ORIGINAL RESEARCHDairy Technology XXX Society UK 1364-0307 1364-727X International Journal Ltd IDT Oxford,of Publishing of Blackwell Dairy

Technology 2008

O R I G I NAL R E SEARCH

Potentially probiotic aa yogurt

MAR IO H B DE ALM E IDA,1 S IDNE Y S Z OE L L NE R ,1 ADR IANO G DA C RUZ,1 * MIR IAM R L M OUR A,2 L UC IA M J DE C ARVAL HO,3 MAR IA C R IS TINA J F R E IT AS 3 and ANDE R S ON DE S S ANT ANA 4
1 Curso de Farmcia, Universidade Estcio de S, RJ Rua do Bispo, 83 Rio Comprido, CEP: 20261-063 Rio de Janeiro, Brazil, 2Departamento de Produtos Naturais e Alimentos, Faculdade de Farmcia, Universidade Federal do Rio de Janiero, Rio de Janeiro, Brazil, 3Departamento de Nutrio Experimental, Faculdade de Nutrio, Universidade Federal do Rio de Janiero Cidade Universitria, Ilha do Fundo, CEP: 21941590 Rio de Janeiro, Brazil, and 4Departamento de Cincia de Alimentos, Faculdade de Engenharia de Alimentos, UNICAMP Cidade Universitria Zeferino Vaz, s/n Campinas, CEP 13083-862 So Paulo, Brazil

The aim of this work was to evaluate the suitability of yogurt containing aa pulp as a food carrier of probiotic cultures. Probiotic yogurts containing increasing amounts of aa pulp (3, 5 and 7%) were processed and submitted to a physicochemical analysis and viable microbial count during refrigerated storage. In general, all the physicochemical parameters showed variations proportional to the amount of aa pulp in the product formulation. Probiotic activity was veried throughout refrigerated storage for all the products, although there was a fall of one logarithmic cycle for both micro-organisms during this period (107108 CFU/mL).
*Author for correspondence. E-mail: food@globo.com

Keywords Aa pulp, Probiotic bacteria, Yogurt.

I N T RO D U C T I O N Yogurt is a product obtained by the lactic fermentation of whole, skimmed or standardized milk by the action of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus, and can be accompanied by other lactic bacteria, which, for their part, contribute to the characteristics of the nal product (Brazil 2007). Yogurt is widely consumed throughout the world for its sensory and nutritional benets. It can be made from milk with high solid content, a lactic culture and sugar, and can be enriched with milk powder, proteins, vitamins, minerals or fruits. Aa (Euterpe oleracea), a palm fruit native to the Amazon Region, has recently captured international interest, not only due to its perceived novelty and exotic avour but also due to the potential health benets associated with its phytochemical composition (Pacheco-palencia et al. 2007). It has been consumed on the beaches of the Brazilian cities in the form of ice-cream, sherberts and fruit cocktails and from there it started to conquer the American continent. It is an abundant source of anthocyanins400 mg/100 g fruit pulpwhich shows its antioxidant properties. Other potential physiological properties of anthocyanins are said to include radiation protective-, chemoprotective-, vasoprotective- and anti-inammatory agents (Linden 2005).

Probiotics are biopreparations containing live cells or their metabolites, which stabilize the autochthonous microbial ora colonizing animal and human intestines, showing a stimulatory effect on the digestive and immune systems of their hosts. Various authors (Salminem 2000; Saarela et al. 2000) have documented the clinical and therapeutic effects of probiotics on human health and the benets attributed to the ingestion of food products containing cultures of this type. Aa stirred yogurt containing probiotic cultures has a unique possibility to produce a dairy product with all the benets provided by this class of micro-organisms, plus the pharmacological benets provided by the fruit. This food product does not exist in the Brazilian dairy market. Thus, the aim of this research was to develop a fermented dairy product, aa probiotic whipped yogurt, and evaluate its probiotic potential and physicochemical characteristics. M AT E R I A L S A N D M E T H O D S

*Author for correspondence. E-mail: food@globo.com 2008 Society of Dairy Technology

Yogurt processing The products were processed in the Laboratory Complex of the Institute of Nutrition of the Federal University of Rio de Janeiro, using four processing procedures: control (Yc with no aa pulp); yogurt 1 (Y3 with 3% aa pulp), yogurt 2 (Y5 with 5% aa pulp) and yogurt 3 (Y7 with 7% aa pulp). For

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all the formulations, with and without aa pulp, the following basic mixture was used: 10 L type C pasteurized milk (CCL, So Paulo, Brazil), 4% skimmed milk powder (Eleg, Rio Grande do Sul, Brazil) and 10% sugar (Guarani, So Paulo, Brazil). This mixture was heated to 45C, submitted to homogenization for 15 min. After this period, the temperature was reduced to 40C, and 2% w/v lactic starter containing L. delbrueckii ssp. bulgaricus and S. thermophilus (Cris Hansen, Valinhos, Brazil) and 2% w/v probiotic culture including Lactobacillus acidophilus and Bidobacterium (Cris Hansen, Valinhos, Brazil) were added. Yogurt fermentation was maintained at 40C and the pH was monitored every 30 min until reaching a pH close to 4.6. The mixture was then immediately cooled to room temperature (25C) by immersion in a 15-L container lled with constantly changed cold water. This basic mixture was then divided into 4 2.5 L portions, adding aa pulp at the concentrations of 3, 5 and 7% w/v, respectively. Each mixture was homogenized using an upright blender (Black and Decker, Hunt Valley, MD, USA), lled into 0.1 L polyethylene cups and stored at 5C.

Carbohydrate content was calculated by difference to achieve 100 g/100 g of total contents. Analytical procedures followed the appropriate standard methods including the energy value of the probiotic yogurts. (Instituto Aldolfo Lutz 2006).

Probiotic bacteria count The yogurts were analysed after 1, 7, 14, 21, and 28 days of storage at 4C. The samples were mixed; appropriate dilutions were made with sterile tryptone diluent (0.1% w/v) and subsequently plated in duplicate onto selective media. Lactobacilus acidophilus was performed on MRS-bile agar (International Dairy Federation 1995) and Bidobacterium bidum was performed on MRS- LP agar (Merck, So Paulo, Brazil) (Lapierre et al. 1992). All the analyses were performed in duplicate. Statistical analysis The results were submitted to a variance analysis (F-test), and the means were compared using the Tukey test at 5% level of signicance using version 6.0 of the Windows Statistica program. All experiments were repeated three times.
R E S U LT S A N D D I S C U S S I O N Table 1 shows the results for the physicochemical analyses (pH and titratable acidity) of the probiotic aa yogurts. The probiotic yogurts containing aa pulp showed signicant variation in relation to the control yogurt (without aa) for both parameters during refrigerated storage. The values for pH varied from 4.37 (Yc) to 3.90 (Y5), reaching values of 4.37 (Yc), 3.93 (Y7), 3.91 (Y3) and 3.90 (Y5) in 21 days. For titratable acidity, the variation was from 1.01 (Y3) to 1.22% (Y5) of lactic acid values of 1.14% (Yc), 1.02 (Y3), 1.20 (Y5) and 1.08 (Y7) being observed after 21 days. These values are in agreement with the values specied by Brazilian legislation, which establishes a range varying from 0.6 to 1.5 g lactic acid/100 g for yogurts (Brazil 2007).

Physicochemical analysis The pH values of yogurts were determined on duplicate samples using a pH meter Micronal B375 (Micronal, So Paulo, Brazil) equipped with a penetration electrode model. Titratable acidity was determined on duplicate samples according to the appropriate standard methods and expressed in terms of acidity in normal. Moisture content was determined by drying at 70C under vacuum (Micronal, So Paulo, Brazil) for 24 h. The mean composition of aa probiotic yogurts (ash, fat and protein content) was determined in the nal product (after 1 day of storage at 5C) in triplicate. Ash was determined gravimetrically by heating the 2 g sample at 550C, until completely ashed. Protein was estimated by measuring the nitrogen content of yogurts by the Kjeldahl method and multiplying by a conversion factor (6.38). Fat was determined following the Gerber method.
Table 1 Physicochemical analyses of probiotic aa yogurts

pH Yogurts Days 0 7 14 21 Yc 4.23a 4.37b 4.37b 4.37b Y3 3.92a 3.93a 3.96b 3.91a Y5 3.96a 3.93ab 3.94a 3.90a Y7 3.95b 3.90a 3.94b 3.93b

Titratable acidity (% lactic acid) Yogurts Yc 1.17a 1.17a 1.18a 1.14a Y3 1.07b 1.08b 1.01a 1.02a Y5 1.20a 1.20a 1.22a 1.20a Y7 1.02a 1.08b 1.07b 1.08b

Means followed by the same letter and in the same column do not differ signicantly (P > 0.05); C = control; Y3 = 3% aa pulp, Y5 = 5% aa pulp, Y7 = 7% aa pulp.

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Table 2 Viable microbial counts for the probiotic micro-organismsd Lactobacillus acidophilus (UFC/mL) Yogurts Days Yc 0 7 14 21
d

Bidobacterium bidum (UFC/mL) Yogurts

Y3 1.70 108a 1.45 108ab 5.50 108a 1.60 108b

Y5 4.35 108a 5.55 107a 6.75 106a 1.20 107ab

Y7 6.00 108a 1.60 108b 4.60 107a 1.50 108c

Yc 5.10 108a 5.25 108a 4.15 108a 5.00 107bc

Y3 5.1 108a 1.65 108a 1.90 108a 2.60 107abc

Y5 2.75 108a 4.45 108a 4.00 108a 1.95 107a

Y7 2.30 108a 4.00 108a 3.00 108a 5.50 107c

3.70 108a 2.50 107b 3.75 107a 1.50 107a

Means followed by the same letter and in the same column do not differ signicantly (P > 0.05); Yc = control; Y3 = 3% aa pulp, Y5 = 5% aa pulp, Y7 = 7% aa pulp.

Titratable acidity is an important quality indicator in yogurts, since its decreaseand consequent increase in pHcan suggest inadequate refrigerated storage conditions, which could create conditions favouring the development of pathogenic and deteriorative micro-organisms, compromising product quality and safety. The results indicate that the growth of the probiotic cultures was not inhibited by the low pH values during the storage time. Post-acidication, a phenomenon frequently occurring in yogurts, characterized by the excessive production of hydrogen peroxide and lactic acid by L. bulgaricus (Lourens-Hattingh and Viljoen 2001), exerted no inuence on the characteristics of the yogurts with added aa pulp. Antunes et al. (2005) reported slight postacidication during the processing of probiotic yogurts elaborated with whey protein concentrate. The pH values varied between 4.35 and 6.5 and there was a slight increase in titratable acidity attributed to the lower concentration of L. bulgaricus added to the product formulation. These authors found that the probiotic potential was maintained during the 21 days of refrigerated storage; counts greater than 105 colony-forming unit (CFU)/mL have been observed in all the yogurts. Although it is well known that the sensitivity of probiotic cultures is affected by low pH values, this sensitivity is extremely dependent on the microbial strain, especially for Bidobacterium sp. (Kailasapathy and Rybka 1997; Lourens-Hattingh and Viljoen 2001), resulting in diverse counts for this micro-organism. A possible interaction between the aa pulp (rich in salts) and the food matrix has been suggested, characterized by a buffer effect caused by the former. Additional studies are required to better understand this fact. Oliveira and Damin (2002) considered that pH variations below 0.12 during storage were slight decreases, and that variations between 0.14 and 0.32 were perceptible decreases. Thus, the pH variations observed in the present study, that is, 0.12 (Yc), 0.05 (Y3), 0.06 (Y5) and 0.05 (Y7), could 180
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be characterized as slight decreases, which, therefore, did not inuence the product composition. Table 2 shows the viable counts for the probiotic micro-organisms during refrigerated storage. The counts for L. acidophilus and B. bidum varied from 6.00 108 CFU/mL (Y7) to 1.20 107 CFU/ mL (Y5) and from 5.25 108 CFU/mL (Y3) to 1.95 107 CFU/mL (Y5), respectively. Signicant differences were observed in all the yogurts, independent of the aa pulp content (P > 0.05), for both micro-organisms, as from the third week of storage for B. bidum and already in the rst week for L. acidophilus. Although a fall of one logarithmic cycle was observed during refrigerated storage (107108 CFU/mL), all the yogurts maintained probiotic activity according to Brazilian legislation, which establishes a minimum of 106 CFU/mL in the product for therapeutic activity, and the International Dairy Federation (IDF), which suggests a minimum of 107 for L. acidophilus and 106 CFU/mL for Bidobacteria, when these are adjunct cultures in fermented products such as yogurt (Champagne and Gardner 2005; Brazil 2007). Kneifel et al. (1993) veried the probiotic potential of yogurts commercialized in Austria. The L. acidophilus counts varied from 4 105 to 2 108 CFU/mL and the B. bidum counts from 4 106 to 2 108 CFU/mL, respectively, during the shelf life, showing maintenance of the therapeutic functions of the products. The titratable acidity of the products increased, on average, by 22.3% during the same period. Shah et al. (1995), on analysing commercial Australian yogurts containing Lactobacillus acidophilus and B. bidum, found pH values between 4.07 and 4.36 soon after cooling (following fermentation), and between 3.8 and 4.26 after 33 days of storage. Dave and Shah (1997b) veried the effectiveness of adding ascorbic acid (0, 50, 150 and 250 mg/kg yogurt mix) as an oxygen scavenger in probiotic yogurts processed with different starter cultures. The authors recorded no signicant differences in pH or titratable acidity, but the levels of dissolved

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oxygen and the redox potential varied inversely with the levels of ascorbic acid added, although a gradual increase was observed during storage, approaching that of the control yogurt. The addition of ascorbic acid was only useful during the rst 10 15 days of storage, when the indexes of dissolved oxygen were maintained at low levels. This can be explained by the permeability of the plastic packages to dissolved oxygen. With respect to the probiotic counts, greater viability of L. acidophilus values above 106 CFU/gwas observed during the storage period, independent of the starter culture used, while the Bidobacteria exhibited the opposite behaviour, which could be related to a characteristic of the strain used. Dave and Shah (1997a) analysed the physicochemical characteristics and probiotic potential of yogurts made with four different commercial starter cultures. Differences found in the pH and titratable acidity values were related to the nature of the cultures used. The probiotic cultures showed different behaviours: L. acidophilus showed a decrease of three logarithmic cycles, showing values below 106 CFU/mL after 25 days storage in the yogurts prepared with L. bulgaricus, but the absence of this inoculum resulted in viable counts after 35 days of storage; while B. bidum presented a viable count in the presence or absence of this micro-organism, suggesting an antagonistic effect of its presence on the probiotic cultures. The excess of hydrogen peroxide produced was shown to be toxic to the rst culture, while its proteolytic nature favoured growth of the second. Shin et al. (2000) analysed the Bidobacteria count in two commercial brands of yogurtA and Bin the USA from 1, 2 to 3 weeks after the shelf life of the products. For both brands, counts above 106CFU/mL were recorded up to 2 weeks after the expiry date indicated by the manufacturer, the value falling after the third week. However, the reductions in viable microbial counts were different for the two brands during the study, being 64% for brand A and 88% for brand B. In addition, the yogurt pH values fell from 4.23 to 4.18 (A) and from 4.20 to 4.17 (B), this decrease being signicant. The authors attributed these differences to the initial amounts of inoculum, processing conditions and strains used by the manufacturers. Thus, the selection of acid-tolerant strains is a principle of absolute importance in obtaining satisfactory results for the survival of probiotic cultures in fermented milks. This is especially so in yogurts, since the survival of the probiotic cultures may be inuenced by metabolites formed by the starter cultures, such as lactic acid, hydrogen peroxide and bacteriocins (Mattila-Sandholm et al. 2002). In fact, exposure to dissolved oxygen during processing and storage is highly signicant for
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B. bidum and L. acidophilus. In contrast to aerobic micro-organisms, which completely reduce oxygen to water, the oxygen absorption system is of low activity or even completely absent in probiotic ones. The absence of an electron transport chain results in incomplete reduction of the oxygen to hydrogen peroxide. In addition, they do not possess catalase, an enzyme essential for the decomposition of hydrogen peroxide, consequently causing an accumulation of toxic metabolic derivatives in the cell, such as the superoxide anion (O 2 ), the hydroxyl radical (OH ) and hydrogen peroxide (H2O2), culminating in death of the micro-organism. This suggests that probiotic strains can be affected by the H2O2 produced by other cultures in the reaction medium, demonstrating the need for various studies aimed at searching for ways to minimize such negative effects, such as the addition of antioxidants like ascorbic acid and eliminating peroxide-producing strains (Champagne and Gardner 2005). Bomba et al. (2002) reported that some chemical elements, including magnesium, were capable of increasing the viability of probiotic cultures. Nevertheless, considering that aa pulp contains appreciable amounts of magnesium (Rogez 2000), and comparing the formulations containing aa pulp with the control, the results did not suggest any increase in the viability of B. bidum in the probiotic yogurts containing aa pulp, in relation to the control yogurt. Kailasapathy (2005) observed an increase in the populations of L. acidophilus and B. lactis of one and two logarithmic cycles during the shelf life of probiotic yogurts, by using microencapsulated cultures. Table 3 shows the mean results for the proximate composition of the probiotic yogurts. In general, all the parameters showed variations proportionally related to the amounts of aa pulp in the product formulation. Extreme values were observed for the moisture and total solids contents in yogurts Y3 and Y7, being 13.78 and 15.63%, respectively; the

Table 3 Centesimal composition of probiotic yogurtsd Yogurts Yc Total solids Protein Fat Ash Carbohydrate Energy value
d

Y3
a

Y5
b

Y7
c

11.78 4.00a 2.50a 0.83c 5.83a 61.82a

13.78 4.01a 2.51a 0.79b 6.74b 65.62b

15.08 4.02a 2.49a 0.78b 7.79c 69.66c

15.63d 4.02a 2.51a 0.72a 8.41d 72.14d

Means followed by the same letter and in the samec olumn do not differ signicantly (P > 0.05); Yc = control; Y3 = 3% aa pulp, Y5 = 5% aa pulp,Y7 = 7% aa pulp.

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variation is signicant (P < 0.05). The energy value showed signicant variation, directly proportional to the aa pulp concentration65.62 kCal (Y3) and 72.14 (Y7) kCal, values similar to and sometimes lower than those found in yogurts normally commercialized on the Brazilian market. Micanel et al. (1997) veried the quality of low caloric value yogurts, with and without fat, immediately after production and during refrigerated storage at 4C and 10C for 6 weeks. Great variation in the probiotic culture counts was observed, and zero counts were recorded for Bidobacteria and L. acidophilus in some samples after 2 weeks. The pH values decreased slightly, showing nal values from 3.9 to 4.2. The increase in storage temperature and fat content did not inuence the viable counts of the micro-organisms. The results found in this study were similar to those found in previous experiments, with the exception of the absence of additional resources to maintain the probiotic potential of the products, such as, for example, oxygen-scavenging elements such as ascorbic acid, or the use of microencapsulation of the micro-organisms. However, process optimization can be carried out to improve product identity, comparing the growth rates of the micro-organisms. CONCLUSIONS The probiotic yogurts containing aa pulp were within the parameters required by Brazilian legislation, with the physicochemical characteristics and minimal viable counts necessary for a benecial effect on the human organism to be suitable for commercialization. Thus, stirred yogurt containing aa pulp could be used as a satisfactory carrier of probiotic bacteria. REFERENCES
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