1 Introduction

Button mushrooms are reportedly known to have several health promoting effects conferred by their intrinsic antioxidants chiefly, the phenolic compounds (e.g., gallic acid, p-coumaric acid, cinnamic acid, rutin, abscisic acid, protocatechuic acid, 2-hydroxy cinnamic acid, ferulic acid, syringic acid, chlorogenic acid) and the amino acid ergothioneine [1, 2]. Besides these, button mushrooms also house important biotherapeutic antioxidant molecules such as serotonin (an indoleamine monoamine neurotransmitter), melatonin (indole hormone) and their precursor L-tryptophan [3,4,5], an essential amino acid. Serotonin regulates a myriad of processes in the body, especially our mood and sleep [6]. Melatonin the ‘hormone of darkness’ is also responsible for several physiological functions such as preservation of mitochondrial homeostasis, and regulation of -circadian rhythms, locomotor activity, food intake, mood, sleep and body temperature, among many others [7,8,9]. Several researchers have quantified the presence of this antioxidant triad (L-tryptophan-serotonin-melatonin) in mushrooms of the genus Basidiomycetes but only one report by Muszyńska et al. [10] has documented their levels in button mushrooms (3.92 µg/g d.w.b of L-tryptophan, 52.1 µg/g d.w.b of serotonin and 1.1 µg/g d.w.b of melatonin).

Although literature reports exist on the antioxidant potencies of button mushroom-based food products such as noodles, pasta and bread [11,12,13], to name a few; the molecular identities of the antioxidants in the antioxidant-rich food products have not been investigated. This encouraged us to develop an antioxidant-rich, value-added functional food [14] viz. button mushroom-based gluten-free noodles (unpublished work) and to ascertain the molecular identities of the antioxidants, namely L-tryptophan, serotonin and melatonin, in the same. In our laboratory, freshly harvested button mushrooms were utilized for the development of the designer noodles with judicious usage of food additives and under minimal processing conditions to avert the losses of the heat sensitive molecules (namely, serotonin and melatonin) while preserving the natural food (antioxidant) synergy [15, 16] w.r.t the antioxidant triad. For brevity, the consortium of the three antioxidants L-tryptophan-serotonin-melatonin has been abbreviated as TSM.

Although the newly developed noodle was rich in TSM, it cannot be conjectured that the said antioxidants would be truly bioavailable during metabolism. Bioavailability is described as the fraction of an ingested nutrient that reaches the systemic circulation and the specific sites where it can exert its biological effect [17]. The current investigation therefore aims to evaluate the release of the aforesaid molecules from the starch-protein-gel noodle matrix in vitro under simulated gut conditions and also in vivo through feeding trials in male Sprague Dawley rats. Hattori et al. and Reiter et al. have conducted significant research on the bioavailability of melatonin such as the enhancement of melatonin in chicks [18] after feeding a melatonin-rich diet (meal comprising of corn, milo, beans and rice), and in rats after feeding walnuts [19]. Delgado et al. [20] reported on the enhancement of melatonin and serotonin in young and aged rats and ring doves after feeding a ‘Jerte Valley cherry-based product mix’; and in continuation of the same study, Garrido et al. [21] reported a significant increase in urinary 6-sulfatoxymelatonin levels and also actual sleep duration in human volunteers post consumption of the product twice daily for 5 days. Bravo et al. [22] reported on the increase of these two molecules in 35 human volunteers (aged 55–75 years) who consumed tryptophan-enriched cereals for breakfast and dinner for 3 weeks. However, there is a lack of literature on the bioavailability of the antioxidant triad (TSM) in an animal model from a processed food product developed using a widely consumed farm product (button mushrooms).

It is imperative that the bioavailabilities of the above-mentioned biomolecules would involve metabolism of the newly designed noodles. The liver has the central processing and distribution role in the metabolism of food, while the glucose available after metabolism of food is absorbed by the brain and gut [23]. Therefore, in order to assess whether the liver and the peripheral muscles plays central roles in the metabolism of the mushroom noodles, the current study determined the blood glucose and insulin levels in the animals at specific time intervals post-consumption of the designer noodles, and the data were fitted to in vitro non-invasive predictive iHOMA2 (the Homeostasis Model Assessment) models (which predict the effects of consumption on fasting glucose and insulin as well as on β-cell function and insulin sensitivity), according to the concept and methodology described by Hill et al. [24].

Therefore the specific objectives of the present study were to investigate—the release kinetics of L-tryptophan, serotonin and melatonin from the new designer noodles by in-vitro dissolution assays;—in vivo release of the biomolecules in the blood serum of rodent (rats) models after consumption of the noodles; and finally, to ascertain the role(s)—of the liver and/or periphery as the functional centralities of metabolism,—and of the primary site (brain and/or gut) of glucose uptake during metabolism by non-invasive iHOMA2 predictive modelling. This endeavor will enable us to comprehensively evaluate whether the new designer mushroom noodles is truly a functional food product based on its antioxidant value.

2 Materials and methods

2.1 Materials

Button mushrooms (Agaricus bisporus) were procured from BSK, Agro Farm, Barasat, North 24 Parganas, West Bengal, India. The mushrooms were organically cultivated at 22° 4391′ N and 88° 3968′ E, at about 9 m height from sea level in the eastern Gangetic plains of India, in a mix compost bed (having compost, paddy straw and chicken manure) in the pH range of 7.5–7.7, at a temperature of 17 ℃ (for fruiting body development) to 25 ℃ (for vegetative cultivation) and 85–89% RH. The procured mushrooms were authenticated from the Department of Botany, University of Calcutta, Kolkata, India. White button mushrooms, free from browning, wilting, black spots, and fungal infestation were harvested and utilized for the development of the designer noodles, immediately after procurement.

Specialty chemicals including melatonin, serotonin, and L-tryptophan standards were purchased from M/s Merck (Mumbai, India) and Folin-Ciocalteu reagent, DPPH, HPLC-grade acetonitrile, acetic acid, water and all other AR grade chemicals were procured from M/s HiMedia (Mumbai, India). ELISA kit for melatonin (with assay sensitivity 2 pg/mL) was procured from M/s SunLong Biotech Co. LTD, Zhejiang, China; the ELISA kits for serotonin (with assay sensitivity 230 pg/mL) and L-tryptophan (with assay sensitivity 4.18 pg/mL) were purchased from M/s Bioassay Technology Laboratory, Zhejiang, China and the ELISA kit for insulin (with assay sensitivity 3.75 pg/mL) was purchased from Elabscience, Texas, USA. A blood glucose measuring device of M/s. AccuSure Simple glucometer was used in the study. Walnut (Chile walnut kernels) was procured from Spencer’s Retail Ltd., Kolkata, India. Food grade chemicals for the formulation of noodles were purchased online from Amazon.in.

2.2 Methods

2.2.1 Formulation of a TSM-rich gluten-free button mushroom noodles

The button mushroom based, gluten-free noodles was developed by judicious usage of food additives such as guar gum, xanthan gum and CMC (as hydrocolloids), lecithin (as an emulsifier), and TiO2 (as a food colouring agent) under minimal processing conditions, to ensure that the noodle was rich in TSM and concomitantly, the consortium of the three antioxidants was present in a synergistic harmony (Sect. 2.5, vide infra). The ingredients and minimal processing conditions used for the formulation of the designer noodles were optimized using a multi-criteria decision-making tool, taking into account the sensory and physicochemical properties of the product (unpublished work). The noodles (Fig. 1) formulated under the optimized conditions was exhaustively characterised for its safety in terms of its bioburden (microbiological assays of total plate counts of bacteria and molds) and presence of heavy metals, if any (by energy-dispersive X-ray analysis); sensory attributes (by a human panel using a 9-point hedonic scale); physical properties (colour lightness in terms of L* value; textural parameter such as firmness; dough rheology; cooking properties; thermogravimetric properties; surface morphology; and degree of crystallinity/amorphicity); chemical properties (proximate composition including reducing sugar content); and phytochemical properties (antioxidant activity in terms of DPPH radical scavenging activity, FRAP value, total phenolic content and reducing powder). The presence of agaritine (toxin) in the designer noodles was also analysed by ESI–MS. Figure 2 summarizes the product development and its physicochemical characterization.

Fig. 1
figure 1

Newly designed mushroom noodles

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Fig. 2
figure 2

Overview of development and physicochemical characterization of the mushroom noodles

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2.2.2 Quantification of TSM in the newly designed noodles

The TSM content in the designer mushroom noodles was quantified by extracting the said biomolecules using the QuEChERS-SPE method followed by HPLC–PDA analysis of the extract, in accordance with the procedure developed in our laboratory [25].

2.2.3 Assessment of antioxidant (TSM) synergy in the designer noodles

“The cooperative action of individual antioxidants displays a greater antioxidant effect than the sum of that of the individuals acquired independently. This is termed antioxidant synergy” [16]. Prior to assessment of bioavailabilities of TSM from the designer noodles, it was necessary to ascertain whether their presence in the same was in antioxidant harmony without perturbation of the natural food synergy, w.r.t the antioxidant triad.

The presence of a few more peaks in the HPLC–PDA chromatogram of the QuEChERS-SPE extract of the noodles (due to the presence of constituents other than TSM) necessitated cross-validation of the presence of TSM in the newly developed mushroom noodles. The same extract used for the quantification of TSM by the HPLC–PDA method, was therefore injected into the ESI-TOFF-MS with Xero-G2-Xs-QT, equipped with an ADC-magnetron detector (M/s. WATERS, Milford, Massachusetts, USA) and analyzed with the programme we used in our previous study to detect the mass spectra of L-tryptophan, serotonin and melatonin in button mushrooms [25]. The TSM-rich noodle was also analyzed for its antioxidant (the triad) synergy by determining its synergistic effect (SE) value (which, if greater than unity signifies antioxidant synergism), in accordance with the methods described by Liu et al. [26], and Chakraborty and Bhattacharjee [27].

2.2.4 Studies on release kinetics of L-tryptophan, serotonin and melatonin from the formulated noodles

To ascertain bioavailabilities of the three antioxidants in vivo, it is imperative to first determine whether they are released in vitro in simulated gut buffers. Based on preliminary experiments (performed with a magnetic stirrer), dissolution of the designer noodles in simulated oral, gastric, intestinal and rectal buffers were conducted (see details provided vide infra). With the exception of the gastric buffer, all buffers were maintained at neutral pH (7), which is conducive to the stability of serotonin and L-tryptophan which have been reported to be more stable at neutral pH than at acidic pH [28, 29]. The stability of melatonin does not depend on the pH of the buffer [30] and therefore its release can be well studied in gut simulated buffers of different pH [29].

Investigations on phenolics and antioxidant activity of a bamboo leaf soup by in vitro digestion method revealed no significant differences in antioxidant activity when the soup was subjected to digestion with and without enzymes (i.e., treated with buffers of different pH), attesting to the fact that the release of antioxidants from the food product is more dependent on the pH of the dissolution buffer than on the enzymatic digestion of the same [31]. It is hereby conjectured that if the target biomolecules are ‘released’ unaided by enzymes (which indeed occurred, vide infra), then this will certainly be the case in the presence of digestive enzymes in vivo.

Therefore, in the current investigation, in vitro dissolution studies have been performed to evaluate the release of the target molecules in four different buffer systems viz. oral, gastric, small intestinal (duodenum) and rectal buffers. Since it is reportedly known that the maximum absorption of melatonin occurs in the rectum of male Sprague Dawley rats [32] and pig-barrow [33], the other regions of the small and large intestines were not considered in the present study. Moreover, corroboration of these findings with the actual gut absorption of the said biomolecules would necessitate not only animal sacrification but also detailed histological examination of the tissues of each section of the intestine [32], which has not been conducted here in sync with the current global legislation based on “3R” principle of “Refining, Reducing and Replacing” that emphasizes on reduction of animal studies [34].

In the preliminary experiments, the release kinetics study was performed using simulated gut buffers (gently agitated using a magnetic stirrer) without the use of enzymes. Simulated phosphate saline buffers (PBS) such as simulated salivary buffer (SSB), simulated gastric buffer (SGB) and simulated intestinal buffer (SIB) with multiple electrolytes rendering strong buffering capacities were prepared according to the composition described by Piexeto et al. [35] with few modifications. Their corresponding pH values were set to 7, 1 and 7.4, to simulate the digestive environment in the mouth, stomach and intestine, respectively. Phosphate buffer with minimal buffering capacity and pH 7.4 [36] was used as the simulated rectal buffer (SRB).

The newly designed raw noodles were boiled and mashed to simulate the process of mastication inside the mouth. The pre-treated noodles (5 g) were added to 100 mL SSB in a 250 mL beaker and stirred constantly at 75 rpm at 37 °C [37] for 10 min using a heat controlled magnetic stirrer (IKA RCT Basic, M/s. IKA-Werke, Staufen, Germany). Aliquots (5 mL of buffer) were withdrawn from the beaker at regular intervals (an equal volume of fresh buffer was replaced each time) of 2, 5, 7 and 10 min. This prolonged dissolution time in the oral phase has been considered as this is the first time the release of the target biomolecules from the complex food matrix has been studied. A long residence time of 10 min in the oral phase for 50–90 g noodles (vide supra) was investigated considering the fact that elderly and geriatric populations who often suffer from dysphagia [38] may require longer time to chew and swallow the noodles. The aliquots were centrifuged using a cold centrifuge (C-24BL, M/s Remi, Mumbai, India) at 12300 × g, at 4 °C for 10 min. The supernatant was collected, filtered through a 0.22 µ syringe filter and stored at − 20 °C prior to HPLC–PDA analysis. Subsequently the release of the above biomolecules in SGB, SIB and SRB was determined in a similar manner, except that aliquots were withdrawn at time intervals of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 and 120 min. Analyses of free L-tryptophan (and not bound tryptophan as no enzymatic digestion of the protein matrix of the mushroom noodles was performed), serotonin and melatonin contents in the buffers revealed that only very small amounts were released (data in supplementary file SI) in SGB and SIB after 60 min.

Although the release of the aforementioned biomolecules from the food matrix was confirmed in this study, a scale-up study was conducted using a standard tablet dissolution unit equipped with a paddle agitator (Inspire 8 Basic, M/s. Electrolab, Mumbai, India) and a buffer volume of 500 mL, to better simulate the conditions in the gut. In this method, three different batch sizes of pre-treated noodles (50 g, 70 g and 90 g of raw noodles, based on commercially available batch /serving size of wheat noodles) were subjected to dissolution [39] inside the standard 8-vessel dissolution unit (equipped with a paddle agitator) containing 500 mL of buffer(s) per vessel, with continuous stirring at 37 °C (maintained by a thermostatically controlled water bath).

The paddle speed was set to the maximum limit of 75 rpm for SSB, SGB and SIB and 60 rpm for SRB since the batch sizes were larger than those in typical pharmaceutical formulations and also because the feed contained relatively dense particles which could otherwise turn lumpy [40]. The aliquots (5 mL buffer) were then withdrawn at a regular interval (as above), centrifuged (as above), and stored at − 20 °C. A similar procedure was followed during dissolution of the food product in SGB and SIB except that aliquots were withdrawn at 10, 20, 30, 40, 50 and 60 min (the end time chosen based on the findings discussed above). Analysis of serotonin, melatonin and L-tryptophan levels in the buffers revealed that their amounts did not follow any specific trend in SGB after 50 min and in SIB after 60 min possibly due to analytical uncertainties arising from the presence of co-extractants and/or interfering pigments (confirmed by the presence of additional peaks in the HPLC–PDA chromatogram) and chemical absorption/physical adsorption of the analytes with the food matrix. A similar procedure was followed for SRB, except that the aliquots were withdrawn at 10, 20, 30, 40, 50 and 60, 70, 80, 90, 100, 110 and 120 min.

Three different serving sizes of noodles was selected primarily to determine the serving size that contributed to the maximum release of the biomolecules mentioned. The batch sizes for noodles were 50 g, 70 g and 90 g (vide supra) raw noodles. It was evident that the smallest serving portion allowed the maximum release of the target biomolecules (data provided in supplementary file SI: Figs S1 and S2). Therefore 50 g of mushroom noodles was selected as the batch size of mushroom noodles for the investigation of their release kinetic models.

The aliquots withdrawn were filtered through double syringe filtration systems (0.45 and 0.22 µm) and then subjected to HPLC–PDA analysis to quantify the amounts of L-tryptophan, serotonin and melatonin, individually. The cumulative percentage release of each molecule in the buffer(s) was plotted against time and the data were fitted to standard kinetic equations, namely, zero order (Qt = K0·t), first order (lnQ = K1·t), sub-types of first order subtypes such as the Higuchi model (Qt = KH·t1/2), Hixson-Crowell’s cube root model (Q1/3 = KHC·t) and Korsmeyer-Peppas (Qt = Kk·tn) equations, where Qt is the amount of the biomolecule released at time t and Q is the amount of the same in the noodles, K is the constant for each model, and n is the diffusion or release exponent of each biomolecule. The kinetic model with the highest regression coefficient (R2) was considered the best model for representing the release of the target biomolecules from the mushroom noodles.

2.2.5 Assessment of antioxidant activity of the designer noodles in the simulated gut buffers

The antioxidant activity of the TSM-rich designer noodles after 10 min (from SSB), 50 min (from SGB), 60 min (from SIB) and 100 min (from SRB) of dissolution in the respective buffers were assayed to ascertain change(s) in antioxidant activity, if any. During in vitro dissolution, the buffer aliquots were subjected to phytochemical characterisation involving analyses of total phenolic compounds, DPPH radical scavenging activities and FRAP values, following the methods described by Camelo-Mendez et al. [41] and Goulas and Hadjisolomou [42]; and the data thus obtained were compared with the antioxidant potency of the noodles prior to dissolution.

2.2.6 Assessment of in vivo bioavailabilities of L-tryptophan, serotonin and melatonin from the designer noodles

The in vitro release kinetics of the said biomolecules in simulated gut buffers (without digestive enzymes) does not truly mimic in vivo gut digestion involving a myriad of enzymes. This necessitated assessment of the release of the target biomolecules from the designer noodles post absorption in actual gut conditions and thus feeding trials with male Sprague Dawley rats and subsequent periodic assessment of serum levels of the three biomolecules were performed.

The protocol for the animal studies was approved by the Institutional Animal Ethical Committee (IAEC) of NSHM Knowledge Campus, Kolkata, West Bengal, India. The approved guidelines and regulations of -IAEC (registration no. NCPT/IAEC-012/22023), EU Directive 2010/63/EU and -Committee for the Purpose of Control and Supervision of Experiments of Animals (CPCSEA) were followed in all animal experiments, to ensure well-being of the animals and their personal hygiene.

Two-month-old male Sprague–Dawley rats (n = 30) with an average body weight of 175–200 g were procured from M/s. Saha Enterprise, Kolkata, India. This part of the work was undertaken in March–April 2023 under the supervision of professional veterinary staff at the facility of NSHM Knowledge Campus, Kolkata, West Bengal, India. The detailed study design (including grouping of animals and timing of blood sampling) has been presented in Fig. 3. The frequency of blood sampling (Table 1) and the volume of blood collected were set according to the IACUC guidelines [43] for blood collection from rats, which state that a maximum of 10% of total blood volume can be withdrawn from each rat in 2 weeks.

Fig. 3
figure 3

Experimental design for in vivo study of bioavailabilities of L-tryptophan, serotonin and melatonin in male Sprague–Dawley rats

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Table 1 Time of blood withdrawal from male Sprague Dawley rats
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In the first week, the animals [two rats were kept in a polyacrylic cage (435 × 290 × 160 mm)] were acclimatized under standard environmental conditions (17–23 °C, 60 ± 5% RH) with 12 h: 12 h dark–light cycle (180–200 lx at daytime) and had free access to standard rat chow diet (a mixture of wheat flour, Bengal gram flour, milk powder, salt and distilled water, on weight basis) and fresh water.

Post acclimatization, each rat was randomly selected (as all rats were healthy, active and of similar body weights) and assigned to one of the five groups (control group, positive control group and test group, control group for iHOMA2 and test group for iHOMA2). Each group consisted of six male rats. The control group, the positive control group and the test group were used for experiments related to serum L-tryptophan, serotonin and melatonin levels; while the other two groups were used for experiments related to iHOMA2 studies (vide infra).

At the end of 7 days of acclimatization with the standard rat diet, the endogenous levels of the three biomolecules in rat serum were measured using apposite standard ELISA kits (in conjunction with an ELISA microplate reader- Merilyzer Eiaquant, M/s. Meril, Vapi, India). Blood was withdrawn from the tail vein of one rat (randomly selected) from each group, serum was separated by cold centrifugation (at 4 ℃ at 1000 × g for 20 min) and stored at − 20 ℃ for further analysis. Thereafter, instead of the standard rat diet (due to the presence of L-tryptophan and melatonin in the same), all animals in the five groups received TSM-lean raw noodles (prepared in our laboratory from tapioca flour, hydrocolloids, salt, oil and water) and water ad libitum for three consecutive days after which the target biomolecules in the blood serum were re-analysed at 9 am and 3 am. The animals were then kept fasting for 24 h, followed by immediate blood sampling to analyse the L-tryptophan, serotonin and melatonin levels.

Subsequently, the rats of the control group and the control group designated for iHOMA2 were fed TSM-lean raw noodles; the rats of the positive control group were fed a melatonin-rich nut, namely walnuts [19]; and the rats of the test group and the test group designated for iHOMA2 with the newly developed mushroom noodles (pre-treated for swallowable consistencies, vide supra) by gavaging (at 9 am) which has been reported to be a reliable method of administering substances into the GI tract [44]. Two additional positive control groups for feeding trials with serotonin-rich or L-tryptophan-rich food/food products were not included in the study in absence of commercially available apposite food products (although several food/food products are reportedly known to be L-tryptophan and/or serotonin-rich).

Blood was withdrawn from the control, the positive control and the test groups at 30, 50 min and 2 h post-feeding and then at a regular time interval of 6 h up to 24 h. After 6 h of feeding, all rats were provided with TSM-lean raw noodles and water ad libitum for the next 18 h. Six hour was fixed based on the studies of Padmanabhan et al. [45] and Munakata et al. [46], who reported this time span to be the average GI transit time in 129SvEv mice and rats. The details of the gavaging procedure and blood sampling have been elaborated in Table 1.

Concomitantly, the behavioural (salivation, lethargy, sleep, convolutions) and physical (locomotion, tremor, diarrhoea, and mortality) wellness parameters of the rats were systematically recorded at 30 min, 50 min, 2 h, 6 h, 12 h and 24 h post feeding with the designer noodles; and otherwise once regularly throughout the entire experimental period, according to the procedures elaborated by us in our previous publication [47]. This entire experimental scheme of assessments of the bioavailabilities of L-tryptophan, serotonin and melatonin was duplicated with new groups of animals in each experimental scheme, following similar procedures and guidelines, as described above.

2.2.7 Food safety assessment of the designer noodles in terms of L-tryptophan, serotonin and melatonin intake

The NOAEL values of L-tryptophan and melatonin are reportedly known to be 779 mg/day/kg body weight and 5 mg/day/kg body weight, respectively [48, 49]. Their ADI values were calculated using Eq. 1 [50]:

$${\text{ADI }} = {\text{ NOAEL value }}\left( {{\text{mg}}/{\text{day}}/{\text{kg body weight}}} \right) \, /{\text{ safety factor}}$$
(1)

where the safety factor is 100 for the extrapolation from animal to human. The ADI values thus obtained were compared with the concentrations of the target biomolecules present in the serving size of the designer noodles (50 g), to assess their safe levels of intake by an adult human being (of 60 kg body weight). A similar endeavour could not be conducted for serotonin in absence of its NOAEL value.

2.2.8 Assessment of effects of consumption of the designer noodles on β-cell function, insulin sensitivity, and glucose uptake using iHOMA2

In the present study, the in vitro non-invasive predictive iHOMA2 modelling was used to evaluate the effects of consumption of the designer noodles on the insulin sensitivity and glucose uptake in the rats without sacrificing the animals. Since the liver plays a central processing role in metabolism involving few other extrahepatic or peripheral tissues and organs [23], it was necessary to adjudge whether the metabolism of the designer noodles involved the functional centralities of the liver. Therefore, the breakdown and release of glucose from the protein-starch matrix of the noodles and its consequent impact on glucose uptake (primarily in the liver and periphery) and/or modulation of insulin sensitivity need to be investigated for this new designer noodles. Additionally, since circulating melatonin is rapidly metabolised in the liver [51], assessing the involvement of the liver with or without it’s peripheral tissues and organs is of paramount importance and was therefore conducted in the current study using iHOMA2.

Post feeding of the TSM-lean raw noodles and the designer noodles to the control group for iHOMA2 and the test group for iHOMA2, respectively, the animals were allowed to completely metabolise the same for 6 h (vide supra). Glucose and insulin values were assayed just prior to feeding, and then at 2 h, 4 h, and 6 h post feeding of the respective noodle. In this study, %S and %β were calculated from experimental insulin (using standard ELISA kits) and glucose (using glucose strips) values using iHOMA2 software in predictive mode [24, 52, 53]. Six predictive models were considered based on the possible sites of metabolism as has been described in our previous work on the antidiabetic effects of 1,8 cineole-rich and melatonin-rich extracts of small cardamom seeds and mustard seeds, respectively [47]. Bland–Altman plots were generated to assess whether there were good agreements between the experimental and predicted levels of blood glucose and that of insulin. Six predictive models based on possible sites of metabolism were considered, and the predicted values of glucose and insulin were analysed by F-test statistic to obtain the best fit model [24, 47].

2.2.9 Statistical analysis

In vitro analyses were conducted in triplicate and the data are expressed as means ± SD of three independent experimental data sets. The results of the in vivo experiments have been expressed as mean ± SD of data obtained from the blood serum of rats of the same group. Duncan’s multiple range test was conducted to determine significant differences among -the antioxidant potencies of the noodles pre and post dissolution in the buffers; -mean of the concentrations of the target biomolecules present in the rat serum and mean of the concentrations of the glucose (blood) and insulin present in blood serum. Value of p ≤ 0.05 was considered significant to establish differences. F-value was determined to predict the best fit model obtained from iHOMA2. All statistical tests were performed by STATISTICA 8.0 software (M/s Statsoft, Oklahoma, USA).

3 Results

3.1 L-tryptophan, serotonin and melatonin contents of the button mushroom based designer noodles

The button mushrooms currently studied contained 16.15 µg/ g d.w.b, 12.83 µg/ g d.w.b and 1.83 µg/ g d.w.b, of L-tryptophan, serotonin and melatonin respectively, as was found by HPLC–PDA analysis of their QuEChERS extract; while the designer noodles prepared with the same contained 122.54 µg/ g d.w.b, 77.98 µg/ g d.w.b, 7.25 µg/ g d.w.b of the said molecules, respectively. Based on these findings, the L-tryptophan, serotonin, and melatonin contents in the best serving size (50 g) of the designer noodles (vide infra) were calculated to be 3899.15 µg/ g d.w.b, 6127 µg/ g d.w.b, and 362.78 µg/ g d.w.b, respectively.

3.2 Antioxidant synergy of TSM in the designer noodles

Based on the ESI-TOF–MS results, it was cross-validated that the three antioxidant molecules were indeed present in the extract of the designer mushroom noodles (unpublished data). The SE value (SE = 1.14) of the TSM consortium in the designer noodles was greater than unity, demonstrating the fact that the antioxidant triad was present in the noodles as a synergistic consortium [15]. Thus the noodles was truly TSM-rich, and can therefore be designated to be a truly antioxidant-rich gluten-free product.

3.3 In vitro release kinetics models of L-tryptophan, serotonin and melatonin from the designer noodles in standard dissolution apparatus

Figure 4a represents the percentage release of the three target biomolecules from the designer noodles in SSB. The amounts of free L-tryptophan, serotonin and melatonin released in 500 mL of SSB immediately after 2 min of dissolution were 1868.17, 1103.65 and 2.1 µg, respectively; whereas, after 10 min the said amounts were 3413.82, 1734.64 and 47.53 µg, respectively; which corresponded to 60.61%, 44.43% and 13.10% release of free L-tryptophan, serotonin and melatonin, respectively, of their original content(s) present in the noodles.

Fig. 4
figure 4

Release profiles of L-tryptophan, serotonin and melatonin from mushroom noodles during dissolution (a) in simulated salivary buffer (SSB) and (b) in simulated gastric buffer (SGB) (c) in simulated intestinal buffer (SIB) (d) in simulated rectal buffer (SRB)

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Figure 4b represents the percentage release of the target biomolecules in SGB. The amounts of free L-tryptophan, serotonin and melatonin released in 500 mL SGB were 1877.92, 981.11 and 20.31 µg after 10 min of dissolution and after 50 min were 6017.55, 3273.14 and 98.89 µg, respectively; corroborating to appreciable releases of free L-tryptophan (98.21%) and serotonin (83.03%). However, a comparatively low percentage of melatonin (27.5%) was released in vitro.

Figure 4c represents the percentage release of the target biomolecules in SIB. The amounts of free L-tryptophan, serotonin and melatonin released in 500 mL SIB were 757.5, 275.9 and 10.5 µg after 10 min of dissolution and after 60 min were 6646.3, 2597.4 and 159.9 µg, respectively; suggesting a considerable release of free L-tryptophan (96.67%) and serotonin (82.48%) and melatonin (44.11%). Figure 4d represents the percentage release of the target biomolecules in SRB. The amounts of free L-tryptophan, serotonin and melatonin released in 500 mL SIB were 154.55, 76.77 and 21.42 µg, respectively, after 10 min of dissolution and after 100 min were 4660, 991.75 and 162.57 µg, respectively; this corresponds to a release of 67.82% of free L-tryptophan 31.49% of serotonin and 44.85% of melatonin. For model fitting, the final time (50 min for SGB, 60 min for SIB and 100 min for SRB) coincided with the time after which the amounts of the biomolecules decreased significantly (possibly owing to instabilities of the molecules upon prolonged dissolution in the buffers).

Post fitting of the cumulative percentage release kinetics data to different models (vide supra), it was found that the release of free L-tryptophan and serotonin from the mushroom noodles in the oral phase (SSB) followed zero-order model (with R2 value of 0.98 and 0.99, respectively); melatonin, on the other hand, followed first order (0.96) release kinetics in the oral phase. All the three molecules followed Korsmeyer’s-Peppas release kinetics model (R2 = 0.99, 0.99 and 0.95 for free L-tryptophan, serotonin, and melatonin, respectively) in the gastric and intestinal (R2 = 0.96, 0.98 and 0.91 for free L-tryptophan, serotonin, and melatonin, respectively) phases. In the rectal phase, the release of free L-tryptophan and serotonin from the mushroom noodles in SRB followed the Korsmeyer’s-Peppas model (with R2 value of 0.98 for L-tryptophan and 0.91 for serotonin); melatonin however followed first (0.97) order model of release kinetics.

3.4 Antioxidant potency of the designer noodles post dissolution in the simulated gut buffers

Table 2 displays the antioxidant activity of the mushroom noodles (in terms of TPC, DPPH radical scavenging activity and FRAP values) prior to dissolution and also after 10, 50, 60, 100 min dissolution in SSB, SGB, SIB and SRB buffers, respectively. It was evident that the antioxidant activity (the values of the above-mentioned antioxidant properties) of the noodles in SIB was significantly (p < 0.05) higher vis-à-vis the same in the other buffers. However, antioxidant activity of the noodles post dissolution in in each of the four buffer systems was significantly (p < 0.05) lower than that of the noodles per se.

Table 2 Antioxidant activities of mushroom noodles before and after dissolution in gut simulated buffers
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3.5 Bioavailabilities of L-tryptophan, serotonin and melatonin from then designer noodles in Sprague Dawley rats

Immediately after acclimatization, the mean blood melatonin level of 44.38 ± 0.36 pg/mL was found in three randomly chosen animals at 3 am. The serum levels of L-tryptophan and serotonin at the same clock time were 275.3 ± 1.98 pg/mL, 8930 ± 23 pg/mL, respectively. Figure 5a–c show the alterations in L-tryptophan, serotonin and melatonin levels, respectively, in the blood serum of the rats during the entire experimental period of 6 days (post-acclimatization). At the end of the 3-day of consumption of the TSM-lean diet, serum levels of the above-mentioned molecules decreased significantly (p < 0.05) on day 10, although the natural trends of increase and decrease in serum serotonin and melatonin levels at different time of the day (when measured at 9 am in the morning and 3 am at midnight) remained unperturbed [54, 55]. The wellness parameters also did not reflect abnormalities in physiological, neurological, and behavioural patterns indicating that the rats remained healthy and active throughout the study period.

Fig. 5
figure 5

Alterations in serum levels of (a) melatonin, (b) serotonin and (c) L-tryptophan in Sprague–Dawley rats with time- before feeding L-tryptophan-serotonin-melatonin-lean noodles, after feeding L-tryptophan-serotonin-melatonin-lean noodles, after fasting and after gavaging the mushroom noodles [TSM- L-tryptophan-serotonin-melatonin]

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The changes in serum melatonin levels during the experimental period (6 days post-acclimatization) are shown in Fig. 5a. Serum melatonin levels in the positive control group followed the natural trend of high and low (vide supra) throughout the experimental period, except on the day of feeding. Post-feeding walnuts, it was observed that serum melatonin level was significantly (p < 0.001) increased after 30 min vis-à-vis that of the control (i.e., the endogenous level) group. Post-gavaging of the designer noodles, a significant increase (p < 0.001) in serum melatonin levels was observed after 30 min compared to the melatonin level in the control group of rats. The blood melatonin levels (Fig. 6a) of the test group of rats after 30 min was significantly higher (p < 0.001) than the melatonin level of the same group before feeding (with noodles), followed by a significant (p < 0.001) decrease after 50 min. Thereafter serum melatonin levels decreased significantly after 120 min (p < 0.001) and also at 3 pm (p < 0.001). Subsequently from 9 pm, the serum melatonin levels increased significantly (p < 0.001) in the test group of rats and reached its peak (p < 0.001) level at 3 am (18 h post consumption). The serum melatonin levels of the positive control and the test groups of animals remained higher than those of the control group at the said time intervals studied (except for serum melatonin levels obtained after 120 min and at 3 pm).

Fig. 6
figure 6

Serum level of (a) melatonin after 30 min, (b) serotonin after 50 min, (c) L-tryptophan after 50 min of feeding

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Figure 5b depicts the alterations in the serotonin levels in the blood of the control, the positive control, and the test groups of rats during the experimental period, post-acclimatization. No effect of feeding was observed in the control group and the walnut-fed positive control group of rats; rather the changes in serum serotonin levels in these two groups followed the natural physiological cycle of the animals [50]. It was observed that after feeding, the test group showed significant increases (p < 0.001) in serum serotonin levels after 30 min vis-à-vis the endogenous serotonin levels in the control group, and also compared to the test group before feeding. Again, a significant increase (p < 0.001) in serum serotonin (Fig. 6b) level was observed in the test group after 50 min. Thereafter the serum serotonin levels decreased significantly after 120 min (p < 0.001) and at 3 pm (p < 0.001). However, compared to that at 9 pm (p < 0.001), the blood serotonin level in the test group of rats increased significantly at 3 am (p < 0.001) and also at 9 am (p < 0.001).

From Fig. 5c it can be observed that the control group showed no elevation in serum L-tryptophan levels during the entire experimental period. However, the same increased sharply in the apposite groups of animals immediately after feeding walnuts and the designer noodles. In the positive control and the test groups alike, the serum levels of L-tryptophan increased up to 50 min (Fig. 6c) and remained elevated for 12 h after consumption; thereafter the levels were similar to those in the control group. However, the test group showed a higher (p < 0.05) increase in serum L-tryptophan levels vis-à-vis that of the positive control group. The control group showed no elevation in L-tryptophan levels throughout the experimental period. The release of L-tryptophan in the rat blood serum of the test group could be due to the content of ‘free L-tryptophan’ and ‘bound L-tryptophan’ (which is released post digestion of the protein in the mushroom noodle matrix) present in the button mushrooms and thus in the noodles.

3.6 Calculated safe levels of L-tryptophan, serotonin in human post-consumption of the designer noodles

The calculated ADI values for L-tryptophan and melatonin were 0.779 mg/day/kg of body weight and 0.05 mg/day/kg of body weight, respectively, which corresponds to 46.74 mg L-tryptophan and 3 mg melatonin for an adult human (60 kg body weight).

3.7 Effects of consumption of noodles on serum glucose and insulin levels and model fitting by non-invasive iHOMA2

Alterations in serum insulin levels and blood glucose levels of the rats together with their respective %S and %β values 2 h, 4 h, and 6 h after feeding with the mushroom noodles are presented in Table 3. The serum insulin levels were lower in the rats fed with TSM-lean diet (control); while they were higher in the rats fed with the noodles (test group). From the obtained F-values, it can be concluded that the models based on the first hypothesis (increased insulin sensitivity in the liver and periphery) and the fifth hypothesis (glucose uptake in the gut) fitted well with the experimental data of glucose and insulin levels in the noodle-fed rats (Table 4). The Bland–Altman plots (Supplementary file SI, Figs S3 and S4) showed good agreement between the experimental and predictive values for both glucose and insulin levels (indicating good agreement between the methods) in rat serum.

Table 3 Blood glucose level, serum insulin level, insulin sensitivity and β-cell function of rats
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Table 4 Results of iHOMA2
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4 Discussion

The release kinetics of the target biomolecules (L-tryptophan, serotonin and melatonin) in SSB reflect their moderate release from the food matrix. Vakkuri [56] and Vakkuri et al. [57] have reported a release of 4.835 × 104 pg/mL of melatonin human saliva after 60 min of ingestion of 100 mg synthetic melatonin (present in a single gelatin capsule). Based on these reports, it could be hypothesised that melatonin will be inadvertently released from the designer noodles in the human saliva within 10 min of residence time in the oral cavity. There is no existing report on similar lines for L-tryptophan and serotonin to support our findings.

The findings of the in vitro dissolution study of the mushroom noodles revealed that the target biomolecules were first bioavailable from the noodles within 10 min of dissolution. In the oral phase containing SSB (pH 7), L-tryptophan and serotonin followed zero order kinetics indicating their continuous release from the noodle matrix, regardless of the concentration of the biomolecule in it. The release of melatonin in SSB from the mushroom noodles followed the first order kinetic model indicating that the release of melatonin (water-soluble biomolecule) from the food product is concentration dependent. There is no existing literature on the release of the said biomolecules from starch-protein gel matrices to substantiate these findings for simulated gut buffers. However, the release of pharmaceutically active ingredients from hydrogels followed similar models such as the release of aceclofenac from a citric acid-cross-linked-hydrogel prepared from Salvia spinosa seeds followed zero order kinetics in phosphate buffer at pH 6.8 [58]; and the release of carvacrol from carboxymethyl-chitosan-sodium alginate hydrogel followed first order kinetics at pH 7 [59].

In the gastric phase containing SGB, the three biomolecules obeyed a single model of release kinetics, viz., the Korsmeyer Peppas model, indicating non-linear trends and controlled-cum-anomalous diffusion of the biomolecules from the noodles matrix. Similarly, Treenate and Monvisade, [60] studied the release of paracetamol from a hydrogel composed of hydroxyethylacryl chitosan and sodium alginate cross-linked with various ionic cross-linkers such as Ca2+, Zn2+ and Cu2+ in SGF (pH 1.2) and found that the release profile of paracetamol followed the Peppas model.

Although the pH of SSB and SIB was similar, different release kinetic models were obtained for each of the biomolecule studied here, possibly owing to differences in the final time of analysis (dissolution time of 10 min in SSB and 60 min in SIB). Similar to the gastric phase, the target biomolecules in the intestinal phase (SIB) obeyed the Korsmeyer Peppas model. Similarly, Treenate and Monvisade, [60] who investigated the release of paracetamol (vide supra) in SIF (pH 7.4) found that the release kinetics conformed to the Peppas model.

In the rectal buffer (pH 7.4), the release of melatonin was found to be similar (p ≤ 0.05) to that in the intestinal buffer, contrary to the established reports of the highest absorption of melatonin in the rectum of rats [32] and pigs [33]. In SRB, the release of serotonin and L-tryptophan was lower (p ≤ 0.05) than in SGB and SIB, which could be attributed to the fact that the presence of salts especially HCO3¯ in the intestinal buffer was essential for the release of the same from the starch-protein noodle matrix (having gums as binders); and therefore in the absence of salts in the rectal buffer [36], the same was hindered. In addition, the presence of phosphate salts in SRB may have facilitated salting out of the proteins in the noodle matrix and thereby impeded the release of the target biomolecules in SRB. The release of L-tryptophan and serotonin followed Korsmeyer Peppas kinetics, while the release of melatonin in SRB from the mushroom noodles followed the first order kinetics model, similar to that in the oral phase buffer. The release of melatonin was also significantly lower vis-à-vis the release of L-tryptophan and serotonin which is probably due to the fact that melatonin as a bulkier molecule (MW = 232.27 g/mol) could have been strongly retained in the protein-starch gel network (with characteristic functional properties).

The precise explanation of the release kinetics models thus obtained is intriguing possibly owing to a complex interplay of several factors, such as differences in the macro and micro-structure of the food matrix (varying surface area exposed to the dissolution buffers with time), changes in viscosity and consistency of the dissolution buffers with the time of dissolution, the abundance of the said biomolecules in the processed food matrix, the interactions among the food components (biomolecules with starch-protein interactions in noodles) and with additives, and the relative bulkiness of the food constituents with respect to buffer (and hence the diffusional resistances). Since no in vitro protein digestion was performed, the release of L-tryptophan from the noodles could be attributed solely to the free form of the amino acid in the processed food product and not to that resulting from protein breakdown.

The trends in the antioxidant activity obtained for mushroom noodles post dissolution in the gut simulated buffers perfectly matched with the findings of Camelo-Méndez et al. [41] who reported the highest values of antioxidant activity of gluten-free pasta (prepared with unripe plantain, chickpea, and blue maize flours) in the intestinal buffer, compared to the same values in the oral and gastric phase buffers. This congruence could be possibly attributed to the similarities in the pH regimes examined in both the studies.

The antioxidant activity in SIB (60 min) was found to be significantly (p < 0.05) higher vis-à-vis that in the other buffers. Similar trends of higher values of these have been reported for gluten-free pasta (prepared with immature plantains, chickpeas and blue maize flour) post dissolution in the intestinal buffer, compared to their corresponding values in the oral and gastric phase buffers [41].

The trend towards increased serum melatonin post-feeding of the noodles was possibly owing to the fact that the biomolecule being amphiphilic [61] was readily absorbed by the body when taken orally. These findings are in consonance with the findings of Hattori et al. [18] in which blood melatonin levels in chicks were measurably enhanced after consumption of melatonin-rich meal of corn, milo, beans, and rice; and with another study by Reiter et al. [19] in which the serum melatonin-level in male Sprague–Dawley rats significantly increased after consuming walnuts. It is reportedly known that melatonin when administered orally in its pure form requires 45 min for its absorption in the GI tract and its release in the blood circulation in significant amount is aided by the presence of chemical stimulants particularly L-tryptophan [18, 62]. Although the average GI transit time in rats is 6 h, an increase in serum melatonin was evident post 30 min of consumption, possibly owing to rapid transit time to the propulsive phase that reportedly occurs in fasted condition in rats [63]. The decline in the melatonin concentration after 30 min of feeding is in sync with the well-known fact that melatonin is a short-lived molecule with an average lifespan of about 20–40 min in the bloodstream [62].

Moreover, the routine peak of melatonin occurred at 3 am (in the acrophase) the following day signifying perfect hormonal homeostasis in the animals [64]. However, this peak in the melatonin levels did not induce lethargy, sleep or impaired locomotion activities (video provided in File SI2). The rats showed no abnormalities in physiological, neurological, and behavioural patterns post-consumption of the noodles, which was possibly owing to an interplay of the presence of dopamine in the mushroom noodles identified in the ESI-TOF–MS spectra (data not shown); presence of serotonin above its endogenous level (vide infra); and due to the presence of melatonin (vide infra) possibly at a level that did not perturb the sleep–wake cycle.

An increase in blood serum melatonin level was observed in human volunteers (18–25 years male) after consuming two whole peeled bananas (ripeness, type/species of the fruit and amount consumed were not specified by the researchers); orange juice and pineapple juice (amounts consumed not known) obtained from one kg of each fruit, when each food product was consumed individually with intermittent wash-out periods [65]. The mushroom noodles were able to release the same within 30 min in Sprague Dawley rats. This difference could be attributed to the fact that processing possibly had rendered the food matrix more bio-amenable for release of melatonin, notwithstanding the fact that animal to human extrapolation involves uncertainties owing to several biochemical factors.

The release of serotonin from the newly designed food product is highly promising since serotonin does not have a synthetic drug counterpart and its deficiency is known to cause neural complications (such as ADHD). This designer noodles could be a novel food source of serotonin. The tapioca-based-TSM-lean diet fed to the rats prior to feeding them the designer noodles is known to house serotonin elicitors such as zinc and magnesium [66] which could have aided in the bioavailability of serotonin from the designer noodles [3, 67]. Therefore, it can be logically inferred that this minimally processed designer noodles (with minimal food additives) could not only be a nutraceutical product for melatonin delivery, but for serotonin as well.

The nutraceutical powder mix of Jerty valley cherries was found to augment blood serotonin levels (230 × 103 ~ to ~ 280 × 103 pg/mL) at 7 pm and melatonin levesl (~ 200 to ~ 280 pg/mL) in the acrophase in young (6–7 months) male Wistar rats when fed continuously for 10 days along with their standard diet [20]; and also increased the urinary 6-sulfatoxymelatonin levels in human volunteers post consumption of the same twice daily for 5 days [21]. Interestingly, the designer noodles augmented the levels of the said hormones within 30 min of consumption.

The absorption of L-tryptophan was highest in the test group of rats and can be attributed to the fact that the newly designed noodles was richest in L-tryptophan. The absorption of L-tryptophan from the noodles are in consonance with the reports of Emori et al. [68], who observed increases in blood L-tryptophan levels post oral administration of synthetic L-tryptophan to a mutant strain of Sprague–Dawley rats. There is no literature to directly corroborate this result with the findings of feeding trials of tryptophan-rich foods. Researchers such as Bravo et al. [22] have monitored levels of serotonin-melatonin derivatives (and not of tryptophan) in the urine of human volunteers after being served with tryptophan-enriched cereals; similar work has also been carried out by Garrido et al. [21] (vide supra), to cite a few. Since the diet provided to the rats (prior to feeding of the designer noodles) had no L-tryptophan, serotonin and melatonin, it can be concluded that the alterations in the blood levels of these three target biomolecules in the rats are due to the consumption of walnuts (in the positive control group) and the designer noodles (in the test group).

Previous studies have reported that the amount of melatonin secretion at night is largely dependent on the amount of tryptophan intake in the morning (first meal) and exposure to bright light [69, 70]. In the present study, the levels of serum melatonin on the consecutive day of feeding at 3 am (in the acrophase) were higher in the test and positive control groups of rats than in the control group of rats (endogenous level), indicating the combined effect of the consumption of an L-tryptophan-rich diet (leading to the production of melatonin via serotonin) and the timing of feeding i.e., in the morning. The results of the current study reiterated that there exists a strong correlation between the timing of consumption of the TSM-rich diet, and the exposure to bright light on the increase of melatonin levels in the blood serum of rats.

Thus from the animal feeding trials, it could be concluded that enhancement of ~ 95%, 20% and 44% of L-tryptophan, serotonin and melatonin, respectively, occurred in the rat blood serum within 30 min of consumption of the designer noodles. This study therefore unambiguously established molecular nutrition [71] of the antioxidant triad from the designer noodles in animal models and holds promise in delivery of the same in human.

The ADI values for L-tryptophan and melatonin in adult human are much higher than their respective amounts present in the serving size of noodles and thus consumption of the designer noodles will not invite amino-acid antagonism [72], and would also safely leave provision for the individual to consume L-tryptophan from other food sources, confirming the complete safety of the designer noodles.

The findings of the in vitro non-invasive predictive model of iHOMA2 implied that post-consumption, the mushroom noodles either enhance hepatic insulin sensitivity in the liver and periphery or glucose uptake in the gut or both. The increase in %β was owing to increase in glucose uptake in the gut; and %S was owing to increase in insulin sensitivity in the liver and periphery. The former contributes to proper absorption of glucose from the designer noodles, which is reportedly known to activate the peripheral insulin-sensitive glucose transporters (predominantly the adipose tissue, muscle, and liver). This had led to increase in insulin sensitivity in the liver, thereby allowing systematic regulation of glucose [73] and also caused stimulation of glucose uptake by the liver and periphery. Therefore, these findings confirm our proposition that the metabolism of the designer noodles involves the functional centralities of the liver.

The first and fifth hypotheses of the iHOMA2 model confirmed actual involvement of the liver, periphery and gut in the metabolism of designer noodles and thus the post-consumption increases in serum L-tryptophan, serotonin and melatonin levels are not invalid metabolic panaceas [74].

The formulated noodles, despite containing tapioca starch, did not cause excessive increase in blood glucose or serum insulin levels post consumption (vide supra). It is reportedly known that the glycemic index value of tapioca starch is high, thus the replacement of tapioca starch by freeze-dried mushroom powder in the current study would perhaps render the noodles suitable for consumption even by the diabetic population. Moreover, the boiled noodles have a swallowable consistency, hence it can be easily consumed by the geriatric population. From the study, it can be inferred that the gluten-free mushroom noodles can provide molecular nutrition with important antioxidants not only for animals but also possibly for human.

4.1 A processed food product as a vehicle of L-tryptophan, serotonin and melatonin: promises and concerns

The new designer mushroom noodles could be a promising food vehicle for serotonin and/or melatonin deficient populations. The gluten-free mushroom noodles for molecular nutrition [71] of the antioxidant triad (TSM) is unique and would be suitable for consumers of all ages.

It is reportedly known that under conditions of enhanced insulin in the blood, tryptophan is largely transported to the brain where it stimulates the synthesis of serotonin [75]. This raises concern for the designer noodles which during its metabolism has inadvertently enhanced blood insulin levels and therefore could potentially boost serotonin production- and therefore may lead to perturbation in the hormonal homeostasis if the noodles are consumed over a prolonged period of time. Furthermore, since greater amount of melatonin resides in the GI tract than in the pineal gland especially under high serum tryptophan levels [3], it necessitates execution of continuous animal feeding trials to assess the adverse effects of prolonged consumption of the designer noodles, prior to conducting human trials.

To summarize, the present study unambiguously established that the designer mushroom noodles (gluten-free) is truly rich in antioxidants since it can deliver appreciable amounts of the antioxidant triad viz. L-tryptophan, serotonin and melatonin in simulated gut conditions (SSB, SGB, SIB, SRB). The release of L-tryptophan (98.21%) and serotonin (83.03%) was highest in SGB post 50 min of dissolution, while the release of melatonin (44.85%) was also highest in SRB post 100 min of dissolution. After model fitting, it was evident that L-tryptophan and serotonin followed zero order release kinetics in SSB; while melatonin followed first order release kinetics in SSB and SRB, respectively. However, all three biomolecules followed the Korsmeyer’s- Peppas model kinetics in SGB and SIB; L-tryptophan and serotonin too followed the same release kinetics model in SRB. The antioxidant potency of the mushroom noodles post dissolution in simulated gut buffers was highest in SIB. From the present study, it was also evident from the feeding trials in male Sprague Dawley rats that the antioxidant triad was successfully absorbed within 30 min of consumption. From the findings of the non-invasive iHOMA2 model fitting, it was further ascertained that the noodles did involve the centralities of metabolism i.e., the liver and the periphery and promoted glucose uptake in the gut.

5 Conclusion

The designer mushroom noodles is truly a functional food since the same is a unique food source for molecular nutrition of important antioxidant molecules in animals and holds promise of delivery of the same to serotonin/melatonin compromised populations (especially for the geriatric populations owing to its swallowable consistency). Moreover, the noodle did not increase the blood glucose level post-consumption and therefore could be part of a diabetic diet. However, more detailed investigations on the mechanism of action of the designer noodles as a functional food; clinical nutrition involving elucidation of glycemic index; and metabolic changes and physiological effects in human subjects, are warranted to render the newly developed noodles suitable for consumers en masse. Nevertheless, the detailed methodology described in this work can be safely extrapolated to assessment of bioavailabilities of the said biomolecules from other processed food products.