1. Introduction
Diet–Gut Microbiome Interplay is a hot-topic relationship that, for the last decade, the scientific community has struggled to pinpoint. Although we still lack a clear picture of what a “normal microbiota” looks like, we do know that the digestive tract plays a vital role in the maintenance of host homeostasis and accomplishes many roles, including its normal development, digestion, vitamin synthesis, anti-infectious defense, immunity, and angiogenesis [
1,
2]. The host–microbe communication is influenced by age, diet, medication (mostly antibiotics), physical exercise, and associated diseases (such as diabetes or metabolic syndrome) [
3]. Beyond the postnatal period, long-term dietary patterns have a strong influence on the composition of the gut microbiome. Therefore, given its importance for health, maintaining the balance of microbiota composition by different interventions, including diet-based ones, is critical [
4]. A healthy diet shapes the gut microbiota composition, thereby influencing host–microbe interactions as well as homeostasis and disease processes [
5].
Nowadays, consumers have gained more and more confidence in non-Western or natural, alternative products with beneficial effects on health, due to accessibility, few adverse reactions or contraindications, and the possibility of being combined with drug treatment (synergistic adjuvants—prebiotics). The development of microbiota-modulating products, such as prebiotics, is emerging to be of paramount importance, as it can be used as a supplement in food and nutraceutical applications. Nutraceuticals can be used as dietary supplements, as they are food ingredients or sourced from food products that, apart from their basic original nutritional value, provide extra benefits for the host including microbiota modulation [
6]. For instance, dietary fibers alleviate several diseases by increasing the abundance and diversity of some microbes present in the gut [
7]. Keeping in mind that, in the field of nutrition, plants and their products have significant importance not only for providing basic nutrients, but also for the prevention of various maladies. They indeed improve quality of life throughout the globe (plant-based traditional medicines have been in use since immemorial times). Consequently, dietary intervention with nutraceuticals and functional ingredients as dietary boosters has opened wide a window of opportunity for gut health nowadays.
The administration of nutraceuticals as well as of various supplements, such as fibers, antioxidant vitamins, omega-3 fatty acids, and herbs, has been employed to improve various diseases associated with dysbiosis [
8]. In a recently published paper [
9], we highlighted that research into the possible role of functional foods and nutraceuticals in mitigating immune function and actively sustain gut health is still in its infancy, especially in the Romanian market.
Therefore, for this study we envisioned the evaluation of the effectiveness of a novel developed nutraceutical to be conducted in correlation with associated health claims approved by Regulation (EC) No 1924/2006 about nutrition and health claims made on foods, (EU) No 432/2012 made on vitamins and minerals, and EFSA regulations regarding the health claims of botanicals, keeping a strong focus on potential health benefits, duplicity effects, and the modulation of gut microbiota.
3. Results
According to Regulation (EC) No 1924/2006 nutritional profiling of novel formulated dietary supplements/nutraceuticals constitutes the starting point in pinpointing quality parameters, as well as drawing conclusions for eventual nutritional benefits.
Therefore, we evaluated the total protein content (%), total lipidic content (%), ash insoluble in 10% HCl (%), moisture content (%), and dry matter (%) for all ingredients that were added into the NN formula, as well as their fingerprints in the final product (
Table 1).
According to previously reported data, the absorption spectra of high-protein products show specific peaks, i.e., Amide I (about 1628 cm
−1) and Amide II (about 1535 cm
−1) bands. The former arises primarily from the C=O stretching vibration, and the latter is attributed to the N-H bending and C-N stretching vibrations of the peptide backbone. The band with the maximum absorption at about 3283 cm
−1 was assigned to Amine A. The absorption band at 3000–2875 cm
−1 corresponds to the C-H stretching vibration, while the low intensity band at about 1076 cm
−1 is due to the C-O stretching (
Figure 1).
Peaks specific for phenolic compounds have also been identified. So, the bands 1233 cm
−1 are a function of the protonation of phenolic acids and are assigned to the stretching vibration of C-OH in the carboxylic group [
25]. The peak at 1390 cm
−1 is characteristic of COO stretching [
26]. Aromatic C-C extending to ~1520 (peak overlapping with the specific Amide II peak) and ~1447 cm
−1 is related to phenolic compounds [
27]. The band at ≈2900 cm
−1 corresponds to the C-H stretching vibration that reflects the organic content in general [
28]. Several peaks located in the region of 1070–1150 cm
−1 are mainly attributed to stretching vibrations of C-O and C-C [
29]. The absorptions at 1230 cm
−1 have been assigned to phenol hydroxyl group [
30]. The stretching of ether linkage, C-O-C, is indicated by the absorption at 1009 cm
−1 [
31].
The high protein content (80.97%) of the final formulation is a result of using high proportions of hydrolyzed collagen (90.30%) and egg white (82.05%), two important sources of high-quality proteins, which might contribute considerably to the recommended human daily protein allowance. Taking into account that the general recommendation for daily protein requirements is a minimum of 0.8 g protein/kg body weight/day, the protein intake provided by the new nutraceutical formula represents 27% of the recommended daily dose for adults.
Due to the fact that ingredients with a high content of good-quality protein were used, the amount of ash insoluble in 10% HCl obtained for the final supplement formulation is very low (0.27%). The water activity measurement is also described, since it parallels the measurement of the total moisture as an important stability and quality factor. The dry matter that remains after moisture removal is commonly referred to as total solids; this was 7.37% for the final formulation. The moisture content of suppliants is significant for their shelf life, with better storage stability maintained by lower moisture contents of the supplements [
32].
The fiber content for the new nutraceutical formula (2.1%) was calculated based on data provided by the producer for the two fiber-containing ingredients: pollen (13%) and cherry powder (3%). The carbohydrate content, determined by difference, was 8.42%, while the energy values are 365 kcal/100 g.
The assay used for the determination of microminerals, hazardous toxic metals, and other potential harmful metals showed good linearity for all elements in the selected ranges with correlation coefficients greater than 0.999.
The concentrations of the metals in the sample are showed in
Table 2 and are expressed as the mean value of the triplicate analysis, followed by the standard deviation. Toxic metals such as Cd, Pb, and Hg were compared with the maximum levels according to Commission Regulation (EC) No. 1881/2006. Lead, cadmium, and mercury are the only metals that are specifically regulated for dietary supplements, with maximum levels of 3 mg/kg for Pb, 1 mg/kg for Cd, and 0.1 mg/kg for Hg. Arsenic, on the other hand, is not specifically regulated for dietary supplements, but we took in consideration the smallest value that is specified in (EC) No. 1881/2006, respectively, 0.1 mg/kg [
33].
As we can see from
Table 2, our sample does not harbor alarming values for heavy metals. Although arsenic, cadmium, and lead are present in the samples in concentrations of 0.0406 µg/g, 0.0266 µg/g, and 0.0534 µg/g, respectively, these values do not exceed the maximum regulated limits, while mercury has not been detected in any form, being below the instrumental detection limit.
As for the rest of the analytes, microminerals are not present in quantities that might exceed the daily population reference intakes (PRI) established by the EFSA [
34]. The requirements for these microminerals vary from grams per day, for elements such as Na or K, to milligrams per day, for elements such as Fe and Zn, and even to micrograms per gram (Cu, Se, Mn). The most abundant species found in our sample are iron, manganese and aluminum, with concentrations of approximately 23 µg/g, 17 µg/g and 17 µg/g, followed by zinc and rubidium with a concentration of ~8 µg/g and 6 µg/g.
All ingredients in terms of phenolic, flavonoid, and anthocyanin content were analyzed. Among the ingredients used in the formulation of the food supplement, pollen had the highest polyphenols and flavonoids. A higher value for Vitamin C was actually generated by the reduced capacity of the Folin-Ciocalteu reagent (
Table 3). The present method is not suitable for the determination of the total phenolic content unless interfering substances are considered or removed. Moreover, the application of this method for the determination of the antioxidant capacity of food samples is proposed for the evaluation of the contribution from phenolic and other reducing substances [
35]. By evaluating the percentage of phenolic and flavonoid compounds quantified in the final formulation compared to the cumulative value of all ingredients from formulation, it was observed that some of the compounds are denatured by the technological process. No significant changes were observed in the case of anthocyanins. The higher values of the electuary and orange flavors for phenolic and flavonoids content were given by the precipitation with alcoholic media. For the final quantification, we took account of these aspects.
In the case of antioxidant activity, a significant difference was observed between the cumulative effects of the ingredients compared with the finished product for the TEAC and CUPRAC variants (
Table 4), but the synergistic effect between the components was relatively low. In the case of DPPH and FRAP, even if the value of
p < 0.05, according to the model these are not significant due to the high values of the standard deviations. So, we consider that the ingredients were not antagonistic, but the effect for TEAC and CUPRAC was synergistic, while for DPPH and FRAP it was additive [
36].
The evaluation of potential microbial hazards results obtained from the microbiological analysis of the NN powder revealed absence of contamination. The total number of aerobic bacteria and the total amount of yeast and mold were expressed in CFU/g. The recorded results indicate for both assays <10 CFU/g (
Table 5), implying that the sample presents no microbiological risk, therefore the quality of the finished products was not affected.
Both mesophilic aerobic bacteria and fungi are common spoilage microorganisms that contaminate the technological flow under conditions of inadequate hygiene. In order to prevent the contamination of finished foodstuffs with microorganisms, a rigorous inspection of the raw materials, working materials, staff hands, the environment in which the product is produced, and the processing method must be performed. These are potentially contaminating elements of the technological flow.
Water activity is the most important parameter of water in terms of food safety. The value of water activity (aw) for food is an essential criterion for the microbiological control of products. Water activity is defined as follows: when a hygroscopic material is placed in a closed chamber, a balance will be achieved between the material and the air above it. Relative humidity, which occurs at a constant air temperature, corresponds to the value of water activity multiplied by 100 (aw = relative humidity (%)/100). A water activity above 0.95 will provide sufficient moisture to support the growth of bacteria, yeast, and mold. The samples tested showed a low value of water activity.
The evaluation of the supposed putative duplicity effect of NN with a prebiotic role was performed by addressing the dose-response on the viability of both the probiotic and pathogenic bacteria that are specific to gut microbiota. The results obtained indicate a promotion of viability for probiotic bacteria and an inhibitory effect on pathogenic bacteria, which reinforce our hypothesis that prebiotic formulas have a duplicity effect (
Figure 2). This finding wisely emphasizes the concept of Duplibiotics, which was recently introduced into the scientific literature and mentioned by Rodríguez-Daza M.C. et.al. [
37].
Lb. rhamnosus strains are present in various probiotic products and are associated with multiple beneficial effects on human health. The active compounds present in the NN product stimulate 10 times the growth of Lb. rhamnosus strains. The new product has inhibitory activity against E. coli, Ent. Faecalis, and Lc. Lactis; the number of bacterial cells is being reduced by the bioactive compounds present in the NN product. In contrast, nutraceuticals have no effect on Sal. Typhimurium, Ent. Aerogenes, and S. boulardii growth. Our results showed that NN exerts a selective effect on the growth of the bacterial strains present in the intestine.
In our study, we selected colorimetric assays for cytotoxicity assessment. The MTT assay is a very popular and widely used colorimetric assay in the in vitro evaluation of cytotoxicity [
38], as it provides beneficial aspects such as rapidity, reliability, and significant knowledge regarding the metabolic activity of eukaryotic cells. The LDH assay provides information about cellular damage (lysis of cells membranes) after cell treatment; it complements the MTT assay in drawing conclusions about the potential mechanism of action.
The NN sample was analyzed at three different concentrations of 200 mg/mL (NN 1:2), 100 mg/Ml (NN 1:4) and 50 mg/Ml (NN 1:6). The results obtained revealed that high concentrations of NN (200 mg/mL) induce a significant cellular viability reduction (
Figure 3a) (up to 56% viability loss), followed by the 100 mg/mL concentration (with 39% viability loss). The greatest percentage of viability was obtained for the 50 mg/mL concentration (with only 15% viability loss). In this case, at 50 mg/mL the NN powder had a beneficial effect on HT-29 cells and acted as a nutritive substrate (viability rates similar to control—untreated cells).
Even though the tested formulations affected the cell proliferation at 200 mg/mL and 100 mg/mL (as indicated by the MTT assay), they did not exhibit cytotoxic effects on the intestinal cells at 50 mg/mL (viability rates being similar to control cells). Moreover, NN 1:6 showed LDH values similar to the control (unstimulated cells).
Bacterial adherence to monolayers of HT-29 cells in the presence of NN sample extract resulted in index values between 82% and 92%. The most significant inhibitory effect has been noticed for the
Escherichia coli ATCC 25922 strain (
Table 6).
The inhibition of bacterial adhesion, invasion, and intracellular survival significantly limits the pathogenicity of microbial agents and provides a beneficial background for the prevention and control of associated infections [
39,
40]. For this assay, we selected a
Campylobacter jejuni NCTC 81-176 strain, a Gram-negative spirally curved microaerophilic bacterium that is recognized as a significant cause of human enteritis (34) (campylobacteriosis), being one of the most widespread infectious diseases of the last century. The incidence and prevalence of campylobacteriosis have increased in both developed and developing countries over the last 10 years [
41]. Therefore, we tested the effect of NN at a concentration of 200 mg/mL against bacterial adhesion and invasion.
The results obtained (
Figure 4) indicate that the total number of bacterial cells adhering to the epithelial cells is significantly lower in the sample in which the NN product was added, while the new product had no effect on the
C jejuni invasion.
4. Discussion
We started this study by asking ourselves if nutraceuticals are a gut friend or a foe. In trials for deciphering this conundrum, we came across various controversies over a specific definition and set of regulations to define a nutraceutical compound. We understand now that the term is recognized and accepted for use for health-enhancing products that improve the mental and physical activities of the body [
42,
43]. Straightforwardly, a nutraceutical product can be defined as “a food or part of a food that provides benefits to health in addition to its nutritional content” [
44].
The general health status of a human host presents an interdependence alliance with the gut microbial ecosystem. The microbiota mediate interactions between the internal and external environments in the gut, forming an intimate partnership among intestinal epithelial cells and dietary nutrients. Scientific findings support the correlation between long-term dietary patterns and their influence on the composition of gut microbes [
45]. Not surprisingly, diet and gut microbes are two critical factors linked with the pathogenesis of gastrointestinal diseases and systemic conditions such as metabolic and cardiac disorders [
45]. Therefore, promoting the use of nutraceuticals is a tempting intervention in terms of how to manipulate diet to promote a healthy microbiota.
Dietary intervention via nutraceuticals opened wide a window of opportunity for gut health. Given its importance for health, maintaining the balance of microbiota composition, by different interventions including diet-based ones, is critical [
46,
47]. A healthy diet shapes the gut microbiota composition, thereby influencing host–microbe interactions as well as homeostasis and disease processes [
5].
According to Regulation (EC) No 1924/2006 on nutrition and health claims made on foods, (EU) No 432/2012 on vitamins and minerals, and EFSA regulations regarding health, claims of botanicals, ingredients used in our NN formula have the following recognized functions: Prunus emarginata—antioxidant activity, protects the general health by antioxidant activity ID4507; bee pollen—helps to improve immunity ID3136, enhances appetite ID3135, supports and promotes good functioning of the hearts, blood vessels and a balanced level of blood lipids ID4648, is a natural source of compounds with antioxidative action that helps maintain the optimum antioxidant status of the body ID4659; vitamin C—contributes to the normal function of the immune system ID4321, contributes to the protection of cells from oxidative stress ID 3331, contributes to the reduction of tiredness and fatigue ID2622, contributes to normal energy-yielding metabolism ID3196. As for the product per se, the following claims align with (EC) No 1924/2006: good source of proteins and fibers, sugar-free, and saturated fat-free.
The nutritional profiling of the final formulation indicates a high protein content (80.97%) owed to the main ingredient of the formula, hydrolyzed collagen, and to the egg whites. Collagen plays a supportive role in rebuilding and strengthening the lining of our digestive tract as it contains the amino acids—particularly glycine and glutamine—that are essential for its repair [
48]. Furthermore, egg whites are a good source of protein and a reasonable option for individuals affected by diabetes, high cholesterol, or cardiovascular disease [
49]. Due to several formulation tryouts, we succeeded in obtaining a low level of fats; out of all ingredients, the highest-ranking one was the bee pollen, which more than reasonably provided important health benefits that counterbalanced the final product. The calorific value of the final product is 365 kcal/100 g and 73 kcal for a single-serving portion.
In terms of phenolic and flavonoid content, bee pollen presents the highest amount; these results effectively support its antioxidant potency and correlate with other authors findings [
50]. According to Oroian et al. (2020) [
51], among the phenolic compounds, the majority compounds are 5-O-caffeoylquinic acids and caffeic acid from the phenolic acids group, quercetin 3-O-galactoside and isorhamnetin 3-O-glucoside from the flavonols group, and luteolin and apigenin from the flavones group. It is well known that the chemical composition of pollen differs significantly depending on the plant species, whether it is uniflora or multiflora, the climatic-geographical conditions [
52], and its processing methods and storage environment [
53,
54]. Due to the fact that a higher value for Vitamin C was generated by the reduced capacity of the Folin-Ciocalteu reagent, the results obtained for it should not be taken into account for the total phenolic and flavonoid content. Moreover, an influence of the technological process selected for product formulation was observed; however, it did not affect the potency of ingredients in a major way, but, nevertheless, it should be considered for further improvement. The anthocyanin content obtained for sour cherries was relatively high (165.95 µg/g); their antioxidant activity values are similar to other reported results [
55], mainly due to the presence of polyphenol but also to the anthocyanin fractions as well.
The evaluation of the antioxidant activity of the final product indicates that the combination of ingredients does not lead to antagonistic effects, the effect for TEAC and CUPRAC was synergistic, while for DPPH and FRAP it was additive. The DPPH values are also influenced by organic solvent-soluble antioxidant compounds [
56], while FRAP values are strongly influenced by the pH of the reaction medium, some antioxidant compounds may undergo a modified antioxidant effect [
57]. The FRAP method has been used successfully on a large scale to determine the antioxidant activity of anthocyanins in various matrices, so the highest value obtained for sour cherries was that obtained using the FRAP method [
58]. In the case of vitamin C, the results obtained correlate with the mechanism proposed by Liu et al. [
59]; higher values being given by the methods that have as HAT mechanism of action (TEAC and DPPH) and lower values for the methods, SET ones (FRAP and CUPRAC) [
60]. Among the ingredients used, the antioxidant activity is given mainly by Vitamin C, pollen, and sour cherries.
The potential hazards of microbiological and chemical origin were evaluated. The results obtained for the total number of aerobic bacteria and total number of yeasts and fungi were in compliance with values indicated as safe for consumption by the ISO 4833-1/2014 and ISO 21527-2:2009 standards.
As for heavy metal content, our sample does not show alarming values. The values obtained for arsenic, cadmium, and lead do not exceed the maximum regulated limits, while mercury has not been detected, as it was below the instrumental detection limit. Iron, manganese, and aluminum were the most abundant microminerals determined in the final product, but their values did not exceed the daily population reference intakes (PRI) established by the EFSA [
34].
The putative duplicity effect of a NN with a prebiotic role was addressed by evaluating the dose-response on probiotic and pathogenic bacteria that are specific and important to the gut ecosystem. A 10× stimulatory response was obtained for the
Lb. rhamnosus strains. This species is appraised for its resistance to acid and bile, good growth characteristics, and adhesion capacity to the intestinal epithelial layer, antimicrobial activity factors that contribute to its probiotic status [
61]. Since dietary supplementation with probiotics in various health-affecting conditions (infections, diarrhea, etc.) is not necessarily a fit-all solution, this result is of significant importance, because it promotes the growth of a self-bacterial population. Moreover, our product presented an inhibitory effect against
E. coli, Ent. Faecalis, and
Lc. lactis strains, with the reduction in bacterial cells number being significant. In contrast, on
Sal. Typhimurium, Ent aerogenes, and
S. boulardii strains, the nutraceutical presented no effect on growth. Although the number of tested strains was limited, the selective effect which was observed reinforces its supposed duplicity effect and encourages us to extend the study to more clinically isolated strains in order to confirm the hypothesis.
Evaluation of the in vitro cellular response plays an important role in safety assessments and assurances. Both the EFSA and FDA regulate finished dietary supplement products (such as nutraceuticals/prebiotics) in order to ensure safe consumption before the product is placed on the market. Compliance with the aforementioned regulations is mandatory. For this study, a safety assessment was performed on a cell line representative for the human gut (HT-29). We addressed the viability, proliferation, and cytotoxicity using MTT and LDH assays. The obtained results indicated viability rates similar to the control (untreated cells), especially for the NN concentration of 50 mg/mL; this result may be due to the high percentage of collagen hydrolysate (48.5%), which is usually associated with beneficial effects on cell proliferation by providing a nutritive substrate [
62,
63,
64].
For an assessment of the influence on the inhibition of bacterial adhesion and invasion, we selected as a model a strain of
Campylobacter jejuni NCTC 81-176. Its structural characteristics and especially the binding proteins CadF and FlpA facilitate the invasion and intracellular survival [
65]. Moreover, due to its capacity to cause one of the most widespread infectious diseases of the last century, we considered it more than relevant for this assay. While no effect on invasion capacity was observed, the bacterial cells adhering to the epithelial cells were significantly lower in the sample in which the NN product was added.
As we walked through every testing phase, we demonstrated that our product shows good prebiotic effects by providing a balanced nutritional profile by promoting a duplicity effect through the growth enhancement of probiotic bacteria and the growth inhibition of pathogenic strains, and by interfering with the adherence mechanisms of
C. jejuni, therefore exhibiting an overall health beneficial impact. Keeping in mind that various factors including dysbiosis play a critical role in pathologies such as type 2 diabetes and metabolic syndrome, microbiome modulation via prebiotics is considered a therapeutic approach [
66,
67] targeted for the alleviation of metabolic outcomes. For the general population (healthy subjects), this type of prebiotic/nutraceutical acts as an adjuvant, boosting immunity by its antioxidant potential and contributes to the reduction of tiredness and fatigue and to normal energy-yielding metabolism.