Kefir Cells Research Articles

Preliminary Evaluation of the Use of Thermally-Dried Immobilized Kefir Cells in Low Alcohol Winemaking

Low alcohol wines (≤10.5% vol) are novel products that have gradually been gaining the consumers’ and market’s interest over the last decade. Taking into account the technological properties of immobilized cell systems alongside with the commercial need for dry cultures, the aim of the present study was to assess the suitability of thermally-dried immobilized kefir cells on DCM, apples pieces, and grape skins in low alcohol wine production. Storage of thermally-dried kefir culture in various temperatures (−18, 5, and 20 °C) resulted in high viability rates for immobilized cells (up to 93% for yeasts/molds immobilized on grape skins and stored at −18 °C for 6 months). Fermentation activity was maintained after storage in all cases, while high operational stability was confirmed in repeated batch fermentations for a period of 6 months. Principal Component Analysis (PCA) revealed that the fermentation temperature rather than the state of kefir culture affected significantly volatiles detected by Head Space Solid-Phase Microextraction Gas Chromatography–Mass Spectrometry analysis. Notably, all new products were of high quality and approved by the sensory panel.

Winemaking using immobilized kefir cells on natural zeolites

The present study investigated the use of natural zeolites, an inorganic support with multiple technological advantages, for kefir culture immobilization and subsequent evaluation in winemaking at various temperatures (5–45 °C). Cell immobilization was confirmed by Scanning Electron Microscopy and immobilized kefir cells biodiversity was studied by Next Generation Sequencing. Simultaneous alcoholic and malolactic wine fermentations were monitored in repeated batch fermentations for a period >3 months, indicating a high operational stability, even at 5 °C. Alcohol content up to 12.5% vol, malic acid conversion up to 69.2% and ethanol productivity values several fold higher than those observed in traditional practice were reported in fermentations with immobilized cells on natural zeolites. PCA applied to minor volatiles detected by HS-SPME GC/MS showed that mostly the fermentation temperature affected the profile of the aroma-related compounds, while the high total ester concentrations detected at 20 οC ascertained the high quality of the products. Importantly, all new wines were accepted by the tasting panel. The results indicated the high potential of immobilized kefir culture on natural zeolites in scale-up industrial applications and suggested their exploitation in the development of low alcohol wines and wines with a semi-sweet character.

Abating whey organic load through ethanol and lactic acid production by kefir

The extent to which selected ethanol and lactic acid production bioprocesses contribute to whey waste abatement was examined. Alcoholic fermentation of whey was carried out by kefir cells immobilized on grape stalks, delignified cellulosic materials, or brewer's spent grains. Lactic acid fermentation was also performed by free kefir cells with or without addition of brewer's spent grains as promoting material. Since whey fermentation rate is affected by the lactose uptake rate, 14C-labeled lactose was used to study the fermentation ability of kefir. The highest reductions in biochemical oxygen demand and chemical oxygen demand of whey, about 68% and 52%, respectively, were achieved by lactic acid fermentation in 6 h at 37 °C and pH 5.5, in the presence of 120 g brewer's spent grains. Additionally, at the same conditions, the highest 14C-labeled lactose uptake rate by kefir and consequently the highest alcoholic fermentation rate were also recorded. However, greater reductions in biochemical oxygen demand and chemical oxygen demand of whey are required prior to final disposal.

Whey decontamination by kefir immobilized on grape stalks

Purpose – The purpose of this paper is to evaluate the possibility of a new biocatalyst prepared by kefir cells immobilization on grape stalks (GS) to reduce quickly and efficiently the Biochemical Oxygen Demand (BOD) and Chemical Oxygen Demand (COD) of the waste whey during its fermentation producing potable ethanol. Design/methodology/approach – Many batch whey fermentations were carried out in order the effect of various conditions (pH, temperature) on 14C-labeled lactose uptake rate by the GS-immobilized kefir cells and consequently on fermentation rate as well as on ethanol production and whey BOD and COD reduction to be determined. Findings – It has been illustrated that GS-biocatalyst was suitable for whey BOD and COD reduction by about 32 and 25 percent, respectively during whey alcoholic fermentation at 30°C and pH 5.5 in only ten hours, producing about 3.30 percent w/v of ethanol. Originality/value – The findings of this research may enhance the existing literature on whey exploitation, for the first time focussing on the use of cheap and abundant GS as support for kefir immobilization during whey fermentation, which is potentially acceptable by industries in order to reduce fast and easily the whey polluting load and produce ethanol.

Study of whey fermentation by kefir immobilized on low cost supports using 14C-labelled lactose

Brewer's Spent Grains (BSG) and Malt Spent Rootlets (MSR) were used as supports for kefir cells immobilization and the role of lactose uptake rate by kefir in the positive activity of produced biocatalysts during whey fermentation was investigated. Lactose uptake rate by the immobilized cells was recorded using (14)C-labelled lactose and the effect of various conditions (pH, temperature and kind of support) on it and consequently on fermentation time and ethanol production was examined. The results showed that lactose uptake rate was correlated to fermentation rate and increased as temperature was increased up to 30°C at pH 5.5. The same results have been recently noticed by using biocatalysts with Delignified Cellulosic Materials (DCM) and Gluten Pellets (GP), but fermentation time of about 7h by kefir immobilized on DCM and BSG resulted to two fold lower than that on GP and MSR. The highest alcohol concentration was observed by MSR.

Kefir Immobilized on Corn Grains as Biocatalyst for Lactic Acid Fermentation and Sourdough Bread Making

The natural mixed culture kefir was immobilized on boiled corn grains to produce an efficient biocatalyst for lactic acid fermentation with direct applications in food production, such as sourdough bread making. The immobilized biocatalyst was initially evaluated for its efficiency for lactic acid production by fermentation of cheese whey at various temperatures. The immobilized cells increased the fermentation rate and enhanced lactic acid production compared to free kefir cells. Maximum lactic acid yield (68.8 g/100 g) and lactic acid productivity (12.6 g/L per day) were obtained during fermentation by immobilized cells at 37 °C. The immobilized biocatalyst was then assessed as culture for sourdough bread making. The produced sourdough breads had satisfactory specific loaf volumes and good sensory characteristics. Specifically, bread made by addition of 60% w/w sourdough containing kefir immobilized on corn was more resistant regarding mould spoilage (appearance during the 11(th) day), probably due to higher lactic acid produced (2.86 g/Kg of bread) compared to the control samples. The sourdough breads made with the immobilized biocatalyst had aroma profiles similar to that of the control samples as shown by headspace SPME GC-MS analysis.

Surface proteins from Lactobacillus kefir antagonize in vitro cytotoxic effect of Clostridium difficile toxins

In this work, the ability of S-layer proteins from kefir-isolated Lactobacillus kefir strains to antagonize the cytophatic effects of toxins from Clostridium difficile (TcdA and TcdB) on eukaryotic cells in vitro was tested by cell detachment assay. S-layer proteins from eight different L. kefir strains were able to inhibit the damage induced by C. difficile spent culture supernatant to Vero cells. Besides, same protective effect was observed by F-actin network staining. S-layer proteins from aggregating L. kefir strains (CIDCA 83115, 8321, 8345 and 8348) showed a higher inhibitory ability than those belonging to non-aggregating ones (CIDCA 83111, 83113, JCM 5818 and ATCC 8007), suggesting that differences in the structure could be related to the ability to antagonize the effect of clostridial toxins. Similar results were obtained using purified TcdA and TcdB. Protective effect was not affected by proteases inhibitors or heat treatment, thus indicating that proteolytic activity is not involved. Only preincubation with specific anti-S-layer antibodies significantly reduced the inhibitory effect of S-layer proteins, suggesting that this could be attributed to a direct interaction between clostridial toxins and L. kefir S-layer protein. Interestingly, the interaction of toxins with S-layer carrying bacteria was observed by dot blot and fluorescence microscopy with specific anti-TcdA or anti-TcdB antibodies, although L. kefir cells did not show protective effects. We hypothesize that the interaction between clostridial toxins and soluble S-layer molecules is different from the interaction with S-layer on the surface of the bacteria thus leading a different ability to antagonize cytotoxic effect. This is the first report showing the ability of S-layer proteins from kefir lactobacilli to antagonize biological effects of bacterial toxins. These results encourage further research on the role of bacterial surface molecules to the probiotic properties of L. kefir and could contribute to strain selection with potential therapeutic or prophylactic benefits towards CDAD.

PH Effect on Lactose Uptake Rate by Kefir Cells Immobilized on Delignified Cellulosic Material, Using 14C-Labelled Lactose during Whey Fermentation

pH Effect on Lactose Uptake Rate by Kefir Cells Immobilized on Delignified Cellulosic Material, Using 14C-Labelled Lactose during Whey Fermentation

Scale-up of Thermally Dried Kefir Production as Starter Culture for Hard-Type Cheese Making: An Economic Evaluation

This paper concerns the effect of thermal-drying methodology on the investment cost for dried kefir cells production in order to be used as starter culture in cheese manufacturing. Kefir cells were produced at pilot plant scale using a 250-L bioreactor and whey as the main substrate. Kefir cells were subsequently dried in a thermal dryer at 38 degrees C and used as a starter culture in industrial-scale production of hard-type cheeses. The use of thermally dried kefir as starter culture accelerated ripening of cheeses by increasing both lipolysis and fermentation rate as indicated by the ethanol, lactic acid, and glycerol formation. Additionally, it reduced coliforms and enterobacteria as ripening proceeded. This constituted the basis of developing an economic study in which industrial-scale production of thermally dried kefir starter culture is discussed. The industrial design involved a three-step process using three bioreactors of 100, 3,000, and 30,000 L for a plant capacity of 300 kg of thermally dried kefir culture per day. The cost of investment was estimated at 238,000 euro, which is the 46% of the corresponding cost using freeze-drying methodology. Production cost was estimated at 4.9 euro/kg of kefir biomass for a 300-kg/day plant capacity, which is the same as with the corresponding cost of freeze-dried cells. However, the estimated added value is up to 10.8 x 10(9) euro within the European Union.

Thermally-dried free and immobilized kefir cells as starter culture in hard-type cheese production

In an attempt to seek for suitable dried cultures, thermally-dried kefir was employed as starter in hard-type cheese production and tested in cheeses ripened at 5, 18 and 22 °C. Both free and immobilised on casein kefir cells were used and compared to cheese made without starter culture. Cheese products made with free cells of kefir culture were characterized by longer preservation time, improved aroma, taste, texture characteristics and increased degree of openness. Volatile profiles obtained by GC/MS analysis revealed a 216% increase in total concentration of esters, organic acids, alcohols and carbonyl compounds between cheeses prepared with and without kefir culture.

Production of Hard-Type Cheese Using Free or Immobilized Freeze-Dried Kefir Cells as a Starter Culture

This study provides a contribution to hard-type cheese starter culture production through the use of a freeze-dried culture in the ripening of hard-type cheeses. The effect of initial cell concentration, ripening temperature, and cell immobilization of kefir on the degree of openness, mold spoilage, microbial associations, physicochemical characteristics, and aroma-related compounds was studied. Use of kefir starter cultures resulted in cheese with an increased shelf life and resistance to spoilage as compared to control cheeses without kefir inoculants. Furthermore, the freeze-dried kefir culture improved aroma, taste, and texture characteristics while increasing the degree of openness in comparison to traditional hard-type cheese products. The kefir culture resulted in an increase in counts of total aerobic bacteria, yeasts and molds, lactococci, and lactobacilli until the 15th day of ripening. From then on, only lactobacilli counts increased, reaching levels up to 9.17 log CFU/g in cheeses ripened at 5 degrees C using freeze-dried kefir cells immobilized on casein. SPME-GC/MS analysis revealed major differences in volatile composition, especially with regard to alcohols (up to 75%), carbonyl compounds (up to 75%), and esters (up to 64%) between cheeses made with kefir cells and cheeses made without kefir inoculants.

Production of Lactobacillus kefir cells for asymmetric synthesis of a 3,5-dihydroxycarboxylate

An efficient fedbatch process for the production of Lactobacillus kefir DSM 20587 cells was developed. An improvement in space time yield of 270% (3.7 g(DCW) l(-1)-day(-1)) and in final enzyme activity of 440% (9.1 U/ml) was achieved on a 150 l scale by controlling the oxygen transfer rate to 7--9 mmol l(-1) h(-1). The cells exhibited good and highly stereoselective reducing activities against tert-butyl 6-chloro-3,5-dioxohexanoate. tert-Butyl (3R,5S)-6-chloro-dihydroxyhexanoate, a chiral building block for HMG-CoA reductase inhibitor synthesis, was produced with 47.5% yield and >99% ee at C(3)3 and C(5)5 in a simple batch biotransformation process.