Changes of bioactive compounds in barley industry by‐products during submerged and solid state fermentation with antimicrobial Pediococcus acidilactici strain LUHS29

Abstract In this study, changes of bioactive compounds (crude protein (CP), crude fat (CF), dietary fiber (DF), fatty acids (FAs), free amino acids (FAAs), phenolic compounds (PCs), biogenic amines (BAs), lignans, and alkylresorcinols) in barley industry by‐products (BB) during submerged and solid state fermentation (SSF) with Pediococcus acidilactici were analyzed. It was established that both fermentation conditions reduce the CP and CF content in BB (by 25.8% and 35.9%, respectively) and increase DF content (on average by 25.0%). Fermentation increases the oleic, arachidic, eicosadienoic, behenic, and lignoceric FA in BB samples. The highest total BA content was found in untreated samples (290.6 mg/kg). Solid state fermentation increased the content of the alkylresorcinol C19:0. Finally, collecting data about the changes of these compounds during technological processes is very important, because according to the specific compounds formed during fermentation, further recommendations for by‐product valorization and uses in food, pharmaceutical, or feed industries can be suggested.


| INTRODUC TI ON
The food processing industry in most countries across the world generates a huge quantity of by-products, which have limited use and create considerable environmental pollution (Radenkovs, Juhnevica-Radenkova, Górnaś, & Seglina, 2018). This waste causes a serious disposal problem for the environment. New approaches to use of these by-products have become a very important issue, because most of them are an excellent source of various bioactive compounds: polyphenols, flavonoids, caffeine, carotenoids, creatine, polysaccharides, etc., which are beneficial for human, as well as animal, health (Wang et al., 2018). Cereal bran is regarded as an unavoidable by-product of the milling industry with little commercial value, and so far, its main use is as a supplement for animal feed (Prückler et al., 2015). One of the major challenges is finding efficient technologies for valorization of the by-products in an environmentally friendly manner. A way to add to the functionality of cereal by-products is fermentation using lactic acid bacteria (LAB).
Our previous study showed that fermentation with selected technological strains could be a promising solution for cereal by-product valorization (Bartkiene et al., 2017). The use of LAB starter The fermentation process can be performed under solid state (SSF), submerged (SMF), or liquid conditions, according to the specific product. Solid state fermentation, the growth of microorganisms on an adequately moistened nonsoluble medium in the absence or near absence of free-moving water and air, is an area of interest to add value to by-products using an inexpensive process (Ozmihci, 2017). The SSF process consists of maintaining conditions such as humidity and temperature closer to natural ones, without the need for drastic corrections in the substrate for fungus growth (Wang et al., 2018). For this reason, SSF has many advantages over SMF. From this point of view, SSF is presented as a promising technology for by-product valorization through bioconversion into high value-added products. Bran fermentation increases the content and bioavailability of several functional compounds, total free phenols, and soluble fiber (Manini et al., 2014).
Most of the studies, about the cereal by-products valorization, are focused on the main nutritional compound changes (carbohydrates, protein, and fat profiles); however, studies about the changes of BAs, lignans, and alkylresorcinols during the cereal technological processes are scarce. However, abovementioned compounds can possess strong (desirable, as well as undesirable) physiological activities in vivo. The novelty of this study is based on possible cereal by-products valorization by applying more sustainable technology-SSF. Also, versatile results, about the both desirable and undesirable compounds formation, in cereal by-products can be useful for the further, such type of products industrialization in food/feed/nutraceutical sectors.
In this study, changes of the bioactive compounds (fatty acid and amino acid profile, phenolic compounds, BAs, lignans, and alkylresorcinols) in barley industry by-products during SMF and SSF with Pediococcus acidilactici strain LUHS29, which shows antimicrobial properties, were analyzed.

| Barley crop industry by-products and their fermentation with P. acidilactici strain LUHS29
The grounded barley by-products were obtained from local mill (Ustukiu malunas Ltd., Pasvalys, Lithuania) in 2018. Cereal by-products samples were collected according to standard procedures described in ISO 24333:2009(Cereals and cereal products-Sampling, ISO, 2009. Samples were collected during barley milling process and stored in the dark at ambient temperature until experiment. The P. acidilactici strain LUHS29, previously isolated from spontaneous fermented cereals and which showed broad antimicrobial activity against opportunistic and pathogenic strains (Bartkiene et al., 2019), was stored at −80°C and cultured at 30°C for 48 hr in MRS broth (CM0359, Oxoid Ltd) with the addition of 40 mmol/L fructose and 20 mmol/L maltose prior to use. The fermentation of barley byproducts was performed with a multiplied P. acidilactici strain (3% by volume of the pure P. acidilactici strain, diluted in MRS broth added to cereal/water mass). Three parallel replicates of fermented samples were prepared, and each fermented sample analysis was repeated three times. The water content of the end-product for SSF was 450 g/ kg; for SMF, it was 650 g/kg. Fermentation was carried out for 72 hr at 32 ± 2°C. Unfermented barley by-products were used as a control.

| Methods for evaluating the content of crude protein, fat, ash, fiber, and their fractions (NDF, ADF, and ADL) in barley crop industry by-products
Ash content was determined by calcinations at 900°C (ICC1990/1990900°C (ICC1990/ :, 1990. Determination of ash in cereals and cereal products). Nitrogen content was determined using Kjeldahl method with a factor of 5.7 to determine protein content (ICC2001/2001(ICC2001/ :, 2001. Determination of crude protein in cereals and cereal products for food and for feed).
The total lipid content was determined by extraction in the Soxhlet apparatus ("Boeco") with hexane technical grade (Fisher Scientific) (ICC1984:, 1984. Cereals and cereal products-Determination of total fat content). Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were analyzed according to Van Soest, Robertson, and Lewis (1991). Analysis of NDF, ADF, and acid detergent lignin (ADL) was carried out using an ANKOM 200 Fiber Analyzer Unit (ANKOM Technology Corp.). Neutral detergent fiber was assayed with the use of alpha amylase and sodium sulfite in the NDF. Both NDF and ADF were expressed without residual ash (AOAC, 2000).

| Evaluation of the fatty acid (FA) and free amino acid (FAA) profile in fermented and nonfermented barley by-products
The FA profile of the barley fat fraction was determined using gas chromatography-flame ionization detection (GC-FID; Agilent 6890N Gas Chromatograph, Agilent Technologies), according to the procedure described by .

| Evaluation of phenolic acids (PAs) in barley by-products
Stock solutions of the standard acids were prepared at a concentration of 1.0 g/100 ml in pure methanol. The working solutions of samples were prepared at a concentration of 1.0 g/100 ml in methanol. The acid mixtures were separated on a Shimadzu LC-9A model HPLC equipped with a manual injector, a programmable wavelength photodiode array UV detector (200-400 nm), and column packing with modified silica gel (C 18 column). Extraction and evaluation of PAs were performed according to method described by Tüzen and Özdemir (2003).

| Evaluation of BA content in barley by-products
Sample preparation and determination of BAs in barley by-products were performed according to the method of Ben-Gigirey, Sousa, Villa, and Barros-Velazquez (1999), with some modifications described by Bartkiene, Bartkevics, Rusko, et al. (2016). Perchloric acid (0.4 M, 10 ml) containing a known amount of 1,7-diaminoheptane used as an internal standard was added to 3 g of sample, and the mixture was homogenized with Ultra-Turrax (IKA Labortechnik) and centrifuged (3,000 g, 4°C, 10 min). The residue was extracted again with an equal volume of 0.4 M perchloric acid. Both supernatants were combined, and the final volume was adjusted to 30 ml with 0.4 M perchloric acid. The extract was filtered through Whatman paper No. 1. One milliliter of extract or standard solution was mixed with 200 μL of 2 M sodium hydroxide and 300 μL of saturated sodium bicarbonate. A 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride reagent) (10 mg/ml, 2 ml) prepared in acetone was added to the mixture and incubated at 40°C for 45 min. Residual dansyl chloride was removed by the addition of 100 μL of 25% ammonium hydroxide. After incubation at room temperature for 30 min, the mixture was adjusted to 5 ml with acetonitrile. Finally, the mixture was centrifuged (3,000 g, 5 min), and the supernatant was filtered through 0.2-μm filters (Millipore Co.) and was kept at −25°C until HPLC analysis. An Agilent 1200 HPLC (Agilent) equipped with DAD detector and Chemstation LC software was employed. A Chromolith C18 HPLC column (100 mm·4.6 mm·4 μm, Merck KGaA/ EMD Chemicals) was used. Ammonium acetate (0.1 M) and acetonitrile were used as the mobile phases by a flow rate of 0.45 ml/min. The sample volume injected was 10 μL, and the amines were monitored by 254 nm. The detection limits for standard amine solutions were approximately 0.1 mg/kg.

| Analysis of alkylresorcinol and lignan concentration in barley crop industry by-products
For the determination of alkylresorcinols, barley by-product samples were placed in 50 ml tubes and extracted by continuous shaking for 24 hr at room temperature (20°C, or rotation), with 40 ml of ethyl acetate containing 0.5 mg (or 0.500 μg/mL → 200 μL) of methyl behenate internal standard and centrifuged for 10 min at 1,500 g (~6,000 rpm, r = 4 cm). Portions (4 ml) of the extract were transferred to 5 ml test tubes and then dried by evaporation in vacuo using a centrifuge evaporator for 40 min. Ethyl acetate (200 μL) was added, and the samples were mixed and filtered through 0.45-μm filters (GHP Acrodisc) then transferred to GC vials for analysis (Annica, Andersson, Åman, Wandel, & Frølich, 2010). The GC/MS analysis was performed according to method described by Bartkiene et al. (2015) on an HP 5890 II gas chromatograph coupled to a TRIO-1000 mass spectrometer with a LAB-BASE data system (version R2.10; Fision Instruments).
For the determination of lignans in barley by-product samples, extraction was performed according to the methods described by Krajčová, Schulzová, Hajšlová, and Bjelková (2009). The analysis was performed according to the method described by Bartkiene et al. (2017). Defatted (n-hexane, 2 hr at 60°C) samples (0.5 g) were mixed with 12 ml of 0.3 M NaOH in methanol/water (70/30, v/v) and incubated for 1 hr at 60°C. The hydrolysate was neutralized with glacial acetic acid and centrifuged (10 min, 3,000 g). An aliquot of 0.5 ml was evaporated to dryness, dissolved in 3 ml of sodium acetate buffer (0.1 M, pH 5.0) with 400 μL of β-glucuronidase/sulfatase enzyme (from Helix pomatia), and incubated overnight at 37°C. The enzymatic hydrolysate was extracted twice with 3 ml of diethyl ether, and the two organic phases were combined and evaporated to dryness (under nitrogen with gentle heating, max 55°C, on a water bath). The dried sample was dissolved in 0.5 ml of methanol. The β-glucuroni-

| Statistical analysis
The results were expressed as the mean value of measurements ± standard deviation. Three parallel replicates of fermented samples were prepared, and each fermented sample analysis was repeated three times. In order to evaluate the effects of the different fermentation conditions (SSF and SMF, as well as different durations of fermentation), the data were analyzed by the analysis of variance (IBM SPSS Statistics, ver. 22). Results were recognized as statistically significant at p ≤ .05.

| Content of crude protein, fat, fiber, and their fractions (NDF, ADF, and ADL) in barley industry byproducts
The chemical composition of the untreated and fermented barley by-products (BB) is given in Table 1. In all cases, fermentation reduced the crude protein content in BB compared with untreated samples: A crude protein content lower by 29.4%, 26.8%, and 21.2% was established in SSF samples after 24, 48, and 72 hr, respectively.
As well as that, a crude protein content lower by 26.9%, 26.7%, and 25.2% was found in SMF samples after 24, 48, and 72 hr, respectively. Lactic acid fermentation significantly reduces the protein content, and a slight decrease in protein content could be explained by the capability of LAB to degrade proteins in fermentable substrates (Gardini, Özogul, Suzzi, Tabanelli, & Özogul, 2016), as LAB are capable of producing proteinases. However, comparing SSF samples, the greatest decrease of crude protein was found after 24 hr of fermentation (compared with untreated samples, decreased by 29.4%); comparing crude protein content after 48 and 72 hr of fermentation with protein content after 24 hr of SSF, it was higher in samples fermented for longer by 0.44% and 1.38%, respectively. It could be that the main proteolytic enzyme activity of LAB occurred during the 24 hr and, after that, activity was reduced by inhibiting LAB cells; the small increase in crude protein can be explained by the presence of bacterial cell proteins in the SSF substrate.
Also, in fermented BB samples, crude fat content was reduced compared with control samples in SSF samples by 39.5%, 36.7%, and 39.5% after 24, 48, and 72 hr, respectively, and in SMF samples by 35.7%, 32.6% and 31.4%, respectively. Lactic acid bacteria can contribute to the formation of free FAs, which can be precursors of characteristic aroma compounds and lactones in some fermented products (Gardini et al., 2016). However, LAB are generally advantageous for use as a starter culture in conditions of low lipolytic activity (Yalçınkaya & Kılıç, 2019). Opposite to the changes observed for crude protein and fat, an increase of crude fiber was established in fermented samples after 24, 48, and 72 hr in SSF samples by 22.7%, 17.8%, and 36.6%, respectively, and in SMF samples by 14.5%, 27.5%, and 31.0%, respectively. Lactic acid bacteria increase the concentrations of NDF, ADF, ADL, and hemicelluloses in fermentable substrates (Wang et al., 2018). In this study, the same tendencies were established, as NDF, ADF, and ADL fractions were higher in fermented BB samples than in untreated samples.
Compared with untreated BB, in SSF and SMF samples, NDF, ADF, and ADL were found to be higher by 27.8% and 28.5%, 19.4% and 22.5%, and 23.4% and 13.5%, respectively. The experimental results are in agreement with Li, Zhou, Zi, and Cai (2017) who determined that LAB can improve fermentation quality, chemical composition, and bioavailability (inhibiting protein degradation and promoting fiber degradation), thus having great potential as an additive for bran fermentation.

| FA composition of the fermented barley byproducts
The FA profile of the BB is given in

| FAA profile of the barley by-products
The FAA profile of the untreated and fermented BB is given in  Khan et al. (2018). This decrease could be attributed to the consumption of FAAs during microbial metabolism and enzymatic conversion, and significant variation in regard to different FAAs throughout fermentation can be established (Khan et al., 2018). Arginine can be metabolized into ornithine and free ammonia TA B L E 1 Changes in the content of crude protein, crude fat, crude fiber, and their fractions (NDF, ADF, and ADL) in barley by-products (BB) fermented with a Pediococcus acidilactici strain under submerged (SMF) and solid state (SSF) fermentation conditions through the arginine deiminase pathway during lactic acid fermentation whereas glutamine can be transformed to γ-aminobutyric acid by glutamate decarboxylase produced by LAB, because groups of FAAs, such as branched-chain (leucine, isoleucine, valine), aromatic (phenylalanine, pyramine), sulfuric (cystine), and acidic (asparagine), are converted into flavor compounds under the effect of aminotransferases generated by LAB so the overall levels tend to be lower.
Moreover, threonine can be transformed into acetaldehyde under the catalysis of threonine aldolase, while glycine can be formed simultaneously, and a promotion in the concentration of glycine can be detectable in the later stages of fermentation (Wang et al., 2018).

| Changes of PA content in barley by-products
The PA content in untreated and fermented BB is shown in industrially important compounds such as ferulic acid, 4-ethyl phenol, vanillic acid, vanillin, and vanillyl alcohol. Additionally, p-coumaric acid can be decarboxylated to the corresponding vinyl derivatives under the effect of a PA decarboxylase generated by LAB (Filannino, Cagno, & Gobbetti, 2018). However, bacterial cell growth is inhibited by hydroxytyrosol, oleuropein, tyrosol and vanillic, p-hydroxybenzoic, sinapic, syringic, protocatechuic, and cinnamic acids at high concentrations (Dey et al., 2016). The increased bioaccessibility of p-hydroxybenzoic and vanillic acids could be due to microbial enzymatic activity.
Free p-coumaric acid may be decarboxylated or reduced by L. plantarum PA decarboxylases or reductases to the corresponding phenol or vinyl derivatives. Therefore, these microbial metabolic pathways could explain the reduction of soluble p-hydroxycinnamic compounds (Filannino et al., 2018).

| Formation of BAs in barley by-products during their treatment with P. acidilactici LUHS29
The BA content in BB samples is given in

| Alkylresorcinol and lignan content in barley byproducts
The alkylresorcinol (ARs) and lignan content in barley BB are presented in Figure 1a, C23:0 homologue was not determined in most of the fermented samples (except in 24 hr SMF); however, in untreated samples, its content was 3.35 ± 0.03 µg/g. It has been reported that fermentation only changes the alkylresorcinol content slightly, and only minor changes are induced by LAB metabolism (Prückler et al., 2015). Our results are in agreement with Zhao et al. (2017) who explained that LAB produce acid in fermentation, and acidification leads to a decrease of ARs. Bran plays a key role in the overall health benefits of whole grains. Clinical trials and epidemiological studies have shown that the consumption of foods high in fiber is linked with a reduced risk of diseases such as colon cancer, diabetes, obesity, and cardiovascular disease, probably due to the phytochemicals (PAs, sterols, alkylresorcinols, vitamin E, and minerals) and fiber, which are embedded in the bran (Prückler et al., 2015). For this reason, collection of data about the changes of these compounds during technological processes is very important.
The lignan content in barley BB is presented in Figure 1b.

| CON CLUS IONS
Finally, both fermentation conditions reduced crude protein and crude fat content in BB; however, they increased dietary fiber con-

ACK N OWLED G M ENTS
This research is funded by the LUHS Science Foundation, support no. 0202/010302. The authors gratefully acknowledge the COST Action CA18101 "SOURDOugh biotechnology network toward novel, healthier and sustainable food and bIoproCesseS."

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
Human and animal studies were not included in this experiment.