Document Type

Dissertation - Open Access

Award Date

2021

Degree Name

Doctor of Philosophy (PhD)

Department

Dairy Science

First Advisor

Sanjeev Anand

Keywords

microencapsulation, probiotics, whey protein, whole genome, sequencing

Abstract

In this study, a novel value-added dairy-based health formulation was developed using whey protein hydrolysate and probiotic organisms. In the first part of the study, different forms of whey proteins, concentrate WPC80, isolate WPI90, and hydrolysates WPH10, WPH15 and WPH20 were screened for bioactivities (antimicrobial, antioxidant, and antihypertensive activity). Hydrolysate WPH10, exhibiting the highest bioactivities was conjugated with maltodextrin. A batch of 2L conjugated solution was spray dried in a Niro drier with an inlet and outlet temperature of 200⁰C and 90⁰C, and alternatively, freeze dried at −80 °C under 50 mTorr vacuum. The bioactivities of the conjugated samples were then assessed. Overall, the hydrolysates showed significantly higher bioactivities as compared to concentrate and isolate. The conjugated WPH10 solution demonstrated higher antimicrobial, and antioxidant activity, whereas no significant difference in the antihypertensive activity was observed, as compared to WPH10 alone. Subsequent spray and freeze drying of the conjugate solution exhibited even higher antimicrobial, and antioxidant activities, while retaining the antihypertensive activity. Overall, the results indicate the ability of the WPH-maltodextrin to retain the bioactivities after conjugating with a reduced carbohydrate. In the second part of the first study, conjugated whey protein hydrolysate (WPH10), was used as an encapsulant to entrap probiotic cultures; Bifidobacterium animalis subsp. lactis ATCC27536 and Lactobacillus acidophilus ATCC4356, through a spray drying process. Each culture was added in the conjugated solution at ratio of 1:1, with spiking level of 10 log CFU/mL. The mixture was spray dried in 2L batches using a Niro drier with an inlet and outlet temperature of 200⁰C and 90⁰C, respectively. Following drying, scanning electron microscopy was used to observe the particle structure of the WPH10 conjugate. Following, the dried formulation was stored in airtight bottles for 16 weeks at 4°, 25° and -18°C, to monitor cell viability, moisture, and functionality. From the micro images it was observed that subsequent drying, nonconjugated WPH10 presented link bridges between the powder particles, whereas in case of conjugated WPH10, the particles presented a round surface with pores on it. The mean counts in conjugated WPH10 matrix before and after drying were 10.59 log CFU/g and 8.98 log CFU/g, respectively. At the end of 10 weeks, the counts were 7.18 log CFU/g at 4°C and 7.87 log CFU/g at -18°C, whereas at 25°C the viability significantly decreased to 3.97 log CFU/g. At this point, the wetting time increased from 47min to 61min, and solubility decreased from 90% to 82%. In the second study, whole-genome sequencing was done to determine the genotypic variants and associate with phenotypic expression in Listeria strains. Genomic DNA was extracted from the strains (Listeria innocua 634-2; Listeria innocua 634-34-S- 5; Listeria innocua 634-34-S-6; Listeria welshimeri 634-3; Listeria welshimeri 634-253- S-5, and Listeria monocytogenes 315-S-1), isolated from the dairy processing environment and run through Whole-genome sequencing on the Ilumina MiSeq and Nanopore sequencing platform. The genome was fed into the RAST database to develop the annotations. The results revealed 13 types of phenotypic responses related to the genotypic variants. Overall, the genotypic variants varied with the species and within the strains. Some of the common variants and features identified in the isolates were virulence (DNase, phage terminase, tRNA-Arg-ACG), cell signaling (NAG-IIA, NAGIIB), phage immunity (CRISPR proteins), osmotic stress (CadA), oxidative stress (YRKL), and antibiotic resistance (BaiE, Lde). L. monocytogenes was positive for all the phenotypic characteristics, allowing it to be more persistent, whereas L. innocua and L. welshimeri isolates lacked variants for motility (ActA), biofilm formation (AgD), and acid tolerance (AdiA), making them more sporadic strains.

Number of Pages

206

Publisher

South Dakota State University

Available for download on Sunday, May 15, 2022

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