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.
Novel Combination of Prebiotics Galacto-Oligosaccharides and Inulin-Inhibited Aberrant Crypt Foci Formation and Biomarkers of Colon Cancer in Wistar Rats.
Qamar, T. R., Syed, F., Nasir, M., Rehman, H., Zahid, M. N., Liu, R. H. & Iqbal, S. (2016). Nutrients, 8(8), 465.
The selectivity and beneficial effects of prebiotics are mainly dependent on composition and glycosidic linkage among monosaccharide units. This is the first study to use prebiotic galacto-oligosaccharides (GOS) that contains β-1,6 and β-1,3 glycosidic linkages and the novel combination of GOS and inulin in cancer prevention. The objective of the present study is to explore the role of novel GOS and inulin against various biomarkers of colorectal cancer (CRC) and the incidence of aberrant crypt foci (ACF) in a 1,2-dimethyl hydrazine dihydrochloride (DMH)-induced rodent model. Prebiotic treatments of combined GOS and inulin (57 mg each), as well as individual doses (GOS: 76–151 mg; inulin 114 mg), were given to DMH-treated animals for 16 weeks. Our data reveal the significant preventive effect of the GOS and inulin combination against the development of CRC. It was observed that inhibition of ACF formation (55.8%) was significantly (p ≤ 0.05) higher using the GOS and inulin combination than GOS (41.4%) and inulin (51.2%) treatments alone. This combination also rendered better results on short-chain fatty acids (SCFA) and bacterial enzymatic activities. Dose-dependent effects of prebiotic treatments were also observed on cecum and fecal bacterial enzymes and on SCFA. Thus, this study demonstrated that novel combination of GOS and inulin exhibited stronger preventive activity than their individual treatments alone, and can be a promising strategy for CRC chemoprevention.
14-3-3γ regulates lipopolysaccharide-induced inflammatory responses and lactation in dairy cow mammary epithelial cells by inhibiting NF-κB and MAPKs and up-regulating mTOR signaling.
Liu, L., Lin, Y., Liu, L., Bian, Y., Zhang, L., Gao, X. & Li, Q. (2015). International Journal of Molecular Sciences, 16(7), 16622-16641.
As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1Β (IL-1β) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-κB (IκB) phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of Β-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPARγ). These results suggest that 14-3-3γ was able to attenuate the LPS-induced inflammatory responses and promote proliferation and lactation in LPS-induced DCMECs by inhibiting the activation of the NF-κB and MAPK signaling pathways and up-regulating mTOR signaling pathways to protect against LPS-induced injury.
Production of impure prebiotic galacto-oligosaccharides and their effect on calcium, magnesium, iron and zinc absorption in Sprague-Dawley rats.
Maawia, K., Iqbal, S., Qamar, T. R., Rafiq, P., Ullah, A. & Ahmad, M. (2016). PharmaNutrition, 4(4), 154-160.
Prebiotic galacto-oligosaccharides (GOS) are important “functional foods” of current scenario and used for various health benefits including improved mineral absorption. In the present study, it was hypothesized that novel GOS mixture, produced through transgalactosylation, with significant amount of mono and disaccharides may enhance mineral absorption in Sprague-Dawley rats. The non-purified GOS having β-(1 → 6) and β-(1 → 3) glycosidic linkage, were evaluated for apparent absorption of calcium, magnesium, iron and zinc. The rats were divided into two main groups (n = 12 per group, 6 male/6 female) fed on control and GOS (5 g/100 g) diet. The feces were collected after 7 days interval for 28 days. The weight gain, feed and water intake were statistically similar (p < 0.05) in both groups irrespective of gender. Similarly, the absorption of minerals was statistically not different in both genders during whole study. The GOS diet significantly (p < 0.05) improved absorption of Ca (34.55–39.93%), Mg (51.22–58.05%) and Fe (31.58–39.21%) as compared to control diet at the end of study. However, no impact on Zn absorption was observed during the whole study. It can be inferred that the use of non-purified GOS for 3–4 weeks may enhance Ca, Mg and Fe absorption.
Epigenetic regulation of miR‐29s affects the lactation activity of dairy cow mammary epithelial cells.
Bian, Y., Lei, Y., Wang, C., Wang, J., Wang, L., Liu, L., Liu, L.,.Gao, X. & Li, Q. (2015). Journal of Cellular Physiology, 230(9), 2152-2163.
Milk is important for human nutrition, and enhanced milk quality has become a major selection criterion for the genetic improvement of livestock. Epigenetic modifications have been shown to be involved in mammary gland development; but the mechanisms underlying their effects remain unknown. MicroRNAs are involved in the regulation of milk synthesis and in mammary gland development. Our study is the first to investigate the roles of miR-29s and epigenetic regulation in dairy cow mammary epithelial cells (DCMECs). Our results show that miR-29s regulate the DNA methylation level by inversely targeting both DNMT3A and DNMT3B in DCMECs. The inhibition of miR-29s caused global DNA hypermethylation and increased the methylation levels of the promoters of important lactation-related genes, including casein alpha s1 (CSN1S1), E74-like factor 5 (ElF5), peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element binding protein-1 (SREBP1), and glucose transporter 1 (GLUT1). The inhibition of miR-29s reduced the secretion of lactoprotein, triglycerides (TG) and lactose by DCMECs. Moreover, the treatment of DCMECs with 5-aza-2′-deoxycytidine (5-Aza-dC) decreased the methylation levels of the miR-29b promoter and increased the expression of miR-29b. The link between miR-29s and DNMT3A/3B enhances our understanding of the roles of miRNAs in mammary gland function, and our data will inform more experimentally oriented studies to identify new mechanisms of regulating lactation. We present new insights regarding the epigenetic regulation of lactation performance. Improved understanding of the molecular basis of lactation will aid in the development of strategies for optimizing milk quality in dairy cows and modifying the lactation performance of offspring.
Bistability and Nonmonotonic Induction of the lac Operon in the Natural Lactose Uptake System.
Zander, D., Samaga, D., Straube, R. & Bettenbrock, K. (2017). Biophysical Journal, 112(9), 1984-1996.
The Escherichia coli lac operon is regulated by a positive feedback loop whose potential to generate an all-or-none response in single cells has been a paradigm for bistable gene expression. However, so far bistable lac induction has only been observed using gratuitous inducers, raising the question about the biological relevance of bistable lac induction in the natural setting with lactose as the inducer. In fact, the existing experimental evidence points to a graded rather than an all-or-none response in the natural lactose uptake system. In contrast, predictions based on computational models of the lactose uptake pathway remain controversial. Although some argue in favor of bistability, others argue against it. Here, we reinvestigate lac operon expression in single cells using a combined experimental/modeling approach. To this end, we parameterize a well-supported mathematical model using transient measurements of LacZ activity upon induction with different amounts of lactose. The resulting model predicts a monostable induction curve for the wild-type system, but indicates that overexpression of the LacI repressor would drive the system into the bistable regime. Both predictions were confirmed experimentally supporting the view that the wild-type lac induction circuit generates a graded response rather than bistability. More interestingly, we find that the lac induction curve exhibits a pronounced maximum at intermediate lactose concentrations. Supported by our data, a model-based analysis suggests that the nonmonotonic response results from saturation of the LacI repressor at low inducer concentrations and dilution of Lac enzymes due to an increased growth rate beyond the saturation point. We speculate that the observed maximum in the lac expression level helps to save cellular resources by limiting Lac enzyme expression at high inducer concentrations.
Effect of calcium reduction on the properties of half-fat Cheddar-style cheeses with full-salt or half-salt.
McCarthy, C. M., Wilkinson, M. G. & Guinee, T. P. (2017). International Dairy Journal, In press.
Standard-calcium (SCa) and reduced-calcium (RCa) half-fat (16%) Cheddar-style cheeses with full-salt (1.9%) or half-salt (0.9%) were made in triplicate, ripened for 270 d, and analysed for composition and changes in lactose metabolism, pH, proteolysis, water-sorption, fracture properties and heat-induced flowability during maturation. The pressing load applied to the moulded cheese was modified to ensure equal moisture in all cheeses despite the differences in salt and calcium levels. The RCa cheeses were characterized by higher primary proteolysis (αs1-casein degradation, pH 4.6-soluble N development), lower secondary proteolysis (concentration of free amino acids), higher water-holding capacity on reducing relative humidity from 85 to 5%, lower fracture stress and strain, and more extensive flow on heating. Overall, the use calcium reduction, when used in conjunction with moisture normalization, proved an effective means of counteracting the adverse effects of fat reduction on texture and cooking properties in half-fat, half-salt cheese.