Ice-cream as a probiotic food carrier

Food Research International 42 (2009) 1233–1239

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Food Research International journal homepage: www.elsevier.com/locate/foodres

Review

Ice-cream as a probiotic food carrier Adriano G. Cruz a,*, Adriane E.C. Antunes b, Ana Lúcia O.P. Sousa c, José A.F. Faria a, Susana M.I. Saad c a b c

Departamento de Tecnologia de Alimentos, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, Caixa Postal 6121, 13083-862 Campinas, São Paulo, Brazil Faculdade de Ciências Aplicadas, Universidade Estadual de Campinas, R. Pedro Zacarias, 1300, 13484-350 Limeira, SP, Brazil Departamento de Tecnologia Bioquímico-Farmacêutica, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 580, 05508-000 São Paulo, SP, Brazil

a r t i c l e

i n f o

Article history: Received 1 January 2009 Accepted 30 March 2009

Keywords: Ice-creams Processing Probiotics Lactobacillus Bifidobacterium

a b s t r a c t Ice-creams are food products showing potential for use as probiotic vehicles, with the added advantage of being appreciated by people belonging to all age groups and social levels. However, the development of ice-creams containing probiotic bacteria requires the overcoming of certain technological intrinsic requirements related to their processing stages. The aim of the present paper was to review the technological parameters involved in the production of probiotic ice-creams. Although the application of probiotics in cheeses, and especially in fermented milks, has been widely explored in the literature, ice-cream is a relatively innovative matrix for the application of probiotics, and thus a review about its potential as probiotic food carrier could be very helpful. Ó 2009 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3.

4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Probiotic ice-cream processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technological hurdles for the incorporation of probiotic bacteria into ice-cream . . . . 3.1. Fruit pulp or juice as an ingredient of probiotic ice-creams . . . . . . . . . . . . . . . 3.2. Addition of probiotic cultures to ice-cream. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Stability of the probiotic cultures during ice-cream storage . . . . . . . . . . . . . . . 3.4. Overrun and probiotic microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Influence of storage conditions on the characteristics of probiotic ice-creams. Sensory features of probiotic ice-creams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The development of new food products turns out to be increasingly challenging, as it has to fulfill the consumer’s expectancy for products that are simultaneously relish and healthy. Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer health benefits on the host (Food and Agriculture Organization of United Nations; World Health Organization – FAO/WHO, 2001). Probiotic food is defined as a food product that contains viable probiotic microorganisms in sufficient populations * Corresponding author. Tel.: +55 19 35214016. E-mail address: [email protected] (A.G. Cruz). 0963-9969/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2009.03.020

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incorporated in a suitable matrix (Gibson & Roberfroid, 1995; Saxelin, Korpela, & Mayara-Makinen, 2003). This means that their viability and metabolic activity must be maintained in all the steps of the food processing operation, from their production up to their ingestion by the consumer, and also that they must be able to survive in the gastrointestinal tract (Sanz, 2007). The information available concerning the concentration of probiotic microorganisms needed for biological effects leads to the conclusion that it will vary as a function of the strain and the health effect desired (Champagne, Gardner, & Roy, 2005). Nevertheless, populations of 106–107 CFU/g in the final product are established as therapeutic quantities of probiotic cultures in processed foods (Talwalkar, Miller, Kailasapathy, & Nguyen, 2004),

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reaching 108–109 CFU, provided by a daily consumption of 100 g or 100 mL of food, hence benefiting human health (Jayamanne & Adams, 2006). In Brazil, the present legislation states that the minimum viable quantity of probiotic culture should be between 108 and 109 CFU per daily portion of product and that the probiotic population should be stated on the product label (ANVISA, 2008). Several health benefits are attributed to the ingestion of foods containing probiotic cultures, some of them proven scientifically and others still requiring further studies in humans. Some of the main health benefits related to probiotics are: anti-microbial activity, prevention and treatment of diarrhea, relief of symptoms caused by lactose intolerance, anti-mutagenic and anti-carcinogenic activities, and stimulation of the immune system (Shah, 2007). It is important to emphasize that the effects on health promotion are strain-dependent and cannot be predicted for a determined specie of microorganisms, and that there is no single probiotic strain capable of providing all the benefits mentioned above (Shah, 2007). The dairy industry, in particular, has found probiotic cultures to be a tool for the development of new functional products (Champagne et al., 2005). Yoghurts and fermented milks are the main vehicles for probiotic cultures. However, new products are being introduced in the international market, such as milk-based desserts, powdered milk for newborn infants, ice-creams, butter, mayonnaise, various types of cheese, products in the form of capsules or powders to be dissolved in cold drinks, and fermented foods of vegetable origin (Champagne et al., 2005; Komatsu, Buriti, & Saad, 2008; Saad, 2006). There is thus an increased variety of products available in the market and consumers are getting more used to the probiotic concept, with a consequent increased demand for such products. Consequently, it turned out to be necessary to acquire knowledge of the various operations currently used in the processing of a specific food product, and the level of influence – positive or negative – on the survival of these microorganisms. The aim of the present paper was to review the technological parameters involved in the production of probiotic ice-creams. Although the application of probiotics in cheeses and especially in fermented milks has been widely explored in the literature, ice-cream is a relatively innovative matrix for the application of this microbial group, and thus a review about its potential as probiotic food carrier could be very helpful.

2. Probiotic ice-cream processing Ice-cream is a frozen mixture of a combination of components, such as milk, sweeteners, stabilizers, emulsifiers and flavoring agents (Marshall, Goff, & Hartel, 2003). This category includes several related products, such as plain ice-cream, reduced fat, low fat, nonfat, fruit, and nut ice-creams, puddings, variegated, mousse, sherbet, frozen yoghurt, besides other frozen products (Marshall & Arbuckle, 1996). Freezing involves vigorously agitating to incorporate air, thus conferring the desirable smoothness and softness of the frozen product (Marshall et al., 2003). Ice-cream is highly accepted product by children, adolescents, and adults, as well as by the elderly public. Due to its rather refreshing features, it is more consumed in the summer, but some people have the habit of consuming it throughout the year. In 2003, 5333 million liters of ice-cream were produced in the USA. Therefore, 10% of the total milk production and 16% of the processed milk were used for this purpose. In Canada, 380 million liters of milk were used to produce desserts, of which 79% was used for the production of full-fat or low-fat icecream (Goff & Griffiths, 2006). In 2006, although an overall decrease in the product sales was registered in the American supermarkets, the sale of light and diet ice-creams increased by 15%

from January to June (International Dairy Foods Association, 2007). In Brazil, a 7.6% increase in the product sales between 2005 and 2006 was observed, when the annual per capita consumption varied from 1202 to 1252 L (Hegg, 2007). These figures summarize an incredible low consumption for a country with such large area and where the majority of the states present high temperatures during the whole year. The ice-cream matrix might be a good vehicle for probiotic cultures, due to its composition, which includes milk proteins, fat and lactose, as well as other compounds. Moreover, the fact that it is a frozen product certainly contributes. However, the product should have relatively high pH values – from 5.5 up to 6.5, which leads to an increased survival of the lactic cultures during storage; also, the lower acidity results in increased consumer acceptance, especially by those who prefer mild products. Even though many ice-cream formulations are rich in sugar and fat, ice-cream is generally considered as a nutritive food, since it contains milk, and sometimes fruit, in its formulation. The addition of probiotic cultures to ice-creams, in addition to adding value to the product, provides it with the advantage of being functional. Besides, the regular consumption of probiotic ice-cream containing Bifidobacterium lactis Bb-12 was reported to decrease the viable counts of salivary streptococci and lactobacilli, caries-associated microorganisms in the oral cavity, probably due to an adherence to the oral mucosa and to the dental tissues, as part of a biofilm, and therefore competing with oral pathogens (Çaglar et al., 2008). During probiotic ice-cream production, each process stage ought to be optimized, aiming at an increased survival of the probiotic bacteria, so as to guarantee the product functional properties. This means that the main challenges involved in the production of conventional ice-cream nowadays must be also taken into consideration during the development of probiotic icecream. These challenges include: the contributions for the microstructure and colloidal properties provided by the ingredients and/or components present in the formulation; the knowledge and control of the ice crystallization; the choice of appropriate stabilizers and, finally, the understanding and control of the fat destabilization and the emulsifier functionality (Goff, 2008). In other words, the incorporation of probiotic bacteria into an ice-cream formulation must not affect the product global quality. Therefore, the physical–chemical parameters involved in the quality control of this product, such as the melting rate, and the sensory features, ought to be the same or even better, when compared to a conventional ice-cream. Overall, the general steps of probiotic ice-cream processing are: reception/weighing of the ingredients involved (milk, emulsifiers, stabilizers, milk powder, sugar); mixing; pasteurizing; cooling to a temperature of around 37–40 °C, for the addition of the freeze-dried starter cultures (usually yoghurt cultures) and the probiotic cultures (adjunct cultures); subsequent fermentation to a pH of 4.8–4.7, or the addition of a previously fermented inoculum containing both types of lactic cultures; cooling to 4 °C and keeping the mixture at this temperature (4 °C) for 24 h for the maturation. The processing steps up to this point lead to the production of the ice-cream mix. The mix is subsequently beaten/frozen, in order to produce the final product, which is packaged and maintained frozen throughout transport, commercial distribution and maintenance, and storage for consumption. During all these steps after freezing, the temperature of the frozen product should be strictly controlled. 3. Technological hurdles for the incorporation of probiotic bacteria into ice-cream The incorporation of probiotic bacteria into ice-creams is highly advantageous since, in addition to being a rich food from the nutritional point of view, containing dairy raw material, vitamins and

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Table 1 Technological hurdles faced during the processing of ice-cream containing probiotic cultures. Step

Problem

Solutions

Adequate choice of the formulation ingredients (fruit pulp/juice)

High acidity of the final product may lead to decreased probiotic survival, in addition to frequently be disadvantageous for its sensory performance Some ingredients may have inhibitory activity against probiotic strains

UPreferably, use of fruit pulp/juices with a lower natural acidity

Preparation of the fermented inoculum with the probiotic cultures

Probiotic bacteria show some loss in viability at low pH values

UControl of the pH during the fermentative process UIncreased inoculum concentration USelection of strains tolerant to low pH values

Beating

Oxygen represents a factor of toxicity for probiotic bacteria, which present anaerobic and/or micro-aerophilic metabolism

USelection of oxygen-tolerant strains

Storage

Stress induced by freezing reduces the viability of probiotic bacteria by at least 1 log cycle

UIncreased inoculum concentration

UCheck for inhibitory activity of the food ingredients to be used against the probiotic strains to be employed

UAvoid temperature oscillations during storage of the product

minerals in its composition, it is usually consumed by everybody, being well accepted by the public. In the specific case of probiotic ice-cream, this is a concrete challenge as, most of the times, icecream is not consumed daily by most of the consumers, and this frozen dessert is more frequently consumed during the summer in most of the countries, and it is hence considered as an occasional food. Therefore, the launching of ice-creams supplemented with probiotic cultures should be accompanied by educational campaigns aiming to encourage consumers towards a more constant consumption and showing them the benefits provided by this change of behavior. The fact that consumers are not aware of the benefits caused by consuming foods containing probiotic bacteria was recently reported (Viana, Cruz, Zoellner, Silva, & Batista, 2008). However, the technology used to transform this food into a vehicle for probiotic microorganisms must be well understood and studied simultaneously with the knowledge of the metabolism of the added cultures. This should be done so as to provide sufficient viability of the microorganisms in the product throughout its shelf life, thus resulting in therapeutic activity for the consumer, without any negative influence on the sensory acceptance of the product and on the functional properties. Overall, it is observed that the growth and survival of probiotic bacteria is strain-dependent (Homayouni, Ehsani, Azizi, Razavi, & Yarmand, 2008b). The technological hurdles which take place in the existing processing steps with respect to the development of ice-creams containing probiotic bacteria should be known and optimized (Table 1). As a general rule, the addition of probiotics strains into a food matrix implies the need to assure the viability of the probiotic culture at high levels during the storage period, without altering its sensory characteristics (Stanton et al., 2003). In the particular case of frozen products like ice-cream, this implies overcoming intrinsic hurdles which take place in the processing of ice-creams, such as the beating step, where air is incorporated – known as overrun – and storage under freezing temperatures, which affects survival of the probiotic microorganisms during storage, and also the way in which the bacterial inoculum is added to the product. In addition, great attention should be given to the correct choice of the other ingredients to be used in the product, especially any fruit pulp/juice, which will give the final flavor to the product. 3.1. Fruit pulp or juice as an ingredient of probiotic ice-creams Fruits, in the form of juice or pulp, are widely used as flavorings in ice-creams, being commercially available in the form of pasteurized juices or frozen pulps, representing the most well known source of flavor for this product (Papademas & Bintsis, 2000). Fruits

or their derivatives with a pronounced acidic character should be avoided in ice-creams containing probiotic cultures, since this attribute could influence their sensory acceptance and also decreased the viability of the cultures (Fávaro-Trindade, Balieiro, Dias, Sanino, & Boschini, 2007; Fávaro-Trindade, Bernadi, Boldini, & Balieiro, 2006) as its addition decrease pH values. Acid pH tolerance in probiotic bacteria is strain dependent, and Bifidobacteria strains are more sensitive than Lactobacillus strains (Godward et al., 2000). Even though this specific mechanism is still not well established, the influence of a particular enzyme, H+-ATPase, was reported (Lankaputhra, Shah, & Britz, 1996; Takahashi, Xiao, Miyaji, & Iwatsuki, 2007). The development of strategies to enhance their survival in an adverse environment is essential. Firstly, the simplest measure is to select strains resistant to low pH values, which means a continuous dialogue with the culture supplier. However, it is important to take into consideration the fact that, although the numbers of researches covering the development of probiotic dairy foods supplemented with probiotic bacteria are increasing, there are still few strains with proven health benefits being marketed by each supplier. This finding limits the options for commercially available probiotic strains, and the easier step is to develop mechanisms of low pH tolerance in a laboratory scale with the probiotic strains available. It is also appropriate to remember that in vitro evaluation of probiotic bacteria acid tolerance may not reproduce a good performance of the in vivo behavior, since poor correlation between them was reported (Morelli, 2007). However, the application of sub-lethal stress conditions, as prolonged exposures of probiotic bacteria to very low pH values (2.0– 3.0) for a determined time were reported to present good results, leading to the generation of a stable and highly acid resistant strain (Collado & Sanz, 2005; Maus & Ingham, 2003). This treatment also introduces phenotypic changes which improve biological properties, as higher fermentative ability and enzymatic activities, although an individual evaluation for each strain is needed (Sanz, 2007). A recent study suggests the addition of chemical compounds – carbonate, and citrate salts – at acceptable levels before or during the incubation, in order to eliminate acidic stress through chemical reactions. The resultants products would be metabolized in a subsequent step, with a consequent provision of a favorable condition for probiotic bacteria growth (Zhao & Li, 2008). This finding deserves more investigation. In a practical and economic vision, fruits and/or flavorings additives with mild features (low acidity values) ought to be used, and should be acquired from processing plants which adopt an assurance quality system (HACCP) to avoid microbial and chemical con-

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tamination, However, it is important to point out that certain fruits, like passion fruit, may inhibit probiotic microorganisms, resulting in loss of viability in the product, due the presence of intrinsic factors linked to their composition (Buriti, Komatsu, & Saad, 2007). 3.2. Addition of probiotic cultures to ice-cream Independently of the moment of their addition during icecream processing, probiotic bacteria play the role of adjunct cultures, which means that starter cultures – yogurt cultures, in most of the cases – will frequently be present. In case fermentation of ice-cream is desirable (frozen yoghurt ice-cream), this co-culture (starter plus probiotic) turns out to be more fitted to an industrial vision, as it decreases fermentation time. Cultures may be added to ice-creams in two ways, considering that they are of the DVS (Direct Vat Set) type, for the direct addition to the product during its manufacture: either adding them directly to the pasteurized mix or using the milk as a substrate for fermentation, producing, in the latter case, frozen yoghurt icecream. In the second case, the pH must be closely controlled during the fermentative process from the moment of obtaining the inoculum, and also the temperature during storage, so that any undesirable reactions do not occur during this period. In addition to the increased sensibility of probiotic microorganisms to low pH values (4.0–4.5), negative effects on sensory acceptance of the product may arise. These changes might be undesirable, since ice-creams are not traditionally characterized as high acidic food products. One alternative is to stop the fermentation at pH values ranging from 5.0 to 5.5 (Vardar & Öksüz, 2007). Probiotic ice-creams using the fermentation as a regular step for their production are naturally more acid, due to the production of lactic acid during the fermentative process (Basßyg˘it, Kuleasßan, & Karahan, 2006). Many lactic acid bacteria show a stationary phase with little growth, but also suffer a rapid loss of cell viability if stored at temperatures below 20 °C (Naidu, Bidlack, & Clemens, 1999). In general, probiotics are added to yogurts and fermented milk that are stored at temperatures of 5–10 °C. Such products may be more susceptible to loss of culture viability than icecreams, because of the lower storage temperature of the latter, which may result in cell injury due to freezing. 3.3. Stability of the probiotic cultures during ice-cream storage The viability of probiotics in a delivery system (food matrix) depends, among many other factors, on the strain selected, the interactions between the microbial species present, the production of hydrogen peroxide due to bacterial metabolism and the final acidity of the product (Vasiljevic & Shah, 2008). Studies carried out in various parts of the world have shown that probiotic cultures were capable of maintaining their stability in frozen food products, having minimum loss of viability. Akalin and Erisßir (2008) investigated the effect of the addition of prebiotic ingredients (inulin and oligofructose) on the quality parameters, as well as on the probiotic cultures survival (Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12). Significant differences in viscosity of the samples were observed, besides higher overrun values for the ice-cream mix with inulin and also less changes in melting properties. The prebiotic ice-creams also showed to be firmer during storage. The authors also reported an increased probiotic survival during storage, mainly for ice-cream containing oligofructose as functional ingredient. They concluded that the higher overrun rate values observed for ice-cream containing inulin might be a hurdle for probiotic survival, mainly B. animalis Bb-12. Basßyg˘it et al. (2006) investigated the survival rates of human probiotic bacteria (L. acidophilus, Lactobacillus agilis and Lactobacil-

lus rhamnosus) in the production of two different ice-cream formulations, containing sucrose and aspartame, during 6 months storage. The samples were stored at 20 °C and the viability rate measured monthly. The probiotic cultures were also submitted to tests of resistance to bile salts, antibiotics and to hydrochloric acid, and were shown to be highly resistant to all these conditions. According to the authors, the results showed that none of the ice-cream formulations tested, prepared with the addition of different types of sweeteners, caused undesirable effects on the survival of the probiotic bacteria, and the concentrations remained constant during storage. Alamprese, Foschino, Rossi, Pompei, and Savani (2002) carried out a study with ice-cream supplemented with Lactobacillus johnsonii La1, simulating industrial and retail conditions. Four formulations containing different amounts of fat (5% and 10% w/v) and sugar (15% and 22% w/v) were prepared. For each formulation, ice-creams with and without La1 were prepared, and stored at temperatures of 16 °C and 28 °C. The fresh cells of La1 and those sampled after freezing were submitted to preliminary tests of resistance to bile, antibiotics, SDS and acidity. The microorganisms were shown to be resistant to the majority of such stress factors, leading the authors to exclude the possibility of finding cells sublethally injured during freezing. The authors observed high survival rates of the probiotics during storage of the ice-creams for 8 months, with no decrease in the population inoculated initially (7 log CFU g 1). Also, the addition of the probiotic microorganism did not cause any modification in the overrun or any alteration in firmness of the finished product. Alamprese, Foschino, Rossi, Pompei, and Corti (2005) carried out another experiment under the same conditions tested before, but using L. rhamnosus GC. Similar results were obtained with this microorganism, which presented populations of 108 CFU g 1 after one year of storage, with no influence on the physical properties of the ice-cream. Hekmat and McMahon (1992) determined the survival of L. acidophilus and Bifidobacterum bifidum in ice-cream to be used as a probiotic food product. The probiotic ice-cream was prepared after the fermentation of the ice-cream base mix by both cultures, and then submitted to frozen storage. The survival of the cultures and the activity of ß-galactosidase were monitored for a period of 17 weeks in the product during frozen storage at 29 °C. Bacterial counts were made immediately after freezing of the fermented mix, and counts obtained were, respectively, 1.5  108 and 2.5  108 CFU mL 1. After seventeen weeks of storage, these populations had decreased to 4  106 and to 1  107 CFU mL 1, respectively. During the same period, the activity of ß-galactosidase decreased from 1800 to 1300 units mL 1. Sometimes, more additional efforts might be needed, which include the use of the microencapsulation of cultures and the supplementation with prebiotics (Akalin & Erisßir, 2008; Akyn, Akyn, & Kirmaci, 2007; Godward & Kailasapathy, 2003; Kailapasathy & Sultana, 2003). Nevertheless, these formulation improvement increases the products’ cost. Also, the products ought to be submitted to a sensory evaluation, in order to check the impact of microcapsules and their sensory and textural features, as sometimes they might lead to rejection by the consumers (Homayouni, Azizi, Ehsani, Yarmand, & Razavi, 2008a). 3.4. Overrun and probiotic microorganisms Overrun is defined as the ice-cream percentage of volume increase in relation to the liquid mix used to make it, and is related to the amount of air incorporated during the manufacturing process. This feature defines the structure of the final product, since the presence of air gives the ice-cream an agreeable light texture and influences the physical properties of melting and hardness of the final

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product (Sofjan & Hartel, 2004). In addition, it results in the partial coalescence and destabilization of the fat present in the mixture, with the formation of an internal lipid structure, capable of imprisoning air bubbles (Bolliger, Goff, & Tharp, 2000). The Brazilian legislation stipulates the maximum degree of overrun that ice-creams can show as 450 g L 1, independently of the type of ice-cream, and refers to this parameter as apparent density (Brasil, 1999). The melting time determined in the melting test is related to the product structure stability after the overrun, which, for its part, is influenced by the type of emulsifier used in the process. It thus indicates the extent of stabilization and partial coalescence occurring during the product manufacture (Correia, Pedrini, & Magalhães, 2007). Oxygen tolerance in probiotic bacteria is also strain-dependent (Kawasaki, Mimura, Satoh, Takeda, & Nimura, 2006), and bifidobacteria strains are more susceptible than L. acidophilus (Vasiljevic & Shah, 2008). Exposure to oxygen during manufacture and storage of dairy products is highly significant for probiotic bacteria. Most of the Lactobacillus and Bifidobacterium spp. are gut-derived organisms and they are microaerophilic and anaerobic, respectively, and therefore unable to synthesize ATP by respiratory means, depending strictly on a fermentative mode of metabolism (Talwalkar & Kailasapathy, 2004b). These microorganisms oxygen-scavenging system is reduced or completely absent, which results in the incomplete reduction of oxygen to hydrogen peroxide. Consequently, accumulation of toxic oxygen metabolites occurs – O2 , OH and H2O2 – in the cell, eventually leading to its death (Champagne et al., 2005; Talwalkar & Kailasapathy, 2004a; Vasiljevic & Shah, 2008). High levels of intracellular H2O2 block fructose6-phosphofructoketolase, a key enzyme in the sugar metabolism of bifidobacteria (Shah, 1997). A correlation between the levels of two enzymes – NAD-oxidase and NAD-peroxidase – and the oxygen susceptibility of bifidobacteria was reported; the former enzyme gives rise to H2O2, prompting the latter to scavenge this compound and prevent cell death (Roy, 2005). The selection of oxygen-resistant strains is essential to succeed in maintaining the viability of the culture in probiotic ice-creams, since the overrun operation cannot be eliminated from the processing procedure. Salen, Fathi, and Awad (2005) reported that the differences in the overrun step of probiotic ice-creams were related to the acidifying capacity of the probiotic cultures, which affects the nature of the proteins, causing denaturation, and also affects the product freezing point. A potential alternative to be considered when dealing with this inherent hurdle is to microencapsulate the probiotic bacteria, so as to overcome their oxygen sensitivity and extended the product shelf-life. Microencapsulation is a process in which the cells are retained within an encapsulating membrane to reduce cell injury or cell loss (Krasaekoopt, Bhandari, & Deeth, 2003). Homayouni et al. (2008a) investigated the effect of microencapsulation on the survival of two probiotic strains added in a synbiotic ice-cream. Two types of synbiotic ice-cream containing 1% of resistant starch with free and encapsulated Lactobacillus casei (Lc-01) and B. lactis (Bb-12) were manufactured. Their survival was monitored during the product storage for 180 days at 20 °C. The probiotic viability was 5.1  109 and 4.1  109 CFU/ mL at day one, and 4.2  106 and 1.1  107 CFU/mL after 180 days of storage at 20 °C, respectively, to the former and to the latter in the free state in the ice-cream mixture. The microencapsulation at probiotic bacteria in calcium alginate beads increased their survival rate about 30% during the same period of storage at the same temperature. According to the authors, these findings reinforce the encapsulation as a useful alternative to increase the survival rate of probiotic bacteria in ice-cream. No significant effect on the sensory properties of non-fermented ice-cream in which the resistant starch was added as a prebiotic compound was observed.

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3.5. Influence of storage conditions on the characteristics of probiotic ice-creams The development and viability of probiotic microorganisms, as for other microorganisms, are influenced by the environmental temperature around them. They can be lethally injured by damage to their cell walls or rupture of their membranes caused by the ice formed in the external medium, or the cells may be ruptured by ice formed inside the cell and by concentration of solutes in the extracellular medium (Gill, 2006). The velocity of dehydratation of microbial cells depends on the permeability of the cell membrane and on the surface area in relation to its volume, which depends on the cell shape and size. However, the size of the ice crystals decreases with increasing freezing rates, and larger intracellular ice crystals cause greater damage to the cells. (Gill, 2006). Ice-creams containing probiotic cultures are frozen foods. Thus, rapid freezing of the mix obtained after inoculating with the microorganisms, associated to a rigorous temperature control throughout storage of the product, contributes with the maintenance of the populations of these microorganisms in the recommended doses, capable of providing therapeutic and/or preventive activity. Preliminary tests should be carried out before and immediately after freezing of the product, so as to have an idea of the inoculum level of probiotic cultures to be added, and also of their survival rate, consequently arriving at an optimized level of probiotic cultures to be added to the product during its manufacture. Some studies reported that the transformation of mix into ice-cream influenced on the viability of the probiotic cultures (Haynes & Playne, 2002; Magariños, Selaive, Costa, Flores, & Pizarro, 2007). In summary, the decline in bacterial populations during freezing is caused by injury as a result of the process to which they were submitted, causing their death. In addition, mechanical stress caused by agitation and the incorporation of air might result in a smaller population of viable cells (Akin, 2005).

4. Sensory features of probiotic ice-creams Davidson, Duncan, Hackney, Eigel, and Boling (2000) reported that, in general, ice-creams produced with probiotic cultures present a less intense aroma and yoghurt flavor than the product prepared with traditional cultures. Thus, the production of probiotic ice-creams with high sensory acceptance is a difficult task, requiring technical knowledge by the processors, as sometimes functional ice-cream presents poor sensory performance, when compared to a conventional ice-cream (Aryana & Summers, 2006; Fávaro-Trindade et al., 2006; Hekmat & McMahon, 1992). However, it is possible to develop probiotic ice-cream with good sensorial quality (Christianesen, Edelten, Kristiansen, & Nielsen, 1996; Vardar & Öksüz, 2007). Probiotic cultures do not modify the sensory properties of the products to which they are added intensely (Champagne et al., 2005; Saxelin et al., 1999). L. acidophilus, as well as the microorganisms used as starter cultures in yoghurt manufacture – Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus – are homofermentative and produce lactic acid from lactose metabolism, without gas production. Bifidobacteria produce acetic and lactic acids in the proportion of 3:2. The flavor and aroma of acetic acid provide extremely undesirable off-flavors to dairy products and might require the use of flavoring agents, so as to minimize or mask this defect, known as ‘‘probiotic flavor”. An innovative and effective alternative to overcome this possible undesired consequences caused by the presence of these cultures is the addition of microencapsulated cells of probiotic cultures to food products such as yoghurts (Arai, Sakaki, &

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Sugimoto, 1996). Nevertheless, it is important to point out that products like non-fermented probiotic ice-cream do not normally present problems resulting from the microbial metabolism, since they are stored at very low temperatures (< 18 °C), minimizing the probiotic microorganisms biochemical reactions. Consumers are generally used to products fermented with yoghurt cultures, in such a way that the aroma and flavor of these products are familiar to them. Considering that the probiotic cultures use different metabolic pathways than the classic cultures employed to ferment milk, the consumers may find the flavor of the products prepared with the addition of these cultures rather unusual. Vardar and Öksüz (2007) reported that artisan strawberry ice-cream supplemented with L. acidophilus showed good sensory acceptance and that the incubation of the mix at pH 5.6 lead to better results in terms of flavor and taste. The authors suggested that adding highly acidic fruit to ice-cream might be useful in masking the sour taste resulting from the metabolism of probiotic cultures. However, the results should have not generalized, as opposite findings were reported by Fávaro-Trindade et al. (2006) for probiotic ice-cream with acerola fruit. Therefore, preliminary tests with different inoculation levels and different probiotic microorganisms are recommended in order to optimize the sensory properties of the product, mainly with respect to the product acidity and sensory performance. 5. Perspectives Ice-creams are food products showing great potential for use as vehicles for probiotic cultures, with the advantage of being foods consumed by all age groups. However, several factors in their processing stages ought to be optimized, in order to maintain the microorganisms in viable doses capable of providing therapeutic activity to consumers. In order to achieve this goal, several factors should be strictly controlled, including: the appropriate selection of cultures to be used, the inoculum concentration, the appropriate processing stage for the cultures to be added, and the strict control of the processing procedures and of the transport and storage temperatures. In conclusion, probiotic cultures do not modify the sensory features of ice-creams and frozen desserts strongly and normally present good viability during the product storage period. Even though several studies have shown adequate viability of the probiotic cultures during storage of ice-creams, more clinical studies on the consumption of probiotic ice-creams are recommended. Also, it is important to confirm if, after long storage periods, the probiotic cultures are still able to confer the same health benefits already observed in other foods with shorter shelf-lives and higher storage temperatures, such as yoghurts and fermented milks. Moreover, the launching of ice-creams supplemented with probiotic cultures should be accompanied by educational campaigns aiming to encourage consumers towards a more constant consumption and showing them the benefits provided by this change of behavior. Acknowledgements The authors thank to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support and scholarships. References Akalin, A. S., & Erisßir, D. (2008). Effects of inulin and oligofructose on the rheological characteristics and probiotic culture survival in low-fat probiotic ice-cream. Journal of Food Science, 73(4), 184–188.

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