Viability of probiotic micro-organism Lactobacillus acidophilus in dairy chocolate dessert and its action against foodborne pathogens

Viabilidade de microrganismo probiótico Lactobacillus acidophilus em sobremesaLáctea de chocolate e ação sobre patógenos alimentares

Luciana Justo Beserra Rosa Luciana Maria Ramires Esper Julia do Prado Lima Guimarães Cabral Robson Maia Franco Marco Antônio Sloboda Cortez About the authors

ABSTRACT:

The ability to produce antimicrobial factors is considered an important feature of probiotic microorganisms. Bacteriocins, hydrogen peroxide, acetic acid and lactic acid are examples of these substances. The present research aimed to develop probiotic dairy desserts (DD) with Lactobacillus acidophilus and evaluate the viability of this strain, as well as its action on food pathogens. Treatments with and without interactions between L. acidophilus and pathogenic Gram-negative bacteria (Salmonella sp. and Escherichia coli O157:H7) and Gram positive (Bacillus cereus and Staphylococcus aureus) were produced. The products were stored at a temperature of 8°C and analyzed at the times 24, 48, 72 hours, 7 days and 28 days (at 28 days, only T1 was analyzed because the other products were deteriorated). In an analysis of the potential for development of new products, the dairy dessert with L. acidophilus was considered a probiotic product. Assessment of the counts of pathogens in dairy desserts with or without L. acidophilus showed different behaviors of these products in response to pathogens, which could be justified by a possible action of bacteriocins or microbial competition, but there has been no overall reduction or reduction up to a safe level. It is concluded that the probiotic products developed reduced significant food pathogens, but not up to safe levels. Thus, we emphasize the importance of the use of quality tools in the development and monitoring of dairy desserts.

Key words:
probiotics; dairy dessert; Lactobacillus acidophilus.

RESUMO:

A capacidade de produzir fatores antimicrobianos é considerada uma importante característica dos microrganismos probióticos. Bacteriocinas, peróxido de hidrogênio, ácido acético e ácido lático, são exemplos destas substâncias. Com o presente trabalho, objetivou-se desenvolver sobremesas lácteas (SL) probióticas, acrescidas de Lactobacillus acidophilus e avaliar a viabilidade desta cepa, além da ação frente a patógenos alimentares. Foram produzidos tratamentos com e sem interações entre o L. acidophilus e bactérias patogênicas Gram negativas (Salmonella sp e Escherichia coli O157:H7) e Gram positivas (Bacillus cereus e Staphylococcus aureus). Os produtos foram armazenados em temperatura de 8ºC e analisados nos tempos 24, 48, 72 horas, 7 dias e 28 dias (apenas T1 por deterioração dos demais produtos neste tempo). A sobremesa láctea com L. acidophilus foi considerada produto probiótico, verificando o potencial de desenvolvimento de um novo produto. Analisando as contagens dos patógenos nas sobremesas lácteas com e sem adição de L. acidophilus, observaram-se diferentes comportamentos diante dos patógenos, o que poderia ser justificado por uma possível ação de bacteriocinas ou competição microbiana, porém não houve uma redução total ou até um nível considerado seguro. Conclui-se que os produtos probióticos desenvolvidos reduziram os patógenos alimentares de importância, porém não a níveis considerados seguros. Dessa forma, ressalta-se a importância de ferramentas de qualidade no desenvolvimento e monitoramento de sobremesas lácteas.

Palavras-chave:
probióticos; sobremesa láctea; Lactobacillus acidophilus.

INTRODUCTION:

According to FAO's/WHO's (2002FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS, WORLD HEALTH ORGANIZATION. Evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Córdoba, 2001. 34p. ) reports, probiotics are defined as living microorganisms that, when administered in adequate amounts, confer benefits to host health, through a positive action on the intestinal microbiota. Additionally, the probiotics are associated to reduction of lactose intolerance, cancer, allergies, hepatic disease, Helicobacter pylori infections, urinary tract infections, hyperlipidemia and increased immunity (EJTAHED et al., 2011EJTAHED, H.S. et al. Effect of probiotic yogurt containing Lactobacillus acidophilus and Bifidobacterium lactis on lipid profile in individuals with type 2 diabetes mellitus. Journal of Dairy Science, v.94, n.7, p.3288-3294, 2011. Available from: <Available from: <http://www.ncbi.nlm.nih.gov/pubmed/21700013 >. Accessed: Apr. 01, 2015. doi: 10.3168/jds.2010-4128. 2011.
http://www.ncbi.nlm.nih.gov/pubmed/21700...
; LOLLO et al., 2013LOLLO, P.C.B. et al. Probiotic yogurt offers higher immune-protection than probiotic whey beverage. Food Research International, v.54, n.1, p.118-124, 2013. Available from: <Available from: <http://www.sciencedirect.com/science/article/pii/S096399691300330X >. Accessed: Apr. 06, 2015. doi: 10.1016/j.foodres.2013.06.003.
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; NABAVI et al., 2014NABAVI, S. et al. Effects of probiotic yogurt consumption on metabolic factors in individuals with nonalcoholic fatty liver disease. Journal Dairy Sciience, v.97, p.7386-7393, 2014. ).

Moreover, probiotics have possible mechanisms of action that produce a response to pathogens. This is justified by the competition for binding sites, forming a physical barrier to pathogens, competition with pathogens for nutrients, inactivation of toxins and receptors through the stimulation of phagocytosis and of specific and nonspecific immune responses against pathogens (WENDLING & WESCHENFELDER, 2013WENDLING, L.K.; WESCHENFELDER, S. Probiotics and fermented dairy foods - a review. Revista do Instituto de Laticínios Cândido Tostes, v.68, p.49-57, 2013. Available from: <Available from: <http://www.revistadoilct.com.br/rilct/article/viewFile/50/56 >. Accessed: Dec. 15, 2014. doi: 10.5935/2238-6416.20130048.
http://www.revistadoilct.com.br/rilct/ar...
). The capacity to produce antimicrobial factors is another possible characteristic of probiotic microorganisms. Bacteriocins, hydrogen peroxide, acetic acid and lactic acid are examples of these substances (RASTALL et al., 2005RASTALL, R.A. et al. Modulation of the microbial ecology of the human colon by probiotics, prebiotics and synbiotics to enhance human health: an overview of enabling science and potential applications. FEMS Microbiology Ecology, v.52, n.2, p.145-152, 2005. Available from: <Available from: <http://onlinelibrary.wiley.com/doi/10.1016/j.femsec.2005.01.003/full >. Accessed: Dec. 12, 2014. doi: 10.1016/j.femsec.2005.01.003.
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). PUUPPONEN-PIMIÄ et al. (2002PUUPPONEN-PIMIÄ, R. et al. Development of functional ingredients for gut health. Trends in Food Science and Technology, v.13, n.1, p.3-11, 2002. Available from: <Available from: <http://www.sciencedirect.com/science/article/pii/S0924224402000201?np=y >. Accessed: Dec. 15, 2014. doi: 10.1016/S0924-2244(02)00020-1.
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) suggest that the use of probiotics in the diet reduces the proliferation of potentially harmful bacteria, enhancing natural defense mechanisms of the host.

The main probiotic foods are dairy products, especially yogurts and fermented milks (EJTAHED et al., 2011EJTAHED, H.S. et al. Effect of probiotic yogurt containing Lactobacillus acidophilus and Bifidobacterium lactis on lipid profile in individuals with type 2 diabetes mellitus. Journal of Dairy Science, v.94, n.7, p.3288-3294, 2011. Available from: <Available from: <http://www.ncbi.nlm.nih.gov/pubmed/21700013 >. Accessed: Apr. 01, 2015. doi: 10.3168/jds.2010-4128. 2011.
http://www.ncbi.nlm.nih.gov/pubmed/21700...
; LOLLO et al., 2013LOLLO, P.C.B. et al. Probiotic yogurt offers higher immune-protection than probiotic whey beverage. Food Research International, v.54, n.1, p.118-124, 2013. Available from: <Available from: <http://www.sciencedirect.com/science/article/pii/S096399691300330X >. Accessed: Apr. 06, 2015. doi: 10.1016/j.foodres.2013.06.003.
http://www.sciencedirect.com/science/art...
; KANMANI et al., 2013KANMANI, P. et al. Probiotics and its functionally valuable products: a review. Critical Reviews in Food Science and Nutrition, v.53, n.6, p.641-658, 2013. Available from: <Available from: <http://www.ncbi.nlm.nih.gov/pubmed/23627505 >. Accessed: Mar. 10, 2015. doi: 0.1080/10408398.2011. 553752.
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; NABAVI et al., 2014NABAVI, S. et al. Effects of probiotic yogurt consumption on metabolic factors in individuals with nonalcoholic fatty liver disease. Journal Dairy Sciience, v.97, p.7386-7393, 2014. ; MAGANHA et al., 2014MAGANHA, L.C. et al. Viability of probiotic bacteria in fermented skim milk produced with different levels of milk powder and sugar. International Journal of Dairy Technology, v.67, n.1, p.89-94, 2014. Available from: <Available from: <http://onlinelibrary.wiley.com/doi/10.1111/1471-0307.12087 >. Accessed: Mar. 15, 2015. doi: 10.1111/1471-0307.12087.
http://onlinelibrary.wiley.com/doi/10.11...
). CRUZ et al., (2012CRUZ, A.G. et al. Probiotic yogurts manufactured with increased glucose oxidase levels: postacidification, proteolytic patterns, survival of probiotic microorganisms, production of organic acid and aroma compounds. Journal of Dairy Science, v.95, n.5, p.2261-2269, 2012. Available from: <Available from: <http://www.ncbi.nlm.nih.gov/pubmed/22541455 >. Acesso em: Apr. 10, 2015. doi:10.3168/jds.2011-4582.
http://www.ncbi.nlm.nih.gov/pubmed/22541...
) developed yogurts with glucose oxidase enzyme, as a potential alternative to increase the survival of probiotic bacteria in fermented milks, minimizing oxidative stress in probiotic yogurts. Cheeses are less frequently used as sources of probiotic products, e.g. Minas Frescal, Feta and curd cheeses, despite their advantages such as higher pH and fat, as well as firmness, providing a favorable environment for the survival of probiotic bacteria in the gastrointestinal tract (GOMES et al., 2011GOMES, A.A. et al. Effect of the inoculation level of Lactobacillus acidophilus in probiotic cheese on the physicochemical features and sensory performance compared with commercial cheeses. Journal of Dairy Science, v.94, n.10, p.4777-4786, 2011. Available from: <Available from: <http://www.journalofdairyscience.org/article/S0022-0302(11)00499-1/pdf >. Accessed: Mar. 20, 2015. doi: 10.3168/jds.2011-4175.
http://www.journalofdairyscience.org/art...
; KARIMI et al., 2012KARIMI, R. et al. Incorporation of Lactobacillus casei in Iranian ultrafiltered Feta cheese made by partial replacement of NaCl with KCl. Journal of Dairy Science, v.95, n.8, p.4209-4222, 2012. Available from: <Available from: <http://www.journalofdairyscience.org/article/S0022-0302(12)00409-2/pdf >. Accessed: Mar. 22, 2015. doi: 10.3168/jds.2011-4872
http://www.journalofdairyscience.org/art...
). In studies on alternative products such as dairy desserts, many researchers have stressed that they are a vehicle for the incorporation of probiotic strains and other functional ingredients (ARAGON-ALEGRO et al., 2007ARAGON-ALEGRO, L.C. et al. Potentially probiotic and symbiotic chocolate mousse. LWT-Food Science and Technology, São Paulo, v.40, n.4, p.669-675, 2007. Available from: <Available from: <http://www.sciencedirect.com/science/article/pii/S002364380600051X >. Accessed: Dec. 10, 2014. doi: 10.1016/j.lwt.2006.02.020.
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; CARDARELLI et al., 2008CARDARELLI, H.R. et al. Effect of inulin and Lactobacillus paracasei on sensory and instrumental texture properties of functional chocolate mousse. Journal of the Science of Food and Agriculture, v.88, p.1318-1324, 2008. Available from: <Available from: <http://onlinelibrary.wiley.com/doi/10.1002/jsfa.3208 >. Accessed: Apr.15, 2015. doi: 10.1002/jsfa.3208.
http://onlinelibrary.wiley.com/doi/10.10...
; MAGARIÑOS et al., 2008, FERNANDES et al., 2013FERNANDES, M.S. et al. On the behavior of Listeria innocua and Lactobacillus acidophilus co-inoculated in a dairy dessert and the potential impacts on food safety and product's functionality. Food Control, v.34, p.331-335, 2013. Available from: <Available from: <http://www.sciencedirect.com/science/article/pii/S0956713513002247 >. Accessed: Dec. 10, 2014. doi: 10.1016/j.foodcont.2013.04.040.
http://www.sciencedirect.com/science/art...
). Consumer preferences regarding texture and flavor are essential in the elaboration of a dairy product and, consequently, impact the acceptability of a given product (TARREGA & COSTELL, 2006TÁRREGA, A.; COSTELL, E. Effect of inulin addition on rheological and sensory properties of fat-free starch-based dairy desserts. International Dairy Journal, v.16, n.9, p.1104-1112, 2006. Available from: <Available from: <http://www.sciencedirect.com/science/article/pii/S0958694605001810 >. Accessed: Jan. 24, 2013. doi: 10.1016/j.idairyj.2005.09.002.
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). The technologies used in the dairy industry and the innovative ingredients have stimulated the production of desserts with new sensory characteristics and higher nutritional value (NIKAEDO, 2004NIKAEDO, P.H.L. et al. Caracterização tecnológica de sobremesas lácteas achocolatadas cremosas elaboradas com concentrado protéico de soro e misturas de gomas carragena e guar. Revista Brasileira de Ciências Farmacêuticas, v.40, n.3, p.397-404, 2004. Available from: <Available from: <http://www.revistas.usp.br/rbcf/article/view/43991 >. Accessed: Dec. 10, 2014. doi: 10.1590/S1516-93322004000300016.
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). The influence of probiotic microorganisms on the behavior of foodborne pathogens has been little studied, given that each pathogen may act differently depending on the type of food. (MADUREIRA et al., 2011MADUREIRA, A.R. et al. Incorporation of probiotic bacteria in whey cheese: decreasing the risk of microbial contamination. Journal of Food Protection, v.74, n.4, p.1194-1199, 2011. Available from: <Available from: <http://www.ncbi.nlm.nih.gov/pubmed/21740724 >. Accessed: Apr. 16, 2015. doi: 10.4315/0362-028X.JFP-10-217.
http://www.ncbi.nlm.nih.gov/pubmed/21740...
). Besides, more in-depth studies are needed on the viability of probiotic microorganisms (CARDARELLI et al., 2008CARDARELLI, H.R. et al. Effect of inulin and Lactobacillus paracasei on sensory and instrumental texture properties of functional chocolate mousse. Journal of the Science of Food and Agriculture, v.88, p.1318-1324, 2008. Available from: <Available from: <http://onlinelibrary.wiley.com/doi/10.1002/jsfa.3208 >. Accessed: Apr.15, 2015. doi: 10.1002/jsfa.3208.
http://onlinelibrary.wiley.com/doi/10.10...
). Therefore, the present study aimed to develop probiotic dairy desserts with addition of Lactobacillus acidophilus, and assess the viability of this strain at the times 24, 48, 72 hours, 7 days and 28 days after preparation, as well as the competition in response to important foodborne pathogens.

MATERIAL AND METHODS:

The lyophilized probiotic culture used in the preparation of the dairy dessert was Lactobacillus acidophilus (LA-05, direct vat setDVS, CHR HANSEN, Juiz de Fora, Minas Gerais, Brasil). The pre-activation was performed according to a methodology described by BURITI et al. (2007BUTIRI, F.C.A. et al. Activity of passion fruit (Passiflora edulis) and guava (Psidium guajava) pupls on Lactobacillus acidplhilus in refrigerated mousses. Brazilian Journal of Microbiology, v.38, p.350-317, 2007. Available from: <Available from: <http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822007000200025 >. Accessed: Apr. 02, 2015. doi: 10.1590/S1517-83822007000200025.
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) with adaptations. It was done as follows: 0.02g of LA-05 DVS probiotic culture were added to 40mL of reconstituted skimmed milk (m/v). Then the culture was incubated at 37ºC for 150 minutes.

The following ingredients were used in the preparation of the probiotic dairy dessert: 81.5% UHT whole milk, 8.5% sucrose, 4% cocoa powder, 4% Global Food(r) formulation and 2% skimmed milk powder. After weighing, the dry ingredients were mixed and UHT milk was added. The product was homogenized during 30 minutes in mix (Pérola Plus Britânia) and heated to 80ºC for 3 minutes. Subsequently, the temperature was reduced to 40ºC to incorporate the milk enriched with the probiotic strain L. acidophilus (SILVA et al., 2012SILVA, A.S. et al. Viabilidade de Lactobacillus casei em flan de chocolate e sua sobrevivência em condições gastrintestinais simuladas. Semina: Ciências Agrárias, v.33, n.2, p.3163-3170, 2012. Available from: <Available from: <http://www.uel.br/revistas/uel/index.php/semagrarias/article/view/13672 >. Accessed: Dec. 21, 2014. doi: 10.5433/1679-0359.2012v33Supl2p3163.
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with modifications).

The Gram positive and Gram negative pathogens used were Escherichia coli O 157:H7 (CDC EDL-933), Salmonella sp. (ATCC 00150), Staphylococcus aureus (ATCC 00358) and Bacillus cereus (ATCC 14579). The identification of these microorganisms was confirmed by biochemical, morphological tests and growing on selective and differential media. The microorganisms were maintained at -80°C, in Eppendorf tube containing Brain Heart Infusion ( BHI, Difco) broth with addition of glycerol (80:20). In each experiment, the cultures were activated and subcultured twice in succession in 10mL of TSB broth and incubated at 35±1°C for 18 hours. Decimal dilutions were performed so that the final concentration of pathogens in each dessert was 6log C/g. The concentrations were confirmed in all the experiments by plating in specific media The study was composed of nine treatments: Treatment (T)1: Dairy dessert (DD) with addition of L. acidophilus, T2: DD with E. coli O157:H7, T3: DD with Salmonella sp., T4 DD with S. aureus, T5: DD with B. cereus, T6: DD with L. acidophilus and E. coli O157:H7, T7: DD with L. acidophilus and Salmonella sp., T8: DD with L. acidophilus and S. aureus, T9: DD with L. acidophilus and B. cereus. The products were transferred to plastic containers with lids, then stored at a temperature of 8ºC, analyzed at the times 24, 48, 72 hours, 7 days and 28 days (at 28 days, fonly T1 was analyzed because the other products were deteriorated).

pH determination was performed with digital pH meter (Micronal, B-375) by direct insertion of the electrode in the sample (MARSHALL, 1993MARSHALL, R.T. Standard methods for examination of dairy products. Whashington: American Public Health Association, 1993. 546p.). Titratable acidity was determined by the method described by BRASIL (2006BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Instrução Normativa n.68, de 14 de dezembro de 2006. Oficializa os Métodos Analíticos Oficiais Físico-Químicos, para Controle de Leite e Produtos Lácteos, em conformidade com o anexo desta Instrução Normativa, determinando que sejam utilizados nos Laboratórios Nacionais Agropecuários. Diário Oficial da UniãoBrasília, 14 de dezembro 2006, Seção 1, p.8. Available from: <Available from: <http://extranet.agricultura.gov.br/sislegis-consulta/consultarLegislacao.do?operacao=visualizar&id=17472 >. Accessed: Apr. 05, 2015.
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) and expressed in lactic acid in g 100g-1 of the product. Viability of the probiotic L. acidophilus was determined according to a methodology recommended with the use of Agar medium De Man Rogosa and Sharpe (MRS) with addition of 0.15% of bile (VINDEROLA; REINHEIMER, 1999VINDEROLA, C.G.; REINHEIMER, J.A. Culture media for the enumeration of Bifidobacterium bifidum and Lactobacillus acidophilus in the presence of yoghurt bacteria. International Dairy Journal, v.9, n.8, p.497, 505, 1999. Available from: <Available from: <http://www.sciencedirect.com/science/article/pii/S095869469900120X >. Accessed: Dec. 10, 2014. doi: 10.1016/S0958-6946(99)00120-X.
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) and pathogen count was performed according to the recommendations: E. coli O157:H7 (ZADIK et al., 1997ZADIK P.M. et al. Use of tellurite for the selection of verocytotoxigenic Escherichia coli O157. Journal of Medical Microbiology, v.39, n.2, p.155-158, 1993. Available from: <Available from: <http://jmm.sgmjournals.org/content/39/2/155.abstract?ijkey=dc61d48776cdaab7734a4c11d33f260f007da8a7&keytype2=tf_ipsecsha >. Accessed: Mar. 02, 2015. doi: 10.1099/00222615-39-2-155.
http://jmm.sgmjournals.org/content/39/2/...
), S. aureus (LANCETTE; BENNETT, 2001LANCETTE, G.A.; BENNETT, R.WStaphylococcus aureus and staphylococcal enterotoxins. In: APHA (American Public Health Association). Compendium of methods for the microbiological examination of foods. Washington: APHA, 2001. V.4, p.387-403. ), Salmonella spp. (ANDREWS et al., 2001ANDREWS, H.W. et al. Salmonella. In: APHA (American Public Health Association). Compendium of methods for the microbiological examination of foods. 4.ed. Washington: APHA, 2001. Cap.37, p.357-380.), B. cereus (BENNETT; BELAY, 2001BENNETT, R.W.; BELAY, N. Bacillus cereus. In: APHA (American Public Health Association). Compendium of methods for the microbiological examination of foods. 4.ed. Washington: APHA, 2001. Cap.32, p.311-316. The results were expressed in log CFU g-1.

Statistical analyzes

Base 10 logarithms were used for normal distribution of the data obtained with the counts. Three independent experiments were performed in duplicate, and statistical treatment was obtained by analysis of variance (ANOVA) in entirely randomized design, with significance level of 5% (P<0.05). Tukey's multiple comparison tests were used for mean comparison, through the software Prism 5.01 GraphPad (GraphPad Software, San Diego, CA, EUA).

RESULTS AND DISCUSSION:

Regarding the dessert with addition of L. acidophilus (T1), on the first day of storage, the count was 8.25 log CFU 25g-1 and at the subsequent times they ranged from 8log CFU 25g-1 (24 hours) to 8.70 CFU 25g-1 (28 days). Thus, the final count of the probiotic strain, considering the daily dose of the product of 120 grams, (BRASIL, 2003BRASIL. Resolução RDC n.359, de 23 de dezembro de 2003. A Diretoria Colegiada da ANVISA/MS aprova o regulamento técnico de porções de alimentos embalados para fins de rotulagem nutricional. Diário Oficial da União, Brasília, 26 dezembro de 2003. (251):28; Seção 1. Available from: <Available from: <http://portal.anvisa.gov.br/wps/content/Anvisa+Portal/Anvisa/Inicio/Alimentos/Publicacao+Alimentos/Rotulagem+de+Alimentos+2 >. Accessed: Apr. 08, 2015.
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), is in accordance with the Brazilian legislation. Probiotic products or products claimed to be probiotic can be considered those that have microorganisms, e.g. L. acidophilus, at minimum concentrations of 108 to 109CFU (8 to 9log CFU) of probiotic microorganisms per portion of the product (BRASIL, 2008BRASIL. AGÊNCIA NACIONAL DE VIGILÂNCIA SANITÁRIA (ANVISA). Alimentos. Comissões e Grupos de Trabalho. Comissão Tecnocientífica de Assessoramento em Alimentos Funcionais e Novos. Alimentos Alimentos com Alegações de Propriedades Funcionais e ou de Saúde, Novos Alimentos/ Ingredientes, Substâncias Bioativas e Probióticos. Atualizado em: julho de 2008. IX- Lista de alegações de propriedade funcional aprovadas. Available from: < Available from: < http://www.anvisa.gov.br/alimentos/comissoes/tecno_lista_alega. htm > Accessed: Dec. 10, 2015.
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). In their experiment of production of a dairy dessert with addition of L. acidophilus, FERNANDES et al. (2013FERNANDES, M.S. et al. On the behavior of Listeria innocua and Lactobacillus acidophilus co-inoculated in a dairy dessert and the potential impacts on food safety and product's functionality. Food Control, v.34, p.331-335, 2013. Available from: <Available from: <http://www.sciencedirect.com/science/article/pii/S0956713513002247 >. Accessed: Dec. 10, 2014. doi: 10.1016/j.foodcont.2013.04.040.
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) and VASCONCELOS et al. (2013VASCONCELOS, C.M. et al. Desenvolvimento e avaliação sensorial de sobremesa láctea potencialmente simbiótica. Revista Instituto de Laticínios Cândido Tostes, v.68, n.391, p.11-17, 2013. Available from: <http://www.revistadoilct.com.br/rilct/article/download/16/16>. Accessed: Dec. 10, 2014. doi: 10.5935/2238-6416.20130015.), reported similar results for microbial counts.

For desserts T2, T3, T4, T5, T6, T7, T8 and T9, the analyzes were performed until the time 7 days, because at the time 28 days the products showed markedly altered sensory characteristics such as color, smell and general aspect, being considered deteriorated, and, so, it was not possible to carry out the analyzes (Figure 1).

Figure 1:
Microbial counts of L. acidophilus in dairy desserts.

In their study with probiotic dairy dessert (LA-05) with coconut flavor and assessment of the behavior of this strain in response to Listeria monocytogenes, FERNANDEZ et al. (2013) did not observe deterioration after 28 days of storage. However, it should be noted that the ingredients and pathogen used in the referred study are different from those in the present study. The pH values of the desserts ranged from 6.3 to 6.81 conferring low acidity to the culture (Figure 2). The pH values of the desserts are not characterized as a factor associated to the inhibition of pathogens, as demonstrated by several authors (PEREIRA & GÓMES, 2007; WENDLING & WESCHENFELDER, 2013WENDLING, L.K.; WESCHENFELDER, S. Probiotics and fermented dairy foods - a review. Revista do Instituto de Laticínios Cândido Tostes, v.68, p.49-57, 2013. Available from: <Available from: <http://www.revistadoilct.com.br/rilct/article/viewFile/50/56 >. Accessed: Dec. 15, 2014. doi: 10.5935/2238-6416.20130048.
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) in studies with fermented milks.

Figure 2:
Changes in pH values of dairy desserts up to day 7.

According to the counts of pathogens in dairy desserts with and without the addition of L. acidophilus, it can be seen that for all Gram negative pathogens, there was no significant difference at 24 hours of storage (Figures 3 and 4). Except for E. coli that showed no significant difference at the time 48 hours, there were significant differences (P<0.05) at all the other times between the pathogens in the presence or absence of the probiotic. Also, the difference in the behavior of E. coli at the last time assessed compared to Salmonella sp. deserves mention: both showed reduction, but it was more significant for E. coli (Figure 3). This can be due to the differences between genera and other factors that deserve more in-depth studies.

Figure 3:
Microbial counts of Gram negative pathogens in dairy dessert (DD) and probiotic dairy dessert (probiotic DD).

In Gram positive pathogens, Bacillus cereus showed a significant difference at all the analyzed times. As for S. aureus only the time 48 hours showed a significant difference (P<0.05) between the pathogens in the presence of absence of the probiotic. Also, bacteriocins, active proteins produced by some microorganisms, can have antimicrobial action, as well as some lactic bacteria with bactericidal action (ZACHAROF & LOVITTB, 2012ZACHAROF, M.P.; LOVITTB, R.W. Bacteriocins produced by lactic acid bacteria. APCBEE Procedia, v.2, p.50-56, 2012. Available from: <Available from: <http://www.sciencedirect.com/science/article/pii/S2212670812000814 >. Accessed: Apr. 02, 2015. doi: 10.1016/j.apcbee.2012.06.010.
http://www.sciencedirect.com/science/art...
; YANG et al., 2012YANG, E. et al. Antimicrobial activity of bacteriocin-producing lactic acid bacteria isolated from cheeses and yogurts. AMB Express, v.2, p.48, 2012. Available from: <Available from: <http://www.amb-express.com/content/2/1/48 >. Accessed: Apr. 10, 2015. doi: 10.1186/2191-0855-2-48.
http://www.amb-express.com/content/2/1/4...
; PEREZ et al., 2014PEREZ, R.H. et al. Novel bacteriocins from lactic acid bacteria (LAB): various structures and applications. Microbial Cell Factories, v.13, n.1, p.2-13, 2014. Available from: <Available from: <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4155820/ >. Accessed: Apr. 02, 2015. doi: 10.1186/1475-2859-13-S1-S3.
http://www.ncbi.nlm.nih.gov/pmc/articles...
), and that also compete for nutrients. Some bacteriocins have already shown to be active against several microorganisms such as Bacillus cereus, Clostridium perfringens, Listeria monocytogenes and S. aureus (CLEVELAND et al., 2001CLEVELAND, J. et al. Bacteriocins: safe, natural antimicrobials for food preservation. International Journal of Food and Microbiology, v.71, p.1-20, 2001. Available from: <Available from: <http://www.sciencedirect.com/science/article/pii/S0168160501005608 >. Accessed: Dec. 10, 2014. doi:10.1016/S0168-1605(01)00560-8.
http://www.sciencedirect.com/science/art...
).

In their experiment with a dessert with L. acidophilus La-05 and Listeria innocua,FERNANDES et al. (2008FERNANDES, M.S. et al. On the behavior of Listeria innocua and Lactobacillus acidophilus co-inoculated in a dairy dessert and the potential impacts on food safety and product's functionality. Food Control, v.34, p.331-335, 2013. Available from: <Available from: <http://www.sciencedirect.com/science/article/pii/S0956713513002247 >. Accessed: Dec. 10, 2014. doi: 10.1016/j.foodcont.2013.04.040.
http://www.sciencedirect.com/science/art...
) demonstrated that the count of both strains increased at the end of the storage of the product. This can be caused by the mutual benefit between these microorganisms. It should be stressed that the probiotic strain may have benefited from metabolites released by L. innocua, such as peptides and aminoacids.

Brazilian legislation RDC 12/2001 (BRASIL 2001BRASIL. Agência Nacional de Vigilância Sanitária. Resolução RDC, n.12 de 02 de janeiro de 2001. Aprova o regulamento técnico sobre padrões microbiológicos para alimentos. Diário Oficial da União, Brasília, Seção 1. Available from: <Available from: <http://portal.anvisa.gov.br/wps/wcm/connect/a47bab8047458b909541d53fbc4c6735/RDC_12_2001.pdf?MOD=AJPERES >. Accessed: Apr. 05, 2015.
http://portal.anvisa.gov.br/wps/wcm/conn...
) that establishes the Microbiological Standards for Food Health, has the following parameters: B. cereus, Coliforms at 45ºC, coagulase-positive staphylococci and Salmonella sp. for dairy desserts, which justifies the selection and importance of these microorganisms used in the present study. Of all the pathogens analyzed, only E. coli O157:H7, both in the non-probiotic and probiotic products, was reduced at th 7th day, though without significant differences between the treatments.

CONCLUSION:

It is concluded that dairy chocolate dessert allows the development of probiotics and has great economic potential. However, it also allows the development of pathogens, with or without the presence of probiotics. Probiotic products developed reduced important foodborne pathogens but not up to safe levels. Therefore, we emphasize the importance of the use of quality tools in the development and monitoring of dairy desserts.

REFERENCES:

  • 1
    CR-2014-1864.R1

Publication Dates

  • Publication in this collection
    3 Nov 2015
  • Date of issue
    Feb 2016

History

  • Received
    24 Dec 2014
  • Accepted
    21 June 2015
  • Reviewed
    10 Sept 2015
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