Multifunctional Nutraceutical Composition Based on Fermented Spirulina, Apple Cider Vinegar, Jerusalem Artichoke, and Bovine Colostrum

The main purpose of this experiment was to develop a multifunctional nutraceutical composition based on ingredients of different origins (Spirulina powder (SP), bovine colostrum (BC), Jerusalem artichoke powder (JAP), and apple cider vinegar (ACV)) which possess different health benefits through their different mechanisms of action. In order to improve the functional properties of Spirulina and bovine colostrum, fermentation with the Pediococcus acidilactici No. 29 and Lacticaseibacillus paracasei LUHS244 strains, respectively, was carried out. These LAB strains were chosen due to their good antimicrobial properties. The following parameters were analysed: for Spirulina (non-treated and fermented)—pH, colour coordinates, fatty acid profile, and contents of L-glutamic and GABA acids; for bovine colostrum (non-treated and fermented)—pH, colour coordinates, dry matter, and microbiological parameters (total LAB, total bacteria, total enterobacteria, Escherichia coli, and mould/yeast counts); for the produced nutraceuticals—hardness, colour coordinates, and overall acceptability. It was established that fermentation reduced the pH of the SP and BC and affected their colour coordinates. Fermented SP contained a greater concentration of gamma-aminobutyric and L-glutamic acids (by 5.2 times and 31.4% more, respectively), compared to the non-treated SP and BC. In addition, the presence of gamma-linolenic and omega-3 fatty acids was observed in fermented SP. Fermentation of BC reduces Escherichia coli, total bacteria, total enterobacteria, and total mould/yeast counts in samples. The obtained three-layer nutraceutical (I layer—fermented SP; II—fermented BC and JAP; III—ACV) demonstrated a high overall acceptability. Finally, our finding suggest that the selected nutraceutical combination has immense potential in the production of a multifunctional product with improved functionality and a high acceptability.


Introduction
A nutraceutical is a biologically active compound, dietary supplement, or food that delivers health advantages, including the ability to prevent and treat illnesses [1]. The public interest in natural nutraceutical items is significantly increasing because natural items are preferred by consumers, and the consumption of these products does not require a prescription from a doctor [2][3][4]. It was reported that the global market for nutraceuticals is expected to reach USD 578.23 billion by 2025 [5].
The development of a nutraceutical in the form of a gummy confection has great potential because this type of product is attractive to consumers due to its confectionary appearance and taste [6]. The chewable gummy market in the United States is expected to reach USD 5.8 billion by 2029 [7]. Formulations of gummy candies or chewable tablets with health-promoting ingredients have already been reported [6,[8][9][10]. However, these ingredients induce various effects on the product's physicochemical characteristics and acceptability; therefore, the demand for new formulations with better sensory properties is still increasing. There are many potential ingredients for the development of nutraceutical combinations, e.g., Spirulina powder, bovine colostrum, Jerusalem artichoke powder, apple cider vinegar, etc.
Bovine colostrum is the milk produced from a cow's mammary glands immediately after calving. A distinctive combination of nutrients and bioactive substances can be found in bovine colostrum [14]. Colostrum contains immunoglobulins, αand β-lactalbumin, growth factors, oligosaccharides and glycosylated proteins (which act as prebiotics), and vitamins A, D, E, and B-group [15]. The immunoglobulins, bioactive oligosaccharides, lactoperoxidase, lysozyme, and lactoferrin in colostrum induce antimicrobial effects, whereas it scytokines, leukocytes, and colostrinin are involved in immune function [14]. The numerous immunomodulating components that bovine colostrum contains, which uphold and restore the health of the gastrointestinal tract, are primarily responsible for its favourable effects on the prevention and treatment of gastrointestinal illnesses (inflammatory bowel disease, diarrhoea, constipation, short bowel syndrome, and colon issues) [16]. It is safe for humans to consume and there are no contraindications, and only several side effects have been reported [15].
Jerusalem artichoke (Helianthus tuberosus L.) contains the bioactive compound inulin and essential amino acids such as leucine, methionine + cystine, isoleucine, lysine, histidine valine, threonine, etc. [17]. Inulin can act as a prebiotic and has been shown to be an antimicrobial and anti-inflammatory agent and a wound healer [18]. Some proteins in H. tuberosus are important for the control of Alzheimer's, Huntington's, and Parkinson's diseases, while others (Kunitz-type and serine hydroxy-methyltransferase proteins) possess antimicrobial and anticancer effects [17]. Fructooligosaccharides found in H. tuberosus have nutraceutical properties, boost immunity, and improve intestinal flora, blood lipids, and bone health [19].
Apple cider vinegar, which is used to preserve and flavour foods, contains organic acids (acetic and malic acids), flavonoids (quercetin, kaempferol, gallic, ferulic, caffeic acids, and catechin), polyphenols, minerals, and vitamins [20]. Due to its antimicrobial, antioxidant, and antibiotic actions, and its ability to maintain the acid-base balance in blood, apple cider vinegar plays an important part in regulating weight loss and preventing body and joint pains, cancer, diabetes, and cardiovascular diseases [21].
The above-mentioned benefits make these ingredients valuable materials for nutraceutical production. However, since nutraceuticals can be concentrated versions of food, it is likely that the level of some unwanted compounds (e.g., pesticides, mycotoxins, heavy metals, etc.) could be increased in a product after the extraction process [22]. Therefore, the bioconversion, e.g., fermentation with lactic acid bacteria (LAB), of separate ingredients could not only enhance their functional properties but also increase their safety [23]. LAB, which has a GRAS status, possess antimicrobial and enzymatic activities, improves sensory properties, prolongs the storage duration of products, and acts as a probiotics In addition to the synthesis of exopolysaccharides, organic acids, bacteriocins, etc., LAB can produce γ-amino butyric acid (GABA) [24]. The glutamate decarboxylase in LAB cells is in charge of GABA synthesis, and many LAB with this enzyme activity have been identified, including Lactobacillus, Pediococcus, and Lactococcus strains [25]. The precursor of GABA is L-glutamic acid, which is a flavour enhancer and neurotransmitter [26]. GABA is a key inhibitory neurotransmitter and has already been recognized as antidepressant, immunity booster, regulator of blood pressure, and an anti-hypertension, anti-diabetic, and anticarcinogenic agent [25]. However, GABA levels in fermented foods depend on the conditions of the fermentation process [27]. Our previous studies showed that fermentation with selected LAB strains is suitable technology for increasing the concentration of GABA in Spirulina [28]. Additionally, our previous studies showed that fermenting bovine colostrum improves it safety characteristics and functional properties, including its antimicrobial properties [29,30].
Finally, taking into consideration that (I) Spirulina shows detoxifying, antioxidant, immunostimulatory, anti-obesity, anti-diabetic, anti-aging, neuroprotective, anti-inflammatory, and antimicrobial effects, (II) bovine colostrum could help to restore gastrointestinal tract functions, (III) H. tuberosus L. shows antimicrobial and anti-inflammatory properties, boosts immunity, and improves intestinal flora, and (IV) apple cider vinegar, due to antimicrobial and antioxidant actions, plays an important role in the prevention of many diseases the combination of these ingredients has many prospects for the development of a nutraceutical formula. Therefore, the main purpose of this experiment was to develop a multifunctional nutraceutical composition based on ingredients of different origins (Spirulina powder, bovine colostrum, Jerusalem artichoke powder, and apple cider vinegar) that possess different health benefits through their different mechanisms of action. In order to improve the functional properties of Spirulina and bovine colostrum, fermentation with Pediococcus acidilactici No. 29 and Lacticaseibacillus paracasei LUHS244 strains, respectively, was carried out. These LAB strains were chosen due to their good antimicrobial properties, which were analysed in our previous work [31]. The following parameters were analysed: for Spirulina (non-treated and fermented)-pH, colour coordinates, fatty acid profile, and contents of Lglutamic and GABA acids; for bovine colostrum (non-treated and fermented)-pH, colour coordinates, dry matter, and microbiological parameters (total LAB, total bacteria, total enterobacteria, Escherichia coli, and mould/yeast counts); for produced nutraceuticals-the hardness, colour coordinates, and overall acceptability.

Materials Used for Multifunctional Nutraceutical Preparation
Lyophilized Spirulina (Arthrospira platensis) powder (content per 100 g: 1.1 g of sodium, 30.3 g of total carbohydrates, 60.6 g of proteins, 151.5 mg of calcium, 1.7 mg of potassium, and 48.5 mg of iron) was provided by Now Foods Company (Bloomingdale, IL, USA). Apple cider vinegar was obtained from the agricultural company Auseklis (Ardiskis, Lithuania).
Jerusalem artichoke powder (content per 100 g: <0.01 g of sodium, 81.7 g of total carbohydrates (comprising 27.6 g of sugar, 13.3 g of dietary fibre, and 46.1 g of inuline), 10.0 g of protein, 160 mg of calcium, 73.0 mg of magnesium, 3.26 mg of iron, and 0.3 mg of vitamin B6) was obtained from the Ltd. Urban Food (Kėdainiai, Lithuania) and producer JSC Kvalitetas (Dotnuva, Lithuania).
The bovine colostrum samples were collected from the Bentnoriaus agricultural company (Paliepiu village, Lithuania). This agricultural company maintains Lithuanian blackand-white (Holstein) dairy cows. Bovine colostrum was collected during the spring period of the year 2022. At the agricultural company, bovine colostrum was collected within 2 h of calf delivery and kept frozen at −18 • C before use. A total of 3 mL of fresh Pediococcus acidilactici, strain No. 29, grown in MRS broth (at an average cell concentration of 8.6 log 10 CFU/mL), was inoculated in 100 mL of Spirulina/water mixture (Spirulina/water ratio of 1:2, w/w) and fermented at 30 • C under anaerobic conditions for 24 h.

Spirulina Fermentation and Analysis
In the later stages of the experiment, Spirulina parameters (pH, colour coordinates, L-glutamic and GABA concentrations, and fatty acid profile) were evaluated.
The pH of the Spirulina/water mixture was evaluated using a pH meter (Inolab 3, Hanna Instruments, Venet, Italy) by inserting the pH electrode into the samples. The colour coordinates of the Spirulina were evaluated on the sample surface using the International Commission on Illumination (CIE) L*a*b* colour space system (CromaMeter CR-400, Konica Minolta, Marunouchi, Tokyo, Japan).
Analyses of the concentrations of L-glutamic acid (L-Glu) and GABA in the Spirulina were carried out using a TSQ Quantiva MS/MS coupled to a Thermo Scientific Ultimate 3000 HPLC instrument (Thermo Scientific, Waltham, MA, USA). All methods are described by Tolpeznikaite et al. in detail [28]. An analysis of the fatty acid (FA) profile of the Spirulina samples was performed using a gas chromatograph GC 2010 Plus (Shimadzu Europa GmbH, Duisburg, Germany) equipped with a mass spectrometer, GC-MS QP2010 (Shimadzu Europa GmbH, Duisburg, Germany). All methods are described by Tolpeznikaite et al. in detail [32].

Bovine Colostrum Fermentation and Analysis Methods
The LUHS244 strain of Lacticaseibacillus paracasei was chosen according to its proper antibacterial and antifungal properties [31]. Before the experiment, Lc. paracasei was stored at −80 • C in a Microbank system (Pro-Lab Diagnostics, UK) and grown in de Man, Rogosa, and Sharpe (MRS) broth (CM 0359, Oxoid, Hampshire, UK) at 30 • C for 48 h prior to use. An amount of 3 mL of MRS broth containing multiplied Lc. paracasei (at an average LAB cell concentration of 9.2 log 10 CFU/mL) was inoculated into 100 mL of defrosted (at 24 ± 2 • C for 12 h) bovine colostrum, followed by fermentation in a CO 2 incubator (Memmert GmbH + Co. KG, Schwabach, Germany) for 24 h at 30 • C.
The pH of the bovine colostrum was evaluated using a pH meter (Inolab 3, Hanna Instruments, Venet, Italy) by inserting the pH electrode into the samples. The colour coordinates of the bovine colostrum were evaluated on the sample surface using the International Commission on Illumination (CIE) L*a*b* colour space system (CromaMeter CR-400, Konica Minolta, Marunouchi, Tokyo, Japan). The dry matter (DM, %) of the bovine colostrum samples was measured with a Pal-3 refractometer (Atago, Japan).
The microbiological parameters of the bovine colostrum were also evaluated (total LAB, total bacteria, total enterobacteria, Escherichia coli, and mould/yeast counts). For the evaluation of the total LAB count, 10 g of bovine colostrum was homogenized with 90 mL of saline (9 g/L NaCl solution). Serial dilutions of 10 −4 -10 −8 with saline were used for the sample preparation. Sterile MRS (Man, Rogosa, and Sharpe) agar (CM0361, Oxoid) with a thickness of 5 mm was used for bacterial growth on Petri dishes. The dishes were separately seeded with the sample suspension using surface sowing and incubated under anaerobic conditions at 30 • C for 72 h. The total bacteria count was determined using plate count agar (PCA), and the bacteria were incubated under aerobic conditions at 32 • C for 24-48 h (CM0325, Oxoid, UK). MacConkey (Oxoid Ltd., Basingstoke, United Kingdom) and Tryptone Bile X-glucuronide agar (Oxoid Ltd., Basingstoke, United Kingdom) were used for the determination of the total number of enterobacteria and Escherichia coli (at 35-37 • C for 18-24 h). The mould and yeast were determined on chloramphenicol agar (CM0549, Oxoid, UK) after incubation at 25 ± 2 • C for 5 days. The number of microorganisms was counted and expressed as log 10 of colony-forming units per millilitre (CFU/mL).

Nutraceutical Preparation and Analysis Methods
Gelatine (Klingai, Lithuania) was used for nutraceutical texture formation. Xylitol (Natur Hurtig, Nuremberg, Germany), citric acid (Sanitex, Kaunas, Lithuania), and sugar (Nordic Sugar Kėdainiai, Kedainiai, Lithuania) were purchased at a local market (JSC Maxima LT, Kaunas, Lithuania). Grapefruit (Citrus paradise, producer JSC Zolotonošskaja PKF, Komunarovskaja, Ukraine) essential oil was obtained from JSC Gintarine vaistine (Kaunas, Lithuania) and was used as an odour-masking agent for the nutraceuticals with a Spirulina formulation. The formulas of the nutraceutical groups are shown in Table 1. Furthermore, in the formulation of the nutraceuticals, sugar was exchanged for xylitol. The principal scheme of the experiment is shown in Figure 1.  15 15 For the preparation of the nutraceuticals, gelatine powder was first soaked in water for 30 min and then melted by heating for 15 min at 90 • C. Sugar or xylitol was added and dissolved in the mixture while boiling. The obtained mixture was further heated to 90 • C under stirring. According to the provided recipes, other ingredients (citric acid, fermented Spirulina, essential oil, fermented bovine colostrum, Jerusalem artichoke, and apple cider vinegar) were incorporated into the nutraceutical mass at the end of the process (mass temperature 40 • C). The mass obtained after mixing was poured into a cast, and the nutraceuticals were dried at 22 ± 2 • C for 24 h to achieve a hard-gel form.

Statistical Analysis
The fermentation of the Spirulina samples was performed in duplicate, and all analytical experiments were carried out in triplicate (n = 6). Bovine colostrum analyses were carried out in triplicate (n = 3). The preparation of the nutraceuticals was performed in duplicate, and analyses of the colour coordinates and texture hardness were carried out in triplicate (n = 6). The overall acceptability of the samples was evaluated by thirty panellists. The mean values were calculated using the statistical package IBM ® SPSS ® for Windows (v28.0.1.0 (142), SPSS, Chicago, IL, USA). The data were compared using Duncan's multiple range test, with significance defined at p ≤ 0.05. A linear Pearson correlation was used to quantify the strength of the relationship between the variables. The results were recognized as statistically significant at p ≤ 0.05.

Parameters of Spirulina (pH, Colour Coordinates, L-glutamic and Gamma-Aminobutyric Acids Concentrations and Fatty Acid Profile)
The colour coordinates (L*, a* and b*) and pH values of the non-treated Spirulina and the Spirulina fermented with P. acidilactici are provided in Figure 2. The L* (lightness) and The hardness of the separate layers of the nutraceuticals was evaluated by the texture profile analysis (TPA), using a Texture Analyser TA.XT2 (StableMicro Systems Ltd., Godalming, UK) (compression force-0.5 N; test speed-0.5 mm/s; post-test speed-2 mm/s; distance-6 mm). The colour coordinates of the separate nutraceutical layers were evaluated on the sample surfaces using the International Commission on Illumination (CIE) L*a*b* colour space system (CromaMeter CR-400, Konica Minolta, Marunouchi, Tokyo, Japan). A sensory analysis of the separate nutraceutical layers and the whole product was carried out according to the ISO 6658 method [33]. Thirty panellists evaluated the overall acceptability (OA) of the nutraceuticals using the hedonic scale from 0 (dislike extremely) to 10 (like extremely).

Statistical Analysis
The fermentation of the Spirulina samples was performed in duplicate, and all analytical experiments were carried out in triplicate (n = 6). Bovine colostrum analyses were carried out in triplicate (n = 3). The preparation of the nutraceuticals was performed in duplicate, and analyses of the colour coordinates and texture hardness were carried out in triplicate (n = 6). The overall acceptability of the samples was evaluated by thirty panellists. The mean values were calculated using the statistical package IBM ® SPSS ® for Windows (v28.0.1.0 (142), SPSS, Chicago, IL, USA). The data were compared using Duncan's multiple range test, with significance defined at p ≤ 0.05. A linear Pearson correlation was used to quantify the strength of the relationship between the variables. The results were recognized as statistically significant at p ≤ 0.05.

Parameters of Spirulina (pH, Colour Coordinates, L-Glutamic and Gamma-Aminobutyric Acids Concentrations and Fatty Acid Profile)
The colour coordinates (L*, a* and b*) and pH values of the non-treated Spirulina and the Spirulina fermented with P. acidilactici are provided in Figure 2. The L* (lightness) and b* (yellowness) coordinate values were significantly higher (by 4 and 32.9% on average, respectively) in the fermented Spirulina when compared to the non-treated samples. On average, the value of the a* coordinate (redness) was 8.6 times lower in the fermented Spirulina than the non-treated samples. A decrease in pH was observed in the fermented Spirulina, and its pH value was lower by 23.5% on average when compared to the nontreated Spirulina. and blueness [34]. The changes in the colour coordinates of the fermented Spirulina could be related to the fact that bound pigments are released as a result of the decrease in pH and enzyme activity during the fermentation process [23]. Spirulina contains fermentable sugars such as rhamnose, glucose, galactose, etc., which serve as a good substrate for the growth of LAB [35]. The bacterial synthesis of organic acids, especially lactic acid, causes a drop in pH during fermentation [23]. Similar results of the pH drop in fermented Spirulina were also found by de Marco Castro et al. and Bao et al. [23,36]. A non-essential amino acid called L-Glu can be found in a wide range of foods in both its free and protein-bound forms [37]. It is mainly produced through microbial fermentation, and the proteolytic enzyme L-glutaminase is involved in the hydrolysation of L-glutamine to produce L-Glu [38]. Our results showed an increase in the content of L-Glu after Spirulina fermentation that is probably related to the proteolytic activity of P. acidilactici. GABA is thought to be a powerful bioactive agent with a wide range of physiological effects, including anti-stress, pain-relieving, antioxidant, and anti-insomnia effects [39]. Microorganisms (bacteria, yeasts, and moulds) produce this compound from L-Glu by glutamate decarboxylase, but the ability of various species to generate GABA differs considerably [25]. The glutamate decarboxylase is found in a variety of LAB species, including Pediococcus, Lactobacillus, and Lactococcus. Anggraini et al. [40] reported GABA Chlorophyll, which predominates in Spirulina, gives the samples their dark green colour, while carotenoids and C-phycocyanin may be related with the presence of redness and blueness [34]. The changes in the colour coordinates of the fermented Spirulina could be related to the fact that bound pigments are released as a result of the decrease in pH and enzyme activity during the fermentation process [23]. Spirulina contains fermentable sugars such as rhamnose, glucose, galactose, etc., which serve as a good substrate for the growth of LAB [35]. The bacterial synthesis of organic acids, especially lactic acid, causes a drop in pH during fermentation [23]. Similar results of the pH drop in fermented Spirulina were also found by de Marco Castro et al. and Bao et al. [23,36].
The concentrations of GABA and L-Glu in the Spirulina samples (non-treated and fermented) are provided in Figure 3. The fermented Spirulina had greater concentrations of both GABA and L-Glu (by 5.2 times and 31.4%, respectively) compared to the non-treated samples.
A non-essential amino acid called L-Glu can be found in a wide range of foods in both its free and protein-bound forms [37]. It is mainly produced through microbial fermentation, and the proteolytic enzyme L-glutaminase is involved in the hydrolysation of L-glutamine to produce L-Glu [38]. Our results showed an increase in the content of L-Glu after Spirulina fermentation that is probably related to the proteolytic activity of P. acidilactici. GABA is thought to be a powerful bioactive agent with a wide range of physiological effects, including anti-stress, pain-relieving, antioxidant, and anti-insomnia effects [39]. Microorganisms (bacteria, yeasts, and moulds) produce this compound from L-Glu by glutamate decarboxylase, but the ability of various species to generate GABA differs considerably [25]. The glutamate decarboxylase is found in a variety of LAB species, including Pediococcus, Lactobacillus, and Lactococcus. Anggraini et al. [40] reported GABA production by Pediococcus acidilactici DS15 using tofu water and palm sugar as sources of nitrogen and carbon. The GABA-producing capacity of Pedioccocus pentosaceus MN12, isolated from fermented fish sauce, was also examined [41]. The results of our study also indicated the ability of P. acidilactici No. 29 to produce GABA during the fermentation of Spirulina. However, factors such as acidity, L-Glu content, duration of fermentation, temperature, and medium composition strongly influence the rates of GABA production [27]. production by Pediococcus acidilactici DS15 using tofu water and palm sugar as sources of nitrogen and carbon. The GABA-producing capacity of Pedioccocus pentosaceus MN12, isolated from fermented fish sauce, was also examined [41]. The results of our study also indicated the ability of P. acidilactici No. 29 to produce GABA during the fermentation of Spirulina. However, factors such as acidity, L-Glu content, duration of fermentation, temperature, and medium composition strongly influence the rates of GABA production [27]. The fatty acid (FA) profile and classification of the non-treated and fermented Spirulina samples are presented in Figures 4 and 5. It was found that palmitic (C16:0) acid composed half of the total FA content in all samples. Palmitic acid, gamma linolenic (C18:3ɣ), and linoleic (C18:2) acids were the predominant FAs in both the non-treated and fermented Spirulina. On average, the fermented samples contained 2.4%, 3%, and 11.1% less palmitoleic (C16:1), cis-and trans-9-oleic (C18:1 cis, trans), and linoleic (C18:2) acids, respectively, than the non-treated Spirulina. However, these samples had increased levels of stearic (C18:0) and alpha-linolenic (C18:3α) acids by 22.8% and 17.8%, respectively, compared to the non-treated samples. The presence of gamma-linolenic (C18:3ɣ) acid was observed after fermentation with P. acidilactici, while the content of C16:0 was similar in both types of samples. The main components of the entire FA profile were saturated (SFA), polyunsaturated (PUFA), and omega-6 fatty acids. Non-treated and fermented Spirulina had similar levels of SFA (on average, 54%), monounsaturated (MUFA) (on average, 7%), PUFA (on average, 39%), omega-6 (on average, 39%), and omega-9 (on average, 7%) fatty acids. However, the presence of omega-3 fatty acids was observed after the fermentation of Spirulina.
Spirulina is a great source of PUFA and contains high concentrations of essential FAs, particularly γ-linolenic acid. In the literature, the data on the FA profile of Spirulina varies due to different Spirulina species, countries of origin, growing conditions, and development stages at the time of harvest [42]. One study revealed that the amounts of SFA, PUFA, MUFA, and omega-6 in Arthrospira platensis accounted for 62.9-80.3%, 10.6-24.5%, 9-13%, and 10-24%, respectively, of the total FAs, and these numbers were influenced by the Spirulina's country of origin [43]. Compared to these results, we found slightly higher levels of PUFA and omega-6, lower level of SFA, and a similar level of MUFA in the non-treated Spirulina. Moreover, similar to our results, that research study did not detect omega-3 or find it in small amounts. It was reported that the percentage of palmitic acid (16:0) in The fatty acid (FA) profile and classification of the non-treated and fermented Spirulina samples are presented in Figures 4 and 5. It was found that palmitic (C16:0) acid composed half of the total FA content in all samples. Palmitic acid, gamma linolenic (C18:3γ), and linoleic (C18:2) acids were the predominant FAs in both the non-treated and fermented Spirulina. On average, the fermented samples contained 2.4%, 3%, and 11.1% less palmitoleic (C16:1), cisand trans-9-oleic (C18:1 cis, trans), and linoleic (C18:2) acids, respectively, than the non-treated Spirulina. However, these samples had increased levels of stearic (C18:0) and alpha-linolenic (C18:3α) acids by 22.8% and 17.8%, respectively, compared to the non-treated samples. The presence of gamma-linolenic (C18:3γ) acid was observed after fermentation with P. acidilactici, while the content of C16:0 was similar in both types of samples. The main components of the entire FA profile were saturated (SFA), polyunsaturated (PUFA), and omega-6 fatty acids. Non-treated and fermented Spirulina had similar levels of SFA (on average, 54%), monounsaturated (MUFA) (on average, 7%), PUFA (on average, 39%), omega-6 (on average, 39%), and omega-9 (on average, 7%) fatty acids. However, the presence of omega-3 fatty acids was observed after the fermentation of Spirulina. Arthrospira platensis is 25% of the lipids [44]. However, we observed a higher content of C16:0 in the non-treated Spirulina. The changes in the FA profile after fermentation could be related to the ability of LAB to utilize lipids and synthesize secondary metabolites in a fermented substrate (e.g., FAs) [45,46]. Similar to our results, in the study by Dewi and Amalia, Spirulina was fermented with Lactiplantibacillus plantarum (FNCL 0127); palmitic acid (45% on average) was found to be the main FA, followed by stearic (20% on average), linoleic (17% on average), and alpha-linolenic (13% on average) acids, while the proportion of SFA was found to be the highest among others [47].

Parameters of Non-Treated and Fermented Bovine Colostrum
The pH value, dry matter, and colour coordinates of the bovine colostrum are provided in Figure 6. The dry matter of the non-treated and fermented colostrum was similar Spirulina is a great source of PUFA and contains high concentrations of essential FAs, particularly γ-linolenic acid. In the literature, the data on the FA profile of Spirulina varies due to different Spirulina species, countries of origin, growing conditions, and development stages at the time of harvest [42]. One study revealed that the amounts of SFA, PUFA, MUFA, and omega-6 in Arthrospira platensis accounted for 62.9-80.3%, 10.6-24.5%, 9-13%, and 10-24%, respectively, of the total FAs, and these numbers were influenced by the Spirulina's country of origin [43]. Compared to these results, we found slightly higher levels of PUFA and omega-6, lower level of SFA, and a similar level of MUFA in the non-treated Spirulina. Moreover, similar to our results, that research study did not detect omega-3 or find it in small amounts. It was reported that the percentage of palmitic acid (16:0) in Arthrospira platensis is 25% of the lipids [44]. However, we observed a higher content of C16:0 in the non-treated Spirulina. The changes in the FA profile after fermentation could be related to the ability of LAB to utilize lipids and synthesize secondary metabolites in a fermented substrate (e.g., FAs) [45,46]. Similar to our results, in the study by Dewi and Amalia, Spirulina was fermented with Lactiplantibacillus plantarum (FNCL 0127); palmitic acid (45% on average) was found to be the main FA, followed by stearic (20% on average), linoleic (17% on average), and alpha-linolenic (13% on average) acids, while the proportion of SFA was found to be the highest among others [47].

Parameters of Non-Treated and Fermented Bovine Colostrum
The pH value, dry matter, and colour coordinates of the bovine colostrum are provided in Figure 6. The dry matter of the non-treated and fermented colostrum was similar and reached 27.3% on average (Figure 6a). Fermentation with L. paracasei decreased the pH of the colostrum by 33.4%. The lightness (L*) values of the non-treated and fermented colostrum were similar (on average, 85.6 NBS) (Figure 6b). However, the values of the b* and a* coordinates were lower by 12.7% and 5.4 times, respectively, compared to the non-treated samples.
higher levels of carotenoids (lutein, all-trans β-carotene, and cis-13 β-carotene), whil redness may occur due to the presence of red blood cells [48]. It was also reported over time, the value of the L* coordinate increases and the b* and a* coordinates dec in bovine colostrum. Bound pigments may be released because of the pH drop an creased enzyme activity during the fermentation process, which may account fo changes in the colour coordinates of fermented colostrum [23]. The microbiological parameters of bovine colostrum, both non-treated and mented with Lacticaseibacillus paracasei LUHS244, are provided in Figure 7. The total counts between the non-treated and fermented colostrum were similar. However, fermentation, the Escherichia coli, total bacteria, total enterobacteria, and total mould/ counts were significantly lower, on average, by 13.2-, 20-, 67-, and 57-fold, respective Microorganisms in bovine colostrum may appear from the mammary gland or ing the collection, manipulation, and storage of colostrum [51]. Our findings for the treated colostrum slightly contrast with those of Santos et al., who found that the enterobacteria, and yeast counts of bovine colostrum from three different comme dairy farms in Brazil accounted for 4.5-5.1, 1.4-3.5, and 1.3-1.7 log10 CFU/mL, respect [52]. However, these differences between studies may arise because of variances in g raphy, climate, technology, management practices, and degree of hygiene [51]. The ence of LAB in colostrum is common, and the growth rate of these bacteria rapidl creases due to unfit storage conditions and temperature [52]. LAB break down lactose reduce pH, while their probiotic properties are also beneficial to the animal immune tem. The presence of Enterobacteria is usually related to the lack of hygiene before, du and after colostrum milking [52]. Bovine colostrum may contain a diverse range of E The pH of bovine colostrum ranges from 6.0 to 6.61, and it is suggested that the higher levels of dihydrogen phosphate, citrate, protein, and carbon dioxide contribute to the low pH value [48]. In this study, the obtained pH and dry matter results of the nontreated bovine colostrum are similar with those reported in other studies [49,50]. During fermentation, a decrease in pH is brought on by the bacterial production of organic acids, particularly lactic acid [23]. The yellowness in the colour of bovine colostrum is elicited by the higher levels of carotenoids (lutein, all-trans β-carotene, and cis-13 β-carotene), while the redness may occur due to the presence of red blood cells [48]. It was also reported that over time, the value of the L* coordinate increases and the b* and a* coordinates decrease in bovine colostrum. Bound pigments may be released because of the pH drop and increased enzyme activity during the fermentation process, which may account for the changes in the colour coordinates of fermented colostrum [23].
The microbiological parameters of bovine colostrum, both non-treated and fermented with Lacticaseibacillus paracasei LUHS244, are provided in Figure 7. The total LAB counts between the non-treated and fermented colostrum were similar. However, after fermentation, the Escherichia coli, total bacteria, total enterobacteria, and total mould/yeast counts were significantly lower, on average, by 13.2-, 20-, 67-, and 57-fold, respectively.
Microorganisms in bovine colostrum may appear from the mammary gland or during the collection, manipulation, and storage of colostrum [51]. Our findings for the nontreated colostrum slightly contrast with those of Santos et al., who found that the LAB, enterobacteria, and yeast counts of bovine colostrum from three different commercial dairy farms in Brazil accounted for 4.5-5.1, 1.4-3.5, and 1.3-1.7 log 10 CFU/mL, respectively [52]. However, these differences between studies may arise because of variances in geography, climate, technology, management practices, and degree of hygiene [51]. The presence of LAB in colostrum is common, and the growth rate of these bacteria rapidly increases due to unfit storage conditions and temperature [52]. LAB break down lactose and reduce pH, while their probiotic properties are also beneficial to the animal immune system. The presence of Enterobacteria is usually related to the lack of hygiene before, during, and after colostrum milking [52]. Bovine colostrum may contain a diverse range of E. coli strains, but just a few strains are harmful, and others do not elicit infections [53]. The process of fermentation with LAB allows for the biological preservation of bovine colostrum [53]. The synthesis of numerous antibacterial substances by Lactobacillus strains is well-known, and their antimicrobial properties are influenced by their environment of growth [54]. In our study, the reduced contamination of fermented bovine colostrum could be related to the ability of LAB to produce inhibitory compounds (acetic and lactic acids, hydrogen peroxide, and others) and to synthesize antimicrobial peptides (bacteriocins) [55]. The high antimicrobial activity of Lacticaseibacillus paracasei LUHS244 was already reported in our previous research [31].
strains, but just a few strains are harmful, and others do not elicit infections [53]. Th cess of fermentation with LAB allows for the biological preservation of bovine colo [53]. The synthesis of numerous antibacterial substances by Lactobacillus strains is known, and their antimicrobial properties are influenced by their environment of g [54]. In our study, the reduced contamination of fermented bovine colostrum cou related to the ability of LAB to produce inhibitory compounds (acetic and lactic hydrogen peroxide, and others) and to synthesize antimicrobial peptides (bacteri [55]. The high antimicrobial activity of Lacticaseibacillus paracasei LUHS244 was al reported in our previous research [31].

Overall Acceptability, Colour, and Texture Parameters of the Separate Nutraceutical La and Overall Acceptability of the Whole Nutraceutical
Images of the produced nutraceutical layers and the final product are provid Table 2.
The colour coordinates and texture hardness of the separate nutraceutical laye provided in Table 3. The values of the layer colour coordinates depended on the col the main ingredients (fermented Spirulina, fermented bovine colostrum, Jerusalem choke, and apple cider vinegar) used for each layer. However, the colour coordina layers with same main ingredients were not influenced by the type of sweetener (su xylitol), except for the L* coordinate of the V and VI layers (with apple cider vinegar the b* coordinate of the I and II layers (with fermented Spirulina). The lowest val the L*, a*, and b* coordinates were found for layers containing fermented Spirulin mulations I and II). The highest lightness and yellowness were determined for layer apple cider vinegar (formulations V and VI). Coordinate a* was the highest for layer bovine colostrum and Jerusalem artichoke (formulations III and IV).
The analysis of texture hardness showed that layers with apple cider vinegar the softest, while layers with bovine colostrum and Jerusalem artichoke (formulatio had the highest hardness. According to the evaluation of the overall acceptability of the separate layers, with fermented Spirulina received the highest scores, while the lowest acceptabilit found for layers with apple cider vinegar ( Table 3). The final nutraceuticals were eval the same as layers with fermented Spirulina and received the highest score. The ac bility of layers with the same main ingredients were not influenced by the type of s ener (sugar or xylitol).
The lower hardness of layers with apple cider vinegar could be explained by th that the acids in vinegar induce gelatine hydrolysis and reduce its ability to gel in

Overall Acceptability, Colour, and Texture Parameters of the Separate Nutraceutical Layers and Overall Acceptability of the Whole Nutraceutical
Images of the produced nutraceutical layers and the final product are provided in Table 2.
The colour coordinates and texture hardness of the separate nutraceutical layers are provided in Table 3. The values of the layer colour coordinates depended on the colour of the main ingredients (fermented Spirulina, fermented bovine colostrum, Jerusalem artichoke, and apple cider vinegar) used for each layer. However, the colour coordinates of layers with same main ingredients were not influenced by the type of sweetener (sugar or xylitol), except for the L* coordinate of the V and VI layers (with apple cider vinegar), and the b* coordinate of the I and II layers (with fermented Spirulina). The lowest values of the L*, a*, and b* coordinates were found for layers containing fermented Spirulina (formulations I and II). The highest lightness and yellowness were determined for layers with apple cider vinegar (formulations V and VI). Coordinate a* was the highest for layers with bovine colostrum and Jerusalem artichoke (formulations III and IV).
The analysis of texture hardness showed that layers with apple cider vinegar were the softest, while layers with bovine colostrum and Jerusalem artichoke (formulation III) had the highest hardness.
According to the evaluation of the overall acceptability of the separate layers, layers with fermented Spirulina received the highest scores, while the lowest acceptability was found for layers with apple cider vinegar ( Table 3). The final nutraceuticals were evaluated the same as layers with fermented Spirulina and received the highest score. The acceptability of layers with the same main ingredients were not influenced by the type of sweetener (sugar or xylitol). Table 2. Images of the separate nutraceutical layers and whole product.

Nutraceutical Formulations Images
Formulation I (sugar + gelatine + citric acid + fermented Spirulina + water + C. paradise essential oil) dies [8]. Kumkong et al. formulated gummies with a whey protein concentrate and freezedried aril and pulp from Gac fruit, which is natural colourant and is beneficial to health [6]. Niam et al. proposed a chewable gummy formulation of Bastard Cedar leaves (Guazuma Ulmifolia), Senna leaves (Cassia Angustifolia) and lime extracts for a low-calorie diet [9]. Rani et al. produced chewable gummy tablets with Moringa oleifera leaf powder, which possess high antioxidant activity, using two types of gelling agents [10]. Table 2. Images of the separate nutraceutical layers and whole product.

Nutraceutical Formulations Images
Formulation I (sugar + gelatine + citric acid + fermented Spirulina + water + C. paradise essential oil) Formulation II (xylitol + gelatine + citric acid + fermented Spirulina + water + C. paradise essential oil) Formulation III (sugar + gelatine + citric acid + fermented bovine colostrum + Jerusalem artichoke powder + water) Formulation II (xylitol + gelatine + citric acid + fermented Spirulina + water + C. paradise essential oil) dies [8]. Kumkong et al. formulated gummies with a whey protein concentrate and freezedried aril and pulp from Gac fruit, which is natural colourant and is beneficial to health [6]. Niam et al. proposed a chewable gummy formulation of Bastard Cedar leaves (Guazuma Ulmifolia), Senna leaves (Cassia Angustifolia) and lime extracts for a low-calorie diet [9]. Rani et al. produced chewable gummy tablets with Moringa oleifera leaf powder, which possess high antioxidant activity, using two types of gelling agents [10]. Table 2. Images of the separate nutraceutical layers and whole product.

Nutraceutical Formulations Images
Formulation I (sugar + gelatine + citric acid + fermented Spirulina + water + C. paradise essential oil) Formulation II (xylitol + gelatine + citric acid + fermented Spirulina + water + C. paradise essential oil) Formulation III (sugar + gelatine + citric acid + fermented bovine colostrum + Jerusalem artichoke powder + water) Formulation III (sugar + gelatine + citric acid + fermented bovine colostrum + Jerusalem artichoke powder + water) dies [8]. Kumkong et al. formulated gummies with a whey protein concentrate and freezedried aril and pulp from Gac fruit, which is natural colourant and is beneficial to health [6]. Niam et al. proposed a chewable gummy formulation of Bastard Cedar leaves (Guazuma Ulmifolia), Senna leaves (Cassia Angustifolia) and lime extracts for a low-calorie diet [9]. Rani et al. produced chewable gummy tablets with Moringa oleifera leaf powder, which possess high antioxidant activity, using two types of gelling agents [10]. Table 2. Images of the separate nutraceutical layers and whole product.
The lower hardness of layers with apple cider vinegar could be explained by the fact that the acids in vinegar induce gelatine hydrolysis and reduce its ability to gel in water [56]. Moreover, the incorporation of milk proteins affects the hardness of gelatine gels by stabilizing them [7]. That is why layers with bovine colostrum had the highest hardness in our study. However, it was reported that functional foods such as yogurts, curd, cheese, ice creams, kefir, milk-based beverages, and candies with the inclusion of the bovine colostrum showed sensory acceptance and nutritional benefits [16,57,58]. Spirulina is mainly incorporated into cereal-based products (biscuits, pasta, and bread), snacks, dairy products, and as a potential meat substitute [59]. However, the high level of Spirulina inclusion in foods can cause undesirable changes in physicochemical quality parameters and texture and a lower acceptance [59]. Fermentation with LAB influences the taste and odour of foods and therefore improves the sensory properties of fermented food [60]. During fermentation, lipolysis and proteolysis are induced and small peptides, free fatty acids, and amino acids are generated, which act as flavour precursors [61]. As flavour is a key element in consumer acceptance, this could probably explain the high acceptability scores of the nutraceuticals produced in our study. To the best of our knowledge, compositions of nutraceuticals such as those included in our study have not been tested by other researchers. However, other bioactive ingredients were used for functional gummy preparation. Paternina et al. prepared gummies enriched with Spirulina biomass and freeze-dried acai pulp and observed that the acceptability index was satisfactory for these candies [8]. Kumkong et al. formulated gummies with a whey protein concentrate and freeze-dried aril and pulp from Gac fruit, which is natural colourant and is beneficial to health [6]. Niam et al. proposed a chewable gummy formulation of Bastard Cedar leaves (Guazuma Ulmifolia), Senna leaves (Cassia Angustifolia) and lime extracts for a low-calorie diet [9]. Rani et al. produced chewable gummy tablets with Moringa oleifera leaf powder, which possess high antioxidant activity, using two types of gelling agents [10].

Conclusions
In this study, the formulation of multifunctional nutraceuticals based on Spirulina powder, bovine colostrum, Jerusalem artichoke powder, and apple cider vinegar was proposed. The nutraceuticals comprised three layers: (I) a layer with fermented Spirulina; (II) a layer with fermented bovine colostrum and Jerusalem artichoke powder; (III) a layer with apple cider vinegar. The fermentation of Spirulina and bovine colostrum with the strains Pediococcus acidilactici No. 29 and Lacticaseibacillus paracasei LUHS244, respectively, which was carried out before nutraceutical preparation, significantly influenced most of the tested parameters of these ingredients. In comparison to the untreated Spirulina, the fermented samples had higher concentration of gamma-aminobutyric and L-glutamic acids (by 5.2 and 31.4%, respectively). The fatty acid profile of the fermented Spirulina showed increased alpha-linolenic, gamma-linolenic, and omega-3 fatty acid contents. The fermentation of bovine colostrum significantly improved its microbiological safety. The colour coordinates of the separate nutraceutical layers depended on the colour of the main ingredients. The hardest layers were those made with Jerusalem artichoke and bovine colostrum, whereas the softest layers were those made with apple cider vinegar. The results of the current study indicate a significant potential of the suggested combination for a nutraceutical with enhanced functionality and a high rating for acceptability.