Proximate, minerals, carotenoid and trypsin inhibitor composition in the exoskeletons of seafood gastropods and their potentials for sustainable circular utilisation

Periwinkle shells of Tympanotonus fuscatus, Pachymelania aurita, and Thais coronata were analyzed for their proximate composition, nutritionally significant minerals, trypsin inhibitors, and carotenoids. The mean values obtained were compared using an ANOVA test. The results showed that T. fuscatus had the highest mean moisture content of 0.96 ± 0.14% and a mean value of 0.49 ± 0.13% for crude fibre but was not significantly different (P > 0.05) from P. auritus. The crude protein and fibre content of T. fuscatus was significantly higher (P < 0.05) than other periwinkle samples. T. coronata had the highest mean total ash content and was significantly different (p < 0.05) from other periwinkle samples. T. fuscatus had the highest mean value for Mg (0.32 ± 0.03 mg/kg) and differed significantly (P < 0.05). The mean Ca content of P. aurita was not significantly different (P > 0.05) from that of T. coronata. The mean values of CaCO3 in T. fuscatus, P. aurita, and T. coronata were 57.20 ± 2.46, 59.50 ± 3.23, and 62.36 ± 1.56 (mg/kg), respectively. T. coronata was significantly different (P < 0.05) from other periwinkle samples. The mean values of carotenoids in T. fuscatus, P. aurita, and T. coronata were 7.17 ± 2.14, 18.00 ± 5.27, and 11.20 ± 3.60 (mg/kg), respectively, and P. aurita was significantly different (P < 0.05) from other periwinkle samples. T. fuscatus and P. aurita had shells with significant amounts of trypsin inhibitor (23.30 ± 4.50 mg/kg and 22.90 ± 14.10 mg/kg, respectively), making them less suitable for livestock feed. In contrast, T. coronata had a lower mean value of 11.80 ± 7.19 mg/kg for trypsin inhibitor, making it an excellent addition to livestock feed. The low crude fibre and fat contents of the periwinkle samples in this study make them suitable for processing complementary foods, especially for hypertensive patients. The high percentage of CaCO3 in periwinkle shells makes them a probable source used in the production of slurry for chromatography. The findings suggest that periwinkle shells contain specific minerals that can be applied in numerous industries. Increased use of these gastropod shells will result in successful application in product creation and a sustainable bio-circular economy.


Materials and methods
Study area.The gastropod samples used for this study were bought from retailers who collected them from the Lagos Lagoon complex.On the West African coast, the Lagos Lagoon is the most extensive Lagoon system in the Gulf of Guinea (Fig. 1).It runs 257 km from Cotonou, Benin, to the Niger Delta, Nigeria.The Lagos Lagoon lies between longitudes 3°22′E′ and 3°4′E′ and latitudes 6°17′N′ and 6°28′N′ 28 .Except for the channels, which are occasionally dredged, the Lagoon is shallow, with an average depth of 1.5 m 29 .It opens into the Gulf of Guinea through Lagos Harbor, which serves as the major extrance to the only seaport for Nigeria's western Lagoons.The sediments in Lagoons include mud, muddy sand, and sand 30 .The two main elements that govern the physicochemical parameters and biology of the Lagoon area are seawater associated with a semidiurnal tidal regime and land drainage from surrounding wetlands.Lagos Lagoon encounters brackish conditions due to seasonal fluctuations in freshwater drainage from land and seawater invasion, particularly noticeable during the dry season.

Sample collection and description. 30 samples of Tympanotonus fuscatus, Pachymelania aurita, and
Thais coronata were collected monthly for three months (March-May in 2021 to reflect the end of the dry season and the beginning of the wet season) using a fish net.The samples were stored in a cooler that contains ice blocks to avoid decomposition during transportation.Afterwards, these species were identified based on species identification sheets from the Food and Agriculture Organization 32 , prepared by Fisher and Bianchi.T. fuscatus is known as periwinkle and has a single species but comprises two varieties.Tympanotonus fuscatus var.fuscatus and Tympanotonus fuscatus var radula inhabit quiet waters and muddy areas, and the substratum is rich in decaying organic matter.T. fuscatus var.fuscatus is differentiated by the presence of turreted, granular and spiny shells with tapering ends.In contrast, T. fuscatus var.radula is characterized by the absence of tiny tubercles on the shell, differentiating it from the other variety.They primarily feed on algae and diatoms.T. fuscatus possesses an elongated shell with a regular, increasing whorl; the ribs are weakly V curved with much fine striation, and blackish brown stripes are visible on the shell 8,33 .P. aurita is morphologically different from T. fuscatus.The shell of P. aurita is characterized by sharp spines, and it has a broader aperture; In P. aurita, the sharpness of the spine solidly depends on the organism's age; as the organism becomes older, the spine of the shell becomes blunt and thicker, and it is one of the most common molluscs in the Lagos Lagoon.The genus Pachymelina has been said to be endemic to West Africa 34,35 .Thais coronata is also a univalve gastropod.It can be found in estuaries, brackish water creeks, and mangrove swamps in many freshwater bodies across Nigeria.The length of the shells was adequately accessed and measured.The edible parts were carefully removed from the shells with a drilling pin before washing them in distilled water to remove dirt and debris before measurement and drying.After that, the samples were sent to the University of Ibadan, Oyo State Laboratory for analysis.

Proximate analysis. Determination of crude fat content using the soxhlet extraction method.
Well-blended samples (5.00 g) were weighed into the thimbles and placed with cotton wool to prevent the sample from pouring out during extraction.The round bottom flask was dried in an oven at 60 °C, and the initial empty weight was recorded.80 ml of hexane was poured into the flask, and the thimble containing the sample was fitted/placed into the extractor.The heating mantle was switched on, and water was set to run through the condenser for cooling.The extraction was allowed to continue its reflux for 2 h after it was discontinued.The flask was then dried in the oven to eliminate all hexane.The amount of crude fat or oil (%) present in the sample was calculated by subtracting the weight of the empty flask from the final weight.

Determination of ash.
Empty crucibles were dried in an oven at 130 °C for 30 min to remove the moisture present on the crucibles, transferred into a desiccator and allowed to cool at room temperature for approximately 20 min.Then, the weight of the empty crucibles was taken and recorded as W 0 .The samples were then blended into powder using a mortar and pestle to increase the surface area.We weighed 1.0000 g of the sample using an analytical balance into the crucibles and recorded it as W 1 , then ashed in the furnace at 500 ± 15 °C for 5-6 h 36 .The crucibles containing the samples were allowed to cool for approximately 30 min in the furnace.Then, with the crucible tongue, the crucibles were transferred into the desiccator and allowed to cool at room temperature for approximately 45 min.The final weight of the crucible and content was then taken and recorded as W 2.

Determination of protein content according to the Kjeldahl method.
A total of 1.00 g of wellprepared sample was weighed to an accuracy of 0.1 mg into a 250 ml digestion tube, and 2kjeltabs Cu 3.5 (alternatively 7 g K 2 SO 4 and 0.8 g CuSO 4 ⋅5H 2 0) from Finlab Nigeria limited was added.Then, 12 ml of concentrated H 2 SO 4 was carefully added and gently shaken to wet the sample with acid.The exhaust system was then added to the digestion tubes in the rack, and the water aspirator was set to full effect.The samples were digested for 1 h at 420 °C.The racks of tubes were removed, placed on a stand and allowed to cool for 10-20 min.The tubes were then inserted into the distillation unit, and the safety door closed; 80 ml of deionized water was carefully added to the tubes.Then, 25-30 ml receiver solution was added to the conical flask and placed into the distillation unit.
The platform was placed, so the distillate outlet was submerged in the receiver solution.50 ml of 40% NaOH was dispensed into the tube and distilled for approximately 5 min.Then, the distillate was titrated with standardized 35% HCl of 0.1 ml until the blue-gray endpoint was achieved.A blank was run through all the steps above.W 1 = sample weight (mg), T = titration volume of sample (ml), B = titration volume of blank (ml), N = normality of acid to 4 decimal places, F = conversion factor for nitrogen to protein = 6.25 for food and feed.
Determination of fibre using fibretec.Samples (2.0 g) were digested in 20 ml of 1.25% H 2 SO 4 , and the mixture was boiled for 30 min and then filtered and washed with hot water to reduce the acidity.This process was tested with pH paper; the residue was again digested in 20 ml of 1.25% NAOH 37 .The mixture was heated for 30 min, filtered and washed with hot water, dried in an oven, transferred to a platinum crucible, weighed (W 1 ), heated in a furnace at 550 °C and weighed again (W 2 ).
Determination of moisture content.Empty porcelain crucibles were dried in an oven at 105 ± 50 °C for 30 min to remove moisture from the dishes.Then, the porcelain crucibles were transferred into a desiccator and allowed to cool at room temperature for 20 min; the weight of the empty porcelain crucibles was taken and recorded as W 0. The samples were blended into powder using a mortar and pestle to increase the surface area; 1.00 g of sample was weighed into porcelain crucibles (recorded as W 1 ) and dried in an oven at 105 ± 50 °C for 4 h.The porcelain crucibles containing the samples were allowed to cool for approximately 10 min in the oven with the use of a crucible tongue.The porcelain crucibles were transferred into a desiccator and allowed to cool at room temperature for approximately 30 min.The final weight of the porcelain crucibles and content was recorded as W 2 .
Nitrogen free extract (N.F.E).The N.F.E.which is the non-evaluated proximate composition parameters such as digestible carbohydrate and soluble organic compounds, was determined by subtracting the sum (%) of all other evaluated proximate composition parameters from 100.The equation below was adopted 38 .

Determination of minerals.
The nitric-perchloric acid digestion method is followed to determine Ca, K, Mg, P, and CaCO 3 from the periwinkle shell.For this digestion, 1.00 g of periwinkle shell sample was weighed into a digestion tube or a conical flask, and 10 ml of H 2 SO 4 and 30 ml of nitric acid were added.It was placed on a hot plate in a fume cupboard and digested until the digest became clear and then diluted to 100 ml, after which it was taken to AAS for mineral determination.According to the method 37 , the absorbance was determined by atomic absorption spectroscopy (AAS) (UV visible U-2900 Hitachi, Tokyo, Japan) at wavelengths of 285.2 nm (Mg), 766.5 nm (K), 422.7 nm (Ca), 213.62 nm (P) and 2155.12nm (CaCO 3 ).AAS standard solutions (1000 mg/l in 5% HNO 3 ) (Merck, Germany) were used for calibration.Finally, lanthanum and cesium chloride (0.1%) were added to the samples and standards to mitigate interferences.The AAS siphoning hose was then placed into the digested sample.After running, the standards for the mineral/metal were determined, and the concentration of the mineral/metals in the solution displayed on the computer screen was read.
(2) The supernatant was brought up to a volume of 10 ml using 80% acetone according to a previously described method 39 .Then, the optical density (OD) (absorbance) was read at a wavelength of 450 nm using spectrophotometer (UV visible U-2900 Hitachi, Tokyo, Japan).Note: Similar AAS machine was used for absorbance determination of mineral parameters and carotenoid but was adjusted at different wavelengths.The reason is because the UV 2900 version can be used for multifunctional analyses.
Determination of trypsin inhibitor.1 g of properly blended died sample was measured into a flask, and 50 ml of 0.5 M NaCl was added.The mixture was quivered for about 30 min and centrifuged at 1500 rpm for 5 min.10 ml of decanted filtrate was pipetted into another flask; then, 2 ml of standard trypsin solution of 2 mg/l was added to it.The absorbance was caliberated at 410 nm while 10 ml of the filtired sample was used as blank.Standard trypsin inhibitors (1, 2, 4, 6, 8, and 10 mg/l) were prepared with N-alpha-benzoyl-dl-argininep-nitroanilide (BAPNA), and their absorbencies were measured at 410 nm.An accurate graph which illustrated the absorbance against the concentration was drawn and extrapolated, by tracing the absorbance to the concentration axis and the trypsin inhibitor content was obtained.DF: Dilution factor.If not diluted, then DF = 1.

Statistical analysis.
Microsoft Excel version 16.0 was used to evaluate the mean ± standard deviation of the raw data and descriptive computation.One way Analysis of variance (ANOVA) at significance level at 5% (P < 0.05) and the correlation coefficients were done using SPSS IBM, 15.0.The disparity of mean between samples was achieved through Duncan multiple post HOC test.
Figure 2 shows a graphical representation of the sample's proximate constituents in March.The amount of moisture was highest in T. fuscatus at 1.09% and lowest in T. coronata at 0.3%.T. fuscatus was also found to be the highest in crude protein, while T. coronata was found to be the lowest in crude protein and crude fat at 1.18% and 0%, respectively, while T. fuscatus was the highest in crude fat at 1.50%.P. aurita has the highest crude fibre content at 0.74%, and T. coronata has the lowest at 0.38%; for the ash content, T. coronata was found to have the    www.nature.com/scientificreports/highest content at 97.38% and the lowest T. fuscatus at 94.08%, while the nitrogen-free extract was highest in T. coronata at 0.76% and lowest in P. aurita.Figure 3 depicts the proximate contents of the samples in April.The moisture content of P. aurita had the highest value at 0.99%, with T. coronata having the lowest value at 0.40% (Fig. 3).T. fuscatus was also found to have the maximum protein and fat at 3.21% and 1.08%, respectively, while T. coronata was also the lowest in crude protein and crude fat at 2.00% and 0.50%, respectively.P. aurita has the highest value of crude fibre at 0.61% and nitrogen-free extract at 1.75%, while T. fuscatus has the lowest ash content at 93.89%.
Figure 4 revealed the sample's proximate composition in the month of May.T. fuscatus had the highest moisture and crude fibre values at 0.99% and 3.95%, while P. aurita had the highest values in crude fat and crude fibre at 0.60% and 0.48%, respectively.T. coronata had the highest ash content at 96.74% and T. fuscatus the lowest at 93.12%, while T. coronata had a negative value in the nitrogen-free extract at -1.71%.
The proximate analysis results expressed as mean value and standard deviation were found to be significantly different as shown in Table 1.Basically, the nutritional constituents of finfish and shellfish vary, due to differences in the type of species, individuals, part (such as tissue, shell or flesh) evaluated, sex differences, season 40 , the morphological status of such species, and the ecological and geographical structure of the ecosystem where such species were extracted.Awareness of the moisture constituent of any aquatic species is crucial because it provides valuable information on the quality and vulnerability of organisms to microbial spoilage and fungal infection 40,41 .It helps stabilize the movement of organisms.Organisms with low moisture content are less susceptible to microbial spoilage 42,43 and have the ability to prolong their shelf life and retain their nutritive content for a considerable period of time during postharvest.T. fuscatus had the highest mean moisture content at 0.96 ± 0.14% and was significantly different (P < 0.05) from other samples, while T. coronata had the lowest mean moisture level at 0.46 ± 0.20%.The present research does not agree with the report of Oyawoye et al. 44 whose work was on  T. fuscatus shell from Nembe, Rivers' state.The results contradict the values obtained from this study, such that the moisture content value obtained in the periwinkle shell was 2.5%, compared to 0.96 ± 0.14% in this study.The result obtained for moisture is considerably similar to that obtained in the research carried out by Etim 26 ; Omotoso 45 ; and Elegbede et al. 43 .
Generally, the types of proteins present in shellfish are basically rich and provide essential amino acids needed for body growth, reproductive processes, and antioxidant synthesis, particularly in living organisms 46 .This agrees with the assertion of Okuzumi and Fujii 47 that proteins are large biomolecules that are naturally significant and beneficial to living organisms and are needed for sustenance.Protein constituents are found abundantly in several shellfishes, such as oysters and bivalves, compared to finfishes 43,48 .The presence of proteins in the human body helps to repair damaged tissues and other important biochemical and anatomical [49][50][51] .The highest percentage of mean crude protein was found in T. fuscatus at 3.23 ± 0.70% and showed a significant difference (P < 0.05), and the lowest percentage of crude protein was found in T. coronata at 2.18 ± 1.06%.Akpan and Oscar 18 recorded a protein content of 22.52% for T. fuscatus from the Calabar River, which was proportionally higher than the 3.23% obtained in this study.Similarly, the result obtained for protein content in this study was lower than the values of 46.51%, 51.30%, and 58.45% recorded in blended marrows of T. fuscatus, P. aurita, and T. coronata, respectively,  by Inyang et al. 19 .A high protein content of 49.54% by Adebayo-Tayo et al. 12 and 48.62% by Job and Ekanem 21 was recorded in the marrow of P. aurita.
Just like protein, fat is also an essential molecule that helps in the body's skeletal formation, allows free movements and distributions of soluble vitamins, aids the retention of protein from food 43 , and is involved in cell biological processes in the human body.The fat content recorded in this study ranged from 0.36 to 1.02%, and a significant difference (P < 0.05) exists.P. aurita was found to have the highest level of crude fibre at 0.61 ± 0.13% but showed no significant difference (P > 0.05) to T. fuscatus, and the lowest level of crude fibre was found in T. coronata at 0.36 ± 0.01%.The mean value for crude fibre in T. fuscatus (0.49%) corresponds with the 0.043% registered by Oyawoye et al. 44 for similar species.The fat contents recorded in samples of this study were relatively lower than those by Inyang et al. 19 for three shellfishes and also lower compared to those obtained in similar species by Job and Ekanem 21 , and Ehigiator and Oterai 52 .Differences in several proximate constituents of the species in our study and those obtained by previous researchers could be attributed to the exoskeletons evaluated in this study compared to marrow or meat evaluated by previous researchers 43 .The low-fat level of these exoskeletons means they could be processed into powders and subsequently added to foods as a supplement for people who are hypertensive and individuals who are suffering from lipid-related diseases such as arteriosclerosis 19,43 .
The total ash, which serves as a signal that indicates the mineral availability in any organism, also helps in proper body formation 43,51 .High total ash contents in periwinkle exoskeletons (T.coronata at 97.0 ± 0.32% was the highest and the lowest in T. fuscatus at 93.6 ± 0.51%) mean the presence of macro-minerals, which include P, Mg, Ca, and their derivative compounds 53 .Although our result was higher than those recorded by 12,19,21,52 .The extremely high amount of total ash in all periwinkle shell samples could be due to chitin availability in shells, enhanced by Ca and its derivative 54 , or by the presence of high inorganic adulterants or activated carbon obtained following calcination.
Low crude fibre was obtained in all samples, ranging from 0.36% in T. coronata to 0.49% in T. fuscatus at P < 0.05.Adebayo-Tayo et al. 55 and Lah et al. 56 also observed variations in crude fat in shellfish.The results of this study were similar to those obtained by 19,21,52 for similar species.Inyang et al. 19 affirmed that the low crude fibre in periwinkle makes it suitable for processing complementary foods.Table 1.Mean value of proximate analysis (%) of the sample periwinkles.Values are given as the mean ± standard deviation of the samples analyzed in monthly triplicates using ANOVA.In each row, the mean with common superscripts is not significantly different.

Parameters (%)
T Correlation matrixes between the proximate components for T. fuscatus, P. aurita, and T. coronata were recorded (Tables 2 and 3).There was a robust positive correlation between the nitrogen-free extract and crude protein and a strong negative correlation between the nitrogen-free extract and crude fat (Table 2).
A robust positive correlation was observed between crude fibre and crude fat, and a strong negative correlation was observed between nitrogen-free extract and total ash (Table 3).
There was a strong positive correlation between the nitrogen-free extract and total ash and a strong negative correlation between the nitrogen-free extract and crude protein (Table 4).
Figure 5 depicts the mineral contents of the exoskeleton samples in March.The mineral composition of the samples for March shows that P. aurita was found to be higher in Ca at 58.78 mg/kg, and T. fuscatus was found to be higher in Mg, K, and P at 0.035 mg/kg, 0.022 mg/kg, and 0.71 mg/kg, respectively, while CaCO 3 was found to be highest in T. coronata at 63.75 mg/kg.
Figure 6 shows the mineral constituents of the samples in April.The mineral composition of the samples recorded Ca to be higher in P. aurita in April at 55.10%.T. fuscatus was the lowest at 53.72 mg/kg.T. fuscatus was found to have the maximum Mg, K and P at 0.31 mg/kg, 0.017 mg/kg, and 0.065 mg/kg, respectively, while T. coronata was found to have the highest CaCO 3 at 62.65 mg/kg.
Figure 7 illustrates the mineral contents of the samples in May.The mineral composition of the samples for May shows T. coronata to have the highest Ca contents of 52.58 mg/kg, while T. fuscatus has the highest values of Mg, K, and P at 0.296 mg/kg, 0.014 mg/kg, and 0.058 mg/kg, respectively (Fig. 7).T. coronata was recorded to have the highest CaCO 3 content with 60.67 mg/kg.
The phytochemical composition of the samples in March showed that T. fuscatus had the highest trypsin inhibitor content at 25.39 mg/kg, while P. aurita had the highest carotenoid content at 16.87 mg/kg (Fig. 8).The phytochemical composition of the samples in April showed that P. aurita had the highest trypsin inhibitor content at 38.77 mg/kg, T. coronata had the highest carotenoid content at 14.81 mg/kg, and T. fuscatus had the highest carotenoid content at 9.43 mg/kg (Fig. 9).The phytochemical composition of the samples for May shows that T. fuscatus had the highest value of trypsin inhibitor at 18.21 mg/kg, while P. aurita had the highest value of carotenoids at 23.76 mg/kg (Fig. 10).
The mean level of minerals in exoskeletons was presented, and a significant difference (P < 0.05) was also established (Table 5).The major mineral in natural periwinkle shells as obtained in our study is basically CaCO 3 , with mean contents of 57.20 ± 2.46 mg/kg, 59.50 ± 3.23 mg/kg, and 62.36 ± 1.56 mg/kg in T. fuscatus, P. aurita, and T. coronata, respectively, as shown in Table 5.The obtained results are in consonance with the typical mineral compositions registered by Orji et al. 57 in their findings.Periwinkle shell grains comprising mineral components of over 85% are sufficient for the production of sandpaper abrasives 58 .Having a higher percentage of Ca composition, Oyawoye et al. 44 also suggested that the shells will be suitable for use in soda ash production, sugar refining, and flue gas desulfurization due to the high demand for limestone.It's so surprising that the major macrominerals believed to generally enhance the morphological development of calcareous exoskeletons were obtained in extremely high amounts, which contradicts the assertion of Davies and Jamabo 59 that shells generally contain macrominerals in extremely high quantities.Na and its derivative element NaCO 3 are another mineral whose absence was not expected since they both play good roles in ensuring the compactness of shells and help  43 .The absence of Na and its derivative element NaCO3 corroborates the finding of Elegbede et al. 43 .The highest mean value for Ca was found in P. aurita at 55.20 ± 3.44 mg/kg.T. fuscatus had the highest mean levels of Mg, K, and P (0.32 ± 0.03 mg/kg, 0.017 ± 0.004 mg/kg, and 0.06 ± 0.006 mg/kg, respectively).Trypsin inhibitor was found to be high in T. fuscatus at 23.3 ± 4.50 mg/kg, while carotenoids were lowest in T. coronata at 11.2 ± 3.60 mg/kg .The CaCO 3 content ranged from 57.2 to 62.36 mg/kg, and the mean highest Ca content was found in T. coronata (Table 5).Due to the high content of CaCO 3 in periwinkle shells, previous research 43 has reported that the periwinkle shell could be applied in designs, the pearl button industry, and the construction industry.Shells and gravel are used for building roads, and lime from the shell is a vital component in the production of concrete and plaster 60 .In this study, the mean value of CaCO 3 in the shells of T. fuscatus, P. aurita, and T. coronata was 57.2 mg/kg, 59.5 mg/kg, and 62.36 mg/kg, respectively, making T. coronata the species with the highest CaCO 3 content at 62.36 mg/kg.The report of Orji et al. 61 on the valorization of shells based on mineral components denotes a possible utilization potential of periwinkle shells as adsorbent materials in production thin layer chromatography plates.The high percentage of CaCO 3 in periwinkle shells makes them a probable source of CaCO 3 , demonstrating that the powdered shells of T. fuscatus and L. littorea may be used in producing slurry for thin-layer chromatography.T. fuscatus in this study has a high amount of CaO compared to work done by Malu and Bassey 62 .CaCO 3 in periwinkles can also serve as supplements added to the cultivation medium to derive the optimal pH that is favorable for the fungus's growth 60 .Shells of periwinkle can also be a suitable replacement for asbestos in brake pad production 58 .Azor et al. 63 illustrated the potential of melon and snail shell waste mixtures as a supplementary source of carbohydrates that enhance the compactness and hardness of mild steel.Periwinkle (T.fuscatus) shells can serve as an alternative source of lime for the glass industry 62 .Ademola et al. 64 asserted that periwinkle shell with a high mineral component can be valorized and used as a supplementary material in the production of cooking utensils; it can be utilized directly for the treatment of measles; and it can also be used as a bioindicator of toxic metals in the aquatic ecosystem 65 .The high availability of Ca and its derivative compound (CaCO 3 ) makes the periwinkle shells of this study important to the biomedical industry in that they can be utilized as an alternative to porcelain used for the production of dental implant materials such as artificial teeth 43,51 , which will not only reduce the expenses involved but also help to alleviate the environmental concern associated with the indiscriminate dumping of these shells.The essentiality of Ca in human nutrition and body functions cannot be overemphasized.Ca could be obtained from both animal and plant sources, and typical examples include dairy products such as milk, broccoli, e.t.c 43 .Elegbede et al. 43 also mentioned the significance of bivalve shells in agriculture, such that blended shells could be incorporated into animal-formulated diets.Scientific trials on the successful utilization of shells in several applications, particularly cosmetics, have been conducted, although none of these have yielded any positive outcomes.However, Azor et al. 66 investigated the suitability of periwinkle shells as carburizing materials for the surface hardness improvement of low-carbon steel.Shell waste has been successfully tested as a suppressant of specific toxic metals present in wastewater and other industrial effluents [67][68][69][70] .Oyster shell powder can supplement the sawdust medium to increase the calcium content of mushrooms.The mean content of phytochemicals in the exoskeleton samples is depicted in Table 5. T. fuscatus had the highest trypsin inhibitor content at 23.30 ± 4.50 mg/kg, while P. aurita had the highest carotenoid content at 18.00 ± 5.27 mg/kg (Table 5).Carotenoids are phytochemicals naturally occurring in plants, vegetables, fruits, and seeds.Some carotenoids are also synthesized artificially for food coloring, such as b-carotene 71 .Carotenoids can be found in various plants, such as citrus fruits, soybean meal, and vegetables, including green leafy vegetables, spinach, pumpkins, and potatoes, among others 72 .Carotenoids in the human diet help lower chronic illnesses connected to coronary heart disease (CHD) and help prevent bronchial and uterine-related issues and prostate and colon cancers.Trypsin inhibitors are phytochemicals naturally found in legumes, soybeans, and other natural products.Trypsin inhibitors are anti-nutritional factors that inhibit the digestion of protein when consumed, resulting in growth deficiency in livestock 73 .No research has been conducted on the presence of trypsin inhibitors in the periwinkle shell in the Lagos Lagoon.Generally, animals, such as mollusc, do not synthesize carotenoids de novo as well as trypsin inhibitors.However, the presence of carotenoids and trypsin inhibitors in periwinkle samples in this study might be due to the presence of these phytochemicals in the food they ingested, which subsequently bioaccumulate in their bodies.Cohen et al. 74 affirmed that trypsin plays a major role in the decomposition of several proteins, specifically in humans, animals with only one chambered stomach, and young ruminants.Modification of metabolic processes might also be largely responsible for the presence of carotenoids and trypsin inhibitors in them [75][76][77] .Several studies have been conducted to investigate the presence of carotenoids in periwinkle shells 72 .One such study is the report of Withnall et al. 78 , on Raman spectra of carotenoids in natural products, where carotenoids were discovered in the shell of the periwinkle Littorina littorea.Ituen 79 mentioned that mollusc shells are used in livestock feeding.Hence, this study investigated the presence of trypsin inhibitors in periwinkle shells and showed that the shells of T. fuscatus and P. aurita possess a considerable mean percentage of trypsin inhibitors (Table 5).These results may render them less suitable for livestock feeding.T. corona has a lower mean value of 11.80%, which projects it as a suitable addition to livestock feed.Nonetheless, shells are known for their hardiness and compactness attributes; this means that shells can be partially utilized as a supplementary filler in the production of materials in the cement industry, such that it helps to enhance the ability of cement to moisten and aids the hardness and compactness of the cement during mixtures 80 .Kozminsky and Lezin 81 also researched the distribution of pigments in the gastropod Littorina obtusata from Kandalaksha Bay, the White Sea, and the results showed that varieties of carotene pigments were discovered in the shell.
The mineral composition in T. coronata shows a strong positive correlation between K and Mg and a strong negative correlation between the trypsin inhibitor and P (Table 8).The correlation matrixes between the mineral components of periwinkle exoskeletons are presented in Tables 6, 7, 8.A robust positive correlation was observed between CaCO 3 and P, and a strong negative correlation was observed between carotenoids and Mg (Table 6).The mean values of mineral contents of the periwinkle (Littorea littorina) evaluated were 11.83 mg/kg for Mg, 0.40 mg/kg for K, and 0.92 mg/kg for P.Although these values are low, they are significantly higher than the The mineral composition of P. aurita shows a strong positive correlation between P and K and a strong negative correlation between carotenoids and trypsin inhibitors (Table 7).
The mineral composition of T. coronata shows a strong positive correlation between K and Mg and a strong negative correlation between the trypsin inhibitor and P (Table 8).

Conclusion
This study is the first documented report that will serve as a reference point for future research on the proximate composition, mineral content, and phytochemical composition of periwinkles from the Lagos Lagoon complex and their potential for a sustainable circular economy.This present study uses three commercially essential gastropod shells, Tympanotonus fuscatus, Pachymelania aurita, and Thais coronata, in the Lagos Lagoon.The study results showed variations among species based on their proximate, mineral, and phytochemical components.For nutritional purposes, T. fuscatus was the best due to its high fat and protein content and low ash and crude fibre content.The low crude fibre and fat content obtained in all samples makes them suitable for processing complementary foods, especially for those who are hypertensive and whose systolic and diastolic blood pressures are above 160 mmHg and 95 mmHg, respectively, and those suffering from fat-related diseases.The mineral compositions (Ca and CaCO 3 ) of the three gastropods were high.This situation indicates that the Lagoon is in good shape for some mollusc in terms of food availability and abundance.They may have devised a method for dealing with and adapting to constant pollution in the environment.Periwinkle shells can be beneficial for various purposes, as they possess certain minerals that can be utilized in various fields.Sustainable incorporation of these shells in several applications after processing will help to enhance the usage of these essential, less expensive, and readily available materials while also promoting a circular bio-economy.Periwinkle shell applications will contribute to the valorization of the economy, create employment, and pave the way for waste management through a proper recycling system.However, an urgent examination of the Lagos Lagoon's environmental conditions is needed, as some of the samples in this study are not favorable in reference to their nutritional contents.

Figure 3 .
Figure 3. Variations in the proximate composition of Tympanotonus fuscatus, Pachymelania aurita and Thais coronata in April.

Figure 4 .
Figure 4. Variations in the proximate composition of Tympanotonus fuscatus, Pachymelania aurita and Thais coronata in May.

Figure 5 .
Figure 5. Variations in the Minerals of Tympanotonus fuscatus, Pachymelania aurita and Thais coronata in March.

Figure 6 .
Figure 6.Variations in the Minerals of Tympanotonus fuscatus, Pachymelania aurita and Thais coronata in April.

Figure 7 .
Figure 7. Variations in the Minerals of Tympanotonus fuscatus, Pachymelania aurita and Thais coronata in May.

Figure 8 .
Figure 8. Variations in the phytochemical contents of Tympanotonus fuscatus, Pachymelania aurita and Thais coronata in March.

Figure 9 .
Figure 9. Variations in the phytochemical contents of Tympanotonus fuscatus, Pachymelania aurita and Thais coronata in April.

Figure 10 .
Figure 10.Variations in the phytochemical contents of Tympanotonus fuscatus, Pachymelania aurita and Thais coronata in May.
0 : weight of empty crucible, W 1 : weight of crucible containing samples, W 2 : weight of crucible containing ashed content.

Table 2 .
Correlation coefficient of the proximate composition in T. fuscatus.C.Pro: crude protein, C.Fat: crude fat, C.Fibre: crude fibre, NFE: nitrogen free extract.

Table 5 .
Mean value of mineral composition (mg/kg) of the periwinkle samples.Values are given as the mean ± standard deviation of the samples analyzed in monthly triplicates using ANOVA.In each row, the mean with common superscripts is not significantly different.

Table 7 .
Correlationwww.nature.com/scientificreports/mean values registered for Pachymelania aurita.This finding indicates that the mean value for minerals derived in this study is relatively low compared to other periwinkle shells.