Plants and Lactic Acid Bacteria Combination for New Antimicrobial and Antioxidant Properties Product Development in a Sustainable Manner.

In this study, nutraceuticals based on antimicrobial ingredients (Artemisia absinthium water extract and essential oil (EO), Lactobacillus uvarum LUHS245 strain cultivated in a whey media, and blackcurrants juice (BCJ) preparation by-products were developed. In addition, two texture forming agents for nutraceutical preparations were tested (gelatin and agar). The developed nutraceutical ingredients showed antimicrobial properties: Artemisia absinthium EO (concentration 0.1%) inhibited methicillin-resistant Staphylococcus aureus, Enterococcus faecium, Bacillus cereus, Streptococcus mutans, Staphylococcus epidermidis, and Pasteurella multocida; LUHS245 strain inhibited 14 from the 15 tested pathogenic strains; and BCP inhibited 13 from the 15 tested pathogenic strains. The best formulation consisted of the Artemisia absinthium EO, LUHS245, and BCP immobilised in agar and this formulation showed higher TPC content (by 2.1% higher), as well as higher overall acceptability (by 17.7% higher), compared with the formulation prepared using gelatin.


Introduction
The global nutraceutical market has grown in the last few years and this growth is expected to continue. For this reason, the challenges related to high prices for nutraceutical compounds became very real [1]. One of the solutions to reduce prices, as well as ensure sustainability of the nutraceuticals analysis. Blackcurrants (Ribes nigrum, variety "Ben Alder") BY were obtained from the Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry (Babtai, Kaunas distr., Lithuania) in 2018. Blackcurrants BY were dried by lyophilization (at −40 ± 2 • C). For lyophilization, a sublimator 3 × 4 × 5 (ZIRBUS Technology GmbH, Germany) was used. The condenser temperature was −85 • C and the vacuum was 2 × 10 −6 MPa. The samples were frozen at −40 ± 2 • C in a laboratory freezer and then left in a freeze dryer for 72 h. Whey (lactose 4.0%, protein 0.8%, lactic acid 0.5%, pH value 4.45, titratable acidity 0.22, minerals 0.6%, total solids 6.5%) was purchased from JSC "Pieno zvaigzdes" (Kaunas, Lithuania) and used as a media for a Lactobacillus uvarum LUHS245 strain biomass growth. The experiment used LAB strain Lactobacillus uvarum LUHS245, which previously demonstrated good antimicrobial activities against a variety of pathogenic and opportunistic microorganisms, which was provided by Lithuanian University of Health Sciences collection (Kaunas, Lithuania). The LUHS245 strain was stored at −80 • C in a Microbank system (Pro-Lab Diagnostics, Birkenhead, UK) and grown in MRS broth (CM 0359, Oxoid, Hampshire, UK) at 30 • C for 48 h prior to use. The agar powder (Gelidium sesquipedale algae, Rapunzel, Germany) was used as a polymer with mucoadhesive properties for nutraceuticals forming. In addition, gelatin was also tested (Klingai, Lithuania).

Grown of the Lactobacillus uvarum LUHS245 in Whey Media
For Lactobacillus uvarum LUHS245 biomass growth, whey media was used and for growth parameters optimisation glucose (GLU), yeast extract (Y) and saccharose (SA) additives were tested. Prior to fermentation, the whey was sterilised, cooled to 30 • C, and different concentrations of Y, GLU, and SA (0.5; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0; 4.5; 5.0%) were added, with the purpose of improving the growth of the LUHS245. The optimal quantities of the Y, GLU, and SA, as well as the optimal fermentation time (the LAB count in the whey media was analysed every 3 h), were selected. Lyophilisation of the LUHS245 cultivated in the dairy production BY was performed in a sublimator 3 × 4 × 5 (ZIRBUS Technology GmbH, Harz, Germany). The parameters of the process were the same as described for blackcurrants BY lyophilisation. The LAB count in the lyophilised powder was determined.

Lactic Acid Bacteria Count in Fermented Lyophilised Dairy Production By-Products Media
The lactic acid bacteria (LAB) count in fermented lyophilised dairy production BY was evaluated according to ISO 15214:1998 [23]. The petri plates, with sterile MRS agar (CM0361, Oxoid) of 5 mm of thickness, were separately seeded with the LAB diluted suspension using sowing in surface and were incubated under anaerobic conditions at 30 • C for 72 h. The number of LAB colonies on the surface of the MRS agar plate was calculated and expressed as log 10 of colony forming units per gram (CFU g −1 ). strain against a variety of pathogenic and opportunistic bacterial strains. Agar well diffusion assay was used for the antimicrobial activities testing of the Artemisia absinthium water extract, LUHS245, and blackcurrants juice preparation by-products. For this purpose, 0.5 McFarland Unit density suspension of each pathogenic bacteria strain was inoculated onto the surface of cooled Mueller Hinton Agar (Oxoid, UK) using sterile cotton swabs. Wells of 6 mm in diameter were punched in agar and filled with 50 µL of the tested compounds. The antimicrobial activities against the tested bacteria were determined by measuring the diameter of the inhibition zones (mm). The experiments were repeated three times and the average of the inhibition zones was calculated.
In addition, the antimicrobial activity of the Artemisia absinthium water extracts and EO against the abovementioned pathogenic and opportunistic strains were determined in liquid media [25]. Antimicrobial activity was defined as the Artemisia absinthium water extract (10%, 20%, and 30% concentrations) and/or EO (0.1% concentration) inhibiting visible microbial growth. For this purpose, water extracts and EO were placed into 1 mL of Mueller Hinton Broth (Oxoid, UK). Then, 10 µL of 0.5 McFarland density of the pathogenic and opportunistic bacterial strain, initially cultivated on Mueller Hinton Agar (Oxoid, UK), was added, mixed, and incubated at 35 • C for 24 h. After incubation, the viable pathogenic and opportunistic bacterial strains were controlled by plating them on an appropriate universal medium (Tryptic Soy Agar, Oxoid). The results were interpreted as (−) if the pathogens did not grow on universal medium and (+) if the pathogens grew on universal medium. Growth control without extracts and EO was carried out.
Experiments were performed in triplicate.

Nutraceuticals Formulation
The main ingredients, formulations, and technological steps of the separate layers of the nutraceuticals based on Artemisia absinthium EO, Lactobacillus uvarum LUHS245 cultivated in a whey media, and blackcurrant juice preparation BY, as well as the whole nutraceutical-product, are shown in Scheme 1. Two formulations of the three layer nutraceuticals based on agar and gelatin were prepared.
The culture supernatant was filtered through a 0.2 mm sterile Millipore filter (Merck KGaA, Darmstadt, Germany) to remove all cells and adjusted to pH 6.5 with 5 mol L −1 NaOH to eliminate the organic acids effect. Supernatants were used for the determination of the antimicrobial activities of LUHS245 strain against a variety of pathogenic and opportunistic bacterial strains. Agar well diffusion assay was used for the antimicrobial activities testing of the Artemisia absinthium water extract, LUHS245, and blackcurrants juice preparation by-products. For this purpose, 0.5 McFarland Unit density suspension of each pathogenic bacteria strain was inoculated onto the surface of cooled Mueller Hinton Agar (Oxoid, UK) using sterile cotton swabs. Wells of 6 mm in diameter were punched in agar and filled with 50 µL of the tested compounds. The antimicrobial activities against the tested bacteria were determined by measuring the diameter of the inhibition zones (mm). The experiments were repeated three times and the average of the inhibition zones was calculated.
In addition, the antimicrobial activity of the Artemisia absinthium water extracts and EO against the abovementioned pathogenic and opportunistic strains were determined in liquid media [25]. Antimicrobial activity was defined as the Artemisia absinthium water extract (10%, 20%, and 30% concentrations) and/or EO (0.1% concentration) inhibiting visible microbial growth. For this purpose, water extracts and EO were placed into 1 mL of Mueller Hinton Broth (Oxoid, UK). Then, 10 µL of 0.5 McFarland density of the pathogenic and opportunistic bacterial strain, initially cultivated on Mueller Hinton Agar (Oxoid, UK), was added, mixed, and incubated at 35 °C for 24 h. After incubation, the viable pathogenic and opportunistic bacterial strains were controlled by plating them on an appropriate universal medium (Tryptic Soy Agar, Oxoid). The results were interpreted as (−) if the pathogens did not grow on universal medium and (+) if the pathogens grew on universal medium. Growth control without extracts and EO was carried out.
Experiments were performed in triplicate.

Nutraceuticals Formulation
The main ingredients, formulations, and technological steps of the separate layers of the nutraceuticals based on Artemisia absinthium EO, Lactobacillus uvarum LUHS245 cultivated in a whey media, and blackcurrant juice preparation BY, as well as the whole nutraceutical-product, are shown in Scheme 1. Two formulations of the three layer nutraceuticals based on agar and gelatin were prepared.

The Evaluation of Total Phenolic Compounds (TPC) Content in Separate Layers and the Whole Formulation of the Prepared Nutraceuticals and Their Antioxidant Activity
The TPC content in separate layers and the whole formulation of the prepared nutraceuticals was determined by the spectrophotometric method, as reported elsewhere [26]. The absorbance of samples was measured at 765 nm using spectrophotometer J.P. SELECTA S.A. V-1100D (Barcelona, Spain). Antioxidant activity of the separate layers and the whole formulation of the prepared nutraceuticals was evaluated according to the method reported by Zhu et al. [27].

Evaluation of Nutraceuticals Colour Coordinates, Texture, and Overall Acceptability
The color characteristics were evaluated using a CIE L * a * b * (L * = lightness; a * = redness or −a * = greenness; b * = yellowness or −b * = blueness) system (CromaMeter CR-400, Konica Minolta, Japan). The hardness parameter was evaluated using a Brookfield texture analyser (Middleboro, MA, USA). Samples were compressed to 10% of their original height at a crosshead speed of 10 mm/s. The resulting peak force of compression was reported as nutraceutical hardness. Three replicates from three different sets of preparation were analyzed and averaged. The overall acceptability was carried out by 50 judges according to International Standards Organisation 8586-1 method [28], using a 140 mm hedonic line scale, ranging from 140 (extremely like) to 0 (extremely dislike).

Statistical Analysis
Statistical analysis was performed using multivariate analysis of variance (ANOVA). For data processing, software packages STATISTICA 7.1 (StatSoft Inc., Tulsa, USA) and SPSS 20.0 (IBM Corp., Armonk, NY, USA) were used. Statistical significance was set at p ≤ 0.05.

Antimicrobial Properties of the Artemisia Absinthium Water Extract and Essential Oil (EO), Lactobacillus uvarum LUHS245, and Blackcurrant Juice Preparation By-Products
Inhibition of the growth of pathogenic bacteria by Artemisia absinthium water extract, L. uvarum LUHS245, and blackcurrant juice preparation BY is shown in Table 1. Artemisia absinthium water extract showed antimicrobial activity against Pasteurella multocida (20.4 ± 4.1 mm). Lactobacillus uvarum LUHS245 strain inhibited 14 from the 15 tested pathogenic strains, and the highest inhibition zones against Pasteurella multocida and Bacillus cereus 18 01 were found (22.0 ± 0.2 and 21.5 ± 0.3 mm, respectively). Blackcurrant juice preparation BY inhibited 13 from the 15 tested pathogenic strains, and the highest inhibition zones against Pasteurella multocida, Bacillus cereus 18 01, and Streptococcus mutans were found (30.7 ± 0.5, 21.4 ± 0.2, and 20.1 ± 0.1 mm, respectively). According to the results obtained, preparation of the nutraceuticals outer layer by using blackcurrant juice preparation BY can be very promising as a dental caries prophylaxis because antimicrobial activity against Streptococcus mutans was established. Streptococcus mutans can cause dental caries and it has been reported that more than 90% of the population is suffering from it [29].
Antimicrobial activities of the Artemisia absinthium EO (concentration 0.1%) and Artemisia absinthium water extract (concentrations 10%, 20%, and 30%) against pathogenic opportunistic microorganisms in liquid medium are shown in Table 2. Artemisia absinthium water extract does not inhibit pathogenic opportunistic microorganisms in liquid medium, however Artemisia absinthium EO (concentration 0.1%) inhibited methicillin-resistant Staphylococcus aureus M87fox, Enterococcus faecium 103, Bacillus cereus 18 01, Streptococcus mutans, Staphylococcus epidermidis, and Pasteurella multocida. According to the results obtained, for nutraceuticals preparation, Artemisia absinthium EO was selected.    Natural plant ingredients are usually characterized as less toxic compared to synthetic ones [30]. Nowaday, EOs, as potential alternatives to synthetic antimicrobials, have become very popular [31]. It was published that terpenes are predominant compounds of the Artemisia absinthium EO [32] and trans sabinyl acetate is a predominant compound in the A. absinthium EO [32]. The lipophilicity of monoterpenes and sesquiterpenes were indicated as the main antimicrobial mechanism of action of the A. absinthium [33]. This characteristic leads to diffusion through microorganism membranes and inhibition of the synthesis processes in bacterial cells [34]. Earlier reports suggested that A. absinthium possesses antibacterial properties [35,36]. Also, it was published that the extracts inhibit pathogenic bacteria, but not LAB [37,38].

Inhibition Zones of the Growth of Pathogenic Bacteria by Blackcurrant Juice Preparation By-products, mm
However, in our study, it was established that the Artemisia absinthium water extract inhibits the growth of Pasteurella multocida (inhibition zone 20.0 ± 4.1 mm), as well as EO (concentration of EO 0.1%) inhibited Methicillin-resistant Staphylococcus aureus M87fox, Enterococcus faecium 103, Bacillus cereus 18 01, Staphylococcus epidermidis, and Pasteurella multocida, Table 2; Table 3). According to the obtained results, EO, as a higher antimicrobial activity component, in comparison with Artemisia absinthium water extract, was selected for nutraceuticals preparation.

Grown and Stabilization of the Lactobacillus uvarum LUHS245 Strain in Dairy Production By-Products Media
Lactic acid bacteria growth after 12, 24, and 48 h of cultivation in whey (WHE) is presented in Table 3. After 12 h of incubation, the highest count of LAB was found in WHE with 4.0% of the yeast extract (Y) (~7.9 log 10 CFU mL −1 ). After 24 h of incubation, the LAB count in the non-enriched WHE was reduced by 15.0%. Opposite tendencies were found in most of the WHE with glucose (GLU), Y, and saccharose (SA), and in many cases, the additives had a positive influence on the LAB growth. The highest LAB count was found in WHE with 2.0% of the Y (8.11 ± 0.14 log 10 CFU mL −1 ). After 48 h of incubation, the LAB count was the same or decreased, except in samples enriched with SA, and the highest LAB count was found in WHE with 0.5% of SA (8.11 ± 0.13 log 10 CFU mL −1 ). The LAB count was significantly influenced by the additives and their quantity, as well as fermentation time (p ≤ 0.0001), and the interaction of the analysed factors was significant (p ≤ 0.0001). The optimal Foods 2020, 9, 433 9 of 16 quantities of the additives were 2.5% of GLU, 2.0% of Y, and 0.5% of SA. The results of the LAB growth in WHE with the optimum concentration of selected additives are presented in Figure 1. The optimal time for LUHS245 incubation in the WHE with 2.5% GLU, 2.0% Y, and 0.5% SA is 28 h. The highest LAB count in WHE with 2.0% of Y was observed after 36 h of incubation. The addition of GLU and SA had a lower influence on the growth of LAB, compared with the addition of Y. LUHS245 strain was incubated in the WHE with 2.5% GLU, 2.0% Y, and 0.5% SA for 28 h (optimal incubation time) and lyophilised. After lyophilisation, a viable LAB count in the obtained powder was 8.90 ± 0.12 log 10 CFU g −1 . The uses of technologically functionalized (e.g., dehydrated) starter cultures is more attractive, due to higher technological microorganisms survival and viability under environmental conditions [58]. Viability of LAB increasing for convenient industrial uses can be performed in a sustainable manner. From an environmental, as well as economical point of view, public awareness for food waste and by-products recycling has recently increased, and the dairy industry generates huge amounts of by-products [59,60]. Also, LAB biomass preparation by using by-products as nutrient media can lead to a profitable process [61]. Finally, whey is perspective sustainable media for LAB cultivation, as well as dehydratation.
Lyophilisation is the most popular process used to preserve LAB because of its ability to protect from spoilage, as well as its long viability during storage [62,63]. During freezing, bacterial cells are exposed to mechanical stress because of intra-and extra-cellular ice crystal formation and increased osmotic pressure caused by solutes in the remaining unfrozen fraction. This process can lead to the destruction of bacterial membranes and cause harmful damage [64]. Moreover, during lyophilisation, the removal of water by sublimation additionally increases osmotic pressure and can be harmful for membranes and surface proteins [65]. For this reason, cryprotectants are usually added to maintain the viability of microorganisms [64]. Different polymers, sugars, milk, honey, polyols, and amino acids have been tested for their protective effect during lyophilisation [64][65][66]. Disaccharides, such as maltose, sucrose, and trehalose, are able to induce shrinkage of the cells by osmosis-derived dehydration before freezing, thereby reducing intracellular ice formation [64]. Compounds mixing with different protective mechanisms can have a positive influence on the higher protection of microorganisms during freezing, as well as drying compared with single-component application, due to components' synergic protective effects [64]. Skim milk, containing a mixture of lactoalbumine and casein, as well as saccharides, is a selected cryoprotectant for many LAB due to its ability to prevent cellular damage by stabilizing the cell membrane and providing a protein-protective coating for the cells [67]. Moreover, different sugars have high levels of protection of LAB during freeze-drying, due to their ability to replace structural water in membranes after dehydration [68]. It was reported that skim milk powder with lactose or saccharide contained the best cryoprotectans for lactobacilli [67]. The uses of technologically functionalized (e.g., dehydrated) starter cultures is more attractive, due to higher technological microorganisms survival and viability under environmental conditions [58]. Viability of LAB increasing for convenient industrial uses can be performed in a sustainable manner. From an environmental, as well as economical point of view, public awareness for food waste and by-products recycling has recently increased, and the dairy industry generates huge amounts of by-products [59,60]. Also, LAB biomass preparation by using by-products as nutrient media can lead to a profitable process [61]. Finally, whey is perspective sustainable media for LAB cultivation, as well as dehydratation.
Lyophilisation is the most popular process used to preserve LAB because of its ability to protect from spoilage, as well as its long viability during storage [62,63]. During freezing, bacterial cells are exposed to mechanical stress because of intra-and extra-cellular ice crystal formation and increased osmotic pressure caused by solutes in the remaining unfrozen fraction. This process can lead to the destruction of bacterial membranes and cause harmful damage [64]. Moreover, during lyophilisation, the removal of water by sublimation additionally increases osmotic pressure and can be harmful for membranes and surface proteins [65]. For this reason, cryprotectants are usually added to maintain the viability of microorganisms [64]. Different polymers, sugars, milk, honey, polyols, and amino acids have been tested for their protective effect during lyophilisation [64][65][66]. Disaccharides, such as maltose, sucrose, and trehalose, are able to induce shrinkage of the cells by osmosis-derived dehydration before freezing, thereby reducing intracellular ice formation [64]. Compounds mixing with different protective mechanisms can have a positive influence on the higher protection of microorganisms during freezing, as well as drying compared with single-component application, due to components' synergic protective effects [64]. Skim milk, containing a mixture of lactoalbumine and casein, as well as saccharides, is a selected cryoprotectant for many LAB due to its ability to prevent cellular damage by stabilizing the cell membrane and providing a protein-protective coating for the cells [67]. Moreover, different sugars have high levels of protection of LAB during freeze-drying, due to their ability to replace structural water in membranes after dehydration [68]. It was reported that skim milk powder with lactose or saccharide contained the best cryoprotectans for lactobacilli [67]. For instance, L. lactis CIDCA 8221 lyophilised using milk and sucrose as a cryoprotective medium had a high survival rate and recovery ability [69]. Different additives can improve the viability of LAB in different matrices, as well as dehydratation processes, and increases the stability of strains, thus alleviating production on an industrial scale.

The Total Phenolic Compounds Content and Radical Scavenging Activity of Nutraceuticals
The total phenolic compounds (TPC) content and antioxidant activity of the different layers, as well as whole nutraceutical, are given in Table 4. When comparing Artemisia absinthium EO+agar (Aex+Ag) and Artemisia absinthium+gelatin (Aex+G) layers, higher TPC content and antioxidant activity by the (Aex+G) layer was found (by 5.1 and 14.6% higher, respectively). Different tendencies of the second layer antioxidant properties were obtained, as a higher TPC content of the layer prepared with gelatin was found (by 4.8% higher), however higher antioxidant activity of the layer prepared with agar was established (by 7.2% higher). In comparison to whole nutraceutical formulations, higher TPC content was shown by products prepared with gelatin (by 2.1% higher), however antioxidant activity of the gelatin and agar prepared nutraceuticals was similar (with gelatin prepared, it was 75.15 ± 3.9% and with agar prepared, 81.96 ± 4.5%). Significant influence of the gelatin/agar uses on the antioxidant properties of the nutraceuticals was not established.
Free radicals are very reactive in biological systems and an overproduction can lead to various life threatening disorders like atherosclerosis, inflammatory diseases, and cancer [69]. A. absinthium extract possesses antioxidant and free radical scavenging activities in vitro, as well as showing cytotoxic and antitumor activity against different malignancies [69]. Finally, A. absinthium extracts can be novel active drug compounds, as they have antiproliferative, antioxidant, and radical scavenging properties, however additional studies need to be undertaken to carry out the in vivo pharmacological experimentation of this plant [22]. Antioxidants, molecules with a radical scavenging capacity, are considered to exercise a defensive consequence against free radical injury and these molecular components may prevent many chronic diseases like cancer, hepatitis, asthma, atherosclerosis, arthritis, heart disease, and diabetes [70,71]. Currently, many synthetic antioxidants are also used, however these antioxidants have been thought to damage the liver and cause cancer [72]. Due to side effects and toxicity to health, their utilization is restricted [73]. The consumption of plant based additives as antioxidants increases day by day [72]. Plant based antioxidant demand continues to increase in the market, as they are used in food, cosmetic, and pharmaceutical industries to replace synthetic antioxidants. For this reason, plant based by-products can be a valuable source for these substances. Among natural antioxidants, phenolic substances have been of special interest because they are found extensively in plants [72]. Natural products containing phenolic substances have antioxidant potential, mostly because of their redox ability, which enables them to behave as hydrogen donors, single oxygen quenchers, and reducing agents as well as they have the potential to chelate metals [72]. The phenolic substances have taken part in body cell protection from harm by H 2 O 2 , lipid peroxide, unsaturated fatty acid, neutralizing, and absorbing free radicals [74]. However, it is not just plants based antioxidants that are promising for nutraceuticals formulations, the antioxidant properties of LAB were also published [75]. Finally, compositions were developed from different origins of antioxidant properties, showing that compounds can be very attractive for nutraceuticals formulations, as well as can lead to dose reducing and other activities, such as antimicrobial, increasing. Aex-Artemisia absinthium essential oil; Ag-agar; G-gelatin; 245-Lactobacillus uvarum LUHS245 strains cultivated in dairy by-products; Bl-blackcurrant juice preparation by-products; TPC-total phenolic compounds content; L*-lightness; a*-redness; b*-yellowness. Data expressed as means (n = 3) ± SD; SD-standard deviation. a-f Mean values with different letters are significantly different (p ≤ 0.05).

Nutraceuticals Colour Coordinates, Texture, and Overall Acceptability
Nutraceuticals contain different biologically and chemically active compounds. Various processes are applied for nutraceutical ingredients technological functionalization. In this study, all the compounds were analysed in accordance with their antimicrobial properties, however especially plant compounds can possess antioxidant properties as well. However, for the product's success in the market, it is not enough to provide only the desirable functional properties; colour, texture, and its related overall acceptability are also very important. Nutraceuticals colour coordinates, texture, and overall acceptability are shown in Table 4. When comparing Artemisia absinthium EO mmobilized in agar and gelatin, gelatin increased the lightness and yellowness of the first layer (by 58.0 and 68.7%, respectively), however agar or gelatin selection was not significant on the redness coordinates. When comparing the colour coordinates of the second layer, in all the cases, the layer prepared with gelatin showed higher lightness (by 9.8% higher), redness (by 83.6% higher), and yellowness (by 17.7% higher). Gelatin or agar selection for the outer layer preparation was not significant on the lightness and yellowness coordinates, however higher redness by using agar was found (by 8.6% higher).
When comparing the hardness of the texture, significant differences of the first layer texture hardness were not established, however harder texture of the second and outer layers by using agar was found (by 23.3% and 81.8%, respectively, higher). When comparing the overall acceptability of the separate layers, as well as whole formulation, it can be stated that the blackcurrant juice preparation by-products are very promising antimicrobial ingredients that are acceptable for consumers and can mask other ingredients for which acceptability is not very high. Between colour coordinates (lightness, redness, yellowness) and overall acceptability of the prepared nutraceuticals, negative moderate (r = −0.6632), positive very strong (r = −0.9001), and negative moderate (r = −0.6514) correlations were found. When comparing whole nutraceuticals formulation prepared with agar and gelatin, higher overall acceptability of the nutraceuticals prepared with agar was established (by 17.7% higher), however for between nutraceuticals texture and overall acceptability, a very low positive correlation (r = 0.2271) was established.

Conclusions
It can be summarized that antimicrobial and antioxidant properties showing nutraceuticals formulation in chewing tablets form, based on Artemisia absinthium EO (concentration ≤ 0.1 inhibited methicillin-resistant Staphylococcus aureus M87fox, Enterococcus faecium 103, Bacillus cereus 18 01, Streptococcus mutans, Staphylococcus epidermidis, and Pasteurella multocida), Lactobacillus uvarum LUHS245 encapsulated in a whey media (inhibited 14 from the 15 tested pathogenic strains), and blackcurrant juice preparation by-products (inhibited 13 from the 15 tested pathogenic strains) can be prepared in a sustainable manner. The best formulation consists of the Artemisia absinthium EO, Lactobacillus uvarum LUHS245 encapsulated in a whey media, and blackcurrant juice preparation by-products immobilized in agar as this formulation showed higher TPC content (by 2.1% higher), as well as higher overall acceptability (by 17.7% higher), compared with the formulation prepared with gelatin. Finally, compositions were developed from different origin antioxidant properties, showing that compounds can be very attractive for nutraceuticals formulations, as well as can lead to the dose reducing and other activities increasing, e.g. antimicrobial activity. Further experiments are planned to evaluate the antimicrobial activities of Artemisia absinthium extracts and LUHS245 strain against non-pathogenic bacterial strains with the aim to evaluate their influence on normal microbiota.