Black tea water kefir beverage : a thesis submitted in partial fulfilment of the requirement for the degree of Master of Food Technology, Massey University, Albany, New Zealand

Fermented foods and beverages play an important role in the human diet as they provide essential nutrients as well as contribute towards prevention of diseases. Lactic acid bacteria and yeasts are a major group of microorganisms associated with fermented products. Some of the microorganisms, known as probiotics, confer health properties to human health. Thus, many different types of fermented foods and beverages containing probiotics are produced around the world to support wellness and health. In recent years, there has been increased interest in the development of fermented functional plant-based foods and beverages due to a surge in scientific research of the products. Further, there is evidence that probiotic microorganisms can grow well in plant-based substrates. Water kefir is a sparkling fermented beverage with an acidic, sweet, slightly alcoholic taste, and a yeasty flavour. Water kefir fermentation can be achieved by the inoculation of water kefir grains as a starter culture into a solution containing sugar. Kefir grains consist of a symbiotic starter culture of lactic acid bacteria (LAB) and yeasts contained in a polysaccharide matrix. Microorganisms present in kefir grains are recognized as probiotics. The majority of previous studies have focused on the isolation and identification of water kefir cultures responsible for fermentation. There is, therefore, scanty information on the fermentation of plant-based water kefir beverages. The main objective of this study was to develop fermented black tea beverage using water kefir grains as a starter culture. Fermentation of black tea infusions as single and mixed substrate with carrot juice using water kefir grains were investigated. Microflora of water kefir grains used consisted of symbiotic starter culture of lactic acid bacteria (Lactococcus spp. and Lactobacillus spp.) and a yeast (Saccharomyces cerevisiae). The study was conducted in three main phases. The first phase investigated the effect of sucrose concentration (5% and 10%) and fermentation temperature (25°C and 30°C) in black tea water kefir fermentation for 72 h. Meanwhile, the effect of added carrot juice (5%, 10%, and 15%) on kefir beverage during secondary fermentation (24 h) at 25°C was investigated in the second phase. The stability of the final black tea water kefir beverage formulation during storage (4°C) for four weeks was investigated in phase three. Samples of black tea water kefir beverages were subjected to various analyses during fermentation and storage (4°C) for 4 weeks: titratable acidity, total soluble solids (°Brix), colour, viable cell counts of constituent starter culture, sensory evaluation, sugars, organic acids, antioxidants, and pH was also measured. Results showed that fermentation temperature, sugar concentration, and carrot juice concentration contributed to the physico-chemical and microbiological characteristic as well as sensory properties of the product. In phases one and two, pH and total soluble solids (°Brix) decreased, while titratable acidity and cell counts of LAB and yeasts increased during fermentation of the products. LAB and yeasts were able to grow in black tea and addition of carrot juice into the beverages slightly increased their growth. The best fermentation conditions based on physico-chemical and sensory properties were kefir beverage containing sugar (10%) and carrot juice (10%) fermented at 25°C for 96 h. In phase three, the growth and survival of Lactococcus spp. and Lactobacillus spp. were low during storage of the product (4°C) while Saccharomyces cerevisiae maintained high cell numbers (7.03±0.07 log cfu/ml) at the end of storage (28 days). Results showed the possibility to produce low sugar water kefir beverage containing 0.08±0.01% (w/v) sucrose, 1.55±0.04% (w/v) glucose, and 2.93±0.20% (w/v) fructose. The fermented kefir beverage also contained 0.202±0.02% (w/v) lactic acid, 0.114±0.03% (w/v) acetic acid and some antioxidants (gallic acid, ECG, EGC, EGCG, theobromine and caffeine) which may be beneficial to human health. There was significant difference (p<0.05) in the colour (L*, a*, b*) of the fermented beverages during storage (4°C). Black tea water kefir beverage containing 10% sugar and 10% carrot juice fermented at 25°C for 96 h was well-liked by consumer sensory panellists

Sequencing-Based Analysis of the Bacterial and Fungal Composition of Kefir Grains and Milks from Multiple Sources

peer-reviewedKefir is a fermented milk-based beverage to which a number of health-promoting properties have been attributed. The microbes responsible for the fermentation of milk to produce kefir consist of a complex association of bacteria and yeasts, bound within a polysaccharide matrix, known as the kefir grain. The consistency of this microbial population, and that present in the resultant beverage, has been the subject of a number of previous, almost exclusively culture-based, studies which have indicated differences depending on geographical location and culture conditions. However, culture-based identification studies are limited by virtue of only detecting species with the ability to grow on the specific medium used and thus culture-independent, molecular-based techniques offer the potential for a more comprehensive analysis of such communities. Here we describe a detailed investigation of the microbial population, both bacterial and fungal, of kefir, using high-throughput sequencing to analyse 25 kefir milks and associated grains sourced from 8 geographically distinct regions. This is the first occasion that this technology has been employed to investigate the fungal component of these populations or to reveal the microbial composition of such an extensive number of kefir grains or milks. As a result several genera and species not previously identified in kefir were revealed. Our analysis shows that the bacterial populations in kefir are dominated by 2 phyla, the Firmicutes and the Proteobacteria. It was also established that the fungal populations of kefir were dominated by the genera Kazachstania, Kluyveromyces and Naumovozyma, but that a variable sub-dominant population also exists.The Alimentary Pharmabiotic Centre is a research centre funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan. The authors and their work were supported by SFI CSET grant APC CSET 2 grant 07/CE/B1368

Influence of Milk Thistle Shot on Quality Parameters of the Sour-milk Beverage

Modern complicated ecological conditions cause a general necessity in improving the food structure of the population due to improving the quality, biological value and taste characteristics of products. Just that is why, the aim of the work was to study the influence of milk thistle shot on quality parameters of the sour-milk beverage – kefir. It was established, that it had the homogenous consistence with a broken clot and color from white to creamy with shot particles. The increase of the milk thistle shot dose to 3 % and 4 % results in the taste with the brightly expressed milk thistle smack and brown color with the expressed milk thistle content. The viscosity of kefir with fms 2,5 % changes during seven days of storage, although remains rather high at 8 day, namely 47 s. The increase of the viscosity of kefir with milk thistle shot is explained by its hygroscopic properties, in which result free moisture of the product is bound. The analysis of microbiological parameters in the process of storage of kefir with milk thistle shot allows to make a conclusion about the satisfactory sanitary condition of the new product and its harmlessness for consumer's health. In the kefir with milk thistle shot the general amount of amino acids grew by 11,6 %, including irreplaceable ones – by 10,1 %, replaceable – by 12,6 %, that indicates its biological value.Addition of milk thistle shot to kefir didn't cause changes of the biological value of the protein component of the combined product. It is testified by the mean value of amino acid scores of control (129,3 %) and experimental (127,9 %) samples of kefir. Some growth of the valine score (3,6 %) can be noted.So, the combined product is characterized by the balanced amino acid composition. Due to adding milk thistle shot, consumption of amino acids, necessary for synthesis of proteins and essential number of compounds, vitally important for the human organism, grew

A Big World in Small Grain: A Review of Natural Milk Kefir Starters

Milk kefir is a traditional fermented milk product whose consumption is becoming increasingly popular. The natural starter for kefir production is kefir grain, which consists of various bacterial and yeast species. At the industrial scale, however, kefir grains are rarely used due to their slow growth, complex application, bad reproducibility and high costs. Instead, mixtures of defined lactic acid bacteria and sometimes yeasts are applied, which alter sensory and functional properties compared to natural grain-based milk kefir. In order to be able to mimic natural starter cultures for authentic kefir production, it is a prerequisite to gain deep knowledge about the nature of kefir grains, its microbial composition, morphologic structure, composition of strains on grains and the impact of environmental parameters on kefir grain characteristics. In addition, it is very important to deeply investigate the numerous multi-dimensional interactions among different species, which play important roles on the formation and the functionality of grains

The Microbiota and Health Promoting Characteristics of the Fermented Beverage Kefir

peer-reviewedKefir is a complex fermented dairy product created through the symbiotic fermentation of milk by lactic acid bacteria and yeasts contained within an exopolysaccharide and protein complex called a kefir grain. As with other fermented dairy products, kefir has been associated with a range of health benefits such as cholesterol metabolism and angiotensin-converting enzyme (ACE) inhibition, antimicrobial activity, tumor suppression, increased speed of wound healing, and modulation of the immune system including the alleviation of allergy and asthma. These reports have led to increased interest in kefir as a focus of research and as a potential probiotic-containing product. Here, we review those studies with a particular emphasis on the microbial composition and the health benefits of the product, as well as discussing the further development of kefir as an important probiotic product.The authors are funded through the Teagasc Walsh Fellowship Scheme(2014025)and internal Teagasc funding(RMIS6486). BW is supported by the Canada Research Chairs Program and research in the Cotter laboratory is funded by SFI through the PI award “Obesibiotics”(11/PI/1137)and in the form of a center grant (APC Microbiome Institute Grant Number SFI/12/RC/2273)

Apoptotic effect of a novel kefir product, PFT, on multidrug-resistant myeloid leukemia cells via a hole-piercing mechanism.

We examined the apoptotic effect of a novel Probiotics Fermentation Technology (PFT) kefir grain product; PFT is a natural mixture composed primarily of Lactobacillus kefiri P-IF, a specific strain of L. kefiri with unique growth characteristics. The aim of this study was to examine the apoptotic effect of PFT on human multidrug-resistant (MDR) myeloid leukemia (HL60/AR) cells in vitro and explore the mechanistic approach underlying its effect. HL60/AR cells were cultured with PFT (0.6-5.0 mg/ml) for 3 days. The apoptotic effect of PFT was assessed through examination of percent apoptosis, caspase 3 activation, Bcl-2 expression levels and changes in mitochondrial membrane potential (MMP). PFT induced apoptosis in HL60/AR cells in a dose-dependent manner which was maximal at 67.5% for 5 mg/ml. Induction of apoptosis was associated with activation of caspase 3, decreased expression of Bcl-2 and decreased polarization of MMP. In addition, PFT showed a unique characteristic of piercing holes in HL60/AR cells, as indicated by AFM studies. This hole induction may be responsible for the apoptotic effect on cancer cells. These results suggest that PFT may act as a potential therapy for the treatment of MDR leukemia

Fermented beverages with health-promoting potential: Past and future perspectives

peer-reviewedFermentation is an ancient form of food preservation, which also improves the nutritional content of foods. In many regions of the world, fermented beverages have become known for their health-promoting attributes. In addition to harnessing traditional beverages for commercial use, there have recently been innovative efforts to develop non-dairy probiotic fermented beverages from a variety of substrates, including soy milk, whey, cereals and vegetable and fruit juices. On the basis of recent developments, it is anticipated that fermented beverages will continue to be a significant component within the functional food market

Comparison of the Antibacterial Activity of Cow Milk Kefir and Goat Milk Kefir Against Bacteria Bacillus Cereus

Background: Kefir is fermented milk and comes from the Caucasus. Kefir is made by inoculating cow milk, goat or sheep with kefir grain. Kefir contains 0.5–1.0% alcohol and 0.9 to 1.1% lactic acid. This product is very popular in the Soviet Union, where the consumption of kefir reach 4.5 kg per capita per year. Kefir made from pasteurized milk and fermented with kefir grain, kefir grain is white seeds from bacteria colony, such as Streptococcus sp., Lactobacilli and some types of yeast/yeast apatogen. As a probiotic drink, kefir contains bacteria of lactic acid and acetic acid bacteria in kefir are giving acidity and produces secondary metabolites that bacteriocins that kill pathogenic bacteria such as Bacillus cereus.Method: This study used cow milk and goat milk kefir as a starting material, to produce a cow milk kefir and goat milk kefir. Cow milk kefir and goat milk kefir were compared antibacterial activity against Bacillus cereus. Previously each made 3 variations of the concentration of 2%, 4%, and 6% kefir grains were added cow milk and goat milk. Antibacterial activity tested using Kirby Bauer method so that the observation a clear zone formed on the media was the inhibition activity of the bacterium Bacillus cereus. The results were analyzed by ANOVA, Mann Whitney, and Tukey with a significance of 95%.Result: The results showed that goat milk kefir has better antibacterial activity against Bacillus cereus compared to cow milk kefir. Goat milk kefir with kefir seeds concentration of 6% has the best antibacterial activity with inhibition zone diameter of 6 mm

Development of a sheep's milk kefir using species isolated from kefir products : a thesis presented in partial fulfilment of the requirements for the degree of Masters in Food Technology at Massey University, Palmerston North, New Zealand

The aim of the research described in this thesis was to develop a good tasting kefir using sheep’s milk. Kefir is a refreshing and effervescent milk beverage fermented with bacteria and yeast. Different combinations of bacteria, yeast and treatment result in different qualities of Kefir. An optimal kefir has a pleasant slightly sour flavour and is slightly effervescent. It is drunk chilled. To arrive at preferred or optimal kefir the following steps occurred: Yeast and bacteria were isolated using MRS, M17, and DRGB agar from five commercial and three homemade kefir products. 54 isolates were identified using 16s rDNA PCR for bacteria, and 26s rDNA for yeast. The commercial yeasts were: Saccharomyces cerevisiae, and Torulaspora delbrueckii, the commercial bacteria were: Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides, Lactobacillus plantarum, Lactococcus lactis subsp. cremoris, and Leuconostoc pseudomesenteroides, and Leuconostoc pseudomesenteroides. The homemade yeast were: Kluyveromyces marxianus, Kazachstania unispora, Pichia membranifaciens, and Clavispora lusitaniae, and the homemade bacteria were: Lactobacillus delbrueckii, Lactobacillus kefiranofaciens, and Lactobacillus kefiri. Streptococcus thermophiles was the only isolate found in both homemade and commercial kefir. One isolate of each species identified was used to form a starter culture and grown in gamma sterilized sheep’s milk, allowing the assessment of the growth characteristics required for kefir. The characteristics assessed were; cell counts, pH, textural properties, effervescence, and the levels of lactose, glucose, galactose, ethanol, lactic acid, acetic acid, and diacetyl using HPLC. The results from these tests were used to determine the optimum mix of species for a sheep’s milk kefir. Four different mixes of 5-6 isolates were chosen based on the results of the individual isolates and grown in sheep’s milk and tested for the same characteristics as the pure isolates as well as taste tested. This optimised kefir was made by inoculating 1x106cuf/mL of each isolate to sheep’s milk, sealed in the final container and fermented at 30°C for 24 hours. After cooling to 4°C the final product has a refreshing sour taste and effervescence, with a pH of 4.6, and a cell count above 3x109cfu/mL which decreases to above 9x108cfu/mL after five weeks, which is over 106cfu/mL required for labelling purposes