Lactose fermentation by Kombucha – a process to obtain new milk–based beverages.
Iličić, M., Kanurić, K., Milanović, S., Lončar, E., Djurić, M. & Malbaša, R. (2012). Romanian Biotechnological Letters, 17(1), 7013-7021.
This paper focuses on fermentation of lactose from a model system (black tea) and from two types of milk (0.9% w/w and 2.2% w/w of fat) by application of Kombucha. Quantities of the applied Kombucha starter were 10% v/v and 15% v/v. All fermentations were performed at 42°C. The process to achieve a desirable pH=4.5 was slower in the model system (16 h) than in milks (9 - 10 h). Regarding starter quantity, 10% v/v proved the optimal. Regarding types of milk, higher fat content guarantees shorter fermentation and higher yield of metabolites. Utilization of lactose was found at a level of ≈20% and ≈30% in milks with 0.9% w/w and 2.2% w/w of fat, respectively. This was correlated with an appearance of intermediates and/or products. Glucose underwent further transformations almost entirely, while galactose showed much lower reactivity. Seven to twelve times higher contents of lactic acid were found compared to acetic acid. Milk-based beverage from the reduced fat sample, inoculated with 10% v/v of Kombucha starter, has the best physical characteristics (syneresis and water holding capacity). It also developed a good texture (especially cohesiveness and index of viscosity). Milk lactose fermentation was a process that could have been used for obtaining new milk-based products.
The effect of transglutaminase on rheology and texture of fermented milk products.
Iličić, M. D., Milanović, S. D., Carić, M. Ð., Vukić, V. R., Kanurić, K. G., Ranogajec, M. I. & Hrnjez, D. V. (2013). Journal of Texture Studies, 44(2), 160-168.
The aim of this study was to investigate the effect of transglutaminase (TG) addition on rheological properties, textural characteristics and microstructure of fermented milk products manufactured by different starters (probiotics and kombucha inoculum). Rheological analysis revealed that all manufactured fermented milk products had higher storage modulus than loss modulus and exhibited thixotropic and a typical shear thinning behavior. The addition of TG in milk increased approximately 10.5% hysteresis loop area, 39% firmness and 48% consistency in sample produced with probiotic starter and had more firm and stable gel structure than kombucha fermented milk products. The scanning electron microscopy micrographs showed that casein matrix of fermented milk products containing TG is continuous and uninterrupted except for void spaces occupied by milk serum and starter culture cell.
Molecular characterization and solution properties of enzymatically tailored arabinoxylans.
Pitkänen, L., Tuomainen, P., Virkki, L. & Tenkanen, M. (2011). International Journal of Biological Macromolecules, 49(5), 963-969.
Two α-L-arabinofuranosidases with different substrate specificities were used to modify the arabinose-to-xylose ratio of cereal arabinoxylans: one enzyme (AXH-m) removed the L-arabinofuranosyl substituents from the monosubstituted xylopyranosyl residues and the other (AXH-d3) the (1 → 3)-linked L-arabinofuranosyl units from the disubstituted xylopyranosyl residue. In this study, we noticed that not only the arabinose-to-xylose ratio but also the position of the arabinofuranosyl substituents affects the water-solubility of arabinoxylans. The AXH-d3 treatment had no significant effect on the solution conformation of arabinoxylans, but the density of the arabinoxylan molecules decreased in DMSO solution after AXH-m modification. The possible heterogeneity of arabinoxylans complicated the interpretation of data describing the macromolecular properties of the enzymatically modified samples.
Analysis of the arabinoxylan arabinofuranohydrolase gene family in barley does not support their involvement in the remodelling of endosperm cell walls during development.
Laidlaw, H. K., Lahnstein, J., Burton, R. A., Fincher, G. B. & Jobling, S. A. (2012). Journal of Experimental Botany, 63(8), 3031-3045.
Arabinoxylan arabinofuranohydrolases (AXAHs) are family GH51 enzymes that have been implicated in the removal of arabinofuranosyl residues from the (1,4)-β-xylan backbone of heteroxylans. Five genes encoding barley AXAHs range in size from 4.6 kb to 7.1 kb and each contains 16 introns. The barley HvAXAH genes map to chromosomes 2H, 4H, and 5H. A small cluster of three HvAXAH genes is located on chromosome 4H and there is evidence for gene duplication and the presence of pseudogenes in barley. The cDNAs corresponding to barley and wheat AXAH genes were cloned, and transcript levels of the genes were profiled across a range of tissues at different developmental stages. Two HvAXAH cDNAs that were successfully expressed in Nicotiana benthamiana leaves exhibited similar activities against 4-nitrophenyl α-L-arabinofuranoside, but HvAXAH2 activity was significantly higher against wheat flour arabinoxylan, compared with HvAXAH1. HvAXAH2 also displayed activity against (1,5)-α-L-arabinopentaose and debranched arabinan. Western blotting with an anti-HvAXAH antibody was used to define further the locations of the AXAH enzymes in developing barley grain, where high levels were detected in the outer layers of the grain but little or no protein was detected in the endosperm. The chromosomal locations of the genes do not correspond to any previously identified genomic regions shown to influence heteroxylan structure. The data are therefore consistent with a role for AXAH in depolymerizing arabinoxylans in maternal tissues during grain development, but do not provide compelling evidence for a role in remodelling arabinoxylans during endosperm or coleoptile development in barley as previously proposed.
Bacterial nanocellulose‐reinforced arabinoxylan films.
Stevanic, J. S., Joly, C., Mikkonen, K. S., Pirkkalainen, K., Serimaa, R., Rémond, C., Toriz, G., Gatenholm, P., Tenkanen, M. & Salmén, L. (2011). Journal of Applied polymer science, 122(2), 1030-1039.
There is an increasing interest in substituting today's films for food packaging applications with films based on renewable resources. For this purpose, rye arabinoxylans, unmodified and enzymatically debranched, were studied for the preparation of neat films and composite films reinforced with bacterial cellulose (BC). Mixing in a homogenizer produced optically transparent, uniform films. Physical and mechanical characteristics of such films are here reported. Debranching of the arabinoxylan caused an increase in its crystallinity of 20%. Debranching as well as reinforcement with BC resulted in a decrease of the moisture sorption of the films. The debranching also resulted in a reduced breaking strain while the reinforcement with BC increased stiffness and strength of the films.
Innovative Caciocavallo cheeses made from a mixture of cow milk with ewe or goat milk.
Niro, S., Fratianni, A., Tremonte, P., Sorrentino, E., Tipaldi, L., Panfili, G. & Coppola, R. (2014). Journal of Dairy Science, 97(3), 1296-1304.
This study assessed and compared the physicochemical, microbiological, and sensorial characteristics of Caciocavallo cheeses, made from cow milk and a mixture of cow with ewe or goat milk, during ripening. Different cheese-making trials were carried out on an industrial scale following the standard procedure of pasta filata cheeses, with some modifications. The percentage of the different added milk to cow milk influenced compositional and nutritional characteristics of the innovative products, leading to a satisfactory microbiological and sensorial quality.
Simultaneous uptake of lignocellulose‐based monosaccharides by Escherichia coli.
Jarmander, J., Hallström, B. M. & Larsson, G. (2014). Biotechnology and Bioengineering, 111(6), 1108-1115.
Lignocellulosic waste is a naturally abundant biomass and is therefore an attractive material to use in second generation biorefineries. Microbial growth on the monosaccharides present in hydrolyzed lignocellulose is however associated with several obstacles whereof one is the lack of simultaneous uptake of the sugars. We have studied the aerobic growth of Escherichia coli on D-glucose, D-xylose, and L-arabinose and for simultaneous uptake to occur, both the carbon catabolite repression mechanism (CCR) and the AraC repression of xylose uptake and metabolism had to be removed. The strain AF1000 is a MC4100 derivative that is only able to assimilate arabinose after a considerable lag phase, which is unsuitable for commercial production. This strain was successfully adapted to growth on L-arabinose and this led to simultaneous uptake of arabinose and xylose in a diauxic growth mode following glucose consumption. In this strain, a deletion in the phosphoenolpyruvate:phosphotransferase system (PTS) for glucose uptake, the ptsG mutation, was introduced. The resulting strain, PPA652ara simultaneously consumed all three monosaccharides at a maximum specific growth rate of 0.59 h-1, 55% higher than for the ptsG mutant alone. Also, no residual sugar was present in the cultivation medium. The potential of PPA652ara is further acknowledged by the performance of AF1000 during fed-batch processing on a mixture of D-glucose, D-xylose, and L-arabinose. The conclusion is that without the removal of both layers of carbon uptake control, this process results in accumulation of pentoses and leads to a reduction of the specific growth rate by 30%.
Galactose can be an inducer for production of therapeutic proteins by auto-induction using E. coli BL21 strains.
Xu, J., Banerjee, A., Pan, S. H. & Li, Z. J. (2012). Protein Expression and Purification, 83(1), 30-36.
Recently lactose mediated auto-induction in Escherichia coli has gained a lot of interest because higher protein titer could be achieved without the need to monitor growth and add inducer at the proper time. In this study a high level therapeutic protein production by auto-induction was observed in E. coli BL21 using either T7 or tac promoters in the modified Luria Bertani (mLB) medium containing soy peptone instead of tryptone in Luria Bertani (LB) medium. Based on medium analysis and spiking experiments it was found that 0.4 mM galactose from the soy peptone caused the auto-induction. E. coli cultures induced by galactose can saturate at considerably higher density than cultures induced by IPTG. Galactose is not consumed by E. coli BL21. Finally it has been demonstrated that auto-induction can be effectively used in fed-batch fermentation for the industrial production of a therapeutic protein. The principle of galactose mediated auto-induction should be able to apply to high throughput microplates, shake flasks and fed-batch fermentors for clone screening and therapeutic protein expression in E. coli gal- strains such as most commonly used BL21.
Production of an 18% protein liquid micellar casein concentrate with a long refrigerated shelf life.
Amelia, I. & Barbano, D. M. (2013). Journal of Dairy Science, 96(5), 3340-3349.
Our objective was to develop a process to produce a high-concentration liquid micellar casein concentrate (18% protein, MCC18) with a long refrigerated shelf life. The MCC18 is a novel milk protein ingredient produced by fractionating skim milk using microfiltration (MF). To achieve a long refrigerated shelf life, the processing of MCC18 was designed to maximize the removal of low-molecular weight compounds [e.g., lactose and nonprotein nitrogen (NPN)] that can be easily metabolized by microbes, while minimizing the microbial count in the final product. The production of MCC18 was done over a period of 5 d. The experiment was replicated 3 times in different weeks with a different batch of raw milk. Raw whole milk was pasteurized and separated to produce skim milk. Skim milk was ultrafiltered (UF) to remove more than half of the lactose and NPN. The UF milk retentate was diluted with water and then MF in 3 stages to remove approximately 95% of the serum protein and further remove lactose and NPN. The retentate from the last stage of MF was UF to concentrate the protein to 18% and batch pasteurized. The MCC18 was collected immediately after processing in sterile plastic vials and stored at 4°C. The average MCC18 contained 21.78% total solids, 18.27% true protein, 0.31% NPN, and 0.13% lactose. The MCC18 at the day of processing contained a mean aerobic bacterial count of 2.1 log cfu/mL and mean aerobic spore count of 2.3 log cfu/mL. The MCC18 formed a solid gel at temperatures <22°C, but the MCC18 reverted back to a liquid when warmed from 4°C temperature to >22°C. This provides a unique opportunity in ingredient handling and packaging and eliminates the challenges encountered in reconstitution of dried milk protein ingredients. The MCC18 produced in this study maintained a bacteria count <20,000 cfu/mL for 16 wk when stored at a refrigeration temperature of 4°C. Further study is needed to determine if changes occur in the organoleptic and functional properties of MCC18. We envision that the conversion of skim milk to MCC and its coproducts (serum protein concentrate and lactose concentrate) could be used as an alternative to the production of nonfat dry milk to balance milk production seasonality, specifically the components of skim milk portion.
Production of bioethanol from effluents of the dairy industry by Kluyveromyces marxianus.
Zoppellari, F. & Bardi, L. (2013). New Biotechnology, 30(6), 607-613.
Whey and scotta are effluents coming from cheese and ricotta processing respectively. Whey contains minerals, lipids, lactose and proteins; scotta contains mainly lactose. Whey can be reused in several ways, such as protein extraction or animal feeding, while nowadays scotta is just considered as a waste; moreover, due to very high volumes of whey produced in the world, it poses serious environmental and disposal problems. Alternative destinations of these effluents, such as biotechnological transformations, can be a way to reach both goals of improving the added value of the agroindustrial processes and reducing their environmental impact.
In this work we investigated the way to produce bioethanol from lactose of whey and scotta and to optimize the fermentation yields. Kluyveromyces marxianus var. marxianus was chosen as lactose-fermenting yeast. Batch, aerobic and anaerobic, fermentations and semicontinuous fermentations in dispersed phase and in packed bed reactor were carried out of row whey, scotta and mix 1:1 whey:scotta at a laboratory scale. Different temperatures (28–40°C) were also tested to check whether the thermotolerance of the chosen yeast could be useful to improve the ethanol yield.
The best performances were reached at low temperatures (28°C); high temperatures are also compatible with good ethanol yields in whey fermentations, but not in scotta fermentations. Semicontinuous fermentations in dispersed phase gave the best fermentation performances, particularly with scotta. Then both effluents can be considered suitable for ethanol production. The good yields obtained from scotta allow us to transform this waste in a source.