IMPACT OF PROCESSING ON THE CONTENT AND COMPOSITION OF EXTRACELLULAR VESICLES IN BOVINE MILK

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1024313
• Grant No.: 2020-67018-33315
• Cumulative Award Amt.: $199,229.00
• Proposal No.: 2020-03933
• Project Start Date: Oct 1, 2020
• Project End Date: Sep 30, 2023
• Grant Year: 2021
• Program Code: [A1364]- Novel Foods and Innovative Manufacturing Technologies

Recipient Organization
CHAPMAN UNIVERSITY
ONE UNIVERSITY DRIVE
ORANGE,CA 92866

Performing Department
Food Science & Nutrition

Non Technical Summary
Most newborns in the US consume their mother's milk for nutrition and this practice is widely recommended by healthcare professionals. However, more than 30% of newborns already start to consume infant formula made from cow's milk within two days of birth. This is important to understand because newborns that consume their mother's milk have better health outcomes than newborns to consume infant formula. There are 2 possible reasons why newborns that consume mother's milk are healthier than those that consume formula: either (1) human breastmilk has better nutritional quality than cow's milk, or (2) the food processing steps used to treat cow's milk that is used to make infant formulas reduces its nutritional quality. In the absence or reduction of breastfeeding from mothers, we want to be able to improve the nutritional quality of infant formulas made from cow's milk. To do this we will (1) compare the composition of human milk and cow's milk, and (2) understand how food processing affects the nutritional quality of cow's milk. By understanding how processing affects the nutritional quality of cow's milk, we will be able to provide new information that manufacturers can use to improve infant formula products. Once these products are improved, newborns consuming formulas made from cow's milk will start to have improved health outcomes. The results from this study will provide information on optimizing how we process cow's milk to ensure it is safe, has high nutritional quality, and improves health outcomes of newborns that consume infant formulas made from cow's milk.

Animal Health Component

10%

Research Effort Categories

Basic

80%

Applied

10%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
502	3450	1010	100%

Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
3450 - Milk;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

baby formula

bovine milk

extracellular vesicle

food

human milk

infant formula

milk

processing

Goals / Objectives
GOAL: To determine howbovine milk extracellular vesicles are affected by food processing techniques.OBJECTIVES:To determine the effect of food processing on bovine milk EV content (number, size, population distribution).To determine the effect of food processing on bovine milk EV composition (protein, miRNA, lipid).To compare the content and composition between bovine and human milk EVs.

Project Methods
Ethics approval for this research study will be obtained from Chapman University Research Ethics Board.Procurement and processing of milk. Before pasteurization or freezing, expressed mature (>6 months lactation) human milk will be obtained and pooled from 10 donors at Prolacta Bioscience (City of Industry, CA; see Letter of Collaboration from Dr. Adam Sun, Vice President of Research and Development). Raw bovine milk will be obtained from Danone/WhiteWave (City of Industry, CA). At Chapman University, the milk will be subject to the following treatments, individually and in combination, to determine the effect on EV properties and their lipid profile. Processing parameters will be the same as those used commercially. Human milk will serve as the reference and comparator.Homogenization: milk will be homogenized using an APV Gaulin 15MR-8TA homogenizerPasteurization: will be performed under two conditions in a Microthermics UHT/HTST pasteurizer. 1) High Temperature Short Time (HTST) at 71.5 for 15 seconds and 2) UHT processing at 135 °C for 1-2 seconds.Sterilization: Milk will be packaged in cans and sterilized in an All American Pressure Sterilizer at 121 °C for 20 minutes.Spray drying: Following concentration, milk will be spray dried in a Buchi B-290 mini spray dryer.For processing treatments that affect changes in EV content or composition, the processing parameters (time and intensity) will be varied to create dose response curves.Isolation of EVs from milk. After processing treatments, EVs will be isolated from milk using ExoQuick (SBI, Palo Alto, CA) reagent according to manufacturer instructions (See Letter of Collaboration from Lynn Lehman, Associate Director at System Biosciences).EV verification. Three procedures must be performed to verify successful isolation of EVs, according to guidance from the International Society of Extracellular Vesicles (Théry et al, 2018). I] Protein concentration of EVs will be assessed by Qubit fluorometer and known EV protein markers (CD9, CD63, CD81) will be assayed by immunolot and visualized (BioRad ChemiDoc XRS+) colorimetrically by reacting substrate solution with HRP-conjugated secondary antibody. II] Markers for tight junction proteins (occludin, zonula occludens-1) present in the tissue of origin (lactocytes) and absent from EVs will also be assayed by immunoblot. Isotype-matched negative control antibodies will be used to determine sensitivity and specificity of the assays. The presence of CD protein markers and absence of tight junction proteins will confirm successful isolation of EVs and absence of contaminating cells or other nanostructures from which the EVs originated. III] Scanning electron microscopy (Zeiss Gemini Sigma 300) will be used to visualize EVs and Nanosight NS300 NTA will be used to obtain data on the number and size distribution of EVs from samples from which different populations of EVs can be distinguished: exosomes, apoptotic bodies, microvesicles. EV samples will be stored at -20 ºC prior to compositional analysis.Analysis of miRNA in milk EVs. After isolation from bovine and human milk, EVs will be incubated with QIAzol Lysis Reagent (Qiagen) and total miRNA will be extracted using an miRNeasy Mini Kit (Qiagen). Qubit 4 Flourometer will be used to measure miRNA quality and concentration from processed, control, and reference milk samples.Lipid analysis of milk EVs. Milk EVs will be analyzed qualitatively and quantitatively using a lipidomic profiling platform on a gas chromatography/mass spectrometry-mass spectrometry (GC/MS-MS) system from University of California San Diego Lipidomics Core. The relative proportions of fatty acids (saturated, unsaturated, polyunsaturated) and lipid classes (phospholipid, cholesterol, sphingolipid) will be compared among processed, control, and reference milk samples. The PI has used this service and published associated findings in the proceedings of the American Society of Nutrition (2020; see Letter of Collaboration from Caroline Steele RD, Children's Hospital of Orange County).StatisticsForty-two (42) measures will be performed for each experimental outcome measure: (2 types of milk) x (4 processing techniques + 1 positive control + 1 negative control + 1 blank) x (3 technical replicates). Descriptive statistics will be calculated from data obtained. Non-parametric mean comparison test (Tukey) will be used to compare EV content (size, count, population distribution) and composition (total protein, miRNA, lipid concentration) between processing treatment and control groups and between bovine and human milk sources. A statistically significant difference is designated as alpha <0.05 in two-tailed testing and a Bonferroni correction will be applied for multiple means comparison testing.

Progress 10/01/20 to 09/30/23

Outputs
Target Audience:Target audiences include dairy product manufacturers, food safety & regulatory agencies, and healthcare professionals. Efforts include news & media outlets, laboratory and classroom instruction, thesis defense, manuscript publication, scientific abstract writing and presentation, presentation to Southern California IFT, and outreach to populations underrepresented in food and nutrition sciences according to national survey data. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? IMPACT Although most newborns in the US receive human milk as nutrition in accordance with best practices and recommendations,more than one-third are introduced to a bovine milk based formula within the first two days of birth. Newborns then are reliant on bovine milk nutrition since continued feeding of formula persists until weaning. Extracellular vesicles (EVs) are newly identified bioactive compounds present in milk that impact growth and development processes such as regulation of immune function. Processing steps in commerical infant formula production such a pasteurization and homeogenization can affect the content, composition, and functionality of EVs in milk. It is the goal of this research to understand how processing techniques for bovine milk affect the content and composition of EVs. This research aims to 1) provide an understanding of how processing affects the nutritional value of newly identified bioactive compounds in milk, and 2) provide new information that manufacturers of formulas can use to narrow the compositional gap between human and bovine milks in order to achieve optimal health of newborns. The results from this study will provide baseline data that can be used to optimize processing parameters to improve nutrition while maintaining food safety; and inform the larger project goal of evaluating how EVs from processed bovine milk impact intestinal cell physiology and the gut microbiome in an in vivo model. Ultimately, the significance of this research resides in decreasing the disparity in health outcomes between newborns fed infant formula and those fed human milk. For corresponding objectives, this is the progress: 1. Completed in previous reporting period. 2. We have determinedthe effects of processing (pasteurization, homogenization) on lipid (oxylipin) content in extracellular vesicles from bovine milk. Data analysis is ongoing. In the previous reporting period, we completed the determination of milk processing on the content of protein and miRNA in EVs from bovine milk. 3. We have determined the effects of processing (Holder pasteurization) on lipid (oxylipin) content in extracellular vesicles from human milk for the purpose of comparing to bovine milk. Data analysis is ongoing. The main outcoe/impact/significance of progress to date is a change in knowledge. We have come to understand that conventional processing techniques used for bovine milk food safety may decrease the content of bioactive extracellular vesicles. The effect is specific to subtypes of vesicles with lower membrane protein contet.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Extracellular vesicles from bovine milk mitigate the LPS-induced reduction in gut barrier integrity in C2BBe1 cells. Skolnick J, Miklavcic JJ. 2023. ISEV Annual Meeting. Seattle WA.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Homogenization and thermal processing reduce the concentration of extracellular vesicles in bovine milk. Colella A, Prakash A, Miklavcic JJ. Food Science & Nutrition. 2023; 00: 1-10. DOI: 10.1002.fsn3.3749.

Progress 10/01/21 to 09/30/22

Outputs
Target Audience:Target audiences include dairy product manufacturers, food safety & regulatory agencies,and healthcare professionals. Efforts include laboratory instruction, experiential learning opportunities, thesis defense, scientific abstract writing and presentation, and outreach to populations underrepresented in food and nutrition sciences according to national survey data. Changes/Problems:Developing a lipidomics platform for the analysis of oxylipins has been challenging. Therefore, the aid of an expert collaborator in lipidomics profilingat UC Davis has been engaged to help conduct this aspect of the research study. As a result, we will not have to change any of the proposed objectives. What opportunities for training and professional development has the project provided?Training activities. One-on-one mentorship of twograduate students with mentor (PD). Onegraduate studentslearned how to use food processing equipment: microthermics, pasteurizer, milk composition analyzer, milk particle size analyzer. Professional development. One graduate thesis student has visited a milk processing facility. Student has also attended and presented research findings an annual Institute of Food Technologists meeting. Two students presented research at Research Day hosted by the Children's Hospital of Orange County. How have the results been disseminated to communities of interest?Two research abstracts havebeen presented at a Research Dayhosted by the Children's Hospital of Orange County. The research has allowed for two new Material Transfer Agreements to be set up between industry members and the PD. For these new partnerships, the PD has been provided with an enriched lipid milk product and human milk samples for compositional analysis and human clinical trials. What do you plan to do during the next reporting period to accomplish the goals?Analyze the oxylipin composition of extracellular vesicles from human and bovine milk after industrial processing has been applied (homogenization, pasteurization). Isolate extracellular vesicles from processed milk. Characterize oxylipins in theextracellular vesicles isolated frommilk. Analyze data, synthesizeresults. ?Report findings in two scientific publications. Share knowledge with stakeholders.

Impacts
What was accomplished under these goals? IMPACT Although most newborns in the US receive human milk as nutrition in accordance with best practices and recommendations, more than one-third are introduced to a bovine milk based formula within the first two days of birth. Newborns then are reliant on bovine milk nutrition since continued feeding of formula persists until weaning. Extracellular vesicles (EVs) are newly identified bioactive compounds present in milk that impact growth and development processes such as regulation of immune function. Processing steps in commerical infant formula production such a pasteurization and homeogenization can affect the content, composition, and functionality of EVs in milk. It is the goal of this research to understand how processing techniques for bovine milk affect the content and composition of EVs. This research aims to 1) provide an understanding of how processing affects the nutritional value of newly identified bioactive compounds in milk, and 2) provide new information that manufacturers of formulas can use to narrow the compositional gap between human and bovine milks in order to achieve optimal health of newborns. The results from this study will provide baseline data that can be used to optimize processing parameters to improve nutrition while maintaining food safety; and inform the larger project goal of evaluating how EVs from processed bovine milk impact intestinal cell physiology and the gut microbiome in an in vivo model. Ultimately, the significance of this research resides in decreasing the disparity in health outcomes between newborns fed infant formula and those fed human milk. For corresponding objectives, this is the progress: 1. Completed in previous reporting period. 2. We are actively determining the effects of processing (pasteurization, homogenization) on lipid (oxylipin) content in extracellular vesicles from bovine milk. We have developed a method for qualitiative and quantitative oxylipin analysis. We have liaised with an expert in oxylipin metabolism at UC Davis to aid in analysis and interpretation.In the previous reporting period, we completed the determination of milk processing on the content of protein and miRNA in EVs from bovine milk. 3. We are actively determining the effects of processing (Holder pasteurization) on lipid (oxylipin) content in extracellular vesicles from humanmilk for the purpose of comparing to bovine milk.We have developed a method for qualitiative and quantitative oxylipin analysis. We have liaised with an expert in oxylipin metabolism at UC Davis to aid in analysis and interpretation. The main outomce/impact/significance of progress to date is a change in knowledge. We have come to understand that conventional processing techniques used for bovine milk food safety may decrease the content of bioactive extracellular vesicles.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: "Oxylipin Content of Extracellular Vesicles in Human Milk." Estrada I, Prakash A, Miklavcic J. CHOC Research Day.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: "Extracellular vesicles from bovine milk mitigate the LPS-induced reduction in gut barrier integrity in C2BBe1 cells." Skolnick J, Prakash A, Miklavcic J. CHOC Research Day.
• Type: Theses/Dissertations Status: Published Year Published: 2021 Citation: Colella, A.P. (2021). The impact of processing on the content and composition of bovine milk extracellular vesicles. Master's thesis, Chapman University. https://doi.org/10.36837/chapman.000309
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Isolation and verification of extracellular vesicles from raw bovine milk. Colella A, Prakash A, Miklavcic JJ. Journal of Dairy Science.

Progress 10/01/20 to 09/30/21

Outputs
Target Audience:Target audiences include dairy product manufacturers and healthcare professionals. Efforts include laboratory instruction, and experiential learning opportunities, thesis defense, scientific abstract presentation. Changes/Problems:Objectives of the research stated that we are investigating how common industry food processing techniques impact the content and composition of extracellular vesicles in bovine milk. Although extracellular vesicles are simple of isolate from raw bovine milk, industrial processing alters fat particle size and results in compositional changes to the milk that appear as debris and interfere with the isolation of extracellular vesicles. We will require procuring larger volumes of bovine milk to complete the project, and are confident to complete the project within the originally specified timeline. What opportunities for training and professional development has the project provided?Training activities. One-on-one mentorship of 3 graduate students with mentor (PD). Two graduate thesis students have learned how to use food processing equipment: microthermics, pasteurizer, milk composition analyzer, milk particle size analyzer. Professional development. One graduate thesis student has visited a milk processing facility. Student has also attended and presented research findings an annual Institute of Food Technologists meeting. How have the results been disseminated to communities of interest?A graduate thesis has been published in the Chapman University Leatherby libraries and a research abstract has been presented at the annual Institute of Food Technologists meeting. What do you plan to do during the next reporting period to accomplish the goals?Analyze the content and composition of EVs from bovine milk after industrial processing has been applied (homogenization, heat). 1. obtain raw bovine milk 2. process milk in pilot scale plant. 3. isolate EVs from processed milk. 4. characterize EVs in milk. 5. analyze data, synthesis results. 6. report findings in scientific publication.

Impacts
What was accomplished under these goals? Although most newborns in the US receive human milk as nutrition in accordance with best practices and recommendations, more than one-third are introduced to a bovine milk based formula within the first two days of birth. Newborns then are reliant on bovine milk nutrition since continued feeding of formula persists until weaning. Extracellular vesicles (EVs) are newly identified bioactive compounds present in milk that impact growth and development processes such as regulation of immune function. Processing steps in commerical infant formula production such a pasteurization and homeogenization can affect the content, composition, and functionality of EVs in milk. It is the goal of this research to understand how processing techniques for bovine milk affect the content and composition of EVs. This research aims to 1) provide an understanding of how processing affects the nutritional value of newly identified bioactive compounds in milk, and 2) provide new information that manufacturers of formulas can use to narrow the compositional gap between human and bovine milks in order to achieve optimal health of newborns. The results from this study will provide baseline data that can be used to optimize processing parameters to improve nutrition while maintaining food safety; and inform the larger project goal of evaluating how EVs from processed bovine milk impact intestinal cell physiology and the gut microbiome in an in vivo model. Ultimately, the significance of this research resides in decreasing the disparity in health outcomes between newborns fed infant formula and those fed human milk. OBJECTIVES: To determine the effect of food processing on bovine milk EV content (number, size, population distribution). To determine the effect of food processing on bovine milk EV composition (protein, miRNA, lipid). To compare the content and composition between bovine and human milk EVs. For corresponding objectives, this is the progress: 1. Determined the contentof EVs in raw bovine milk. Collected data, and obtained results on EV number and size. 2. Determined the composition of EVs in raw bovine milk.Collected data, and obtained results on EV protein and miRNA content. 3. Determined the content of EV in human milk.Collected data, and obtained results on EV number,size, protein and miRNA content. The main outomce/impact/significance of progress to date is a change in knowledge: the validation of a methodologic technique to isolate EVs from raw bovine milk.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: "The content and composition of extracellular vesicles in raw bovine milk. Colella AP, Prakash A, Miklavcic JJ. 2021. IFT21, Chicago IL.
• Type: Theses/Dissertations Status: Accepted Year Published: 2021 Citation: Colella, A.P. (2021). The impact of processing on the content and composition of bovine milk extracellular vesicles. Master's thesis, Chapman University. https://doi.org/10.36837/chapman.000309
• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Isolation and verification of extracellular vesicles from raw bovine milk. Colella A, Prakash A, Miklavcic JJ. Journal of Food Science.