Effect of lactic acid fermentation of red beetroot juice on volatile compounds profile and content

The study aimed to evaluate the effect of lactic acid fermentation on the profile and content of volatile organic compounds (VOCs) in red beetroot juice. Red beetroot juice was fermented by three different amounts (1%, 2%, and 2.5%) of three various Lactobacillus strains (L. acidophilus ATCC 8014, L. brevis Lbbr 12A, L. plantarum ATCC 3543) for 24 h. The head-space micro-extraction (HS-SPME) with gas chromatography–mass spectrometry (GC–MS) was implemented to qualify and quantify the VOCs presence in samples: fresh juice, two controls incubated at 30 and 37 °C, and nine fermented juice samples. A total number of 100 volatiles were identified in the analyzed juice samples, which were classified into 18 chemical groups. Fermented juice samples were characterized by a greater variety of VOCs than unfermented juice samples. In fermented juice samples, 17 to 38 compounds were identified, while in unfermented juice samples, it was 13–15 compounds. The highest number of VOCs was found in juice fermented by L. plantarum (33–38 volatiles) and the lowest in fresh juice (13 volatiles). Total relative content of VOCs ranged from 84.00 (fresh juice) to 881.31 µg/mL (control, 30 °C). On the other hand, the highest relative content of VOCs among fermented juice samples was noticed in the juice fermented by a 1% addition of L. plantarum. According to this study, it can be said that 24-h lactic acid fermentation of red beet juice allows forming a significant amount of volatilized molecules. Therefore, the relatively high content of volatiles can increase potentially the aroma attractiveness of red beetroot juice.


Introduction
For centuries, lactic fermentation has been used to preserve food with minimal use of natural resources [1,2]. Lactic fermentation is a desirable process of biochemical modification of basic food products by microorganisms and their enzymes. It is a biological conversion of sugars into lactic acid (1:2 ratio) in anaerobic conditions [3,4]. A fermentation system consists of three elements, i.e., microorganisms, substrate, and environmental conditions. This process is based on the action of the substrate together with microorganisms (bacteria).
The fermentation process can be divided into spontaneous and forced fermentation (inoculation). [3]. In food industries, the production of fermented foods is based on the use of starter cultures-forced fermentation [5]. These cultures initiate rapid acidification of the raw material environment by producing organic acids, mainly lactic acid [5,6]. Starter cultures are very often isolated from spontaneously fermented products. Then they are cleaned and selected [7]. In contrast, spontaneous fermentation highly depends on naturally occurring lactic acid bacteria (LAB) in food. This process is obtained from the action of native microorganisms (living in the natural environment). Lactic fermentation occurs naturally when substrate availability is adequate and environmental conditions are optimal for the growth of fermentative microorganisms. It is most often used in households [3,7]. Lactic fermentation is considered a method with great potential to improve functional properties [1,2]. Using LAB increases the product's microbiological safety, nutritional properties, and health benefits and contributes to a formation of pleasant sensory attributes of the final product [6,8,9].
During the fermentation process, volatile organic compounds (VOCs) are formed, reducing the undesirable aroma and taste in the final product [9]. LAB causes three main metabolic pathways (glycolysis, lipolysis, and proteolysis), which are involved in the production of fermented food and shaping its, taste and smell [10,11]. Glycolysis is the main metabolic activity during fermentation, and this process leads to the acidification of food through the production of lactic acid from carbohydrates [10,12,13]. In addition, beneficial compounds are formed during the fermentation of sugars, i.e. organic acids (e.g. acetic acid), carbon dioxide, alcohols (e.g. aldose ethanol), isomerases, bacteriocins, antifungal substances, esters (e.g. acetate), exopolysaccharides [2,10,14,15]. On the other hand, proteolysis is caused by hydrogen ions (H +) of lactic acid, which coagulate proteins, cell plasmas, and protein parts of enzymes, thus contributing to inhibiting plant life processes and bacterial cells. During lactic fermentation, decomposition of amino acids contributes to forming aroma compounds [13,15]. The degradation of amino acids allows obtaining alcohols, aldehydes, acids, esters, and sulfur compounds, contributing to the desired aroma of the final product. In addition, citrates are metabolized into peptides, in which volatile compounds, such as diacetyl, acetoin, and butanediol, are formed [15,16]. The metabolism of protein gives the post-fermentation products with different flavors, amino acids-sweet and umami, oligopeptides-bitter, and organic acids-sour tastes [17]. During lipolysis, fats are broken down into glycerol and fatty acids. This process also allows the metabolism of long-chain fatty acids into short-chain fatty acids [18]. Moreover, the degradation of fats could lead to formation of polyhydric alcohols, short-chain and long-chain fatty organic acids, and vitamins [5,18,19]. Therefore, the study aimed to investigate the profile and relative content of VOCs in red beetroot juice fermented by the three different LAB.

Red beetroot juice preparation
Fresh red beetroots of Czerwona Kula (Beta vulgaris L. subsp. vulgaris) variety were purchased from a local commercial market in Olsztyn, Poland. Then, the roots were washed in distilled water and manually diced. To obtain the red beetroot juice, a juice extractor was used (Waring Commercial Juice Extractor WJX50, China). To determine the composition of volatiles, a sample volume (100 mL) was taken from the obtained red beet juice; the sample was not diluted or sweetened.. Next, the rest of the red beetroot juice was transferred to the sterile containers (previously sterilized at 121 °C for 15 min) and pasteurized (80 °C for 15 min) to eliminate vegetative microflora.

Red beetroot juice fermentation
Juice was subjected to fermentation using of three microorganisms (Lactobacillus plantarum ATCC 8014, Lactobacillus brevis Lbbr 12A and Lactobacillus acidophilus ATCC 3543) from the strain collection of the Department of Industrial and Food Microbiology, Faculty of Food Sciences of the University of Warmia and Mazury in Olsztyn (Poland). The pasteurized juice (100 mL) was inoculated in amounts of 1, 2, or 2.5% (v/v) of an active culture of the strains grown in optimal media (MRS broth sticks). Fermentation was conducted for 24 h at a temperature of 37 °C for L. acidophilus or 30 °C for L. plantarum and L. brevis. Pasteurized juice samples with no inoculation treated under the same conditions were considered as the control. Three independent fermentation experiments were carried out. After fermentation, all the samples including the control were stored at -24 °C until further analysis.

Microbial testing
All samples were subjected to microbial analysis and tested for cell counts. The cell density of LABs before and after fermentation was determined by the MRS-agar surface method (Merck, Darmstadt, Germany). Incubation was anaerobic and carried out at 30 °C for 72 h. The number of yeasts was determined by the YGC-agar surface method (Merck, Darmstadt, Germany). Incubation was carried out aerobically at 25 °C for 96 h. The most probable number of spore-forming bacilli from Clostridium sp. strain was determined by the liquefied bars of meat-liver agar surface. Incubation was carried out aerobically at 37 °C for 48 h. The limit of detection for the used plate count methods was below 10 CFU/mL.

The analysis of VOCs
The headspace solid-phase micro-extraction gas chromatography-mass spectrometry (HS-SPME GC/MS), previously described by Starowicz et al. [20], was used to determine the VOCs in fermented red beetroot juice. Therefore, 1.5 mL of red beetroot juice was pipetted into a 20-mL vial and closed with seal with PTFE/silicone septa. The vials were placed on an Eppendorf agitator/heater (Eppendorf, Germany). Samples were shaken with 600 rpm and heated at 50 °C for 45 min. Then, volatilized compounds were allowed to absorb onto the SPME fiber for 15 min at 50 °C (without shaking).
A 50/30 µm stable DVB/CAR/PDMS fiber (Supelco, Bellefonte, PA, USA) was selected to be used in this specific analysis. The injection was done manually. Thus, the SPME fiber was introduced into the chromatograph injection port (splitless mode), where the analytes were desorbed at 250 °C for 5 min and transferred on a capillary column (DB-WAX, 30 m, 0.25 mm × 0.50 µm). The analyses were performed on a gas chromatograph (Agilent Technologies 7890A GC system, USA) coupled to a mass spectrometer (Agilent Technologies 5975C VL MSD, USA). The temperature was initially set to 40 °C and held for 5 min. Then, it was increased to 240 °C and held for 5 min. In the method, helium was used as the carrier gas with a flow rate of 1 ml min −1 . The flow was kept constant. Mass spectra were recorded in an electron impact mode of 70 eV in a scan range 33-333. All analyses were repeated in triplicate.

Statistical analysis
The data are presented as mean values ± standard deviations of triplicate measurement. The differences between samples were analyzed by a one-way ANOVA with Tukey test (p < 0.05). Principal component analysis (PCA) was carried out to show differences between juice samples. The statistical analysis was performed using STATISTICA 13.0 software (StatSoft Inc., Tulsa, OK, USA).

Microbial analysis
The analyzed samples were tested for count of microbials. Fresh beetroot juice (FJ), juice after pasteurization (before inoculation), control juice samples incubated at 30 and 37 °C (C1 and C2, respectively), and juice fermented with LAB were analyzed. The fresh juice samples were characterized by a significant count of fungi (3.39 ± 0.08 log CFU/ mL) and Bacillus sp. (2.26 ± 0.08 log CFU/ mL) with a total count of microbes at 5.84 ± 0.03 log CFU/ mL. The pasteurization process reduced the total count of microorganisms to 3.38 ± 0.02 log CFU/ mL. However, in the C1 and C2 samples, an increase in the total count of microorganisms was observed (Table 1). After inoculation by the LAB strains, the initial count was 5.80-6.66 log CFU/mL. After 24 h of fermentation, a significant increase in the concentration of the LAB was noted, resulting in a final count of 9.10-9.89 log CFU/mL of the fermented red beetroot juice samples ( Table 1). The highest count increase was characterized juice fermented by 1% of L. brevis (Lb1%), while 2% of L. acidophilus (La2%) showed the lowest values. According to the literature data, in a variety of retail fermented foods, the live LAB count is between 10 5 and 10 9 CFU/g or mL [21]. Therefore, obtained data suggested that red beetroot juice was a valuable nutrient source for bacteria growth and multiplication.

Profile of VOCs
There is only scarce information about the VOCs profile in fermented beetroot products. Most studies have been devoted to profile of volatiles in fermented flour, apple juice, oat and almond drinks, and watermelon juice [5,22,23]. Thus, this is the first study to present the volatiles composition in red beetroot-based juice fermented by three different amounts of Lactobacillus strains.
The relative content of the main volatiles (nonanoic acid, acetic acid, 2-methyl-4-vinylphenol and benzaldehyde) varied between the analyzed juice samples. The higher content of nonanoic acid in La1% sample was detected ( Table 3). The relative content of this volatile was higher from eightto twenty-one-fold than in La2% and Lb2.5% samples.      Moreover, more than forty-and fifty-eight-fold lower content of nonanoic acid was noted in the FJ and C2 samples, compared to the juice fermented by 1% of L. acidophilus. The higher relative content of 2-methyl-4-vinylphenol (193.50 ± 14.90 µg/mL) in the Lb1% sample was noted. Also, 2-methyl-4-vinylphenol dominated in the La2.5%, Lb2%, Lb2.5%, and Lp1%. Furthermore, a high content of this compound was also detected in control sample incubated at 30 °C (C1). This phenomenon may suggest that the combination of incubation temperature and used suitable strains may affect the high contribution of 2-methyl-4-vinylphenol in fermented juice samples, which can contribute to the formation of roasted peanut-like, apple-like, spicy aroma. What's more, the highest contents of this compound were observed in juice samples fermented at 30 °C, but a higher concentration of the L. plantarum strain decreased the content of 2-methyl-4-vinylphenol. In the case of acetic acid, a higher relative content of this volatile was detected in the Lp1% sample. However, no statistical differences were found between the Lb1%, Lb2%, and Lb2.5% samples (p < 0.05). Also, a significant content of acetic acid was noted in other fermented red beetroot juice samples. The high contribution of acetic acid is related to LAB metabolism. To obtain energy, LAB breaks down simple sugars into lactic acid and other by-products, such as carbon dioxide, ethanol, and acetic acid [10]. Benzaldehyde, a compound which, apart from acetic acid, is present in all analyzed samples, has its highest concentration in the C1 sample (Table 3). In the other samples, benzaldehyde was present in the much lower concentration. According to the available data, benzaldehyde production can be only due to some specific LAB. Formation of this compound has been previously reported only for Lactobacillus casei, L. acidophilus, L. plantarum, L. mesenteroides, and yeasts [23,24,27]. However, in the cited studies, a control sample was not presented that could explain the formation of benzaldehyde. According to our study, benzaldehyde may be created mostly by the impact of temperature than activity of LAB. The presence of the three VOCs (nonanoic acid, acetic acid, and 2-methyl-4-vinylphenol) was shown in the highest concentration in the fermented juice samples, apart from the prominent role in the creation of sensory properties of the final product, may also have an impact on the human body. Acetic acid, apart from bactericidal and antimicrobial properties and extending the shelf life of food [28], plays a crucial role in the trophism of the intestinal mucosa, accelerates metabolism, has a positive effect on the intestinal barrier (reduces inflammation) [29]. 2-methoxy-4-vinylphenol, a compound belonging to the group of phenols has a proven potent antioxidant and anti-inflammatory effect [30].
Eighteen different chemical classes of volatile compounds were identified in the samples tested (Fig. 1). In unfermented samples, the number of chemical groups ranged from seven         (C2) to nine (FJ), while in fermented juice samples, from six (La2%, Lb1%) to twelve (Lp2.5%). It is worth nothing than in the aroma profiles of red beetroot juice samples, 3 groups of VOCs were shared by all samples, namely acids, aldehydes, and phenols. The dominant group of chemical classes in the FJ sample had furans (48.0% of the total number of identified volatile compounds), followed by phenols (21.7%), acids (8.6%), pyrazines (7.1%), alcohols (4.0%), and alkaloids (3.2%). In addition, amides, aldehydes, and lactones were also detected in fresh juice. On the other hand, compounds from the group of alkaloids were identified only in the FJ sample. These data suggest that processes, such as fermentation and incubation, affect the presence of volatile compounds from the alkaloid group. The study by Kasprowicz-Potocka et al. [31] observed that the fermentation process not only improves the microbiological quality of food but also reduces the content of anti-nutrients, i.e., alkaloids, raffinose family oligosaccharides, or phytate content. In addition, the FJ sample was characterized by a 91.1% higher percentage of furans than the sample with the second highest content of this class of compounds (Lp1%-4.3%). Furans were totally absent in C2, La2.5%, Lb1%, and Lb2%. VOCs of alkenes, alkanes, cresols, cyanines, diols, esters, ketones, pyridines, and thiophenes groups were not identified in FJ.
The dominant contribution of ketones was observed in sample C1 (66.9%). The negligible content of ketones was noted in Lb2% (0.5%); additionally, compounds from this group were not detected in the remaining juice samples fermented by L. brevis. This may indicate that L. brevis bacteria degraded ketones into other compounds during fermentation. Moreover, L. brevis and alcohol dehydrogenase (ADH) nowadays are used for the enzymatic reduction of ketones. L. brevis bacteria and ADH have been shown to be versatile catalysts for reducing all ketones (from aliphatic ketones to benzylic or propargylic ketones) [32,33]. Sample C1 emitted a high percentage of phenols (18.3%) and pyridines (5.2%). At a lower level, we determined aldehydes (2.9%), furans (2.4%), thiophenes (2.0%), acids (1.2%), and cresols (1.2%). A high contribution of ketones (29.8%) in the overall aroma of C2 sample was identified. In addition, acids, alcohols, pyridines, aldehydes, and diols were noted at lower levels in this sample. The highest content of acids was found in juice samples fermented by L. acidophilus. These samples were characterized by the contribution of acids at 79.1, 57.6, and 50.1%, respectively, for La1%, La2%, and La2.5%. In addition, juice samples fermented with L. acidophilus were characterized by a high content of phenols (La1%-10.1%, La2%-18.4%, La2.5%-19.5%). Also, other identified components in these samples were ketones, alcohols, and aldehydes. In addition, the La1% and La2.5% samples were characterized by a negligible amount of esters. An insignificant relative content of furans was noted in the La1% and La2% samples. In the juice samples fermented by L. acidophilus, an increase in the percentage of phenols, ketones, and alcohols was observed, along with an In other reports, the effect of lactic acid fermentation on the VOCs profile in various plant-based juice samples/ beverages observed the same trend. In a study by Cele et al. [34], in mango juice fermented with the L. plantarum strain, six main chemical classes were noticed, i.e., alkanes, ketones, esters, aldehydes, alcohols, and terpenes. Cele et al. [34], shown that adding these bacteria significantly improved the profile of volatiles. In addition, dominant chemicals identified in mango juice samples were alcohols (from 14.7 to 33.1%, depending on the variety of mango) [34]. Ricci et al. [35], who investigated the effect of lactic fermentation on the profile and content of VOCs in cherry juice, observed the increase of number and concentration of volatile compounds. They determined that the main identified chemical classes in fermented cherry juice were ketones, alcohols, aldehydes, terpenes, terpene derivates and norisoprenoids, acids, and esters. Quan et al. [36] showed that the dominant groups of compounds in orange juice inoculated with L. acidophilus and L. plantarum were terpenes (monoterpenes and sesquiterpenes) and alcohols. In addition, minor amounts of acids, esters, aldehydes, and ketones were found after fermentation of orange juice samples. Dąbrowski et al. [22] found that the almond beverage before fermentation was characterized by a higher contribution of aldehydes, esters, and lactones, whereas in the oat drink, aldehydes, esters, and organic acids dominated. After fermentation with two different strains of L. plantarum (ATCC 8014 and PK1.1) in the almond drink, esters and aldehydes emitted in the dominant amount, with lower levels being alcohols, lactones, and organic acids. After fermentation of oat drink, the greatest content of esters, alcohols, aldehydes, and lactones was determined. Huang et al. [37] proved that L. brevis addition is positively correlated with acids and alcohols in fermented rice wine. In addition, the research team of Di Renzo et al. [38] in the study of fermented doughs also noted that doughs fermented with L. brevis were characterized by a low content of ketones. Figure 2 shows the sum of volatile compounds in the tested samples, which ranged from 84.00 to 881.31 µg/ mL. The highest sum of VOCs was determined in the C1 sample (881.31 ± 75.56 µg/mL), while the lowest value in the FJ sample (84.00 ± 3.46 µg/mL) was observed. In the case of fermented juice, the lowest sum of VOCs was characteristic for La2% and La2.5% samples. There were no significant differences between these three juice samples (FJ, La2% and La2.5%) at p < 0.05. Moreover, the sum of VOCs in sample C1 was about 90.5, 76.5, and Sum of volatile compounds Sample 77.6% higher compared to samples FJ, La2% and La2.5%, respectively. On the other hand, the highest sum of VOCs in fermented juice samples was found in the Lp1% sample, that was comparable to C2. Red beetroot juice samples fermented by L. brevis were characterized by the slightest difference in the content of VOCs. There were no statistical differences between these samples (Fig. 2). However, it was observed that the incubation temperature influenced the sum of VOCs. In this case, the lower incubation temperature (30 °C) was characterized by the highest sum of VOCs. Moreover, the concentration of VOCs decreases with an increase of the amount of strains usage. In comparison, the sum of VOCs in mango juice fermented by L. plantarum 75 was lower than in our fermented beetroot juice samples and ranged from 244.4 to 543.0 µg/mL [34]. It means that beetroot juice is volatile-rich fermented product, and thus, could have high consumer acceptance.

Conclusion
As already mentioned, this is the first study to show the effects of lactic fermentation on the content and profile of VOCs in beetroot juice. The research showed that each red beetroot juice had its unique profile of VOCs compounds, and its sum differed between individual samples. The lowest concentration of volatile compounds was found in fresh juice Lp2.5% I II III (84.00 µg/mL), while the highest concentration was found in the control sample, incubated at 30 °C (881.31 µg/mL). However, the highest number of VOCs was found in juice samples fermented by L. plantarum, while juice samples fermented by L. brevis were characterized by a lower number of volatiles. Nonanoic acid, 2-methoxy-4-vinylphenol, and acetic acid were the most abundant compounds in fermented juice samples. Due to this experiment, it can be said that the volatile compound profile and content during fermentation by Lactobacillus strains are affected by several factors, such as incubation temperature, amount and species of added strain. Among all used strains, L. plantarum was a major producer of volatiles in cases of number and content of these compounds. According to these studies, 24-h lactic fermentation of beetroot juice allows the formation of a rich profile of volatiles. In addition, their relatively high content may increase the attractiveness of the aroma of red beetroot juice.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Data availability All data included in this manuscript are available upon request by contacting with the corresponding authors.

Conflict of interest The authors declare no conflict of interest.
Compliance with ethics requirements This article does not contain any studies with human or animal subjects.
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