2.1. Gracilaria sp. biomass production and harvesting
Gracilaria sp. biomass was grown in a 700 L polyvinylchloride (PVC) tank with running seawater and continuous aeration at Israel Oceanographic and Limnological Research (IOLR), Haifa, Israel, in 2021. Twice a week, 0.1 mM PO4 and 1.0 mM NH4 were added to the seaweed tank. The biomass utilized as a stock was 1.5 kg FW m-3 and harvested after it grew to 3-4 kilogram FW m-3 in around 5 weeks. The harvested biomass was cleansed in a 20L tank filled with clean tap water. A batch centrifuge was used to remove surface water and the "fresh weight" (FW) biomass of Gracilaria sp. was obtained.
2.2 Description of the high voltage PEF system
The electrical circuit of the High Voltage Pulse Generator (HVPG) used in this investigation is shown in Figure 1a and was described in detail in our previous publications (Levkov et al. 2020; Prabhu et al. 2020; Prabhu et al. 2019). The sliding Press-Electrode Device (Figure 1b) was created especially for handling biomass while applying pressure during PEF-induced electroporation. To exert this pressure, one electrode is allowed to move freely and vertically while being driven by the weight placed on the load-receiving platform.
The sliding Press-Electrode Device consists of an Electroporation Cell (EPC), an Electroporation Cell Retainer (EPCR), a load-receiving platform, a node of the moving electrode, a holder, and a sensor for detecting the inter-electrode gap. Guides at the base of the holder ensure precise alignment of the installed EPCR. The EPC (Figure 1c), which consists of a cylinder with one electrode at the bottom and another at the lower end of the sliding electrode assembly's rod, acts as the working volume for electroporation and pressing. The liquid fraction can exit the cylinder through slit-like apertures in the side walls, and a ring-shaped groove renders it convenient to drain the liquid component into a storage container. The high-voltage input of the HVPG is attached to the operational electrode of the EPC via a male connector.
2.3 Experimental design
Using a spinner, 20 g of FW Gracilaria sp. biomass was dewatered until less than 1 g of surface water remained. The PEF apparatus described above was then used to treat 20 g of FW biomass in three replicates using the following parameters: frequency=3 Hz; pulse duration=50 µs; number of pulses=200; pulse voltage=1000 V; inter-electrode gap was varied in the range of 3.9 to 4.4 mm; the 20 g of biomass was treated with 10 PEF cycles by feeding 2 g/cycle (step 1 in Figure 2). The PEF treatment steps were not used in the control group.
Following PEF, the treated biomass was suspended in 100 mL of distilled water, agitated at 32°C and 150 RPM overnight in a shaking incubator (Benchmark Scientific, Incu-shaker small, USA), and then separated with a batch centrifuge (Yingtai Instruments, TGL 18, China) at 3750 g for 10 min (steps 2 and 3 in Figure 2). The biomass (solids 3b, Figure 2), was further treated with different pH treatments: 1) sequential pH 12 to 1 extraction, 2) sequential pH 1 to 12 extraction (Table 1, steps 4 and 5 in Figure 2). The compositions of buffers used appear in Table 2. To establish the total solutes (dry matter) released and protein released, all the liquid fractions from the sequential extractions were combined. The residual biomass was dried in an oven at 40°C (step 6, Figure 2).
Table 1. Protein extraction from Gracilaria sp. biomass using various process steps; process description based on the experimental design shown in Figure 2.
Process stage
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Equipment
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Process parameters
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Control (without PEF)
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1) Pressing
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5 kg weight, gravitational PEF device
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20 g of biomass was pressed with 10 cycles by feeding 2 g/cycle, no PEF applied.
|
2) Water-mediated extraction
|
Shaker incubator
|
Condition 1: 32°C, 150 RPM for 1 hr
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3 and 5) centrifugation
|
Batch centrifuge
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3750 xg for 10 min
|
4) pH treatment
|
Shaker incubator
|
Conditions 2 and 3: Sequential pH 1 to 12 and 12 to 1 treatments: 2 g FW biomass from step 3 (b), Figure 2 was treated with solutions at pH levels ranging from 1 to 12 (condition 2) and 12 to 1(condition 3).
The same biomass was transferred from one falcon tube to another falcon tube with 10 mL of solution of a different pH and incubated at 32°C and 150 RPM for 1 hr at each pH. Then it was centrifuged, and separated. The protein content of all liquid (supernatant) fractions was combined for each condition.
|
6) Drying
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Oven
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40°C
|
PEF treatment (with electric fields)
|
1) Pressing
|
5 kg weight, gravitational PEF device
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Fresh biomass (20 g) was treated with 10 PEF cycles by feeding 2 g/cycle. PEF parameters: Frequency=3 Hz, pulse length=50 µs, number of pulses=200, pulse voltage=1000 V, inter-electrode gap varied from 4.4 to 3.9 mm because of the biomass compression and liquid extraction.
|
2) Aqueous extraction
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Shaker incubator
|
Condition 4: 32°C, 150 RPM for 1 hr
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3 and 5) centrifugation
|
Batch centrifuge
|
3750 xg for 10 min
|
4) pH treatment
|
Shaker Incubator
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Conditions 5 and 6: Sequential pH 1 to 12 and 12 to 1: 2 g FW biomass from step 3 (b) was treated with solutions at pH levels ranging from 1 to 12 (condition 5), and from 12 to 1 (condition 6)
The same biomass was transferred from one falcon tube to another falcon tube with 10 mL of different pH solution and incubated at 32°C and 150 RPM for 1 hr in each pH. Then it was centrifuged, and separated. The protein content of all liquid (supernatant) fractions was combined for each condition.
|
6) Drying
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Oven
|
At 40°C
|
Table 2. Volumes and molar strengths of the salts, acids, and bases used for preparation of pH solution 1 to 12 used in the current study.
pH solution
|
Composition of the solution
|
Molar Concentration (M)
|
pH1
|
50 mL of 0.2 M KCl + 134 mL of 0.2 M HCl
|
3.68
|
pH2
|
50 mL of 0.2 M KCl + 13 mL of 0.2 M HCl
|
1.26
|
pH3
|
100 mL of 0.1 M C8H5KO4 + 44.6 mL of 0.1 M HCl
|
1.44
|
pH4
|
100 mL of 0.1 M C8H5KO4 + 0.2 mL of 0.1 M HCl
|
1.02
|
pH5
|
100 mL of 0.1 M C8H5KO4 + 45.2 mL of 0.1 M NaOH
|
1.45
|
pH6
|
100 mL of 0.1 M KH2PO4 + 11.2 mL of 0.1 M NaOH
|
1.11
|
pH7
|
100 mL of 0.1 M KH2PO4 + 58.2 mL of 0.1 M NaOH
|
1.58
|
pH8
|
100 mL of 0.1 M KH2PO4 + 93.4 mL of 0.1 M NaOH
|
1.93
|
pH9
|
100 mL of 0.1 M tris (hydroxymethyl) amino methane + 11.4 mL 0.1 M HCl
|
1.11
|
pH10
|
100 mL of 0.05 M NaHCO3 + 21.4 mL of 0.1 M NaOH
|
0.62
|
pH11
|
100 mL of 0.05 M NaHCO3 + 45.4 mL of 0.1 M NaOH
|
0.96
|
pH12
|
50 mL of 0.2 M KCl + 12 mL of 0.2 M NaOH
|
1.04
|
2.4 Protein quantification
The protein content in the initial biomass was quantified after cell wall and membrane disruption by bead-beating under alkaline conditions, and after drying in an oven at 40°C until reaching a constant weight. Gracilaria sp. dry biomass (15 mg) and 1.5 mL of 2 M NaOH were mixed. Zirconium beads (2mm, Sarstedt) were added to one-third of the bead-beater tubes containing a mixture of biomass and 2 M NaOH. The tubes containing the mixture were then put into a Mini Bead-Beater-16, Model 607 EUR, Biospec, OK, and subjected to three cycles of 60 seconds each, separated by 10-minute intervals, at 25°C. After bead beating, the tubes were centrifuged at 19,500 g for 20 min in an Eppendorf 5424 centrifuge (Germany), and protein was quantified in the supernatant (Prabhu et al. 2020).
The protein content of the various liquid fractions collected throughout the investigation was determined using Lowry's technique. To create the linear calibration curve, several dilutions corresponding to concentrations ranging from 0-500 µg/mL were prepared, using a bovine serum albumin (BSA) stock solution of 1 mg/mL (Lowry et al. 1951). To measure protein concentration, 100 µL of samples or standard (BSA, 0-500 g/mL) were mixed with 200 µL of Biuret reagent (500 µL of 1% cupric sulfate, 500 µL of 2% sodium potassium tartrate, in 50 mL of 2% sodium carbonate in 0.1 N NaOH). After thoroughly blending, the mixture was equilibrated at room temperature for 15 min. 20 µL of 1 N Folin and Ciocalteu's reagent (Sigma-Aldrich, St. Louis, USA) were added to the reaction mixture and it was incubated for 30 min at room temperature. After the incubation, absorbance at 650 nm was measured using a microplate reader (TECAN Infinite 200-Pro, Tecan, Switzerland).
2.5 Amino acid profiling
The protein was extracted from the PEF-treated samples and control from step 3a) and step 5a) (Figure 2) in aqueous solution and following sequential pH changes from 1 to 12 and from 12 to 1. The protein-rich sediment was precipitated using 35% and 85% ammonium sulfate salting out processes (Duong-Ly and Gabelli 2014). The precipitated proteins were then dialyzed using a SnakeSkin™ dialysis bag (3.5 kDa, ThermoFisher Scientific, USA). The dialyzed proteins were then lyophilized and analyzed for amino acid profile through high-pressure anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) as follows. One mg of biomass was added to 1 mL of 6 M HCl and heated at 112 °C for 16 h in a dry bath (Bio-Base, China). The samples were then dried using nitrogen. The dried samples were suspended in 1 mL of ultrapure ion chromatography grade water and left at rest for 1 h (Kazir et al. 2019). The samples were then filtered using a 0.22 μm syringe filter. The filtered samples were separated through HPAED-PAD Dionex ICS-5000 (Dionex, Thermo Fischer Scientific, MA, USA). Amino-pack 10 analytical grade column was used for amino acid separation. Signal detection was done using a gold AAA electrochemical detector with an AgCl reference electrode. Separation was achieved with gradient elution of the following parameters: 0.250 mL/min, column temperature 30°C, auto-sampler temperature 5°C, and a 25 μL injection loop volume. An amino acid standard mix (AAS18, Sigma-Aldrich, MO, USA) with the following dilutions 1/5, 1/10, 1/15, 1/20, and 1/25 was used to validate the separation program. The following amino acids were detected in the amino acid mix: alanine, arginine, aspartate, cysteine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine. The calibration curve formed from these dilutions (R2>99% for each amino acid) was then incorporated into the program to quantify the amino acids in the unknown samples.
2.6 Release yield and content calculations.
Solute (dry matter) release yield (DMRY %) was calculated according to Equation 1:
![](data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAq4AAABgCAYAAADcgvDrAAAaGUlEQVR4Ae2cDbXjxrJGh0IwhEI4BEIwhEIYhEEYBEEQhEAIhEE4zFt77vvm1q10SfI5ssfW2b2WR3b/VFftllufyjrz6bNFAhKQgAQkIAEJSEACL0Dg0wv4qIsSkIAEJCABCUhAAhL4rHD1JJCABCQgAQlIQAISeAkCCteXWCadlIAEJCABCUhAAhJQuHoOSEACEpCABCQgAQm8BAGF60ssk05KQAISkIAEJCABCShcPQckIAEJSEACEpCABF6CgML1JZZJJyUgAQlIQAISkIAEFK6eAxKQgAQkIAEJSEACL0FA4foSy6STEpCABCQgAQlIQAIKV88BCUhAAhKQgAQkIIGXIKBwfYll0kkJSEACEpCABCQgAYWr54AEJCABCUhAAhKQwEsQULi+xDLppAQkIAEJSEACEpCAwtVzQAISkIAEJCABCUjgJQgoXF9imXRSAhKQgAQkIAEJSEDh6jkgAQkcIvDjjz9+/vTp0/L1ww8/fP75558///nnn7u2ug3GbZV//vnn8/fff/91Xt7/9ddfW0Nsk4AEJCCBixJQuF50YQ1LAmcTQEBGvCJUU/7+++/Pv//+++fvvvvui7jcE6L0r+IVm1vl119//dqfOfDDIgEJSEACH5OAwvVjrrtRS+BNBBCliM6VOCULGvH6yy+/bNqvwpUM6lQicmP3p59+mrpaLwEJSEAC7yTAPr7a34+aZe8/8svbUXurfgrXFRXrJCCBJYFkXMmwrgr1EaWIzlUhYxrxm76rftQxH8IWwUrf3377bepqvQQkIAEJvIMA+zeidfpVi3r2YPbkLXGKDX4pu1dRuN6LrHYlcEECEZrTM6bJkNJvErd//PHHFxHKxhd7q40yIrj2m+a9IGpDkoAEJPAwAuy302Nb7OsI0fzyxb69JVxxmmTDezK3W4ErXLfo2CYBCXwlEAHJ5rVVIkanO+48s8pmmI2wb4IIWdrY/DIvdi0SkIAEJHAugSQTtn4lY8aamOh7dvcoe/g9fiXzStBp+1kCElgSYANCPE535Rm0J1zz8z/9eU9/Ns5aeE4K4Zo7/SPz1vG+l4AEJCCBfQIRmEezo9nf94QrM9ckxb4nx3soXI+zsqcEPjSBPGc6ZVIDJxvb1I92bFFWNpNhzZ16xO1kL/N6lIAEJCCB2whEXPbkwWQl+/sR4ZoM7VFRPM3Z6xWunYifJSCBJYH8rL+1wWWjYnNb9euiNJtm/V8I+K+26n+3lXmPbJRLx62UgAQkIIF/EUi2lf36aLlFuGKTP+RiDNeGs8pxb8+aUTsSkMDLEaiClM1uKojVbGyrjSpCNX9klT/AyuMHvZ1+sbc17+SP9RKQgAQksCaQ/bcmCtY9/1ub/fhoIiG/mOUXtP9aevs7hevb2TlSAh+GQDa4rf9zFRj8JMTGNm2EPBpABjUlGVg2N4QubTX7mudqJ3ux41ECEpCABG4jkP06iYMjo28VrklG3DLHnh8K1z1CtktAAl8F6dazSjUri9BdFYRpnm+lnSxqNkLqEcY1s5qNtYrZld0z6yLSqx9n2n+rrfBdPYLxVpuOk4AEPi6BZEMRl0dL9uujGdcIV8adVc6zdJZH2pGABJ6OABlPNp6tn3siMqesbH727zayEXLsoizPR01C+GxQiFXmfMYMb/6Q7dkE9dlroD0JSOAxBPL3A/cUrvnVTOH6mDV1FglIoGVF82xqB5NnW9kIpz7ZwPqdekRxzcRiPxlGNjzeTwV7EXX0JYtQxR2iF7941bnJ4tI/mzbHCGXq80o7/tMeO8yBDT7Tt/vIXMlo0I6w53MX7sSFLdrr/MkywzaM4hNHbFkkIAEJvJVA9pPscUfsZEzdS7fG0S9jtvrd0mbG9RZa9pXAByQQUcrmsyq0RxjyfioRl70dAcb4Lvzyk/00L3YiHCMGMyafsYl9hCGiMOKYcfTlWH3O+FrHPNQjLLGDP2z0fEak04btWmInYzI/Y/uGjw3iR5zib/oyNoV6xkbMpt6jBCQggbcSyE33PYUreyl7F6+zynmWzvJIOxKQwFMRQECx6dQMH+KKDYk62hBdU6aVYOpd91a/BB7xlg1vNSYbYhWZqUM49hKRS59JAFLPnF1ExxZ+0Y5QXflEv4jMiOSMDat8zhF22MN2Cn0jvqnbiitjPEpAAhK4hUD2pGk/XNnKntxvwFd9qUMUZ8zU59Z6heutxOwvgQ9EIFnSbDz1iOBC1FbhuEJTRWvG72166VePXSgi9th4KbQh9JK5rCIwPkX8ZUzq6zEistbV94llK0MRod/FL7b73MnMroR2nXdPUNe+vpeABCRwhED2qr4vbY3Nnry3h8dGhOstc2TsdFS4TmSsl4AEnpYAQjUbKEdELCJ7SwBmzJboxBab+VSyCXdRWvtHPNe6ZGp7ZiM3BiuhXcez6ROjRQISkMBZBHIzz0310ZJ996hwzR63te8enTv9FK4h4VECEngZAsl87mV7a0BsoIjKSZjGZv2Jvo7nPQISG1uFjb1nF5JZ7f7S78hFA5v4b5GABCRwJgH2M/aXvZtn5sweSf+jQpT9jf5bN/u3xnMX4YqT04uNmqzDXhAVUGxtZVMIPBmV9GeuvhjJmKRPPwIZ//o4sh29b4edn/NqP3xK4aIVO5wsPfuSfvVIDPTd41XHPMt71ot499Yt/sInJzkMeX/kywHj3NUxjjkRJ30NMw/Hj7YWNfYrvM/+0IUg50L9ziVWxCjnEucJ59WqZG/Adko/hzi/+E5uld6H726+9/17jK3uD3PWGBJr/S50v7b8sU0CEpDARCD7Xt9La//sQext/VX3yzqG9+xTfT/sfd7y+S7Clc05Kr6KMzbjQCKYPUFDe4VUN+5VsFwE0n/r4lIFZr2Q4F8EEBeaenGoMTFHHRdf6B/hRfx1QRNLYs7nyid2cuRiy1xbGaD0faYjseVCjf+Vw+RnfdaGLxCvsJwyZNgKR+bjPXNlDRlf1zBzZwzHauOKa5GYr3bk+8e5xVqzxrxYT86DbMB8n9kHqKcfJd8p2nhfv1tp44g9zru65zCGOWvdiivf/ewfjMFO9hzsMj7nXs77fEc4ct7WOTJvjWFrf1v5ZJ0EJCCBFQH2JPasrevsatyRuuypK710ZPzU5y7ClckiXLJBVweo4wLAKxeZ2p73bOLpx3ELbGxGMNcLUuzlmL74uCrxvdvIIuDLVBiLD1xsaqGeC1ItEWarRc3J1MfU8c/2Hq7EyaveROSiPPmbdWZcLWEAb2z3Uts7w8y/Omc+wlp0Vlf8zN7BWnJ+ZOOt5wHttEXcwoBzJt+7fqNCW84b7PZzLt//rT2LORiXfYjzD7uc49TxqvsKbfiHn/F19X2J8GU8opZxFglIQAJnEMg1uO6fZ9hlH6373Rk2sTErsHfOkI14AsGGTh8Cm0ouPOk7ZRnYxNnQ6ccRu1041jnY+OlD/1XJxavPRyyJa3XhyIWtX/ByUvQLZfzo/fEpF7OtOFa+f6s6eCAIaixhtboQVz/Du45Ne9Z+dZ6E32odsZX56zn4EdYi7DxKQAISkIAEjhDgmsk1fKVtjozvfdA7q2tz7/eWz3cRrhEHiMipTMKi9k+WIfam7CNwmCtCd2te7EcoTXcCaV9BR0AhiLrIQhwxbxenzJdYEVq1RHj1+sS7slXHP/v7CEfimUq9GagCM/2zptjq7dNaMJYvX+ava/VR1yI8PUpAAhKQgARWBLg+onveK17RVvfUL3cRrhFkeSZrBSjiDHExCRsEJOIkz3jRt5fYAVTmZdxWiaBZZTOr4Fn9JBgx3UUtonqKN351gbqqZ35i5vXek2eLwSPawnlaX3yIkFytLe117et6VME72edGArv1C7Rizjyr+iutxSPW2zkkIAEJSOC1CXBtrdfMW6NBi03X5FttTf3/rQSnnjfU52fuLtSqiQhOhMUUJG0RiLzn1cUcgjGZ2GRKj8w7ZWXj+yR+4zfCMoVFxofuW9pZSHzvfq3EUuqqSIudtxzDJPxuOU4MjvqRuab1xU7ipe9UYqfyyzrQNtlP7DWOb7kWU3zWS0ACEpCABCRwjMCsFo6NX/ZKpmsSFAzaEx5pR2hQVjYjepI5jcDZmjdjanYUwYlQjNBBiE4itPrCvIzDt/jwxdn2T2LpdzHxJf4mu1h9a6Zu/piYwuaWYxV8N0/MA9T/f7OR+FY2wiA3H6s+sUPflDClbVqrxF7jyLhHrkVixNcaQ2LxKAEJSEACEpDAMQKnC9eIry1BgWv1J+KV8MjFPoIwIiSZSNLZCMYIkDrvVujJqEYM1SO2+nOUK1tkgRlHf3yIT6u+1BEf/bs4wxfGJ35i5HN84Fj9ZfzeXJMP36I+bI8I1youu6+xU0VfBChtU8k5U21/i7XIuYyvNYbJb+slIAEJSEACElgTmK/66/67tRGkXaT1gYg+LuT1J/faJyIudRGLufDTXp8DzU/Ae/MiDJk3YgohE9sRwZlzOibGW4RI4k0GOcIr8cT/tOMX8REPApbPEbHxffLvWerhU1mv/IqoY12mEjthRb/woy03N338SrjS59FrkRjxtcbQ/eVzYvX4n3NHDq/JYXVuWycBCUjgDAKnC9eIQI5bBVHGRWkSiwgZhFpKLv4cyToytmYfI+ome9iZsrIIw1wgk+3MvKtj5mfMLQXfI5yJPyIGUUp9Fd0RsjXG+Fm53DL/o/uG6ZbQzrpusYydiHriqMJ1sh/huuL1rGuRWD2+pmBz3f6zbo/ea5xPAhL4OARuU14HuESQkpWcSs1YroRiBGYVKhGLiDvm6GIk81ah1+ePGKwCMX0yPmIy9atjMnYIozNKRHfNHEZ0IWpryYWx1j3r+/g6CUv8zprQd1WIf2Un5whtk33WmfYja5q5r7oWic+jBCQgAQlI4JUJrNXCGyOqIqOKsG4uInHKykbMVBs1w0Z2sgreZCIRKbW+z5ts8CorG8HSBXG3wee3CKKVHeoSV/cpwrWPm+p7v3xO/4i/W46MfU/JXJOwxHbip++qX22fRPwkTDP/1s1MjS9z3Wst6ly+l4AEJCABCUjgdgKnCtdkRRGWU0m2EvHXhUjGRETmM8cqimsmlrZkcBHEWyWCeSVkED8InT0b1Y+V0Nqav7dhi/ng1VlEcPYxU33vl8/pHxF3y/ERwhU/iR+/+rrSlvNldUOR82TVduRcDCOOj1iLOp/vJSABCUhAAhK4ncCpwjUiYxI8EYeI1ppN7W4jZFY2EDeM7SWZ1NWY9K1Z2ZXgTLaNObaythFE9HtvCY+VkCYW5uiCFja8XqHgP69VfNX/cEDE13gjJrGxt2b9fIqoxfaREh9Wvl5hLY4wsI8EJCABCUjg2Qm8X32VCJPRrGIBEUhGNG0IiipOyvAvbyMguojp/fIZwRKBNI1hvggZ+q4ye9iLnep/5uGInTwmQN8ulmrfvffxexLbuQmoQirie3rEYm/OR7XDKesIJ5ht3QxUrvQlZl5hPa0X8eSmBTHPeYbAzVpzPFKuvBZH4rePBCQgAQlI4FUInCJcESURGRF/9Yg4Q4jtCb0qdjJ+C2TNkqb/KhuZjFn6cFyJ09qPeHqp4/O+Csvef+szc+HrJOiopx0/eI+4Q6RRt8dxa957tq3WL5xyJO5VidjNDQ796XuEL4K1nn+8X63val7qrrgWU6zWS0ACEpCABF6ZwCnC9ZUBfAvfySAizPbEFQIVURXRRwbxWUXrt+B4xpzPvBarG7OcC9zAcG7gP6J/q6xuKKYbptgJl8zHjadFAhKQgAQk8K0JKFy/9Qo4vwQ2CJBxjnis2ef6SMSRLHweqYgtxk8FIYzN9N27wZrsWC8BCUhAAhI4m4DC9Wyi2pPAiQTIjEZArrKkEaQIzVV7XOlZVx6vmEoeScm8ZvknUtZLQAISkMCjCShcH03c+SRwA4E8LsCzv1PJc8EIzqnkj/0idKcsauZLfwSxRQISkIAEJPAsBBSuz7IS+iGBBYFkSrf+h4SI0S1xyx+s8Uxs7E3PrGKj9tuad+GuVRKQgAQkIIG7ElC43hWvxiXwPgL54zz+WGoqEaP8tD8V2hC4+aMr7PYSO/WPAqmzSEACEpCABJ6FwHylexYP9UMCH5hA/khq64+pIjgn4Zqf//P/3NKPDGwtPB/LXMnE0ofX1rx1vO8lIAEJSEACjyCgcH0EZeeQwBsIkPmMgNwavidc0469ySYZWB4T4H8UiNBlbosEJCABCUjgmQh4ZXqm1dAXCRQC+Vm/Z0dLly9v84zr1A9RWv/IKmI4//9r/sut/HdbmXf1OEGf288SkIAEJCCBRxJQuD6StnNJ4AYCEaT5+X4aimBFjNJ/VWirf2SV/4WAzGr+z9baznvGTPPyyAHtW/+l1sqPXvceO8TA69lL1nDrvyp79hj0TwISkMAzEVC4PtNq6IsECoEIzC2BiCBCRPJKxrSY+PpoQP3jLjKp9Ee4Ik7JxlZhledqV/awfZYYe6udxDwJ9Rr/9D6PT8Chspn6pz7swi/10/FVBPbkv/USkIAEno2AwvXZVkR/JPD58xchiTjiVUVlhxPxN2Uf87M/z7amIFaxm8xqFW4RhbTnUYKMe+SReKaM71l+hMOtf4CWDPdZfmhHAhKQgASOE1C4HmdlTwk8jECeO63PpvbJq8icsrKI026jZhv7c7H5+X4Swt2He3yO2J4yvmfNGeF6qz2Y+fzvrdTsLwEJSOAcAgrXczhqRQKnEoioqs+e9gmS+dv6yRzR2kVWRDFZ1ZqJxX4yuCubEbWMq9lQMpbxJdlbjohf+iKUa5nsIMTrT/GMzSvjY5P6VUYYX6oN4uBz/Iodjm8RoMyZmGCXuJmj+hOO9F0JcPrCJfFw7DcfYcgaEhdjcl5QF7vY4TNzreIkVthWn/CXz6vzizliD5vEWLPS+JFsPe28evyVs+8lIAEJnElA4XomTW1J4CQCEURd9GG+CgcEBJ9XJf/1VbeBCEFsVPGZ8RFSfUzaI5wimhBE+IoPHBEw9ImAShwZn2O3k3qO+MC4VWGeiKneHkGMIKMfr8RThRfjYmfFoNutnys7xmKHWPEpMad/YqdPLXymjVf8gjc2wpVYEgf1tMOW/vE9rDOGWBGcvXAeUM98rBclwrOvMzHRNzc0YRo/GRvfE1cEcZ/XzxKQgATuQUDheg+q2pTAOwggLhArvKpgQEwgNJIN66KjTomoiDjheKREgDHvNIY5aY8AqnYRNPhWM4eIK/r3MtmJKEMMTQV7vT3Mut/T/Ig97FRfp/lqffyu88fn1dyIyV7iU2eIP91GbGMnYhJ79OUV0UodduHfC2N5YSslcdTzizZssI618LmOZd4q+GnrY+p430tAAhI4k8C/ryhnWteWBCRwEwGECCIjwqQeESUICwRmFz19koijjOfzVomQSX+OXUQxHjv4tyqM6fPQdyVqJjt7ghKhxTxdcCbr17kwd/cJ3xFe2On9V3HVOmyxDlXIYQNbVcwyZlWH+FzVp3/3NfGyPinMjY3el7q+ZsmYdl6JPzZzTD2x1BjTzhGmMKg+1XbfS0ACErgnAYXrPelqWwIXI7ASR4QYgVV/Lo+gq9m54JjsREBPgjLtNfuITYRUF8gReKv5I0Djz9EjfneBGrFdhdyKB3PE/5oppT79u6/pX3lkvmoj4yt/7CJk8bmLUFh1XmGQOWHaBS99sBW73Jj0tYgdjxKQgATuQUDheg+q2pTABQlM4ohQI3bokzJl+7bs7AnKqR1x1jOQmb8KvPg2Cee0r47JlnZxmGxvFXDhUeuwmfrKifpkOrtQXMUbG1XMpq7bZTyx1rJ1Q5F+9EHYMrbHkD7MhbjlZZGABCTwKAL/u6M9albnkYAEXo4Agg0h08URgawEUgRdFVj03bKD/SpAe6YQkVTbA7GPY848ctHnx3/6I/ZuKSu/sY2t/hN9MpLd/kpgEiNxrR7B6HFhj/h73xX/9MVGLenbRXLvF05Zb46dfeKp9n0vAQlI4J4E/ndHu+dM2paABF6aQEQKAgbRU4XfSlBGIBE0IjZCac8Oogwxxzx5H3ARcrRjP8I2wo/PZAiZL1lM6pgz82MXO3zmfRedmasfI0aTcWVsfnKPHxnTY6cvJXPnkQCELzbwv2c2+YyflTM2qOs+M19YEXvmi8/YwkfaIsDJRPPClwjwzJW++JXY4MXcsR3fmdsiAQlI4FEEFK6PIu08EnhxAogfhAyvCBxCisCKGEuYiCL6Iqgi9tJ/ZYc2xBFtCCREFuKoFupoQywxb0ofh9hCYGWeOj9t+RkcexFmsTUdiYMYOeIDtqfxEXn07Y8q0NZt9DjxoQrM+BThW/nTNs0Ho8wVZsSbOuLhM/PTHvbEh+itjImDPrTtxR9/PUpAAhI4m4DC9Wyi2pOABCQgAQlIQAISuAsBhetdsGpUAhJ4NQI1m5isYo6vFov+SkACErgqAYXrVVfWuCQgAQlIQAISkMDFCChcL7aghiMBCUhAAhKQgASuSkDhetWVNS4JSEACEpCABCRwMQIK14stqOFIQAISkIAEJCCBqxJQuF51ZY1LAhKQgAQkIAEJXIyAwvViC2o4EpCABCQgAQlI4KoEFK5XXVnjkoAEJCABCUhAAhcjoHC92IIajgQkIAEJSEACErgqAYXrVVfWuCQgAQlIQAISkMDFCChcL7aghiMBCUhAAhKQgASuSkDhetWVNS4JSEACEpCABCRwMQIK14stqOFIQAISkIAEJCCBqxJQuF51ZY1LAhKQgAQkIAEJXIyAwvViC2o4EpCABCQgAQlI4KoEFK5XXVnjkoAEJCABCUhAAhcjoHC92IIajgQkIAEJSEACErgqAYXrVVfWuCQgAQlIQAISkMDFCChcL7aghiMBCUhAAhKQgASuSkDhetWVNS4JSEACEpCABCRwMQIK14stqOFIQAISkIAEJCCBqxL4P+i3Q18MxYhEAAAAAElFTkSuQmCC)
where the dry matter (DM(g)) of the initial biomass was measured by drying in the oven at 400C to constant weight and the DM (g) of the extract was measured by drying all the extracts combined for each of the conditions in Table 1 in the oven at 400C to constant weight.
Protein release yield (PRY%) was calculated using Equation 2:
![](data:image/png;base64,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)
where protein (g) in the extracts (Proteinextract) was measured by Lowry and protein in the initial biomass (Proteininitial_biomass)was measured by Lowy after bead beading in alkaline solution. PRY% of the pH shift experiments is the combined yield of water and all pH extracts (1 to 12) in the specific experiment.
Essential Amino Acid content (EAA %) was calculated using Equation 3:
![](data:image/png;base64,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)
Where YEAA(g) is the content of essential amino acids detected with HPIC in the Proteinextract and YTAA are the total content of amino acids detected in the Proteinextract. EAA determined were histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine and total amino acids (TAA) determined were alanine, arginine, aspartate, cysteine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine.
Branched Amino Acid content (BCAA%) was calculated using Equation 4:
![](data:image/png;base64,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)
Where Y(g) is the content of amino acids detected with HPIC in the Pextract. BCAA determined were leucine, isoleucine, and valine.
2.7 Statistical analysis
Student’s t-test was performed to analyze the significance of the difference between data sets obtained in the study. The p-values were calculated using the Student’s t-test in a paired manner with 0.05 as the maximal p value indicating statistical significance.