Recipient Organization
IMVELA CORP.
141 FLUSHING AVE BLDG 77 STE 907
BROOKLYN,NY 112051338
Performing Department
(N/A)
Non Technical Summary
In this USDA SBIR Phase I project, we will develop a proof-of-concept natural preservative that extends the shelf life of bread and other baked goods, by preventing or slowing the growth of mold. This will help reduce food waste: baked goods account for 4% of food waste nationally, or >$6 billion in economic loss, and this food waste contributes to environmental harms such as greenhouse gas emissions. Current artificial chemical preservatives (e.g., benzoates and sorbates) are increasingly subject to scrutiny by consumers and regulators, and may have unintended health consequences particularly on the gut microbiome. Existing natural, bioprotection solutions do not provide protection to levels required by food manufacturers, and there is an urgent market need for higher performance natural alternatives. We will follow an innovative approach in which we precisely and intentionally combine the natural byproducts (called supernatants or fermentates) from individual natural microbial cultures (yielding a new ingredient which we call a "Superculture"). Existing natural, bioprotectant products are based on individual strains and work through single mechanisms of actions; our project will identify and utilize proprietary synergies between a variety of bacterial cultures to replace chemical preservatives while maintaining or improving preservative performance. To do this, we will use state of the art computer model along with extensive screening of potential solutions in our lab. The output of this Phase I effort will be validated Superculture prototypes at the benchtop scale that would motivate a larger effort to optimize and scale a new product with significant commercial impact and societal and environmental implications.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
100%
Goals / Objectives
The goal of this project is to develop a natural antifungal bioprotectant solution capable of eliminating fungal spoilage in processed baked goods, leading to significant decreases in food waste and achieving a key USDA aim (Objective 8.5). This technological innovation will be achieved using Imvela Corp.'s (publicly facing name Kingdom Supercultures) novel platform for microbial isolation, characterization, and combinatorial optimization.The core technical questions that our work plans address center around the technical feasibility of a Superculture-based bioprotection solution for baking ingredients. First we will determine if we can identify natural microbial culture fermentates with natural antifungal properties. Next we will strive to combine these natural fermentates with the aim of producing synergistic antifungal properties that outperform state of the art single compound bioprotection approaches. Finally, we will prototype a Superculture bioprotection solution that demonstrates increased performance in a proof of concept study at benchtop scale.The steps outlined above will inform our core technical objectives (TOs). We have determined these objectives through extensive customer discovery efforts with potential customers - some of the leading brands in this space. If successful, we believe a prototype meeting these specifications would enable further R&D in a Phase II SBIR towards commercialization.TO1: Development of a powder that is soluble/miscible in dough matrix and heat stable in typical baking conditions (450F, 60 minutes).TO2: shelf-life extension that outperforms current bioprotection solutions. >20% in shelf-life in challenge study with commercial bioprotection solution (natamcyin and propionic acid based, at recommended dosing) in baked good matrixTO3: Dosing compatible with manufacturer needs. Added to finished product between 0.01% to 1% w/wTO4: No perceptible sensory properties. No perceptible sensory differences when added to product as determined through blinded sensory differentiation test.TO5: Compatible with GRAS ingredient pathway and clean label. Product is based on food-derived microorganisms with standard food culture properties; can be labeled as cultured dextrose or similar. These objectives will be accomplished by the resources outlined in our budget and budget justification. Over the course of this 8 month project, our Chief Scientific Officer, Ravi Sheth, will spend a quarter of his time advising and contributing to the objectives. The other two senior scientists on this project will spend half of their time on this project, carrying out the experimental/wet lab procedures as well as the computational and bioinformatic aspects addressed above.Commercially, the goal for this SBIR Phase I project is to provide proof of concept that a natural superculture can outperform existing bioprotection solutions. We have identified strong commercial demand for bioprotectant approaches that prevent spoilage through clean-label means (natural, non-synthetic, and non-GMO). Currently, chemical preservatives used in baked goods (sodium benzoate and potassium sorbate) face increasing consumer scrutiny and existing commercial bioprotectant solutions (e.g. MicroGARD, bioprox) fail to match the performance of chemical preservatives, thus better bioprotectants are urgently needed to reduce food waste. We will systematically expand on previous research, emphasizing key aspects of commercial product development: extensive in situ validation of benchtop hits in food-like matrices and close collaboration with potential commercial partners, to ensure that our solution can be produced at scale and easily integrated into existing food production pipelines.Within the 8 month span of this project, our commercial objective is to sign a contract that demonstrates serious interest in our solution, as evidenced by either payment for R&D or a deposit on commercial supply of a solution, with at least one customer in the baked goods space. As indicated by our letter of support, we have a strong relationship with ADM, a global leader in ingredients for human and animal nutrition. ADM is a major player in the flour and grain space, and could be a great partner to validate market demand for our natural biopreservative solution and potentially eventually reach a large supply scale. In 2021, we also had a number of conversations with Clif Bar & Company about extending the shelf-life of their baked goods product line. As part of this project in 2022, we hope to continue these conversations and determine if Clif could be a suitable pilot customer.
Project Methods
Our proposed efforts build on the promise of existing, in-market bioprotection solutions and apply our unique "Superculture" design platform that allows us to intentionally combine multiple biological mechanisms of action for synergistic output. We hypothesize that multiple biological mechanisms of action, when combined together, may lead to synergistic and higher performance outcomes. Below are the types of "Efforts" we will use across a variety of "Evaluation Studies" (ES).Effort #1: Discovery of bioprotectant activity from our biobankTo determine if we can identify natural microbial culture fermentates with natural antifungal properties, we will conduct a primary screen for antifungal activity in our biobank. First we will algorithmically select top strains in biobank for bioprospecting of antifungal activity (ES 1.1). Then we will screen for antifungal activity on selection of 3-5 fungi associated with spoilage of baked goods. Strains will be arrayed in broad range media and fermented for 48 hours to produce individual chemical environments containing putative antifungal bioactives. Cell-free supernatants of each strain will be prepared using filtration plates, subjected to heat inactivation (to simulate baking), and dosed in a 1:1 ratio with seed cultures of target spoilage organisms such as Rhizopus nigricans, Rhizopus stolonifer, Penicillium expansum, and Aspergillus niger. From there we will determine the vitality of spoilage organisms (ES 1.2). To normalize results, each plate will contain a negative control condition in which the spoilage organism is grown with no additive (designated as 0% response) and a positive control condition in which the spoilage organism is grown with a known biopreservative at industry relevant concentration (designated as 100% response). Hits within the plate will be determined as described in ES 1.3 below.Evaluation Studies for Effort #1?1.1 Downselection from ~17,000 strains to 768 strains based on taxonomic identity and phylogenetic relatedness as well as antifungal production potential, as measured by presence of biosynthetic gene clusters and peptides with antimicrobial-like characteristics.1.2 Measuring vitality of spoilage organisms at multiple timepoints (1 hr to 72 hrs) using canonical fluorescent assays in which ATP, reducing potential, or other direct measures of cell metabolism are quantified (Promega).1.3 Hits within the plate will be classified as any supernatant that produces a response >75%. Hits from our screen will be validated using an 8-point dose-response curve using the same vitality assay.Effort #2: Assessment of synergistic bioprotectant mechanisms of actionNext, we will investigate whether these natural fermentates can be precisely combined to lead to synergistic antifungal properties that outperform state of the art single compound bioprotection approaches. First we will conduct a coarse delineation of antifungal compound class according to molecular and physical characteristics. Top candidates will be selected according to ES 2.1 below. Banked crude extracts of primary hits will be subject to rounds of coarse purification to determine general molecular class, then retested as described in 1.2 above. This will simplify potential compounds of interest and increase identification efforts. In brief, extracts will be subject to solid phase microextraction along various 96-well column beds that differentiate polarity (reversed phase), charge (ion exchange), or molecular size (gel filtration or size exclusion). In order to improve empirical characterization of the diversity of antifungal compounds identified in the initial screen, the primary screen will be repeated with a reduced number of candidate bioprotectant strains and an expanded number of fungal targets (selected via 2.2 below). Next we will use computational methods to generate promising combinations of microbes based on an expanded screen of antifungal activity and fractionation of molecular class. We will use the bases identified by NMF (2.3), the coarse delineation of distinct molecular classes, and simple algorithmic approaches to combine strains belonging to distinct molecular classes and distinct decompositional bases: these supercultures are most likely to combine multiple distinct modes of antifungal activity with potential for synergistic interactions. The output of this computational step will be the generation of 3-5 Superculture prototypes expected to broadly and effectively limit growth of fungal contaminant organisms.Evaluation Studies for Effort #22.1 Top candidate bioprotectant strains will be selected from our primary screen based on summed reduction in fungal growth, with a secondary consideration of identifying a set of strains cumulatively able to impact all initially targeted fungal organisms.2.2 We aim to produce an expanded panel of approximately 20 fungal organisms, The expanded set of fungal targets will be sourced from our biobank of fungal organisms, and constructed to maximize taxonomic diversity.2.3 Non-negative matrix factorization (NMF) is an algorithmic approach that aims to decompose a matrix of positive values exhibiting potentially complex structure into a low-rank composable set of bases. In the context of antifungal activity, these decomposed bases can be hypothesized to delineate independent mechanisms of antifungal activity, with potential for synergistic effects on reduction of fungal growth when combined.Effort #3: Demonstration of efficacy against state-of-the art solutionsTo determine whether a Superculture bioprotection solution can demonstrate increased performance in a proof of concept study at benchtop scale, we will need to evaluate the product efficacy in realistic conditions. To validate the antifungal efficacy of our 3-5 lead prototypes, we will generate material and test efficacy in more rigorous, commercially relevant conditions. First, prototype material will be generated at pilot scales. Next, the fermentate will be dosed at 1% and 0.1% against a negative control (no addition) and positive control (MicroGard or equivalent commercially available bioprotection agent) in a mock benchtop trial. Briefly, a basic bread recipe will be utilized and followed per industry standard with addition of the experimental or control bioprotection agent(s). The recipe will be prepared, baked, and then transferred to a sterile hood. After inoculation, the sliced bread will be placed into a controlled incubator, and will be imaged every 3 days over the course of 30 days. Supercultures will be evaluated according to ES 3.1 below.To ensure compatibility with commercial expectations we will subject the lead supercultures to additional validation testing to understand performance specifications. In particular, we will specifically investigate the impact of the Superculture candidates on key functionalities of the material (ES 3.2). In addition, we will also investigate the inherent safety qualities of the culture components (ES 3.3) and will concurrently test application of the Lead Superculture at a variety of dosing ranges as well as in various food matrices to understand the scalability of the product.Evaluation Studies for Effort #33.1 Mold will be scored with a binary metric (present/not present) and the time to first appearance of mold will be tracked and compared to controls. Supercultures with performance >20% than the positive control will be deemed successful.3.2 Assess viscosity, visual appearance, and mechanical properties of dough and other finished products.3.3 Safety evaluation: Lead cultures will be subject to a preliminary safety screen to identify potential issues. In particular the cultures will be subjected to whole genome sequencing and screened for presence of undesirable functions (including horizontally transferable antimicrobial resistance genes, biogenic amine production, or potential toxin genes).