SENSORY COMPARISON OF LOW-PROTEIN RECIPES WITH GLYCOMACROPEPTIDE-

CONTAINING BETTERMILK™ AND LIQUID NON-DAIRY COFFEE CREAMER

AMONG ADULTS WITH PHENYLKETONURIA

by

AMELIA FOSTER

KRISTI CROWE-WHITE LINDA KNOL RANI SINGH

A THESIS

Submitted in partial fulfillment of the requirements for the degree of Master of Science

in the Department of Human Nutrition and Hospitality Management in the Graduate School of

The University of Alabama

TUSCALOOSA, ALABAMA

2014

26

Copyright Amelia Foster 2014 ALL RIGHTS RESERVED

26

ABSTRACT

Phenylketonuria (PKU) results from deficiency of phenylalanine hydroxylase enzyme

which is responsible for converting the essential amino acid phenylalanine to the non-essential

amino acid tyrosine. PKU is treated with a low-protein diet and medical food/ formula providing

supplemental protein without excess phenylalanine.

Naturally phenylalanine-free, Glycomacropeptide (GMP) used in PKU medical foods/

formulas contains four essential amino acids and is fortified with methionine, leucine, histidine,

tryptophan, and tyrosine to provide a near complete protein. Although sensory research has been

conducted on GMP-based foods, sensory evaluation of commonly utilized low-protein recipes

substituted with GMP-based formulas is undocumented. Such research is critical to development

of appealing means for formula consumption. Study objectives were to compare sensory

attributes (taste, aroma, texture, and overall acceptability) and preferences for low-protein cream

of tomato soup, lemon pudding, and vanilla ice cream containing either a GMP-based formula or

unflavored liquid non-dairy coffee creamer as the primary liquid ingredient and to solicit medical

formula/food procurement information among adults with PKU.

Data were subjected to paired t-tests with the Bonferroni correction (p< 0.0125) and

analysis of variance. Thirty-one adults (80% female, 93% white) completed the study.

Significantly higher scores (p<0.01) were reported for aroma, taste, and overall

acceptability of the control soup and for all four sensory attributes of the control pudding.

Control ice cream was ranked significantly higher (p<0.01) in taste and overall acceptability. No

ii 26

significant differences existed among scores of the four sensory attributes within each control or

experimental recipe. Control samples were preferred over experimental samples for all recipes.

Experimental samples for each recipe were on average scored in the “like” range. Only

23% of the sample reported they frequently consume medical foods, while equal numbers of

participants reported they were/were not interested in using a medical food in preparation of low-

protein recipes. These data may partially explain why control samples were preferred.

Additionally, lower sensory scores for taste and aroma of the experimental soup, pudding, and

ice cream may be attributed to the probiotics and docosahexaenoic acid within the GMP-based

formula as both of these functional ingredients are capable of imparting strong flavors and

aromas to foods. Additional research is needed to investigate the use of GMP-based formulas

without added functional ingredients and isolated GMP, not GMP-based formulas, in low-protein

recipes in order to expand dietary offerings for individuals with PKU.

iii 26

LIST OF ABBREVIATIONS AND SYMBOLS

g Gram, a unit of measure of mass

kcal Kilocalories, a unit of measure of energy

mg Milligram, a unit of measure of mass

< Less than

> Greater than

≥ Greater than or equal to

= Equal to

± Plus or minus

% Percent

iv 26

ACKNOWLEDGEMENTS

I would like to first express my sincere appreciation to Dr. Kristi Crowe-White for her

constant encouragement and unwavering commitment to the success of this project. I would also

like to thank my thesis committee, which includes Dr. Kristi Crowe-White, Dr. Linda Knol, and

Dr. Rani Singh for their suggestions and ideas in the formation of this thesis. Without the

expertise and guidance of this committee, this project would not have been possible.

I would like to thank Cambrooke Therapeutics, Inc. for providing the BetterMilk™ for

the study, as well as the nutrition facts panels which correlate to each recipe. I sincerely

appreciate the staff of Emory University’s Metabolic Camp for their willingness to accommodate

whatever needs I had in preparation for sensory testing, including providing dry ice for

maintaining the temperature of ice cream samples. I would also like to thank the National PKU

Alliance for allowing me the opportunity to conduct research as part of their event.

Thank you to my supportive husband and my best friend, Kevin, and to our family and

friends for their prayers. Most of all, I would like to express my thanks to God for allowing me

to be among those born with PKU after newborn screening was implemented. It is my goal to use

the potential I have been given to use nutrition to improve the lives of others living with inborn

errors of metabolism.

v 26

CONTENTS

ABSTRACT……………………………………………………………………………….......….ii

LIST OF ABBREVIATIONS AND SYMBOLS………………………………………….....…..iv

ACKNOWLEDGEMENTS…………………………………………………………………....…v

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

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

1 INTRODUCTION………………….……………………………………………….……..…....1

2 LITERATURE REVIEW……………………………………………………………….…........3

3 METHODS………………………………………………………………………….………....15

4 RESULTS…………………………………………………………………………….………..21

5 DISCUSSION………………………………………………………………………….…........26

REFERENCES…………………………………………………………………………………..31

APPENDICES…………………………………………………………………………………...35

vi 26

LIST OF TABLES

Table 1: Protein and Phenylalanine Content of Recipes Per Standard Serving According to the

United States Department of Agriculture National Nutrient Database…………………….…….20

Table 2: Comparative Analyses of Sensory Attribute Scores for Experimental and Control

Recipes……………………………………………………………………………………….…..23

Table 3: Demographics…………………………………………………………………………..23

Table 4: Medical Food Procurement and Use……………………………………..…………….24

vii 26

LIST OF FIGURES

Figure 1: Nine-point Hedonic Scale for Sensory Testing……………………………..…………20

Figure 2: Participant Responses for not Consuming all Daily Prescribed Medical Food……….25

viii 26

CHAPTER 1

INTRODUCTION

As of October 2000, one in every fifteen thousand individuals in the United States is

living with an inborn error of metabolism called phenylketonuria (PKU).1 This disease results

from a genetic anomaly which causes the affected individual to lack the enzyme which converts

the essential amino acid phenylalanine to tyrosine, the non-essential amino acid product of

phenylalanine hydrolysis. The resulting accumulation of phenylalanine is toxic to the brain and

can cause severe mental and developmental abnormalities.2

Since the time that newborn screening for birth defects was implemented in the early

1960’s, PKU has been treated with a lifelong low-protein diet (low-phenylalanine diet). Success

of dietary intervention in individuals with PKU requires consumption of an amino acid-based

formula and adherence to a low-protein diet. This formula serves as a protein supplement,

providing all essential amino acids except phenylalanine, along with the amino acid essential for

individuals with PKU, tyrosine. Formulas or other products containing supplemental protein

which serve the same function have recently been referred to in the product development realm

as medical foods for PKU.3 Although these products are important to the health of individuals

with PKU, they can be expensive and subsequently difficult for patients and caregivers to

obtain.4

Over the past six years, medical food companies have been experimenting with

glycomacropeptide (GMP), incorporating it into medical formulas and products for PKU diet

therapy.5 GMP is a protein extracted from cheese whey which is naturally free of phenylalanine.6

1 26

However, commercially available GMP contains a small amount of phenylalanine from

contamination by other whey proteins during extraction and processing.6 In one study, significant

decreases in blood phenylalanine levels of individuals with PKU were observed through

replacement of amino acid-based medical foods with medical foods containing GMP.2 GMP-

based medical foods may contribute to satiety more than amino acid-based medical foods,

causing individuals with PKU to consume less phenylalanine from “regular” food sources.7

Additionally, the low-protein diet is often high in fat and carbohydrate and, thus, higher in total

calories than a standard diet. Therefore, individuals with PKU may gain excess weight

throughout their lifespan. In contrast, GMP-based medical foods may promote needed weight

control by contributing to earlier satiety due to the near complete protein characteristics of

GMP.7 Of great importance, GMP-based medical foods have been shown in sensory testing to

have a higher acceptability than traditional amino acid-based medical formulas.5 These factors

create an identity for GMP as a seemingly ideal alternative to standard amino acid formulas for

treatment of PKU.

Gaps in the research on GMP use in medical foods and formulas for PKU exist. To date,

incorporation of GMP-based medical foods or formulas into widely-utilized low-protein recipes

is undocumented.8 Since PKU patients rely on both low-phenylalanine protein formulas and low

protein recipes, incorporation of GMP-based products and formulas should be investigated in a

variety of recipe types to assist in dietary compliance and maintenance of this inborn error of

metabolism. If these formulas are sensorially acceptable in low protein recipes, this would yield

an alternative means of formula consumption which may lead to greater therapeutic compliance.

2 26

CHAPTER 2

LITERATURE REVIEW

The following section provides a review of the scientific literature regarding PKU

including implications of the disease along with daily challenges regarding protein restriction

and subsequent amino acid supplementation. Also, the functional and chemical properties of

GMP are reviewed as well as evidence suggesting the positive role that GMP may play in PKU

management. Since GMP is an emerging ingredient in medical foods and formulas for PKU,

sensory testing of these products will be addressed. The literature review will conclude with a

discussion of costs, procurement methods, and reimbursement for PKU medical foods.

PKU AND ITS MANAGEMENT

PKU is an inborn error of metabolism which was first identified in 1934 by Norwegian

physician Dr. Asbjorn Folling, when a desperate mother asked him to examine her mentally

challenged children in order to determine a cause for their condition.9 Following the discovery of

PKU, Dr. Horst Bickel introduced the concept of a diet low in phenylalanine for PKU therapy in

the 1950’s.10 However, the diet was limited in its impact, since differentiation of PKU from

general mental/physical disabilities in infancy was a challenge.

In 1962, Dr. Robert Guthrie created a screening test for PKU. Prior to the availability of

this screening test, many children with the disease rapidly became severely mentally disabled

and were subsequently institutionalized for the duration of their lives.11 Since the development of

this screening test, it is now required by law in the United States, as well as many other nations

for newborns to be screened between 48 to 72 hours of age. For example, the heel of a newborn

3 26

is pricked and the blood is pressed to a piece of filter paper. A hole is punched in the dried blood

spot, yielding a small disc of the sample. This disc is then analyzed for phenylalanine content. A

positive test for PKU is a blood phenylalanine level of 6-80 milligrams per deciliter.12 If this

screening test is positive, subsequent testing of blood and urine samples will be conducted in

order to confirm a PKU diagnosis. This system for early detection has resulted in significant

improvement in the prognoses of individuals with PKU.

The primary goal of PKU diet therapy is to maintain blood phenylalanine levels within

the recommended range of two to six milligrams per deciliter.12 This may be achieved through

adherence to a prescribed low-phenylalanine diet, along with consumption of a phenylalanine-

free or low-phenylalanine medical food. The prescribed diet is based on the specific

phenylalanine tolerance of the PKU patient, since variances in phenylalanine tolerance exist

among individuals with PKU. The average phenylalanine tolerance of individuals with PKU is

250 milligrams per day in Classical PKU (the rarest and most restrictive type) and 200-450

milligrams per day in moderate PKU. 13 Factors which may cause differences in phenylalanine

tolerance among individuals with PKU include genetics, age, lifestyle, and medication. As such,

there is no standard prescribed diet for PKU.14 Each PKU patient requires a diet of phenylalanine

restriction and supplemental protein provision which is specifically tailored to meet the needs of

the individual. Similarly, every PKU patient also has unique protein needs. Factors which

influence protein needs in PKU are no different from those which influence protein needs in

individuals without PKU. Such factors include age, gender, body mass index (BMI), and activity

level among others. Each gram of dietary protein allotted in the low-phenylalanine diet for PKU

4 26

is correlated with roughly 50 milligrams of phenylalanine.15 For example, a phenylketonuric

patient whose phenylalanine tolerance is 400 milligrams and protein needs are 45 grams per day

would be said to have a dietary protein allotment of eight grams of protein per day. The

remaining protein needs of this patient would be 37 grams which would be met through

supplementation of a low-phenylalanine diet with a PKU medical formula.

Restriction of phenylalanine in the diet translates into dietary protein restriction, since

nearly all dietary proteins contain phenylalanine.16 As a result, protein supplementation is a

necessity for meeting the nutritional needs of individuals with PKU. The supplemental protein

required in the PKU diet is either phenylalanine-free or low in phenylalanine. These

supplemental formulas or foods provide all essential amino acids except phenylalanine, along

with tyrosine, the non-essential amino acid product of phenylalanine hydrolysis.

In the early years of PKU treatment, it was generally believed that the low-phenylalanine

diet for PKU could safely be discontinued once the brain had completely developed

(approximately age four).17 However, it has been found that individuals with PKU can

experience memory and attention impairments, among other problems, when blood

phenylalanine levels are poorly controlled at any age.18 As such, the current consensus for PKU

treatment, pioneered by Dr. Richard Koch in the late 1970’s, is a life-long low-phenylalanine

diet.19

Poor dietary compliance is common among individuals with PKU. Noncompliance issues

may be the result of the restrictive nature of the low-phenylalanine diet. Such issues may be

caused by length of time on such restrictive diets or the fact that amino acid-based medical foods

5 26

and formulas often possess generally offensive sensory characteristics including unsatisfactory

taste and aroma.20 In fact, it has been noted that the taste of amino acid formulas was a major

deterrent for individuals with PKU to adhere to the low-phenylalanine diet.21 Of interest, it has

been documented that compliance to the PKU diet may be particularly poor in adolescence and

early adulthood.22 In contrast, one study evaluating dietary compliance as it relates to quality of

life in individuals with PKU over three years of age suggested that dietary compliance was

unrelated to age in PKU.23 Additionally, this study found no significant relationship between diet

adherence and quality of life in subjects.23 To date, it is unknown whether phenylketonuric

individuals of a certain gender are more likely to adhere to the low-phenylalanine diet for PKU.

GMP: AN ALTERNATIVE INGREDIENT IN PKU MEDICAL FOODS

GMP requires supplementation with five essential amino acids including tryptophan,

methionine, leucine, histidine, and tyrosine (which is an essential amino acid for individuals with

PKU) in order to be a near complete protein minus phenylalanine.7 Both GMP- and amino acid-

based medical foods and formulas are available in formulations of varying protein quantities.

Many of these contain 10 or 15 grams of protein per serving. Individuals with PKU may

consume any combination of these products in order to meet their phenylalanine-free or low-

phenylalanine protein needs. However, metabolic dietitians typically ask PKU patients or

caregivers to commit to a certain amount of each type of medical food or formula per day, if the

PKU patient chooses to use more than one type of product. This ensures that PKU patients are

meeting, but not exceeding, protein needs. This also applies to other nutrients provided by many

medical foods for PKU such as vitamins, minerals, sugar, total carbohydrate, and fat.

6 26

The functional properties of GMP allow for variety in GMP-based medical foods and

formulas as these products are more stable to high temperatures and among acidic ingredients

compared to amino acid-based medical foods for PKU.6 As a result of such stability, GMP offers

versatility of consumption in various product forms. At present, GMP-based medical foods are

available in meal replacement bars, sports drinks, formulas, cereals, and puddings supplemented

with additional amino acids. One such formulation, Glytactin (Cambrooke Therapeutics, Inc.,

Ayer, MA) is a formulation containing GMP, vitamins and minerals, probiotics, omega-3 fatty

acids, and the limiting amino acids required for GMP to be considered a nearly complete protein

minus phenylalanine. Products containing Glytactin include BetterMilk which is a formula

powder resembling milk when reconstituted with water, Restore which is an electrolyte-

containing sports drink, Swirl which is a powder consumed as either a pudding or a smoothie

depending on the amount of added water, Complete which is a meal replacement bar, and

Ready-to-Drink (RTD) which is a concentrated, prepared formula.

In addition to the variety of GMP-based medical foods available, certain medical food

companies which manufacture these formulas make recipes available to consumers for

incorporation into various recipes.24 Given the stability of GMP and its versatility of use, GMP-

based medical foods and formulas for PKU may prove to be considerably more attractive to

individuals with PKU compared to amino acid-based formulas. This is due to the fact that amino

acid-based formulas lack heat stability and are not compatible with a range of food ingredients

and recipe formulations. As such, amino acid-based formulas can rarely be used as an ingredient

7 26

in low-phenylalanine recipes. Additional research is needed to demonstrate sensory acceptability

of low-protein recipes using GMP-based medical foods and formulas.

CURRENT RESEARCH ON GMP

GMP is a natural constituent of whey protein from bovine milk. It is isolated by the

enzyme rennin acting on the bond between the amino acids phenylalanine and methionine.25

GMP in itself does not contain phenylalanine; however, commercially-available GMP contains

trace amounts of phenylalanine due to contamination from other whey proteins during the

extraction process.26 GMP does not contain five of the nine essential amino acids including

tyrosine, tryptophan, methionine, histidine, and leucine. The addition of these amino acids, along

with certain vitamins and minerals, in GMP-based medical foods and formulas allows for

marketing as nutritionally-appropriate replacements for amino acid-based medical foods and

formulas for PKU.27

Isolated GMP possesses many functional attributes in food systems which are attributed

to its protein content. Although its functions may differ from a truly complete protein, GMP may

impact gel formation, water-binding ability, emulsification, foaming abilities, and bioactivity

upon consumption.28, 29 Also of importance, GMP is relatively stable to heat and acidic

ingredients, unlike amino acid-based formulas.6

In a crossover study design to evaluate the safety and impact of GMP-based medical

foods for PKU on blood amino acid concentrations, 11 patients with PKU ages 11-31 years were

provided a four-day amino acid medical food diet and a subsequent four-day GMP-based

medical food diet.30 No significant differences were observed in mean postprandial blood

8 26

phenylalanine levels of subjects after GMP-based medical food use versus amino acid-based

medical food use. However, ten of the 11 subjects preferred GMP-based medical foods in

supplementation of the low-protein diet as opposed to their usual amino acid-based medical

foods.

Preference for GMP-based medical foods over traditional amino acid-based medical

foods for PKU is not uncommon. For example, it was found in sensory studies conducted among

individuals with PKU that products containing isolated GMP (a sports drink, a chocolate drink, a

pudding, a fruit leather, and snack crackers) resulted in higher overall acceptability than similar

products which were either amino acid-based medical foods or products modified to be low in

protein.5 The phrase modified to be low in protein refers to food products substituted for

“regular” foods which are often too high in protein to be included in the PKU diet.31 Such foods

commonly include rice, pasta, and bread.

In another study, sensory characteristics of a GMP-based drink for PKU diet therapy

were investigated.32 Children and adult subjects were asked to rate the drink as ‘very good’,

‘good’, ‘acceptable’, or ‘not acceptable’ in each of the categories of appearance, color, odor,

flavor, and overall acceptability. The experimental drink was found to be acceptable in color, and

good in appearance, odor, flavor, and overall acceptability. Although sensory research has been

conducted on marketed food products formulated with GMP, there is a void in sensory research

evaluating commonly relied-upon low-protein recipes substituted with GMP-based formulas in

place of liquid low-phenylalanine ingredients. Information regarding sensory acceptability of

low-phenylalanine recipes incorporating GMP-based formulas and foods would expand the use

9 26

of formulas while identifying more appealing means for formula consumption. For this reason,

the proposed study aims to analyze the sensory characteristics of low-protein recipes prepared

with a GMP-based formula versus those prepared with unflavored liquid non-dairy creamer as

the primary liquid low-phenylalanine ingredient.

Given the preference for and overall acceptability of GMP-containing products, it has

been demonstrated that these products may positively impact satiety.7 For example, one study

conducted with healthy adults ages 18 to 40 years noted a positive correlation between GMP

intake and satiety.33 However, this did not cause a decrease in food intake among participants. In

another study, a significant (p = 0.01) decrease in appetite and suppression of postprandial

plasma ghrelin levels immediately after mealtime was noted in 11 phenylketonuric individuals

ages 11 to 31 years after consuming GMP-containing breakfasts.7 Plasma ghrelin, the hormone

which arouses hunger, is increased during energy deficit to initiate food consumption.27 This

outcome is particularly attractive, considering that individuals with PKU often encounter weight

management difficulties associated with the low-phenylalanine diet.34

As a result of the proposed effects of GMP on satiety, GMP has been investigated in

studies for weight reduction. For example, in one study investigating the effect of GMP-based

meal replacements on weight loss in adults without PKU, subjects in the intervention group

received daily meal replacements containing 27 g GMP for the first six months of the study and

daily meal replacements in the last six months of the study containing 13.5 g GMP.35 Subjects in

the control group received corresponding amounts of skim milk powder. Results suggest that

daily meal replacements with GMP had no significantly different effect than the control on

10 26

weight loss among 127 overweight or obese adults ages 20 to 70 years. Additionally, no

significant differences were noted between weight loss in subjects while receiving daily meal

replacements with either 27 g GMP or 13.5 g GMP.

Additionally, GMP is being investigated for its cholesterol- and inflammation-lowering

properties by reducing production of the hormone leptin as well as cytokines which contribute to

inflammation.36 In a study evaluating the effect of GMP on obesity, obese rats were divided into

four groups, receiving a high-fat diet with either no GMP, a small amount of GMP (100 mg/kg

body weight), a marginal amount of GMP (200 mg/kg body weight), or a high amount of GMP

(400 mg/kg body weight). Following six weeks of dietary intervention, obese rats on a high-fat

diet receiving any amount of GMP had significantly lower weight adipose tissue (p < 0.05) as

well as lower cholesterol levels and markers of inflammation. Subjects consuming the high-fat

diet with a high amount of GMP were found to have significantly decreased hepatic lipid

accumulation (p < 0.05) compared to all other groups. Body weight of subjects at the end of the

study was significantly lower (p < 0.05) in rats receiving a GMP-supplemented diet compared to

those receiving no GMP. In fact, body weight of subjects receiving GMP was decreased in a

dose-dependent fashion.

PROCUREMENT OF MEDICAL FOODS FOR PKU

Financial inability to acquire needed dietary supplements has been identified as a primary

reason for poor compliance among individuals with health issues which require dietary

supplements, including PKU.1,37 These individuals along with their caregivers may encounter

financial burdens in order to acquire medical foods. For example, in 2000, the last recorded

11 26

average annual cost of medical foods for PKU in the United States was $4,000 per patient per

year.38 Average medical food costs have risen considerably. For example, in 2012, it was

reported that the average annual cost of medical foods for individuals with PKU ages 14 and

older was over $7,000.39

In thirty-eight states, however, legislation is in place which requires insurance coverage

of medical foods for individuals with PKU.40 Unfortunately, many of these states allow

insurance providers to discontinue provision of medical food coverage benefits once insurance

customers with PKU reach eighteen years of age. This could potentially present problems in

medical food acquisition for individuals with PKU entering adulthood, since medical foods

continue to be vital to the success of PKU nutrition therapy during and past the age of eighteen.31

In the twelve states (including Alabama) which have never enacted legislation requiring

insurance reimbursement for PKU medical foods, families of children with PKU may be unable

to acquire the medical foods required for healthy growth and development of the affected child.

Costs encountered by individuals affected with PKU in states which either do not cover PKU

medical foods or which discontinue PKU medical foods coverage at the age of eighteen may be

much higher. For example, caregivers of individuals with PKU may encounter medical costs in

hospitalization or institutionalization exceeding those which would have been incurred in

covering medical foods for PKU as a maintenance treatment of the disease.37 In addition to the

impact of inadequate medical food acquisition to the affected individual, poor dietary

compliance or lack of medical foods in pregnant women with PKU will likely cause serious birth

defects in their children.41

12 26

In some states which do not require insurance reimbursement for PKU medical foods,

there may be alternative procurement methods in place for individuals and families affected. For

example, patient assistance programs may be offered by medical food companies which provide

discounted rates on medical foods based on income. Patient assistance programs may also serve

PKU families without health insurance. To their detriment, some individuals with PKU will not

receive medical foods due to insurance not reimbursing the cost, the lack of health insurance, or

inability to qualify for patient assistance programs.

Although not adopted into law, the “Medical Foods Equity Act of 2013” would have

required insurance coverage of medical foods for the entire lifespan of individuals with PKU.31

This would have increased the availability of medical foods and allowed individuals with PKU to

choose medical foods which would best suit their lifestyles regardless of cost. The opportunity

for consumption of desirable medical foods which are superior in taste and convenience versus

those which are more affordable, require preparation, and are often offensive in taste and aroma,

would potentially have improved adherence to the low-protein diet in individuals with PKU

regardless of age or financial status. Since these products are often significantly more expensive

than their amino acid-based counterparts, enactment of this bill as a law might have been

beneficial in increasing the availability of GMP-based medical foods for PKU. Additionally, the

National PKU Alliance (the primary non-profit organization serving individuals with PKU in

America) advocated for inclusion of medical foods for inborn errors of metabolism as a health

benefit under the “Affordable Care Act” for two years; however, the United States Department

13 26

of Health and Human Services finally decided to allow each state to determine which health

benefits it would provide its citizens.

14 26

CHAPTER 3

METHODS

INTRODUCTION

This study comparatively evaluated the sensory characteristics of low-protein recipes

containing a GMP-based formula, BetterMilk, to those containing unflavored non-dairy liquid

coffee creamer as the primary liquid low-phenylalanine ingredient. The following section will

discuss the methods of the study including participant selection criteria, recruitment, sensory and

nutritional analyses of tested recipes, and statistical analysis of all results.

RECIPE DEVELOPMENT

Study recipes were adapted from the PKU cookbook Low Protein Cookery for PKU

Third Edition in order to represent a variety of temperatures, flavors, and pH ranges in low-

protein recipes (Appendix A).8 Low-phenylalanine recipes of cream of tomato soup, lemon

pudding, and vanilla ice cream were prepared with unflavored non-dairy liquid coffee creamer as

the primary liquid low-phenylalanine ingredient in control recipes. BetterMilk served as the

main liquid ingredient in experimental recipes. A complete ingredient list and nutritional

information is provided for both BetterMilk (Appendix B) and Rich’s® Coffee Rich®, which

was the unflavored liquid non-dairy coffee creamer used in the control recipes (Appendix C).

BetterMilk is a GMP-based medical formula in the form of a powder which resembles

milk when mixed with water. The Glytactin formulation serving as its base is composed of

isolated GMP which does contain some phenylalanine (23 mg per packet) due to processing

contamination. The Glytactin formulation also contains methionine, leucine, histidine, and

15 26

tryptophan in addition to GMP, yet BetterMilk is not considered an amino acid-based medical

food due to the near complete protein nature of GMP.

Development and pilot sensory testing of the control and experimental vanilla ice cream

recipes were conducted with a direct (1:1) substitution of the experimental ingredient. In the

experimental cream of tomato soup and lemon pudding, sensory attributes were inferior to those

of control samples, thus alteration was required. For example, when BetterMilk™ was

substituted for unflavored non-dairy liquid coffee creamer in the cream of tomato soup, the

experimental recipe was too sweet. The experimental cream of tomato soup was improved by

omission of sugar, use of additional tomato juice instead of water, and preparation of

BetterMilk™ with tomato juice instead of water.

Upon testing the lemon pudding recipes, the experimental recipe was comparatively too

thin and formula-flavored. Sweetening and thickening were attempted using various amounts and

combinations of sugar, agar, tapioca, and cornstarch. Recipe modification which was found to

yield comparable sensory characteristics included use of instant lemon pudding mix instead

cook-type, omission of sugar, addition of whipped topping, and refrigeration for 24 hours prior

to serving.

SUBJECTS

The sample size calculation needed for statistical significance in this study was based on

a mean standard deviation between sensory characteristics (SD) of 2.0 using a 9-point hedonic

scale (Figure 1). Power level was set at 90% with α = 0.05. According to this calculation, the

16 26

required sample size for the study was 49. Prior to initiation, the study was approved by the

University of Alabama Institutional Review Board (see Appendix D).

Participants were recruited through informational flyers (Appendix E) posted at Emory

University Department of Human Genetics Metabolic Camp and at the National PKU Alliance

(NPKUA) Annual Conference. Sensory testing was conducted in the summer of 2014 on the

campus of Emory University in Atlanta, Georgia, and at the NPKUA Conference in Salt Lake

City, Utah.

Healthy individuals ages 19 and over previously diagnosed with PKU and without known

food allergies were recruited to taste and rate six low-phenylalanine samples as well as answer a

participant questionnaire. All study subjects were self-identified, thus participating on a

voluntary basis. An “explain and repeat back” method was used to determine the understanding

of potential subjects to willfully participate in the study. After reviewing the consent form

(Appendix F), potential participants were asked to briefly explain the purpose of the study and

identify their role in the study.

SAMPLE PREPARATION

Samples were prepared at sensory testing sites and stored in thermal carafes and coolers.

A digital thermometer was used to assess product temperature upon arrival at each testing facility

and every 30 minutes of testing to ensure samples were safe for consumption and appropriate for

sensory testing. Table1 provides information on the protein and phenylalanine content of control

and experimental recipes. This nutrition information is of particular interest to individuals with

PKU, since the goal of PKU diet therapy is to meet protein needs without exceeding

17 26

phenylalanine restrictions. Complete nutritional analyses for each prepared recipe are provided in

Appendix G.

SENSORY EVALUATION

Each participant was individually seated at a table and provided with a cup of water,

paper napkin, and pen. In accordance with standard sensory testing procedures, all samples were

coded with a randomly-generated numeric code, thus blinding the participants to control and

experimental samples. Samples were individually distributed in 2-tablespoon portions in

translucent testing bowls. The size of each sample was chosen in order to provide an amount

suitable for sensory testing while preventing sensory fatigue from an excessive serving size.

Upon providing consent, participants tested two samples per recipe evaluated. The

recipes were presented in the following order of decreasing acidity and temperature to prevent

sensory desensitization: cream of tomato soup, lemon pudding, and vanilla ice cream. Individual

samples of the control and experimental recipes were distributed to participants in random order

within recipe type. Participants rated each sample on a 9-point hedonic scale, where 1= dislike

extremely and 9= like extremely (Figure 1). The 9-point hedonic scale is the most frequently

used of the hedonic scales and has been widely used and accepted as an effective method for

determining acceptance of food products.42 Sensory attributes evaluated for each recipe included

taste, aroma, smell, and overall acceptability.

A sensory testing form (Appendix H) was provided with each sample. Participants were

asked to cleanse the palate between samples. Once both testing samples of each recipe had been

rated, participants were asked to indicate preference for one of the two samples on the second

18 26

sensory testing form per item using the following question: “Did you prefer the first or second

sample?”

The participant questionnaire was filled out following sensory analysis (Appendix I). The

participant questionnaire was developed to solicit basic demographic information (age, gender,

and race) through multiple-choice and fill-in-the-blank questions. Additionally, self-image of

healthiness of weight and diet status was assessed through multiple-choice questions, as was

individual preference and procurement of medical foods for PKU. Based on a 5-point Likert

scale where 1= strongly disagree and 5= strongly agree, participants responded to statements of

reasons they may not consume their full-prescribed daily amount of medical food.

STATISTICAL ANALYSIS

Descriptive statistics were used to determine means, standard deviations, and frequencies.

The Bonferroni correction (p< 0.0125) was determined based on the previous significance level

of 0.05 divided by 4 for each of the sensory characteristics compared. Paired t-tests with the

Bonferroni correction were used to determine differences in sensory attributes of control and

experimental recipes. Differences in the scores of sensory characteristics within recipes were

compared using analysis of variance (ANOVA). Data analysis was conducted using Statistical

Analysis Software, version 9.2 (SAS Institute, Cary, NC).

19 26

Figure 1: Nine-point Hedonic Scale for Sensory Testing

Table 1: Protein and Phenylalanine Content of Recipes Per Standard Serving According to the United States Department of Agriculture National Nutrient Database 42

Ice cream (1/2 c) Soup (1 c) Pudding (1/2 c) Control 0.8g, 29mg 1.4g, 40mg 0.5g, 18mg Experimental 5.8g, 32mg 8.6g, 62mg 6.7g, 20mg

20 26

CHAPTER 4

RESULTS

Sensory attribute scores (mean ± SD) for each recipe are provided in Table 2. In

comparative analysis of experimental and control samples, significantly higher scores were noted

for aroma (p=0.0002), taste (p=0.0002), and overall acceptability (p=0.0001) of the control

cream of tomato soup. The control lemon pudding received a significantly higher mean score for

aroma (p=0.0092), texture (p=0.0051), taste (p=0.0001), and overall acceptability (p=0.0001)

compared to the experimental lemon pudding. The control vanilla ice cream was ranked

significantly higher than the experimental vanilla ice cream in taste (p=0.0001) and overall

acceptability (p=0.0001). The greatest mean difference in sensory attribute scores for each recipe

was as follows: overall acceptability of the cream of tomato soup (1.61±1.89), taste of the lemon

pudding (2.71±2.15), and taste of the vanilla ice cream (2.23±2.33). As for preference, 22%, 9%,

and 16% of subjects favored the experimental cream of tomato soup, the experimental lemon

pudding, and the experimental vanilla ice cream, respectively. Thus, for each recipe evaluated,

the control recipes were preferred over experimental recipes. No significant differences were

observed among the scores of the four sensory attributes within each control or experimental

sample for any of these recipes. Thus, no one aspect of each sample appeared to be the

determining factor of preferential scoring.

Thirty-one participants completed the study. The mean age of the study participants was

30.2 years ± 9.2 with over 80% of the participants of female gender. Ethnicities were self-

reported. The complete demographic breakdown of the study population is provided in Table 3.

21 26

According to results of the participant questionnaire (Table 4), study subjects acquire medical

foods in many different ways. For example, 35.5% stated that they use other methods for medical

food procurement, 29% of subjects stated health insurance covers the entire cost, 25.8% of

subjects reported that a health insurance covers part of their medical food cost, 6.5% of subjects

declared that they participate in patient assistance programs, and 3.2% reported that they do not

consume medical foods. Also of interest, over 90% of subjects reported that if they were able to

obtain a medical food they preferred over the medical food they currently take, they would take

the full-prescribed daily amount every day. Figure 2 depicts responses given in the participant

questionnaire as to reasons individuals may not consume all of their prescribed medical food

daily. Among those who do not consume all of the prescribed formula daily, taste or smell of

formula impacts this decision for over 40% of participants. However among all participants, a

mean score of 2.5 ± 1.5 was given in response to the statement “I do not like how my formula

tastes” based on a 5-point Likert scale where 1= strongly disagree and 5= strongly agree,

whereas a mean score of 2.7 ± 1.6 was given in response to the statement “I do not like how my

formula smells”. Of particular importance to study outcomes, only 38.7% of the participants

reported that if they were able to obtain a formula product which were versatile enough to be

used in recipes that they would use it every day whereas an equal number (38.7%) of subjects

responded that they would never or occasionally use such a product in low-phenylalanine

recipes.

22 26

Table 2: Comparative Analyses of Sensory Attribute Scores

for Experimental and Control Recipes Control Experimental Cream of Tomato Soup

Aroma 7.55±1.34* 6.42±1.78 Texture 7.71±1.10 7.29±1.47

Taste 7.61±1.26* 6.0±2.24 Overall Acceptability 7.77±1.12* 6.13±2.14

Lemon Pudding Aroma 7.48±1.36* 6.48±1.81

Texture 7.87±0.92* 7.13±1.88 Taste 8.13±0.96* 5.42±2.33

Overall Acceptability 8.19±0.83* 5.74±2.27 Vanilla Ice Cream

Aroma 7.16±1.51 6.68±1.85 Texture 7.42±1.52 6.77±2.12

Taste 7.87±1.26* 5.65±2.47 Overall Acceptability 7.84±1.21* 5.84±2.42

mean ± standard deviation *Scores reflect the statistical difference between experimental and control samples (p<0.0125)

Table 3: Demographics Characteristic Amount Represented

Mean ± standard deviation

Age (y) 30.2 ± 9.2

n (%)

Sex Female 25 (80.6)

Male 6 (19.4) Ethnicity

White 29 (93.5) American Indian or Alaska Native 1 (3.2) Hispanic or Latino 1 (3.2)

23 26

24 26

Always consume all

30%

Dislike taste 21%

Dislike smell 23%

Too busy 26%

Figure 2: Participant Responses for not Consuming all Daily Prescribed Formula

25 26

CHAPTER 5

DISCUSSION

The aim of the present study was to comparatively assess the sensory attributes of low-

phenylalanine recipes made with either a GMP-containing formula, BetterMilk or unflavored

liquid non-dairy coffee creamer among adults with PKU. It was hypothesized that the recipes

containing BetterMilk™ would receive statistically similar sensory scores as the recipes prepared

using unflavored liquid non-dairy coffee creamer. The results of this study do not support this

hypothesis.

The experimental recipes were on average scored in the “like” range on the hedonic

scale; however, experimental samples of each recipe did not receive mean scores as high as the

control samples for each sensory attribute of each recipe. Additionally, control samples were

preferred over all experimental samples. Overall, not all sensory attributes of control samples

were scored significantly higher. For example, texture was not scored significantly differently

between the experimental and control cream of tomato soup. This may be attributed to the

temperature stability of BetterMilk™ to withstand extreme heat without causing recipe

ingredients to separate out of suspension, thus yielding an experimental recipe with similar

texture to the control. Likewise, texture of the experimental and control vanilla ice cream

samples was not rated significantly different. At low temperatures, BetterMilk™ does not

crystallize out of suspension, demonstrating the stability of GMP in extreme temperatures.6 As

such, the GMP content of BetterMilk lends the opportunity for greater application and usage in

prepared products than traditional amino acid-based formulas. In contrast, the texture of the

26 26

control lemon pudding was scored significantly higher than the experimental lemon pudding

recipe as was expected, considering the difficulty encountered in producing a comparably-

thickened lemon pudding containing BetterMilk. This may be explained by the hydration or

water-binding ability of complete proteins compared to GMP, which is not a complete protein as

it lacks tryptophan, histidine, leucine, methionine, and some phenylalanine. Because of the

addition of these amino acids to BetterMilk, the formula is considered elemental in form, thus

lacking the three-dimensional structure of an intact protein. In summary, the elemental parts may

not recreate the functionality of a whole protein, specifically the hydration capacity of proteins.

Another likely explanation for textural differences in the lemon pudding recipes may

have arisen from the fact that unflavored liquid non-dairy coffee creamer contains carbohydrate

as a primary macronutrient, whereas BetterMilk consists of incomplete proteins and amino

acids as primary macronutrients. The carbohydrate base of unflavored liquid non-dairy coffee

creamer may deem it more successful as a thickening agent as thickening is a functional role of

carbohydrates in food systems.

With regard to low sensory scoring of taste and aroma for the experimental soup,

pudding, and ice cream, it should be acknowledged that BetterMilk contains probiotics and

docosahexaenoic acid (DHA), both of which are capable of imparting strong flavors and aromas

to food products.44,45 Since taste is a combination of the senses, including smell, the aroma

imparted to BetterMilk by its added functional compounds may have caused the significantly

lower scores for both aroma and taste of the experimental recipes. In comparison to previous

27 26

sensory studies reporting the sensory acceptability of GMP-based products among individuals

with PKU, those results may be attributed to the fact that isolated GMP and not a formula with

added amino acids and functional ingredients was used in these tested products.5,32

According to the participant questionnaire, 54.8% of the subjects only occasionally

consumed all of their formula. This could have influenced the results of sensory testing such that

subjects may have been disinclined to accept medical foods in any form. Over thirty-eight

percent of subjects reported that they would not often use a formula product in cooking. This

may be attributed to the characteristic taste and aroma of medical foods, which has been noted as

a primary barrier to compliance with PKU diet therapy.26

In the present study, subjects reported neutrality or indifference toward either positive or

negative taste and smell attributes of the medical food they currently consume. Thus, subjects

appear acclimated to the taste and aroma of their current formula. As such, participants may have

been more scrutinizing in sensory testing of experimental recipes containing formula different

from their usual formula. If these same participants also indicated that they were not interested in

trying or obtaining a different formula product from the one currently consumed, these results

may reflect a complete aversion to a new formula, even if it could be one used in low-

phenylalanine recipes. Although subjects in the present study were blinded to the samples tested,

the knowledge that a recipe contains a medical food may be sufficient to cause an aversion to

experimental recipes among study participants. In most cases, eating is an experience which

individuals with and without dietary restrictions go to great lengths to make pleasurable.

Individuals with PKU may not be willing to allow the taste, smell, or thought of a medical food

28 26

to disrupt their sensory expectations but may rather wish to keep formula separate from other

dietary intake. Nevertheless, just as taste is subjective, person-to-person variation exists in

preference for medical foods and their use.

The present study has some limitations. In this pilot sensory study, a mean standard

deviation between sample characteristics of 2.0 using the 9-point hedonic scale was employed in

order to determine the smallest level at which data were statistically significant. According to the

sample size power calculation, the required sample size for statistical significance using the

aforementioned metric was 49; however, only 31 participants met eligibility criteria. Study

results were determined to be statistically significant, although results were not significant at the

level the study aimed to attain. Effort was made to prevent this limitation through data collection

at events with both national and international audiences. Since PKU is a rare disease (prevalence

of one in fifteen thousand live births in the United States)1, recruitment for the study was

conducted at events where large groups of individuals with PKU would be present. Another

limitation of the study is the fact that the sample population was not an accurate representation of

the population of individuals with PKU, which is equally prevalent among males and females. In

the present study, over 80 percent of subjects were female.

In conclusion, experimental samples for each recipe were on average scored in the “like”

range using a 9-point hedonic scale. Additionally, only 23% of the study sample reported they

frequently consume medical foods, while equal numbers of participants reported they were/were

not interested in using a medical food in preparation of low-protein recipes. These results may

partially explain why participants preferred the control samples over samples prepared with

29 26

GMP-containing BetterMilk™. In summary, this pilot study provides critical data foundational

to understanding the application and acceptability of GMP-based formulas while also guiding

future research. For example, additional research is needed to investigate the use of GMP-based

formulas without added functional ingredients in low-protein cooking, which may be beneficial

to the nutritional management of PKU among interested individuals. Also, future research should

be conducted among individuals with PKU to examine the acceptability of low-protein recipes in

which unflavored liquid non-dairy coffee creamer has been only partially replaced with a GMP-

containing formula. This would also decrease the phenylalanine and protein content per serving

which would be beneficial for individuals with PKU who have low phenylalanine tolerances.

Lastly, future research should also evaluate the use of isolated GMP, not GMP-based formulas,

in low-protein cooking. Such research is needed to expand the dietary offerings for individuals

limited by PKU.

30 26

REFERENCES

1. National Institutes of Health Consensus Development Panel. National Institutes of Health consensus development conference statement: phenylketonuria: screening and management, October 16-18 2000. Pediatrics 2001;108:972-82. 2. Anastasoaie V, Kurzius L, Forbes P, Waisbren S. Stability of blood phenylalanine levels and IQ in children with phenylketonuria. Mol Genet Metab. 2008;95(1-2):17-20. 3. van Calcar SC, Ney DM. Food products made with glycomacropeptide, a low-phenylalanine whey protein, provide a new alternative to amino acid-based medical foods for nutrition management of phenylketonuria. J Acad Nutr Diet. 2012;112:1201-1210. 4. Camp KM, Lloyd-Puryear MA, Huntington KL. Nutritional treatment for inborn errors of metabolism: Indications, regulations, and availability of medical foods and dietary supplements using phenylketonuria as an example. Mol Genet Metab. 2012;107(1-2):3-9. 5. Lim K, van Calcar SC, Nelson KL, Gleason ST, Ney DM. Acceptable low-phenylalanine foods and beverages can be made from glycomacropeptide from cheese whey for individuals with PKU. Mol Genet Metab. 2007;92(1-2):176-178. 6. Etzel MR. Manufacture and use of dairy protein fractions. J Nutr. 2004;134(suppl 4):S996-S1002. 7. MacLeod EL, Clayton M, van Calcar SC, Ney DM. Breakfast with glycomacropeptide compared with amino acids suppresses plasma ghrelin levels in individuals with phenylketonuria. Mol Genet Metab. 2010;100(4):303-308. 8. Schuett V. Low Protein Cookery for PKU, Third Edition. Madison, WI: The University of Wisconsin Press; 1997. 9. Folling A. About excretion of phenylpyruvic acid in the urine as metabolic anomaly in connection with imbecility. Ztschr Physiol Chem 1934;227(1-4):169-181. 10. Bickel H, Gerrard JW, Hickmans EM. Influence of phenylalanine intake on phenylketonuria. Lancet 1953; 2:169. 11. Guthrie R, Susi A. A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics. 1963;32:338-43.

31 26

12. MediResource, Inc. (Toronto, Ontario). National PKU Alliance. PKU and the brain: new research and therapies- a current review of published clinical research. Available at: npkua.org/portals/0/pdfs/pkubrain/PKUBrain-1-Introduction.pdf. Accessed March 24, 2014. 13. Blau N, van Spronsen FJ, Levy HL. Phenylketonuria. Lancet. 2010;376:1417-1427. 14. Marcason W. Is there a standard meal plan for phenylketonuria? J Acad Nutr Diet. 2013;113(8):1124. 15. Sweeney AL, Roberts RM, Fletcher JM. Dietary protein counting as an alternative way of maintaining metabolic control in phenylketonuria. J Inherit Metab Dis. 2012;3:131-9. 16. University of Utah Health Sciences. Phenylketonuria. University of Utah Genetic Science Learning Center website. 2014. Available at: http://learn.genetics.utah.edu/content/disorders/singlegene/pku/. Accessed March 3, 2014. 17. Koch R, Azen CG, Friedman EG, Williamson ML. Paired comparisons between early treated PKU children and their matched sibling controls on intelligence and school achievement test results at eight years of age. J Inherit Metab Dis. 1984;7(2):96-90. 18. ten Hoedt AE, deSonneville M, Francois B, et al. High phenylalanine levels directly affect mood and sustained attention in adults with phenylketonuria: A randomized, double-blind, placebo-controlled, crossover trial. J Inherit Metab Dis. 2011;34(1):165-171. 19. Azen CG, Koch R, Friedman EG, et al. Intellectual development in 12-year-old children treated for phenylketonuria. Arch Pediatr Adolesc Med. 1991;145(1):35-39. 20. Monch E, Herrmann ME, Brosicke H, Schoffer A, Keller M. Utilisation of amino acid mixtures in adolescents with phenylketonuria. Eur J Pediatr. 1996;155:S115-S120. 21. Schuett VE, Brown ES, Michals K. Reinstitution of diet therapy in PKU patients from twenty-two US clinics. Am J Pub Health. 1985;75(1):39-42. 22. MacLeod EL, Ney D. Nutritional management of phenylketonuria. Ann Nestle Eng. 2010; 68(2):58-69. 23. Cotungo G, Nicolo R, Cappelletti S, Goffredo BM, Dionisi Vici C, Di Ciommo V. Adherence to diet and quality of life in patients with phenylketonuria. Acta Paediatr. 2011; 100(8):1144-9.

32 26

24. Recipes and Tips. Cambrooke Foods website. Available at: http://www.cambrookefoods.com/recipes_and_tips/. Accessed March 3, 2014. 25. Tullio LT, Karkle EL, Candido LB. Review: Isolation and purification of milk whey glycomacropeptide. Curitiba. 2007;25(1):121-132. 26. Ney DM, Gleason ST, van Calcar SC, et al. Nutritional management of PKU with glycomacropeptide from cheese whey. J Inherit Metab Dis. 2009;32(1):32-39. 27. Cummings DE, Purnell JQ, Frayo RS, Schmidova K, Wisse BE, Weigle DS. A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes. 2001;50(8):1714-1719. 28. Kovacova R, Sedlarik M, Mihulova M, Sirmerova M, Curda L, Stetina J. The functional properties of casein glycomacropeptide as stabilising agent for emulsion based food products. J Biotech. 2010;150S:326-327. 29. Neelima SR, Rajput YS, Mann B. Chemical and functional properties of glycomacropeptide (GMP) and its role in the detection of cheese whey adulteration in milk: a review. Dairy Sci Technol. 2013;93(1):21-43. 30. van Calcar SC, MacLeod EL, Gleason ST, et al. Improved nutritional management of phenylketonuria by using a diet containing glycomacropeptide compared with amino acids. Am J Clin Nutr. 2009;89:1068-77. 31. U.S. House of Representatives. 113th Congress, 1st session. H.R. 3665, Medical Foods Equity Act of 2013. GPO Access. Available at: https://www.govtrack.us/congress/bills/113/hr3665. Accessed January 7, 2014. Updated December 5, 2013. 32. Abdel-Salam AM, Effat LK. Preparation and evaluation of a novel therapeutic dairy-based drink for phenylketonuria. N Am J Med Sci. 2010;2(2):66-70. 33.Chung Chun Lam SMS, Moughan PJ, Awati A, Morton HR. The influence of whey protein and glycomacropeptide on satiety in adult humans. Phys & Behav. 2009;96:162-168. 34. Rocha JC, MacDonald A, Trefz F. Is overweight an issue in phenylketonuria? Mol Genet Metab. 2013;110:S18-24. 35. Keough JB, Clifton P. The effect of meal replacements high in glycomacropeptide on weight loss and markers of cardiovascular disease risk. Am J Clin Nutr. 2008;87:1602-5.

33 26

36. Xu SP, Mao XY, Cheng X, Chen B. Ameliorating effects of casein glycomacropeptide on obesity induced by high-fat diet in male Sprague-Dawley rats. Food Chem Toxicol. 2013;56:1-7. 37. Olsson GM, Montgomery SM, Alm J. Family conditions and dietary control in phenylketonuria. J Inherit Metab Dis. 2005;28:627-37. 38. Dolan BE, Koch R, Bekins C, Schuett V. Diet Intervention Guidelines for Adults with Untreated PKU. National PKU News website. Updated August 2000. Available at: http://www.pkunews.org/adults/guide.htm. Accessed February 25, 2014. 39. Camp KM, Lloyd-Puryear MA, Huntington KL. Nutritional treatment for inborn errors of metabolism: Indications, regulations, and availability of medical foods and dietary supplements using phenylketonuria as an example. Mol Genet Metab. 2012;(107):3-9. 40. Huntington K, Buist NRM. Medical food treatment for inborn errors of metabolism and state legislative mandates. Topics in Clin Nutr. 2009;(24):289-306. 41. Levy HL. Historical background for the maternal PKU syndrome. Pediatrics. 2003;112:1516-8. 42. Lawless HT, Heymann H. Sensory Evaluation of Food: Principles and Practices. New York, NY: Kluwer Academic/Plenum Publishers; 1999. 43. U.S. Department of Agriculture, Agricultural Research Service. USDA National Nutrient Database for Standard Reference, Release 25. Updated February 7, 2014. Available at: http://www.ars.usda.gov/ba/bhnrc/ndl. Accessed March 15, 2014. 44. Kolanowski W, Swiderski F, Berger S. Possibilities of fish oil application for food products enrichment with omega-3 PUFA. Int J Food Sci Nutr. 1999;50(1):39-49. 45. Anjum N, Maqsood S, Masud T, Ahmad A, Sohail A, Momin A. Lactobacillus acidophilus: characterization of the species and application in food production. Crit Rev Food Sci Nutr. 2014;54(9):1241-51.

34 26

APPENDIX A

LOW-PHENYLALANINE RECIPES FOR SENSORY TESTING

35 26

Cream of Tomato Soup*

2 tablespoons margarine or butter ¾ cup water

2 tablespoons cornstarch ½ teaspoon salt

24 oz. tomato juice 2 teaspoons granulated sugar

1 cup unflavored liquid non-dairy coffee creamer**

In a large saucepan, melt the margarine. Remove the margarine from the heat. Add the

cornstarch and stir until the mixture is smooth. Add ½ cup tomato juice and stir constantly over

medium heat, until the mixture thickens. Add the remaining tomato juice, water, salt, sugar, and

unflavored liquid non-dairy coffee creamer. Heat the mixture thoroughly without boiling.

Yield: 4 ½ cups

Per recipe: 164 mg phenylalanine, 6.5 g protein, 796 calories

Per ½ cup serving: 18 mg phenylalanine, 0.7 g protein, 88 calories

*Recipe adapted from Low Protein Cookery for PKU Third Edition8

**For testing purposes, the recipe was prepared using Rich’s Coffee Rich®.

BetterMilk Variation

Substitute 1 cup BetterMilk (prepared using 2 packets + 8 oz. tomato juice) for unflavored

liquid non-dairy coffee creamer. Omit sugar and replace water with tomato juice. Prepare the

recipe as directed.

Per recipe: 310 mg phenylalanine, 35.7 g protein, 866 calories

Per ½ cup serving: 34 mg phenylalanine, 3.97 g protein, 96 calories

36 26

Lemon Pudding*

1 package (4-serving size) Lemon 1 cup whipped topping

Instant JELL-O® Pudding and 1 cup unflavored liquid non-dairy

Pie Filling mix coffee creamer**

Prepare the recipe according to the package instructions. Chill.

Yield: 2 cups

Per recipe: 80 mg phenylalanine, <1 g protein, 880 calories

Per ½ cup serving: 20 mg phenylalanine, 0 g protein, 220 calories

*Recipe adapted from Low Protein Cookery for PKU Third Edition8

**For testing purposes, the recipe was prepared using Rich’s Coffee Rich®.

BetterMilk Variation:

Substitute 1 cup BetterMilk (prepared using 2 packets + 8 oz. water) for the 1 cup unflavored

liquid non-dairy coffee creamer. Prepare the recipe as directed.

Per recipe: 94 mg phenylalanine, 30 g protein, 880 calories

Per ½ cup serving: 24 mg phenylalanine, 3 g protein, 220 calories

37 26

Vanilla Ice Cream (Churned)*

2 cups unflavored liquid non-dairy coffee creamer** 2 to 3 teaspoons vanilla

Two 8 oz. cartons whipped topping 1/8 teaspoon salt

¾ cup granulated sugar 4 drops yellow food coloring

Place the freeze bowl in the freezer and let it remain there for at least 15 hours. Attach the

freeze bowl, drive assembly, and dasher to the mixer. Pour the mixed ingredients into the freeze

bowl and set the mixer speed to “STIR”. Mix for 20-30 minutes. Detach the freeze bowl, and

remove the dasher. Transfer the ice cream to a plastic freezer container and freeze until firm.

Yield: 6 cups

Per recipe: 88 mg phenylalanine, 1.4 g protein, 2,547 calories

Per ½ cup serving: 7 mg phenylalanine, 0.1 g protein, 212 calories

*Recipe adapted from Low Protein Cookery for PKU Third Edition8

**For testing purposes, the recipe was prepared using Rich’s Coffee Rich® in the ice cream

maker attachment for a KitchenAid® stand mixer.

BetterMilk Variation:

Substitute 2 cups BetterMilk (prepared using 4 packets + 16 oz. water) for unflavored liquid

non-dairy coffee creamer. Prepare the recipe as directed.

Per recipe: 92 mg phenylalanine, 60 g protein, 2,499 calories

Per ½ cup serving: 8 mg phenylalanine, 5 g protein, 208 calories

38 26

APPENDIX B

BETTERMILK™ SPECIFICATION SHEET

39 26

40 26

APPENDIX C

RICH’S® COFFEE RICH® INGREDIENTS AND NUTRITION FACTS PANEL

41 26

42 26

APPENDIX D

UNIVERSITY OF ALABAMA INSTITUTIONAL REVIEW BOARD STUDY APPROVAL

43 26

44 26

APPENDIX E

RECRUITMENT FLYER

45 26

Healthy men and women with PKU

ages 18 and older needed to participate in

TASTE TESTING

of

LOW-PROTEIN ICE CREAM, PUDDING, AND SOUP!

If interested, please attend a taste testing session at

DATE, TIME, LOCATION (TBA)

Study conducted by students and researchers

at the University of Alabama

PI Contact – Amelia Foster, RD

46 26

APPENDIX F

CONSENT FORM

47 26

48 26

49 26

50 26

51 26

APPENDIX G

NUTRITIONAL COMPOSITION OF LOW-PHENYLALANINE RECIPES

52 26

Control Vanilla Ice Cream Phenylalanine: 19 mg, Protein: 0.34 g

53 26

Experimental Vanilla Ice Cream with BetterMilk Phenylalanine: 23 mg, Protein: 15 g

54 26

Control Lemon Pudding Phenylalanine: 42 mg, Protein: 0.8 g

55 26

Experimental Lemon Pudding with BetterMilk Phenylalanine: 13 mg, Protein: 7.52 g

56 26

Control Cream of Tomato Soup Phenylalanine: 40 mg, Protein: 1.5 g

57 26

Experimental Cream of Tomato Soup with BetterMilk Phenylalanine: 58.3 mg, Protein: 9.63 g

58 26

APPENDIX H

SENSORY TESTING FORMS

59 26

60 26

61 26

62 26

63 26

64 26

65 26

APPENDIX I

PARTICIPANT QUESTIONNAIRE

66 26

67 26

68 26

69 26