Location: Dairy and Functional Foods Research
2024 Annual Report
Objectives
Objective 1: Use process simulation to develop cost-effective processes for valorization of waste dairy streams to reduce environmental impact and reuse water.
Objective 2: Investigate technologies for large-scale production of edible coatings, made from predominately dairy proteins and waste dairy streams; their storage stability; and, as films for secondary packaging.
Objective 3: Develop fermentation technologies for utilizing dairy coproducts or waste streams to produce antimicrobial peptides by lactic acid bacteria, and assess their activity against bacterial pathogens and spoilage microorganisms.
Objective 4: Develop a process for recovering food-grade, allergen-free fat from frozen dessert waste.
Approach
Dairy processing generates waste streams ranging from those that are mostly water to packaged products that did not meet a manufacturer’s quality standards. This project aims to utilize the watery acid whey streams generated from Greek-style yogurt (GSY) manufacture, cheese whey, permeates, and wasted products such as ice cream through computer simulation and laboratory and pilot plant research. Computer simulation of an actual GSY plant will be used to investigate ways to recover and reuse water from the waste streams and for optimizing extraction of nutrients with economic value. The model will be customized by choosing conditions for straining based on the properties of a finished GSY with properties that consumers appreciate using knowledge-based systems and advanced simulation methods. Laboratory and pilot plant experiments will examine the manufacture of edible films and coatings from the watery streams for food and nonfood uses by standardizing the feed material used for each type of film. Other research will determine if the dairy waste streams or byproducts can be utilized as fermentation substrates for the production of antimicrobial peptides by dairy lactic acid bacteria. Preliminary studies will seek to optimize antimicrobial peptide production by varying substrate composition and fermentation parameters. Additionally, molecular techniques will be investigated for developing bacterial strains capable of producing higher concentrations of antimicrobial peptides, and fermentates will be assessed for novel antimicrobial peptides generated via the degradation of native milk proteins. Fermentation technologies will be assessed for pilot-scale production of antimicrobial peptides and resulting fermentates will be investigated for food safety and functional food applications. Finally, research will determine whether allergen free butterfat can be recovered from waste ice cream using a cost-appropriate process. This will be accomplished by studying the stages of fat separation and refining individually, and at each stage testing a variety of technologies for efficacy and projected cost. Pilot plant implementation of the chosen technologies will be used along with techno-economic analysis to generate results that enable ice cream manufacturers to evaluate the process for possible adoption. This project will introduce several sustainable approaches for reducing energy and water use, and recovery of process wastes with added value for food or nonfood uses.
Progress Report
Objective 1. Process simulations were completed for the yogurt plant. A manuscript has been prepared. Process simulations were also conducted for a generic ice cream plant. Energy use, water use, and greenhouse gas emissions can be calculated. The plant will be used to conduct process improvement trials for minimizing waste as operating data becomes available from partners.
Objective 2. Formulations for use with a newly purchased 3D bioprinter have been developed. Rheological tests indicate that the new formulations are shear-thinning, so that a more liquid consistency is achieved by flow or shear. Upon stopping of flow as in right after extrusion, the overall stiffness (measured by the complex modulus, G*) recovers and shape is retained. pH conditions were optimized for increased printability, shape retention, and resilience of dried structures when placed in water. The formulation thus has applications where contact with water over the course of up to 2 hours does not result in loss of structure integrity or considerable swelling. An invention disclosure was submitted.
Objection 3. Research addressing the 36-month milestones for Sub-Objective 3a investigated the effect of modifying fermentation conditions, including temperature (30°C, 37°C and 42°C) and length of fermentation (8h, 24h and 96h), on the production of bacteriocins within a waste ice cream (WIC) by-product. Previous work showed that S. thermophilus B59671 could produce thermophilin 110 within the WIC by-product, but this work included additional bacteriocin producing LAB, including a nisin-producing strain of Lactococcus lactis; a pediocin-producing strain of Lactobacillus plantarum, and an enterocin-producing strain of Enterococcus durans. Results showed that prolonged fermentation (96 h) did not result in fermentates with higher antimicrobial activities. Fermentation temperature affected bacterial growth and bacteriocin-production, with S. thermophilus being able to produce its bacteriocin at 42°C, but production was similar at 37°C. All strains were capable of producing their bacteriocins at 37°C. The goal of producing various bacteriocins is to allow for a blending of fermentates to generate a final product containing multiple antimicrobials to increase their efficacy and prevent antimicrobial resistance to a single bacteriocin. Additionally, a collaborative study with ARS food safety labs and the University of Virginia was initiated to combine bacteriocins and additional antimicrobial hurdles to disrupt Listeria monocytogenes biofilms; and collaborative studies with Carnegie Mellon University continued to characterize a novel bacteriocin-encoding gene cluster in S. thermophilus ST134. For the later project, gene knock out mutants were generated and screened for antimicrobial activity against other S. thermophilus strains, and a thermophilin-resistant mutant was isolated and will be used to identify the bacteriocin receptor on sensitive cells. Finally, in addition to the antimicrobial work, WIC by-product was studied as a fermentation substrate for production of ethanol using yeast species. Results showed that the addition of a protease during fermentation increased the ethanol yield, which reached ~13%. This concentration is expected to be sufficient to allow for subsequent distillation to produce a novel beverage alcohol.
Additional studies addressing Sub-Objective 3a, involved the computational analysis of 20 sequenced genomes from S. thermophilus strains. This work characterized the Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas systems present in the S. thermophilus strains to identify an endogenous system that can be used to perform gene editing techniques without compromising the food-grade status of S. thermophilus. Potential guide RNAs have been identified and plasmid design has begun. Gene editing techniques will initially focus on making modifications to promoters upstream of genes within the bacteriocin-like peptide gene cluster to increase production.
Research to address milestones for Sub-objective 3b focused on the characterization of a novel milk-derived peptide generated following the fermentation of skim milk with Weissella viridescens. Antimicrobial activity was only observed when W. viridescens was grown in commercial skim milk but was not observed using autoclaved or UHT skim milk. The antimicrobial was lost after exposure to proteases or boiling, further confirming the antimicrobial was a protein or peptide. Work has begun to identify the protein responsible for this unique antimicrobial activity using sodium dodecyl sulfate-polyacrylamide gel electrosphoresis (SDS-PAGE) analysis and mass spectrometry.
Objective 4, churning was further developed as a practical and efficient process for the recovery of fat from waste ice cream (WIC). An important tradeoff between fat recovery rate and recovered fat moisture was studied, and it was shown that this tradeoff is governed by churning temperature. Separately, a treatment to speed the churning of WIC was developed; this treatment is attractive because it requires only the addition of more of an emulsifier that is already used in ice cream. Experiments were also conducted in a much larger scale format to confirm that the results would hold up in commercial application.
The enzyme and centrifuge based free fat method that was discovered last year was investigated in scale up tests with industry relevant equipment, although it was not successful in producing relevant quantities of free fat. Additionally, industrial techniques such as flocculation and dissolved air flotation were investigated as a means to cheaply concentrate fat and remove water as a first step in processing. While the dissolved air flotation was not effective, current work on flocculation has shown promise in quickly dewatering waste ice cream.
Accomplishments
Review Publications
Miller, A.L., Renye Jr, J.A., Oest, A.M., Liang, C., Garcia, R.A., Plumier, B.M., Tomasula, M.M. 2024. Bacteriocin production by lactic acid bacteria using ice cream by-product as the fermentation substrate. Journal of Dairy Science. https://doi.org/10.3168/jds.2023-24249.
McAnulty, M.J., Guron, G.P., Oest, A.M., Miller, A.L., Renye Jr, J.A. 2024. The quorum sensing peptide BlpC regulates the transcription of genes outside its associated gene cluster and impacts the growth of Streptococcus thermophilus. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2023.1304136.
Plumier, B.M., Akkurt, S., Bonnaillie, L., Yee, W.C., Ashby, R.D., McAnulty, M.J., Garcia, R.A., Renye Jr, J.A., Tomasula, M.M. 2023. Effect of alkaline pH values on physical and functional properties of edible films made from calcium caseinates. International Dairy Journal. 151:105851. https://doi.org/10.1016/j.idairyj.2023.105851.
