BR102015021165A2 - yogurts enriched with red propolis extract, process of obtaining yogurts and using them. - Google Patents

Abstract

The present invention belongs to the field of dairy products, more specifically to yogurts enriched with red propolis extract with a functional food characteristic, intended for human consumption with a focus on disease prevention and cure. It is proposed to obtain yoghurts enriched with red propolis extract (IEEPV), compositions of yoghurts enriched with red propolis extract and stabilizers with whole milk or whey with characteristics of peptides and functional amino acids. Additionally, the present application deals with dairy compositions containing the so-called IEEPV, with natural flavorings that mask the bitter and strong flavor of the red propolis extract. IEEPVs have specific biological activities in the prevention of diseases of the gastrointestinal tract, metabolic syndrome, menopause, osteoporosis and arthritis that will provide sensory, nutritional, functional characteristics appropriate to the dairy product.

Description

ENRICHED YOGHURES WITH RED PROPOLIS EXTRACT, PROCESS OF OBTAINING YOGURTES AND USE THEM. FIELD OF THE INVENTION

[1] The present invention belongs to the field of dairy products, more specifically to yogurts and milk drinks with characteristics of nutritional and functional food, intended for human consumption with a focus on disease prevention, improving body aesthetics and increasing performance of athletes high yield. It is proposed a composition of yoghurts containing milk or whey powder enriched with standardized extract of red propolis (EPV) and flavoring that will be used for industrial production of yoghurts with red propolis, compositions of milk yoghurts containing EPV, compositions of whey yoghurts of milk containing EPV and uses of it. The production of these yoghurts enriched with red propolis extract is also linked to the use of lactic acid bacteria in combination with Lactobacillus delbruekii spp bulgaricus and Streptococcus thermophilus in a specific mixture, as well as other ingredients, called flavorings, used to mask the strong flavor of the propolis extract. red. In addition, this application addresses the uses of yoghurts enriched with red propolis that has specific biological activities and will provide sensory, nutritional and functional characteristics suitable for the dairy product with application in the dairy industry for the production of dairy products, especially yoghurts and dairy drinks.

BACKGROUND OF THE INVENTION

[2] Fermented milks, specifically yogurt, originated many years ago in the Middle East or India. The nomads, when storing milk always in the same containers, were selecting a microbiota that fermented the milk and produced a food with a pleasant taste, hence the origin of fermented milk. By definition, fermented milks are prepared in dairy products in which milk of different species undergoes a fermentative process, modifying their sensory properties (ORDOÑEZ. JA Tecnologia de Alimentos. Foods of Animal Origin, vol. 2. Porto alegre: Artmed, 2005.) .

[3] For Brazilian legislation, fermented milks are products added or not with other food substances, obtained by coagulation and lowering the pH of milk or reconstituted milk, whether or not added with other dairy products, by lactic fermentation through the action of crops of specific microorganisms (BRAZIL. Ministry of Health. National Health Surveillance Agency. Normative Instruction No. 46, of October 23, 2007. Adopts the Technical Regulation on Identity and Quality of Fermented Milk. Official Gazette, Brasilia, 24 October 2007.). Thus, yogurt is fermented milk obtained by the cultivation of microorganisms Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, milk fermented with thermophilic lactic bacteria, by coagulation and lowering the pH of the milk, which must be viable, active and abundant in the final product during its shelf life. The activity of these crops contributes to the determination of the characteristics of the final product (FORSYTHE, S. Microbiology of food safety. Artmed: Porto Alegre, 2002; CODEX ALIMENTARIUS COMMISSION. Codex Standards for fermented milk. Codex Stan 243-2003. Adopted in 2003 Revision 2008, 2010. Available at: <http://www.codexalimentarius.net/download/standard/400/CXS _243e.pdf> Accessed Jan 31, 2012 .; BRAZIL, Ministry of Health, National Health Surveillance Agency. Normative Instruction No. 46, of October 23, 2007. Adopts the Technical Regulation on Identity and Quality of Fermented Milk (Official Gazette, Brasília, October 24, 2007.).

[4] Milk is the largest constituent of yogurt, however the addition of some ingredients is allowed, in order to meet the requirements of the different segments in the market (MONTEIRO, AA; PIRES, ACS; ARAÚJO, EA Tecnologia de Produção de derived from milk Viçosa, MG: Editora UFV, 2011.). In this sense, pasteurized concentrated milk, pasteurized whole or partially skimmed milk, pasteurized whole or partially skimmed concentrated milk, with or without the addition of pasteurized cream, whole, semi-skimmed or skimmed milk powder, whey powder, milk proteins and / or others from the fractionation of milk can be used for its production (ORDONEZ. JA Tecnologia de Alimentos. Foods of Animal Origin, vol. 2. Porto alegre: Artmed, 2005.).

[5] According to Brasil (2007) the yogurt can present a firm, pasty, semi-solid or liquid consistency, white color or according to the food substance and / or added color. Its odor and taste must be characteristic or according to the food substance and / or flavoring / flavoring substance added. It is a highly nutritious food, rich in proteins, calcium and phosphorus, recommended for pregnant and lactating women, in addition to elderly people who need calcium replacement. Its nutritional value is similar to the milk used in its preparation, added by the nutrients metabolized by the microorganisms involved in the fermentation process (TAMINE, AY; ROBINSON, RK Yogur - Science and technology. Zaragoza: Acribia, 1991, p. 368 .; apud MARTIN, AF Storage of commercial yogurt and the effect on the proportion of lactic acid bacteria 2002. 62 f. Dissertation (Master in Food Science and Technology). University of São Paulo, Piracicaba, São Paulo, 2002.).

[6] Among all fermented milks, it is the most popular and most consumed in Brazil, mainly for its organoleptic characteristics, for its nutritional qualities and possible therapeutic properties (AQUARONE, E. et al. Industrial biotechnology. Biotechnology in food production , vol. 4. São Paulo: Edgard Blücher LTDA, 2001.).

[7] The technology for making yogurt is well defined, even so, aiming to meet consumers' demands for more nutritious foods and with health claims, research has been carried out observing the incorporation of new ingredients into the formulation. In this context, several authors, such as Santos (2012), Bastiane (2009) (BASTIANE, MID Yogurt added with whey protein concentrate and flaxseed: Development, nutritional and sensory quality. 2009. 115 f. Thesis ( PhD in Food Science and Technology), Federal University of Viçosa, Minas Gerais, 2009.) and Espírito Santo et al. (2012) (ESPÍRITO SANTO, AP et al. Fibers from fruit by-products enhance probiotic viability and fatty acid profile and increase CLA content in yoghurts. Food Science and Technology., São Paulo, Brazil. V. 154, p. 135- 144, 2012.) carried out studies that are related to the quality, processing and technological innovations of yogurts, demonstrating the interest of the scientific community for this food. In these and other studies, the effect of adding other food substances to yogurt such as fruit fibers, antioxidants from other products such as grape seed extracts, grape marc, and fortified with polyphenols obtained from black currant (CHOUCHOULI, V .; et al. Fortification of yoghurts with grape (Vitis vinifera) seed extracts.Food Science and Technology., Athens, Greece. Vol. 53, p. 522 - 529, 2013 .; TSENG, A .; ZHAO, Y. Wine grape pomace as antioxidant dietary fiber for enhancing nutritional value and improving storability of yogurt and salad dressing. Food Chemistry. Corvalles, USA. N. 138, p. 356-365, 2013 .; SUN-WATERHOUSE, D .; ZHOU, J .; WADHWA, SS Drinking yoghurts with berry polyphenols added before and after fermentation. Food Control. N ° 32 p. 450-460, 2013.).

[8] The addition of reconstituents to yogurt aims to improve its rheological characteristics, one of which is the texture. Research using whey or whey protein concentrate in the manufacture of yogurt has been developed to verify its effects on sensory, nutritional and rheological characteristics (LIMA. SCG; GIANT, ML; ALMEIDA, TCA Effect of adding different types and concentrations of solids on the sensory characteristics of firm type yogurt Revista Brasileira de Produtos Agroindustriais, Campina Grande, v.8, nl, p.75-84, 2006 .; BASTIANI, MID Yogurt added with whey protein concentrate and flaxseed flour : Development, nutritional and sensory quality, 2009. 115 f. Thesis (Doctorate in Food Science and Technology), Federal University of Viçosa, Minas Gerais, 2009.).

[9] Thus, in the preparation of yogurt, food substances of animal and vegetable origin can be added (BRAZIL. Ministry of Health. National Health Surveillance Agency. Normative Instruction No. 46, of October 23, 2007. Adopts the Technical Regulation of Identity and Quality of fermented milk (Diário Oficial da União, Brasilia, October 24, 2007.). A product of plant origin that has many bioactive compounds and has the potential to be a natural food additive, acting as an antioxidant and for its antimicrobial activity as well as in the form of a functional ingredient is propolis. The compounds present in propolis can bring many health benefits, among these compounds, the flavonoids with potential antioxidant action stand out (ACKERMAN ACKERMAN, T. Fast Chromatrographic Study Propolis Crudes. Food Chemistry. Vol. 42. p. 135-138. Israel, 1991 .; COSTA, NM B; ROSA, COB Functional foods. Bioactive components and physiological effects. Rubio. Rio de Janeiro, 2010.).

[10] Whey has been widely used in several food formulations. Its use occurs in several forms, such as solid state, pasteurized fluid or in the form of protein concentrates. In order to verify the use of whey, Vidigal et al. (2010) (VIDIGAL, MCTR; et al. Viscoelasticity of yogurt containing whey protein concentrate. In Anais ... XXI Brazilian Congress of Food Science and Technology. Salvador: 2010.) added Whey Protein Concentrate (CPS) in yoghurts, in different concentrations to replace fat, such an addition resulted in the alteration of the rheological properties of yogurt, giving it greater elasticity. The authors concluded that the addition of CPS at levels of 3.0% and 4.5% was effective as a fat substitute. Silva K. et al. (2004) (SILVA, K .; BOLINI, HMA; ANTUNES, AJ Different products of bovine whey in ice cream. Alim. Nutr., Araraquara, v. 15, n. 2, p. 187-196, 2004.) , highlighted its use as an ingredient in ice cream and concluded that there were improvements in their physical, chemical and sensory characteristics. Viotto et al. (2010) (VIOTTO, WH; LOIOLA, LS; GONÇALVES, MC; Effect of the addition of whey protein concentrate on the functional properties of creamy curd. In Anais ... IV Brazilian Milk Quality Congress. Florianópolis: 2010.) studied the effect of the addition of whey protein concentrate, in the processing of creamy curd and found an increase in nutritional value and yield. Where the addition of 0.5% CPS to creamy curd was accepted by consumers.

[11] When using pasteurized whey (65 ° C / 30 min.) In drink, Silva T. et al. (2010) (SILVA, TMS; et al. Whey-based drink. In Anais ... XXI Brazilian Congress of Food Science and Technology. Salvador: 2010.) observed that the flavor of the whey was masked by the concentrates of fruits, obtaining satisfactory viscosity and high acceptance of the product.

[12] Ferrari et al. (2010) (FERRARI, AS; AZAREDO, EMC; BALDONI, NR; Sensory analysis of products made from whey. In Anais ... XXI Brazilian Congress of Food Science and Technology. Salvador: 2010) acceptance for all elaborated products (cake, chocolate milk drink and fruit jam (guava frappe). Bastiani, (2009) (BASTIANI, MID Yogurt added with whey protein concentrate and flaxseed: Development, nutritional quality and sensorial. 2009. 115 f. Thesis (Doctorate in Food Science and Technology). Federal University of Viçosa, Minas Gerais, 2009.) and Lima et al. (2006) (LIMA. SCG; GIGANTE, ML; ALMEIDA, TCA Effect of adding different types and concentrations of solids on the sensory characteristics of firm type yogurt (Revista Brasileira de Produtos Agroindustriais, Campina Grande, v.8, nl, p.75-84, 2006.) developed yoghurts added with whey protein concentrate of milk for r verification of its effects on the sensory and nutritional characteristics of this food.

[13] The whey can, due to its technological properties previously mentioned, be used in other non-dairy foods promoting desirable characteristics of the product. Other scientific research highlights the advantages of using whey in yogurt due to the properties of the functional peptides present in whey against obesity, hypertension, cancer, phenylketonuria, skin aging and stimulating the physiology of the gastrointestinal tract (Seema Patel, Emerging trends in nutraceutical applications of whey protein and its derivatives, Journal of Food Science and Technology, June 2015. DOI: 10.1007 / sl3197-015-1894-0 .; Mamta Rathi, Neelam Upadhyay, RS Dabur, Ankit Goyal, Formulation and physico-chemical analysis of whey-soymilk dahi, Journal of Food Science and Technology, Volume 52, Issue 2, pp 968-975, February 2015 .; K. Shiby Varghese, K. Radhakrishna, AS Bawa, Moisture sorption characteristics of freeze dried whey-grape beverage mix, Journal of Food Science and Technology, Volume 51, Issue 10, pp 2734-2740, October 2014 .; Nayereh Sabokbar, Faramarz Khodaiyan, Characterization of pomegranate juice and whey based novel bever age fermented by kefir grains, Journal of Food Science and Technology, Volume 52, Issue 6, pp 3711-3718, June 2015.).

[14] YOGURT AS FUNCTIONAL FOOD: The various factors that have contributed to the development of functional foods are innumerable, one of which is the increase in the awareness of consumers, who wishing to improve the quality of their lives, choose healthy habits (MORAES, FP; COLLA, LM Functional and nutraceutical foods: definitions, legislation and health benefits. Electronic pharmacy journal. V. 3. p. 109-122, 2006.). The attributes of functional foods include, among other health benefits, reduced risk of cardiovascular disease, cancer, diabetes, obesity, osteoporosis and other chronic non-communicable diseases. Interest in functional foods is growing and has attracted the attention of consumers and the food industry, as many foods contain bioactive ingredients that, in addition to their functions of nourishing, bring health benefits (COSTA, NM B; ROSA, COB Functional foods. Bioactive components and physiological effects (Rubio, Rio de Janeiro, 2010.). Despite this, there are many benefits of yogurt (HUERTAS, R. A. P. Yogur en la salud humano. Lasallista Investig. Vol.9, n.2, p. 162-177. 2012.). Among these are the prevention of colon cancer, lowering cholesterol, improving the intestinal flora, effects on the immune system, reducing diarrhea in children, in addition to the synthesis and bioavailability of nutrients, improving both the quantity and availability of nutrients in the diet. . Research carried out by the National Institute of the Higher Council for Scientific Research in Spain (CSIC) reports on the benefits of yogurt, stating that the individual who consumes it usually secretes substances that benefit the immune system, which remains afterwards, even if consumption is suspended (MILKPOINT. Spanish researcher highlights the benefits of consuming yogurt. 2001. Available at: <http://www.milkpoint.com.br/cadeia-do-leite/giro-lacteo/pesquisadora-espanhola-ressalta-os-beneficios -do-consumption-of-yogurt-12629n.aspx> Accessed: 20/09/2014 às 14h.).

[15] A food can be considered functional if it is demonstrated that it can beneficially affect one or more target functions in the body, in addition to having adequate nutritional effects, in a way that is both relevant to well-being and health and to reducing the risk of a disease (ROBERFROID, M. Functional food concept and its application to prebiotics. Digestive and Liver Disease, v. 34, Suppl. 2, p. 105-10, 2002.).

[16] Yogurt stands out among the foods widely publicized for health claims. Several studies recognize its nutritional characteristics and the presence of a series of factors that imply the promotion of human health (MORAES, MN Physical-chemical and rheological characterization of yogurts made with different fat substitutes. 2011. f. 91. Dissertation (Master in Food Science and Technology). Federal University of Viçosa, Minas Gerais.). Despite this, only yogurts made with probiotic bacteria can be called functional yogurts (ANTUNES, AEC Influence of whey protein concentrate and probiotic cultures on the properties of skimmed natural yoghurts. 4002. 240 f. Thesis (PhD in food engineering) Department of Food and Nutrition, Campinas, 2004.). Among the probiotic microorganisms approved by Anvisa that contribute to the balance of intestinal flora, there are: Lactobacillus casei shirota, Lactobacillus casei variety rhamnosus, Lactobacillus casei variety defensis, Lactobacillus paracasei, Lactococcus lactis, Bifdobacterium bifidium bifidum bifidum Lactis), Bifdobacterium longum and Enterococcus faecium (BRAZIL. Ministry of Health. National Health Surveillance Agency. Normative Instruction No. 46, of October 23, 2007. Adopts the Technical Regulation on Identity and Quality of Fermented Milk. Official Gazette of the Union , Brasilia, October 24, 2007.). As noted, yeast fermenting microorganisms Lactobacillus bulgaricus and Streptococcus thermophillus are not included in this list, as they do not have scientifically proven probiotic effects and are necessary for their processing. However, these varieties of lactobacillus have been widely used in the production of functional yogurts. The invention patent (WO 2011/161085 Al) uses the Lactobacilus bulgaricus strain PIM-1966 and Streptococcus thermophilus strain CHCC-11977 for the production of yoghurts and other milk drinks. Another patent (PI 9202246 A) develops yoghurts containing mutant lactobacillus bulgaricus with fragmented DNA for a part of the beta-galactosity gene and the streptococus thermophilus.

[17] The literature provides some studies that fortify yogurts with bioactive compounds or compounds from other foods, giving the characteristic of "functional food" or simply with health claims or even with higher nutritional value in relation to other yogurts without the supplement . In the study by Espírito Santo et al ,. (2012) (ESPÍRITO SANTO, AP et al. Fibers from fruit by-products enhance probiotic viability and fatty acid profile and increase CLA content in yoghurts. Food Science and Technology., São Paulo, Brazil, v. 154, p. 135- 144, 2012.) the effect of supplementing yogurts with fruit fibers was evaluated. The authors monitored pH, titratable acidity, post-acidification, lactic acid count and fatty acid profile in yoghurts made from skimmed milk and fermented by strains of probiotic bacteria. It was found that fruit fibers improved the fatty acid profile of probiotic yogurts, thus obtaining a product with high added value. In this study, the authors combined fruit fibers with probiotic yoghurts, thus obtaining yogurt with a probiotic and prebiotic function. Probiotic is the term used for living microorganisms that can be added as supplements in the diet, beneficially affecting the development of microbial flora in the intestine. They are also known as biotherapeutic, bioprotective and bioprophylactic and are used to prevent enteric and gastrointestinal infections (REIG, ALC; ANESTO, JB Prebióticos y probióticos, una Relación Beneficiosa. Instituto de Nutrición e Hiene de los Alimentos. Revista Cubana de Comida e Nutrição v. 16, n. 1, p. 63-8, 2002.). While prebiotics are non-digestible but fermentable oligosaccharides whose function is to change the activity and composition of the intestinal microbiota with a view to promoting the health of the host. Dietary fibers and non-digestible oligosaccharides are the main substrates for the growth of microorganisms in the intestines. Prebiotics stimulate the growth of endogenous groups of microbial population, such as Bifidobacteria and Lactobacillus, which are said to be beneficial to human health (BLAUT, M. Relationship of prebiotics and food to intestinal microflora. European Journal of Nutrition. V. 41, supplement 1, p.1-16, 2002.). For a substance (or group of substances) to be defined as such, it must meet the following requirements: be of plant origin; form part of a heterogeneous set of complex molecules; not be digested by digestive enzymes; be partially fermented by a colony of bacteria and be osmotically active (RODRÍGUEZ, MBS; MEGÍAS, SM; BAENA, BM Alimentos Funcionales y Nutrición optimal. Revista da Espanha de Salud Pública. v. 77, n. 3, p. 317-331 , 2003.).

[18] Chouchouli et al. (2013) (CHOUCHOULI, V .; et al. Fortification of yoghurts with grape (Vitis vinifera) seed extracts. Food Science and Technology., Athens, Greece. Vol. 53, p. 522 - 529, 2013.) enriched yogurts with grape seed extracts (Vitis vinífera) aiming to give antioxidant action to whole and skimmed yogurts. The authors evaluated color, viability of Lactobacillus ssp., PH and grape seed compounds such as epicatechin and total polyphenols. The extracts were added to yoghurts acquired in the local market and after adding the extracts, the samples were followed during 32 days of storage. The extracts were obtained from two grape varieties from the Central Greece region, in Attica, and these varieties were used in the production of red and white wine. Simple polyphenol compounds and oleanolic acid were identified in the yogurt samples. Total phenolic compounds, epicatechin, radical scavenging activity, reduced ferric ion power, were detected over three weeks. The addition of 5-10mg of grape seed extract to yogurt did not interfere with the growth of Lactobacillus ssp. and did not mischaracterize the product. In the study by Bastiane (2009) (BASTIANE, MID Yogurt added with whey protein concentrate and flaxseed flour: Development, nutritional and sensory quality. 2009. 115 f. Thesis (PhD in Food Science and Technology). Federal University de Viçosa, Minas Gerais, 2009.) in addition to whey protein concentrate, flaxseed flour was added to yogurts. Through these ingredients, yogurts acquired higher protein quality and an excellent source of a-linolenic fatty acid, containing dietary fiber and low in sodium. Flax seeds are rich in proteins, fibers, ash and minerals, in addition to amino acids with high rates of aspartic acid, glutamic acid, leucine and arginine, featuring a complete protein and with effects on the body's immune functions. It is the main plant source of α-linolenic fatty acid (OOMA, B. D. Flaxseed as a functional food source. Journal of the Science of food and agriculture. V. 81. p. 889-894, 2001.). In this study, yogurt formulations containing 2% and 3% flaxseed flour were classified as an excellent source of α-linolenic fatty acid, while yoghurts added with 1% were classified as a good source. The formulations were well accepted by consumers. However, the acceptability of the product was lower as the percentage of flaxseed flour to yogurt increased.

[19] Sun-Waterhouse; Zhou; Wadhwa (2013) (SUN-WATERHOUSE, D .; ZHOU, J .; WADHWA, SS Drinking yoghurts with berry polyphenols added before and after fermentation. Food Control. N ° 32 p. 450-460, 2013.) studied the effect of addition of purified polyphenols and from blackcurrant extract in yogurt, before and after fermentation. The yogurts were stabilized with powdered milk and high and low methoxylation pectin. The chemical, rheological and microbiological properties of yoghurts were studied. The addition of pectins modified the rheological properties, obtaining firmer gel in yogurts formulated with high methoxylation pectins. Blackcurrants have a high content of anthocyanins and their use in yoghurts aimed to add value for the antioxidant action as well as to give flavor. The chromatographic results detected typical currant anthocyanins with proven beneficial health properties.

[20] New scientific publications are attesting to the use of additives derived from fruits and / or seeds to impart nutritional, functional, organoleptic characteristics, different flavor to yogurts and other dairy drinks, among the functional additives we mention the candied chestnut, coconut milk , plant extracts and resins enriched with phytosteroids, astaxanthin, phenolic compounds (Shabboo Amirdivani, Ahmad Salihin Hj Baba, Green tea yogurt: major phenolic compounds and microbial growth, Journal of Food Science and Technology, Volume 52, Issue 7, pp 4652- 4660, July 2015 .; Vasiliki Schoina, Antonia Terpou, Gialleli Angelika-Ioanna, Athanasios Koutinas, Maria Kanellaki, Loulouda Bosnea, Use of Pistacia terebinthu s resin as immobilization support for Lactobacillus casei cells and application in selected dairy products, Journal of Food Science and Technology, November 2014. DOI: 10.1007 / sl3197-014-1627-9; Zahra Izadi, Ali Nasirpour, Ghasemali Ali Garoosi, Fardin Tamjidi, Rheological and physical properties of yogurt enriched with phytosterol during storage, Journal of Food Science and Technology, Volume 52, Issue 8, pp 5341-5346, August 2015 .; Meenakshisundaram Selvamuthukumaran, Farhath Khanum, Optimization of seabuckthorn fruit yogurt formulation using response surface methodology, Journal of Food Science and Technology, Volume 52, Issue 2, pp 831-839, February 2015 .; S. Cid-Ortega, JA Guerrero-Beltrán, Roselle calyces (Hibiscus sabdariffa), an alternative to the food and beverages industries: a review, Journal of Food Science and Technology, April 2015. DOI: 10.1007 / sl3197-015-1800- 9 .; Pedro Cerezal Mezquita, Blanca E. Barragán Huerta, Jenifer C. Palma Ramírez, Claudia P. Ortíz Hinojosa, Milks pigmentation with astaxanthin and determination of color stability during short period cold storage, Journal of Food Science and Technology, Volume 52, Issue 3, pp 1634-1641, March 2015 .; Shana, R. Sridhar, B. S. Roopa, M. C. Varadaraj, S. V. N. Vijayendra, Optimization of a novel coconut milk supplemented dahi - a fermented milk product of Indian subcontinent, Journal of Food Science and Technology, April 2015. DOI: 10.1007 / sl3197-015-1825-0. Melike Sakin-Yilmazer, S. Nur Dirim, Davide Di Pinto, Figen Kaymak-Ertekin, Yoghurt with candied chestnut: freeze drying, physical, and rheological behavior, Journal of Food Science and Technology, Volume 51, Issue 12, pp 3949-3955 , December 2014.).

[21] Seeking comparative data on the state of the art, 16 patents of invention (PI) and a utility model (MU) related to yogurt were detected in Brazil. Of these, the patent entitled "Yogurt and milk drink" (PI 0302340-0 A), published in March 2005, refers to a yogurt and milk drink sweetened with brown sugar complemented with fruits (apple, peach, banana, strawberry, papaya, etc.), vegetables (pumpkin, carrot, beet, xuxu, avocado, etc.) and alternatively with a soy derivative. The intention was to add nutritional value to beverages derived from nutrients from fruits and vegetables as well as brown sugar as it contains a higher content of nutrients and is rich in iron compared to the others.

[22] One of the patents consulted, Chocolate-Yogurt with Malt (MU 88005712-U2), refers to a utility model (MU). The objective was to unite two ingredients already on the market through a suitable container in which the chocolate with malt was disposed in one container and the yogurt in another, coupling them before consumption, mixing the two ingredients. This utility model suggests that yogurt has been the focus of some research that involves not only its production technology, but also utility models.

[23] The patent (PI 0403377-9 A) refers to a process for obtaining frozen natural yogurt from a delicacy obtained through a process of mixing ingredients and differs in the state of the art in terms of flavor. It arose from the need to offer a different delicacy in different commercial establishments for food and beverages. Additionally, an order with similar characteristics was requested for frozen diet yogurt (PI 0403378-7 A).

[24] An invention patent for yogurt (PI 0606220-2A) that offers isoflavones in its composition from soy refers to a yogurt made from "fermentable soy bean milk" that gradually had the formulation improved obtaining a product in a creamy state that has been sweetened from fruits with high sugar content. This product was used to prevent degenerative diseases such as bronchitis.

[25] Invention patent involving yogurt in its formulation is entitled frozen composition based on yogurt and fruit (PI 0620307-8 A2). As described, this is not a yogurt, but a product that features yogurt in its formulation. It is a composition of one or more fruits in the form of puree and / or juice and refers to a process for its manufacture and use. Another invention patent (PI 0802311-5 A2) presents composition and process for obtaining yogurts containing broad beans (Vicia faba) and hibiscus (rosa sinensis).

[26] Invention patent (PI 0503145-1 A) shows the composition and process for obtaining deactivated yogurt containing enzyme responsible for the reduction of lactose. Invention patents (PI 0810125-6 A2 and PI 0810127-2 A2) use pinolenic acid, linoleic acid and derivative to produce functional yogurts promoting a feeling of fullness.

[27] Invention patent (WO 2008/092458 Al) presents a new composition of yogurts enriched with casein proteins and whey proteins in specific proportions that gives the drink organoleptic characteristics and yogurt flavor. In this processing, the coagulation step is absent. Invention patent (WO 96/18310) presents a new process for the manufacture of yoghurts using carbon dioxide stream during preparation to impart adequate viscosity with a semi-solid preparation characteristic to dairy drinks including yoghurts, puddings, gelatines, ice creams and creams.

[28] In all researched patent documents, related to yogurt, research similar to the proposed patent was not found. According to Proudlove (1996), red propolis, due to its chemical composition, rich in flavonoids, has a strong and bitter taste and due to this characteristic its use in food is difficult. The production of a fermented milk (yogurt and milk drink) containing red propolis extract differs in the state of the art among all the patent documents consulted in terms of technological innovation, nutritional enrichment, processing and flavor.

[29] Propolis constituents have been identified worldwide (Pietta, P .; Gardana, C .; Scaglianti, M .; Simonetti, P., Journal of Pharmaceutical and Biomedical Analysis, V. 45, p. 390-399 , 2007). Flavonoids, phenolic acids and terpenes are the main substances found and used to track quality and, in some cases, to demonstrate the authenticity of propolis from some geographic regions (Volpi, N .; Bergonzini, G., Journal of Pharmaceutical and Biomedical Analysis, V. 42, pp. 354-361, 2006). Among the flavonoids and phenolic acids most commonly used as markers, the following can be mentioned: quercetin, canferol, naringenin, chrysin, pinocembrine, galangin (flavonoids); gallic acid, caffeic acid, p-coumaric acid, ferulic acid, cinnamic acid and derivatives (phenolic acid), clerodanes (diterpenoids) (Rosalen, PL; Castro, ML; Cury, JA, Quimica Nova, V. 30, N. 07 , 1512-1517, 2007); (Yao, L .; Jiang, Y .; Singanusong, R., Datta, N .; Raymont, K., Food Chemistry, V. 86, p.169-177, 2004); (Weston, R. J., Mitchell, K. R., Allen, K. L., Food Research International, V. 37, p. 166-174, 2004).

[30] The increasingly popular use of propolis and its derivatives with antimicrobial action (Sforcin, JM; Fernandes Jr., A .; Lopes, CAM; Bankova, V. And Funari, SRC, J. Ethnopharmacology, V. 73, p. 243-249, 1998), anti-inflammatory (Khayyal, MT; el-Ghazaly, MA; el-Khatib, AS, Drug Experimental and Clinical Research, V. 19, p. 197-203, 1993), antiviral ( Vynograd, N .; Vynograd, I .; Sosnowisky, Z., Phytomedicine, V. 7, p. 1-6, 2000), anticarcinogenic (Bazo, AP; Rodrigues, MAM; Sforcin, JM; Camargo, JLV, Ribeiro, JL; Salvadori, DMF, Teratogenis, Carcinogenis and Mutagenis, V. 22, p. 183-194, 2002) and immunomodulatory (Sforcin, JM, Kanemo, R. Funari, SRC, Journal of Venemous Animal and Toxins, V. 8, p. 19-29, 2002); (Sá-Nunes, A .; Faccioli, L.H., Sforcin, J. M., Journal of Ethnopharmacology, V.83, p. 93-97, 2003) has been demonstrating the great therapeutic power of propolis to replace conventional synthetic drugs.

[31] The characterization of all these biological activities associated with the tendency to use natural products has resulted in an increased demand for propolis and products containing propolis, such as extracts, creams, ointments, tablets, capsules or powders (H. Menezes, M. Bacci Jr., SD Oliveira, FC Pagnocca, Antibacterial properties of propolis and products containing propolis from Brazil, Apidologie, V. 28, p. 71-76, 1997).

[32] In the case of red propolis, it was classified as the 13th subtype of Brazilian propolis found in the mangroves, lagoons, rivers and beaches of northeastern Brazil between the states of Sergipe, Alagoas, Pernambuco and Paraíba by the researcher (Andreas Daugsch, Cleber S. Moraes, Patricia Fort and Yong K. Park, Brazilian Red Propolis — Chemical Composition and Botanical Origin, eCAM 2008; 5 (4) 435-441 doi: 10.1093 / ecam / nem057). The main botanical origin of red propolis is due to the Dalbergia ecastophyllum plant, locally known as Rabo-de-Bugio, which has a red resinous exudate released from its sap.

[33] National and international researchers with expertise in propolis have been identifying the constituents of red propolis. 11 isoflavonoids and 1 chalcone of Cuban red propolis were identified and among these substances we can mention: Formononetine (Biochanina B), Bichanina A, vestitol, ortho-methyl vestitol, medicarpine, homopterocarpine, other pterocarpan derivatives, liquiritigenin and isoliquiritigenin (chalcone) (Anna) Piccinelli et al J. Agric. Food Chem. 2005, 53, 9010-9016). Other subclasses of flavonoids have also been identified, including new types of flavonoids from the red propolis of the Northeast region, which can be mentioned: rutin, quercetin, luteolin, pinocembrine and biochanin A, formononetine, daidzein, liquiritigenin, pinobanksina, dalbergine (neoflavugs) (Andreas , Cleber S. Moraes, Patricia Fort and Yong K. Park, Brazilian Red Propolis — Chemical Composition and Botanical Origin, eCAM, Evidence-based complementary and alternative medicine, 2008; 5 (4) 435-441 doi: 10.1093 / ecam / nem057 ).

[34] Researchers studying the red propolis from the mangrove region of Marechal Deodoro-Alagoas, identified the flavonoid chrysin and quercetin, new isoflavonoids in the red propolis of Alagoas, vestitol, 2'4'hydroxyethoxy flavana, isoprenylated benzophenones, as well as ferulic acid and phenolic esters, methoxyugenol, methyleneugenol, guiacol, dimethyl esters of butanenedioic acid, hexadecanoic acid methyl esters (Bruno B. Silva, Pedro L. Rosalen, Jaime A. Cury, Masaharu Ikegaki, Vinícius C. Souza, Alessandro Esteves and Severino M. Alencar Chemical Composition and Botanical Origin of Red Propolis, a New Type of Brazilian Propolis, eCAM, evidence based complementary and alternative medicine 2008; 5 (3) 313-316, doi: 10.1093 / ecam / nem059).

[35] In addition, researchers identified 14 substances in the red propolis of the mangroves of Alagoas, including triterpenes (cycloartenol, lupeol, elemicin, α-amyrin and β-amyrin), phenolic acids (anethol, eugenol and methyleneugenol), isoflavonoids (isosativan, medicarpina) (Boryana Trusheva, Milena Popova, Vassya Bankova, Svetlana Simova, Maria Cristina Marcucci, Patricia Laguna Miorin, Flavia da Rocha Pasin and Iva Tsvetkova, Bioactive Constituents of Brazilian Red Propolis, eCAM, evidence-based complementary and alternative medicine 2006; 3 (2) 249-254, doi: 10.1093 / ecam / nel006).

[36] With a view to discovering molecules and their therapeutic targets for curing diseases, red propolis has been subjected to several detailed studies on its biological activities. Research on the identification and structural elucidation of new substances continues to be carried out in the identification of new substances present in the red propolis extract. A new chalcone, 3, 4, 2 ', 3' tetrahydroxchalcona and a new C-glycosylated flavonoid, narigenin-8-C-hexoside, were identified, in addition to (3S) -vestitol and (3S) -7-O-methylvestitol (Adne A Righi, Thiago R Alves, Giuseppina Negri, Lucas M Marques, Henrique Breyer and Antonio Salatino, Brazilian red propolis: unreported substances, antioxidant and antimicrobial activities, J Sci Food Agric 2011/91: 2363-2370, DOI 10.1002 / jsfa .4468). Biological research with the ethanol extract of red propolis and isolated substances, vestitol and neovestitol, demonstrated anti-inflammatory activity in the concentration of 10mg / kg and antimicrobial activity in concentrations between 6 to 100μg / mL (Bruno Bueno-Silva, Severino M. Alencar, Hyun Koo, Masaharu Ikegaki, Gil VJ Silva, Marcelo H. Napimoga, and Pedro L. Rosalen, Anti-Inflammatory and Antimicrobial Evaluation of Neovestitol and Vestitol Isolated from Brazilian Red Propolis, J. Agric. Food Chem. 2013, V. 61, 4546-4550, dx.doi.org/10.1021/jf305468f).

[37] The presence of various flavonoids and phenolic acids in propolis extracts and tinctures shows that these substances act synergistically in cytostatic / cytotoxic, anti-inflammatory, healing, antimicrobial and antioxidant action. In this way, the cocktail of combined substances, even in low concentrations, will promote a potent action against pathogens. The development and production of herbal and herbal medicines has been regulated in the country with the resolutions that establish quality control methodologies for these products. Standardization studies of phytotherapics and opotherapies recommend tests in order to guarantee the quality of these classes of drugs from collection, phytochemical screening, qualitative and quantitative determination of phytochemical markers, authenticity test, processing, production, stability study, validation of analytical methodologies , packaging and product quality control.

[38] Due to its different characteristics in terms of chemical composition and multiple biological activities, as well as for presenting characteristics of production and standardized management in the mangrove areas of Alagoas-Brazil, respecting a policy of environmental preservation and conservation of the Atlantic Forest areas and mangroves, the production of fresh red propolis and hydroalcoholic extract is part of a production chain of opotherapeutic products that promotes the sustainable development of the mangrove and lagoon region and is awarded the geographical indication seal with the designation of origin.

[39] In general, propolis extracts present in their composition several active substances of different phytochemical classes, namely: flavonoids, phenolic acids, terpenes, clerodanes, xanthones, benzophenones, among others, which may present unacceptable organoleptic characteristics in a functional food. In this sense, it is necessary to develop food preparation techniques that can be more acceptable to the consumer, maintaining the functional properties of bioactive extracts of red propolis.

[40] Still, as an essential differential in view of the state of the art, functional yogurts containing pasteurized milk or whey, fruit syrup and standardized red propolis extract combined in the same functional composition proposed in the present invention, can be prepared in compositions stable without destroying or reducing the viability of the lactobacillus present in yogurt during the proposed shelf life. The yogurts proposed here use bioactive substances that will promote benefits for the gastrointestinal tract, prevent cancer of the gastrointestinal tract, menopausal discomfort. In this way, the dairy composition disclosed here can be used to solve industrial technical problems in the areas of functional dairy foods as well as some applications for use as nutraceutical food.

SUMMARY OF THE INVENTION

[41] The present invention features yoghurts enriched with standardized red propolis extract (IEEPV), consisting of yogurt produced with pasteurized milk or whey powder, a specific mixture of lactobaillus delbruekii ssp bulgaricus and Streptococcus thermophilus, standardized red propolis extract and fruit syrup. Such a semi-solid emulsive and gelatinous system was obtained by fermenting milky bacteria with pH control, followed by the addition of bioactive propolis extract and fruit syrup. The compositions were stable during the shelf life of the yogurts without drastic reduction or destruction of milk bacteria present. The flavonoids and isoflavonoids from the red propolis extract were identified by HPLC-UV. The acceptability of the compositions was also characterized by sensory analysis. Finally, uses of yoghurts enriched with red propolis extract and fruit syrup are presented for nutritional, functional and / or nutraceutical purposes to combat certain diseases of the gastrointestinal tract.

BRIEF DESCRIPTION OF THE FIGURES

[42] The modality of the invention, together with its additional advantages, can be explained and understood by reference to the attached figures and the following description:

[43] The attached Figure 1 shows the flowchart of the preparation and characterization process of milk yoghurts and whey yoghurts enriched with red propolis extract and fruit syrup.

[44] The attached Figure 2A shows Principal Component Analysis (PCA) of milk yogurt compositions enriched with red propolis extract and different flavors (strawberry syrup, guava syrup, honey and no flavor) after sensory analysis. Figure 2B shows Hierarchical Cluster Analysis (HCA) of milk yogurt compositions enriched with red propolis extract and different flavors (strawberry syrup, guava syrup, honey and no flavor) after sensory analysis.

[45] The attached Figure 3A shows Principal Component Analysis (PCA) of whey powder yogurt compositions enriched with red propolis extract and different flavors (strawberry syrup, guava syrup and no flavor) after sensory analysis. Figure 3B shows Hierarchical Cluster Analysis (HCA) of powdered whey yogurt compositions enriched with red propolis extract and different flavors (strawberry syrup and guava syrup and without flavor) after sensory analysis.

[46] The attached Figure 4 shows chromatograms of flavonoid patterns present in the red propolis extract (A), IEEPVO (B), IEEPVO + strawberry syrup (C) and final composition of IEEPV1 0.3% (D).

[47] The attached Figure 5 shows chromatograms of flavonoid patterns present in the red propolis extract (A), IEEPV6 (B), IEEPV6 + strawberry syrup (C) and IEEPV4 0.3% final composition (D).

DETAILED DESCRIPTION OF THE INVENTION

[48] The processes and compositions described in the present invention can be better detailed and understood by reference to the figures present in this document and the following description:

[49] The milked raw milk went to the pasteurization stage at the Federal Institute of Education of Alagoas, campus Satuba-AL.

[50] Milk yogurts enriched with red propolis extract and fruit syrup, as well as whey yoghurts also enriched with red propolis extract and fruit syrup were prepared with industrial milk yeast composed of lactobacillus delbruekii ssp bulgaricus and Chr Hansen Streptococcus thermophilus (Yoflex®, YF-L812).

[51] Red propolis extract was acquired by beekeepers from the city of Marechal Deodoro-Al, specifically from the Primavera apiary.

[52] Fruit syrups obtained as flavorings were obtained from the Selecta top® brand and the Nutrimel® brand honey in local stores. Whey powder and milk powder were purchased from the manufacturer Natville®. Cereal alcohol from sucroquímica®.

[53] Sensory analysis performed with pasteurized milk and whey yogurts, both enriched with red propolis extract and fruit syrup, were approved by the research ethics committee of the Federal University of Alagoas, under number CAAE: 10166113.0.0000.5013.

- Preparation of Red Propolis Extract and Tincture

[54] A 200 g sample of fresh red propolis is subjected to an extraction process by maceration in 300 to 450 mL of cereal alcohol 60 to 90 ° GL, preferably at 80 ° GL, for a period of 12 to 120 hours, preferably 72 hours. The extraction process is repeated 2 more times, which is sufficient for the complete extraction of the active substances.

[55] After the complete extraction of the active substances, the material is concentrated in a rotary evaporator, coupled to a vacuum pump, using a rotation speed of 80rpm, in a water bath in a temperature range of 35 to 55 ° C and pressure between 550 to 700mmHg.

[56] A dark red solid mass, with a yield between 50 to 70%, will be obtained with a percentage of solvent between 0.01 to 15%, being called crude extract of red propolis. The crude extract, preferably, should have a percentage of solvent between 0.01 to 3%.

[57] The red propolis tincture was obtained from the crude red propolis extract, weighing 10g of the dark red solid mass in a 200mL beaker and adding 80 mL of cereal alcohol and with the aid of an ultrasonic bath was completely solubilized and transferred to a 100 mL flask to obtain a standardized tincture of red propolis at a concentration of 10% (w / v) after checking the flask as cereal alcohol itself. A great advantage of the extract proposed here, for the preparation of IEEPV is that it is obtained directly with ethanol without the presence of toxic or harmful solvents to human health and without using the extract washing step.

- Preparation of Milk Yogurts and Whey Yoghurt powder enriched with red propolis extract and fruit syrup (IEEPV)

[58] The IEEPV1 has in its composition the ingredients: Pasteurized milk in a proportion between 70% to 95% preferably between 80% to 90%, milk yeast composed of lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus in a proportion between 0.05% to 5 % and preferably between 0.06% and 2.50%, red propolis extract in a proportion between 0.05% to 0.50% preferably between 0.10% and 0.30%, crystal and refined sugar in a proportion between 3 at 8% and preferably between 4% and 6%, whole milk powder in a proportion between 0.01 and 4% and preferably between 0.05 and 1.5%, strawberry fruit syrup in a proportion between 3 and 10% and preferably between 6 to 8%.

[59] One embodiment of the present invention, an IEEPV2 yogurt, has a composition of: pasteurized milk in a preferential proportion between 79 to 90%, milk yeast composed of lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus in a preferential ratio between 0.06 to 2.50%, red propolis extract in a proportion between 0.05% to 0.50% preferably between 0.10% and 0.30%, crystal and refined sugar in a proportion between 3 to 7%, whole milk powder between 0.01 to 2.5% and guava fruit syrup in a proportion between 6 to 8%.

[60] Another modality of the proposed invention, an IEEPV3 yogurt, has the following composition: Pasteurized milk in a proportion between 78 to 90%, milk yeast composed of lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus in a proportion between 0.06 to 2, 5%, red propolis extract in a proportion between 0.05% to 0.50% preferably between 0.10% and 0.30%, crystal and refined sugar in a proportion between 3 to 7%, whole milk powder in a proportion between 0.01 to 2.5% and honey in a proportion between 6 to 12%.

[61] Another embodiment of the proposed invention, an IEEPV0 yogurt, is presented in the following composition: Pasteurized milk in a proportion between 86 to 90%, milk yeast composed of lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus in a proportion between 0.6 to 2, 5%, red propolis extract in a proportion between 0.05% to 0.4% preferably between 0.10% and 0.30%, crystal and refined sugar in a proportion between 3 to 7%, whole powdered milk in a proportion between 0.01 to 2.5% and without flavoring.

[62] Another embodiment of the invention, an IEEPV4 yogurt, has the following composition: Pasteurized milk in a proportion between 80 to 90%, milk yeast composed of lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus in a proportion between 0.06 to 1.5 %, red propolis extract in a proportion between 0.05 to 0.5% preferably between 0.10% and 0.30%, crystal and refined sugar in a proportion between 3 to 7%, whey powder in a proportion between 0 , 05 to 2.0%, and strawberry syrup in a proportion between 4 to 10% and preferably between 6 to 8%.

[63] Another embodiment of the invention, an IEEPV5 yogurt, is presented in the following composition: Pasteurized milk in a proportion between 80 to 90%, milk yeast composed of lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus in a proportion between 0.06 to 1.5 %, red propolis extract in a proportion between 0.05 to 0.50% preferably between 0.10% and 0.30%, crystal and refined sugar in a proportion between 4 to 6%, powdered whey in a proportion between 0 , 05 to 2.0%, and guava syrup between 4 to 10% and preferably between 6 to 8%.

[64] Another modality of the proposed invention, an IEEPV6 yogurt, is presented in the following composition: Pasteurized milk in a proportion between 86 to 90%, milk yeast composed of lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus in a proportion between 0.05 to 2, 5%, red propolis extract in a proportion between 0.1% to 0.5%, crystal and refined sugar in a proportion between 3 to 7%, whole whey powder in a proportion between 0.05 to 2.5% and without flavoring.

[65] The contents of the envelope containing commercial yeast (Yoflex®, YF-L812) were previously diluted in 1000mL of UHT milk in a frozen and sterile container. Then, a pre-established amount of 11.0 mL of the reconstituted yeast was used for an amount of 5,000mL of yogurt. The reconstituted yeast was stored in a freezer to avoid uncontrolled fermentation.

[66] Raw milk, recently obtained in the milking process, was cooled to 4 ° C, transported to the Sensory Analysis Laboratory of the IFAL-Campus Satuba-AL. Raw milk was added to 1% whole milk powder and sugar, then preheated and homogenized. Pasteurization at 85 ° C followed for 15 minutes. Subsequently, the milks were cooled to 42 ° C and inoculated with lactic yeast (Yoflex®, YF-L812) in a specific amount.

[67] Fermentation was carried out for approximately 4 hours, establishing the pH 4.6 fermentation as the end of fermentation and then stored in a cold chamber at a temperature of approximately 5 ° C until the following day. The curd was broken, the red propolis tincture was preferably added between 0.1 and 0.3%, followed by homogenization and the syrup with the desired flavor was added (strawberry syrup, or guava syrup, honey) followed by homogenization. The yogurts were stored at a temperature of 5 ° C until beginning sensory analysis procedures, studies of physico-chemical and microbiological characterization. The yogurts were stored at a temperature of 5 ° C for a maximum period of 28 days (Figure 1).

[68] In the modalities of the invention, all IEEPV yogurt compositions are prepared by controlling the fermentation process by adjusting the pH to 4.6 and the amount of lactic acid between 0.70 and 0.8, where maximum coagulation occurs. .

[69] IEEPV yogurt compositions are prepared by reducing the temperature after the pasteurization step (85 ° C for 15 minutes), and it presents itself as an optimum temperature around 42 ° C for maximum replication of milk bacteria in a shorter period of time. which may vary depending on the scale of production of IEEPV yoghurts.

[70] The addition of red propolis extract in the form of red propolis tincture between 0.05 to 0.05% and preferably between 0.1 to 0.3%, as well as fruit syrup to the yogurt obtained after the process The fermentation of lactic acid bacteria occurs under constant agitation and without undergoing any pressurization process for a period between 10 to 30 minutes. The addition will occur in small quantities at regular intervals (every 2 minutes) in order to obtain yoghurt enriched with red propolis extract and uniform fruit syrup (Figure 1).

- IEEPV Acceptability Study through Sensory Analysis

[71] Yogurt acceptance tests were conducted at the Sensory Analysis Laboratory of the Federal Institute of Alagoas (IFAL), Campus Satuba-AL. Sensory analyzes were conducted in a balanced complete block design using a subjective analytical method. The treatments were evaluated individually on four different days, one day for each flavor and always by a group of 50 untrained judges composed of students and servers of the Campus, habitual consumers of yogurt. The judges evaluated the samples of yoghurts enriched with red propolis extract and fruit syrup or yogurt without fortification in individual booths under white light.

[72] In each sensory analysis, the judges received a tray with three yogurt treatments of the same flavor, with different percentages of red propolis tincture, served in disposable 50mL cups, encoded with random three-digit numbers containing approximately 25g of the IEEPV sample. The judges also received a water and salt biscuit, in addition to 200mL of water to wash the palate, as well as the evaluation form containing the nine-point structured hedonic scale anchored at the ends of "disgusted a lot" (grade 1) lowest grade , "disliked a lot" (note 2), "disliked moderately (note 3)," disliked slightly "(note 4), neither liked / disliked (note 5)," liked it slightly "(note 6)," liked it moderately " (note 7), "I liked it a lot" (note 8) and "I liked it a lot" (note 9), the highest note, through which they expressed global acceptance.

[73] Tukey's test analysis (p ≤ 0.05) with a 95% significance level was performed using the SAS® version 9.0 statistical program. Multivariate analysis (Principal Component Analysis, PCA; Hierarchical Cluster Analysis, HCA) were performed using the Statistica 12.0 test version software.

- Physico-chemical characterization of yogurts enriched with red propolis extract (IEEPV)

[74] The physical-chemical analyzes carried out on the milk occurred according to the methodology described by the Adolfo Lutz Institute (2008).

[75] Titratable acidity: To determine the titratable acidity in lactic acid, 10 ml of milk was transferred to a 100mL Becker with the aid of a pipette, adding 3 drops of the phenolphthalein solution (1%). The solution was titrated with 0.1N sodium hydroxide until a pink color appeared. To verify the result, the volume of the spent sodium hydroxide solution was observed by submitting this value to the following formula:
A = (Ci x fc x 9 x V) / Vg
Where: A = Acidity in% lactic acid; Ci = Concentration of sodium hydroxide; fc = correction factor for the sodium hydroxide solution; v = volume of spent hydroxide; g = grams of the sample.

[76] pH: The pH was determined by direct introduction of the electrode to the sample in a previously calibrated digital pot from Gehaka, model PG 1800.

[77] Density: To determine the density, a previously homogenized amount of milk was transferred to a 250mL beaker, introducing the thermolactodensimeter. The milk level was read in the upper meniscus and the density was read with the aid of the correction table at 15 ° C, expressed in g / ml.

[78] Lipids: The percentage of lipids was determined using the Gerber butyrometer, in duplicate. For each butyrometer, 10mL of sulfuric acid was transferred and then 11 ml of milk and 1 ml of isoamyl alcohol were added slowly. After corking, it was weighed and stirred vigorously until completely dissolved. The butyrometers were taken to the Cap Lab® model 8BT centrifuge at 1200 rpm for 5 minutes in the vertical position with the cork positioned downwards. After centrifugation, they were submitted to a water bath at 65 ° C for 3 to 5 minutes. The direct reading, in percentage, was made within the graduated scale of the Gerber butyrometer.

[79] Total dry extract: The total dry extract was determined using the Ackermann calculating disc. The disk was rotated until the density value (g / mL) coincided with the fat value (%) of the sample in the respective scales and the point indicated by the cursor was read in the dry extract scale, in% ( m / m).

[80] Humidity: The moisture percentage was obtained using the gravimetric method, by drying on an infrared scale to determine moisture, model MOC-120H from the Shimadzu brand. A quantity of 5 grams of the sample was dried at a temperature of 120 ° C until moisture loss of less than 0.01% was obtained.

[81] Lactose: Lactose was determined using lactose-reducing glycids. 10mL of the sample was transferred to a 100 ml volumetric flask, 50 ml of water, 2 ml of the 30% zinc sulphate solution and 2 ml of the 15% potassium ferrocyanide solution were added, mixing well after each addition. Methylene blue was used as an indicator. After sedimentation for 5 minutes, the volume was made up with water and stirred. This was followed by filtering on 300mL Erlenmeyer filter paper. 10mL of each of Fehling's solutions were transferred, added with 40 mL of water to a 300 mL flat-bottomed flask, which was heated to boiling in a hot plate. The filtrate was transferred to a 25 mL burette, and dropwise adding to the solution of the boiling flask, under agitation, until the color changes from blue to colorless, with the presence of a brick red residue at the bottom of the flask. The result was expressed through the formula:
Lactose = (V x 0.068 x 100) / (L xv)
Being: Factor 0.068 = amount in g of lactose that corresponds to 10 mL of Fehling's solution; v = volume in mL of the sample solution, spent on the titration; L = volume in mL of the sample; V = volume in mL of the sample dilution (100 mL).

[82] Ashes: The ashes were determined by muffle incineration at 550 ° C. 5 grams of the sample were weighed in a porcelain capsule previously heated in a muffle for two hours, cooled and weighed. The sample was previously oven dried at 105 ° C until drying and then incinerated for approximately 4 hours.

-Chromatographic characterization of isoflavonoids present in yogurts enriched with red propolis extract

[83] Sample preparation: Samples of red propolis extract, strawberry syrup, yogurt enriched with red propolis extract and strawberry syrup, yogurt without fortification were pipetted (1.0 ml) and transferred to 1.5 cm eppendorf tubes mL and centrifuged in a mini spin eppendorf mini-centrifuge using a rotation speed of 10,000 rpm for a period of 15 minutes.

[84] This clean-up process was used to decant proteins and other high molecular weight interferents in yogurt, as well as lipid / lipoprotein precipitation and other high molecular weight interferents.

[85] After centrifugation, aliquots of the aqueous supernatant (0.7 mL) were filtered in 0.45μm filter units and transferred to the autoinjector vials where 20 μL were injected.

[86] CLAE-UV conditions: The chromatograms were obtained in a Shimadzu chromatographic system containing high pressure pump (model LC-20AT), auto-injector (model SIL 20A), oven for column conditioning (model CTO-20A), detector diode array (model SPD-M20A) and a controller (CBM-20A) with interfacing to the computer controlled by an LC-solution software from Shimadzu.

[87] In the Conditions of the chromatographic method, a mobile phase consisting of H2O, Acetonitrile (A: B), both acidified with 0.1% formic acid, was used. The mobile phase was pumped with a flow of 1 mL / min to the Phenomenex column (C18 Jupiter 250 x 4.6 mm; 5μm) and Phenomenex pre-column (C18 4.0 x 3.0 mm; 5μm;) stabilized at a temperature of 35 ° C and the Diode Array Detector was programmed to detect at wavelength 265, 280 320 and 370 nm. The injection volume was 20 μL.

[88] The chromatographic column received a gradient elution system from the mobile phase, which consisted of the following change in the proportion of solvents (A: B), in which it started with 29% solvent B in time 0 minutes, maintaining until the time of 3 minutes; change of B percentage to 37 minutes in 8 minutes, change of B percentage to 41% in 10 minutes; changing the percentage of B to 42.5% in 12 minutes; keeping in isocratic mode for up to 14 minutes; changing the percentage of B to 47% in 16 minutes; changing the percentage of B to 55% in 18 minutes; changing the percentage of B to 78% in 27 minutes; changing the percentage of B to 87% in 30 minutes; change of the percentage of B to 100 in 38 minutes remaining in isocratic mode until 41 minutes and returning the initial condition of 29% of B in 42 minutes and maintaining in isocratic mode until 46 minutes.

[89] Analytical standards acquired from sigma-aldrich (catechin, caffeic acid, ferulic acid, p-coumaric acid, daidzein, genistein, pinobanksina, dalbergine, formononetine, isoliquiritigenina, Biochanina A, pinocembrina and crisina) and Extrasynthesis (Liquiritigenina) e exactly 2.0 mg were weighed and transferred to 10 mL volumetric flasks to obtain a concentration of 200μg / mL. The identification of the flavonoids was carried out using retention times comparative to the analytical standards using the same analytical conditions as the working day.

- Microbiological characterization of IEEPV through a viability study of lactic acid bacteria

[90] The yogurts were packaged and stored in the refrigerator after preparation and evaluated microbiologically during their useful life as well as characterized by chemical and physical-chemical methods. A 28-day deadline has been established. Yogurts were evaluated in three storage times: 1.15 and 28 days.

[91] The microbiological analyzes carried out were: determination of the Most Probable Number (NMP) of coliforms at 45 ° C, mold and yeast count, viability of total lactic acid bacteria and culture confirmation test.

[92] For microbiological analyzes, serial dilutions were performed with the removal of a 25mL aliquot of yogurt added to 225mL of sterile peptone water (APT), obtaining the first 10-1 dilution. From that, five successive dilutions were made, always removing 1mL aliquots from the previous dilution, making dilutions up to 10-6.

[93] Coliforms at 45 ° C: For analysis of coliforms at 45 ° C, the Most Probable Number (NMP) technique was used, using series of three tubes for each dilution with a 95% confidence interval. 1 ml aliquots of the 10-1, 10-2 and 10-3 dilutions were transferred to the tubes containing the Lauryl Sulfate Tryptose (LST) broth which were incubated at 35 ° C for 48 hours. After the incubation time, the positive tubes were separated, those with turbidity and gas production inside the Durham tube. One loop of each positive LST tube was transferred to tubes containing Escherichia coli broth (EC), incubated in a water bath at 45 ° C for 48 hours. Positive tubes were selected and the results were obtained in NMP / per ml of yogurt.

[94] Molds and Yeasts: For the counting of molds and yeasts, 0.1 ml of each dilution was inoculated, plating on the surface in plates previously prepared and dried with the medium Agate Potato Dextrose (BDA) spreading the inoculum with the handle of Drigalski . The plates were incubated at 30 ° C for five days. After incubation, the total mold and yeast count in CFU / mL of yogurt was determined.

[95] Total Lactic Bacteria Count: The total lactic bacteria count was performed on MRS Agar (Difco ™) culture medium, consisting of: peptone, meat extract, yeast extract, dextrose, polysorbate, ammonium citrate, acetate sodium, manganese sulfate, magnesium sulfate and dipotassium phosphate. To count lactic acid bacteria, 1mL of the 10-3, 10-4, 10-5 and 10-6 dilutions were inoculated in depth (pour-plate) with overlay. The MRS plates were incubated at 36 ± 1 ° C for 72 hours in anaerobic jars (Gaspak® Systems).

[96] Cultivation confirmation test: For the observation of their biochemical characteristics, three colonies were transferred from a plate, from each treatment, to a glass slide and a smear was performed, stained by the Gram method, obtaining a positive result. In the catalase test, three colonies on the same plate were subjected to 10% hydrogen peroxide. As the bacteria counted is catalase negative, the samples did not show effervescence. After the tests, the cultures showed proof of negative catalase and the presence of bacilli and cocci, Gram positive, being then confirmed as lactic acid bacteria.

- Uses of IEEPV dairy compositions

[97] In view of the biological activities of the red propolis extract combined with a dairy product containing lactobacillus with probiotic action, yoghurts enriched with red propolis extract can be used to improve the physiological functions of the gastrointestinal tract (GIT) and avoid certain pathologies with constipation , traveller's diarrhea, diarrhea associated with the use of antibiotics, elimination of certain viruses and rotavirus that cause diarrhea, in addition to diarrhea caused by the side effect of certain antibiotics.

[98] The combination of red propolis extract and proteins, peptides and functional amino acids from milk and whey that can be applied in a wide range of metabolic diseases with hypertension, diabetes, cardiovascular diseases, obesity, phenylketonuria, cancer, stimulate bowel functions and assist in protecting the skin from solar radiation.

[99] The use of propolis as an active ingredient in the prevention and treatment of various diseases is already widely described. However, red propolis has a different composition from other propolis already widely described in the state of the art, which indicates varied biological activities. the red propolis extract containing isoflavonoids, chalcones, isoflavans and guttiferones will inhibit the growth of pathogenic bacteria in the gastrointestinal tract (GIT): stomach (H. pylori) and intestine (E. coli) due to food infections, infections due to problems with contamination of drinking water and joint action with probiotic lactobacillus will regulate the intestinal flora, restoring normal TGI functions.

[100] Due to their proven biological antioxidant, anti-inflammatory and anticancer properties, yoghurts enriched with red propolis extract can be used as supplementary food in the treatment of stomach and bowel cancer, helping to improve the patient's quality of life. as a differential compared to that already exposed by the state of the art.

[101] The use of yogurt with functional characteristics with disease prevention characteristics combined with isoflavonoids and other flavonoids and phenolic compounds present in the red propolis extract have several biological activities, including anti-inflammatory, anti-allergic, anthelmintic, antioxidant, hepatoprotective , control of the effects of menopause (hot flashes), osteoporosis and arthritis making this new composition a combined functional and nutraceutical food.

EXAMPLES IEEPV Acceptability Study through Sensory Analysis

IEEPV1 - Iogurtes enriquecidos com extrato de própolis vermelha e calda de morango; IEEPV2 - Iogurtes enriquecidos com extrato de própolis vermelha e calda de goiaba.[102] Table 1. Frequency of grades attributed to yogurts (IEEPV1 and IEEPV2) made with whole milk powder enriched with red propolis extract and flavoring.

IEEPV1 - Yogurts enriched with red propolis extract and strawberry sauce; IEEPV2 - Yogurts enriched with red propolis extract and guava syrup.

IEEPV3 - Iogurtes enriquecidos com extrato de própolis vermelha e mel; IEEPV0 - Iogurtes enriquecidos com extrato de própolis vermelha sem flavorizante.
Tabela 3 - Valores médios do teste de aceitabilidade dos iogurtes com leite em pó integral, flavorizante e diferentes percentuais de extrato de Própolis Vermelha (EPV) .

Médias com letras em comum na mesma coluna não difere entre si a p ≤0,05 pelo teste de Tukey. 1DMS = Diferença Mínima Significativa.[103] Table 2. Frequency of grades attributed to yogurts (IEEPV3 and IEEPV0) made with whole milk powder enriched with red propolis extract and flavoring.

IEEPV3 - Yogurts enriched with red propolis extract and honey; IEEPV0 - Yogurts enriched with red propolis extract without flavoring.
Table 3 - Average values of the acceptability test of yogurts with whole milk powder, flavoring and different percentages of propolis extract (EPV).

Means with common letters in the same column do not differ from each other after ≤0.05 by the Tukey test. 1DMS = Minimum Significant Difference.

[104] Table 3 shows averages of acceptability according to the statistical test performed, it was observed that there was a significant difference (p ≤ 0.05) in relation to the different percentages of red propolis extract for some treatments of guava flavored yogurt (IEEPV) 2) as well as for flavorless yogurt (IEEPV3). However, for strawberry (IEEPV1) and honey (IEEPV3) yogurts there was no significant difference, being equally accepted by the judges obtaining averages between 6.30 and 7.16, equivalent to slightly liked and moderately liked according to the hedonic scale.

[105] Yogurts with whole milk powder had good percentages of acceptability, the highest percentage being attributed to guava yogurt (88%) despite the fact that it received greater variability in grades compared to strawberry yogurt (87.3%) . The honey yogurt obtained 74.6% of acceptance while the "natural" flavorless one received 76% of acceptance.

[106] Propolis has a strong and bitter taste, and this characteristic is related to the presence of a large number of flavonoid compounds. And in the production of yogurt the presence of this strong flavor can be masked by the addition of a certain flavoring agent, mainly strawberry syrup and guava syrup, a characteristic observed in this study through figures 2 using PCA and HCA analysis.

[107] Yogurts containing whole milk powder were subjected to Principal Component Analysis (PCA). Although the yogurts contain different flavorings and different proportions of propolis tincture, there was a certain similarity in the scores attributed by the 50 judges. In this way, multivariate analysis was used to try to differentiate these dairy compositions. Significant differences can be observed in the scores between guava yoghurts (group located in the first quadrant) in relation to strawberry yogurt (group located in the second quadrant) and honey yogurt (group located in the third quadrant). However, natural yogurt (without flavoring) showed scores close to guava yogurt and strawberry yogurt (grouping located between the first and the second quadrant). This leads us to an important conclusion, the judges perceived the taste of strawberry flavoring differently, in relation to guava and honey flavoring and at the same time masking in their perception the bitter taste of red propolis extract (Figure 2A).

[108] In the hierarchical analysis of Clusters (HCA), it is possible to interpret the homogeneity or heterogeneity between the compositions of different flavorings, mainly using the complete bonding method, in which the highest level of aggregation is the least similar (the base tree diagram) and using the simple link method in which the distance between the two clusters (clusters) is determined by the distance of two closest objects (closest neighbors) in the different groups. This hierarchical tool will establish a classification system among the analyzed samples in which each composition, depending on its Euclidean distance (<shortest distance), will present greater similarity in relation to the group. the HCA diagram made it possible to identify that IEEPV1 strawberry yogurts (0.1 to 0.3% EPV) had an Euclidean connection distance <13 (~ 12.5) and, therefore, more similar, whereas guava yogurts ( 0.1 to 0.3%) had a binding distance of 13.5, followed by IEEPV0 flavorless yoghurts (0.1 to 0.3% EPV) with a binding distance of 14.9 and IEEPV3 honey yoghurt ( 0.1 to 0.3% EPV) with Euclidean connection distance of 15.4 which were the most dissimilar to each other. We can also highlight, through the HCA analysis, that IEEPV1 strawberry yogurts (0.1 to 0.3% EPV) and IEEPV2 guava yoghurts (0.1 to 0.3% EPV) showed some similarity within the same group. Thus, it is possible to observe that the different percentages of red propolis tincture did not interfere in the global acceptance of yogurts, however the flavor of the flavoring agent (strawberry syrup and guava syrup) used has a strong influence on acceptance and were more similar in the same group and different between groups (Figure 2B).

[109] Yogurts made with whey with stabilizer in different whey percentages (0.5%, 1.0%, 1.5% and 2.0%) were evaluated to establish the ideal whey percentage. milk and compared to yogurts containing red propolis extract and fruit syrup.

IEEPV4 - Iogurtes de soro de leite em pó enriquecidos com extrato de própolis vermelha e calda de morango; IEEPV5 -Iogurtes de soro de leite em pó enriquecidos com extrato de própolis vermelha e calda de goiaba.
Tabela 5. Valores médios do teste de aceitabilidade dos iogurtes de soro de leite, flavorizantes e diferentes percentuais de extrato de própolis vermelha (EPV).

Médias com letras em comum na mesma coluna não difere entre si a p ≤0,05 pelo teste de Tukey. ¹Diferença Mínima Significativa.[110] Table 4. Frequency of grades attributed to yogurts (IEEPV4 and IEEPV5) made with powdered whey enriched with red propolis extract and flavoring.

IEEPV4 - Whey powder powder enriched with red propolis extract and strawberry sauce; IEEPV5 -Whey powder powder enriched with red propolis extract and guava syrup.
Table 5. Average values of the acceptability test of whey yoghurt, flavoring and different percentages of red propolis extract (EPV).

Means with common letters in the same column do not differ from each other after ≤0.05 by the Tukey test. ¹Significant Minimum Difference.

[111] Whey and fruit pulp have acidic properties that aid in the pH and acidity requirements of the yogurt contributing to the formation of the gelatinous consistency (increase in viscosity) of this new whey yogurt composition, in addition to providing shine, pleasant taste and aroma to this new composition. This physicochemical property of whey added to the new composition promoted improvements in the composition, reflected in its acceptability characteristics during studies of sensory analysis.

[112] Yogurts made with different whey percentages (0.5, 1.0, 1.5 and 2.0%), as well as yogurts made with 1.0% whey containing strawberry syrup or guava syrup (IEEPV 4 or IEEPV5) (Table 4), fortified with red propolis extract (0.1%, 0.2% and 0.3%) were analyzed using Principal Component Analysis (PCA) (Figure 3A ). Differences were observed between the scores for the whey yoghurts in different percentages mentioned above, demonstrating a great variability in the scores between the composition that presented 0.5% of serum in relation to the others. The composition containing 1% of whey showed values of scores very close to the whey yogurt with 1.5% and greater proximity in the scores with the whey yoghurts containing strawberry syrup enriched with propolis extract (IEEPV4) and demonstrated little proximity to yogurts containing whey with guava syrup (IEEPV5). Yogurt with whey had good percentages of acceptability, with the highest percentage attributed to strawberry yogurt (80.7%) and guava yogurt (78.7%).

[113] Cluster hierarchical analysis (Figure 3B), using the complete bonding method, showed little similarity for formulations containing whey. The formulations containing strawberry syrup (IEEPV4) had a Euclidean bonding distance of 13.2, while the formulations containing guava syrup (IEEPV5) had a Euclidean bonding distance of 15.9. Thus, it can be said that whey formulations with strawberry syrup showed greater similarity within the group (IEEPV4). Yoghurts containing whey were also able to mask the bitter and strong taste of EPV.

- Physico-chemical characterization of yogurts (IEEPV)

[114] The physicochemical assessments of yogurts (IEEPV) using lactobacillus delbruekki spp bulgaricus and Streptococcus thermophilus bacteria and enriched with red propolis extract and fruit syrup for antioxidant, antifungal, anti-inflammatory and antimicrobial purposes. The results obtained are shown in table 6 and demonstrate that the yogurts showed stable pH and titratable acidity and consistent with these compositions containing fermenting bacteria at pH 4.6. The presence of red propolis extract and fruit syrup that contains phenolic acids and flavonoids in their compositions, did not change the physical-chemical stability of these dairy compositions.

[115] For yoghurts containing un enriched whole milk powder (value of lactose 2.83), yogurt with whole milk powder and enriched with propolis extract and strawberry sauce (lactose value 3.61) and elaborated yogurts with whole milk powder and strawberry syrup presented the highest values (lactose value 4.35). For yogurts made with powdered whey, lower lactose values were obtained and ranged between 2.73 (non-enriched powdered whey yogurt), 3.29 (whole powdered milk yogurt enriched with extract of propolis and strawberry syrup) and 3.58 (yogurt enriched with strawberry syrup extract). The red propolis extract is helping to reduce the sugar content in yogurts (Table 6).

Valores expressos em médias ± desvio padrão para os tratamentos de iogurtes de morango analisadas em triplicatas. * Acidez em % de ácido lático. IEEPV0 (não enriquecido); IEEPV0* (enriquecido apenas com calda de morango); IEEPV1 (enriquecido com Extrato de própolis e calda de morango); IEEPV6(não enriquecido); IEEPV6* (enriquecido apenas com calda de morango); IEEPV4 (enriquecido com Extrato de própolis e calda de morango).[116] Table 6 shows the physicochemical profile of yogurts made with powdered milk (IEEPV1) or powdered whey (IEEPV4) enriched with red propolis extract and strawberry syrup.

Values expressed as means ± standard deviation for strawberry yogurt treatments analyzed in triplicates. * Acidity in% of lactic acid. IEEPV0 (not enriched); IEEPV0 * (enriched only with strawberry syrup); IEEPV1 (enriched with Propolis extract and strawberry sauce); IEEPV6 (not enriched); IEEPV6 * (enriched only with strawberry syrup); IEEPV4 (enriched with Propolis extract and strawberry sauce).

[117] Yogurts made with powdered whey showed lower lactose values compared to yogurts made with whole milk powder, resulting in healthier compositions. The improvement in the digestibility of this disaccharide occurs due to the lactase activity conferred by the presence of lactic cultures (Table 7).

IEEPV0 (não enriquecido); IEEPV0* (enriquecido apenas com calda de morango); IEEPV1 (enriquecido com Extrato de própolis e calda de morango); IEEPV6(não enriquecido); IEEPV6* (enriquecido apenas com calda de morango); IEEPV4 (enriquecido com Extrato de própolis e calda de morango) . *(% Ácido láctico). Médias com letras em comum na mesma coluna não difere entre si a p ≤0,05 pelo teste de Tukey.[118] Table 7. Post-acidification stability study of yogurts with whole milk powder (IEEPV0 and IEEPV 1) and yogurts with powdered whey (IEEPV 6 and IEEPV 4) without fortification or enriched with red propolis extract and strawberry syrup during storage period.

IEEPV0 (not enriched); IEEPV0 * (enriched only with strawberry syrup); IEEPV1 (enriched with Propolis extract and strawberry sauce); IEEPV6 (not enriched); IEEPV6 * (enriched only with strawberry syrup); IEEPV4 (enriched with Propolis extract and strawberry sauce). *(% Lactic acid). Means with common letters in the same column do not differ from each other after ≤0.05 by the Tukey test.

[119] Post-acidification stability studies are a significant decrease in pH and a significant increase in acidity during storage, under refrigeration. This is because the lactic acid bacteria continue their metabolic activities even after the fermentation conditions have ended (DEETH, HC, TAMIME, AY Yogurt: nutritive and therapeutic aspects. Journal of Food Protection, v. 44, n. 1, p. 78- 86, 1981.). The compositions (IEEPV0 * and IEEPV1; IEEPV6 * and IEEPV4) that were enriched only with strawberry syrup or enriched with strawberry syrup and red propolis extract showed a small reduction in pH values and titratable acidity, however there was no significant difference between the samples from day 1, day 15 and day 28, demonstrating that all compositions were stable for the period of 28 days and no breakage of the emulsion or phase separation was observed in this period.

- Chromatographic characterization of yogurts (IEEPV)

[120] The chromatographic profile of the flavonoids present in the red propolis, obtained from the tincture used to fortify the yoghurts. The presence of 15 chemical compounds is observed. It was possible to separate some phenolic compounds present in red propolis, in particular liquiritigenin (6.80 min.), Isoliquiritigenin (18.5 min), formonontein (19.0 min) and biochanin A (20.0 min) (Figure 4A). The chromatographic profile of the strawberry pulp (figure 4B) used as a flavoring in the yogurt compositions also showed some phenolic compounds in the range between 3.0 and 6.0 minutes. Generally, the most commonly found phenolic compounds in strawberries are catechins and anthocyanins, which have high polarity characteristics and are easily eluted by the reverse phase C18 column. The powdered milk yogurt and strawberry pulp (IEEPV 0 *) presented a chromatographic profile very similar to the whey yogurt and strawberry pulp with protein elution over 40 minutes and elution and protein in 2.5 minutes (Figure 4C ). Powdered milk yogurt containing red propolis extract and strawberry pulp (IEEPV 1) (Figure 4D) showed chromatographic peaks of catechins and isoflavonoids, as well as proteins in the same retention time range similar to the isolated components.

[121] Figure 5A shows the profile of yogurt made with whey (IEEPV 6) without flavoring and without red propolis tincture. There was an absence of peaks in the range mentioned for the flavonoids of red propolis and for the phenolic compounds of strawberry pulp. Some chromatographic peaks eluting in 2.5 minutes and 02 peaks over 40 minutes identify sugars and proteins, respectively that are present in yogurt. In figure 5D it is different, of the 15 compounds detected in the extract, 10 are present in strawberry yoghurts made with whey and enriched with 0.3% of red propolis extract (IEEPV 4). The compounds not identified in this yogurt were: pinobanksina, luteonina and pinocembina.

- Microbiological characterization of yogurts (IEEPV)

[122] The data in table 8 are in accordance with Resolution No. 5 of 2007 of the Ministry of Agriculture, Livestock and Supply (MAPA) which establishes the identity and quality standards of fermented milks, the count of lactic bacteria in yogurt until its final expiration date must be a minimum of 107 CFU / mL.

* Padronizado de acordo com instruções do fabricante de fermento para iogurte. Tratamentos: IEEPV0 e IEEPV6 (amostras não enriquecidos e elaboradas com leite em pó integral e elaboradas com soro de leite em pó, respectivamente);IEEPV0* (Iogurte elaborado com leite em pó integral e calda de morango); IEEPV1(Iogurte elaborado com leite em pó e calda de morango, e enriquecido com extrato de própolis vermelha) ; IEEPV6* (Iogurte elaborado com soro de leite em pó e calda de morango); IEEPV6 (Iogurte elaborado com soro de leite em pó, calda de morango, enriquecido com extrato de própolis vermelha).[123] Table 8. Microbiological stability study during storage of strawberry yogurts stabilized with whole milk powder or whey powder enriched with red propolis extract and strawberry syrup.

* Standardized according to yogurt maker's instructions. Treatments: IEEPV0 and IEEPV6 (samples not enriched and made with whole milk powder and made with powdered whey, respectively); IEEPV0 * (Yogurt made with whole powdered milk and strawberry sauce); IEEPV1 (Yogurt made with powdered milk and strawberry sauce, and enriched with red propolis extract); IEEPV6 * (Yogurt made with powdered whey and strawberry syrup); IEEPV6 (Yogurt made with powdered whey, strawberry syrup, enriched with red propolis extract).

[124] For 15 days, yogurts obtained counts within the limits established by legislation and, at 28 days (life time), the results showed the minimum recommended value for all samples, 107 CFU / mL.

[125] In general, it was found that yoghurts enriched with red propolis extract behaved similarly to non-enriched yogurts both for indicators of hygienic-sanitary conditions and for counting total lactic acid bacteria. In microbiological terms, there were no differences between yogurts made with powdered whey or whole milk powder.

Claims (40)

Composition of yogurt enriched with red propolis extract characterized by the fact that it comprises cultures of lactic bacteria Lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus. Composition of yoghurt enriched with red propolis extract according to claim 1, characterized by the fact that it is obtained by the fermentative process of pasteurized milk. Composition of yogurt enriched with red propolis according to claim 1, characterized by the fact that it is supplemented with standardized extract of Brazilian red propolis between 0.05% to 0.5% and preferably between 0.1% to 0.3%. Composition of yogurt enriched with red propolis extract according to claims 1 and 2, characterized by the fact that it is supplemented with strawberry fruit syrup as a flavoring in a preferential concentration between 6% and 8%. Composition of yogurt enriched with red propolis extract according to claims 1 and 2, characterized by the fact that it is supplemented with guava fruit syrup as a flavoring in a preferential concentration between 6% and 8%. Composition of yogurt enriched with red propolis extract according to claims 1 and 2, characterized by the fact that it is supplemented with honey as a flavoring in a preferential concentration between 6% and 12%. Composition of yogurt enriched with red propolis extract according to claim 1, characterized by the fact that it is added with whole milk powder as a stabilizer for lactic acid bacteria in a concentration preferably between 0.50% and 2.00% during the fermentation process. Composition of yogurt enriched with red propolis extract according to claim 1, characterized by the fact that powdered whey is added as a stabilizer for lactic acid bacteria, preferably in a concentration between 0.05 and 2.00% during the fermentation process. Composition of yogurt enriched with red propolis extract according to claims 1 to 3, characterized by the fact that it consists of organoleptic and sensory properties of taste and aroma due to the yogurt due to the presence of lactic bacteria and flavoring of strawberry syrup that mask the bitter taste of red propolis extract. Composition of yoghurt enriched with red propolis extract according to claims 1 to 3, characterized by the fact that it consists of organoleptic and sensory properties of flavor and aroma proper to yogurts due to the presence of lactic bacteria and flavoring of guava syrup that mask the bitter taste of red propolis extract. Composition of red propolis extract-enriched yogurt according to claims 1 to 3, characterized by the fact that it comprises sensory properties of acceptable taste and aroma proven through studies of sensory analysis and multivariate analysis for yogurts stabilized with whole milk powder and syrup strawberry or guava sauce. Composition of red propolis extract enriched yogurt according to claims 1 to 3, characterized by the fact that it comprises of acceptable sensory properties of taste and aroma proven through studies of sensory analysis and multivariate analysis for yogurts stabilized with powdered whey and strawberry syrup or guava syrup. Composition of yogurt enriched with red propolis extract according to claims 1 to 3, characterized by the fact that it is stable for 28 days by physical-chemical analysis through a post-acidification test. Composition of yogurt enriched with red propolis extract according to claims 1 to 3, characterized by the fact that it is stable for a period of 28 days by microbiological analysis by testing total lactic bacteria. Composition of yogurt enriched with propolis extract according to claims 1 to 3, characterized by the fact that it contains isoflavonoids and phenolic compounds determined by high performance liquid chromatography. Composition of yoghurts enriched with red propolis extract characterized by the fact that it comprises the use of cultures of lactic bacteria Lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus combined in the production of natural yoghurts, as well as lactic beverages, more preferable for the production of artisanal and industrialized yoghurts. Compositions of yoghurts enriched with red propolis extract, according to claim 15, characterized by containing, among its components, lactic bacteria Lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus and derived compounds preferably standardized extract of red propolis, to assist treatment of diseases of the gastrointestinal tract such as colds, as well as improving the physiological functions of GIT. Compositions of yoghurts enriched with red propolis extract, according to claim 15, characterized by containing, among its components, lactic bacteria Lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus and derived compounds preferably standardized extract of red propolis, to assist treatment of diseases of the gastrointestinal tract such as diarrhea, traveller's diarrhea, diarrhea caused by the use of antibiotics, as well as diarrhea caused by certain viruses. Compositions of yoghurts enriched with red propolis extract, according to claim 15, characterized by containing, among its components, lactic bacteria Lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus and derived compounds preferably standardized extract of red propolis, to assist treatment of diseases of TGI as in growth inhibition of pathogenic bacteria in the stomach (H. pylori) and intestine (E. coli) due to food infections, infections due to drinking water contamination problems and joint action with probiotic lactobacillus will regulate the intestinal flora restoring functions normal TGI. Compositions of yoghurts enriched with red propolis extract, according to claim 15, characterized by containing, among its components, lactic bacteria Lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus and derivative compounds preferably standardized extract of red propolis, to assist treatment of metabolic diseases with hypertension, diabetes, cardiovascular diseases, obesity, cancer, phenylketonuria, stimulate bowel functions and help protect the skin against solar radiation. Compositions of yoghurts enriched with red propolis extract, according to claim 15, characterized by containing, among its components, lactic bacteria Lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus and derivative compounds preferably standardized extract of red propolis, to assist treatment of diseases as an anti-inflammatory, anthelmintic, antioxidant, hepatoprotective, control of the effects of menopause (hot flashes), osteoporosis and arthritis making this new composition a combined functional and nutraceutical food. Process of obtaining yoghurts enriched with red propolis extract characterized by the fact that it comprises steps of:

• a) Selection of raw milk;
• b) Addition of whole powdered milk or commercial powdered whey;
• c) Pasteurization of raw milk with milk or whey at a temperature of 85 ° C for 15 minutes;
• d) Cooling of the added and pasteurized milk to a temperature of 42 ° C;
• d) Addition of lactic bacteria culture reconstituted with UHT milk as a stabilizing agent;
• e) Fermentation of additive and pasteurized milk in
• temperature of 42 ° C for a period between 3.5 to 6 hours until the pH of the milk drops to 4.6;
• f) Cooling of fermented milk to a temperature of 25 ° C;
• g) Breaking of the curd to form yogurt;
• h) Enrichment of yogurt with red propolis extract;
• i) Flavoring of yogurt enriched with extract of
• red propolis and fruit sauce;
• j) Homogenization and filling of yoghurts enriched with red propolis extract;
• k) Storage at a temperature of 5 ° C;
• l) IEEPV acceptability study through sensory analysis;
• m) Physical-chemical tests of IEEPV;
• n) IEEPV chromatographic tests;
• o) IEEPV microbiological tests.

Process of obtaining yogurt enriched with red propolis extract, according to claim 21, characterized by the fact that, in step "d)" the addition of lactic bacteria with culture of Lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus in a concentration between 0.06 to 1.5%, and preferably between 0.5 and 1.3%, and more preferably between 0.8% and 1.2%. Process of yogurt enriched with red propolis extract, according to claim 21, characterized by the fact that, in step "e)" the fermentation time may vary the reconstitution of the culture of lactic bacteria called Lactobacillus delbruekii SSP bulgaricus and Streptococcus thermophilus can occur between 3 , 5 to 4 hours in semi-industrial processes and between 4 to 6 hours in industrial processes. Process of obtaining yogurt enriched with red propolis extract, according to claim 21, characterized by the fact that, in step "h)" the enrichment of yogurt with red propolis extract occurs in a concentration between 0.05% to 0.5% and preferably between 0.1% to 0.3%. Process of obtaining yogurt enriched with red propolis extract, according to claim 21, characterized by the fact that, in step "i)" the flavoring of yogurt enriched with red propolis extract occurs with strawberry fruit syrup, or fruit syrup from guava, or with honey. Process of obtaining endogenous milk yeast in stabilized powder, according to claim 21, characterized by the fact that, in step "k)" the yoghurts enriched in red propolis extract will be stored for a period of 28 days. Process of obtaining stabilized endogenous milk yeast powder, according to claim 21, characterized by the fact that, in step "1)" the yoghurts enriched in red propolis extract, whole milk powder and strawberry syrup as flavoring showed acceptability of 87, 3% by the tasters. Process of obtaining endogenous milk yeast in stabilized powder, according to claim 21, characterized by the fact that, in step "1)" the yoghurts enriched in red propolis extract, whole milk powder and guava syrup as flavoring showed acceptability of 88, 0% by the tasters. Process of obtaining stabilized powdered endogenous milk yeast, according to claim 21, characterized by the fact that, in step "1)" the yoghurts enriched in red propolis extract, whole milk powder and honey as flavoring presented acceptability of 74.6% by the tasters. Process of obtaining endogenous milk yeast in stabilized powder, according to claim 21, characterized by the fact that, in step "1)" the yogurts enriched in red propolis extract, whey and strawberry syrup as flavoring showed 80.7% acceptability by tasters. Process of obtaining stabilized powdered endogenous milk yeast, according to claim 21, characterized by the fact that, in step "1)" the yogurts enriched in red propolis extract, whey and guava syrup as flavoring showed an acceptability of 78.7 % by the tasters. Process of obtaining endogenous milk yeast in stabilized powder, according to claim 21, characterized by the fact that, in step "m)" the yogurts enriched in red propolis extract, whole powdered milk or whey with stabilizer and strawberry syrup as flavoring showed physical-chemical stability through the post-acidification test for a period of 28 days and without phase separation of the emulsive system. Process of obtaining endogenous milk yeast in stabilized powder, according to claim 21, characterized by the fact that, in step "n)" the yogurts enriched in red propolis extract, whole powdered milk or whey with stabilizer and strawberry syrup as flavoring showed isoflavonoids present in the red propolis extract in chromatographic analysis. Process for obtaining endogenous milk yeast in stabilized powder, according to claim 21, characterized by the fact that, in step "o)" the yogurts enriched in red propolis extract, whole powdered milk or whey with stabilizer and strawberry syrup as flavoring showed microbiological stability by testing total lactic bacteria for a period of 28 days. Use of yogurt enriched with red propolis extract, according to claims 1 to 3, characterized by being either alone or as an adjuvant agent, for treating diseases of the gastrointestinal tract such as colds, as well as improving the physiological functions of GIT. Use of yogurt enriched with red propolis extract, according to claims 1 to 3, characterized by being alone or as an adjuvant agent, for the treatment of diseases of the gastrointestinal tract such as diarrhea, traveller's diarrhea, diarrhea caused by the use of antibiotics, as well as diarrhea caused by certain viruses. Use of yogurt enriched with red propolis extract, according to claims 1 to 3, characterized by being either alone or as an adjuvant agent, for treating diseases such as inhibiting the growth of pathogenic bacteria in the stomach (H. pylori) and intestine (E. coli) and joint action with probiotic lactobacillus will regulate the intestinal flora, restoring normal TGI functions. Use of yogurt enriched with red propolis extract, according to claims 1 to 3, characterized by being alone or as an adjuvant, for treating metabolic diseases with hypertension, diabetes, cardiovascular diseases, obesity, cancer, phenylketonuria, stimulating bowel and auxiliary functions in protecting the skin from solar radiation. Use of yogurt enriched with red propolis extract, according to claims 1 to 3, characterized by being isolated or as an adjuvant agent, for treating diseases such as an anti-inflammatory, anthelmintic, antioxidant, hepatoprotective, control of the effects of menopause (hot flashes) , osteoporosis and arthritis making this new composition a combined functional and nutraceutical food.

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