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Ciência e Tecnologia de Alimentos ISSN The influence of different combinations of probiotic bacteria and fermentation temperatures on the microbiological and physicochemical characteristics of

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Ciência e Tecnologia de Alimentos ISSN The influence of different combinations of probiotic bacteria and fermentation temperatures on the microbiological and physicochemical characteristics of fermented lactic beverages containing soybean hydrosoluble extract during refrigerated storage Influência de diferentes combinações de bactérias probióticas e temperaturas de fermentação nas características microbiológicas e físico-químicas de bebidas lácticas fermentadas contendo extrato hidrossolúvel de soja sob estocagem em refrigeração Original Ângela Maria FIORENTINI 1 *, Cristiano Augusto BALLUS 1, Marlon Leonardo de OLIVEIRA 1, Márcio Ferraz CUNHA 2, Vera Maria KLAJN 3 Abstract Lactic beverages containing probiotics were prepared with whole UHT milk, whey of Mozzarella cheese, soybean hydrosoluble extract and sugar. Three formulations were studied, each one containing a different combination of probiotic/starter bacteria, fermented at two different temperatures (37 and 45 C). The aim of this work was to verify the influence of these variables on the viability of probiotic microorganisms and on the physicochemical stability of lactic beverages during storage under refrigeration (21 days at 7 C). The results indicated that the fermentation temperature had a significant effect on the viability of probiotic bacteria. Counts for Lactobacillus acidophilus were affected by storage time, resulting appropriate after 21 days only for the beverage fermented at 37 C. Physicochemical parameters did not exhibit drastic variations - proving the stability of formulations during storage. Cells of Bifidobacterium spp. showed high survival ability, probably due to the presence of growth promoters from soybean and cheese whey. The fermentation temperature of 37 C allowed counts above the minimum limit for all the studied microorganisms, being preferred to the temperature of 45 C. The inclusion of soybean hydrosoluble extract, a prebiotics source, resulted in a symbiotic product with more benefits to the health of consumers. Keywords: Bifidobacterium spp.; Lactobacillus acidophilus; lactic beverages; soybean; probiotics; functional foods. Resumo Bebidas lácticas contendo probióticos foram elaboradas com leite UHT integral, soro de queijo Mozarela, extrato hidrossolúvel de soja e açúcar. Três formulações foram estudadas, cada uma contendo diferentes combinações de bactérias probióticas/starters e fermentadas em duas temperaturas (37 e 45 C). O objetivo foi verificar a influência destas variáveis sobre a viabilidade de microrganismos probióticos e sobre a estabilidade físico-química das bebidas lácticas durante armazenamento sob refrigeração (21 dias a 7 C). Os resultados indicaram que a temperatura de fermentação foi significativa para a viabilidade de bactérias probióticas. A contagem de Lactobacillus acidophilus foi influenciada pelo tempo de armazenamento, resultando adequada ao final de 21 dias apenas para a bebida fermentada a 37 C. Os parâmetros físico-químicos não variaram drasticamente, demonstrando a estabilidade das formulações durante a estocagem. As células de Bifidobacterium spp. apresentaram elevada capacidade de sobrevivência, possivelmente pela presença de promotores de crescimento provenientes da soja e do soro de queijo. A temperatura de fermentação de 37 C possibilitou contagens acima do limite mínimo para todos os microrganismos estudados, sendo preferida ao invés de 45 C. A inclusão de extrato hidrossolúvel de soja, uma fonte de prebióticos, resultou em um produto simbiótico, com mais vantagens para a saúde do consumidor. Palavras-chave: Bifidobacterium spp.; Lactobacillus acidophilus; bebidas lácticas; soja; probióticos; alimentos funcionais. 1 Introduction One of the oldest methods used for milk conservation is fermentation, a process that has been used for thousands of years. Nowadays, this process is still of high importance for developing new dairy products, by combining artisanal features and scientific knowledge of different areas (chemistry, biochemistry, microbiology, enzimology, physics and engineering) (TAMIME, 2002; ORDÓÑEZ et al., 2005). Besides, modern consumers worry about personal health, increasing the demand of foodstuffs capable of supplying not only nutritional aspects, but also healthy benefits, the so-called functional food. A well established market of functional foods is of probiotic yoghurts and probiotic dairy products (MATTILA- SANDHOLM et al., 2002). Received 9/5/2009 Accepted 4/4/2010 (004190) 1 Departamento de Biologia e Química, Universidade Regional do Noroeste do Estado do Rio Grande do Sul UNIJUÍ, RS 344, Km 39, Bairro Timbaúva, CP 489, CEP , Santa Rosa, RS, Brasil, 2 Departamento de Nutrição, Universidade de Uberaba - UNIUBE, Av. Guilherme Ferreira, 217, Centro, CEP , Uberaba, MG, Brasil 3 Faculdade de Agronomia Eliseu Maciel, Universidade Federal de Pelotas - UFPEL, Campus Capão do Leão, FAEM; Av. Eliseu Maciel, s/n, CEP , Capão do Leão, RS, Brasil *Corresponding author Ciênc. Tecnol. Aliment., Campinas, 31(3): , jul.-set Different combinations of probiotic bacteria in lactic beverages containing soybean extract According to FAO (Food and Agriculture Organization of the United Nations) and WHO (World Health Organization), scientific evidences indicate that there is potential for probiotic foods in providing health benefits and that specific strains are safe for human usage (OTIENO; ASHTON; SHAH, 2005). Bifidobacterium spp. and Lactobacillus acidophilus are the most frequently probiotic bacteria employed in the production of probiotic dairy products (GOMES; MALCATA, 1999). There are several health benefits in consuming probiotics: balancing out temporary instabilities of the intestinal flora; inhibition of the invasion and colonization by pathogenic microorganisms; decrease of the concentration of ammonia, indols and other putrefactive substances; lowering of rotavirusassociated diarrhea in a study with 55 hospitalized children; stimulation of the immune system; inhibition of the growth and division of cancer cells in vitro; cholesterol-lowering effect (probably by deconjugation of bile salts) (MÖLLER; DE VRESE, 2004). However, it is necessary to point out that the regular intake of probiotic viable cells must be of 10 6 CFU.g 1 of a product in order to provide these beneficial effects (SHAH, 2000; VINDEROLA; BAILO; REINHEIMER, 2000; TALWALKAR; KAILASAPATHY, 2004; ZACARCHENCO; MASSAGUER- ROIG, 2004; THAMER; PENNA, 2006). A probiotic strain needs to fulfill some selection criteria to be able to exert its beneficial effects. It is essential to take into account some safety aspects, such as origin (healthy human gastrointestinal tract) and non-pathogenicity. Functional aspects include tolerance to acid and human gastric acid; bile tolerance; adherence to epithelial surfaces and persistence in the human gastrointestinal tract; immunostimulation; antagonistic activity against pathogens; antimutagenic and anticarcinogenic properties. Some technological aspects are good sensory properties; phage resistance; viability during processing; stability in the product and during storage (SANDERS; KLAENHAMMER, 2001; MATTILA-SANDHOLM et al., 2002; CORCORAN et al., 2005; SCHILLINGER; GUIGAS, HOLZAPFEL, 2005; BEGLEY; HILL; GAHAN, 2006; MARAGKOUDAKIS et al., 2006). Some strains of Streptococcus thermophilus are able to synthesize exopolysaccharides (EPS) that act as natural biothickeners capable of improving texture of fermented foods. This EPS production in situ, during milk fermentation, results in an obvious advantage for the food industry (SVENSSON et al., 2005). Streptococcus thermophilus strains that produce EPS can reduce syneresis and enhance product texture and viscosity. Thus, these types of cultures are commonly used as a substitute for commercial stabilizers in yoghurt manufacture (BROADBENT et al., 2003). Moreover, studies showed that EPS can have beneficial effects on human health, such as cholesterollowering ability, prebiotic effects, immunomodulating and antitumoral activities (RUAS-MADIEDO; REYES-GAVILÁN, 2005). Some species of Bifidobacterium spp. genus can be stimulated by compounds called prebiotics. Prebiotic is a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of a limited number of bacteria in the colon. Lactulose, fructooligosaccharides, inulin, resistant starch and soybean oligosaccharides are some examples of prebiotics. Soybean oligosaccharides, as well as raffinose and stachiose, correspond to the flatulence factors, which limit the soybean consumption (RIBEIRO; MORAES; ROIG, 1987; HOU; YU; CHOU, 2000). Since these compounds are not assimilated by humans, they can stimulate and promote the growing of probiotic microorganisms such as bifidobacteria - so being considered a prebiotic (TAMIME; MARSHALL; ROBINSON, 1995; HOU; YU; CHOU, 2000). When a product contains both, probiotics and prebiotics, it is defined as symbiotic (SCHREZENMEIR; DE VRESE, 2001; ITSARANUWAT; SHAL-HADDAD; ROBINSON, 2003). Soybean hydrosoluble extract showed better sensorial quality after lactic fermentation, when the characteristic beany flavor is masked and, at the same time, the nutritional value is improved (BEHRENS; ROIG; SILVA, 2001; MORAES et al., 2006). In this way, new products, such as soybean cheese and several types of fermented beverages were obtained (CHOU; HOU, 2000). According to Champagne, Gardner and Roy (2005), the fermentation of soybean hydrosoluble extract with probiotic bacteria provide some benefits like reduction of flatulence sugars and improvement of product conservation, as well as contribution to consumer s health. Wang, Yu and Chou (2004) verified a decrease in flatulence oligosaccharides in soybean hydrosoluble extract fermented by mixed cultures of lactic and probiotic bacteria. For most Brazilian cheese manufacturers, cheese whey is considered a residue being disposed inappropriately in water courses, what can lead to serious contamination problems for aquatic ecosystems (TEBALDI, 2005). Cheese whey represents 85-90% of the milk volume used for cheese manufacture, retaining about 55% of the milk nutrients. Thus, the use of cheese whey in lactic beverages becomes a rational way of employing this secondary product with excellent nutritive value (ALMEIDA; BONASSI; ROÇA, 2001). Almost all aminoacid types present in the sweet cheese whey overcome the minimum daily doses of nutrients recommended by the World Health Organization (FAO/WHO) (MACEDO, 1997). Considering lactic beverages manufacture, the addition of high quantities of cheese whey and soybean hydrosoluble extract reduces product costs. For dairy industries, cheese whey conversion into fermented beverages is one of the most attractive options, when using cheese whey for human consumption, due to the process simplicity - equipment commonly used in milk processing, as well as to the excellent functional properties of whey proteins (MACEDO, 1997). In the present work, formulations of lactic beverages included milk, soybean hydrosoluble extract (soymilk) and cheese whey from Mozzarella manufacture. The aim of this study was to elaborate fermented lactic beverages (using dairy and non-dairy substrates) with different combinations of probiotic/ starter bacteria and fermentation temperatures in order to verify the influence of these combinations on the viability of probiotic microorganisms and on the physicochemical stability of lactic beverages during refrigerated storage. 598 Ciênc. Tecnol. Aliment., Campinas, 31(3): , jul.-set. 2011 Fiorentini et al. 2 Materials and methods 2.1 Starter and probiotic cultures Lyophilized cultures of Lactobacillus acidophilus La-5, Bifidobacterium bifidum BB-12 and ABT-4 (containing Streptococcus thermophilus, Lactobacillus acidophilus, Bifidobacterium lactis and Bifidobacterium bifidum) were purchased from Christian Hansen Ltda. (Valinhos, Sao Paulo, Brazil). Cultures of Bifidobacterium lactis BL LYO and Streptococcus thermophilus EPS producing strain were kindly provided by Danisco (Dangé-Saint-Romain, France) and ISP Brasil (Cabreuva, Sao Paulo, Brazil), respectively. Each culture was aseptically diluted in 240 ml of sterile UHT milk (Elegê, Rio Grande do Sul, Brazil), which was subsequently divided into twelve equal portions of 20 ml. The cultures were then stored at 80 C until use. with the combination of single bacteria cultures, according to each formulation. Counts of viable lactic bacteria and physicochemical analysis were carried out at 0, 7, 14 and 21 days of refrigerated storage. The analyses of 0 day were carried out after overnight cold storage of the lactic beverages. 2.4 Physicochemical analysis Samples of each lactic beverage formulation were submitted to the following physicochemical analyses: ph, by using a digital phmeter (Digimed, model DM-20, Brazil) with a combined glass electrode and temperature probe, previously calibrated with fresh ph 4.0 and 7.0 standard buffers; 2.2 Soybean hydrosoluble extract (soymilk) and cheese whey powder The soybean hydrosoluble extract was obtained according to the procedure described by Prudencio and Benedet (1999), using organic soybean (Glycine max) purchased at local supermarkets. Whey powder from Mozzarella cheese, supplied by a local dairy industry (Elegê, Rio Grande do Sul, Brazil), was dissolved in water at a ratio of 1:12 (whey powder:water), immediately before use. 2.3 Lactic beverage preparation and storage Preliminary tests were performed in our laboratory to determine the most appropriate concentration of each substrate in lactic beverage formulation, considering physicochemical and sensorial properties (data not published yet). Lactic beverages were prepared mixing 40% of UHT whole milk (Elegê, Rio Grande do Sul, Brazil), 30% of Mozzarella cheese whey, 30% of soybean hydrosoluble extract and 10% of sugar (sucrose) in a pilot-scale yoghurt fermented (Tecnoinox, Rio Grande do Sul, Brazil). Then, this mixture was heat-treated (90 C for 5 minutes) and subsequently cooled to fermentation temperature. Two different temperatures were evaluated: 37 and 45 C. An inoculum of 20 ml of each culture (according to the formulation) was used to inoculate 6 L of lactic beverage, and the incubation was carried out until ph reached After the end of fermentation, the lactic beverages were cooled to 20 C, homogenized and distributed into 1 L high-density polyethylene plastic bottles. These bottles were then stored at 7 C ± 1 C, for 21 days. Figure 1 presents a flowchart for the lactic beverage preparation. Three formulations were studied: BL1 (fermented only by probiotic bacteria: Lactobacillus acidophilus, Bifidobacterium lactis and Bifidobacterium bifidum); BL2 (fermented by starter and probiotic bacteria: Streptococcus thermophilus, Lactobacillus acidophilus, Bifidobacterium lactis and Bifidobacterium bifidum); and BL3 (fermented by Streptococcus thermophilus EPS producing strain with probiotic bacteria: Lactobacillus acidophilus, Bifidobacterium lactis and Bifidobacterium bifidum). BL2 formulation was inoculated with ABT-4, while BL1 and BL3 were inoculated Figure 1. Flowchart for lactic beverage elaboration. Ciênc. Tecnol. Aliment., Campinas, 31(3): , jul.-set Different combinations of probiotic bacteria in lactic beverages containing soybean extract Titratable acidity (expressed as lactic acid content), by an acid-basis reaction between lactic acid and NaOH N, with the end point visualization related to color change of phenolphthalein indicator (INSTITUTO ADOLFO LUTZ, 1985); Proteolysis, by measuring liberated tyrosine (amino acid), according to spectrophotometric method described by Almeida, Bonassi and Roça (2001). In this method, 5 ml of lactic beverage samples were added to a test tube, and subsequently mixed with 10 ml of trichloroacetic acid 0,72 N and 1 ml of distilled water; after 10 min, the mixture was filtered, and 5 ml of the permeate was added to a 50 ml volumetric flask; then, 10 ml of sodium carbonate/sodium hexametaphosphate solution (at 40 C) and 3 ml of Folin-Ciocalteu phenol reagent (1:2 aqueous solution) were added; absorbance readings at 650 nm were performed using a double-beam UV- Visible Spectrophotometer (Perkin Elmer, model Lambda 2S, USA), after 5 minutes; the quantification of liberated tyrosine was done from the standard curve obtained using tyrosine solutions with pre-determined concentrations; and Syneresis, according to Hashimoto and Antunes (1995), in which 100 ml of lactic beverage sample were added to a 100 mesh cloth, supported by a funnel inserted at a graduated cylinder, used to collect the permeate. This set was kept at 10 C for 2 hours, and then, the collected permeate was read as syneresis in 100 ml of sample. All chemicals used were of analytical grade, and determinations were made in duplicate. 2.5 Enumeration of viable lactic bacteria Lactic beverages samples (1 ml) were decimally diluted in test tubes with 9 ml of sterile peptone water (0.1% w/v) (Acumedia, USA), and 0.1 ml aliquot was placed over each culture media. For Streptococcus thermophilus selective counts, the ST (Streptococcus thermophilus) medium with aerobic incubation at 30 C for 48 hours (DAVE; SHAH, 1996; modified by ZACARCHENCO, 2004) was used; For selective enumeration of Lactobacillus acidophilus, MRS (De Man, Rogosa & Sharp) agar (Acumedia, USA) added with 0.15% (w/v) of bile (Vetec, Brazil) (Bile- MRS) and aerobic incubation at 37 C for 72 hours (VINDEROLA; REINHEIMER, 1999; ZACARCHENCO, 2004) was employed; Selective enumeration of Bifidobacterium spp. was performed in MRS agar (Acumedia, USA) added with 0.5 g.l 1 of lithium chloride (Ecibra, Brazil) and 0.75 g.l 1 of sodium propionate (Sigma, Germany) Lithium Chloride- Sodium Propionate Agar (Agar LP MRS), with incubation in anaerobic conditions (Anaerocult A, Merck, Germany) at 37 C for 72 hours (LAPIERRE; UNDELAND; COX, 1992; modified by ZACARCHENCO, 2004). Colonies identification was initially checked through Gram stain, and all counts were realized in duplicate. 2.6 Experimental design and statistical analysis The experimental design was completely randomized, with factorial arrangement (formulations fermentation temperatures storage time). The experiment was carried out in triplicate, resulting in 72 experimental units. Data were analyzed using Statistica 6.0 software (StatSoft Inc., USA, 2001), applying ANOVA with 95% confidence level (P 0.05). Differences between media were detected by Tukey s test. 3 Results and discussion The mathematical model for a factorial arrangement at a completely randomized design with three replicates is (Equation 1): Y ijkm = μ + F i + T j + S k + (FT) ij + (FS) ik + (TS) jk + (FTS) ijk + E ijkm (1) (i = 1, 2, 3; j = 1, 2; k = 1, 2, 3, 4; m = 1, 2, 3), where: Y ijkm = observed value for the variable that received level i of factor F, level j of factor T, and level k of factor S, in the m replicate; μ = experiment general medium; F i = effect of formulation i; T j = effect of fermentation temperature j; S k = effect of storage time k; (FT) ij = effect of interaction between formulation i and fermentation temperature j; (FS) ik = effect of interaction between formulation i and storage time k; (TS) jk = effect of interaction between fermentation emperature j and storage time k; (FTS) ijk = effect of interaction among formulation i, fermentation temperature j and storage time k; and E ijkm = experimental error associated with each observation. To investigate violations of the basic assumptions of ANOVA and model adequacy, an examination of residuals was performed. The normality of residuals assumption was checked by the construction of a normal probability plot (if the error distribution is normal, this plot will be similar to a straight line), while the assumption of variances homogene
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