Physicochemical, Nutritional, and Organoleptic Characterization of a Skimmed Goat Milk

16 Pages·2017·2.12 MB·English

nutrients
Article
Physicochemical, Nutritional, and Organoleptic
Characterization of a Skimmed Goat Milk Fermented
with the Probiotic Strain Lactobacillus plantarum C4
MiriamMoreno-Montoro,MiguelNavarro-Alarcón* ID,TrianaBergillos-Meca,
RafaelGiménez-Martínez,SilviaSánchez-HernándezandManuelOlalla-Herrera
DepartmentofNutritionandFoodChemistry,FacultyofPharmacy,UniversityofGranada,
E-18071Granada,Spain;[email protected](M.M.-M.);[email protected](T.B.-M.);[email protected](R.G.-M.);
[email protected](S.S.-H.);[email protected](M.O.-H.)
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:12April2018;Accepted:16May2018;Published:17May2018
Abstract: The benefits of goat milk, fermented milks, and probiotics for the humans are well
documented. In this study, a novel fermented goat milk was manufactured with the putative
probioticstrainLactobacillusplantarumC4togetherwithL.bulgaricusandStreptococcusthermophilus.
Ultrafiltration was chosen as the skimmed milk concentration method because it produced the
best viscosity and syneresis and a high casein content. The viability rate of all bacterial strains
was>107 cfu/mL,evenafter5weeksofstorageorafterinvitrogastrointestinaldigestion,which
is especially important for exertion of the probiotic strain functionalities. This fermented milk is
also a good source of nutrients, having a low lactose and fat content, high protein proportion,
andgoodmineralconcentration. Accordingtothesedataandtheoverallacceptabilitydescribedby
panelists,thisfermentedmilkisahealthydairyproductcomparablewithcommerciallyavailable
fermentedmilks.
Keywords: fermented goat milk; Lactobacillus plantarum C4; milk ultrafiltration; probiotic;
physicochemical;nutritionalandorganolepticcharacterization
1. Introduction
Thereisincreasedinterestinfoodswithapositiveeffectonhealthbeyondtheirnutritionalvalue,
and considerable attention has focused on probiotic products. Fermented milks, especially when
probioticsarepresent,havebeenattributedwithnumerousproperties,including: animprovementin
lactoseabsorption,increasesinproteinandfatdigestibilityandinantibacterialactivity[1],immune
system stimulation, preventive action against digestive system cancer, anticholesterolemic action,
andtheenhancementofmineralbioavailability,amongothers[2,3]. Consequently,fermentedmilk
consumptionhasbeenrecommendedforlactoseintolerance,diarrhea,constipation,Helicobacterpylori
treatment,prevention/improvementofrespiratoryandgastrointestinaltractinfections,strengthening
oftheimmunesystemimprovement,andatopiceczema,amongotherconditions[4,5]. Inaddition,
pre-andpro-biotic-containingfermentedinfantformulaearefrequentlyusedtorelieveorprevent
symptomsofgastrointestinaldiscomfortinyounginfants[6].
Fermented milk is widely used to carry probiotic strains because the bacteria are kept alive,
and its daily intake is recommended. However, cheese is in general the best carrier due to its
buffercapacityandhigherpH.Therehasbeenalongandsafehistoryofdairyproductscontaining
the genus Lactobacillus [5]. Specifically, supplementation with Lactobacillus plantarum has been
showntohavemultiplebenefits. Thus,long-termsupplementationwithL.plantarumTWK10may
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increasemusclemass,enhanceenergyharvesting,promotehealth,enhanceperformance,andcombat
fatigue [7]. In another study, a significant decrease in blood pressure was observed in DOCA-salt
hypertension-inducedVaDratsafteroraladministrationofL.plantarumTWK10-fermentedsoymilk
extract[8].ThepresentinvestigationwasdesignedtotesttheputativeprobioticstrainL.plantarumC4in
thiscontext.Thisstrainfulfillsinvitrocriteriafortheselectionofpotentiallyeffectiveprobioticbacteria
andhasdemonstratedantimicrobial,microbiota-modulatingandimmunomodulatingproperties[9,10].
Itcouldthereforebeahighlyvaluablestrainforthedairyindustryandforhealthcare. Skimmedmilk
waspreviouslyfoundtobeanappropriatevehiclefortheintragastricadministrationofthisprobiotic
straintomice[11].
Inapreviousstudyontheantioxidant,ACE-inhibitoryandantimicrobialactivityoffermented
goatmilkswiththeclassicalstarterbacteria(St: L.bulgaricusandStreptococcusthermophiles)orwith
theStplustheL.plantarumC4probioticstrain,nodifferencesinbiologicalactivitieswereobserved
betweenthetwofermentedmilks[1]. Only,someantimicrobialactivityagainstE.coliwasobservedfor
thefermentedmilkcontainingtheprobiotic,probablybeduetosomepeptidesbeingreleasedbythe
probioticstrain[1]. Additionally,intheinvitroevaluationofthefermentationpropertiesandpotential
probioticactivityofL.plantarumC4[10]itwasconcludedthatthisprobioticstraincouldmodulate
theintestinalmicrobiotainvitro,promotingchangesinsomenumericallyandmetabolicallyrelevant
microbialpopulationsandshiftsintheproductionofsmallshortfattyacids(SCFA)[10].
Goat milk has shown better digestibility, mineral bioavailability, and protein and fat profiles
in comparison to cow milk [3], and the consumption of probiotic fermented goat milk has been
attributed with certain therapeutic properties [12]. However, the production of goat set-yoghurts
mustovercomesometechnicalchallenges,becausethelowbufferingcapacityofgoatmilkincreases
theriskofover-acidification,anditslowα -caseincontentandhighcaseinmicelledispersioncan
s1
lead to the formation of an almost semi-liquid gel [13]. The low firmness and high surface whey
separation that can affect this type of yoghurt can be reduced by rigorous monitoring of the heat
treatment,incubationtemperature,andconcentrationsoffat,proteinandtotalsolids,amongother
measures[14–16]. Amongdifferentmilkconcentrationmethods,ultrafiltrationmainlyconcentrates
caseinsandmineralsboundtothem[17]. Inaddition,becausenoadditionalheattreatmentisrequired,
the properties of the milk are preserved, unlike in other traditional methods such as powdered
caseinatesorskimmedmilkaddition.
Oneofthemosttypicalsensorialcharacteristicsofgoatmilkisthecaprineflavorderivedfromthe
presenceofshortandmediumchainfattyacids,whichisgenerallyconsideredanegativeorganoleptic
attribute[3,18]. Itisessentialtoreducethisflavorinfermentedgoatmilk.
The objective of this study was to develop an appropriate procedure to manufacture a novel
probiotic fermented goat milk (PFM) with good physicochemical, nutritional, and organoleptic
properties,selectingandstandardizingthemilkconcentrationmethod,evaluatingbacterialinteractions
andviability(L.plantarumC4,L.bulcaricusandS.thermophilus),andcarryingoutphysicochemical,
nutritional,andorganolepticanalyses.
2. MaterialsandMethods
2.1. RawandSkimmedMilks
RawmilkfromMurciano–GranadinagoatswascollectedandfrozeninGranadaoveroneyear
to avoid seasonal variations. The absence of β-lactam antibiotics (penicillins and cephalosporins),
sulfamides,andtetracyclineswastestedwithmilkTriSensorandHeatsensorkits(Unisensor,Liège,
Belgium), incubating at 40 ◦C. The raw goat milk (RM) was skimmed in a skimming centrifuge
(Suministros químicos Arroyo, Santander, Spain) at 30–35 ◦C and then pasteurized for 30 min
at 80 ◦C, determining the efficacy of the process by measuring phosphatase (Phosphatesmo MI,
Macherey-Nagel, Germany) and lactoperoxidase (Peroxtesmo MI, Macherey-Nagel) activities. In
addition,themicrobiologicalqualityoftherawandpasteurizedmilkwasevaluatedbycountingcolony
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formingunits(CFUs)ofmesophilicaerobicmicroorganismsandtestingtheabsenceofEnterobacteriaceae
inthepasteurizedmilk[10,11].
2.2. StandardizationofProbioticFermentedGoatMilkManufacturing
Themilkconcentrationmethod,fermentationconditions,andbacterialinteractionsandviability
wereevaluatedinordertostandardizemanufactureofthisprobioticfermentedgoatmilk.
2.2.1. SelectionoftheConcentrationMethod
Theinfluenceofthemilkconcentrationmethodonthefermentedmilksyneresisandviscosity
wasstudied[19],comparingbetweentheadditionofpowderedskimmedgoatmilk(2%or4%)and
ultrafiltration(Figure1). Theskimmedgoatmilkwasconcentratedbyultrafiltrationasdescribedby
Bergillos-Mecaetal. (2015)[20],filteringthrougha50kDacut-offmembrane(Vivaflow2000,Sartorius
Stedim)withaperistalticpump(Masterflex®L/S,EconomyDrive,ColeParmer®)at2barpressure,
uptoa12±0.5%ofdryextract,consideredanadequatetotalsolidcontentforthemanufactureof
fermentedmilkswithagoodconsistency[21]. Thefollowingfermentedmilksweremanufactured
and analyzed: SY (made with skimmed milk: SM), SYP2 and SYP4 (with skimmed milk plus 2%
and4%ofpowderedskimmedmilk, respectively), andUFY(withskimmedmilkconcentratedby
ultrafiltration: UFM).Thedifferentmilkswereinoculatedwithclassicalfreeze-driedstarterbacteria
(St), L. bulgaricus plus S. thermophilus (YO-MIX® 350, Dupont™ Danisco, Barcelona, Spain) at the
concentrationrecommendedbythemanufacturer. Afterinoculation,samplesweredistributedinto
sterileglasspotsandincubatedat42◦C.TheincubationwasstoppedatpH4.7or4.2,beingtheusual
pH in yoghurt manufacture and the isoelectric point of goat milk proteins, respectively. Samples
were then stored under refrigeration at 4 ◦C. Three samples of each type of fermented milk were
manufacturedandanalyzedintriplicate.
Figure1.Selectionoftheconcentrationmethod.SY:Yoghurtmadewithskimmedmilk(SM)fermented
withtheclassicalstarterbacteria(St);SYP2:YoghurtmadewithSMplus2%ofpowderedSMfermented
withSt;SYP4:YoghurtmadewithSMplus4%ofpowderedSMfermentedwithSt;UY:Yoghurtmade
withskimmedgoatmilkconcentratedbyultrafiltration(UFM)fermentedwithSt; PFM:Probiotic
fermentedmilkmadewithUFMfermentedwithStplusLactobacillusplantarumC4.
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2.2.2. ViabilityandInteractionsoftheFermentingBacteria
InteractionsamongL.bulgaricus,S.thermophilesandL.plantarumC4wereinvestigatedusingthe
spot test on Tryptone Soy Agar (TSA) (Difco™, Becton, Dickinson and Company; Madrid, Spain),
ManRogosaSharpe(MRS)agar(Difco™,Becton,DickinsonandCompany;Madrid,Spain)andL.
plantarumselectivemedium[22](LPSM)agarplates,accordingtoBergillos-Mecaetal. (2013a)[23].
The fermentation was also standardized in terms of time, temperature, and viable probiotic
bacteria. ForSt,theinoculationwascarriedoutasdescribedabove. FortheinoculationofL.plantarum
C4,thestrainwasrecoveredafterovernightgrowthinMRSbroth(Difco™,Becton,Dickinsonand
Company; Madrid, Spain) and re-suspended in the pasteurized milk after washing with sterile
phosphatebufferedsaline(PBS;Sigma-Aldrich,Steinheim,Germany).
First,theappropriatefermentationtemperaturewasselected. ThemilkwasfermentedwithSt
andL.plantarumattheirknownoptimumgrowthtemperature(42◦CforStand37◦CforL.plantarum.
Second,thefermentationtimeandprobioticbacteriacountwerestandardizedbymanufacturing
sixdifferenttypesoffermentedgoatmilk: SMandUFMfermentedfor8hwithSt,L.plantarumC4
orStandL.plantarumC4togetheratselectedtemperatures,inoculatingthesameconcentrationsof
L.plantarumC4andSt(106cfu/mL).Themicroorganismcountwasdoneatdifferenttimepointsduring
thefermentationprocessbyplatingserialdilutionsonTSA,MRSandLPSMagarandincubatingfor
24–48hat37◦C[10,11,23].
2.3. AnalysisoftheStandardizedProbioticFermentedGoatMilk
Forthisanalysis,8batchesofprobioticfermentedgoatmilksweremanufacturedatdifferenttime
periodsweeksoveroneyearandthreesamplesofeachbatchwereanalyzed.
All analyses were done in triplicate, and blanks were prepared and analyzed following the
sameprocedures.
2.3.1. ViableBacteriaafterFermentationandStorage
ViablemicroorganismsinthefinalPFMwerecountedbyplatingserialdilutionsofthesampleon
TSA,MRSandLPSMagarplatesandincubatingfor24–48hat37◦C.Inordertoassaytheirviability
duringstorage,theywerecountedweeklyuntil6weeksaftertheirmanufacture.
2.3.2. ViableBacteriaafterInVitroGastrointestinalDigestion
Samples underwent simulated gastrointestinal digestion in duplicate as described by
Bergillos-Mecaetal.,2013b[24]. Briefly,20gofeachfermentedmilkwerehomogenizedwith80mLof
bidistilleddeionizedwater. ForgastricdigestionthepHwasadjustedto2.0with6MHCl. ThepH
wascheckedafter15minandifnecessaryreadjustedto2.0,thenanamountoffreshlypreparedpepsin
solution,sufficienttoyield0.02gpepsin/sample,wasadded. Thesamplewasincubatedinashaking
waterbathat37◦Cand120strokes/minfor2h. Thedialysisassaycomprisedtheperviousgastricstep
followedbyanintestinalstepwheredialysiswasincluded(dialysisbag: molecularweight12–14kDa;
Visking45mm × 27mm, MedicellInternational, London, UK).Dialysistubing, containing25mL
ofbidistilleddeionizedwaterandanamountofNaHCO equivalenttotitratableaciditymeasured
3
previously,wereplacedintheflaskstogetherwith20-galiquotsofthepepsindigestandincubatedin
theshakenbathat37◦Cfor30min. Anamountoffreshlypreparedpancreatin-bileextractmixture
(0.001gpancreatinand0.006gbilesalts/samples)wasaddedtotheflaskandtheincubationcontinued
upto2h. Dialyzableandnon-dialyzablefractionswereweighted,freezedried,andstoreduntilthe
assay[24].
Aftergastricandintestinalsteps,serialdilutionsofthedigestedproductwereplatedonLPSM
andMRStocountviablemicroorganisms[10,11,23].
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2.3.3. Viscosity
TheviscositywasdeterminedusingaBrookfieldDV-II+Viscosimeter(Brookfield,Harlow,UK)
equippedwitha21-spindlefollowingapreviouslyreportedprocedure[19]. Thesamplestresswas
measuredinthePFMsandacommercialprobioticskimmedfermentedgoatmilk(GFM;withStanda
probioticBifidobacteriumstrain)usingdifferentrpmvaluesat20◦C.
Fermentedgoatmilksampleswerealsodistributedinto100mLvolumetriccylindersforweekly
measurement during one month of the volume of whey separated from the curd, as described by
Moreno-Montoroetal. (2013)[19]. Thesamplestresswasmeasuredinthemanufacturedfermented
milksandacommercialprobioticskimmedfermentedgoatmilk(withStandaprobioticBifidobacterium
strain)usingdifferentrpmvaluesat20◦C.
2.3.4. SpontaneousSyneresis
Fermentedgoatmilksampleswerealsodistributedinto100mLvolumetriccylindersforweekly
measurement during one month of the volume of whey separated from the curd, as described by
Moreno-Montoroetal. (2013)[19].
2.3.5. pHandAcidity
ThepHandacidityofthefermentedgoatmilksweremeasuredaccordingtotheAssociationof
OfficialAnalyticalChemists(AOAC,2006)procedure[25].
2.3.6. D/L-LacticAcidTest
D-andL-lacticacidisomersweremeasuredwiththeBoeringerMannheimD-Lactic/L-lactictest
(R-Biopharm,Darmstadt,Germany)usingacertifiedstandardsuppliedinthekitforthevalidation;
recoveryof99.7%wasobtained.
2.3.7. DryExtract
ThedryextractcontentwasmeasuredaccordingtotheAOACmethod(2006)[25].
2.3.8. LactoseandGalactose
Lactose and D-galactose levels were measured with the Megazyme enzymatic kit (Wicklow,
Ireland),usingacertifiedstandardsuppliedinthekitforthevalidation;recoveryof98%wasobtained.
2.3.9. Proteins
Total protein concentration was measured by the Kjeldahl method according to Olalla et al.
(2009)[26]butweighing3goffermentedmilk. Bovineserumalbumin(Sigma-Aldrich,Steinheim,
Germany)wasusedforthevalidation;recoveryof97.5%wasobtained.
2.3.10. Fat
Fat concentration was determined using the Gerber method adapted for fermented milks
accordingtotheAOACmethod(2006)[25].
2.4. SensorialAnalysis
Threesampleswereselectedforanalysisbytentrainedjudges: (1)PFM;(2)GFM;and(3)CFM:
CommercialskimmedcowyoghurtfermentedwithSt. Thescreeningtestwasinterpretedaccording
to ASTM [27]. In each season, four tablespoons of each sample were presented to the panelists in
plasticplatesthatwererandomlycodedusingthree-digitnumbersandtwoletters. Table1exhibitsthe
responseformdesignedforthisstudyandtheparametersanalyzed[3,28–31].
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Table1.Parametersanalyzedintheresponseform.
Evaluation Score Parameter Description
Color Fromgrey/yellow-whitetopurewhite
Scaleof
0–5 Syneresis Amountofwateronthesamplesurface
perception
Smoothness Smoothappearance,freeofirregularities
Visual Curdhomogeneity
Floury
Presence/absence Fermentedmilkresemblesflour
Lumps
Bubbles
Scaleof 1–8 Oralviscosity Yoghurtresistancetoflowinmouth
Texture perception Degreetowhichthesamplesticksor
1–4 Stickiness
adherestoteethandpalate
Aromafineness Naturalyoghurt-likearoma
Scaleof
0–5 Aromaintensity Perceivedstrengthofthearoma
perception
Aromapersistency Perceiveddurationofthearoma
Aroma
Acetaldehyde Yoghurt-likearoma
Presence/absence Diacetyl Butter-likearoma
Goatboiledmilk
Tastefineness Naturalyoghurt-liketaste
Tasteintensity Howstrongthetasteisperceived
Scaleof
0–5 Sweetness Sweettaste
perception
Taste Acidity Acidtaste
Bitterness Bittertaste
Presence/absence Goat,astringent,metallic,salty,insipid,boiledmilk
Overallacceptability Finalimpressionoftheyoghurt
2.5. StatisticalAnalysis
ThehomogeneityofvariancewasassessedusingtheLevenetestandthenormaldistribution
of the samples with the Shapiro-Wilk test. The Student’s t-test was used to analyze parametric
dataandtheKruskall–Wallistesttoanalyzenon-parametricdata. Finally,therelationshipbetween
assays was evaluated by computing the correlation coefficient by Pearson linear correlation (for
normallydistributedvariables)orSpearmanlinearcorrelation(fornon-normallydistributedvariables).
The significance level was set at 5% (p < 0.05) in all tests. SPSS 15.0 for Windows (IBM SPSS Inc.,
Chicago,IL,USA)wasusedfordataanalyses.
3. ResultsandDiscussion
3.1. StandardizationofProbioticFermentedGoatMilkManufacture
3.1.1. SelectionofConcentrationMethod
Thephysicalpropertiesofset-yoghurtareveryimportantforconsumeracceptance. Spontaneous
syneresisisthecontractionofagelwithnoexternalforceapplicationandisrelatedtoinstabilityof
thegelnetworkandconsequentlossoftheabilitytoentrapalloftheserumphase[14]. Thereisno
standardizedmethodforitsquantification, hamperingthecomparisonofresults. Amongvarious
factors related to viscosity and syneresis in fermented milks, we focused on the concentrations of
proteinandsolids.
Insummary,asdetailedbelow(inSections3.2.3and3.2.4),ultrafiltrationprovedtobesuperiorto
powderedmilkadditionasagoatmilkconcentrationmethod,withincreasedwheyretentioninthe
finalproductandbetterviscosity.
3.1.2. ViabilityandInteractionsoftheFermentingBacteria
Considerable research attention is being paid to the selection of bacterial strains for efficient
fermentation[32],L.plantarumC4wastheonlystrainshowingnochangeingrowthinthespotteston
MRSagar,whileinhibitionwasonlyobservedonL.bulgaricusbyL.plantarumC4inthetestonTSA,
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whichmaybeduetotheacidproductionfromthehighdextroseconcentrationinMRS.Theactionof
L.plantarumC4onL.bulgaricusinTSAmaybeattributabletobacteriocin-likesubstancesproduced
byL.plantarumC4. Nevertheless,whenthethreestrainswereco-culturedingoatmilk,Stgrowth
wasnotinhibitedbyL.plantarumC4. Oneexplanationforthedifferencemaybethatmicroorganisms
use dextrose in MRS but lactose in milk. These data underscore the importance of environmental
factors,suchasthenatureandconcentrationofsugars. Thus,itiswelldocumentedthatthegrowth
andfermentationratesofprobioticsdependonthecarbohydratespresentduringfermentation[33].
With respect to the fermentation temperature, whereas the probiotic bacteria L. plantarum C4
showedhardlyanygrowthat42◦C,theygrewbyalmostoneexponentialunitat37◦C.Ontheother
hand,thegrowthofstartercultureswasnotaffectedbythefermentationtemperature.Thefermentation
temperaturewasthereforesetat37◦Ctoobtainthemaximumconcentrationofprobioticbacteriain
thefinalproduct.
WhenL.plantarumC4wasusedalone,thegrowthwashigherbutnochangeinpHwasobtained;
therefore,itwasusedtogetherwithSttoobtainthefermentedmilk. Althoughsomeinteractionhas
beendescribedbetweenprobioticstrainsandStmicroorganisms[32],growthofbothmicroorganisms
presentinStgrewtothesamedegreeduringthemilkfermentationwhenusedaloneandwhenused
withtheprobioticstrain,aspreviouslydemonstratedfortheprobioticbacteriaL.helveticusR0050[33].
ThisexemplifiesthevariabilityinrelationshipsandinteractionsbetweenStandprobiotics, which
requirefurtherresearchtodeterminewhethertheyresultfromsubstratecompetition,theproductionof
inhibitorycompounds,proteolysis,orsimplyfromthechangesinpHduringfermentation. Whenused
togetherwithSt,L.plantarumC4grew~0.90Logcfu/mLat6h,anditsgrowthwasnotinfluenced
by the milk used, reaching around 6.75 Log units. Therefore, in order to obtain a high load of
viable probiotics in the final fermented milk, the initial probiotic concentration in the milk was
108–109cfu/mL,highenoughtoexertitshealthyproperties. Nonegativeinteractionswereobserved
amongthefermentingstrains.
Basedontheabovefindings,theoptimumfermentationconditionswereinoculationoftheprobiotic
strainat109cfu/mLfollowedbyfermentationat37◦Cforaround6h,whenpH4.2isreached.
Oncefermentationconditionswereoptimized,thefollowingmanufacturingprocesswascarried
out: afterUFMpasteurization,milkwasquicklycooledandtheninoculatedwithStplusL.plantarum
C4,obtainingPFM.Theinoculatedmilkwasdistributedinsterile200mLglasspotsandincubatedat
37◦CuptoapHof4.2. Finally,thefermentedmilkswererapidlycooledandstoredat4◦C.
3.2. AnalysisoftheStandardizedProbioticFermentedGoatMilk
3.2.1. ViableBacteriaafterFermentationandStorage
The mean concentration ± SD of viable bacteria in PFM was 8.98 ± 0.32 Log cfu/mL for
L. plantarum C4 and 8.72 ± 0.31 Log cfu/mL for St. Concentrations always exceeded 107 cfu/mL,
theminimumrequiredtomanufacturefermentedmilk,andwerewithinthereportedrangeforstarter
cultures[34].
Anonsignificantincreasewasobservedinallviablebacteriaupto4weeksofstorageat4◦C.
AnonsignificantslowdecreasebeganinL.plantarumC4atweek4andinStatweek5(Figure2a).
Nevertheless,thepercentageviabilitywashigherthan107cfu/mLafter6weeksofcoldstorage.
Nutrients2018,10,633 8of16
Figure2.Numberofviablebacteriaintheprobioticfermentedgoatmilk(PFM)manufacturedwith
skimmedmilkconcentratedbyultrafiltrationandfermentedwithSt(classicalstarterbacteria)and
LactobacillusplantarumC4bythestandardizedprocedure. (a)Duringthestorageat4◦C;(b)After
invitrogastrointestinaldigestion.St:classicalstarterbacteria(L.bulgaricusplusS.thermophilus).a,b,c,d
Meanvalues±SDwiththesamesuperscriptdenotestheexistenceofsignificantdifferences:*p<0.001,
**p<0.05.
3.2.2. ViableBacteriaafterInVitroGastrointestinalDigestion
Invitro gastric digestion produced a significant fall (p < 0.001) of almost one Log unit in the
L.plantarumC4countandofmorethantwoLogunitsintheStcount(Figure2b). However,Stand
L.plantarumC4showedasimilarresistancetoinvitrointestinaldigestion. Thus,theconcentration
ofviableL.plantarumC4wasaround108cfu/mLafterinvitrogastricdigestion,aresistanceof90%,
whichiswithintherangerecommendedbysomeauthorsforaprobioticstraintoexertitsfunctionand
deliveritsbenefitstotheconsumer[35]. Withregardtothestarterbacteria,resistancetolowpHof
between0and48%hasbeenreportedforL.bulgaricusandbetween0and45%forS.thermophilus[36],
lowerthanobservedforStinthepresentstudy(Figure2b). Withrespecttotheintestinalstage,despite
highresistancetobilesaltsforL.plantarumC4,mediumresistancehasbeenreportedforS.thermophilus
and high vulnerability for L. bulgaricus, while all of the bacteria showed high resistance (99%) in
our study [11,36]. This difference could be due to a potential matrix protective effect, differences
amongstrains,and/orinteractionsduetotheirjointpresenceinPFM,whichrequiresevaluationin
futurestudies.
In summary, the viability of all bacteria was maintained at an acceptable concentration
during fermentation and after 5 weeks of storage. They also proved to be resistant to invitro
gastrointestinaldigestion.
Nutrients2018,10,633 9of16
3.2.3. Viscosity
The best viscosity was observed for UFY and the other fermented milks when fermentation
was stopped at pH 4.2. Therefore, the pH 4.2 was chosen for stopping fermentation. Specifically,
theviscosityofPFMandSYP4(skimmedgoatmilk+4%ofpowderedskimmedgoatmilk)wassimilar
tothatoftheGFMandsignificantlydifferent(p<0.05)fromthatofSYP2(skimmedfermentedgoat
milk+2%ofpoweredskimmedgoatmilk)(Figure3).
Figure3.ViscositycurvesofthegoatmilksfermenteduntilpH4.2,comparedwithacommercialone.
SYP2andSYP4:Yoghurtmadewithskimmedmilk(SM)and2%or4%,respectively,ofcommercial
powderedSM,fermentedwiththeclassicalstarterbacteria(St);GFM:Commercialprobioticskimmed
fermentedgoatmilk(StandBifidobacterium);PFM:Probioticfermentedgoatmilkmadewithskimmed
milkconcentratedbyultrafiltration,fermentedwithStandL.plantarumC4.
3.2.4. Syneresis
Syneresis diminished with addition of greater dry extract and with ultrafiltration (Figure 4a).
Improvementinrheologicalbehaviorwithhighersolidcontenthaspreviouslybeenreported[15].
In addition, when the final pH was 4.2, a lower final syneresis was found (Figure 4b). However,
thebestwheyretentionwasobservedwhenultrafilteredmilkwasused,withnosignificantvariation
atdifferentpHvalues(Figure4a). Theadditionofpowderedskimmedmilkledtosimilarviscosity
butgreatersyneresisincomparisontoultrafiltration. Inthisregard,distinctrheologypropertieshave
beendescribedforfermentedmilksconcentratedbydifferentmethods[37]. Thesedifferencesmaybe
attributabletocaseinalterationbythespray-dryingprocessortotheincreaseofallmilkcompounds
in the same proportion with the addition of powdered skimmed milk, whereas ultrafiltration of
milkmainlyconcentratescaseins,whichareresponsibleforformationoftheyoghurtcoagula[17,38].
Syneresis in fermented milks may be dependent on the pH because the isoelectric point of caprin
caseins(atwhichtheystarttoaggregate)isreachedatpH4.2,formingthegelnetwork. AtpH4.7,
theisoelectricpointofcowcaseins,thereisalesseraggregationofcaseins,formingagelnetworkwith
ahighertendencytosyneresis[16].
PFMshowedaverylowmeansyneresisvalue(0.20±0.25%),denotinggoodcoagulaformation
withadequatewheyretention. Inthisregard,thereisalessertendencytosyneresiswhenthewater
isbetterretainedintheproteinnetwork. However,ithasbeenreportedthatotherparametersare
alsoimportantinthisprocess,suchastotalsolidandproteincontent,concentrationsofCa2+andfat,
pH,fermentationtemperature,andpreheatingofthemilk[38]. Thus,Bergillos-Mecaetal. (2015)[20]
demonstratedhigherCa,P,MgandZnconcentrationsinfermentedgoatmilksmanufacturedwith
ultrafiltration-concentratedmilk,whichcouldfavortheimprovedgelformationinPFM.
Nutrients2018,10,633 10of16
Figure4.(a)Dryextractcontentandsyneresisafter21daysofstorageat4◦C;(b)Syneresisevolution
over21daysofstorageinfermentedmilksconcentratedbydifferentmethods. SY(4.2)andSY(4.7):
Yoghurtmadewithskimmedmilk(SM)fermentedwiththeclassicalstarterbacteria(St)whenthe
fermentationwasstoppedatpH4.2or4.7,respectively;SYP2(4.2)andSYP2(4.7):Yoghurtmadewith
SMplus2%ofpowderedSMfermentedwithStwhenthefermentationwasstoppedatpH4.2or4.7,
respectively;SYP4(4.2)andSYPA(4.7):YoghurtmadewithSMplus4%ofpowderedSMfermented
withStwhenthefermentationwasstoppedatpH4.2or4.7,respectively;UY(4.7):Yoghurtmadewith
skimmedgoatmilkconcentratedbyultrafiltration(UFM)fermentedwithStwhenthefermentation
wasstoppedatpH4.7;PFM(4.2):ProbioticfermentedmilkmadewithUFMfermentedwithStplus
L.plantarumC4whenthefermentationwasstoppedatpH4.2. Infigure(a),significantdifferences
betweenthesyneresisofeachsampleatpH4.7and4.2weresignaledasfollows:*p<0.001.
TheadditionofL.plantarumC4isalsoofinterestbecauseitdidnotchangethetexturalproperties
of the final product and could contribute to a healthier final product due to its probiotic effects,
includingtheangiotensin-I-converting-enzymeinhibitoryandantioxidantactivitiesreportedforthe
smallandnon-basicbioactivepeptidesinthisPFM[1,39].