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Dow Specialty Surfactants in Emulsion Polymerization
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Dow Specialty Surfactants in Emulsion Polymerization
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Dow Specialty Surfactants in Emulsion Polymerization
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Original articleChemical composition and sensory analysis of cheese whey-based beverages using kefir grains as starter cultureKarina Teixeira Magalha˜es,1,2Disney Ribeiro Dias,3Gilberto Vinicius de Melo Pereira,1Jose ´Maria Oliveira,2Lucı´lia Domingues,2Jose´Anto ´nio Teixeira,2Joa ˜o Batista de Almeida e Silva 4&Rosane Freitas Schwan 1*1Department of Biology,Federal University of Lavras (UFLA),Campus Universitario,37.200-000,Lavras,MG,Brazil 2IBB –Institute for Biotechnology and Bioengineering,Centre of Biological Engineering,Universidade do Minho,Campus de Gualtar,4710-057,Braga,Portugal3Department of Food Science,Federal University of Lavras (UFLA),37.200-000,Lavras,MG,Brazil 4Department of Biotechnology,Engineering School of Lorena,University of Sa o Paulo (USP),12.602-810,Lorena,SP,Brazil(Received 12August 2010;Accepted in revised form 9January 2011)SummaryThe aim of the present work was to evaluate the use of the kefir grains as a starter culture for tradicionalmilk kefir beverage and for cheese whey-based beverages production.Fermentation was performed by inoculating kefir grains in milk (ML),cheese whey (CW)and deproteinised cheese whey (DCW).Erlenmeyers containing kefir grains and different substrates were statically incubated for 72h at 25°ctose,ethanol,lactic acid,acetic acid,acetaldehyde,ethyl acetate,isoamyl alcohol,isobutanol,1-propanol,isopentyl alcohol and 1-hexanol were identified and quantified by high-performance liquid chromatography and GC-FID.The results showed that kefir grains were able to utilise lactose in 60h from ML and 72h from CW and DCW and produce similar amounts of ethanol ( 12g L )1),lactic acid ( 6g L )1)and acetic acid ( 1.5g L )1)to those obtained during milk fermentation.Based on the chemical characteristics and acceptance in the sensory analysis,the kefir grains showed potential to be used for developing cheese whey-based beverages.KeywordsCheese whey beverage,GC-FID,HPLC,kefir,lactic acid bacteria,sensory evaluation,yeasts.IntroductionCheese whey (CW)is the major by-product of the dairy industry and its disposal without expensive sewage treatments represents a major source of environmental pollution.Considerable efforts have been made over the past years to explore new outlets for CW utilisation and reduce environmental pollution (Magalha es et al.,2010a).Besides potable ethanol production by lactose converting microorganisms (Guimara es et al.,2010)and the production of distilled beverages (Dragone et al.,2009)and kefir-like CW beverages (Magalha es et al.,2010a)this by-product has also been suggested as an alternative for industrial residue utilisation which may reduce environmental pollution.Traditionally kefir grains have been used in many countries,especially Eastern Europe,as a natural starter in the production of kefir,a unique self-carbonated dairy beverage.Kefir differs from other fermented milks in its starter,which exists in the form of grains (Simova et al.,2002).Kefir grains contain lactic acid bacteria(LAB)including Lactobacillus,Lactococcus,Leuconos-toc and Streptococcus spp.and yeasts (Kluyveromyces,Torula,Candida and Saccharomyces spp.).Both the bacteria and yeast are surrounded by a polysaccharide matrix,called kefiran,which is a water-soluble branched glucogalactan (Magalha es et al.,2010b).The production of a functional beverage produced upon whey fermentation by kefir grains could be an interesting alternative for CW utilisation.CW fermenta-tion by kefir microrganisms could decrease the high lactose content in CW,producing mainly lactic acid and other metabolites such as aroma compounds contributing to the flavour and texture and increasing carbohydrate solubility and sweetness of the end product.Manufacture of beverages through lactic fermentations can provide desirable sensory profiles and have already been consid-ered an option to add value to CW (Pescuma et al.,2008).Recently,Magalha es et al.(2010a)set out to cha-racterise kefir-associated microbiota by using two unre-lated techniques,DNA analysis [by denaturing gradient gel electrophoresis (DGGE)]and optical microscopy (combining fluorescence staining with confocal laser microscopy).The composition of microbiota was related to Lactobacillus kefiranofaciens subsp.kefirgranum,*Correspondent:Fax:+553538291100;e-mail:rschwan@dbi.ufla.brInternational Journalof Food Science and Technology 2011,46,871–878871doi:10.1111/j.1365-2621.2011.02570.xÓ2011The Authors.International Journal of Food Science and Technology Ó2011Institute of Food Science and TechnologyLactobacillus kefiranofaciens subsp.kefiranofaciens,an uncultured bacterium related to the genus Lactobacillus, Kluyveromyces marxianus,Saccharomyces cerevisiae and Kazachstania unispora.No differences were found in the community structure detected in the analysed beverage and Brazilian kefir grains,showing that microbiota of kefir grains is highly stable along the fermentations carried out in different substratum.However,this characterisation was restricted to the microbiota and until recently,we were not aware of any reports concerning the chemical and sensorial characterisation of these beverages.Therefore,the aim of the present work was to evaluate the use of the kefir grains as a starter culture for traditional milk kefir beverage and for CW-based beverages production,besides evaluating the biochem-ical changes,organic acids production and volatile compounds formation during fermentation process. Materials and methodsCW-based and milk fermentation mediaThree different substrates containing lactose concentra-tion of46g L)1were used as fermentation media: pasteurised full cows milk(ML),CW and deproteinised cheese whey(DCW).CW powder,obtained from a regional dairy industry(Lactogal,Porto,Portugal),was dissolved in sterile distilled water until the desired lactose concentration.DCW was made by autoclaving at115°C for10min the CW solution,followed by aseptic centrifugation(2220g for20min)to remove cream.Confirmation of CW deproteinisation was accomplished for Kjeldahl method.Kefir beverages productionBrazilian kefir grains were employed in the present study.The grains(12.5g)were washed with sterile distilled water and inoculated in250mL of ML,CW and DCW.The milk is used commonly for kefir beverage,therefore was used to compare the fermenta-tion with CW kefir beverages.Erlenmeyers containing kefir grains were statically incubated for72h at25°C. Samples of the kefir beverages were aseptically taken every12h for analysis of organic acids,ethanol,sugars and volatileflavour substances.Determination of total reducing sugars was used to assess the substrate consumption.The pH of the fermented beverages was measured using a Micronal B474pH meter.Two replicates were done in each fermentation batch. Analytical methodsChemicals1-Hexanol and ethyl acetate were purchased from Aldrich Chemistry(Munich,Germany).1-propanol,2-methyl-1-propanol,2-methyl-1-butanol,3-methyl-1-butanol were purchased from Fluka Analyticals(Seelze, Germany).Ethyl acetate,Acetaldeyde lactose,were purchased from Sigma-Aldrich(St Louis,MO,USA) and acetic acid,lactic acid,ethanol,methanol were purchased from Merck(Darmstadt,Germany). Organic acids,sugars and ethanolLactose and ethanol were quantified by high-perfor-mance liquid chromatography(HPLC),using a Jasco chromatograph equipped with a refractive index(RI) detector(Jasco830-RI,Madrid,Spain).Lactic acid and acetic acid were also quantified by HPLC,using a Jasco chromatograph equipped with UV–Visible detector (Jasco870-UV-visible).A Chrompack column(300·6.5mm)at60°C,using5mM sulphuric acid as the eluent,at aflow rate of0.5mL min)1and a sample volume of20l L was used.Volatileflavour substancesIn order to identify the volatile compounds,the kefir beverages were analysed directly without any previous treatment according to Fraile et al.(2000).A Chrompack CP-9000gas chromatograph equipped with a Split⁄Split-less injector,aflame ionisation detector,and a capillary column(50m·0.25mm i.d.,0.2l mfilm thickness; Chrompack)coated with CP-Wax57CB was used.The temperature of the injector and detector was set to250°C. The oven temperature was held at50°C for5min,then programmed to run from50°C to220°C at3°C min)1and then held at220°C for10min.Helium was used as the carrier gas at125kPa,with a split vent of15mL min)1. Injections of1l L were made in the splitless mode(vent time,15s);4-nonanol(internal standard)was added to the sample to afinal concentration of122.05mg L)1.The volatile compounds were identified by comparing the retention times of the samples with those of standard compounds.Quantification of volatile compounds was performed with Varian Star Chromatography Worksta-tion software(Version6.41)and expressed as4-nonanol equivalents,after determining the detector response factor for each compound.Sensory evaluationThefinal kefir beverages(ML,CW and DCW)were evaluated by twenty-five untrained tasters,males and females,25–35years of age(students of the Centre of Biological Engineering,University of Minho,Campus Gualtar,Braga,Portugal).Randomised,refrigerated (10°C)samples of10mL were served(containing 1.0mg of sucrose)in clear,tulip-shaped glasses with a volume of50mL;these were marked with three digit random numbers and covered with Petri dishes.Dis-tilled water was provided for rinsing of the palate during the testing.Tasters were asked to indicate how much they liked or disliked each product on a9-point hedonicChemical and sensory analysis of CW-based kefir beverages K.T.Magalha˜es et al.872International Journal of Food Science and Technology2011Ó2011The AuthorsInternational Journal of Food Science and TechnologyÓ2011Institute of Food Science and Technologyscale(9=like extremely;1=dislike extremely)according to colour,odour,aroma,appearance,tasteand overall acceptability characteristics.Statistical analysisStatistical analysis was carried out with Statistica soft-ware version9.0(StatSoft Inc.,Tulsa,OK,USA).Principal component Analysis(PCA)was used tosummarise the information in a reduced number ofprincipal components.A one-way anova was performedfor chemical parameters,concentration of volatile com-pounds values to determine significant differences(P<0.05)by using the Duncan’s multiple range testusing the spss version10.0.Results and discussionMicrobial metabolitesMilk and CW-based kefir beverages were monitoredduring the72h fermentation period by determining theacidity.During the72h of incubation,pH values of thefermented milk kefir and whey-based beverages rangedfrom6.1to3.9,notfinding significant differences for allthe substrates(P<0.05)(data not shown).These pHvalues were similar to those previously reported for kefirbeverage(Magalha es et al.,2010b).pH is an importantfactor that can strongly affect the quality of a beverage(Sharma et al.,2009).Furthermore,pH values of thefermentation broth significantly influence the fermenta-tion time of lactose and the levels of volatile compounds,reflecting possible variations in the sensory characteris-tics of thefinal product(Athanasiadis et al.,2004).High performance liquid chromatography(HPLC)was used to analyse organic acids,ethanol e sugars inthe produced kefir beverages.The Figs1–3show theconcentration of sugars,organic acids and ethanolobtained by ML,CW and DCW fermentation.Theproduction process of organic acids and alcohol wasfollowed by the lactose consumption in kefir beverages(ML,CW and DCW).The total lactose consumptionwas observed in60h in the ML fermentation and72hin the CW and DCW fermentation(Figs1–3).Thislikely reflects an adaptation period of the microbialcommunity to the whole and deproteinised CW as kefirgrains are fermented in milk ctose readilydegraded to galactose and glucose by Group N strep-tococci,Lactobacillus and by some strains of Kluyver-omyces(Gu zel-Seydim et al.,2000).In the present work,the lactic acid content increasedduring the72h of fermentation process in kefir bever-ages,reaching maximum value of6.35g L)1,6.34g L)1and6.81g L)1in ML,CW and DCW kefir beverages,respectively(Figs1–3).The fermentation of lactose by LAB present in kefir culture can be associated with the increase in lactic acid production through the hydrolysis of sugars released from the glycomacropeptide of casein as well as the glycoproteins associated with the fat globule membrane(Rynne et al.,2007).Acetic acid was also formed during the fermentation process of kefir beverages(ML,CW and DCW),reaching maximum value of 1.5g L)1(Figs1–3).The acetic acid was formed probably by heterolactic bacteria,previously identified in Brazilian kefir beverages(Magalha es et al., 2010b).These results are of great importance since lactic acid and acetic acid provides pleasant taste and inhibits the development of undesirable or pathogenic microor-ganisms,due to the substrate acidity increase(Ma-galha es et al.,2010b).Ethanol concentration increased during the kefir fermentation process in all three kefir beverages,reach-ing maximum concentration of12.26,12.72and 11.86g L)1in ML,CW and DCW kefir beverages, respectively(Figs1–3).Saccharomyces cerevisiae,previ-ously identified in kefir beverages(Magalha es et al., 2010a),which exhibits strong fermentative metabolism and tolerance to ethanol,is primarily responsible for the alcohol production(Pereira et al.,2010).However,some bacteria from the genus Lactobacillus also have the ability to produce ethanol,since they have alcohol-dehydrogenase activity,an enzyme able to convert acetaldehyde to ethanol(Magalha es et al.,2010b).The content of alcohol should be enough to give kefir the flavour of a light alcoholic beverage that is typical of traditional(ancient)kefir of the Caucasus and the yeast aroma ensures the specificity of this type of fermented beverage(Beshkova et al.,2003).Aroma-related compoundsLAB present in kefir grains starter cultures produce a plethora of enzymes that contribute to the formation of volatiles via proteolysis,lipolysis and carbohydrate degradation during ripening.Such enzymes are peptid-ases which are involved in the transformation of casein into free amino acids which are further degraded to volatile aroma compounds.Other enzymes are esterases and lipases that hydrolyse triglycerides of dairy of fat in free fatty acids(Dragone et al.,2009).GC⁄FID analysis was employed to determine volatile com-pounds in kefir beverages(ML,CW and DCW)during 72h fermentation process.The Table1shows the results of the following aroma forming compounds produced in the kefir beverages.Isoamyl alcohol(3-methyl-1-butanol),isobutanol(2-methyl-1-propanol), 1-propanol,isopentyl alcohol(2-methyl-1-butanol) and1-hexanol were the alcohols found in the kefir beverages(ML,CW and DCW)(Table1).The iden-tified ester is represented for ethyl acetate,while amongst the aldehyde group,acetaldehyde was found in kefir beverages.According to some authors(Apos-tolopoulou et al.,2005),ethyl esters(mainly ethyl acetate),alcohols with three or more carbon units,and acetaldehyde,are the major agents responsible for the flavour of fermented beverages.Ethyl acetate has a significant effect on the organo-leptic characteristics of fermented beverages.The pres-ence of this ester results in a pleasant aroma with fruity properties,but can turn vinegary at levels above 150mg L)1,adding spoilage notes to the beverage (Falque et al.,2001).Thus,the ethyl acetate concentra-tion in kefir beverages(8.18,8.27and8.38mg L)1in ML,CW and DCW,respectively)was found at a level suitable to confer a pleasantflavour.Normally,in-creased ethyl acetate concentrations are indicative of long term storage of the raw material and probable acetic bacterial spoilage.1-propanol can also be an indicator of bacterial spoilage.The lowfinal concentra-tion of1-propanol in kefir beverages(1.97mg L)1for ML,2.44mg L)1for CW and2.38mg L)1for DCW) can be compared with levels of other beverages,such as whiskies and cider brandies(Apostolopoulou et al., 2005).The concentration of2-methyl-1-propanol in kefir beverages(Table1)can also be well compared with levels in other beverages(Apostolopoulou et al.,2005; Dragone et al.,2009).Amyl alcohols(3-methyl-1-butanol and2-methyl-1-butanol)are formed during fermentation by deamina-tion and decarboxylation reactions from isoleucine and leucine,respectively(Dragone et al.,2009).Such com-pounds constitute quantitatively the greater fraction of the alcohols in most fermented beverages(Soufleros et al.,2004).In the kefir beverages produced in our study they were found in thefinal concentration of 8.80mg L)1for2-methyl-1-butanol and 5.80mg L)1 for3-methyl-1-butanol(Table1).Increased concentra-tion of amyl alcohols can contribute negatively to the aroma of the beverages(Falque et al.,2001).The1-hexanol alcohol was also found in kefir beverages.This alcohol has a positive influence on the aroma of the fermented beverage when it occurs in concentrations up to20mg L)1.On the contrary,increased concentration of1-hexanol,seriously impairs the organoleptic charac-teristics of the beverage(Falque et al.,2001).The low 1-hexanolfinal concentration found in kefir beverages (0.5mg L)1for ML,0.57mg L)1for CW and0.58 mg L)1for DCW)can be considered to affect positively theflavour of the product.Methanol was not found in kefir beverages,that is benefic since a highly toxic effect has been reported for this compound(maximum legal limit1000g hL)1of100%vol.ethanol–Council Regulation(EEC)No.1576⁄89,1989)(Geroyiannaki et al.,2007).The absence of methanol in kefir beverages is probably due to the lack of pectin in milk and CW.Chemical and sensory analysis of CW-based kefir beverages K.T.Magalha˜es et al.874International Journal of Food Science and Technology2011Ó2011The AuthorsInternational Journal of Food Science and TechnologyÓ2011Institute of Food Science and TechnologyT a b l e 1C o n c e n t r a t i o n o f v o l a t i l e s c o m p o u n d s p r e s e n t i n k e fir b e v e r a g e s s p i r i t b y G C -F I DK e fir b e v e r a g e s f e r m e n t a t i o n C o m p o u n d s v o l a t i l eA c e t a l d e h y d e E t h y l a c e t a t e 1-P r o p a n o l 2-M e t h y l -1-p r o p a n o l (i s o b u t y l a l c o h o l )2-M e t h y l -1-b u t a n o l (i s o p e n t y l a l c o h o l )3-M e t h y l -1-b u t a n o l (i s o a m y l a l c o h o l )1-H e x a n o lM i l k (m g L )1)0h n .d n .d n .d n .d n .d n .d n .d 12h 2.16±0.02a c d e n .d n .d n .d n .d 0.08±0.04b c d e n .d 24h 2.17±1.12a c d e 2.88±1.22b c d e n .d 5.14±1.56d e 3.97±1.26d e 3.46±1.58b c d e n .d 36h 2.68±0.75a c d e 3.71±0.86b c d e n .d 5.49±1.96d e 4.20±1.67d e 4.24±0.47b c d e n .d 48h 3.38±0.65a c d e 3.77±1.30b c d e n .d 6.82±0.35d e 4.44±0.90d e 4.40±1.79b c d e n .d 60h 5.08±0.82a c d e 8.18±1.67b c d e 1.97±0.29a b 10.89±2.15d e 8.65±1.94d e 5.89±1.73b c d e 0.50±0.02a b c 72h5.08±0.82a c d e 8.18±1.67b c d e 1.97±0.29a b 10.89±2.15d e 8.65±1.94d e 5.89±1.73b c d e0.50±0.02a b cC h e e s e w h e y (m g L )1)0h n .d n .d n .d n .d n .d n .d n .d 12h 1.56±0.01b c d e 1.06±0.08c d e 0.15±0.07a b c d n .d n .d n .d n .d 24h 2.92±0.16b c d e 2.35±0.46c d e 2.54±1.22a b c d 3.99±1.10b c d e 3.84±0.14d e 5.62±0.26d e n .d .36h 3.16±0.95b c d e 2.56±1.98c d e 2.46±0.57a b c d 4.43±1.76b c d e 4.15±2.14d e 5,45±0.40d e n .d .48h 3.23±0.98b c d e 2.56±1.84c d e 2.15±0.78a b c d 4.21±0.30b c d e 4.07±0.07d e 5.73±0.05d e n .d .60h 5.97±0.11b c d e 6.42±0.19c d e 2.37±0.13a b c d 8.88±2.03b c d e 8.15±0.95d e 5.75±0.71d e 0.57±0.02a b c 72h5.98±0.17b c d e 8.27±0.37c d e 2.44±0.18a b c d 10.51±0.41b c d e 8.88±0.23d e5.91±0.16d e0.57±0.06a b cD e p r o t e i n i s e d c h e e s e w h e y (m g L )1)0h n .d n .d n .dn .d n .d n .d n .d 12h 1.39±0.11b c d e 0.98±0.03b c d e 0.10±0.01a b c d n .d .0.13±0.04e 0.27±0.24b c d e n .d .24h 2.81±0.01b c d e 2.32±0.50b c d e 2.34±0.93a b c d 3.61±0.55b c e 3.62±0.17e 5.40±0.57b c d e n .d .36h 2.80±0.04b c d e 2.56±0.57b c d e 2.31±0.04a b c d 3.94±1.07b c e 3.70±0.42e 5.37±0.52b c d e n .d .48h 3.12±0.82b c d e 2.75±0.35b c d e 2.35±0.5a b c d 4.04±0.07b c e 4.01±0.02e 5.80±0.15b c d e n .d .60h 5.95±0.08b c d e 6.30±0.36b c d e 2.39±0.16a b c d 8.22±1.10b c e 7.74±0.37e 5.83±0.74b c d e 0.59±0.01a b c 72h 5.98±0.61b c d e 8.38±0.25b c d e 2.38±0.25a b c d 10.60±0.28b c e 8.89±0.16e 5.82±0.02b c d e0.58±0.04a b c O d o u r t h r e s h o l d (m g L )1)25n §12.3m †266n 750o §–7m *à7m *à0.592n *àD e s c r i p t o r sW h e y n W h e y nW h e y n –C h e e s e m ,W h e y n C h e e s e mW h e y nD a t a a r e a v e r a g e v a l u e s o f d u p l i c a t e ±s t a n d a r d d e v i a t i o n .n .d .-n o t d e t e c t e d .*O l f a c t o r y p e r c e p t i o n t h r e s h o l d i n h y d r o -a l c o h o l i c s o l u t i o n .†O l f a c t o r y t h r e s h o l d i n m o d e l w i n e .àO l f a c t o r y p e r c e p t i o n t h r e s h o l d i n w a t e r .§O l f a c t o r y d i f f e r e n c e t h r e s h o l d i n b e e r .m E s c u d e r o e t a l .(2004).n D r a g o n e e t a l .(2009).a –e =T h e a v e r a g e s o f t h e c o l u m n s w i t h d i f f e r e n t l e t t e r a r e s i g n i fic a n t l y d i f f e r e n t (P <0.05).Chemical and sensory analysis of CW-based kefir beverages K.T.Magalha˜es et al.875Ó2011The AuthorsInternational Journal of Food Science and Technology 2011International Journal of Food Science and Technology Ó2011Institute of Food Science and TechnologyLow molecular mass carbonyl compounds such asaldehydes and ketones are normally found in fermented beverages as by-products of yeasts fermentation,inter-mediates in the formation of fusel oil and as a result of alcohol oxidation at various stages of beverage produc-tion.Nevertheless,their presence is not desirable because some of them are responsible for unpleasant organoleptic properties (Dragone et al.,2009).In the present study,acetaldehyde was the only carbonylated compound identified amongst the major volatile com-pounds,but its concentration value (5.08mg L )1for ML and 5.98mg L )1for CW and DCW)was low when compared to other beverages,such as tsipouro or grappa (Apostolopoulou et al.,2005).This low concen-tration value is interesting,because elevated acetalde-hyde concentrations give a pungent irritating odour to the beverage,and can be health hazards (Geroyiannaki et al.,2007).The results obtained for the volatile compounds (Table 1)were submitted to PCA to obtain a more simplified view of the relationships amongst the volatile compounds analysed (Fig.4).The first principal com-ponent accounted for 76.85%of the total variation,while PC2and PC3explained 13.29%and 7.86%of the total variation,respectively.A plot of the results shows the formation of four groups (Fig.4).Two of the groups are located on the first factor (X -axis positive,Y -axisnegative and Z -axis positive or negative),and includes A1,B1,C1and A2,A3,A4samples.These groups corresponded to the early times of fermentation and were far plotted of the 1-propanol,isoamyl alcohol and isopentyl alcohol point,suggesting that these volatiles compounds made significant contributions to separation between these.The third group is closely related on the second or third part of the axis (X -axis positive,Y -axis positive and Z -axis positive or negative),and includes the samples B2,B3,B4,C2,C3,and C4.These times of fermentation were mainly associated with larger con-centrations of isoamyl alcohol and 1-propanol.The following group is also closely related on the second or third part of the axis (X -axis negative,Y -axis negative and Z -axis positive or negative)and includes the samples of end fermentation (A5,A6,B5,B6,C5and C6).The final concentration of volatiles revealed only little variation amongst in the samples of beverages produced (ML,CW and DCW),confirming results (Table 1).Our results indicated a significant contribution of kefir culture in volatile compounds as the composition of organic acids,esters,acetaldehyde and alcohols.The compounds identified in in milk and CW ⁄based kefir beverages are similar to those present in other beverages,like sake (Teramoto et al.,2002),mouro (Soufleros etal.,2004).,or mescal (Leon-Rodrıguez et al.,2006)produced from Agave salmiana ,for example.Dragone et al.(2009)also found similar compounds in CW alcoholic beverage.Sensory analysisThe kefir beverages were subjected to sensory analysis to assess its acceptance.(Table 2).For all attributes assessed the beverages showed good acceptance (at least 5points or 50%of acceptance).The likeness in sensory analysis found amongst these three beverages analysed here might be the result of the similar chemical and volatile compounds compositions of these final products (Figs 1–3and Table 1).Table 2Sensory colour,appearance,taste and overall acceptabilityscores on a 9-point hedonic scale (9=like extremely;1=dislike extremely)of kefir beveragesKefir beverages Sensory propertiesColour Aroma Appearance Taste Overallacceptability Milk6.02 6.11 5.82 6.17 6.16Cheese whey 5.78 6.01 5.69 6.08 6.01Deproteinised cheese whey5.515.925.545.825.89Figure 4Principal Component Analysis (PCA)3D plot of concentra-tion of volatiles compounds of kefir beverages.Chemical and sensory analysis of CW-based kefir beverages K.T.Magalha˜es et al.876International Journal of Food Science and Technology 2011Ó2011The AuthorsInternational Journal of Food Science and Technology Ó2011Institute of Food Science and TechnologyThe traditional milk kefir beverage is commonly manufactured in different countries and is known for its organoleptic characteristics assessed.The potential for use of CW as a medium for manufacturing products with a sensory profile similar to that of fermented milk beverages was demonstrated by the results of this study,i.e.there was no evidence that,when using the kefir grains,adversely effect the substrate(CW or milk) on volatile compounds and sensory(Figs1–3and Table1).Assadi et al.,(2008)tested the technological potential of various ratios of lactic bacteria,yeasts and acetic acid bacteria isolated from kefir grains starter culture for CW fermentation.The best results were obtained when all the starter cultures were combined,i.e.bacterial mixed yeast fermentation.The potential of starter culture in production of healthy beverage from CW was found and the beverage produced good organoleptic quality and presented taste of artificial butter milk and naturally carbonated.These results confirm the good acceptable of the beverages produced in this study by kefir grains containing mixed bacterial and yeast cultures. ConclusionsKefir grains were able to reduce the lactose concentra-tion in CW producing volatile compounds for good quality of the beverages.GC-FID revealed the presence of volatile compounds in the kefir beverages.Most of these compounds are similar to those reported for other fermented beverages,although the concentration values are different.Higher alcohols(mainly isobutyl alcohol and isopentyl alcohol)and ethyl esters(mainly ethyl acetate)were the most dominant compounds present,contributing thus for the greatest proportion of the total aroma.The results of this study indicate that novel beverages of acceptable organoleptic char-acter can be produced by CW-based fermentation by kefir grains.The proposed technology in this study is environmen-tally significant due to the fact that a very polluting liquid industrial waste is employed to produce products of nutritional value,including the use of probiotic kefir grains as alternative.The one key point for industrial application of the proposed technology is the promotion of fermentation by kefir of granular biomass which provides the possibility of eliminating the use of centrifugal separators that have a high energy demand and require high industrial investment. AcknowledgmentsThe authors acknowledge thefinancial support from Coordenac a o de Aperfeic oamento de Pessoal de Nıvel Superior(CAPES),CAPES-GRICES and Lactogal for supplying CW powder.ReferencesApostolopoulou, A.A.,Flouros, A.I.,Demertzis,P.G.&Akrida-Demertzi,K.(2005).Differences in concentration of principal volatile constituents in traditional Greek distillates.Food Control, 16,157–164.Assadi,M.M.,Abdolmaleki,F.&Mokarrame,R.R.(2008).Appli-cation of whey in fermented beverage production using kefir starter culture.Nutrition&Food Science,38,21–127.Athanasiadis,I.,Paraskevopoulou,A.,Blekas,G.&Kiosseoglou,V. (2004).Development of a novel whey beverage by fermentation with kefir granules.Effect of various treatments.Biotechnology Progress, 20,1091–1095.Beshkova, D.M.,Simova, E.D.,Frengova,G.I.,Simov,Z.I.& Dimitrov,Zh.P.(2003).Production of volatile aroma compounds by kefir starter culture.International Dairy Journal,13,529–535. Dragone,G.,Mussatto,S.I,Oliveira,J.M.&Teixeira,J.A.(2009). Characterisation of volatile compounds in an alcoholic beverage produced by whey fermentation.Food Chemistry,112,929–935. Escudero,A.,Gogorza, B.,Melus,M.A.,Ortin,N.,Cacho,J.& Ferreira,V.(2004).Characterization of the aroma of a wine from Maccabeo.Key role played by compounds with low odor activity values.Journal of Agricultural and Food Chemistry,52,3516–3524. Falque,E.,Ferna ndez,E.&Dubourdieu,D.(2001).Differentiation of white wines by their aromatic index.Talanta,54,271–281. Fraile,P.,Garrido,J.&Ancın,C.(2000).Influence of a Saccharo-myces selected strain in the volatile composition of Rose wines. Evolution during fermentation.Journal of Agricultural and Food Chemistry,48,1789–1798.Geroyiannaki,M.,Komaitis,M.E.,Stavrakas,D.E.,Polysiou,M., Athanasopoulos,P.E.&Spanos,M.(2007).Evaluation of acetal-dehyde and methanol in greek traditional alcoholic beverages from varietal fermented grape pomaces(Vitis vinifera L.).Food Control, 18,988–995.Guimara es,P.M.R.,Teixeira,J.A.&Domingues,L.(2010).Fermen-tation of lactose to bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey.Biotechnology Advances,28,375–384.Gu zel-Seydim,Z.B.,Seydim, A.C.,Greene, A.K.&Bodine, A.B. (2000).Determination of organic acids and volatileflavor substances in kefir during fermentation.Journal of Food Composition and Analysis,13,35–43.Leo n-Rodrıguez,A.,Gonza lez-Herna ndez,L.,De la Rosa,A.P.B., Escalante-Minakata,P.&Lo pez,M.G.(2006).Characterization of volatile compounds of mezcal,an ethnic alcoholic beverage obtained from Agave salmiana.Journal of Agricultural and Food Chemistry, 54,1337–1341.Magalha es,K.T.,Pereira,M.A.,Nicolau,et al.(2010a).Production of fermented cheese whey-based beverage using kefir grains as starter culture:evaluation of morphological and microbial variations. Bioresource Technology,101,8843–8850.Magalha es,K.T.,Pereira,G.V.M,Dias, D.R.&Schwan,R.F. (2010b).Microbial communities and chemical changes during fermentation of sugary Brazilian kefir.World Journal of Microbiol-ogy and Biotechnology,26,1241–1250.Pereira,G.V.M,Ramos,C.L.,Galva o,C.,Dias,E.S.&Schwan,R.F. (2010).Use of specific PCR primers to identify three important industrial species of Saccharomyces genus:Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces pastorianus.Letters in Applied Microbiology,51,131–137.Pescuma,M.,Heberta,E.M.,Mozzia,F.&Valdez,G.F.(2008).Whey fermentation by thermophilic lactic acid bacteria:evolution of carbohydrates and protein content.Food Microbiology,25,442–451. Rynne,N.M.,Beresford,T.P.,Kelly,A.L.&Guinee,T.P.(2007). Effect of milk pasteurisation temperature on age-related changes in lactose metabolism,pH and the growth of non-starter lactic acid bacteria in half-fat cheddar cheese.Food Chemistry,100, 375–382.Chemical and sensory analysis of CW-based kefir beverages K.T.Magalha˜es et al.877Ó2011The Authors International Journal of Food Science and Technology2011 International Journal of Food Science and TechnologyÓ2011Institute of Food Science and Technology。

第４８卷第１０期东　北　林　业　大　学　学　报Ｖｏｌ．４８Ｎｏ．１０２０２０年１０月ＪＯＵＲＮＡＬＯＦＮＯＲＴＨＥＡＳＴＦＯＲＥＳＴＲＹＵＮＩＶＥＲＳＩＴＹＯｃｔ．２０２０１）江苏省科技厅农业重点研发项目（ＢＥ２０１８３２１）；江苏高校优势学科建设工程资助项目（ＰＡＰＤ）。

第一作者简介：郑琰邁，女，１９６８年１２月生，南京林业大学期刊部，副编审，Ｅ－ｍａｉｌ：ｘｕｅｂａｏ＠ｎｊｆｕ．ｃｏｍ．ｃｎ。

通信作者：朱咏莉，南京林业大学生物与环境学院，研究员。

Ｅ－ｍａｉｌ：ｌｙｌｙ１２６２０１１＠１２６．ｃｏｍ。

收稿日期：２０２０年５月２７日。

责任编辑：张　玉。

醋糟基质栽培的南方红豆杉苗光合日变化特征１）郑琰邁　朱咏莉　王宇　高飞（南京林业大学，南京，２１００３７） 摘　要　以南方红豆杉苗为栽培对象，分析醋糟基质栽培的苗木光合指标及光合特性的变化，探讨醋糟基质对南方红豆杉苗生长的影响。

结果表明：不同栽培基质处理的红豆杉苗光合速率不同，除Ｖ（锯末）∶Ｖ（土壤）为８０∶２０的处理外，所有含有醋糟的处理都高于土壤（ＣＫ１），但低于泥炭的值，其中纯醋糟处理与泥炭比较接近，说明醋糟可以作为南方红豆杉苗的栽培基质。

醋糟基质上，供试南方红豆杉１～２年生苗，５月份的光合速率整体较低，光合日变化呈单峰曲线；３～４年生苗，５月份光合日变化较大，晴天苗木的光合速率高于阴天，而且两者的变化曲线不同，晴天波动比较大，有“光合午休”现象，阴天呈单峰曲线，说明南方红豆杉在醋糟基质上的生长符合其生物学特性。

南方红豆杉苗光合速率与其光合有效辐射、胞间ＣＯ２摩尔分数均呈显著的正相关关系，但与气孔导度、蒸腾速率的线性关系未达到显著水平。

关键词　醋糟基质；南方红豆杉；光合日变化分类号　Ｓ７９１．４９；Ｓ７１８．４３ＤｉｕｒｎａｌＶａｒｉａｔｉｏｎＣｈａｒａｃｔｅｒｉｓｔｉｃｓｏｆＰｈｏｔｏｓｙｎｔｈｅｓｉｓｏｆＴａｘｕｓｗａｌｌｉｃｈｉａｎａｖａｒ．ｍａｉｒｅｉＳｅｅｄｌｉｎｇｓｕｎｄｅｒＶｉｎｅｇａｒＲｅｓｉ ｄｕｅＳｕｂｓｔｒａｔｅＣｕｌｔｉｖａｔｉｏｎ／／ＺｈｅｎｇＹａｎｙｉ，ＺｈｕＹｏｎｇｌｉ，ＷａｎｇＹｕ，ＧａｏＦｅｉ（ＮａｎｊｉｎｇＦｏｒｅｓｔｒｙＵｎｉｖｅｒｓｉｔｙ，Ｎａｎｊｉｎｇ２１００３７，Ｐ．Ｒ．Ｃｈｉｎａ）／／ＪｏｕｒｎａｌｏｆＮｏｒｔｈｅａｓｔＦｏｒｅｓｔｒｙＵｎｉｖｅｒｓｉｔｙ，２０２０，４８（１０）：２５－２９．ＴａｋｉｎｇｔｈｅｓｅｅｄｌｉｎｇｓｏｆＴａｘｕｓｗａｌｌｉｃｈｉａｎａｖａｒ．ｍａｉｒｅｉａｓｔｈｅｃｕｌｔｉｖａｔｉｏｎｏｂｊｅｃｔ，ｔｈｅｃｈａｎｇｅｓｏｆｐｈｏｔｏｓｙｎｔｈｅｔｉｃｉｎｄｅｘｅｓｏｆｓｅｅｄｌｉｎｇｓｗｉｔｈｄｉｆｆｅｒｅｎｔｐｒｏｐｏｒｔｉｏｎｓｏｆｖｉｎｅｇａｒｒｅｓｉｄｕｅｓｕｂｓｔｒａｔｅｗｅｒｅａｎａｌｙｚｅｄ，ａｎｄｔｈｅｅｆｆｅｃｔｓｏｆｄｉｆｆｅｒｅｎｔｐｒｏｐｏｒｔｉｏｎｓｏｆｖｉｎｅｇａｒｒｅｓｉｄｕｅｓｕｂｓｔｒａｔｅｏｎｔｈｅｇｒｏｗｔｈｏｆＴ．ｗａｌｌｉｃｈｉａｎａｖａｒ．ｍａｉｒｅｉｓｅｅｄｌｉｎｇｓｗｅｒｅｄｉｓｃｕｓｓｅｄ．ＴｈｅｒｅｓｕｌｔｓｓｈｏｗｅｄｔｈａｔｔｈｅｐｈｏｔｏｓｙｎｔｈｅｔｉｃｒａｔｅｏｆＴ．ｗａｌｌｉｃｈｉａｎａｖａｒ．ｍａｉｒｅｉｓｅｅｄｌｉｎｇｓｔｒｅａｔｅｄｗｉｔｈｄｉｆｆｅｒｅｎｔｓｕｂｓｔｒａｔｅｓｗａｓｄｉｆｆｅｒｅｎｔ．ＥｘｃｅｐｔｆｏｒｔｈｅｔｒｅａｔｍｅｎｔｏｆＶ（ｓａｗｄｕｓｔ）∶Ｖ（ｓｏｉｌ）ｏｆ８０∶２０，ａｌｌｔｒｅａｔｍｅｎｔｓｃｏｎｔａｉｎｉｎｇｖｉｎｅｇａｒｌｅｅｓｗｅｒｅｈｉｇｈｅｒｔｈａｎｔｈｏｓｅｏｆｓｏｉｌ（ＣＫ１），ｂｕｔｌｏｗｅｒｔｈａｎｐｅａｔ．Ｔｈｅｔｒｅａｔｍｅｎｔｏｆｐｕｒｅｖｉｎｅｇａｒｌｅｅｓｗａｓｃｌｏｓｅｔｏｐｅａｔ．ＴｈｉｓｒｅｓｕｌｔｉｎｄｉｃａｔｅｄｔｈａｔｖｉｎｅｇａｒｌｅｅｓｃｏｕｌｄｂｅｕｓｅｄａｓｃｕｌｔｉｖａｔｉｏｎｓｕｂｓｔｒａｔｅｏｆＴ．ｗａｌｌｉｃｈｉａｎａｖａｒ．ｍａｉｒｅｉｓｅｅｄｌｉｎｇｓ．Ｗｉｔｈｔｈｅｔｒｅａｔｍｅｎｔｏｆｖｉｎｅｇａｒｌｅｅｓ，ｔｈｅｐｈｏｔｏｓｙｎｔｈｅｔｉｃｒａｔｅｏｆ１－２ ｙｅａｒ ｏｌｄｓｅｅｄｌｉｎｇｓｉｎＭａｙｗａｓｌｏｗｅｒ，ａｎｄｔｈｅｄｉｕｒｎａｌｖａｒｉａｔｉｏｎｏｆｐｈｏｔｏｓｙｎｔｈｅｓｉｓｐｒｅｓｅｎｔｅｄａｓｉｎｇｌｅｐｅａｋｃｕｒｖｅ．Ｔｈｅｐｈｏｔｏｓｙｎｔｈｅｔｉｃｒａｔｅｏｆ３－４ ｙｅａｒ ｏｌｄｓｅｅｄｌｉｎｇｓｃｈａｎｇｅｄｇｒｅａｔｌｙｉｎＭａｙ，ａｎｄｔｈｅｐｈｏｔｏｓｙｎｔｈｅｔｉｃｒａｔｅｏｆｓｅｅｄｌｉｎｇｓｉｎｓｕｎｎｙｄａｙｓｗａｓｈｉｇｈｅｒｔｈａｎｔｈａｔｉｎｃｌｏｕｄｙｄａｙ．Ｔｈｅｉｒｖａｒｉａｔｉｏｎｃｕｒｖｅｓｗｅｒｅｄｉｆｆｅｒｅｎｔ．Ｔｈｅｒｅｗａｓａｐｈｅｎｏｍｅｎｏｎｏｆ“ｍｉｄｄａｙｄｅｐｒｅｓｓｉｏｎｏｆｐｈｏｔｏｓｙｎｔｈｅｓｉｓ”ｉｎｓｕｎｎｙｄａｙ．Ｔｈｅｃｕｒｖｅｏｆｓｉｎｇｌｅｐｅａｋａｐｐｅａｒｅｄｉｎｃｌｏｕｄｙｄａｙ．ＴｈｉｓｉｎｄｉｃａｔｅｄｔｈａｔｔｈｅｇｒｏｗｔｈｏｆＴ．ｗａｌｌｉｃｈｉａｎａｖａｒ．ｍａｉｒｅｉｏｎｖｉｎｅｇａｒｒｅｓｉｄｕｅｓｕｂｓｔｒａｔｅｗａｓｃｏｎｓｉｓｔｅｎｔｗｉｔｈｉｔｓｂｉｏｌｏｇｉｃａｌｃｈａｒａｃｔｅｒｉｓｔｉｃｓ．ＴｈｅｐｈｏｔｏｓｙｎｔｈｅｔｉｃｒａｔｅｏｆＴ．ｗａｌｌｉｃｈｉａｎａｖａｒ．ｍａｉｒｅｉｓｅｅｄｌｉｎｇｓｓｈｏｗｅｄｓｉｇｎｉｆｉｃａｎｔｌｙｐｏｓｉｔｉｖｅｃｏｒｒｅｌａｔｉｏｎｗｉｔｈＰＡＲａｎｄｉｎｔｅｒｃｅｌｌｕ ｌａｒＣＯ２ｃｏｎｃｅｎｔｒａｔｉｏｎ，ｂｕｔｔｈｅｌｉｎｅａｒｒｅｌａｔｉｏｎｓｈｉｐｗｉｔｈｓｔｏｍａｔａｌｃｏｎｄｕｃｔａｎｃｅａｎｄｔｒａｎｓｐｉｒａｔｉｏｎｒａｔｅｗａｓｎｏｔｓｉｇｎｉｆｉｃａｎｔ．Ｋｅｙｗｏｒｄｓ　ＶｉｎｅｇａｒＲｅｓｉｄｕｅＳｕｂｓｔｒａｔｅ；Ｔａｘｕｓｗａｌｌｉｃｈｉａｎａｖａｒ．ｍａｉｒｅｉ；Ｄｉｕｒｎａｌｖａｒｉａｔｉｏｎｏｆｐｈｏｔｏｓｙｎｔｈｅｓｉｓ 醋糟是生产食醋过程中产生的有机废弃物。

ORIGINAL ARTICLESynthesis,Surface and Thermodynamic Properties of Substituted Polytriethanolamine Nonionic SurfactantsNabel A.Negm •Ahmed F.El-Farargy •Salah M.Tawfik •Ahmad M.Abdelnour •Hassan H.Hefni •Manal M.KhowdiaryReceived:7May 2012/Accepted:1October 2012/Published online:20October 2012ÓAOCS 2012Abstract Three series of nonionic surfactants derived from polytriethanolamine containing 8,10,and 12units of triethanolamine were synthesized.Structural assignment of the different compounds was made on the basis of FTIR and 1H-NMR spectroscopic data.The surface parameters of these surfactants included critical micelle concentration (CMC),surface tension at the CMC (c CMC ),surfactant concentration required to reduce the surface tension of the solvent by 20mN m -1(p C 20),maximum surface excess (C max ),and the interfacial area occupied by the surfactant molecules (A min )using surface tension measurements.The micellization and adsorption free energies were calculated at 25°C.Keywords Nonionic ÁSurface tension ÁEffectiveness ÁEfficiency ÁSurface pressureIntroductionThere have been recent moves to reduce the use of certain classes of surfactants polluting the environment and to replace them with new ones [1–3].Accordingly,efforts are being made to develop new classes of surfactants with reduced foaming capacity and milder action,which operate at a lower temperature with synergistic action of their com-ponents [4–6].Such surfactants should be environmental friendly and cost efficient.Alkyl glycosides [7],sugar-based [8],natural product surfactants [9]have been used in the production of detergents in recent years [6].The use of environmentally friendly surfactants for the production of detergents shows promise not only from the efficiency standpoint but also in protection of the environment [10].Environmentally friendly surfactants are characterized by high biodegradability resulting from being manufactured from natural raw materials.At the same time,these are mild,low toxic surfactants providing no skin irritation.In recent years,the cost of surfactants has steadily declined.In our previous works,we synthesized several types of surfactants which are environmentally friendly [11–14].In the contin-uation of our research in surfactant science,we synthesized and studied the surface activity of nine nonionic surfactants obtained from the polymerization of triethanolamine.The results were then compared with those obtained for the known anionic and nonionic surfactants.Synthesis of Polytriethanolamine (EP,TP,and WP)Polytriethanolamine was prepared by condensation poly-merization of triethanolamine in the presence of NaOH as a catalyst at 245°C for different time intervals of 3,4,and 6h to produce EP,TPs and WP,where E,T,and W refer to 8,10,and 12units of triethanolamine in the polymericElectronic supplementary material The online version of this article (doi:10.1007/s11743-012-1412-6)contains supplementary material,which is available to authorized users.N.A.Negm (&)ÁS.M.Tawfik ÁH.H.Hefni ÁM.M.KhowdiaryPetrochemicals Department,Egyptian Petroleum Research Institute,Nasr City,Cairo,Egypt e-mail:nabelnegm@A.F.El-FarargyChemistry Department,Faculty of Science,Zagazig University,Zagazig,EgyptA.M.Abdelnour ÁM.M.Khowdiary Khalda Petroleum Company,Cairo,EgyptJ Surfact Deterg (2013)16:333–342DOI 10.1007/s11743-012-1412-6chain,ing a three-neckedflask equipped with a mechanical stirrer,nitrogen bubbler,and Dean–Stark apparatus,1mol of triethanolamine was charged in the presence of0.5mol of NaOH as a catalyst.The reac-tion was operated by heating the reaction mixture at245°C until the water of the reaction needed to produce polytri-ethanolamine EP,TP,and WP was isolated[15,16]. Scheme1represents the synthetic steps of the different polytriethanolamine polymers.After the removal of the desired amount of water,the reaction vessel was removed from the heat and allowed to cool.Then,the viscous layer containing the produced polymers was separated and washed by petroleum ether to separate the catalyst used (NaOH)(Scheme1).The molecular weights of the pro-duced polymers were determined using GPC chromatog-raphy,and polyethylene glycol(Mw=1,000,2,000g mol-1)was used as a standard(Table1).Synthesis of Polyethylene Glycol Monooleate-Maleic Anhydride Ester(OM)Amounts of0.21mol of polyethylene glycol(Mw=400g mol-1)and0.2mol of oleic acid(28.2g)were esterified individually under reflux conditions in the presence of 100mL of xylene as a solvent and0.1%by weight of p-toluene sulphonic acid as a dehydrating agent.The reac-tion was stopped when the theoretical amount of the water produced from the reaction(3.6mL)was obtained.Then,the solvent was removed under reduced pressure.The reaction product was dissolved in petroleum ether(250mL),and then 100mL of distilled water was added.The aqueous layer which contains the catalyst and the excess polyethylene glycol was separated with a separating funnel and the ether layer was evaporated to obtain the polyethylene glycol/mo-nooleate ester[17].An amount of0.2mol of polyethylene glycol-monooleate was reacted by0.2mol of maleic anhy-dride in the presence of150mL of benzene as a solvent under continuous stirring.The reaction was followed by IR spectroscopy and was deemed complete at the point of the disappearance of the anhydride linkage at1,850cm-1 (Scheme2).The reaction product was dissolved in200mL (50:50vol.)of ethyl acetate/diethyl ether mixture.The ethyl acetate layer contained the diester and the unreactants dis-solved in the diethyl ether layer.The lower layer was sepa-rated in a funnel,and the solvent was evaporated under reduced pressure to obtain the desired ester[18].Table1Expected and experimental molecular weights of polytriethanolamine polymersPolymer Triethanolamine added H2O produced Expected molecularweight(g mol-1)Obtained molecular weight(g mol-1)EP14915.61,0661,060 TP14916.21,3281,320 WP14916.71,5901,600Synthesis of the Nonionic Surfactants POM yPolymerized triethanolamine polymers(EP,TP,and WP) were esterified individually by different molar amounts of polyethyleneglycol-oleic-maleic anhydride ester in a suf-ficient amount of xylene and0.1%by weight of p-toluene sulphonic acid under reflux condition until the theoretical amount of water was obtained(see Table2for reacted amounts and the esters produced).The product was washed with a hot supersaturated sodium chloride solution.Then, the organic layer which contains the products was sepa-rated and the solvent was distilled off under vacuum[19].Elemental AnalysisMicroelemental analysis of the synthesized surfactants was performed using a Vario Elementar instrument(USA).IR SpectraInfrared spectroscopic analysis of the synthesized surfac-tants was performed by the KBr disk method using a Fourier transform infrared spectrophotometer(FTIR).1H-NMR Spectra1H-NMR spectra were recorded using Varian NMR-300 Mercury300MHz spectrometer with TMS as an internal standard and DMSO-D6as a solvent.Gel Permeation Chromatography(GPC)GPC experiments were carried out using a Supremamax 3000column(Polymer Standard Service,Mainz,Germany) with water as eluent(1mL min-1).The system comprised a pump(Hitachi,Darmstadt,Germany),an auto sampler device(Merck Hitachi model AS-2000A),and a vacuum in-line degreaser.The amount of injected sample volume per run was40l L.The samples were analyzed with a differential refractive index(RI)detector RI-71(Merck). Molecular weights were calculated using Astra software (Wyatt Technology).The standard compounds(Mw of 1,000,2,000,and3,000g mol-1)were used for calibration [20].The average molecular weights(Mwt GPC)of the synthesized compounds were obtained relative to the standard compounds and are listed in Table1.Surface Tension Measurements(c)Surface tension measurements were obtained using a De-Nou¨y Tensiometer(Kruss-K6type;Germany)with a platinum ring device.A freshly prepared aqueous solution of the synthesized polytriethanolamine derivatives in deionized water was prepared with a concentration range of 0.01–0.00001M at25°C.The ring was washed twice after each reading,first by ethanol then by distilled water.The apparent surface tension was measured5times for each sample within a2-min interval between each reading[17, 21].Structure of SurfactantsThe synthesis of the nonionic surfactants was performed through three main reactions including synthesis of the polytriethanolamine throughout polymerization of trietha-nolamine from three polymers containing8,10,and12 triethanolamine units,respectively.The products of the polymerization reaction were abbreviated as EP,TP,and WP,Scheme1.The structure of the three polymers were confirmed by FTIR spectra which showed the following absorption bands(EP polymer was considered as repre-sentative sample for the synthesized three polymers): 3,500–3,100cm-1(stretching of H–O groups),2,948, 2,877cm-1(stretching of H–C groups),1,455–1,360cm-1 (scissoring and bending of H–C groups),1,115cm-1 (stretching of C–O groups),and1,037cm-1indicating stretching of C–N groups(Fig.1in supplementaryfile). The molecular weights of the obtained polymers were confirmed by gel permeation chromatography and the results showed comparable values between the expected and experimental values within0.5%.The synthesis of the polyethylene glycol monooleate-maleic anhydride ester(OM)was obtained through Scheme2.The chemical structure of the obtained OM compound was confirmed by FTIR spectra which showed the following absorption bands:3,600–3,200cm-1 (stretching of H–O groups),2,960,2,850cm-1(stretching of H–C groups),1,470,1,350cm-1(scissoring and bend-ing of H–C groups),1,734cm-1(stretching of C=O group),1,640cm-1(stretching of C=C group),1,249cm-1 (stretching of C–O of seter group),and1,105cm-1 (stretching of C–O of ether groups)(Fig.2in supplemen-taryfile).The structures of the nonionic surfactants(Scheme3) were confirmed using FTIR spectra(EPOM4was considered as representative sample for the other surfactants)which showed the following absorptions:3,100–3,600cm-1(stretching of H–O groups),2,923,2,856cm-1(stretching of C–H groups),1,459,1,350cm-1(scissoring and bending of C–H groups),1,735cm-1(stretching of C=O groups), 1,648cm-1(stretching of C=C groups),1,250cm-1 (stretching of C–O ether groups),and1,111cm-1(stretch-ing of C–N groups)(Fig.1a).The1H-NMR spectra of EPOM4showed the signals at d=0.83ppm(t,12H,CH3), 1.24ppm(dd,104H,CH2)n),1.96ppm(t,48H,CH2N), 3.66ppm(t,16H,COOCH2), 4.16ppm(q,132H (CH2CH2O)n),4.3ppm(s,8H,CH=CH)and5.28ppm(d, 8H,COOHC=CHCOO)(Fig.1b).The molecular weights of the surfactants were measured using GPC chromatography and the results were in good agreement with the calculated values.The above discussion confirms the structure of the synthesized surfactants as represented in Scheme3. Surface Tension and Critical Micelle ConcentrationThe relationship between the surface tension and-log C of the synthesized nonionic surfactants is plotted in Fig.2as representative for the synthesized surfactants at25°C. Two conclusions can be obtained from Fig.2.First,the gradual increase in the surfactant concentration of the different surfactants considerably decreases the surface tension values.Second,within the homologue series of EPOM,TPOM,or WPOM,increasing the number of the polyethylene glycol oleate chains attached to the polymer chain decreases the surface tension values.Increasing the number of polyethylene glycol oleate chains in the mole-cule increases their hydrophobic characters,which increa-ses their tendency to adsorb at the air/water interface, which considerably decreases their surface tension.The critical micelle concentration values(CMC)of these surfactants were determined by the intercept of the pre-and post-micellar trends in Fig.2.These values are listed in Table2.As a general observation,CMC values depend on the number of polyethylene glycol oleate chainsTable2Amounts of reactants and molecular weights of the obtained nonionic surfactantsSurfactants abbreviations Polytriethanolamine(P n)expressed in grams Nonionic ester(OM)expressed in grams Molecular weight(g mol-1) Number ofmolesUsed weight Number of moles Used weight Expected MeasuredEPOM20.005 5.330.017.362,5022,474 EPOM40.005 5.330.0214.723,9383,895 EPOM60.005 5.330.0322.085,3745,315 TPOM30.005 6.640.01511.043,4823,444 TPOM50.005 6.640.025 4.9184,9184,864 TPOM70.005 6.640.03525.766,3546,284 WPOM40.0057.950.0214.724,4624,413 WPOM70.0057.950.03525.766,6166,543 WPOM90.0057.950.04533.128,0527,963attached to the different molecules and the number of the triethanolamine molecules polymerized in the surfactant molecules.The gradual increase of the polyethylene glycol oleate chains in the polytriethanolamine molecules con-siderably decreases CMC values from2.72to1.41mM for the EPOM series.Also,that behavior is predominant in the TPOM and WPOM series due to the increase of the hydrophobic characteristics of the molecules.The same behavior was observed by increasing the triethanolamine units in the surfactant molecules and this was in good agreement with the previous study[18].The increase of the polymerized units from8to10units decreases the CMC values by a factor of0.1.Also,the CMC values of WPOM are decreased by a factor of0.1 more than TPOM derivatives while the effect of the polyethylene glycol oleate chain number within the same homologue has a minor effect on their CMC values (Table3).That is to be expected from the increase in hydrophobicity of the molecules as the number of methy-lene groups increases by increasing the degree of poly-merization[19,22].This decrease is linear as is seen in Fig.3.This usually happens in a homologous series of anionic[23],cationic[24,25]and nonionic[26] surfactants.Efficiency and Effectiveness of Surfactant Adsorption The performance of a surfactant in lowering the surface tension of a solution can be discussed in terms of(1)the concentration required to produce a given surface tensionreduction,and(2)the maximum reduction in surface ten-sion that can be obtained regardless of the concentration. These are referred to as the surfactant efficiency and effectiveness,respectively.A good measure of the surfactant adsorption efficiency is the concentration of surfactant required to produce a 20mN m-1reduction in the surface tension compared to distilled water.This value of the surfactant concentrationis Fig.1a FTIR spectra and b1H-NMR spectra of the EPOM4surfactantclose to the minimum concentration needed to produce maximum adsorption at the interface.There is a regular decrease in c CMC with increasing the number of substituents attached to the different polytri-ethanolamine chains,as expected[2,19,22–25].The decrease in c CMC values of the synthesized surfactants is attributed to the increase in the hydrophobic characters of these molecules.The data in Table3revealed that the highest depression in the surface tension at the critical micelle concentration is accompanied by the highest value of PO/nTEA.The increase in the hydrophobicity is due to the increase in the ratio of the nonpolar chains of the oleic acid chains to the total number of triethanolamine units in each chain in the molecules[27–29].Values of C max(the surface excess concentration of surfactant)and A min(interfacial area occupied by the surfactant molecules)were calculated using Gibbs equa-tions(1and2):C max¼12:303RTo co log Cð1ÞA min¼À1014N C maxð2Þwhere dc/d log C is the maximum slope in surface tension versus-log C profile,R=gas constant(8.314J mol-1 K-1),N is Avogadro’s number,and T is the absolute temperature(K).By inspection of the data of C max,it can be concluded that the C max decreases by increasing the number of polyethylene glycol oleate chains attached to the surfactant molecules. This means that the increased number of these chains leads to a decrease in the adsorbed number of surfactant molecules at the air/water interface and also to an increase in the area occupied by each molecule at that interface.It is clear from the data listed in Table3that there is a gradual decrease in C max values by increasing the molecular weights of the synthesized surfactants,which is a direct result of increasing the degree of polymerization of the triethanolamine units.The maximum surface excess values ranged between 4.68910-10and9.46910-10mol cm-2,which is in good agreement with the published data for the nonionic surfactants[18,30].The area occupied by each surfactant molecule at the air/water interface was calculated using Eq.2.The data showed the increasing trend of the area by increasing the molecular weight.That can be attributed to the increasing of the attachment points in the surfactant molecules at the air/water interface.The increase of these points is due to the increase of the hydroxyl groups, polyethylene glycol chains,and tertiary nitrogen atoms.Hydrophilic–Lipophilic BalanceThe hydrophilic–lipophilic balance(HLB)of a surfactant is a measure of the degree to which it is hydrophilic orTable3Surface and thermodynamic properties of the synthesized surfactants at25°CSurfactants CMC(molL-1)c cmc(mN m-1)p cmc(mN m-1)C max910-10(mol cm-2)pC20(ML-1)A min(nm2molecule-1)D G mic(kJ mol-1)D G ads(kJ mol-1)HLB PO/nTEAEPOM2 2.72910-337.534.39.46 1.0910-5176-14.64-18.277.10.25 EPOM4 2.60910-336.335.68.78 2.9910-6189-14.94-18.809.00.75 EPOM6 2.41910-334.637.28.32 6.3910-7200-14.94-19.4210.00.75 TPOM3 2.51910-440.031.88.99 1.9910-6185-20.54-24.097.70.30 TPOM5 1.93910-439.332.5 6.99 1.7910-7238-21.19-25.859.00.50 TPOM7 1.68910-437.234.6 6.26 3.2910-8265-21.54-27.0610.00.77 WPOM4 1.18910-43833.8 6.12 2.2910-8272-22.41-27.958.00.33 WPOM7 6.68910-53734.8 4.77 1.9910-8348-23.82-31.129.40.58 WPOM9 3.65910-53635.8 4.68 1.3910-8355-25.32-32.9810.00.75lipophilic,and is determined by calculating values for the different regions of the molecule,as described by Griffin [31,32].Other methods have been suggested,notably by Davies[33].Griffin’s method for nonionic surfactants as described in [35]works as follows:HLB¼ð20ÂM hÞ=Mð3Þwhere M h is the molecular mass of the hydrophilic portion of the molecule and M is the molecular mass of the whole molecule,giving a result on an arbitrary scale of0–20.An HLB value of0corresponds to a completely hydrophobic molecule and a value of20would corre-spond to a molecule made up completely of hydrophilic components.The HLB value can be used to predict the surfactant properties of a molecule as follows:a value\10:lipid soluble(water insoluble);and a value[10:water soluble. HLB values of the synthesized surfactants range between7 and10which describes these compounds as water in oil emulsifiers.The calculated HLB values of the synthesized compounds using Eq.3showed high solubility of the synthesized compounds in water(aqueous phase)and also their tendency to dissolve in the oil phase.Thermodynamic AspectsThe thermodynamic characteristics of the synthesized nonionic surfactants were studied using the calculated values of adsorption and micellization free energies,D G ads and D G mic,respectively(Table3.These data were calcu-lated using the thermodynamic equations4and5[34,35].D G mic¼2:303RT log CMCðÞð4ÞD G ads¼D G micÀð0:06Âp cmcÂA MinÞð5ÞThe micellization free energies of the targeted surfactants in their aqueous media showed a negative sign,indicating a spontaneously micellization process.The driving force of micelle formation is the repulsion occurring between the hydrophobic chains and the polar medium.The stabilization of the formed micelles occurred due to the hydrogen bonds which were formed between the polyethylene glycol groups and the water molecules[21]. Obviously,increasing the number of the polymerized triethanolamine units and the number of attached PO groups in these chains increases the negativity of the D G mic values and consequently their micellization tendency.The gradual increase in the negativity of D G mic agrees with the trend of the critical micelle concentration values as listed in Table3.On the other hand,the adsorption free energy change of the studied surfactants showed the exact response of D G mic by changing these two variables.The negativity of both D G ads and D G mic indicates that the two processes occurred spontaneously.The two values of D G mic and D G ads are comparable and refer to the equi-librium between the two phases of the surfactant molecules (adsorbed and micellized phase).Also,the increase of D G ads values may be ascribed to the tendency of the molecules to adsorb at the air/water interface until there is complete surface coverage.Beyond this,the molecules diffuse to the bulk of their solution to form micelles. 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Deterg7:23–3037.Varka EM,Argyropoulou EC,Infante MR,Pegiadou S(2004)Synthesis,characterization,and surface properties of phenylala-nine-glycerol ether surfactants.J Surf Deterg7:409–414 Author BiographiesNabel A.Negm received his PhD from Ain Shams University in 2000.He spent6months as a research fellow at the Max Planck Institute of Colloids and Surfaces,Golm,Germany.He is currently an assistant professor in the petrochemicals department of the Egyptian Petroleum Research Institute.He is interested in several surfactants applications,including detergency,emulsification,solubilization, corrosion inhibition of metals,and oilfield chemicals.Also,he is interested in the biological activity of surfactants and their metal complexes towards bacteria,fungi,and yeast.His recent R&D was focused on mixed surfactants and mixed surfactant–protein systems. Ahmed F.El Farargy is a professor of organic chemistry and ex-head of the chemistry department,Faculty of Science,Zagazig University.His major interests are focused on synthesis of hetero-cyclic compounds and their applications as biocides for bacteria and fungi.He is an expert in spectroscopy and its use in elucidation of the chemical structures of the novel compounds.Salah M.Tawfik is a research assistant in the petrochemicals department at the Egyptian Petroleum Research Institute.In his MSc thesis at Helwan University,his interest was in the synthesis and application of novel surfactants containing heterocyclic rings and their application as corrosion inhibitors and biocides.Ahmad M.Abdelnour is a MSc student,and working as a chemist at the Khalda Petroleum company.He is a specialist in corrosion protection of petroleum equipment.。