VALIDATION OF AN ALTERNATIVE METHOD TO GENERATE PREBIOTICS FROM LACTOSE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1001158
• Project Start Date: Oct 1, 2013
• Project End Date: Sep 30, 2017
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Microbiology

Non Technical Summary
The complex interaction between diet, normal intestinal microbiota, and wellbeing has encouraged the development of strategies to promote the selective proliferation of beneficial microorganisms into the gastrointestinal track of humans. Probiotics are microorganisms that positively affect human health with attributed powerful antipathogenic and anti-inflammatory properties. Years of probiotic research indicate that a selective modification of the intestinal microbiota and its associated biochemical activities can be promoted by selective prebiotics. Prebiotics are non-digestible oligosaccharides (NDOs) that have a dual ability. First they reduce the intestinal colonizing efficiency of harmful bacteria and second they act as selective substrate to promote the growth and thereby increasing the number of specific probiotic bacteria. In addition, an increasing number of studies have shown that probiotics work best when combined with prebiotics. This combined form of delivery is known as a synbiotic. Galacto-oligosaccharides (GOS) are considered one of the preferred choices of prebiotics and in the gastrointestinal tract, GOS are resistant to enzymes and transit though the small intestine without being digested, but in the large intestine GOS are fermented and can activate growth of intestinal bifidobacteria as well as Lactobacillus acidophilus and L. casei, hence acting as a prebiotic.

Animal Health Component

33%

Research Effort Categories

Basic

34%

Applied

33%

Developmental

33%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
502	5010	1020	100%

Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
5010 - Food;

Field Of Science
1020 - Physiology;

Keywords

galactooligosacharides

prebiotics

hesoxyl-transferase

Goals / Objectives
To producea bioactive recombinant B Hexosyl Transferase byPichia pastoris and tooptimize its expresion To validate a method for GOS production using complex substrate mixture lactose-rich To test for lack of toxicity of the obtained products

Project Methods
Heterologous expression of optimized gene versions for improved transgalactosylation in Pichia pastoris. The yeast optimized version of Bht (op-Bht) will be clonedusing standard methodsand introduced in Pichia pastorisby electroporation. Transformants will be confirmed by PCRanalysis using standard procedures. Screening of other strains of Sporobomolomyces sp. for galactosidase-like activitiy. Strains will be phenotypically characterized by standard microbiology protocols (carbon source assimilation, optimal growth temperature, and pH). Genetic characterization of strains will be carried out by cariotypic and restriction enzyme mitochondrial DNA analysis. In addition, analysis of 18S and/or 26S genes will be used for strain identification. Methods to detect and identify fermentation products will include biochemical conventional methods such as, thin layer chromatography (TLC) or high performance liquid chromatography (HPLC). Standard protocols (SDS polyacrylamide gels, enzymatic, and colorimetric assays) will be carried out to characterize the proteins. Fermentation and NDO generation. Homologous and/or heterologous production of NDOs will be assessed using 500 mL to 5 L capacity fermentors and in-line sensors developed in-house by Navarro et al. (european patent number P200101757). Products and/or enzymes involved in the generation of NDOs will be purified using fluidized bed columns (Pharmacia) and the resin adequate for each product. The commercial software Matlab (http://www.mathworks.com/) will be utilized to adjust data to kinetic models previously characterized (as the model that describes the physiological behavior of the yeast Saccharomyces cerevisiae) and to control the process evolution. Propagation growth experiments will be conducted in a 2 l model BIOSTAT B+ equipped with PID control units for pH, temperature, oxygen and agitation speed. Experiments will start with a working volume of 700 ml at 30°C. Initial pH will be maintained constant during the batch or continuous phase the reactor by the automatic addition of 42.5% H3PO3 or 1 M NaOH. The reactor will be continuously fed with medium by a type-501 peristaltic pump (Watson-Marlow, Falmouth, UK) at the desired flow rate. Dissolved oxygen, measured with an electrode (Mettler-Toledo, US), will be maintained above 20% by a PID control system that allowed the automatic modification of the agitation speed between the range limits of 300 to 500 rpm. Cell growth will be followed by measuring the OD600. Cell dry weight determination will be carried out by cell centrifugation at 5000 g for 10 min, wash with distilled water and drying the cells at 80°C until constant weight. Analytical procedures DNOs, sugars, acetate, lactate, ethanol and other bioproducts will be measured by HPLC. Separation will be carried out at 65°C on a Phenomenex (Torrance, USA), Spherisorb ODS-2.5 m (250 x 4.6 mm) with a Spherisorb C8 10 m (50 x 4.6 mm) pre-column at an eluent flow-rate of 1.0 ml min-1, (NaH2PO4.H2O/H3PO4, pH 3.0); 20 µl samples will be injected through a loop. Components will be identified and quantified by refractive index measurements with suitable standards.

Progress 10/01/13 to 09/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?While this project did not train professionals until this point, we believe future grant proposals and collaborations from the generated new research findings will strengthen our opportunities for future student and professional training. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? There is a strategic opportunity with regard to health and bioremediation to convert commodity sugars such as lactose into commercially viable high value added products. These products should be able to reduce the overall process cost and improve the overall food processing economy. Despite the multiple independent uses of lactose in nutrition and medicine and its moderate market value, its derivatives galacto-oligosaccharides (GOS) are highly valued functional food ingredients able to mimic Human Milk Oligosaccharides properties. Previously, we designed and patented and licensed a method by which lactose can be enzymatically converted into galactosyl-lactose derivatives called galacto-oligosaccharides (GOS) (Dagher et al., 2013). The main catalyst to improve the proposed process is the partially characterized membrane protein β-hexosyl-transferase (BHT) from the eukaryote Sporobolomyces singularis. We have achieved protein purification to homogeneity of the small quantities secreted by our initial clones (reviewed in (Dagher et al., 2013)) Those amounts were sufficient to evaluate industrial process viability and confirmed that this enzyme is one of the most promising catalysts in the field of glycobiology due to its high stability and particular enzymatic properties. Moreover, BHT has been known for a long time but major rate-limiting steps have restricted its biotechnological utilization; the level of homologous expression (limited by its endogenous promoter), and the historic inability to generate extracellular soluble, stable, and bioactive heterologous BHT. Since, BHT is expressed and later localized into the cell membrane facing outside the cell. We achieved surprisingly cost-effective levels of soluble BHT generating a chimeric gene by replacing the native leader for an alternative secretory sequence (Dagher & Bruno-Barcena, 2016)). GOS are one of the most extensively evaluated prebiotics, and systematic evidence shows that GOS are able to stimulate the growth and activity of beneficial bacteria in the digestive system, and are widely used in food products such as, nutritional supplements, yogurts, baked goods, and animal feed. Additionally, GOS are the most inexpensive alternative and often added to infant and follow-on formulas to mimic the beneficial effects of the human milk oligosaccharides (HMOs). Furthermore we demonstrated that our preparations lack of toxicity and in fact stimulate bifidogenic microbiota (Monteagudo-Mera A, et al 2016).

Publications

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audience for this period include only a industry producer of GOS. There has been an exclusivity agreement in place extended to 2016 between the company and NCSU Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?The proposed goals have been acomplished.In the future we will try to improve the production levels of the enzyme

Impacts
What was accomplished under these goals? Once validated,designed and patented the method by which lactose can be enzymatically converted into galactosyl-lactose derivatives called galacto-oligosacchatides (GOS). During this period we tested the lack of toxicity using mice models

Publications

• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: A Monteagudo-Mera, Arthur JC, Jobin C, Keku TO, Bruno-B�rcena JM, and Azcarate-Peril MA (2016) Enriched galacto-oligosaccharides (GOS) enhance specific Bifidobacterium species and their metabolic activity in the mouse gut microbiome. Beneficial Microbes. 11;7(2):247-64.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The target audience for this period include only a industry producer of GOS. There has been anexclusivity agreement in place between the company and NCSU Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We would like to demostrate a positive impact of this active components on the gut microbiota. Therefore simultaneously testing safety

Impacts
What was accomplished under these goals? We designed and patented a method by which lactose can be enzymatically converted into galactosyl-lactose derivatives called galacto-oligosacchatides (GOS)

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: SF Dagher, Bruno-B�rcena JM (2015) A novel N-terminus region of the membrane ?-hexosyltransferase; its role in secretion of soluble protein by Pichia pastoris. Microbiology 11/2015; DOI:10.1099/mic.0.000211

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: The target audience for this communication are the dairy, food and pharmaceutical industries having interest in new methods of prebiotic production impacting the marketplace overall final product purity. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? This technology has generated multiple rounds of IP negotiation and strategies for licensing during 2014 What do you plan to do during the next reporting period to accomplish the goals? We would like to demonstrate novel biological industrial process to generate GOS in which sugar transformation can occur while using existing inline processes

Impacts
What was accomplished under these goals? We have met the milestone for obtaining economically efficient amounts of soluble enzyme to catalyze the reactions

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Heterologous expression of a bioactive ?-hexosyltransferase, an enzyme producer of prebiotics, from Sporobolomyces singularis.Dagher SF, Azcarate-Peril MA, Bruno-B�rcena JM. Appl Environ Microbiol. 2013 Feb;79(4):1241-9