Morus alba L. leaves modulate sweet (TAS1R) and bitter (TAS2R) taste in the studies on human receptors – is it a new perspectives of white mulberry leaves utilization in food production?

doi:10.21203/rs.3.rs-3197405/v1

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Morus alba L. leaves modulate sweet (TAS1R) and bitter (TAS2R) taste in the studies on human receptors – is it a new perspectives of white mulberry leaves utilization in food production?

https://doi.org/10.21203/rs.3.rs-3197405/v1

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From the nutritional point, the main direction for white mulberry (Morus alba L.) elements utilization so far is to create dietary supplements or functional foods for diabetics or over-weight patients. Leaves are widely known as a valuable source of bioactive compounds responsible for its antioxidant and antidiabetic effects, both in animals and humans. We found that processed leaves can be also considered as a bitter and/or sweet taste modulators – important aspect in designing of new functional foods. The aim of the study was to validate the inhibitory effect of Morus alba L. on the TAS2R3 and TAS2R13 bitter taste and TSA1R2/TSA1R3 receptors, and determine the changes that conditioning process caused in such receptors. Effect on the receptors was evaluated in specially transfected HEK293T cells, and the inhibition ratio was measured using the calcium release test. Moreover, the stability of phenolic acids and flavonols in the simulated intestinal in vitro digestion process was determined. Results showed that the tested Morus alba leaf extracts were rich in gallic, chlorogenic and caffeic acids together with rutin and quercetin 3-(6-malonyl)-glucoside, while the conditioning process positively affected on their amount. Mostly identified phenolics were reduced as a result of in vitro digestion. In taste receptors test, it was found that the phytochemicals from conditioned Morus alba leaf extract enhanced sweet taste, together with reduction of bitter taste receptors activity in some cases. To conclude, we found Morus alba, especially conditioned for 4 hours, seems to be valuable modulator of taste, what should be considered in future as a crucial reason for its new utilization.

Morus alba

TAS2R receptors

bitter taste

taste modulator

In the past decade much research has focused on mulberry antioxidant status, related with antidiabetic, anticholesterolemic or against obesity activity. White mulberry (Morus alba L.) products have been analyzed because of its utilization for nourishing blood, improving kidney function, and treating various ailments such as weakness, fatigue, urinary incontinence, dizziness, constipation and even possibly anticancer properties [1–7]. There were found a lot of nutrients in all morphological elements of white mulberry, belonging into groups of organic acids, chalcons, coumarins, flavonoids, phenolic acids, stilbens, catechins, aldehydes, terpenoides, steroids, alkaloids and other valuable components [8, 9]. There are investigation on white mulberry usage as a bioactive component of dietary supplements, as well as food products [10]. Molecularly, much attention is currently being paid to the effects of active compounds in the context of normal glycemia. For prevention retinol-binding protein 4 and haptoglobinone downregulation in diabetic patients was shown as one of responsible mechanism [11].

All of these areas are undeniably relevant for the society, in the era of galloping diabetes incidences. Nevertheless, having above in mind, it remains unclear in what direction can be further pursued for a broader or other application of white mulberry's capabilities, including the technological aspects – so important in the effective food production.

In the area of food sciences, technologists together with nutritionist found that the modulation of bitter taste is important for consumers, when introducing some nutritional food products with ex. dark cocoa, into diet. Moreover, it can be valuable aspect of newly design functional foods. From the nutritional point of view, appropriate taste increases the probability that patients will incorporate a health-promoting product into their diet. From the consumer's point of view, favorable taste helps break down the natural fear barrier to novelty.

In diet-depended diseases, such as overweight, diabetes mellitus etc., the use of such plant-origin ingredients is often combined with the addition of sugar or sugar substitutes, in order to obtain a functional product with adequate, acceptable palatability. The simplest way for manufacturers seems to be usage of synthetic or semisynthetic, mostly cheap, sweeteners however, their safety for sensitive consumers (ex. children, elderly, pregnant) may be questioned from a health standpoint [12]. The search for molecular inhibitors of bitter taste receptors, especially from natural sources like high-nutritional plants [13–15], is a new interesting way to improve the quality of many new created food products for such consumers.

There are scientific proofs, that natural flavonols may inhibit such bitter perception in food [16]. Therefore, in this study Morus alba is considered as such plant. We have already conducted that the composition of biological compounds, including flavonols, in white mulberry leaves is changed or positively improved as a result of some technological processes [2, 17, 18]. Therefore, the key objective of this study was to assess the overall impact of Polish Morus alba leaves (conditioned and non-conditioned) on the activity of taste receptors (mask the bitter taste and enhance the perception of sweetness). The additional objective was to check whether the valuable components (phenolic acids and flavonols) from Morus alba are stable after the simulated in vitro digestion process. So far, no research has been studied in the direction of taste modulation in the presence of white mulberry leaves in any form.

2.1 Material and reagents

The main plant material were white mulberry (Morus alba L.) leaves, Polish var. żółwińska wielkolistna (WML) picked manually from mulberry tree farm (Institute of Natural Fibres & Medicinal Plants, Pętkowo, central Poland).

Standards for phenolic acids and flavonols (gallic acid, protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, coffee acid, chlorogenic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid, rutin, isoquercitrin, quercetin 3-(6-malonyl)-glucoside, astragalin, myricetin, quercetin, kempferol, isoramnetin) were purchased from Sigma Aldrich, Germany. Reagents for receptors test were purchased from: IDT, Belgium (sequence coding G16-gust44), Invitrogen USA (pcDNA 3.1 vector), Biowest, USA (DMEM, Gibco, USA (Gltamax, PenStrep, sodium puryvate), VWR, Poland (FBS), GenScript, USA, Sigma Aldrich, ATCC, USA (HEK293T, TAS2R13, TAS2R3). Other chemicals were of analytical grade or chromatographic grade, purchased from POCH, Gliwice, Poland or Merck, Darmstadt, Germany.

2.2 Morus alba leaves – conditioning process

WML were processed at the Institute of Agricultural and Food Biotechnology in Poznań, Poland (IAFB) in semi-technological scale. The fresh, whole leaves were 1) shredded with a chopper-crusher (Stihl, Viking GE 103, Tengen, Germany) into small particles (ca. 50–70 mm x 10–20 mm), 2) divided into batches, 3) placed on wooden sieve trays in prisms (100 cm long, 40 cm wide, 10–15 cm high), and 4) conditioned for 4 hours (industrial dryer - SSO, Izoterma, Łany, Poland) at the temperature of 32.0˚C ± 3.0 (WML/4). One part of chopped leaves was not conditioned (sample: WML/0). Next, each part of WML was air-dried at inlet temperature of 90˚C and outlet temperature of 60˚C (tunnel dryer - IAFB, Poznań, Poland), and grounded (400 rpm, 15 s, laboratory mill - Retsch, GM200, Haan, Germany) to 0.8–0.08 mm powder.

2.3 Extract preparation

Following water extraction was used in the studies: 10.0 g ± 0.2 of WML was extracted for 15 minutes with using boiling distilled water (100 mL and 40 mL) (EM, Thermo Scientific, Waltham, MA, USA) according to Przeor et al. [17].

2.4 Effect of Morus alba leaves on receptors

In the study two extracts were used: 1) the water extract of WML (WML₁), and 2) the water extract of WML diluted with PBS buffer (10-fold) (WML_1:10) (Biowest, USA). The analysis was based on three steps according to Szczepaniak et al. [19]:

1) Transfection with TAS2R13 and TAS2R3 genes – synthetic sequence, coding G16-gust44 (IDT, Belgium) - amplification matrix; insert into the pcDNA 3.1 vector (Invitrogen, USA); transfection of HEK293T cells; cell cultures dissolved in supplemented DMEM medium (Biowest, USA); incubation for 3 weeks; transfection with the lipofection method with genes coding bitter taste receptors (TAS2R13, TAS2R3) (GenScript, USA); separation with electrophoresis;

2) Transfection with TAS1R genes - genes coding sweet taste receptors (TAS1R2/TAS1R3) cloned into the PSF-CMV-CMV-SBFI-UB-PURO—DUAL CMV vector (Sigma Aldrich, Poland), transfection to the HEK293T cell (ATCC, USA) with the lipofection method;

3) Calcium release test - release of Ca²⁺ determined by using the Fluo-4 Direct™ Calcium Assay Kit (ThermoFisher) and then fluorescence measurement (Spectra-Max i3x, Molecular Devices, USA); results were normalized with the results of the MTT test each time after the experiment; positive control samples: 10 mM of sucralose for TAS1R2/TAS1R3, 30 mM of chlorokin for TAS2R3, 30 mM of denatonium for TAS2R13.

The result was presented as ΔF/ F0 (fluorescence growth of cells with WML / intensity of fluorescence of control sample).

2.5 Digestion process

Simulation of the digestion process was carried out using a static in vitro digestion model. A 1 dm³ Biostat B Plus laboratory bioreactor (Sartorius Stedim Biotech) equipped with an Easy Ferm plus PHI K8 160 electrode (Hamilton) was used. Intestinal microflora (30 ml, incubated at 37˚C for 24 hours prior to administration), distributed in BHI (Merck KGaA, Germany) was used to simulate conditions in the intestinal section. 1M HCl and 1M NaOH (Merck, Germany) were used to adjust the pH of the environment. The different stages of the tube were ensured by the presence of pepsin, pancreatin, bile acid salts (Sigma-Aldrich). To ensure aseptic conditions, the equipment was sterilized (121˚C, 15 min, Sanyo Techno Solutions Tottorico sterilizer, Japan), kept under a laminar chamber (Safe 2020, ThermoScientific), and 72% ethanol solution (POCH, Poland) was applied to the surfaces. During the process, successive sections of the gastrointestinal tract were simulated: oral cavity, stomach, duodenum, small intestine, large intestine). The entire process took 24 hours. Digested samples of WML were stored as frozen.

2.6 Phenolic acids and flavonoids content

The content of phenolic acids and flavonols in the samples was determined by high-performance liquid chromatography with a diode array detector (HPLC/DAD) technique on a Agilent Infinity 1290 chromatograph. Separation was performed on Agilent Technology's Zorbax SB C18 column (150 mm x 3.9 mm ID, 5 µm) based on method described by Przeor et al. [3].

Detection of separated phenolic acids was performed at λ = 260 nm: gallic acid, protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, coffee acid, and at λ = 310 nm: chlorogenic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid. Flavonols were detected at λ = 370 nm: rutin, isoquercitrin, quercetin 3-(6-malonyl)-glucoside, astragalin, myricetin, quercetin, kempferol, isoramnetin. Tests were performed in duplicates.

3.1. Effect of Morus alba leaves on taste TAS2R receptors

The key aim of this study was to observe the response of taste sweet TAS1R2/ TAS1R3) and bitter (TAS2R13, TAS2R3) receptors to the Morus alba leaf extract. The results are summarized in the graphs below (Fig. 1, 2, 3). The final results of the experiment was presented as the ratio of the increase in fluorescence of cells exposed to the tested extracts (not diluted WML₁, diluted WML_1:10 ) with reference to the control sample.

The study used bitter taste receptors type 2 which belong to a subgroup of G protein-coupled receptors (GPCRs) of family A. They are crucial in the perception of bitterness, and are increasingly finding therapeutic use. These chemosensory GPCR receptors are found in the plasma membrane of type II taste receptor cells (TRCs) in the taste buds of the tongue, pharynx, larynx, soft palate and several other tissues throughout the body such as the intestinal epithelium (in enterocytes, tuft cells, cup cells, Paneth cells, microfollicular cells) [20].

The final product of the entire project carried out was to identify a substance that adequately modulates sweet taste (preferably enhancing), which would enhance the functional activation effect for sucralose by at least several times. As an added value, it was assumed that such a substance from a natural source could simultaneously inhibit (reduce) the perception of bitter taste.

The study showed that from both WML variants, which were selected based on our previous research [3], the closest to achieve basic criterion in its effect on TSA1R2/TSA1R3 sweet taste receptors were conditioned leaves (WML/4). At basal concentration (WML/4₁) it enhanced sweet taste over three times (3.35-fold), and at 10-fold dilution (WML/4_1:10) – 3.8-fold, comparing to sucralose, after 1 min of interaction. A similar effect was observed after 2 min of interaction, but to a lesser extent (Fig. 1).

Simultaneously, it was observed that conditioned mulberry leaf extract (WML/4) deactivated the bitter receptor TAS2R13, both after 1 min and 2 min (Fig. 2), but enhanced bitter taste in TAS2R3 receptor (stronger in samples WML_1:10) (Fig. 3).

Ligands from WML/0 samples also enhanced sweet taste, stronger after 2 min of interaction with TSA1R2/TSA1R3 than those from WML/4 sample (Fig. 1). When comparing both bitter taste receptors, mulberry extracts activated TAS2R3 (with chlorokin as positive) receptor better than TAS2R13 (with denatonium as positive).

In the context of the above results, it is worth bearing in mind that powdered mulberry leaf extracts are themselves characterized by a relatively bitter taste. However, the substances they contain react with taste receptors to some extent. The food industry points to the bitter taste of the leaves precisely as a major problem in their industrial processing. This bitter taste of mulberry leaves is difficult to eliminate even as a result of technological processing. Despite some similarity, the mechanism of bitterness formation in mulberry leaves is quite different from that in tea leaves. It was showed that intensity is a result of the interaction of many health-promoting compounds (amino acids, flavonoids, phenolic acids, alkaloids) on one hand, and sugar alcohol metabolites on the other hand [21].

A similar study to ours was conducted by Szczepaniak et al. [19] where authors recorded several times lower effects against receptors for the cornelian cherry, than the white mulberry leaves studied here. This raises hopes that white mulberry leaves in this area provide an interesting solution for the palatability of food products.

3.2. Stability of phenolic acids and flavonols from Morus alba L. leaves during in vitro digestion process.

According to the research model, the digestion process of WML/0 and WML/4 was simulated, mapping the human digestive tract. The digested samples were collected sequentially at six stages (from the stomach to the large intestine) of the process and analyzed qualitatively and quantitatively for the content of phenolic acids and flavonols, as presented in Table 1.

Table 1

Average amount (± standard deviation) of phenolic acids and flavonols in white mulberry leaves (digested samples) at each step of *in vitro* digestion.
[µg * ml^− 1]	WML/0						WML/4
[µg * ml^− 1]	A	B	C	D	E	F	A	B	C	D	E	F
rutin	103,88 b	100,08 b	84,95 b	76,84 b	51,01 b	0,68 a	33,39 a	39,12 a	25,91 a	26,83 a	33,60 a	11,51 b
rutin	± 0,39	± 0,16	± 4,80	± 0,24	± 0,49	± 0,11	± 0,49	± 0,24	± 0,03	± 0,82	± 4,46	± 0,19
isoquercitrin	32,60 b	29,91 b	30,33 b	32,30 b	21,09 b	1,28 a	11,00 a	14,63 a	11,58 a	12,25 a	16,99 a	18,39 b
isoquercitrin	± 0,93	± 1,64	± 2,18	± 1,32	± 1,30	± 0,55	± 0,09	± 0,03	± 0,03	± 0,04	± 1,34	± 0,19
quercetin 3-O-(6”-O-malonyl)-β-D-glucoside	82,12 b	71,73 b	83,36 b	92,47 b	61,34 b	41,88 b	30,61 a	37,39 a	31,26 a	35,29 a	44,18 a	24,75 a
quercetin 3-O-(6”-O-malonyl)-β-D-glucoside	± 0,21	± 0,06	± 1,81	± 1,11	± 2,90	± 0,15	± 0,07	± 0,06	± 0,01	± 0,04	± 1,27	± 0,14
astragalin	11,98 b	11,10 b	11,20 b	12,87 b	6,93 b	5,07 a	4,58 a	6,23 a	5,06 a	5,14 a	4,79 a	6,86 b
astragalin	± 0,16	± 0,08	± 2,31	± 0,66	± 0,54	± 0,16	± 0,12	± 0,01	± 0,01	± 0,04	± 0,10	± 0,09
myricetin	13,919 b	13,90 b	15,00 b	19,86 b	15,70 b	1,61 b	5,35 a	6,44 a	5,49 a	6,26 a	8,00 a	2,43 a
myricetin	± 0,09	± 0,08	± 3,19	± 0,63	± 3,11	± 0,31	± 0,09	± 0,01	± 0,01	± 0,00	± 1,01	± 0,96
quercetin	0,33 b	0,30 a	0,37 b	0,82 b	0,81 b	0,63 b	0,26 a	0,31 a	0,33 a	0,27 a	0,32 a	0,58 a
quercetin	± 0,00	± 0,00	± 0,07	± 0,02	± 0,06	± 0,03	± 0,00	± 0,01	± 0,00	± 0,00	± 0,02	± 0,02
kaempferol	0,18 a	0,17 a	0,19 a	0,32 b	0,41 b	0,16 a	0,16 a	0,17 a	0,19 a	0,16 a	0,17 a	0,20 b
kaempferol	± 0,02	± 0,00	± 0,02	± 0,03	± 0,09	± 0,01	± 0,00	± 0,00	± 0,00	± 0,00	± 0,01	± 0,01
isorhamnetin	0,58 a	0,58 a	0,59 a	0,60 b	0,58 b	0,58 a	0,58 a	0,59 a	0,57 a	0,56 a	0,55 a	0,55 a
isorhamnetin	± 0,04	± 0,00	± 0,01	± 0,01	± 0,01	± 0,01	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,02
TOTAL amount of flavonols	245,59	227,77	225,98	236,08	157,87	51,89	85,94	104,89	80,38	86,76	108,61	65,27
gallic acid	7,96 b	7,35 b	7,42 b	7,32 b	4,59 b	2,72 a	2,43 a	3,29 a	2,79 a	3,09 a	3,09 a	4,2 b
	± 0,03	± 0,07	± 0,02	± 0,00	± 0,01	± 0,02	± 0,00	± 0,07	± 0,01	± 0,02	± 0,02	± 0,01
protocatechuic acid	0,59 b	0,54 b	0,46 b	0,41 b	0,35 b	0,13 b	0,23 a	0,28 a	0,20 a	0,22 a	0,20 a	0,05 a
	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00
4-hydroxybenzoic acid	0,03 b	0,04 a	0,03 b	0,04 b	0,03 b	0,08 b	0,02 a	0,03 a	0,02 a	0,02 a	0,02 a	0,01 a
	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00
vanillic acid	0,03 b	0,04 b	0,05 b	0,07 b	0,07 b	0,09 b	0,02 a	0,02 a	0,02 a	0,02 a	0,02 a	0,01 a
	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00
chlorogenic acid	2,56 b	2,40 b	1,68 b	1,41 b	0,55 a	0,00 a	0,71 a	0,98 a	0,77 a	0,72 a	0,62 b	1,07 b
	± 0,02	± 0,01	± 0,55	± 0,01	± 0,00	± 0,00	± 0,00	± 0,00	± 0,24	± 0,01	± 0,00	± 0,00
caffeic acid	2,18 b	1,88 b	1,80 b	1,40 b	0,78 b	0,58 a	0,60 a	0,83 a	0,55 a	0,63 a	0,56 a	0,97 b
	± 0,02	± 0,00	± 0,03	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00
syringic acid	0,04 b	0,03 b	0,02 b	0,01 a	0,00 a	0,00 a	0,02 a	0,02 a	0,01 a	0,01 a	0,00 a	0,00 a
	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00
p-coumaric acid	0,01 a	0,01 a	0,01 b	0,01 b	0,01 b	0,004 b	0,01 a	0,01 a	0,00 a	0,00 a	0,00 a	0,00 a
	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00
ferullic acid	0,03 b	0,03 b	0,03 b	0,03 b	0,02 b	0,01 b	0,01 a	0,01 a	0,01 a	0,01 a	0,01 a	0,01 a
ferullic acid	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00
sinapic acid	0,02 b	0,01 a	0,01 a	0,02 b	0,01 b	0,00 b	0,01 a	0,01 a	0,01 a	0,01 a	0,00 a	0,00 a
sinapic acid	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00	± 0,00
total content of phenolic acids	13,44	12,32	11,51	10,69	6,41	3,63	4,05	5,48	4,37	4,72	4,51	6,34
WML/0 – non-conditoned white mulberry leaves; WML/4 - conditoned white mulberry leaves; A - initial stage in stomach, B - final stage in stomach, C - stage in duodenum (pH 6.0), D - stage in small intestine (pH 7.4), E - initial stage in large intestine, F - final stage in large intestine; a, b – different letters indicate statistically significant differences in a ANOVA test.

Table 1. Average amount (± standard deviation) of phenolic acids and flavonols in white mulberry leaves (digested samples) at each step of in vitro digestion.

/table attached/

The gastrointestinal tract plays the role of a gateway to the human body for ingredients obtained from the meal. Given that taste receptors have been identified in the extraoral area of the gastrointestinal tract, checking the stability of bioactive compounds after the digestion process from white mulberry can determine the real strength of this effect. After all, the presence of receptors plays a key role in the protection of the human organism, including the induction of appropriate motor and secretory responses, elimination of dangerous products (vomiting, diarrhea, temporary anorexia) [20].

Quantitative changes in different sections of the simulated gastrointestinal tract were evident for phenolic acids present in model. With reference to retention times and UV-VIS spectra, ten phenolic acids were identified. Gallic acid predominated at all stages of the digestion process, while chlorogenic acid together with caffeic acid were also measured in quite high amount. Thus, this confirmed the previously identified relationships for phenolic acids in the characterization of Polish variety white mulberry leaves [3].

Statistically higher contents of each phenolic acids were determined in WML/0 than in WML/4 samples. Moreover, the quantitative changes in these compounds were more intense in the case of WML/0. The WML/ 0 variant showed also highest total phenolic acids content. This was clearly evident at stages A, B, C, and D, where, relative to analogous digested WML/4 samples, 2–4 times as many phenolic acids were recorded.

The in vitro digestion process resulted in fairly regular changes in the amounts of flavonols. Starting from the small intestine stage (D or E), the amounts were gradually reduced, the greatest at the terminal stage of the large intestine. For many flavonols, the decreasing content over the course of the process was preceded by an initial increase in their content at the A-D sections. Among the eight identified flavonols, rutin and quercetin 3-(6-malonyl)-glucoside were found in the highest amounts at the beginning of the digestion process. In addition, smaller amounts of isoquercitrin, astragalin and myricetin were found. The amounts of quercetin, kaempferol and isoramnetin were at levels lower than 1 µg*ml^− 1 at all stages of digestion. The smallest variations in content between stages were recorded for isoramnetin and quercetin (WML/0). Leaves conditioned for 4 hours (WML/4) also showed the lowest contents of each flavonols, and there were almost 3 times less total amount of flavonols comparing with WML/4 samples. The greatest quantitative losses of total flavonols were observed at stage F.

The observed different trends (increase or decrease) in the content of individual polyphenols at the initial stages of the simulated digestion process are supported by the literature. Some studies have pointed to the loss of polyphenols during the task of oral and gastric conditions [22], while in others, authors have emphasized the stability of compounds during the passage through these stages. This was explained by too short exposure of the samples to the acidic environment of the stomach, which did not involve hydrolysis and release of polyphenols from cellular structures. In addition, it is speculated that low pH has a protective effect on polyphenol structures [23, 24].

After incubation under conditions simulating the stomach, the intestinal stage was conducted in a bioreactor. At the duodenal stage, pancreatic extract and bile acid salts were administered while increasing the alkalinity of the environment. A further pH increase to 7.4 corresponded to conditions in the small intestine, while holding at pH 8.0 for 18 hours – to conditions in the large intestine. The greatest reduction in phenolic acids and flavonols was observed after F stage (large intestine). Polyphenols when reaching the colon are intensively transformed into smaller forms by the intestinal microflora. Their presence can also affect the growth of major strains of intestinal bacteria [25, 26]. It is increasingly emphasized that the antioxidant capacity of plant-derived foods is evidenced not only by polyphenol content, but also by the activity of phenolic metabolites of bacterial origin, high concentrations of which are recorded precisely within the colon [27].

Polyphenols are generally poorly absorbed during digestion, as they are converted through the action of digestive enzymes and intestinal microflora to lower molecular weight compounds. Animal and human studies showed that relation to many polyphenols, including chlorogenic acid, caffeic acid, ferulic acid and rutin [28].

Our previous research on white mulberry leaves mainly was focused on finding the best processing conditions for obtaining highly polyphenolic extracts, from a nutritional point of view. However, it turned out that conditioning for 4 hours in most analyses resulted in the strongest reduction of bioactive compounds. Fortunately, this work found a new direction in the use of this variant of conditioned leaves - as a taste modulator. It seems that the track taken towards such conditioned mulberry leaves may have broad technological perspectives and should be continued, ex. in matrix of specific food products, also in humans studies.

Funding

This research was co-funded by the National Centre for Research and Development in Poland (www.ncbr.gov.pl), grant POIR.04.01.02-00-0059/17 and Poznań University of Life Sciences as the research program „First grant”, no. 14/2022/Pierwszy Grant. The APC was funded by Poznań University of Life Sciences as the research program „First grant”, no. 14/2022, and by Department of Gastronomy Science and Functional Foods, Faculty of Food Science and Nutrition, Poznań University of Life Sciences (statutory fundings).

Author Contributions

M.P. and M.J.: resources, data curation, investigation; M.P.: conceptualization, methodology, validation, writing, review, editing, supervision, funding acquisition; M.J.: visualization. All authors contributed to reviewing and editing the final manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethics Approval

Not applicable.

Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Acknowledgments

Authors would thank Professor Joanna Kobus-Cisowska and Oskar Szczepaniak, PhD for support given during research, and Wojciech Woźny, PhD from PORT Polish Cen-ter for Technology Development, Wrocław, Poland, for his organizational help.

Data Availability

The datasets of the current study are available from the corresponding author on reasonable request.

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Morus alba L. leaves modulate sweet (TAS1R) and bitter (TAS2R) taste in the studies on human receptors – is it a new perspectives of white mulberry leaves utilization in food production?

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Material and reagents

2.2 Morus alba leaves – conditioning process

2.3 Extract preparation

2.4 Effect of Morus alba leaves on receptors

2.5 Digestion process

2.6 Phenolic acids and flavonoids content

3. Results and discussion

3.1. Effect of Morus alba leaves on taste TAS2R receptors

4. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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