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Food Control 32 (2013) 450e460

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Food Control

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Drinking yoghurts with berry polyphenols added before and after fermentation

Dongxiao Sun-Waterhouse*, Jing Zhou, Sandhya S. WadhwaThe New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland 1020, New Zealand

a r t i c l e i n f o

Article history:Received 4 October 2012Received in revised form8 January 2013Accepted 15 January 2013

Keywords:AnthocyaninsDrinking yoghurtFermentationPectin stabiliserStarter culture growthRheological properties

* Corresponding author. The New Zealand InstitutLimited, 120 Mt Albert Road, Mt Albert, Private BagZealand. Tel.: þ64 9 925 7230.

E-mail addresses: Dongxiao.Sun-Waterhouse@waterhouse@auckland.ac.nz, dxsun72@hotmail.com (

0956-7135/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2013.01.011

a b s t r a c t

Health-promoting berry polyphenols (PPs) can be added in the form of berry fruit juice, or an extractfrom berry materials (which has a much higher PP concentration), to yoghurt products either pre- orpost-fermentation to add flavour and antioxidant functionality. This study compared the effects of addingpurified PP (cyanidin 3-o-b-glucopyranoside chloride, Cyanidin) or a blackcurrant PP extract (BPE) beforeor after fermentation on the chemical, rheological and microbiological properties of drinking yoghurtsformulated with low or high methoxyl (LM or HM) pectin. The control yoghurt (in the absence of addedPPs) and PP-enhanced yoghurts were subjected to total extractable PP content (TEPC) analysis, PPprofiling by High Performance Liquid Chromatography (HPLC), rheological examinations, and micro-biological testing for the survival and growth of starter cultures. Results show that adding BPE beforefermentation led to the presence of small phenolic molecules (e.g. phenolic acids) in the yoghurts anda TEPC that was 3.5e3.9 times greater than BPE added after fermentation. Fermentation influenced thePP profiles of yoghurt. BPE and Cyanidin added before fermentation affected differently the colonynumber and appearance of starter cultures Streptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus, as well as the elastic property and viscosity of the resultant yoghurts. Addition of pectinsmodified the rheological properties of the yoghurts, with the HM pectin yoghurts having a stronger gelstructure than the LM pectin yoghurts. The effects of added PPs on the starter cultures were diverse,depending on yoghurt formulation (e.g. type of pectin stabiliser), starter culture type, PP type, andapproach for PP addition. Such effects can be tailored to maximise the TEPC and desirable PPs as me-tabolites of initially incorporated PPs, offering both the probiotic effects of starter cultures and theproven health benefits of blackcurrant PPs. We conclude that the pre- and post-fermentation approachesfor adding PPs to drinking yoghurts are both feasible, but present different product processing challengesand functionality advantages.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The recommended daily intake of polyphenols (PPs) isapproximately 1 g (Georgé, Brat, Alter, & Amiot, 2005). Smallberries such as blackcurrants (Ribes nigrum spp.) are good sourcesof PPs including anthocyanins, proanthocyanidins, hydrox-ybenzoic acids, hydroxycinnamic acids (e.g. m-coumaric acid),flavonols (e.g. quercetin), flavanols (e.g. catechins) (da Costa,Nelson, Margolis, & Horton, 1998; Häkkinen et al., 1999; Landbo& Meyer, 2001; Stevenson, Wibisono, Jensen, Stanley, & Cooney,2006). Blackcurrants have a high anthocyanin content contain-ing cyanidin-3-o-glucoside, cyanidin-3-o-rutinoside, delphinidin-

e for Plant & Food Research92169, Auckland 1025, New

plantandfood.co.nz, dx.sun-D. Sun-Waterhouse).

All rights reserved.

3-o-glucoside and delphinidin-3-o-rutinoside (da Costa et al.,1998). All these PPs possess potential health-promoting prop-erties such as antioxidant activity, increasing peripheral bloodflow and reducing muscle fatigue (Ghosh, McGhie, Fisher, &Joseph, 2007; Lyall et al., 2009; Matsumoto et al., 2005; Skrede,Larsen, Abby, Jorgensen, & Birkeland, 2004). Thus, it is desirableto increase the intake of these PPs, especially those from naturalsources such as blackcurrants.

The use of PPs in popular foods is a convenient and viableapproach to increase the daily intake of PPs (Sivam, Waterhouse,Zujovic, Perera, & Sun-Waterhouse, 2011; Sun-Waterhouse, Zhou,& Wadhwa, 2011). Fermented dairy foods have high consumerpopularity and positive health benefits, due to ingested live mi-croorganisms (probiotics) (Douaud, 2007; Stanton, Ross, Fitzgerald,& Sinderen, 2005). Drinking yoghurts are one of the fastest growingfermented products in the functional food market, because of theirmulti-nutrient content, and health-promoting properties related toboosting the immune response, HDL-cholesterol increase and ulcer

D. Sun-Waterhouse et al. / Food Control 32 (2013) 450e460 451

prevention (Douaud, 2007; Fabian & Elmadfa, 2006; Fortes et al.,2000; Gill, Rutherfurd, & Cross, 2001; Meyer, Micksche, Herbacek,& Elmadfa, 2006; Wang et al., 2004). Yoghurt drinks enhancedwith value-added ingredients gain positive consumer perception(Allgeyer, Miller, & Lee, 2010).

Yoghurt consists of a casein network aggregated through iso-electric precipitation by lactic acid bacteria (Tamime & Robinson,1999). Milk fermented with multiple species of starter cultures re-sults in a stronger gel structure than fermented with single speciesstarter culture (Skriver, Baek-Madsen, & Jelle, 1997). Gram-positivebacteria Streptococcus salivarius subsp. thermophilus (called Strepto-coccus) and Lactobacillus delbrueckii subsp. bulgaricus (called Lacto-bacillus) are commonly used to metabolise lactose into lactic acidduring fermentation (Tamine, 2006). Streptococcus appears likechains of spherical shape while Lactobacillus occurs in long slenderstraight rods, short but coccoid rods or pleomorphic cells (Tamine,2006). Streptococcus grows more rapidly at the start of fermenta-tion, producing formic acid and reducing oxygen that stimulates thegrowth of Lactobacillus. Lactobacillus generates peptides, stimulatingthe growth of Streptococcus (Tamime & Robinson, 1999). Stabiliserssuch as pectins are often used to incorporate small fat globules intomilk protein network with reduced synaeresis, increased viscosityand gel strength (Ares et al., 2007; Teles & Flores, 2007). HM and LMpectins have different methoxyl contents, which influence theirchemical reactions and rheological behaviour in various foodmatrices, e.g. sensitivity to the changes of soluble solid content, pH,ions like Ca2þ (Imeson, 1997; Phillips & Williams, 2000).

PPs can be added to yoghurt via two approaches (Sun-Waterhouse, Zhou, et al., 2011): via the pre-fermentation approach(adding PPs before fermentation as part of the yoghurt ingredientmixing), or via the post-fermentation approach (adding PPs afterfermentation as part of the usual practice for imparting flavour andcolour agents). Sun-Waterhouse, Zhou, et al. (2011) explored thesetwo approaches when adding apple PPs to yoghurts, and found thepre-fermentation approach introduced some favourable changes interms of promoting growth of starter cultures. Blackcurrant andapple have very different PP profiles. These differenceswere found toimpart different effects on the properties of PP-enhanced finishedfoods such as bread (Sun-Waterhouse, Sivam, et al., 2011; Sivam,Sun-Waterhouse, Perera, & Waterhouse, 2011). In this study, black-currant PPs were added to drinking yoghurts either pre- or post-fermentation, with two different yoghurt stabilisers, to examinethe effects, through comparing the results of this study with those ofSun-Waterhouse, Zhou, et al. (2011), on the chemical, rheologicaland microbiological properties of drinking yoghurts. The differentimpacts of blackcurrant and apple PPs on the yoghurts (which pro-duced using similar formulation and processing methods), could beexplored. PP-spiked drinking yoghurts were also prepared throughadding a purified PP compound (cyanidin 3-o-b-glucopyranosidechloride, termed “Cyanidin” hereafter) to yoghurt to help elucidatethe structureefunctionality relationship of blackcurrant PPs in thedrinking yoghurt matrix.

2. Materials and methods

2.1. Materials and chemicals

Pams� instant skimmilkpowder andwhite table sugar (Chelsea�,Auckland, New Zealand) were purchased from Foodtown, St Lukes,Auckland, New Zealand. Pectins, classic AB 901 (low methoxyl con-tent, LM) and classic AU 201 USP (high methoxyl content, HM) werepurchased from Herbstreith & Fox KG (D-75305 Neüenburg, Swit-zerland). Starter culture, Yo-MixTM 401 LYO containing Streptococcusthermophilus and L. delbrueckii subsp. bulgaricuswas purchased fromDanisco (Niebüll, Germany). Blackcurrant PP extract (BPE, Anthomix

30, 30% anthocyanin content) was sourced from Just the Berries Ltd(Palmerston North, New Zealand). Cyanidin 3-o-b-glucopyranosidechloride (Cyanidin) was from the Polyphenols Laboratories AS(Hanaven, Sandnes, Norway).

Methanol, n-hexane, formic acid and iodine crystals were pur-chased from Ajax Finechem (Auckland, New Zealand). Potassium io-dide was obtained from the May & Baker (Dagenham, England).Epicatechin, catechin, ferulic acid, salicylic acid, protocatechuic acid,caffeic acid, p- and o-coumaric acids, and FolineCiocalteu phenol re-agentwere fromSigmaeAldrich (St Louis,MO,USA). Crystal violetwaspurchased from the Coleman & Bell (Norwood, USA). Ammoniumoxalate and safranin O (for Gram staining) were from Gurr Certistain(BDH, Poole, England). Ethanol absolute GR was from Merck (Darm-stadt, Germany). HPLC grade acetone was from Burdick & Jackson(Muskegon,MI,USA), andacetonitrilewas from JTBaker (Phillipsburg,NJ, USA). Microscopic glass slides (75 � 25 � 1 mm) were obtainedfrom Marienfeld (Lauda-Königshofen, Germany). Disposable Loop-last� inoculation loops were purchased from LP Italiana SPA (Milan,Italy). Milli-QPLUS water was used for the preparation of all reagents.

2.2. Yoghurt preparation

Theproductionof drinking yoghurtswith PPs addedbefore or afterfermentation followed the method of Sun-Waterhouse, Zhou, et al.(2011) with some modifications. The control yoghurt (in the absenceof added PPs) was formulated with skim milk powder (14.6%w/w),stabiliser (HM or LM pectin, 0.01%w/w), table sugar (1%w/w), Yo-Mix� 401 starter culture (0.002%w/w) and water. Before inoculationwith the starter culture in a Gelman Laminar flow hood (Woodlands,Singapore), the mixture of milk powder, pectin, sugar and water wassubjected to homogenisation (using a Silverson mixer, X screen,200 rpm; Silverson Machines Inc., East Longmeadow, MA, USA), pas-teurisation (95 �C for 10 min) and cooling to 40 �C. Incubation wasconducted at 40 �C in aMIR-162 Sanyo incubator (Osaka, Japan), untilthe pH reached 4.4 (when the yoghurt was stored at 4 �C). The co-agulumwas broken andmixed with water (50 mL per 200 g yoghurt,pasteurised at 95 �C for 10 min) simultaneously using the Silversonmixer (6000 rpm). The resultant drinking yoghurt was stored at 4 �Cbefore chemical, physical and microbiological analyses, as well aspreliminary evaluation on product attributes. For the PP-enhancedyoghurts, the same quantity of BPE (4 g) or purified Cyanidin (0.3 g)was added to 200 g yoghurt before or after fermentation. When PPswere added pre-fermentation, BPE or purified Cyanidinwas added tothemix of milk powder, stabiliser and sugar ingredients at the start ofyoghurt making. When PPs were added post-fermentation, BPE orpurified Cyanidinwas first mixed with water, and the resultant watersolutionwas thenpasteurisedat 95 �C for10minandadded toyoghurt(in theplaceof pasteurisedwater)during the coagulum-breaking step.

2.3. Rheological examination

The rheological properties of the yoghurts were examined at4 �C in triplicate, following the method of Sun-Waterhouse, Zhou,et al. (2011). An amplitude sweep and analyses of storage mod-ulus G0 and viscosity were performed using a shear rate-controlledrheometer (Anton Parr Physica MCR301, Anton Parr GmbH, Graz,Austria), equipped with a Peltier temperature control device,20mm serrated parallel plates, and a humidity chamber (to preventwater loss). Datawere acquired and processed with Rheoplus V2.66(Anton Paar GMBH, Graz, Austria).

2.4. Microbiological tests

The spread plating, total bacterial count and bacteria cell mor-phology examinations were conducted following the method

Fig. 1. Total extractable polyphenol contents of the control and polyphenol-enhancedyoghurts. Error bars are the standard deviation of the mean. LM and HM refer to lowand high methoxyl pectins, respectively. BPE and Cyanidin refer to blackcurrant pol-yphenol extract and cyanidin 3-o-b-glucopyranoside chloride, respectively.

D. Sun-Waterhouse et al. / Food Control 32 (2013) 450e460452

described in Sun-Waterhouse, Zhou, et al. (2011). MRS and M17mediawere used spread plating. The Streptococcus and Lactobacilluswere identified on the basis of colony type and confirmed bymicroscopic examination using a Nikon Eclipse E600 microscope(Nikon Instech Co. Ltd, Kanagawa, Japan), after being grown ina MIR-162 Sanyo incubator at 37 �C for 48 h. The number of bac-terial colonies of replicate plates was generated using equation (1):

Number of cfu=ml yoghurt ¼ 1Plate Factor� Dilution Factor� Average number of colonies

(1)

The “Plate Factor” and “Dilution Factor” refer to the amount ofsample pipetted onto the agar plate and the dilution series of theyoghurt sample, respectively. The morphology of the colonies wasexamined via gram staining (Harrigan & McCance, 1976) undera Nikon Eclipse E600 microscope at two magnifications (60� and100�).

2.5. Polyphenol content and composition analyses

The extraction of PPs from the yoghurts was performed using anAccelerated Solvent Extractor (ASE, 300, Dionex, Sunnyvale, CA). Aquantity (5 g) of yoghurt sample was mixed with 8.5 g Celite�(diatomaceous earth; Manville Service Corporation, USA), beforebeing transferred into the ASE Dionex 33 mL stainless steelextraction cells. Three cycles of extractions (with 5 min heating and10 min static time) were carried out at 40 �C and 1500 psi under N2using n-hexane (100%), acetone (100%) and methanol (100%). Thesolvent containing extracted PPs was then collected, concentratedusing the Ultra-Low Cold Trap Centrivap� (Model 78100-01, Lab-conco Corp., Kansas City, MO), and dried using a freeze drier (TelstarCryodos-80, Telstar Industrial SL, Spain). The extracts were keptat �80 �C and reconstituted in 25% methanol for FolineCiocalteuassay and HPLC analysis.

The total extractable PP content (TEPC) was analysed using themethod of Singleton, Orthofer, and Lamuela-Raventos (1999) andexpressed as catechin equivalent (CtE). The absorbance at 760 nmwas detected using a microplate reader (SpectraMax Plus 384,Molecular Devices, Sunnyvale, California, USA).

The High Performance Liquid Chromatography (HPLC) profilingof individual PPs was performed following the methods ofStevenson et al. (2006), using a Shimadzu analytical HPLC equippedwith a diode-array detector (SPD-M10AVP), a Synergi� Polar-RPether-linked column (250 � 4.6 mm, 4 mm particle size, 80 �Aether-linked column; Phenomenex, Auckland, New Zealand). Themobile phases (A) acetonitrile þ 0.1% formic acid, and (B) aceto-nitrile:water:formic acid (5:92:3) were pumped at 1.5 mL/min at45 �C. Injection volume was 40 mL. Individual PPs were identifiedbased on their retention time and absorbancemaximum (lmax), andthrough comparing to the BPE anthocyanidin peaks that wereidentified in our previous published papers (Slimestad & Solheim,2002; Sun-Waterhouse, Sivam, et al., 2011; Sivam, Sun-Waterhouse, Waterhouse, Quek, & Perera, 2011). External stan-dards used include epicatechin, catechin, ferulic acid, salicylic acid,protocatechuic acid, caffeic acid, p- and o-coumaric acids, andCyanidin.

2.6. Statistical analysis

At least three replicate determinations were obtained for eachdatum point. Data were analysed using two-way ANOVA (Minitab15, State College, PA, USA), as described in Sun-Waterhouse, Zhou,et al. (2011).

3. Results

3.1. Preliminary evaluation on product attributes

The drinking yoghurts with added BPE had a dominant darkpurple colour, and minimal difference was observed in the colourintensity between the yoghurts with BPE added before and afterfermentation. The yoghurt with BPE added pre-fermentationappeared to be more viscous than the control and the yoghurtwith BPE added post-fermentation in the case of using HM pectin,whereas, the yoghurt with BPE added post-fermentation was themost viscous in the case of using LM pectin. The yoghurts with BPEadded post-fermentation had detectable chalky and gritty mouth-feel, especially for the HM pectin formulation. The yoghurts withBPE added via either approach had a distinct blackcurrant flavourand pleasant bitterness.

3.2. Total extractable polyphenol content

Adding BPE before yoghurt fermentation led to about 4 times asmuch TEPC as did adding BPE after fermentation (Fig. 1), for thesame quantity of BPE. This suggests that these PPs added pre-fermentation might be broken down into smaller PP forms thatare more extractable or stable during fermentation. PPs addedpost-fermentation may not have broken down, or conjugated tonon-PP yoghurt components, thereby reducing their extractabilityand lowering the TEPC. The detected TEPC values of control yo-ghurts resulted from the phenolic compounds originally present inmilk such as estrogens (Andersson & Skakkebaek, 1999; Pape-Zambito, Roberts, & Kensinger, 2010). Interestingly, no significantdifference (p < 0.05, for LM pectin formulations) or marginal dif-ference (p < 0.05, for HM pectin formulations) in TEPC values wasdetected between the Cyanidin-yoghurts produced using pre- andpost-fermentation approaches.When the same PP additionmethodwas applied, LM and HM pectins had a marginal effect on the TEPCvalues, suggesting that the methoxy content of pectin had little tono influence on PP extractability and PP stability during yoghurtprocessing.

PPs can be extracted to different extents by different extractionmedia from food systems (Bravo, 1998; Murphy, Barua, & Hauck,2002; Schieber, Ullrich, & Carle, 2000; Sun-Waterhouse, Wen,Wibisono, Melton, & Wadhwa, 2009). ASE is an automated

D. Sun-Waterhouse et al. / Food Control 32 (2013) 450e460 453

extraction technique that is suitable for extracting temperature-sensitive components (Richter, Jones, Ezzell, & Porter, 1996). Thedifference in PP extraction efficiency depends on the polarity of theextraction medium, and the strength and type of interaction be-tween PPs and other food components (Shahidi & Naczk, 2004).Chemical and physical effects such as protection by the gel struc-ture of yoghurt (Rozman & Gasperlin, 2007), binding to amphi-pathic yoghurt peptides (Papadopoulou & Frazier, 2004; Plet,2006), or complexation with proteins and polysaccharides(Haslam, 1998, p. 422; Rawel, Rohn, & Kroll, 2003; Renard, Baron,Guyot, & Drilleau, 2001), may influence PP extractability.

3.3. High Performance Liquid Chromatography (HPLC) polyphenolprofiling

Differences in the type and quantity of BPE PPs were detectedbetween the drinking yoghurts with BPE added before or after

Fig. 2. HPLC chromatograms (l ¼ 280 nm) of A) control and polyphenol-enhanced yoghurtextract. Peak 1, protocatechuic acid; peak 2, catechin derivative; peak 3, delphinidin-3-o-gcyanidin-3-o-rutinoside; peak 7, ferulic acid; peak 8, salicylic acid and/or o-coumaric acid;

fermentation (Fig. 2A). Either of the BPE-enhanced drinking yo-ghurts (produced using pre- or post-fermentation approach) con-tained delphinidin-3-o-rutinoside, delphinidin-3-o-glucoside,cyanidin-3-o-rutinoside and cyanidin-3-o-glucoside, with theamount of cyanidin-3-o-rutinoside being the highest (Fig. 2A andTable 1). These detected anthocyanins are the typical PP antioxi-dants in blackcurrant (da Costa et al., 1998; Häkkinen et al., 1999;Nielsen, Haren, Magnussen, Dragsted, & Rasmussen, 2003) andhave been proven to possess health-promoting properties (Ghoshet al., 2007; Lyall et al., 2009; Matsumoto et al., 2005; Skredeet al., 2004). The BPE ingredient mainly contains delphinidin-3-o-glucoside (20.6 mg/mg BPE ingredient), delphinidin-3-o-rutinoside(91.8 mg/mg BPE ingredient), cyanidin-3-o-glucoside (13.1 mg/mgBPE ingredient) and cyanidin-3-o-rutinoside (99.9 mg/mg BPEingredient), with very small amounts of other PP compounds suchas ferulic acid (2.2 mg/mg BPE ingredient) (Fig. 2B). Thus, the re-covery of anthocyanins added pre-fermentation was 28.8% for LM

B

A

s, B) BPE ingredient (in 25% aqueous methanol). BPE refers to blackcurrant polyphenollucoside; peak 4, delphinidin-3-o-rutinoside; peak 5, cyanidin-3-o-glucoside; peak 6,peak 9, unknown (possibly catechin).

Table 1Individual anthocyanin content in blackcurrant extract-enhanced yoghurts.

Yoghurt formulation Delphinidin-3-o-glucoside(mg/200 g yoghurt)

Delphinidin-3-o-rutinoside(mg/200 g yoghurt)

Cyanidin-3-o-glucoside(mg/200 g yoghurt)

Cyanidin-3-o-rutinoside(mg/200 g yoghurt)

LM pectin Pre-fermentation 11.25 � 0.06Da 44.82 � 0.10Ba 26.60 � 0.09Ca 175.6 � 0.1Aa

Post-fermentation 9.10 � 0.06Dc 22.75 � 0.08Bc 9.37 � 0.05Cc 46.23 � 0.09Ac

HM pectin Pre-fermentation 10.89 � 0.06Db 43.35 � 0.06Bb 25.73 � 0.07Cb 169.8 � 0.1Ab

Post-fermentation 7.82 � 0.05Dd 19.56 � 0.03Bd 8.06 � 0.05Cd 39.73 � 0.07Ad

Data expressed as mean � standard deviation of replicate measurements. “LM or HM pectin” refers to low or high methoxyl pectin. Different uppercase superscript letters(within the same entire row) indicate statistically significant differences at p < 0.05. Different lowercase superscript letters (within the same entire column) indicate sta-tistically significant differences at p < 0.05.

A

B

D. Sun-Waterhouse et al. / Food Control 32 (2013) 450e460454

pectin yoghurt and 27.6% for HM pectin yoghurt, respectively,whilst the recovery of anthocyanins added post-fermentation was9.7% for LM pectin yoghurt and 8.3% for HM pectin yoghurt,respectively. The yoghurt polymer network and gel matrix mighthave protected the PPs of BPE to some extent, through reducing theoxidation rates of these sensitive components (Rozman &Gasperlin, 2007). The methoxyl content of pectin slightly affectedthe individual anthocyanin contents. In general, the LM pectinformulation had a higher anthocyanin content when the same PPaddition approach was used.

The yoghurt with BPE added pre-fermentation was found tocontain a significant amount of small polar molecules with shortretention times (high polarity) such as protocatechuic acid (13.6and 13.2 mg/200 g yoghurt for LM or HM pectin yoghurt, respec-tively) and a catechin derivative (9.7 and 9.4 mg/200 g yoghurt forLM or HM pectin yoghurt, respectively), as well as those witha longer retention time (lower polarity) such as ferulic acid (14.3and 13.8 mg/200 g yoghurt for LM or HM pectin yoghurt, respec-tively) and salicylic acid or o-coumaric acid (6.7 and 6.5 mg/200 gyoghurt for LM or HM pectin yoghurt, respectively) (Fig. 2A). Bycomparison, such small molecule phenolic acids were not detectedby HPLC for yoghurts with BPE added post-fermentation, and themethoxyl content of pectin had much lesser impact on the contentsthese molecules than on anthocyanin contents.

The differences in the HPLC profiles (i.e. PP peak area andretention time) between the yoghurts with Cyanidin added beforeand after fermentation were insignificant (p < 0.05). The HPLCchromatograms of both Cyanidin-yoghurts were dominated anintense peak that was identified as Cyanidin (chromatograms notshown). This explained the identical TEPC values of these two yo-ghurts (Fig. 1). The fermentation process appeared to have littleimpact on cyanidin-3-o-glucoside present in both Cyanidin-enhanced yoghurts. These results suggest that either no inter-action between Cyanidin and starter cultures occurred during fer-mentation, or that Cyanidin played no role in the increased PPextractability observed. The acidity of yoghurt could have inducedacid hydrolysis of PPs, so it was not surprising that no flavonolcompounds like quercetin were detected in the yogurts(Kapasakalidis, Rastall, & Gordon, 2006). The yields of hydrox-ycinnamic acids and flavonols detected by HPLC and the FolineCiocalteu assay might be dependent on the extractability of thesePPs in different product matrices (Sun-Waterhouse et al., 2009;Wibisono, Zhou, & Sun-Waterhouse, 2009).

Fig. 3. Storage modulus as a function of strain of the control and polyphenol-enhancedyoghurts A) low methoxyl pectin formulations; B) high methoxyl pectin formulations.BF and AF refer to pre- or post-fermentation approach, respectively. BPE and Cyanidinrefer to blackcurrant polyphenol extract and cyanidin 3-o-b-glucopyranoside chloride,respectively.

3.4. Rheological tests

Yoghurt behaves as a non-Newtonian, shear thinning andpseudoplastic fluid (Rohm & Schmidt, 1993). G0 indicates the elasticproperty of yoghurt. The type of PPs (BPE extract or purified Cya-nidin), the type of pectin (LM or HM) and PP addition approach (PPadded pre- or post-fermentation) all influence the elastic property

of yoghurt (Fig. 3). The G0 values for all the control and PP-enhanceddrinking yoghurts were very low (mostly around 1 Pa, except forthe yoghurt with BPE added pre-fermentation which was higherand in the range of 1e10 Pa).

For the LM pectin formulations (Fig. 3A), the G0 values appearedto be quite constant until the strain increased to 1%, after which itdecreased significantly (p < 0.05) with increasing strain whena strain > 1% was applied. Thus, the imposed strain exerteda greater effect on the elastic component G0 at >1% strain. The G0 of

B

A

Fig. 4. Viscosity as a function of shear rate of the control and polyphenol-enhancedyoghurts A) low methoxyl pectin formulations; B) high methoxyl pectin formula-tions. BF and AF refer to pre- or post-fermentation approach, respectively. BPE andCyanidin refer to blackcurrant polyphenol extract and cyanidin 3-o-b-glucopyranosidechloride, respectively.

D. Sun-Waterhouse et al. / Food Control 32 (2013) 450e460 455

yoghurt with BPE added pre-fermentation behaved very differentlyfrom those of other yoghurt formulations, and had a much highervalue than other formulations when the strain was �10%, whichsuggests a stronger structured yoghurt (Renan et al., 2009). The G0

of the control yoghurt and the yoghurt with BPE added post-fer-mentation changed according to the same pattern. The G0 of theyoghurts with Cyanidin added pre- or post-fermentation, werealmost the same as those of the control and the yoghurt with BPEadded post-fermentation, when the strain was <1%. Above the 1%strain, the G0 values differed, decreasing in the order of yoghurtwith Cyanidin added before fermentation > yoghurt with Cyanidinadded after fermentation > control yoghurt and the yoghurt withBPE added after fermentation.

For the HM pectin formulations (Fig. 3B), the G0 values exhibiteda similar pattern to the LM pectin formulations (Fig. 3A) (a plateaufollowed by a steady drop with a turning point around 1% strain).But the magnitude of the G0 values was different between the LMand HM pectin formulations. Moreover, the difference in the G0

values of the HM pectin formulations became more significant inthe strain range between 1.0 and 46.4% (Fig. 3B). Otherwise all theG0 values were similar. The G0 values of control yoghurt and theyoghurt with Cyanidin added post-fermentation were the highestand second highest, respectively. The G0 of the yoghurt containingBPE added prior to fermentation was the lowest. For the sameadded PP ingredient (BPE or Cyanidin), the G0 of the yoghurt usingpost-fermentation addition was higher than those prepared by thepre-fermentation PP addition approach (p < 0.05).

The G0 values will likely be a net reflection of the number andstrength of bonds between casein particles and the distribution ofcasein. The drinking yoghurts in this study were weakly structuredmaterials. Adding PP before fermentation might enable PPs tointeract with the starter cultures. For the post-fermentation PPaddition approach, the added PPs would not be involved in thegrowth of starter culture during fermentation, but instead will stillinteract with milk proteins (Haslam, 1998, p. 422; Papadopoulou &Frazier, 2004; Rawel et al., 2003). The impact of the methoxylcontent of pectin is obvious. The methoxyl content of pectininfluenced yoghurt’s G0, i.e. the control yoghurt with HM pectin(Fig. 3B) exhibited a higher G0 value than that of LM pectin (Fig. 3A)(p < 0.05). The addition of BPE or Cyanidin generally increased theG0 values of LM pectin yoghurts (compared to the control yoghurt),but caused a decrease in G0 of the HM pectin yoghurts. This suggeststhat the use of LM or HM pectin as a stabiliser impacted on therheological and organoleptic attributes of the yoghurts (Gaonkar,1995; Jawalekar, Ingle, Waghmare, & Zanjad, 1993; Shukla & Jain,1991; Shukla, Jain, & Sekhon, 1988). The effects may or may notbe complementary to the effects exerted by added PPs on the col-loidal calcium phosphate cross-linking and casein dispersion(Tamime & Robinson, 1999).

Viscosity is a quality attribute of drinking yoghurts, and can beaffected by formulation composition (e.g. total soluble solid contentand stabiliser), type of starter cultures, heat treatment and processingmethods (Aguirre-Ezkauriatza et al., 2008; Becker & Puhan, 1989;Guirguis, Broome,&Hickey,1984; Skriveret al.,1997). In this study, theviscosity of the LMpectin formulations ranked in the decreasingorderof theyoghurtwithCyanidinaddedafter fermentation>yoghurtwithBPE added after fermentation > yoghurt with BPE or Cyanidin addedbefore fermentation and the control yoghurt, when the shear rateincreased from 0.1 s�1 (Fig. 4A). The viscosity of the yoghurt withCyanidin added post-fermentation remainedmuch higher than otherLM pectinyoghurts when a shear rate larger than 0.2 s�1 was applied.For the HM pectin formulations (Fig. 4B), the yoghurt viscositybehaved in a different manner. The viscosity of the yoghurt withCyanidin added after fermentation was greater than the controlyoghurt and theyoghurtswithCyanidin addedbefore fermentationor

BPE added after fermentation, when the shear rate was �1 s�1. Theviscosity of the yoghurt with BPE added before fermentation droppedrapidly from a very high initial value (10 times higher than the otheryoghurts) and remained thehighest until the shear ratewas increasedto 2.2 s�1, after which it decreased at a lower rate until 100 s�1. Theviscosity results in the shear rate range of 30e60 s�1 indicate thedetected viscosity in the humanmouth (Wood & Goff, 1973). For boththe LMandHMpectin formulations, the yoghurtwith Cyanidin addedafter fermentation would possibly have tasted the most viscous. Theyoghurt with BPE added before or after fermentationwould likely beperceived as the second most viscous for the HM and LM pectinyoghurt, respectively.

The chemical structure of the PPs present in the BPE ingredientand Cyanidin are shown in Fig. 5: The structural difference betweendelphinidin-3-o-rutinoside and delphinidin-3-o-glucoside, or be-tween cyanidin-3-o-rutinoside and cyanidin-3-o-glucoside, relateto the attached sugar moieties (rutinose is the disaccharide con-taining 6-o-L-rhamnosyl-D-glucose); The difference between del-phinidin-3-o-rutinoside and cyanidin-3-o-rutinoside, or betweendelphinidin-3-o-glucoside and cyanidin-3-o-glucoside, lies in theabsence or presence of the third hydroxyl group attached to the Bphenyl group at the 2-position of the C ring; The difference be-tween cyanidin-3-o-glucoside and Cyanidin lies in the neutralisa-tion of the positive charge on the O atom at the 1-position of thecyanidin-3-o-glucoside by the chloride ions. Therefore, there

Fig. 5. Chemical structure of the four major anthocyanins in blackcurrant extract ingredient (delphinidin-3-o-rutinoside, delphinidin-3-o-glucoside, cyanidin-3-o-rutinoside andcyanidin-3-o-glucoside), and purified polyphenol cyanidin 3-o-b-glucopyranoside chloride (Cyanidin).

D. Sun-Waterhouse et al. / Food Control 32 (2013) 450e460456

existed more hydroxyl groups and positive charges on the PPs inthe BPE anthocyanidins, compared to the Cyanidin ingredient.Pectins used as the yoghurt stabiliser are anionic polysaccharides.The pectin chains are networked together by hydrogen bridges andinteractions between methylester groups, and acid would inhibitthe dissociation of pectin’s free carboxyl groups. HM and LM pec-tins differ in their charge densities, the amount of Ca2þ required forgelation, pH and soluble solid concentration (Imeson, 1997; Phillips& Williams, 2000). The PP addition would lead to an increasedgelation pH, ionic strength and total soluble solids, and the degreeby which these parameters increased would depend on the PPingredient. This, in turn, would impact on the aggregation of caseinnetwork in yoghurts via iso-electric precipitation, and on theresistance for the yoghurt matrix to flow (Becker & Puhan, 1989;Guirguis et al., 1984; Rice-Evans, Miller, & Paganga, 1996; Tamime &Robinson,1999). Here, PPs were added before or after fermentation.During fermentation the pH of the yoghurt system decreased,during which different interactions with LM or HM pectin haveoccurred, which caused the observed variations in the yoghurtrheological properties. Hydrophobic interactions, hydrogen bond-ing and electrostatic interactions were possible during the yoghurtprocessing when inter-particle bonds and gel structure were

formed or reinforced (Renan et al., 2009). The specific inter-molecular interactions among yoghurt components responsible forthe changes in G0 and viscosity need to be investigated further.

3.5. Microbiological tests

Streptococcus and Lactobacillus in all the yoghurt formulationsexhibited normal morphology (Figs. 6 and 7). Streptococcus andLactobacillus appeared as chains of spherical and rod shaped sub-units, respectively. Microscopic examinations (Fig. 6) showed thatadding BPE before fermentation to the HM pectin yoghurt formu-lations, resulted in an increased growth of Streptococcus and Lac-tobacillus. Adding Cyanidin before fermentation caused reducedgrowth of Lactobacillus (little difference between the HM pectincontrol and Cyanidin-HM pectin yoghurts). In the case of using LMpectin, adding BPE before fermentation facilitated Streptococcusand Lactobacillus growth, and changed the pattern of Streptococcusstrains (i.e. smaller clusters, compared to the control) (Fig. 7).Adding Cyanidin before fermentation remarkably altered theappearance of Streptococcus (i.e. forming aggregates) and did notsupport Lactobacillus growth. Therefore, adding BPE to yoghurtbefore fermentation might promote the fermentative activities of

Fig. 6. Micrographs of Streptococcus and Lactobacillus starter cultures in the control and polyphenol-enhanced yoghurts formulated with high methoxyl pectin. BPE and Cyanidinrefer to blackcurrant polyphenol extract and cyanidin 3-o-b-glucopyranoside chloride, respectively.

D. Sun-Waterhouse et al. / Food Control 32 (2013) 450e460 457

the two starter culture bacteria. Purified Cyanidin might be detri-mental to the survival and/or growth of the two starter cultures.

The mean viable counts of Streptococcus and Lactobacillus in thecontrol yoghurts and the yogurts with BPE or Cyanidin addedbefore fermentation are shown in Fig. 8. The Streptococcus countwas much larger (40e50 times) than that of Lactobacillus. AddingBPE to the HM pectin formulations pre-fermentation had led toa significant increase (p < 0.05) in the number of Lactobacillus butlittle change in the number of Streptococcus. Adding BPE to the LMpectin formulation pre-fermentation did not cause a significantchange in the count number of both Streptococcus and Lactobacillus.Adding Cyanidin to either the LM or HMpectin formulations causeda small decrease (p< 0.05) in the count number of Lactobacillus, butminimal change in the number of Streptococcus. Under the condi-tions used, Streptococcus appeared to be more stable than Lacto-bacillus, a result that is consistent with the findings of Altieri,Bevilacqua, D’Amato, Del Nobile, and Sinigaglia (2008).

4. Discussion

For a functional yoghurt, maximal retention of added PPs,desired nutrients, and count number and metabolic activities ofstarter cultures, is desired (Chandan, White, Kilara, & Hui, 2006;Kailasapathy, Harmostorf, & Phillips, 2008; Stanton et al., 2005).Ideally, the added PPs stimulate starter culture activity and do notinhibit their survival and growth. PPs and their metabolites by thestarter cultures, may exert different effects on the growth of startercultures. It is possible that PPs are transformed into more activederivatives (e.g. aglycones) under certain yoghurt matrix condi-tions, which might enhance starter culture activity. The results ofthis study suggest that the PPs present in the BPE ingredient did not

contain components that inhibited starter culture activity andgrowth, and these PPs were not degraded or metabolised by thestarter cultures into compounds detrimental to the starter cultures.

The effects of Cyanidin and BPE on the starter cultures werediverse, and dependent on the type of PPs, type of starter culturesused, formulation composition and processing method. One con-tributing factor to the loss of cell viability was the decreased pHcaused by the accumulation of small organic acids in yoghurt(Donkor, Heriksson, Vasiljevic, & Shah, 2005; Kneifel, Jaros, &Erhard, 1993). PPs possess different levels of antibacterial or anti-microbial effects (Rauha et al., 2000; Välimaa et al., 2007), soadding PPs pre-fermentationmay have positive or negative impactson the growth of bacteria. Varied effects of added bioactive in-gredients on different microorganismswere reported, which can beassociated with the difference in chemical structure and anti-oxidant activity of PPs (Duda-Chodak, Tarko, & Statek, 2008) andbacteria type (Alberto, Farias, & Manca De Nadra, 2001; Lee, Jenner,Low, & Lee, 2006). For example, catechin was found to have noinfluence on growth of Clostridium sp. but stimulated Lactobacillusand Bifidobacterium (Lee et al., 2006). A Pycnogenol extract did notaffect the growth of Streptococcus and Lactobacillus in yogurt(Ramchandran & Shah, 2008), whilst isoflavones and phytosterolsslightly decreased the count of these probiotic bacteria (Awaisheh,Haddadin, & Robinson, 2005). Thus, the interactions betweenadded bioactives andmicroorganisms might vary on a case-by-casebasis.

Themode of PPmetabolism by the yoghurt starter cultures duringfermentation, may include flavonoid glycoside hydrolysis or C-ringcleavage, and generation/release of phenolics with simpler structureand smaller molecular mass such as phenolic acids (Bingham, 2006;Braune, Engst, & Blaut, 2005;Winter,Moore, Dowell, & Bokkenheuser,

Fig. 8. Colony count of Streptococcus and Lactobacillus starter cultures in the controlyoghurt and the yoghurts with polyphenols added before fermentation. Error bars arethe logarithmic upper and lower values of colony count. LB and ST refer to Lactobacillusdelbrueckii ssp. bulgaricus and Streptococcus thermophilus, respectively. LM and HMrefer to low and high methoxyl pectins, respectively. BPE and Cyanidin refer toblackcurrant polyphenol extract and cyanidin 3-o-b-glucopyranoside chloride,respectively.

Fig. 7. Micrographs of Streptococcus and Lactobacillus starter cultures in the control and polyphenol-enhanced yoghurts formulated with low methoxyl pectin. BPE and Cyanidinrefer to blackcurrant polyphenol extract and cyanidin 3-o-b-glucopyranoside chloride, respectively.

D. Sun-Waterhouse et al. / Food Control 32 (2013) 450e460458

1989). Such transformations may lead to deactivation of bioactivecompounds or activation of inactive compounds, for example, hy-drolysis of glycosides into their aglycones with higher potential forradical scavenging, or breakdown of procyanidins toflavan-3-ols or tosmall phenolic acids (Okuda, 1999; Rice-Evans, 1999). PPs present infoods are mostly in the form of esters, glycosides, or polymers, whichcannot be absorbed directly and must be hydrolysed by intestinalenzymes or colonic microflora. Only aglycones and some glycosidescan be absorbed in the small intestine (Hollman & Katan, 1997;Manach et al.,1995). Direct interactions such as bindings between PPsand milk proteins or pectic polysaccharides, are possible(Papadopoulou & Frazier, 2004; Plet, 2006; Renard et al., 2001; Rohn,Rawel, & Kroll, 2004; Serafini, Ghiselli, & Ferro-Luzzi, 1996). Amphi-pathic peptides released from polymeric proteins during the fer-mentation of milk (Korhonen & Pihlanto, 2003; Smacchi & Gobbetti,2000), may interact with added PPs. Metabolic processes of startercultures decreased the pH of yoghurt, which in turn influenced PPextractability and the rheological properties of yoghurt. At acidic pHs,proteins could undergo partial or complete hydrolysis, releasingpeptides that can bind PPs (Lin, Tornatore, & Weinberger, 2001;Papadopoulou & Frazier, 2004; Plet, 2006; Zubarev, Chivanov,Håkansson, Sundqvist, & Ens, 1994).

The influence of the type of added PPs on the chemical, physicaland microbiological properties of drinking yoghurt can be dem-onstrated directly through comparing the results of this currentstudy in which a blackcurrant PP extract (BPE) was used, with ourrecent study (Sun-Waterhouse, Zhou, et al., 2011) inwhich an applePP extract (APE) was used. The PP profile of the BPE used in this

D. Sun-Waterhouse et al. / Food Control 32 (2013) 450e460 459

study is very different to that of the APE used in our previous study(Sun-Waterhouse, Zhou, et al., 2011). Accordingly, the nature of theblackcurrant PP-yoghurt component interactions would be con-siderably different from the interactions between the apple PPs andyoghurt components. The difference in TEPC between the two PPaddition approaches for the BPE yoghurts were 2.6 times greaterthan for the APE yoghurts. In either case (BPE or APE addition), thepre-fermentation approach always led to a higher TEPC than thepost-fermentation approach. The presence of small molecules, suchas phenolic acids, in the yoghurts with PPs added before fermen-tationwas a common feature observed in this current study and ourprevious study (Sun-Waterhouse, Zhou, et al., 2011). For HM or LMyoghurts produced via the pre-fermentation approach, the differ-ence in the colony count of Streptococcus or Lactobacillus strain wasinsignificant (p < 0.05) between the BPE and APE yoghurts. Suchinterplay between microorganisms and food PPs was explored inprevious studies (Bock & Ternes, 2010; Gross et al., 2010; Tabascoet al., 2011; Torras, Faura, Schonlau, & Rohnelwald, 2005), e.g. PPsselectively modified the growth of microorganisms while thestrains may metabolise the PPs (e.g. flavan-3-ol monomers) intosmaller phenolic molecules such as gallic acid, pyrogallol and cat-echol by the microorganism-generated enzymes such as galloyl-esterase, decarboxylase and benzyl alcohol dehydrogenase. Thus,it is possible that the metabolite profiles vary, depending onmicrobial bioconversion of dietary PPs.

5. Conclusions

The BPE ingredient containing a relatively high PP concentrationcan be successfully incorporated into drinking yoghurts via eithera pre- or post-fermentation approach. For either approach, the fourmajor blackcurrant anthocyanins, which are well-known for theirproven health benefits, were significantly retained in the finalyoghurt product. However, these two approaches influenced dif-ferently the stability and extractability of PPs, and the PP profilesand rheological properties of the yoghurt products. Adding BPEbefore fermentation had resulted in PP metabolism to small phe-nolic molecules (primarily phenolic acids) in the yoghurts and 3.5e3.9 times the TEPC value of yoghurts obtained by adding BPE afterfermentation. Adding PPs (in the form of BPE or Cyanidin) influ-enced the microbiological properties of the starter cultures (i.e. theappearance, survival and growth of Streptococcus and Lactobacillusbacteria observed). The specific effects depended on the type andexisting form of PPs, and other ingredients in yoghurt formulation(e.g. the methoxyl content of pectin stabiliser). Results of this studyindicate an opportunity to optimise yoghurt processing and pro-duction to maximise synergies between PPs, yoghurt starter cul-tures and milk components. Future work should be directedtowards the validation of gut health-promoting properties of suchdrinking yoghurts, as well as the difference in bioavailability of theblackcurrant PPs that are added to yogurts pre- or post-fermentation.

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