Formulation of sustainable gluten-free beer from rice malt and potato processing residue with a view to a circular economy

Formulation of sustainable gluten-free beer from rice malt and potato processing residue with a view to a circular economy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Formulation of sustainable gluten-free beer from rice malt and potato processing residue with a view to a circular economy Francesco Canino, Ignazio Maria Gugino, Angela Maffia, Mariateresa Russo, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7036247/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract This study explores the development of a sustainable, gluten-free beer using regionally sourced ingredients, including rice, potato peels, and native forest botanicals (St. John's Wort, Juniper, and Helichrysum). The research aimed to optimize the fermentation of rice-based wort at a lab-scale by incorporating potato peels and various amylolytic enzymes to improve brewing efficiency and sugar profile complexity. Rice malt was produced at the experimental facilities of KU Leuven (Belgium), and fermentation trials were conducted using both malted and unmalted rice. The study employed different mashing techniques, including traditional decoction mashing, an innovative proprietary enzyme, and a combination of commercial enzyme preparations. Results demonstrated that the novel enzyme significantly enhanced starch hydrolysis, reducing mash times by approximately 30 minutes while improving overall fermentation efficiency. Additionally, the integration of potato peels contributed to a more balanced sugar composition, mitigating glucose dominance and increasing the diversity of fermentable sugars, which could influence yeast metabolism and flavor profile. These findings suggest that enzymatic innovations and agro-industrial byproducts, such as potato peels, could play a key role in advancing sustainable brewing practices while maintaining desirable fermentation characteristics in gluten-free beer production. gluten-free beer potato peel amylase enzymes fermentation efficiency Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The growing demand for diverse flavors and aromas, coupled with the desire for beverages that not only offer organoleptic pleasure but also provide health benefits, has contributed to the continuous expansion of the craft beer market [ 1 ]. The increasing prevalence of gluten-related disorder such as celiac disease and non-celiac gluten sensitivity diseases, led worldwide to a rising demand for gluten-free food products, including beer. This trend has driven extensive research and innovation in brewing science to develop high-quality gluten-free alternatives [ 2 , 3 ]. However, global regulations for gluten-free beer labelling vary, with the Codex Alimentarius establishing a threshold of less than 20 ppm gluten to classify a product as gluten-free [ 4 ]. The increasing interest in healthier gluten-free products is driving brewers and researchers toward new production frontiers, exploring the use of alternative raw materials to create beverages suitable for individuals with gluten intolerance [ 5 , 6 ]. Industrial gluten-free beers made from deglutinized barley malt exist, but they are not completely free of gluten [ 7 ]. As a viable alternative to traditional brewing cereals like barley, wheat, and spelt, rice malt can be used to produce entirely gluten-free beer with characteristics similar to traditional beer. The use of gluten-free cereals and pseudocereals in brewing has become increasingly common, primarily due to their complete absence of gluten [ 8 ].Beyond their gluten-free nature, these raw materials exhibit distinct properties that impact the brewing process, including gelatinization temperature, starch composition, enzymatic activity, and the ratio of amylose to amylopectin. A key challenge associated with these alternatives is their higher gelatinization temperature, which often exceeds the optimal range for amylolytic enzyme activity [ 9 ]. As a result, the breakdown of starch into fermentable sugars is less efficient, leading to a higher proportion of non-fermentable sugars in the final wort. Studies confirmed the feasibility of producing malt from rice; however, the enzymatic processes involved differ from those of barley malt, affecting certain aspects of the brewing process [ 10 ]. However, the unique starch composition of rice often requires supplementary enzymatic treatments or adapted brewing techniques to enhance starch conversion and maximize fermentable sugar yield [ 11 , 12 ]. Implementing targeted enzymatic strategies can help overcome these challenges, ensuring efficient saccharification and improving overall brewing performance. In barley malt, α- and β-amylase enzymes, which are crucial for starch breakdown, are measured by diastatic power [ 13 ]. Rice malt contains lower levels of these enzymes but is rich in other amylolytic enzymes that, in synergy with α- and β-amylase, facilitate the conversion of starch into fermentable sugars. One such enzyme, limit dextrinase, is present in much higher activity in rice malt compared to barley malt[ 14 , 15 ]. Interestingly, potatoes also contain α- and β-amylase, as well as limit dextrinase, suggesting that the combination of rice malt and potato peels could enhance diastatic enzymatic activity, leading to more efficient starch degradation [ 16 ]. Potato peels, which are rich in starch (52 g per 100 g dry weight), non-starch polysaccharides, lignin, polyphenols, proteins, and some lipids, provide an excellent substrate for fermentation processes [ 17 – 19 ]. These insights underscore the potential of utilizing rice malt and potato peels in brewing to produce gluten-free beers that meet consumer demands for flavor diversity and health benefits. Further research into optimizing the enzymatic synergy between these substrates could pave the way for innovative brewing practices that align with contemporary market trends. One of the most important steps in brewing is mashing. This process generally consists of mixing milled malted cereals, most commonly barley, with hot water to solubilize the grains’ starch and protein content. The hypothesis driven this research was based on the supposition that the combination of rice malt and potato peels, along with optimized enzymatic treatments, could enhance starch degradation efficiency and fermentation performance, leading to the production of a high-quality, gluten-free beer with improved sugar balance and sensory characteristics [ 20 ]. Thus, the main aim of this research was to investigate the feasibility and effectiveness of using rice malt and potato peels as alternative brewing ingredients, optimizing their enzymatic interactions that is crucial for optimizing starch hydrolysis to enhance fermentation efficiency and develop a sustainable, gluten-free beer with characteristics comparable to traditional barley-based beers. Building on this premise, investigation at lab-scale on fermentation of rice with the addition of potato peels and various amylolytic enzymes to enhance fermentation efficiency in the brewing process have been carried on. The rice malt was produced at the experimental facilities of KU Leuven (Belgium), where experimental parameters were established. Fermentation trials were conducted using both malted and un-malted rice, employing either the decoction mashing technique, a novel enzyme, or a combination of commercial enzymes to determine the most effective procedure for maximizing rice fermentation efficiency. Materials and methods Malting Plant The rice malting process was conducted at the pilot malting facility of KU Leuven, located at the Technology Campus in Ghent. Whole grain rice, supplied by a Dutch mill, was used. The first stage involved soaking the rice, followed by transferring the germinated malt to a germination tank. This tank was equipped with advanced climate control, allowing for precise regulation of temperature, humidity, airflow, and circulation, which was also used during the drying phase. For small-scale research, the soaking and germination tanks were not filled with bulk grains but with metal buckets containing 3.0 kg of rice. It was essential to limit the amount of grain in the buckets, as they expand significantly during the malting process due to moisture absorption. The rice was stirred manually every 8 hours, both in the morning and evening, starting from the beginning of germination. This process was crucial to separate the grains and prevent the formation of a compact root mass, which could hinder proper moisture absorption. These stirring moments also allowed for a more representative sample of moisture content, compared to simply sampling the surface of the bucket. This ensured optimal conditions for the malting process by adjusting the grain’s moisture if necessary. Experiments were conducted to determine the optimal germination and soaking times for the final quality of rice malt. Initially, 3.0 kg of rice were placed in metal buckets and submerged in the soaking tank. For each malting program, three buckets were used, totalling six buckets to cover two different programs. The soaking tank was filled with water to completely submerge the buckets. Continuous aeration was applied throughout the soaking period, with air blown from the bottom of the tank, maintaining the water temperature at 21°C. The soaking period lasted 8 hours, followed by a 16-hour dry resting phase. During the dry rest, air was aspirated to prevent the buildup of CO2. After two days of soaking, three of the six buckets were removed from the tank, completing two wet soaking phases and two dry resting phases, which was designated as "rice malt 1." The other three buckets, designated "rice malt 2," remained in the tank for an additional day, completing an extra soaking and resting phase. The buckets were then manually transferred to the germination tank, where the temperature was maintained at 20°C and the humidity of the air blown during germination was kept at 98%. Since the buckets were processed together, the drying phase began simultaneously for both groups of malt. "Rice malt 1" underwent a germination period of 5 days, while "rice malt 2" germinated for 4 days [ 21 – 23 ]. Gelatinization Curves To achieve optimal starch degradation during mashing, it is essential to know at what temperature the starch gelatinizes. Gelatinization curves were determined using a viscometer, as starch gelatinization causes a peak in viscosity. Seventy-five grams of malt (DLFU2) were finely ground using a laboratory disk mill and transferred to a 250 mL beaker along with 180 mL of demineralized water. It was crucial to homogenize the solution thoroughly to ensure that the malt did not stick to the walls or bottom of the beaker. A magnetic bar was then inserted into the beaker. If the malt was not adequately distributed in the solution, the magnetic bar would struggle to fully mix the liquid during analysis, compromising result accuracy. The viscometer, along with the spindle and spindle guard, was then inserted into the solution, ensuring proper mixing. The viscometer was set to rotate the spindle at 50 RPM. The water bath was filled to the level of the liquid in the beaker, and the water was heated to approximately 80°C, with a heating rate of 1.5°C/min. When the bath temperature reached 30°C, viscosity was measured every 2 minutes for the first 10 minutes. For the remainder of the analysis, viscosity was monitored every minute until reaching 80°C. Each viscosity measurement was recorded along with the corresponding temperature to later plot the gelatinization curves. Enzymatic Activity The enzymatic activity of the malt and rice was measured using various kits specific to each enzyme involved. The Endo-protease kit (TPRAK-200T) was used to evaluate proteolysis efficacy. For the hydrolysis of α-(1→6) bonds in dextrins and pullulan, the Pullulanase/Limit Dextrinase kit (LOT: 190622-2) from Megazyme was used. The activity of α-amylase and β-amylase was measured using the Malt Amylase Assay Kit, a standardized method widely used for these analyses. For α-amylase, the Ceralpha® method was employed, while β-amylase activity was monitored using the Betamyl-3® kit, which allowed for the breakdown of complex sugars into fermentable sugars. All kits were purchased from Megazyme (Bray, Ireland) [ 24 ]. Lab-Scale Mash Trials Several trials were conducted to evaluate the diastatic activity of enzymes in both rice malt and unmalted rice. Lab-scale mash trials were performed using the LB Electronic Mashing Device (Lochner Labor) to simulate standard mashing conditions, allowing for multiple tests while maintaining realistic parameters on a smaller scale. Each trial was carried out in triplicate to ensure repeatability and accuracy. Finely ground malted and unmalted rice (DLFU2) was prepared using a Laboratory Disk Mill, maintaining a grind-to-water ratio of 1:3. Distilled water, supplemented with calcium chloride (CaCl₂) to enhance amylolytic enzyme efficiency and optimize sugar extraction, was used for the mashes. The water pH was adjusted to match the requirements of each malt type, with the goal of achieving a mash pH around 5.3. A range of temperature steps was employed to optimize sugar extraction and wort quality, with the mash temperatures tailored to the enzymatic characteristics of the malted and unmalted rice. In some trials, decoction mashing was utilized, where a portion of the mash was removed, heated to 80°C, and then returned to the main mash. This technique facilitated rice starch gelatinization, making it more accessible to enzymes and improving the extraction of fermentable sugars during the mashing process. The trials named Trial 1 were all performed using the decoction technique, except for Trial 1.3. Specifically, in Trial 1, 70% of the malt was heated to 85°C using 50% of the water intended for mashing, with the goal of achieving starch gelatinization. Cold water was then added to lower the temperature to 45°C, reaching a 1:3 malt-to-water ratio. Once the desired temperature was achieved, the remaining 30% of the malt was added to avoid degradation of the present enzymes. In Trials 1.1 and 1.2, 80% and 90% of the malt, respectively, was heated to 85°C for the decoction process. Trial 1.3 was conducted using a classic multi-step mashing program to assess whether there were significant differences compared to decoction. All trials that showed interesting results were supplemented with potato peels provided by a Belgian company specialized in potato processing. In the trials labeled P10, 10% potato peels were added, while P20 trials included 20%, with the objective of assessing whether this addition could favor the fermentation process. This was hypothesized due to the presence, albeit limited, of enzymes in the peels, particularly limit dextrinase. Furthermore, the potato peels used provided pre-gelatinized starch, which was easily utilized by the malt enzymes thanks to the steam-peeling process employed by the supplying company. For Trial 2, in addition to rice malt, a new enzyme not yet available on the market, which we will call Enzyme X, was used. The mash was performed with a brief initial step at 65°C, during which the enzyme was added, followed by a 2-hour phase at 85°C. This temperature was chosen because it coincides with the gelatinization temperature of the rice malt produced at KU Leuven – Campus Gent. Thanks to the enzyme's thermostability, it was possible to conduct the mash directly at the optimal temperature for gelatinization without compromising the enzyme's effectiveness, a crucial aspect that allowed for process optimization. Trials 2.1 and 2.2 differ only in the duration of the 85°C step, which lasted 1 hour for Trial 2.1 and 30 minutes for Trial 2.2. All UM trials were conducted using unmalted whole grain rice, with the aim of comparing the fermentation efficiency and sugar composition in the wort with the trials that used rice malt. However, as in UM Trial 2, the steps and procedures followed were identical to those used for Trial 2. In Trial 3, three different enzymes were used: Termamyl, Ceramix Flex, and Attenuzym Core. Trial 4 was conducted using barley malt, to compare the results with those from the rice malt-based trials. The wort obtained at the end of the mash-out was hot-filtered to remove solid residues and then diluted to reach a Plato degree of 12, thereby standardizing all samples. This step is important to ensure that the sugar content of each sample was homogeneous and comparable. After filtration and dilution, the samples were boiled for one hour. After boiling, the wort was cooled to reach the optimal temperature for yeast inoculation. Once cooled, yeast was inoculated into the samples, initiating the fermentation process. The goal of this procedure was to monitor and later evaluate fermentation efficiency by measuring the conversion of fermentable sugars in the wort and comparing the results obtained across different samples. After the designated fermentation time, the samples were filtered and analyzed using the Anton Paar instrument, yielding the following parameters: ethanol concentration, density, extract (apparent and real). Determination of Sugar Profile The sugar profile was determined using high-performance liquid chromatography with a refractive index detector (HPLC-RI). Before the HPLC analysis, proteins were removed from the beer samples through precipitation with Carrez-1 reagent (106 g of K₄Fe(CN)₆·3H₂O dissolved in 1,000 mL of demineralized water) and Carrez-2 reagent (220 g of Zn(CH₃COO)₂·2H₂O and 30 mL of glacial acetic acid, diluted to 1,000 mL with demineralized water). After centrifugation at 11,000 × g for 5 minutes, the supernatant was prepared for manual injection into the HPLC system. For the sugar analysis, each sample was injected twice into the HPLC system, with the eluent injected prior to the separation column (EC 250/4 Nucleodur 100-5 NH2-RP) under very high pressure. This high-pressure condition ensures smooth and efficient transmission of the sample through the column, avoiding mixing with the eluent. The column used is reverse-phase, meaning that polar compounds pass through the column first. When the sugars elute from the column, they are detected by a Waters 2414 refractive index detector, which generates a signal proportional to the sugar concentration in the sample, allowing the results to be visualized in a chromatogram [ 25 ]. Statistical analysis Analysis of variance was carried out for all the data sets. One-way ANOVA with Tukey's Honestly. Powerful Statistical Analysis and Graphics Software for Windows 7, was used for all the statistical analyses. Effects were considered significant at p ≤ 0.05. To explore relationships among different techniques of fermentation on beer parameters, datasets have been analyzed using Principal Component Analysis (PCA) with XLStat. Results and discussion Malting Plant and gelatinization curves The results indicated that the enzymatic activity of Rice Malt 2 was not significantly different from that of Rice Malt 1 (Fig. 1 ). However, the over-modification of Rice Malt 2 led to excessive degradation of the cell walls, making the grain more fragile and prone to breaking easily during milling. This produced finer particles, which posed a risk of clogging the filters during wort filtration, slowing down the process and potentially causing operational challenges [ 26 ]. In contrast, Rice Malt 1 maintained better structural integrity due to its lower degree of modification, while only showing a slightly lower enzymatic activity. Given that the difference in enzymatic activity between the two malts was minimal and did not justify the operational risks associated with Rice Malt 2, subsequent trials were conducted using Rice Malt 1. This choice ensured a smoother mashing process, better control over wort filtration, and maintained the quality of the final product. Regarding the gelatinization curves results showed that in Rice Malt 1, viscosity significantly increased from 70°C, reaching a peak of 65 cP at 76.7°C, indicating that the starch gelatinized and the granules lost their crystalline structure. A similar behavior was observed in Rice Malt 2, with a peak around 78°C. This suggested a difference in starch composition, indicating that Rice Malt 2 contained a higher percentage of amylose in respect to Rice Malt 1 which tended to gelatinize at higher temperatures [ 27 ]. These peaks suggested that the optimal range for gelatinization was between 76°C and 78°C, although these temperatures could denature the amylolytic enzymes. The subsequent step included the use of decoction and thermostable enzymes that work at temperatures above 80°C, improving mash efficiency. At these temperatures, gelatinization and saccharification occur faster, reducing process times and increasing starch-to-sugar conversion. Enzymatic Activity Enzymatic analyses revealed that rice exhibited strong limit dextrinase activity, with values of 2.27 U/g for Rice Malt 1 and 2.15 U/g for Rice Malt 2, both significantly higher than those reported for barley (0.349–0.800 U/g) (Fig. 2 ). The α-amylase activity in rice malt was measured at 25.27 Ceralpha U/g for Rice Malt 1 and 25.25 Ceralpha U/g for Rice Malt 2, though this was notably lower than the 239.5 Ceralpha U/g observed in barley malt. Similarly, β-amylase, which plays a key role in generating fermentable sugars, was significantly lower in rice malt, with values of 0.56 U/g for Rice Malt 1 and 0.31 U/g for Rice Malt 2, compared to 13.79 U/g in barley malt [ 28 ]. In terms of proteolysis, endo-protease activity in Rice Malt 1 and 2 measured 2.31 and 2.60 U/g, respectively, similar to the 2.42 U/g found in barley malt. Although rice malt has a lower capacity for starch degradation compared to barley, it still provides essential nutrients for yeast metabolism during fermentation. Despite its lower amylolytic activity, rice can serve as a viable starch source in brewing, especially when supplemented with external enzymes to compensate for its reduced fermentable sugar production. Mash Lab-Scale The results revealed significant variability in fermentation efficiency (RDF) across the different trials (Table 1 and Table 2 ). Decoction-based trials, such as Trial 1, exhibited lower RDF values, likely due to partial enzyme inactivation caused by high temperatures and the limited enzyme content in rice malt. In contrast, Trial 2 demonstrated high fermentability, achieving RDF values above 78%, largely due to the use of Enzyme X. Even in Trial 2.2, which had the shortest mash time, a high RDF was still achieved, highlighting the enzyme's effectiveness despite the reduced processing time. Overall, trials using unmalted rice (UM) consistently produced the best RDF results. Particularly in UM Trial 2, the addition of potato peels in samples P10 and P20 further boosted fermentation efficiency, a trend not observed in other trials (Fig. 3 ). Table 1 Fermentation parameters assessed across the studied trials SAMPLE Alcohol Er Ea Oe RDF ADF TRIAL 1 4.01 ± 0.3 bc 5.92 ± 0.44 a 4.46 ± 0.33 a 11.96 ± 0.41 a 52.12 ± 3.1 ba 62.67 ± 2 c P10 TRIAL 1 3.75 ± 0.13 c 6.5 ± 0.23 a 5.15 ± 0.18 a 12.13 ± 0.09 a 47.98 ± 4.0 b 57.54 ± 1.78 c P20 TRIAL 1 3.43 ± 0.18 c 6.58 ± 0.34 a 5.33 ± 0.28 a 11.73 ± 0.66 a 45.5 ± 7.8 b 54.56 ± 4.93 c TRIAL 1.1 3.28 ± 0.49 c 6.59 ± 0.19 a 5.4 ± 0.26 a 11.52 ± 0.73 ab 44.16 ± 3.9 b 52.95 ± 4.67 c TRIAL 1.2 3.08 ± 0.09 c 7.37 ± 0.2 a 6.26 ± 0.17 a 11.99 ± 0.02 a 40.00 ± 2.3 b 47.8 ± 1.5 c TRIAL 1.3 3.57 ± 0.08 c 6.43 ± 0.15 a 5.14 ± 0.12 a 11.8 ± 0.00 a 47.02 ± 2.6 b 56.42 ± 1.01 c P10 TRIAL 1.3 3.47 ± 0.07 c 6.93 ± 0.15 a 5.68 ± 0.12 a 12.13 ± 0.04 a 44.41 ± 3.3 b 53.16 ± 0.94 c P20 TRIAL 1.3 3.45 ± 0.14 c 6.91 ± 0.27 a 5.67 ± 0.22 a 12.08 ± 0.03 a 44.35 ± 4.7 b 53.1 ± 1.97 c TRIAL 2 5.92 ± 0.26 b 2.25 ± 0.05 b 0.1 ± 0.12 b 11.35 ± 0.37 b 81.09 ± 0.86 a 99.12 ± 1.04 b TRIAL 2.1 5.86 ± 0.17 b 2.53 ± 0.17 b 0.4 ± 0.16 b 11.52 ± 0.32 ab 79.11 ± 1.06 a 96.57 ± 1.37 b TRIAL 2.2 5.86 ± 0.3 b 2.61 ± 0.09 b 0.48 ± 0.05 b 11.59 ± 0.52 ab 78.56 ± 0.42 a 95.87 ± 0.49 b P10 TRIAL 2.2 5.79 ± 0.12 b 2.98 ± 0.06 b 0.88 ± 0.02 b 11.83 ± 0.21 ab 75.99 ± 1.41 a 92.56 ± 0.26 b P20 TRIAL 2.2 5.5 ± 0.21 b 2.99 ± 0.11 b 0.99 ± 0.04 b 11.42 ± 0.6 b 74.98 ± 2.54 a 91.36 ± 0.31 b UM TRIAL 2 6.39 ± 0.24 a 2.11 ± 0.08 c -0.21 ± 0.01 c 11.91 ± 0.08 a 83.21 ± 2.01 a 101.75 ± 0.08 a P10 UM TRIAL 2 6.36 ± 0.29 a 1.67 ± 0.08 c -0.64 ± 0.03 c 11.48 ± 0.17 b 86.16 ± 1.14 a 105.58 ± 0.18 a P20 UM TRIAL 2 6.55 ± 0.13 a 1.71 ± 0.04 cc -0.66 ± 0.02 c 11.78 ± 0.2 a 86.22 ± 1.1 a 105.61 ± 0.05 a TRIAL 3 6.47 ± 0.14 a 1.88 ± 0.01 c -0.46 ± 0.06 c 11.82 ± 0.21 a 84.88 ± 0.34 a 103.9 ± 0.4 a P10 TRIAL 3 6.10 ± 0.1 a 2.08 ± 0.04 bc -0.15 ± 0.01 c 11.46 ± 0.28 b 82.77 ± 1.23 a 101.25 ± 0.04 a P20 TRIAL 3 6.53 ± 0.22 a 2.2 ± 0.07 b -0.16 ± 0.01 c 12.21 ± 0.31 a 82.9 ± 1.68 a 101.31 ± 0.04 a UM TRIAL 3 6.83 ± 0.18 a 1.25 ± 0.04 b -1.23 ± 0.04 c 11.79 ± 0.09 a 89.96 ± 1.07 a 110.42 ± 0.22 a P10 UM TRIAL 3 6.71 ± 0.54 a 1.35 ± 0.11 c -1.09 ± 0.09 c 11.7 ± 0.24 b 89.1 ± 1.75 a 109.32 ± 0.54 a P20 UM TRIAL 3 6.64 ± 0.17 a 1.45 ± 0.04 c -0.96 ± 0.03 c 11.68 ± 0.09 a 88.26 ± 1.29 a 108.24 ± 0.13 a TRIAL 4 6.44 ± 0.13 a 2.4 ± 0.2 b 0.07 ± 0.17 bc 12.25 ± 0.36 81.47 ± 1.01 a 99.47 ± 1.34 a Alcohol (% v/v); Er , real extract (% w/w); Ea , apparent extract (% w/w); Oe , original extract (% w/w); RDF , real degree of fermentation (% w/w); ADF , apparent degree of fermentation (% w/w). Different letters in the same column indicate significant differences between means (Tukey’s test, p < 0.05) Table 2 Summary of fermentation parameters categorized by brewing methods SAMPLE Alcohol Er Ea Oe RDF ADF Decoction 3.5 ± 0.33 c 6.65 ± 0.46 a 5.39 ± 0.53 a 11.92 ± 0.38 a 45.69 ± 4.92 c 54.78 ± 4.72 c Enzyme x 5.79 ± 0.24 b 2.67 ± 0.31 b 0.57 ± 0.35 b 11.54 ± 0.4 a 77.95 ± 2.59 b 95.1 ± 2.98 b UM + enzyme x 6.43 ± 0.22 a 1.83 ± 0.22 c -0.5 ± 0.22 c 11.72 ± 0.23 a 85.2 ± 1.98 a 104.31 ± 1.93 ab Enzyme mix 6.37 ± 0.24 a 2.05 ± 0.14 b -0.26 ± 0.16 c 11.83 ± 0.4 a 83.52 ± 1.47 a 102.15 ± 1.33 a UM enzyme mix 6.73 ± 0.31 a 1.35 ± 0.1 c -1.09 ± 0.13 c 11.72 ± 0.14 a 89.11 ± 1.42 a 109.33 ± 0.99 a Alcohol (% v/v); Er , real extract (% w/w); Ea , apparent extract (% w/w); Oe , original extract (% w/w); RDF , real degree of fermentation (% w/w); ADF , apparent degree of fermentation (% w/w). Different letters, in the same column, indicate significant differences between means (Tukey’s test, p < 0.05) Sugar Profile The analysis of sugar composition revealed that enzyme addition across various trials significantly enhanced the conversion of complex carbohydrates into fermentable sugars, particularly glucose and maltose (Fig. 4 ). Notably, in trials where Enzyme X was utilized, including Trial 2 and its sub-trials, a higher maltose concentration was observed compared to other enzyme-treated trials. This suggests that Enzyme X facilitated a more controlled hydrolysis of starches, likely due to its specific selectivity for α-1,4 glycosidic bonds, leading to a more balanced ratio between monosaccharides and disaccharides. Such enzymatic control can influence fermentation kinetics, yeast metabolism, and final beer composition, as a higher maltose-to-glucose ratio may support a more gradual and stable fermentation process [ 29 ]. In contrast, Trial 3, which incorporated a combination of Termamyl, Ceramix Flex, and Attenuzym Core, exhibited near-complete hydrolysis of complex carbohydrates, yielding a sugar profile overwhelmingly dominated by glucose (Fig. 4 ). The synergistic action of these thermostable amylases and amyloglucosidases resulted in extensive starch breakdown, promoting rapid yeast assimilation but potentially leading to higher ethanol yields at the expense of flavor complexity [ 30 ]. This suggests that while such enzymatic blends enhance brewing efficiency, they may alter the balance of residual sugars, affecting mouthfeel and overall beer character. An intriguing pattern emerged in sub-trials P10 and P20 of Trial 2.2, where the addition of potato peels led to a less glucose-dominant sugar composition. In these cases, maltose and other oligosaccharides were more pronounced, suggesting that potato peels contributed to a more moderated enzymatic hydrolysis. This effect is likely attributed to pre-gelatinized starches and the presence of limit dextrinase in potato peels, which influenced the progressive breakdown of dextrins into fermentable sugars [ 16 , 31 ]. The observed shift in sugar composition has notable implications for fermentation dynamics, as maltose-rich worst tend to promote smoother yeast attenuation and more complex aromatic profiles, particularly in lager and ale fermentations [ 32 ]. Generally, the use of non-barley cereals in brewing results in beers with sensory profiles that differ from traditional counterparts, influencing consumer perception and acceptance. This innovative methodology with the use of potato peels can enhance the appeal of gluten-free beers. These findings highlight the potential of targeted enzymatic treatments and agro-industrial byproducts in optimizing sugar composition, ultimately influencing fermentation performance, ethanol yield, and sensory attributes in gluten-free beer production [ 33 ]. Conclusions This study demonstrated the potential of rice malt and potato peels as alternative brewing ingredients for producing gluten-free beer, providing valuable insights into the impact of enzyme selection and alternative starch sources on gluten-free brewing efficiency, sugar composition, and fermentation dynamics. The use of novel enzyme is a promising approach for producing gluten-free beer, even if further studies occur to investigate the optimization of this technique for industrial-scale production. Further research should focus on refining enzymatic strategies to enhance the sensory profile and stability of gluten-free beers, while also exploring the influence of these ingredients on yeast performance, attenuation, and flavor development. The integration of sustainable brewing practices using agro-industrial byproducts represents a promising avenue for innovation in the craft beer industry, aligning with consumer demand for health-conscious and environmentally friendly alternatives . Declarations Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment This research was carried on in the doctoral programme of Agricultural, Food and Forestry Science – XXXVI Cycle, Mediterranea University of Reggio Calabria Italy. This work was funded by the Next Generation EU—Italian NRRP, Mission 4, Component2, Investment 1.5, call for the creation and strengthening of ‘Innovation Ecosystems’, building ‘Territorial R&D Leaders’ (Directorial Decree n. 2021/3277)—project Tech4You—Technologies for climate change adaptation and quality of life improvement, n. ECS0000009. This work reflects only the authors’ views and opinions, neither the Ministry for University and Research nor the European Commission can be considered responsible for them. Data Availability The datasets used in the current study are available from the corresponding author on reasonable request. Compliance with ethics requirements The research did not involve Human Participants and/or Animals, no informed consent was necessary. Nevertheless, the research was conducted in accordance with institutional ethical standards and the principles of good scientific practice. Human Ethics and Consent to Participate declarations Not applicable. The research did not involve human participants or animals, and no informed consent was required. References Habschied K, Živković A, Krstanović V, Mastanjević K (2020) Functional Beer—A Review on Possibilities. 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Front Nutr 5:116. https://doi.org/10.3389/fnut.2018.00116 Watson HG, Decloedt AI, Hemeryck LY, et al (2021) Peptidomics of an industrial gluten-free barley malt beer and its non-gluten-free counterpart: Characterisation and immunogenicity. Food Chemistry 355:129597. https://doi.org/10.1016/j.foodchem.2021.129597 Baillière J, Laureys D, Vermeir P, et al (2022) 10 unmalted alternative cereals and pseudocereals: A comparative analysis of their characteristics relevant to the brewing process. Journal of Cereal Science 106:103482. https://doi.org/10.1016/j.jcs.2022.103482 Zarnkow M, Keßler M, Back W, et al (2010) Optimisation of the Mashing Procedure for 100% Malted Proso Millet (Panicum miliaceum L.) as a Raw Material for Gluten-free Beverages and Beers. Journal of the Institute of Brewing 116:141–150. https://doi.org/10.1002/j.2050-0416.2010.tb00410.x Marconi O, Sileoni V, Ceccaroni D, Perretti G (2017) The Use of Rice in Brewing. In: Li J (ed) Advances in International Rice Research. InTech Gasiński A, Kawa-Rygielska J, Spychaj R, et al (2023) Production of gluten-free beer brewing from sorghum malts mashed without external enzyme preparations. Journal of Cereal Science 112:103693. https://doi.org/10.1016/j.jcs.2023.103693 Ledley AJ, Elias RJ, Cockburn DW (2023) Impact of mashing protocol on the formation of fermentable sugars from millet in gluten-free brewing. Food Chemistry 405:134758. https://doi.org/10.1016/j.foodchem.2022.134758 Charmier LMJ, McLoughlin C, McCleary BV (2021) Diastatic power and maltose value: a method for the measurement of amylolytic enzymes in malt. J Inst Brew 127:327–344. https://doi.org/10.1002/jib.665 Jnawali P, Kumar V, Tanwar B (2016) Celiac disease: Overview and considerations for development of gluten-free foods. Food Science and Human Wellness 5:169–176. https://doi.org/10.1016/j.fshw.2016.09.003 Mayer H, Ceccaroni D, Marconi O, et al (2016) Development of an all rice malt beer: A gluten- free alternative. LWT - Food Science and Technology 67:67–73. https://doi.org/10.1016/j.lwt.2015.11.037 Bethke PC, Jansky SH (2008) The Effects of Boiling and Leaching on the Content of Potassium and Other Minerals in Potatoes. Journal of Food Science 73:. https://doi.org/10.1111/j.1750-3841.2008.00782.x Al-Weshahy A, Rao VA (2012) Potato Peel as a Source of Important Phytochemical Antioxidant Nutraceuticals and Their Role in Human Health - A Review. In: Rao V (ed) Phytochemicals as Nutraceuticals - Global Approaches to Their Role in Nutrition and Health. InTech Arapoglou D, Vlyssides A, Varzakas TH, et al (2009) Alternative ways for potato industries waste utilisation. In: Proceedings of Proceedings of the 11th International Conference on Environmental Science and Technolo gy, Chaina, Crete, Greece. pp 3–5 Wu Z-G, Xu H-Y, Ma Q, et al (2012) Isolation, identification and quantification of unsaturated fatty acids, amides, phenolic compounds and glycoalkaloids from potato peel. Food Chemistry 135:2425–2429. https://doi.org/10.1016/j.foodchem.2012.07.019 Calcio Gaudino E, Colletti A, Grillo G, et al (2020) Emerging Processing Technologies for the Recovery of Valuable Bioactive Compounds from Potato Peels. Foods 9:1598. https://doi.org/10.3390/foods9111598 Owusu-Mensah E, Oduro I, Sarfo KJ (2011) STEEPING: A WAY OF IMPROVING THE MALTING OF RICE GRAIN: IMPROVING THE MALTING OF RICE GRAIN. Journal of Food Biochemistry 35:80–91. https://doi.org/10.1111/j.1745-4514.2010.00367.x Ceppi ELM, Brenna OV (2010) Experimental Studies To Obtain Rice Malt. J Agric Food Chem 58:7701–7707. https://doi.org/10.1021/jf904534q Usansa U, Sompong N, Wanapu C, et al (2009) The influences of steeping duration and temperature on the α‐and β‐amylase activities of six Thai rice malt cultivars (Oryza sativa L. Indica). Journal of the Institute of Brewing 115:140–147 Evans DE, Stewart S, Stewart D, et al Profiling Malt Enzymes Related to Impact on Malt Fermentability, Lautering and Beer Filtration Perfo De Rouck G, Jaskula B, De Causmaecker B, et al (2013) The Influence of Very Thick and Fast Mashing Conditions on Wort Composition. Journal of the American Society of Brewing Chemists 71:1–14. https://doi.org/10.1094/ASBCJ-2013-0113-01 Zhang G, Li C (2009) Genetics and improvement of Barley Malt quality. Zhejiang University Press, Hangzhou Schepper CFD, Courtin CM (2023) Intrinsic and extrinsic factors drive differences in the gelatinisation behaviour of barley and malt starch. Food Research International 167:112653. https://doi.org/10.1016/j.foodres.2023.112653 Guimaraes BP, Schrickel F, Rettberg N, et al (2024) Investigating the Malting Suitability and Brewing Quality of Different Rice Cultivars. Beverages 10:16. https://doi.org/10.3390/beverages10010016 Okolo BN, Amadi OC, Moneke AN, et al (2020) Influence of malted barley and exogenous enzymes on the glucose/maltose balance of worts with sorghum or barley as an adjunct: Malted barley and exogenous enzymes and the glucose/maltose balance of worts with sorghum or barley. J Inst Brew 126:46–52. https://doi.org/10.1002/jib.598 Stewart GG (2017) Brewing and Distilling Yeasts. Springer International Publishing, Cham Hu S, Dong J, Fan W, et al (2014) The influence of proteolytic and cytolytic enzymes on starch degradation during mashing: The influence of proteolysis and cytolysis on amylolysis during mashing. J Inst Brew n/a-n/a. https://doi.org/10.1002/jib.172 Gibson BR, Storgårds E, Krogerus K, Vidgren V (2013) Comparative physiology and fermentation performance of Saaz and Frohberg lager yeast strains and the parental species Saccharomyces eubayanus . Yeast 30:255–266. https://doi.org/10.1002/yea.2960 Cadenas R, Caballero I, Nimubona D, Blanco CA (2021) Brewing with Starchy Adjuncts: Its Influence on the Sensory and Nutritional Properties of Beer. Foods 10:1726. https://doi.org/10.3390/foods10081726 Additional Declarations No competing interests reported. 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(Italy","correspondingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"","lastName":"Canino","suffix":""},{"id":484698450,"identity":"31784c6c-1083-4acc-82e8-b86e7f76e92c","order_by":1,"name":"Ignazio Maria Gugino","email":"","orcid":"","institution":"University of Palermo","correspondingAuthor":false,"prefix":"","firstName":"Ignazio","middleName":"Maria","lastName":"Gugino","suffix":""},{"id":484698452,"identity":"68e3cdc2-46cf-41f7-ad35-355c5896d9a7","order_by":2,"name":"Angela Maffia","email":"","orcid":"","institution":"Department of AGRARIA- Mediterranea University of Reggio Calabria, Località Feo di Vito, 89122 Reggio Calabria. (Italy","correspondingAuthor":false,"prefix":"","firstName":"Angela","middleName":"","lastName":"Maffia","suffix":""},{"id":484698453,"identity":"8f99626c-334e-4cc9-8a75-9825b8243f01","order_by":3,"name":"Mariateresa Russo","email":"","orcid":"","institution":"Department of AGRARIA- Mediterranea University of Reggio Calabria, Località Feo di Vito, 89122 Reggio Calabria. (Italy","correspondingAuthor":false,"prefix":"","firstName":"Mariateresa","middleName":"","lastName":"Russo","suffix":""},{"id":484698455,"identity":"9bbc4bfc-74fb-43b6-99a7-2f2d0be8d889","order_by":4,"name":"Gert De Rouck","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Gert","middleName":"","lastName":"De Rouck","suffix":""},{"id":484698456,"identity":"34c3ae24-b7d6-4b9f-bf06-6c7e7f71298f","order_by":5,"name":"Adele Muscolo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYPACCwZ+IHkAiHmI1SLBINkA1UKsHgkGgwNQJkEt/NPOGH/4USEhZ3wj9+DhihoGGXuCxt/OMZPsOSNhbHYjL+HgmWPEOAyohZmxTSJx240cg4ONDURokb+dY/wZpGXzDGK1GNzOMZAGadkgQawWw9tpZWC/SJx5Y3Cw4ZgED88BAlrkbidvBoaYjRx/e47xx4YaG3v2BkLWoAEJEtWPglEwCkbBKMAKAEudOT/arjffAAAAAElFTkSuQmCC","orcid":"","institution":"Department of AGRARIA- Mediterranea University of Reggio Calabria, Località Feo di Vito, 89122 Reggio Calabria. (Italy","correspondingAuthor":true,"prefix":"","firstName":"Adele","middleName":"","lastName":"Muscolo","suffix":""}],"badges":[],"createdAt":"2025-07-03 09:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7036247/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7036247/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86709057,"identity":"6466076c-ec7f-4cd4-8a79-9ab83700fc3c","added_by":"auto","created_at":"2025-07-14 18:04:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":28524,"visible":true,"origin":"","legend":"\u003cp\u003eChange in gelatinization of rice malt 1 and 2 in respect to different temperatures\u003c/p\u003e","description":"","filename":"drawingimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7036247/v1/f19e66e5dfd31f2e8c1733f6.png"},{"id":86709702,"identity":"328a696a-b635-4200-aa87-eeedadeeb0ec","added_by":"auto","created_at":"2025-07-14 18:12:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":14197,"visible":true,"origin":"","legend":"\u003cp\u003eEnzymatic activities in Rice and Rice malt 1 and 2\u003c/p\u003e","description":"","filename":"drawingimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7036247/v1/42803fe3105e325eb40e2ec3.png"},{"id":86709059,"identity":"df705679-00da-4c42-b4e8-0fbf3d1cb934","added_by":"auto","created_at":"2025-07-14 18:04:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":14725,"visible":true,"origin":"","legend":"\u003cp\u003eReal degree of different percentage of potato peel fermentation (RDF)\u003c/p\u003e","description":"","filename":"drawingimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7036247/v1/dc816ad2b416af7e1067d065.png"},{"id":86709841,"identity":"c0e73359-cae0-4b6b-8ba3-53c103e15603","added_by":"auto","created_at":"2025-07-14 18:20:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18724,"visible":true,"origin":"","legend":"\u003cp\u003eWort sugars in the different trials with different percentage of potato peels\u003c/p\u003e","description":"","filename":"drawingimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7036247/v1/23aed4ef88bf618f9664563d.png"},{"id":86710310,"identity":"48d613f0-49f8-456a-a76d-c7368dc340c2","added_by":"auto","created_at":"2025-07-14 18:28:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":895945,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7036247/v1/7c3e70c9-ab77-44b8-9446-ac6d5c821ffe.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Formulation of sustainable gluten-free beer from rice malt and potato processing residue with a view to a circular economy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe growing demand for diverse flavors and aromas, coupled with the desire for beverages that not only offer organoleptic pleasure but also provide health benefits, has contributed to the continuous expansion of the craft beer market [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The increasing prevalence of gluten-related disorder such as celiac disease and non-celiac gluten sensitivity diseases, led worldwide to a rising demand for gluten-free food products, including beer. This trend has driven extensive research and innovation in brewing science to develop high-quality gluten-free alternatives [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, global regulations for gluten-free beer labelling vary, with the Codex Alimentarius establishing a threshold of less than 20 ppm gluten to classify a product as gluten-free [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The increasing interest in healthier gluten-free products is driving brewers and researchers toward new production frontiers, exploring the use of alternative raw materials to create beverages suitable for individuals with gluten intolerance [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIndustrial gluten-free beers made from deglutinized barley malt exist, but they are not completely free of gluten [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As a viable alternative to traditional brewing cereals like barley, wheat, and spelt, rice malt can be used to produce entirely gluten-free beer with characteristics similar to traditional beer. The use of gluten-free cereals and pseudocereals in brewing has become increasingly common, primarily due to their complete absence of gluten [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].Beyond their gluten-free nature, these raw materials exhibit distinct properties that impact the brewing process, including gelatinization temperature, starch composition, enzymatic activity, and the ratio of amylose to amylopectin. A key challenge associated with these alternatives is their higher gelatinization temperature, which often exceeds the optimal range for amylolytic enzyme activity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. As a result, the breakdown of starch into fermentable sugars is less efficient, leading to a higher proportion of non-fermentable sugars in the final wort. Studies confirmed the feasibility of producing malt from rice; however, the enzymatic processes involved differ from those of barley malt, affecting certain aspects of the brewing process [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the unique starch composition of rice often requires supplementary enzymatic treatments or adapted brewing techniques to enhance starch conversion and maximize fermentable sugar yield [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Implementing targeted enzymatic strategies can help overcome these challenges, ensuring efficient saccharification and improving overall brewing performance.\u003c/p\u003e\u003cp\u003eIn barley malt, α- and β-amylase enzymes, which are crucial for starch breakdown, are measured by diastatic power [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Rice malt contains lower levels of these enzymes but is rich in other amylolytic enzymes that, in synergy with α- and β-amylase, facilitate the conversion of starch into fermentable sugars. One such enzyme, limit dextrinase, is present in much higher activity in rice malt compared to barley malt[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eInterestingly, potatoes also contain α- and β-amylase, as well as limit dextrinase, suggesting that the combination of rice malt and potato peels could enhance diastatic enzymatic activity, leading to more efficient starch degradation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Potato peels, which are rich in starch (52 g per 100 g dry weight), non-starch polysaccharides, lignin, polyphenols, proteins, and some lipids, provide an excellent substrate for fermentation processes [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These insights underscore the potential of utilizing rice malt and potato peels in brewing to produce gluten-free beers that meet consumer demands for flavor diversity and health benefits. Further research into optimizing the enzymatic synergy between these substrates could pave the way for innovative brewing practices that align with contemporary market trends. One of the most important steps in brewing is mashing. This process generally consists of mixing milled malted cereals, most commonly barley, with hot water to solubilize the grains\u0026rsquo; starch and protein content. The hypothesis driven this research was based on the supposition that the combination of rice malt and potato peels, along with optimized enzymatic treatments, could enhance starch degradation efficiency and fermentation performance, leading to the production of a high-quality, gluten-free beer with improved sugar balance and sensory characteristics [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Thus, the main aim of this research was to investigate the feasibility and effectiveness of using rice malt and potato peels as alternative brewing ingredients, optimizing their enzymatic interactions that is crucial for optimizing starch hydrolysis to enhance fermentation efficiency and develop a sustainable, gluten-free beer with characteristics comparable to traditional barley-based beers. Building on this premise, investigation at lab-scale on fermentation of rice with the addition of potato peels and various amylolytic enzymes to enhance fermentation efficiency in the brewing process have been carried on. The rice malt was produced at the experimental facilities of KU Leuven (Belgium), where experimental parameters were established. Fermentation trials were conducted using both malted and un-malted rice, employing either the decoction mashing technique, a novel enzyme, or a combination of commercial enzymes to determine the most effective procedure for maximizing rice fermentation efficiency.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eMalting Plant\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe rice malting process was conducted at the pilot malting facility of KU Leuven, located at the Technology Campus in Ghent. Whole grain rice, supplied by a Dutch mill, was used. The first stage involved soaking the rice, followed by transferring the germinated malt to a germination tank. This tank was equipped with advanced climate control, allowing for precise regulation of temperature, humidity, airflow, and circulation, which was also used during the drying phase.\u003c/p\u003e\u003cp\u003eFor small-scale research, the soaking and germination tanks were not filled with bulk grains but with metal buckets containing 3.0 kg of rice. It was essential to limit the amount of grain in the buckets, as they expand significantly during the malting process due to moisture absorption. The rice was stirred manually every 8 hours, both in the morning and evening, starting from the beginning of germination. This process was crucial to separate the grains and prevent the formation of a compact root mass, which could hinder proper moisture absorption. These stirring moments also allowed for a more representative sample of moisture content, compared to simply sampling the surface of the bucket. This ensured optimal conditions for the malting process by adjusting the grain\u0026rsquo;s moisture if necessary.\u003c/p\u003e\u003cp\u003eExperiments were conducted to determine the optimal germination and soaking times for the final quality of rice malt. Initially, 3.0 kg of rice were placed in metal buckets and submerged in the soaking tank. For each malting program, three buckets were used, totalling six buckets to cover two different programs. The soaking tank was filled with water to completely submerge the buckets. Continuous aeration was applied throughout the soaking period, with air blown from the bottom of the tank, maintaining the water temperature at 21\u0026deg;C. The soaking period lasted 8 hours, followed by a 16-hour dry resting phase. During the dry rest, air was aspirated to prevent the buildup of CO2.\u003c/p\u003e\u003cp\u003eAfter two days of soaking, three of the six buckets were removed from the tank, completing two wet soaking phases and two dry resting phases, which was designated as \"rice malt 1.\" The other three buckets, designated \"rice malt 2,\" remained in the tank for an additional day, completing an extra soaking and resting phase. The buckets were then manually transferred to the germination tank, where the temperature was maintained at 20\u0026deg;C and the humidity of the air blown during germination was kept at 98%. Since the buckets were processed together, the drying phase began simultaneously for both groups of malt. \"Rice malt 1\" underwent a germination period of 5 days, while \"rice malt 2\" germinated for 4 days [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eGelatinization Curves\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo achieve optimal starch degradation during mashing, it is essential to know at what temperature the starch gelatinizes. Gelatinization curves were determined using a viscometer, as starch gelatinization causes a peak in viscosity.\u003c/p\u003e\u003cp\u003eSeventy-five grams of malt (DLFU2) were finely ground using a laboratory disk mill and transferred to a 250 mL beaker along with 180 mL of demineralized water. It was crucial to homogenize the solution thoroughly to ensure that the malt did not stick to the walls or bottom of the beaker. A magnetic bar was then inserted into the beaker. If the malt was not adequately distributed in the solution, the magnetic bar would struggle to fully mix the liquid during analysis, compromising result accuracy.\u003c/p\u003e\u003cp\u003eThe viscometer, along with the spindle and spindle guard, was then inserted into the solution, ensuring proper mixing. The viscometer was set to rotate the spindle at 50 RPM. The water bath was filled to the level of the liquid in the beaker, and the water was heated to approximately 80\u0026deg;C, with a heating rate of 1.5\u0026deg;C/min.\u003c/p\u003e\u003cp\u003eWhen the bath temperature reached 30\u0026deg;C, viscosity was measured every 2 minutes for the first 10 minutes. For the remainder of the analysis, viscosity was monitored every minute until reaching 80\u0026deg;C. Each viscosity measurement was recorded along with the corresponding temperature to later plot the gelatinization curves.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnzymatic Activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe enzymatic activity of the malt and rice was measured using various kits specific to each enzyme involved. The Endo-protease kit (TPRAK-200T) was used to evaluate proteolysis efficacy. For the hydrolysis of α-(1\u0026rarr;6) bonds in dextrins and pullulan, the Pullulanase/Limit Dextrinase kit (LOT: 190622-2) from Megazyme was used.\u003c/p\u003e\u003cp\u003eThe activity of α-amylase and β-amylase was measured using the Malt Amylase Assay Kit, a standardized method widely used for these analyses. For α-amylase, the Ceralpha\u0026reg; method was employed, while β-amylase activity was monitored using the Betamyl-3\u0026reg; kit, which allowed for the breakdown of complex sugars into fermentable sugars. All kits were purchased from Megazyme (Bray, Ireland) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eLab-Scale Mash Trials\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSeveral trials were conducted to evaluate the diastatic activity of enzymes in both rice malt and unmalted rice. Lab-scale mash trials were performed using the LB Electronic Mashing Device (Lochner Labor) to simulate standard mashing conditions, allowing for multiple tests while maintaining realistic parameters on a smaller scale. Each trial was carried out in triplicate to ensure repeatability and accuracy.\u003c/p\u003e\u003cp\u003eFinely ground malted and unmalted rice (DLFU2) was prepared using a Laboratory Disk Mill, maintaining a grind-to-water ratio of 1:3. Distilled water, supplemented with calcium chloride (CaCl₂) to enhance amylolytic enzyme efficiency and optimize sugar extraction, was used for the mashes. The water pH was adjusted to match the requirements of each malt type, with the goal of achieving a mash pH around 5.3.\u003c/p\u003e\u003cp\u003eA range of temperature steps was employed to optimize sugar extraction and wort quality, with the mash temperatures tailored to the enzymatic characteristics of the malted and unmalted rice. In some trials, decoction mashing was utilized, where a portion of the mash was removed, heated to 80\u0026deg;C, and then returned to the main mash. This technique facilitated rice starch gelatinization, making it more accessible to enzymes and improving the extraction of fermentable sugars during the mashing process.\u003c/p\u003e\u003cp\u003eThe trials named Trial 1 were all performed using the decoction technique, except for Trial 1.3. Specifically, in Trial 1, 70% of the malt was heated to 85\u0026deg;C using 50% of the water intended for mashing, with the goal of achieving starch gelatinization. Cold water was then added to lower the temperature to 45\u0026deg;C, reaching a 1:3 malt-to-water ratio. Once the desired temperature was achieved, the remaining 30% of the malt was added to avoid degradation of the present enzymes.\u003c/p\u003e\u003cp\u003eIn Trials 1.1 and 1.2, 80% and 90% of the malt, respectively, was heated to 85\u0026deg;C for the decoction process. Trial 1.3 was conducted using a classic multi-step mashing program to assess whether there were significant differences compared to decoction.\u003c/p\u003e\u003cp\u003e All trials that showed interesting results were supplemented with potato peels provided by a Belgian company specialized in potato processing. In the trials labeled P10, 10% potato peels were added, while P20 trials included 20%, with the objective of assessing whether this addition could favor the fermentation process. This was hypothesized due to the presence, albeit limited, of enzymes in the peels, particularly limit dextrinase. Furthermore, the potato peels used provided pre-gelatinized starch, which was easily utilized by the malt enzymes thanks to the steam-peeling process employed by the supplying company.\u003c/p\u003e\u003cp\u003eFor Trial 2, in addition to rice malt, a new enzyme not yet available on the market, which we will call Enzyme X, was used. The mash was performed with a brief initial step at 65\u0026deg;C, during which the enzyme was added, followed by a 2-hour phase at 85\u0026deg;C. This temperature was chosen because it coincides with the gelatinization temperature of the rice malt produced at KU Leuven \u0026ndash; Campus Gent. Thanks to the enzyme's thermostability, it was possible to conduct the mash directly at the optimal temperature for gelatinization without compromising the enzyme's effectiveness, a crucial aspect that allowed for process optimization. Trials 2.1 and 2.2 differ only in the duration of the 85\u0026deg;C step, which lasted 1 hour for Trial 2.1 and 30 minutes for Trial 2.2.\u003c/p\u003e\u003cp\u003eAll UM trials were conducted using unmalted whole grain rice, with the aim of comparing the fermentation efficiency and sugar composition in the wort with the trials that used rice malt. However, as in UM Trial 2, the steps and procedures followed were identical to those used for Trial 2. In Trial 3, three different enzymes were used: Termamyl, Ceramix Flex, and Attenuzym Core. Trial 4 was conducted using barley malt, to compare the results with those from the rice malt-based trials.\u003c/p\u003e\u003cp\u003eThe wort obtained at the end of the mash-out was hot-filtered to remove solid residues and then diluted to reach a Plato degree of 12, thereby standardizing all samples. This step is important to ensure that the sugar content of each sample was homogeneous and comparable. After filtration and dilution, the samples were boiled for one hour. After boiling, the wort was cooled to reach the optimal temperature for yeast inoculation. Once cooled, yeast was inoculated into the samples, initiating the fermentation process. The goal of this procedure was to monitor and later evaluate fermentation efficiency by measuring the conversion of fermentable sugars in the wort and comparing the results obtained across different samples.\u003c/p\u003e\u003cp\u003eAfter the designated fermentation time, the samples were filtered and analyzed using the Anton Paar instrument, yielding the following parameters: ethanol concentration, density, extract (apparent and real).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of Sugar Profile\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe sugar profile was determined using high-performance liquid chromatography with a refractive index detector (HPLC-RI). Before the HPLC analysis, proteins were removed from the beer samples through precipitation with Carrez-1 reagent (106 g of K₄Fe(CN)₆\u0026middot;3H₂O dissolved in 1,000 mL of demineralized water) and Carrez-2 reagent (220 g of Zn(CH₃COO)₂\u0026middot;2H₂O and 30 mL of glacial acetic acid, diluted to 1,000 mL with demineralized water). After centrifugation at 11,000 \u0026times; g for 5 minutes, the supernatant was prepared for manual injection into the HPLC system.\u003c/p\u003e\u003cp\u003eFor the sugar analysis, each sample was injected twice into the HPLC system, with the eluent injected prior to the separation column (EC 250/4 Nucleodur 100-5 NH2-RP) under very high pressure. This high-pressure condition ensures smooth and efficient transmission of the sample through the column, avoiding mixing with the eluent. The column used is reverse-phase, meaning that polar compounds pass through the column first. When the sugars elute from the column, they are detected by a Waters 2414 refractive index detector, which generates a signal proportional to the sugar concentration in the sample, allowing the results to be visualized in a chromatogram [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAnalysis of variance was carried out for all the data sets. One-way ANOVA with Tukey's Honestly. Powerful Statistical Analysis and Graphics Software for Windows 7, was used for all the statistical analyses. Effects were considered significant at p\u0026thinsp;\u0026le;\u0026thinsp;0.05. To explore relationships among different techniques of fermentation on beer parameters, datasets have been analyzed using Principal Component Analysis (PCA) with XLStat.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cb\u003eMalting Plant and gelatinization curves\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe results indicated that the enzymatic activity of Rice Malt 2 was not significantly different from that of Rice Malt 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, the over-modification of Rice Malt 2 led to excessive degradation of the cell walls, making the grain more fragile and prone to breaking easily during milling. This produced finer particles, which posed a risk of clogging the filters during wort filtration, slowing down the process and potentially causing operational challenges [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn contrast, Rice Malt 1 maintained better structural integrity due to its lower degree of modification, while only showing a slightly lower enzymatic activity. Given that the difference in enzymatic activity between the two malts was minimal and did not justify the operational risks associated with Rice Malt 2, subsequent trials were conducted using Rice Malt 1. This choice ensured a smoother mashing process, better control over wort filtration, and maintained the quality of the final product. Regarding the gelatinization curves results showed that in Rice Malt 1, viscosity significantly increased from 70\u0026deg;C, reaching a peak of 65 cP at 76.7\u0026deg;C, indicating that the starch gelatinized and the granules lost their crystalline structure. A similar behavior was observed in Rice Malt 2, with a peak around 78\u0026deg;C. This suggested a difference in starch composition, indicating that Rice Malt 2 contained a higher percentage of amylose in respect to Rice Malt 1 which tended to gelatinize at higher temperatures [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These peaks suggested that the optimal range for gelatinization was between 76\u0026deg;C and 78\u0026deg;C, although these temperatures could denature the amylolytic enzymes. The subsequent step included the use of decoction and thermostable enzymes that work at temperatures above 80\u0026deg;C, improving mash efficiency. At these temperatures, gelatinization and saccharification occur faster, reducing process times and increasing starch-to-sugar conversion.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnzymatic Activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEnzymatic analyses revealed that rice exhibited strong limit dextrinase activity, with values of 2.27 U/g for Rice Malt 1 and 2.15 U/g for Rice Malt 2, both significantly higher than those reported for barley (0.349\u0026ndash;0.800 U/g) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The α-amylase activity in rice malt was measured at 25.27 Ceralpha U/g for Rice Malt 1 and 25.25 Ceralpha U/g for Rice Malt 2, though this was notably lower than the 239.5 Ceralpha U/g observed in barley malt. Similarly, β-amylase, which plays a key role in generating fermentable sugars, was significantly lower in rice malt, with values of 0.56 U/g for Rice Malt 1 and 0.31 U/g for Rice Malt 2, compared to 13.79 U/g in barley malt [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn terms of proteolysis, endo-protease activity in Rice Malt 1 and 2 measured 2.31 and 2.60 U/g, respectively, similar to the 2.42 U/g found in barley malt. Although rice malt has a lower capacity for starch degradation compared to barley, it still provides essential nutrients for yeast metabolism during fermentation. Despite its lower amylolytic activity, rice can serve as a viable starch source in brewing, especially when supplemented with external enzymes to compensate for its reduced fermentable sugar production.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMash Lab-Scale\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe results revealed significant variability in fermentation efficiency (RDF) across the different trials (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Decoction-based trials, such as Trial 1, exhibited lower RDF values, likely due to partial enzyme inactivation caused by high temperatures and the limited enzyme content in rice malt. In contrast, Trial 2 demonstrated high fermentability, achieving RDF values above 78%, largely due to the use of Enzyme X. Even in Trial 2.2, which had the shortest mash time, a high RDF was still achieved, highlighting the enzyme's effectiveness despite the reduced processing time. Overall, trials using unmalted rice (UM) consistently produced the best RDF results. Particularly in UM Trial 2, the addition of potato peels in samples P10 and P20 further boosted fermentation efficiency, a trend not observed in other trials (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFermentation parameters assessed across the studied trials\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSAMPLE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAlcohol\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEr\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEa\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOe\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eRDF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eADF\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTRIAL 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e52.12\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003csup\u003eba\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e62.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP10 TRIAL 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e47.98\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e57.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP20 TRIAL 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e45.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e54.56\u0026thinsp;\u0026plusmn;\u0026thinsp;4.93\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTRIAL 1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e44.16\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e52.95\u0026thinsp;\u0026plusmn;\u0026thinsp;4.67\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTRIAL 1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e40.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e47.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTRIAL 1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e47.02\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e56.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP10 TRIAL 1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e44.41\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e53.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP20 TRIAL 1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e44.35\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e53.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTRIAL 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e81.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e99.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTRIAL 2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e79.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e96.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTRIAL 2.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e78.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e95.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP10 TRIAL 2.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e75.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e92.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP20 TRIAL 2.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e74.98\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e91.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUM TRIAL 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e83.21\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e101.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP10 UM TRIAL 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e86.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e105.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP20 UM TRIAL 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ecc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e86.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e105.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTRIAL 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e84.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e103.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP10 TRIAL 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e82.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e101.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP20 TRIAL 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e82.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e101.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUM TRIAL 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e89.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e110.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP10 UM TRIAL 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e89.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e109.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP20 UM TRIAL 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e88.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e108.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTRIAL 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e81.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e99.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAlcohol\u003c/b\u003e (% v/v); \u003cb\u003eEr\u003c/b\u003e, real extract (% w/w); \u003cb\u003eEa\u003c/b\u003e, apparent extract (% w/w); \u003cb\u003eOe\u003c/b\u003e, original extract (% w/w); \u003cb\u003eRDF\u003c/b\u003e, real degree of fermentation (% w/w); \u003cb\u003eADF\u003c/b\u003e, apparent degree of fermentation (% w/w). Different letters in the same column indicate significant differences between means (Tukey\u0026rsquo;s test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of fermentation parameters categorized by brewing methods\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSAMPLE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAlcohol\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEr\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEa\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOe\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eRDF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eADF\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDecoction\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e45.69\u0026thinsp;\u0026plusmn;\u0026thinsp;4.92\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e54.78\u0026thinsp;\u0026plusmn;\u0026thinsp;4.72\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnzyme x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e77.95\u0026thinsp;\u0026plusmn;\u0026thinsp;2.59\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e95.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.98\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUM\u0026thinsp;+\u0026thinsp;enzyme x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e85.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e104.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.93\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnzyme mix\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e83.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e102.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUM enzyme mix\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e89.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e109.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAlcohol\u003c/b\u003e (% v/v); \u003cb\u003eEr\u003c/b\u003e, real extract (% w/w); \u003cb\u003eEa\u003c/b\u003e, apparent extract (% w/w); \u003cb\u003eOe\u003c/b\u003e, original extract (% w/w); \u003cb\u003eRDF\u003c/b\u003e, real degree of fermentation (% w/w); \u003cb\u003eADF\u003c/b\u003e, apparent degree of fermentation (% w/w).\u003c/p\u003e\u003cp\u003eDifferent letters, in the same column, indicate significant differences between means (Tukey\u0026rsquo;s test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSugar Profile\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe analysis of sugar composition revealed that enzyme addition across various trials significantly enhanced the conversion of complex carbohydrates into fermentable sugars, particularly glucose and maltose (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Notably, in trials where Enzyme X was utilized, including Trial 2 and its sub-trials, a higher maltose concentration was observed compared to other enzyme-treated trials. This suggests that Enzyme X facilitated a more controlled hydrolysis of starches, likely due to its specific selectivity for α-1,4 glycosidic bonds, leading to a more balanced ratio between monosaccharides and disaccharides. Such enzymatic control can influence fermentation kinetics, yeast metabolism, and final beer composition, as a higher maltose-to-glucose ratio may support a more gradual and stable fermentation process [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn contrast, Trial 3, which incorporated a combination of Termamyl, Ceramix Flex, and Attenuzym Core, exhibited near-complete hydrolysis of complex carbohydrates, yielding a sugar profile overwhelmingly dominated by glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The synergistic action of these thermostable amylases and amyloglucosidases resulted in extensive starch breakdown, promoting rapid yeast assimilation but potentially leading to higher ethanol yields at the expense of flavor complexity [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This suggests that while such enzymatic blends enhance brewing efficiency, they may alter the balance of residual sugars, affecting mouthfeel and overall beer character.\u003c/p\u003e\u003cp\u003eAn intriguing pattern emerged in sub-trials P10 and P20 of Trial 2.2, where the addition of potato peels led to a less glucose-dominant sugar composition. In these cases, maltose and other oligosaccharides were more pronounced, suggesting that potato peels contributed to a more moderated enzymatic hydrolysis. This effect is likely attributed to pre-gelatinized starches and the presence of limit dextrinase in potato peels, which influenced the progressive breakdown of dextrins into fermentable sugars [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The observed shift in sugar composition has notable implications for fermentation dynamics, as maltose-rich worst tend to promote smoother yeast attenuation and more complex aromatic profiles, particularly in lager and ale fermentations [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Generally, the use of non-barley cereals in brewing results in beers with sensory profiles that differ from traditional counterparts, influencing consumer perception and acceptance. This innovative methodology with the use of potato peels can enhance the appeal of gluten-free beers. These findings highlight the potential of targeted enzymatic treatments and agro-industrial byproducts in optimizing sugar composition, ultimately influencing fermentation performance, ethanol yield, and sensory attributes in gluten-free beer production [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrated the potential of rice malt and potato peels as alternative brewing ingredients for producing gluten-free beer, providing valuable insights into the impact of \u003cb\u003eenzyme selection and alternative starch sources\u003c/b\u003e on gluten-free brewing efficiency, sugar composition, and fermentation dynamics. The use of novel enzyme is a promising approach for producing gluten-free beer, even if further studies occur to investigate the optimization of this technique for industrial-scale production.\u003c/p\u003e\u003cp\u003eFurther research should focus on refining enzymatic strategies to enhance the sensory profile and stability of gluten-free beers, while also exploring the influence of these ingredients on yeast performance, attenuation, and flavor development. The integration of \u003cb\u003esustainable brewing practices using agro-industrial byproducts\u003c/b\u003e represents a promising avenue for innovation in the craft beer industry, aligning with consumer demand for \u003cb\u003ehealth-conscious and environmentally friendly alternatives\u003c/b\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was carried on in the doctoral programme of Agricultural, Food and Forestry Science \u0026ndash; XXXVI Cycle, \u003cem\u003eMediterranea\u003c/em\u003e University of Reggio Calabria Italy. This work was funded by the Next Generation EU\u0026mdash;Italian NRRP, Mission 4, Component2, Investment 1.5, call for the creation and strengthening of \u0026lsquo;Innovation Ecosystems\u0026rsquo;, building \u0026lsquo;Territorial R\u0026amp;D Leaders\u0026rsquo; (Directorial Decree n. 2021/3277)\u0026mdash;project Tech4You\u0026mdash;Technologies for climate change adaptation and quality of life improvement, n. ECS0000009. This work reflects only the authors\u0026rsquo; views and opinions, neither the Ministry for University and Research nor the European Commission can be considered responsible for them.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used in the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethics requirements\u003c/strong\u003e The research did not involve Human Participants and/or Animals, no informed consent was necessary. Nevertheless, the research was conducted in accordance with institutional ethical standards and the principles of good scientific practice.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eHuman Ethics and Consent to Participate declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. The research did not involve human participants or animals, and no informed consent was required.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHabschied K, Živković A, Krstanović V, Mastanjević K (2020) Functional Beer\u0026mdash;A Review on Possibilities. Beverages 6:51. https://doi.org/10.3390/beverages6030051\u003c/li\u003e\n\u003cli\u003eYang D, Gao X (2022) Progress of the use of alternatives to malt in the production of gluten-free beer. Critical Reviews in Food Science and Nutrition 62:2820\u0026ndash;2835. https://doi.org/10.1080/10408398.2020.1859458\u003c/li\u003e\n\u003cli\u003eCela N, Condelli N, Caruso MC, et al (2020) Gluten-Free Brewing: Issues and Perspectives. Fermentation 6:53. https://doi.org/10.3390/fermentation6020053\u003c/li\u003e\n\u003cli\u003eSingh Deora N, Deswal A, Dwivedi M (2022) Challenges and Potential Solutions in Gluten-Free Product Development. 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In: Proceedings of Proceedings of the 11th International Conference on Environmental Science and Technolo gy, Chaina, Crete, Greece. pp 3\u0026ndash;5\u003c/li\u003e\n\u003cli\u003eWu Z-G, Xu H-Y, Ma Q, et al (2012) Isolation, identification and quantification of unsaturated fatty acids, amides, phenolic compounds and glycoalkaloids from potato peel. Food Chemistry 135:2425\u0026ndash;2429. https://doi.org/10.1016/j.foodchem.2012.07.019\u003c/li\u003e\n\u003cli\u003eCalcio Gaudino E, Colletti A, Grillo G, et al (2020) Emerging Processing Technologies for the Recovery of Valuable Bioactive Compounds from Potato Peels. Foods 9:1598. https://doi.org/10.3390/foods9111598\u003c/li\u003e\n\u003cli\u003eOwusu-Mensah E, Oduro I, Sarfo KJ (2011) STEEPING: A WAY OF IMPROVING THE MALTING OF RICE GRAIN: IMPROVING THE MALTING OF RICE GRAIN. Journal of Food Biochemistry 35:80\u0026ndash;91. https://doi.org/10.1111/j.1745-4514.2010.00367.x\u003c/li\u003e\n\u003cli\u003eCeppi ELM, Brenna OV (2010) Experimental Studies To Obtain Rice Malt. J Agric Food Chem 58:7701\u0026ndash;7707. https://doi.org/10.1021/jf904534q\u003c/li\u003e\n\u003cli\u003eUsansa U, Sompong N, Wanapu C, et al (2009) The influences of steeping duration and temperature on the \u0026alpha;‐and \u0026beta;‐amylase activities of six Thai rice malt cultivars (Oryza sativa L. Indica). Journal of the Institute of Brewing 115:140\u0026ndash;147\u003c/li\u003e\n\u003cli\u003eEvans DE, Stewart S, Stewart D, et al Profiling Malt Enzymes Related to Impact on Malt Fermentability, Lautering and Beer Filtration Perfo\u003c/li\u003e\n\u003cli\u003eDe Rouck G, Jaskula B, De Causmaecker B, et al (2013) The Influence of Very Thick and Fast Mashing Conditions on Wort Composition. 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Beverages 10:16. https://doi.org/10.3390/beverages10010016\u003c/li\u003e\n\u003cli\u003eOkolo BN, Amadi OC, Moneke AN, et al (2020) Influence of malted barley and exogenous enzymes on the glucose/maltose balance of worts with sorghum or barley as an adjunct: Malted barley and exogenous enzymes and the glucose/maltose balance of worts with sorghum or barley. J Inst Brew 126:46\u0026ndash;52. https://doi.org/10.1002/jib.598\u003c/li\u003e\n\u003cli\u003eStewart GG (2017) Brewing and Distilling Yeasts. Springer International Publishing, Cham\u003c/li\u003e\n\u003cli\u003eHu S, Dong J, Fan W, et al (2014) The influence of proteolytic and cytolytic enzymes on starch degradation during mashing: The influence of proteolysis and cytolysis on amylolysis during mashing. J Inst Brew n/a-n/a. https://doi.org/10.1002/jib.172\u003c/li\u003e\n\u003cli\u003eGibson BR, Storg\u0026aring;rds E, Krogerus K, Vidgren V (2013) Comparative physiology and fermentation performance of Saaz and Frohberg lager yeast strains and the parental species \u003cem\u003eSaccharomyces eubayanus\u003c/em\u003e. Yeast 30:255\u0026ndash;266. https://doi.org/10.1002/yea.2960\u003c/li\u003e\n\u003cli\u003eCadenas R, Caballero I, Nimubona D, Blanco CA (2021) Brewing with Starchy Adjuncts: Its Influence on the Sensory and Nutritional Properties of Beer. Foods 10:1726. https://doi.org/10.3390/foods10081726\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-food-research-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [European Food Research and Technology](https://link.springer.com/journal/217)","snPcode":"217","submissionUrl":"https://submission.springernature.com/new-submission/217/3","title":"European Food Research and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"gluten-free beer, potato peel, amylase enzymes, fermentation efficiency","lastPublishedDoi":"10.21203/rs.3.rs-7036247/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7036247/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study explores the development of a sustainable, gluten-free beer using regionally sourced ingredients, including rice, potato peels, and native forest botanicals (St. John's Wort, Juniper, and Helichrysum). The research aimed to optimize the fermentation of rice-based wort at a lab-scale by incorporating potato peels and various amylolytic enzymes to improve brewing efficiency and sugar profile complexity. Rice malt was produced at the experimental facilities of KU Leuven (Belgium), and fermentation trials were conducted using both malted and unmalted rice. The study employed different mashing techniques, including traditional decoction mashing, an innovative proprietary enzyme, and a combination of commercial enzyme preparations. Results demonstrated that the novel enzyme significantly enhanced starch hydrolysis, reducing mash times by approximately 30 minutes while improving overall fermentation efficiency. Additionally, the integration of potato peels contributed to a more balanced sugar composition, mitigating glucose dominance and increasing the diversity of fermentable sugars, which could influence yeast metabolism and flavor profile. These findings suggest that enzymatic innovations and agro-industrial byproducts, such as potato peels, could play a key role in advancing sustainable brewing practices while maintaining desirable fermentation characteristics in gluten-free beer production.\u003c/p\u003e","manuscriptTitle":"Formulation of sustainable gluten-free beer from rice malt and potato processing residue with a view to a circular economy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 18:04:50","doi":"10.21203/rs.3.rs-7036247/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-07T10:52:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-03T20:52:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-01T18:56:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-01T17:06:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"260682318924589813899401186146030361210","date":"2025-07-14T11:29:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8934826182529439200158364521942310038","date":"2025-07-14T09:35:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"155801491967346398926360627170473982788","date":"2025-07-13T15:23:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125405019900965152341170580500207961895","date":"2025-07-11T17:52:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-10T18:55:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-07T09:42:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-07T09:40:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Food Research and Technology","date":"2025-07-03T09:11:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-food-research-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [European Food Research and Technology](https://link.springer.com/journal/217)","snPcode":"217","submissionUrl":"https://submission.springernature.com/new-submission/217/3","title":"European Food Research and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a8572d4b-9cd3-47a0-8f36-581ffd6dd4de","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-06T11:38:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-14 18:04:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7036247","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7036247","identity":"rs-7036247","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}