Sustainable Processing of Brewers’ Spent Grain for Plant-Based Yogurt Alternatives

Abstract

During the preparation of beer wort, significant amounts of waste raw materials, such as brewers’ spent grain (BSG), are generated. In line with the zero-waste approach, a processing technology for BSG was developed to valorize this by-product. The developed method involves obtaining a BSG extract (plant-based milk), followed by filtration to remove insoluble residues and subsequent fermentation to produce vegan BSG-based yogurt-like products, with and without the addition of sucrose, as well as pectin, guar gum, and konjac gum as stabilizers. The samples were analyzed for pH, moisture and protein content, water activity (Aw), color, viscosity, and syneresis, and were also subjected to an organoleptic evaluation. Fermentation with starter cultures yielded BSG-based yogurt-like products with an optimal pH (~4.0), which, combined with Aw values below 0.95, ensures microbiological safety by inhibiting the growth of pathogenic and spoilage microorganisms. Due to phase separation, the use of stabilizers was necessary to achieve a yogurt-like texture. Their application also contributed to a reduction in syneresis—sometimes even preventing its occurrence—and led to an increase in viscosity, which ranged from 0.162 to 0.463 Pa·s, depending on the stabilizer used. The moisture content of fermented BSG extracts ranged from 88.2% to 91.7%. All samples showed similar protein content, approximately 50% on a dry matter basis. Furthermore, organoleptic assessment (5-point scale) revealed that sensory characteristics varied depending on the stabilizer and sugar used. The yogurt-like variant formulated with 0.5% pectin and 1% sucrose received the highest acceptance score (4.0), indicating good sensory quality.

1. Introduction

Facing growing challenges related to climate change, environmental degradation, and increasing food demand, the implementation of sustainable production and consumption strategies has become crucial. The conception of the circular economy (CE) is one of the solutions that can help reduce the negative impact of human activities on the ecosystem. The circular economy involves minimizing waste through reuse, recycling, or the implementation of more effective and innovative technologies [1]. In the context of food production, the introduction of the CE in food technology requires a transition from a linear “produce-use-disposal” model to a closed-loop system. This conversion is difficult to apply in such a specific industry, but a procedure where waste products are valuable resources can be successfully implemented [2]. The utilization of by-products from the agri-food industry and their reintegration into the production chain can help reduce food waste, lower greenhouse gas emissions, and improve the economic efficiency of the agri-food sector.

Sustainable development and food safety are expected to be two key pillars of the food industry [3]. Food market analysts anticipate growing interest in plant-based alternatives, which are evolving in quality as consumers increasingly opt for less processed products made from natural ingredients. Alongside staples like tofu and tempeh, ingredients such as nuts and lentils are becoming more prevalent, enriching the variety of flavors and nutritional benefits. Food companies are embracing simpler recipes and locally sourced ingredients to enhance authenticity and sustainability. Plant-based alternatives are no longer merely substitutes for meat but have become a distinct category catering to health-conscious consumers who prioritize taste, nutrition, and environmental responsibility.

Plant-based food is a small but expanding category, and as more innovations and products become available, consumer interest broadens. The trend of replacing dairy and meat products with non-dairy and meatless alternatives is expanding globally. Consumers adopt plant-based diets for a variety of reasons, including health benefits, environmental sustainability, animal welfare considerations, and religious or spiritual beliefs [2,4]. Those consuming only plant-based products believe that they contribute to reducing the greenhouse gas emissions associated with livestock production. Regarding consumer health, aside from the fact that plant-based products provide beneficial compounds such as antioxidants and dietary fiber [5,6], individuals who are lactose intolerant or allergic to milk proteins particularly benefit from these alternatives, as they offer a safe and nutritious option without triggering adverse reactions. However, this shift presents a challenge for the modern food industry, as dairy products are a primary source of essential nutrients, particularly protein, vitamin D, and calcium. Even those who cannot consume dairy still need to meet their daily protein requirements. To ensure adequate nutrition, it is crucial to find suitable alternative protein sources, such as soybeans, cashews, almonds, and brewer’s spent grain. These alternatives are already being incorporated into a variety of plant-based products, including milk, cheese, and yogurt, providing nutritious options for consumers with dietary restrictions.

Plant-based product sales are growing across the globe [6]. The demand for non-dairy milk and cheese alternatives in the United Kingdom has been increasing at a consistently high compound annual growth rate; since 2018, sales of plant-based cheese and milk have risen by 165% [7]. Meat alternatives, such as chicken and beef substitutes, accounted for only 1% of total meat sales in the United States in 2024. However, this segment is constantly evolving, with certain regions showing significantly higher adoption rates [8]. Circana data analysis shows that plant-based sales across European “Big 5” countries (Germany, France, UK, Spain, and Italy) reached €5.4 billion in 2023, 5.5% more than in 2022 [9]. This demonstrates the increasing consumption of plant-based foods (such as fruits, vegetables, nuts, seeds, and legumes) among consumers following plant-based diets.

However, it is important to recognize that animal-based products and their plant-based alternatives differ not only in terms of raw materials but also in their macronutrient and micronutrient profiles. While plant-based alternatives aim to replicate the taste and texture of animal-derived products, their nutritional composition—particularly in terms of protein quality, vitamin content, and bioavailability of essential nutrients—can vary significantly. In 2015, over 130 variants of different plant-based milks were available on the European market [10]. Non-dairy milk has grown in popularity as a plant-based alternative to conventional dairy milk. Additionally, these plant-based drinks are not only consumed as beverages but have also become key ingredients in non-dairy cheeses and yogurts [11].

Various dairy milk and plant-based milk alternatives available in the local market (Bydgoszcz, Poland) have become the subject of a review of selected nutritional properties, focusing on their key characteristics such as calorie content, fat composition, carbohydrate levels, protein amounts, and calcium concentrations (Table S1 in the Supplementary Material). The comparison includes dairy milk with different fat contents, as well as plant-based drinks derived from ingredients like pea, bean, hemp, coconut, almond, oat, rice, rye, and soybean.

Milk is an excellent source of protein, containing approximately 3.0–3.3 g per 100 g in its standard version. Protein-enriched milk has an even higher protein content, reaching 8 g per 100 g, making it an ideal choice for individuals with increased protein needs, such as athletes [12]. Dairy milk provides higher protein content than most plant-based alternatives. However, plant-based options offer a variety of nutritional profiles, catering to different dietary needs. For those looking for higher protein content, soybean and bean-based drinks are the best alternatives, while oat and rice-based options are richer in carbohydrates. Almond milk, on the other hand, is the lowest in calories and protein, making it a lighter option. Most plant-based alternatives are fortified with calcium to match dairy milk, ensuring similar calcium intake for consumers choosing plant-based options (Table S1). Manzoor et al. [13] compared soybean and cashew milk, finding that both offer a balanced alternative for those seeking a dairy-free option, as they are low in fat and carbohydrates while providing a moderate amount of protein.

Plant-based drinks derived from various raw materials can be used to produce vegan yogurts or other fermented products. Bai et al. [14], in their work, proved that fermentation significantly increases the content of free amino acids, peptides, and dietary fiber in plant-based yogurt-like soy products, but this phenomenon also refers to other fermented plant products. When evaluating their quality and health benefits, it is essential to consider their protein, fat, and carbohydrate content. A variety of fermented plant-based products are available on the market, made from ingredients such as soy, rice, cashews, coconut, almonds, peas, beans, broad beans, and oats [6,10,11,15]. These alternatives undergo fermentation processes similar to those used in traditional dairy products, which enhance their texture, flavor, and nutritional profile [10,15]. As a result, they provide a valuable option for consumers seeking dairy-free choices while still benefiting from probiotics and essential nutrients.

An emerging and innovative addition to this category of fermented products is brewers’ spent grain (BSG)-based fermentation. BSG is the primary by-product of the brewing process, accounting for approximately 85% of all brewing by-products. It is generated during beer production, where malt and water are used. The primary purpose of malting is the conversion of malt starches into simple sugars, which serve as an energy source for yeast during fermentation. After this process, the remaining solid residue—BSG—is separated from the wort and is often discarded [16,17]. With global beer production ranging between 1.91 and 1.97 billion hectoliters, approximately 12 million tons of BSG are generated annually [18]. The composition of BSG depends largely on the type of malt used, resulting in variations in its nutritional profile. Brewer’s spent grain is primarily composed of fiber, making up 59.1% to 73.2% of its dry mass (DM). It also contains a moderate amount of protein, ranging from 14.2% to 24.7%, making it a potential protein source. The fat content varies between 6.0% and 13.0%, while the ash content, which represents the mineral component, ranges from 1.2% to 4.6% [18]. Given its high fiber and protein content, BSG presents a promising raw material for the development of novel fermented food products, contributing to sustainability and waste valorization in the food industry.

The incorporation of BSG into yogurt production has been shown to significantly impact its chemical composition. Research indicates that adding 10% BSG increases protein content by 32.62% and carbohydrate content by 43.16% [19]. Such findings highlight the potential of BSG as a functional ingredient that enhances both the nutritional value and sustainability of fermented plant-based products. These innovative approaches to vegan yogurt production reflect a broader trend in the food industry toward healthier, more environmentally friendly products that align with consumers’ growing demand for plant-based alternatives.

In addition to its basic composition, BSG is also a rich source of micro- and macroelements and vitamins, mainly from the B and E groups [16,17,18]. However, due to its significant nutritional value, brewers’ spent grain has the potential to be more than just a by-product or a functional additive—it can serve as a valuable ingredient in the development of innovative and sustainable food products. Brewer’s spent grain may be used in a variety of creative ways since it is high in proteins, fiber, and vital amino acids. Thanks to the presence of many antioxidants, mainly phenolics, but also flavonoids and tannins [18], BSG is an attractive raw material for food and pharmaceutical technology. Moreover, the low market price and stable annual availability of BSG favor the use of this by-product for further transformation into food, feed, and pharmaceutics [20].

Nowadays, BSG is mostly used to supplement animal feed, especially for farm animals, fish feed, or as a fertilizer in agriculture; some is sent to landfills [18,21,22,23]. Due to the high moisture of BSG and transport costs, its use is limited, and it is usually disposed of in this way by farms located near the brewery. On the other hand, as a source of many health-beneficial compounds, BSG is the subject of research for applications in human nutrition, focusing on extracting proteins or phenolics from BSG [24,25,26,27]. A broad review on BSG application in food products was made by Hejna et al. [18], and brewer’s spent grain was utilized as a substitute for the most commonly used wheat flour. Besides this, there are many patented products obtained from brewer’s spent grain, such as BSG protein powder or fiber-rich formulation [28,29,30,31]. Protein powders from BSG are characterized by a high PDCAAS (protein digestibility corrected amino acid score) value, low phytic acid level, and antioxidant qualities. Those properties make BSG protein extracts the perfect addition to a variety of food items to improve their nutritional profile. Despite this, it is difficult to find products on the market that contain BSG. For now, everything remains a concept developed by scientists and a small-scale production.

The purpose of this research was to describe a novel process for making plant-based yogurt using brewer’s spent grain (BSG) as a key ingredient. This study aimed to explore the potential of BSG, a by-product of the brewing industry, as a sustainable and nutritious component in plant-based yogurt production. By using BSG, this research sought to enhance the nutritional profile of plant-based yogurt, particularly in terms of protein content, while also addressing the growing demand for environmentally friendly food solutions. Additionally, this study aimed to develop a production method that ensures desirable texture and taste, making the final product a viable alternative to traditional dairy-based and existing plant-based yogurts. Through this investigation, this research contributes to the valorization of food industry by-products and promotes the development of innovative, sustainable, and nutritious plant-based dairy alternatives.

2. Materials and Methods

2.1. Materials

Crushed pilsner malt (Viking Malt, Strzegom, Poland) was the raw material for beer production. Extract for further processing was obtained from the brewer’s spent grain used in beer production.

2.2. Brewing Process

For brewing, the G30 Grainfather Advanced Brewery Setup (Bevie Handcraft NZ Ltd., Auckland, New Zealand), including the G30v2 Brewing System (with the tank of volume 30 L, the control box, the grain basket, and the pump), the GC4 Glycol Chiller, and the GF30 Conical Fermenter, was used. Brewing steps are presented in the scheme below (Figure 1). Steeping the malt, the starch source, in water resulted in the obtaining of brewer’s spent grain, a by-product, and wort, a sweet liquid, for fermentation with yeast. The mashing process was conducted using a malt-to-water ratio of 1:3, with each trial utilizing 1.5 kg of malt. A two-step infusion mashing protocol was applied: the first rest at 62 °C for 40 min, followed by a second rest at 72 °C for 30 min. The duration of the second rest was adjusted based on the results of the iodine test, which was employed to monitor starch conversion. To perform the iodine test, a 5 cm³ sample of wort was collected and cooled to room temperature. A drop of iodine solution (iodine dissolved in potassium iodide) was added to the sample on a white spot plate. The appearance of a dark blue or black coloration indicated the presence of residual starch, signifying incomplete saccharification. In such cases, the mash was maintained at 72 °C for an additional 10 min before repeating the test. This process was continued until a negative iodine test result was obtained, characterized by no significant color change, confirming the completion of starch conversion. The mash was subjected to a mash-out step. This involved heating the mash to approximately 78 °C to inactivate residual enzymatic activity, particularly that of α- and β-amylases, thereby stabilizing the carbohydrate composition of wort. Subsequently, the solid and liquid fractions were separated to obtain brewer’s spent grain (BSG).

2.3. BSG Processing

After separation from the wort, BSG was mixed with water in a 1:2 ratio and ground using a Grindomix GM 200 laboratory knife mill (Retsch, Haan, Germany). The knife speed was 15,000 rpm, and the time of grinding was 5 min. In each trial, one part of brewer’s spent grain (100 g) was combined with two parts of water (200 g) and ground to form a homogeneous slurry. The mixture was then sieved to remove particles larger than 1 mm. The resulting extract was pasteurized at 95 °C for 30 min to ensure microbial safety and stability. This protocol was developed based on preliminary experiments conducted on-site. The scheme of BSG processing is presented in Figure 2.

2.4. BSG Extract Fermentation

The pasteurized BSG extract was the basis for the production of fermented non-dairy products, i.e., yogurt analogs. BSG extract was mixed with starter culture of bacteria for fermentation of vegan fermented product, yogurt alike, Beaugel Soja1 (Streptococcus thermophilus, Lactobacillus bulgaricus and L. casei) (Ets Coquard, Villefranche-sur-Saône, France). Optionally, sugar (sucrose) in a concentration of 1% and stabilizers, guar gum 5000–5500 CPS (Hortimex Sp. z o.o., Konin, Poland), konjac gum (Pixelamber, Gdynia, Poland), or low methoxyl pectin (LM) NECJ A2 (Agnex, Białystok, Poland), in concentrations of 0.5%, were added. The concentration of added stabilizers was determined on the basis of preliminary tests, and their results are included in the Supplementary Material (Figures S1 and S2). In the case of guar gum and pectin, BSG extracts were mixed at room temperature and then stirred and heated up to a temperature of 60 °C using a laboratory hot plate magnetic stirrer (C-MAG HS 7 with thermometer ETS-D5, IKA GmbH & Co. KG, Staufen im Breisgau, Germany). Konjac gum does not require heat activation to exhibit its thickening properties. Once the additives had dissolved, the mixtures were cooled to fermentation temperature and other ingredients were added. The mixture of BSG extract, bacteria, and optional sugar and stabilizers was transferred into an incubator with a temperature of 37 °C set for 24 h. After fermentation, samples were placed in a refrigerator set at a temperature of 4 °C for 24 h. Depending on the composition, 8 variants of fermented products were prepared: fermented plant-based beverage with/without sugar (FS, FP), fermented plant-based beverage with guar gum and with/without sugar (FPG, FSG), fermented plant-based beverage with pectin and with/without sugar (FPP, FSP), fermented plant-based beverage with konjac gum and with/without sugar (FPK, FSK).

2.5. Moisture Determination

The moisture of fermented products and BSG extract samples was measured by using the loss on drying (LOD) method with a moisture balance (model MA 50.R, RADWAG, Radom, Poland) at 120 °C until a constant weight was attained [32,33]. For each sample, three measurements were made.

2.6. pH Determination

The pH of the fermented BSG extracts was determined using a pH meter (model CP-505, Elmetron, Zabrze, Poland) equipped with a glass spear combination probe (Corning, Corning, NY, USA). To measure the pH of plant-based yogurt-like products, the glass probe was inserted into a beaker containing the sample [34]. Three measurements were taken for each sample.

2.7. Protein Determination

Approximately 1 g of each sample was accurately weighed (±0.1 mg) into a digestion tube. To facilitate the digestion process, two Kjeldahl catalyst tablets (comprising a mixture of heavy metal catalysts and sulfate salts) were added, followed by 20 mL of sulfuric acid (98%). The digestion was carried out using a Büchi Digestion Unit K-438 (Flawil, Switzerland) connected to a Büchi Scrubber B-414 (Flawil, Switzerland), ensuring efficient removal of fumes. This step converts organic nitrogen into ammonium sulfate. Post-digestion, the solution was cooled and rendered alkaline by the addition of sodium hydroxide, liberating ammonia gas. The liberated ammonia was then distilled using a Büchi Distillation Unit K-360 (Flawil, Switzerland) equipped with an autosampler. The distillate was captured in a boric acid solution, forming an ammonium–borate complex. Subsequent titration with standardized hydrochloric acid allowed for the quantification of nitrogen content [35]. Each sample was analyzed in triplicate to ensure accuracy and reproducibility. The nitrogen content obtained was converted to crude protein content using a conversion factor of 6.25 [36]. Results are reported on both wet and dry matter bases.

2.8. Water Activity Measurement

The water activity (Aw) of fermented samples was measured after storage at a temperature of 4 °C for 24 h. When samples achieved room temperature, they were transferred into a Rotronic sample cup (40 mm deep) placed in the sample holder and covered by the measurement (HC2-AW-USB, Rotronic AG, Bassersdorf, Switzerland). The Aw of each sample was measured in triplicate.

2.9. Colorimetric Measurements

The color of the BSG extract and fermented products was evaluated using the Commission Internationale de l’Eclairage (CIE) L*a*b* color model. A sample of approximately 25 g was carefully placed in the measurement dish, ensuring no free space remained. The L* (lightness), a* (red-green), and b* (yellow-blue) values were recorded using a colorimetric spectrophotometer (Chroma Meter CR-410, Tokyo, Japan). The total color difference (ΔE) was then calculated as follows [37]:

ΔE = [(ΔL*)² + (Δa*)² + (Δb*)²]^1/2

(1)

Total color difference (ΔE) was used to evaluate the different treatments and composition influence between the BSG extracts fermented samples. Three measurements were made for each sample.

2.10. Viscosity Measurement

The viscosity of fermented products was measured using a rheometer (Brookfields R/S+ Rheometer, Toronto, Canada) with coaxial cylinders measuring system (CC-25). The sample of plant yogurt (17 cm³) was first transferred to the outer cylinder and then mounted. The apparent shear viscosity curves were recorded at a shear rate of 50 s⁻¹. The measurement was carried out for 2 min, at a temperature of 20 °C ± 1 °C and a data activation time of 2 s⁻¹. Before the analysis, samples were relaxed in the rheometer for 2 min. The obtained results were presented as apparent viscosity curves, initial and final viscosity values, and viscosity Δ (the difference between initial and final viscosity). The results and calculations were recorded using software Rheo3000 (Brookfield, Toronto, ON, Canada). Three measurements were made for each sample.

2.11. Organoleptic Tests

The organoleptic tests were carried out by 10 participants (5 women and 5 men aged between 20–45 years old) trained for this purpose. Additionally, the participants were nonsmokers and without sensory disorders. The organoleptic tests were assessed around 1 pm, about 2 h after a meal and drinking beverages (other than water). Selecting, training, and monitoring individual panelists was guided by PN-EN ISO 8586:2014-03 [38]. The methodology of organoleptic tests was based on PN-ISO 4121:1998 [39]. The tests were carried out by using the point method. Quality features such as appearance, consistency, smell, and taste were defined on the basis of features description [40]. Additionally, the overall organoleptic quality was calculated based on the quality features and weight factors as follows: appearance—0.3, consistency—0.2, smell—0.2, taste—0.3.

2.12. Syneresis Measurement

For the observation of the fermented BSG extracts’ syneresis, a Turbiscan Lab® Thermo (Microtrac Retsch GmbH, Haan, Germany) was used. Syneresis was followed via analysis of the transmitted or backscattered light proportion versus the sample’s height. When the syneresis process progresses, concentration changes can be observed, like sedimentation near the bottom of the sample dish and samples becoming more transparent at the top, where fluid (supernatant) is accumulated. Preparation of the samples for determination of the transmitted and backscattered light proportion consisted in placing the BSG extract with the start culture and optional sugar and stabilizer in glass vials with a capacity of 50 cm³ immediately after the mixing. The measurements were taken at 0, 1, and 24 h during fermentation (the temperature of the measurement cell was set at 37 °C). An additional measurement was performed after 5 days of refrigerated storage. The results are presented as % of changes in transmitted and backscattered light. Syneresis percentage was calculated using the following equation:

$$Syneresis \left[\%\right]=\frac{Height of a leakage zone}{Height of a sample}·100\%$$

2.13. Statistical Analysis

Averages are presented as values with standard deviation (average ± SD). To evaluate significant statistical differences among the studied parameters and storage periods, a one-way variance analysis ANOVA was used. The comparison of averages was performed using the Student’s t-test. Statistical calculations were performed in Statistica 13. Significance was defined as p < 0.05.

3. Results and Discussion

3.1. Moisture Determination

In all analyzed samples, the moisture of fermented products changed in comparison to BSG extract (Figure 3), but it was not statistically significant. Additionally, the changes depended on the stabilizer used and sugar addition. Moisture increase was observed for fermented samples without added stabilizer. In the case of all remaining samples, moisture was lower after fermentation in comparison to fermented samples without added stabilizer, which was expected due to the addition of solids. Fermentation lowered moisture only in the case of yogurt-like samples with guar gum, both with and without sugar (FSG and FPG), and the yogurt-like sample with konjac but without sugar added (FPK). For BSG fermented extracts, moisture content ranged between 88.2 and 91.7%, while non-dairy yogurts made from soy, almond, coconut, and oat available in the market ranged between 53.8 and 58.2% [41]. These differences might result from ingredients, e.g., protein powder, acidity regulator, and stabilizers, present in non-dairy yogurts, since BSG yogurts had a simple formula including BSG extract, stabilizer, sugar, and bacteria.

3.2. pH Determination

The change in pH is a common occurrence during fermentation, as bacteria produce a variety of organic acids. It is caused by the conversion of sugars present in milk or, as in the case of plant-based products, in the extract, into organic acids (especially lactic acid, depending on the starter microorganisms used) [42]. During the fermentation process, the pH value of all samples decreased (Figure 4). The pH value of fermented products for both variants (without and with sugar) decreased in the following order: samples without stabilizer > samples with pectin > samples with guar gum > samples with konjac gum. Therefore, it can be assumed that the addition of stabilizers influenced the pH of fermented BSG extract. However, none of the stabilizers used should have a great impact on the pH. Both guar and konjac gum are classified as non-ionic polysaccharides, which is why their addition should not lower the pH significantly. In the case of pectin, the one used in this study was amidated. During the amidation process, some carboxyl groups are changed into amide groups [43] and do not dissociate, and therefore do not release H⁺ ions and do not lower the pH significantly.

3.3. Protein Determination

There were no statistically significant differences in protein concentration between the fermented products for both dry matter (DM) and wet matter (WM) (Figure 5a,b). Prior to processing for BSG extract preparation, the protein content in BSG was approximately 54.92% DM and 12.63% WM. After extraction, the remaining solid residue (BSG okara) contained about 25.16% and 8.90% protein for DM and WM, respectively. This indicates the efficiency of the protein extraction process and provides a basis for considering either the potential applications of the okara by-product or ways to enhance the extraction process to yield a protein-rich extract. It can be estimated that approximately 30% of the protein (based on wet matter) was successfully transferred to the BSG extract. Furthermore, the protein yield could likely be improved through process optimization, as proteins are commonly associated with fermented dairy products, and thus are expected to be present in fermented alternatives as well.

The concentration of the proteins in the typical plant-based dairy product alternatives is mostly low, ranging from 0.2 (for coconut yogurt) to 4.0 (for soybean yogurt) [41]. Therefore, only some of them are suitable as a protein-rich dairy alternative. With the increasing interest in and demand for plant-based dairy alternatives, there has been growing discussion regarding their composition and ability to meet nutritional requirements [41]. The nutritional profile of these products is largely determined by the primary plant-based raw materials used in their production. BSG contains protein, ranging from 14.2% to 24.7% [18], but many plant-derived ingredients utilized in dairy alternatives are deficient in protein while being rich in carbohydrates. This imbalance may have negative implications for the nutritional status of individuals who rely on these products as a staple component of their diet. Both BSG extract and fermented products appear to be promising solutions to address this gap and meet protein demand, while also enabling the utilization of a brewery by-product.

3.4. Water Activity Measurement

The addition of stabilizers had a slight impact on water activity (Aw) (Figure 6). Notably, the presence of sugar led to an increase in water activity only in the samples containing konjac gum, where Aw rose from 0.88 to 0.93. Water activity is a key parameter influencing microbial growth and, consequently, the microbiological stability and shelf life of food products. In traditional dairy yogurts, Aw typically ranges between 0.97 and 0.99 [44], while in commercially available plant-based yogurt alternatives, it is reported to be between 0.98 and 0.99 [41]. The relatively lower Aw observed in the BSG-based fermented products may be attributed to the high content of suspended particles and water-binding compounds derived from BSG. These components can reduce the amount of free water available in the system, thus lowering the Aw. In general, the higher the purity of water—and correspondingly, the lower the concentration of suspended or dissolved substances—the higher the Aw.

From a microbiological perspective, lower water activity (Aw) values can significantly inhibit the growth of spoilage microorganisms and pathogens, thereby enhancing product safety and stability. Most microorganisms are unable to grow in environments where Aw falls below 0.85; however, some xerophilic molds and osmophilic yeasts can still proliferate at Aw levels as low as 0.60 [45]. A decrease in water activity not only reduces microbial viability but also slows down the metabolic activity of microorganisms, extending the lag phase—the period before rapid microbial growth begins— and thus further enhancing the microbiological stability of the product.

Since the BSG-based fermented samples demonstrated Aw values as low as 0.88, particularly in the absence of added sugar or when certain stabilizers were used, it can be inferred that these products may possess improved microbiological stability in comparison to conventional dairy or plant-based yogurts, which generally have higher Aw levels. Based on these Aw values, it is reasonable to expect that the shelf life of the BSG-based fermented products could be extended under appropriate storage conditions, especially refrigeration. However, to make precise and reliable shelf-life predictions, further studies involving microbial challenge tests and real-time storage experiments would be essential. Nonetheless, the relatively low Aw observed in these samples provides a promising indication of enhanced stability and reduced susceptibility to microbial spoilage.

3.5. Colorimetric Measurements

For all samples analyzed, fermentation resulted in an increase in color parameters L*a*b* (

Table 1. Change in color coordinates during fermentation and storage.

Color Coordinate	Sugar Addition	BSG Extract	FP	FPG	FPP	FPK
L*	Plain	50.35 a ± 0.02	55.56 eB ± 0.02	54.43 dA ± 0.24	51.98 bA ± 0.06	53.77 cB ± 0.02
	With Sugar	-	55.36 dA ± 0.01	54.05 cA ± 0.14	51.90 aA ± 0.010	53.49 bA ± 0.015
a*	Plain	5.79 b ± 0.01	7.03 dA ± 0.01	6.15 cA ± 0.09	6.26 cB ± 0.02	5.76 aB ± 0.01
	With Sugar	-	7.12 dB ± 0.02	6.03 bA ± 0.02	6.20 cA ± 0.01	5.63 aA ± 0.01
b*	Plain	13.83 a ± 0.02	18.29 eA ± 0.04	16.64 dA ± 0.24	15.45 bA ± 0.04	15.84 cB ± 0.02
	With Sugar	-	18.44 dB ± 0.01	16.31 cA ± 0.07	15.43 aA ± 0.02	15.56 bA ± 0.01

a, b, c, d, e—samples with the same letter in the row do not differ significantly; A, B—samples with the same letter in the column do not differ significantly.

). Sugar addition caused only slight changes in color coordinates, which also depend on the stabilizer used. However, both the fermentation process and the type of stabilizer used resulted in a significant increase in all coordinates. Fermentation and the presence of stabilizers increased the brightness (L*) and the b* coordinate value responsible for the yellow color of the samples in the following order: FPP < FPK < FPG < FP. The color of food products is highly bound to the raw materials and ingredients used during the production process. Milk, a raw material for fermented products, has a characteristic creamy-white color; therefore, especially in the case of the L* coordinate, it can reach over 90 [41]. As it was expected, the color of BSG products (extract and fermented yogurt-like products) was characterized by different color coordinate values than dairy products. Montemurro et al. [46] tested yogurt-like products with the inclusion of hemp flour in the formulation. Due to the dark green color of the hemp flour, all the supplemented products exhibited a green hue. In the case of BSG products, depending on the malt used for brewing, they can have different color parameters. BSG used in this study, obtained from pilsner malt, had a yellowish to brown color; therefore, BSG-based fermented products had color coordinates similar to BSG. Adding food colorants can improve the color of plant-based yogurt-like products. Dias et al. [47] evaluated the color stability of betalain- and anthocyanin-rich extracts in a soybean dairy-free alternative to achieve a pleasant pink color in the product and enhance its overall value through potential health benefits.. Besides the visual aspect of plant-based yogurt-like products, which results in increasing consumer acceptance, the nutritional value was higher because the additives used were characterized by antioxidant properties. Considering that consumers are increasingly craving functional foods, BSG fermented products can also be improved by incorporating food additives which, apart from correcting the appearance, will present health-beneficial effects.

Even if differences in color coordinate values were small, they affect the total color differences, and when samples are compared together, depending on ΔE, the observer does not or does notice the difference, or even gets the impression of two different colors. According to

Table 2. The total color differences (ΔE) and sample colors calculated from color parameters.

Sample Name	BSG Extract	FP	FS	FPG	FSG	FPP	FSP	FPK	FSK
BSG extract	0.0 +
FP	7.0 +++	0.0 +
FS	6.9 +++	0.3 +	0.0 +
FPG	5.0 ++	2.2 +	2.2 +	0.0 +
FSG	4.5 ++	2.7 +	2.7 +	0.5 +	0.0 +
FPP	4.8 ++	3.7 ++	3.5 ++	3.0 +	2.9 +	0.0 +
FSP	4.9 ++	3.8 ++	3.6 ++	3.1 ++	3.0 +	0.2 +	0.0 +
FPK	4.0 ++	3.3 ++	3.3 ++	1.1 +	0.6 +	3.1 ++	3.2 ++	0.0 +
FSK	3.6 ++	3.7 ++	3.7 ++	1.5 +	1.0 +	3.2 ++	3.3 ++	0.4 +	0.0 +
Color

⁺ the observer does not notice the difference; ⁺⁺ the observer notices the difference; ⁺⁺⁺ the observer gets the impression of two different colors.

, when samples were compared to BSG extract only in the case of fermented BSG extracts without added stabilizers, the total differences were bigger than 5, which is classified as samples with two different colors. This is a result of the syneresis of fermented samples without stabilizers in their composition. Leakage causes changes in the structure of those samples and makes them not homogeneous. That is why the color is a result of the color of different sample sections, where syneresis (brighter) and sedimentation/agglomeration (darker) are observed. Samples with added stabilizer combined with BSG extract are characterized by ΔE values smaller than 5 and bigger than 3, which means that the observer can notice differences but does not get the impression of two different samples.

3.6. Viscosity Measurement

Both the addition of stabilizers and sugar affected the samples’ viscosity (

Table 3. Initial viscosity [Pa·s] of BSG fermented products.

Sugar Addition	Sample Name
	FP	FPG	FPP	FPK
Plain	0	0.328 bB ± 0.003	0.407 cA ± 0.022	0.208 aB ± 0.026
With sugar	0	0.267 bA ± 0.004	0.463 cA ± 0.035	0.162 aA ± 0.008

a, b, and c—samples with the same letter in the row do not differ significantly; A and B—samples with the same letter in the column do not differ significantly.

and

Table 4. Final viscosity [Pa·s] of BSG fermented products.

Sugar Addition	Sample Name
	FP	FPG	FPP	FPK
Plain	0	0.295 cA ± 0.003	0.273 bA ± 0.007	0.156 aA ± 0.025
With sugar	0	0.240 bA ± 0.003	0.294 cA ± 0.021	0.120 aA ± 0.005

a, b, and c—samples with the same letter in the row do not differ significantly; A—samples with the same letter in the column do not differ significantly.

). The viscosity of the samples without stabilizers was similar to the viscosity of pure water, and the used measuring system did not detect the viscosity of these samples; therefore, the obtained values were 0 Pa·s. However, the chosen measuring system was successfully used for the precise determination of the viscosity of samples with stabilizers. In the case of typical cow milk yogurts, a decrease in the pH during fermentation causes protein gelation and formation of a characteristic gel-like network structure. This is in contrast with plant-based fermented yogurt-like products, where pH decrease causes the destabilization of the plant protein structure and phase separation. Therefore, obtaining plant-based yogurt-like products with both visual and sensory acceptance requires the addition of stabilizers, which affect (create) structure and viscosity [46]. The highest initial viscosity was observed for samples with pectin. Additionally, sugar slightly increased the measured viscosity. However, in the case of these samples, the viscosity was also the most unstable one (Figure 7) and decreased during the experiment (by over 0.1 Pa·s). Samples with konjac and guar gums incorporated had lower (both initial and final) viscosity, which also decreased during the experiment (Table 3 and Table 4). However, the decrease was not as high as for samples with pectin (about 0.03 and 0.05 for guar and konjac gums, respectively). Also, the initial and final viscosity of samples containing sugar and guar or konjac gum were reduced. It can be assumed that the samples with guar and konjac gums were more stable, resistant to shearing structures (

Table 5. Viscosity difference (Δ) [Pa·s] of BSG fermented products.

Sugar Addition	Sample Name
	FP	FPG	FPP	FPK
Plain	0	0.034 aB ± 0.004	0.134 cA ± 0.026	0.051 bA ± 0.006
With sugar	0	0.028 aA ± 0.001	0.168 cA ± 0.020	0.043 bA ± 0.004

a, b, and c—samples with the same letter in the row do not differ significantly; A and B—samples with the same letter in the column do not differ significantly.

). The viscosity of yogurts and plant-based yogurt-like products highly depends on the amount and type of proteins, pH, and the type and concentration of stabilizers used. The decrease in pH can reduce the stability of some gums used. Like in the case of BSG-based fermented products, fermentation results in phase separation and creates watery products with low viscosity. Therefore, stabilizers are the main factor affecting viscosity and rheological properties. This is one of the reasons why the results of different studies indicate different viscosity values. Marlapati et al. [41], who analyzed the viscosity of commercially available dairy and plant-based yogurts, obtained results in the range of 1.6 to 3.3 Pa·s (for oat and dairy yogurt). On the other hand, the viscosity of soy yogurts was over 34,000 cP (over 34 Pa·s) [48], and for the phytosterol-enriched dairy yogurts and control, it ranged from 27,400 to 34,500 cP (27.4 and 34.5 Pa·s, respectively) [49]. Additionally, viscosity can change during storage as a result of the retrogradation process and syneresis, which results in the release of water from the protein and/or stabilizer matrix.

3.7. Organoleptic Tests

During the organoleptic assessment, four different features were rated (Figure S3). The sugar addition did not cause statistically significant differences in the score of each property. On the other hand, stabilizer addition had a significant impact on the assigned scores. For appearance, consistency, and taste, FP samples were rated as the worst; for smell, they were rated similarly to the others. For the taste, all samples except FP were rated above 3.0, without statistically significant differences among them.. For other features, FPP samples were rated with the highest score, which was about 4.0 or even up to 5.0 for consistency. Based on the result and calculations, the overall organoleptic quality was determined as the highest for FPP samples and the lowest for FP (

Table 6. Overall organoleptic quality.

Sugar Addition	Sample Name
	FP	FPG	FPP	FPK
Plain	1.76	2.92	3.80	2.82
With sugar	2.00	2.44	4.00	3.28

). For food products, organoleptic tests and acceptance by consumers play a crucial role in the products’ future [50]. One of the main issues in the sensory properties of plant-based yogurt-like products is their consistency and appearance (color). Dairy yogurts are characterized by a milky-white color and a creamy, firm, and smooth homogenous structure. Due to the different types of proteins and phase separation during fermentation, the consistency of products without stabilizers was rated as the worst, which correlates with the viscosity results. In the case of appearance, it mostly depends on the color, syneresis, and homogeneity. As mentioned above, color parameters depend on the raw material used and its color. In the case of the BSG-fermented products, stabilizers did not affect the color but had a great impact on the homogeneity and water leakage. That is why the consistency of the products with stabilizers was rated higher than FP and FS.

3.8. Syneresis Measurement

Syneresis is an undesirable phenomenon where liquid is expelled from a large number of solid products, e.g., fermented dairy products [51]. Excessive syneresis can negatively affect the texture of the product, which is why in the food industry, stabilizers, thickeners, and gelling agents are used [52]. Those compounds can represent different chemical structures and have different origins, but they are used with the same purpose: to alter the food texture [53,54]. To prevent syneresis and to ensure the desired texture, three food additives were used, i.e., guar gum, pectin, and konjac gum. Concentrations of the gelling agent were selected on the basis of the stability studies of BSG extracts with added compounds at concentrations from 0.1 to 1% (Figures S1 and S2 in the Supplementary Material). The syneresis of BSG fermented products was analyzed with the use of Turbiscan Lab® Thermo (Microtrac Retsch GmbH, Haan, Germany). The device provides information on the percentage of transmission (%T) and the proportion of backscattered light (%BS). These data allow for the assessment of occurring changes, particularly syneresis, sedimentation, and agglomeration. On the basis of the proportion of %T in function of height, the extent of the leakage layer was calculated (

Table 7. Extent of syneresis in fermented BSG extracts over time.

Sample Name	Leakage During Fermentation and Storage [%]
	0 h	1 h	24 h	120 h
FP	0.0	0.4	24.4	29.4
FS	0.0	3.2	26.6	30.6
FPG	0.0	0.0	1.0	2.1
FSG	0.0	0.0	0.0	0.0
FPP	0.0	0.0	0.0	0.0
FSP	0.0	0.0	0.0	0.0
FPK	0.0	0.0	0.0	0.2
FSK	0.0	0.0	0.0	7.5

). During fermentation and storage of BSG extracts without any stabilizers, phase separation and leakage on the top of the sample can be observed. Already in the first hour of fermentation, leakage appeared, greater for the BSG extract with sugar: 0.1% without sugar and 2.4% with sugar. While samples with stabilizers did not show any major changes in structure after fermentation, the samples without additives were already characterized by syneresis at the level of 25%. On the 4th day of storage (5 days after preparation), leakage was observed only in the case of FPG and FSK among the samples with stabilizers. Still, it was 4 to 15 times smaller than in the case of BSG extracts without added gelling agents. The addition of stabilizers helps to reduce leakage in BSG yogurt-like products. Vareltzis et al. [55], instead of texture additives, supplemented dairy yogurts with proteins, i.e., whey protein concentrate, albumin, and sodium caseinate, and analyzed their influence on sebum separation. The control sample was characterized by high syneresis at a level around 25%, while adding 5% albumin and 5% of the protein mixture exhibited the least syneresis, due to gel formation.

The Turbiscan Stability Index (TSI) is a parameter that evaluates quantitatively the stability of any formulation and is used to predict the stability of products, e.g., dairy yogurts [56]. TSI values of samples with pectin, guar, or konjac gums are similar (Figure 8), while FP and FS are characterized by 5 to 7 times higher values of TSI. TSI of samples with gelling agents added ranged from 2.0 to 2.8 and from 2.8 to 3.6 after 1 and 5 days, respectively. The observed small increase in TSI during storage suggests that samples are stable due to the formation of the gel network [57].

4. Conclusions

Brewers’ spent grain (BSG), a by-product of the brewing industry, presents a promising raw material for the production of non-dairy products, especially beverages and fermented foods. Given its high protein content, BSG has the potential to serve as a new source for yogurt-like assortments. Studies confirm the significant potential of BSG for use in plant-based dairy alternatives.

Fermented BSG extracts contain approximately 5% protein, which is higher than that of many plant-based yogurt-like products currently available on the market. Moreover, the protein content can be further increased by optimizing the BSG extraction process, since the extraction resulted in liberating 30% of the protein from BSG. The fermentation conditions and starter cultures applied in this study enabled the production of yogurt-like products characterized by an optimal pH range of 4.0 to 4.3, which corresponds with the pH of fermented dairy products. Syneresis analysis indicated the necessity of stabilizers, which effectively minimized or eliminated this undesirable phenomenon. Additionally, viscosity measurements demonstrated the need for texturizing agents to achieve a gel structure similar to that of dairy yogurts. Organoleptic tests showed positive consumer acceptance of the yogurt-like variant formulated with 0.5% pectin and 1% sugar. According to the panelists, the overall rating of this BSG yogurt-like product was 4.0, indicating good quality. The highest scores were awarded for consistency, appearance, and the absence of syneresis confirming the effectiveness of pectin addition.

The proposed utilization of BSG allows for at least partial valorization of this industrial by-product. This innovative approach aligns with current trends in the circular economy, waste management, and sustainable food production while addressing the growing demand for high-nutritional-value vegan products. Future research should focus on exploring the use of stabilizer combinations to further optimize the texture, consistency, and overall sensory appeal of the fermented BSG-based yogurt-like products. Additionally, the incorporation of various flavor variants could enhance consumer acceptance and support the development of a market-ready product suitable for large-scale production. These efforts would contribute to the creation of a functional, sustainable plant-based product with clear commercial potential. Moreover, further studies could investigate the bioavailability and functionality of bioactive compounds and amino acids present in BSG after fermentation, potentially uncovering new health-promoting properties. Overall, this research opens promising avenues not only for upcycling brewing by-products but also for expanding scientific knowledge in the field of sustainable food innovation.