BR102013015842A2 - production of kefir grain-fermented sheep's milk with antimicrobial activity - Google Patents

Abstract

production of sheep's milk fermented by kefir grains with antimicrobial activity. This document proposes the manufacture of a low-cost functional food rich in minerals, proteins, low lactose, added with multiple probiotic strains, with antimicrobial substances capable of killing and / or inhibiting clinically relevant pathogenic microorganisms. your shelf life. In the manufacture of this fermented milk, low cost and short time raw materials are used, enabling industrial production and minimizing costs. The process of obtaining a new ke? r fermented sheep's milk with antimicrobial activity is included in the field of applied biotechnology and food science and technology.

Description

REPORTING DESCRIPTION OF THE INVENTION Patent PRODUCTION OF KEFÍR GRAIN-FERMED SHEEP

ANTIMICROBIAN

This document proposes the production of a functional food with the production of low cost, rich in minerals, proteins, low inactivity, added probiotic microorganisms, with and antimicrobial substances capable of killing and / or inhibiting pathogenic microorganisms. clinical relevance. In the manufacture of this fermented milk, low cost and short time raw materials are used, enabling industrial production and minimizing costs. The process of obtaining a new Kefir-fermented sheep's milk with antimicrobial activity is included in the field of applied biotechnology and food science and technology. The high level of resistance of bacterial clinical isolates results in decreased efficiency of conventional antibiotics and further studies are needed to identify alternative strategies for infectious disease prevention and control (Chifiriuc et al., 2011).

Because of this, efforts have been made to develop and study new compounds that are not part of conventional antibiotic therapy. These include new organic compounds, new isolated peptides and probiotic strains. A probiotic can be a single strain or a mixture of different organisms and are used to improve well-being through immunomodulatory responses and barrier against pathological process activities (Ferreira, 2012). Kefír is an example of a mixture of probiotics (Rodrigues et al., 2005).

References from a recent study show that cow's milk fermented with kefir grains beyond its probiotic potential showed antibacterial, immunostimulating, antitumour activity! and lower cholesterol levels (Chifiriuc et al., 2011), thus differing from conventional probiotics, proving to be a great functional food.

Functional foods, which promise to help cure or prevent disease, are the new trend of the powerful food market at the beginning of the 21st century. Yoghurts and fermented milks promise to help cure or prevent diseases such as cardiovascular disease, certain cancers, allergies, intestinal problems, etc. (Raud, 2008). The growing consumption power of Brazilians in recent years, coupled with the concern to live longer and better, has been consolidating a promising scenario for the functional food market, estimated by the consultancy Euromonitor at US $ 4 billion a year in the country. These are products such as nutraceuticals, food-medicine hybrids, such as omega-3 fish oil capsules, or functional foods themselves, such as yogurts, cereals or margarines, enriched with fiber, vitamins or probiotics (D AT AMAR K, 2012). The world market for functional food is expected to grow 38 percent by 2017 to $ 207 billion. In 2011, this business earned $ 150 billion (DATAMARK, 2012).

A major stumbling block in scientific research on probiotic fermented milk is the lack of research by other bioactivities. In addition to the fact that most of the research carried out involves the manufacture, evaluation of physicochemical quality and sensory analysis of fermented milk is described in many Brazilian studies using different known strains, we can mention the use of Lactobacillus buigarícus and Streptococcus thermophifus (Soares et al. al., 2011); Lactobacillus acidophiíus and Weissel / a confused (Viegas et al., 2010); Streptococcus thermophilus, Bifidobacterium longum and Lactobacillus acidophius (Zacarchenco & Massaguer-Roig, 2006). Fermented milk resulting from the addition of kefir grains has a double fermentation, because kefir through its microbiological and chemical composition can be considered a complex probiotic product, as it has in its microorganism composition as: yeasts, lactic acid bacteria and acetic acid bacteria, in symbiotic association and surrounded by a matrix of polysaccharides referred to as kefirans, capable of improving the intestinal microbial balance producing beneficial effects on the health of the individual who consumes it (Wrobiewska et al,} 2009).

During the fermentation process kefir grains produce a variety of ingredients that give it the original flavor and texture, as well as other bioactive ingredients (Farnworth, 2005; Wiííhuhn et al., 2005). Commercial-scale production of kefir-fermented milk already exists in some countries by Danone® in Russia and Saudi Arabia (GROUPE DANONE, 2007), however, most industries use only a few bacterial samples isolated from kefir grains. kefir for beverage production, which means that many of the natural properties found in the beverage with a complex microbiota are not yet found in commercial products. Weschenfelder et al. (2011) commented that, although not yet industrialized in Brazil, kefir has been gaining adherents in various regions of the country. Its preparation, only in artisanal scale, results in a product with physicochemical, sensory and microbiological characteristics not yet well defined. There are few studies regarding the bioactivity of kefir fermented milk here in Brazil, since research is related to the mode of manufacture, sensory analysis and microbiological content of this fermented milk, as an example we have the work of Montanuci et al. (2010) who analyzed eight formulations of kefir fermented wite and sensorially analyzed these. Magalhães et al. (2010) studied the production of fermented milk with the addition of whey using kefir grains and evaluated the microbiological variation of this milk. In 2011 Magalhães et al. published a study on biochemical changes and the formation of volatile compounds during the production of new fermented milk added with whey.

Machado et al. (2012) conducted a technological monitoring related to the production of fermented milk with kefir grains, to evaluate the world panorama of process and product protection, investigating patent documents filed by country of origin, in this study among the 527 documents that make reference Regarding the use of kefir, Brazil presented only 8 patents filed, so research with kefir grains should be directed and qualified.

One of kefir's main bioactivities is its ability to inhibit the growth of microorganisms that are not part of its microbiota. Therefore pathogenic microorganisms can be inhibited by peto kefir (OtBes and Cagindí, 2003). The well diffusion antimicrobial activity of molasses-grown kefir was confirmed in the work of Rodrigues et al. (2005) who evaluated the healing and antimicrobial activity of kefir and kefiran extract against the microorganisms Staphylococcus aureus ATCC 6536, Streptococcus salivaríus ATCC 39562, Streptococcus pyogenes ATCC 17568, Pseudomonas aeruginosa ATCC 27853, Salmonelia tyCCimier 4857 and Escheríchia coli ATCC 8739. In this study all microorganisms used were inhibited by kefir use and the zone of inhibition of kefir suspension was slightly larger than the zones of the drugs used (amptyline, azyiromycin, ceftriaxone and oxalicine).

In the study by Chifiriuc et al. (2011) Cow's milk was fermented by kefir for 24h and for 48h and preserved for 7 days at 4-8 ° C and the well diffusion antimicrobial activity of these samples was tested against the bacteria Bacífus subtilis spp. spízizenií ATCC 6633, Staphylococcus aureus ATCC 6538, Enerococcus faecatis ATCC 29212, Escheríchia coli ATCC 8739, Salmonelia enteritidis ATCC 13076, Pseudomonas aeruginosa ATCC 9027. Antimicrobial activity of 24h as well as 48h of fresh-fermented preservation after 4-7 days 8 ° C was similar and observed against B. subtilis, S. aureus, E. coli, E. faecatis and S. enteritídis. For E. coli, S. enteritidis and B. subtilis the antimicrobial activity was superior to control antibiotics and for E. faecatis and S. aureus superior to an antibiotic. The products tested showed no activity against P. aeruginosa. Our work showed antimicrobial activity of fermented sheep's milk against the microorganisms Micrococcus iuteus, Escherichia coli ATCC 25922, Klebisiefla pneumonae ATCC 29665, Pseudomonas aeruginosa ATCC 27853, Listaria innocua, Baciilus subtilis ATCC 6633 and Staphylococcus 6538.

Our activity was similar to some strains already tested in the work of Chifiriuc eí af. (2011) who used cow's milk, but we evaluated whether this activity was bactericidal or bacteriostatic, as well as testing the antimicrobial activity during storage time at ~ 1h, 7 days, 14 days, 21 days and 28 days, another positive factor was that Our activity was carried out in liquid medium where there is also the dispersion of the fermented on pathogenic microorganisms differing from the evaluation in solid medium.

Since the 1990s, companies have been focusing on functional products to reinforce their positioning in the “healthy” niche. Scientific research and technological applications have enabled the launch of products that have regained their central place in consumers' daily diets, this time thanks to the addition of ingredients that claim specific health benefits. Following the unexpected success of the launch of Yakult on the European market in 1994, several European companies, such as Danone and Nestlé, began offering yoghurt and fermented milks focused on intestinal health. Within a few years, a real revolution has occurred in the European dairy market, based on technological innovation and a new concept: the daily dose that ensures digestive comfort and well-being (Raud, 2008).

Currently in our market are consumed yoghurts and fermented milk with probiotic capacities produced by major companies: Yakult®, Danone® and Nestlé®, and the drinks consumed: * Yakult® Fermented Milk is a lactobacillus-fermented skim milk-based food. selected, the unique probiotics Lactobaciiius casei Shirota, which resist the acidity of the stomach like no other and come alive more in the gut to help regulate bowel functions and protect the digestive system. • Yakult 40® contains 40 billion Lactobacillus casei Shirota probiotics as active ingredients that help adults maintain a balanced intestinal microbe. Very important factor due to decreased resistance and increase of harmful bacteria in the intestines as the individual ages. Poor diet, medication (especially antibiotics and laxatives), and alcohol associated with a busy life interfere with proper bowel function. • Activia® is a probiotic food as it is a fermented dairy product with traditional yogurt bacterial strains combined with a specific culture: DanRegularis, named for the probiotic Bifidobacterium animalis DN173010. Activia is considered a functional food as it promotes beneficial effects on human health and helps keep the intestines in rhythm. • Actimei® is a fermented milk with Lactobacillus casei defensis, a unique lactobacillus that arrives alive in the intestinal flora, where most of its defenses are. • Molico Total Calcium® with Probiotic Action is a new light yogurt rich in vitamins A and D with light and tasty strawberry and plum fruit pulp. With just 2 servings, you meet 100% of your daily calcium requirement and help balance the intestinal flora. Bifídobacterium animalis (subspecies B. lactis) contributes to the balance of intestinal flora. Its consumption must be associated with a balanced diet and healthy lifestyle habits. • Chamyto® Fermented Milk is good for health as it contains Nestlé® exclusive live lactobacilli. As mentioned above the probiotic market is growing and when further studies on the bioactivity of this product are added it adds more value to these foods. and health to consumers.

Our product differs from products already marketed in that it has: * Multiple probiotic strains; • Sheep milk compared to cow's milk has the same lactose content and the levels of fat, protein and some minerals are much higher, thus making a food rich in nutrients; • Besides having bactericidal and bacteriostatic activity against pathogenic strains of relevance in food and hospital infections; * Antimicrobial activity throughout storage.

In Activia® fermented milk produced in Turkey there are some strains of Kefsr yeast and bacteria, this product is consumed on a regular basis every day and helps to regulate the digestive system in a natural way and offers beneficial health effects of kefir. The big difference from our work is that this fermented milk is produced with isolated strains and not with kefir grains, and has no other confirmed bioactivity besides being probiotic.

In the literature the use of fermented milk as a probiotic is quite extensive, but used as an antimicrobial agent there are few published data. The potential of the present patent is to place a low cost functional food on the market, capable of improving the intestinal microflora, because besides being probiotic its metabolites act quickly killing pathogens of clinical interest. In its production process it does not require additional costs, it does not require sophisticated machinery and specialized labor demand, thus being a technologically viable production.

In addition to this product is produced with sheep's milk that contain nutritional factors that influence the composition of dairy products. The processing of sheep's milk derivatives, focusing on milk fat and protein concentrations, contains bioactive compounds, aromatic compounds that improve organoleptic characteristics. The high concentration of fat in the milk is marked by higher levels of fatty acids such as conjugated oleic acid and omega-3, which is very healthy for the human diet, as they reduce the amount of bad cholesterol in the body (Pulina et a !, 2006).

The formation of eating habits is a complex action that suffers interference from several variables, so continuous studies aim to add benefits to products that are part of the consumer's food routine. The development of new products is also considered of great importance, since the diet-health relationship represents a challenge for food science and technology, especially when it comes to functional foods (Soares et al., 2011).

Dairy products have a large part of the functional food market, particularly in their own manufacturing process dairy products involve the addition of lactic acid bacteria that are known to have probiotic properties or that produce secondary metabolites associated with health promoting effects (Miliis). et al., 2011). Kefir-fermented milk is a unique-flavored refreshing creamy drink * containing lactic acid bacteria (Lactobaciífus, Lactococcus, Leuconostoc), acetic acid bacteria and symbiosis yeasts, blended into a complex matrix composed of complex sugars and caseins surrounded by a matrix of the kefiran polysaccharide (Irigoyen et al, 2005).

The antimicrobial properties of kefir-grained sheep's milk have not been studied.

In the present invention, fermented milks are produced by fermentation of the milk of crossbred Bergamacia sheep. The fermented milk production process and its antimicrobial activity use kefir grains that were kept in 3% fat whole UHT cow's milk, the whole process being divided into three stages: ieite quality evaluation, fermented milk production and evaluation of antimicrobial activity.

In the first stage, sheep's milk is aseptically milked, kept at a temperature of 4 to 10 ° C and 50mL of this raw milk was separated for analysis of centesimal composition and somatic cell count. These analyzes were performed in the quality laboratory. of milk PROGENE, from the Department of Animal Science - UFRPE.

For fermentation, the raw ieite is pasteurized at 63-65 ° C for 30 minutes, then cooled to 30 ° C in an ice bath and then subjected to analysis of the peroxidase and phosphatase enzymes. The second step is performed when it is verified that the already pasteurized milk has the presence of peroxidase enzymes and absence of phosphatase enzymes, this milk is then deposited in a sterile glass container with 5% kefir grains (w / v). The vial is then left at a temperature of 20 ° C (± 2) for 45-48 h until reaching pH 4.5.

The kefir grains are removed through a sieve and preserved in 3% fat whole UHT cow's milk. During storage of fermented milk in the refrigerator ± 4 ° C, aliquots taken after the times: -1 h, 7 days, 14 days, 21 days and 28 days are frozen at -20 ° C. The third step is performed after obtaining all aliquots, these samples are then thawed and centrifuged at 10,000 rpm for 20 minutes at 4 ° C, the supernatant from each sample is filtered at 0.22 pm membrane and stored in sterile tubes, this liquid It is called the fermented milk supernatant (SLF).

For the evaluation of antimicrobial activity the pre-inoculum of the microorganisms Micrococcus luteus, Escherichia coli ATCC 25922, Klebisiella pneumonae ATCC 29665, Pseudomonas aeruginosa ATCC 27853, Listeria innocua, Bacillus subtillis ATCC 6633 and Staphyiococcus aureus Hu- midos. (Hmedia®) at 37 ° C for 18 ~ 24h.

Evaluation of the antimicrobial activity of fermented milk supernatant (SLF) by kefir grains was performed according to (NCCLS, 2006) in a 96-well microplate. The microorganisms were standardized according to the Mac Fariand scale at 1x108 cfu / mL and diluted to the concentration of 1x107 cfu / mL.

In each well of the microplate were added 45pL of the Müller Hinton broth (Hmedia®), 5pL of the standardized microorganisms and 50pL of the SLF to be tested. In the growth control we exchanged the SLF for 50pL of water. Only difference in growth control is the absence of microorganisms, this microplate is then incubated at 37 ° C for 18-24h.

After 24h growth at 37 ° C, each well of the microplate was streaked into Petri dishes containing Müller Hinton Agar (Hidmedia®), these plates were incubated at 37 ° C for 18-24h. Antimicrobial activity was performed in duplicate with each sample. Bactericidal activity is confirmed by the absence of cell growth in the petri dish where the microorganism was striated. The bacteriostatic activity is confirmed by the absence of turbidity of the striated microplate well, but that in the Petri dish there was cell growth of few colony forming units (in this work we considered up to 5ufc per striatum).

Among the results, it was observed that the bacteriostatic potential of the fermented milk supernatant was confirmed in the microorganisms Micrococcus luteus (time 1 h), Klebisielia pneumonae (time 14 days) and Bacillus subtillis (time 14 days). SLF was bactericidal for the microorganisms Micrococcus luteus (times 7 days, 14 days, 21 days and 28 days), Escherichia coli (times 1h, 7 days, 14 days, 21 days and 28 days), Klebisielia pneumonae (times 1h, 7 days, 21 days and 28 days), Pseudomonas aeruginosa (times 1h, 7 days, 14 days, 21 days and 28 days), Bacilfus subtillis (times 1h, 7 days, 21 days and 28 days), Staphylococcus aureus (times 1h , 7 days, 14 days, 21 days and 28 days) and Listeria innocua (times 1h, 7 days, 14 days, 21 days and 28 days). The production of fermented sheep's milk is evidenced by the low pH 4.5-4.7, measured by the pH meter (HI2221 HANNA®). To reach the optimal concentration of microorganisms (kefir grains) we tested at concentrations of 0.2%, 1%, 5% and 10% (Gronnevik et al., 2011; Irigoyen et al., 2005; Simova et al. ., 2006) using temperatures ranging from 17-23 ° C and always allowing fermentation to occur between 36 - 48h, under these conditions points were taken every 6 hours of fermentation and for each point the acidity and pH were measured.

When tests with 0.2% kefir were performed, it was found that this time was short for fermentation due to the low concentration of kefir.

Fermentations with concentrations of 1%, 5% and 10% were performed simultaneously. In 10% fermentation, the pH dropped from 6.8 to 3.97 in 36h, this concentration was discarded due to the large number of microorganisms used. The 1% fermentation reached the ideal pH in 36h, and with 5% reached the ideal pH range with approximately 33h of fermentation.

Other fermentations were performed with 1% and 5%, but there was no reproducibility at 36h and at 1% concentration.

We achieved reproducibility with the temperature set at 22 ° C (± 2) for 45-48h and 5% kefir concentration.

After fermentations the grains are removed with a fine sieve and the fermented milk is stored in the refrigerator at 4 ° C.

For the evaluation of antimicrobial activity aliquots were taken at storage times ~ 1h, 7 days, 14 days, 21 days and 28 days, to see if this milk maintains its antimicrobial potential throughout its shelf life, these aliquots were kept at - 20 ° C.

After all aliquots were obtained, they were thawed and centrifuged at 10,000 rpm for 20 minutes at 4 ° C, the supernatant from each sample was filtered on 0.22 pm membrane and stored in sterile tubes. Evaluation of antimicrobial activity was performed with the microorganisms Micrococcus luteus, Escherichia coli ATCC 25922, Klebisiella pneumonae ATCC 29665, Pseudomonas aeruginosa ATCC 27853, Listaria innocua, Baciiius subtillis ATCC 6633 and Staphylococcus aureus ATCC 6538. (2006). The tests were initially performed in unicata, after confirmation of the activity the tests were repeated in duplicate. The disadvantage of this product lies in the variation of microorganisms present in each fermented milk, being justified by the variation in the composition of strains in kefir grains. The present use of kefir grains fermented sheep's milk promoted the inhibition of growth of pathogenic strains of relevance. in the areas of hospital and food infection. Fermented milk can be tested in vivo in mice and in humans infected with these tested bacteria.

After confirmation of the results can be marketed and is therefore indicated for the immunological strengthening of patients in hospitals and for children and adults who are concerned with disease prevention.

In addition, the product is an innovation because for the food industries, the key to success in the functional food market is innovation, which is a powerful barrier to entry for new businesses.

References CHIFÍRIUC, M, C .; CIOACA, A. B .; LAZAR, V. in vitro assay for antimicrobial activity of kephír against bacteria! and fungai strains. Anaerobe, v.17, p. 433-435, 2011. DANONE BRAZIL. 2013. Available at: vwww.danone.com.br Access on: 25. jan. 2013. DATAMARK. 2012. Anvisa's rule to sell functional food is missing. Available at: www.datamark.com.br/noticias/2012 Accessed: 26. Feb. 2013. FARNWORTH, E.R. Kefir - a complex probiotic. Food Science and Technology Buiietim Functional Foods, v. 2, n.1, p.1-17, 2005. FERREIRA, C. L. L. F. Prebiotics and Probiotics: Update and Prospecting - Célia Lúcia de Luces Fortes Ferreira (2012), Rubio Publisher, p. 248. GRONNEVIK, H .; FALSTAD, M .; NARVHUS, J. A. Microbiological and Chemical properties of Norwegian kefir during storage. International Dairy Journal, v. 21, p.601-606, 2011. GROUPE DANONE. 2007. Available at: www.danone.com. Access on: 17.nov.2007 IRÍGOYEN, A .; ARANA, L; CASTIELLA, M .; TORRE, P .; IBÁNEZ, F.C. Microbiological, physicochemical, and sensory characteristics of kephyr during storage. Food Chemistry, v.90, p. 613-620, 2005. MACHADO, B.A.S .; REIS, J.H.O .; PIRES, E.A .; SANTOS, F.L. Technology Mapping of kefir patents. Prospectus Notebooks, v. 5, no. 2, p. 86-97, 2012. MAGALHÃES, K.T .; DRAGONE, G .; PEREIRA, G. V. M .; OLIVEIRA, J.M .; DOMINGUES, L; TEIXEIRA, J. A .; SILVA, J.B.A .; SCHWAN, R.F. Comparative study of the biochemical changes and volatile compound formations during the production of novel whey-based kefir beverages and traditional milk kefir. Food Chemistry, v.126, p.249-253, 2011. MAGALHÃES, K.T .; PEREIRA, M.A .; NICOLAU, A .; DRAGONE, G .; DOMINGUES, L .; TEIXEIRA, J. A .; SILVA, J.B.A .; SCHWAN, R.F. Production of fermented whey-based cheese using kefir grains as starter cuiture: Evaluation of morphologica! and microbia! variation. Bioresource Technology, v.101, p. 8843-8850, 2010. MILLS, S .; ROSS, R.P .; HILL, C .; FITZGERALD, G.F .; STANTON, C. Mifk intelllgence: Mining milk for bioactive substances associated with human heatth. International Dairy Journal, v. 21, p. 377-401, 2011. MONTANUCí, F.D .; GARCIA, S .; PRUDENCfO, S.H. Sensory characterization and acceptance of skimmed whole sugar and skimmed with insulin. Brazilian Journal of Food Technology, 6th SENSIBER, p.79-90, 2010. NESTLÉ BRAZIL. 2013. Available at: www.nestle.com.br Access on: 25. jan. 2013. of milk proteins and sensory quality of kefir, Food Hydrocolioids, v. 23, p. 2434-2445, 2009. OTBES, S. & CAGINDI, O. Kefir: a probiotic dairy-composition, nutritional and therapeutic aspects. Pakistan Journal of Nutrition, v. 2, no. 2, p. 54-59, 2003. PULINA, G .; NUDDA, A .; BATTACONE, G .; CANNAS, A. Effects of nutrition on the contents of fat, protein, somatic cells, aromatic compounds, and undesirable substances in sheep milk. Animal Feed Science and Technology, v.131, p. 255-291, 2006. RAUD, C. Functional foods: the new frontier of the food industry analysis of Danone and Nestlé strategies in the Brazilian yoghurt market. Journal of Political Sociology, v, 16, n. 31, p. 85-100, 2008. RODRIGUES, K. L; CAPUTO, L. R. G .; CARVALHO, J. C. T .; EVANGELIST, J .; SCHNEEDORF, J.M. Antimicrobial and bealing activity of kefir and kefíran extract. Internationa! Journal of Antimicrobial Agents, v. 25, p. 404-408, 2005. SIMOVA, E .; SIMOV, Z .; BESHKOVA, D .; FRENGOVA, G .; Dimitrov, Z .; SPASOV, Z. Amino acid profiles of icactic acid bacteria, isoiated from kefir grains and kefír starter made from them. International Journal of Food Microbiology, v.107, p.112 - 123, 2006. SOARES, D.S .; FAI, A.E.C .; OLIVEIRA, A.M .; PIRES, E.M.F .; STAMFORD, T.L.M. Utilization of cheese whey for probiotic yogurt production. Brazilian Archives of Veterinary Medicine and Animal Science, v, 63, n.4, p.996-1002, 2011. VIEGAS, R.P .; SOUZA, M.R .; FIGUEIREDO, T.C .; RESENDE, M.F.S .; PENNA, C.F.A.M .; CERQUEIRA, M.M.O.P. Quality of functional fermented milks made from lactic acid bacteria isolated from rennet cheese. Brazilian Archives of Veterinary Medicine # Zootechnics, v.62, n.2, p.460-467, 2010. WESCHENFELDER, S .; PEREIRA, G.M .; OAK, WIEST, J.M.

Physicochemical and sensorial characterization of traditional kefir and derivatives. Brazilian Archives of Veterinary Medicine and Animal Science, v.63, n.2, p.473-480, 2011. WITTHUHN, R.C., LSCHOEMAN, T .; BRITZ T.J. Characterization of the microbial popuJation there different stages of Kefsr production and Kefir grain mass cultivation. internationai Dairy Journal, v.15, p. 383-389, 2005. WRÓBLEWSKA, B .; KOTAKOWSKI, P .; PAWLIKOWSKA, K .; TROSZYNSKA, A .; KAUSZEWSKA, A. influence of the addition of transglutaminase on the immunoreactivity YAKULT. 2013 Available at: www.yakult.com.br Access on: 25. jan. 2013. ZACARCHENCO, P. B. & MASSAGUER-ROIG, S. Properties of Streptococcus thermophilus fermented miik containing varying concentrations of Bifidobacterium longum and Lactobacillus acidophilus. Braziüan Journal of Microbiology, v. 37, p. 338-344, 2006.

KEFIR GRAIN-FERMED SHEEP MILK PRODUCTION WITH ANTIMICROBIAN ACTIVITY

Claims (7)

1. Process for obtaining a new ewe's milk fermented by Kefir grains that have great antimicrobial activity against antibiotic resistant pathogens. Product according to Claim 1, characterized by the use of its cell free supernatant as an antimicrobial agent for use in dairy foods. Product according to Claim 1, characterized in that it has antimicrobial activity throughout its shelf life. Product according to Claim 1, characterized by the nutritional use of a large amount of protein, fat and minerals, adding value to the food of those who consume it. Product according to Claim 1, characterized by probiotic use, recommended to improve the intestinal microflora of the user. Product according to claim 1, characterized as a dairy derivative containing high calcium content and may be indicated for persons with a deficiency of this mineral. Product according to Claim 1, characterized in that it is low in factose and may be recommended for people who have an intolerance to this type of sugar.