Characterization of Beer Produced with the Addition of Brown Macroalgae Fucus virsoides

Featured Application

The concept of this investigation aimed to formulate a beer enriched with functional ingredients originating from sea macroalgae Fucus virsoides.

Abstract

Marine macroalgae are organisms rich in bioactive compounds such as polysaccharides, polyphenols, and various minerals. Macroalgae are increasingly being added to the human diet precisely because they contain useful compounds that can also be used in the pharmaceutical industry. Previous research describes their addition to meat products, yogurt, bread, and baby food. However, data on the addition of algae to beer have been scarce. The goal of this work was to produce beer with the addition of brown macroalgae (Fucus virsoides) from the Adriatic Sea. In addition, the basic physical–chemical parameters (color, pH, ethanol, extract, and polyphenols) were determined. The most important premise is the transfer of selenium (Se) to beer, since Se is deficient in human food chain. The transfer of different metals, namely, S (sulfur), Mg (magnesium), P (phosphorus), K (potassium), Ca (calcium), Cr (chromium), Mn (manganese), Fe (iron), Co (cobalt), Ni (nickel), Cu (copper), Zn (zinc), As (arsenic), Se (selenium), Mo (molybdenum), Cd (cadmium), Hg (mercury), and Pb (lead), from algae to beer was determined using inductively coupled plasma–mass spectrometry (ICP−MS). The results, however, were not satisfactory regarding metal transfer. In particular, Se was detected in beer, but other metals such as As, Cd, and Pb were not. Alga addition contributed to extract values, and the original extract reached 14.3 °P in wort with alga addition, as opposed to 12.8 °P in the control sample. Such high extract content, however, resulted in beer with low alcohol content, <4% v/v for both beers. This could be explained by the high levels of unfermentable extract. pH values showed statistical difference between samples, meaning that the addition of algae significantly affected the pH value of beer, reducing acidity by almost 5%.

1. Introduction

Algae can be designated as heterogeneous flora widely distributed in different parts of the world. As reported by Mac Monagail et al. [1], about 10,000 species of algae are found around the globe, and many species are used by people [2]. Marine algae are proven to be abundant in proteins, carbohydrates, fiber, amino acids, vitamins, and fatty acids. All these components act positively on the human body. Algae can be marketed in different forms (e.g., natural forms, powder, and extracts), with various bioactive functions [3,4,5]. Countries that traditionally use marine sources of food, such as Japan, have the world’s longest life expectancy and this has been related to their dietary patterns, which include the regular consumption of macroalgae [5]. Thus, marine macroalgae are designated as plant-based food, a trend that is slowly taking over the market. This is the trend of the future, which is spreading among the Western civilization [6].

Fucus is a widespread genus of brown, perennial seaweeds. It can thrive in cold-temperate waters from the littoral and sublittoral regions along the rocky shorelines of the northern hemisphere [7]. The genus Fucus includes 66 currently accepted taxonomical species with a greenish brown trisected thallus, which is composed of a holdfast, a small stipe, and flattened dichotomously branched blades with terminal receptacles that swell during the reproductive season [8,9]. One of the algae belonging to the genus Fucus is Fucus virsoides J. Agardh. This is an endemic brown alga that grows only in the Adriatic Sea [10,11]. Some members of this genus can exhibit anticancer potential (F. vesiculosus L.) [12]. Besides the huge range of beneficial effects of the brown alga F. virsoides, the increased anthropogenic impact on the marine environment causes an accumulation of heavy metals in algal biomass.

Beer is made from mash/wort obtained by extracting sugars from crushed malt during mashing with water and then boiling with hops. F. virsoides contains certain amounts of carbohydrates, but the most interesting compounds are actually metals, especially the ones designated as deficient in human nutrition, such as selenium (Se), which are abundant in alga biomass. Nearly every European country is being classified as a low-selenium region, meaning that the dietary intake of this metal is insufficient. This is due to the low concentration of Se in soils, water, food, and feed [13]. Se is the active center of many proteins, thus being an essential micronutrient for living organisms [14]. Oxidative stress is a major trigger for other health issues related to Se deficiency [15]. For this reason, Se supplementation is widely stipulated. Such supplements can be derived from microorganisms like microalgae and can be implemented in food or feed. The biofortification of soil with Se in order to obtain Se enriched crops is also possibility [16,17,18]. Yeast is also a great biotransformation pod of inorganic Se to an organic form [19], so it can be utilized by higher organisms. Since beer is such a widespread medium, there have been several attempts to enrich beer with Se via biofortified barley/malt, or even with the direct addition of sodium selenite during the beer fermentation process [20,21,22]. Thus, the intention of this investigation is to provide a Se-rich beer.

The hypothesis of this work was that adding the alga F. virsoides into the boil, together with hops, and then subjecting it to fermentation can result in beer with valuable compounds such as Se. The additional carbohydrates could potentially enhance fermentation, resulting in higher alcohol content. This kind of beer could be utilized as a food supplement. In other words, the aim was to assess the transfer of metals from the alga to beer and to check if fermentation obstructs the concentrations of these compounds in the final beer. Physical–chemical properties were also determined in the final beer. Additionally, Se and other heavy metals were analyzed in the spent yeast which can be re-utilized or used for other purposes.

2. Materials and Methods

2.1. Alga Collection

Fucus virsoides J. Agardh, 1868, was collected in the southwest Novigrad sea area, located in the Adriatic Sea (44°1200200 N, 15°2805100 E), Croatia. A representative sample was collected using a single-point sample collection from a depth of 0.5 m at a sea temperature of 12 °C. Fresh F. virsoides was then freeze-dried using a laboratory freeze dryer (CoolSafe PRO, Labogene, Denmark). The freeze-drying was performed under a high vacuum (0.13–0.55 hPa) for 24 h, with 30 °C and 20 °C as the primary and secondary drying temperatures. The freeze-dried alga was further used in beer production.

2.2. Beer Production

For this purpose, porter beer style was brewed in a homebrewing unit BrewTaurus (40 L). Twenty L of wort was prepared containing 2.5 kg of pale ale malt (Simpsons Malt, Northumberland, UK), 5.0 kg DRC^® malt (Simpsons Malt, Northumberland, UK), and 0.1 kg of chocolate malt (Paul’s Malt, Bury Saint Edmunds, United Kingdom). Malt was purchased from the local brewing and malting equipment store. Reverse-osmosis water was adjusted for porter production by adding 2.5 g calcium chloride, 2 g Epsom salt, and 1 g gypsum. Mashing was carried out at 65 °C for 60 min, and mash-out was performed at 75 °C for 10 min. Boiling was carried out at 100 °C for 60 min. Then, 25 g of dry algae was added during boiling time (60 min), alongside hops: 5 g Chinook (12.0% α-acids) and 5 g of Galaxy (17.9% α-acids) were added at the beginning of the boil. An additional 5 g of Columbus hops (15.0% α-acids) was added 20 min before the end of the boil. Cooling was conducted using a coil chilled with tap water, so the temperature was reduced to 25 °C. Then, 20 L of wort was separated in two fermentation vessels, 10 L each, marked as “A”. Inoculation was performed with Safale US-05 yeast (Fermentis, Marcq en Baroeul, France). One bag (11.5 g) of dried yeast was used for 20 L of wort.

Control beer was produced in the same way, using the same ingredients, but without the addition of algae during boiling. The corresponding fermenter was marked as “C”. Upon inoculation, vessels were stored at 21 °C (in a cooling chamber). At the end of fermentation, beer was transferred to kegs, carbonated, and stored in a cool place (4 °C). Brewing was conducted in duplicate for each batch (control and with the addition of algae).

2.3. Physical–Chemical Analysis of Beer

Before the inoculation of wort, samples were analyzed using an Anton Paar Beer Analyser (Anton Paar GmbH, Graz, Austria). Fermentation was analyzed via a portable EasyDens Anton Paar (Anton Paar GmbH, Graz, Austria). Polyphenol, color, and bitterness analysis was performed according to MEBAK [23], and the final beers were analyzed using an Anton Paar Beer Analyser (Anton Paar GmbH, Graz, Austria). All analyses were performed in duplicate for both fermentation vessels.

2.4. Measurement of Metals with ICP-MS

Alga samples were dried in a laboratory dryer at 70 °C to a constant mass, and the dry sample was ground to powder using an ultra-centrifugal grinding mill without heavy metal residues (Retsch ZM 200, Haan, Germany). All samples of algae, yeast, and beer were digested with a wet microwave technique (CEM Mars 6, Charlotte, NC, USA) according to the procedure described in [24,25,26]. Briefly, the samples were quantitatively transferred into a Teflon cuvette, and 9 mL of 65% HNO₃ and 2 mL of 30% H₂O₂ were added. After digestion, the solutions were filtered into volumetric flasks, transferred to a 50 mL polypropylene tube, and filled up to the mark with deionized water. The concentrations of analyzed elements (S (sulfur), Mg (magnesium), P (phosphorus), K (potassium), Ca (calcium), Cr (chromium), Mn (manganese), Fe (iron), Co (cobalt), Ni (nickel), Cu (copper), Zn (zinc), As (arsenic), Se (selenium), Mo (molybdenum), Cd (cadmium), Hg (mercury), and Pb (lead)) were measured directly in solution using inductively coupled plasma–mass spectrometry (ICP–MS, Agilent Technologies 7800 ICP-MS, Santa Clara, CA, USA). Each batch of samples run on the ICP was analyzed with an internal pooled plasma control and with the different certified reference materials (Institute for Reference Materials and Measurements BCR 129; Institute of Nuclear Chemistry and Technology (INCT/ICHTJ) INCT-SBF-4) prepared in the same way as were the other samples. Recovery values, LOD and LOQ values are visible in Supplementary Materials as Table S1. Recovery values for analyzed metals and Table S2. LOD and LOQ values of analyzed metals.

2.5. Statistical Analysis

Analysis of variance (ANOVA) and Fisher’s least significant difference test (LSD) were conducted, with the least statistical significance set to p < 0.05. Statistica 13.1. (TIBCO Software Inc., Palo Alto, CA, USA) was the software of choice for this data set.

3. Results and Discussion

The results of this research are presented in

Table 1. Basic physical–chemical indicators of produced beers.

Quality Indicator	Unit	Algae Beer	Control Beer
Specific gravity	mg/L	1.02935 a ± 0.00012	1.02495 b ± 0.00010
Real extract	%	8.70 a ± 0.1	7.60 b ± 0.2
Original extract (wort)	°P	14.30 a ± 0.5	12.80 b ± 0.3
Apparent extract	%	7.40 a ± 0.1	6.30 b ± 0.1
Alcohol content	%	3.80 a ± 0.45	3.55 b ± 0.23
Polyphenol content	mg/L	513 b ± 1	590 a ± 1
pH		4.25 a ± 0.02	4.07 b ± 0.05
Bitterness	IBU	42 a ± 1	42 a ± 1
Color	EBC	100 a ± 2	98 a ± 1

Means ± SD; a, b within rows with different superscripts are significantly different (p < 0.05); IBU—international bitterness unit.

and

Table 2. Results of metal analysis in alga F. virsoides dry matter.

Metal	S	Mg	P	K	Ca	Cr	Mn	Fe	Co	Ni	Cu	Zn	As	Se	Mo	Cd	Hg	Pb
Beer/Unit	mg/L	mg/L	mg/L	mg/L	mg/L	µg/L	mg/L	mg/L	µg/L	µg/L	mg/L	mg/L	µg/L	µg/L	µg/L	µg/L	µg/L	µg/L
Control	113.45 b	148.55 b	300.05 b	903.10 b	98.22 b	1.03 a	1.01 b	0.23 b	0.19 b	7.69	0.07 a	0.07 a	0.42 b	1.72 a	28.71 a	0.19 b	<LOD	0.98 a
Algae	179.9 a	188.40 a	361.05 a	1140.50 b	149.05 a	1.67 a	1.26 a	0.72 a	2.30 a	15.52	0.03 b	0.49 b	77.94 b	1.77 a	26.98 b	0.91 a	<LOD	1.05 a

. Table 1 shows the basic physical–chemical indicators determined in the produced beers. When looking at the basic parameter, such as the specific gravity, it can be noted that wort with the addition of algae had a somewhat higher extract content, 1.02935 mg/L, in comparison to the control sample, which had 1.02495 mg/L.

This indicates that alga addition contributed to extract values, due to its content of carbohydrates. This was reflected on the original extract which reached 14.3 °P in wort with alga addition, as opposed to 12.8 °P in the control sample. Such high extract content, however, resulted in beer with low alcohol content, <4% v/v for both beers. This could be explained by the high levels of unfermentable extract that is unavailable to yeast to conduct fermentation. The pH values are showing statistical difference between samples, meaning that the addition of algae significantly affected the pH value of beer, reducing the acidity by almost 5%. This is a significant change in pH and would affect the sensory properties. Such significant change could be related to a higher alcohol content in beer with the addition of algae (3.80% v/v) in comparison to the control sample which contained 3.55% v/v. Bitterness did not undergo any changes with the addition of algae, but it was the same for both beers, 42 IBU, meaning that algae do not contribute to bitterness. Color was slightly affected with the addition of algae, and was a bit higher in beer with the addition of F. virsoides. This is expected since F. virsoides is a brown alga. Since there are not many data about this topic, it is hard to compare the obtained results with published materials. There are some research studies in which microalgae such as Arthrospira platensis (spirulina) or Tetraselmis chui were added into the brew [27,28], but macroalgae have not yet been described as a potential brewing adjunct, even though they possess health-beneficiary ingredients such as fucoidan and carrageenan [29]. The results of wort in the original extract, in the research conducted by Beisler and Sandmann [27], are in accordance with the data collected in this research. The pH value was higher in their research and did not show a decline with the addition of the microalga spirulina. Also, alcohol content was higher than in this research, above 4.5 % v/v for all samples. The lower alcohol content was probably a result of the high content of DRC^® malt. To avoid astringent and bitter aromas and taste, native to dark ales, and to bring about the alga aromas, DRC^® malt was used. Small amounts of hops (bitter and aromatic) were used too. This combination aimed to reduce the influence of raw materials such as malt and hops to beer brewed with algae. The high content of DRC^® malt also contributed to the beer color, reducing the influence of alga addition. Heavier notes of dark ale matched the heavy notes of algae during boiling.

The lower polyphenol content in beer with the addition of algae could be caused by the presence of carrageenan in alga biomass. Specifically, carrageenan is used as a fining agent during boiling. Fining agents are usually added to the wort approximately 10 min before the end of the boil. They tend to speed up the aggregation and sedimentation of the protein–protein and protein–polyphenol complexes during cooling [30]. Carrageenan reacts mainly with high-molecular-weight proteins [31]. As algae were added to the boiling phase in the brew, it is possible that some of the polyphenolic substances were precipitated with proteins during cooling. Thus, lower polyphenolic levels were determined in beer with alga addition. However, this is yet to be investigated.

The results of metal analysis in the samples are presented in Table 2,

Table 3. Results of metal analysis in produced beer.

Metal	S	Mg	P	K	Ca	Cr	Mn	Fe	Co	Ni	Cu	Zn	As	Se	Mo	Cd	Hg	Pb
Sample/Unit	mg/kg	mg/kg	mg/kg	mg/kg	mg/kg	µg/kg	mg/kg	mg/kg	µg/kg	µg/kg	mg/kg	mg/kg	µg/kg	µg/kg	µg/kg	µg/kg	µg/kg	µg/kg
Algae (dry matter)	23,855.00	9167.00	1290.50	24,035.00	18,305.00	572.70	72.40	86.97	1215.00	2685.00	2.44	19.59	25,390.00	36.32	264.12	484.54	4.31	328.68

and

Table 4. Results of metal analysis in yeast biomass after fermentation (spent yeast).

Metal	S	Mg	P	K	Ca	Cr	Mn	Fe	Co	Ni	Cu	Zn	As	Se	Mo	Cd	Hg	Pb
Yeast/Unit	mg/kg	mg/kg	mg/kg	mg/kg	mg/kg	µg/kg	mg/kg	mg/kg	µg/kg	µg/kg	mg/kg	mg/kg	µg/kg	µg/kg	µg/kg	µg/kg	µg/kg	µg/kg
Control	535.65 a	530.95 a	2421.50 a	3298.50 a	813.35 a	4.99 b	2.51 a	41.80 b	23.84 a	113.90 b	2.88 a	34.46 a	14.46 b	13.38 a	225.17 a	27.73 a	<LOD	11.82 b
Algae	375.00 b	167.15 b	489.70 b	780.80 b	655.00 b	13.30 b	1.57 b	22.54 b	24.59 a	142.25 a	2.15 b	13.47 b	44.78 a	4.37 b	165.61 b	8.74 b	<LOD	32.66 a

. Metals were determined in alga dry matter (Table 2) and in beer and spent yeast (Table 3 and Table 4). Macroalgae are known to retain heavy metals such as chromium, cadmium, copper, nickel, arsenic, mercury, lead, and zinc, which are globally the most commonly detected metals [32,33]. Some of them are crucial for the normal functioning of metabolism, but in higher concentrations, they are surely toxic to humans and animals [34]. Concentrations of heavy metals, and trace metals, can be used as a bioindicator of the pollution of an ecosystem [35]. However, heavy metals exhibit a harmful impact on algae by initiating oxidative stress [36]. The results of this research indicate that alga biomass contains high concentrations of different metals, or “potentially toxic elements”, as they are referred to in studies [37]. Alga biomass contained extremely high concentrations of K, 24,035.00 mg/kg, then S, 23,855.00 mg/kg, and Ca with 18,305.00 mg/kg. Mg, Ni, P, and Co showed high concentrations in alga biomass, over 1000.00 mg/kg. Alga dry matter contained significant amounts of As (25,390.00 µg/kg), Ni (2685.00 µg/kg), and Co (1268.00 µg/kg). Such high concentrations of heavy metals are not surprising since algae are an excellent bioremediation medium [38].

Cr, Cd, Pb, and Mo were below 500 µg/kg. The most surprising finding in alginic biomass was the low Se concentration (<20 µg/kg).

The beer produced with the addition of alga showed higher levels of all metals in comparison to the control sample (Table 2). Beer with F. virsoides addition had approximately 25% higher levels of all elements than control beer. However, only a small portion of heavy metals transferred from alga biomass to the brew. As levels were determined to be high, reaching 77.94 µg/L, which is significantly higher than the tolerable intake level (TIL) of 3 µg/kg body weight per day, recommended by the World Health Organization (WHO) [39]. By drinking 0.5 L of such beer, one would take over 35 µg of As in the system. Thus, the idea of sensory analysis was dropped. On the other hand, the levels of As in control beer were significantly lower, reaching 0.42 µg/L. Zn showed a certain deviation. Specifically, its concentration was significantly lower in beer produced with F. virsoides. Zn is necessary for yeast growth and metabolism, and it was probably utilized by yeast biomass during fermentation [40]. Alcohol dehydrogenase is the terminal enzyme of the fermentation process and it is a zinc-dependent enzyme [41]. Its job is to reduce acetaldehyde to ethanol during glucose fermentation [42].

Yeast samples were also tested for heavy metals. The results are shown in Table 3. These results show a deviation in heavy metal accumulation. Specifically, spent yeast obtained after fermentation with alga showed lower levels of macroelements such as S, Ca, Mg, K, P, Mn, Cu, and Zn than yeast from the control sample. Microelements As, Pb, Cr, Co, and Ni showed higher levels in beer with the addition of alga than in the control beer. This could indicate that dry alga biomass could have absorbed heavy metals from beer/alga. Recent studies showed that dead (or dried) algal biomass is a good absorbent of heavy metals or other xenobiotics from wastewater due to their complex mucilaginous polysaccharide-composing cell walls which exhibit a high affinity for di- and trivalent cations [43,44,45]. Brown algae have a higher capacity for binding metals than red and green algae due to alginate content which has a high affinity for bivalent metals [46,47,48]. Spent alga biomass was discarded with hops after boiling, and thus there are no results for this by-product of brewing. In that sense, further investigation should be conducted to elucidate the amount of metals left in spent alga biomass.

The addition of F. virsoides changed the wort content, resulting in a higher extract. Subsequently, this beer contained higher alcohol levels and higher real extract, which would give beer a sweeter taste than the control. However, the planned sensory analysis was avoided due to the subsequent analysis of heavy metals. In particular, the high concentrations of metals in beer were considered a potential health hazard, and thus the sensory analysis was halted at smell, and tasting was not performed. The smell of the brew during boiling was indigenous to dry algae/seaweed, very heavy, and took over the malty odors. After fermentation, the smell was reduced, and finer notes of chocolate and caramel arose in the beer smell. After two weeks, the alginic smell was even more reduced, and just a hint of seaweed could be smelled during this part of the sensory analysis.

4. Conclusions

The results of this research show that the addition of alga F. versoides contributes to the extract content and subsequently to higher alcohol content in beer. Lower polyphenol levels were determined in beer with the addition of alga F. virsoides, probably due to the fining properties of alga biomass through the presence of carrageenan. Lower pH was detected in the control sample. This could be related with the higher alcohol content in beer with algae. In any case, alga addition to beer could be useful and could result in a functional alcoholic beverage. However, the heavy metals which can be detected in algae end up in the brew and could present a hazard to humans. Perhaps, this risk could be reduced by adding the alga extract to wort during boiling or even at the end of cooling. Nevertheless, the extract should also undergo heavy metal analysis prior to using it in beer production. Even though beer with alga addition did not exhibit any negative odors, it changed over time, resulting in a less alginic odor. Another benefit of using extract would be the reduced alginic odor during boiling and consequently in beer. A potential practical benefit of alga addition to beer could be the reduction in high-molecular-weight proteins, which would improve and extend the shelf life of beer [31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49]. However, this is yet to be investigated, preferably on a lighter beer style.