THESIS 

 

 

CHARACTERIZATION OF PROTEIN-POLYPHENOL INTERACTIONS BETWEEN 

NOVEL PLANT PROTEINS (PEA AND HEMP) AND BLUEBERRY POLYPHENOLS WITH 

RESPECT TO POLYPHENOL BINDING AND DELIVERY 

 

 

Submitted by 

Bianca Chima 

Department of Food Science and Human Nutrition 

 

 

In partial fulfillment for the requirements 

For the Degree of Master of Science 

Colorado State University 

Fort Collins, Colorado 

Spring 2022 

 

Master’s Committee: 

Advisor: Charlene Van Buiten 

Sarah Johnson 

Jessica Prenni 

 



  

 

 

 

 

 

 

 

 

 

 

 

Copyright by Bianca Chima 2022 

All Rights Reserved



 ii 

 

ABSTRACT 

 

 

 

CHARACTERIZATION OF PROTEIN-POLYPHENOL INTERACTIONS BETWEEN 

NOVEL PLANT PROTEINS (PEA AND HEMP) AND BLUEBERRY POLYPHENOLS WITH 

RESPECT TO POLYPHENOL BINDING AND DELIVERY 

 

 

 

Despite the numerous health benefits associated with polyphenols, dietary intake of this 

class of compounds is low in the United States due to low intake of fruits and vegetables. It has 

been shown that dairy foods (i.e. milk, yogurt) increase polyphenol bioavailability due to 

polyphenols interacting with whey protein, enhancing polyphenol stability and uptake 

throughout digestion. However, increasing concerns for sustainability and health have introduced 

a variety of novel plant-based proteins as dairy alternatives. This study aimed to investigate the 

abilities of edible pea and hemp protein isolates to form complexes with blueberry polyphenol 

extract (BPE) and characterize the physical and biological functionalities of these complexes 

compared to whey proteins. Protein/polyphenol solutions were analyzed using UV-Vis 

spectroscopy to determine if complexation occurred. Secondary structures and binding affinities 

were analyzed by far-UV CD Spectroscopy and fluorimetry, respectively. In vitro digestion was 

performed to determine whether the protein profile changed in the presence of BPE via SDS-

PAGE and determination of free amino acids using the ninhydrin method. Protein isolates from 

pea and hemp successfully formed complexes with BPE with binding affinities for the compound 

similar to whey protein. Relative helicity of the hemp protein was higher than the other protein 

sources and increased upon complexation with BPE. Furthermore, the SDS-PAGE profiles of all 

the proteins were the same whether BPE was present or not and the free amino acid content 



 iii 

 

increased after digestion for the protein and protein/polyphenol solutions. Overall, complexation 

of BPE with plant proteins was successful. Fluorescence quenching and changes to the secondary 

structure of the proteins in the presence of BPE indicate that polyphenols were bound but the 

mechanisms and structures responsible for complexation seem to vary between proteins. More 

research is needed to determine the interactions that cause binding between the polyphenols and 

the proteins and whether the bioavailability of the compounds will increase when bound to the 

proteins in cell model and/or clinical study. This study provides a foundation for exploring the 

effects of plant-based proteins on phytochemical functionality in complex, “whole food” 

matrices. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 iv 

 

ACKNOWLEDGEMENTS 

 

 

 

Many people have helped me throughout this process which I will be forever grateful for. 

Firstly, I would like to thank my advisor Dr. Charlene Van Buiten for all of her insight and 

support and for giving me the opportunity to work on a project that so closely aligns with my 

future goals. I would also like to thank my committee members Dr. Sarah Johnson and Dr. 

Jessica Prenni for their assistance with materials, instrumentation and encouragement. 

 

Special thanks to Scott Morgan, Emily Nock and Paul Matthews for their work on the 

turbidity, purification and circular dichroism experiments which I appreciate greatly considering 

it saved me many extra hours of research. 

 

Thank you to my family, specifically my mother Augustina Chima, who has worked 

extremely hard to allow me to have the opportunities I currently have and has supported me 

throughout everything reminding me that I am capable of achieving any goals that I set for 

myself. Truly, I do not believe I would be where I am without her. 

 

Lastly, I would like to thank all the friends that I have made inside and outside of the 

department that have made this journey much easier and were always willing to support me 

through trials and tribulations of graduate school.  

 

 

 



 v 

 

TABLE OF CONTENTS 

 

 

 

ABSTRACT ...................................................................................................................................ii 

ACKNOWLEDGEMENTS ...........................................................................................................iv 

LIST OF TABLES ........................................................................................................................vii 

LIST OF FIGURES .....................................................................................................................viii 

CHAPTER I – LITERATURE REVIEW .......................................................................................1 

1.1 Introduction ...................................................................................................................1 

1.2 Health Benefits of Consuming Blueberry Polyphenols ................................................1 

1.3 Scope of Polyphenol Consumption in the US ...............................................................4 

1.4 Bioavailability of Polyphenols ......................................................................................5 

1.5 Strategies for Improving the Bioavailability of Polyphenols .......................................6 

1.6 Whey Protein as a Strategy for Delivering Polyphenols ..............................................7 

1.7 Popularity and History of Consuming Fruit and Dairy .................................................8 

1.8 History, Popularity and Consumer Reasons for Consuming Plant-Based Dairy 

Alternatives .........................................................................................................................9 

CHAPTER II – STATEMENT OF PURPOSE ............................................................................12 

CHAPTER III – MANUSCRIPT .................................................................................................14 

3.1 Introduction .................................................................................................................14 

3.2 Materials & Methods ..................................................................................................16 

 3.2.1 Polyphenol Extraction ..................................................................................16 

  3.2.1.1 Crude Blueberry Polyphenol Extraction .......................................16 

   3.2.1.2 C18 Solid Phase Extraction ..........................................................17 

   3.2.1.3 Determination of Total Phenolic Content .....................................17 

 3.2.2 Protein Purification ......................................................................................18 

  3.2.2.1 Fat Extraction using the Soxhlet Method ......................................18    

   3.2.2.2 Alkaline Extraction-Isoelectric Point Precipitation ......................18                        

   3.2.2.3 Determination of Protein Content .................................................19 

 3.2.3 Sample Preparation ......................................................................................19 

 3.2.4 Turbidity Measurements ..............................................................................19 

 3.2.5 Circular Dichroism (CD) .............................................................................20 

 3.2.6 Fluorescence Spectroscopy ..........................................................................20 

3.2.7 Analysis of the Digestibility of Pea, Hemp and Whey Protein with 

Blueberry Polyphenol Extract ...............................................................................21 

3.2.7.1 In Vitro Digestion .........................................................................21 

 3.2.7.2 Determination of Free Amino Acids .............................................21 

3.2.7.3 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis 

(SDS-PAGE) .............................................................................................22 

  3.2.8 Statistical Analysis .......................................................................................22 

 3.3 Results .........................................................................................................................23 

3.3.1 Physical and Chemical Characterization of the Protein-Polyphenol 

Complexes .............................................................................................................23 

3.3.2 Polyphenol Binding Affects Hemp Protein Structure ..................................25 

3.3.3 Binding Affinities Do Not Differ Between Protein Sources .......................26 



 vi 

 

3.3.4 Assessment of the Digestibility of the Protein-Polyphenol Complexes ......28  

 3.4 Discussion ...................................................................................................................31 

CHAPTER IV – CONCLUSIONS AND FUTURE DIRECTIONS ............................................38 

REFERENCES .............................................................................................................................40 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 vii 

 

LIST OF TABLES 

 

 

 

Table 1. Stern-Volmer constant (Ksv), binding affinity (Ka) and Hill coefficient (n) for 

interactions between each protein and BPE, calculated based on Figure 4 graphs ......................28 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 viii 

 

LIST OF FIGURES 

 

 

 

Figure 1. (A) Turbidity of pea, whey and hemp protein in the presence and absence of BPE 

expressed as average absorbance at 600 nm. (B) Difference in turbidity between the whey, pea 

and hemp samples with or without BPE added ............................................................................24 

 

Figure 2. Turbidity of pea, whey and hemp protein in the presence and absence of BPE at pH 

values related to digestion and food production expressed as average absorbance at 600 nm .....24 

 

Figure 3. (A) Smoothed CD spectra for hemp, pea, and whey protein in the presence and absence 

of BPE and the comparisons of the protein secondary structures to the other proteins in the 

CAPITO database (B) Differences in relative helicity between the protein types and the proteins 

in the presence of BPE ..................................................................................................................25 

 

Figure 4. Quenching effect of BPE on protein fluorescence intensity in whey-BPE (A), pea-BPE 

(B) and hemp-BPE (C) complexes as a function of BPE concentration .......................................26 

 

Figure 5. (A) Stern-Volmer plot for the fluorescence quenching of each protein by BPE. (B) 

Modified form of a Stern-Volmer plot to calculate Ka (C) Calculation of Hill coefficient reveals 

the number of binding sites for each protein ................................................................................27  

 

Figure 6. SDS-PAGE of pea, hemp (A) and whey (B) proteins in the presence and absence of 

BPE after in-vitro digestion ..........................................................................................................29 

 

Figure 7. Free amino acid content expressed as mM leucine equivalents of protein and protein-

polyphenol complexes before and after in vitro digestion ............................................................31 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 1 

 

CHAPTER I 

LITERATURE REVIEW 

 

 

 

1.1 Introduction 

Polyphenols are a class of biologically active compounds found in plant foods such fruits, 

vegetables, coffee and chocolate. The main constituents of polyphenols are phenolic rings which 

are attached in various ways to create the different classes. Some of the major classes of 

polyphenols are phenolic acids, stilbenes and flavonoids (M. Abbas et al., 2016). Polyphenols are 

not essential to human life like vitamins and minerals but are believed to contribute many 

benefits to health. The quantity and type of polyphenols found in these foods contribute greatly 

to their beneficial effects. Berries such as blueberries have been found to have high numbers of 

polyphenols and are popular among many consumers making them valuable to study for their 

benefits (Kang et al., 2015). One major complication with polyphenols is that they are not highly 

bioavailable which limits the ability of the compounds to improve health. The aim of this review 

is to discuss the health benefits of blueberry polyphenols and their bioavailability. Also, it 

addresses the current solutions being studied to improve polyphenol bioavailability and why food 

proteins specifically plant proteins, may be adequate to solve the issue naturally based on how 

they are customarily consumed as a part of a food matrix. 

 

1.2 Health Benefits of Consuming Blueberry Polyphenols  

Polyphenols, which are a class of secondary plant metabolites, have gained interest in recent 

years for providing additional health benefits from consuming fruits and vegetables. Some of the 

most notable benefits have been the ability of these compounds to acts as antioxidants, reduce 



 2 

 

the risk for metabolic syndrome, obesity, cardiovascular disease and inflammation. In general, 

polyphenols consumed as a part of a Mediterranean diet were found to reduce the risk of 

mortality in Spanish individuals by 37% as a part of a parallel-group, randomized, controlled 

feeding trial that occurred over the course of about five years (Tresserra-Rimbau et al., 2014). 

Moreover, various types of foods and drinks such as tea and berries have been shown to provide 

these effects. Blueberries have been studied for their potential health effects because of their 

popularity with consumers and high nutritional value (Kang et al., 2015). 

 Obesity is currently a major health concern in America. In 2015, approximately 75% of 

Americans were overweight or obese, 41% being obese and these values are projected to grow 

(Wang et al., 2020). Research continues to expand in this area to find solutions to this epidemic. 

Blueberry polyphenols have been found to potentially reduce the risk for obesity and negative 

outcomes associated with the disease. One study conducted on rats that consumed a high fat diet 

in addition to a blueberry polyphenol extract for four weeks found that weight gain was reduced 

by 6.7% in comparison to the high fat diet only group. Additionally, LDL cholesterol levels were 

significantly decreased by the addition of polyphenols to the diet while HDL cholesterol 

increased. Furthermore, factors of lipid metabolism such as PPAR, FAS and SREBP-1 were 

downregulated as a result of consumption of the polyphenols (Jiao et al., 2019). Similarly, a 

study conducted in rats, found that blueberry proanthocyanidins reduced visceral obesity and 

weight gain (Morissette et al., 2020). 

Researchers have investigated the effects of cranberries which have similar polyphenolic 

constituents to blueberries, on conditions related to obesity in vivo. Cardiovascular disease 

(CVD) and type II diabetes mellitus are diseases typically related to obesity that are also being 

targeted for treatment. The study showed that consumption of cranberry polyphenols in humans 



 3 

 

was able to increase insulin sensitivity which would reduce the risk for type II diabetes. Also, 

HDL cholesterol increased, and the presence of C-reactive protein decreased which can dimmish 

the potential for developing cardiovascular disease (Chew et al., 2019). Vascular function was 

also found to improve in vivo after the consumption of a blueberry drink in healthy men. Flow 

mediation dilation increased after intake, which could lead to decreased blood pressure and 

reduced risk for CVD (Rodriguez-Mateos et al., 2013). Nonalcoholic fatty liver disease is 

another condition typically associated with metabolic diseases such as obesity. Blueberry 

polyphenols were found to reduce the production of triglycerides which could prevent or prolong 

the first stage of the disease (Liu et al., 2011). 

 Polyphenolic compounds have also been cited to reduce inflammation in a number of 

diseases including cancer and act as antioxidants to prevent some inflammatory responses from 

occurring in the first place. Several studies have discussed the ability for blueberry polyphenols 

to reduce the presence of inflammatory cytokines such as IL-6, TNF-, IL-1 in vitro (ben 

Lagha et al., 2015; Kang et al., 2015). Also, nitric oxide synthase and cyclooxygenase, enzymes 

involved in lipopolysaccharide induced inflammation, have been found to be reduced in vitro 

after treatment with blueberry polyphenol extract (Lau et al., 2009).  

 Studies have shown that blueberry polyphenols have the potential to reduce oxidative 

stress in cardiomyocytes, erythrocytes and after exercise (Kang et al., 2015; Louis et al., 2014; 

McAnulty et al., 2004). Furthermore, inflammation and oxidative stress are conditions associated 

with the development of all cancers, therefore the disease may be mediated by the consumption 

of blueberry polyphenols. One study found that crude fractions of blueberry polyphenols reduced 

tumor growth in rats. Also, breast cancer cell metastasis was reduced as well as the severity of 

lung tissues damage showing that blueberry polyphenols may be able to slow down the 



 4 

 

progression of the disease (Yang et al., 2021). Despite the numerous benefits of blueberry 

polyphenols discussed, many of these outcomes may not actually be observed in humans due to 

the low intake and low bioavailability of these compounds. 

 

1.3 Scope of Polyphenol Consumption in the United States (US) 

 Fruit and vegetables contain numerous health promoting compounds such as fiber, 

vitamins, minerals and polyphenols. Although these benefits are well known, consumption of 

fruits and vegetables remains low in the United States. The United States Department of 

Agriculture recommends consuming 5 to 9 servings of fruit and vegetables a day, which many 

Americans fall below (Tkacz et al., 2021). The National Health and Nutrition Examination 

Survey (NHANES) from 2013-2016 found that about 70% of US consumers take in 4 servings or 

less of fruit and vegetables on any given day and 37% consume 2 servings or less (Katherine 

Hoy et al., 2020). As a result of low intake of fruits and vegetables, polyphenol consumption also 

remains low in the US.  

Polyphenol intake has been estimated to be around 800 to 1600 mg per day (Burkholder-

Cooley et al., 2016; Huang et al., 2020). This would be equivalent to around two to four servings 

of fruit or vegetables per day, but this is not how many Americans obtain their daily polyphenol 

intake (Zhong et al., 2017). Most polyphenol consumption comes from non-alcoholic beverages, 

the main source being coffee. Coffee was found to make up around 40 to 65 percent of the 

consumption of polyphenols, while fruits and vegetables made up only 15 to 20 percent 

(Burkholder-Cooley et al., 2016; Huang et al., 2020). Furthermore, older, educated, non-

Hispanic white females with a normal BMI between 18.5-24.9 typically had higher intakes of 

polyphenols than other groups (Huang et al., 2020). This parallels with groups that traditionally 



 5 

 

have greater access to these foods and more education on nutrition (Gundersen et al., 2011). 

Access to fruits and vegetables is limited for many individuals, which would hinder their ability 

to be able to consume polyphenols despite their many health benefits. 

 

1.4 Bioavailability of Polyphenols 

 The bioavailability of all polyphenols has been found to be between 5 to 10 percent in the 

gastrointestinal tract, which indicates that most of the polyphenols consumed are not being fully 

absorbed (Fang & Bhandari, 2010). In a clinical trial where participants consumed a blueberry 

drink containing polyphenols equivalent to two servings of blueberries, only 1% of anthocyanins 

were present in the plasma (Zhong et al., 2017). Likewise, another study in which human 

subjects consumed a blueberry juice with similar phenolic composition, found that anthocyanin 

content was also low in the plasma after intake. The absorption rate for two types of 

anthocyanins, malvidin-3-glucoside and petunidin-3-glucoside, was measured in a cell model 

and found to be 0.5% and 0.6%, respectively (Kuntz et al., 2015).  

The lack of bioavailability of polyphenols in general is believed to result from pH changes, 

enzymes and fluids throughout the gastrointestinal tract which impacts their solubility. Many 

polyphenols tend have low solubility or permeability through the cell membrane. Their intestinal 

impermeability may be due to lack of receptors to transport polyphenols which makes only low 

molecular weight polyphenols able to diffuse through the membrane. Another factor that affects 

the bioavailability of polyphenols is the transformation of these compounds by gut 

microorganisms. During phase I and II metabolism of polyphenols, gut microorganisms alter the 

structure of polyphenols which leads to them being excreted in the urine rather than being 

transported to the small intestine where they can be absorbed (Annunziata et al., 2020). All of 



 6 

 

these factors contribute to the lack of bioavailability of these compounds, but various strategies 

are being investigated to improve their absorption. 

 

1.5 Strategies for Improving the Bioavailability of Polyphenols 

 Nanodelivery systems have been the main strategy for trying to enhance the 

bioavailability of polyphenols. These systems include the use of nanoemulsions, micelles, 

microencapsulation, and lipid, polymeric, and silica nanoparticles (Z. Zhang et al., 2021). Fiber 

has been a material researched to be used to form these delivery systems. One type of fiber that 

has been studied is chitosan, which comes from the outer skeleton of shellfish, and has been 

found to enhance the bioavailability and bioactivity of polyphenols such as quercetin and 

curcumin in nanodelivery systems (Tang et al., 2020). Pectin, a fruit fiber, in a 

nanoencapsulation system has been found to increase the release of bilberry anthocyanins into 

the gastrointestinal tract (Oidtmann et al., 2012). Furthermore, chitin, a fiber found in the cell 

wall of fungi and arthropods, were used to create microspheres that enhanced the amount of 

anthocyanins delivered to the GI tract. When the polyphenols were also coated in ethyl cellulose, 

it increased their delivery to up to 85% (Wang et al., 2017). Synthetic polymers including 

poly(lactide-co-glycolide), polyethelene gycol, polycaprolactone and polylactide have also been 

used to encapsulate resveratrol and found to increase uptake of the compound 7-fold in a cell 

model. Another type of delivery system that has been used are liposomes which have also been 

shown to increase bioavailability of polyphenols (Annunziata et al., 2020).  

Many of the strategies currently being studied to enhance the bioavailability of polyphenols 

are addressing the pharmaceutical and supplement industries, targeting methods such as 



 7 

 

nanoencasuplation. Therefore, few approaches have been studied on whether the food matrix can 

be used improve the bioavailability of these compounds.  

 

 

1.6 Whey Protein as a Strategy for Delivering Polyphenols  

 Various types of proteins have been found to interact with polyphenols to be used in 

nanodelivery systems. Some of these proteins include ferritin, gelatin, albumin, and collagen but 

studies focusing on the effects of these compounds on bioavailability as a part of the food matrix 

are limited (Z. Zhang et al., 2021). Whey protein is one of the most popular proteins currently 

studied for its potential to increase polyphenol bioavailability. Several studies have shown that 

whey proteins interact with many different types of polyphenols spontaneously (Meng & Li, 

2021; Ni et al., 2020; Schneider et al., 2016). The complexes that are formed have been found to 

have the same antioxidant activity as the polyphenols alone (Ming et al., 2020; Schneider et al., 

2016). One study even found that radical scavenging ability of chlorogenic acid was greater 

when bound to whey protein than just chlorogenic acid alone (Jiang et al., 2018). Another study 

that assessed the complexes as a part of a food matrix, found that cookies containing freeze dried 

whey protein and blackcurrant complexes had greater antioxidant activity than the control 

cookies. Furthermore, the cookies containing the polyphenol-protein complexes exhibited a 

lower glycemic response which could help with blood glucose control in those with poor 

regulation (Wu et al., 2021).   

The complexation of whey protein and polyphenols has been found to improve 

bioavailability and bioaccessibility in vitro. In an intestinal cell model, the binding of apple 

polyphenols to whey protein increased the bioavailability of the polyphenols by approximately 



 8 

 

15% (Li et al., 2022). Another study found that blueberry polyphenols and whey protein 

aggregates would be able to deliver approximately two servings worth of polyphenols (Diaz et 

al., 2020). Similarly, the bioaccessibility of sea buckthorn polyphenols complexed to whey 

protein increased by 20% which indicates that the polyphenols would be more available for cell 

processes (Ashwar & Gani, 2021). 

Not only do whey protein-polyphenol complexes serve the potential to be beneficial to health 

but they can also improve the functional properties of the foods that they are used in. Several 

studies have shown that whey protein and polyphenol aggregates can act as food ingredients to 

improve emulsification properties, solubility, stability and foaming capacity of a product (Meng 

& Li, 2021; Thongkaew et al., 2014; Tian et al., 2021). Soy protein, one of the most well-known 

and well researched plant proteins, also has exhibited similar outcomes when complexed with 

polyphenols. Bioaccessibility of soy protein bound to polyphenols from a plant commonly 

known as Russian tarragon, increased by 15% and the polyphenols were found to be eight times 

more bioavailable (Ribnicky et al., 2014). Soy protein and polyphenols aggregates were also 

found to maintain their antioxidant activity and produced hypoglycemic affects in rats (Djuardi 

et al., 2020; Roopchand et al., 2013). All in all, whey protein is traditionally consumed as a part 

of a dairy and fruit food matrix, which makes it a good candidate to help improve the 

bioavailability of fruit polyphenols in its natural form.  

 

1.7 Popularity and History of Consuming Fruit and Dairy 

Yogurt is a popular way to consume dairy due to its convivence and nutritional value. The 

yogurt industry is estimated to reach 9.1 million dollars by the end of 2021 and is expected to 

grow another 2.5% (US Yogurt and Yogurt Drinks Market Report 2021, n.d.). Furthermore, one 



 9 

 

of the most popular varieties of yogurt are fruit flavored and fruit containing yogurts which are 

produced by adding fruit puree to the bottom or stirring it throughout (Chandan et al., 2017). 

Since the introduction of yogurt, consumers have had a strong interest in consuming fruit 

flavored yogurt. Nearly 50% of consumers were found to have a preference for purchasing a fruit 

flavored yogurt beverage (Ryan et al., 1984). Fruit inclusions are preferred by US consumers in 

their yogurt to offset the tart flavors present from lactic acid (Das et al., 2019). Additionally, 

epidemiological studies have shown that consuming fruit and yogurt can reduced the risk for 

obesity, type II diabetes, and cardiovascular disease which is believed to be caused by the 

symbiotic affects from probiotics and fiber found in fruit but could potentially be due to other 

compounds as well (Fernandez & Marette, 2017).  

Smoothies are another popular way to consume dairy and fruit. In 2020, the global smoothie 

market was worth 12.1 billion and is expected to reach just over 17 billion dollars by 2025 

(Cano-Lamadrid et al., 2020). Smoothies have gained popularity due to consumer preferences for 

ready to eat and on the go options (Tkacz et al., 2021). Also, smoothies are a convenient way to 

increase the intake of fruits and vegetables considering one smoothie can have more than one 

fruit or vegetables. Both of these food options have been customarily prepared with dairy, but in 

recent years there has been an increase in consumption of plant-based alternatives for a multitude 

of reasons. 

 

1.8 History, Popularity and Consumer Reasons for Consuming Plant-Based Dairy 

Alternatives 

The plant-based food industry has grown drastically in the last decade. From 2017 to 2019, 

the market for these food products grew 29% and is estimated to reach a market value of nearly 5 



 10 

 

billion dollars (McClements & Grossmann, 2021). Plant-based dairy is one of the largest sections 

of the plant-based foods market, making up approximately 40%. The market consists of milk, 

yogurt, cheese, creamers and ice cream alternatives produced using a variety of different plant 

sources such as almond, soy, and coconut (Alcorta et al., 2021). The consumption of plant-based 

dairy dates back thousands of years. Soymilk has been estimated to be produced in China for the 

past 2000 years, but commercial soymilk production began within the last 100 years (Mendly-

Zambo et al., 2021). Since its introduction, plant-based dairy has become a staple in many 

households all over the world. In a 2020 survey of nearly a thousand households, it was 

determined that 40% of households consume plant-based dairy on a regular basis. On the other 

hand, dairy milk sales have decreased nearly 3 billion from 2013 to 2018 (Wolf et al., 2020). 

People consume plant-based dairy for numerous reasons including health, ethical, and 

environmental considerations. A global survey in 2019 found that 40% of consumers are trying 

to reduce their consumption of animal protein. In the US, the number of vegans increased from 

nearly 4 million in 2014 to 19.6 million in 2017. (Alcorta et al., 2021). Lactose intolerance and 

milk allergy prevalence have also increased over the years. It is estimated worldwide that 75% of 

people are lactose intolerant. Lactose intolerance results in gastrointestinal symptoms such as 

bloating, flatulence, and abdominal pain after consumption of dairy products. Asian, Black, and 

Hispanic populations are affected at a greater rate and the only solution is to avoid consumption 

of lactose containing dairy products (Mäkinen et al., 2015).  

Consumers have a perception of plant-based milk being more sustainable than cow’s 

milk. Almond-based products, which are preferred by many consumers, contradict this 

assumption due to irrigation issues caused by the use of large amounts of water in the production 

of almond milk. However, legume production has been shown to produce less greenhouse gas 



 11 

 

emissions than other agricultural sectors. Likewise, emerging plant protein sources, such as 

microalgae, require less land for protein production in comparison to pork, chicken, and beef. 

Even the Academy of Nutrition and Dietetics considers plant-based diets more environmentally 

sustainable than other diets due to its use of fewer natural resources and decreased environmental 

damage. In general, plant-based industries offer more environmentally friendly practices than 

animal-based ones (Alcorta et al., 2021).  

Lastly, one of the major reasons why people consume plant-based alternatives is for their 

health. Reducing cholesterol is one the benefits most desired from the consumption of non-dairy 

milk, and studies have shown that consumption of cow’s milk has been associated with increased 

risk of certain cancers (Vanga & Raghavan, 2017). A review of plant-based diets has also found 

that they were associated with decreased risk of cardiovascular disease (Satija & Hu, 2018). 

Furthermore, a review of the nutritional content of four types of plant-based milks: almond, soy, 

rice, and coconut found that overall, the milks were lower in calories, lack cholesterol, and have 

a comparable mineral content to normal milk. The reduction of LDL cholesterol levels from 

almond and soy milk is attributed to monounsaturated fatty acids which have a host of other 

health benefits as well (Vanga & Raghavan, 2017). Overall, a healthful plant-based diet has been 

associated with decreased risk of disease, which is why many consumers are moving towards 

these products increasing the need for more research in this area. 

 

 

 

 

 



 12 

 

CHAPTER II 

STATEMENT OF PURPOSE 

 

 

 

Polyphenols have been shown to have positive effects on human health. One of the major 

pitfalls associated with their consumption, is that despite these benefits, many polyphenols are 

not highly bioavailable (Fang & Bhandari, 2010). Numerous techniques have been studied to 

help improve the bioavailability of these compounds, but few have taken into account how these 

foods are consumed as a part of a food matrix. Polyphenols can interact and form complexes 

with different types of dietary proteins which has led to improved bioavailability and 

bioaccessibility (Z. Zhang et al., 2021). There is evidence to support that fruit and vegetables are 

often consumed in mixed meals rather than as a snack or side on their own (Katherine Hoy et al., 

2020). Specifically, dairy and fruit have been consumed together in many food products such as 

yogurt and smoothies. Therefore, assessing the interactions between dairy proteins and 

polyphenols would be a viable option to try to investigate the potential for these proteins to aid in 

the delivery of polyphenols when consumed as a part of a whole food. Consumers are moving 

towards plant based dairy alternatives for health and ethical reasons (Alcorta et al., 2021)which 

is why it is important to establish these relationships between plant proteins in addition to dairy 

proteins. 

The objective of this study was to determine whether commercially available protein 

powders from whey and novel plant protein sources (pea and hemp) would form complexes with 

blueberry polyphenol extract. Moreover, if complexation occurred, we sought to investigate the 

affinities of each protein for polyphenols, and how complexation would affect the digestibility 

and structural characteristics of the proteins. We hypothesized that all three protein types would 



 13 

 

form complexes with blueberry polyphenols and that the proteins would remain digestible in the 

presence of the compounds. Additionally, we expected the protein structure to change in the 

presence of the phenolics and that the different protein sources would exhibit similar binding 

affinities. The characterization of these interactions will act as a basis for future studies to 

determine whether the bioavailability of blueberry polyphenols is improved by its complexation 

with these proteins in a transepithelial cell model. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 14 

 

CHAPTER III 

MANUSCRIPT 

 

3.1 Introduction 

 Polyphenols are a class of phytochemicals that have been shown to have to confer 

beneficial effects on health. Berries such as blueberries (Vaccinium cyanococcus) contain high 

levels of polyphenols specifically anthocyanins. Polyphenols present in blueberries have been 

found to have protective effects against various diseases such as CVD, obesity, and cancer 

(Chew et al., 2019; Jiao et al., 2019; Yang et al., 2021). Blueberries are second-most consumed 

berry by Americans which makes them an important berry to study for their health effects (Kang 

et al., 2015). Even though blueberry consumption is high, studies have shown that overall 

consumption of polyphenols is low in Americans due to their low consumption of fruits and 

vegetables (Huang et al., 2020). Furthermore, the functional foods market is growing as a result 

of consumers being more interested in health-promoting foods, so research is also increasing in 

this area to find ways to maximize the health benefits of certain foods (Devcich et al., 2007). 

Although polyphenols have many health benefits, the bioavailability of many of these 

compounds is typically low. The bioavailability for anthocyanins has been found to be between 

0.7 and 1.1 %, which means most of the polyphenols consumed are not readily absorbed (Zhong 

et al., 2017). Strategies for increasing intake have been explored, including incorporation of 

polyphenol-rich foods into mixed meals. It has been shown that binding of these compounds to 

macromolecules such as proteins in dairy foods may be able to improve their absorption.  

Smoothies are a popular way to consume fruits and vegetables since consumers are able to reap 

the benefits of these foods without the hassle of preparation. In 2020, the global smoothie market 

was worth 12.1 billion and is expected to reach just over 17 billion dollars by 2025 (Cano-

Lamadrid et al., 2020). Smoothies are typically made of fruit and some liquid (juice or dairy 



 15 

 

milk) blended together and is a common way to consume dairy foods (Teleszko & Wojdylo, 

2014). Additionally, yogurt, another dairy product typically flavored with fruit has gained 

popularity in recent years for its beneficial effects on gut health. The spoonable and drinkable 

yogurt industry is estimated to reach 9.1 million dollars by the end of 2021 and is expected to 

grow another 2.5% (US Yogurt and Yogurt Drinks Market Report 2021, n.d.).  

As these dairy foods become more popular, non-dairy alternatives are also being created 

as consumers look for more health promoting and sustainable foods. A global survey in 2019, 

found that 40% of consumers are trying to reduce their consumption of animal protein (Alcorta 

et al., 2021). Moreover, some of the most commonly consumed non-dairy products are sourced 

from oat, soy, almond and coconut. Although, there are several plant protein options on the 

market, innovative plant sources are being studied considering the allergenicity and low 

functionality of the types of proteins available currently. Pea and hemp proteins are novel plant 

proteins being considered for use in these products due to their sustainability, lack of 

allergenicity and high nutritional value (Gao et al., 2020; Mamone et al., 2019).  

It has been established on multiple accounts that dairy protein specifically whey protein 

can form complexes with polyphenols (Baba et al., 2021). Despite the fact that polyphenols have 

been found to have a low digestibility and can demonstrate anti-nutritional effects against 

proteins (Samiya et al., 2020), studies have shown that when bound to whey protein there is no 

effect on the protein digestibility. A study conducted by de Morais et al. demonstrated that when 

whey protein was digested with caffeic acid and epigallocatechin gallate (EGCG), there was no 

profound difference in protein digestibility (de Morais et al., 2020). However, this phenomenon 

has not been studied in the context of plant proteins with specifically blueberry phenolics which 



 16 

 

can provide a new perspective on how to improve the bioavailability of these compounds 

maximizing their potential health benefits.   

The goal of this study was to determine whether commercially available plant protein 

powders from novel plant protein sources (pea and hemp) would form complexes with blueberry 

polyphenol extract similarly to what has been observed for whey protein. Upon successful 

complexation of the proteins and polyphenols, we aim to assess the affinities for the polyphenols 

to bind the proteins and how binding would affect protein structure and digestibility. We 

hypothesized that the pea and hemp proteins would form complexes with blueberry polyphenols 

and that the proteins would remain digestible when bound to the compounds. Furthermore, we 

expected the protein structure to change in the presence of the phenolics and that all three protein 

sources would exhibit similar binding affinities. This research will guide future studies that 

determine whether the bioavailability of blueberry polyphenols is improved by its complexation 

with these proteins in a transepithelial cell model. 

 

3.2 Materials & Methods 

3.2.1 Polyphenol Extraction 

3.2.1.1 Crude Blueberry Polyphenol Extraction   

Powdered blueberries were purchased from Bulk Supplements (Henderson, NV) and 

purified. The crude blueberry powder was mixed with 50% ethanol at a ratio of 1:5 (blueberry 

powder: 50% ethanol). The pH of the solution was lowered to 3 using concentrated sulfuric acid 

and then mixed for two hours. The ethanol was removed using rotary evaporation at 80°C. Once 

cooled, the samples were frozen at -20°C for further processing.  

 



 17 

 

3.2.1.2 C18 Solid Phase Extraction  

C18 solid phase extraction was performed to remove residual sugars and polysaccharides 

from solution. The purified blueberry solution was diluted 1:1 using acidified water (1% acetic 

acid in water). C18 solid phase extraction columns were conditioned with methanol then 

acidified water, twice for each solution. The diluted blueberry solution was added to the column 

and the solution was moved through the column using a vacuum. The column was washed thrice 

with acidified water and then dried in preparation for elution. The column was eluted twice using 

acidified methanol (1% acetic acid in methanol). The solvent was removed using rotary 

evaporation at 80°C. The blueberry polyphenol extract (BPE) was diluted with ultrapure water 

and frozen at -20°C. The samples were freeze dried and stored at -20°C for further analysis.  

 

3.2.1.3 Determination of Total Phenolic Content  

Total phenolic content of BPE was determined using the Folin-Ciocalteau assay. Freeze-

dried blueberry polyphenol extract was used to create a 1 mg/mL solution. A standard curve was 

produced using gallic acid with concentrations ranging from 0 to 500 µg/mL. 10 µL of sample 

and standard solutions were added to 790 µL of ultrapure water and 50 µL of Folin reagent 

(Sigma-Aldrich, St. Louis, MO) and shaken for 5 minutes. After mixing, 150 µL sodium 

carbonate solution at a concentration of 200 g/L was added to each sample and the plate was 

incubated at 37 °C. After incubation, absorbance was measure using a Bio-Tek UV-Vis plate 

reader (Winooski, VT) at 765 nm.  

 

 

 



 18 

 

3.2.2 Protein Purification  

3.2.2.1 Fat extraction using the Soxhlet Method   

Pea protein powder (Bulk Supplements, Henderson, NV) and hemp protein powder (Fit 

Hemp LLC, Melbourne, FL) were defatted using Soxhlet extraction with hexane. Whey protein 

isolate (Nutricost, Vineyard, UT) contained no fat, therefore did not require this procedure. The 

Soxhlet extraction was performed for 8 hours. Rotary evaporation was used to determined how 

much fat was extracted.   

 

3.2.2.2 Alkaline Extraction-Isoelectric Point Precipitation  

The protein extraction method was adapted from a by Shen et al (Shen et al., 2020). 

Defatted pea and hemp protein powders underwent an alkaline extraction and isoelectric point 

precipitation to isolate the protein and remove insoluble fiber. The whey protein isolate 

contained no other ingredients, therefore did not require this procedure. The defatted protein 

powders were dissolved in ultrapure water at a ratio of 1:15 (protein powder: water). The pH was 

adjusted to 10 for the hemp protein and 9.5 for pea using 2 M NaOH. The solutions were then 

stirred for 2 hours at 600 rpm. After stirring, the solutions were centrifuged at 4150 rpm for 20 

minutes and the pellet was discarded. The collected supernatant was adjusted to a pH of 5 for the 

hemp and 4.5 for the pea using 1.2 M HCl. The adjusted solutions were centrifuged once again 

for 10 minutes at 4150 rpm. The precipitates were collected and resuspended in ultrapure water. 

The pH was adjusted to 7 using 2 M NaOH, frozen at -20°C, and then freeze dried for further 

analysis.  

 

 



 19 

 

3.2.2.3 Determination of Protein Content  

Protein Concentration was determined using the BCA assay with the Pierce BCA Protein 

Assay kit from Thermo-Scientific (Waltham, MA). All samples were prepared at a concentration 

of 1 mg/ml and solubilized in 0.1 M NaOH. Assay was performed according to instructions 

provided with the kit. A standard curve was generated using bovine albumin serum (BSA) with 

concentrations ranging from 0 to 2 mg/mL. Absorbances were measured using a BioTek UV-Vis 

plate reader at 562 nm.  

 

3.2.3 Sample Preparation  

Samples were prepared to mimic the conditions of a beverage with a single serving of 

protein supplement (15 g protein) and the polyphenolic equivalent of 175 g of highbush 

blueberries (1 cup; 490 mg polyphenols from BPE). Samples were prepared to contain pea, whey 

or hemp protein alone or the forementioned proteins in addition to polyphenols. Samples had a 

protein concentration of 1 mg/mL protein, unless otherwise stated and subsequently a 

concentration of 33 µg/mL of blueberry polyphenol extract, when the samples contained 

polyphenols, to match the desired ratio of protein to polyphenols (30:1). All samples used a pH 

6.8 10 mM phosphate buffer for the solvent, unless otherwise stated.  

 

3.2.4 Turbidity Measurements  

A Jasco V-730 UV-Vis Spectrophotometer (Talbot County, MD) was used to measure 

the turbidity of whey, pea and hemp protein solutions and the respective solutions containing 

blueberry polyphenols. Turbidity measurements were also taken over a range of different pH 

values (2.0, 4.6, 6.8, 7.4) to assess the changes in complexation at food production and digestion 



 20 

 

related values. The values correlated to gastric, yogurt, duodenal/protein shake, and ileac pH, 

respectively. Absorbance of the solutions were measured at a wavelength of 600 nm.  

 

3.2.5 Circular Dichroism (CD)  

A Jasco J-1100 Circular Dichroism Spectrophotometer (Talbot County, MD) was used to 

measure the changes in secondary protein structure of whey, pea and hemp protein in solution 

and when bound to blueberry polyphenols. The samples were prepared with a protein 

concentration of 0.1 mg/mL and the protein and polyphenol. Measurements were taken at a 

wavelength from 190 to 260 nm. The data was interpreted using the CD Plotting and Analysis 

tool (CAPITO) (Wiedemann et al., 2013).  

 

3.2.6 Fluorescence Spectroscopy  

Fluorescence spectroscopy was performed to determine the binding affinities for 

blueberry polyphenols binding to pea, whey and hemp proteins. Protein solutions were created at 

a concentration of 0.5 mg/mL in the presence or absence of polyphenols. BPE controls were 

produced for all polyphenol concentrations. The protein concentration remained the same in all 

solutions, but polyphenol concentrations ranged from: 0.066 mg/mL, 0.033 mg/mL, 0.017 

mg/mL (ideal ratio, 30:1), 0.008 mg/mL, 0.004 mg/mL. An Edinburgh Instruments 

Spectrofluorometer FS5 (Livingston, UK) was used to measure the samples and the Flouracle 

program was used for the data output. An emission scan was run with an excitation wavelength 

of 280 nm and emission spectrum from 300 to 450 nm. The following emission scan parameters 

were applied: dwell time 0.2, steps: 1 nm, # of scans: 3, all corrections applied. BPE controls 



 21 

 

were subtracted from each spectrum. Data was analyzed using Stern-Volmer plots and 

calculation of Hill coefficients to determine the number of binding sites for each protein source.  

 

3.2.7 Analysis of the Digestibility of Pea, Hemp and Whey Protein with Blueberry 

Polyphenol Extract  

3.2.7.1 In Vitro Digestion  

An in vitro digestion was performed with solutions of pea, whey, and hemp protein in the 

presence and absence of blueberry polyphenol extract. The pH of the solutions was adjusted to 

approximately 2 using 1 M HCl to mimic the conditions of the stomach. Pepsin was added at a 

concentration of 0.3 mg/mL and the samples were incubated at 37°C for two hours. After the two 

hours, the pH was adjusted to 7.4 using 2 M NaOH which mimics the conditions of the small 

intestine. Trypsin was added at a concentration of 0.3 mg/mL and the samples were incubated at 

37°C for another four hours. Once the digestion was complete, the samples were boiled for 30 

minutes to inactivate the enzymes. Aliquots of the of the pure solution and supernatant of the 

solution were taken at the beginning and end of the digestion. The samples were stored at -20°C 

until further analysis.   

 

3.2.7.2 Determination of Free Amino Acids  

The ninhydrin method adapted from Moore and Stein and Reyanaud et al. was used to 

determine the free amino acid content of the digested samples (Moore & Stein, 1954; Reynaud et 

al., 2020). Trione ninhydrin reagent (Pickering Laboratories, Mountain View, CA) was mixed 

with the supernatant of the digested samples in a ratio of 1:1. The samples were heated for 16 

minutes at 95°C. Once cooled to below 30°C, 50% ethanol was added at a ratio of 1:5 (ninhydrin 



 22 

 

reagent: 50% ethanol). Absorbance was measured using a BioTek UV-Vis Plate Reader at a 

wavelength of 570 nm. L-leucine with concentrations ranging from 0 to 4 mM was used to 

produce a standard curve.   

 

3.2.7.3 Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis (SDS-PAGE)  

Digested samples were analyzed using SDS-PAGE. Samples were mixed with 2X Tris 

glycine loading buffer (Invitrogen, Waltham, MA) containing 5% beta-mercaptoethanol. The 

prepared loading buffer and sample were added to a tube at a ratio of 1:1 and boiled for 10 

minutes. The gel box was loaded with 1X tris glycine running buffer. Tris-glycine gels 

(Invitogen, Waltham, MA) were used and 5 µL of Novex Sharp Pre-Stained Protein Standard 

(Invitogen, Waltham, MA) was added to one well. The prepared samples were added at a volume 

of 15 µL to the other wells. The gel was run at 165 V. After the gel finished running, it was 

stained using Coomassie brilliant blue for an hour. The gel was then destained for four hours 

using a solution of 50% methanol and 10% acetic acid. A second destaining step was performed 

to rehydrate the gel for imaging using a solution of 20% methanol and 10% acetic acid 

overnight. The gel was imaged using the UVP Bioimaging System (Upland, CA).  

 

3.2.8 Statistical Analysis  

All results are expressed as an average of triplicate measurements, unless otherwise 

stated. Values are presented as the mean ± standard deviation. Variance analysis was performed 

using two-way ANOVA with Šídák’s multiple comparisons to compare the protein samples 

without polyphenols to those containing polyphenols. One-way ANOVA with Tukey’s multiple 

comparisons was used to compare the protein types to one another. GraphPad Prism v 9.3.1 



 23 

 

(GraphPad Software, Inc., San Diego, CA, USA) was used to perform all statistical analysis and 

significant statistical difference was calculated at a p<0.05 level. 

 

 

3.3 Results 

3.3.1 Physical and Chemical Characterization of the Protein-Polyphenol Complexes 

The total polyphenol content of the blueberry extract was determined to be 260.1 ± 1.8 

mg gallic acid equivalents (GAE) per g extract. The protein content of the hemp, pea and whey 

protein powders were determined to be 73.8%, 50.4% and 69.7% respectively. The first analysis 

that was performed were turbidity measurements to determine whether the proteins did form 

complexes with the blueberry polyphenols. The addition of blueberry polyphenols to purified 

proteins resulted in the formation of protein-polyphenol aggregates. The absorbance of all three 

protein sources increased significantly in presence of blueberry polyphenol extract indicating 

complexation. The difference in absorbance between the proteins alone and the proteins in the 

presence of polyphenols is not equivalent to the polyphenol blank alone indicating that the 

differences observed are as result of the polyphenols binding to the proteins. Moreover, hemp 

protein increased the turbidity of the solutions when bound to polyphenols to a greater degree 

than the pea and whey proteins.  

Aggregate formation was found to be pH dependent for the pea and hemp proteins. At pH 

4.6, pea protein exhibited the highest increase in turbidity indicating increased complexation 

with BPE. At pH 6.8, the turbidity of the pea protein-BPE complexes decreased significantly due 

to decreased complex formation. In contrast, the hemp protein exhibited the highest levels of 

complexation at pH 6.8 and 7.4. Whey protein did not exhibit any pH-dependent changes in 

complex formation. 

 



 24 

 

  

 

 

Figure 1. (A) Turbidity of pea, whey and hemp protein in the presence and absence of BPE 

expressed as average absorbance at 600 nm. Values are expressed as mean ± SD. Asterisks 

indicate significant differences (p<0.05) based on 2-way ANOVA with Šídák’s multiple 
comparisons. (B) Difference in turbidity between the whey, pea and hemp samples with or 

without BPE added. Asterisks indicate significant differences (p<0.05) based on one-way 

ANOVA with Tukey’s multiple comparisons. 
 

 

Figure 2. Turbidity of pea, whey and hemp protein in the presence and absence of BPE at pH 

values related to digestion and food production expressed as average absorbance at 600 nm. 

Values are expressed as mean ± SD. Different letters in a column indicate significant differences 

(p<0.05) based on one-way ANOVA with Tukey’s multiple comparisons. 
 

 

2.0 4.6 6.8 7.4

0.0

0.1

0.2

0.3

0.4

pH

A
6
0
0

Whey

Whey+BPE

Pea

Pea+BPE

Hemp

Hemp+BPE

b

d

ab

b

b
b

d

e

bc

c

b
b

a

ab

d

e

b
b

a

b

d

e

a

b

   A                                                      B 



 25 

 

3.3.2 Polyphenol Binding Affects Hemp Protein Structure 

 

Far UV-Vis circular dichroism was used to determine the secondary protein structures of 

pea, hemp and whey protein isolates and how it changed when bound to blueberry polyphenols. 

The relative helicity of the hemp protein increased significantly when bound to polyphenols. 

Also, hemp protein exhibited a higher relative helicity when compared to pea and whey protein. 

This indicates that hemp protein has a greater proportion of alpha-helices in its secondary 

structure than the other protein types. In contrast, both pea and whey proteins did not show 

significant changes in relative helicity when bound to BPE. The content of other secondary 

protein structures such as beta sheets, beta-turns, and random coils was not measured but it can 

be implied that other secondary structures were more prevalent in the whey and pea proteins. 

Whey, pea and hemp proteins were found to adopt primarily unfolded structures based on 

comparisons to the CAPITO circular dichroism database. This information could give more 

insight on how the protein-polyphenol interactions could occur. 

  
Figure 3. (A) Smoothed CD spectra for hemp, pea, and whey protein in the presence and 

absence of BPE and the comparisons of the protein secondary structures to the other proteins in 

the CAPITO database (B) Differences in relative helicity between the protein types and the 

proteins in the presence of BPE. Different letters in a column indicate significant differences 

(p<0.05) based on one-way ANOVA with Tukey’s multiple comparisons. 
 

Whey Pea Hemp

0.0

0.5

1.0

1.5

2.0

2.5

R
e
la

ti
v
e
 H

e
li
c
it

y

(q
2
2
2
/q

2
0
8
)

Protein Protein + BPE

a
a

a a

b

c

A                                                                     B 



 26 

 

3.3.3 Binding Affinities Do Not Differ Between Protein Sources 

 

Fluorescence spectroscopy was used to determine the binding affinities for all three 

protein sources and whether any significant differences could be observed in their affinity for 

binding blueberry polyphenols. All three protein sources showed fluorescence quenching when 

in the presence of BPE. Fluorescence intensity decreased as the ratio of BPE to protein decreased 

(figure 3) indicating increased binding of the extract to the protein. No shifts in peak 

fluorescence emission were observed due to the binding of the blueberry polyphenols to the 

proteins. Figure 4 shows the Stern-Volmer plot used to calculate the Stern-Volmer coefficient 

(Ksv), modified Stern-Volmer to calculate binding affinity (Ka), and a plot to calculate the hill 

coefficient (n). The binding affinity, hill coefficient and Stern-Volmer coefficient for pea, whey 

and hemp proteins are displayed in table 2. There are no significant differences in binding 

affinity or in the hill coefficient which indicates number of binding sites for the polyphenols to 

the proteins.  

 

  A                                                               B 

300 350 400 450

0

100000

200000

300000

400000

Wavelength (nm)

F
lu

o
re

s
c
e
n

c
e
 I
n

te
n

s
it

y

Whey Protein Extract
120:1 (Protein:BPE)
60:1
30:1

15:1
7.5:1

300 350 400 450

0

50000

100000

150000

200000

250000

Wavelength (nm)

F
lu

o
re

s
c
e
n

c
e
 I
n

te
n

s
it

y Pea Protein Extract
120:1 (Protein:BPE)
60:1
30:1
15:1
7.5:1



 27 

 

 
 

 

Figure 4. Quenching effect of BPE on protein fluorescence intensity in whey-BPE (A), pea-BPE 

(B) and hemp-BPE (C) complexes as a function of BPE concentration. 

 

 

 
 

 

Figure 5. (A) Stern-Volmer plot for the fluorescence quenching of each protein by BPE. (B) 

Modified form of a Stern-Volmer plot to calculate Ka (C) Calculation of Hill coefficient reveals 

the number of binding sites for each protein. 

 

300 350 400 450

0

100000

200000

300000

400000

500000

Wavelength (nm)

F
lu

o
re

s
c
e
n

c
e
 I
n

te
n

s
it

y Hemp Protein Extract
120:1 (Protein:BPE)
60:1
30:1

15:1
7.5:1

1 2 3

-1.0

-0.5

0.0

0.5

1.0

log[BPE]lo
g

(F
0
/F

0
-F
)

C                                                   

 A                                                                  B 

C                                                   

0 100 200 300 400 500

0.0

2.0

4.0

6.0

8.0

10.0

BPE (m M GAE)

F
0
/F

Whey

Pea

Hemp

0.00 0.01 0.02 0.03 0.04 0.05

0

2

4

6

1/[BPE]

F
0
/F

0
-F



 28 

 

Table 1. Stern-Volmer constant (Ksv), binding affinity (Ka) and Hill coefficient (n) for 

interactions between each protein and BPE, calculated based on Figure 4 graphs. Different letters 

in a column indicate significant differences (p<0.05) based on one-way ANOVA with Tukey’s 
multiple comparisons. 

Protein Source Ksv (104  L·mol−1) Ka (104  L·mol−1) n 

Whey 1.319 ± 0.0002 3.026 ± 0.516 0.92 ± 0.008 

Pea 1.860 ± 0.0004 5.141 ± 2.163 0.99 ± 0.009 

Hemp 1.211 ± 0.0006 4.842 ± 1.543 0.94 ± 0.001 

 

3.3.4 Assessment of the Digestibility of Protein-Polyphenol Complexes 

SDS-PAGE was used to identify the protein profile of the hemp, pea and whey purified 

protein powder with and without BPE. Also, this technique was used to determine how the 

protein profile changed after an in vitro digestion and if it was similar to when the proteins are 

digested with polyphenols. The pea and hemp proteins digestion displayed a wide array of 

different sized proteins ranging from 100 kDa to less than 10 kDa. The pea protein isolate had 

bands for legumin (40-50 kDa), convillicin (~ 70 kDa) and lipoxygenase (~ 100 kDa) which are 

some of the main types of proteins typically present in peas (Gao 2020). Similarly, the SDS-

PAGE of hemp protein isolate showed bands for edestin (20-35 kDa), villicin (40-50 kDa) and 

albumin (~20 kDa) which are distinctive protein subunits typically observed (Mamone 2019).  

The whey protein displayed characteristic bands for α-lactalbumin, β-lactoglobulin, and bovine 

serum albumin between 60 and 70 kDa, slightly above 15 kDa, and between 10 and 15 kDa 

respectively (Xu 2019). When polyphenols were added to the samples, they exhibited same SDS-

PAGE protein profile. After the digestion, there were only faint bands for low molecular weight 

proteins visible for all three proteins between 10 to 15 kDa. The samples containing BPE 

appeared to have similar bands to the samples without. The supernatant of the samples 



 29 

 

containing BPE were also run on the gel to see how much of the protein formed complexes with 

the blueberry polyphenols. It appears that there is much more protein available than can complex 

with BPE considering that the supernatant and suspension appear to have similar band density. 

Hemp protein appears to have more complexation due to the fainter image of the supernatant but 

this could also be due to the low solubility of the protein. 

 

 

 

A                                                   



 30 

 

 

Figure 6. SDS-PAGE of pea, hemp (A) and whey (B) proteins in the presence and absence of 

BPE after in-vitro digestion. Abbreviations: Pre: pre-digestion, Post: Post digestion, PP: Pea 

Protein Isolate, HP: Hemp Protein Isolate, WP: Whey Protein Isolate Susp: protein-polyphenol 

suspension, Sup: protein-polyphenol supernatant. Samples containing only protein are taken 

from the suspension. 

 

The free amino acid concentration of the protein and protein-polyphenol samples were 

measured before and after in vitro digestion to quantitatively assess whether digestion was 

successful and how BPE affected the digestibility of the proteins. After digestion, the free amino 

acid content of the protein and protein-polyphenol complexes increased significantly. The pre-

digestion free amino acid concentration was approximately 0.5 mM leucine equivalents for the 

pea and whey solutions and around 0.4 mM leucine equivalents for the hemp solutions. After 

B                                                  



 31 

 

digestion, the free amino acid ranged between 1.5 mM and nearly 3 mM leucine equivalents. The 

pea protein solutions in the presence and absence of blueberry polyphenols experienced the 

greatest release of amino acids after incubation with the digestive enzymes while hemp protein 

experienced the least. Furthermore, the free amino acid content is not significantly different 

between the protein with or without polyphenols, demonstrating that the presence of polyphenols 

does not affect digestion when applied at these ratios.  

 
 

Figure 7. Free amino acid content expressed as mM leucine equivalents of protein and protein-

polyphenol complexes before and after in vitro digestion. Asterisks indicate significant 

differences (p<0.05) based on 2-way ANOVA with Šídák’s multiple comparisons. Number of 

asterisks indicate significance of: **** (p<0.0001), ***(p<0.001), ** (p<0.01).  

 

 

3.4 Discussion 

 

The aim of this study was to establish and characterize whether pea and hemp proteins 

can interact with blueberry polyphenols similarly to whey proteins. Also, the digestibility of all 

three proteins in the presence of blueberry polyphenol was assessed to determine whether these 

proteins could successfully act as delivery matrices to improve the bioavailability of these 

compounds. Pea and hemp protein were found to successfully form complexes with BPE using 

UV-Vis spectroscopy. The digestibility of the proteins was not impacted by the complexation 



 32 

 

with blueberry polyphenols because the release of free amino acids after in vitro digestion were 

not significantly different between the samples with or without polyphenols. 

To begin, powdered blueberry and commercially available pea and hemp protein powders 

were extracted to create the polyphenol extract and protein isolates. The polyphenol content in 

other papers that utilized solvent extraction to purify blueberry powder ranged greatly. Total 

phenolic content was reported to be as low as 1.22 mg GAE per g and up to 702 mg/g GAE 

(Grace et al., 2009; Jiao et al., 2018). The blueberry polyphenol extract used in this experiment 

falls within this range at approximately 260 mg per gram GAE. The discrepancies in total 

phenolic content may be due to slight changes in extraction protocol such as using microwave or 

ultrasound assisted extraction and the use of different SPE or resin columns. Additionally, the 

region, variety and season in which the blueberries were harvested in could contribute to the 

diversity of the values. Moreover, the determined values for the pea and hemp protein powders 

were lower than what was reported in the studies in which the extraction method this was 

adapted from. However, there has been a wide range of protein yield reported in studies that have 

used this method. For the hemp protein, isoelectric point precipitation has been reported to 

produce isolates with a protein content of as low as 44.3% and as high as 94.6% (Malomo et al., 

2014; Teh et al., 2014). The pea protein also has had values reported protein isolate prepared 

using this method to be around 80 to 85% (Boye et al., 2010; Gao et al., 2020; Stone et al., 

2015). These differences may be attributed to different sources of the proteins being used to 

perform the extractions as well as different protein determination techniques. In this paper, 

commercially available protein powders were used rather than the raw plant ingredient which 

could have impacted yield. Also, many other studies used the Khejdhal method to determine total 



 33 

 

nitrogen in the protein isolates and therefore the percent protein whereas in this study a 

colorimetric protein assay was used for protein determination. 

To determine whether protein-polyphenol complexation occurred, turbidity of the 

solutions was measured and found to increase when the proteins were in the presence of BPE. 

Increased turbidity of a solution containing protein and polyphenols has been associated with the 

formation of protein-polyphenol complexes numerous accounts (Seczyk et al., 2019; Ye et al., 

2013). In this study, the same phenomenon was observed. The hemp protein exhibited a greater 

degree of change in turbidity when the polyphenols were bound. This may be due to the hemp 

proteins forming a greater number of complexes with BPE in comparison to the other protein 

sources. Although this may be possible, it does not appear to be the case considering that all 

three proteins had similar affinities for binding the blueberry polyphenols as determined in the 

fluorescence spectroscopy experiments. 

Various factors can contribute to the likelihood for proteins and polyphenols to form 

complexes. In this study, it was found that pea and hemp protein complex to BPE in a pH 

dependent manner. This phenomenon was not observed for whey which is supported by other 

studies which found that binding of green tea polyphenols to whey protein was not affected by 

pH (Belščak-Cvitanović et al., 2015). Pea protein and other legumes such as soy have been 

shown to have the strongest protein-polyphenol interactions near, usually slightly above their 

isoelectric point (Cuevas-Bernardino et al., 2018; Kosińska et al., 2011; X. Zhang et al., 2020). 

The isoelectric point for pea protein is approximately 4.5 which may explain why increased 

complexation was visible at pH 4.6 (Gao et al., 2020). The isoelectric point of hemp protein is 

closer to pH 5 which might explain why the highest levels of complexation were observed above 

this pH (Shen et al., 2020). Additionally, differences in amino acid composition and protein 



 34 

 

structure can result in various effects of pH on different proteins (Ozdal et al., 2013). The 

secondary protein structures of pea and hemp were not similar according to the CD analysis 

which may also be responsible for the differences in complexation at the varying pH values. This 

data provides preliminary evidence on how polyphenol complexation with these proteins may be 

altered throughout digestion and effect the delivery of the compounds as well as how they may 

act functionally in a food product. 

The proteins all exhibited a change in secondary structure as a result of binding of the 

polyphenols also indicating complexation. Alpha helices appear to be involved in the binding of 

blueberry polyphenols to hemp proteins, but other secondary structures seem to be involved for 

pea and whey. This is supported by other papers which have reported that whey and pea protein 

isolates contain a higher percentage of beta-sheets than alpha-helices which may be the main 

structures responsible for binding polyphenols as well has a high proportion of random coils 

(Abd El-Maksoud et al., 2018; Yan et al., 2020). Also, legumes such as pea and hemp, have been 

found to have predominantly beta-sheets in their secondary structure (Malomo & Aluko, 2015; 

Shen et al., 2020) although this has been reported to be pH-dependent for hemp protein (Malomo 

et al., 2014) which could be why the hemp proteins in this study exhibited a change in relative 

helicity in the presence of BPE.  Furthermore, the presence of more beta sheets may indicate the 

presence of a more open protein structure. Based on the CAPITO database, all three proteins 

exhibited an unfolded protein structure. This supports the idea that all three proteins contain 

more beta sheets in their secondary structures leading to a more open protein conformation. The 

unfolded structure can increase the potential for proteins to bind to other molecules which may 

be why these proteins tend to spontaneously bind to polyphenols (Garbuzynskiy et al., 2004). 



 35 

 

Additionally, due to the highly alkaline environment present when purifying the pea and hemp 

proteins, there is potential that this structural conformation is due to denaturation. 

The intrinsic fluorescence of all three proteins was quenched by the blueberry 

polyphenols which is indictive of complexation. Intrinsic fluorescence of the proteins has been 

attributed to the major fluorophore tryptophan (Cao & Xiong, 2017) and the binding of the 

polyphenols to this amino acid is likely responsible for the quenching affect. No shifts were 

visible mostly likely because the protein structure was already unfolded as determined by the 

CAPITO analysis. Therefore, the tryptophan residues were already exposed allowing the 

polyphenols to bind easily without requiring the protein structures to change. Traditionally, shifts 

are observed due to the fluorophores being exposed as a change in polarity of the environment 

alters the protein structure exposing those residues. Environmental factors such as pH and 

temperature can cause these changes to occur (S. A. Abbas et al., 2013). The number of binding 

sites for all three protein sources were close 1 indicating that there is most likely one type of 

binding site responsible for binding BPE (Dai et al., 2019). Moreover, all three proteins appear to 

have similar abilities to bind blueberry polyphenols and can bind a large range of concentrations 

of blueberry polyphenols. Studies that have been conducted on the binding affinity of EGCG to 

bind whey protein isolate, have reported slightly higher Ksv values (2.62 and 1.9 L·mol−1) (Cao 

& Xiong, 2017; Chen et al., 2019) than these findings, so these proteins may have even higher 

affinities for binding other types of phenolics as well. Lastly, at a ratio lower than 7.5:1 (protein: 

polyphenols) the proteins may not be able to bind more polyphenols considering the fluorescence 

intensity was extremely low at this ratio. 

Whey, hemp and pea proteins were found to be fully digested after in-vitro digestion 

visually through the analysis of SDS-PAGE which was later confirmed quantitatively. The 



 36 

 

decrease in higher molecular weight bands for all three proteins indicates that the proteins were 

successfully digested. Additionally, the similarity between the protein profiles of the protein 

samples in the presence and absence of BPE indicates that the polyphenols are not inhibiting 

digestion at the ratio that they are being used in this paper. Moreover, comparable amounts of 

free amino acids were released after digestion of the proteins in the presence and absence of 

BPE. It was imperative that the protein digestibility was not affected by the polyphenols 

considering polyphenols have been shown to inhibit digestion of other proteins. This is believed 

to be as a result of the ability of polyphenols to inhibit the activity of digestive enzymes 

(Cirkovic Velickovic, 2018). Other studies have shown that whey protein digestibility decreases 

significantly when bound to polyphenols. In these studies, a ratio of 1:0.5 (whey protein: 

polyphenols) is used which may be why these effects observed (de Morais et al., 2020; Xu et al., 

2019). This ratio of protein to polyphenols would be much greater than how much of these 

compounds are customarily consumed together. In this study, a ratio of 30:1 (protein: 

polyphenols) was used which is more similar to how these compounds would be consumed in a 

food matrix.  

Pea protein seems to have a comparable digestibility to whey protein whereas hemp 

protein may require more time to be fully hydrolyzed. One study found that in beverages 

formulated with milk or pea proteins, both proteins were digested to similar amounts. The release 

of free amino acids from both protein drinks after in-vitro digestion were not significantly 

different from one another (Štreimikytė et al., 2020). Similarly, Hernandez et al. found that 

garden pea and grass pea isolates had a similar free amino acid release after in vitro digestion to 

whey protein. Furthermore, hemp protein which was found to be less digestible than pea protein 

in this study has been reported to have a protein digestibility‒corrected amino acid score 



 37 

 

(PDCAAS) value of 0.5 to 0.6. This is much lower than the score for animal proteins such as 

whey which is about 1 (Shen et al., 2021). Despite this, there is potential for the protein 

digestibility to be increased. When hemp and pea protein were used together in a protein 

beverage, the protein digestibility increased significantly from the control indicating that the use 

of various proteins could enhance its digestibility and nutritional value (Manus et al., 2021). The 

results are promising that polyphenol consumption at levels present in a serving of berries would 

not impact digestibility of the proteins which could allow for the proteins to be used as a delivery 

mechanism for blueberry polyphenols. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 38 

 

CHAPTER IV 

CONCLUSIONS & FUTURE DIRECTIONS 

 

 

 

In this study, the physical and chemical characteristics of whey, hemp and pea protein 

and blueberry polyphenol complexes were assessed. Firstly, the hemp and pea protein isolates 

were found to successfully formed complexes with the blueberry polyphenol extract similarly to 

whey protein which has been established to occur in many other studies. Then, the change in 

secondary structure in the presence of BPE was examined. Pea and whey protein displayed 

similar changes in secondary structure due to complexation with BPE whereas hemp protein 

exhibited a shift in its alpha helical structure. Furthermore, the binding affinities for the 

polyphenols to all three proteins were not significantly different which indicates that they would 

all have similar affinities for the compound. After the in-vitro digestion of the proteins with or 

without polyphenols, the free amino acid content of all the solutions increased significantly 

compared to before the digestion. This confirmed the success of the in-vitro digestion which also 

visualized using SDS-PAGE. Also, release of free amino acid of the protein-polyphenol 

complexes were similar to the proteins alone indicating that the polyphenols were not hindering 

digestion, which provides evidence that these proteins can be used to improve polyphenol 

bioavailability. 

One major limitation of this study is that the purification of the protein powders and 

blueberry powder do not fully represent how these compounds would interact as a part of a 

whole food matrix. For future studies, it would be important to assess the whole protein powder 

and the whole berry to see if the effects would be the same but for these preliminary studies it 

was helpful to see how the protein and the polyphenols alone interact. Also, further processing of 



 39 

 

the circular dichroism would give more information on how the secondary structures change for 

the protein as a result of polyphenol binding. Beta-sheets are a major structural component of all 

three proteins so it would be important to assess how those structures change as well.  

All in all, these results indicate that novel plant proteins may be effective delivery 

mechanisms for blueberry polyphenols in whole food matrices. These current findings suggest 

that due to the spontaneous nature of these interactions, when these foods (plant proteins and 

blueberry polyphenols) are consumed together as a part of a meal or snack, there is potential for 

these interactions to occur and in turn more polyphenols could be delivered to the small intestine 

and absorbed. Future directions include assessing the bioavailability of blueberry polyphenols in 

transepithelial cell model since this study has provided evidence that the delivery of polyphenols 

to the small intestine could be enhanced as a result of binding to plant proteins. Eventually, 

bioavailability will also be assessed in clinical trials by providing the protein/polyphenol 

complexes as a part of a whole food such as a smoothie rather than through supplementation. 

 

 

 

 

 

 

 

 

 

 

 



 40 

 

REFERENCES 

 

 

 

Abbas, M., Saeed, F., Anjum, F. M., Afzaal, M., Tufail, T., Bashir, M. S., Ishtiaq, A., Hussain, 

S., & Suleria, H. A. R. (2016). Natural polyphenols: An overview. 

Http://Dx.Doi.Org/10.1080/10942912.2016.1220393, 20(8), 1689–1699. 

https://doi.org/10.1080/10942912.2016.1220393 

Abbas, S. A., Gaspar, G., Sharma, V. K., Patapoff, T. W., & Kalonia, D. S. (2013). Application 

of Second-Derivative Fluorescence Spectroscopy to Monitor Subtle Changes in a 

Monoclonal Antibody Structure. Journal of Pharmaceutical Sciences, 102(1), 52–61. 

https://doi.org/10.1002/JPS.23354 

Abd El-Maksoud, A. A., Abd El-Ghany, I. H., El-Beltagi, H. S., Anankanbil, S., Banerijee, C., 

Petersen, S. v., Pérez, B., & Guo, Z. (2018). Adding functionality to milk-based protein: 

Preparation, and physico-chemical characterization of β-lactoglobulin-phenolic conjugates. 

Food Chemistry, 241, 281–289. https://doi.org/10.1016/J.FOODCHEM.2017.08.101 

Alcorta, A., Porta, A., Tárrega, A., Alvarez, M. D., & Pilar Vaquero, M. (2021). Foods for Plant-

Based Diets: Challenges and Innovations. Foods 2021, Vol. 10, Page 293, 10(2), 293. 

https://doi.org/10.3390/FOODS10020293 

Annunziata, G., Jiménez-García, M., Capó, X., Moranta, D., Arnone, A., Tenore, G. C., Sureda, 

A., & Tejada, S. (2020). Microencapsulation as a tool to counteract the typical low 

bioavailability of polyphenols in the management of diabetes. Food and Chemical 

Toxicology, 139, 111248. https://doi.org/10.1016/J.FCT.2020.111248 

Ashwar, B. A., & Gani, A. (2021). Noncovalent Interactions of Sea Buckthorn Polyphenols with 

Casein and Whey Proteins: Effect on the Stability, Antioxidant Potential, and 

Bioaccessibility of Polyphenols. ACS Food Science & Technology, 1(7), 1206–1214. 

https://doi.org/10.1021/ACSFOODSCITECH.1C00103 

Baba, W. N., McClements, D. J., & Maqsood, S. (2021). Whey protein–polyphenol conjugates 

and complexes: Production, characterization, and applications. Food Chemistry, 365, 

130455. https://doi.org/10.1016/J.FOODCHEM.2021.130455 

Belščak-Cvitanović, A., Dordević, V., Karlović, S., Pavlović, V., Komes, D., Ježek, D., 
Bugarski, B., & Nedović, V. (2015). Protein-reinforced and chitosan-pectin coated alginate 

microparticles for delivery of flavan-3-ol antioxidants and caffeine from green tea extract. 

Food Hydrocolloids, 51, 361–374. https://doi.org/10.1016/J.FOODHYD.2015.05.039 

ben Lagha, A., Dudonné, S., Desjardins, Y., & Grenier, D. (2015). Wild Blueberry (Vaccinium 

angustifolium Ait.) Polyphenols Target Fusobacterium nucleatum and the Host 

Inflammatory Response: Potential Innovative Molecules for Treating Periodontal Diseases. 

Journal of Agricultural and Food Chemistry, 63(31), 6999–7008. 

https://doi.org/10.1021/ACS.JAFC.5B01525/SUPPL_FILE/JF5B01525_SI_002.PDF 

Boye, J. I., Aksay, S., Roufik, S., Ribéreau, S., Mondor, M., Farnworth, E., & Rajamohamed, S. 

H. (2010). Comparison of the functional properties of pea, chickpea and lentil protein 

concentrates processed using ultrafiltration and isoelectric precipitation techniques. Food 

Research International, 43(2), 537–546. https://doi.org/10.1016/J.FOODRES.2009.07.021 

Burkholder-Cooley, N., Rajaram, S., Haddad, E., Fraser, G. E., & Jaceldo-Siegl, K. (2016). 

Comparison of polyphenol intakes according to distinct dietary patterns and food sources in 



 41 

 

the Adventist Health Study-2 cohort. British Journal of Nutrition, 115(12), 2162–2169. 

https://doi.org/10.1017/S0007114516001331 

Cano-Lamadrid, M., Tkacz, K., Turkiewicz, I. P., Clemente-Villalba, J., Sánchez-Rodríguez, L., 

Lipan, L., García-García, E., Carbonell-Barrachina, Á. A., & Wojdyło, A. (2020). How a 
Spanish Group of Millennial Generation Perceives the Commercial Novel Smoothies? 

Foods 2020, Vol. 9, Page 1213, 9(9), 1213. https://doi.org/10.3390/FOODS9091213 

Cao, Y., & Xiong, Y. L. (2017). Binding of Gallic Acid and Epigallocatechin Gallate to Heat-

Unfolded Whey Proteins at Neutral pH Alters Radical Scavenging Activity of in Vitro 

Protein Digests. Journal of Agricultural and Food Chemistry, 65(38), 8443–8450. 

https://doi.org/10.1021/ACS.JAFC.7B03006 

Chandan, R. C., Gandhi, A., & Shah, N. P. (2017). Yogurt: Historical Background, Health 

Benefits, and Global Trade. Yogurt in Health and Disease Prevention, 3–29. 

https://doi.org/10.1016/B978-0-12-805134-4.00001-8 

Chen, W., Wang, W., Ma, X., Lv, R., Balaso Watharkar, R., Ding, T., Ye, X., & Liu, D. (2019). 

Effect of pH-shifting treatment on structural and functional properties of whey protein 

isolate and its interaction with (−)-epigallocatechin-3-gallate. Food Chemistry, 274, 234–
241. https://doi.org/10.1016/j.foodchem.2018.08.106 

Chew, B., Mathison, B., Kimble, L., McKay, D., Kaspar, K., Khoo, C., Chen, C. Y. O., & 

Blumberg, J. (2019). Chronic consumption of a low calorie, high polyphenol cranberry 

beverage attenuates inflammation and improves glucoregulation and HDL cholesterol in 

healthy overweight humans: a randomized controlled trial. European Journal of Nutrition, 

58(3), 1223–1235. https://doi.org/10.1007/S00394-018-1643-Z/TABLES/4 

Cirkovic Velickovic, T. D. (n.d.). The Role of Dietary Phenolic Compounds in Protein Digestion 

and Processing Technologies to Improve Their Antinutritive Properties. 

https://doi.org/10.1111/1541-4337.12320 

Cuevas-Bernardino, J. C., Leyva-Gutierrez, F. M. A., Vernon-Carter, E. J., Lobato-Calleros, C., 

Román-Guerrero, A., & Davidov-Pardo, G. (2018). Formation of biopolymer complexes 

composed of pea protein and mesquite gum – Impact of quercetin addition on their physical 

and chemical stability. Food Hydrocolloids, 77, 736–745. 

https://doi.org/10.1016/J.FOODHYD.2017.11.015 

Dai, T., Chen, J., McClements, D. J., Hu, P., Ye, X., Liu, C., & Li, T. (2019). Protein–
polyphenol interactions enhance the antioxidant capacity of phenolics: analysis of rice 

glutelin–procyanidin dimer interactions. Food & Function, 10(2), 765–774. 

https://doi.org/10.1039/C8FO02246A 

Das, K., Choudhary, R., & Thompson-Witrick, K. A. (2019). Effects of new technology on the 

current manufacturing process of yogurt-to increase the overall marketability of yogurt. 

LWT, 108, 69–80. https://doi.org/10.1016/J.LWT.2019.03.058 

de Morais, F. P. R., Pessato, T. B., Rodrigues, E., Peixoto Mallmann, L., Mariutti, L. R. B., & 

Netto, F. M. (2020). Whey protein and phenolic compound complexation: Effects on 

antioxidant capacity before and after in vitro digestion. Food Research International, 133, 

109104. https://doi.org/10.1016/J.FOODRES.2020.109104 

Devcich, D. A., Pedersen, I. K., & Petrie, K. J. (2007). You eat what you are: Modern health 

worries and the acceptance of natural and synthetic additives in functional foods. Appetite, 

48(3), 333–337. https://doi.org/10.1016/J.APPET.2006.09.014 



 42 

 

Diaz, J. T., Foegeding, E. A., & Lila, M. A. (2020). Formulation of protein–polyphenol particles 

for applications in food systems. Food & Function, 11(6), 5091–5104. 

https://doi.org/10.1039/D0FO00186D 

Djuardi, A. U. P., Yuliana, N. D., Ogawa, M., Akazawa, T., & Suhartono, M. T. (2020). 

Emulsifying properties and antioxidant activity of soy protein isolate conjugated with tea 

polyphenol extracts. Journal of Food Science and Technology, 57(10), 3591–3600. 

https://doi.org/10.1007/S13197-020-04391-9/FIGURES/5 

Fang, Z., & Bhandari, B. (2010). Encapsulation of polyphenols – a review. Trends in Food 

Science & Technology, 21(10), 510–523. https://doi.org/10.1016/J.TIFS.2010.08.003 

Fernandez, M. A., & Marette, A. (2017). Potential Health Benefits of Combining Yogurt and 

Fruits Based on Their Probiotic and Prebiotic Properties. Advances in Nutrition, 8(1), 155S-

164S. https://doi.org/10.3945/AN.115.011114 

Gao, Z., Shen, P., Lan, Y., Cui, L., Ohm, J. B., Chen, B., & Rao, J. (2020). Effect of alkaline 

extraction pH on structure properties, solubility, and beany flavor of yellow pea protein 

isolate. Food Research International, 131, 109045. 

https://doi.org/10.1016/J.FOODRES.2020.109045 

Garbuzynskiy, S. O., Lobanov, M. Yu., & Galzitskaya, O. v. (2004). To be folded or to be 

unfolded? Protein Science : A Publication of the Protein Society, 13(11), 2871. 

https://doi.org/10.1110/PS.04881304 

Grace, M. H., Ribnicky, D. M., Kuhn, P., Poulev, A., Logendra, S., Yousef, G. G., Raskin, I., & 

Lila, M. A. (2009). Hypoglycemic activity of a novel anthocyanin-rich formulation from 

lowbush blueberry, Vaccinium angustifolium Aiton. Phytomedicine, 16(5), 406–415. 

https://doi.org/10.1016/J.PHYMED.2009.02.018 

Gundersen, C., Kreider, B., & Pepper, J. (2011). The Economics of Food Insecurity in the United 

States. Applied Economic Perspectives and Policy, 33(3), 281–303. 

https://doi.org/10.1093/AEPP/PPR022 

Huang, Q., Braffett, B. H., Simmens, S. J., Young, H. A., & Ogden, C. L. (2020). Dietary 

Polyphenol Intake in US Adults and 10-Year Trends: 2007-2016. Journal of the Academy of 

Nutrition and Dietetics, 120(11), 1821–1833. https://doi.org/10.1016/j.jand.2020.06.016 

Jiang, J., Zhang, Z., Zhao, J., & Liu, Y. (2018). The effect of non-covalent interaction of 

chlorogenic acid with whey protein and casein on physicochemical and radical-scavenging 

activity of in vitro protein digests. Food Chemistry, 268, 334–341. 

https://doi.org/10.1016/J.FOODCHEM.2018.06.015 

Jiao, X., Li, B., Zhang, Q., Gao, N., Zhang, X., & Meng, X. (2018). Effect of in vitro-simulated 

gastrointestinal digestion on the stability and antioxidant activity of blueberry polyphenols 

and their cellular antioxidant activity towards HepG2 cells. International Journal of Food 

Science & Technology, 53(1), 61–71. https://doi.org/10.1111/IJFS.13516 

Jiao, X., Wang, Y., Lin, Y., Lang, Y., Li, E., Zhang, X., Zhang, Q., Feng, Y., Meng, X., & Li, B. 

(2019). Blueberry polyphenols extract as a potential prebiotic with anti-obesity effects on 

C57BL/6 J mice by modulating the gut microbiota. The Journal of Nutritional 

Biochemistry, 64, 88–100. https://doi.org/10.1016/J.JNUTBIO.2018.07.008 

Kang, J., Thakali, K. M., Jensen, G. S., & Wu, X. (2015). Phenolic Acids of the Two Major 

Blueberry Species in the US Market and Their Antioxidant and Anti-inflammatory 

Activities. Plant Foods for Human Nutrition, 70(1), 56–62. https://doi.org/10.1007/S11130-

014-0461-6/FIGURES/2 



 43 

 

Katherine Hoy, M., Clemens, J. C., Martin, C. L., & Moshfegh, A. J. (2020). Fruit and Vegetable 

Consumption of US Adults by Level of Variety, What We Eat in America, NHANES 2013–
2016. Current Developments in Nutrition, 4(3). https://doi.org/10.1093/CDN/NZAA014 

Kosińska, A., Karamać, M., Penkacik, K., Urbalewicz, A., & Amarowicz, R. (2011). Interactions 

between tannins and proteins isolated from broad bean seeds (Vicia faba Major) yield 

soluble and non-soluble complexes. European Food Research and Technology, 233(2), 

213–222. https://doi.org/10.1007/S00217-011-1506-9/FIGURES/6 

Kuntz, S., Rudloff, S., Asseburg, H., Borsch, C., Fröhling, B., Unger, F., Dold, S., Spengler, B., 

Römpp, A., & Kunz, C. (2015). Uptake and bioavailability of anthocyanins and phenolic 

acids from grape/blueberry juice and smoothie in vitro and in vivo. British Journal of 

Nutrition, 113(7), 1044–1055. https://doi.org/10.1017/S0007114515000161 

Lau, F. C., Joseph, J. A., McDonald, J. E., & Kalt, W. (2009). Attenuation of iNOS and COX2 

by blueberry polyphenols is mediated through the suppression of NF-κB activation. Journal 

of Functional Foods, 1(3), 274–283. https://doi.org/10.1016/J.JFF.2009.05.001 

Li, D., Yang, Y., Yang, X., Wang, Z., Yao, X., & Guo, Y. (2022). Enhanced bioavailability and 

anti-hyperglycemic activity of young apple polyphenols by complexation with whey protein 

isolates. Journal of Food Science. https://doi.org/10.1111/1750-3841.16062 

Liu, Y., Wang, D., Zhang, D., Lv, Y., Wei, Y., Wu, W., Zhou, F., Tang, M., Mao, T., Li, M., & 

Ji, B. (2011). Inhibitory Effect of Blueberry Polyphenolic Compounds on Oleic Acid-

Induced Hepatic Steatosis in Vitro. Journal of Agricultural and Food Chemistry, 59(22), 

12254–12263. https://doi.org/10.1021/JF203136J 

Louis, X. L., Thandapilly, S. J., Kalt, W., Vinqvist-Tymchuk, M., Aloud, B. M., Raj, P., Yu, L., 

Le, H., & Netticadan, T. (2014). Blueberry polyphenols prevent cardiomyocyte death by 

preventing calpain activation and oxidative stress. Food & Function, 5(8), 1785–1794. 

https://doi.org/10.1039/C3FO60588D 

Mäkinen, O. E., Wanhalinna, V., Zannini, E., & Arendt, E. K. (2015). Foods for Special Dietary 

Needs: Non-dairy Plant-based Milk Substitutes and Fermented Dairy-type Products. 

Https://Doi.Org/10.1080/10408398.2012.761950, 56(3), 339–349. 

https://doi.org/10.1080/10408398.2012.761950 

Malomo, S. A., & Aluko, R. E. (2015). A comparative study of the structural and functional 

properties of isolated hemp seed (Cannabis sativa L.) albumin and globulin fractions. Food 

Hydrocolloids, 43, 743–752. https://doi.org/10.1016/J.FOODHYD.2014.08.001 

Malomo, S. A., He, R., & Aluko, R. E. (2014). Structural and Functional Properties of Hemp 

Seed Protein Products. Journal of Food Science, 79(8), C1512–C1521. 

https://doi.org/10.1111/1750-3841.12537 

Mamone, G., Picariello, G., Ramondo, A., Nicolai, M. A., & Ferranti, P. (2019). Production, 

digestibility and allergenicity of hemp (Cannabis sativa L.) protein isolates. Food Research 

International, 115, 562–571. https://doi.org/10.1016/j.foodres.2018.09.017 

Manus, J., Millette, M., Uscanga, B. R. A., Salmieri, S., Maherani, B., & Lacroix, M. (2021). In 

vitro protein digestibility and physico-chemical properties of lactic acid bacteria fermented 

beverages enriched with plant proteins. Journal of Food Science, 86(9), 4172–4182. 

https://doi.org/10.1111/1750-3841.15859 

McAnulty, S. R., McAnulty, L. S., Nieman, D. C., Dumke, C. L., Morrow, J. D., Utter, A. C., 

Henson, D. A., Proulx, W. R., & George, G. L. (2004). Consumption of blueberry 

polyphenols reduces exercise-induced oxidative stress compared to vitamin C. Nutrition 

Research, 24(3), 209–221. https://doi.org/10.1016/J.NUTRES.2003.10.003 



 44 

 

McClements, D. J., & Grossmann, L. (2021). The science of plant-based foods: Constructing 

next-generation meat, fish, milk, and egg analogs. Comprehensive Reviews in Food Science 

and Food Safety, 20(4), 4049–4100. https://doi.org/10.1111/1541-4337.12771 

Mendly-Zambo, Z., Powell, L. J., & Newman, L. L. (2021). Dairy 3.0: cellular agriculture and 

the future of milk. Https://Doi.Org/10.1080/15528014.2021.1888411, 24(5), 675–693. 

https://doi.org/10.1080/15528014.2021.1888411 

Meng, Y., & Li, C. (2021). Conformational changes and functional properties of whey protein 

isolate-polyphenol complexes formed by non-covalent interaction. Food Chemistry, 364, 

129622. https://doi.org/10.1016/J.FOODCHEM.2021.129622 

Ming, Y., Chen, L., Khan, A., Wang, H., & Wang, C. (2020). Effects of tea polyphenols on 

physicochemical and antioxidative properties of whey protein coating. Food Science and 

Biotechnology, 29(12), 1655. https://doi.org/10.1007/S10068-020-00824-5 

Moore, S., & Stein, W. H. (1954). A MODIFIED NINHYDRIN REAGENT FOR THE 

PHOTOMETRIC DETERMINATION OF AMINO ACIDS AND RELATED 

COMPOUNDS. Journal of Biological Chemistry, 211, 907–913. 

https://doi.org/10.1016/S0021-9258(18)71178-2 

Morissette, A., Kropp, C., Songpadith, J. P., Moreira, R. J., Costa, J., Mariné-Casadó, R., Pilon, 

G., Varin, T. v., Dudonné, S., Boutekrabt, L., St-Pierre, P., Levy, E., Roy, D., Desjardins, 

Y., Raymond, F., Houd, V. P., & Marette, A. (2020). Blueberry proanthocyanidins and 

anthocyanins improve metabolic health through a gut microbiota-dependent mechanism in 

diet-induced obese mice. American Journal of Physiology - Endocrinology and Metabolism, 

318(6), E965–E980. 

https://doi.org/10.1152/AJPENDO.00560.2019/ASSET/IMAGES/LARGE/ZH1005208340

0008.JPEG 

Ni, H., Hayes, H., Stead, D., Liu, G., Yang, H., Li, H., & Raikos, V. (2020). Interaction of whey 

protein with polyphenols from salal fruits (Gaultheria shallon) and the effects on protein 

structure and hydrolysis pattern by Flavourzyme®. International Journal of Food Science 

& Technology, 55(3), 1281–1288. https://doi.org/10.1111/IJFS.14394 

Oidtmann, J., Schantz, M., Mäder, K., Baum, M., Berg, S., Betz, M., Kulozik, U., Leick, S., 

Rehage, H., Schwarz, K., & Richling, E. (2012). Preparation and Comparative Release 

Characteristics of Three Anthocyanin Encapsulation Systems. Journal of Agricultural and 

Food Chemistry, 60(3), 844–851. https://doi.org/10.1021/JF2047515 

Ozdal, T., Capanoglu, E., & Altay, F. (2013). A review on protein–phenolic interactions and 

associated changes. Food Research International, 51(2), 954–970. 

https://doi.org/10.1016/J.FOODRES.2013.02.009 

Reynaud, Y., Lopez, M., Riaublanc, A., Souchon, I., & Dupont, D. (2020). Hydrolysis of plant 

proteins at the molecular and supra-molecular scales during in vitro digestion. Food 

Research International, 134, 109204. https://doi.org/10.1016/j.foodres.2020.109204 

Ribnicky, D. M., Roopchand, D. E., Poulev, A., Kuhn, P., Oren, A., Cefalu, W. T., & Raskin, I. 

(2014). Artemisia dracunculus L. polyphenols complexed to soy protein show enhanced 

bioavailability and hypoglycemic activity in C57BL/6 mice. Nutrition, 30(7–8), S4–S10. 

https://doi.org/10.1016/J.NUT.2014.03.009 

Rodriguez-Mateos, A., Rendeiro, C., Bergillos-Meca, T., Tabatabaee, S., George, T. W., Heiss, 

C., & Spencer, J. P. E. (2013). Intake and time dependence of blueberry flavonoid–induced 

improvements in vascular function: a randomized, controlled, double-blind, crossover 



 45 

 

intervention study with mechanistic insights into biological activity. The American Journal 

of Clinical Nutrition, 98(5), 1179–1191. https://doi.org/10.3945/AJCN.113.066639 

Roopchand, D. E., Kuhn, P., Krueger, C. G., Moskal, K., Lila, M. A., & Raskin, I. (2013). 

Concord Grape Pomace Polyphenols Complexed to Soy Protein Isolate Are Stable and 

Hypoglycemic in Diabetic Mice. Journal of Agricultural and Food Chemistry, 61(47), 

11428–11433. https://doi.org/10.1021/JF403238E 

Ryan, J. M., White, C. H., Gough, R. H., & Burns, A. C. (1984). Consumer Acceptance of Fruit-

Flavored Yogurt Drinks. Journal of Dairy Science, 67(7), 1369–1375. 

https://doi.org/10.3168/JDS.S0022-0302(84)81450-2 

S eczyk, L., Swieca, M., Kapusta, I., & Gawlik-Dziki, U. (2019). Protein–Phenolic Interactions 

as a Factor Affecting the Physicochemical Properties of White Bean Proteins. Molecules 

2019, Vol. 24, Page 408, 24(3), 408. https://doi.org/10.3390/MOLECULES24030408 

Samtiya, M., Aluko, R. E., & Dhewa, T. (2020). Plant food anti-nutritional factors and their 

reduction strategies: an overview. Food Production, Processing and Nutrition 2020 2:1, 

2(1), 1–14. https://doi.org/10.1186/S43014-020-0020-5 

Satija, A., & Hu, F. B. (2018). Plant-based diets and cardiovascular health. Trends in 

Cardiovascular Medicine, 28(7), 437–441. https://doi.org/10.1016/j.tcm.2018.02.004 

Schneider, M., Esposito, D., Lila, M. A., & Foegeding, E. A. (2016). Formation of whey 

protein–polyphenol meso-structures as a natural means of creating functional particles. 

Food & Function, 7(3), 1306–1318. https://doi.org/10.1039/C5FO01499A 

Shen, P., Gao, Z., Fang, B., Rao, J., & Chen, B. (2021). Ferreting out the secrets of industrial 

hemp protein as emerging functional food ingredients. Trends in Food Science & 

Technology, 112, 1–15. https://doi.org/10.1016/J.TIFS.2021.03.022 

Shen, P., Gao, Z., Xu, M., Ohm, J. B., Rao, J., & Chen, B. (2020). The impact of hempseed 

dehulling on chemical composition, structure properties and aromatic profile of hemp 

protein isolate. Food Hydrocolloids, 106, 105889. 

https://doi.org/10.1016/J.FOODHYD.2020.105889 

Stone, A. K., Karalash, A., Tyler, R. T., Warkentin, T. D., & Nickerson, M. T. (2015). 

Functional attributes of pea protein isolates prepared using different extraction methods and 

cultivars. Food Research International, 76(P1), 31–38. 

https://doi.org/10.1016/J.FOODRES.2014.11.017 

Štreimikytė, P., Keršienė, M., Eisinaitė, V., Jasutienė, I., Lesauskaitė, V., Damulevičienė, G., 
Knašienė, J., & Leskauskaitė, D. (2020). Formulating protein-based beverages for the 

dysphagia diets of the elderly: viscosity, protein quality, in vitro digestion, and consumers 

acceptability. Journal of the Science of Food and Agriculture, 100(10), 3895–3901. 

https://doi.org/10.1002/JSFA.10431 

Tang, H. Y., Fang, Z., & Ng, K. (2020). Dietary fiber-based colon-targeted delivery systems for 

polyphenols. Trends in Food Science & Technology, 100, 333–348.