Tropomyosin micelles are the main components contributing to the white color of boiled shellfish soups
The materials used
manila clam (C. japonica), blood clams (Anadara bringonii), Asiatic clam (Meretrix lusoria), small Japanese neck (Ruditapes philippinarum), Japanese oysters (Crassostrea nippona), clam (mercenaria mercenaria), Japanese scallop (Mizuhopecten yessoensis), Mediterranean mussel (Mytilus galloprovincialis), banana shrimp (Fenneropenaeus merguiensis) and snow crab (Chionoecetes opilio) were obtained from a supermarket in Shimane Prefecture, Matsue City.
Cloudy liquid formation
To study the optimal extraction temperature, 100 g of the clam samples placed in 200 ml of water were heated at 30, 50, 60, 70, 80 and 90°C for 30 min. To study the optimal cooking time, 100 g of the clam samples placed in 200 ml of water were boiled for 0, 1, 2, 3, 4 and 5 min. To study the optimal number of clams needed to obtain the highest degree of cloudiness in the soup, 12.5, 25, 50, 100 and 166 g of clam samples were placed in 100 ml of boiling water for 3 min. . Treated samples were filtered using filter paper (Qualitative Filter Papers No. 2; Advantec, Tokyo, Japan) and centrifuged at 8000×g for 10 min at 4°C.
Processed samples were subjected to ultrafiltration using an Amicon Pro purification system with a cut-off of 100 kDa (Merck Millipore, Tokyo, Japan) during centrifugation at 8000×g for 90 min at 4°C.
Equal volumes of 1% ninhydrin solution (Fujifilm Wako Pure Chemical, Osaka, Japan) were added to the obtained treated samples, heated in a boiling water bath for 10 min and then allowed to cool to 25°C. Color formation was examined.
Precipitation of trichloroacetic acid (TCA)
Sample and 20% TCA were mixed in equal amounts, vortexed, placed on ice for 10 min and centrifuged at 10,000xg for 10 min at 4°C.
A laser pointer (635 nm, 200-LPP029; Sanwa Supply Inc., Okayama, Japan) was used to observe the Tyndall effect.
The boiled soup was stained using a negative staining method. Briefly, a drop of the soup was placed on a microgrid covered with a carbon support film and hydrophilized by ion sputtering. The microgrid was allowed to absorb the sample for 10 min; then a 2% uranyl acetate solution was added on the microgrid and excess solution was removed using filter paper. The microgrid was air-dried and imaged using a transmission electron microscope (EM-002B; TOPCON Co., Ltd., Tokyo, Japan) at an accelerating voltage of 80 kV.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and enzymatic proteolysis
Protein concentrations were measured using Bradford’s method, with bovine serum albumin (Fujifilm Wako Pure Chemical, Osaka, Japan) used as a standard. Proteins (5 µg) were separated on a 10% (w/w) polyacrylamide gel at a constant current of 25 mA/gel and stained for 60 min with Coomassie Brilliant Blue R-250 (Fujifilm Wako Pure Chemical) and an acetic acid solution for 60 min. Depending on the volume of the gel pieces, 2-5 µL of trypsin (50 ng/µL) (Promega Madison, WI, USA) was added for protein digestion at 37°C for 16 h.
Matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MALDI-TOF–MS/MS)
Trypsin digested samples were diluted 1:5 in 0.1% trifluoroacetic acid (TFA) dissolved in water. The reaction mixture was then loaded onto C18 pipette tips (ZipTip; Millipore, USA) and passed 10 times. The reaction mixture was then washed with 0.1% TFA in water, and the peptide fractions were eluted with 50% acetonitrile solution. The eluted fractions were then mixed with α-cyano-4-hydroxycinnamic acid (Fujifilm Wako Pure Chemical) in 60% (v/v) acetonitrile and 1% (v/v) TFA, and applied to a mass spectrometry (MS) target. plate. MS data was obtained using a 5800 MALDI-TOF/TOF Analyzer (ABSciex, Concord, Canada) according to the manufacturer’s instructions. A monoisotopic precursor for MS/MS was interpreted via automatic precursor selection using the DynamicExit algorithm (AB Sciex). MS/MS data were analyzed using ProteinPilot™ software (version 3.0) and the Paragon Protein Database (AB Sciex) search algorithm. Each MS/MS spectrum was searched using the database built by AB Sciex.
The ultraviolet-visible absorption spectrum was obtained in the 200-300 nm range using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Cleveland, OH, USA).
Element Estimation Using Inductively Coupled Plasma (ICP)-MS Analysis
The boiled soup was filtered through a 100 kDa ultrafiltration spin column and precipitated using TCA; the pellet was treated with nitric acid and then analyzed by ICP-MS. The concentration of S, Na, Mn, Ti, As, Fe, Ni, Cu, Li, Mg, Al, Si, P, K, Ca, Co, Zn, Rb and Cs was determined by ICP-MS (8800 Triple Quadrupole ICP-MS; Agilent, CA, USA), following the manufacturer’s instructions.
Analysis of fatty acids by gas chromatography-mass spectrometry (GC–MS)
The fatty acids in the soup were extracted using chloroform. All fatty acids were derivatized into volatile methyl esters using esterification kits (Nacalai Tesque Inc.), following the manufacturer’s instructions. Gas chromatography analysis of methylated fatty acids was performed on a Shimadzu QP2010 quadrupole gas-mass spectrometry (GC-MS) instrument equipped with a carbowax capillary column (30 m × 0.25 mm ID; 0.25 mm film thickness) (intercut DB5MS; Agilent Technologies, CA, USA).
Isolation of the gene encoding tropomyosin from C. japonica
Total RNA was extracted from C. japonica using ISOSPIN Plant RNA Kit (Nippon Gene, Tokyo, Japan) and digested with DNase I (NipponGene Co., Ltd., Tokyo, Japan) according to manufacturer’s instructions. A 3 μg aliquot of total RNA was used to synthesize single-stranded cDNA using ReverTra Ace qPCR RT Master Mix (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer’s instructions. For PCR amplification, the cDNA was denatured at 98°C for 3 min in the first cycle and then for 10 s in subsequent cycles. Primer annealing and template extension were performed at 55°C for 5 s and 72°C for 10 s, respectively. Thirty cycles of PCR were carried out using the following oligonucleotide primers: 5′-GCCATCAAGAAGAAGATGCAGGCCAATGG-3′ and 5′-CCAGCCAATTCAGCAAA-3′. PCR products were fractionated on a 1% (w/v) agarose gel and DNA fragments from agarose gel slices were purified using a DNA fragment purification kit. DNA (MagExtractor, Toyobo Co., Ltd.). Purified reverse transcription-polymerase chain reaction (RT-PCR) products were directly sequenced using the BigDye ver 3.1 Termination Kit (Applied Biosystems, Foster City, CA, USA).
Construction of recombinant tropomyosin in Escherichia coli
The C. japonica The tropomyosin gene was amplified by PCR using appropriate primers (5′-CATCATCATCATCATATGGATGCCATCAAGAAGAA-3′ and 5′-TATCTAGACTGCAGGTTAATACCCAGCCAATTCAG-3′). Amplicons were cloned into the pColdII expression vector to create pColdII–tropomyosin with a His-tag using In-fusion® Snap Assembly Master Mix (Takara Bio Inc., Shiga, Japan) according to manufacturer’s instructions. The construct was sequenced for confirmation and then transformed into E.coli BL21 (DE3) cells for His production6-tagged (N-terminal) C. japonica tropomyosin.
Purification of recombinant proteins
Transformants were grown at 37°C in Luria-Bertani medium containing ampicillin (50 µg/L) (Fujifilm Wako Pure Chemical) to OD600 was about 0.5. Then, the temperature was lowered to 15°C to induce cold shock promoters, and incubation was continued for 24 h. Cells were harvested by centrifugation (6000×g, 10 min, 4 °C) and suspended in 1 ml protein extraction reagent (Integral Co., Tokushima, Japan). A microliter of 1000 U/mL of recombinant DNase I (Takara Bio Inc. Shiga, Japan) and 0.2 mg/mL of lysozyme (Fujifilm Wako Pure Chemical) were added to each sample and incubated for 15 min at room temperature. Then, the samples were centrifuged (13,000×g20 min, 4 °C) and recombinant proteins were isolated using the His-Spin Protein Mini-Prep Kit (Zymo Research Co., Irvine, CA, USA), following the manufacturer’s instructions.
Elemental analysis of tropomyosin by ICP-MS
The boiled soup was purified using an Amicon Pro purification system with a cutoff of 100 kDa (Merck Millipore. MA, USA). Samples were collected after TCA precipitation. Then nitric acid was added to the pellet and the mixture was incubated at 100°C for 12 h. The degraded product was suspended in 0.1 N nitric acid solution and analyzed by ICP-MS (8800 Triple Quadrupole ICP-MS; Agilent, CA, USA).
Amino acid composition of all human proteins
Amino acid sequence data of all human proteins were downloaded from Uniprot (https://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/reference_proteomes/Eukaryota/UP000005640/UP000005640_9606. fasta.gz). Only canonical entries were used for the following analysis. Amino acid composition was calculated using the Perl script (https://www.nntp.perl.org/group/perl.beginners/2010/12/msg114941.html) with slight modification (Supplementary Data 1 ).