Original article
Chemical composition and nsory analysis of chee whey-bad beverages using kefir grains as starter culture
Karina Teixeira Magalha
˜es,1,2Disney Ribeiro Dias,3Gilberto Vinicius de Melo Pereira,1Jo ´Maria Oliveira,2Lucı´lia Domingues,2Jo
´Anto ´nio Teixeira,2Joa ˜o Batista de Almeida e Silva 4&Rosane Freitas Schwan 1
*1Department of Biology,Federal University of Lavras (UFLA),Campus Universita
rio,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,Portugal
3Department 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 revid form 9January 2011)
Summary
The aim of the prent work was to evaluate the u of the kefir grains as a starter culture for tradicional
milk kefir beverage and for chee whey-bad beverages production.Fermentation was performed by inoculating kefir grains in milk (ML),chee whey (CW)and deproteinid chee whey (DCW).Erlenmeyers containing kefir grains and different substrates were statically incubated for 72h at 25°C.Lacto,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 utili lacto 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 tho obtained during milk fermentation.Bad on the chemical characteristics and acceptance in the nsory analysis,the kefir grains showed potential to be ud for developing chee whey-bad beverages.
Keywords
Chee whey beverage,GC-FID,HPLC,kefir,lactic acid bacteria,nsory evaluation,yeasts.
Introduction
Chee whey (CW)is the major by-product of the dairy industry and its disposal without expensive wage treatments reprents 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 lacto 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 ud in many countries,especially Eastern Europe,as a natural starter in the production of kefir,a unique lf-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 (Kluyver
omyces,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 decrea the high lacto 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 nsory profiles and have already been consid-ered an option to add value to CW (Pescuma et al.,2008).Recently,Magalha es et al.(2010a)t out to cha-racteri 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 lar microscopy).The composition of microbiota was related to Lactobacillus kefiranofaciens subsp.kefirgranum,
*Correspondent:Fax:+553538291100;e-mail:rschwan@dbi.ufla.br
International Journal
of Food Science and Technology 2011,46,871–878871
doi:10.1111/j.1365-2621.2011.02570.x
Ó2011The Authors.International Journal of Food Science and Technology Ó2011Institute of Food Science and Technology
Lactobacillus 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 analyd 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 nsorial characterisation of the beverages.Therefore,the aim of the prent work was to evaluate the u of the kefir grains as a starter culture for traditional milk kefir beverage and for CW-bad beverages production,besides evaluating the biochem-ical changes,organic acids production and volatile compounds formation during fermentation process. Materials and methods
CW-bad and milk fermentation media
Three different substrates containing lacto concentra-tion of46g L)1were ud as fermentation media: pasteurid full cows milk(ML),CW and deproteinid chee whey(DCW).CW powder,obtained from a regional dairy industry(Lactogal,Porto,Portugal),was dissolved in sterile distilled water until the desired lacto concentration.DCW was made by autoclaving at115°C for10min the CW solution,followed by aptic centrifugation(2220g for20min)to remove cream.Confir
吸血鬼日记4mation of CW deproteinisation was accomplished for Kjeldahl method.
Kefir beverages production
Brazilian kefir grains were employed in the prent study.The grains(12.5g)were washed with sterile distilled water and inoculated in250mL of ML,CW and DCW.The milk is ud commonly for kefir beverage,therefore was ud 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 aptically taken every12h for analysis of organic acids,ethanol,sugars and volatileflavour substances.Determination of total reducing sugars was ud to asss 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 methods
Chemicals
1-Hexanol and ethyl acetate were purchad from Aldrich Chemistry(Munich,Germany).1-propanol,2-methyl-1-propanol,2-methyl-1-butanol,3-methyl-1-butanol were purchad from Fluka Analyticals(Seelze, Germany).Ethyl acetate,Acetaldeyde lacto,were purchad from Sigma-Aldrich(St Louis,MO,USA) and acetic acid,lactic acid,ethanol,methanol were purchad from Merck(
Darmstadt,Germany). Organic acids,sugars and ethanol
Lacto 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 ud.
Volatileflavour substances
In order to identify the volatile compounds,the kefir beverages were analyd 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 ud.The temperature of the injector and detector was t 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 ud 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 tho of standard compounds.Quantification of volatile compounds was performed with Varian Star Chromatography Worksta-tion software(Version6.41)and expresd as4-nonanol equivalents,after determining the detector respon factor for each compound.
Sensory evaluation
Thefinal 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).Randomid,refrigerated (10°C)samples of10mL were rved(containing 1.0mg of sucro)in clear,tulip-shaped glass with a volume of50mL;the 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 hedonic
Chemical and nsory analysis of CW-bad kefir beverages K.T.Magalha˜es et al.
872
International Journal of Food Science and Technology2011Ó2011The Authors
International Journal of Food Science and TechnologyÓ2011Institute of Food Science and Technology
zikascale(9=like extremely;1=dislike extremely)
according to colour,odour,aroma,appearance,taste
and overall acceptability characteristics.
Statistical analysis
Statistical analysis was carried out with Statistica soft-
ware version9.0(StatSoft Inc.,Tulsa,OK,USA).
Principal component Analysis(PCA)was ud to
summari the information in a reduced number of
principal components.A one-way anova was performed
for chemical parameters,concentration of volatile com-
pounds values to determine significant differences
(P<0.05)by using the Duncan’s multiple range test
using the spss version10.0.
Results and discussion
Microbial metabolites
Milk and CW-bad kefir beverages were monitored
during the72h fermentation period by determining the
acidity.During the72h of incubation,pH values of the
fermented milk kefir and whey-bad beverages ranged
from6.1to3.9,notfinding significant differences for all
the substrates(P<0.05)(data not shown).The pH
values were similar to tho previously reported for kefir
beverage(Magalha es et al.,2010b).pH is an important
factor that can strongly affect the quality of a beverage
(Sharma et al.,2009).Furthermore,pH values of the
fermentation broth significantly influence the fermenta-
jennifer lawrencetion time of lacto and the levels of volatile compounds,
reflecting possible variations in the nsory characteris-
tics of thefinal product(Athanasiadis et al.,2004).
High performance liquid chromatography(HPLC)
was ud to analy organic acids,ethanol e sugars in
the produced kefir beverages.The Figs1–3show the
concentration of sugars,organic acids and ethanol
obtained by ML,CW and DCW fermentation.The
production process of organic acids and alcohol was
followed by the lacto consumption in kefir beverages
(ML,CW and DCW).The total lacto consumption
was obrved in60h in the ML fermentation and72h
in the CW and DCW fermentation(Figs1–3).This
likely reflects an adaptation period of the microbial
community to the whole and deproteinid CW as kefir
grains are fermented in milk commonly.Lacto readily
degraded to galacto and gluco by Group N strep-
tococci,Lactobacillus and by some strains of Kluyver-
omyces(Gu zel-Seydim et al.,2000).
In the prent work,the lactic acid content incread
during the72h of fermentation process in kefir bever-
ages,reaching maximum value of6.35g L)1,6.34g L)1
and6.81g L)1in ML,CW and DCW kefir beverages,
董小姐原唱respectively(Figs1–3).The fermentation of lacto by LAB prent in kefir culture can be associated with the increa in lactic acid production through the hydrolysis of sugars relead from the glycomacropeptide of cain 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).The 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 increa(Ma-galha es et al.,2010b).
Ethanol concentration incread 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-dehydrogena 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 compounds
过去
LAB prent 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-as which are involved in the transformation of cain into free amino acids wh
ich are further degraded to volatile aroma compounds.Other enzymes are esteras and lipas that hydroly 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 reprented 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-cread
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).
moderate是什么意思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).Incread 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,incread concentration of1-hexanol,riously 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)hanol–Council Regulation(EEC)No.1576⁄89,1989)(Geroyiannaki et al.,2007).The abnce of methanol in kefir beverages is probably due to the lack of pectin in milk and CW.
Chemical and nsory analysis of CW-bad kefir beverages K.T.Magalha˜es et al.
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International Journal of Food Science and Technology2011Ó2011The Authors
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T 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 D
K 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 e
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M 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 72h
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
±1.73
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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 72h
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±0.23d e
5.91
±0.16d e
0.57±0.06a b c
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n .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 e
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仁爱英语八年级上册Chemical and nsory analysis of CW-bad kefir beverages K.T.Magalha
倒廪倾囷
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