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    Immobilization of Baker's Yeast in The Alginate-Based Hydrogels

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    Immobilization of baker's yeast in the alginate-based hydrogels

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    Foud Boseence 42 (2021) 101143 Contents lists avalable nt ScienceDiceet es i Food Bioscience ELSEVIER journal homepage: wwww.clsevier comocatortbio iy Immobilization of baker’s yeast in the alginate-based hydrogels to impart sensorial characteristics to frozen dough bread ‘A, Mihaly Cozmuta‘*’, A. Jastrzebska", R. Apjok", M. Petrus”, L, Mihaly Cozmuta®, A. Peter", G. Nicula* Tal Cert of Napoca, Dagan of Chr Bl, Ba Moe, Rana Wana Une of Taal, Fay of Maria Slee on gr, Wa, Pend ARTICLE INFO ABSTRACT a ee = ee == ee ere eee erie rere lated in CAY ane CASY. The Sensory evaluation of bread bake from bh esha frozen doughs revealed that the highest scores forest color and fumes, ast, smell and overall acceptably were awarded to bread Inked using ough containing CAY encapsulated yeast char was subjected to 3 months sarage. The sores ob- ‘tained for eruni texture and specif volume (61 and 45, respectively) fo frozen dough bead containing CA. capsule yeast were sins tthe highest scores, which were awarded o bread mae fom es dough with fee yeast els (363 and 4.84, respectively). Mhese ess indicate that the nmobzaton of baker's eas in an alginate based tyogel could representa viable solution fr overcoming yeast cyosenstvy, allowing for {he podeton of rozen dough bread with improved sexsoralearacteisties, whic would be highly acceptable by coasmers and opening new avec forthe eonomic str of fozen baked god progies 1. Introduction siicrowaving took less than $0 min to thaw while those thawed at ambient and refrigerated temperatures required about 100 min and Frozen dough bread is obtained through the application of free ‘ing-frozen storage thawing cycle to dough pleces, followed by proof. Ing and baking. The production of fozen dough bread products has Increased rapidly daring recent years, an the market for frozen dough ‘read products is predicted to grow ata compound annual growth rate (CAGR) of 4.3% during the forecast period of 2020-2025 (Mordor In teligence Report, 2020). The inereased demand for frazen dough bread is associated with convenionce-rlated factors nt bakeries and rest rants shortening the time required prior to baking; allowing forthe use ‘of highly mechanized processes to produce dough, which can reduce production costs; ineressing the Ukelthood of obsining a uniform ‘quality av all times; felltating and improving transportation logistics; ‘and removing the necesity of night work at bakeries (Rosell & Gone, 2007), The frozen doughs need to be thawed before proofing, the thawing methods significantly influence the thawing times, The work of ‘Yang eal (2020) indicates that the frozen doughs subjected (0 * Gonespning shot. Ema eres scstilyconn@ gui (A Mihaly Cae), bups/doog/10.1016/.io-2021.101143 220 min, respectively. The largest barrier 1o the development and growth of the frozen dough bread market isthe reduced sensorial ‘haraeteristis associated with frozen dough bread compaed with those offiesh dough bread (wiarker Dain Forecast, 2020). The freezing fozen storage thawing cycle results in the disruption of the gluten network due tothe release of reducing compounds (especialy glutathione), che depolymerization of gluenin aggrogates, the formation of ice crystal, ‘and the modification of strc (Fn eta, 2020). Yeast cells viability it flso daniaged during the cycle due to an increase in osotie presse, oxidative stress, and the damaging effects of ce (La eal, 2020; Omedi cecal, 2019), likely resulting inthe reduction of yeast population in the thinwed dough. The changes result in the production af bread with poor fermentation, low specific volunes, surfaces that are prone to cracking, taneven internal structures, increased hardness and chewiness, and decreased springiness and resilience (Peng eal, 20205 He eal, 2020; ‘Wang el, 2017), The addon of oxidizing agents (Such as organic Receive 21 January 2021; Reeived in revised form 14 May 2021; Accepted 17 May 2021, ‘Available online 31 May 2021 22124292/0) 2001 Hever Ld Al igs veserved. A: iat Cooma acids and thelr salts) eam proteet the gliten nexwork in frozen doug, Increasing gluten strength and resulting in bread with higher specific volumes. The addition of yeasts at levels 20%-100% above normal levels stil sults insignificant losses o fermentation eapacty der the damaging effects of frozen storage. Various approaches have been pro posed to improve yeast exyotolerance, such as increasing their vitality ‘with trehalose and e-poly lysine (Let al, 2020), or optimizing the freezing rate (Fao etal, 2016), but further investigations to find new "approaches renin necessary Several authors have reported the ability to overcome some of the Inurdls associated with the bread.making process through the applica tion of the encapsulation technique (Aliairano-Foril etal, 20125 Pasija et al, 20155 Bryszewsks ex all, 2019). However, the available sclentific iteraure exploring the application of encapsulation tech niques to the refrigerated and frozen dough sectors scare. The shelf fe ‘of refigerated dough was succesflly extended through the immobil zation of yeast into algnate/gelatin microbeads, whieh allowed for the ‘complete arrest of fermentation at temperatures below 10 °C, with the resumption of leavening as the temperature inereased (Gusti ct al. 2008) To the best of our knowledge, no report in the literature have ‘examined the application of encapsulated yeast cells to improve the sensorial cherscteristies of frozen dough bread. Our study was per formes 10 examine the sensorial atibutes of bread baked from frozen ovigh containing immobilized baker's yeast encapsulated in either alginate or alginate-starch beads, “The study was organized into two levels.) First beads containing calcium alginate yeast (CAY) and calehum alginate-stare-yeast(CASY) ‘were prepared and characterized. Alginate was selected due to its ad ‘vantages over other polymers, including the formation of water based ‘matrix, room-temperature gelation, andthe rapid release of embedded ‘compounds de tothe high porosity of ges. Starch was used as fille. ‘One type of ench system wth the best enempaulation parameters vas Pot cn 42 (2021 10119 selected, stored frozen for one and theee months, and the morphology, struenite, and cryotolerance ofthe encapsulated yeast cells were char acterized compared with those of free yeast eels. (i) Second, bread Joaves were baked fom ether fesh or frozen doughs containing fre oF encapsulated yeast, and thei physial and sensorial characteristics were examine. 2, Materials and methods 2.1. Preparation and storage of alginate based beads ‘The extemal gelation method was wed to prepare GAY and CASY beads Beads were prepared by dissolving 2g of sodium alginate (Sign Aldril, St Louis, USA, with a viscosity of 25.7 eps at 25 °C), either lone (for GAY) or wth diferent aniount of commercial com starch (for CCASY, Tobie 1, Desert, Bucegi, Romania), in 100 mi of eistilled water, which was stirred for 1h at 25°C (Heidolph Instruments, Schwabach, Germany) and sonicated for 20 min (Sonorex Super, Bandelin Elec tronic, Berlin, Germany) a 35 kHz Different amounts of commercial éry yeast (Saccharomyces cerevisiae, DY, Pakmaya, Pascani, Romani) ‘ging from 11010 , were addled tothe prepared sodium alginate and sodium alginate-starch gels (ble), followed by homogenization for 30 min. The solutions were fed through a perstlie pump (PMOS ARS Laboratoire, Bz, France) at 50 rpm and extruded throngh a capillary tube ameter 1 mn) int a bath contsining 500 miLof 0 mol/L CaCl, (Merck, Darmstadt, Germany), in which the Deads were maintained for 15 min to harden. After filtration, the beads were rinsed with distilled water to remove exces cle ions, To vod dansnging the yeas cells, the beads were environmentally dried (22.°C) until they reached @ constant weight. DY and CASY and CAY beads were stored for one and 8 months at ~18 °C in a conventional freezer (Zanussi Chest Freezer, Pordenone aly), thawed for 20 min at 25°C, and submitted to annie. ‘Table Encapsulation parameters. Sample code ‘Compan’ (we Ledafiadr wh vinleyeant Dey mater fads napanion Amount af ied bende quali ~ ‘cl sto "ei owen licen, ype, pot bende gD ‘Calcium algite yeast Alginatestarch: 037 400% 82 + 0138 592 + 095 1746 +031 ‘iniwestrch: = 18 son"! samesos mesos’ arosaaat 1a tome oumnsost® —— gnaetnast ‘num ago en so.ta sealsasi® —sisataas* ——aspsoart Alpmesarc: 1.764009" 20 = 041 movsas? 380 Alsmecsarc: 06 002" sais coast sasrtoat sami" Alsmicsurch: 042 LOOM suas aria’ tsat bose Alsmicsare: 115 L008 ouaosoam —eiastias! ssp aac ‘CRY 201-0, CASY 2:11100 the agen of bends ld or osc prope, probaly du od high Muyo the slatons CASY 22125100 the fora ‘was nor prepted because the ain was to investigate the ado f stale tothe open formula of CAV: 2.05:100 wil incsese the encapsulation eleney,CASY 22:7:100 high viscosity ofthe gel olded vs indicates the beads fous that result in opti patamneters in emi of Lond of tds with vinble ya cell, Encapalation eficency and Laweat amount of dried besds equivalent 1g of iy Yeas. ‘Average value + standard deviation SD (a ~ atleast 3). Ineach column, mean ves with even ters are igndfeanly fren at p< 0.0, ‘The diferenition was made bas on the Tey test post hoe ANOVA (BM SPSS Statistics Version 2). A: iat Cooma ‘nfrozen DY and freshly prepared CASY, and CAY were also considered In te investigations. 22. Eneapslaion parameters ‘The moisture content (6) was measured by subjecting DY and CASY ‘and CAY beads to 105 °C until they reached a constant weight and was calculated asthe ratio between the dry weight andthe initial weight of each sample ‘The numbers of viable yeast cells in DY and beads were counted ‘according to the procedure deseribed in 180 6887-4:2017 method. Five ‘grams of sample was shaken vigorously in. 45 mL sterile buffered peptone water (0.1% w/w) in astomacher, and 0.1 ml-of stil dilutions ‘of each suspension, up to 10, were transferred onto Pet dishes con taining dichloran Rose Bengal’ chloramphenicol (DRBC) agar (Oxoid, LTD, England). The plates were incubated for 5 day at 25 °C, and the developed colonies were counted. The encapsulation efficiency was calculated according othe following equation: BS. 92100/y @ where: E Is the encapsulation efficiency (4); Ay Is the number of yeast cells add to the gels during bead preparation (in colony forming units, ‘GPU and Asis the number of yeast eels entrapped in the beads (CFU). ‘The amount of CAY or CASY beads equivalent (0 1 g of DY was calculated according 0 the following equation Avelig=(NvsDMy)/(NaeDMa) ® where: Ais the amount of beads equivalent 1 1 g of DY (in g beads/1 g DY}; Ny and No are the numbers of viable yeast cells in 1g of DY and |g ‘of beads, respectively (in CFU/g); and DMy and DMy are the dry mx values of DY and beads, respectively (3, w/). 2.8. Morphology and structure Optical microscopy and a digital image processing technique (Gmaged computer software, Nasional Institutes of Heald and the Lab ‘oratory for Optical and Computational instrumentation LOCT, University ‘of Wisconsin) were employed to measure the convex hull area, convex hull perimeter, area, nd perimeter ofa least 100 beads ofeach type. To describe the beads shape, equivalent diameter, ert's diameter, cre larity, convexity, and spherical factor (SF) were calculated, according to Fig. 1. SM and equation (1).SM in the Supplementary Material (340, “The surface morphologies of yenst cells and beads were observed "sing scanning eleeton microscope (SEM, HITACHI $3500, Tokyo, Japan). Because ofthe non conduetive nature of the investigated ma terials, the samples were placed onto condietive carbon tape and further sprayed witha conductive gold nanoparticle layer, Fourier sransform infrared spectroscopy (FTIR) was used to Invest ‘gate structural changes oceusred in the yeast cells and beads duxing freezing frozen storage thawing eyele, FTIR spectra were acquired using 4 PerkinElmer Spectrum BX IR spectrophocometer (Waltham, USA) in the range of 600-4000 en ata 4 em” optical resolution and 28 sans per analysis. 24, Cryotoerance of fre and inmobitized yeut Frozen DY, CASY, and CAY capsules were thawed for 30 min 25°C, ‘andthe survival of yeast cells was investigated according to the method ‘eseribed in section 2.2. Te exyotolerance index was eaeulated the percent of viable yeast cell counted in the thawed sample relative ro the ‘umber in unfeozen contal saps. 2.5, Frozen dough bread preparation and physical and sensorial analysis Dovighs were prepared that contained the lowest equivalent yeast Pot cn 42 (2021 10119 antounts using GASY and GAY beads; dough containing DY was used as & reference, The dough containing DY was prepared using the following formule 500g white wheat flour (Banease, Bucharest, Romania), 5g DY (per the maataetarer’s recommendation, Paknaya,Pascani, Reman), 8 g sale (Salrom, Slane, Romania), 0.25 g ascorbic acid (to proteet the sluten network, Merck, Darmstadt, Germany), and 350 ma. water (the hydration capacity of white flour; detals can be found in SM). Another ‘neo dough samples were obtained by replacing the DY with the lowest equivalent amounts of bends extracted fron Table 1, namely 19 g CASY beads or 28.5 g.CAY beads The ingredients were Dende for 10min ina laboratory mixer for optimal dough development. Dough pieces, measuring 810 g each, were wrapped in plas foil and frozen at 18°C (Zanussi Chest Freezer, Pordenone, Italy), at an average freezing rate of 3.2 “C/min. Frozen samples were collected afer one and three months ‘and allowed to thaw for 2 until the dough pieces reached 22 °C, at whieh temperature the yeast becomes active. They were round shaped fd proofed at 32°C and 75% relive humidity in & proofing chan ber (Matina D 6040, Bucharest, Romania). After 60 min, the doughs were hand kneaded for 2 min, shaped, placed into baking trays (28 em « 10 em x 6 em), and proofed for an ditional 60 min. The pieces were baked in preheated oven (iron, Cadoneghe, lay) for 1 81180 °, implementing steam during the frst 10min, The bread loaves were removed from the trays, allowed f0 cool to room temperatnre (22°C), and analyzed. The Toa specific volume was measured sing the rapeseed displacentent method (Wily Cozauta et al, 2015) Crust firmness was tested at a minimum of 15 points using @ Wagner FDKIO force dial penetrometer (Wagner Instruments, Vernon Hills, USA), and average values are reported. The height (Hand diameter (D) ofeach bread loaf was measured in 10 replicates (at different positions of the bread) using a caliper, and an H/D index was calculated using the average values. Crist moistire was measured according to the ICC “Meth (199) in five replicates. Freshly prepared breed samples that so contained DY, CAY and CASY were als investigated as references. ‘The sensorial study was condicted within 5 hater baking under conditions of room temperature (22 °C) and daylight, using a group of 50 panelists (25 women and 25:men, with ages fom) 181060 years) who were regular read consumers. The study was approved by the Fihies Committee of the Center forthe Scientific Research into Environment, Food and Health Safety, Technical University of Chi Napoca, Romania, ‘and complies withthe principles of the Declaration of Helsinki as revised {n2013, The consumers were previonsy informed regarding the sins of the study and provided writen informed consent, Sliced bread samples, 2.em thick, were cut inte four pieces, coded, and randomly presented to the panelists, The attributes of crust color an firnness, crumb aspect, bread volume, smell, taste, and overall acceptability were evaluated using @ fivepoint ‘hedonic scale CLdisike extremely 10 Slike extremely), The overall sensory score ofeach ateibuce was computed as an average ofthe individual seores provided by the panelists. 2.6, stavisical analysis All investigations were performed at last in tiplicate, and the re sults are expressed as the mean + standard deviation. The differentiation between values was made based on the Tukey test posthoc ANOVA (p< 0.05) by using IBM SPSS Statistics Version 24. The boxplot mualsis was used to reveal the existence of potential outliers associated to the values of whose standard deviations are not displayed. 3. Results and discussion 4, Bneapsulation parameters Tolle 1 shows thatthe best encapsulation parameters for DY in CAY beads were achieved for 5 g DY. The yeast cell load and encapsulation ciency of CAY beads inereased 3,73 fold and 1.97 fold reference co the values obtained for CAY beads prepared with 2 g of yeast. The A Mhaty Comat ea Pod ne 422021 101189 CASY beads CAY beads Wet ‘components Dried components 1-month frozen storage months frozen storage (option on next page) A: iat Cooma Pot cn 42 (2021 10119 Fig. 1, Plesres and SEM iniges 40; x500) of re yes cols and yeast cells immobilize in gina ane alginate arch beds: Petes ‘A~ fies prepared CASY beads B - fies prepared CAY bonds; © dy yest els dred CASY beads; E ~ died CAY beads, 1H SeM image fered GAY beads (4); I~ SEM image of ry yeas cells (500), J SEM image of dried CASY beads (500); K-— SEM image of ered CAY beads (2500) sea geo ry yest ester I-month of frozen orage (3500); M - SEM nage of hel CASY bead afer 1 month of froen storage 500); N SEM nnage of ‘vet CAY bead ifr mont of ine stone 9500); 0 SEM ine of ry yeast el ter 3 months of faze trie (500); P SEM nage of ed CASY ends afer Soni of frozen storage (2500; SEM image of died CAY bead afer 3 months of frozen storage (500 ‘numberof capsules equivalent to 1 g of DY was reduced by 3.71 fold when the amount of DY mixed with alginate ineteased from 2 gt0 5 . ‘The Further increase ofthe DY ratio by 40%, from 5 g £0 7 g did not result in significant improvements in the encapsulation parameters Perneica etal. 204) reported immobilization yields of 58.73% and |56.43% for two strain ofS. cerevisiae in solutions containing 296 soda Alginate and 2 of yeast. The work of lt el. (2017) indeates the yeasts to gellan rai of 5:1 (W/) as the optimum in the yeast immo Diizntion corresponding toa vale higher than 90% of the light tans mitanee throngh the alcoholic medium in which the beads were suspended. An encapsulation effcieny of about 39-40% was obtained by immobilization of the yeast clls in water soluble polyacrylamide ‘nanofibers by electrospining method (et, 2021). Different other ‘matrices in terms of oak chips and cellulose powder, bacterial cellulose, {ape skins, sweet sorghum stalks, plactoglobulin and sodium alginate were also reported to provide high encapsulation yields of yerst ces Gariyajaoenwong et a, 2016 Besbeyal etal, 20195 Genisheva et al 2014 Nguyen et al, 2020; Zywicka e al, 2019), Instant Yeast NA (2157) (AB Mauri USA), SAF instant yeast (Leste Yeest Corporacon USA), ProBlif QA23, ProRestart and ProDessert Proenol Portugal and Lallemand Canada) are some ofthe commercial encapsulated currently in use in baked: goods and brewery industries characterized by stability, consistency, high fermentative capacity and extended shel life. “The addition of 1 gstarch in CASY bends enhanced the eneapsulaton parameters (p < 0.05) compered with the corresponding amount of DY In AY beads (bie 1). For instance, the CASY bess generated using an Alginate'starch:yeastwater ratio of 21:5:100 demonstrated a yeast load ‘and encapsulation efficiency 1,22 fold and 1.26old, respectively, those ‘of the corresponding parameters for CAY beads prepared using. an alginaestarelzyeaswater ration of 20:5:100. Moreover, the amount of ‘GASY beads equivalent (01 g of DY (8.80 g/1 g of DY) was 1.24 fold lover than that of CAY beads (4.70 9/1 of DY). The CASY beads pre pared asing 2 g of starch did na exhibit improved encapsulation pa rameters compared with CASY beads prepared using 1 gof starch, Our work is supported by dhe study performed by Cordoba otal. (2013), who ‘reported the improved encapsulation efficiency of polyphenols derived from yerba mate following the adition of starch. similar to CAY beads, ‘no significan differences in the encapsulation parameters of CASY boads were observed following the incorporation of greater than 5% DY. The ‘entrapment of yeast eel in the alginate matrix ean be explained by the ‘establishment of hydrogen bond between the protein and carbohydrate ‘components of the yeast cell walls ad the polysaccharide grows inthe Alginate, The favorable effect of stareh on the eneapsulation efficiency ‘an be ascribed tothe formation of additional hydrogen bonds between the compounds in the yeast cell walls andthe amylose and anaplopectin ‘components of starch, which was indicated by the FTIR analysts, “The results shown in Toble 1 indicate that CASY and CAY beads ‘generated using alginatestarch:yeastwater ratlos of 2:1:5°100 and 2:08:100, respectively, displayed dhe best characteristics in tems of yeas load, encapsulation efficiency, and lowest beads quantity equiv alent (o 1 of DY. These two encapsulation systems were selected for further investigation, 8.2, Reads morphology Wer CASY and GAY beads both appeared whitish an glossy, and the color tumed brown during drying (is. 1). During experiments, the tendency of dried CASY beads to aggregate was noticed due 10 the Dining properties of starch, That may ease dhe beads o disperse het erogeneously in the dough, easing uneven fermentation that result in bread faults, such as ieregular volume, crust cracks, and large and Srregular pores in the crumb. As shown in Fig 1 (> 40), the outer to pographies of dried CASY and CAY beads exhibic relatively spherical shapes with concavities obtained during water evaporation and the farther shrinkage ofthe droplets. At higher magnification ( 900), the dried beads displayed compact morphologies with large numbers of yeast cells embeded in their matrixes. The yeast cells were attenuated n the CASY beads due 10 the presence of starch, which occupied the trata Dead space, whetexs the yeast cells were more prominent in the CAY beads. No faenites were observed onthe srfaces of died CASY and CAY beads, indicating slow rate of water evaporation, The study by Tonia. Selo etal, (2017) reported dat alginate-based beads that wore subjected 10 feewze drying and vacuum drying displayed large pores and cracks. Freezing and frozen storage afected both types of beads, with longer frozen storage times resulting in more severe impact. After one month of frozen storage, the impact on CAY beads was fatty advanced, with large cracks and slits observed. In contrast, after one ‘ionthof frozen stage, the CASY beads deplayed a corrgated surface with no visible pores or fractures. These findings inleste the structural suppor provides y starch, which likely reduced matrix collapse ding the freezing-frozen storage-thaving eyele, The prolongation of storage ‘o three months significandy inereased the roughness ofthe CASY bead Likely, as those dense beads becane more bite, the necwork collapsed, resulting inthe formation of dep fractures and voids. ‘The characteristic shape parameters of alginate-based beeds and {hee relative changes are shown in Tobie 2. Wet CAY beads displayed significantly higher values for equivalent and Ferer's diameters compared with those of wet CASY beads, This outcome may be due to the increase in the viscosity of the alginate-starch solution and the changes that occur inthe surface tension and viscosity of the resultant froplets, in addition tothe establishment of new hydrogen bond in the CCASY bead structure, as demonstrated by FTIR analysis, resulting tn & tighter stricture. However, the statistical analysis did nor revel sig ificant differences between the above mentioned parameters. Aer drying, the equivalent diameter values isble2)indiaued the shrinkage ofthe wer beads by 50.46% for CASY beads and 56.21% for CAY beads duet water evaporation, resulting in the rearrangement of thehydrogel network structure. In addition to the establishment of new hydrogen bonds berween the bead matrix and yeast cells, the beed size influences the encapsulation parameters, which are indireetly correlated with the loading capacity and encapsulation effieteney. According to Busi ct a (2018), smaller and firmer eapsules can form a more stable alginate “egg box" structure, resulting in a high entrapment effiieney. similar finding was also observed in our study, as the CASY gel displayed @ higher viscosity and resulted in frmer,staller beads compared with the CCAY gel (able 2, sigesting tha the presence of starch is favorable to pea hardness. However, CASY beads originated fom formula Alginate: A: iat Cooma Pod ne 422021 101189 ‘able? Morploga characters of CASY ad CAY bens Sno Taka Ta Pac Ganaey am Mimi er anae a SR oer Gonealy Crea oe cae on = tant tet 2 oo coe tow tan {eth ote ape one co = tow ane Stet fo ee oon coe . tour tae a oer on = tor tao a con cou = toa uae ae ten aes os cost - tout tas Stohr srg comm oon a osm aaa “Average value= standard deviation SD (n= at leas 100), Statistical analysis was performed Separaely for CASY and CAY beads Ineach colin, ean ves with ifevent letters ae significantly ferent at p= 0.05 “he dexeatition was made Bas on te Tukey test posthoc ANOVA (IM SPSS States Version 2. ‘No satay significant ferences wete fan between the corresponding vals of CASY std CAY Dea ‘nthe ese of shteiity fern, Dolo analysts did no eval the existence of oer. (a) () ry 7 Co . oo we | | vase = (c) (4) eae \ ws os : | ore ji i wo] a —— 7 Fig. 2. FTIR specu of aw matevas, fe a hozen dough made wit ie and inmobiied yeast cell. ‘2-8 materials eae alginate, sodiun alginate, starch), 1 -DY., DY. 1, DY. fee yeas cells, es (0) and subjected of month (1) and 3:months (3) fozensoxage; {¢-CASY 0, CAS 1, CASYS: CASY beds, es (0) and subjected of Lon (1) and $ oats (3) frozen storage; 4 CAY., CAV: CAY 3: CAY bend, fe (0) and subjected of moadh (1) and $month 3) fozensoxege. A: iat Cooma Stareh:veast Water 2:1:5:100 (w:wew:v) entrapped yeast cells better than CAY beads exjginated from formula Alginate:Stach:Yeasc Water 2:05:100 (w:wew) (Tuble 1, During three months of frozen storage, ho significant changes were observed in the equivalent diameters of dried CASY and CAY beads Wer CASY and CAY beads were nearly spherical in shape (Fis. 1, “Table 2, with beads designated as sperial if the SF value < 0.05 (cas), ‘os, 2000), According tothe four basle types of beads deseribed by ‘can etal. (2009), wet CASY and CAY bosds can be categorized as ‘gg shape (ellipsoidal). Upon drying, freezing and frozen storage the sphericity, convexity and circularity of wet CAY and CASY beads were not significant modied (Fable 2) 8.8. PTR spectroscopy ‘The FTIR spectra of the raw materials ae spayed in ig. 2 and the main characterise bands were identified according to theHteratre. ‘he spectra of sodium alginate displays the following characteristic tortion band (Dec Sia, 2012) sueteing Vibrations the “OH group at 3198 em™s stretching vibrations ofthe -CH aliphatic group at 2912 cm asymmetric ad symmetric stetching vibrations of ‘mbosylate salt ons at 1592 em and 1402 cm, respectively and the (60 stretching vibrations of the pyranosy ring and the €-0 stretehing ‘sssocinted withthe deformntion of €-C- Hand €-0-H.at 1022em- and 1934 en. The 3000-8600 cm sbsorption regon was aseribed to the stetchin vibrations of 0-1 bonds, which appeared tobe narrower for Calcium lpinate compared with tbat for sodlum alginate The pari pation of hydroxy] ad carboxylate groups in alginate, whieh form the egg box" svete in the presence of elem fons, reduces the iydrogen bonding betwen yop groups, casing the FTIR spectra band to naow. The asymmeuie suetching vibration ofthe (C0 group shied tothe lower value of 1584 ex de tothe higher tonic weights and charge densities ofthe clean ons that replaced the sodium fons in alginate, In the spectrum of starch, the band stretching around $400 em comesponds othe vibration sets of the free, inter, and inramcaculer bound hydroxyl groups, which constitute the whole souetre of starch (inl, 2013). Absorption bands ac 1156 en, 1074 an, nd 996 em aze also present, asso slated with the C-0 hood stetching and the anhydrogliease ring ©-0 stretch, respectively. FTIR spectroscopy has een extensively used In the exploration of fesh and frozen yen cells dehydration Kneis, Moet i the amide T(ahelx and sew) and amide 1 band i the secondary pro tein suctre, shift of symmeuse C-H stretching vibration of the Cy ‘group upon a higher wavenumber were corelated with increased sma membrane egy and os fell viaility (Carve, 30005 Nguyen eal, 20175 Nguyen et, 2020. In our study, Fg. 2 depiets the FTIR spectra of fresh and frozen cerevisiae cells, for which the following regions have been identified {The broad absorption band e 3000-3600 ea" coresponds to the vibrations of fe, inter, ad intramoleeue ~OH groups inthe Yeast cells The downshift of the-OH seeing band poston from the value 1013400 em! ported inthe iterate 2017) the vale of $201 cn! in our study can be explained by alteration ofthe Inydrogen bonds present inthe fresh yeast ells during drying. They continue 1 deteriorate daring feezing and fozen storage, and the ‘owns in the -OH srtching vibrations contined tobe observed in both month and $month tozen yeast cells Gi). The reglon 31000-2800 en-"is primarily assigned othe lipids and fatty aes ofthe yeast plasma membrane (Sesnz Moree al, 201°). The peaks ‘eownd 2920 en? and 2849 em* describe the symmetry note streching ofthe methylene and mexyl groups (ili e9, 2017). In this region, the sift in the symmetric CH. vibration fom 2847 cu" in unfrozen yeast 10 2841 ent" and 2836 em i the {month and 3-month froaen yest, respectively, ere observed likely ssscited wid the stfening of dhe cll membranes nnd the loss of Pot cn 42 (2021 10119 membrane integrity dive co changes in the lipid ehains (Pssor eal 2019) i) The spectral region 1800-1500 em? is ascribed to amide 1 (1658-1637 ene) and amide 1 (1380-1550 em™) groups, whieh deseribe the stretching vibrations speife to the C0, N-#, and C-N bonds in proteins and peptides (la Cruz Gavia tal, 201% Nguyen fecal, 2015). The band 1500-1200 em! was assigned tothe amide I region and depicts overtone vibrations of the N-H bending end C-N stretching in tide bonds and the vibration wagging ofthe CH, groupe (de la Cruz Gavia et al, 2018). Nguyen et al. (2017) reported two vibrational bans around 1652 em» (Stretching vibrations oF C-O) and 1547 cot [a combination of the N-H bending and n (C-N) stretching ‘vibrations, whieh were assigned tothe acheicand sheet, respectively, re components of the secondary protein sruetre. In ou study, and 1535 emt, with difer ences in the studied yeast strains likely responsible for these observed shits, Significant downshifs ofthe ahelix and sheet vibrations to 1629 en and 1525 emt respectively, were observed after one month of frozen storage, and after an additional two months offczen storage, the bands shifted to 1625 cm and 1518 ca. The los of intensities for the qhelix and fsheetstructres following frozen storage indicated disruptions in the conformational strictures of proteins, which may be ssocated with yeast cells Isis. (lv) The region 1200-900 em* was attibnced to carbolydrates(P-lueans, glyeogen, and mannans), with !inor contributions from phosphate groups associated with DNA, RNA, and phospholipids (Serie Moret et al, 2018), Reductions i these absorption bands were observed for yews subject o freezing forone and three months compared with unfrozen yeast, inieating the hydrolysis of lycosidic linkages in frozen yeast. The absorption band around 1079 en reflects the DNA content (Beuedoti et a, 1989), core sponding to the symmetric PO; (P0,~) stretching vibrations of the phosphate groups associated with the DNA double-stranded backbone (Benedettl et a, 1990; Gasparri & Muzio, 200% Liu & Mant, 2001), the 0-0 stretching vibrations of nucle sei polysaccharides, and the 16-0-P sietching vibration of phosphorylated lipids (Gide et al 200% Jamin etl, 2008), Another wavenumber associated with nucleic rcids was loctted at 1048 em"?, which was atebted to the 2-0 stretching (Gautam tal. 2012) of deoxyribose (Gao e al, 2015), RNA, tnd ribose (Ganidenzi et, 2009). The decreased band intensities around 1079 em and 1048 em? during frozen storage indicated the loss of nucleic aid contents due to DNA damage (iso el, 2017). Thus, foe yeast cells showed 71.25% and 73.76% decrease in the in tensites of the 1079 em"? and 1048 cm? absorption bands, respec tively, after one month of frozen storage, and additonal dacreased intensities of 9.91% and 10.44% after another two months of frozen storage. ‘The FTIR spectra of the feesh GAY and CASY bends ae displayed in ig. 2e and d Specific bands corresponding to the alginate, yeast, and starch can be observed in the CAY and GASY bead. spectra, which confirm the successful loading of S. cereislae into the alginate and alginate-starch beads, respectively. A wide band, widh peaks. at 3259 em and 3256 cm, indicated the establishment of intermolee ‘lar hydrogen bonds between the hydrophilic groups ofthe rave mute Fiols. A downshift of the-OH stretching band position could be observed for CASY beads duc to the establishment of nev hydrogen bonds following the starch sddition. The insmobilization of yeast cells in the beads was indicated by the absorption bands atoured at 2850 em? and the specific components of nucleic acids, associated with Bands at and 1025 em, The alginate macrix appeared at 2910 ent 8/1410 ext"? and 1292 ent, Signals around 1156 ex", and 1019 ent“ indented the presence of starch inthe CASY. beads (Cércoba etal, 2013), "The changes that occured in CAY and CASY beads during freezing and frozen storage were also revealed by the FTIR spectra (Fs. 2e and 4). Significant downshift behavior was observed for the regions ‘8600-8000 em aad 3000-2800 em after the fist month of frozen storage due to the disorganization ofthe yeast plasiia membrane (es A: iat Cooma ‘cae Gavia et aly 2018}. ‘The amide 1 band, inthe region 1500-1800 ent, showed higher intensities for the ehelix and p-sheet ‘components in the CAY and CASY beads conspared with those observed for free yeast calls, suggesting that changes in protein structures ‘occurred in the immobilized yeast cells during freezing and frozen storage compared with those observed in free yest cells, The carboky- rates band (1800-1200 em) decreased after frozen storage, in ‘cating a weakening ofthe glycosidic linkages, The DNA integrity ofthe ‘encapsulated yeast cells was also disrupted during freezing and frozen, storage, but to lesser extent than observed for fee yeast cells ‘Thus, yeas cells in GAY beads showed 59.64% and 57.84% decrease Inthe intensities ofthe 1079 em and 1048 ent-® absorption bands, respectively, after ane month of frozen storage, and decreased teasiies of 1.8796 and 2.419 after another two months of fozen stor age, Improved exyoprotection was also observed for yeast DNA in the ‘CASY bead matrix, with reductions in the intensities ofthe 1079 emt-* ‘and 1048 cm bands of 4.12% and 57.50%, respectively, after one month of frozen storage and of 5.91% and 0.5496 after another two months of frozen storage. A doveushft in the characteristic band in tensiies of. cerevisiae microencapsulated with complex coacervate was also reported following the freezing process (ce In Cri. Gavia et al. 2018) Comparisons in the intensities of characteristic absorption bands ‘81079 em and 1048 em. fr DY and CASY and CAY beads berween ‘one month and three months of frozen storage siggested that freezing ‘and one month of frozen storage had the strongest impacts on yeast cells abi 34. Cryotoerance indexes “Tolle 3 sts yeast cells viability in the investigated samples andthe ‘calculated cryotolerance indexes. The freeing frozen storage- thawing cycle affected yeast cells viability in all samples, and the eryotolerance Indexes decreased to varying extents depending on the storage time. Signfieant diferences (p * 0.05) in yeast cells viability were ‘observed during $ months of frozen storage for DY, CAY and CASY beads. The highest impacts were noticed for DY, which showed a 70.06% decline in population after one month of frozen storage, ‘compared with desliies of 61.9088 and 69.04% CASY and CAY beads, respectively. Three months of frozen storage had an even nore severe ‘Table a ‘he viabilty of fice and immobilized yeast calls afer feeng-ozen stor age-thavelng eel ‘Gonpositon Sample Granta Gout vable cawaleance Stree, eshoangle, one rae ‘Average value= standard deviation SD (= at eas). In each column, ean values with fete letters are lalicanly fren at p

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