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luckypunklemonade · 2 months ago

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Temptation Greets You

| Curiosity killed the cat but satisfaction brought it back. Stiles comes across a small dosage of pollen out in the woods.

[smut MDNI 18+; sex pollen; 3k words] Stiles Stilinski

This work belongs to me, luckypunklemonade (Minte_Condition on AO3). I do not give anyone permission to distribute or share my work without consent.

He was so fucking stupid. “You guys are so fucking stupid.”

Going out into the Beacon Hills Wildlife Preserve was a regular occurrence for Stiles, but you always expected them to be at least a little bit careful. You’d have thought that he’d turn back and plan for more research when Stiles stumbled upon a freshly abandoned coven ritual ground with suspicions of black magic. No, he needed to poke around and take notes without checking his surroundings or prioritizing his own safety. Stiles picked up a mortar and pestle, underestimating the powdery substance left inside, and brought it up to look closer. The wind picked up the powder, blowing it into Stiles’s face. After the initial panic, he called to tell you. The threat of a powdered drug was skeptical as the powder was a deep blue. You rambled off a thousand excuses or hopes of what it could be. Less harmful things like spirulina or a kind of dried starch, something weird but harmless that witches use. Stiles had only inhaled a small amount, but you knew that amount of a particular drug could kill in minutes. You didn’t want to risk it.

Your car was haphazardly parked halfway off of the trail. Your coat was halfway on as you rushed over the uneven ground, imagining the grief tonight would be with a broken or sprained ankle. Stiles was sitting on a tree stump, elbows resting on his knees. You step closer to get a good look at Stiles. It had been 20 minutes. That’s how long it took for you to get to the wildlife reserve in your car. He looked fine. A little tired, but he rolled his eyes as you rotated his head in your hands. “I’m okay. I feel fine. You were probably right.”

“We have to get you to a hospital. It could be-“

“It was probably that stuff you said.”

“Spirulina.”

“Yeah, witches use stuff like that all the time.”

You tried to get a better look into his eyes, but he pulled away from your touch. You thought his pupils looked blown. You fussed over him to get him up and walking. “We’re going to the hospital.”

“No-“

You grabbed his keys from his pocket before he could finish and herded him out of the woods toward the trail. Stiles went silent, walking in front of you awkwardly. He really did look fine.

He only started having trouble breathing when you were on the trail back to the main road. You cursed and sped up, headed to the hospital, grabbing your phone to call Melissa when his hand grabbed your wrist.

“Pull over.”

“We’re going to the hospital, it’s—“

“Pull over.”

“No, you could be-“

“It’s not a drug, not like that. I knew it wasn’t- I shouldn’t have let you- I didn’t think it was enough.”

“Stiles, if it wasn’t a drug, what the hell was it?”

“Please just pull over.”

“It’s not fatal? Will it hurt you?”

“Please.”

The crack in his voice and the way he pushed his head against the headrest and squeezed his eyes closed shifted your attitude from panicked to empathetic. You pulled the jeep off the trail and parked it, still buzzing with anxiety as his breath came out almost violently. He breathed like that in between fragments of his thoughts.

“I did some research about the coven.”

He kept his eyes closed, and his hand gripped the Jeep’s door handle tight.

“The blue powder wasn’t a drug. It- I couldn’t tell….”

He seemed beyond embarrassed; his ears were red. “It’s an aphrodisiac derived from a mix of herbs and flowers. Pollen. The members of the coven use it for various holistic purposes but mostly in rituals to gods of fertility or prosperity. They mix it with water to create a paste and-“

He winces, leaning forward and harshly thumping his head against the dash and resting it there. You reach forward out of pity.

“Please don’t.” He takes a deep breath and continues, “They make markings, they…fuck, whatever. You need to- I should…”

He doesn’t finish his sentence. He opens the Jeep door and stumbles out. You yell his name, but he turns around. “Just leave the woods for a few hours. Come back at dusk. I should be better.”

“Stiles, if it’s an aphrodisiac, why is it hurting you?”

“This- God, it’s not- aphrodisiac is a simplification; if I don’t get some sort of…of release, it’s gonna keep hurting. It’s gonna get worse. I’m not gonna be in my right mind. I need you to just go home and come back in a few hours, okay?”

“I’m not gonna leave you out here, hurting in the cold woods for a few hours, Stiles.”

Stiles actually whimpers. It’s like the sound of you saying his name hurts. Or something else.

“I won’t be able to think straight, just drive. I’ll be okay. Don’t call anyone. Don’t tell Scott or Isaac. I’ll figure it out. Just go.” The words are starting to seem taxing, hard even to say. He begins to walk into the trees. You think about how hard it’ll be to find him in the huge wildlife preserve. For a minute, you consider leaving him. He’s done the research; he’s been out in these woods alone before and gotten home okay, but he wasn’t under the influence of anything then.

You get out of the Jeep and walk around to the passenger side, where he had walked off the trail. As soon as he hears the door shut, he turns, the sight of you out of the vehicle making him throw his head back in frustration. He yells, and it stops you in your tracks. “Get back in the Jeep, just get back in the fucking Jeep!”

And he’s walking toward you. He’s almost scary, his voice a little unrecognizable in such a low register. You do; you get back in and yelp a little as he reaches the passenger door. He presses his palm to it, almost hitting the metal. He looks utterly desperate, upset, frustrated, scared, even. “Please, just…just stay inside. Just..”

Stiles leans his forehead against the door, shoulders heaving up and down. His pleas fade. Before they become silent, though, his tone shifts from “Please stay inside” to “Please help me.” He mutters softly, no longer even speaking to himself and, if you weren’t listening hard enough, to no one. “I need..” he says breathily, pathetically.

He moves to lean against the front bumper, resting his forehead against the cold metal. You crank the window down just slightly and speak in a small voice, “Stiles?”

He doesn’t move. He just keeps sucking in air and huffing it out. You’re worried now. It’s not what you thought. He looked like he couldn’t breathe, and his fists were clenched, his head buried in his arms on the cold hood of his Jeep.

“It’s cold, Stiles. I can’t leave you, just…just get in, okay?”

“I can’t- not you.”

“What? Stiles, if you need help, we can figure it out. I’ll find a witch or something, just-“

“You can’t help me. The only thing…”

You remember his words. He needs some kind of release, aphrodisiac, understatement, et cetera. “Stiles. Let me help you figure this out. I understand. Please get back in the Jeep.”

And then, you unlock the vehicle and pop the door open. It swings wide, and he looks up. He looks a little scared, a little worried. His eyebrows are knitted together painfully, his shoulders heaving up and down, hot breath leaving his airways visibly in the cold. Stiles shakes his head.

“I understand. Let me…let me help. I can-“

He’s slowly walking around the door, hands still in clenched fists and breath labored. “I can’t.”

“It’s okay, I can help. I know what you need, and I know it hurts. Just let me help you.”

“Not you…Not like this.”

“Come here.”

His eyes flutter, and he takes a few steps toward the car, toward you. Your hand touches his shoulder, and he whimpers. His hands moved quickly, grabbing the seat lever and pulling it back. The back of the seat fell, lying flat, catching your attention until you looked back at Stiles. He was hefting himself up into the Jeep toward you. You didn’t realize he was herding you into the back until you were sitting on the reclined back of the passenger seat. His arms, after reaching to shut the door, spanned wide. One hand was on the driver's seat headrest, the other bracing himself against the interior. It made him look bigger. He looked hesitant. “You don’t know what you’re doing…I don’t even-”

“It’s okay. I can…help. I can help.” Your hand reached up to bring his down from the headrest.

“I can’t…not to you.”

“Well, I’m not leaving you, and I want to help.”

His eyes scanned the Jeep, taking in the cramped space. His eyes seemed unfocused, the effects worsening. He didn’t speak as he pushed your shoulders down, cradling your head as you lowered. He planted his knees on the sides of the seat, hooked his arms around your thighs, and pulled you down by your hips. He looked desperately up at you, trying to convey an ask but failing and giving you the most pleading look you’d ever seen. You breathlessly nodded, and he dramatically dropped his head onto your chest in relief. His hands immediately gripped your hips, but they were restless and curious, and needy. He watched as his own hand relaxed and kneaded the denim-trapped skin of your hips as if he didn’t know what his perverted hands would do next. His other palm leveled, and his fingers parted, relaxed from fists, and moved slowly, experimentally up your ribcage under your shirt. The entire time, his face looked guilty—upset, even.

“I should’a brought you.” His voice is distracted, but he means it. “You’re so careful.” His eyes are glued to the way his hands line your curves, up and down, but his words are genuine. When his short-lived need to just touch you was gone, he closed his eyes in shame. He needed more.

He presses himself against you, eyes hardening in intense and conflicted emotions, and his hands squeezing and grabbing at you mindlessly. It would be pathetic if he weren’t so completely driven. He still wasn’t saying much, kneeling between your legs as he bent them at the knees and pushed them up and out wide to make room for himself. Once he does, he’s pressed into you again. You think the warmth between you is only something you notice, but Stiles groans and rests his forehead back against your chest again, even if it takes some craning of his neck downward. His forehead was burning up, and the sheen of sweat, while thin, made him almost glow in the early-setting winter sun. You can feel him hardening in his jeans. It was impossible to ignore, especially when he started rocking against you.

His hands retreated from your hip, from the seat beneath you, holding him up, to unzip his jeans. His mind is foggy, you can tell because his hands shake and struggle between his zipper and yours, not knowing which to focus on first. You softly reach down and unzip his jeans. His hands pause, and you look up, making debilitating eye contact, and you think he might pass out. He’s sweating, shaking, silent. All things your Stiles isn’t usually—or at least not all at once. Of course, he’s never seen you like this, conversely. Underneath him and willing to let him, rather, wanting him to touch you.

You jumped when he scrambled to get your jeans undone and yanked down—or up. Up your legs in your position. Everything about this made Stiles want to whine. You, how close you are, how long he’s wanted you, the position he’s got you in, what you’re gonna let him do, where you’re letting him do it. In his Jeep, out in the woods off a secluded trail. He wanted to speak, to make this perfect like he wanted. How he imagined when he was in his room mumbling your name and pumping his hand as if it was yours. But he was so damn impatient and it hurt and you were so willing. He spoke softly and barely intelligibly as he pulled his jeans and boxers down just enough and your underwear to the side, “M’gonna make it up to you, ‘kay? When I don’t need it so bad, I’ll make it good.”

You couldn’t respond before he’s got his tip pressed against you, and he’s pushing forward. It’s too late to stop him to ask for the mercy of preparation or caution and the sound you let out is proof. It’s an open-mouthed, guttural gasp as if he knocked the wind out of you. He stops once he can feel you completely envelop him, and he almost collapses on top of you. He felt a sense of relief, a scratch to the itch of the substance, but he could feel the discomfort of it saturating again. Two times worse now that the release was wrapped warmly around him. He was still, though. He stopped as soon as you made a noise of pain, even though his hips twitched. He looked up after telling himself that if you looked the least bit upset when your eyes found him, he’d stop even if it killed him.

But before he could find your pretty eyes, you clenched around him. It was an encouragement. More so torture when he was so sensitive. He moaned and resorted to broken rambles, some of which were not even audible. “Please-‘

You told him to move and he didn’t rock back as you had expected, only forward more, burying himself fully into you. His breath fanned across your ear as he was so close on top of you, the sound of his breathing laced with whines and moans as you satisfied the twinge in his stomach. Then, he set a slow pace. Somewhere, his need to be close intensified, and he looped his arm around your head, ushering your face softly into his shoulder as he pushed his hips into yours, trying not to focus on how quickly the release would come if he didn’t care about hurting you. Eventually, he couldn’t stop himself from speeding up, his other hand holding your hip down. The hand cradling your head held you tighter, more secure as if you’d break. You made small noises that punctuated his thrusts, each of them gripping his conscience as harder than he should be handling you.

“I’m so sorry,” He choked out against your hair. “Feels so fucking good, I’m sorry.”

He was unsure of how much pain he was causing. All he could tell was you were taking away his pain. You just moaned into his shoulder as he sped up against his better judgment.

”I was so irresponsible, I was so stupid, I’m sorry-“ He moaned as he drove himself deeper. It matched the one you breathed out in response as well.

He couldn’t be sure at first, but he thought he felt you press your lips against the fabric stretched across his shoulder. As he felt the vibration from your lips as you strained out another moan, he was certain. The small act being something he was too impatient and hazy to do, but something he wanted to give you made him squeeze his eyes shut, his climax on the horizon. Chasing harder, his words were almost cries, “Thank you.’

”For letting me do this- thank you.” He moved his head so he wasn’t facing away from you and began sucking messy kisses into your neck. They were somewhat controlled, but he needed to communicate his gratitude in a way that wouldn’t betray how unintelligible his thoughts were.

“I’ll be more careful. I’m so sorry, I’m- Thank you. Fuck-“ he’s cut off as you clench around him again. He doesn’t know why, but you do. The sounds of his whines sent you nearer and nearer to your ruin. In your ear, mixed with his moans and utterly desperate. Amplified by his need from the pollen.

His breathing stuttered in time with his hips, and his fingers tightened in your hair. He frantically pulled himself away from you, pulling out. The last rational decision he could make before tipping over the edge. When he came, his whole body tensed, and he let out a breathy whine. He breathed heavily and desperately in your ear, whining out, “Thank you.” Due to the amount of the pollen he’d consumed, he felt satisfied. Relieved. It felt as if he’d been waiting weeks for it. His arm gave out, and he laid himself on top of your chest.

“Thank you,” He pressed a long kiss to your collarbone, lazily letting his head fall down. “I’m so sorry, I should’ve-“

He’s too tired. Presumably crashing hard from the powder. Your hand comes up to his hair, gently running your nails over the shorter hair at the nape of his neck. “Mh- Stiles, look up at me.”

Your focus was his eyes, but it was too dark to see. He sits up at your command and pulls his jeans up quickly, turning to help you. He pushes your hands away, tugging your pants up and buttoning them back, his hands gentle.

He’s crying. You hop down from the jeep. The sun had set, and the temperature quickly dropped. You awkwardly get out, turning back with your phone flashlight to check on him, and he’s silently crying. You gently tilt his head toward you, focused on his health first. Once you’ve made sure his eyes look fine, you hold his head to look at you without the light. “Hey.”

He looks down, shaking his head. “I’m so sorry.”

“It’s not your fault. You didn’t know.”

“But I did, as soon as I-“

“Well, it happened, okay? How do you feel?” Your hands brush tears away from his eyes.

He describes how he feels, coming down from the sort of ‘high.’ His eyes fill with new tears but you lean forward to his confused surprise and kiss him, assuring him with a new, soft tone of voice. “I’m glad it happened.”

#stiles stilinski #dylan obrien #dylan o’brien #teen wolf stiles #teen wolf #fanfic #stiles stilinksi fanfiction #sex pollen #smut #stiles stilinski x you #stiles stilinski x reader #✰lucky writes

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helen0086 · 2 months ago

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Efficient Preparation of Analytical Grade Phycocyanin from Spirulina

Abstract: In order to produce analytical grade C-Phycocyanin (C-Phycocyanin) with low cost and high efficiency, this paper used Spirulina obtusususus alginatus powder as the raw material, and extracted C-Phycocyanin with ultrasound coupled with high-pressure homogenization with high efficiency, and purified the extracted C-Phycocyanin through salting out, dialysis and glucose gel column G-75, so that the purity of the extracted C-Phycocyanin could reach the analytical grade, and the changes of the components in the purified extract were analyzed with SDS-PAGE and UV-absorbance. At the same time, SDS-PAGE and UV-absorbance were used to analyze the compositional changes in the purified extracts. The experimental results showed that the high pressure homogenization coupled with ultrasound method could effectively improve the yield of alginate blue protein, and the optimal combination of extraction conditions was as follows: 390 W ultrasound for 2 min, ultrasound 2 s/interval of 2 s, 60 MPa for 3 times, and the yields and purities of alginate blue protein in the crude extracts were 131.2 mg - g- 1 and 0.69, respectively, which increased 51.07% and 51.07% compared with those of the high pressure homogenization and ultrasound alone, respectively. The yield and purity (A620/A280 ) were 131 mg - g- 1 and 0.69, respectively, which were higher than those of high-pressure homogenization and sonication alone by 51.07% and 58.15%, respectively, and higher than those of the previous reports. After two steps of ammonium salting out (12% ammonium sulfate and 50% ammonium sulfate) and dialysis, the purity of alginate could be increased to 1.09 (＞0.7), and finally purified by 25-min glucose gel G-75 chromatography, the yields and purities of alginate (A620/A280) were 107.65 mg-g-1 and 4.23 (＞4.0), which were up to the standard of analytical grade, and the recoveries reached 82.05%, and the yields were higher than those reported in the previous report. The recoveries were 82.05%, and the L ∗ a ∗ b ∗ values of the analytical grade alginate were 58.07, - 15.44 and - 17.86, which were mainly related to the purity, concentration and the source of the raw materials. The use of SDS-PAGE gel electrophoresis provides technical support for the production of analytical grade algal blue protein at low cost.

Because of its high nutritional value, Spirulina is widely used in human and animal health supplements, such as food and feed, pharmaceuticals and personal care, etc. Among all the functional components, the protein content is the highest (up to 60% ~ 70% of the dry weight), and the content of essential amino acids reaches up to 8%, which makes Spirulina an all-natural protein food source with comprehensive nutrition[1-2] . According to the difference of absorption spectrum, the algal bile protein in Spirulina is mainly divided into C-phyco cyanin (C-phyco cyanin), A-phyco cya- nin (A-PC) and R-phyco cyanin (R-PC), of which the algal cyanin accounts for 20% of the dry weight of Spirulina, and can be used as a natural pigment in food, cosmetics, etc.[3] . It can be used as a natural coloring for food, cosmetics and so on[3] . Phycocyanin is a kind of multi-chain protein, mainly composed of α-subunit (two cysteine and two methionine residues) and β-subunit (three cysteine and five methionine residues), in which each subunit contains 160 ~ 180 amino acid sequence, and α3 β3 cyclic triplet and (α3 β3 )2 hexamer are the main forms of phycocyanin[4] . Meanwhile, the chromophore of phycocyanin is mainly derived from phycocyanin (linear tetrapyrrole compound), which is connected to the carrier protein through the thioether bond. C phycocyanin solution shows cobalt blue in water, while A-PC solution is bright water blue, and the maximum absorption wavelengths of C phycocyanin and A-PC are 620 and 652 nm, respectively[5] .

C phycocyanin is a kind of natural blue compound of algal blue protein which has been widely exploited, according to the purity grade of the ratio of the characteristic peak absorbance at 620 nm to the absorbance of the protein at 280 nm, when A620/A280 ≥ 0.7, the CPC is the food grade; when A620/A280 is 0.7~3.9, the C phycocyanin is the reagent grade; when A620/A280 ≥ 4.0, the C phycocyanin is the analytical grade[6] . When A620/A280 ≥4.0, CPC is analytical grade[6] . The higher the purity of C-cyhalocyanin, the higher its commercial value, and the price of food-grade C-cyhalocyanin is in the range of 0.9 Yuan-mg-1 , while the price of analytical-grade C-cyhalocyanin is about 105 Yuan-mg-1 [7]. In addition, it was previously reported in the literature that high-purity phycocyanin has bioactivities such as anti-inflammatory, antioxidant, anti-tumor, and immunofluorescent properties, and can be used as a pharmaceutical ingredient in healthcare as a natural ideal substance without toxic side effects [8-9], therefore, large quantities of high-purity phycocyanin have become an urgent need for realizing the high-value applications of phycocyanin at the present time.

Currently, physical, chemical and biological methods have been reported for the extraction of cyanobacterial proteins from Spirulina, such as ultrasonication, high pressure homogenization, repeated freezing and thawing, chemical solvents and enzyme processing[10-13] . Wanida[14] and others used Spirulina powder as raw material, and ultrasonic cell crushing method, 10 mmol -L-1 phosphate buffer, material-liquid ratio of 1:15, power of 750 W, time of 5 min, the final yield and purity of C albicans were 60 mg-g-1 and 0.52 mg-g-1, respectively. Tavanandi[15] et al. used Spirulina powder as raw material to extract algal blue protein by freeze-thawing method under the following conditions: 0.1 mol -L-1 phosphate buffer, 1:8 ratio of material to liquid, soaking for 4 h, freeze-thawing for 4 h, thawing for 1 h, and the yields and purities of C algal blue protein were 73.73 mg - g- 1 and 0.66, respectively.

Currently, there are some shortcomings in the methods of C alginate extraction, such as the use of ultrasonic cell fragmentation or freeze-thawing alone, the yield and purity of C alginate are relatively low, and freeze-thawing is more time-consuming, and chemical and enzymatic methods are more costly. The main purification techniques for C alginate include salting out[16] , column chromatography[17] , dual-phase extraction[15] , and membrane filtration[18] . Marina[19] and others used 50 ml of water for the analysis of the membrane filtration. Marina[19] and others utilized 50 kDa polyethersulfone membrane to ultrafiltrate the crude extract of cyanobacterial blue protein, and the purity of C cyanobacterial blue protein was increased to 1.5, and then after passing through an ion exchange column, the final purity of C cyanobacterial blue protein was 3.9, and its recovery was 79.7%. Ravi[20] et al. extracted the crude extract of C alginate from ultrasonic cell crushing, after 20%, 70% ammonium sulfate two-step starching, and then by DEAE-Cellulose anion-exchange column chromatography, the purity of analytical-grade C alginate was 4. 03. The existing technology of C alginate separation and purification has the problems of instability, unsuitable for large-scale application and low recovery rate[21] , in order to promote the deep processing of C alginate and its high-value application, low-cost, efficient and rapid production of high-purity C alginate has become the main direction of the current research.

Therefore, the aim of this thesis is to identify the molecular weight and color of the analytical grade C alginate obtained by ultrasonication-coupled high-pressure extraction-P,H, and, at the same time, to identify the molecular weight and color of the analytical grade C alginate obtained by SDS-PAGE and color analysis, so as to provide technological support for the low-cost and high efficiency of production of spirochaetal alginate and its high-value applications.

1 Materials and Methods

1.1 Raw materials, reagents and instruments

Raw material of Spirulina dried algae powder (Jiangxi Zhongzhao Bio-technology Co., Ltd.); Ammonium sulfate, sodium chloride, barium chloride, etc. are analytically pure (Guangzhou Xilan Science Co., Ltd.); Dialysis bag (8 000 ~ 14 000 Da, Beijing Soleilbao Science and Technology Co., Ltd.); Dextran gel column G-75 (Beijing Soleilbao Science and Technology Co., Ltd.); Electrophoresis kit (Beijing Soleilbao Science and Technology Co., Ltd.); Low-molecular-weight Marker (Beijing Soleilbao Science and Technology Co., Ltd.); Low-molecular-weight Marker (Beijing Soleilbao Science and Technology Co., Ltd.). Molecular Weight Marker (Beijing Soleilbao Technology Co., Ltd.); Electrophoresis Kit (Beijing Soleilbao Technology Co., Ltd.)

UV-visible spectrophotometer (UV-9000, Shanghai Yuan Analytical Instrument Co., Ltd.); desktop high-speed freezing centrifuge (H1850R, Hunan Xiang Instrument Laboratory Instrument Development Co., Ltd.); computerized ultraviolet chromatography (HD-3001, Shanghai Jiapeng Technology Co., Ltd.); high-pressure homogenizer (GJJ-0.06/100, Shanghai Taichi-Tongyi Light Industry Equipment Co., Ltd.); ultrasonic cell pulverizer (Ningbo Xinzhi Biotechnology Co., Ltd.); electrophoresis system (Bio-BAD); gel image analyzer (WD-9400, Ningbo Xinzhi Bioscience and Technology Co. Ltd.); ultrasonic cell pulverizer (Ningbo Xinzhi Bio-technology Co., Ltd.); electrophoresis system (Bio-BAD Co., Ltd.); gel image analyzer (WD-9413C, Beijing Liuyi Bio-Technology Co., Ltd.); colorimeter (TS7700, Shenzhen Sanyanshi Technology Co., Ltd.). 1.2 Optimization of extraction method and conditions of algal blue protein

1.2. 1 High-pressure homogenized extraction

50.0 g of spirulina powder was taken, and 1.0 L of ultrapure water was added to prepare spirulina solution, which was then processed in a high-pressure homogenizer for three times consecutively at the pressures of 0, 20, 40, 60 and 80 MPa, and the treated algal slurry was centrifuged for 10 min at 8000 r-min-1. The supernatant was subjected to a full-wavelength scanning (200-800 nm) and the absorbance at 620 and 280 nm was determined, The absorbance at 620, 280 and 652 nm was measured, and the yield and purity of the extracted algal blue protein were calculated at different pressures, and the above experiments were repeated three times.

1.2.2 Ultrasonic extraction

50.0 g of Spirulina powder was taken in a 2.0 L beaker and 1.0 L of ultrapure water was added. The cells were broken at 390 W for 1, 2, 3, 4, 5 and 6 min after 2 s of ultrasonication, and the ultrasonic cell breakage was carried out in a water bath at 4 ℃ to prevent overheating. After sonication, the samples were centrifuged at 4 ℃ for 10 min at 8000 r-min-1, and the supernatant was subjected to ultraviolet scanning at full wavelength (200-800 nm), and the absorbance at 620, 280 and 652 nm was measured to calculate the yield and purity of the algal cyanine protein obtained from different sonication times, and the above experiments were repeated three times.

1.2.3 Ultrasound-coupled high-pressure homogenization method for extraction of algal blue protein

The optimal conditions for the extraction of algal blue protein by ultrasonic crushing were 390 W, 2 s of ultrasonic power, 2 s of ultrasonic intervals, and a total processing time of 2 min; the optimal processing pressure for the extraction of algal blue protein by high-pressure homogenization was 60 MPa for three times. 50.0 g of Spirulina powder was taken into a 2.0 L beaker, and 1.0 L of ultrapure water was added and stirred well, then the cells were broken under the optimal conditions of ultrasonic crushing/high-pressure homogenization, and the broken solution was centrifuged at 4 ℃ for 10 min under the condition of 8,000 r - min-1, and the supernatant was subjected to the full-wavelength scanning (200-800 nm) and the absorbance at 620, 280, and 652 nm was measured. The absorbance at 620, 280 and 652 nm was measured, and the yield and purity of alginate obtained were calculated[22] , and the above experiments were repeated three times.

1.2.4 Determination of alginate (C alginate) content and purity

The absorbance at 620, 280 and 652 nm was measured, and the concentration, yield and purity of phycocyanin were calculated according to Eqs. 1~3[13] : Purity = A620 /A280

(1) where C:A- n,2 ,Ag6 21n∗ protein solution ()

The absorbance at 620,280,652 nm, C is the concentration of phycocyanin by volume (mg - mL-1 ), m is the mass of Spirulina powder (g), and V is the volume of phycocyanin solution (mL).

1.3 Salting and dialysis of algal blue proteins

The algal slurry obtained from the treatment was centrifuged in a freezing centrifuge at 4 ℃ and 8 000 r - min- 1 speed for 10 min, and the supernatant was taken, and the ammonium sulfate was added to 10%, 11%, 12%, 13%, 14%, 15% ammonium sulfate, and then stored in the refrigerator at 4 ℃ for 6 h, and then centrifuged at 8 000 r - min- 1 speed for 10 min, and then the supernatant was further supplemented with ammonium sulfate up to 50%, and then stored in a dialysis bag (8 000 ~ 14 000 Da) for 6 h at 4 ℃, and centrifuged at 4 ℃, 8 000 r - min- 1 for 10 min. The supernatant was further replenished with ammonium sulfate to 50%, stored at 4 ℃ for 6 h and centrifuged at 4 ℃ for 10 min at 8 000 r - min- 1. The resulting precipitate was dialyzed by dialysis bag (8 000 ~ 14 000 Da) for 12 h, and the endpoint of dialysis was examined by barium chloride.

1.4 Algae blue protein purification by column chromatography

A dextran gel column G-75 was used to further purify the dialyzed desalted samples. The dried dextran gel powder was soaked in distilled water for 24 h. The column was shaped as 2 cm × 50 cm with a bed height of 30 cm. The dextran gel bed was rinsed with PBS buffer pH 7.0 to bring the packing material into anionic and cationic equilibrium. The dialyzed samples were eluted with 0.1 mol-L-1 NaCl in a gradient (elution rate: 0.5 mL-min-1 ), and the samples were collected in tubes every 5 min. The absorbance at 620, 280 and 652 nm of the collected samples was measured and the concentration and purity of phycocyanin were calculated. The fraction with the highest purity was then scanned at full wavelength from 200 to 800 nm.

1.5 SDS-gel electrophoresis

The mass fraction of the separator was 15% and that of the concentrator was 5%. The electrophoresis voltage in the concentrated gel was 80 V for 30 min, and then 120 V for 120 min. After electrophoresis, the color was stained with Coomassie Brilliant Blue for 30 min, and the color was removed by decolorizing solution of acetic acid:dot:water = 1:1:8 until the color was removed.

1.6 Determination of color

The color was measured by TS7700 colorimeter, the light source for color measurement was D65, the spot diameter was 8 mm, 5 measurement points were selected evenly, and the colorimeter was calibrated with black and white boards, then the L ∗ a ∗ b ∗ value of each measurement point was measured and averaged, where L ∗ represents the brightness of the sample.

1.7 Experimental processing and statistical methods of data

The experimental data were analyzed using Origin Pro 9.0 and SPSS 9.0 software. All data were analyzed by one-way analysis of variance (ANOVA), and each group of experiments was repeated more than three times to ensure the accuracy of the data. Different letters represent significant differences between the data (P<0.05).

2 Results and analysis

2.1 High-pressure homogenization extraction of algal blue protein

High-pressure homogenization is a rapid method for cell breakage, which is mainly used to break the cell membrane by high pressure, and finally, the intracellular components are released[24] . Figure 1A shows the yield and purity of phycocyanin obtained from 50 g-L-1 Spirulina solution after three consecutive treatments at different pressures. The results showed that the C phycocyanin yield increased significantly with the increase of treatment pressure and reached a maximum value of 88.15 mg-g-1 at 60 MPa, and then there was no significant difference as the pressure continued to increase. In addition, the purity of C phycocyanin (A620/A280) increased significantly with increasing pressure, and was maintained at 0.63-0.66 (<0.7) from 20 to 60 MPa, and decreased to about 0.53 as the pressure was further increased to 80 MPa.

The main reason may be that with the increase of the treatment pressure, the damage to the cell wall and cell membrane was gradually increased, resulting in the dissolution of more C-protein in the cells, and finally the C-PC production was increased[15] . At 0~20 MPa, with the increase of pressure, the dissolution of phycocyanin in the cell increased significantly, and the purity of C-PC was also increased to the maximum value, and then with the increase of pressure to 80 MPa, the destruction of the cell wall was further increased, and the nucleic acid, sugars, lipids and other substances in the cell were released, and the purity of C-PC was decreased. Figure 1B shows the ultraviolet scanning map of Spirulina C alginate obtained at 60 MPa (200-800 nm), which showed that C alginate had the maximum absorption peak at 620 nm, which was basically consistent with the characteristic peaks of the previously reported alginate, indicating that the treatment of Spirulina C at 60 MPa had no effect on the structure of the alginate. Therefore, 60 MPa was selected as the optimum pressure for the extraction of C alginate from Spirulina sp. by high pressure homogenization method, taking into account the cost-effectiveness of extraction energy consumption.

Fig. 1 Effect of different treatment pressures on the yield and purity of C alginate and UV scan (60 MPa).

2.2 Ultrasonic extraction of algal blue protein

Ultrasonic cell disruption through ultrasound to break the cell wall of biological tissues, promote the release of extractable compounds within the cell wall, enhance the solvent from the continuous phase into the cell, increase the release of compounds within the cell to increase the yield of extracted intracellular material[25] . Figure 2A shows the effect of sonication time on the yield and purity of phycocyanin. The results showed that the yield of C alginate increased significantly to 81.29 mg-g-1 in the first 2 min, then there was no significant difference in the yield in 2~4 min, and the yield decreased significantly to 68.62 mg-g-1 with the further extension of the time to the end of 6 min. Due to the cavitation and mechanical effects of ultrasound, the cell wall can be broken and the intracellular substances can be outflowed in a very short time[26] , which can increase the solubility of C-cell cyanobacterial proteins and thus increase the yield of C-cell cyanobacterial proteins. Under the condition of 2~4 min, ultrasonic waves had no significant effect on the cell wall disruption with the increase of time, and the yield was almost unchanged.

With the increase of sonication time (4~6 min), the system may produce too much heat, which may lead to the denaturation of C alginate, resulting in the decrease of the yield. At the same time, with the increase of ultrasonication time, the intracellular impurities continued to be leached out, and the purity of C alginate (A620/A280) decreased significantly from 0.6 to 0.48 (Fig. 2A). Figure 2B shows the ultraviolet scanning map (200-800 nm) of Spirulina C alginate obtained by ultrasonication for 2 min. The results showed that C alginate had the maximum absorption peak at 620 nm, which was basically consistent with the characteristic peaks of the previously reported alginate, and indicated that ultrasonication for 2 min had no effect on the structure of the alginate. Therefore, 2 min was chosen as the optimum time for ultrasonic extraction of algal cyanobacterial proteins, taking into account the cost-effectiveness of extraction energy consumption.

2.3 Extraction of algal blue proteins by ultrasound and high pressure homogenization

Fig. 3 shows the effect of high-pressure homogenization coupled with ultrasonication on the extraction of cyanobacterial proteins from Spirulina under the optimal conditions of high-pressure homogenization and ultrasonication. As shown in Fig. 3A, the yield of C-PC obtained by high pressure homogenization coupled with ultrasonication was significantly higher than that obtained by high pressure homogenization or ultrasonication, but the purity of C-PC was significantly lower than that obtained by high pressure homogenization or ultrasonication. The maximum yield of C- PC obtained by high pressure homogenization coupled with ultrasonication was 131.2 mg-g-1 , followed by high pressure homogenization + ultrasonication (124.3 mg-g-1 ), high pressure homogenization (86.85 mg-g-1 ), and ultrasonication (82.96 mg-g-1 ), which increased the yields of C- PC by 51.07% and 58.15% compared with that obtained by high pressure homogenization and ultrasonication, respectively. The yields were 51.07% and 58.15% higher than those obtained by high pressure homogenization and ultrasonication respectively.

The main reason for this is that the destruction of cell walls and cell membranes by the HPHMA coupled ultrasound method is much higher than that of the individual treatment, and the cell contents such as proteins, nucleic acids, chlorophylls and polysaccharides were dissolved in large quantities, which finally led to a significant increase in the algal blue protein content and the lowest purity (0.69)[15] . The results showed that the efficiency of alginate extraction by this coupling method was much higher than that of previous literature reports, such as Tavanandi[15] and others, who used four freeze-thawing methods for 4 h to extract alginate with a yield of 73.73 mg-g-1 ; Pan-utai[26] and others, who utilized ultrasonication to extract the alginate with a yield of 60 mg-g-1 after 5 min extraction; Ilter[27] and others, who used homogenization to obtain C-alginate with a yield of 0.69 mg-g-1 ; and others, who used the homogenization method to obtain C-alginate with a yield of 0.69 mg-g-1 . Ilter[27] et al. obtained a yield of 67.61 mg-g-1 of C alginate by homogenization; Kaferbock[28] et al. obtained a yield of 119.48 mg-g-1 of C alginate by pulsed electric field treatment for 7 h. Therefore, in order to obtain a higher yield of C alginate, it is important to use the same method as that used for the extraction of C alginate by ultrasonic extraction. Therefore, in order to obtain a higher yield of cyanobacterial protein, ultrasonication + high pressure homogenization was used as the most suitable extraction method for C alginate in the subsequent experiments.

Figure 3B shows the ultraviolet full-wavelength scanning results of C phycocyanin obtained by the four methods, and the results show that the samples obtained by the four extraction methods have four absorption peaks at 280, 400~450, 620, and 670 nm, respectively. Previous studies have shown that 280 nm is the characteristic absorption peak of protein, 400~450 nm is the characteristic absorption peak of carotenoid, 620 nm is the characteristic absorption peak of phycocyanin, and 670 nm is the characteristic absorption peak of chlorophyll[29] . From 3B, it can be seen that the samples extracted by the four methods have higher absorption peaks at 280 and 620 nm, indicating that the main substance in the extract is phycocyanin; and the absorption peaks of the sample extracted by ultrasonication + high-pressure homogenization are higher than those of the other three curves, indicating that this extraction method has the highest yield of C phycocyanin, but it contains more impurities, and its purity is lower than that of the other three methods, which is in agreement with the results of Fig. 3A. This is consistent with the results of Fig. 3A.

2.4 Ammonium sulfate salting out and dialysis purification of algal blue proteins

Purification of C alginate using ammonium sulfate is based on the principles of salting out and salting out. At low concentrations, ammonium sulfate surrounds the protein molecules, which are salted (solubilized). At high concentrations, the salt ions are very strong and bind more easily to water molecules. The binding of salt to water molecules leads to an increase in the attraction between protein molecules, which results in hydrophobic interactions and a decrease in the solubility of the protein molecules, leading to the formation of a precipitate[30] . From Fig. 4, it can be seen that the purity of C alginate increased and then decreased with the increase of ammonium sulfate concentration. When the concentration of ammonium sulfate was 12%, the purity of C-PC (A620/A280) reached the maximum value (1.09), and then decreased to 0.76. The purity of C-PC (A620/A280) reached the maximum value (1.09), and then decreased to 0.76. In addition, with the increase of ammonium sulfate concentration, the recovery of C-PC in the supernatant remained unchanged at about 90% from 10% to 12% of ammonium sulfate concentration, and then decreased significantly to about 69.5% with the increase of ammonium sulfate concentration to 15%.

The main reason may be due to the principle of salt solubilization, at the beginning of ammonium sulfate can make impurities (chlorophyll, carotenoids, etc.) precipitated, and the target product will not be lost, so in the supernatant of C alginate recovery remains unchanged and the purity of the increase. With the increase of ammonium sulfate concentration (>12%), too much ammonium sulfate can make the C alginate precipitate down and the target product will be lost, so the recovery of C alginate in the supernatant decreased. Therefore, in this experiment, 12% ammonium sulfate was used as the first step of salting-out to dissolve the cyanobacterial protein, and then the concentration of ammonium sulfate was increased to 50% to precipitate C alginate completely for subsequent purification.

2.5 Glucan Gel G-75 Purification of Algal Blue Protein

Gel column chromatography (GCC) is a method to separate and purify proteins based on their relative molecular masses. Previous studies have shown that C alginate is structurally composed of two peptide chains, i.e., α-unit (13-20.5 kDa) and β-unit (11-24.4 kDa)[30] . The separation range of dextran gel G-75 is about 3~80 kDa, therefore, dextran gel G-75 was chosen for the purification of C alginate cyanobacteria.

Figure 5a shows the elution curves of C-PC at the absorbance of 280 and 620 nm. At 25 min elution time, the elution peaks appeared at 280 and 620 nm, and the absorbance value at 620 nm was much larger than that at 280 nm, and the purity of A620/A280 reached the maximum value of 4.20 (analytical grade ＞4.0), which may be due to the fact that the heteroprotein with a larger amount of molecules needed shorter time to pass through the dextran gel column G-75, and the heteroprotein with a smaller amount needed longer time to be separated from C-protein [Figure 5a]. The reason for this may be that it takes a shorter time for the larger heteroproteins to pass through the dextran gel column G-75, while the smaller heteroproteins take a longer time to separate from C alginate[29] , resulting in a decrease in the absorption peak at 280 nm. The purity of C-PC can be improved by separating the heteroproteins from C alginate by column analysis. Therefore, 25 min is the optimal time for the purification of phycocyanin by dextran gel G-75 column.

Figure 5b shows that the UV scan of the algal blue protein collected after 25 min of dextran gel G-75, the absorbance at 620 nm showed a large increase, and the absorbance at 280 nm showed a slight decrease, which was mainly due to the removal of heteroproteins after dextran gel G-75, and the decrease in absorbance at 280 nm, 300~400 nm, and the weak peak at 300~400 nm may be caused by the absorption of disulfide bonds in the protein. The weak peak at 300~400 nm may be caused by the absorption of disulfide bonds in the protein. After the dextran gel G-75, the purity of C alginate was further increased from 0.85 to 4.2, which reached the analytical purity.

2.6 SDS-PAGE and color of analytical grade algal blue protein

After two steps of salting out, dialysis and 25 min column chromatography, the purified sample was freeze-dried, and the yield of analytical alginate was 107.65 mg-g-1 with a purity of 4.23, and the recovery rate reached 82.05%. Figure 6 shows the SDS-PAGE and color of the analytical grade alginate. From Fig. 6a, it can be seen that the electrophoretic lane of alginate dialyzed after salting out had more bands and the bands were not obvious, which indicated that the sample obtained had more impurities and lower purity. On the other hand, the purified phycocyanin by dextran gel G-75 column chromatography showed fewer and clearer bands, indicating that most of the impurities were removed by column chromatography. Thus, the electrophoretic bands showed the target protein and the purification result was satisfactory, which was consistent with the purity results of phycocyanin obtained in Figure 4 and Figure 5a. In addition, the molecular weight of C alginate was assessed to be about 17 kDa by comparison with the Marker sample. This is basically consistent with the molecular weight reported in previous papers and others[31] . Figure 6b shows the color values of phycocyanin obtained after column chromatography purification, with a brightness (L ∗ value) of 58.07, a redness (a ∗ ) of - 15.44, and a yellowness off[32] .

3 Conclusion

In this paper, we investigated the separation and purification process of analytical grade C alginate from Spirulina, in order to produce analytical grade C alginate with low cost and high efficiency. Firstly, the pressure of high pressure homogenization and the ultrasonic time of ultrasonic method were optimized respectively, and the optimal conditions of the two methods were used together to improve the high knotting of C alginate by 51.07% and 58.15% compared with different treatment methods and higher than that of the previous report. The salt concentration of ammonium sulfate on alginate was optimized(1) . After purification by column chromatography on an ammonium-pressure, min-glucose gel column G-75, the yield and purity of alginate were 107.65 mg-g-1 and 4.23 (> 4.0), respectively, and the recovery rate reached 82.05%. In addition, the purity and molecular weight (17 kDa) of the purified C alginate were verified by SDS-PAGE, and the results were basically consistent with those of previous reports. In this paper, we improved the traditional extraction and purification process to produce analytical grade C alginate with high efficiency and low cost, which can provide technical support for the wide application of analytical grade C alginate in Spirulina.

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#Phycocyanin #Spirulina #cPhycocyanin #Phycocyaninpowder

#healthy food #healthy eating #veganfood

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