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Probiotics must be consumed at a level of 10 CFU/mL for successful colonization of the gut. In yogurts containing beneficial cultures, the survival of probiotic strains can quickly decline below this critical concentration during cold storage . The inclusion in yogurt of beta-glucan, a possible prebiotic for bifidobacteria als o known to have heart-healthy effects, would increase the healthfulness of yogurt. We h ypothesized that betaglucan would increase the viability of bifidobacteria strains in yogurt during cold storage. Yogurts were produced containing 0.44% beta-glucan (concentrated or freeze-dr ied) extracted from whole oat flour and/or 1.33% corn starch, and bifidobacteria ( B. breve or B. longum) at a concentration of at least 10 9 CFU/mL. All yogurts were stored at 4°C. Bifidobacteria and yogurt cultures, S. thermophilus and L. bulgaricus, were enumerated from undisturbed aliquots before fermentation, after fermentation, and once a week for five weeks. S. thermophilus and L. bulgaricus maintained a concentration of at least 10 8 CFU/mL in 27 yogurts containing concentrated or freeze-dried beta-glucan regardless of starch addition, and in the control with no added beta-glucan or starch. Similarly, the probiotic, B. breve, survived above a therapeutic level in all treatments. The addition of beta-glucan pr olonged the survival of B. longum at a concentration of at least 10 7 CFU/mL by up to two weeks on average beyond the control. Further, the inclusion of concentrated beta-glucan in yog urt improved survival of B. longum above 10 CFU/mL by one week longer than did freezedried beta-glucan. Study results suggest that beta-glucan has a protective e ffect on bifidobacteria in yogurt when stressed by low-temperature storage. The c ombined benefits of the heart-healthy effects from beta-glucan and of the gut-health effect s from bifidobacteria should provide the food industry with information needed to formulate more healthful yogur t products. INTRODUCTION Probiotics are defined as “a preparation of or a product containing viable, defined microorganisms in sufficient numbers, which alter the microflora (by implant atio or colonization) in a compartment of the host and by that exert beneficial health effec ts in this host” (Schrezenmeir and de Vrese 2001). Bifidobacteria, known to act as probiotics, naturally occur in the gut of humans, and predominate in the guts of infants; however, as infants age their gut flora are soon joined by a variety of other bacteria suc h a E. coli, lactobacilli and clostridia that eventually outnumber bifidobacteria (Candy a others 2008). Gut colonization by bifidobacteria falls in adults by 1000-fold after age 60 (van T ongeren and others 2005). This drop in bifidobacteria is associated with an increase in less de sirable enterobacteriaceae. Many strains of bifidobacteria have been investigate d for possible health 28 benefits, with some of the better documented health benefits including decreased f qu ncy and duration of antibiotic-associated diarrhea, as well as positive immunomodulatory activity. Also, colonization in the human gut of the bifidobacteria is postulated to incre ase mineral absorption, prevent hypercholesterolemia, enhance immunity and provide anticarcinogenic activity (Fuller 1989; Schrezenmeir and de Vrese 2001). Indeed, t he role of bifidobacteria as a part of intestinal flora in maintaining overall health and w ell-being is not fully understood; however, it is recognized that probiotic cultures must be consumed at a level of at least 10 7 CFU/mL to provide the therapeutic benefits listed previously (LourensHattingh and Viljoen 2001). When probiotics are incorporated into food products, culture viability often decreases as a result of low pH, cold temperature stress dur ing storage, lack of nutrients, and oxidative stress; thus, it can be a challenge to maintain proper levels of th bacteria in food products so the food can deliver the desired amount. The addition of a prebiotic to foods may reduce these stressors and increase probiotic viability duri ng sto age (Bruno and others 2002; Capela and others 2006; Corcoran and others 2004) . Prebiotics are defined as “a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limit ed number of bacteria in the colon” (Gibson and Roberfroid 1995). Currently, the predominate prebiotic used in high fiber foods is the oligofructan, inulin. Oat beta-glucan has potential advanta ges over inulin as a prebiotic. Oat beta-glucan is a soluble dietary fiber that reduce s serum cholesterol, decreases insulin response and can provide prolonged satiety (DeVries 2001; Bell and others 1999) The Food and Drug Administration (FDA) recognizes oat-bet a glucan for its cholesterol-lowering effects with a health claim (CFR 21, Section 1018 ). According 29 to the FDA, the amount of beta-glucan needed to achieve health benefits in yogurt is 0.75g/ 6 oz serving which equates to 0.44% w/w beta-glucan. In addition, oat beta-glucan has a unique high viscosity that can provide desirable textural properties to yogurt (Ske ndi and others 2003). As a prebiotic, beta-glucan is postulated to improve probiotic survival in foods, such as yogurt, however little is known about the relationship between bifidobacte ria and beta-glucan in a food system during cold storage (Vasiljevic and others 2007) . Additionally, modified corn starch is often added to yogurts to improve texture. In particular, corn starch greatly improved yogurt viscosity and syneresi s when oat beta-glucan was included in the formulation (White and Yao 2007) . The impact of corn starch on bifidobacteria survival in yogurt was not evaluated. This study was undertaken to ex plore the relationship between oat beta-glucan, modified corn starch, and bifidobacteria surviv l in yogurt during refrigerated storage. The objectives of this study were to investigate the effects of adding oat bet a-glucan and/or corn starch on the viability of two strains of bifidobacteria in a set-sty l yogurt during 5 wk of refrigerated storage. MATERIALS AND METHODS Propagation of Cultures Pure strains of Bifidobacterium breve R0070, Bifidobacterium longum R0175, Bifidobacterium infantis R0033, and Bifidobacterium bifidum R0071 (Institut Rosell Inc., Montreal, QC) were grown anaerobically in de Man Rogosa and Sharpe (MRS broth) br oth (Fisher Scientific, Pittsburgh, PA) supplemented with 0.22 μm-filter steri lized 0.05% L30 cysteine (Sigma-Aldrich, St. Louis, MO) for 24 h at 37 °C for two consecutive passes . The cultures were then concentrated, 20% glycerol (v/v) was added, and samples were stored in cyrovials at -75 °C. Preliminary results eliminated the use of B. infantis and B. bifidum in this study because of their profound fragility during storage at 4 °C. Yogurt start er culture strains, Streptococcus thermophilus and Lactobacillus bulgaricus (YC-180, Chr. Hansen, Milwaukee, WI), were stored at -20 °C. Prior to yogurt production both bifidobacteria strains were inoculated and propagated twice in MRS broth at 37 °C for 24 h. Beta-glucan extraction An experimental oat line (N979-5-2-4), developed at Iowa State University to have a high beta-glucan concentration (7.88%), was grown and harvested at the Agronomy and Agricultural Engineering Field Research Center near Ames, Iowa in 2006 (Kim and White 2009). The oats were dried and shipped to the Quaker Oats Inc. pilot plant in Cedar Rapids, Iowa where they were steamed for one min at 80 °C and rolled to a flake thickness of 0.61 mm followed by grinding in a Hammer Mill though a 0.56 mm screen. Beta-gluca n was extracted from the whole oat flour (Yao and White 2008) and the supernatant collected.. Extracted beta-glucan was concentrated in a rotary evaporator at 75 °C and 200 mbar unti l the solution reached one-third its starting mass or freeze-dried (-20 °C pre-c hill, -26 °C for 24 h followed by 22 °C for remainder of drying, 80 mbar). The beta-glucan concentration of both treatments was determined by using the Megazyme mixed-linkage beta-gl ucan assay procedure (AACC method 32-23, AOAC method 995.16). 31 Yogurt Preparation Yogurt containing 1.5% fat and 11.5% milk-solids not-fat (MSNF) provided the base for the treatments (Table 1). Yogurt treatments containing beta-glucan r e eived a final concentration of 0.44% beta-glucan (w/w). Yogurt treatments containing modifie d waxy corn starch (Thermflo®, National Starch, Bridgewater, NJ) received a fi nal concentration of 1.33% starch (w/w). The ingredients were combined, homogenized, and heated at 85 °C for 30 min. The yogurt was then cooled to 43 °C and inoculated with yogurt starter cultur e (200 mg/L). The bifidobacteria strains, B. breve, and B. longum, were added, individually, to each yogurt treatment at a concentration of at least 10 9 CFU/mL. Each treatment was divided into 10-mL aliquots and fermented at 42 °C until reaching a pH <4.6, cooled to 4 °C, and stored for 5 wks. Culture viability determination To determine cell viability, cell counts were made from aliquots taken prior t o fermentation (day 0), the day following fermentation (day 1), and once every w e k for five total weeks (days 7-35), each from previously undisturbed yogurt. Each sample wa s s rially diluted in 0.1% peptone water, plated in duplicate on modified MRS agar containing Xα-Gal (5-bromo-4-chloro-3-indolylα-galactopyranoside) and incubated anaerobically at 37 °C for 48 h. The plates were then incubated aerobically an additional 24 h at 22 °C to allow for the bifidobacteria color change reaction to occur. Bifidobacteria (blue) and yogurt culture (white) colonies were counted after 72 h (Chevalier and others 1991). 32 Experimental Design For each bacterial strain ( B. longum, B. breve, no bifidobacteria added), the experiment was conducted as a three-way factorial, with factors (and level s) beta-glucan (present, absent), starch (present, absent) and time

Viability of bifidobacteria in yogurts containing oat beta-glucan and/or corn starch during cold storage