Edible mycelium as proliferation and differentiation support for anchorage-dependent animal cells in cultivated meat production.

Cultivated meat production requires bioprocess optimization to achieve cell densities that are multiple orders of magnitude higher compared to conventional cell culture techniques. These processes must maximize resource efficiency and cost-effectiveness by attaining high cell growth productivity per unit of medium. Microcarriers, or carriers, are compatible with large-scale bioreactor use, and offer a large surface-area-to-volume ratio for the adhesion and proliferation of anchorage-dependent animal cells. An ongoing challenge persists in the efficient retrieval of cells from the carriers, with conflicting reports on the effectiveness of trypsinization and the need for additional optimization measures such as carrier sieving. To surmount this issue, edible carriers have been proposed, offering the advantage of integration into the final food product while providing opportunities for texture, flavor, and nutritional incorporation. Recently, a proof of concept (POC) utilizing inactivated mycelium biomass derived from edible filamentous fungus demonstrated its potential as a support structure for myoblasts. However, this POC relied on a model mammalian cell line combination with a single mycelium species, limiting realistic applicability to cultivated meat production. This study aims to advance the POC. We found that the species of fungi composing the carriers impacts C2C12 myoblast cell attachment-with carriers derived from Aspergillus oryzae promoting the best proliferation. C2C12 myoblasts effectively differentiated on mycelium carriers when induced in myogenic differentiation media. Mycelium carriers also supported proliferation and differentiation of bovine satellite cells. These findings demonstrate the potential of edible mycelium carrier technology to be readily adapted in product development within the cultivated meat industry.

Multi-objective Bayesian algorithm automatically discovers low-cost high-growth serum-free media for cellular agriculture application.

In this work, we applied a multi-information source modeling technique to solve a multi-objective Bayesian optimization problem involving the simultaneous minimization of cost and maximization of growth for serum-free C2C12 cells using a hyper-volume improvement acquisition function. In sequential batches of custom media experiments designed using our Bayesian criteria, collected using multiple assays targeting different cellular growth dynamics, the algorithm learned to identify the trade-off relationship between long-term growth and cost. We were able to identify several media with >100% more growth of C2C12 cells than the control, as well as a medium with 23% more growth at only 62.5% of the cost of the control. These algorithmically generated media also maintained growth far past the study period, indicating the modeling approach approximates the cell growth well from an extremely limited data set.

Assessing Edible Filamentous Fungal Carriers as Cell Supports for Growth of Yeast and Cultivated Meat.

The growth and activity of adherent cells can be enabled or enhanced through attachment to a solid surface. For food and beverage production processes, these solid supports should be food-grade, low-cost, and biocompatible with the cell of interest. Solid supports that are edible can be a part of the final product, thus simplifying downstream operations in the production of fermented beverages and lab grown meat. We provide proof of concept that edible filamentous fungal pellets can function as a solid support by assessing the attachment and growth of two model cell types: yeast, and myoblast cells. The filamentous fungus Aspergillus oryzae was cultured to produce pellets with 0.9 mm diameter. These fugal pellets were inactivated by heat or chemical methods and characterized physicochemically. Chemically inactivated pellets had the lowest dry mass and were the most hydrophobic. Scanning electron microscope images showed that both yeast and myoblast cells naturally adhered to the fungal pellets. Over 48 h of incubation, immobilized yeast increased five-fold on active pellets and six-fold on heat-inactivated pellets. Myoblast cells proliferated best on heat-treated pellets, where viable cell activity increased almost two-fold, whereas on chemically inactivated pellets myoblasts did not increase in the cell mass. These results support the use of filamentous fungi as a novel cell immobilization biomaterial for food technology applications.

Spent media analysis suggests cultivated meat media will require species and cell type optimization.

Cell culture media design is perhaps the most significant hurdle currently facing the commercialization of cultivated meat as an alternative source of dietary protein. Since media optimization for a specific culture system requires a significant amount of effort and investment, a major question remaining is whether media formulations can be easily shared across multiple production schemes for cells of different species and lineages. Here, we perform spent medium analysis to compare the specific nutrient utilization of primary embryonic chicken muscle precursor cells and fibroblasts to the murine C2C12 myoblast cell line. We demonstrate that these related cell types have significantly different nutrient utilization patterns collectively and on a per-cell basis, and that many components of conventional media do not appear to be depleted by the cells. Namely, glucose was not consumed as rapidly nor as completely by the chicken muscle precursors compared to other cells overall, and there were significant differences in specific consumption rates for several other key nutrients over the first day of culture. Ultimately, our results indicate that no one medium is likely ideal and cost effective to culture multiple cell types and that novel methods to streamline media optimization efforts will be important for the industry to develop.

Multi-information source Bayesian optimization of culture media for cellular agriculture.

Culture media used in industrial bioprocessing and the emerging field of cellular agriculture is difficult to optimize due to the lack of rigorous mathematical models of cell growth and culture conditions, as well as the complexity of the design space. Rapid growth assays are inaccurate yet convenient, while robust measures of cell number can be time-consuming to the point of limiting experimentation. In this study, we optimized a cell culture media with 14 components using a multi-information source Bayesian optimization algorithm that locates optimal media conditions based on an iterative refinement of an uncertainty-weighted desirability function. As a model system, we utilized murine C2C12 cells, using AlamarBlue, LIVE stain, and trypan blue exclusion cell counting assays to determine cell number. Using this experimental optimization algorithm, we were able to design media with 181% more cells than a common commercial variant with a similar economic cost, while doing so in 38% fewer experiments than an efficient design-of-experiments method. The optimal medium generalized well to long-term growth up to four passages of C2C12 cells, indicating the multi-information source assay improved measurement robustness relative to rapid growth assays alone.

Metabolic flux sampling predicts strain-dependent differences related to aroma production among commercial wine yeasts.

BACKGROUND: Metabolomics coupled with genome-scale metabolic modeling approaches have been employed recently to quantitatively analyze the physiological states of various organisms, including Saccharomyces cerevisiae. Although yeast physiology in laboratory strains is well-studied, the metabolic states under industrially relevant scenarios such as winemaking are still not sufficiently understood, especially as there is considerable variation in metabolism between commercial strains. To study the potential causes of strain-dependent variation in the production of volatile compounds during enological conditions, random flux sampling and statistical methods were used, along with experimental extracellular metabolite flux data to characterize the differences in predicted intracellular metabolic states between strains. RESULTS: It was observed that four selected commercial wine yeast strains (Elixir, Opale, R2, and Uvaferm) produced variable amounts of key volatile organic compounds (VOCs). Principal component analysis was performed on extracellular metabolite data from the strains at three time points of cell cultivation (24, 58, and 144 h). Separation of the strains was observed at all three time points. Furthermore, Uvaferm at 24 h, for instance, was most associated with propanol and ethyl hexanoate. R2 was found to be associated with ethyl acetate and Opale could be associated with isobutanol while Elixir was most associated with phenylethanol and phenylethyl acetate. Constraint-based modeling (CBM) was employed using the latest genome-scale metabolic model of yeast (Yeast8) and random flux sampling was performed with experimentally derived fluxes at various stages of growth as constraints for the model. The flux sampling simulations allowed us to characterize intracellular metabolic flux states and illustrate the key parts of metabolism that likely determine the observed strain differences. Flux sampling determined that Uvaferm and Elixir are similar while R2 and Opale exhibited the highest degree of differences in the Ehrlich pathway and carbon metabolism, thereby causing strain-specific variation in VOC production. The model predictions also established the top 20 fluxes that relate to phenotypic strain variation (e.g. at 24 h). These fluxes indicated that Opale had a higher median flux for pyruvate decarboxylase reactions compared with the other strains. Conversely, R2 which was lower in all VOCs, had higher median fluxes going toward central metabolism. For Elixir and Uvaferm, the differences in metabolism were most evident in fluxes pertaining to transaminase and hexokinase associated reactions. The applied analysis of metabolic divergence unveiled strain-specific differences in yeast metabolism linked to fusel alcohol and ester production. CONCLUSIONS: Overall, this approach proved useful in elucidating key reactions in amino acid, carbon, and glycerophospholipid metabolism which suggest genetic divergence in activity in metabolic subsystems among these wine strains related to the observed differences in VOC formation. The findings in this study could steer more focused research endeavors in developing or selecting optimal aroma-producing yeast stains for winemaking and other types of alcoholic fermentations.

Optimization of muscle cell culture media using nonlinear design of experiments.

Optimizing media for biological processes, such as those used in tissue engineering and cultivated meat production, is difficult due to the extensive experimentation required, number of media components, nonlinear and interactive responses, and the number of conflicting design objectives. Here we demonstrate the capacity of a nonlinear design-of-experiments (DOE) method to predict optimal media conditions in fewer experiments than a traditional DOE. The approach is based on a hybridization of a coordinate search for local optimization with dynamically adjusted search spaces and a global search method utilizing a truncated genetic algorithm using radial basis functions to store and model prior knowledge. Using this method, we were able to reduce the cost of muscle cell proliferation media while maintaining cell growth 48 h after seeding using 30 common components of typical commercial growth medium in fewer experiments than a traditional DOE (70 vs. 103). While we clearly demonstrated that the experimental optimization algorithm significantly outperforms conventional DOE, due to the choice of a 48 h growth assay weighted by medium cost as an objective function, these findings were limited to performance at a single passage, and did not generalize to growth over multiple passages. This underscores the importance of choosing objective functions that align well with process goals.

Nitrogenous Compound Utilization and Production of Volatile Organic Compounds among Commercial Wine Yeasts Highlight Strain-Specific Metabolic Diversity.

Genetic background and environmental conditions affect the production of sensory impact compounds by Saccharomyces cerevisiae. The relative importance of the strain-specific metabolic capabilities for the production of volatile organic compounds (VOCs) remains unclear. We investigated which amino acids contribute to VOC production and whether amino acid-VOC relations are conserved among yeast strains. Amino acid consumption and production of VOCs during grape juice fermentation was investigated using four commercial wine yeast strains: Elixir, Opale, R2, and Uvaferm. Principal component analysis of the VOC data demonstrated that Uvaferm correlated with ethyl acetate and ethyl hexanoate production, R2 negatively correlated with the acetate esters, and Opale positively correlated with fusel alcohols. Biomass formation was similar for all strains, pointing to metabolic differences in the utilization of nutrients to form VOCs. Partial least-squares linear regression showed that total aroma production is a function of nitrogen utilization (R2 = 0.87). We found that glycine, tyrosine, leucine, and lysine utilization were positively correlated with fusel alcohols and acetate esters. Mechanistic modeling of the yeast metabolic network via parsimonious flux balance analysis and flux enrichment analysis revealed enzymes with crucial roles, such as transaminases and decarboxylases. Our work provides insights in VOC production in wine yeasts. IMPORTANCE Saccharomyces cerevisiae is widely used in grape juice fermentation to produce wines. Along with the genetic background, the nitrogen in the environment in which S. cerevisiae grows impacts its regulation of metabolism. Also, commercial S. cerevisiae strains exhibit immense diversity in their formation of aromas, and a desirable aroma bouquet is an essential characteristic for wines. Since nitrogen affects aroma formation in wines, it is essential to know the extent of this connection and how it leads to strain-dependent aroma profiles in wines. We evaluated the differences in the production of key aroma compounds among four commercial wine strains. Moreover, we analyzed the role of nitrogen utilization on the formation of various aroma compounds. This work illustrates the unique aroma-producing differences among industrial yeast strains and suggests more intricate, nitrogen-associated routes influencing those aroma-producing differences.

Considerations for the development of cost-effective cell culture media for cultivated meat production.

Innovation in cultivated meat development has been rapidly accelerating in recent years because it holds the potential to help attenuate issues facing production of dietary protein for a growing world population. There are technical obstacles still hindering large-scale commercialization of cultivated meat, of which many are related to the media that are used to culture the muscle, fat, and connective tissue cells. While animal cell culture media has been used and refined for roughly a century, it has not been specifically designed with the requirements of cultivated meat in mind. Perhaps the most common industrial use of animal cell culture is currently the production of therapeutic monoclonal antibodies, which sell for orders of magnitude more than meat. Successful production of cultivated meat requires media that is food grade with minimal cost, can regulate large-scale cell proliferation and differentiation, has acceptable sensory qualities, and is animal ingredient-free. Much insight into strategies for achieving media formulations with these qualities can be obtained from knowledge of conventional culture media applications and from the metabolic pathways involved in myogenesis and protein synthesis. In addition, application of principles used to optimize media for large-scale microbial fermentation processes producing lower value commodity chemicals and food ingredients can also be instructive. As such, the present review shall provide an overview of the current understanding of cell culture media as it relates to cultivated meat.

Downshifting Yeast Dominance: Cell Physiology and Phospholipid Composition Are Altered With Establishment of the [GAR +] Prion in Saccharomyces cerevisiae.

Establishment of the [GAR +] prion in Saccharomyces cerevisiae reduces both transcriptional expression of the HXT3 hexose transporter gene and fermentation capacity in high sugar conditions. We evaluated the impact of deletion of the HXT3 gene on the expression of [GAR +] prion phenotype in a vineyard isolate, UCD932, and found that changes in fermentation capacity were observable even with complete loss of the Hxt3 transporter, suggesting other cellular functions affecting fermentation rate may be impacted in [GAR +] strains. In a comparison of isogenic [GAR +] and [gar -] strains, localization of the Pma1 plasma membrane ATPase showed differences in distribution within the membrane. In addition, plasma membrane lipid composition varied between the two cell types. Oxygen uptake was decreased in prion induced cells suggesting membrane changes affect plasma membrane functionality beyond glucose transport. Thus, multiple cell surface properties are altered upon induction of the [GAR +] prion in addition to changes in expression of the HXT3 gene. We propose a model wherein [GAR +] prion establishment within a yeast population is associated with modulation of plasma membrane functionality, fermentation capacity, niche dominance, and cell physiology to facilitate growth and mitigate cytotoxicity under certain environmental conditions. Down-regulation of expression of the HXT3 hexose transporter gene is only one component of a suite of physiological differences. Our data show the [GAR +] prion state is accompanied by multiple changes in the yeast cell surface that prioritize population survivability over maximizing metabolic capacity and enable progeny to establish an alternative adaptive state while maintaining reversibility.

A combined phenolic extraction and fermentation reactor engineering model for multiphase red wine fermentation.

Red wine production begins with a simultaneous fermentation and solid-phase extraction process. Red wine color and mouthfeel is the result of the extraction of phenolics from grape skins and seeds during fermentation, where extraction is a strong function of temperature and ethanol concentration. During fermentation, grape solids form a porous "cap" at the top of the fermentor, resulting in a heterogeneous fermentation system with significant temperature and concentration gradients. In this work, we present a spatial, time-variant reactor engineering model for phenolic extraction during red wine fermentation, incorporating fermentation kinetics, mass transfer, heat transfer, compressible fluid flow, and phenolic extraction kinetics. The temperature and ethanol concentration profiles predicted by this model allow for the calculation of phenolic extraction rates over the course of fermentation. Phenolic extraction predictions were validated against prior experimental data to good agreement and compared to a well-mixed model's predictions to show the utility of a spatial model over well-mixed models.

Impact of Temperature, Ethanol and Cell Wall Material Composition on Cell Wall-Anthocyanin Interactions.

The effects of temperature and ethanol concentration on the kinetics of anthocyanin adsorption and desorption interactions with five cell wall materials (CWM) of different composition were investigated. Using temperatures of 15 °C and 30 °C and model wine with ethanol concentrations of 0% and 15% (v/v) over 120 min, the adsorption and desorption rates of five anthocyanin-glucosides were recorded in triplicate. Small-scale experiments were conducted using a benchtop incubator to mimic a single berry fermentation. Results indicate that more than 90% of the adsorption occurs within the first 60 min of the addition of anthocyanins to CWM. However, desorption appears to occur much faster, with maximum desorption being reached after 30 min. The extent of both adsorption and desorption was clearly dependent not only on temperature and ethanol concentration but also on the CWM composition.

A Mechanistic Model for the Extraction of Phenolics from Grapes During Red Wine Fermentation.

Phenolic extraction is a critical part of red wine making. Though empirical models of phenolic extraction kinetics exist, the current level of mechanistic understanding does not allow for accurate predictions. In this work, we propose a mechanistic model for the extraction of phenolics from grape skins and seeds as a function of temperature and ethanol. This model examines the release of phenolics, the adsorption of phenolics onto grape material, and the disappearance of anthocyanins from solution. Additionally, we performed epifluorescence microscopy to explore our finding that seed tannins' release rate appears independent of concentration, and found that the grape seed appears to ablate over fermentation. We also determined the activation energy of anthocyanin disappearance, in good agreement with similar systems. The proposed model results in an excellent fit, and increases the understanding of phenolic extraction and the ability to predict and optimize product outcome in red wine making.

Creation and validation of a reactor engineering model for multiphase red wine fermentations.

Red wine fermentations are performed in the presence of grape skins and seeds to ensure the extraction of color and other phenolics. The presence of these solids results in two distinct phases in the fermentor, as the solids float to the top to form a "cap." Modeling of red wine fermentation is, therefore, complex and must consider spatial heterogeneity to predict fermentation kinetics. We have developed a reactor-engineering model for red wine fermentations that includes the fundamentals of fermentation kinetics, heat transfer, diffusion, and compressible fluid flow. To develop the heat transfer component of the model, the heat transfer properties of grapes were experimentally determined as a function of fermentation progression. COMSOL was used to solve all components of the model simultaneously utilizing a finite element analysis approach. Predictions from this model were validated using prior experimental work. Model prediction and experimental data showed excellent agreement. The model was then used to predict spatial profiles of active yeast cell concentration and ethanol productivity, as well as liquid velocity profiles. Finally, the model was used to predict how these gradients would change with differences in initial bioavailable nitrogen concentration, a key parameter in predicting fermentation outcome in nitrogen-limited wine fermentations.

Conversion of cassava leaf to bioavailable, high-protein yeast cell biomass.

BACKGROUND: Cassava leaves are an abundant global agricultural residue because the roots are a major source of dietary carbohydrates. Although cassava leaves are high in protein, the protein is not bioavailable. This work aimed to convert cassava leaves to a bioavailable protein-rich animal feed ingredient using high-protein yeasts. RESULTS: The structural proteins (ca 200 g kg-1 d.b.) from sundried cassava leaves were solubilized by mild alkali pretreatment, and the resulting cassava leaf hydrolysate (CLH) was used to screen for growth of 46 high-protein yeasts from 30 species. Promising candidates from the initial screen cultivated at a 10 mL scale demonstrated increases in relative abundance of essential amino acids over that of CLH. In particular, lysine, growth-limiting for some livestock, was increased up to 226% over the CLH content. One yeast, Pichia kudriavzevii UCDFST 11-602, was grown in 3 L of CLH in a bioreactor to examine the scale-up potential of the yeast protein production. While glucose was completely consumed, yeast growth exited log phase before depleting either carbon or nitrogen, suggesting other growth-limiting factors at the larger scale. CONCLUSIONS: High-value animal feed with enriched essential amino acid profiles can be produced by yeasts grown on agricultural residues. Yeasts convert structural protein solubilized from cassava leaves to essential amino acid-enriched, digestible protein. The low carbohydrate content of the leaves (ca 200 g kg-1 d.b.), however, necessitated glucose supplementation for yeast growth. © 2018 Society of Chemical Industry.

Impact of cold soak duration on Cabernet Sauvignon fermentation and phenolic composition.

BACKGROUND: Cold soak is a prefermentative maceration technique believed to enhance grape skin extraction. Studies show variable results depending on cold soak and winemaking conditions. To investigate the effect of cold soak more fully, systematic and highly reproducible Cabernet Sauvignon fermentations with increasing cold-soak durations were performed. RESULTS: Phenolic extraction during cold soak and fermentation showed significant differences among all treatments for monitored phenolics at the end of the cold soak. At the end of alcoholic fermentation only gallic acid, (-)-epicatechin, and the flavonols were significant, and only (-)-epicatechin was significant after bottle ageing. Descriptive analysis of the bottled wines showed that the 4- and 7-day treatments were significantly higher in caramelized/vanilla/browned flavor compared to the 1-day treatment and lower levels of bitterness were observed up to 2 days of cold soak. While oligosaccharide content increased with increasing cold-soak duration, differences were not large enough to result in sensory differences. CONCLUSION: While increased cold soak duration led to differences in phenolic extraction during early fermentation, these differences did not last through to the end product. Thus, under the conditions of this study, cold-soak duration had little overall impact on Cabernet Sauvignon wine composition and style. © 2018 Society of Chemical Industry.

Extracellular fungal polyol lipids: A new class of potential high value lipids.

Extracellular fungal glycolipid biosurfactants have attracted attention because productivities can be high, cheap substrates can be used, the molecules are secreted into the medium and the downstream processing is relatively simple. Three classes of extracellular fungal glycolipid biosurfactants have provided most of the scientific advances in this area, namely sophorolipids, mannosylerythritol lipids and cellobioselipids. Polyol lipids, a fourth class of extracellular fungal glycolipid biosurfactants, comprise two groups of molecules: liamocins produced by the yeast-like fungus Aureobasidium pullulans, and polyol esters of fatty acids, produced by some Rhodotorula yeast species. Both are amphiphilic, surface active molecules with potential for commercial development as surfactants for industrial and household applications. The current knowledge of polyol lipids highlights an emerging group of extracellular fungal glycolipid biosurfactants and provides a perspective of what next steps are needed to harness the benefits and applications of this novel group of molecules.

Simultaneous production of intracellular triacylglycerols and extracellular polyol esters of fatty acids by Rhodotorula babjevae and Rhodotorula aff. paludigena.

Microbial oils have been analyzed as alternatives to petroleum. However, just a handful of microbes have been successfully adapted to produce chemicals that can compete with their petroleum counterparts. One of the reasons behind the low success rate is the overall economic inefficiency of valorizing a single product. This study presents a lab-scale analysis of two yeast species that simultaneously produce multiple high-value bioproducts: intracellular triacylglycerols (TG) and extracellular polyol esters of fatty acids (PEFA), two lipid classes with immediate applications in the biofuels and surfactant industries. At harvest, the yeast strain Rhodotorula aff. paludigena UCDFST 81-84 secreted 20.9 ± 0.2 g L-1 PEFA and produced 8.8 ± 1.0 g L-1 TG, while the yeast strain Rhodotorula babjevae UCDFST 04-877 secreted 11.2 ± 1.6 g L-1 PEFA and 18.5 ± 1.7 g L-1 TG. The overall glucose conversion was 0.24 and 0.22 g(total lipid) g (glucose)-1 , respectively. The results present a stable and scalable microbial growth platform yielding multiple co-products.

Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria.

UNLABELLED: Milk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-ß-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intact N-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 released N-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-released N-glycans supported the rapid growth of Bifidobacterium longum subsp. infantis (B. infantis), a species that grows well on human milk oligosaccharides, but did not support growth of Bifidobacterium animalis subsp. lactis (B. lactis), a species which does not. Conversely, B. infantis ATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed that B. infantis consumed 73% of neutral and 92% of sialylated N-glycans, while B. lactis degraded only 11% of neutral and virtually no (<1%) sialylated N-glycans. These results provide mechanistic support that N-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, released N-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety of N-glycans released from bovine milk glycoproteins suggests that they may serve as novel prebiotic substrates with selective properties similar to those of human milk oligosaccharides. IMPORTANCE: It has been previously shown that glycoproteins serve as growth substrates for bifidobacteria. However, which part of a glycoprotein (glycans or polypeptides) is responsible for this function was not known. In this study, we used a novel enzyme to cleave conjugated N-glycans from milk glycoproteins and tested their consumption by various bifidobacteria. The results showed that the glycans selectively stimulated the growth of B. infantis, which is a key infant gut microbe. The selectivity of consumption of individual N-glycans was determined using advanced mass spectrometry (nano-liquid chromatography chip-quadrupole time of flight mass spectrometry [nano-LC-Chip-Q-TOF MS]) to reveal that B. infantis can consume the range of glycan structures released from whey protein concentrate.

Kinetic characterization of a novel endo-ß-N-acetylglucosaminidase on concentrated bovine colostrum whey to release bioactive glycans.

EndoBI-1 is a recently isolated endo-ß-N-acetylglucosaminidase, which cleaves the N-N'-diacetyl chitobiose moiety found in the N-glycan core of high mannose, hybrid and complex N-glycans. These N-glycans have selective prebiotic activity for a key infant gut microbe, Bifidobacterium longum subsp. infantis. The broad specificity of EndoBI-1 suggests the enzyme may be useful for many applications, particularly for deglycosylating milk glycoproteins in dairy processing. To facilitate its commercial use, we determined kinetic parameters for EndoBI-1 on the model substrates ribonuclease B and bovine lactoferrin, as well as on concentrated bovine colostrum whey. Km values ranging from 0.25 to 0.49, 0.43 to 1.00 and 0.90 to 3.18 mg/mL and Vmax values ranging from 3.5×10(-3) to 5.09×10(-3), 4.5×10(-3) to 7.75×10(-3) and 1.9×10(-2)to 5.2×10(-2) mg/mL×min were determined for ribonuclease B, lactoferrin and whey, respectively. In general, EndoBI-1 showed the highest apparent affinity for ribonuclease B, while the maximum reaction rate was the highest for concentrated whey. EndoBI-1-released N-glycans were quantified by a phenol-sulphuric total carbohydrate assay and the resultant N-glycan structures monitored by nano-LC-Chip-Q-TOF MS. The kinetic parameters and structural characterization of glycans released suggest EndoBI-1 can facilitate large-scale release of complex, bioactive glycans from a variety of glycoprotein substrates. Moreover, these results suggest that whey, often considered as a waste product, can be used effectively as a source of prebiotic N-glycans.