An integrated bioprocess to recover bovine milk oligosaccharides from colostrum whey permeate
Introduction
Human milk oligosaccharides (HMO) are indigestible sugars with complex structures that act as selective growth substrates for beneficial bacteria in the infant’s gastrointestinal tract and possess anti-pathogenic and immunomodulatory activities (Zivkovic and Barile, 2011). Increasing interest in HMO has prompted the search for suitable sources of HMO-like oligosaccharides and for the development of economically-viable scalable processes for the production of bioactive oligosaccharides for in vitro and human studies, and as therapeutic ingredients (Lange et al., 2014).
Our research group has demonstrated that bovine milk and dairy streams such as whey permeate contain bovine milk oligosaccharides (BMO) that are structurally similar to HMO in that both contain branched oligosaccharides with glucose, galactose, N-acetylglucosamine, sialic acid and to a certain extent, fucose decorations (Barile et al., 2009, Aldredge et al., 2013). BMO are more similar to HMO than the currently available oligosaccharides in commercial prebiotics, which possess simpler structures and may not selectively stimulate the growth of Bifidobacterium longum subspecies infantis (B. infantis) in the infant gut to the same extent as HMO (Ninonuevo and Bode, 2008).
Whey permeate, a by-product of the recovery of whey protein by membrane filtration, has generally been considered a problematic and abundant stream leading to environmental pollution if disposed of inappropriately. Owing to recent research findings, whey permeate is now considered an attractive source of bioactive oligosaccharides with many applications in human nutrition. Considering the presence of bovine milk oligosaccharides (Barile et al., 2009) and the global production of whey, exceeding 200 million tons per year (Affertsholt, 2009), the development of large-scale processing techniques and high-throughput analytics will enable the production of large quantities of high-value compounds that will ameliorate both environmental and economic issues associated with dairy processing (Cohen et al., 2015).
Current techniques to isolate oligosaccharides from milk rely on the combination of lactose hydrolysis and membrane filtration, typically followed by diafiltrations to increase the purity of the recovered components (Sarney et al., 2000, Nordvang et al., 2014, Altmann et al., 2015). However, isolating oligosaccharides in milk remains challenging due to the high lactose concentration and lower concentration of target oligosaccharides. This imbalance complicates recovery of the target bioactive oligosaccharides at high purity.
Recently, several nanofiltration membranes were evaluated for the recovery of oligosaccharides from bovine milk (Altmann et al., 2015). Experiments at industrial scale (1000 L) using a 300 Da spiral wound membrane at 5–10 bar and concentration factor (CF = volume of feed/volume of retentate) of 10 have enabled the recovery of 70–97% of three oligosaccharides present in bovine milk (3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL), and N-acetylgalactosaminyl-lactose), with a very low degree of purity (1.4%). The use of diafiltration increased the purity of the retained oligosaccharides from 1.4 to 10.6% at the expense of reducing their retention from between 70 and 97% down to 32 to 56%.
Thus far, oligosaccharides have been isolated at a high recovery yield at the expense of their purity, and vice versa. High purity is critical for assessing their biological activity. In addition, owing to the low concentration of bovine milk oligosaccharides, a high recovery yield corresponds with maximal process feasibility and economics.
The overall objective of this study was to develop an integrated process to maximize the recovery yield and purity of bovine milk oligosaccharides. The process relies on optimized conditions for maximum lactose hydrolysis, complete fermentation of monosaccharides released from the hydrolysis of lactose followed by concentration by nanofiltration membrane. The specific objectives of this work were to: a) optimize monosaccharide fermentation at laboratory-scale; b) scale-up monosaccharide fermentation to pilot-scale; c) evaluate the effects of transmembrane pressure on the retention of biologically important sialyloligosaccharides (3′-siallylactose (3′-SL), 6′-siallylactose (6′-SL), and 6′-Sialyl-N-acetyllactosamine (6′-SLN)) and permeate flux; and d) demonstrate the proof-of-concept of the integrated process at pilot-scale.
Section snippets
Bovine colostrum whey permeate
Bovine colostrum whey permeate was kindly provided by La Belle Colostrum (Bellingham, WA, USA). Industrial production of colostrum whey includes an initial defatting step via cream separators followed by the removal of caseins by enzymatic precipitation. The obtained whey was pasteurized at 63 °C for 30 min. Whey proteins were removed by ultrafiltration (10 kDa membrane) under continuous diafiltration to produce whey permeate (de Moura Bell et al., 2016). Lactose and oligosaccharide content of
Optimization of monosaccharide fermentation at laboratory-scale
The interaction of the reaction parameters as well as their individual effects on the fermentation of monosaccharides and their optimum levels were investigated using a central composite rotatable design. The effects of reaction time (h) and amounts of active dry yeast and yeast extract (g L−1) on the fermentation of monosaccharides arising from the lactose hydrolysis step are shown in Table 2. Maximum fermentation (100%) was observed in 9 out of the 17 experimental runs (3, 4, 7, 8, 10, 12,
Conclusions
A novel approach based on the integration of three different processing techniques (enzymatic hydrolysis, yeast fermentation and membrane filtration) has been developed and validated at pilot-scale to produce a purified complex oligosaccharide fraction free of digestible sugars such as lactose, glucose, and galactose. This work shows that bovine colostrum represents a practical source of bioactive milk compounds for use in clinical trials and eventually in therapeutics. Additionally, a
Acknowledgements
This research was supported by the Bill and Melinda Gates Foundation, National Institutes of Health Awards R01AT007079 and R01AT008759, UC Davis Peter J. Shields Endowed Chair in Dairy Food Science and Sustainable AgTech Innovation Center (Award No. 07 79 06923-SATIC). This research was partially supported by an industry/campus supported fellowship under the Training Program in Biomolecular Technology (T32-GM008799) at the University of California, Davis and by the Coordination for the
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2021, Journal of Functional FoodsCitation Excerpt :The quantity of oligosaccharides in mature bovine milk as mentioned is low, meaning that extremely efficient enrichment procedures are necessary. Concentrating oligosaccharides from bovine colostrum may be a solution and de Moura Bell et al. (2018) to this end recently developed a novel pilot-scale approach for the recovery of highly pure oligosaccharides, from colostral bovine whey permeate. Another consideration relating to certain bovine sialylated oligosaccharides is that humans lack the ability to synthesize the sialic acid, N-glycolylneuraminic acid (Neu5Gc), which is commonly produced in bovine milk (Padler-Karavani & Varki, 2011) and this structure is suggested to play a role in chronic inflammation-mediated diseases (Okerblom & Varki, 2017).
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Present address: Department of Food Science and Technology, University Federal Fluminense, Niteroi, Rio de Janeiro, 24230340, Brazil.
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Present address: Department of Food Science and Technology, Dong-Eui University, Busanjin, Busan 47340, Republic of Korea.
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Present address: Department of Agricultural Systems Management, Food and Forestry University of Firenze, 50144 Firenze, Italy.