M.S. - Food Science

Permanent URI for this collectionhttps://hdl.handle.net/10125/2061

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Effects of ultrasonication on germination and bioactive content of red rambo radish microgreens (Raphanus sativus)
(University of Hawai'i at Manoa, 2025) Nakamura, Taryn; Ho, Kacie; Food Science
Radish microgreens are a high-value commodity due in part to their rapid growth and nutrientdensity. Ultrasonication, the application of sound waves, has been shown to improve the germination rates, but it is not clear how it impacts nutritional quality. The aim of this study was to evaluate the effects of ultrasonication (0, 3, 6, 9, 12, 15 min, 20 kHz) on germination and bioactive content (carotenoids and polyphenols) in Red Rambo radish microgreens. The effects of ultrasonication on Red Rambo radish microgreens were assessed pre-harvest by treating seeds prior to germination and post-harvest by treating the microgreen plant. The percent of seeds that germinated was recorded every eight hours over a 128-hour period. Bioactive compounds were extracted with 0.1% BHT in hexane and acetone. Carotenoids were quantified using high- performance liquid chromatography equipped with a diode array detector and a spectrophotometric method. The total polyphenolic and anthocyanin compounds were quantified through the Folin-Ciocalteu method and pH differential method, respectively. Results showed a significant increase (P < 0.05) of germination percentage in the ultrasonicated samples compared to the control group. Differences were not observed (P > 0.05) in the carotenoid profile for radish microgreens treated pre-harvest and post-harvest. Ultrasonication did not have a significant impact (P > 0.05) on the total monomeric anthocyanin content for microgreens for both pre- and post-harvest treatments. Ultrasonication can potentially stimulate the production of secondary metabolites and induce oxidative stress but has not been shown to be stimulated in Red Rambo radish microgreens. Overall, findings indicate that ultrasonication improves germination and but not health promoting compounds of Red Rambo radish microgreens. Conducting research in this area can potentially add value to existing crops and improve growth efficiency. This exploration can expand to include various types of microgreens and their mature counterparts.
NUTRITIONAL AND PHYTOCHEMICAL COMPOSITION OF PROCESSED HAWAIIAN SEAWEED
(University of Hawai'i at Manoa, 2024) Xie, Dongjun; Ho, Kacie; Food Science
Limu, referring to various types of Hawaiian seaweed, like limu ogo (red algae) and limu manauea (green algae), is known for its rich profile of minerals, carotenoids, and vitamin E. However, certain limu species may contain potentially harmful heavy metals such as arsenic and copper. This research seeks to understand how various cooking techniques, namely boiling and soaking, influence the retention of beneficial compounds like carotenoids. Drawingfrom existing literature, cooking methods can enhance the bioaccessibility of carotenoids and reduce harmful heavy metal content but may degrade essential nutrients. While extensive research exists on various seafood, studies on edible seaweed, in particular those from Hawaii, remain limited. Thus, the objectives of this work were to: 1) quantify the primary carotenoids in seaweeds grown in Hawaii and 2) estimate the effects of cooking (soaking and boiling) on carotenoid and chlorophyll retention. Various red and green seaweeds (Gracilaria parvispora, Graciliara tikvahiae, Halymenia formosa, Ulva expansa, and Ulva ohnoi) were generously donated from aquaculture facilities on the island of Hawaii and Oahu. Fresh or cooked (soaked for 24 hrs, boiled for 10 minutes, or boiled for 20 minutes) were freeze-dried, ground into a powder, and stored at -20°C prior to further analysis. Proximate analysis was conducted to estimate protein, crude fat, ash, and moisture content. Total carbohydrates were calculated by taking the difference of protein, crude fat, ash, and moisture from 100%. Lipophilic bioactive compounds were extracted from the samples using a mixture of hexane, acetone, and ethanol (2:1:1 v/v/v) assisted with ultrasonication. Specific carotenoids were quantified using reverse-phase high-performance liquid chromatography (RP-HPLC) equipped with a diode array detector (DAD) and a C-30 column. Specific carotenoids were identified and quantified by comparison with external calibration curves of analytical standards. Total carotenoid, chlorophyll a, and chlorophyll b content was quantified spectrophotometrically. Digestive stability of fresh and cooked seaweed were measured using a three-stage in vitro digestion model. Digestive stability was calculated as the relative % retention of the bioactive compound (total carotenoid or chlorophyll) in the digested fraction compared against a non-digested sample. Statistical analysis was conducted using analysis of variance (ANOVA) and the Tukey-Kramer method for post-hoc comparisons. Statistical significance was established at P<0.05. Proximate analysis indicated that Gracilaria tikvahiae was significantly higher in protein (~17 % of the freeze-dried weight) compared to others (~12.3-13.6%). Ash was the highest in Halymenia formosa (~39% of the freeze-dried weight) compared to all other samples (~12-34%). Additionally, total carbohydrates were statistically highest in Ulva expansa (~70% of the freeze-dried weight) compared to others (~42-51%). Across the seaweeds, provitamin A carotenoids (beta-carotene and beta-cryptoxanthin) were prominent in red seaweeds (Gracilaria parvispora, Gracilaria tikvahiae, and Halymenia formosa). Non-provitamin A carotenoids lutein and zeaxanthin were notable in fresh Ulva ohnoi, but were reduced in all cooked samples (soaked 24hr, boiled 10 min, boiled 20min). Beta-carotene was prominent in raw Gracilaria tikvahiae, but substantially decreased in all cooked samples. In digestive stability tests, total carotenoids were not significantly different in raw vs. boiled (20 and 30 min) samples, indicating that although cooking may reduce specific carotenoids, total carotenoids remained relatively unchanged. However, samples that were boiled for 30 min had a statistically higher carotenoid digestive stability compared to raw samples and those boiled for 20 min, suggesting that there may be some matrix-specific effects with cooking. Chlorophyll a was significantly higher in samples that were boiled (20 and 30 min) compared to raw samples, likely due to enhanced release and extraction efficiency, but chlorophyll b was not significantly different across raw and cooked samples. Although differences were observed in digestive stability of chlorophylls, there were no significant differences across raw and boiled samples. Overall, these findings indicate that red and green seaweed in Hawaii can be sources of provitamin A carotenoids and non-provitamin A carotenoids, respectively. In most cases, soaking and boiling appeared to reduce the content of specific carotenoids in seaweeds, although total carotenoid content appeared relatively unchanged. Digestive stability tests suggest that carotenoid content is affected by cooking-induced matrix changes but chlorophyll content is not significantly affected. Overall, these findings guide culinary practices to maximize limu's nutritional benefits and demonstrate the value of both fresh and cooked edible seaweeds in Hawaii.
NON-DESTRUCTIVE DEEP LEARNING APPROACHES FOR FRUIT QUALITY PREDICTION AND CLASSIFICATION: PAPAYA AND AVOCADO
(University of Hawai'i at Manoa, 2024) An, Sujin; Jun, Soojin; Food Science
Artificial intelligence (AI) is a broad discipline that creates systems with abilities to perform a variety of tasks that would normally require human intelligence. Machine learning is a sub-field of artificial intelligence focused on the design of algorithms, giving computers the capability to learn from and analyze decisions based on data. Deep learning is another subset of machine learning that uses many layers of neural networks for modeling complex patterns within big data. Deep learning models are particularly powerful in recognizing patterns and making predictions, making them suitable for a wide range of applications. Consequently, deep learning is applied in various fields, including the food industry. It integrates multiple disciplines and is researched and utilized for various purposes. In the food industry, deep learning holds potential in several aspects, such as quality control and assurance, food safety, and process automation. By leveraging complex neural networks, deep learning can analyze large amounts of data to make accurate predictions and classifications, thus enhancing the efficiency and reliability of processes.Studying and assessing fruit ripening is crucial for ensuring optimal quality, marketability, and consumer satisfaction. This is particularly important for climacteric fruits such as papayas and avocados, which ripen after harvesting. Accurate ripeness assessment can help manage post-harvest handling better, reduce waste, and improve supply chain efficiency. The study aimed to advance the application of deep learning in non-destructive fruit quality assessment. The main goal is to develop an innovative model that can accurately predict physicochemical properties and classify the ripening stages of fruit. Specifically, the focus is on two models: a CNN (Convolutional Neural Network) that predicts the physicochemical properties of papayas through image analysis and weight evaluation, and a multimodal model that classifies avocado ripening stages using image analysis, acoustic impulse response audio signals, and weight. By integrating these methodologies, the paper seeks to demonstrate the feasibility and efficiency of deep learning techniques to improve fruit quality assessment and provide more accurate, non-destructive and automated solutions to the agricultural sector. In the first study (Chapter 3), images of papaya at different stages of ripeness were captured and weighed. From a total of 132 papayas, 528 images were obtained. Through image augmentation, the number of images increased threefold to 1584 images, which were used in the study. Various physicochemical properties such as texture, pH, total soluble solids (TSS), and seed weight were subsequently measured. The data were split into training, validation, and test sets in an 8:1:1 ratio for model training and evaluation. A CNN model was trained using image snapshots and weights as input values to predict physicochemical property values. Model performance was evaluated using the mean squared error (MSE) and the coefficient of determination (R²) as evaluation metrics. The CNN model achieved the high accuracy with MSE values of 0.0284 and 0.1729 and R² values ranging from 0.71 to 0.94 for the training and validation sets, respectively. These findings demonstrated that CNN-based models can provide detailed and quantitative insights to improve the understanding and management of papaya quality and characteristics. The second study (Chapter 4) involved a multimodal approach to classify Hass avocado ripening stages (unripe, ripe, or overripe) using image analysis, audio analysis, and weight measurements. Data points, including 584 images, acoustic impulse response audio signals, and weight measurements, were collected from 146 Hass avocados. The image data underwent image augmentation, resulting in the image data being doubled to 1168 images. Additionally, the audio data were converted to log-Mel spectrograms, and the avocado firmness data were processed using Box-Cox transformation. The collected data were split into training, validation, and test sets in an 8:1:1 ratio for model training and evaluation. A multimodal ripeness classification model integrating image, audio, and weight (i.e. IAWM) was used to predict avocado firmness and classify ripeness. Model performance was validated using accuracy, precision, recall, F1 score, and confusion matrix. The IAWM model was compared to the image-based ripeness classification model (IM) and multimodal ripeness classification model integrating image and audio (IAM). The IAWM achieved an accuracy of 0.949, compared to 0.838 for the IM and 0.897 for the IAM. In addition, the IAWM demonstrated superior performances in terms of the precision, recall, and F1 score. These results indicate that integrating multiple data sources improves the ability of the deep learning model to accurately classify ripeness stages. Both studies show that non-destructive deep learning approaches can significantly enhance fruit quality assessment. The CNN model for papayas and the multimodal model for avocados demonstrate the potential of deep learning to offer accurate, efficient, and practical solutions for predicting physicochemical properties and classifying ripeness stages. These advances promise to improve quality control, reduce waste and increase consumer satisfaction in the agricultural sector. Unified data formats not only improve fruit quality predictive modeling, but also help gain insights and better management formats. This study demonstrates the potential of deep learning approaches in non-destructive fruit quality assessment. By optimizing the parameters of the model and integrating various data sources, the accuracy and efficiency of fruit quality prediction and classification can be significantly improved, providing substantial benefits to the food industry.
Antimicrobial Activity of Coffee Husk Extracts aginst Listeria monocytogenes and Salmonella Typhimurium in Microbiological Media and Milk
(University of Hawai'i at Manoa, 2024) He, Yu; Li, Yong; Food Science
Pathogenic bacteria pose significant challenges to food safety in the United States and globally. Salmonella enterica serovar Typhimurium and Listeria monocytogenes are among the pathogens that frequently trigger foodborne illness outbreaks, leading to substantial economic losses and public health burdens. Consumption of food contaminated by these bacteria, such as raw milk, can result in various gastrointestinal symptoms, ranging from mild discomfort to severe, life-threatening conditions. With the rise of antibiotic-resistant strains, it is crucial to explore alternative antimicrobial agents. Coffee, particularly the species Coffea arabica (yellow) and Coffea liberica (red) grown in Hawaii, represents a significant cash crop. The coffee cherries, the fruit of coffee trees, is considered a superfood due to its skin and juice, rich in bioactive compounds. However, the wet coffee seed comprises only approximately 55% of the fruit, leaving the other half of the coffee fruit (coffee husk) underutilized or considered a by-product of coffee production. This study aimed to assess the antimicrobial efficacy and mechanism of action of coffee husk against S.S. Typhimurium and L. monocytogenes. Coffee husk samples were obtained from the Island of Hawaiʻi and Oʻahu, and bioactive compounds were extracted using 80% acidified methanol. The total phenolic content of the extracts was quantified, and other chemical analyses, such as pH, sugar content, titratable acidity, and anthocyanin content, were conducted. The pH values of red and yellow coffee husk extracts were 4.10 and 4.18, respectively. The sugar and titratable acidity ratios for red and yellow coffee husk extracts were 9.60/1.55 °Brix/% and 8.87/1.41 °Brix/%, respectively. The total phenolic content of red and yellow coffee husk extracts was 5058.27 GAEmg/L and 4600.27 GAEmg/L, respectively. The anthocyanin content of the red coffee husk extract was 22.03 mg/mL, significantly higher than that of the yellow coffee husk extract, which was 0.06 mg/mL. Subsequent evaluations revealed that the red and yellow coffee husk extracts had a minimum inhibitory concentration (MIC) of 12.5 mg/mL and a minimum bactericidal concentration (MBC) of 50 mg/mL against S. Typhimurium and L. monocytogenes. Notably, probiotic strains Lactococcus lactis and Lacticaseibacillus rhamnosus GG grew even in the presence of 50 mg/mL coffee husk extracts. Biofilm assays demonstrated that 6.25 mg/mL (1/2 MIC) red and yellow coffee husk extracts significantly reduced S. Typhimurium levels in formed biofilm by 0.53 log and 0.42 log, respectively. However, coffee husk extracts at this concentration did not affect biofilm formation by L. monocytogenes. Both coffee husk extracts at 12.5 mg/mL (MIC) caused significant damage to the cell membrane of L. monocytogenes and S. Typhimurium. The cell membrane damage to S. Typhimurium caused by 25 mg/mL (2 MIC) red and yellow coffee husk extracts was comparable to the positive control treated with 70% ethanol. Additionally, this study demonstrated significant protein leakage in both L. monocytogenes and S. Typhimurium when treated with red and yellow coffee husk extracts at 12.5 mg/mL. Notably, the intracellular protein leakage caused by 25 mg/mL red coffee husk extract in L. monocytogenes and S. Typhimurium was significantly higher than that caused by 70% ethanol. This correlation between loss of membrane integrity and leakage of intracellular proteins confirmed the detrimental effects of coffee husk extracts on pathogenic bacterial cells. Finally, the antimicrobial properties of coffee husk extracts were evaluated in milk artificially contaminated with L. monocytogenes and S. Typhimurium. Comparing the concentration of bacteria in treated milk with the initial concentration of the control group, neither red nor yellow coffee husk extract at 25 mg/mL showed a significant effect on L. monocytogenes or S. Typhimurium in milk stored at 7°C, whereas 12.5 mg/mL red and yellow coffee husk extracts reduced the growth of these pathogenic bacteria in milk at 21°C. Moreover, both coffee husk extracts at 25 mg/mL completely suppressed L. monocytogenes and S. Typhimurium in milk during storage for up to 72 hours. In conclusion, coffee husk extracts exhibit strong antimicrobial effects on L. monocytogenes and S. Typhimurium but not on tested probiotic bacteria. The extracts can damage the cell membrane of these pathogenic bacteria and cause the leakage of intracellular proteins. They can also reduce biofilm formation by S. Typhimurium. Coffee husk extracts offer a potent and natural alternative to traditional food preservatives for enhancing the microbiological quality and safety of food.
PEA PROTEIN ISOLATE-GREEN TEA CATECHIN PICKERING PARTICLES FOR OIL-IN-WATER (O/W) EMULSION STABILITY
(University of Hawai'i at Manoa, 2023) Vital de Sousa Junior, Wanderley; Ho, Kacie; Food Science
An emerging trend involves replacing animal-derived proteins in food emulsions with plant proteins due to their positive clean label image and potential as sustainable ingredients. Among these plant proteins, pea proteins show promise in stabilizing oil-in-water (O/W) emulsions, but they tend to create insoluble clumps that can affect physical stability. One strategy to mitigate this is to complex or add polyphenols, which may improve the emulsifying properties of proteins. However, the impact on the non-adsorbed fraction, which can greatly affect physicochemical stability, is not clear. Therefore, this study aimed to assess the effects of pea protein and catechin concentration on O/W emulsion stability, while also evaluating the non-adsorbed fraction and lipid oxidation (over 14 days). Various concentrations of green tea catechins (ranging from 0.1 to 2 mg/mL) were complexed with pea protein isolate particles (1.5 g/100 mL) at pH 7. Subsequently, the aqueous phase and 10% canola oil were pre-homogenized and passed through a microfluidizer to create a fine emulsion. Induced heating at 90 °C for 30 min before homogenization was tested for emulsion optimization. Excess non-adsorbed protein was estimated by centrifuging the emulsions and quantifying the protein in the subnatant via a BCA assay. Overall, the study showed that while induced heating resulted in a reduced non-adsorbed protein fraction, samples without heating treatment yielded better outcomes in terms of average droplet size, zeta potential, and polydispersity index. HPLC analysis revealed that (-)epigallocatechin gallate hydrate was the primary catechin in the extract used for the emulsion formulation (85.26% of the total extract). Emulsion samples with higher catechin concentrations (2 mg/mL) potentially influenced protein adsorption at the oil-water interface, resulting in a greater non-adsorbed fraction (70.01 ± 27.22) compared to lower catechin concentrations (0.1-1 mg/mL). Confocal microscopy results indicated that at catechin concentrations of 0-0.5 mg/mL, protein particles encapsulated the oil droplets, while at 1-2 mg/mL, protein particles floated freely in the aqueous phase instead of coating the oil droplets. Moderate catechin concentrations (0.5 mg/mL) slightly reduced lipid oxidation compared to the control (no catechin), whereas lower catechin concentrations (0.1, 0.3 mg/mL) appeared to significantly increase lipid oxidation (P<0.05). These results provide valuable insights for the development of emulsions, indicating that factors such as an excess of protein and polyphenols can have notable effects on PPI-GTC emulsion-based systems. This work has broader industrial implications as it demonstrates strategies to enhance ingredient functionality in emulsion-based systems.
MAGNETIC FIELD-BASED SUPERCOOLING: CELL VIABILITY ENHANCEMENT OF FREEZE-DRIED LACTOBACILLUS SPP. AND INHIBITORY ICE NUCLEATION OF IRONOXIDE NANOPARTICLES IN AN AGAR MODEL SYSTEM
(University of Hawai'i at Manoa, 2023) Joya, Amanda; Jun, Soojin; Food Science
Supercooling is the phenomenon of lowering the temperature of a solution or food material past its equilibrium freezing point without the formation of ice crystals. It is a novel food preservation technology that can extend the shelf-life of fresh and perishable foods, while still maintaining food quality aspects. Freezing can extend the shelf-life of foods for extended periods of time for months and in some cases for up to a year. However, the food quality can be degraded due to the formation of ice crystals, in which the texture and nutrient losses can be comprised. Supercooling preservation can contribute to shelf-life extension without destroying food membranes and tissues by inhibiting the formation of ice crystals. A method to achieve this supercooling status is the use of magnetic fields paired with the conventional freezing methods. Oscillating magnetic fields (OMFs) have been employed with freezing as a novel preservation technique that has been utilized in biomedical and food applications. Additionally, this technology is still in the developing stages and remains controversial. Probiotics are live microorganisms that confer health benefits to the host when taken in adequate amounts. These microorganisms may provide several benefits to overall human health and contribute to the gastrointestinal tract (GIT) as they can aid in digestion, immune health, and various GIT diseases, such as irritable bowel syndrome (IBS), constipation, and diarrhea. Probiotics can be administered in various forms such as fermented foods, beverages, and supplements in the form of powder, capsules, gels, and tablets. Probiotic supplement processing usually involves a drying step to preserve the cell viability and extend the shelf-life. Common drying steps include spray-, freeze-, vacuum-, or fluidized bed-drying; however, these methods can lead to detrimental effects on the probiotic cells due to drastic temperature changes and dehydration, which poses a threat to the viability of these cells and may have an impact to the potential benefits that can be administered to the host. Freeze-drying, also known as lyophilization, is the preferred method of drying since most probiotic strains are heat sensitive and the use of cryoprotectants can be applied to the cells to minimize the detrimental effects caused by the initial freezing step. Cryoprotectants are additives used to maintain high cell viability during freeze-drying processing and are also used as bulking agents for probiotic material. After the initial freezing step, cells undergo a primary drying step, in which water or other solvents are removed via sublimation, allowing ice to directly change from solid to vapor, and a secondary drying step is applied to remove any additional water. This method is highly preferred for long-term preservation and the cells keep their biological and structural integrity. However, the freeze-drying process does have an effect on the cell viability due to the ice formation initial freezing step. In Chapter 3, OMF-assisted freezing was used as a pretreatment to probiotics before freeze-drying to investigate the effects on the cell viability of Lactobacillus acidophilus (L. acidophilus). The OMF-assisted freezing parameters for this experiment were 10 mT for the magnetic field strength and 10 Hz for the frequency and maintained a supercooled status at -5°C for 6 h. It was found that supercooled cells before freeze-drying compared to cells that were not(control) had a lower reduction of cell viability of 0.5 and 1-log reduction, respectively, to the initial cell count, which were cells before freeze-drying. A preliminary study by Wang (2021) found similar results, in which supercooling achieved the highest cell viability of 78% compared to fast and slow freezing, and refrigeration storage, which can be explained by the formation of surface layer proteins (SLPs) that are formed when L. acidophilus was exposed to cold temperatures. The subzero temperatures allow L. acidophilus to strengthen its natural defense mechanism by protecting itself from cold stress. This current research study was a continuation of the preliminary study that changed several parameters to focus on practices that can be implemented for industrial purposes. A case study done with a collaborative partnership company used our supercooling system with OMF as a pretreatment for L. salivarius before freeze-drying to investigate the effects of the supercooling technology on the cell viability and shelf-life after 0-, 3-, and 8 days of storage. Supercooling technology was able to maintain high cell viability of supercooled cells even without the use of cryoprotectants after 8 days of storage. This technology has great potential in maintaining a high cell viability in probiotics, though there still needs to be further research on the effects of different supercooling parameters such as temperatures, magnetic field strengths, frequencies, exposure time, and different probiotic strains. Furthermore, it should be pointed out that OMF-assisted supercooling is still highly controversial and remains a topic of debate and the mechanisms regarding this technology are still unknown. In Chapter 4, the effects of OMF-assisted freezing on the supercooling behavior of magnetic nanoparticles (MNPs), particularly iron-oxide nanoparticles (IONPs) were also investigated. The current literature about this topic of magnetic field freezing is a controversial topic due to the variations in results of many researchers. The mechanisms involved are still not fully understood as this technology is still in the beginning and developing stages. Proposed mechanisms for this technology include the reorientation or vibration of the water molecules within solutions and/or food samples, due to the diamagnetic properties of water, as well as hydrogen bonds breaking between water molecules, allowing for the inhibition of ice crystallization. However, some researchers disagree with this proposed mechanism, and it has been postulated that there is ferromagnetic material naturally present in biomaterials, which could be the reason for supercooling to occur under OMF during freezing without the formation of ice crystals. It is difficult to conclude that supercooling is successful in all foods, since foods are composed of complex matrices with varying constituents of proteins, lipids, carbohydrates, water, and minerals, which lead to the varying and contradictory results reported in the current literature. Due to this reason, food models are employed to exclude the factors that will have an influence on supercooling. Nevertheless, the mechanisms still need to be further investigated to understand the interactions between supercooling and foods and/or biomaterials under magnetic fields. The effects of OMF-assisted freezing on the supercooling behavior of magnetic nanoparticles were investigated in an agar food model system. The IONPs at different concentrations of 3, 6, 12, and 15 mg were used to investigate the supercooling behavior at 10 mT as the magnetic field strength and 10 Hz as the frequency at -8°C for 24 h treatment time. Samples without any nanoparticles were used as a control and compared with 12 mg IONP and 12 mg zinc nanoparticle (ZNP) agar samples, as well. For each sample, a supercooling probability was calculated, which is the number of times that a sample successfully supercooled divided by the total amount of outcomes or trials (n=20). 12 mg IONPs exhibited a high supercooling probability of 90%, while the 3, 6, and 15 mg IONP samples had a supercooling probability of 75%. The control samples had a supercooling probability of 60% and ZNP samples had a 55% supercooling probability, which can possibly be due to the stochastic nature of ice nucleation during the supercooling status. This research demonstrates that magnetic nanoparticles do affect the supercooling behavior under OMF during freezing. This study can contribute to some understanding of the interactions or mechanisms within foods under OMFs during freezing to improve the current technology and systems to maintain supercooling and potentially implement this technology in the industry. In any case, further investigation on the supercooling parameters such as the magnetic field strength, frequency, temperature, and exposure time, as well as different types of NPs and concentrations.
Oscillating Magnetic Field (OMF) And Pulsed Electric Field (PEF)-assisted Supercooling For The Improved Shelf Life Of Fresh Salmon Fillets
(University of Hawaii at Manoa, 2022) Tang, Jinwen; Jun, Soojin; Food Science
In recent years, global fish and seafood markets have continued to expand, and seafood demand and transaction volumes have also increased yearly. Due to increasing demands for protein-rich fish products, countries such as China, India, and Indonesia have high consumption of fresh fish products. Meanwhile, salmon, a type of seafood with a broad market volume and potential, has significant shares in terms of revenue in the North American seafood market. As a result, consumers' inclination toward healthy lifestyles and changing dietary habits drive the demand for fish products.However, seafood is extremely perishable partly due to the presence of amino acids, which can decompose into biogenic amines and ammonia. Seafood is also rich in polyunsaturated fatty acids (PUFA), which is easily oxidized and leads to unpleasant odor, taste, nutrient loss, and color changes. Therefore, proper food processing techniques should be employed to maintain the quality of salmon and extend storage time. Freezing is the most common process for the long-term preservation of food. Immobilization of liquid water by lowering the storage temperature can reduce the quality deterioration rate. Freezing temperatures below 0°C also prevent the growth of microorganisms in the food that cause food spoilage and foodborne illness. However, during the freezing process, the initial freezing step forms ice crystals that damage food structures and reduce food quality. Therefore, there is a great need to preserve food without freezing damage. Supercooling is a new food processing technology that keeps food below the initial freezing temperature without forming ice crystals. It provides a longer shelf life for the food while maintaining the quality of the food. In the supercooling protocol developed by Dr. Jun’s lab, ice nucleation during freezing is prevented by the pulsed electric field (PEF) and oscillating magnetic field (OMF). The fundamental is that electric and magnetic fields have potential interference and rotation effects on water molecules present in the food, ultimately associated with the inhibited aggregation of water molecules. As a result of this, I investigated the effects of supercooling technology based on PEF and OMF on the preservation of salmon to extend salmon's storage time while maintain the freshness. Thiobarbituric acid reactive substances (TBARS) values represent quantitative oxidative changes in meat and meat products during storage. Malondialdehyde (MDA) is one of the most commonly used markers of lipid oxidation secondary products. The color index of frozen and refrigerated samples differed significantly (P<0.05) from the original state. Color differences of frozen, refrigerated, and supercooled samples were 4.2, 5.8 and 2.9, respectively. In addition, the TBARS value of the supercooled sample was about 0.96 MDA/kg, which was close to 1.06 MDA/kg of the frozen/thawed sample, but the value of the refrigerated sample was 2.13 MDA/kg. Meanwhile, the drip loss for the refrigerated samples reached as high as 4% and 2.5%, while the supercooled sample was as low as 0.9%, which was not frozen/thawed or damaged by ice, resulting in little change in drip loss. For the microbial enumeration experiments, after 10 days of experimentation, the microbial counts of all samples increased, the microbial counts of supercooled samples, refrigerated sample, and frozen/thawed samples were 5.4, 6.4, and 5.9 log CFU/ml, respectively. After the experiment of PEF and OMF-assisted supercooling treatment of salmon, our result showed that supercooling treatment could increase the storage time of salmon without quality degradation.
Effects Of Supercooling-assisted Freezing On Physical And Structural Properties Of Agar Gel And Tofu
(University of Hawaii at Manoa, 2022) Wang, Yijing; Jun, Soojin; Food Science
Supercooling is the process of lowering the temperature of the product below its typical freezing point without forming ice crystals. Compared with well-known long-term preservation methods such as freezing, supercooling can slow down changes in food qualities and also shorten the processing time, reduce energy consumption, and potentially carbon emissions. During the supercooling process, the combined electric field and magnetic field treatments could enhance the mobility of water molecules while at freezing temperature ranges. Pulsed electric fields (PEF) have been reported to reorient and realign water molecules because of the electric dipole present in water molecules. Oscillating magnetic fields (OMF) vibrate water molecules due to water diamagnetism, preventing ice crystal nucleation. The purpose of this study was to evaluate the effect of the supercooling pretreatment before freezing on the physical and structural properties of solid model foods.In this experiment, agar gels (2% agar powder & 10% maltodextrin) and firm tofu were treated using four different methods: freezing at -18℃ for 24 hours, freezing at -18℃ for 24 hours with 6 hours of supercooling pre-treatment, freezing at -80℃ for 24 hours, and freezing at -80℃ for 24 hours after 6 hours of supercooling pre-treatment. The supercooling temperature of agar gel is about -6℃, and firm tofu was supercooled about -2℃. Before treatments, all samples were stored in a refrigerator at 4℃. Their syneresis and texture were measured after thawing for 24 hours. Microscopic observation and micro-CT scanning of the samples were committed at the same time. The results showed that compared with control samples, samples after supercooling treatment showed low syneresis, and the peak force was close to fresh samples. By analyzing the structure of the samples treated under different storage conditions, it was found that the ice crystal of the samples stored at -80°C were finer and denser than that of the samples stored at -18°C. This study proved that the supercooling pretreatment before freezing is beneficial in reducing the damage of ice crystals in the internal structure of the sample and can maintain post thawing physical properties as close to fresh samples. The experimental results of agar gels and firm tofu can serve as a foundation for the supercooling and freezing experiments of more complicated food materials in future studies.
Nutritional and Phytochemical Content of Underutilized Taro Cultivars From Hawai‘i
(University of Hawaii at Manoa, 2021) Senga, Kento; Ho, Kacie; Food Science
Taro (Colocasia esculenta) is a starchy root crop that is commonly found in most tropical climates around the world. In Hawai‘i, various taro cultivars exist, each with potentially unique nutritional and phytochemical profiles as a result of their pigmented coloration. However, not much is known about the nutrient and phytochemical properties of these cultivars. The primary purpose of this research is to identify the nutritional and phytochemical aspects in these underutilized taro cultivars from Hawai‘i. The nutrient (proximate and mineral), phytochemical (carotenoid, total phenolic, and total monomeric anthocyanin), and digestibility (bioavailability & bioaccessibility) content of two commercially underutilized taro, Mana Ulu & Pi‘iali‘i, were compared with a commercial variety, Maui Lehua. An immature (colloquially referred to as “Keiki”) Pi‘iali‘i corm was also utilized in this study and compared to the other three. Both raw and cooked forms, through steaming, were utilized in this experiment. Overall findings indicated variation in nutrient and phytochemical content across each taro cultivar. Notable distinctions include a higher carbohydrate content found in the Maui Lehua and Mana Ulu cultivars, 31.92% and 31.76% respectively, while Pi‘iali‘i and the Keiki exhibited a lower content of 16.34% and 15.68%, respectfully. Phytochemical identification determined beta-carotene and lutein as the primary carotenoid compounds across each cultivar. Loss of carotenoid content was observed in all cultivars during their transition from raw to cooked states. Following in vitro digestion, only beta-carotene was retained from the cooked Mana Ulu variety. Bioaccessibility testing indicated a digestive stability of 45.8% and a micellarization efficiency of 13.6% for beta-carotene. Values of total phenolic content reported 83.52, 76.04, 75.44, and 66.86 mg/100 g FW and total anthocyanin content of 10.02, 9.33, 7.32, and 6.93mg/100g FW for Maui Lehua, Mana Ulu, Pi‘iali‘i Keiki, and Pi‘iali‘i, respectively. Cooked taro had significant polyphenol losses with total phenolic contents of 20.55, 17.73, 22.61, 25.93mg/100g FW as well as total anthocyanin contents of 3.19, 2.16, 2.12, and 2.88 mg/100g FW for Maui Lehua, Mana Ulu, Pi‘iali‘i Keiki, and Pi‘iali‘i, respectively. This research can provide a significant insight into the utilization of nutrients in underutilized food crops that would otherwise be absent in conventional crop utilization. This can apply not only to taro cultivars in Hawai‘i but other food sources around the world that have different species varieties.
Enhanced viability of freeze-dried Lactobacillus acidophilus using supercooling pretreatment
(University of Hawaii at Manoa, 2021) Wang, Yu; Jun, Soojin; Food Science
Viability loss during the freezing process remains a big challenge for freeze-dried lactic acid bacteria. Oscillating magnetic field (OMF)-assisted supercooling pretreatment was studied and validated for the improving the viability of freeze-dried L. acidophilus. OMF-assisted supercooling decreased supercooling temperature to -7 °C without ice crystallization, while one of the reference groups without OMF assistance was frozen at -7 °C. Supercooling pretreatment increased surface layer protein (SLP) expression and improved cell viability. The SLP on supercooling pretreated cells was thicker than those without supercooling pretreated cells. Supercooling pretreatment significantly increased the viability of L. acidophilus after freeze-drying compared to those that did not undergo a pretreatment. The highest viability of 78% was observed after freeze-drying with supercooling pretreatment. SLP expression during pretreatment protected cells in the freezing process and helped maintain cell viability. These results suggested that OMF-assisted supercooling pretreatment enhanced the viability of freeze-dried L. acidophilus.
Biochemical changes in the development of alcoholic fermented products from taro (Colocasia esculenta)
(University of Hawaii at Manoa, 1987) Cai, Tiande; Food Science
Biochemical changes in the development of alcoholic fermented taro products were investigated under different fermentation conditions. The products were prepared by fermenting steamed taro grits with powdered commercial Chinese yeast ball (rice ball) . So
Development of Soy Protein Isolate-Isoflavone Pickering Particles for Oil-in-Water (O/W) Emulsion Stability
(University of Hawaii at Manoa, 2021) Pereira, Garinn; Ho, Kacie; Food Science
Many food and beverage systems are colloidal oil-in-water emulsions, which contain dispersed oil droplets throughout an aqueous phase. In particular, Pickering emulsions are stabilized by interfacial solid particles and offer improved stability compared to conventional emulsions via irreversible absorption, which prevents the natural tendency of droplets to coalesce. Polyphenols (plant derived secondary metabolites) are capable of complexing with proteins through structural interactions. Certain polyphenols have shown influence on interfacial properties of proteins, yet the use of isoflavones has not been studied. Thus, the objective of this research is to understand the interaction of isoflavones on particle and emulsion stability. Okara (soybean residue) was initially evaluated to determine potential for protein and isoflavone capabilities. Overall extraction from okara was determined to be insufficient for further testing, thus soy protein isolate and isoflavone supplements was used for particle and emulsion formulation. Soy protein fractions were classified and assessed by SDS-PAGE and BCA test. Main proteins found were β-conglycinin (7S) and glycinin (11S) protein fractions. HPLC analysis and the Folin-Ciocalteu method were performed to identify and quantify the primary isoflavones used for particle formulation. Notable isoflavones identified from HPLC analysis were daidzin and genistin. Varying concentrations of isolated isoflavones were complexed to soy proteins through pH differentiation and induced heating. Physical characteristics (size, zeta potential, polydispersity index, and wettability) of soy protein isolate-isoflavone (SPI-I) particles and emulsion droplets (10% oil fraction) were measured using a Zetasizer Nano ZS and goniometer. The optimal concentration of soluble soy proteins for protein (control) particles was 3.3 mg/mL, which provided the smallest size (nm) while maintaining a low polydispersity (PdI). The introduction of a heating step to develop SPI-isoflavone particles greatly assisted in maintaining the size and polydispersity, especially during emulsion stability assessments. Particles formulated with higher concentrations (0.58mg/mL – 1.25mg/mL) of isoflavones had significantly smaller particle sizes. Though, isoflavone concentrations (0.94 mg/mL – 1.25mg/mL) showed a slight reduction of repulsive energy compared to the control. Moreover, the incorporation of isoflavones had also shown to significantly change the contact angle of sessile drops, moving towards a neutral wettability state. Destabilization of emulsions were assessed via creaming index. Results suggest that the use of isoflavones within emulsions decrease creaming within the first hour of being produced. Emulsion droplets formed by SPI-I particles sustained size and zeta potential over a 7-day evaluation. Microscopic observation indicated that emulsion droplets formed with isoflavones exhibited less flocculation and were more stable. In particular, emulsions formulated with 0.94 mg/mL – 1.25mg/mL isoflavones appeared the most stable as they exhibited well dispersed, small droplets. Collectively, these findings indicate that isoflavone addition improved emulsion stability compared to control particles without isoflavones. Overall, these results demonstrate potential for SPI-I particles as functional ingredient for food emulsion applications.
Effects of inulin, fructooligosaccharide, and breadfruit fiber on biofilm formation, growth, and gastrointestinal survival of probiotic yeast and bacteria
(University of Hawaii at Manoa, 2021) yuen, Beverly; Li, Yong; Food Science
A healthy gut microbiome is critical for digestion and immunity. Probiotics help balance the gut microflora and compete against pathogens for binding sites. Probiotics are live microorganisms that provide health benefits upon ingestion. Prebiotics are non-digestible oligosaccharides that selectively stimulate the survival and growth of probiotics in the colon. Many foods, such as breadfruit, contain fibers that have the potential to be prebiotics. The overall objective of this thesis was to increase the growth, biofilm formation, and gastrointestinal survival of probiotics with inulin, fructooligosaccharide (FOS), and breadfruit fiber. Water soluble breadfruit fiber was extracted from breadfruit and used as a prebiotic in this study. The acid tolerance at pH 2, tolerance to 0.3% bile salt, prebiotic activity score (PAS), and survival under simulated digestion were determined on Lactobacillus rhamnosus, Lactobacillus acidophilus, and Saccharomyces boulardii supplemented with the three prebiotics. The effects of prebiotics on biofilm formation of the probiotics and pathogenic Escherichia coli O157:H7 were also tested. S. boulardii showed a significantly higher survival under acidic conditions than L. rhamnosus and L. acidophilus, and the optimum concentration of breadfruit fiber for the survival of L. rhamnosus was 2.5% and 3.0%. The addition of breadfruit fiber showed a similar effect to glucose on the acid survival of L. acidophilus. Three tested probiotics showed high tolerance to bile salt. Moreover, 1.0% of inulin, FOS, and breadfruit fiber was the optimum concentration for biofilm development of S. boulardii. The addition of breadfruit fiber was as effective as glucose in helping L. rhamnosus form biofilm. With S. boulardii, inulin and FOS exhibited a higher PAS than breadfruit fiber. The PAS of breadfruit fiber was approximately 0.4 for S. boulardii and L. rhamnosus and 0.2 for L. acidophilus. After 24 h of simulated digestion, the addition of breadfruit fiber increased the survival of S. boulardii, L. rhamnosus and L. acidophilus by 0.37,5.45, and 5.59 log CFU/mL, respectively, compared to the negative control. These findings reveal the potential of S. boulardii to be marketed with prebiotics for individuals with gastrointestinal disorders. Moreover, breadfruit fiber is a possible prebiotic alternative to inulin and FOS in Hawai’i due to its sustainability and effectiveness with probiotics.
Relationship Between Antibiotic Resistance And Sanitizer Susceptibility Of Escherichia Coli Isolated From Agricultural Water Used In Hawaii
(University of Hawaii at Manoa, 2020) Nguyen, Yen Pham Thuy; Li, Yong; Food Science
Escherichia coli is notoriously known as a common cause of foodborne infections. Pathogenic E. coli serotypes cause approximately 73,480 illnesses, 2,168 hospitalizations, and 61 deaths annually in the United States. The food industry utilizes sanitizers to minimize the risk of foodborne illness outbreaks. Implementing sanitizers to wash fresh produce and meat has been found effective in reducing pathogens. However, there are concerns about bacteria acquiring resistance against sanitizers. Bacteria may utilize the same resistance mechanisms against antibiotics as they do against sanitizers. Thus, it raises the question of cross-resistance against both antimicrobials and the potential risks to food safety and public health. Antibiotics are used therapeutically to treat bacterial infections and to prevent infections during medical procedures. Unfortunately, overuse and misuse of antibiotics have led to the emergence of antibiotic-resistant bacteria. The concern of antibiotic-resistant bacteria is an ongoing global phenomenon. A key factor that has contributed to the rise of multidrug-resistant bacteria is the spread of resistant genes by mobile genetic elements. These genes are commonly located on plasmids, which are self-replicating DNA that can harbor various functional genes. Antibiotic-resistant bacteria are reservoirs of resistance genes that can be mobilized and transmitted between bacteria. This study aimed to explore the prevalence of antibiotic resistance in E. coli strains isolated from agricultural water used in local farms in Hawaii. Additionally, the susceptibility profiles of tested bacteria were determined against an array of sanitizers. The association between antibiotic resistance and sanitizer susceptibility of the E. coli strains was evaluated. Finally, efforts were made to investigate whether plasmids are involved in the resistance and cross-resistance of tested bacteria against these antimicrobials. Antibiotic and sanitizer susceptibility profiles of 182 E. coli strains were constructed using the Kirby-Bauber disk diffusion method. Inhibition zones of 15 common antibiotics were measured. The antimicrobial effects of three sanitizers, bleach, peroxyacetic acid, and lactic acid, were determined on tested bacteria. Correlation tests were used to evaluate association between the antibiotic resistance and sanitizer susceptibility. Six E. coli isolates that showed cross-resistance to the antibiotics and sanitizers were subjected to plasmid curing using sublethal concentrations of sodium dodecyl sulfate. Afterwards, derived colonies were randomly selected and tested against the antimicrobials agents they were once resistant to. Finally, polymerase chain reaction (PCR) was used to detect antibiotic resistance genes in parent and plasmid cured strains of the E. coli isolates. Of the 15 tested antibiotics, E. coli isolates showed a high prevalence of resistance to cefotaxime (61.5%), ampicillin (42.3%), and erythromycin (41.74%). Among all the isolates, 99 (54.3%) showed resistance against two or more antibiotics. One isolate showed resistance against 11 antibiotics. Most of the tested strains demonstrated higher resistance against peroxyacetic acid than bleach. There was a moderate correlation between tetracycline resistance and peroxyacetic acid resistance, with a correlation coefficient of 0.4. In comparison, the association between resistances against different antibiotics was much stronger. The highest correlation coefficient was 0.83 between sulfamethoxazole/trimethoprim (SXT) and kanamycin. After plasmid curing, the six cross-resistant E. coli isolates showed increased inhibition zones in the disk diffusion assay with some antibiotics and sanitizers. The kanamycin resistance gene aphA1 was detected in both the parent and plasmid-cured strains of isolate 32-2. However, the ampicillin resistance gene ampC was only detected in the parent strain of isolate N1, suggesting the gene is located on the plasmid and lost during plasmid curing. This study has laid the groundwork for understanding the relationship between antibiotic resistance and sanitizer resistance in foodborne pathogens, which pose a severe threat to food safety and public health. To our knowledge, this is the first report illustrating a correlation between tetracycline resistance and peroxyacetic acid resistance in E. coli isolated from water sources. The genetic and physiological characteristics of this kind of cross-resistant strain warrant further investigation.
Effect Of Anthocyanins In Okinawan Sweet Potato On Growth And Physicochemical Properties Of Salmonella Typhimurium And Listeria Monocytogenes
(University of Hawaii at Manoa, 2020) Flores Calle, Andrea; Li, Yong; Food Science
Okinawan Sweet Potato (OSP) is a popular tuber among the Hawaiian Islands and the Japanese archipelago, although it is not native to either of those islands. OSP purple flesh is rich in anthocyanins, which have been proven to have antioxidant activity and a modulatory effect on gut microbiota. Balanced gut microbiota is essential for human health, and its alterations have been linked to an increased likelihood of developing diseases such as schizophrenia, dementia or Alzheimer’s disease. There are several reports on the antioxidant activity of anthocyanins, but their effect on gut microbiome has not been studied in depth yet. This study aimed to determine how anthocyanins present in OSP affect the growth and physicochemical properties of harmful and beneficial bacteria. To do so, OSP powder was extracted with an acidified mix of ethanol and ammonium sulfate. Ethanol was removed using a rotary evaporator at 40ºC. The obtained extract was quantified as cyanidin-3-glucoside equivalents using the pH differential method combined with spectrophotometry at 520 nm and 700 nm. To determine the effect of anthocyanins in OSP on bacteria, inhibition tests were performed using the agar well diffusion assay with Salmonella Typhimurium, Listeria monocytogenes and Lactobacillus rhamnosus GG. After a 24-hour incubation at 37ºC, the plates were observed for the presence of inhibition zones. Minimum inhibitory concentration and minimum bactericidal concentration assays of the anthocyanin extract were also performed in order to determine if the treated cells were dead or only inhibited, by incubating the aforementioned bacteria with the anthocyanin extract and plating them and their negative controls to identify the hypothesized antimicrobial effect. Results showed anthocyanins in OSP could inhibit the growth of pathogenic Salmonella Typhimurium and Listeria monocytogenes. But it did not affect probiotic Lactobacillus rhamnosus GG. Based on the obtained MIC and MBC against Salmonella Typhimurium and Listeria monocytogenes, hydrophobicity, autoaggregation, sedimentation, swimming and swarming, and biofilm assays were performed on the pathogenic bacteria exposed to sublethal concentrations of the anthocyanin extract. With the exception of autoaggregation, the rest of tested bacterial physicochemical properties were significantly affected by anthocyanins in OSP. Our results suggest the obtained extract not only acts as a bactericidal agent on the pathogenic bacteria, but also at lower concentrations reduces their colonization activity. These findings could be applied to the food industry, where anthocyanins may serve as a natural food preservative. Anthocyanins in OSP may also be used to modulate and maintain a healthy gut microbiota.
Curcuminoid Analysis Of Hawaii-grown Turmeric (Curcuma longa) By High-performance Liquid Chromatography And Investigation Of Color Relationship With Curcuminoid Content For Total Curcuminoid Estimation
(University of Hawaii at Manoa, 2020) Calpito, Justin Kekoa Maikai; Bingham, Jon-Paul; Food Science
An HPLC method was designed to analyze turmeric (Curcuma longa) cultivars for curcuminoid contents curcumin, demethoxycurcumin and bis-demethoxycurcumin, and the sum of these were taken as total curcuminoids (TC). Of the Hawaii-grown cultivars, ‘Joy’ contained significantly more TC than all the others, with exception to ‘BKK’, a low yielding but extremely high TC cultivar. ‘Olena’ was the only cultivar to have less TC than what is commercially acceptable. Of the Asian cultivars analyzed, Indian cultivars 18-013, 18-014, and 18-010 had the highest TC content, comparable to ‘BKK’. Further propagation and in field trials will demonstrate their yield potential. The colors of the Hawaii-grown and Asian cultivars were measures using a standardized color chart to design a color grading chart. The yellow to orange relationship with TC content was used to produce approximate colors for different levels of curcuminoid content. These results and future experiments will help guide Hawaii agriculture towards producing high quality and globally competitive turmeric rhizomes.
Nanoengineered Surfaces And Carbon Nanotube Conjugated Microwire Biosensor For Microbial Control And Detection
(University of Hawaii at Manoa, 2019) Lee, Bog Eum; Jun, Soojin; Food Science
Nanotechnology is applied in various fields including the food industry. Nanotechnology integrates several disciplines and uses nanomaterials with size in the range from 1 to 100 nm. In the food industry, nanotechnology has potential to cover many aspects such as product development, food security, and new functional materials. Particularly, nanotechnology is a promising tool to address public food safety concerns by reducing the consumption of contaminated food products. Over the past years, the demand for real-time and sensitive detection of pathogenic bacteria in food has increased significantly. Current detection methods cannot facilitate the needs of food processors due to limitations such as time, cost, and mandatory laboratory settings. Therefore, a biosensor-based detection technology, which has advantages such as high sensitivity and portability, has emerged as an alternative. With the rapid advancement of nanotechnology, various nanomaterials have been integrated into biosensing platforms to address challenges such as sensitivity and rapid response time. In this study, a single-walled carbon nanotube (SWCNT)-based electrochemical impedance immunosensor for on-site detection of Listeria monocytogenes (L. monocytogenes) was developed. The L. monocytogenes immunosensor was functionalized by coating a gold plated tungsten wire with polyethylenimine, SWCNTs, streptavidin, biotinylated L. monocytogenes antibodies, and bovine serum albumin to induce specificity and selectivity. A linear relationship (R2 = 0.982) was observed between the electron transfer resistance measurements and concentrations of L. monocytogenes in the range of 103 - 108 CFU/mL. In addition, the sensor detected L. monocytogenes without significant interference in the presence of other bacterial cells such as Salmonella Typhimurium and Escherichia coli O157:H7. To address the needs of on-site monitoring, the sensor was integrated into a smartphone-controlled biosensor platform. The performance of the smartphone-controlled platform was evaluated with a conventional laboratory instrument. The sensing signals of the sensors immune-reacted with 103 - 105 CFU/mL of L. monocytogenes measured with both devices were not significantly different. The feasibility of the proposed platform for use in real food samples was examined with a lettuce homogenate. The recovery of the lettuce homogenates spiked with 103 - 105 CFU/mL of L. monocytogenes ranged from 90.21% to 93.69%, which proved to be suitable for food samples. Therefore, the developed on-site applicable SWCNT-based immunosensor platform appeared to be a promising tool to be used in field settings for food and agricultural applications. In order to additionally reduce the risk of microbial food contamination, nanotechnology has been extensively utilized to control biofilm formation. Bacterial adhesion on food-contact surfaces results in biofilm formation and imposes a significant challenge to food safety. Current biofilm control strategy is operating routine cleaning using chemical disinfectants. The main limitation of this method is its efficacy is altered by organic materials, pH, and temperature. It has been recognized that surface engineering could mitigate the level of bio-contamination by controlling the topography and physicochemistry of the substrate. As a result, superhydrophobic (SH) surface, which is known to be self-cleanable, has emerged as an alternative. SH surface has a water contact angle (WCA) greater than 150° and can be produced by introducing low surface energy nanoscale roughness on food-contact surface. Although there are many methods to produce SH surface, a combination of electrochemical etching and polytetrafluoroethylene (PTFE) coating has been suggested as an efficient technique due to the possibility of controlling surface morphologies and ease of operation. In this study, surface alterations on stainless steel were performed with electrochemical etching and PTFE film. The substrate was electrochemically etched at various conditions to induce nanoscale roughness and coated with PTFE to lower the surface energy. The nanostructures produced on the stainless steel substrates were characterized by field emission scanning electron microscopy. The stainless steel substrates etched at 10 V for 5 min and 10 V for 10 min with PTFE deposition resulted in an average WCA of 154° ± 4° with pore diameter of 50 nm. The bacterial resistance of these substrates (154° ± 4°) was evaluated by adhering 60 µL of L. monocytogenes (108 CFU/mL) on the substrates for 24 hours. As compared to the bare substrate, these SH surfaces significantly inhibited the bacterial adhesion up to 99%. The anti-biofilm characteristic of the superhydrophobic substrate (10 V 5 min with PTFE) was further evaluated with a CDC biofilm reactor and the bacteria entrapped in the biofilms were reduced by 98.4%. This nanoscale surface modification technique showed the feasibility for use as anti-microbial and anti-biofilm surfaces in the food industry.
Rapid and Sensitive Detection of Pathogenic Bacteria in Chicken Products by Single Tube Nested Real-Time PCR
(University of Hawaii at Manoa, 2019) Wu, Biyu; Li, Yong; Food Science
Salmonella spp. and Campylobacter jejuni are highly infectious and leading causes of human bacterial gastroenteritis throughout the whole world, as well as in Hawaii where the reported cases were approximately 300 and 750 each year, respectively. The diseases associated with these pathogens are a major cause of morbidity, which is a significant public health concern. In the United States, chicken is commonly contaminated by Salmonella spp. and Campylobacter jejuni. Traditional culture-based methods for their detection are time-consuming, cumbersome, and lacking in reliability. Thus, this study aimed to explore a molecular technique named single tube nested real-time polymerase chain reaction (STN-rtPCR) to overcome the drawbacks of culture-based methods and enable rapid detection of Salmonella spp. and Campylobacter jejuni in chicken products. Initially, a single tube nested PCR (STN-PCR) assay was developed for the detection of C. jejuni in the artificially contaminated ground chicken homogenate. Nested primers were designed based on the hippuricase (hipO) genes of C. jejuni. The annealing temperatures and concentrations of nested primers were optimized. The specificity of the established STN-PCR assay was evaluated with thirteen bacterial strains. The sensitivity of the assay was evaluated with a serial dilution of C. jejuni DNA and C. jejuni cells in the artificially contaminated ground chicken homogenate. In addition, the efficacy of the STN-rtPCR assay was compared with standard culture-based methods and conventional rtPCR for identification of C. jejuni in artificially contaminated ground chicken homogenate at different enrichment time. As a result, the optimum annealing temperatures for the outer and inner primers were 65oC and 55oC, respectively. The concentrations of outer and inner primers were chosen as 0.1 pmol and 40 pmol, respectively. No amplicon was generated using tested non-target bacterial strains as templates. The sensitivity was determined to be 10 C. jejuni DNA copies, which was 100 times more sensitive than conventional PCR with inner primers. Furthermore, this assay was able to detect as low as 36 CFU/ml of C. jejuni in artificially contaminated ground chicken homogenate without enrichment. Besides, after 24 h of enrichment, the ground chicken homogenate with an initial inoculum of 0.1 CFU/g of C. jejuni was identified correctly by STN-rtPCR, while it was not tested positive by both culture-based methods and conventional rtPCR until the sample had been enriched for 48 h. Moreover, single C. jejuni cells per gram ground chicken, that was tested positive by the culture-based methods after 48 h of enrichment, was identified correctly by STN-rtPCR after 6 h of enrichment. Moreover, a multiplex STN-rtPCR assay was developed for concurrent detection of Salmonella spp. and C. jejuni. Nested primers for the detection of Salmonella spp. were designed to target the invA gene. The annealing temperatures and concentrations of Salmonella primers were optimized based on the amplification conditions of the STN-rtPCR assay for C. jejuni as described above. The sensitivity and efficacy of established multiplex STN-rtPCR assay were evaluated with pure DNA of S. Typhimurium and C. jejuni. The performance of the developed assay was demonstrated with the artificially contaminated chicken rinse. The results showed the established multiplex STN-rtPCR assay yielded expected amplicons of 226 bp and 173 bp for Salmonella spp. and C. jejuni, respectively, while no amplification products were observed with non-target bacteria. The detection sensitivity was determined to be 1×10-3 ng/µl of Salmonella and C. jejuni DNA, and 102 CFU/ml of Salmonella and C. jejuni in the chicken rinse. Additionally, the assay exhibited a comparable efficiency for co-amplifying 107 to 102 CFU/ml of Salmonella and C. jejuni in chicken rinse. In summary, the developed single tube nested real-time PCR assays displayed a promising approach for simultaneously detecting Salmonella spp. and Campylobacter jejuni in chicken products with reduced time. It showed advantages of rapidity, high sensitivity and specificity, and low riks of cross contamination due to its closed-tube format. Moreover, this study would provide valuable information to food testing institutions and food manufacturers, which is necessary for preventing the spread of diseases and reducing economic losses.
Design And Evaluation Of A Continuous-flow Dielectrophoresis Device To Eliminate Pathogens From Tap Water
(University of Hawaii at Manoa, 2018-12) Chun, Cherisse Kanani; Jun, Soojin; Food Science
Since water is vital to all forms of life, achieving basic water security for all human beings remains a top priority. However, millions of people around the world, especially those living in least developed countries, do not consume water from a reliable potable water source. As a result, contaminated water has been the cause of a wide range of diseases such as hepatitis A, cholera, typhoid, polio, dysentery, and diarrhea. Water stress is brought on by a variety of social, environmental, and economic factors that threaten the health and well-being of a large number of individuals. The World Health Organization predicts that by the year 2025, half of the population will live in water-scarce areas that will need to implement progressive strategies for water re-use and management. Water intended for drinking contains undesirable pollutants such as organic and inorganic contaminants, as well as pathogens like bacteria, viruses, and protozoa. Although water sanitation has improved greatly over the past few decades, the technologies currently being used to enhance the safety of water have a number of disadvantages. For instance, chemical disinfection with the addition of chlorine can result in the production of carcinogenic disinfection by-products that are linked to heart disease, liver and kidney cancer, and sometimes death. In addition, pressure-driven filtration methods, such as reverse osmosis and nanofiltration, are energy inefficient and prone to fouling. These drawbacks have been heavily documented and can lead to steep increases in the general cost to run the system. As an alternative to filtration, a cell manipulation method known as dielectrophoresis (DEP) has garnered interest in the scientific community as a means to remove bioparticles from tap water. DEP is the net translational movement of a polarizable particle that occurs when it is subject to a non-uniform electric field. Positive-DEP occurs when the particle moves towards the region of high electric field, whereas negative-DEP describes the motion of a particle when it moves away from the high field region. There are two dominant forms of separation DEP: migration and retention. In migration DEP, particles are able to migrate to either weak or strong electric field areas; while in retention DEP, DEP forces are made to compete with fluid-flow forces to trap particles in place and prevent them from moving out of the channel. DEP has been shown to separate, trap, and sort a wide range of bioparticles, including bacteria, proteins, viruses, DNA, and blood cells. To test the hypothesis that DEP can influence bacterial cells so that they may be separated from tap water, this thesis describes two different types of DEP devices that were fabricated for bacterial removal from a continuous stream of water. The main objective of this study was to design a single-stage dielectrophoresis device that could separate Escherichia coli K12, a target bacterium and fecal indicator organism, from contaminated tap water. Next, the device fabricated using relatively inexpensive materials and simple procedures was tested for its efficacy using a combination of different experimental parameters such as varying voltages, flow rates, and dielectric bead sizes. The fabrication methods, materials and surfaces, and possible applications for both devices were discussed. The second chapter of this thesis discusses the first DEP device design that was microfluidic and utilized a standard pin-and-plane electrode configuration. Based on a previous design from Dr. Jun’s research lab, this new device was intended to address and resolve issues such as the large channel width and short electrodes. The channel width was decreased since high electric field is only produced in the narrow electrode gap directly near the edges of the electrodes. Consequently, the strength of the DEP force is greatly reduced as the distance from the electrodes increases in a wide channel. Also, by elongating the electrodes, the bacterial cells are subject to a longer period of DEP influence so that they have more time to move across the channel. The device’s Y-junction channel used migration-based DEP to draw the E. coli K12 cells towards one outlet, leaving the other outlet with a reduced microbial load. Polydimethylsiloxane (PDMS) was used to construct the device, and the pin and base electrodes were made out of titanium. A syringe pump created a continuous flow of E. coli solution through the device, and a frequency generator and voltage amplifier were used to produce the non-uniform electric field that was monitored on an oscilloscope. Different voltages and flow rates were applied to see which combination resulted in the highest separation efficiency. The greatest separation yield was 67.3% with the device set at 60V and 1 MHz, paired with a 0.1 mL/min flow rate. This yield was slightly greater than the 47% that was achieved with the former graduate student’s device, but was still under a 1-log reduction. To achieve a greater separation efficiency, a new design approach was taken. The third chapter of this thesis explains how the shortcomings of the first DEP device were resolved using an alternative to traditional DEP, called insulator-based DEP (iDEP). A millifluidic iDEP device was packed with dielectric glass beads that altered the electric field distribution so that there were a greater number of high electric field regions. Instead of migration DEP, retention DEP occurred by trapping the bacteria on the surface of the glass beads, as well as at the contact points, where the electric field was greatest. The device channel was printed on a stereolithography 3D printer and coated with PDMS. Stainless steel plates surrounded both sides of the rectangular channel to form the electrodes. The same experimental set-up used in the second chapter was utilized, and the applied voltages, flow rates, and glass bead sizes were varied. The highest percent reduction of E. coli K12 was 99.9% with a combination of 60 V, 200 µm bead size, and a 1.0 mL/min flow rate. Greater applied voltages, slower flow rates, and smaller bead sizes seemed to lead to a greater reduction of the bacteria. These studies showed that iDEP serves as a practical alternative to traditional DEP that has a limited throughput and is generally intended for microfluidic applications. Since this study acted as a basic proof-of-concept, further research should be conducted to determine the potential for iDEP to be scaled-up and serve as a method of filtration that is not prone to fouling and is more energy efficient. The potential uses of DEP span far beyond the current biomedical applications that are already studied at length. DEP may also have a variety of food processing functions that should be evaluated, including the removal of yeast from beer and contaminants from milk or juice without the application of heat.
Advanced Ohmic Heating for Rice Cooking: Quality Factor Assessment
(University of Hawaii at Manoa, 2018-08) Liu, Junhuang; Food Science
Conventional thermal processing of foods containing particulates significantly relies on several heat transfer steps, including conduction and convection, which usually take longer cooking times for the solid-liquid mixture foods and tend to be overly conservative ensuring microbial safety, thus compromising quality. Rice is one of the world’s biggest cereal crops next to wheat and maize and is one of the most important staple foods for the world population. Asian people consume cooked rice at almost every meal. Existing methods of cooking are about 10-15% thermally efficient. The ever-increasing population will need more amount of energy and water to be spent on rice cooking. Advanced food processing technologies such as ohmic heating and microwave heating have been developed in the last few decades as alternatives to conventional processing methods. The advanced technologies could contribute to shortening processing times, energy savings, and high-quality safe food. The microwave heating has been employed to cook rice and can reduce the cooking time by more than 40% compared to the conventional cooking method. However, the energy consumption was nearly doubled. Therefore, a new concept to use ohmic heating for rice cooking has been extensively evaluated. This technology was an attractive alternative method with high energy transfer efficiency, time savings, and high quality of purpose. In this study, an alternative cooking method that offers both high energy efficiency and short cooking time was developed, and a static ohmic heater was designed and fabricated to heat treat rice-water mixtures. The energy consumption, textural characteristics of rice, and simulation of the electrical field in ohmic heater were investigated. Two types of rice were used in this study: white rice and brown rice respectively. The electrical conductivities of rice-water mixtures at various volume ratios were measured during the rice cooking process. The endpoint of rice cooking by using ohmic heating was identified. The results showed that the rice cooked by the ohmic heating method has significantly different textural properties from rice cooked by an electric rice cooker. The magnitude of texture difference was dependent on the type of rice. The electrical conductivities of white rice, and the brown rice mixture were approximately 0.03-0.08, 0.04-0.1, 0.06-0.12 S/m at volume ratios of 1:0.8, 1:1.2, 1: 1.5 and 0.025-0.16, 0.032-0.2 S/m at volume ratios of 1:1.5 and of 1:2 respectively. The research also found that ohmic heating required a cooking time of around 17-18 min. The estimated amount of energy consumed by the ohmic heating process was about 1/4 of the total energy consumed by electric rice cooker. The developed ohmic heating technique showed a great potential over the conventional electric cooker regarding the high energy efficiency, shorter cooking time, and lower water usage.

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