The Novel Strain of Gluconobacter oxydans H32 Isolated from Kombucha as a Proposition of a Starter Culture for Sour Ale Craft Beer Production

Abstract

Acetic acid bacteria (AAB) has found applications in food technology, including beverages and vinegar. Generally, AAB shows several beneficial properties and has technological usefulness. Properly selected and tested strains of this group of bacteria may constitute a new and interesting solution among starter cultures for functional food. Therefore, the study aimed to develop a sour beer technology, based on the novel strain Gluconobacter oxydans H32. The microbiological, physical-chemical (HPLC method), and sensory (QDP method) quality were determined during 6 months of storage of dark and light beer samples. The AAB count at the beginning of storage was approximately 8 log CFU mL⁻¹, and 6 log CFU mL⁻¹ after 6 months of storage. As a result of the metabolic activity, acetic acid, gluconic acid, and ascorbic acid were detected in the samples. The light beer had a significantly better sensory quality, especially sample BPGL with the addition of G. oxydans H32 starter culture. It was found that it is possible to develop a functional beer with the novel strain Gluconobacter oxydans H32. These Sour Ale craft beers were not only a good source of H32 strain but also its pro-health metabolites.

1. Introduction

According to the data of the World Health Organization (WHO), beer is one of the most consumed alcoholic beverages worldwide. Beer is a malt beverage with an alcohol content greater than or equal to 0.5%, obtained as a result of alcoholic fermentation in which sugars are converted by brewer’s yeast into ethanol, carbon dioxide, and other fermentation products [1,2,3,4]. According to Beer Judge Certification Program [5], craft beers are “ale” and “lager” styles which are in opposition to the popular mass-produced corporate beers. Nowadays, the main trend in the brewing market is to look for various kinds of innovation. Krennhuber et al. [6] suggest that beer-based fitness beverages nowadays are a great alternative to widespread “synthetic” drinks. Rednondo et al. [7] and Horn et al. [8] showed that craft beers had higher values of phenolic content and antioxidant capacity than commercial beers. Unconventional ingredients and new fermentation cultures are increasingly used. To this end, brewers use both historical, once forgotten styles and create new, original compositions [9]. For their production, in addition to traditional ingredients such as malt and hops, unconventional and modern additives are used [3]. For the production of craft beers, traditional and also unconventional fermentation techniques such as co-fermentation by yeast and bacteria are often used, although these methods are not used in mass production. For example, fermentation cultures including yeast species other than Saccharomyces cerevisiae and Saccharomyces pastorianus are also utilized. However, these two types of yeast are mostly used in the production of beer. The selection of the appropriate strain determines the characteristics of the beer obtained. This is due to strain differences in metabolized aromatic compounds. Most often, S. pastorianus yeast and some strains of S. cerevisiae such as US-05 are used for the production of beers with a pure aromatic profile. For beers with a high ester content, Belgian and German yeast S. cerevisiae is more often used [10].

Moreover, bacterial cultures (lactic as well as acetic acid bacteria) may also be used to produce beer with the desirable characteristics [1,11,12]. These beer styles include sour beers, which are produced with the use of bacteria that lower the pH of beer. There are two ways to acidify beer or wort with bacteria. The first method is the traditional method of spontaneous fermentation with the participation of various types of microorganisms. The second method consists of adding selected strains with specific technological properties and conducting the process under standardized conditions. A new approach is the use of starter cultures of bacteria with health-promoting properties, which additionally introduce a characteristic flavour and odor [13]. Usually, research in this area focuses on a known group of lactic acid bacteria (LAB) exhibiting probiotic properties [1]. A new solution is isolating from naturally fermented food and searching for strains of bacteria from outside the LAB group [14,15]. Therefore, designing starter cultures from well-defined and safe AAB strains is an important and future-oriented aspect in the development of the fermented food segment with health-promoting features.

Kombucha is a fermented drink that is much less popular than beer. This beverage is obtained by fermenting a sweetened tea infusion by a symbiotic (AAB, LAB, and yeast) starter culture—SCOBY [16]. The available scientific research points to some health-promoting properties of Kombucha, including anti-stress and anti-cancer properties, supporting immunity, or the biosynthesis of vitamin C and organic acids with beneficial effects on the human body [16]. Therefore, microorganisms isolated from Kombucha and then tested for their technological suitability and also for their health-promoting properties, are valuable materials for the design of new starter cultures for food fermentation.

According to the definition of the Food and Agriculture Organization of the United Nations (FAO), WHO and as amended by the International Scientific Association for Probiotics and Prebiotics (ISAPP), probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host [17,18]. However, these criteria are not law. In the European Union, EFSA (European Food Safety Authority) uses a Qualified Presumption of Safety (QPS) approach [19]. The newly notified taxonomic unit is preliminarily assessed by taking into account aspects such as related knowledge base, history of apparent safe food use, scientific literature as well as clinical aspects, and industrial application. New strains as candidates for food fermentation starters must therefore obtain the status of safe to use and also derive from traditional food ingredients. They should also belong to a species defined as safe, e.g., Gluconobacter or Komagataeibacter [20]. Moreover, Gluconobacer oxydans is species safe in terms of use in food, which was the only AAB classified on the QPS list [19,20,21]. They have found applications in food technology, including for the production of beverages and vinegar. However, AAB have often been considered contaminants in beer. There is no evidence of attempts to use these bacteria in the production of sour beer, except for spontaneous fermentation beers. Nevertheless, the properties of compounds metabolized by some species of AAB may confer the sour-type beer the appropriate sensory characteristics. An example is gluconic acid, which is characterized by a slight sweet and acid flavour note [22]. Such properties of bacterial metabolites allow for the use of AAB in the production of low alcoholic beverages.

Therefore, in this study, we attempted to obtain a beer-like beverage using a novel and safe AAB strain—Gluconobacter oxydans H32. Ultimately, the presented study aimed to develop a novel sour beer technology, based H32 strain and to determine their effects on the microbiological, physicochemical, and sensory properties of the produced beer beverages.

2. Materials and Methods

2.1. Preparation of Gluconobacter oxydans H32 Starter Culture

The strain Gluconobacter oxydans H32 was obtained from the collection of the Technical University of Łódź in Poland. The studied strain meets the safety criteria of origin [19,23] and selected in vitro criteria set for probiotic bacteria by FAO/WHO [17]. Based on the conducted preliminary studies by Neffe-Skocińska et al. [24], that the selected AAB novel strain H32 can survive in the model human gastrointestinal tract (at least 6 log CFU mL⁻¹) and showed very good sensitivity to most of the 12 antibiotics used, including vancomycin. Strain H32 was obtained from a safety food source (Kombucha beverage).

Bacteria were activated from a frozen culture at −80 °C by incubation at 28 °C for 48 h in 5 mL GC broth (Glucose Calcium Carbonate) consisting of 20 g kg⁻¹ glucose (Sigma-Aldrich, Poznan, Poland), calcium carbonate 7 g kg⁻¹ (Sigma-Aldrich, Poznan, Poland) yeast extract 3 g kg⁻¹ (Sigma-Aldrich, Poznan, Poland), casein peptone 3 g kg⁻¹ (Merck, Poznan, Poland) and distilled water 967 g kg⁻¹. After incubation, the tubes were centrifuged for 5 min at 10.000 rpm (laboratory centrifuge MPW-251; MPW MED Instruments, Warsaw, Poland) to separate G. oxydans H32 cells from the medium. The supernatant was replaced with 8.5 g kg⁻¹ saline and the centrifugation procedure was performed thrice to remove the residual growth medium. The saline was then replaced with 10 mL of pasteurized cherry juice or plum juice (Döhler, Kozietuły Nowe, Poland) and incubated for 72 h at 28 °C to achieve H32 count on average 8 log CFU mL⁻¹.

2.2. Beer Technology and Sampling Procedures

The scope of the presented study was wide so as to ensure that the H32 has technological properties and can be used as an innovative starter culture for the production of craft beer beverages.

The study material consisted of two variants of beer beverages (light and dark) fermented under laboratory conditions with or culture. Ingredients (malt and water) were prepared once for light and dark beer and fermented. Light and dark beer were prepared separately. After fermentation, three independent replications of each sample (BJPD, BGD and BPGD in case of dark beer, and BPGL, BGL and BJPL in case of light beer) were prepared. For each sample replication, separate juice samples were used to inoculate the beer. From each sample, beer was poured into eight bottles of 330 mL. Samples for analyses were taken randomly from bottles (Figure 1,

Table 1. Variants of study beer beverages and their composition.

Types of the Base Beer	Designation of Samples			Description of Research Sample	Abbreviation of Research Sample
	Pasteurization Process	Juice Addition	AAB Inoculum
Light “L”	+	-	-	Control 1: pasteurized light beer without juice and AAB	CL
	+	+	-	Control 2: pasteurized light beer only with juice	BJPL
	+	+	+	Pasteurized light beer with juice and AAB	BPGL
	-	+	+	Unpasteurized light beer with juice and AAB	BGL
Dark “D”	+	-	-	Control 1: pasteurized dark beer without juice and AAB	CD
	+	+	-	Control 2: pasteurized dark beer only with juice	BJPD
	+	+	+	Pasteurized dark beer with juice and AAB	BPGD
	-	+	+	Unpasteurized dark beer with juice and AAB	BGD

Explanatory notes: “+” was used; “-” was not used.

). The preparation protocols of samples are described in the following sections of the article.

Microbiological, physicochemical, sensory analyses and pH measurements were conducted directly after the re-fermentation process in the bottle and after the 6 months of storage bottles at 10 °C. Detailed technologies for the production of basic beer beverages and research beer beverages (“AAB Beer” starter culture addition) are described below.

2.2.1. Preparation of Basic Beer Beverage

The beer samples were prepared in light and dark versions. Pale Ale malt (Viking Malt, Strzegom, Poland) was used to produce both types of beer. Additionally, roasted barley (Viking Malt, Strzegom, Poland) was added only to the dark beer. The beer wort was prepared using the single infusion mashing method for one hour at 68 °C in an automatic brewery kettle Coobra CB3 PRO (CBF Drinkit; Möltand, Sweden). Roasted barley was added to the dark beer mash for the last 15 min of mashing. The mash was then heated to 78 °C and held at that temperature for 15 min. Then, the wort was filtered and the draff was sparged with water at 85 °C. The obtained wort was boiled for an hour. The wort was cooled by flow through a sterile Nordic Tec heat exchanger (Szydlowiec, Poland) to 20 °C. The wort was aerated by gravity by flowing freely into the fermentation container. The yeast S. cerevisiae US-05 (Lesaffre; Marcq-en-Barœul, France) was added to the wort according to the manufacturer’s instructions, at a rate of 7 log CFU per 1 mL of the wort. The starting extract of the wort was 100 g kg⁻¹ (10 °C Blg). Fermentation was carried out at a temperature of 16 °C for 10 days until the final extract stabilized at 5 g kg⁻¹ (0.5 °C Blg). The extract was measured with a Balling hydrometer (Alla, Chemillé en Anjou, France). The described fermentation process concerned light and dark beer. Basic beer beverages after pasteurization were used as control samples (CL—control light beer, CD—control dark beer). Pasteurization was carried out at 90 °C for 10 min.

2.2.2. Preparation of AAB Beer

The beer samples were mixed with AAB starter culture prepared in juice in a ratio of 3:1, respectively, and then bottled. The AAB starter culture prepared in cherry juice was combined with light beer (BPGL, BGL) while the plum juice was used to prepare culture when mixed with dark beer (BPGD, BGD). The juices used were adjusted to the type of beer (light or dark). The criterion was that the juice addition does not adversely affect the sensory experience, which was proved under preliminary testing. The samples BJPL and BJPD contained juice without AAB and were fully pasteurized. The beer samples were bottled in 330 mL dark glass capped bottles and re-fermented at 20 °C for 10 days. After the re-fermentation period, samples were stored at a temperature of 10 °C for 6 months.

2.3. Microbiological Analyses

The AAB and yeast count were determined in all variants of beer beverages. Furthermore, 1 mL of the test sample was diluted in 9 mL of sterile peptone water (Biocorp, Warsaw, Poland), following which serial dilutions were made. The number of AAB was determined by the spread plate technique. The growth medium for AAB contained of 3 g kg⁻¹ casein peptone (Merck, Poznan, Poland), 3 g kg⁻¹ yeast extract (Merck, Poznan, Poland), 7 g kg⁻¹ calcium carbonate (Sigma-Aldrich, Poznan, Poland), 20 g kg⁻¹ glucose (Sigma-Aldrich, Poznan, Poland), 20 g kg⁻¹ agar (Sigma-Aldrich, Poznan, Poland), 20 mL kg⁻¹ ethanol 96% (Chempur, Piekary Śląskie, Poland) and 50 mg L⁻¹ natamycin (Sigma-Aldrich, Piekary Śląskie, Poland). The Petri plates with the solidified medium and the serial dilution of the samples were incubated at a temperature of 28 °C for 72 h. The number of yeasts was determined by the spread plate technique. The growth medium was YGC agar (Biokar Diagnostic, Allonne, France). Serial dilutions of the sample were applied to the Petri dishes with the solidified growth medium, spread with a sterile spreader, and incubated for 5 days at a temperature of 25 °C. For each sample, all serial dilutions from the decimal dilution to the 10-millionth dilution were plated to obtain quantifiable microbial growth. After incubation, typical colonies were counted. Microbial colonies were counted in Petri dishes to verify if they were in the range of 30–300 colonies. If the number of colonies was above or below this range, it was not counted.

2.4. Physicochemical Analyses

2.4.1. Determination Content of the Organic Acids

The organic acid composition of the beer beverages was determined according to the method suggested by Flores et al. [25] with slight modifications. For the analysis, 2.5 mL of beverage were extracted with 7.5 mL of water for 30 min (at 25 °C, with stirring at 250 rmp). The mixtures were centrifuged at 15,133 rmp at 4 °C and the supernatants were filtered through a 0.45 µm syringe filter and then passed through a Sep-Pak^® C18 cartridge (Waters, Milford, MA, USA). The cartridge was preconditioned by washing with methanol followed by phosphoric acid solution (1.0 g L⁻¹). The organic acids were removed from the cartridge by a phosphoric acid solution to a volumetric flask for 25 mL. Finally, the samples were filtered with 0.45 µm nylon membrane for HPLC analysis. A Prominence HPLC system (with LC-20AD pump, SPD M20A detector, SIL-20A HT autosampler, degasser, column oven, and LC solution data collection software, Shimadzu, Kyoto, Japan) was utilized for sample analysis. Separation was carried out using a Cosmosil 5C18-PAQ (4.6 mm × 150 mm) column (Waters, Milford, MA, USA) at 25 °C. Additionally, 20 mmol phosphoric acid was used as the mobile phase at a flow rate of 1 mL/min. The injection amount was 20 µL and the organic acids peaks were detected at 210 or 254 nm. The peaks were identified via a comparison of the retention time and UV-vis spectroscopic data with authentic standards. The content of organic acid was expressed as mg per 100 mL of beer beverage.

2.4.2. Determination of Sugars and Ethanol Content

The analysis of sugar and ethanol in beverages was performed according to the procedure described by Usenik et al. [26] and Monošík et al. [27] with modifications. The equipment used was the Prominence HPLC system described above connected with a refractive index detector. Then, 1 mL of beverage was diluted in 9.0 mL of ultrapure water, filtered through a 0.45 µm nylon membrane and a volume of 20 µL was injected into HPLC for analysis. Each sample was diluted in triplicate before HPLC analysis. Column (RezexTM RCM-Monosaccharide Ca+, 300 × 7.8 mm, Phenomenex, Torrance, CA, USA) was thermostated at 65 °C. Sugars (sucrose, glucose, fructose, maltose) and ethanol were eluted with Milli-Q water at a flow rate of 0.6 mL/min. Individual sugars and ethanol were identified by comparison of retention times with authentic standards and their concentration was determined by the external standards methods. The content of sugars and ethanol were expressed as g or mL per 100 mL of beer beverages, respectively.

2.4.3. Determination of Vitamin C Content

Vitamin C (the sum of ascorbic acid and dehydroascorbic acid) content in beer beverages was determined according to the method outlined by Chebrolu et al. [28] with modifications. To analyze the total vitamin C content, dehydroascorbic acid was reduced to ascorbic acid by the addition of dithiothreitol (DTT). A total of 5 mL of the beverage was diluted for an equal volume of 2% metaphosphoric acid, shaken to ensure thorough mixing and centrifuged for 10 min at 15,133× g at 4 °C. The supernatant was filtered on a nylon 0.45 µm pore size membrane filter. For ascorbic acid analysis, the sample (300 µL) was mixed with of equal volume of 2% metaphosphoric acid and immediately injected into HPLC. For total ascorbic acid analysis, the sample (300 µL) was mixed with an equal volume of 10 mmol/L DTT and after 30 min the sample was introduced into HPLC. Separation was carried out using an Onyx Monolithic column (100 × 4.6 mm) with guard cartridge Onyx Monolithic C18 (Phenomenex, Torrance, CA, USA) at 25 °C with a mobile phase of 0.1% phosphoric acid eluted with a flow rate of 1 mL/min. The injection amount was 20 µL and the ascorbic acid peak was detected at 254 nm. The concentration was calculated based on a standard L-ascorbic acid calibration curve and expressed as mg per 100 mL of beverages.

2.4.4. pH Measurement

The acidity of the beer beverages was determined with the use of a pH meter (ELMETRON CP551, Zabrze, Poland) with a precision of up to 0.01.

2.5. Sensory Analyses

A sensory analysis was performed with the Quantitative Descriptive Profile (QDP) method (ISO 13299:2016) [29] after the re-fermentation process. Before the analysis started, a panel, made up of 16 experts, was trained in terms of sour beer standards and extensively and formally tested before being selected, according to the ISO standard (ISO 8586:2012) [30]. The panelists had 4 to 18 years of theoretical and practical experience with sensory procedures and sensory evaluation of different food products using various methods. The assessors’ ability to differentiate product samples by various concentrations of volatile and non-volatile stimuli was verifiedUnstructured linear scale [0–10 conventional units (c.u.)] was used. Firstly, a discussion among experts was performed, after which the discriminants of the beer were defined. During analysis, the following characteristics were assessed: the intensity of beer, fruit, sour, acetic, honey, and other odors. Furthermore, clarity, colour brightness, and carbonation, as well as the intensity of beer, fruit, sour, acetic, tangy, and other flavors together with the overall quality were evaluated. The odor was defined as a sensation that was caused when smelling the sample. The flavour was defined as the sensation after tasting the sample. Flavour had dual effects on the taste and smell senses. For the discriminants related to the intensity of odor and flavour, the boundary terms of the scale were “none” and “very strong”. Appropriate boundary terms have been adjusted for determinants such as clarity and colour brightness. Boundary terms related to the overall quality of products were “low” and “high”. The overall quality in the QDP method is expressed as the harmonization of all product characteristics and is not related to the expression of the hedonic quality. The beverages for analysis were placed in 25 mL transparent plastic containers with a lid. The samples were coded and given for analysis in random order. The analysis was performed under laboratory conditions at a temperature of 20 °C with constant light in separate evaluation rooms. Furthermore, water at room temperature was added between the samples to neutralize the taste.

2.6. Statistical Analysis

Statistical analyses were performed using Statistica 13.3 software (StatSoft, Cracow, Poland). The arithmetic means and standard deviation (SD) were calculated. The analyzed data had a normal distribution which was checked with the Shapiro–Wilk test. The homogeneity of variance was checked using the Brown-Forsythe and Levene tests. A multi-factor analysis of variance ANOVA and Bonferroni post hoc test was used to analyze the data. The results of the sensory analysis were subjected to a principal components analysis (PCA), where a correlation matrix was used. The difference was considered statistically significant when p < 0.05 with regard to the number of microorganisms, the results of chemical analyses, pH value, and the results of sensory evaluation. Error bars in figures and values after “±” in tables represent SD. All the laboratory analyses were performed in triplicate.

3. Results and Discussion

3.1. Microbiological Analyses and Survival of AAB Starter Culture in Beer Beverages

Table 2. Changes in the total count (log CFU mL⁻¹) of yeast, AAB, and pH values in study beer beverages after re-fermentation and after 6 months of storage time.

Sample	Yeast		AAB		pH
	Storage (Months)
	0	6	0	6	0	6
CL	nd aA	nd aA	nd aA	nd aA	4.10 aA ± 0.02	4.12 aA ± 0.03
BJPL	nd aA	nd aA	nd aA	nd aA	3.71 bA ± 0.02	3.75 bA ± 0.04
BPGL	nd aA	nd aA	8.14 bA	6.39 bB	3.54 cA ± 0.04	3.42 cA ± 0.03
BGL	5.45 bA	2.74 bB	8.04 bA	5.06 cB	3.39 dA ± 0.01	3.34 cdA ± 0.03
CD	nd aA	nd aA	nd aA	nd aA	4.06 aA ± 0.03	4.09 aA ± 0.04
BJPD	nd aA	nd aA	nd aA	nd aA	3.75 bA ± 0.01	3.75 bA ± 0.04
BPGD	nd aA	nd aA	8.17 bA	4.20 cB	3.26 dA ± 0.02	3.31 dA ± 0.04
BGD	2.01 cA	nd aB	7.91 bB	7.19 dB	3.48 cA ± 0.04	3.45 cA ± 0.03

Explanatory notes: nd—not detected; means in the same column followed by different lowercase letters of the alphabet represent significant differences between samples, means in columns in the same line followed by different uppercase letters of the alphabet represent statistic differences between the storage time of one sample according to the ANOVA test; the statistical differences in the lower case apply to all samples in one column; statistical differences in capital letters refer to only one sample in terms of one examined parameter (in rows) (p < 0.05); n = 3.

presents the changes in the total count of yeast, AAB, and pH values in the beer beverages after the re-fermentation process and after 6 months of storage. In this research variant of study beer beverages, significant changes in the number of AAB were found between 0 and after 6 months of storage (p < 0.05). These changes were also found between the light (BPGL) and dark (BPGD) types of the study beers. In this case, the count of AAB is related only to the survival of the starter culture G. oxydans H32. In light beer, the number of AAB decreased by two logarithmic levels and was still high after 6 months of storage (approximately 6.39 log CFU mL⁻¹). In dark beer, the total count of AAB decreased by as much as four logarithmic orders and was on average 4.20 log CFU mL⁻¹ at the end of the experiment.

Statistical differences in the number of AAB between the samples of light beer with their addition (BGL—unpasteurized light beer inoculated with the studied starter culture G. oxydans H32 and BPGL—pasteurized light beer inoculated with the studied starter culture G. oxydans H32) were not observed before storage period (p > 0.05). After the re-fermentation process, the AAB count was approximately 8.04 log CFU mL⁻¹ and after 6 months of storage, the number of AAB decreased approximately by about three logarithmic levels in the BGL sample (p < 0,05). Significant differences (p < 0.05) of the total count of AAB were found also in the BGL variant of the studied beer between BPGL after storage time. In the sample, BPGL survived by 1.33 log CFU mL⁻¹ more bacteria than in the BGL sample, whereas there were no statistically significant changes in the number of AAB in the sample BGD of dark beer. The count of bacteria was on average at the level of 7 log CFU mL⁻¹ in the case of this sample (p > 0.05). Based on these results, it can be concluded that the survival rate of G. oxydans H32 differs depending on the presence of live yeast cells, raw materials used in production, and the beer variant. The survival of these bacteria was influenced by the presence of live yeasts and the type of beer (light/dark). Nevertheless, the high survival rate of G. oxydans H32 bacteria was present in all types of beer. Compared to the studies by Calumba et al. [31], the number of bacteria added in our study remained at a similar level. However, in our research, the beer was stored 5 months longer than in the Calumba research. Compared to the research conducted by Calumba et al. [31], the number of added bacteria was similar to that in the presented studies after only 24 days. These results are suggestive of the high survival of AAB in beer despite the lack of access to oxygen. On the other hand, Alcine Chan et al. [1] demonstrated that in an unpasteurized sample of craft beer, a combination of live Saccharomyces cerevisiae S-04 and low temperature significantly enhanced the survival of the probiotic lactic acid bacteria (LAB). According to Silva et al. [13] and Narvhus and Gadaga [32], other intrinsic chemical constituents in beer, including yeast, ethanol, and carbon dioxide may enhance probiotic viability and stability. Although, Suharja et al. [33] observed an enhancement in their probiotic survival in acidic environments such as fermented milk, possibly due to the formation of mixed-species biofilms which protect probiotic bacteria from external stress.

The obtained results of the microbiological analyses were used in determining the total count of AAB and were carried out to confirm the survival ability of a novel starter culture of G. oxydans H32 in the variants of beer beverages. G. oxydans H32 was isolated from the natural microbiota of local Kombucha drink, identified and classified to the AAB group as safe for humans [19,23]. Furthermore, H32 has been subjected to preliminary in vitro tests recommended by FAO/WHO [17]. Despite the fact that the G. oxydans H32 represents some in vitro beneficial properties [28] it can not be included in the probiotic bacteria group, due to a lack of scientific evidence of the health benefits. However, the study strain can be a good candidate for the starter culture. With regard to its metabolic activities, it produces many metabolites with a beneficial effect on human health, as well as creating technological features. The presented research results are intended to confirm the technological use of the tested bacterial strain.

The AABs show high adaptability to the prevailing environmental conditions, unlike most known probiotic strains belonging to LAB, which is an important technological feature in food production. According to the definition of FAO/WHO, the minimum number of probiotic bacteria in a gram or millilitre of the food product is 6 log CFU to confer the product with health-promoting, probiotic properties. The high number of AAB bacteria in the sample of the study BPG beer proves that even after 6 months of storage, a properly selected strain of AAB can adapt to environmental conditions, ensuring that the final product has optimal technological properties. Furthermore, the use of innovative starter culture in beer brewing allows for the expected product quality and the development of a new type of beer beverage [34].

Based on the obtained results, it can be concluded that the conditions created in this type of drink are appropriate for the growth and survival of G. oxydans H32. The factor facilitating the adaptation of starter G. oxydnas H32 to beer-based conditions was the method of preparing the starter culture, i.e., transferring the inoculum of study bacteria into cherry (light beer) and plum (dark beer) juice as the optimal strain carrier. The selection of such a carrier when designing the starter culture for the studied beer beverages was justified by the fact that the natural habitat of Gluconobacter sp. is commonly in the flower and fruit environment, in sweet, sour soft, and low ethanol drinks. Therefore, these microorganisms are also adapted to difficult environmental conditions, i.e., high acidity and a significant concentration of sugars and alcohols [35,36].

3.2. The Content of Organic Acids, Sugars, Ethanol, and Vitamin C

During the mashing process, polysaccharides undergo hydrolysis under the influence of enzymes and acids (

Table 3. Average results of physicochemical analyses (ethanol, vitamin C, and sugars) in study beer beverages after the re-fermentation process.

Sample	Ethanol [mL 100 mL−1]	Vitamin C [mg 100 mL−1]	Glucose	Fructose	Maltose	Sucrose
			[g 100 mL−1]
BJPL	2.82 a ± 0.04	1.03 a ± 0.06	1.13 a ± 0.01	0.83 a ± 0.01	0.27 a ± 0.01	0.18 a ± 0.02
BPGL	2.72 ab ± 0.03	2.83 bf ± 0.06	0.34 b ± 0.02	0.03 b ± 0.01	0.23 b ± 0.01	nd b
BGL	3.14 c ± 0.04	2.03 c ± 0.06	nd c	0.01 cg ± 0.00	0.24 b ± 0.01	0.03 c ± 0.00
CL	3.79 d ± 0.09	nd d	0.05 d ± 0.01	nd cd	0.33 c ± 0.01	nd b
BJPD	2.53 b ± 0.02	1.57 e ± 0.06	0.65 e ± 0.02	0.02 bc ± 0.01	0.39 d ± 0.01	nd b
BPGD	2.94 ac ± 0.03	2.43 f ± 0.06	0.99 f ± 0.02	0.67 e ± 0.01	0.23 b ± 0.01	0.03 c ± 0.00
BGD	3.22 c ± 0.13	2.60 bf ± 0.20	nd c	0.09 f ± 0.01	0.39 d ± 0.01	nd b
CD	3.28 c ± 0.08	nd d	0.03 cd ± 0.01	nd dg	0.52 e ± 0.01	nd b

Explanatory notes: nd—not detected; means in the same column followed by different lowercase letters of the alphabet represent significant differences between the samples according to the ANOVA test; the statistical differences in the lower case apply to all samples in one column (p < 0.05); n = 3.

). Afterwards, the role of yeasts in the fermentation process is to convert sugar into alcohol and carbon dioxide [37,38]. Therefore, at this stage of the experiment, control samples “C” (CL, CD) (CL—control light beer, CD—control dark beer,), i.e., basic beer, where the amount of ethanol was 3.79 mL 100 mL⁻¹ of light beer and 3.28 mL 100 mL⁻¹ of dark beer, this alcohol content is deduced by the mashing parameters used. Additionally, in unpasteurized beer samples with the addition of the AAB starter culture, the amount of ethanol was at a comparable level (Table 3). Significantly lower amounts of ethanol (p < 0.05) were determined in pasteurized beer and inoculated with AAB starter culture. This demonstrates good environmental conditions in BPGL and BPGD samples for the growth of the AAB starter culture, as AAB utilized ethanol to grow. These differences were due to the pasteurization of the samples (BJP and BGP) and the inactivation of the yeast that produces ethanol.

AAB tolerates the active acidity of the environment in the range of 3.6–6.3 [35,36]. In the studied beverage variants, the pH values in samples with the addition of AAB starter culture were significantly lower than in beer without the addition of G. oxydans H32 (p < 0.05). These results are confirmed by the number of AAB in each of the study beverage samples (Table 2). It additionally attests to the metabolic processes carried out by these microorganisms in the studied beers. The main metabolites produced by AAB are organic acids, including acetic acid. It is formed during the oxidation of ethanol, and this reaction is accompanied by enzymes that catalyse the entire process. Alcohol dehydrogenase (ADH), which accompanies the oxidation of ethyl alcohol to acetaldehyde, is responsible for the first stage of its entire production. In the next phase, the formed aldehyde is oxidized by the enzyme aldehyde dehydrogenase (ALDH), which is located on the outer surface of the cytoplasmic membrane, thereby producing the end product. Strains of the Gluconobacter sp. are unable to further process acetic acid due to the non-functional cycle of tricarboxylic acids in this case, and more precisely by two enzymes occurring in it, namely α-ketoglutarate dehydrogenase and succinate dehydrogenase [39]. In the study of beer beverages, a statistically significant difference was observed in the amount of acetic acid produced by the added G. oxydans H32 strain compared to the samples without the addition of starter AAB (p < 0.05). These amounts were 3–4 times higher than the BJP beer sample and the control “C” samples (

Table 4. Average results of physicochemical analyses (organic acids) in study beer beverages after the re-fermentation process.

Sample	Malic Acid	Acetic Acid	Gluconic Acid	Fumaric Acid	Lactic Acid	Pyruvic Acid
	[mg 100 mL−1]
BJPL	242.43 a ± 1.29	9.60 a ± 0.10	nd a	2.50 ad ± 0.10	26.53 ad ± 0.35	6.83 a ± 0.06
BPGL	259.40 b ± 2.36	46.93 b ± 0.65	69.07 b ± 0.45	3.13 b ± 0.06	30.97 b ± 0.50	8.30 b ± 0.10
BGL	257.80 b ± 0.70	37.00 c ± 1.49	44.93 c ± 1.15	3.00 bc ± 0.10	42.50 c ± 0.96	7.50 c ± 0.10
CL	54.13 c ± 1.08	12.93 d ± 0.76	nd a	2.27 af ± 0.12	40.87 cd ± 0.67	10.8 d ± 0.35
BJPD	251.77 d ± 1.42	10.83 ad ± 0.40	nd a	2.67 acd ± 0.15	27.43 a ± 0.57	7.77 bc ± 0.15
BPGD	239.60 a ± 0.85	32.17 e ± 0.59	15.17 d ± 0.38	3.50 e ± 0.10	39.67 d ± 1.06	7.43 ac ± 0.21
BGD	262.93 b ± 4.03	28.07 f ± 0.50	8.60 e ± 0.10	3.33 be ± 0.12	31.63 b ± 1.00	8.13 bc ± 0.25
CD	65.30 e ± 1.20	20.60 g ± 1.05	nd a	2.53 adf ± 0.12	34.67 e ± 1.14	12.73 e ± 0.31

Explanatory notes: nd—not detected; means in the same column followed by different lowercase letters of the alphabet represent significant differences between the samples according to the ANOVA test; the statistical differences in the lower case apply to all samples in one column (p < 0.05); n = 3.

). Gluconobacter species are glucophilic and can oxidize glucose via two alternative pathways. The first operates inside the cell, where oxidation takes place through the pentose phosphate pathway, and the second, outside the cell, which involves the formation of gluconic and ketogluconic acid [40]. Moreover, gluconic acid is a metabolite of AAB with a pro-health effect, including a precursor in the biosynthesis of natural vitamin C. As a substrate for the initiation of this reaction, i.e., obtaining 2-oxo-L-gluonic acid, a precursor of vitamin C, glucose is used. Besides glucose, other possible carbohydrates can be fructose, galactose, as well as also arabinose, rhamnose, xylose, sorbose, or sucrose [41]. In this study of beer beverage variants, significant amounts of gluconic acid (p < 0.05) were determined only in the beers with the addition of AAB starter culture (pasteurized beer BPGL = 69.07 mg 100 mL⁻¹, BPGD = 15.17 mg 100 mL⁻¹ and unpasteurized beer BGL = 44.93 mg 100 mL⁻¹, BGD = 8.60 mg 100 mL⁻¹). Additionally, in these products, the content of vitamin C was the highest (p < 0.05) at BPGL = 2.83 mg 100 mL⁻¹, BPGD = 2.43 mg 100 mL⁻¹ i w BGL = 2.03 mg 100 mL⁻¹, BGD = 2.60 mg 100 mL⁻¹, which, per 500 mL of the study beverages, provides about 10 mg of vitamin C. According to the EFSA recommendation, the average intake of vitamin C among adults aged 35–64 years old in European countries should be 100 mg per day [42]. Moreover, although developed beer samples may serve as a minor source of vitamin C in the diet, vitamin C addition is significantly higher than in commercial beers. The conscious consumer can therefore choose the more nutritious, pro-health product. Similar conclusions were shown from the Horn et al. [8] study, wherein the authors also highlighted the potential benefits to human health, including, among others, a high content of vitamin C and high antioxidant capacity, pointing to new approaches that can increase the interest in craft beer.

The content of malic, fumaric, lactic, and pyruvic acids was at the level characteristic for beers. Their content proves the proper course of fermentation and a lack of contamination with unwanted microorganisms [43]. The addition of fruit juices to beer samples affected the profile of organic acids. Additionally, in this case, the acid content in the products was similar to the literature data on this subject [44,45].

3.3. Sensory Quality

The sensory profiles of the light and dark beer beverages after the re-fermentation process are shown in Figure 2a,b. The relationship between sensory attributes and types of tested products is shown graphically in Figure 3.

This projection shows the correlation of the assessed sensory attributes of the plane, consisting of two selected factors. The first and second principal components represented 41% of the sum of all sensory attributes for a light beer and 42% for dark beer. In this study, the principal component analysis (PCA) allowed us to distinguish the most important discriminants in the conducted sensory assessment. The attributes that significantly (p < 0.05) determined the shape of the sensory profile of the studied variants of beer beverages were acetic, fruit odor and flavor, honey odor, tang flavor. The quality of the tested beverages was also influenced by the degree of saturation with carbon dioxide. The intensity of carbonation was observed to a greater extent in unpasteurized samples (BG) (p < 0.05). This observation is consistent with the results of microbiological analyses and the presence of live yeast cells. It was also observed that the degree of carbonation positively correlates with the intensity of sour and acetic flavors. On the other hand, no significant (p > 0.05) influence on the sensory quality of distinguishing features such as beer and other odors and flavors as well as the sour smell, was observed. Flavor additives used in the production of craft beers include distinct ingredients such as honey, fruits, cassava, pumpkin, herbs, and ginger, among other agents, to cause positive sensory changes on the final product [46,47]. In the presented research, a simple composition (wort, fruit juice), properly selected technological conditions, and, most importantly, a well-designed starter culture ensured a high sensory quality, without the need to add other flavor additives.

When comparing the sensory quality of the two types of studied beer beverages (light and dark), it was found that all kinds of light beer samples, especially trial BPGL with the addition of G. oxydans H32 starter culture, were characterized by a significantly (p < 0.05) better overall sensory quality. Additionally, all kinds of pasteurized and inoculated light beer samples were characterized by the most balanced sensory attributes profile, ultimately shaping the high overall quality (p < 0.05).

Only a few types of research can be found regarding the possibility of using LAB as a starter culture of pro-health and probiotic sour beer production [1,12,13,33]. To the best of our knowledge, no comparable studies have been conducted using AAB as a starter culture with beneficial properties. On the other hand, AAB are often perceived as contaminants in brewing, negatively affecting the final sensory quality of the beer [39,48]. Based on the conducted research, it was shown that a well-selected strain of AAB bacteria is a proper solution for controlling the light sour beer re-fermentation process. It has also been proven that AAB are not only associated with creating beer spoilage but can shape the appropriate sensory characteristics. Furthermore, the suitable AAB strain and method of introducing it into the beer may positively affect the sensory quality of sour beer and also provide health-promoting properties.

4. Conclusions

The conducted research intended to investigate whether the promising AAB Gluconobacter oxydans H32 bacterial strain shows technological suitability. Based on the obtained results, it was found that pasteurized light beer served as a better environment for the development of novel strain G. oxydans H32 than dark beer. The designed beverages are a good source of pro-health organic acids and vitamin C, which are the metabolite compounds of the studied acetic acid bacteria. The designed technology of sour Ale beer production allowed the introduction of a selected AAB strain with potential functional properties into the raw material, thus proving its technological usefulness. Future studies should focus on the investigation of antioxidant capacity, nutritional quality, mineral content, osmolarity, calories, etc., of selected beer samples. This knowledge will provide more information regarding the quality of such drinks and can contribute to the development of the beer market. Moreover, we believe that the novel G. oxydans H32 strain can be subjected to further tests to demonstrate its potential probiotic properties, following the rationale from preliminary research. Thanks to the use of an appropriately selected starter culture for fermentation, it is possible to use AAB to design functional beverages with high sensory, microbiological and physical-chemical qualities.