Elsevier

Bioresource Technology

Volume 101, Issue 22, November 2010, Pages 8843-8850
Bioresource Technology

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

https://doi.org/10.1016/j.biortech.2010.06.083Get rights and content

Abstract

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.

Introduction

Cheese whey is the liquid remaining after the precipitation and removal of milk casein during cheese-making. This byproduct represents approximately 85–90% of the milk volume and retains 55% of milk nutrients. Among the most abundant of these nutrients are lactose (4.5–5.0% w/v), soluble proteins (0.6–0.8% w/v), lipids, and mineral salts (Dragone et al., 2009 and references there in). Cheese whey represents an important environmental problem because of the high volumes produced and its high organic matter content, exhibiting a COD of 60,000–80,000 ppm. Worldwide production of whey is estimated to be in the order of 160 million tonnes per year, showing a 1–2% annual growth rate (Smithers, 2008). The pressure of antipollution regulations together with whey nutritional value challenges the dairy industry to face whey surplus as a resource and not only as a waste problem (Guimarães et al., 2010).

Several methods have been proposed for whey valorization (Guimarães et al., 2010, Koutinas et al., 2009 and references there in). Besides potable ethanol production by lactose converting microorganisms (reviewed by Guimarães et al. (2010)) and genetically-engineered Saccharomyces cerevisiae cells (Domingues et al., 2001, Guimarães et al., 2008, Domingues et al., 2010), the production of alcoholic beverages from whey has also been pointed as an alternative (Holsinger and Posati, 1974), including distilled beverages (Dragone et al., 2009) and kefir-like whey beverages (Paraskevopoulou et al., 2003).

Kefir is made by inoculating milk with kefir grains. These grains are irregular granules that vary in size from 3 to 35 mm in diameter (Güzel-Seydim et al., 2005) contain lactic acid bacteria (Lactobacillus, Lactococcus, Leuconostoc), acetic acid bacteria and yeast mixture coupled together with casein and complex sugars by a matrix of polysaccharides denominated kefiran (Güzel-Seydim et al., 2005). Yeasts are important in kefir fermentation because of the production of ethanol and carbon dioxide. Kefir grains usually contain lactose-fermenting yeasts (Kluyveromyces lactis, Kluyveromyces marxianus and Torula kefir), as well as non lactose-fermenting yeasts (S. cerevisiae) (Farnworth, 2005). This mixed culture of kefir yeast, which ferments lactose, seems to have the potential for beverage production using cheese whey.

Cheese whey utilization by kefir grains has been studied for potable alcohol production (Koutinas et al., 2009) indicating the ability of this biocatalyst to produce high yields in alcoholic fermentations. In addition, the production of kefir-like whey beverages using a cheese whey–milk mixture as substrate has also been reported (Paraskevopoulou et al., 2003). Reports on single cell protein production (using kefir yeasts; Koutinas et al., 2005) and more recently, on starter culture production from whey for use in cheese ripening (Koutinas et al., 2009) can also been found. All these studies show promising perspectives for kefir grains application in whey valorization strategies. Nevertheless, one important aspect has to be clarified for fully application of kefir grains to whey fermentations. Namely, if the microbiota present in the grains change when using whey instead of the traditional milk as substrate. Another relevant issue is whether the kefir probiotic bacteria are present in the beverages. Therefore, the motivation of the present work was to elucidate the stability, organization and identification of the dominant microbiota present in Brazilian kefir grains and correspondent beverages.

Section snippets

Milk and whey-based fermentation media

Three different substrates with a lactose concentration of 46 g/l were used as fermentation media: pasteurized full cows’ milk (M), cheese whey (CW) and deproteinised cheese whey (DPW). Cheese whey powder, obtained from a regional dairy industry (Quinta dos Ingleses, Caíde de Rei, Portugal), was dissolved in sterile distilled water to the desired lactose concentration. Deproteinised cheese whey was made by autoclaving at 115 °C for 10 min the cheese whey solution, followed by aseptic

Kefir fermentation chemical analysis

Table 3 summarizes the main chemical characterization results of kefir beverages fermentation. Lactose was consumed and ethanol was produced during the fermentation. At 48 h the lactose concentration in the milk fermentation was residual while in the whey fermentations a lactose concentration of 15–20 g/l was observed. This likely reflects an adaptation period of the microbial community to the whole and deproteinised cheese whey as kefir grains were preserved in milk. Despite the higher lactose

Conclusions

The present study revealed a consistent grain structure and kefir microbiota when replacing milk with whole/deproteinised cheese whey as fermentation substrate. The dominant microbiota, as revealed by PCR-DGGE, was composed by yeast affiliated to K. marxianus, S. cerevisiae, K. unispora, and bacteria affiliated to the Lactobacillus genus. Interestingly, this dominant bacterial community was also found in the fermented beverages, conferring probiotic label to kefir beverages. In addition, the

Acknowledgements

The authors acknowledge Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), CAPES-GRICES.

References (35)

  • S. Tada et al.

    Fed-batch coculture of Lactobacillus kefiranofaciens with Saccharomyces cerevisiae for effective production of kefiran

    J. Biosci. Bioeng.

    (2007)
  • T.J. White et al.

    Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics

  • J.C. Araújo et al.

    DGGE with genomic DNA: Suitable for detection of numerically important organisms but not for identification of the most abundant organisms

    Water Res.

    (2008)
  • L. Domingues et al.

    Alcohol production from cheese whey permeate using genetically modified flocculent yeast cells

    Biotechnol. Bioeng.

    (2001)
  • L. Domingues et al.

    Metabolic engineering of Saccharomyces cerevisiae for lactose/whey fermentation

    Bioeng. Bugs.

    (2010)
  • D. Ercolini et al.

    Bacterial community structure and location in Stilton cheese

    Appl. Environ. Microbiol.

    (2003)
  • D. Ercolini et al.

    Behavior of variable V3 region from 16S rDNA of important lactic acid bacteria in denaturing gradient gel electrophoresis

    Curr. Microbiol.

    (2001)
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