extrusion on FA profile of corn by-products. Wani et al. [57] reported that FA composition, including PFA and MFA content, of corn-based snacks, was not significantly affected by extrusion. Contrarily, Ramos Diaz et al. [58] found that after extrusion, the content of palmitic, linoleic, oleic, and linolenic acid was reduced in corn-based extrudates, compared to those non-extruded. The slight loss of some lipids during extrusion occurs due to the high temperature, which could also cause the essential reduction in PFA stability, and the formation of amylose-lipid complexes [37,58]. Lower conditions of temperature and moisture in the extrusion process increase the stability of PFA in extruded products during storage [14]. Changes in the FA profile after fermentation could be attributed to the lipolytic activity of LAB [59]. In addition, LAB abilities to generate FA and modify their saturation and desaturation are reported [60].

Antimicrobial Characteristics of Corn By-Products
Diameter inhibition zones (DIZ) of the non-treated and treated corn by-products against pathogenic and opportunistic microorganisms are given in Table 6. Fermented corn by-products C conLpl , C conLu , and C conLpa displayed inhibition properties against 3 of the 10 tested pathogenic/opportunistic bacterial strains: Acinetobacter johnsonii, Staphylococcus aureus, Aeromonas veronii. Sample C conLc showed inhibition properties only against Acinetobacter johnsonii and Staphylococcus aureus. Most of samples, except C con , C ex14 , C ex16 , C ex18 , C ex18Lpl , C ex18Lpa , showed inhibition properties against Acinetobacter johnsonii (DIZ of 12.5 mm on average). No efficiency in inhibiting Salmonella enterica Infantis, E. coli, Bacillus pseudomycoides, Cronobacter sakazakii, Hafnia alvei, Enterococcus durans, Kluyvera cryocrescens was observed by any of the samples. Table 6. Antibacterial properties of non-treated and treated corn by-products.

Aeromonas veronii LT 105
Acinetobacter johnsonii LT 110 Diameter of Inhibition Zones, mm  nd nd nd Table 6. Antibacterial properties of non-treated and treated corn by-products.

Staphylococcus aureus
LT 102 Table 7. Antifungal properties of non-treated and treated corn by-products.

Conclusions
The economic efficiency and environmentally friendly production are important aspects in the sustainable valorization of cereals processing by-products, and this is still challenging. The valorization of corn processing by-products could be designed in a more appropriate and sustainable manner by using whole by-product conversion, and by combining extrusion and fermentation processes, as the latter are common and economically efficient processes in the food and feed industry. This study indicated that fermentation with antimicrobial properties possessing LAB strains or the combined technique of extrusion and fermentation improved the microbiological safety of corn by-products. The latter technique increased the content of certain amino acids (e.g., valine, methionine) in most of the samples. Such biogenic amines as cadaverine and spermidine were not found after treatments of corn by-products, while the lowest content of biogenic amines was found in extruded-fermented (with L. paracasei-LUHS244, moisture 18%) samples. Applied treatments affected the content of most fatty acids. The level of omega 3 was significantly higher in extruded and extruded-fermented corn by-products. However, the contents of saturated, monounsaturated, and polyunsaturated fatty acids were similar between most of the samples. Corn by-products fermented with L. plantarum-LUHS122, L. uvarum-LUHS245, and L. paracasei-LUHS244 showed antibacterial activity against Acinetobacter johnsonii, Staphylococcus aureus, Aeromonas veronii. Extruded (14% moisture) and fermented with L. plantarum-LUHS122 corn by-products inhibited Rhizopus and Aspergillus fumigatus. In sum, combining extrusion and fermentation processes for corn by-product valorization can improve certain parameters and properties of these products, and they can be recommended as safer and more nutritious ingredients for food and feed production.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.