Yeasts from Canastra cheese production process: Isolation and evaluation of their potential for cheese whey fermentation

Canastra cheese is a cheese with geographical indication recognized by the Brazilian National Institute of Industrial Protection under number IG201002. It is produced in seven municipalities in the state of Minas Gerais in a region called Serra da Canastra. In thiswork, samples of milk, “pingo” (natural starter),whey and Canastra cheese were collected on a farm inMedeiros-MG/Brazil to evaluate the yeast microbiota and select yeasts for whey fermentation to produce ethanol and volatile aromatic compounds of relevance in the production of cheese. Thirtynine isolates capable of fermenting lactose in a synthetic medium were identified by MALDI-TOF as Kluyveromyces lactis (29), Torulaspora delbrueckii (7) and Candida intermedia (3). Eleven isolates of K. lactis and three of T. delbrueckii efficiently fermented lactose until 4th day, and due to this reason were selected for cheese whey fermentation with Brix 12, 14 and 18. Generally, the isolates T. delbrueckii B14, B35, and B20 and K. lactis B10 were the most effective regardless of the initial Brix value. The identification of these four isolates by MALDI TOF was confirmed by sequencing of the ITS region. In the fermentation of cheese whey 14 Brix, T. delbrueckii B14 and B35, respectively yielded 24.06 g/L and 16.45 g/L of ethanol, while K. lactis B10 was more efficient in the consumption of lactose. In sequential culture with K. lactis B10 inoculated 48 h after T. delbrueckii B14, 97.82% of the total sugars were consumed resulting in the production of 19.81 g/L ethanol and 39 aromatic volatile compounds. The most abundant compounds were 3-methyl-1-butanol, octanoic acid and ethyl decanoate, which are reported as important for the aroma and flavor of cheeses. Based in our results, B10 isolate inoculated 48 h after B14 isolate is a promising yeast inoculum to be used for fermentation of dairy substrates.The authors thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico do Brasil), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais) for financial support. The authors also thank J.C Amorim for her help in MALDI TOF analysis and Dr. P.S Geraldino for her help in sequences analysis

Profile of sugars comsumption in fruit wines produced by different Saccharomyces cerevisiae

In wine fermentation, the two main soluble sugars present in must (glucose and fructose) are fermented to ethanol, CO2 and other compounds. Wine yeasts have a slight preference for glucose compared to fructose resulting in a difference between glucose and fructose consumption. Fructose is approximately twice as sweet as glucose and its presence as a residual sugar has a much stronger effect on the final sweetness of wine. The objective of this study was to evaluate the profile of sugars consumption by different yeasts during fruit wines production. Different Saccharomyces cerevisiae were used in the fermentation of raspberry must with 16 ºBrix and pH adjusted to 4.0. Batch fermentations were carried out at 22ºC. The concentrations of sucrose, glucose and fructose were determined by HPLC. The total conversion of sucrose into glucose and fructose by yeast UFLA CA11 was the fastest, within the first 8 h of fermentation, while for the yeast UFLA CA1174 the total hydrolysis of sucrose occurred after 32 h of fermentation. For the yeasts UFLA CA15 and UFLA EU 60.1, the sucrose hydrolysis occurred after 12 and 16 h, respectively. As expected, for all yeasts, was confirmed a preferential and fast consumption of glucose in comparison to the consumption of fructose. The highest residual concentrations of glucose and fructose were respectively, 300 and 920 mg/l for yeast UFLA CA11 and 400 and 2100 mg/l for yeast UFLA CA1174. Based on the concentrations of ethanol and residual sugar, the best efficiency of conversion was 93.88% for the yeast UFLA CA15. The other yeasts showed an efficiency of 75.56% (UFLA EU 60.1), 82.08% (UFLA CA11) and 83.69% (UFLA CA1174). It can be concluded that the evaluated yeasts were able to ferment the main sugars present in raspberry must. The yeast UFLA CA15 showed the best performance with higher conversion efficiency, rapid hydrolysis of sucrose and low concentration of residual sugars at the end of fermentation

Profile of higher alcohols in fruit wines produced by different Saccharomyces cerevisiae

Higher alcohols are secondary yeast metabolites, and can have both positive and negative impacts on the aroma and flavour of wine. Concentrations lower than 300 mg/l of higher alcohols contribute to the aroma, while concentrations above 400 mg/l negatively influence the wine quality. The formation of these compounds is influenced by several factors like temperature, presence of nitrogen compounds and the type of yeasts used for the fermentation. The aim of this study was to evaluate the profile of higher alcohols in alcoholic beverage produced from the fermentation of raspberry must by different yeast. The pH of the must was adjusted to 4.0 by the addition of CaCO3 and the initial sugar concentration to 16 Brix (adjusted with sucrose syrup). Batch fermentations were carried out at 22º C. Higher alcohols were determined by gas chromatography (GC­FID). Five higher alcohols (1­propanol, 2­methyl­1­propanol, 2­methyl­1­butano, 3­methyl­1­butanol and 2­phenylethanol) were identified and quantified. 3­methyl­1­butanol was the alcohol found in highest quantity. The highest concentrations were 140.9 and 149.4 mg/l for the yeast UFLA CA1162 and UFLA CA15, respctively. The beverage produced by the yeast UFLA CA155 showed the highest concentration of 2­phenylethanol (29.1 mg/l). 1­propanol was found in similar values (21 mg/l) in raspberry wine fermented by the yeast UFLA CA11 and UFLA CA1174. The highest and lowest total concentrations of higher alcohols were 284.5 and 161.8 mg/l for the beverage produced by the yeast UFLA CA1162 and UFLA CA11, respectively. It can be concluded that raspberry wine with a total higher alcohol concentration similar to that considered desirable (300 mg/l) in alcoholic beverages can be produced by using the yeast UFLA CA1162

Efeito da concentração do mosto e temperatura na fermentação para produção de vinho de frutas

Tradicionalmente o vinho é uma bebida alcoólica, a qual é produzida pela fermentação de uvas frescas, no entanto, diversas frutas apresentam características (teor de açucares, pH) que tornam possível seu uso na produção de vinho. Nos últimos anos vêm crescendo o número de trabalhos de pesquisa relativos ao emprego de outras frutas na produção de vinhos, destacando-se o kiwi, banana, cajá, manga, acerola, jabuticaba, cacau, laranja e gabiroba. Fatores como temperatura de fermentação e concentração de açúcares no mosto exercem grande influência no metabolismo das leveduras e consequentemente influenciam também a qualidade final da bebida produzida. O objetivo deste trabalho foi avaliar o efeito da concentração do mosto (A) e temperatura (B) na conversão do substrato em etanol (Yp/s) utilizando a metodologia de superfície de resposta. O mosto de framboesa foi preparado em 3 diferentes concentrações (14, 18 e 22º Brix), sendo a correção realizada com adição de sacarose. A levedura S. cerevisiae UFLA CA 15 foi inoculada (1 g/L) em 100 mL de mosto e os frascos foram colocados em 3 diferentes temperaturas (17, 22 e 27º C). As concentrações de açúcares e etanol foram determinadas por HPLC. De acordo com a análise estatística, a concentração do mosto apresentou um efeito linear positivo, significativo a 95% de confiança; enquanto que o efeito da temperatura foi do tipo quadrático e negativo, significativo a 99% de confiança. O modelo que melhor descreve a variação da resposta em função das variáveis na faixa de valores estudados pode ser expresso pela equação: 0,49 + 0,019*A 0,045*B2, sendo A e B os valores codificados das variáveis concentração do mosto e temperatura, respectivamente. Os melhores resultados de YP/S foram obtidos a 22º C, empregando um mosto com 22º Brix. Em uma próxima etapa do experimento será avaliado o efeito da concentração do mosto e temperatura no perfil de compostos voláteis (alcoóis, ésteres, aldeídos, cetonas e outros) dos vinhos produzidos

Identificação de microrganimos isolados de processos fermentativos: uma abordagem polifásica que inclui a técnica de MALDI-TOF MS

Ao longo das últimas duas décadas, o Laboratório de Fermentações do Departamento de Biologia da Universidade Federal de Lavras (UFLA) tem desenvolvido um trabalho de bioprospecção de milhares de isolados de bactérias, leveduras e fungos filamentosos provenientes de amostras de fermentação de alimentos indígenas, cacau, café, silagens, etc. As linhagens de microrganismos obtidas nos diferentes projetos em curso, ao longo deste tempo, têm sido usadas em processos fermentativos variados. Contudo, a idenfiticação e caracterização confiável deste material biológico tem sido uma tarefa que envolve muitos custos e mão-de-obra dedicada e qualificada. O MALDI-TOF MS é uma técnica físico-química atualmente difundida internacionalmente no campo da microbiologia. A técnica tem dado um grande contributo para o conhecimento científico acerca da identificação de microrganismos ao nível de espécie e, em alguns casos, ao nível de linhagem. Trata-se de uma ferramenta que já tem sido eficazmente utilizada em testes de identificação rápida em microbiologia clínica, alimentar e ambiental. Recentemente, com a implementação da técnica de MALDI-TOF MS no Laboratório de Fermentações da UFLA, a identificação e caracterização dos microrganismos em estudo foi aprimorada. Uma abordagem polífasica para a identificação rápida e confiável desses microrganismos foi redesenhada e posta em prática. Neste contexto, o presente trabalho tem como objetivo principal apresentar o work-flow, que inclui técnica de MALDI-TOF MS, para a identificação e caracterização de microrganismos provenientes de nichos variados e, alguns deles complexos, em um laboratório de pesquisa que isola milhares de linhagens microbianas durante a execussão dos seus projetos de pesquisa

Comparative analyze of the kefir fermentation process and microbiota, using milk and cheese whey as substrates

Kefir, a mixed culture that ferments lactose, is known for the production of a refreshing fermented beverage popular in EasternEuropean countries by inoculating milk with kefir grains. Kefir grains are gelatinous white or creamcoloured, water insoluble, irregular granules with diameter ranging 0,33,5 cm. They are composed mostly of proteins and polysaccharides in which the complex microbiota is enclosed. The beverage consists of a microbial diversity that includes lactic acid bacteria, yeasts and their metabolites. The aim of this work was to compare the fermentation and the microbiota of kefir , using milk and cheese whey as substrates. The grains were added in the proportion of 5% in 250ml of each substrate. Assays were performed at 25 °C for 48h. The concentrations of lactose, ethanol, lactic acid and acetic acid were quantified by HPLC. To determinate the composition of microbiota in Kefir of fermentation, PCRDGGE analysis was used.The fermentation of milk and cheese whey by kefir grains are observed in this study. It can be observed that the lactose concentration at the end of the milk fermentation was lower in comparison with that obtained at 48 h for cheese whey fermentation. Despite the higher lactose consumption during the fermentation of milk by kefir grains, the concentrations of ethanol, acetic acid and lactic acid did not show significant differences with those obtained during the cheese whey fermentation. No changes in the DGGE profiles in all fermentations were observed to fungal and bacterial communities. It was thus concluded that lactose from cheese whey is converted to products with higher yields than lactose from milk, in addition to showing the same group of microorganisms for both fermentation process

Brazilian kefir: structure, microbial communities and chemical composition

Microbial ecology and chemical composition of Brazilian kefir beverage was performed. The microorganisms associated with Brazilian kefir were investigated using a combination of phenotypic and genotypic methods. A total of 359 microbial isolates were identified. Lactic acid bacteria (60.5%) were the major isolated group identified, followed by yeasts (30.6%) and acetic acid bacteria (8.9%). Lactobacillus paracasei (89 isolates), Lactobacillus parabuchneri (41 isolates), Lactobacillus casei (32 isolates), Lactobacillus kefiri (31 isolates), Lactococcus lactis (24 isolates), Acetobacter lovaniensis (32 isolates), Kluyveromyces lactis (31 isolates), Kazachstania aerobia (23 isolates), Saccharomyces cerevisiae (41 isolates) and Lachancea meyersii (15 isolates) were the microbial species isolated. Scanning electron microscopy showed that the microbiota was dominated by bacilli (short and curved long) cells growing in close association with lemon-shaped yeasts cells. During the 24 h of fermentation, the protein content increased, while lactose and fat content decreased. The concentration of lactic acid ranged from 1.4 to 17.4 mg/ml, and that of acetic acid increased from 2.1 to 2.73 mg/ml. The production of ethanol was limited, reaching a final mean value of 0.5 mg/ml.The authors acknowledge Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) for scholarshipsand and CAARG - Cooperativa Agricola Alto Rio Grande Ltda. (Lavras/MG, Brazil) for the milk supply

Characterization of different fruit wines made from cacao, cupuassu, gabiroba, jaboticaba and umbu

The main aim of this work was to produce fruit wines from pulp of gabiroba, cacao, umbu, cupuassu and jaboticaba and characterize them using gas chromatography–mass spectrometry for determination of minor compounds and gas chromatography-flame ionization detection for major compounds. Ninety-nine compounds (C6 compounds, alcohols, monoterpenic alcohols, monoterpenic oxides, ethyl esters, acetates, volatile phenols, acids, carbonyl compounds, sulfur compounds and sugars) were identified in fruit wines. The typical composition for each fruit wine was evidenced by principal component analysis and Tukey test. The yeast UFLA CA 1162 was efficient in the fermentation of the fruit pulp used in this work. The identification and quantification of the compounds allowed a good characterization of the fruit wines. With our results, we conclude that the use of tropical fruits in the production of fruit wines is a viable alternative that allows the use of harvest surpluses and other underused fruits, resulting in the introduction of new products into the market.Conselho Nacional de Desenvolvimento Científico e Tecnológico do Brasil (CNPq) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Production of fermented cheese whey-based beverage using kefir grains as starter culture : evaluation of morphological and microbial variations

Whey valorization concerns have led to recent interest on the production of whey beverage simulating kefir. In this study, the structure and microbiota of Brazilian kefir grains and beverages obtained from milk and whole/deproteinised whey was characterized using microscopy and molecular techniques. The aim was to evaluate its stability and possible shift of probiotic bacteria to the beverages. Fluorescence staining in combination with Confocal Laser Scanning Microscopy showed distribution of yeasts in macro-clusters among the grain’s matrix essentially composed of polysaccharides (kefiran) and bacteria. Denaturing gradient gel electrophoresis displayed communities included yeast affiliated to Kluyveromyces marxianus, Saccharomyces cerevisiae, Kazachatania unispora, bacteria affiliated to Lactobacillus kefiranofaciens subsp. Kefirgranum, Lactobacillus kefiranofaciens subsp. Kefiranofaciens and an uncultured bacterium also related to the genus Lactobacillus. A steady structure and dominant microbiota, including probiotic bacteria, was detected in the analyzed kefir beverages and grains. This robustness is determinant for future implementation of whey-based kefir beverages.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), CAPES-GRICES