Valorização biotecnológica de compostos bioativos e prospecção de bactérias lácticas presentes no colostro bovino para aplicações em produtos funcionais e nutracêuticos

Orientador : Prof. Dr. Carlos Ricardo SoccolTese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa: Curitiba, 14/11/2013Inclui referências : f. 41-44;92-96;118-122Área de concentração: Saúde humana e animalResumo: O colostro bovino faz parte da cadeia produtiva do leite e consiste de uma secreção glandular obtida das fêmeas durante os primeiros dias após o nascimento do bezerro. Este líquido possui concentração superior de nutrientes, especialmente de proteínas funcionais, em relação ao leite. Considerando que, grande parte do colostro produzido é descartado nas fazendas leiteiras, e que o colostro possui um alto valor biológico, este subproduto pode apresentar grande aplicação no desenvolvimento de novos alimentos nutracêuticos e funcionais. Dessa forma, este trabalho teve por objetivo a agregação de valor do colostro bovino através do desenvolvimento de um produto em pó, contendo componentes funcionais como a imunoglobulina G (IgG) e outros fatores que promovem benefícios a saúde, bem como a prospecção de microrganismos probióticos presentes. Este trabalho foi dividido em quatro capítulos que abordam a caracterização físico-química do colostro bovino, o uso de técnicas moleculares para o isolamento e identificação da microflora láctica do colostro, o estudo de técnicas para a purificação das imunoglobulinas G e estudos de estabilidade e formulação de um produto a base de imunoglobulina G. No capítulo 1, o perfil físico-químico de 15 amostras de colostro de bovinos da raça holandesa, coletadas na região metropolitana de Curitiba, foi definido através de análises de extrato seco, cinzas, proteína, gordura, lactose, densidade e acidez, seguindo as metodologias estabelecidas pela AOAC (Association of Official Analytical Chemists). As maiores variações ocorreram da primeira (após o parto) para a segunda ordenha (12 h pós-parto), sendo a segunda ordenha similar a terceira ordenha (24 h pós-parto). As variáveis mais influenciadas pela ordenha foram o teor de cinzas, proteínas e lactose. A fração lipídica foi extraída e estimada em ésteres metilados de ácidos graxos (FAME). A maioria dos ácidos graxos foi representada pelo ácido palmítico (36,9%), sendo verificada também a presença do ácido linoleico (2,63%). No capítulo 2, o estudo da microflora láctica do colostro foi realizado pelo seqüenciamento do gene 16S rRNA e pelo método de LH-PCR (Length Heterogeneity Polymerase Chain Reaction). No primeiro método, 29 cepas foram isoladas em meios MPCA, M17 e MRS suplementado com cisteína. Dentre elas, 13 espécies cultivadas foram identificadas pelas sequências do gene 16S rRNA. A LH-PCR confirmou a presença de algumas espécies isoladas em placa (L. casei e Staphylococcus sp.), no entanto, outras espécies não recuperadas nos meios de cultivo foram identificadas pelos picos (Lactococcus lactis, Staphylococcus sp., Streptococcus thermophilus e Kocuria kristinae). No capítulo 3, 18 amostras de colostro bovino foram quantificadas quanto ao teor de imunoglobulinas G total, através da técnica de imunodifusão radial. As amostras de colostro apresentaram uma alta concentração de IgG (37,59 a 110,25 g/L). O estudo de diferentes técnicas para a purificação das imunoglobulinas G foi realizado testandose 4 métodos que foram a micro e ultrafiltração, filtração em carvão ativado, precipitação com sulfato de amônio e gel filtração do soro de colostro. Todos os processos levaram a reduções nos teores de proteínas e IgGs, sendo que o maior rendimento em IgGs foi alcançado pela filtração em coluna de carvão (57,2%) e com o uso de membranas de 0,22 ?m, recuperando-se a fração do retentado (45,5%) (Pool 1). A reprodutibilidade dos processos foi avaliada em 3 pools de colostro e verificou-se que o retentado de 0,22 ?m foi o único que não apresentou diferença significativa em relação ao soro dialisado, e este tratamento foi, portanto, selecionado para a elaboração do produto. Todos os tratamentos mostraram a presença das bandas de 25 e 50 kDa, confirmando a recuperação da molécula de IgG. No capítulo 4, os testes de pasteurização e liofilização não indicaram uma redução no teor das IgGs. No entanto, quando os lioprotetores foram testados verificou-se uma maior estabilidade das IgGs no uso de uma solução de exopolissacarídeo (EPS) a 1% (p/v). O produto elaborado apresentou uma alta composição em IgG bovina (78%), compondo 77% das proteínas totais. Testes de estabilidade confirmaram a manutenção da concentração das IgGs em 30 dias de armazenamento a 20-25oC. Concluiu-se, portanto, que o processamento e utilização do colostro bovino em novas formulações têm aplicações relevantes e de interesse para a indústria. O produto desenvolvido requer uma dose diária inferior (menor que 5 gramas) a recomendada para os produtos já disponíveis no mercado e poderia ser destinado a diversos segmentos como na nutrição de pacientes imunodeprimidos, na melhora da performance atlética e como suplemento em alimentos infantis. Palavras-chave: colostro bovino, LH-PCR, imunoglobulina G, proteína do soro, sistema imune, imunodifusão radial.Abstract: The bovine colostrum is part of the production chain of milk and consists of a glandular secretion obtained from females during the first days after birth of the calf. This liquid has higher concentration of nutrientes, especially functional proteins compared to milk. Whereas largely colostrum produced is disposed on dairy farms and, it has a high biological value, this by-product may have a high application in the development of news functional foods and nutraceuticals. Thus, this study aimed to add value to colostrum through the development of a powder product containing functional components such as immunoglobulin G (IgG) and other factors that promote health benefits as well as the prospecting ofpresent probiotic microorganisms. This manuscript was divided into four chapters that approach the physicochemical characterization of bovine colostrum, the use of molecular techniques for the isolation and identification of the lactic microflora of colostrum, the study of techniques for the purification of immunoglobulin G and stability studies and formulation a product based on immunoglobulin G. In chapter 1, the physical-chemical profile of 15 samples of colostrum from Holsteins cows, collected in the metropolitan region of Curitiba, was defined based on analysis of solids, ash, protein, fat, lactose, density and, acidity according to methodology of AOAC (Association of Official Analytical Chemists). The analyzes showed the greatest variation between the first (postpartum) and second milking (12 hours postpartum), where the second milking was very similar to third milking (24 hours postpartum). The variables more influenced by milking were the ash, protein and, lactose content. The lipid fraction was extracted and estimated in fatty acid methylated esters (FAME). Most fatty acids have been represented by palmitic acid (36.9%), and it was also verified the presence of linoleic acid (2.63%). In chapter 2, the study of the lactic microflora of colostrum was performed by sequencing the 16S rRNA and the LH-PCR (Length Heterogeneity Polymerase Chain Reaction). In the first method, 29 strains were isolated in medium MPCA, M17 and MRS supplemented with cysteine. Among them, 13 cultivated species were identified by 16S rRNA gene sequences. LH-PCR confirmed the presence of some species isolated plate (L. casei e Staphylococcus sp.), however, other species could not recovered in culture media were identified by peaks (Lactococcus lactis, Staphylococcus sp., Streptococcus thermophilus and Kocuriakristinae). In chapter 3, 18 samples of colostrum were quantified for immunoglobulin G total content by radial immunodiffusion. Samples of colostrum showed a high concentration of IgG (37.59 to 110.25 g/L). The study of different methods for purification of immunoglobulin G was performed by testing four methods that were micro and ultra filtration, activated carbon filtration, precipitation with ammonium sulfate and, gel filtration of whey colostrum. All processes have led to reduction in levels of proteins and IgG, and the highest IgG yield was achieved by filtration in carbon column (57.2%) and with the use of membranes from 0.22 microns recovering the retentate fraction (45.5%) (Pool 1). The reproducibility of the processes was evaluated in 3 pools of colostrum and it was found that the retentate of 0.22 ?m was the only one that showed no significant difference compared to dialysate colostrum whey, and this treatment was therefore selected for the preparation of the product. All treatments showed the presence of bands of 25 and 50 kDa, confirming the recovery of the IgG molecule. In chapter 4, pasteurization and lyophilization tests did not indicate a reduction in the levels of IgGs. However, when the lyoprotectants were tested there was a higher stability of IgG when exopolysaccharide (EPS) solution 1% (w/v) was used. The final product had a high composition of bovine IgG (78%), comprising 77% of total proteins. Stability tests confirmed the maintenance of the concentration of IgG in 30 days storage at 20- 25oC. It was concluded, therefore, that the processing and use of bovine colostrum in new formulations have relevant applications of interest to the industry. The product developed requires a lower daily dose (less than 5 grams) recommended for products already available on the market and could be directed to different segments such as in the nutrition of immunocompromised patients, the improvement of athletic performance and as supplement in infant foods. Keywords: bovine colostrum, LH-PCR, immunoglobulin G, whey protein, immune system, radial immunodiffusion

Izdvajanje i identifikacija mikroflore iz kravljeg kolostruma tradicionalnim i molekularnim metodama

Colostrum is the first milk produced by the mammary gland during the early postpartum period. In addition to nutrients, antimicrobial and growth factors, bovine colostrum contains valuable microflora, including members of the genera Lactobacillus and Bifidobacterium, which have been used widely in probiotic food. The aim of this work is to identify and quantify the microflora of colostrum samples from two Holstein dairy cows on the first three postpartum days through culture-dependent 16S rRNA gene sequencing and length heterogeneity polymerase chain reaction (LH-PCR) techniques, using an adapted method to extract the total DNA. Twenty-nine strains isolated in synthetic media were identified by their 16S rRNA gene sequences, revealing two potential probiotic strains (Lactobacillus casei and Bifidobacterium pseudolongum). Colostrum samples were subjected to LH-PCR analysis to obtain fingerprint profiles of the microflora. Lactobacillus casei was the main species present in the samples. The probiotic potential of these bacteria for use in fermented and functional foods remains to be evaluated.Kolostrum je prvo mlijeko, koje proizvodi mliječna žlijezda tijekom ranog postpartalnog razdoblja. Osim što sadrži hranjiva, antimikrobne tvari i hormone rasta, kravlji kolostrum sadržava dragocjenu mikrofloru, uključujući sojeve rodova Lactobacillus i Bifidobacterium što se koriste u proizvodnji probiotičke hrane. Svrha je ovoga rada bila identificirati i kvantificirati mikrofloru u uzorcima kolostruma dvaju Holstein mlječnih krava, uzetih tijekom prva tri dana postpartalnog razdoblja. Mikroorganizmi su određeni sekvenciranjem 16S rRNK gena i LH-PCR metodom kojom se ekstrahira ukupna DNK. U sintetičkoj je podlozi izolirano dvadeset i devet sojeva, identificiranih pomoću sekvencija 16S rRNK gena, te su izdvojena dva potencijalno probiotička soja (Lactobacillus casei i Bifidobacterium pseudolongum). Uzorci su kolostruma analizirani LH-PCR metodom kako bi se utvrdio DNK profil mikroflore, te je utvrđeno da u uzorcima prevladava vrsta Lactobacillus casei. Probiotička svojstva ove bakterije za proizvodnju fermentiranih i probiotičkih proizvoda tek treba istražiti

Izdvajanje i identifikacija mikroflore iz kravljeg kolostruma tradicionalnim i molekularnim metodama

Colostrum is the first milk produced by the mammary gland during the early postpartum period. In addition to nutrients, antimicrobial and growth factors, bovine colostrum contains valuable microflora, including members of the genera Lactobacillus and Bifidobacterium, which have been used widely in probiotic food. The aim of this work is to identify and quantify the microflora of colostrum samples from two Holstein dairy cows on the first three postpartum days through culture-dependent 16S rRNA gene sequencing and length heterogeneity polymerase chain reaction (LH-PCR) techniques, using an adapted method to extract the total DNA. Twenty-nine strains isolated in synthetic media were identified by their 16S rRNA gene sequences, revealing two potential probiotic strains (Lactobacillus casei and Bifidobacterium pseudolongum). Colostrum samples were subjected to LH-PCR analysis to obtain fingerprint profiles of the microflora. Lactobacillus casei was the main species present in the samples. The probiotic potential of these bacteria for use in fermented and functional foods remains to be evaluated.Kolostrum je prvo mlijeko, koje proizvodi mliječna žlijezda tijekom ranog postpartalnog razdoblja. Osim što sadrži hranjiva, antimikrobne tvari i hormone rasta, kravlji kolostrum sadržava dragocjenu mikrofloru, uključujući sojeve rodova Lactobacillus i Bifidobacterium što se koriste u proizvodnji probiotičke hrane. Svrha je ovoga rada bila identificirati i kvantificirati mikrofloru u uzorcima kolostruma dvaju Holstein mlječnih krava, uzetih tijekom prva tri dana postpartalnog razdoblja. Mikroorganizmi su određeni sekvenciranjem 16S rRNK gena i LH-PCR metodom kojom se ekstrahira ukupna DNK. U sintetičkoj je podlozi izolirano dvadeset i devet sojeva, identificiranih pomoću sekvencija 16S rRNK gena, te su izdvojena dva potencijalno probiotička soja (Lactobacillus casei i Bifidobacterium pseudolongum). Uzorci su kolostruma analizirani LH-PCR metodom kako bi se utvrdio DNK profil mikroflore, te je utvrđeno da u uzorcima prevladava vrsta Lactobacillus casei. Probiotička svojstva ove bakterije za proizvodnju fermentiranih i probiotičkih proizvoda tek treba istražiti

Mogućnosti primjene probiotika

Probiotics, live cells with different beneficiary characteristics, have been extensivelly studied and explored commercially in many different products in the world. Their benefits to human and animal health have been proven in hundreds of scientific research. Lactobacillus and Bifidobacterium are the main probiotic groups; however, there are reports on the probiotic potential of Pediococcus, Lactococcus, Bacillus and yeasts. Some of the identified probiotic strains exhibit powerful anti-inflammatory, antiallergic and other important properties. Apart from that, the consumption of dairy and non-dairy products stimulates the immunity in different ways. Various food matrices have been used with probiotics, which are briefly documented. In this review, the history of probiotics, their application in the health and food areas and new trends in probiotic products and processes are presented.Probiotici su žive stanice koje imaju različite pozitivne osobine, pa se intenzivno proučavaju i komercijalno primjenjuju u mnogim proizvodima diljem svijeta. U više stotina znanstvenih istraživanja dokazan je njihov pozitivan učinak na zdravlje ljudi i životinja. Glavne su skupine probiotika bakterije vrste Lactobacillus i Bifidobacterium, ali spominju se i probiotička svojstva bakterija vrsta Pediococcus, Lactococcus i Bacillus, te nekih kvasaca. Neki od identificiranih probiotičkih sojeva imaju izraženi protuupalni i antialergijski učinak, pa se konzumiranjem probiotičkih mliječnih i nemliječnih proizvoda povećava imunitet. U radu su ukratko spomenute razne namirnice što se koriste u kombinaciji s probioticima. Također je dan povijesni pregled razvoja probiotika, navedene su mogućnosti njihove primjene u prehrambenoj industriji, a i u zdravstvu, te prikazani novi pravci proizvodnje probiotičkih proizvoda

Recovery and Identification of Bovine Colostrum Microflora Using Traditional and Molecular Approaches

Colostrum is the first milk produced by the mammary gland during the early postpartum period. In addition to nutrients, antimicrobial and growth factors, bovine colostrum contains valuable microflora, including members of the genera Lactobacillus and Bifidobacterium, which have been used widely in probiotic food. The aim of this work is to identify and quantify the microflora of colostrum samples from two Holstein dairy cows on the first three postpartum days through culture-dependent 16S rRNA gene sequencing and length heterogeneity polymerase chain reaction (LH-PCR) techniques, using an adapted method to extract the total DNA. Twenty-nine strains isolated in synthetic media were identified by their 16S rRNA gene sequences, revealing two potential probiotic strains (Lactobacillus casei and Bifidobacterium pseudolongum). Colostrum samples were subjected to LH-PCR analysis to obtain fingerprint profiles of the microflora. Lactobacillus casei was the main species present in the samples. The probiotic potential of these bacteria for use in fermented and functional foods remains to be evaluated

The Potential of Probiotics: A Review

Probiotics, live cells with different beneficiary characteristics, have been extensivelly studied and explored commercially in many different products in the world. Their benefits to human and animal health have been proven in hundreds of scientific research. Lactobacillus and Bifidobacterium are the main probiotic groups; however, there are reports on the probiotic potential of Pediococcus, Lactococcus, Bacillus and yeasts. Some of the identified probiotic strains exhibit powerful anti-inflammatory, antiallergic and other important properties. Apart from that, the consumption of dairy and non-dairy products stimulates the immunity in different ways. Various food matrices have been used with probiotics, which are briefly documented. In this review, the history of probiotics, their application in the health and food areas and new trends in probiotic products and processes are presented