Diry Foods Evlution of Yogurt with Enhnced Cysteine Content S. Bl nd K. A. Schmidt Summry Amino cids re the uilding locks of protein nd ssist with metolism in the ody. In the humn ody, the mino cid cysteine cn e synthesized from methionine y the enzyme Υ-cystthionse. Becuse certin humn supopultions such s those prone to ctrcts hve decresed Υ-cystthionse ctivity, dietry cysteine my e eneficil. Nutritionlly, yogurt mix is one of the est diry food sources of methionine nd cysteine, ut the het tretment used in mnufcturing yogurt decreses the dietry vilility of cysteine. Lst yer, it ws shown tht supplementing yogurt mixes with whey protein isolte (WPI) (>90% protein) nd processing yogurt mixes t lower temperture produced yogurts with incresed cysteine. Becuse the qulity or cysteine content of the yogurt during the expected storge life is unknown, this study ws conducted to determine if comintion of WPI ddition nd non-optiml process conditions could produce yogurt with higher cysteine content nd n cceptle shelf life. In this study, control yogurt mixes were mde with nonft dry milk (NDM) nd processed t 90 o C for 7 minutes, wheres the experimentl yogurt mixes were mde with NDM nd WPI nd processed t 70 o C for 20 minutes. Both mixes were cooled, inoculted, fermented into yogurt, stored t 4 C, nd evluted periodiclly over 60-dy period. The experimentl yogurts hd ~2X more cysteine thn the control yogurt; this trend ws present throughout storge. After 60 dys of storge, the wter-holding cpcity (WHC) nd firmness ws greter nd the syneresis ws less for the experimentl yogurt thn the control yogurt. These results show tht yogurt supplemented with WPI nd processed t less optiml conditions my e good source of the conditionl mino cid cysteine during storge. Key words: yogurt, whey protein isolte, cysteine Introduction In 2011, the USDA reported n 85% increse in yogurt populrity nd sles since 2002. This incresed populrity my e ttriuted to the nutritionl nd helth enefits of yogurt, such s improved digestiility nd lctose utiliztion nd ntgonism towrd enteric pthogens. When psteurized t high tempertures, yogurt mixes hve greter whey protein denturtion (WPD) ut less dietry vilility of cysteine. In milk, cysteine is component of whey proteins. Het cuses whey proteins to unfold, which cuses whey nd csein proteins to ggregte. This ggregtion contriutes to high-qulity yogurt, which is defined s exhiiting firm gel, expressing miniml syneresis, nd mximizing wter-holding cpcity (WHC). Yogurt firmness nd syneresis re therefore relted to protein content nd WPD, ecuse these properties re functions of the numer nd strength of the whey protein-csein interctions. According to the Centers for Disese Control nd Prevention (CDC, 2012), the current estimte is tht nerly 20.5 million (17.2%) Americns who re 40 yers nd older hve ctrct in one or oth eyes; 30.1 million Americns re predicted to hve ctrcts y 2020. Studies of the occurrences nd cuses of ctrcts hve shown tht elderly rts (24 to 26 months) hd less or no Υ-cystthionse (n enzyme tht converts methionine to cysteine) in their eye lenses compred with young rts (5 to 6 months). Other reserchers hve reported tht incresed ctrct formtion ws ssocited with decresed glutthione (GSH) contents in humn eye lenses. 29
Diry Foods One of the sustrtes for GSH synthesis is cysteine (Figure 1); hence, the decresed ctivity of Υ-cystthionse hs een one of the focus in ctrct reserch. These dt suggest tht people prone to ctrcts my enefit from consuming dietry cysteine ecuse it is precursor for GSH synthesis. Most commercil yogurts contin 9 to 14% milk solids, which re derived from the milk se nd dditionl milk solids. Previous reserchers hve reported on the impct of vrious supplements (nonft dry milk [NDM], whey protein concentrtes [WPC], or whey protein isolte [WPI] in yogurt mixes. Typiclly, s WPC concentrtions incresed, yogurt hd significntly greter firmness nd reduced syneresis. Yogurts contining WPI hd n even greter reduction in syneresis nd increse in WHC. In our previous reserch, we formulted yogurts with vrious levels of WPI nd oserved greter firmness (1.4X) nd cysteine contents (~4.5X), ut we used non-optiml process conditions. Although the yogurt ws of good qulity on dy 1, we did not know whether the cysteine or gel qulity would e sustined during the expected yogurt shelf life. Thus, this reserch project ws undertken to: (1) compre protein nd cysteine contents in n experiment yogurt mix contining WPI nd processed t less optiml conditions to control yogurt, nd (2) ssess cysteine contents nd gel qulity of these yogurts during storge (dy 1, 15, 30, 45, nd 60). Experimentl Procedures Low-het NDM, WPI, nd yogurt cultures were otined from commercil suppliers nd mintined t 2 or 10 o C (culture) until usge. Two formultions were mde: control (C) mix consisting of 12.5% NDM nd n experimentl (E) mix consisting of 10% NDM nd 2.5% WPI. Dried diry powders were rehydrted in deionized distilled wter t 22 to 24 o C for 30 minutes. The C mix ws processed t 90 o C for 7 minutes to ensure ~90% WPD, wheres the E mix ws processed t 70 o C for 20 minutes to minimize WPD (nd preserve cysteine). Both mixes were then cooled to 43 o C, inoculted with Lctocillus delrueckii ssp. ulgricus nd Streptococcus slivrius ssp. thermophilus, pckged into sterile continers, incuted until ph 4.6, nd then plced in storge (4 ± 1 o C) for up to 60 dys. Stndrdized, pulished methods were followed for ll nlyses, nd three replictions were performed. Mixes were mde nd nlyzed for protein contents, WPD, nd cysteine contents using rndomized complete lock design. Stored yogurts were evluted for cysteine contents, firmness, syneresis, nd WHC on dys 1, 15, 30, 45, nd 60 using split-plot design. All dt were nlyzed using SAS (SAS Institute, Cry, NC), nd significnt (P < 0.05) mens nd interctions were differentited using Fisher s LSD tests. Results nd Discussion The C yogurt ws formulted nd processed like set-style commercil product. To ensure tht the mixes were of different protein composition, mixes were nlyzed for protein (true protein nd whey) nd WPD (Tle 1). The E mixes hd 32% nd 189% more true protein nd whey protein, respectively, thn the C mixes. Mixes did not significntly differ in csein content (~3.34%). WPD ws ~19X less for E mixes thn C mixes due to the processing difference. Tle 1 lso displys the effects of the mnufcturing steps (formultion [unheted], processed [het tretment], nd fermenttion) on cysteine. The heting step (process) drmticlly ffected cysteine, with the C mix exhiiting 65% loss vs. the E mix exhiiting only 21% loss compred with their respective unheted mixes. Interestingly, fermenttion did not significnt- 30
Diry Foods ly ffect the cysteine content (the cysteine losses during fermenttion were ~3% in oth yogurt smples), ut the E yogurt required 30 minutes more fermenttion time thn the C yogurt. To mke n effective delivery vehicle for compound such s cysteine, the product needs to e cceptle to the consumer not only on dy 1, ut lso throughout storge. On dy 1, ll yogurts hd similr totl solids content (12.35%), ph (4.42), syneresis (6.51%), nd WHC (22.83%), ut the E yogurt ws ~2.1X more firm nd hd ~190% more cysteine thn the control yogurt. Compred with commercil, set-style yogurt (128 g) purchsed t locl grocery store, the E yogurt hd similr firmness (133 g). With time, yogurt exhiits syneresis nd shrinks wy from the pckge. These two qulities, which re directly relted to the whey protein csein interctions, re considered defects y consumers; hence, the storge stility of this yogurt ws evluted. Results showed significnt interctions for gel qulity. Overll, firmness of the E yogurt ws ~2.1X greter thn the C yogurt (Figure 2), nd oth yogurts exhiited significnt increse in firmness (25%) from dy 1 to dy 15. The firmness of C yogurt ws constnt throughout the remining storge period ut the firmness of the E yogurt decresed from dy 30 to dy 45. The E yogurt exhiited 76% less syneresis thn the C yogurt on dy 1, nd oth yogurts decresed in syneresis from dy 1 to dy 15 (Figure 3), ut yogurt syneresis remined constnt therefter. On dy 1, the E yogurt hd greter WHC thn the C yogurt. The WHC of the E yogurt remined constnt throughout storge, ut the C yogurt incresed in WHC (21%) from dy 1 to dy 15 nd eventully decresed to its initil vlue t dy 60 (Figure 4). More importntly, the E yogurt hd greter cysteine content (398.4 mg/l) thn the C yogurt (135.7 mg/l), nd the cysteine content ws not ffected y storge time, which suggests tht greter cysteine content would e stle nd ville throughout the storge life. These results indicte tht WPI supplementtion of yogurt mix comined with less optiml process conditions my produce yogurt tht effectively delivers cysteine. Conclusions In yogurt, cysteine content is function of the type nd mount of milk protein nd the het tretment of the mix. A yogurt mde from mix with WPI nd processed t less optiml conditions ws shown to hve greter cysteine content nd cceptle gel qulity. Further reserch involving sensory properties of this yogurt nd investigtion of how enhnced cysteine in yogurt ffects GSH production in tissue cultures should e pursued. 31
Diry Foods Tle 1. True protein nd whey protein contents, whey protein denturtion (%) of yogurt mixes, nd cysteine contents s function of process tretments (mens ± stndrd error) Composition Control 1 Experimentl 2 Mix True protein % 4.17 ±0.07 5.51 ±0.02 Whey % 0.77 ±0.01 2.23 ±0.02 WPD 3,4 % 70.28 ±0.73 3.45 ±0.88 Mnufcturing step Cysteine (mg/l) Unprocessed mix 306.98 C ±1.65 504.96 A ±2.59 Processed mix 4 138.52 D ±4.21 400.15 B ±12.08 Fermented 135.74 D ±4.64 398.32 B ±19.79, Mens (n=3) within rows with different lower cse superscripts differ (P < 0.05). A-D Mens (n=3) with different upper cse superscripts differ (P < 0.05). 1 Nonft dry milk (NDM) (12.5%). 2 NDM (10%) + whey protein isolte (WPI) (2.5%). 3 Whey protein denturtion. 4 Control processed t 90 o C for 7 minutes nd experimentl processed t 70 o C for 20 minutes. Methionine Υ-cystthionse Cysteine Glutmte Clycine Υ-glutmylcysteine synthetse nd glutthione synthetse Glutthione Figure 1. Glutthione synthesis. 32
Diry Foods Dy 1 200 180 160 140 Dy 15 Dy 30 Dy 45 Dy 60 c Firmness, g 120 100 80 60 e d de d de 40 20 0 Control Experimentl Figure 2. Yogurt firmness during 60 dys of storge. Control: nonft dry milk (NDM) (12.5%) processed t 90 o C for 7 minutes. Experimentl: NDM (10%) + whey protein isolte (2.5%) processed t 70 o C for 20 minutes. -e Brs with different superscripts differ (P < 0.05). Dy 1 Dy 15 8 Dy 30 Dy 45 7 6 Dy 60 Syneresis, % 5 4 3 c c c c 2 1 0 Control Experimentl Figure 3. Yogurt syneresis during 60 dys of storge. Control: nonft dry milk (NDM) (12.5%) processed t 90 o C for 7 minutes. Experimentl: NDM (10%) + whey protein isolte (2.5%) processed t 70 o C for 20 minutes. c Brs with different superscripts differ (P < 0.05). 33
Diry Foods Dy 1 Dy 15 30 Dy 30 Dy 45 25 20 d c c c d Dy 60 WHC, % 15 10 5 0 Control Experimentl Figure 4. Yogurt wter-holding cpcity (WHC) during 60 dys of storge. Control: nonft dry milk (NDM) (12.5%) processed t 90 o C for 7 minutes. Experimentl: NDM (10%) + whey protein isolte (2.5%) processed t 70 o C for 20 minutes. c Brs with different superscripts differ (P < 0.05). 34