The Effect of Replacing 10–30% of the Barley Malt Grist with Wheat Bran and the Addition of Flaxseed or Sunflower Seeds on the Health-Promoting Properties of Non-Alcoholic Beer: A Comparison with Commercial Non-Alcoholic Beer

1. Introduction

Beer is one of the oldest beverages in the world and third most popular, after water and tea. Its refreshing effect is attributed to the presence of carbon dioxide, certain fermentation byproducts, and the aromatic and flavorful components of hops. It replenishes lost minerals, has a high energy value, contains antioxidants, polyphenols, vitamins and essential micronutrients, and the extract’s components are easily digestible, as they are partially in the form of colloids [1,2,3]. In the following study, the authors try to prove that non-alcoholic beer can act as a functional food, being a source of antioxidants, polyphenols, vitamins and minerals.

Antioxidants are a group of bioactive molecules that naturalize the potential effect of free radicals by donating an electron to the free radical [4]. They are very important group of chemicals as reactive oxygen species (ROS) may interact with cellular macromolecules and cause damage of DNA, and other cellular structures [5,6]. Antioxidants include such substances as polyphenols, vitamins, carotenoids or peptides [7,8]. It is reported that Vitamin C shows preventive and therapeutic benefits in various forms of cardiovascular diseases [4]. Due to the fact that while human evolution we lost the ability of producing an enzyme needed to make an ascorbic acid, we need to obtain it from a diet as a vitamin. Vitamin E is a set of eight related tocopherols and tocotrienols which are fat soluble [9].

Polyphenols are of group of approximately 10,000 substances of plant origin [10]. Their activity includes modulating oxidative and inflammatory stress, altering macronutrient digestion and to exerting prebiotic-like effects on gut microbiota. In plants they are involved in the attraction of pollinators, the execution of structural functions, protection of plants against UV exposition and microorganisms causing diseases [11,12]. They are present in most fruits and vegetables, but also in whole grains, chocolates, tea and wine [10,13]. The most commonly occurring dietary polyphenols are flavonoids and phenolic acids [10].

Vitamin B₃, also called niacin or vitamin PP, is involved in many metabolic functions. It is also important for converting food to energy. The function of niacin is as the precursor of the nicotinamide nucleotide coenzymes NAD and NADP, which are involved in oxidation/reduction reactions and associated with both catabolic and anabolic processes [14]. The main sources of niacin are: meat, cereals, legumes, seeds, milk, green leafy vegetables, fish, as well as coffee and tea [15]. Mild niacin deficiency can slow metabolism, and cause headaches and other minor symptoms, but severe deficiency causes the disease pellagra, characterized by diarrhea, dermatitis and other skin disorders, dementia, inflammation of the mouth and tongue, and other symptoms which can be fatal if left untreated [16]. Serious deficiency of niacin may cause cognitive impairment, anemia, cardiovascular disease, neural tube defects, neuropsychiatric disorders, and thromboembolic processes [17].

Vitamin B₆ (pyridoxine) is involved in over 150 biochemical reactions. It is active in the metabolism of carbohydrates, lipids, amino acids, and nucleic acids, and participates in cellular signaling [18]. Even mild deficiency has effects on human metabolism [19]. Its sources are: meat, fish, eggs, soybeans, corn and also many vegetables and fruits [20,21]. Recommended daily intake of vitamin B₆ is 1.3 mg for adults [20]. Deficiency of vitamin B₆ is very rare and it is usually the deficiency of other B-complex vitamins, however it can manifest as naso-lateral seborrhea, glossitis, peripheral neuropathy, and normocytic, microcytic or sideroblastic anemia [19,21].

Magnesium (Mg) is the fourth most abundant cation in human body involved in over 300 enzymatic reactions, including energy production, muscle contraction, nerve transmission, and also synthesis of protein, DNA, and RNA [22,23,24]. Deficiency of Mg causes heart rhythm disturbances or nervous tics (e.g., eyelid twitching), as well as calf cramps, numbness and tingling in the lower limbs, hair loss, and brittle nails [25]. Long-term Mg deficiency may cause numerous complications such as: metabolic syndrome, osteoporosis, asthma or migraines [23]. Recommended daily intake of Magnesium is 300-400 mg and increases while physical exertion. The sources of Magnesium are: pumpkin seeds, wheat bran, cocoa, almonds, buckwheat groats or soy [25].

Potassium (K) is an essential mineral that is necessary for normal cell and membrane function and for maintaining both fluid balance and acid-base balance. Potassium deficiency is rare. It is proved that its intake above 3,500 mg/day helps in reducing risk of stroke and is beneficial for persons with high blood pressure [26]. Recommended daily intake amounts are 2,600 mg for women and 3,400 mg for men [27]. Food rich in potassium includes cooked salmon and chicken, boiled potatoes, bananas and milk [28].

Sodium (Na) plays an important role in human body. It is associated with metabolic and enzymatic processes as a cellular activator and is responsible for cell membrane function. The sodium deficiency is very rare and is associated with taking medicines with diuretic effect. Also diarrhea, vomiting or heavy sweating may cause low sodium levels [29]. The natural sources of sodium are salt, milk, meat, and shellfish. However it is found in processed foods, such as breads, crackers, processed meats and snack foods. The daily intake of Na rages from 200 to 500 mg. It is reported that the average Na consumption is above the recommended one [30].

Calcium (Ca) is one of the most abundant minerals in the human body and it is very important in total body health. It is stored in teeth and bones, but also ensures proper functioning of muscles and nerves and helps in blood clot. The daily recommended intake is 700 mg for adults [31]. Calcium is a biochemical regulator of enzymes as its concentration changes in cells enable them to change their behavior in response to a physiological stimulus such as hormones or neurotransmitters. As a main source of calcium it is considered milk and its preserves, such as cheese. By the 18th century the flour was enriched with chalk and bone powder. The rich plat sources of calcium are soy, sesame seeds, dried figs, almonds, boiled spinach, curly kale, and hazelnuts [32].

Iron (Fe) is a very important mineral that prevents anemia due to production of red blood cells. It is also an activator for enzymes and hormones [33]. The daily intake varies depending on sex. It is 18 mg/day for women and 8 mg/day for men. Women needs more iron in their diet due to menstruation and blood loss [34]. The sources of iron are: animal meat, chicken liver, seafood, eggs, beans and green vegetables [33]. People who do not eat meet need to eat twice as much iron due to the fact that human body does not absorb iron from plants as well as from animal foods [34]. Iron deficiency causes anemia and may lead to cardiovascular diseases, cancer, and impaired development of the brain and the central nervous system [35].

Zinc (Zn) is a component of over 200 enzymes and hormones. It is essential for growth. Zinc also plays an important role in health of bones, teeth, skin, hair, nails but also nerves, and brain function [36]. Recommended daily intake for adults is 8 mg for women and 11 mg for man [37]. The Zinc deficiency results in growth retardation, hypogonadism, rough skin, poor appetite and frequent infections [38]. Food rich in zinc includes: oysters, meat, fish, seafood, eggs, beans, nuts and breakfast cereals [39].

The aim of the study was investigation if adding 20% or 30% wheat bran enriched with linseed with a varied bran and linseed or sunflower seed composition to the malt grist will increase its antioxidant activity, vitamin, micronutrient, and macronutrient content compared to existing products.

Wheat bran is rich in minerals such as iron, zinc, and magnesium. It is also a source of B vitamins, such as B₃ and B₆ [40]. Linseed is a source of calcium and magnesium. It also contains small amounts of zinc, iron, and vitamins B₃ and B₆ [41]. Sunflower seeds are also an excellent source of vitamins and minerals such as vitamin B₆, iron, magnesium, and zinc [42].

Alternatively, adding dried oak chips at the end of the third or fourth stage of mashing, in an amount of 2-4% of the grist, would improve mash filtration parameters without the need for artificial additives and increase the content of polyphenols in the wort, which have health-promoting properties. The aim was also a verification if replacing barley malt with 20% or 30% wheat bran enriched with linseed or sunflower seeds:

• will not affect foam stability; beer made with 20% wheat bran and sunflower or linseed seeds will have the desired foam stability of over 300 seconds, as determined by the NIBEM method.
• will not impair yeast propagation or wort fermentation compared to barley malt.
• will improve the concentration of magnesium, potassium, calcium, and B vitamins in the wort.

All results were compared to the properties of non-alcoholic Żywiec beer which is an industrial beer from Poland.

2. Materials and Methods

Preparation of malt grist – wheat bran (20% or 30%), either alone or enriched with linseed or sunflower seeds, was incorporated into the malt grist. The grist compositions and the corresponding variants involving the addition of oak chips during mashing are presented below.

Tested samples (1-8) and variants in samples (1-4) of additives to malt meal, wheat bran enriched with linseed and oak chips added at the 3rd and 4th stage of mashing:

1. 20% wheat bran addition with linseed, with a structure of 3 bran units, one linseed unit

1.1. First stage of the technological process to which oak chips are applied at 2%1.2. First stage of the technological process to which oak chips are applied at 4%1.3. Second stage of the technological process to which oak chips are applied at 2%1.4. Second stage of the technological process to which oak chips are applied at 4%

2. 20% wheat bran addition with linseed, with a structure of 2 bran units, one linseed unit

2.1. First stage of the technological process to which oak chips are applied at 2%2.2. First stage of the technological process to which oak chips are applied at 4%2.3. The second stage of the technological process, to which oak chips are added at a rate of 2%2.4. The second stage of the technological process, to which oak chips are added at a rate of 4%

3. A 30% addition to the wheat bran filling with added linseed, with a structure of 3 bran units and one linseed unit

3.1. The first stage of the technological process, to which oak chips are added at a rate of 2%3.2. The first stage of the technological process, to which oak chips are added at a rate of 4%3.3. The second stage of the technological process, to which oak chips are added at a rate of 2%3.4. The second stage of the technological process, to which oak chips are added at a rate of 4%

4. A 30% addition to the wheat bran filling with added linseed, with a structure of 2 bran units and one linseed unit.

4.1. The first stage of the technological process, to which 2% oak chips are applied4.2. The first stage of the technological process, to which 4% oak chips are applied4.3. The second stage of the technological process, to which 2% oak chips are applied4.4. The second stage of the technological process, to which 4% oak chips are applied

Variants of malt bran additives enriched with sunflower seeds and oak chips added at the 3rd and 4th stage of mashing

5. 20% addition to the wheat bran filling with the addition of sunflower seeds, with a structure of 3 bran units, one sunflower seed

5.1. First stage of the technological process to which oak chips are applied at 2%5.2. First stage of the technological process to which oak chips are applied at 4%5.3. Second stage of the technological process to which oak chips are applied at 2%5.4. Second stage of the technological process to which oak chips are applied at 4%

6. 20% addition to the wheat bran filling with the addition of sunflower seeds, with a structure of 2 bran units, one sunflower seed

6.1. First stage of the technological process to which oak chips are applied at 2%6.2. First stage of the technological process to which oak chips are applied at 4%6.3. Second stage of the technological process to which oak chips are applied at 2%6.4. Second stage of the technological process to which oak chips are applied at 4%

7. 30% addition to the wheat bran filling with the addition of sunflower seeds with a structure of 3 units of bran and one sunflower seed

7.1. First stage of the technological process to which oak chips are applied at 2%7.2. First stage of the technological process to which oak chips are applied at 4%7.3. Second stage of the technological process to which oak chips are applied at 2%7.4. Second stage of the technological process to which oak chips are applied at 4%

8. 30% addition to the wheat bran filling with the addition of sunflower seeds with a structure of 2 units of wheat bran and one sunflower seed

8.1. The first stage of the technological process, to which 2% oak chips are applied8.2. The first stage of the technological process, to which 4% oak chips are applied8.3. The second stage of the technological process, to which 2% oak chips are applied8.4. The second stage of the technological process, to which 4% oak chips are applied

2.1. Polyphenols Content

Polyphenol scontent analysis was determined by spectrophotometric method according to Analytica EBC (9.11), at a wavelength of λ= 600 nm, using a spectrophotometer DR6000 (Hach Lange, Wroclaw Poland). The analysis results are expressed in mg/L.

2.2. Antioxidant Activity

Analysis of antioxidant activity was conducted with spectrophotometer DR6000 (Hach Lange, Wroclaw, Poland). The beer was degassed by mixing for 10 minutes to remove CO2, and clarified by spinning for 5 minutes at 10.000 rpm. Solution of 7 mmol of ABTS radical cation and 2.45 mmol of potassium peroxodisulfate (Sigma-Aldrich, Steinheim, Germany) left for 16 hours in the dark to produce ABTS radical cation color radicals. Before an analysis the solution was diluted with redistilled water to obtain the value of absorbance of 0.700 for wavelength (λ) of 734 nm. 0.03 of beer was mixed with 3 mL of ABTS radical cation solution. After 6 minutes the absorbance was measured for λ = 734 nm. The reference sample was redistilled water. The analysis results are expressed in mmol Trolox (TE)/L of beer.

2.3. Vitamins Content

The concentrations of vitamins B₃, B₆, and C in beer samples were determined by reversed-phase high-performance liquid chromatography (RP-HPLC) with spectrophotometric detection. The analyses were performed by an external accredited laboratory using a validated analytical procedure.

Prior to analysis, samples were prepared according to the laboratory protocol and analyzed under conditions enabling separation and quantification of the target vitamins. Identification and quantification were based on retention times and calibration curves obtained using external standards.

All results are expressed in mg/L. Vitamins were determined as individual compounds rather than total vitamin B content.

2.4. Minerals Content

Minerals content analysis was determined by flame atomization absorption spectrometry. The concentrations of Mg²⁺, K⁺, Na⁺ Ca²⁺, Fe²⁺, and Zn²⁺, and in the wort samples were determined by flame atomic absorption spectrometry (FAAS) using a Varian AA240FS spectrometer equipped with a Sample Introduction Pump System (SIPS-20) (Agilent Technologies, Inc., Santa Clara, USA). The acetylene and air flow rates were maintained at 3.5 and 14 L min⁻¹, respectively. Prior to analysis, the samples underwent wet digestion in sealed pressure vessels using a Mars Express microwave digestion system (CEM Corporation, Matthews, USA) (170 °C for 15 min) with 5 mL of 65% HNO₃. Absorbance measurements were performed at wavelengths of 285.2 nm for Mg²⁺, 766.5 nm for K+, 589.0 nm for Na+, 422.7 nm for Ca²⁺, 248.3 nm for Fe2+, and 213.9 nm for Zn²⁺.

2.5. Physicochemical parameters

The density, alcohol content, basic wort extract, apparent extract, and real extract of the beer samples (50 mL each) were determined using an Anton Paar analytical system. Prior to analysis, the samples were degassed to remove dissolved carbon dioxide. Measurements were carried out according to standard brewing analytical procedures.

2.6. pH and Bitterness Analysis

The pH of the beer samples was measured using a calibrated pH meter at room temperature. Beer color was determined spectrophotometrically according to the European Brewery Convention (EBC) method and expressed as EBC units.

Bitterness was determined according to the European Brewery Convention (EBC) method and expressed as International Bitterness Units (IBU).

2.7. Energy Value

The energy value (caloric content) of the beer samples was determined from the measured density and the corresponding alcohol and extract values obtained using the Anton Paar analytical system. Energy content was expressed as kcal per 100 mL of beer.

The energy value of beer was calculated according to the following equation:

Energy (kcal/100 mL) = 7 × A + 3.5 × RE × ρ

where:

A = alcohol content (% w/w),

RE = real extract (% w/w),

ρ = density of beer (g/mL).

2.8. Turbidity

Beer turbidity was measured using the Anton Paar beer analysis system equipped with a haze measurement module. The determination was based on nephelometric measurement of light scattered by suspended particles in the sample. Results were expressed in EBC turbidity units.

3. Results

The developed non-alcoholic beer formulations were characterized in terms of their content of bioactive compounds, vitamins, minerals and selected physicochemical parameters in order to evaluate the influence of wheat bran, linseed, sunflower seeds and oak chips on beer quality. The results were compared with those obtained for a commercially available non-alcoholic beer (Żywiec 0%) and are presented in Table 1, Table 2, Table 3 and Table 4.

3.1. Polyphenols, Antioxidants and Vitamins Content

Bioactive compounds and vitamins constitute important indicators of the nutritional quality and functional potential of beer. Therefore, the contents of total polyphenols, antioxidant capacity, vitamin C, vitamin B₃ and vitamin B₆ were determined for all experimental formulations and compared with those of the commercially available non-alcoholic beer (Żywiec 0%). The results are presented in Table 1.

The highest polyphenol content was obtained in variants 6.1-6.4, and in samples 7 and 8. The average content of this parameter in samples 7.1-7.4 and 8.1-8.4 constitutes, respectively, 78.7 and 76.2% of the average of the variants from samples 6.1-6.4. The lowest polyphenol values were obtained in sample variants 3.1-3.4; 4.1-4.4. and 5.1-5.4. The average polyphenol content for beer samples with wheat bran and linseed added to the grist is 16% lower than for samples with wheat bran and sunflower seeds added to the grist. The value of this parameter determined for Żywiec’s non-alcoholic beer represents only 23.4% of the average polyphenol content in samples 6.1-6.4, reaching 53 mg/dm3, compared to 226.75 for samples in variant 6.1-6.4. The average polyphenol content in the tested samples is 3.2 times higher than the value of this parameter determined for Żywiec’s non-alcoholic beer. The sample with the lowest polyphenol content contains 2.62 times more polyphenols than Żywiec’s beer.

The highest antioxidant contents were obtained in trial 1 (variants 1.1-1.4) and trial 7 (variants 7.1-7.4). The average antioxidant content in trial 1 is 22% higher than in trial 7. The lowest antioxidant values were obtained for trials 5, 3, and 4. The highest antioxidant content was obtained for the trial with a 20% addition of wheat bran and linseed to the grist, with a structure of three bran units per linseed unit, and a 4% addition of oak chips at the fourth stage of mashing. The average antioxidant contents in the trials with wheat bran and linseed added to the grist did not differ from the trials with wheat bran and sunflower seeds added to the grist. The value of this parameter determined for the non-alcoholic beer offered by Żywiec constitutes 36% of the average antioxidant content in sample 1 and is 1.37 mmolTrolox/L, compared to 3.78 mmolTrolox/L for the variants from sample 1 and. The highest antioxidant value determined for the beer with 20% wheat bran and linseed added to the grist, with a structure of 3 bran units, one linseed unit, and 4% oak chips added at the fourth mashing stage, is 2.87 times higher than the content of this parameter in Żywiec’s beer. The lowest antioxidant content in beer 5.1 is 1.55 times higher than that determined for Żywiec’s beer.

The highest vitamin C content in beer was obtained in trials 1 and 2 (variants 1.1-1.4 and 2.1-2.4), in which 20% wheat bran and linseed were added to the grist, with a structure of 3 bran units and one linseed unit (trial 1). In trial 2, a 20% addition of wheat bran and linseed was added to the grist, with a structure of 2 bran units and one linseed unit. The lowest vitamin C content was obtained in trial 7 (variants 7.1-7.4).

As the proportion of linseed relative to wheat bran in the grist increases, the vitamin C content in beer increases. Simultaneously, its content in beer decreases with the proportion of wheat bran in the grist. The same relationship occurs in samples where wheat bran and sunflower seeds are added to the grist. At the same time, the vitamin C content in the beer is higher in samples with linseed added than in those with sunflower seeds. On average, the vitamin C content in samples with wheat bran and linseed added to the grist is 1.23 times higher than in samples with sunflower seeds added. The value of this parameter determined for the non-alcoholic beer offered by Żywiec represents 63% of the average vitamin C content in sample 1, reaching 11.86 mg/dm³ compared to 18.73 mg/dm³ for the variants from sample 1.

The highest vitamin B₃ content in the beer was obtained in test 8 (variants 8.1-8.4) and test 4 (variants 4.1-4.4). These values are 3.5 times higher than the milestone value of the fifth stage of the study. The lowest vitamin B₃ content in the beer was obtained in tests 1 and 2 (variants 1.1-1.4 and 2.1-2.4). The average value of this parameter in these variants is 2.73 times higher than the milestone value of the fifth stage of the study. The highest vitamin B₃ content was obtained for the test with a 30% addition of wheat bran and sunflower seeds to the grist, with a structure of two units of wheat bran, one unit of sunflower seeds, and a 2% addition of oak chips at the third stage of mashing. As the proportion of wheat bran, linseed, and sunflower seeds in the grist increases, the beer’s vitamin B₃ content increases. Adding sunflower seeds to the wheat bran increases the beer’s vitamin B₃ content by approximately 8% compared to linseed.

The value of this parameter determined for the non-alcoholic beer offered by Żywiec is 2% of the average vitamin B₃ content in sample 8, reaching 0.42 mg/l, compared to 21.52 mg/l for the variants in sample 8. The lowest vitamin B₃ content in the beer from samples 1 and 2, 5.1, is 68 times higher than that determined for the Żywiec beer.

The highest vitamin B₆ content in beer was obtained in trial 1 (in variants 1.1-1.4). The highest vitamin B₆ content was obtained for the trial with a 20% addition of wheat bran and linseed to the grist, with a structure of 3 bran units and one linseed unit, and a 2% addition of oak chips at the third mashing stage. The lowest vitamin B₆ values were obtained for trial 7.

With increasing wheat bran and linseed content in the grist, the vitamin B₆ content in beer decreased. The highest vitamin B₆ content was obtained with a 20% addition of wheat bran and linseed to the grist, with a structure of 3 bran units and one linseed unit. The same relationship was not observed with the addition of sunflower seeds. The average vitamin B₆ content in samples with wheat bran and linseed added to the grist is 37% higher than in samples with wheat bran and sunflower seeds added to the grist. The value of this parameter determined for the non-alcoholic beer offered by Żywiec constitutes 44% of the average vitamin B₆ content in sample 1, reaching 0.35 mg/l, compared to 0.80 mg/l for the variants from sample 1. The highest vitamin B₆ value determined for the beer with 20% wheat bran and linseed added to the grist, with a structure of three units of bran, one unit of linseed, and 2% oak chips added at the third mashing stage, is 2.74 times higher than the content of this parameter in Żywiec’s beer.

3.2. Minerals Content

The incorporation of wheat bran and oilseeds into brewing formulations may increase the transfer of nutritionally valuable minerals from the raw materials to the final product. To evaluate this effect, the contents of selected macro- and microelements, namely magnesium, potassium, sodium, calcium, iron and zinc, were determined in all experimental beers and compared with those of the commercially available non-alcoholic beer (Żywiec 0%). The obtained results are summarized in Table 2.

Magnesium contents for sample variants 1-5 are similar (average value: 5.95 mg/100 cm³). Their values range from 5.88-6.04 mg/100 cm³. The average content of this parameter for sample variants 6, 7, and 8 is 6.49 mg/100 cm³. They range from 6.45-6.56 mg/100 cm³. The magnesium content for sample variants 1-5 is 92% of the average values for this parameter determined for sample variants 6, 7, and 8.

The highest magnesium content was obtained for the sample with a 30% addition of wheat bran and sunflower seeds to the grist, with a structure of two bran units and one sunflower seed unit, and with a 2% addition of oak chips at the fourth stage of mashing. The average magnesium content in samples with wheat bran and linseed added to the grist is 5.4% lower than in samples with wheat bran and sunflower seeds added to the grist.

The value of this parameter determined for the non-alcoholic beer offered by Żywiec is 76% of the average magnesium content in samples 8.1-8.4, reaching 4.9956 mg/100 cm3, compared to 6.56 mg/100 cm³ for the sample in variants 8.1-8.4.

Potassium contents for sample variants 1-5 and 8 are similar (average value 53.9mg/100cm3) and range from 51.6-55.4mg/100cm³. The highest potassium value was obtained in sample 6 (62.2mg/100cm³). It was slightly lower for sample variants 7 (59.6mg/100cm3), i.e., for samples with a 30% addition of wheat bran and sunflower seeds with a structure of 2 bran units, 1 sunflower seed unit, and 3 bran units, 1 sunflower seed unit, respectively. The highest potassium content of 65.6mg/100cm³ was found in the beer with a 20% addition of wheat bran and sunflower seeds to the grist, with a structure consisting of two units of bran, one unit of sunflower seeds, and 2% oak chips added at the fourth mashing stage. The average potassium content in the samples with wheat bran and sunflower seeds added to the grist was 6.8% higher than in the samples with wheat bran and linseed.

The value of this parameter determined for the non-alcoholic beer offered by Żywiec represents 42% of the average potassium content in variant 6 (samples 61-6.4) and is 25.9 mg/100cm³, compared to 62.2 mg/100cm³ for the average of variants 6.1-6.4.

Sodium contents for samples 1-3 are similar (average value 4.33mg/100cm³) and range from 4.34-4.52mg/100cm³. They are 1/3 higher than the values of this parameter obtained for sample 4 and samples 1-3 of sample 5. The highest sodium value was obtained in sample 7 (6.73mg/100cm³), i.e., a beer with a 30% addition of wheat bran and sunflower seeds to the grist, with a structure of three bran units and one sunflower seed unit, and a 4% addition of oak chips at the third mashing stage. The average sodium contents from samples with wheat bran and linseed added to the grist are 5.1% higher than those with wheat bran and sunflower seeds added to the grist. The value of this parameter determined for the non-alcoholic beer offered by Żywiec is 16% of the average potassium content in variant 7 (samples 7.1-7.4) and is 1.07 mg/100 cm³ compared to 6.73 mg/100 cm³ for the average of variants 7.1-7.4.

The highest calcium contents were obtained for samples 1-4; they are at a similar level (average value: 2.65 mg/100 cm³) and range from 2.46-2.8 mg/100 cm³. These are approximately 17% higher than the values of this parameter obtained for samples 5, 6, and 8. The lowest sodium value was obtained in sample 7 (1.78 mg/100 cm³). The average calcium contents from samples with wheat bran and linseed added to the grist are 27% higher than those with wheat bran and sunflower seeds added to the grist. The highest calcium value determined for variant 2.1 is 37% lower than in the non-alcoholic beer offered by Żywiec and amounts to 3.13 mg/100 cm³ compared to 3.76 mg/100 cm³ for variant 2.1.

Iron content in the tested samples was characterized by significant variation; the highest value of this parameter was determined for variants of trial 3, followed by variants 1, 2, 8, 4, 5, 7, and 6.

The highest iron value was obtained in trial 3.1 (0.25 mg/100 cm³), i.e., beer with a 30% addition of wheat bran and linseed to the grist, with a structure of three units of bran, one unit of linseed, and a 2% addition of oak chips at the third stage of mashing. The average iron content in the samples with wheat bran and linseed added to the grist is 2.06 times higher than in the samples with wheat bran and sunflower seeds added to the grist. The average iron content determined for sample 3, which had the highest iron content (1.673 mg/100 cm³), is at least 6.7 times higher than that of the non-alcoholic beer offered by Żywiec. The lowest iron content determined in sample 6 (0.0387 mg/100 cm³) is at least 1.5 times higher than that of the non-alcoholic beer offered by Żywiec Non-Alcoholic Beer.

Zinc contents in the tested samples, with the exception of samples 1 and 2, are <0.025 mg/100 cm³. This is the value obtained for Żywiec’s non-alcoholic beer. The average content of this parameter in samples 1 and 2 is 1.75 and 1.87 times higher, respectively, than for Żywiec’s non-alcoholic beer.

3.3. Physicochemical Characteristics

Selected physicochemical parameters were determined to assess the technological quality of the developed non-alcoholic beer formulations following the incorporation of wheat bran and oilseed adjuncts. Density, original wort extract, apparent extract, real extract, pH and measurement temperature were measured in all experimental beers and compared with those of the commercially available non-alcoholic beer (Żywiec 0%). The results are presented in Table 3.

Density values of beer variants obtained in the investigation were lower than the value of Żywiec beer. The highest values of investigated beer variants were for trials 7 and were in ranges of 1.00740-1.00744 g/cm³. The average of density values was 0.99854 g/cm³ and it was lower of 0.01809 g/cm³ than for Żywiec beer.

Basic wort extract values were more than twice as high for all samples compared to a value for Żywiec beer (4.75% Plato).

The apparent extract content was much lower in the tested samples compared to Żywiec beer, due to the alcohol content, which was not evaporated at this stage of the research. The only positive values were obtained for variants 7.1-7.4 and they were in the range of 2.37-2.38%. However they were twice as small as the value for Żywiec beer (4.70%) with the alcohol content of 0.02%

The real extract for most samples were in the range of 1.68-1.98%. However for samples 7.1-7.4 its values were similar as for Żywiec beer (4.71%) with values In the range of 4.02-4.04%.

The temperature of the measurements ranged from 21.5 °C for Żywiec beer to 24.4 °C for other samples. Those results could influence other measurement results.

pH results were at a similar level of acidity, ranging from 4.54 to 4.86. The results closest to the pH of Żywiec beer were for all variants of 1, 2, and 8.

3.4. Quality-Related Characteristics

Selected technological and sensory-related quality parameters were determined to assess the effect of wheat bran, linseed, sunflower seeds and oak chip addition on the overall characteristics of the developed non-alcoholic beers. Bitterness, color, caloric value and turbidity were measured in all experimental formulations and compared with those of the commercially available non-alcoholic beer (Żywiec 0%). The results are presented in Table 4.

Bitterness for Żywiec beer was 18.01 IBU. Close results were obtained for samples 1.2, 1.3, and 3.1 (respectively: 18.83 IBU; 18.55 IBU; and 17.86 IBU). The lowest results were obtained for samples were for variants 1.1, 1.4, 2.1-2.4, and 3.2-3.4. All variants of 1, 2, 3 and 7, and also 4.1-4.3, 5.1-5.3, 6.1-6.2, and 8.4 were characterized by mild, balanced bitterness, while 4.4, 5.4, 6.3-6.4, and 8.1-8.3 were with moderate bitterness.

Color value of Żywiec beer was smaller (9.01 EBC) than for other samples (10.94-14.60 EBC). The highest values (over 14 EBC) were obtained for all samples for variants 6 and 7. The lowest values were obtained for samples 5.2 and 5.3, respectively: 10.94 EBC and 10.98 EBC. All beer samples, including the one for Żywiec were gold / yellow blonde which corresponds to the types of beers such as: Ale, Belgian Tripel or Weissbier.

The caloric content of the beer samples tested is similar (37.70 kcal/100 ml). The highest values of this parameter were obtained for samples 6 and 7. They are 3.8% and 5.7% higher, respectively, than the average caloric content of the remaining non-alcoholic beer samples developed by Browar Staropolski. The average content of this parameter in the samples included in the study is 2.23 times higher than for Żywiec’s non-alcoholic beer.

The content of turbidity for the tested samples and variants within the samples is characterized by a very large scatter. The highest levels of turbidity are found in the beer from samples 1, 4, and 2. In the case of sample 2, the contents of this parameter vary significantly, from 24.05 and 23.27 NTU to 10.48 and 7.47 NTU. Lower turbidity values were obtained for samples 3 and 7, the individual variants of which are characterized by a significant scatter of turbidity values (7.91 and 7.35, and 5.62 and 4.33 NTU). Low turbidity values were obtained for samples 6 and 5, i.e., 3.76 and 3.20 NTU, respectively. The variants of test 5, with their low average haze values, are characterized by a wide range of this parameter, from 4.52 and 4.22 to 2.27 and 1.80 NTU. The lowest haze value was obtained for test 8. The determined average haze level for the variants of this test is 10% lower than that of Żywiec’s non-alcoholic beer.

4. Discussion

Alcohol has been a persistent component of the social, cultural, and economic fabric of human societies throughout history, playing important roles in social bonding, religious rituals, economic exchange, and cultural practices [43]. Growing consumer awareness of the benefits associated with reducing alcohol intake has contributed to the increasing popularity of non-alcoholic beer [44]. Beer can contain a range of potentially health improving compounds as an isotonic and may contain a range of botanicals that have potential health benefits and acts as a functional food [45].

The present study demonstrated that partial replacement of malt with wheat bran enriched with linseed or sunflower seeds markedly improved the nutritional and functional value of non-alcoholic beer while maintaining physicochemical characteristics typical of this beverage. The most pronounced changes concerned the concentration of polyphenols, antioxidant capacity, vitamins and selected minerals, indicating that cereal by-products combined with oilseeds may serve not only as sustainable brewing adjuncts but also as effective functional ingredients. These findings are consistent with the current trend towards the valorization of cereal processing by-products within the framework of the circular economy [46] and the development of functional alcoholic and non-alcoholic beverages [47].

One of the most significant observations was the substantial increase in total polyphenol concentration in all experimental beers compared with the commercial reference beer. The highest values were obtained in variants containing 30% wheat bran supplemented with sunflower seeds (220–233 mg/L), whereas the commercial non-alcoholic beer contained only 53 mg/L. Even the formulation with the lowest polyphenol concentration exceeded the reference sample by more than 2.5-fold. These results confirm that wheat bran represents an exceptionally rich source of phenolic compounds, particularly hydroxycinnamic acids such as ferulic, p-coumaric and sinapic acids, which are predominantly bound to arabinoxylans within the cell wall matrix [48,49]. During mashing, endogenous and microbial enzymes partially hydrolyze these structures, facilitating the release of bound phenolics into the wort and consequently increasing their concentration in the final beer [50,51].

The particularly high polyphenol concentrations observed in sunflower-enriched formulations suggest that sunflower seeds contributed additional phenolic compounds and/or promoted their extraction during brewing. Sunflower seeds are recognized as valuable sources of chlorogenic acid, caffeic acid, ferulic acid and other phenolic constituents exhibiting considerable antioxidant activity [52]. Previous studies have demonstrated that the incorporation of oilseeds and other plant-derived materials into brewing formulations may substantially enhance the phenolic profile and antioxidant potential of beer without adversely affecting brewing performance [47]. Therefore, the approximately 16% higher average polyphenol concentration observed in sunflower-containing beers compared with linseed-containing variants most likely reflects differences in the native phenolic composition of these botanical materials rather than differences in brewing efficiency.

Interestingly, the increase in total polyphenol concentration was not always accompanied by a proportional increase in antioxidant activity. The highest antioxidant capacity was determined for beers produced with 20% wheat bran and linseed, particularly the formulation containing a 3:1 bran-to-linseed ratio combined with a 4% addition of oak chips during the fourth stage of mashing. Although sunflower-enriched beers contained higher total polyphenol concentrations, their antioxidant activity remained lower than that of the corresponding linseed formulations. This observation suggests that the antioxidant potential depends not only on the concentration of phenolic compounds but also on their qualitative composition, molecular structure and possible interactions with other antioxidant constituents. The observed differences may additionally reflect the presence of naturally occurring bioactive compounds in linseed, including tocopherols, lignans and other reducing substances, as well as interactions with Maillard reaction products formed during wort production. These findings are consistent with previous studies demonstrating that the antioxidant capacity of beer cannot be explained solely by total phenolic concentration, but is also determined by the chemical composition, structural diversity and synergistic interactions of antioxidant compounds present in the beverage [53,54,55].

Another noteworthy finding concerns the vitamin profile of the developed beers. All formulations containing wheat bran and linseed exhibited considerably higher concentrations of vitamins C and B₆ than the commercial beer, whereas sunflower-enriched variants were characterized by the highest vitamin B₃ concentrations. The vitamin C concentration reached approximately 19 mg/L in beers produced with 20% wheat bran and linseed, representing an increase of more than 50% compared with the commercial reference. Likewise, vitamin B₆ almost tripled in selected formulations. Although conventional beer is generally considered a poor dietary source of vitamin C because ascorbic acid is highly susceptible to oxidative degradation during wort production and beer processing, enrichment with wheat bran and linseed substantially increased its concentration in the experimental beers. This effect may be attributed both to the contribution of plant-derived micronutrients and to the presence of antioxidant compounds capable of limiting oxidative degradation during processing and storage [47,56]. Wheat bran contains appreciable amounts of B-group vitamins retained within the aleurone layer, whereas linseed provides not only additional micronutrients but also antioxidant compounds that may contribute to the improved stability of oxidation-sensitive vitamins during brewing and storage [47,55,57].

The distinct behavior of vitamin B₃ deserves particular attention. In contrast to vitamins C and B₆, the highest niacin concentrations were recorded in beers supplemented with sunflower seeds, especially at the higher proportion of wheat bran. Since sunflower seeds naturally contain higher concentrations of niacin than linseed, their incorporation into the brewing grist most likely contributed to the elevated vitamin B₃ concentrations observed in the finished beers [58]. Thermal processing during mashing may additionally facilitate the release of water-soluble vitamins from plant tissues, increasing their availability in wort, although the extent of this effect depends on processing conditions [56]. These findings indicate that the nutritional profile of beer may be selectively tailored through the appropriate selection of adjuncts, allowing brewers to optimize individual micronutrients according to the intended functional characteristics of the final product [59].

Beyond the improvement in antioxidant compounds and vitamins, the developed beers exhibited a favorable mineral profile, confirming that wheat bran and oilseeds can effectively enhance the nutritional value of non-alcoholic beer. Magnesium and potassium concentrations were consistently higher in all experimental formulations than in the commercial reference beer, while iron and zinc contents also increased in selected variants. These findings are consistent with the well-established nutritional characteristics of wheat bran, as the aleurone layer and outer grain tissues constitute the principal reservoir of minerals, vitamins and other bioactive compounds that are largely removed during flour milling [57,60]. Consequently, the incorporation of wheat bran into brewing formulations facilitates the transfer of nutritionally valuable macro- and microelements into wort and subsequently into the finished beer, thereby improving its overall nutritional quality [59]. The particularly pronounced increase in magnesium, potassium and iron agrees well with the known anatomical distribution of minerals within cereal kernels, where these elements are predominantly localized in the aleurone layer and germ rather than in the starchy endosperm [57,60].

The highest magnesium and potassium concentrations were observed in sunflower-enriched beers, whereas linseed-containing formulations generally contained higher concentrations of iron and calcium. These differences most likely reflect the intrinsic mineral composition of the botanical materials rather than solely technological effects during brewing. Sunflower seeds are recognized as valuable dietary sources of magnesium and potassium, whereas wheat bran is particularly rich in iron, calcium and other minerals concentrated within the aleurone layer and outer grain tissues [57,61]. The more than twofold higher iron concentration observed in linseed-enriched beers compared with sunflower-containing variants suggests that differences in the mineral composition of the adjuncts and their behavior during mashing may influence the transfer of iron into wort. Although beer is not generally regarded as an important dietary source of minerals, the observed improvements contribute to the overall nutritional quality of the beverage and support current efforts to develop cereal-based functional foods with enhanced micronutrient density [59]. The substantially greater variability observed for iron than for magnesium or potassium may indicate that iron transfer is influenced not only by its concentration in the raw materials but also by interactions with cereal constituents and process conditions during mashing.

An important finding of the present study is that the nutritional enhancement of the experimental beers was achieved without substantial deterioration of their physicochemical characteristics. Despite the incorporation of considerable amounts of wheat bran and oilseeds, the pH values of all formulations remained within the range typically reported for non-alcoholic beers, indicating that these adjuncts did not adversely affect acidification during brewing [56]. Maintaining pH within this range is technologically important because it contributes to microbiological stability, flavor balance, protein stability and overall shelf life of beer [62]. The observed results therefore suggest that the applied modifications of the brewing process successfully improved the nutritional profile of the beers without compromising one of the key quality parameters governing product stability. The relatively narrow pH range observed across all experimental variants demonstrates that the applied formulation strategy did not disrupt the acid–base balance of the brewing process, despite the substantial replacement of malt with nutritionally valuable adjuncts.

Similarly, the original wort extract remained relatively consistent among the experimental formulations despite the substantial modification of the grist composition. This observation indicates that partial replacement of malt with wheat bran and oilseeds did not adversely affect extract formation under the optimized mashing conditions applied in the present study. The results suggest that appropriate adjustment of the mashing programme can compensate for the lower starch content of cereal by-products and oilseeds, allowing the production of nutritionally enriched beers without compromising extract characteristics [50]. Similar observations have been reported in previous studies investigating the incorporation of cereal by-products and other plant-based adjuncts into brewing formulations, where moderate substitution levels maintained satisfactory technological performance while improving the nutritional quality of beer [59,63]. The relatively small variation in original wort extract across all experimental formulations demonstrates that the applied process modifications effectively compensated for substantial changes in grist composition, confirming the technological feasibility of producing nutritionally enhanced non-alcoholic beers with high proportions of cereal by-products and oilseeds.

Although color values increased in all enriched beers relative to the commercial reference, the products remained within the color range typical of pale specialty beers. The increase in color intensity can most likely be attributed to several complementary factors, including the higher concentration of phenolic compounds, enhanced Maillard reactions promoted by wheat bran constituents and the extraction of naturally colored phenolic substances from oak chips during mashing [56,64]. Similar increases in beer color have been reported following the incorporation of cereal fiber fractions, alternative cereal adjuncts and wood-derived materials into brewing formulations, where moderate darkening was generally regarded as an acceptable consequence of improving the nutritional and sensory characteristics of the final product [50,59]. Despite the increase in color intensity, all experimental beers remained within the range characteristic of pale specialty beer styles, indicating that nutritional enrichment did not result in excessive darkening that could negatively affect consumer acceptance.

Bitterness also remained within the range commonly reported for pale specialty and ale-style beers despite some variation between formulations. Slightly higher bitterness values observed in beers containing greater proportions of sunflower seeds and oak chips may reflect the combined contribution of hop-derived iso-α-acids and phenolic compounds extracted from the added plant materials and oak wood, both of which are known to influence bitterness perception and overall flavor complexity [56,64]. Importantly, none of the experimental beers exhibited bitterness levels likely to negatively affect sensory acceptance, indicating that nutritional enrichment through wheat bran and oilseed supplementation can be achieved without compromising the characteristic flavor profile of non-alcoholic beer. Similar observations have been reported for beers enriched with botanical ingredients, where moderate increases in bitterness were considered acceptable and did not adversely affect overall sensory quality [59]. Collectively, these findings demonstrate that the incorporation of wheat bran and oilseeds improved the nutritional profile of non-alcoholic beer while maintaining bitterness within the sensory limits expected for this beer category, thereby preserving overall product acceptability.

The observed differences in turbidity deserve particular consideration. Wheat bran is rich in insoluble dietary fiber, proteins and non-starch polysaccharides, particularly arabinoxylans, which are known to promote haze formation through the formation of protein–polyphenol complexes and other colloidal interactions during beer storage [56,62]. Consequently, the incorporation of fiber-rich cereal by-products would generally be expected to increase beer turbidity. However, several experimental formulations, particularly those containing sunflower seeds, exhibited turbidity values comparable to or even lower than those of the commercial non-alcoholic beer. These findings suggest that appropriate selection of adjunct composition, together with optimization of the mashing process, can effectively limit colloidal instability despite the use of high-fiber raw materials. From a technological perspective, this observation is particularly important because colloidal stability remains one of the major quality challenges in the production of fiber-enriched and functional beers [56,59].

Taken together, the present results demonstrate that wheat bran enriched with linseed or sunflower seeds should be regarded as a multifunctional brewing adjunct rather than merely as a partial malt substitute. Depending on the composition of the adjunct, it is possible to selectively enhance different nutritional attributes of non-alcoholic beer. Linseed proved more effective in increasing antioxidant capacity together with vitamins C and B₆, whereas sunflower seeds promoted greater accumulation of polyphenols, vitamin B₃ and selected minerals, particularly magnesium and potassium. Such complementary effects provide brewers with an opportunity to design beverages possessing targeted nutritional properties while simultaneously supporting the sustainable utilization of cereal processing by-products.

The findings of the present study also have broader implications for the brewing industry. Increasing consumer demand for functional foods and beverages, together with growing pressure to improve sustainability and reduce food-processing waste, has stimulated considerable interest in the use of alternative brewing raw materials [Mirabella 2014, Liguori 2022]. Wheat bran is one of the most abundant cereal by-products generated by the milling industry worldwide, yet it remains substantially underutilized in human nutrition despite being a rich source of dietary fiber, phenolic compounds, vitamins and essential minerals [Stevenson 2012]. The results of the present study demonstrate that wheat bran can be successfully incorporated into non-alcoholic beer formulations without compromising key technological and sensory attributes, while simultaneously improving their nutritional value. Consequently, the proposed approach supports the principles of the circular bioeconomy by promoting the valorization of cereal processing by-products into value-added functional beverages, thereby contributing to both environmental sustainability and the development of healthier food products [57,65,66].

Future studies should further characterize the individual phenolic profile using chromatographic techniques, determine the bioaccessibility of minerals and antioxidant compounds after simulated gastrointestinal digestion, and evaluate sensory acceptance during extended storage. Such investigations would provide a more comprehensive understanding of the nutritional and technological potential of wheat bran-based brewing adjuncts and facilitate their application in commercial production of functional non-alcoholic beers.

5. Limitations

There are no restrictions on the use of data outside the country of the study.

6. Conclusions

This study demonstrates that partial substitution of barley malt with wheat bran enriched with linseed or sunflower seeds, combined with controlled addition of oak chips during mashing, is an effective strategy for producing non-alcoholic beer with enhanced functional and nutritional properties. Experimental beers showed significantly higher concentrations of polyphenols, antioxidants, vitamins (C, B₃, and B₆), and essential minerals (Mg, K, Ca, Fe, Zn) compared to a commercial non-alcoholic beer. The strongest enrichment effects were observed in formulations containing sunflower seed-enriched bran, particularly for polyphenols, and magnesium content.

At the same time, modifications in grist composition influenced key physicochemical properties, including turbidity, color, bitterness, and extract composition. Increased haze observed in some variants reflects enhanced retention of bioactive compounds rather than technological instability, which may be considered advantageous in functional beverage development.

Overall, the present results demonstrate that partial substitution of barley malt with wheat bran enriched with linseed or sunflower seeds represents a feasible strategy for developing functional non-alcoholic beer with enhanced nutritional value. Although the enrichment increased turbidity and energy content, these changes were accompanied by substantial improvements in antioxidant capacity and micronutrient composition, suggesting that appropriate formulation can balance technological quality and nutritional benefits.

Overall, the results confirm that wheat bran and oilseed supplementation can be successfully applied in non-alcoholic beer production to enhance nutritional value and antioxidant potential, while maintaining acceptable physicochemical stability. Further research should focus on optimizing formulation balance between functional enrichment, clarity, and sensory acceptability.

This research was funded by National Centre for Research and Development in Poland, grant number: POIR.01.01.01-00-0763/20.