Impact of Lactic Acid Bacteria on Immunoreactivity of Oat Beers

Abstract

The common contamination of oats with gluten cereals represents a problem for celiacs. One way to reduce the level of toxic peptides may be hydrolysis by lactic acid bacteria (LAB). The study examined the influence of the addition of a LAB starter at the grain malting stage on the immunoreactivity of oat beers using enzyme-linked immunosorbent assays with rabbit antibodies and human sera. Immunoblotting was used to identify proteins involved in the immunoenzymatic reaction. The immune response to QQQP and PQQQ sequences was much higher in barley and barley malt (64–76% in relation to wheat) than in oats (20%) and oat malts (below 26%). In the case of anti-QQQPP peptide antibodies, the differences were not so pronounced, mainly due to the high heterogeneity of the oat malt samples. The remaining immunoreactivity was effectively reduced during the technological process of beer production. The mashing process contributed most to the decrease in immunoreactivity, with the wort produced from oat sour malt having an immunoreactivity level of lower than 4%. In the subsequent stages of the beer production process, the immune response was further reduced to below 2% in the resulting beer. Although the level of immunoreactivity of oat sour malt assessed with rabbit antibodies was comparable to that of the regular one, oat sour beers presented significantly weaker immune responses than barley beers, which was not always the case with regular oat beers. This proves the beneficial effect of LAB on reducing the immunoreactivity of the raw material. The analysis performed with human sera confirmed this tendency. Although the immune response to oat beer was strongly dependent on individual sensitivity, the remaining immunoreactivity in oat beers after simulated digestion was only 0.6–2.0%.

1. Introduction

In people with celiac disease, the consumption of gluten-containing products triggers an immune response in the small intestine, which is associated with impaired absorption of nutrients from food. Celiac disease, as well as other forms of gluten intolerance, is not curable and requires a lifelong gluten-free diet, i.e., exclusion of gluten cereals from the menu [1,2,3]. A gluten-free diet is often deficient in nutrients, so the possibility of broadening the range of permitted products with alternative ingredients is constantly sought [4,5,6].

Recent research has shown that oats can be tolerated by the majority of people who suffer from celiac disease [2,7,8]. Oats, unlike wheat, rye, and barley, do not cause symptoms of the disease in most people suffering from celiac disease as there is a significant difference in the chemical composition between oats and gluten cereals [9,10]. The results of numerous clinical studies, which demonstrated the possibility of the safe consumption of oat products by people with celiac disease, resulted in the acceptance of oats as a foodstuff suitable for a gluten-free diet in Europe (2009, Commission Regulation (EC), No. 41/2009) and the USA (2013, FDA regulation) [11,12,13].

Moderate consumption of oats by people with celiac disease is beneficial due to their high nutritional value and health-promoting effects, resulting mainly from their high content of dietary fiber and protein rich in essential amino acids [14]. Oat β-glucan, due to its prebiotic properties, stimulates the growth of lactic acid bacteria in the intestines [8]. Consumption of β-glucans from oats helps to limit the increase in blood glucose levels after a meal and helps to maintain normal blood cholesterol levels [14,15,16].

However, there are cases where after eating oat products, symptoms of intolerance appear, which are associated with contamination of oat products with wheat, rye, and barley [17,18,19]. Numerous studies have shown that products labeled gluten-free, and even more often those naturally gluten-free, may be contaminated [20,21]. The reason for the presence of prolamins of other cereals in oat products is that oats are grown near wheat or barley, which creates the possibility of cross-contamination, and, in addition, the same machines are used to harvest and transport different cereals. Grain is also contaminated during storage and processing at food industry plants [9,19,22]. One of the possibilities of using oat grain in a gluten-free diet is to use it as a raw material for beer production. Malting and lactic acid fermentation are considered very effective in enhancing the functionality of cereal grains [23]. Even though oats are not a conventional raw material for brewing, they have great potential for creating new types of beer that are safe for celiacs [24,25,26]. However, in attempts to produce oat beer, the potential contamination of oat grains with gluten cereals during the processing stage is a real risk, as it is difficult to avoid in large-scale production. Proline- and glutamine-rich gluten proteins responsible for evoking celiac disease are among the major constituents of cereal dietary proteins, which are largely resistant to complete cleavage by the human gastrointestinal tract’s digestive enzymes [27,28]. These digestion-resistant amino acid sequences, consisting mainly of proline and glutamine, cause adverse reactions of varying severity in gluten-intolerant individuals [29,30].

Various strategies are currently being used to find solutions for people with gluten hypersensitivity. The basic recommendation is to follow a restrictive diet consisting of certified products that are naturally gluten-free, which, however, as mentioned, does not always provide a 100% guarantee of safety due to potential contamination. In addition, new products are developed by purifying gluten-containing raw materials via enzymatic treatment. It is also suggested to use selected cultures of lactic acid bacteria as fermentation starters as a way to eliminate the risk of gluten contamination. An innovative approach is to support the digestion of toxic sequences already in the gastrointestinal tract through oral administration of enzyme preparations or by shaping the composition of the intestinal microbiome with the desired metabolic abilities [21,29,31]. So far, none of these solutions is completely satisfactory, but they all aim to effectively eliminate toxic proteins. An alternative may be to combine several of the above methods.

The beer production process itself is associated with a significant reduction in protein content in the product. Both during the malting and mashing stages, the digestion of proteins occurs as a result of the activity of proteolytic enzymes [32]. The mashing process includes a proteolytic rest step at temperatures optimal for protease activity (45–55 °C) to enhance protein degradation. By selecting processing conditions such as malt grind size, temperature, and time, the protein hydrolysis process can be effectively regulated [33]. As a result, only part of the proteins in a soluble form forms the wort extract, because a significant portion of the proteins forms sediments and is separated from the wort during filtration. This precipitation occurs as the temperature is progressively raised from the initial 45 °C [32].

The mashing process is often enhanced by the use of commercially available proteases [32]. This strategy is behind the technologies already implemented on an industrial scale for the production of gluten-free beers from barley. The production of gluten-removed barley beer is based on protein hydrolysis supported by the addition of exogenous enzymes. However, such solutions are still controversial, as there is a high risk that toxic amino acid sequences may remain in the product [34,35].

Another way to support the degradation of gluten peptides in brewing could be the use of lactic acid bacteria. The potential of lactic acid bacteria (LAB) to minimize the immunological response by hydrolyzing alpha-gliadin fragments and reducing the level of gluten is currently often listed as a promising strategy in combating gluten intolerance [21]. The proteolytic mechanism of lactic acid bacteria comprises extracellular and cell wall-associated proteases, which cleave proteins into oligopeptides that are transferred across the cell membrane, and several intracellular peptidases that may contribute to gluten degradation. Highly variable peptidase activity has been observed in different strains (i.e., iminopeptidase, aminopeptidase N, prolidase, prolyl endopeptidyl peptidase, prolinase, tripeptidase, and dipeptidase) [31].

A way to reduce the level of toxic peptides in beer may be the hydrolysis of peptide bonds by LAB used at the malting stage [36,37,38]. The introduction of lactic acid bacteria into malt production is a solution already known in brewing. Lactic acid bacteria are a group of microorganisms naturally occurring on the surface of the grain. The use of LAB starter cultures in the process of malting is primarily intended to reduce the development of unfavorable microbiota. It also improves the technological parameters of so-called sour malts and the quality of the resultant beers [39,40,41,42]. An additional advantage could be the reduction of the risk of unintentional ingestion of toxic gluten by hypersensitive individuals. This beneficial effect may be due to the fact that some bacteria synthesize peptidases capable of degrading immunoreactive peptides [37,43,44,45,46]. Numerous studies confirm the ability of lactobacilli to hydrolyze gluten proteins, reducing their toxicity and inflammatory effect in the intestines [31]. The aim of this study was to compare the immunoreactivity of oat and barley beers and examine the effect of lactic fermentation on reducing immunoreactivity as a result of the proteolytic activity of LAB towards proline-rich peptides.

2. Materials and Methods

2.1. Raw Materials

Oats of the Sławko variety (Plant Breeding Strzelce, Strzelce, Poland) were used in the study, and barley malt (Maltings Soufflet, Poznań, Poland) was used as a reference material. Oats were malted in a micromalting machine of an 8 kg capacity at a Maltings Soufflet plant using procedures previously optimized on a laboratory scale (temperature: 14 °C; steeping: a 4 h wet stand, a 2 h air rest, a 3 h wet stand; 120 h germination). For sour malts production, the Lactobacillus delbrueckii subsp. delbrueckii 20074 (DSMZ, Braunschweig, Germany) culture, previously cultivated at 30 °C for 24 h in MRS broth (BTL, Łódź, Poland) and centrifuged, was added to the grain with steeping water in quantities equivalent to 3 × 10⁸ CFU/g of grains. After germination, green malt was dried at the highest temperature of 84 °C for 1.5 h. All the experiments were carried out in triplicate.

2.2. Brewing

The brewing process was performed on a laboratory scale. The same process conditions were applied for barley and oat malts according to the pattern previously optimized for oat brewing. The mashing process was carried out with a proteolytic rest at 45 °C, a maltose rest at 62 °C, and a saccharification rest at 73 °C (Figure 1). Immediately after the start of the process, enzymatic preparations Filtrase BR (DSM) and Mycolase LV (DSM) were applied in the maximum dose recommended by the manufacturer to reduce wort viscosity. The wort was hopped by 1 h of boiling with the addition of bitter hops Marynka (type 45, 20 BU variety) (Chmiel Polski S.A., Lublin, Poland). The obtained worts were standardized to 12°Blg. When using sour malt, the pH of the wort was 6.11 compared to 6.31 for regular malt. Yeasts Saccharomyces cerevisiae TT (Pure Culture Collection LOCK 105, Łódź, Poland) and Saccharomyces pastorianus W30/78 (Hefebank Weihenstephan, Au in der Hallertau, Germany) were used for top and bottom fermentation, respectively. The bottom fermentation temperature was 10 °C, and the top fermentation temperature was 18 °C. Fermentation was continued until the extract content no longer changed. Maturation was carried out at 1 °C for three weeks, followed by filtration through diatomaceous earth. All the experiments were carried out in triplicate.

2.3. Determination of Protein Immunoreactivity

The indirect non-competitive ELISA method with rabbit antibodies or human primary antibodies was used to determine the immunoreactivity of the samples. In immunological studies, sera containing rabbit polyclonal antibodies directed against amino acid sequences (QQQPP, PQQQ, and QQQP (Eurogentec, Seraing, Belgium)) and human sera containing antigliadin antibodies from patients at the Polish Mother’s Memorial Hospital in Lodz suffering from celiac disease were used. Cereal grains (oats and barley), malts, sweet worts, hopped worts, and beers after the maturation process were analysed. Additionally, the obtained beers were subjected to simulated digestion with pepsin and pancreatin according to Berti et al. [47]. An in vitro digestion test with the enzyme-to-substrate ratio of 1:30 was performed at 37 °C in two steps: first, a 2 h incubation with pepsin (≥250 U/mg) (Sigma-Aldrich, Burlington, MA, USA) at pH 2.0, followed by a 1 h incubation with pancreatin (Nature’s Plus, New York, NY, USA), a commercial mixture of digestive enzymes (amylase, 25,000 USP/1000 mg; protease, 25,000 USP/1000 mg; and lipase, 2000 USP/1000 mg) at pH 8.3. The enzymatic reaction was terminated by incubating the samples in a boiling water bath for 10 min.

Extracts of solid samples (grains and malts) were prepared by incubating 0.5 g of the appropriate sample ground into flour and 5 mL of 0.015 M NaOH for 1 h at room temperature. After a 10-minute centrifugation at 5000× g, the sample extracts were diluted 10-fold with distilled water and then diluted 10-fold with 0.05 M carbonate buffer (pH 9.6). The reference sample was prepared in an identical manner using wheat flour. In the case of worts and beers, the tested sample was used directly and diluted as above. The resulting 100-fold dilutions were used to coat 96-well microtiter plates by overnight incubation at 4 °C. The plates were washed using phosphate-buffered saline (PBS) (pH 7.2) containing 0.1% Tween 20. Unbound sites in the wells were blocked by incubating the plates for 2 h with a 3% skimmed milk powder solution in PBS buffer. After removing the buffer solution, the plates were rinsed four times with PBS buffer. Rabbit sera diluted 20-fold or human sera diluted 100-fold were added to the plates in a volume of 100 μL per well. The plates were incubated for 1 h at room temperature and then washed 4 times with PBS buffer. Monoclonal anti-human IgG antibodies conjugated with an alkaline phosphatase or goat peroxidase-conjugated anti-rabbit immunoglobulin, diluted 1000-fold, were added to the wells in a volume of 100 μL and incubated for 1 h at room temperature. After a subsequent wash of the plates with PBS buffer, p-nitrophenyl phosphate (p-NPP) as a substrate for alkaline phosphatase or 3,3’,5,5’-tetramethylbenzidine (TMB) as a substrate for peroxidase conjugation was alternatively added to the plates in a volume of 100 μL per well. After a 1 h incubation at room temperature, the reaction was terminated by adding 100 μL of 3 M NaOH for p-NPP substrate or 100 μL of 1 M H₂SO₄ for TBM. The absorbance was measured at a wavelength of 405 nm (pNPP) or 450 nm (TBM) with a microplate reader.

The analysis was performed three times for two independently prepared samples. The remaining immunoreactivity was calculated by relating the value of the specific immunoreactivity of the sample to the immunoreactivity of wheat flour, assumed to be 100%.

2.4. Identification of the Protein Fractions Involved in the Immunoenzymatic Reaction

The immunoblotting method was used to identify proteins involved in the immunoenzymatic reaction with antibodies from people suffering from celiac disease. Extracts of cereal grains and malts, as well as worts and beers, were subjected to electrophoretic separation (SDS-PAGE) in polyacrylamide gel. After electrophoretic separation, proteins were transferred from the polyacrylamide gel to the cellulose membrane, followed by an enzyme-linked immunosorbent reaction. The analysis of the cropped blots was carried out using the GelScan program (Kucharczyk TE, Warsaw, Poland) for electropherogram and blot processing.

2.5. Statistical Analysis

Data analysis was performed using the Excel (Microsoft) and Origin 8.0 (OriginLab) programs. The results were presented as arithmetic means and standard deviations. Comparisons between the means were made using Tukey’s test (α = 0.05) for probability values of p < 0.05. A one-way analysis of variance (ANOVA) with Tukey’s tests was performed to determine the significance of differences at p < 0.05.

3. Results and Discussion

In accordance with the main goal of the work, which was aimed at obtaining an oat drink safe for people with gluten intolerance, immunoreactivity tests of raw materials, semi-finished products, as well as the obtained beers were performed. For this purpose, rabbit antibodies directed against peptides rich in proline and glutamine were used, representing the specific sequences appearing in the gluten structure that are considered to be crucial in the pathogenesis of celiac disease. An allergic response could either be induced by the pentapeptide QQQPP or tetrapeptides [27,48,49]. The results of enzyme-linked immunosorbent assays performed using rabbit antibodies directed against specific peptide sequences QQQPP, QQQP, and PQQQ are presented in Figure 2. The results obtained for oat raw materials were compared with the results for barley, which is the basic brewing raw material. The immune response was expressed as a percentage of the immunoreactivity of wheat.

The mean values of the immune response of barley grains determined with rabbit antibodies against PQQQ and QQQP were approximately 76% and 64%, respectively. The mean immunoreactivity values of oats obtained with the use of these antibodies were several times lower than those for barley and reached only 20%. A much higher value was determined using the QQQPP pentapeptide. The pentapeptide QQQPP is considered to be the most toxic to people suffering from celiac disease, and it is also the most active allergen among the low-molecular-weight fraction of wheat proteins [27,50]. In the conducted research, the mean value of immunoreactivity of oats, determined with the use of antibodies directed against the QQQPP pentapeptide, was as high as 62%. Such a strong immune response, with a simultaneous large standard deviation of the results, indicating the heterogeneity of the samples, suggests with a high probability that the raw material used was contaminated with gluten cereal grains. Similar observations were made by Friz and Chen [51], who drew attention to the risk of oat cross-contamination with gluten-containing grains and, at the same time, the large differences in the adverse response to gluten exposure among CD patients. However, the remaining immunoreactivity of oats assessed for the examined samples was still much lower than the value determined for wheat gluten (100%) or for barley, which showed an immune response of 83%.

Similarly, the mean values of the remaining immunoreactivity for the PQQQ and QQQP sequences were much lower in oat malts than in barley malt and did not exceed 26%, with values for barley malt of 76% and 66%, respectively. In the case of anti-QQQPP peptide antibodies, the differences were not so pronounced due to the high heterogeneity of the oat malt samples. Unlike Dostálek et al. [52], who noted an increase in immunoreactivity after the malting process for barley malts, in this study, for both cereal species, the malting process did not cause a statistically significant (p < 0.05) increase in the immune response. Although the germination process may result in the exposure of specific epitopes involved in the immunological reaction as a result of proteolytic hydrolysis of the proteins contained in the grain, at the same time, the products of further hydrolysis formed in the course of the technological process may lose their allergenic properties. In the germination process, not only are the proteolytic enzymes present in the grain activated, but an additional pool of newly synthesized enzymes is created [46,53,54]. Thus, the final result depends on the activity of all the proteases involved in malting.

Although it was expected that the use of a starter culture with steeping water would result in a weakening of the immune response already at the malt production stage due to potential proline endopeptidase activity typical of LAB, the immunoreactivity level of oat sour malt was not significantly lower (p > 0.05) than that of regular malt. The strain used in the study originated from sour grain mash, and this choice was based on its suitability for optimizing the technological parameters of oat malt. Its ability to hydrolyze toxic gluten sequences was not effective enough to demonstrate a significant impact on the immune response at this stage of oat processing. As indicated by numerous studies [31], a broad set of enzymes is necessary for the complete degradation of immunoreactive peptides, which can only be achieved by the simultaneous use of several LAB strains.

In the further part of the study, changes in the level of remaining immunoreactivity in subsequent stages of the oat beer production process were examined. The findings showed that immunoreactivity was effectively reduced in the course of the beer production process, during which most of the toxic protein fractions were removed (Figure 3).

The mashing process contributes most to the reduction in immunoreactivity. In the mashing process, during the proteolytic rest, malt proteins are hydrolyzed into soluble proteins due to the action of endogenous proteolytic enzymes. Most of them are then converted into free amino nitrogen (FAN). A gradual increase in temperature in the later stages of mashing causes further reduction of protein content due to physical factors such as precipitation and adsorption [32,33]. In the post-mashing filtration process, most of the proteins are removed from the beer along with the sediments, and only simple polypeptides pass into the wort [55,56]. This was reflected in a sharp decrease in the immune response determined for sweet wort, which ranged from 2.3% to 3.6% depending on the rabbit antibodies used for testing. In addition, at this stage, the highest values were found for the reaction with the QQQPP pentapeptide. However, some proteins with a strong allergenic nature can still enter beer during the technological process and cause allergic reactions in hypersensitive people [57]. Further reduction of the immune response occurred after boiling the wort with hops and the separation of trub. The fermentation and maturation process of beer also had a positive effect on the weakening of the immunoreactivity of the product. This was the result of the precipitation of certain proteins as a result of the decrease in pH and the adsorption of these compounds onto yeast cells [52,56]. In addition, the beer obtained in this study was filtered using diatomaceous earth after fermentation, which resulted in the removal of another portion of proteins. In the finished oat beers, the immune response was only 1.5% to 1.9% in comparison to wheat flour.

The results of the study justify the statement that the process of mashing and filtration, as well as fermentation and stabilization of beer, has a statistically significant impact (p < 0.05) on the gradual decrease in the immunoreactivity of the tested oat drinks. However, even in the final beverages, no significant differences were found between the types of malt used (regular vs. sour malts) (p > 0.05) or the types of fermentation (bottom- vs. top-fermented beers) (p > 0.05).

To more deeply investigate changes in the immunoreactivity of the samples during the oat beer production process, an immunoblotting technique was used to identify proteins involved in the immunoenzymatic reaction with antibodies from celiac disease sufferers. Electrophoretic separation of proteins was carried out for samples from the subsequent stages of the technological process (grain, malt, sweet wort, hopped wort, bottom- and top-fermented beers) (Figure 4).

For unit processes in the production of oat beer, no significant differences were noted in the patterns of immunoreactive protein fractions during the entire process with regular and sour malts. The use of an LAB starter culture did not cause changes in the composition of proteins in malts, worts, and beers.

The proteins present in oats showed a high immunoreactivity towards the serum containing antibodies against gliadin. Mainly, these were protein fractions with approximate molecular weights of 35 kDa, 30 kDa, 25 kDa, and 22 kDa. In the course of the malting process, some of the proteins were hydrolyzed, as a result of which additional immunoreactive fractions with a molecular weight of 24 kDa and 26 kDa were determined in oat malt, as well as a wide spectrum of proteins with a molecular weight below 10 kDa. Similarly, new immunoreactive fractions were detected in sour malt, identified as 24 kDa and 26 kDa fragments. After the malting process, the protein fraction with a molecular weight of about 25 kDa was no longer present in the malt extract, and it is probable that this fraction was hydrolyzed to low-molecular-weight components.

The process of obtaining sweet wort, followed by the filtration stage, resulted in the removal of a significant part of the proteins from the extract. In sweet wort, protein fractions showing an immune response were characterized by molecular weights of 43 kDa, 26 kDa, 23 kDa, and below 10 kDa. According to Klose et al. [24], in oat mashes, the main protein fractions are those with molecular weights of 6–15 kDa, 22 kDa, and 40 kDa. Comparing these data with the results obtained in the study, it can be concluded that all the protein fractions present in oat worts contain epitopes recognized by the antibodies present in the sera used. Boiling wort with hops did not cause significant differences in the composition of immunoreactive proteins present in the wort.

In turn, after the fermentation process, no immunoreactive fraction of proteins with the lowest molecular weight (below 10 kDa) was detected in the finished beers. Both bottom- and top-fermented beers achieved similar results. The immune response was mainly triggered by proteins with a molecular weight of about 43 kDa, and a much less intense reaction was observed for the fraction with a mass of 26 kDa. According to Steiner et al. [56], in barley, a Z protein weighing about 40 kDa is identified, which is an inhibitor of serine proteinase (so-called serpin). It is present in beer because it is not subject to changes during the beer production process. In oats, the analogue of serpin is a protein with a molecular weight of 43.5 kDa, and it may also be present in beer [24,58,59].

An important stage in the research was the in vitro digestion of the obtained beers with proteolytic enzymes (pepsin and pancreatin). The simulated digestion aimed to draw conclusions about possible changes in the immunoreactivity of oat beers occurring after the hydrolysis of beer proteins in the gastrointestinal tract. To activate celiac disease, a gluten peptide must be (partially) resistant to gastrointestinal digestion. It is evident that only intact gliadin-derived immunogenic peptides that persist throughout digestion can stimulate an immune response [45]. With the use of rabbit antibodies, the immune response of digested bottom- and top-fermented beers obtained from regular and sour oat malt was evaluated and compared with the results for barley beers as a reference sample (Figure 5).

Immunogenic properties of gluten are localized within proline- and glutamine-rich sequences [27,28]. Due to the presence of a high percentage of proline and glutamine in gluten, its digestion by human gastrointestinal digestive enzymes is challenging [38]. However, according to most literature data [60,61,62,63], the products of enzymatic digestion may show a lower immunoreactivity than the initial proteins. On the other hand, according to some findings, this response may also increase, because cereal proteins digested in the gastrointestinal tract can be a source of allergenic peptides [64]. Kushimoto and Aoki [65] found that digestion of gliadins and glutenins with pepsin results in the formation of highly allergenic proteins with a molecular weight of 15–100 kDa. Similar conclusions were reached by Simonato et al. [66] after digestion of wheat products with pepsin and pancreatin. This result is likely due to the enzymatic hydrolysis of high-molecular-weight proteins, such as α, β, ω, and γ gliadins, leading to lower-molecular-weight proteins and peptides [30]. Analysis of the gluten fraction during simulated in vitro digestion [45] showed that immunogenic peptides were absent in the gastric phase but were rapidly released from the food matrix upon entering the intestinal phase. Some of them underwent rapid proteolysis in the intestinal phase, suggesting they may not play a role in celiac disease, but at the same time, the relatively high abundance of peptides indicates a complex digestive mechanism.

In this study, after performing simulated digestion, the remaining immunoreactivity values of all the tested beers, both those made from barley and oat malts, were reduced by approximately 20% in relation to the undigested beers, while maintaining statistically significant differences for barley beer and oat beer made from sour malt (p < 0.05). The immune response of the latter did not exceed 1.5% of the wheat immunoreactivity, while the remaining immunoreactivity of barley beers ranged from 1.7% to 2.7%. Although the level of toxic sequences was reduced in all the samples, the tested beverages still retained their immunogenic potential. The inability of gastric and pancreatic proteases to cleave completely proline–glutamine bonds leads to the formation of peptide fragments capable of crossing the small intestinal epithelial barrier and evoking an adverse immune response [67]. It is worth emphasizing, however, that beers produced from sour oat malt were characterized by a significantly reduced content of gluten epitopes. This suggests that the lactic fermentation process has a positive effect on reducing the immunoreactivity of the raw material, which may be associated with the ability of lactic acid bacteria to produce proline peptidases, which hydrolyze peptide bonds formed with proline. The degradation of these bonds increases tolerance to gluten [5,37,38,43,68]. In vitro and in vivo studies demonstrate that some LAB species are capable of hydrolyzing gluten proteins. A key role in this process is played by bacterial endopeptidases, which digest toxic gluten epitopes [31].

Analogous examination of beers digested in vitro was performed using sera from patients with celiac disease containing antigliadin antibodies (Figure 6).

Analysis of the results obtained proves that the immune response to the tested beers is very diverse and specific to each patient suffering from celiac disease. This is related to individual sensitivity to proteins present in gluten, but also to the inefficiency of intestinal peptidases to degrade immunogenic epitopes [38,69]. The inability of gastric enzymes to cleave proline–glutamine bonds within gluten protein sequences leads to the formation of peptide fragments with a significant immunogenic potential, as they can induce an adverse immune response of varying intensity in genetically susceptible individuals [67]. The amount of gluten that can be tolerated by patients also varies according to individual pathologies. Surprisingly, patients with non-celiac gluten sensitivity may not tolerate even very low amounts of gluten [70].

The remaining, not fully digested gluten fraction can provoke an innate response or engage the immune system through an adaptive response. The innate immune response activated by certain gluten-derived peptides induces the production of interleukin-15 (IL-15), while the adaptive immune response is initiated by T cells recognizing specific gluten epitopes presented by antigen-presenting cells [71]. The assessments conducted with human antigliadin antibodies from CD patients revealed very diverse immune responses to oats, ranging widely from only 12% to 70%, which were specific to each individual. Such large variations in individual gluten hypersensitivity are typical among people with celiac disease. This is one of the reasons why it is difficult to clearly establish a safe dose of gluten for CD patients. According to various sources, the safe level is referred to in the range of 10–100 mg/day, with most specialists indicating a maximum threshold of 50 mg/day [20].

However, similarly to rabbit antibodies, for reactions with human sera, there was a statistically significant (p < 0.05) reduction in the immune response to beer for all types of raw materials used. In oat beers digested with pepsin and pancreatin, the remaining immunoreactivity was as low as 0.6–2.0%. It can therefore be assumed that with such a low immune response, these beers would be well-tolerated by patients. This assumption is supported by reports indicating that patients with moderate gluten intolerance can tolerate occasional consumption of barley beers [20], for which, in this study, the mean value of the remaining immunoreactivity determined for the sera of the most sensitive patients exceeded 3%. What is more, adherence to a strict gluten-free diet is not mandatory for non-celiac gluten sensitivity patients, as sometimes it is sufficient to reduce the amount of gluten in the diet [21].

In addition to very large differences in the immune response to the tested beers among the group of patients subjected to the study, not all sera showed statistically significant differences (p < 0.05) between the samples. Despite the relatively diverse specific characteristics of individual human sera, the changes in immunoreactivity observed with their use had a similar pattern to that seen in the case of rabbit antibodies. As reported by the Working Group on Prolamin Analysis and Toxicity [67], the experimental studies on animal models have demonstrated the effectiveness of certain lactobacilli in degrading immunogenic gluten peptides, thus alleviating small intestinal damage. Therefore, it seems crucial to search for LAB strains that demonstrate the ability to effectively hydrolyze toxic gluten sequences.

4. Conclusions

Summing up the results of the experiments, it can be stated that despite the high risk of oat contamination with other cereals, oats show a much lower immunoreactivity than barley, although the immune response to oat proteins is strongly dependent on individual sensitivity.

The application of a starter culture of lactic acid bacteria with steeping water did not result in the expected attenuation of the immune response at the malting stage to a statistically significant extent. However, in the course of the beer production process, most of the proteins causing the immune reaction were removed, mainly during the production of wort. The remaining immunoreactivity was further reduced in subsequent technological stages. The residual immune reaction in oat beer is caused by a protein fraction with a molecular weight of 38–40 kDa.

The process of simulated digestion in vitro resulted in a further weakening of the immune response. The residual immunoreactivity in oat beer obtained from sour malt was significantly lower than that for barley beers. This indicates the degradation of toxic peptides as a result of the hydrolytic activity of lactic acid bacteria and the beneficial effect of lactic fermentation on the reduction of the immunoreactivity of the oat raw material. The analysis performed with human sera showed a very diverse response of celiac disease patients to oat products. However, despite the high probability of oat contamination with other cereals and the very diverse human sensitivity to gluten, the remaining immunoreactivity in digested oat beers did not exceed 2.0%, which suggests their good tolerance by celiacs. So far, there are no reference values defining the acceptable level of residual immunoreactivity, but it can be assumed that oat beer is safe. First, the remaining immune response indicates the presence of only trace amounts of the original level of gluten contamination. Moreover, unlike bread, beer is consumed occasionally, and its dry matter content is much lower, so the potential dose of unintentionally consumed gluten will not exceed the acceptable threshold.

However, in order to increase the efficiency of gluten cleavage, it is necessary to carefully select LAB starter cultures for the production of sour malt. The search should be focused on strains with the greatest potential to degrade complex secondary structures of gluten, which are resistant to hydrolysis by enzymes in the gastrointestinal tract, and to degrade toxic amino acid sequences.

At the same time, the conducted studies clearly indicate that in the search for solutions ensuring 100% dietary safety of oat beers for people with high gluten sensitivity, a strategy of hurdle technology should be adopted, based on the simultaneous use of several procedures with potentially synergistic effects. In addition to the already mentioned use of sour malts with effectively reduced immunoreactivity, it seems reasonable to optimize the mashing process aimed at further hydrolysis of toxic sequences, which can also be supported by using exogenous enzyme preparations.