Effects of Non-Alcoholic Beer after Running in Three Consecutive Days on Antioxidant Enzyme Activity and Muscle Damage Biomarkers

g recovery is challenging for several body systems and can be improved by nu- tritional focus. Non-alcoholic beer is a widely used post-exercise beverage for its antioxidant and energetic properties. After three consecutive days of 1 h submaximal running (80% HRmax), antioxi- dant enzyme activity (glutathione peroxidase [GPx], glutathione reductase [GR], catalase), lactate dehydrogenase (LDH) activity as a muscle damage blood marker, and lower limb thermographic values were determined in order to observe possible changes in 20 subjects divided into two groups: control (n = 10) and NAB (n = 10). NAB drank 10 mL/kg of non-alcoholic beer post-exercise (both groups drank water ad libitum). Non-alcoholic beer did not show statistically signi cant changes compared to water. Regarding the effect size, the NAB group had a medium increase in thermog- raphy values (15 0 Post-15 0 Pre) on days 1 and 2 compared to the control group; a large increase in LDH activity (both 60 0 Post-0 0 Post and 60 0 Post-Pre) on day 2, and a medium increase (60 0 Post-0 0 Post) on day 3; a medium decrease in

NAB group had a medium increase in thermog- raphy values (15 0 Post-15 0 Pre) on days 1 and 2 compared to the control group; a large increase in LDH activity (both 60 0 Post-0 0 Post and 60 0 Post-Pre) on day 2, and a medium increase (60 0 Post-0 0 Post) on day 3; a medium decrease in GR (60 0 Post-Pre) on days 1 and 3; and a large (60 0 Post-0 0 Post) and medium (60 0 Post-Pre) decrease in GPx on day 3. These ndings support the idea that non-alcoholic beer is not an appropriate recovery beverage after 1 h running for three consecutive days. Keywords: antioxidant capacity; lactate dehydrogenase; spectrophotometry; thermography; aerobic exercise; recovery beverage 1. Introduction Running is one of the most practiced exercises by healthy people in the world [1,2]. Nevertheless, a high incidence of injuries amongst the sports population of runners has been observed [3–5]. In addition to a correct technique and an appropriate workload, dietary supplementation is a factor that can be used as intervention in order to prevent in- juries. For example, the rectus femoris damage level due to exercise was decreased by olive oil supplementation [6], and several indices of aerobic performance were improved thanks to quercetin supplementation [7]. Findings like these led us to think about using other sup- plements, with a more frequent presence in the diet, that may prevent musculotendinous injuries and reduce their incidence rate. Within the common components of a diet, one of the most consumed by people, including athletes, is beer [8,9]. For the last few years, studies with contradictory results about its effects, both harmful and bene cial, on exercise performance, thermoregulation, and hydration have been published [10–12]. Its content, based on sodium, potassium, maltodextrins, vitamins (e.g., B, C, E), minerals, and antioxidant molecules, which allow recovery from hydroelectrolytic leak due to exercise and the replacement of used metabolic substrates, could justify its effectiveness as a rehydration beverage [9,11]. The moderate intake of beer as a post-exercise rehydrating supplement, alternative or complementary Appl. Sci.2023,13, 9795.

on sodium, potassium, maltodextrins, vitamins (e.g., B, C, E), minerals, and antioxidant molecules, which allow recovery from hydroelectrolytic leak due to exercise and the replacement of used metabolic substrates, could justify its effectiveness as a rehydration beverage [9,11]. The moderate intake of beer as a post-exercise rehydrating supplement, alternative or complementary Appl. Sci.2023,13, 9795.

Appl. Sci.2023,13, 9795 2 of 11 to water, has been found in several works [13,14]. In addition, the exercise-produced in ammation after prolonged efforts at high intensity, as well as the respiratory disorder symptoms incidence rate, are both reduced by beer intake, as has been observed [15]. On the other hand, due to the alcohol content, some detrimental effects on performance have been reported in the last decades. Ethanol ingestion increases ATP degradation in the liver and could increase lactic acid blood concentration [16]. Furthermore, impairments in myo brillar protein synthesis, neuromotor function, and fat distribution due to high doses of alcohol have been published, implying suboptimal training adaptations [17–19]. Regarding rehydration characteristics, alcohol negatively affects uid balance restoration due to diuretic overstimulation [20,21], although the alcohol percentage in beer seems to in uence its rehydrating potential [8,14]. Therefore, the selected drinkable supplement to carry out this study was non-alcoholic beer, which has a similar nutrient composition without the negative effects of alcohol, making it a potentially interesting rehydration drink [22,23]. Non-alcoholic beer has been used as a sports beverage for efforts recovery, even increasing the maximum oxygen consumption (VO2max) during the post-exercise recovery period, which may have an in uence on physical performance [24]. In addition, non-alcoholic beer intake has been shown to prevent exercise damage, probably due to the antioxidant molecules in beer, such as polyphenols (e.g., proanthocyanidins, avonoids, and quercetin), which could minimize the oxidative stress induced by acute exercise [25–27]. In fact, some antioxidant and anti-in ammatory avonoid effects have been proven, considering that quercetin is a avonoid with high antioxidant capacity [28,29]. In relation to exercise, this beverage could act on increasing the amount of free radicals from reactive oxygen species (ROS) overproduction, leading to delayed onset muscle soreness (DOMS) prevention, a faster organism predisposition to tolerate subsequent efforts and recovery improvement [30]. When exercising, some practitioners may suffer experiences of discomfort, which could be caused by musculoskeletal ber damage related to the eccentric phase of the activity, since the negative work during muscle lengthening structurally affects the contractile protein elements located deep inside [31].

delayed onset muscle soreness (DOMS) prevention, a faster organism predisposition to tolerate subsequent efforts and recovery improvement [30]. When exercising, some practitioners may suffer experiences of discomfort, which could be caused by musculoskeletal ber damage related to the eccentric phase of the activity, since the negative work during muscle lengthening structurally affects the contractile protein elements located deep inside [31]. For example, acute downhill hiking may induce DOMS, which in turn is associated with muscle damage and in ammation, affecting in this way the muscle bers' structure and the excitation–contraction coupling process [32,33]. In this sense, several biochemical markers have been used to assess the muscle tissue damage level considering the increase in their blood concentrations, such as the enzyme lactate dehydrogenase (LDH), which participates in the pyruvate–lactate conversion [34]. The skeletal muscle is a major source of free radicals and ROS during exercise, due to both contractile activity and mitochondrial oxidation, and their effort-related overpro- duction within muscle bers could also contribute to a perception of DOMS and a feeling of discomfort [35]. The exercise-mediated in ammatory process in muscle may be one of the responsible factors contributing to oxidative stress due to free radical overproduction, especially when exercise is acute, since macrophages and other phagocytic cells invade the area involved [36]. Thus, it has been speculated that in ammation is a responsible process that leads to DOMS [37]. Infrared thermography is a non-invasive and low-cost assessment technique used in the detection of clinical changes, which can be applied for muscle injury prevention, although this function is not extensively known [38]. A good inter-examiner reliability to detect temperature changes in some body areas and in several sports has been described [39]. Body temperature behavior is considered pathological when differences > 6 C appear among contralateral areas. This technique also allows the examiner to focus measurements on a concrete body area called the region of interest (ROI) [40]. Additionally, the adaptation degree to exercise intensity may be determined by this method since temperature decreases in anaerobic exercise but increases in aerobic exercise [41]. Thermography's applicability, just like its limitations, has

> 6 C appear among contralateral areas. This technique also allows the examiner to focus measurements on a concrete body area called the region of interest (ROI) [40]. Additionally, the adaptation degree to exercise intensity may be determined by this method since temperature decreases in anaerobic exercise but increases in aerobic exercise [41]. Thermography's applicability, just like its limitations, has been well reported [42]. In fact, its function has been shown to detect possible injuries in an ef cient way by an infrared thermographic camera, which may be used before exercise to reduce the potential risk of muscle damage [43].

Appl. Sci.2023,13, 9795 3 of 11 Therefore, our aim was to assess the effectiveness of non-alcoholic beer as a rehydrat- ing and recovery supplement post-exercise, based on 1 h submaximal intensity running on several days, by observing possible alterations in the plasma markers of antioxidant capacity (the antioxidant enzyme activity of glutathione peroxidase, glutathione reductase, and catalase) and muscle damage (LDH enzyme activity), as well as changes in the lower limbs' thermographic values as a possible injury risk-predictor. 2. Materials and Methods 2.1. Subjects Twenty healthy adult male volunteers, Sports Sciences Degree students in Univer- sidad San Jorge (Villanueva de G¡llego, Zaragoza, Spain), participated in a controlled trial over three months to assess the effects of non-alcoholic beer as a supplement on re- hydration and post-exercise recovery, registering blood markers of antioxidant capacity and muscle damage after exercise, such as quadriceps and gastrocnemius thermography. Each subject reported a regular sport activity for 3 days/week at least, although none of them were runners. They also reported beer (alcoholic and/or non-alcoholic) consumption less than 500 mL/week. They were randomly distributed, by drawing, into two groups, n = 10/group: control (CON) and non-alcoholic beer supplementation (NAB). The NAB group was instructed to drink 10 mL/kg alcohol-free beer (Ambar, Zaragoza, Spain) immediately after exercise. CON and NAB subjects could drink mineral water (Fontecabras, Zaragoza, Spain) ad libitum. Non-alcoholic beer ingredients were water, barley malt, rice, dietary ber, hops, and scents, having a nutritional content as follows (per 100 mL): 18 kcal energy value, 0 g fats, 4 g carbohydrates (0 g sucrose), 0 g proteins, 1 g dietary ber, and 0 g salt. In addition, water composition was 117 mg/L sulfates, 295 mg/L bicarbonates, 49.5 mg/L chlorides, 90.1 mg/L calcium, 37.5 mg/L magnesium, and 32.6 mg/L sodium. Every subject was informed orally and in writing by a participant information sheet and signed an informed consent by hand prior to his participation in this study. All personal data were recorded in a database speci cally designed for the study and pseudo- anonymized. Age, weight, height, body mass index (BMI), and 1 h run distance were

32.6 mg/L sodium. Every subject was informed orally and in writing by a participant information sheet and signed an informed consent by hand prior to his participation in this study. All personal data were recorded in a database speci cally designed for the study and pseudo- anonymized. Age, weight, height, body mass index (BMI), and 1 h run distance were recorded with regard to descriptive statistics. This study was conducted according to the Declaration of Helsinki principles (2008) and following the note for guidance on Good Clinical Practice in the European Union, CPMP/ICH/135/95. The intervention began after approval by the appropriate regional ethical committee: Comit²de’tica de la Investigaciân de la Comunidad Autânoma de Aragân (CEICA), CP08/2014 (CP-CI-IP13/0175), according to valid legal regulations in Spain (RD 1090/2015). 2.2. Experimental Protocol All participants started with a standardized warm-up and then carried out a 1 h running session for three consecutive days, at 80% maximum heart rate (maxHR) as submaximal intensity, which was calculated by the Karvonen method [44]. Heart rate (HR) was supervised by a Polar Heart Rate Monitor RS300X (Polar Electro Ib²rica, Barcelona, Spain). Exercise protocol intensity was controlled by one researcher. As a running track, a grass soccer eld perimeter was used. After 30 min running in one direction, participants were instructed to carry on in the opposite direction up to 1 h, registering reached distance. Blood samples, 10 mL antecubital vein-removed blood samples, were obtained in three stages: just before exercise (Pre); immediately after exercise although before rehydration (0 0 Post); 1 h after exercise (60 0 Post). Thermography images were taken as described below. 2.3. Thermography Infrared thermography camera shots were used to observe muscle changes. Thermog- raphy was assessed by a FLIR Thermacam E60 infrared camera (FLIR Systems, Wilsonville, OR, USA). On each running day, images were taken 15 min before (15 0 Pre) and 15 min after

Appl. Sci.2023,13, 9795 4 of 11 exercise (15 0 Post), according to the American Academy of Thermography guidelines [45]. Frontal plane images were taken from anterior and posterior views with both lower limbs visible on screen, and 2.5 m camera–participant distance. Quadriceps and gastrocnemius ROI temperature means were selected and analyzed with FLIRTools-Software (device software pack). Thermographic evaluation was carried out in a room at 21 C steady tem- perature with 60% humidity, without metallic equipment to avoid thermographic image distortions. All subjects stood in their underwear on a step to avoid direct contact with the oor. A skin emissivity of 0.98 was assumed [46]. 2.4. Plasma Samples Procedure The antioxidant activity of catalase (Cat), glutathione peroxidase (GPx), and glu- tathione reductase (GR) enzymes were used to determine plasma antioxidant capacity [47]. Muscle damage was evaluated by plasma LDH enzyme activity [46]. Just after drawing them, blood samples were centrifuged at 1000 gfor 10 min in a Biofuge Primo R refriger- ated centrifuge (Thermo Scienti c, Waltham, MA, USA). The 250 L plasma aliquots were stored at 20 C until LDH and antioxidant enzyme measurements were taken. 2.5. Antioxidant Capacity Cat, GPx, and GR enzyme activity was determined with a spectrophotometric com- mercial kit (Cayman Chemical, Ann Arbor, MI, USA) to evaluate plasma antioxidant capacity [36]. Enzyme activity was expressed as enzyme units (U), which represent the enzyme amount required to catalyze the conversion of 1 substrate- mol/min. 2.6. Skeletal Muscle Damage Blood LDH activity was determined to assess the mechanical damage level on the skeletal muscle bers. LDH activity was measured by spectrophotometry with a DGKC Kinetic UV kit (Spinreact, Barcelona, Spain) [48]. This method is based on LDH catalysis on pyruvate reduction by nicotinamide adenine dinucleotide + hydrogen (NADH). In a sample which is measured photometrically, the speed of the NADH concentration decrease is proportional to the LDH catalytic concentration. 2.7. Statistical Analysis Statistical processing was performed using SPSS version 28.0 for Windows (SPSS Inc., Chicago, IL, USA). Descriptive statistics corresponding to participants (age, weight, height, BMI, and 1 h run distance) were presented as mean standard deviation

(NADH). In a sample which is measured photometrically, the speed of the NADH concentration decrease is proportional to the LDH catalytic concentration. 2.7. Statistical Analysis Statistical processing was performed using SPSS version 28.0 for Windows (SPSS Inc., Chicago, IL, USA). Descriptive statistics corresponding to participants (age, weight, height, BMI, and 1 h run distance) were presented as mean standard deviation (SD). The datasets' normality was checked with the Shapiro–Wilk test. Values differences obtained in the “60 0 Post-Pre” and “60 0 Post-0 0 Post” time intervals were calculated to study possible changes in Cat, GPx, GR, and LDH for 1 h after the non-alcoholic beer supplementation moment. The thermographic variable differences in quadriceps (ThQ) and gastrocnemius (ThG) were calculated in “15 0 Post-15 0 Pre” time intervals. Regarding these data, means comparison tests for independent samples were performed (Student'sttest for normal distributed variables and Mann–Whitney U test for non-normal distributed variables) for the 3 running days of the NAB and CON groups. These tests were conducted with a signi cance levelp< 0.05. To assess possible changes in the variables of this study over time depending on the group, a two-way repeated measures ANOVA test was performed (time: day 1, day 2, and day 3; and group: NAB vs. CON). Those changes that reached a signi cance valuep< 0.05 and a statistical power 1-SS > 0.80 were considered as signi cant. The 2value was also reported to assess the effect size in this test. In these comparisons, Cohen's d effect size with 95% con dence interval (ES, 95% CI) was calculated to study possible differences, clinically relevant but statistically not signi cant, establishing d = 0.20 as the minimum relevant clinical effect. The CI results without 0 (zero) value included were considered for discussion; i.e., <5% probability for

Appl. Sci.2023,13, 9795 5 of 11 null/trivial ES. The magnitude of ES was considered small (0.2 |d| < 0.5), medium (0.5 |d| < 0.8), or large (0.8 |d|) [49]. Regarding clinical effect, negative results indicated a harmful effect (bene cial effect when positive) in Cat, GPx, and GR; whereas negative results indicated a bene cial effect (harmful effect when positive) in LDH, ThQ, and ThG. For these calculations, Hopkins spreadsheets for independent samples comparisons were used [50]. 3. Results A total of 20 subjects participated in this study. Their main characteristics (anthropo- metric parameters and 1 h run distance) are shown in Table. No intergroup signi cant differences were observed (p> 0.05), neither in the anthropometry values nor in the run distance for 1 h. Furthermore, there were no intragroup performance differences among the three running days, independent of supplementation post-exercise (=NAB) or without supple- mentation (=CON). Table 1. Anthropometric characteristics and run distance. CON, n = 10; NAB, n = 10. Data are expressed as mean SD. Variable CON NAB Age (years old) 23.40 4.79 21.30 3.27 Body mass (kg) 75.75 10.09 68.47 6.13 Height (cm) 179.50 7.68 178.20 7.60 BMI (kg/m 2 ) 23.57 3.38 21.55 1.21 Day 1 10,035.61 2099.24 8620.70 1332.68 Run distance (m) Day 2 10,218.70 1998.32 8968.80 1563.72 Day 3 10,383.70 1855.97 9382.26 1535.27 CON = control group; NAB = non-alcoholic beer group; SD = standard deviation; BMI = body mass index. None of the variables has a significance valuep< 0.05, a statistical power 1-SS > 0.45, or an effect size 2> 0.25 for the repeated measures ANOVA test. Therefore, it could be concluded that there are no changes in the variables in relation to the three moments of evaluation or to the interaction between the moment of evaluation and the group of belonging. The effects analysis of non-alcoholic beer as a rehydrating beverage immediately post-exercise (NAB group), in comparison to water normal use (CON group), after 1 h submaximal intensity running for three days, is re ected in the following tables: rst day results in Table; second day results in Table; third day

between the moment of evaluation and the group of belonging. The effects analysis of non-alcoholic beer as a rehydrating beverage immediately post-exercise (NAB group), in comparison to water normal use (CON group), after 1 h submaximal intensity running for three days, is re ected in the following tables: rst day results in Table; second day results in Table; third day results in Table. The study variables on each variable are as follows: plasma antioxidant enzyme activity (GPx, GR, Cat) (U), plasma LDH enzyme activity (U), lower limb thermographic register (quadriceps, gastrocnemius) ( C). As can be seen, no intragroup signi cant difference (60 0 Post-Pre interval vs. 60 0 Post- 0 0 Post interval) was produced, neither in CON nor in NAB. There was also no intergroup signi cant difference (CON vs. NAB), neither in the 60 0 Post-Pre interval nor in the 60 0 Post- 0 0 Post interval. This was observed in the three running days. Therefore, based on these results, to drink non-alcoholic beer did not imply statistically signi cant changes regarding the study variables. Considering this, clinical relevance by ES was then investigated, comparing the NAB group in relation to the CON group and establishing a 95% IC, with 0.20 being the minimum relevant value. This threshold value was set to negative for antioxidant enzymes (so that if there is a positive ES, the result = bene cial), and was set to positive for the muscle damage (LDH) and muscle injury risk (thermography) indicators (so that if there is a positive ES, the result = harmful).

This study assesses non-alcoholic beer's effectiveness as a recovery supplement post-exercise.