Nutritional Quality and Safety of Windowpane Oyster Placuna placenta from Samal, Bataan, Philippines ^†

Abstract

The windowpane oyster (Placuna placenta) is common in coastal areas of the Philippines, thriving in brackish waters. Its shells underpin the local craft industries. While its meat is edible, only small amounts are consumed locally, most going to waste. Utilization of this potential nutrient source is hindered by the lack of information concerning its organic and mineral content, the possible presence of heavy metal ions, and the risk of microbial pathogens. We report extensive analysis of the meat from Placuna placenta, harvested during three different seasons to account for potential variations. This comprises proximate analysis, mineral, antioxidant, and microbial analyses. While considerable seasonal variation was observed, the windowpane oyster was found to be a rich source of protein, fats, minerals, and carbohydrates, comparing well with the meats of other shellfish and land animals. Following pre-cooking (~90 °C, 25–30 min), the standard local method for food preparation, no viable E. coli or Salmonella sp. were detected. Mineral content was broadly similar to that reported in fish, although iron, zinc, and copper were more highly represented, nevertheless, heavy metals were below internationally acceptable levels, with the exception of one of three samples, which was slightly above the only current standard, FSANZ. Whether the arsenic was in the safer organic form, which is commonly the case for shellfish, or the more toxic inorganic form remains to be established. This and the variation of arsenic over time will need to be considered when developing food products. Overall, the meat of the windowpane oyster is a valuable food resource and its current (albeit low-level) use should lower any barriers to its acceptance, making it suitable for commercialization. The present data support its development for high-value food products in urban markets.

1. Introduction

Bivalves (Bivalvia) are a widespread class of aquatic molluscs (Phylum Mollusca), which ingest phytoplankton from tidal waters, are adapted to changing temperature and salinity and to wave action in the intertidal zone [1]. They provide valuable nutrition to the human diet, being sources of protein and low in calories and fat, yet exhibiting high vitamin and mineral content, comparing favorably in this regard with other meats. In common with other molluscal shellfish, bivalves are a rich source of vitamin B12, choline, selenium, iron, and zinc [2]. The nutritional value of some shellfish regarding omega-3 fatty acids, iron, selenium, and zinc is superior to that of beef, chicken, and pork [3].

The windowpane oyster (Placuna placenta) is a mollusc which thrives in brackish water up to depths of 100 m (reviewed, [4]). In the Philippines, it is known locally as ‘kapis’, ‘capiz’, ‘pios’, or ‘lamperong’ and is a major fishery product. The oyster is edible but is commercially important principally because of its translucent shell, from which the name windowpane oyster derives. This is a versatile material often used to fashion handcrafted items, providing products that are exported to the USA, Japan, and Europe [4]. The meat, which is currently an under-exploited by-product of the windowpane oyster industry and is often discarded, represents about 25% of its wet weight.

Several studies have been conducted to assess the nutritional value of oysters and other molluscs but very little information pertains to the nutritional benefits of Placuna placenta. In the Philippines, the Food Nutrition and Research Institute of the Department of Science and Technology included windowpane oyster in the Philippine Food Composition Table. Although proximate composition and other nutrient contents of this oyster have been determined [4], the information remains incomplete in terms of its nutritional quality. The minerals assessed only included calcium, phosphorus, iron, and sodium, and there is no information concerning the concentration of metal ions (which can be toxic in nervous tissue, kidneys, liver, and the endocrine system) nor the microbial quality of the windowpane oyster with which to evaluate its safety for human consumption. Moreover, a comprehensive fatty acid profile has not been determined. The oyster meat is a by-product consumed principally by the oyster-harvesting communities. However, it is not exploited commercially more widely and so is not fully integrated into the food system of the Philippines, although its current, albeit limited, use suggests that barriers to its wider acceptance would not be significant. Thus, it would be beneficial to undertake a thorough investigation of the biochemical characteristics of this bivalve to establish its processing potential and safety for human consumption. This is particularly relevant from an economic perspective, as the oyster harvesters reside in the bottom two income deciles [5] and uplifting their income would be highly beneficial. The purpose of this study, therefore, was to determine the nutritional quality of windowpane oyster meat to inform future decisions as to how the oyster could be better utilized as a raw material for the production of higher-value food products. In tandem, the effect of pre-cooking the meat on its nutritional content and safety properties, including the concentration of common heavy metals and persistence of pathogenic bacteria, was also investigated.

2. Materials and Methods

2.1. Sample Collection and Preparation

Fresh windowpane oyster meat was obtained from a shell-processing area in Samal, Bataan at three different times: September 2020, February 2021, and March 2022. The oysters were weighed and shucked. During shucking, the meat was strained to separate the slime and weighed. Afterwards, fresh oyster meat was pre-cooked before cooking to remove sliminess. Traditional methods of pre-cooking involve boiling the meat with salt, ginger, and vinegar and parboiling the meat with just salt and monosodium glutamate or with, salt, ginger, and vinegar [5]. The meat was packaged appropriately and immediately placed on ice to prevent spoilage during transport. The pre-cooked windowpane oyster meat was stored at −10 °C.

2.2. Proximate Analysis of Windowpane Oyster Meat

Windowpane oyster meat samples were subjected to proximate analysis to estimate the macronutrients in this bivalve.

2.2.1. Moisture Determination

Approximately 3 g of chopped material was placed in a tared crucible and oven dried at 105 °C until constant weights were obtained. The oven drying method was performed on three replicates to determine the moisture content of samples.

2.2.2. Ash Determination

Crucibles with the samples from moisture determination were placed in a muffle furnace. The samples were heated at 550–600 °C (2 h) and cooled (50 °C) before being placed in a desiccator. Samples were then dried (1 h) to ensure the absence of water and, following a cooling period (30 min), were weighed. The ash content of the samples was determined for three replicates.

2.2.3. Crude Fat Determination

Approximately 1 g of dried sample was wrapped in filter paper and placed in a thimble. Approximately 50 mL of petroleum ether was added into a tared aluminum cup. The thimble containing the sample and the aluminum cup with the solvent were arranged in a Soxtec Fat Analyzer (ST 243 Soxtec Solvent Extraction System, FOSS, Hilleroed, Denmark). The fat present in the sample was extracted (1 h), after which the aluminum cup containing the solvent and fat were placed on a hotplate and dried until all the solvents had been completely evaporated. The aluminum cup was then dried in an oven (102 °C, 30 min), allowed to cool to 50 °C, placed in a desiccator for 25 min, and then weighed. The crude fat content of the sample was calculated as the percent of oil in relation to the weight of the sample.

2.2.4. Crude Protein Determination

Crude protein content was determined for three replicates using a Kjeltec Autoanalyzer (UDK159, VELP Scientifica, Usmate, Italy) and following the manufacturer’s instructions. Approximately 0.3 g of sample was poured into a 250 mL Kjeltec tube and a Kjeldahl tablet (catalyst) and 5 mL of concentrated sulfuric acid were added. The sample was processed (300 °C, 1 h and 420 °C, 30 min) then allowed to cool until the color had turned from dark green to light blue. Distilled water (25 mL) was then added and the sample was then subjected to an automatic distillation and titration process in the Kjeltec Autoanalyzer. The crude protein content of the sample was computed as follows:

$$Crude protein content \left(\%\right)={\displaystyle \frac{\left(Vs-Vb\right) \times Nacid \times 0.014\times 6.25 \times 100}{Wt of sample}}$$

where:

Vs—Volume of acid used in the sample;

Vb—Volume of acid used in the blank;

Nacid—Concentration of sulfuric acid in normality.

2.2.5. Crude Fiber Determination

The crude fiber content of the windowpane oyster samples was determined using the Weende method [6]. Approximately 1.0 g of defatted sample was weighed, transferred to a fitted glass crucible, and placed into a Fibertec hot extraction unit. Then, 150 mL of pre-heated 1.25% (w/v) NaOH was added to each column and then boiled, filtered, and washed again. The crucible was weighed in an oven at 105 °C over 1 h. The crucible was further incinerated in the muffle furnace (330 °C, 2 h and 500 °C, 3 h). The crucible was allowed to cool, placed inside the desiccator, and weighed. Crude fiber was determined for three replicates.

2.2.6. Available Carbohydrates or Nitrogen-Free Extract Determination

The percentage of available carbohydrates or nitrogen-free extract was calculated by subtracting the sum of moisture, ash, crude fat, crude fiber, and crude protein from 100.

2.3. Chemical Analysis of Fresh Windowpane Oyster

The chemical properties of pre-cooked windowpane oyster meat, comprising antioxidant activity, total carotenoid content, and total phenolic content, were determined.

2.3.1. Extraction of Sample

Approximately 1 g of windowpane oyster sample was transferred into a 15 mL centrifuge tube and extracted at room temperature using 85% (v/v) aqueous methanol (10 mL). The mixture was shaken (12 h) and then centrifuged at 1107× g for 10 min. The supernatant was collected into another 15 mL centrifuge tube and stored at 4 °C for further analysis.

2.3.2. Antioxidant Activity (2,2′-Diphenyl-1-picrylhydrazyl (DPPH) Radical-Scavenging Activity)

DPPH radical-scavenging activity (DPPH-RSA) of the sample extract was evaluated based on the reported method of [7] with some modifications. Mixtures containing 0.5 mL of sample extract/standard and 5 mL freshly prepared 0.1 mM DPPH solution were incubated (1 h). After incubation, the absorbance of the final solution was read at 517 nm against a blank. The DPPH-RSA was calculated as mg Trolox equivalent/g sample using the following formula:

$$DPPH-RSA={\displaystyle \frac{abs 517 \mathrm{n}\mathrm{m}\left(sample-blank\right) \times vol\left(extract\right) \times 0.25029 \left(\mathrm{m}\mathrm{g}/\mathrm{\mu }\mathrm{m}\mathrm{o}\mathrm{l}\right)\times dilutionfactor}{slope \times weight of sample}}$$

where slope is the slope of a standard curve of Trolox.

2.3.3. Total Carotenoid Content

Total carotenoid content of the samples was determined using a successive solvent extraction method. First, 300 mg of sample was mixed with water (1 mL) in a 15 mL centrifuge tube and incubated (60 °C, 10 min). The supernatant was transferred into a new 15 mL centrifuge tube and acetone (2 mL) was added to the residue. The mixture was agitated using a vortex mixer, applied with short pulses of sonication (30 s), and centrifuged at 1107× g for 10 min. The supernatant was collected and 5 mL acetone was added to the residue, mixed using a vortex mixer, and sonicated (30 s). The supernatant was collected, and 1 mL petroleum ether and 1 mL ether were added, mixed, and centrifuged at 1107× g for 5 min. After the successive extraction, the upper yellow phase of the supernatant was transferred to 2 mL Eppendorf tubes. The sample extract was evaporated under controlled pressure using a speed vac apparatus. The dried sample was reconstituted with acetone (2 mL) and the absorbance read at 450 nm using an Agilent Cary 60 UV-Vis spectrophotometer (Agilent, Muntinlupa City, 1770 Metro Manila, Philippines).

2.3.4. Total Phenolic Content (TPC)

The total phenolic content (TPC) of the sample was measured using the Folin–Ciocalteu method [8]. Approximately 500 µL of sample extract was transferred into a test tube and mixed with freshly prepared Folin–Ciocalteu reagent (1:10 dilution, 2.5 mL). After 15 min of incubation, 2 mL of 7.5% (w/v) sodium carbonate was added to the mixture. It was then allowed to stand for 1 h for maximum color development. The absorbance of the resulting blue color was measured at 760 nm against a blank. Gallic acid (GA) was used as the standard and TPC was expressed as mg GA equivalent per gram of sample. The TPC was calculated as follows:

TPC (mg GAE/g) = abs (blank-b) × vol (extract) × dilution factor slope × weight of sample

2.3.5. Heavy Metals and Mineral Analysis

Determination of heavy metals and minerals in pre-cooked windowpane oyster meat was conducted using the Official Methods of Analysis of the Association of Official Agricultural Chemists (AOAC) International 21st edition. The levels of calcium, magnesium, iron, copper, and zinc and the heavy metals, lead and cadmium were determined by flame atomic absorption spectrophotometry. Potassium was assessed by flame atomic emission spectrophotometry, phosphorus and arsenic by colorimetry, and mercury by cold vapor atomic absorption spectrophotometry [9].

2.3.6. Fatty Acid Profile

The fatty acid profile of pre-cooked windowpane oyster meat was analyzed using Official Methods of Analysis of the AOAC International 21st edition, specifically by gas chromatography. The boron trifluoride method was used in the preparation of methyl esters of the fatty acids in fats [10].

2.4. Microbial Analysis of Windowpane Oyster

2.4.1. Aerobic Plate Count

Windowpane oyster samples were blended using a disinfected blender. Serial dilutions of 10⁻¹ to 10⁻⁴ were prepared. Then a 1 or 0.1 mL inoculum was aseptically obtained using a pipette and transferred into sterile Petri dishes in duplicate. Plate count agar was poured onto sterile plates. The agar and the inoculum were mixed thoroughly by carefully rotating and tilting the dishes. After the agar solidified, plates were inverted and incubated for 2–5 days at 32–35 °C. Growth on the plates was examined and colonies were counted on plates with 25 to 250 colonies to allow the colony forming units (cfu) to be calculated.

2.4.2. Detection of Coliform

Pre-cooked windowpane oyster samples and brackish water samples from Samal, Bataan were subjected to microbial analysis to detect coliform by the most probable number (MPN) technique using brilliant green lactose bile broth. Samples were homogenized or mixed using a disinfected blender. Serial dilutions of 10⁻¹ to 10⁻⁴ were prepared. Three tubes of brilliant green lactose bile broth were inoculated with each prepared dilution, together with 3 uninoculated controls. Tubes were incubated for 48 h at 37 °C before observation.

The number of positive tubes at each dilution were determined. Tubes were considered positive if there was gas formation. MPN values were determined and multiplied by the reciprocal of the mid-dilution and reported as ‘coliforms MPN per gram’.

2.5. Detection of Salmonella

Pre-cooked windowpane oyster samples were blended using a disinfected blender and mixed with 99 mL peptone water to obtain a dilution of 10⁻¹. A loopful of the dilution was streaked on Salmonella–Shigella agar plates. The plates were incubated at 37 °C for 48 h before observation on Salmonella–Shigella agar plates. Salmonella colonies appear colorless to pale pink and may be transparent to translucent. Some Salmonella strains produce black-centered colonies.

2.6. Detection of Vibrio parahaemolyticus

2.6.1. Enrichment

Eleven grams of pre-cooked windowpane oyster sample were weighed and chopped. The sample was mixed thoroughly with 99 mL of peptone water and incubated for 4 h.

2.6.2. Isolation

A loopful of dilution was streaked onto thiosulfate–citrate–bile salts–sucrose (TCBS) agar. TCBS agar plates were incubated overnight at 35 °C. V. parahaemolyticus appears as round, green or bluish colonies, 2–3 mm in diameter.

2.7. Statistical Analysis of Data

Statistical analysis of the results of determination of proximate composition, total carotenoids, total phenolics, and antioxidant activity were performed using a t-test to compare if fresh and pre-cooked windowpane oyster meat samples were significantly different.

Means were calculated after assays for minerals, heavy metals, and fatty acid profile. The mean values obtained were not compared because of the importance of documenting the effect of season and other factors.

3. Results

3.1. Sample Collection

As noted, there are some data in the Philippine Food Composition Table and other sources concerning windowpane oyster meat, but this is not nearly as comprehensive as for other edible molluscs. Moreover, there may be seasonal influences on its proximate composition, other nutrient content, as well as contamination by microorganisms and heavy metals. Consequently, samples were collected on three different occasions, covering three major seasons in the Philippines (

Table 1. Information on collection dates of windowpane oysters.

Sampling Date	September 2020	February 2021	March 2022	Reference
Estimated monthly sea surface temperature range of Manila Bay, °C	30.8–31.2	27.6–27.9	28.6–28.5	[11]
Average weather (from international forecast of weather in Manila, Philippines and nearby provinces)	13 days precipitation, light to heavy intensity rain, 16 days cloudy, 1 day sunny	13 days precipitation, light drizzle, 12 days cloudy, 3 days sunny	15 days precipitation, light rain and thunderstorm to moderate rain, 11 days cloudy, 5 days sunny	[12]
Season	Rainy/wet	Cool, dry	Hot, dry	[13]
Rice production activity in Central Luzon	Planting to growing	Growing to harvesting	Growing to harvesting	[14]
Microbial quality of brackish water at the time of collection (coliform most probable number/g)	7.5 × 103	2.8 × 103	4.3 × 103

), to provide some insight into seasonal and time-dependent variation. Rice production activity was also recorded, since run-off from farms might affect the contents of the oysters. The hypotheses that pre-cooking the meat, a common local practice, may influence the nutritional value as well as the presence of common pathogenic microorganisms, were tested.

3.2. Proximate Analysis of Windowpane Oyster Meat

Proximate analysis refers to the quantitative methods of determining macromolecules in both foods and feeds. The procedures of proximate analysis estimate moisture, ash or mineral components, lipids as ether extracts, protein content, the available carbohydrate, and fiber or indigestible components.

Can the Meat from Oysters Provide Nutrition Comparable to Alternative Sources?

The proximate composition of fresh and pre-cooked windowpane oyster meat from Samal, Bataan, Philippines was determined using AOAC procedures. After analysis, it was found that moisture content significantly decreased from 69% (fresh oysters) to 3.9% after pre-cooking. This is primarily the result of evaporation of moisture during pre-cooking and the removal of most of the remaining water by the oven drying step prior to proximate analysis. Proximate analysis of fresh windowpane oyster can determine whether there are significant differences in the content of fresh and pre-cooked meat and also whether the content differs significantly from other sources. The analysis revealed that the bivalve contains 3.3% ash, 1.5% crude fat, 23.2% crude protein, 0.2% crude fiber, and 2.8% available carbohydrates (

Table 2. Proximate Composition of Windowpane Oyster Meat (g/100 g). Values are per dry weight (Section 2.2.2) as mean ± SD of three samples. Estimates of minerals as ash, fat, protein, fiber, and available carbohydrates are per 100 g dried sample. Data are the mean ±SD of three samples. The FCT data are from the Philippine Composition Tables 2019 [15]. The respective values for moisture content, ash, crude fat, crude protein, crude fiber, and available carbohydrates of fresh and pre-cooked meat were compared and were all significantly different (p ≤ 0.05; t-test).

Sample	Moisture Content	Ash	Crude Fat	Crude Protein	Crude Fiber	Available Carbohydrates
Fresh	69 ± 0.54	3.3 ± 1.65	1.5 ± 0.14	23.2 ± 0.36	0.2 ± 0.04	2.8 ± 2.0
Pre-cooked	3.9 ± 0.86	8.4 ± 3.53	8.2 ± 0.2	48.8 ± 0.33	6.5 ± 0.09	24.2 ± 4.23
Fresh [15]	70.2	1.8	1.4	23.3	0	3.3

). These values are generally similar to those in the Philippine Food Composition Table ([15]; summarized in Table 2). However, there are some notable differences. In the samples analyzed here, the content of ash and fiber was higher (Table 2). The source of these differences in proximate composition is not certain but they could be related to the location of the oysters, the season in which the oysters were collected, and their diet.

These findings confirm that windowpane oyster is a good source of low-cost protein, fats, minerals, and even carbohydrates in the form of glycogen. Compared with the oyster Crassostrea madrasensis, windowpane oyster has higher protein, lower fat, higher ash, and similar amounts of carbohydrates. Windowpane oyster also has a higher protein content than clams, mussels, oysters, and scallops [15]. However, it has a lower protein content than M. galloprovincialisis (44.27%) and V. verucosa (70.62%) [16], whereas, compared to the European flat oyster or to the mud oyster Ostrea edulis (protein content of 6.85 to 10.30 g/100 g, fat content of 1.47 to 5.51 g/100 g, and ash 3.42 to 9.45 g/100 g, all per wet weight) [17], the protein content is higher.

Molluscs contain moderate amounts of carbohydrates in their tissue, mainly in the form of glycogen, though small amounts of free sugars are always present as well [18]. While most molluscs are considered to be low in carbohydrates, some have slightly higher levels than observed in the windowpane oyster, e.g., raw scallops, oysters, and mussels contain carbohydrates ranging between 3% and 5% g/100 g dry weight [3]. The Pacific oyster (Crassostrea gigas) has high glycogen levels which contributes to its flavor, quality, and tolerance to low temperatures. The levels of carbohydrates could be related to the maturity of the oysters gathered; when bivalves reach full maturity, they typically accumulate large amounts of carbohydrates during the growing season and deplete these stores throughout the rest of the year [18]. The pre-cooked windowpane oyster meat was expected to have increased ash, crude fat, crude protein, crude fiber, and available carbohydrate content due to the loss of moisture through cooking and then drying. As noted, the value calculated on the basis of dry meat provides a more universal means of comparing these oysters to other molluscs.

3.3. Antioxidant Activity, Total Carotenoids, and Total Phenolics Content of Windowpane Oyster Meat

The DPPH test was used to assess antioxidant activity because of its speed, low cost, and sensitivity [19]. We tested the hypothesis that there might be a difference in these properties between uncooked and cooked oyster meat but no significant difference in the content of antioxidants, carotenoids, or phenolics was observed between fresh and pre-cooked windowpane oyster meat (

Table 3. Antioxidant Activity, Total Carotenoids, and Total Phenolics Content of Windowpane Oyster Meat. Values are per dry weight as mean ± SD of three samples. There was no significant difference between fresh and pre-cooked samples (p > 0.05, t-test).

Sample	Antioxidant Activity (mg Trolox Equivalent)	Total Carotenoids (µg/g)	Total Phenolics (mg GAE/g)
Fresh	7.9 ± 0.48	57.6 ± 0.61	0.85 ± 0.07
Pre-cooked meat	7.3 ± 0.98	54.4 ± 5.82	0.79 ± 0.14

).

The results from the antioxidant assay showed that the extract of windowpane oyster can scavenge free radicals. Dietary antioxidants are important nutritionally and are attractive features of proposed foodstuffs, which will increase their marketability. Marine animals contain structurally diverse carotenoids. There are more than 250 structurally diverse carotenoids in nature such as allenic carotenoids and the acetylenic carotenoids of marine algae and animals, excluding neoxanthin and its derivatives. In animals, carotenoids are accumulated from food and may be modified through metabolic reactions [20]. Table 3 shows the total carotenoids in fresh (57.6 µg/g) and pre-cooked (54.4 µg/g) windowpane oysters. Based on the Philippine Food Composition Table [15] windowpane oyster has no beta carotene, however, does possess 95 µg of vitamin A, retinol activity equivalent. Bivalves obtain fucoxanthin, diatoxanthin, diadinoxanthin, and alloxanthin from dietary microalgae and these may then be modified by their metabolism. Information regarding carotenoids of freshwater or brackish water shellfish is sparse, although, recently, the freshwater clam Corbicula sandai and brackish water clam Corbicula japonica were found to contain corbiculaxanthin, corbiculaxanthin 3′-acetate, 6-epiheteroxanthin, and 7′,8′didehydrodeepoxyneoxanthin [20].

Total phenolics are less often reported in molluscs and marine organisms but, because many bivalves are filter feeders, it is possible that these organisms contain particular phenols and polyphenols. However, since very little is known about windowpane oyster metabolism, whether the phenolics are acquired through feeding or biosynthesis or both remains to be established. In this study, it was found that fresh and pre-cooked windowpane oyster contained 0.85 mg and 0.79 mg phenolic content as gallic acid equivalent/g, respectively. The levels of phenolics in the windowpane oyster may turn out to be modest. Anadara granosa, a bivalve species inhabiting the east coast of South Sumatra, is reported to contain over 10 mg GAE/g phenol [21].

3.4. Minerals and Heavy Metals Analysis

Shellfish are good sources of the minerals iodine, copper, zinc, sodium, potassium, selenium, and inorganic phosphate. In this study, mineral content of pre-cooked windowpane oyster meat was determined. It was found that pre-cooked windowpane oyster meat from the first sample (

Table 4. Minerals and heavy metals content of pre-cooked windowpane oysters. The values are mean ± SD of 3 technical replicates. The values below the limit of detection are reported as ‘less than.’

Minerals	First Sampling/µg/g	Second Sampling/µg/g	Third Sampling/µg/g
Ca	3212 ± 13	794 ± 8	2188 ± 6
Mg	1155 ± 37	234 ± 6	974 ± 6
K	3591 ± 58	122 ± 6	1955 ± 7
Fe	540 ± 19	29 ± 0.5	348 ± 7
P	1968 ± 10	27 ± 0.4	785 ± 6
Cu	29 ± 1.3	Less than 0.05	16 ± 0.3
Zn	226 ± 7.77	17 ± 0.67	155 ± 7.02
Heavy metals
As	Less than 0.25	2 ± 0.07	Less than 0.25
Hg	Less than 0.005	Less than 0.005	Less than 0.005
Cd	Less than 0.03	Less than 0.025	Less than 0.025
Pb	Less than 0.1	Less than 0.1	Less than 0.1

) had 3212 µg/g Ca, 1155 µg/g Mg, 3591 µg/g K, 540 µg/g Fe, 1968 µg/g P, 29 µg/g Cu, and 226 µg/g Zn. The values, however, dropped markedly in the second sample but increased in the third sample. In the Philippine Food Composition Tables 2019 [15], windowpane oyster contains calcium (110 mg), phosphorus (257 mg), iron (17.3 mg), and sodium (467 mg) (values per 100 g meat). The amounts of calcium and phosphorus measured in the first and third samples are much higher than those in the Philippine Food Composition Table, however, they are lower in the second sample. Conversely, the amounts of iron in the three windowpane oyster meat samples were all higher than the Philippine Food Composition Table values. These differences are likely to reflect external factors which may include seasonal factors, including rainfall, and their effect on food supply and mineral run-off from land; the latter perhaps related in part to agricultural practices (Table 1). These data suggest that a more refined time-resolved set of samples would be valuable, as there may be intrinsic differences in oysters sourced from different locations or between oyster beds or differences according to season and perhaps meteorological conditions.

Accumulation of minerals and heavy metals in windowpane oysters is often considered to arise from their filter feeding. Thus, while the consumption of shellfish is generally safe, risks may arise from their exposure to diverse environments and filter feeding, coupled with poor methods of farming and handling [22]. The most prevalent minerals in the oyster meat, calcium, potassium, phosphorus, and magnesium (in that order—taking sums across Table 4), were also those most prevalent in the muscles and livers of two species of fish, M. cephalus and O. regia, sampled off the coast of Peru [23] and, furthermore, some of the values found in the fish samples exceeded those from the oyster meat. It was notable that, of the less prevalent metals, Fe, Zn, and Cu, which also occurred in broadly comparable order in the oyster and the fish samples, were present at considerably higher levels in the oyster meat (comparing Table 4 above with the data in Table 1 of [23]). Although each of these is essential in the diet, zinc is the best tolerated and copper the most toxic at higher levels. The tendency to accumulate these metals, which can cause broad toxicity [24,25], in bivalves compared to fish seems to be borne out by this comparison, albeit limited in scope. However, this propensity to accumulate heavy metals also make bivalves a useful tool for monitoring the concentration of heavy metals in coastal areas due to their tolerance of these contaminants, immobility, and widespread distribution [26].

There was some variation in the heavy metal content across the three samples (Table 4). However, the values are generally all below the maximum limit set by the Food Standards Agency, UK [27], FSANZ [28], and FAOLEX [29] (

Table 5. Maximum permitted level of heavy metal contaminants in bivalve molluscs of different food standard agencies, µg/g.

Organization	As	Hg	Cd	Pb
FSANZ	1	1.5	2	2
Food Standards Agency, UK	-	0.5	1.0	1.5
FAOLEX	-	0.5	1.0	1.0

). The one exception was the As content, which is only present in the FSANZ standard. Inorganic As is considered much more toxic than its organic form which is the form usually associated with seafood [30], which may be why many food standards do not consider As, e.g., FAOLEX and Food Standards Agency, UK (Table 5). However, which of these is responsible for the As levels measured is currently unknown. Oysters in the first and third samples were at acceptable or safe levels based on FSANZ [28], but the second sample had higher levels of As which were above the acceptable limit set by this agency (Table 5). These data suggest that there is time-dependent variation in As, which will need to be considered when preparing food products. This is supported by trends in the levels of Hg, Cd, and Pb. Though these were within safe levels in all samples, they were highest in the second sample. These differences are likely to reflect the seasons in which the samples were collected and the possibility for a greater input of mud or sand contaminated with these heavy metals, either from sea currents during downwelling or from rainwater run-off. However, the former seems less likely, and there is no evidence for major seasonal differences in sea currents in this area, particularly since it is a large bay. Moreover, there may also be regional variations, arising from the differential leaching of As-containing compounds from minerals. These factors are currently unknown, but their analysis would have an important positive impact on the entire fishing industry, relating both to capture and to aquaculture in the Philippines.

The influence of the season and, potentially, also the time of gathering may provide a possible explanation for the observed variation of windowpane oyster mineral and heavy metal content. Fluctuations in the concentrations of all heavy metals such as Cu, Pb, Fe, Cr, and Ni were observed in Donax faba (a saltwater clam) on the southwest coast of the state of Karnataka in India and are attributed to differences in fresh water run-off [31]. Some of the seasonal differences in metal accumulation may be related to food supply. It has been suggested that variations in metal accumulation may be also be associated with seasonal variations in flesh weight during gonadic tissue development [32].

Rice farming in adjacent provinces can also contribute to differences in mineral and heavy metal composition both of coastal waters and of the aquatic organisms therein. Possibly, during the dry season in the Philippines there is an accumulation of heavy metals from rice fields, while leaching of the metals can be expected during the rainy season. It can also be assumed that build-up of heavy metals and other pollutants in aquatic organisms is caused by farming practices. There has been a very substantial increase in the use of chemical fertilizer to increase the yield of rice and other crops in the Philippines, however, typically, the harvest has only doubled or tripled depending on the commodity. This suggests that excessive use of fertilizers and poor management of their application have resulted in a significant loss of fertilizers into the water systems, which may have contributed to the high phosphate content observed in all three samples (Table 4) [33].

3.5. Fatty Acid Content of Windowpane Oyster

Pooling different classes of lipids and fatty acids (

Table 6. Fatty Acid Profile of Pre-cooked Windowpane Oyster. The values are mean ± SD of 3 technical replicates.

Fatty Acids	First Sampling g/100 g	Second Sampling g/100 g	Third Sampling g/100 g
Saturated:
Myristic acid	0.87 ± 0.02	0.37 ± 0.013	0.77 ± 0.005
Pentadecanoic acid	0.10 ± 0.008	0.06 ± 0.002	0.09 ± 0.006
Palmitic acid	4.48 ± 0.201	3.82 ± 0.030	4.25 ± 0.031
Heptadecanoic acid	0.30 ± 0.005	0.28 ± 0.005	0.33 ± 0.009
Stearic acid	0.93 ± 0.017	1.03 ± 0.007	0.95 ± 0.008
Arachidic acid	0.02 ± 0.002	0.03 ± 0.003	0.03 ± 0.006
Heneicosanoic acid	0.05 ± 0.003	0.05 ± 0.003	0.05 ± 0.024
Behenic acid	0.02 ± 0.003	0.04 ± 0.004	0.03 ± 0.004
Lignoceric acid	0.04 ± 0.002	0.02 ± 0.004	0.04 ± 0.011
Total	6.81	5.7	6.54
Monounsaturated:
Palmitoleic acid	1.39 ± 0.019	0.81 ± 0.015	1.37 ± 0.015
Elaidic acid	0.02 ± 0.003	0.04 ± 0.003	0.02 ± 0.004
Oleic acid	0.14 ± 0.002	0.13 ± 0.003	0.13 ± 0.006
Nervonic acid	0.11 ± 0.004	0.28 ± 0.003	0.15 ± 0.005
Total	1.66	1.26	1.67
Polyunsaturated:
Linoleic	0.10 ± 0.01	0.18 ± 0.006	0.12 ± 0.005
γ-linolenic	0.10 ± 0.006	0.16 ± 0.004	0.16 ± 0.008
Linolenic	0.50 ± 0.01	0.65 ± 0.004	0.57 ± 0.009
cis-8, 11, 14 Eicosatrienoic acid	0.44 ± 0.003	0.71 ± 0.006	0.53 ± 0.013
cis-11, 14, 17 Eicosatrienoic acid	0.10 ± 0.003	0.04 ± 0.004	0.08 ± 0.006
Arachidonic acid	1.20 ± 0.008	1.19 ± 0.016	1.20 ± 0.006
cis-5, 8, 11, 14, 17 Eicosapentanoic acid	0.10 ± 0.002	0	0.08 ± 0.003
cis-4, 7, 10, 13, 16, 19 Docosahexanoic acid	0.04 ± 0.01	0.69 ± 0.025	0.28 ± 0.006
Total	2.58	3.62	3.02

) indicated that there was some variation between the three samples, but it was considerably less than observed for minerals and the contaminating heavy metals (Table 4). Previous work [15] established values per 100 g wet weight, so they are not directly comparable to those in Table 6. However, since the crude fat content obtained for fresh windowpane oyster (Table 2) is similar to that in the Food Composition Tables 2019 [15], it can be concluded that windowpane oyster meat is a good source of important nutrients. When compared with bivalves commonly consumed in the Philippines such as Magallana bilineata (cupped or slipper oyster), Crassostrea sp. (Pacific oyster), and Perna viridis (green mussel), windowpane oyster possesses less saturated fatty acid and similar amounts of monounsaturated and polyunsaturated fatty acids, except for Perna viridis which has exceptionally high levels both of saturated and unsaturated fatty acids because green mussels are commercially cultured/farmed in the Philippines.

Although marine molluscs are generally characterized by the majority of essential n-3 polyunsaturated fatty acids, mostly cis-5, 8, 11, 14, 17 eicosapentanoic acid and cis-4, 7, 10, 13, 16, 19 docosahexanoic acid, which usually constitute nearly 50% of all fatty acids [34], windowpane oysters contain more saturated fatty acids. Interestingly, a high proportion of saturated fatty acids, such as arachidic acid, was associated with bivalves growing in habitats rich in organic material and high bacterial loads, in contrast to those mainly subsisting on marine phytoplankton, which primarily consist of (n-3) polyunsaturated fatty acids of 18, 20, and 22 carbons [35].

All fatty acids found in dried windowpane oyster in the first sample were also in the second and third samples except for cis-5, 8, 11, 14, 17 eicosapentanoic acid, which was absent in the second sample (Table 6). Of the saturated fatty acids, palmitic acid was abundant, while pentadecanoic, heptadecanoic, stearic, arachidic, heneicosanoic, behenic, and lignoceric acids were present at almost the same levels in all samples. However, in the case of myristic and palmitic acid, the values obtained in the second sample were distinct. In the case of monounsaturated fatty elaidic and oleic acids, values obtained in the three sampling periods were close to each other, whereas palmitoleic and nervonic acids differed in the second sample, being decreased and increased, respectively. Palmitoleic acid is the most abundant monounsaturated fatty acid in windowpane oyster.

The levels of polyunsaturated linoleic, linolenic, ᵧ-linolenic, arachidonic, and cis-11, 14, 17 eicosatrienoic fatty acids were similar in the three samples (Table 6). However, those for cis-8, 11, 14 eicosatrienoic acid, cis-5, 8, 11, 14, 17 eicosapentanoic acid, and cis-4, 7, 10, 13, 16, 19 docosahexanoic acid differed between samples 1 and 3 and sample 2 (Table 6). Diets of bivalves, which are mostly suspension feeders, consist mainly of bacteria from detritus, zooplankton, heterotrophic flagellates and ciliates, planktonic and benthic microalgae, as well as protozoans. As noted above, this feeding mode and the distribution of dietary constituents have the greatest effect on the composition of the bivalve fatty acids, which are rich in docosahexanoic acid, eicosapentanoic acid, and frequently arachidonic acid [34]. Modest amounts of polyunsaturated linolenic, eicosapentanoic, and docosahexanoic fatty acids among others were present in the windowpane oyster samples analyzed, while arachidonic acid prevailed.

The variation observed in the fatty acid content of pre-cooked oyster could be attributed to the environmental conditions in which the windowpane oysters grow and their diet. In Callista umbonella and other molluscs, such seasonal variations in terms of fatty acid content have been documented [36], with palmitic acid being the major fatty acid, and a relatively low level of polyunsaturated fatty acids was evident.

3.6. Microbial Analysis of Windowpane Oyster

Microbial analysis is necessary to assure consumers that foods and feeds will not be adversely affected by the presence of biological pathogens or disease-causing and toxin-producing microorganisms. In particular, molluscs and other shellfish can accumulate pathogenic bacteria and viruses when grown in contaminated water and can represent a public health risk when consumed raw or only mildly heated. Indeed, analysis of the water demonstrated the presence of coliform bacteria (Table 1). Pre-cooking is a standard procedure by the oyster harvesters of existing food, and potential new products would be based on such material [4]. To assess the safety of pre-cooked windowpane oyster as potential material for the development of future food products and to determine the appropriate preservation technology to be applied to guarantee microbial safety, aerobic plate count, determination of coliform, detection of Vibrio parahaemolyticus, and detection of Salmonella sp. were undertaken.

Aerobic plate counts estimate the number of microorganisms in food by pour plating or spread plating in appropriate agar. After microbial analysis of the pre-cooked sample, it was found that the sample had an aerobic plate count of 3.7 × 10² cfu (

Table 7. Microbial properties of pre-cooked windowpane oyster meat. MPN: most probable number, VRBA: violet red blue agar.

Sample	Aerobic Plate Count	Coliform MPN/g	Coliform Colonies in VRBA	Vibrio parahaemolyticus	Salmonella
Pre-cooked oyster	3.7 × 102	1.59 × 103	8 est. APC (pinkish)	No growth	No growth

). On the other hand, the coliform count using brilliant green lactose bile broth tubes was 1.59 × 10³ MPN/gram, but after plate counting in violet red bile agar plates, it was found that there were eight pinkish coliform colonies. It can be confirmed, however, that the plates were negative for Escherichia coli because there were no dark-red-centered colonies were evident. VRBA is a selective medium that contains bile salts and also crystal violet, which inhibits particular Gram-positive microorganisms, especially Staphylococci. Neutral red is used as a pH indicator, and it changes to red-purple in the event of the accumulation of acid during fermentation. Thus, coliforms fermenting lactose give red colonies surrounded by reddish precipitation of bile salts after 18–24 h at 35 °C under anaerobic conditions. Non-fermenters of lactose and late lactose fermenters produce pale colonies [37].

Additionally, no growth of Vibrio parahaemolyticus and Salmonella sp. was observed in the pre-cooked samples (Table 7). Although it is possible that these bacteria were present in fresh windowpane oyster, they would likely have been destroyed during pre-cooking because of the heat applied, the effect of which could have been augmented by the salt and vinegar added. Thus, this standard procedure for the initial preparation of the oyster meat provides a safe material for the development of food products.

4. Discussion

The harvesting of windowpane oysters in the Philippines currently focuses on the translucent shell. Only a small proportion of the meat is consumed locally and a large amount is either discarded as waste or used as food in aquaculture, a situation which contributes to the limited availability of data relating to windowpane oyster meat in the Philippine Food Composition Table (summarized in Table 1 and described in detail in [4]). The data from the present study provide additional information covering both nutritional and safety aspects of the meat. The analyses demonstrate that windowpane oyster meat is a useful source of protein, as well as fatty acids and antioxidants, with both carotenoids and phenolics contributing to the latter (Table 2, Table 3 and Table 6). Importantly, while the meat is a good source of minerals, it was not found to be contaminated significantly with heavy metals (Table 4). The collection of samples over three seasons (Table 1) identified that the nutritional value of the meat varied considerably; the February 2021 sample stood out as the most distinct. Indeed, it was also in the latter sample that As levels were somewhat above those set by the FSANZ agency. Given that the heavy metals were all well below the most stringent standards (Table 4 and Table 5), the higher value of arsenic in this sample may be particular to the local environment of Manila Bay and/or the season.

In a study of two fish species, M. cephalus and O. regia, off the Peruvian coast [23], high levels of As in some locations were also observed but it is not known, either in this earlier study or in the present work, whether the As was present in the more toxic inorganic form, most likely arising from contaminated water (suggestive of geological origin or human activity), or the less toxic organic form often found in seafood, which is deemed by the WHO to pose less of a health risk [30]. Identifying which form of As—inorganic or organic—is present, collecting further time-dependent data, and, for example, analyzing the major sources of local run-off to identify potential sources of this increased As content will clearly be important. These studies will, in turn, inform future assessments of their significance to the safety of oyster meat and also any potential mitigation strategies.

The nutritional content of the windowpane oyster compares favorably with that of other local shellfish, such as the Philippine slipper oyster, Magallana bilineata [38], which is an important and accepted seafood, and complements existing land-based protein sources. There is potential for introducing windowpane oyster meat into the food system of the Philippines and indeed elsewhere. Such a move would enhance food security, improve nutrition, as well as benefit the economic prospects of those associated with the windowpane oyster industry. Further monitoring of the nutritional content is desirable to enable an assessment of its long-term economic and nutritional viability to be made. In the present study, beyond noting the prevailing conditions (Table 1), no attempt was made to correlate the availability or variability of nutrients or toxins in the area of harvest.

The windowpane oyster beds in the Philippines are in currently decline, and the introduction of the meat into the national food system could provide an important impetus to their improved management, including the enforcement of existing laws regarding harvesting [4].

Pertaining to future commercial development, there are existing dishes prepared locally by some oyster-harvesting families, such as adobo, a marinara style sauce, and a powdered seasoning derived from fermented oyster meat, ‘sisi,’ which can be incorporated readily into prepared foods that are suitable for the urban market. These existing uses of the meat suggest that products based on oyster meat would face a relatively low barrier for acceptance. Indeed, pilot production of three such products has been undertaken and their acceptability to two focus groups was very positive [5]. A thorough study of the commercial and technical feasibility for developing and marketing new products from the oyster meat should now be undertaken. Such developments would support the necessary extension of the present data by undertaking similar analyses at the locations of other major Philippine oyster beds at different times of year as part of food safety standards.