Encapsulation of Benzaldehyde Produced by the Eco-Friendly Degradation of Amygdalin in the Apricot Kernel Debitterizing Wastewater

Ci a ion: Song, L.; Ga cía Ma ín, J.F.; Zhang, Q.-A. Encapsula ion o Benzaldehyde P oduced by he Eco-F iendly Deg ada ion o Amygdalin in he Ap ico Ke nel Debi e izing Was ewa e . Foods 2024, 13, 437. h ps://doi.o g/10.3390/ oods13030437 Academic Edi o : Isabel Bo ás-Lina es Recei ed: 11 Janua y 2024 Re ised: 18 Janua y 2024 Accep ed: 23 Janua y 2024 Published: 29 Janua y 2024 Copy igh : © 2024 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). oods A icle Encapsula ion o Benzaldehyde P oduced by he Eco-F iendly Deg ada ion o Amygdalin in he Ap ico Ke nel Debi e izing Was ewa e Lei Song 1, Juan F ancisco Ga cía Ma ín2and Qing-An Zhang 1,* 1School o Food Enginee ing and Nu i ion Science, Shaanxi No mal Uni e si y, Xi’an 710119, China 2 Depa amen o de Ingenie ía Química, Facul ad de Química, Uni e sidad de Se illa, s/n, 41012 Se ille, Spain; [email p o ec ed] *Co espondence: [email p o ec ed]; Tel.: +86-13572932273 Abs ac : In o de o ully u ilize he by-p oduc s o ap ico ke nel-debi e izing and add ess he chemical ins abili y o benzaldehyde in he ood indus y, benzaldehyde was i s p epa ed by adding he ap ico ke nel powde o deg ade he amygdalin p esen in he ap ico ke nel-debi e izing wa e . Subsequen ly, β -cyclodex in was employed o encapsula e he benzaldehyde, and i s encapsula ion e icacy was e alua ed h ough a ious echniques including Fou ie ans o m in a ed spec oscopy, he mog a ime ic analysis, elease kine ics i ing inhibi o y e ec and he e ec on Bo y is cine ea. Finally, he encapsula ion was explo ed ia molecula docking and molecula dynamics simula ions. The esul s indica e ha he op imal p epa a ion condi ions o he benzaldehyde we e 1.8 h, 53 ◦ C and pH 5.8, and he encapsula ion o benzaldehyde wi h β -cyclodex in (wall–co e a io o 5:1, mL/g) has been e i ied by he decele a ion in he elease a e, he enhanced he mal s abili y and he p olonged inhibi ion e ec agains Bo y is cine ea. The encapsula ion p oceeded spon aneously wi hou s e ic hind ance in he simula ion, which led o a educ ion in he hyd ophobic ca i y o β -cyclodex in. In conclusion, he amygdalin in he debi e izing was ewa e can be deg aded in an eco- iendly way o p oduce benzaldehyde by adding ap ico ke nel powde , which con ains β-glucosidase ; he encapsula ion o benzaldehyde is s able, hus enhancing he u iliza ion o amygdalin in he debi e izing was ewa e o ap ico ke nels. Keywo ds: benzaldehyde; eco- iendly p epa a ion; amygdalin; encapsula ion; molecula simula ion 1. In oduc ion Amygdalin, a cyanogenic glucoside, can hyd olyze in o hyd ogen cyanide (HCN) in he body, which is a po en ial oxic subs ance abundan ly p esen ed in ap ico ke nels [ 1 ]. Thus, he emo al o amygdalin (debi e izing) was a necessa y p ocedu e in ap ico ke nel p ocessing [ 2 ]. A la ge amoun o amygdalin was ans e ed o he debi e izing was ew- a e , so he di ec discha ge o such was ewa e would cause en i onmen al pollu ion and he was e o esou ces [ 3 , 4 ]. Acco ding o p elimina y esea ch, Amygdalin can be deg aded in o benzaldehyde by β -glucosidase, making he ap ico ke nel debi e izing was ewa e a po en ial aw ma e ial o use in benzaldehyde p oduc ion [5]. Due o i s non- oxic na u e, ease o accessibili y, and possession o an ibac e ial and an ioxidan p ope ies, benzaldehyde demons a ed subs an ial po en ial o applica ions in he ood indus y. In addi ion, esea ch has shown ha benzaldehyde is an impo an subs ance o use in an i-co osion and mosqui o- epellen essen ial oils [ 6 – 8 ]. The mos commonly adop ed en i onmen ally iendly me hod o p epa ing benzaldehyde u i- lizes na u al cinnamon oil o cinnamaldehyde as aw ma e ials, bu hese me hods had d awbacks such as equi ing expensi e ca alys s, c ea ing ha sh eac ion condi ions, and showing suscep ibili y o eac ion ailu e [ 9 ]. Compa ed wi h he mos commonly adop ed Foods 2024,13, 437. h ps://doi.o g/10.3390/ oods13030437 h ps://www.mdpi.com/jou nal/ oods Foods 2024,13, 437 2 o 15 me hod, u ilizing amygdalin in debi e izing was ewa e as a aw ma e ial has many ad an- ages, such as lowe cos , he easy a ailabili y o he ca alys , he mild eac ion condi ions and he simple p ocess. The e o e, i is o g ea alue o esea ch he p ocess o deg ading amygdalin in debi e ing was ewa e in o benzaldehyde and ex ac ing i . Howe e , he di ec addi ion o benzaldehyde o ood has a ious d awbacks, including impac ing he ood’s as e due o i s ap ico ke nel la o , quick loss h ough apid ola iliza ion, and ans o ming in o benzoic acid unde he in luence o ul a iole ligh and oxygen. To enhance he applica ion o benzaldehyde in he ood indus y, an encapsula ion echnology needs o be de eloped o educe i s ola ile deg ada ion and con ol i s e- lease a e. As a equen ly u ilized encapsula ion ma e ial, β -cyclodex in can employ gues molecules o co e he ag ance, slow down he elease a e and imp o e he s a- bili y [ 10 , 11 ]. Szej li explained ha β -CD exhibi ed a deg ee o hyd ophobici y due o i s high elec on cloud densi y wi hin he ca i y.; he seconda y hyd oxyl g oup on he su ace o cyclodex in makes i s la ge opening end and ou e wall hyd ophilic. This unique cha ac e is ic o β -CD makes i a e sa ile hos o a ious gues molecules, including o ganic compounds, ino ganic ions, and ine gases, allowing i o enhance hei s abili y and enable con olled elease [ 12 ]. Fu he mo e, Rukmani’s esea ch con i med ha β -CD encapsula ion is among he mos e ec i e me hods o p o ec ac i e compounds om oxida ion, he mal deg ada ion and e apo a ion, and i also se es as an e icien app oach o masking undesi ed odo s o la o s while enhancing solubili y [ 13 ]. These examples ha e demons a ed ha he encapsula ion o benzaldehyde wi h β -cyclodex in is a iable solu ion o add ess he issues associa ed wi h benzaldehyde. Despi e esea ch ocusing on he encapsula ion o β -cyclodex in as a na u al p ese a i e o ood p ocessing ap- plica ions, no wo k has been done on he e ec s and mechanisms o he β -cyclodex in encapsula ion o benzaldehyde. Fu he mo e, he e has been a lack o esea ch on he p oduc ion o sa e and a ailable benzaldehyde h ough he applica ion o β -glucosidase o amygdalin ea men in ap ico ke nel-debi e izing was ewa e . In enhancing he added alue o ap ico ke nel p ocessing, his s udy did no di ec ly eco e amygdalin om debi e izing was ewa e , bu i inno a i ely ans o med i in o benzaldehyde, a compound wi h g ea e ma ke demand and wide applica ions. In e ms o bi e almond deg ada ion me hods, his s udy a oided cos ly mic obial deg ada ion, op ing o he u iliza ion o he complex enzymes p esen in he ap ico ke nel o deg ade he debi e izing was ewa e . In his esea ch, he ap ico ke nel-debi e izing was ewa e was u ilized as a aw ma e ial o p oduce benzaldehyde, and he associa ed pa ame e s we e op imized. Subsequen ly, β -cyclodex in was employed o encapsula e benzaldehyde; hen, he encapsula ion e ec was e alua ed by a ious means, and he encapsula ion mechanism has been explo ed ia molecula simula ions. The main pu pose o his esea ch was o sys ema ically explo e he easibili y o using amygdalin in he debi e izing was ewa e o p epa e benzaldehyde in an en i onmen - iendly way, and using β -cyclodex in o encapsula e benzaldehyde in o de o enhance he added alue o ap ico ke nels in he p ocessing indus y, as well as sol ing he p oblems o he ins abili y and ola ili y o benzaldehyde. 2. Ma e ials and Me hods 2.1. Ma e ials and Reagen s Ap ico ke nels we e pu chased om he No hwes He b Ma ke o Xi’an, Shaanxi P o ince, China. Wi h he impu i ies emo ed, he seeds we e soaked in boiling wa e o se e al minu es, and hen manually peeled. Then, 5 kg o he peeled ap ico ke nels we e pu in a s eam-jacked ke le wi h 50 L wa e (ma e ial o liquid a io o 1:10, kg:L) o debi e izing a he empe a u e o 65 o 75 ◦ C o 6 h, and he debi e izing was ewa e was collec ed and s o ed a 4 ◦C o u he use. Benzaldehyde s anda d was pu chased om Macklin Company (Shanghai, China). β -CD was pu chased om LookChem Company (Zhejiang, China). Me hanol o high- Foods 2024,13, 437 3 o 15 pe o mance liquid ch oma og aphy (HPLC) pu i y was pu chased om OceanPak Com- pany (Tianjin, China). 2.2. P epa a ion o Benzaldehyde The ap ico ke nel powde was added o he debi e izing was ewa e a o he a io (ma e ial o liquid) o 1:125 (g/mL), and hen he pH was adjus ed. Subsequen ly, he eac ion was ca ied ou a a ce ain empe a u e. The eac ion p oduc unde wen s eam dis illa ion, ollowed by ex ac ion wi h 80 mL o n-hexane and subsequen p essu e concen a ion, esul ing in he isola ion o benzaldehyde. 2.3. De e mina ion o Benzaldehyde Amygdalin and Hyd ocyanic Acid Con en De e mina ion o benzaldehyde: An isoc a ic elu ion was applied o he sepa a ion o benzaldehyde wi h he mobile phase o me hanol-H2O (35:65; / ), a 1.0 mL/min low a e, a 35 ◦ C column empe a u e, a 250 nm de ec ion wa eleng h and a 20 mL injec ion olume [ 14 ]. The s anda d cu e was y = 1345.2442x − 0.6348 (R 2 = 0.9998), and he con en o benzaldehyde was calcula ed De e mina ion o amygdalin: We e e ed o Zhang’s me hod and made sligh modi- ica ions. The HPLC wo king pa ame e s we e as ollows: a sample injec ion olume o 20 mL, a 35 ◦ C column empe a u e and a 1 mL/min low a e, wi h he a io o sol en A (me hanol):sol en B (wa e ) ¼ as 28:72 ( / ), and he wa eleng h was 214 nm [ 15 ]. The s anda d cu e was y = 131.90764x − 3.04545 (R 2 = 0.9984), and he con en o amygdalin was calcula ed. De e mina ion o hyd ocyanic acid: The pH o he solu ion was adjus ed using ace ic acid and sodium hyd oxide, ollowed by he addi ion o chlo amine T o he eac ion. Subsequen ly, isonico inic acid-ba bi u ic acid was in oduced o de elop he colo , and he abso bance was measu ed a a wa eleng h o 600 nm [ 16 ]. The s anda d cu e was y = 0.85882x −0.00781 (R2= 0.9991), and he con en o hyd ocyanic acid was calcula ed. 2.4. P ocess Op imiza ion o Deg ading Amygdalin in o Benzaldehyde by Response Su ace Me hodology Based on he esul s o single- ac o es s, h ee independen a iables we e se ( X1- ime ; X 2 - empe a u e; X 3 -pH) wi h he con en o benzaldehyde as he dependen a iable, and he Box–Behnken design o Response Su ace Me hodology (RSM) was em- ployed o op imiza ion. The e we e a o al o 15 es poin s, including 3 axial poin s o assessing expe imen al e o s, and he emaining 12 se s we e used as he analysis poin s. Table 1displays he encoded a iables conce ning he expe imen al ac o s and le els. Design Expe 8.0.6 so wa e was used o design he expe imen s and calcula e he da a. Table 1. Fac o s and hei le els o BBD o he p epa a ion p ocess o benzaldehyde. Independen Va iables Fac o −1 0 1 X1—Time (h) 1.5 2 2.5 X2—Tempe a u e (◦C) 65 55 45 X3—pH 5 6 7 2.5. P epa a ion o he bz-β-CD We e e ed o Aba ca’s encapsula ion p epa a ion p ocess wi h sligh modi ica- ions [ 17 ]. Benzaldehyde was dissol ed in anhyd ous e hanol a a olume a io o 1:30 (mL/mL). The e hanol solu ion o benzaldehyde was added o he aqueous β -CD solu ion o embedding, and he mix u e was le o 2 h. A e wa ds, i was emo ed and allowed o cool o oom empe a u e, and hen i was e ige a ed a 4 ◦ C o e nigh . The inal p oduc , bz-β-CD, was ob ained h ough il a ion and d ying. Foods 2024,13, 437 4 o 15 2.6. Cha ac e iza ion o he bz-β-CD Encapsula ion E ec 2.6.1. De e mina ion o Encapsula ion Ra e The a e (%) o he encapsula ion, indica ing he amoun o benzaldehyde encapsula ed in he β -CD ca i y, was de e mined using he Heyda i me hod [ 18 ]. Fo he de e mina ion o he o al oil con en o mic ocapsules, 0.1 g bz- β -CD was weighed ou , ully imme sed in 20 mL anhyd ous e hanol, ul asonica ed a 300 W, 59 HZ o 30 min, and allowed o s and o 1 h. The con en o benzaldehyde in e hanol was hen de e mined by he me hod shown in Sec ion 2.3. Acco ding o he measu ed su ace oil and he o al oil con en , he embedding a e was calcula ed using Equa ion (1). Encapsula ion a e = (V1−V2)/V1×100% (1) No e—V 1 : he olume o o al oil in mic ocapsules (mL). V 2 : he su ace oil olume o mic ocapsules (mL). 2.6.2. The E ec s o Di e en Wall–Co e Ra ios on Encapsula ion Ra e The bz- β -CD was p epa ed wi h wall–co e a ios (mL/g) o β -CD o benzaldehyde o 10:1, 5:1, 10:3, 5:2, and 2:1. The o al oil and su ace oil olume o he encapsula ion we e measu ed and he encapsula ion a e was de e mined acco ding o he me hod shown in Sec ion 2.6.1. 2.6.3. Fou ie T ans o m In a ed Spec oscopy Analysis (FI-IR) The po assium b omide and he sample we e d ied sepa a ely and hen mixed o g inding. Subsequen ly, hey we e p essed in o able s and scanned in he ange o 400–4000 cm−1 using a VERTEX 70 FT-IR Spec ome e (BRUKER, Ka ls uhe, Ge many) The numbe o scans was 16 and he esolu ion was 4 cm–1. 2.6.4. The mo-G a ime ic Analysis (TGA) The mog a ime ic analysis was pe o med using he he mal analysis equipmen . The low a e o N 2 gas was 20 mL/min, he a e o empe a u e inc ease was 10 ◦ C/min, and he maximum empe a u e was 650 ◦ C. In his way, he inal he mal weigh loss cu e was ob ained. 2.6.5. Pa icle Size Dis ibu ion Analysis The pa icle size and dis ibu ion o he bz- β -CD we e de e mined by lase di ac ion analysis using an LS13320 lase pa icle size dis ibu ion ins umen . Each g oup o samples o be es ed was epea edly measu ed h ee imes, and he D 10 , D 50 , D 90 and Span o he encapsula ion we e measu ed. The ela ionship be ween he ou is exp essed by he ollowing Equa ion (2). Span = (D90 −D10)/D50 (2) No e—Span: he pa icle size dis ibu ion ange o encapsula ion. D 90 : he cumula i e dis ibu ion o pa icles o 90%. D 10 : he cumula i e dis ibu ion o pa icles is 10% o he pa icle size. D 50 : he cumula i e dis ibu ion o pa icles is 50% o he pa icle size used o ep esen he a e age pa icle size o encapsula ion. 2.6.6. Release Kine ics Analysis The bz- β -CD was allowed o s and a 5 ◦ C, 30 ◦ C, and 55 ◦ C o a speci ic du a ion. Samples we e aken on days 0, 1, 2, 3, 4 and 5 o de e mine he o al oil con en ia he me hod in Sec ion 2.6.1, and we calcula ed he change in he benzaldehyde e en ion a e o encapsula ion using he ollowing Equa ion (3): Re en ion (%) = V4/V3×100% (3) Foods 2024,13, 437 5 o 15 No e—V3: The ini ial olume o bz-β-CD (mL). V4: The cu en olume o bz-β-CD (mL). The elease kine ics o bz- β -CD ha e been s udied by i ing he ze o-o de kine ics, i s -o de kine ics and A ami equa ion acco ding o he e en ion a e, using he me hod o Ren [19]. 2.6.7. Inhibi ion Expe imen o Mycelial G ow h o Bo y is cine ea Following he me hod ou lined by Edwa ds, he inhibi o y e ec s o benzaldehyde and bz- β -CD on Bo y is cine ea we e de e mined using he mycelial g ow h a e me hod [ 20 ]. The colony g ow h diame e was measu ed using he c oss me hod, and he g ow h inhibi ion a e o he mycelium was calcula ed. Then, he d ug concen a ion was con e ed in o he loga i hm o he concen a ion, and he mycelium g ow h inhibi ion a e was con e ed in o he inhibi ion a e p obabili y alue. The eg ession equa ion (y = aX + b) and co ela ion coe icien (R 2 ) we e ob ained, and he e ec i e bac e ios a ic concen a ion (EC50) was calcula ed. 2.7. Molecula Simula ion 2.7.1. Molecula Docking Simula ion Molecula docking o he β -CD and benzaldehyde molecules was pe o med using Au oDockTools-1.5.6 so wa e, applying he empi ical ee ene gy unc ion and Lama ckian gene ic algo i hm [ 21 ]. The ini ial s uc u e o he β -CD molecule was ob ained om he Camb idge c ys al da abase (CCDC, h ps://www.ccdc.cam.ac.uk/ (accessed on 9 June 2023)) (CCDC ID: 1235577). Likewise, he ini ial s uc u e o he benzaldehyde molecule was de i ed om he ZINC da abase (h p://zinc.docking.o g/ (accessed on 9 June 2023)) Each docking p ocess consis ed o 50 independen dockings, and he con o ma ion wi h he lowes docking ene gy was selec ed based on ene gy magni ude. 2.7.2. Molecula Dynamics Simula ion Molecula dynamics simula ions we e unde aken ia he p e iously epo ed me h- ods, wi h some modi ica ions made [ 22 ]. The ini ial con o ma ion o bz- β -CD was aken om he p e ious docking esul s, and he opology was c ea ed based on he a ailable GROMACS da abase. GROMACS 2020.6 so wa e was used o simula e β -CD and bz o 50 ns. 2.8. S a is ical Analysis All he abo e measu emen s we e epea ed in iplica e, and he esul s ha e been exp essed as mean ± s anda d de ia ion. The signi icance o he da a was analyzed using SPSS 25 so wa e. p< 0.05 was conside ed as indica ing a signi ican co ela ion, and p< 0.01 was conside ed as indica ing an ex emely signi ican co ela ion. In addi ion, O igin2021 was used o d aw he ele an cha s. SPSS was used o he signi icance analysis. 3. Resul s and Discussion 3.1. Op imiza ion o Benzaldehyde P epa a ion P ocess The esul s o he es a e shown in Table 2, and he e ec s o he h ee ac o s on he con en o benzaldehyde ha e been in e ed om he esul s o he ollowing quad a ic polynomial eg ession equa ion. The independen a iables and he es a iables we e co ela ed using he ollowing second-o de polynomial equa ion: y = 2.12 −0.11 ×X1−0.15 ×X2−1.86 ×10−3×X3−0.014 ×X1×X2+ 0.13 ×X1×X3− 0.041 ×X2×X3−0.18 ×X12−0.42 ×X22−0.14 ×X3(4) The esul s o he analysis o a iance, pe o med ia a eg ession model, a e shown in Table 3. The F alue o he model is 13.95, he p alue is 0.0049, and he p alue o he misma ch e m is 0.2395, indica ing ha he model is signi ican and can be used o op imiza ion. A he same ime, i can be seen om Table 3 ha he linea coe icien (X 1 , X 2 ) and c oss coe icien (X 1 X 3 ) a e signi ican a he p< 0.05 le el. The h ee-dimensional Foods 2024,13, 437 6 o 15 esponse su ace and con ou s o benzaldehyde a e shown in Figu e 1a,c,e. In gene al, an ellip ical con ou ep esen s a signi ican in e ac ion be ween independen a iables, while a ci cula in e ac ion is no signi ican [ 5 ]. F om Table 3and Figu e 1b,d, , we can in e ha he in e ac ion be ween X 1 and X 3 is signi ican , while he in e ac ion be ween X 1 X 2 and X 2 X 3 is no signi ican . This indica es ha he e ec o ime and pH on he p oduc ion o benzaldehyde in ol es a ce ain in e ac ion. Acco ding o he p edic ions o he quad a ic eg ession model, he op imum pa ame e s o benzaldehyde p oduc ion a e: ime 1.82 h, empe a u e 53.34 ◦ C, pH 5.84 and benzaldehyde con en 2.1539 g/L. Fo indus ial ap- plica ions, he op imum o mula can be modi ied as: ime 1.8 h, empe a u e 53 ◦ C, pH 5.8 and benzaldehyde con en 2.1521 g/L. The ela i e e o be ween he p edic ed alue and he measu ed alue is only 0.8%, indica ing ha he model has high accu acy and can simula e he ac ual si ua ion well. Table 2. Box–Behnken expe imen esul s o he p epa a ion o benzaldehyde. Tes X1—Time (h) X2—Tempe a u e (◦C) X3—pH Y—Benzaldehyde (g/L) 1 2 45 5 1.674874309 2 1.5 45 6 1.726983809 3 2.5 45 6 1.662509763 4 2 45 7 1.740413837 5 2.5 55 5 1.496666262 6 1.5 55 5 2.11229181 7 2 55 6 2.169431375 8 2 55 6 2.14237309 9 2 55 6 2.053541632 10 2.5 55 7 1.757337655 11 1.5 55 7 1.848597421 12 2 65 5 1.469880542 13 2.5 65 6 1.302500764 14 1.5 65 6 1.397502106 15 2 65 7 1.514259103 Table 3. Va iance analysis o eg ession equa ion. Sou ce SS d MS F Value pValue Model 1.11 9 0.12 13.95 0.0049 ** X10.099 1 0.099 11.27 0.0202 * X20.18 1 0.18 20.02 0.0066 ** X32.781 ×10−512.781 ×10−53.157 ×10−30.9574 X1X27.647 ×10−417.647 ×10−40.087 0.7801 X1X30.069 1 0.069 7.80 0.0383 * X2X36.618 ×10−316.618 ×10−30.75 0.4258 X120.12 1 0.12 13.13 0.0152 X220.64 1 0.64 72.60 0.0004 X320.073 1 0.073 8.34 0.0343 Residual 0.044 5 8.811 ×10−3 Lack o Fi 0.037 3 0.012 3.33 0.2395 Pu e E o 7.351 ×10−323.676 ×10−3 Co To al 1.15 14 No e: * ep esen s a signi ican di e ence (p< 0.05), ** ep esen s a highly signi ican di e ence (p< 0.01). A e he addi ion o he ap ico ke nel, he amygdalin in he ap ico ke nel-debi e izing was ewa e dec eased om 14.6 o 1.98 g/L, ep esen ing a deg ada ion a e o 86.43%. The con en o benzaldehyde in he inal p oduc was 198 g/L, and he cyanide con en was 4.856 × 10 −6 g/L. These esul s indica e ha his me hod enables en i onmen ally iendly, apid, and mild amygdalin deg ada ion. The benzaldehyde ob ained h ough his me hod exhibi ed high pu i y, while he cyanide con en emained low. The cyanide p oduced by Foods 2024,13, 437 7 o 15 he deg ada ion o amygdalin was en iched in he dis illed liquid h ough s eam dis illa ion, acili a ing he debi e izing was ewa e ea men . O e all, hese indings sugges ha his me hod is a iable and en i onmen ally iendly app oach o benzaldehyde p epa a ion. Foods 2024, 13, x FOR PEER REVIEW 7 o 16 X3 2.781 × 10−5 1 2.781 × 10−5 3.157 × 10−3 0.9574 X1X2 7.647 × 10−4 1 7.647 × 10−4 0.087 0.7801 X1X3 0.069 1 0.069 7.80 0.0383 * X2X3 6.618 × 10−3 1 6.618 × 10−3 0.75 0.4258 X12 0.12 1 0.12 13.13 0.0152 X22 0.64 1 0.64 72.60 0.0004 X32 0.073 1 0.073 8.34 0.0343 Residual 0.044 5 8.811 × 10−3 Lack o Fi 0.037 3 0.012 3.33 0.2395 Pu e E o 7.351 × 10−3 2 3.676 × 10−3 Co To al 1.15 14 No e: * ep esen s a signi ican diffe ence (p < 0.05), ** ep esen s a highly signi ican diffe ence (p < 0.01). Figu e 1. Response su ace plo s and con ou plo s o p epa a ion p ocess o benzaldehyde. No e: (a,c,e): Response su ace plo s o he e ec s o empe a u e and ime, pH and ime, pH and empe a- u e on he con en o benzaldehyde, espec i ely. (b,d, ): Con ou plo s o he e ec s o empe a u e and ime, pH and ime, pH and empe a u e on he con en o benzaldehyde, espec i ely. Figu e 1. Response su ace plo s and con ou plo s o p epa a ion p ocess o benzaldehyde. No e: (a,c,e): Response su ace plo s o he e ec s o empe a u e and ime, pH and ime, pH and empe a- u e on he con en o benzaldehyde, espec i ely. (b,d, ): Con ou plo s o he e ec s o empe a u e and ime, pH and ime, pH and empe a u e on he con en o benzaldehyde, espec i ely. The colo s ep esen he size o he esul . 3.2. Cha ac e iza ion o Benzaldehyde Encapsula ion by β-CD 3.2.1. The E ec s o Di e en Wall–Co e Ra ios on he Encapsula ion Ra e o bz-β-CD I can be seen om Figu e 2a ha he encapsula ion a e inc eased i s , and hen dec eased, wi h he ise in he wall–co e a io. The wall–co e a ios o 5:1 and 10:3 (mL/g) exhibi ed signi ican ly highe encapsula ion a es han he o he h ee a ios. Due o he limi ed capaci y o β -CD o encapsula e benzaldehyde, he excess benzaldehyde will no be encapsula ed, so a high wall–co e a io esul s in a dec ease in he encapsula ion a e. The e o e, in he subsequen expe imen s, a wall–co e a io o 5:1 (mL/g) was chosen as he encapsula ion condi ion o benzaldehyde. Foods 2024,13, 437 8 o 15 Foods 2024, 13, x FOR PEER REVIEW 8 o 16 A e he addi ion o he ap ico ke nel, he amygdalin in he ap ico ke nel-debi e - izing was ewa e dec eased om 14.6 o 1.98 g/L, ep esen ing a deg ada ion a e o 86.43%. The con en o benzaldehyde in he inal p oduc was 198 g/L, and he cyanide con en was 4.856 × 10−6 g/L. These esul s indica e ha his me hod enables en i onmen- ally iendly, apid, and mild amygdalin deg ada ion. The benzaldehyde ob ained h ough his me hod exhibi ed high pu i y, while he cyanide con en emained low. The cyanide p oduced by he deg ada ion o amygdalin was en iched in he dis illed liquid h ough s eam dis illa ion, acili a ing he debi e izing was ewa e ea men . O e all, hese indings sugges ha his me hod is a iable and en i onmen ally iendly app oach o benzaldehyde p epa a ion. 3.2. Cha ac e iza ion o Benzaldehyde Encapsula ion by β-CD 3.2.1. The Effec s o Diffe en Wall–Co e Ra ios on he Encapsula ion Ra e o bz-β-CD I can be seen om Figu e 2a ha he encapsula ion a e inc eased i s , and hen dec eased, wi h he ise in he wall–co e a io. The wall–co e a ios o 5:1 and 10:3 (mL/g) exhibi ed signi ican ly highe encapsula ion a es han he o he h ee a ios. Due o he limi ed capaci y o β-CD o encapsula e benzaldehyde, he excess benzaldehyde will no be encapsula ed, so a high wall–co e a io esul s in a dec ease in he encapsula ion a e. The e o e, in he subsequen expe imen s, a wall–co e a io o 5:1 (mL/g) was chosen as he encapsula ion condi ion o benzaldehyde. Figu e 2. Cha ac e iza ion o bz-β-CD. No e: (a) Effec s o diffe en wall–co e a ios on encapsula- ion a e; (b) FTIR spec a o β-CD, benzaldehyde, and bz-β-CD; (c) TGA cu e o β-CD, benzalde- hyde, and bz-β-CD; (d) Pa icle size dis ibu ion o bz-β-CD. 3.2.2. Fou ie T ans o m In a ed Spec oscopy Analysis o bz-β-CD The in a ed spec um, p esen ed in Figu e 2b, e eals se e al no able abso p ion peaks. Speci ically, a 2732 cm−1, 1709 cm−1, and 1586 cm−1, dis inc abso p ion peaks co - espond o he C–H s e ching o he benzaldehyde aldehyde g oup, he C=O s e ching o he benzaldehyde aldehyde g oup, and he benzaldehyde benzene ing, espec i ely [23]. The abso p ion peaks o bz-β-CD a 2732 cm−1 and 1709 cm−1 we e diffe en om hose Figu e 2. Cha ac e iza ion o bz- β -CD. No e: (a) E ec s o di e en wall–co e a ios on encapsula ion a e; a–c in Figu e 2a a e he esul s o LSD es . Di e en le e s indica e signi ican di e ences in esul s; (b) FTIR spec a o β -CD, benzaldehyde, and bz- β -CD; (c) TGA cu e o β -CD, benzaldehyde, and bz-β-CD; (d) Pa icle size dis ibu ion o bz-β-CD. 3.2.2. Fou ie T ans o m In a ed Spec oscopy Analysis o bz-β-CD The in a ed spec um, p esen ed in Figu e 2b, e eals se e al no able abso p ion peaks. Speci ically, a 2732 cm −1 , 1709 cm −1 , and 1586 cm −1 , dis inc abso p ion peaks co espond o he C–H s e ching o he benzaldehyde aldehyde g oup, he C=O s e ching o he benzaldehyde aldehyde g oup, and he benzaldehyde benzene ing, espec i ely [ 23 ]. The abso p ion peaks o bz- β -CD a 2732 cm −1 and 1709 cm −1 we e di e en om hose o β -CD, bu he same as hose o benzaldehyde, indica ing he encapsula ion o benzaldehyde by β -CD. Addi ionally, bz- β -CD also showed di e en abso p ion peaks a 1586 cm −1 om β -CD, because β -CD lacked a benzene ing and he encapsula ed bz- β -CD con ained a benzene ing. Howe e , he abso p ion peaks o he aldehyde C–H bond and he benzene ing o bz- β -CD a e no ob ious, which may be due o he masking o inhibi o y e ec s o he β -CD ca i y on he abso p ion ib a ion. Qiao obse ed simila phenomena in he in a ed spec um o he HP- β -CD–as axan hin es e inclusion complex, whe eby some cha ac e is ic bands belonging o he six-membe ed ing o as axan hin es e we e missing, p esumably because he six-membe ed ing en e ed he HP- β -CD ca i y and o med a hyd ogen bond wi h -OH on he ca i y [ 24 ]. In conclusion, i was p o en ha benzaldehyde was encapsula ed in β-CD. 3.2.3. The mog a ime ic Analysis o β-CD, Benzaldehyde, and bz-β-CD Acco ding o he TGA cu e shown in Figu e 2c, benzaldehyde unde wen a weigh loss o up o 98.4% in he empe a u e ange o 40 ◦ C o 180 ◦ C, indica ing ha he he mal s abili y o benzaldehyde is poo . In con as , he ini ial weigh loss o bz- β -CD was only 8.74% a 25 ◦ C o 143 ◦ C, associa ed wi h a loss o wa e du ing he hea ing p ocess. The second s age o weigh loss occu ed be ween 260 ◦ C and 370 ◦ C, wi h a no able weigh educ ion o 72.9%, ela ed o he he mal decomposi ion o he composi e in his s age. In he empe a u e ange o 200 ◦ C o 300 ◦ C, bz- β -CD unde wen a deg ee o weigh loss di e en om β -CD, due o he ola iliza ion o benzaldehyde in bz- β -CD. In he case o complexa ion, he in e ac ion be ween he hos and gues enhances he s abili y o ypically ola ile gues molecules wi hin he inclusion complex. A e he o ma ion o bz- β -CD, he mass loss o benzaldehyde occu s a empe a u es anging om 200 ◦ C o 300 ◦ C, encapsu- la ing he benzaldehyde and enabling i o endu e highe empe a u es. In summa y, he Foods 2024,13, 437 9 o 15 he mal s abili y o benzaldehyde is signi ican ly enhanced a e encapsula ion by β -CD. Celebioglu epo ed ha he weigh loss o ee eugenol was comple ed a 50–190 ◦C, and he inclusion complex had wo weigh loss p ocesses, namely, he dehyd a ion p ocess be o e 130 ◦ C and he main deg ada ion p ocess a 330 ◦ C, indica ing ha he he mal s abili y o eugenol was signi ican ly imp o ed a e encapsula ion wi h γ -cyclodex in [ 25 ]. The change in he mal s abili y a e encapsula ion was consis en wi h his esea ch. This phenomenon also p o es ha he encapsula ion o β -CD enhances he mal s abili y, and β-CD can be employed o encapsula ing benzaldehyde. 3.2.4. Pa icle Size Analysis o bz-β-CD As shown in Figu e 2d, he pa icle size dis ibu ion o bz- β -CD is mainly be ween 0.4 and 7.42 µ m, wi h a capsule size o 1.707 µ m. The span measu es 2.72 µ m, indica - ing a ela i ely na ow pa icle size dis ibu ion. These da a collec i ely indica e ha he p epa ed encapsula ion exhibi ed minimal a ia ions in pa icle size and a igh ly concen a ed dis ibu ion. These cha ac e is ics sugges ha bz- β -CD did no unde go signi ican polyme iza ion. 3.2.5. E ec s o Di e en Tempe a u es on he Release Kine ics o bz-β-CD The i ing cu e o encapsula ion elease kine ics is shown in Figu e 3, and he speci ic i ing equa ion and R 2 a e shown in Table 4. Unde condi ions o 5 ◦ C, 35 ◦ C, and 55 ◦ C, he R 2 o each eg ession equa ion was abo e 0.9, indica ing ha A ami’s equa ion had a good i o he elease p ocess o benzaldehyde a di e en empe a u es. The alues o he elease mechanism pa ame e “n” o he encapsula ion a 5 ◦ C, 35 ◦ C, and 55 ◦ C we e de e mined as 0.5467, 0.5982, and 0.6252, espec i ely. These alues all wi hin he ange o 0.54 o 1.00, signi ying ha he elease eac ion kine ics align wi h a egime somewhe e be ween di usion-limi ed kine ics and i s -o de eac ion kine ics. Compa ed wi h ze o- o de kine ic i ing, i s -o de kine ic i ing showed excellen i ing, and was able o mo e accu a ely ep esen he elease beha io o benzaldehyde. A 5 ◦ C, 35 ◦ C and 55 ◦ C, he elease a e cons an s (k) calcula ed by A ami’s equa ion we e 0.1518, 0.1643 and 0.1760, espec i ely, which inc eased wi h he inc ease in empe - a u e. This esul indica ed ha benzaldehyde is indeed encapsula ed wi hin β -CD, and empe a u e exe s a p onounced in luence on i s elease cha ac e is ics. Consequen ly, i is ad isable o s o e bz- β -CD a lowe empe a u es o enhanced con ol o e benzalde- hyde elease. Foods 2024, 13, x FOR PEER REVIEW 10 o 16 Figu e 3. Release kine ics analysis o bz-β-CD. (a) Release cu e o bz-β-CD a h ee diffe en em- pe a u es; (b) ze o-o de i ing; (c) i s -o de i ing; (d) A ami’s i ing. Table 4. Release kine ics pa ame e o bz-β-CD. Tempe a u e (°C) Fi ing Equa ion R2 A ami’s i ing 5 y = 0.54665x − 1.88522 0.91091 30 y = 0.59824x − 1.80598 0.90536 55 y = 0.62519x − 1.73751 0.95358 Ze o-o de i ing 5 y = 3.98832x + 6.76983 0.91297 30 y = 4.65976x + 7.24012 0.92636 55 y = 5.26538x + 7.75528 0.93743 Fi s -o de i ing 5 y = −0.5382x − 0.0598 0.94713 30 y = −0.6676x − 0.05795 0.96374 55 y = −0.7991x − 0.05568 0.97764 A 5 °C, 35 °C and 55 °C, he elease a e cons an s (k) calcula ed by A ami’s equa ion we e 0.1518, 0.1643 and 0.1760, espec i ely, which inc eased wi h he inc ease in empe - a u e. This esul indica ed ha benzaldehyde is indeed encapsula ed wi hin β-CD, and empe a u e exe s a p onounced in luence on i s elease cha ac e is ics. Consequen ly, i is ad isable o s o e bz-β-CD a lowe empe a u es o enhanced con ol o e benzalde- hyde elease. 3.2.6. Effec s o Diffe en Concen a ions o bz and bz-β-CD on An i ungal Ac i i y o Bo y is cine ea As shown in Figu e 4a,b, bz-β-CD and benzaldehyde ha e diffe en capaci ies o inhibi ing he colony g ow h o Bo y is cine ea. I is shown ha he inhibi ion a e o my- celial g ow h ollows a concen a ion-dependen pa e n, wi h an ibac e ial efficacy in- c easing as concen a ion ises. The effec i e inhibi o y concen a ions (EC50) we e calcu- la ed o be 0.2029 µg/mL o benzaldehyde and 0.1929 µg/mL o bz-β-CD, based on linea eg ession equa ions de i ed om diffe en concen a ions and hei co esponding my- celial g ow h inhibi ion a es, as p esen ed in Figu e 4b,d. Because he effec i e inhibi o y Figu e 3. Release kine ics analysis o bz- β -CD. (a) Release cu e o bz- β -CD a h ee di e en empe a u es; (b) ze o-o de i ing; (c) i s -o de i ing; (d) A ami’s i ing.