Postprandial Inflammatory and Metabolic Responses Induced by the Authentic Ladotyri Mytilinis PDO Cheese and Italian Parmesan PDO Cheese
Phase: NA
Conditions: Healthy
Sponsor: University of the Aegean

 
 
 
 
 
 
 
 
 
 
 
 
 
 
Nutritional Intervention-Clinical Trial Protocol 
 
 
 
 
Title: Postprandial Inflammatory and Metabolic Responses Induced by the 
Authentic Ladotyri Mytilinis PDO Cheese and Italian Parmesan PDO Cheese 
 
NCT IDENTIFIER: NCT05788887 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
16 December 2022 

1. Protocol Summary 
 
This is the protocol of a randomized, blinded and controlled nutritional intervention – clinical 
study to investigate the postprandial effect of consuming Authentic Ladotyri Mytilinis, PDO 
cheese on the lipid, glycemic profile and the oxidative status of the blood of healthy volunteers. 
 
The acute nutritional intervention – clinical study is a cross over design (acute study), with two 
axes. The purpose of this study was the investigation of the postprandial metabolic responses, 
after the intake of a traditional, Ladotyri Mytilinis cheese, compared to corresponding 
responses after an Italian Parmesan cheese consumption, in healthy participants. 
 
The study volunteers will be healthy, aged 18-65 years, residing during the intervention in 
Lemnos. The study will take place in the specially designed space of the Human Nutrition Unit, 
University of the Aegean and in voluntary collaboration with the General Hospital of Limnos. 
The duration of the study will be 7 days. 
 
Participants will be randomly divided into two groups. During the first day, the first group will 
consume Italian Parmesan, PDO cheese (control group) and the second group will consume 
Authentic Ladotyri Mytilinis, PDO cheese (intervention group), together with two slices of 
bread, while 1 glass of water will be available for each volunteer [1st test period]. After a 7- 
day washout period, the same volunteers will come to the specially designed area and the 
process will be repeated, with the difference that the control group of the 1st day will belong 
to the intervention group, and the intervention group will belong to the control [2nd test 
period]. 
 
In each test period, blood samples will be taken from all consecutive volunteers in a fasted 
state, as well as 30 minutes, 1.5 and 3 hours after consuming meals. This will be followed by 
analysis of the samples and statistical processing of the results of the analyzes to present 
data regarding the acute effect of meal consumption on the postprandial biomarkers of 
lipemia, glycemia and oxidative stress in healthy volunteers. 
 
2. Theoretical background and study objectives 
 
2.1. Argumentation, Research evidence, Rigor of study 
 
The existing scientific evidence indicates a correlation of the levels of postprandial lipid, 
glycemic biomarkers and oxidative stress markers, with chronic diseases, such as 
cardiovascular diseases, diabetes, etc. (Koutelidakis and Dimou, 2016). Recent nutritional 
interventions by the team in healthy volunteers, but also participants at high metabolic risk, 
have shown a beneficial effect of consuming functional foods, such as a functional spreadable 
cheese, enriched with mountain tea and orange peel, but also a functional refined olive oil, 
fortified with antioxidants components from orange peel, on postprandial markers of lipemia, 
glycemia and oxidative stress (Papagianni et al, 2021). 
 
The findings of previous clinical studies-dietary interventions suggest that the consumption of 
a meal containing cheese can cause a significant increase in the concentration of triglycerides, 
2 hours after taking the meal (Drouin-Chartier et al., 2016). 

Olive oil, the main characteristic of the Mediterranean Diet, thanks to the bioactive 
compounds it has in its composition, has been extensively studied in recent years, in vitro 
and in vivo, highlighting its antioxidant, anti-inflammatory and other actions (Jędrusek- 
Golińska et al., 2020). In vitro evidence shows that cheese enriched with polyphenols may 
contribute to maintaining the integrity of polyphenols and antioxidant activity in the 
gastrointestinal environment (Lamothe et al, 2016). The postprandial effect of consuming 
functional foods rich in polyphenols (e.g. olive oil, grapes) in combination with meals rich in 
fats and carbohydrates has been evaluated with promising results, since they seem to have a 
beneficial effect on metabolic biomarkers and pathways, postprandial (Magrone et al., 
2013). 
 
Based on the aforementioned scientific evidence regarding the possible postprandial effect 
of the bioactive components of olive oil (in which the cheese is extracted for the production 
of the oil cheese) on specific biomarkers, the present nutritional intervention will be 
composed. 
 
2.2. Study Objectives / Research Questions 
 
Purpose 
 
The purpose of this study was the investigation of the postprandial responses on serum total, 
HDL-, LDL- cholesterol, triglycerides, uric acid, glucose and plasma Total Antioxidant Capacity 
(TAC), after the intake of a traditional, Authentic Ladotyri Mytilinis cheese, compared to 
corresponding responses after an Italian Parmesan cheese consumption, in healthy 
participants. 
 
Research Questions 
 
The research questions of this clinical study are the following: 
 
 Which is the acute effect of ALM-PDO cheese consumption, in the framework of a 
high-fat and carbohydrates meal, on the postprandial biomarkers of lipemia, glycemia 
and oxidative stress? 
 Does the postprandial inflammatory and metabolic responses to ALM-PDO cheese 
differs, compared to Italian Parmesan PDO cheese? LDL- cholesterol and triglycerides 
determined at 3h after the meal intake, was set as the primary endpoint of the 
present, pilot study. 
 
3. Summary presentation / study design 
 
3.1. Overview 
 
A nutritional intervention – clinical study of self-comparison design (cross over design: acute 
study) will take place in a sample of healthy volunteers, where the difference in the 
effectiveness of the nutritional intervention will be evaluated in the same volunteer. This 
means that the response of lipemia, glycemia and oxidative stress biomarkers to the 
intervention meal will be compared with their response to the control meal, in the same 
volunteer each time. The specific research will be carried out in the specially designed area of 

the Human Nutrition unit of the University of the Aegean and in collaboration with the hospital 
of Lemnos, with which a collaboration protocol has been concluded with the University of the 
Aegean. 
 
3.2. Study design 
 
In a total sample of approximately 10 healthy volunteers, the number of which was 
determined through a special statistical program for calculating the required sample of the 
research to draw safe and reliable conclusions, a nutritional intervention – clinical study of 
cross-over design will be carried out. The study for each participant will be 7 days long, 
including the washout period. 
 
Each participant during the first test period and in a state of 12-hour fasting, will be asked to 
come in the morning hours of 9 am. - 12:30 p.m., the specially designed blood collection room 
of the Human Nutrition Unit, Department of Food Science and Nutrition, University of the 
Aegean. In this specially designed area, a blood sample (baseline) will be taken by a doctor or 
competent staff of the Lemnos Hospital. This will be followed by a discussion with the healthy 
volunteer, signing of a declaration of participation, filling in a questionnaire and a 24-hour 
recall of food consumption. The questionnaire that each volunteer will fill out on the first day 
of the start of the study concerns their medical history and general information about their 
habits (especially dietary). Afterwards, a meal will be served that will include 2 slices of bread 
(80g- 40g each) and 2 pieces of cheese (100g- 50g each piece), according to the group each 
volunteer belongs to, accompanied in each case by 1 glass of water. The consumption of each 
meal will be completed within the time period of 15 minutes by all participants (controlled). 
Finally, the following blood samplings will be carried out for the volunteers, which will be 
conducted 30 minutes, 1.5 and 3 hours after the completion of the meal. 
 
After a seven-day washout period, on the eighth day (2nd test period) the same participants 
will be asked to come again, in a fasting state, to the specially designed area of the University 
of the Aegean, at the predetermined morning hours to repeat the procedure nutritional 
intervention and blood sampling. In other words, a blood sample will be taken by a doctor or 
competent staff of the Limnos Hospital, followed by a discussion with the volunteer and 
completion of a 24-hour recall of food consumption questionnaire. The questionnaire that the 
participants will complete at the start of the second test period is to record their specific eating 
habits during the washout period (ie the interval between the two periods of the intervention). 
The meals served to the volunteers are determined by the control or intervention group to 
which they belong. After each meal is consumed within the 15-min time period, subsequent 
blood sampling will be done for participants 30, 90, and 180 minutes (min) after the completion 
of the meal . 
 
4. Enrollment of participants and case of study withdrawal 
 
The goal is to gather an initial sample of people from the Greek area in order to achieve the 
collection of a required number of cases for study. The research team will then review the 
inclusion/exclusion criteria and select the participants who will receive the meals in the two 
test periods of the study. 

4.1. Sample size calculation 
 
In order to note a 40% difference in the postprandial response of triglycerides and/or LDL- 
cholesterol and in order to have an 80% chance of a statistically significant difference between 
the studied groups at a significance level of 5%, a necessary sample of 10 subjects was 
calculated. 
 
4.2. Study inclusion criteria 
 
The criteria that must be met by those interested in conducting the study are the following: 
 
 
Age between 18 and 65 years 
 
Willingness to join and complete the nutritional intervention 
 There should be no history of loss of consciousness in previous blood draws as 
well as personal reasons for not being able to donate blood (e.g. fear) 
 Who will sign a written consent to participate in the study 
 
4.3. Exclusion criteria from the study 
 
Participants meeting, at baseline, the following exclusion criteria will be excluded from the 
study for reasons of safety as well as efficacy of the study: 
 
 Inability or unwillingness to provide informed consent 
 Age <18 and >30 years 
 Three-month nutritional supplement 
 Chronic diseases history 
 Hemoglobin A1c - HbA1c > 5.7 % 
 Abnormal Body Mass Index (BMI) (> 25 kg / m2) 
 Abnormal hematological or bio-chemical profile (total cholesterol > 240 mg/dL, 
triglycerides > 250 mg/dL, glucose > 100 mg/dL) 
 Alcohol overdose (> 40 g alcohol / day) 
 Heavy smokers (> 10 cigarettes / day) 
 Concurrent participation in another intervention study or unconsciousness in 
previous blood draws 
4.4. Strategy for participant inclusion and retention 
 
Finding the study participants will be done by the University and/or the wider local community 
of Lemnos. The volunteers will be either students or members of the teaching staff of the 
University of the Aegean or residents of the island. One of the members of the research 
coordinators will undertake the briefing of each healthy volunteer, which will take place in a 
special area of the University. Those who will meet all the criteria of the study in order to be 
accepted and at the same time agree in writing to participate in it, on the days that they will 
come to this special area, they will have the possibility at any time to ask for help either from 
the main researcher or from anyone else research representative to record any difficulties or 
questions they may have. The members of the research team will be responsible for timely 

informing the participants, in writing or by telephone, of the visits and the biological samples 
to be collected. 
 
The scientific manager of the study or his representatives will be responsible for the 
safekeeping of the data and results. The anonymity of the participants will be preserved and 
the safe keeping of the history of the healthy will be ensured, to which only the personnel who 
will be employed in this research will have access. 
 
4.5. Participant withdrawal criteria 
 
Participants are free to withdraw from the study at any time. Accordingly the researcher will 
be able to ask the participant to leave the study if: 
 
o Clinical adverse events, laboratory abnormality, or other medical condition or condition are 
identified that endanger the participant from remaining in the study 
 
o Participants meet one of the aforementioned exclusion criteria (either newly developed or 
not previously identified) that precludes further participation in the study 
 
o Participants appear to not adequately adhere to protocol requirements 
 
4.6. Suspension or early termination of the study 
 
This study may be suspended or terminated early only if there is sufficient cause. A written 
notice documenting the reason for stopping or suspending the study will be provided by the 
person responsible for this decision (research team, hospital). 
 
5. Study Intervention 
 
5.1. Description of intervention 
 
As part of a meal that will include two slices of bread of 80 grams (g), and a glass of 250 ml of 
water, the control group will consume two pieces of Italian parmesan cheese (PDO), total 
weight of 100 grams (g), while the intervention groups will consume corresponding bread 
meal, where the control cheese will be replaced with 100 grams (two pieces) of Authentic 
Ladotyri Mytilinis cheese (PDO). The meals will be prepared at the Nutrition and Public Health 
laboratory of the Human Nutrition Unit, University of the Aegean. 
 
For the selected foods, there does not seem to be any data suggesting that they may have a 
possible risk or negative impact on health, as on the one hand there is no confirmed adverse 
effect of its individual components on health based on the scientific literature and on the other 
hand it appears according to corresponding research in similar foods the potential beneficial 
role of their biofunctional components in health promotion. 
 
5.2. Assessment of participant compliance for administration 
 
The assessment of the compliance of the participants in the study with regard to the 
administration of cream cheese, will be achieved following continuous supervision by a 
member of the research team, of the volunteers who will come to the University site to 
consume all the meals. 

6. Study Procedures 
 
6.1. Questionnaires 
 
A specially designed questionnaire, based on bibliographic data, will be used to collect 
important data regarding the participants of the study. 
 
The questionnaire will concern the clinical condition and habits (especially nutritional) of the 
volunteers of the clinical study and will include the following parts: 
 
1st part 
 
General socio-demographic and anthropometric data of the study population (gender, age, 
anthropometric indicators) 
 
2nd part 
 
Clinical status of each participant (medication, dietary supplements, etc.). 
3rd part 
Dietary and other habits before the start of the intervention, as well as more specific dietary 
habits for the washout period. 
 
6.2. Biological samples 
 
The collection of blood samples, by a doctor or competent staff of the Limnos Hospital, of the 
healthy volunteers of the clinical study will be used to determine indicators of postprandial 
lipemia, glycemia and oxidative stress in the blood (plasma antioxidant capacity, lipid profile 
specifically triglycerides, HDL & LDL cholesterol, glucose, etc.) that will be analyzed using a 
special biochemical analyzer. 
 
6.3. Statistical analysis 
 
After the above analyzes have been carried out using a special biochemical analyzer, the use 
of the statistical package SPSS (Statistical package for social sciences: SPSS) will follow in order 
to record, process and analyze the results of the study. The Kolmogorov–Smirnov test was 
applied to check normality. The main participants’ characteristics will be analyzed by 
descriptive statistics. Differences in baseline values between groups for each biomarker tested, 
will be analyzed using a paired sample two tailed t test. Repeated ANOVA measures with 
Geisser–Greenhouse correction will be conducted, for serum total, HDL-, LDL- cholesterol, 
triglycerides, glucose, uric acid and plasma Total Antioxidant Capacity (TAC). If time or group 
was significant, multiple comparison post hoc Bonferroni test will be performed for the 
comparison of concentrations. To check changes in the metabolic biomarkers from baseline 
to postprandial responses (within-group variation), the Wilcoxon sign-rank test will be carried 
out. 

7. Maintenance of study records 
 
The study files will remain securely registered for a maximum of 3 years from the date of their 
registration in special areas of the Human Nutrition Unit, University of the Aegean. Study 
records should be kept for a maximum of 2 years after the last marketing approval, or longer 
if required by local regulations. 
 
8. Ethics, Consent and Privacy Statement 
 
8.1. Ethical standards 
 
The members of the research team agree that they will provide their professional expertise 
and knowledge to conduct the study in accordance with the requirements of the protocol 
and in accordance with the provisions of the Declaration of Helsinki and the provisions of the 
General Data Protection Regulation. 
 
8.2. Rules of ethics and research ethics 
 
During the conduct of this clinical study, all fundamental rules of ethical, scientific and 
research ethics will be observed. Therefore, an application for the approval of the clinical study 
will be made by the Bioethics and Research Ethics Committee of the University of the Aegean, 
with the research volunteers also being informed about its purpose, the confidentiality of the 
data and the voluntary nature of their participation. Participants will take part in the study 
after signing a consent form to participate in it. 
 
8.3. Formal consent process 
 
Formal consent in the clinical study is a process that will begin before the enrollment of the 
interested parties in the study and will continue throughout their participation in it. Those 
interested will be provided with all the information regarding the benefits and potential risks 
of the study, as well as a consent form where the necessary procedures will be presented. 
Every interested party must read it and review it or, in special cases (e.g. blind) be informed 
by third parties. A copy will be provided to interested parties. The researcher or representative 
will explain the study and its objectives to each participant and address any questions they 
may have. 
 
The rights and care of the subjects will be protected by the fact that the quality of their clinical 
care will not be affected in any way if they refuse to participate in this clinical study. This 
process will take place at the University where this research will take place. 
 
8.4. Privacy of study participants 
 
Confidentiality will be maintained by the researchers or representatives of the research team. 
Confidentiality will support the conduct and study of biological samples and genetic tests, as 
well as any study information concerning participants. The study protocol, documentation, 
data, as well as other information will be conducted with absolute confidentiality, prohibiting 
their disclosure to third parties. 

9. References 
 
Koutelidakis, A., Dimou, C. (2016). The effects of functional food and bioactive compounds on 
biomarkers of cardiovascular diseases. In: Martirosyan D (ed) Functional Foods Text Book, 1st 
edn. USA, pp. 89-117. 
 
Papagianni, O., Loukas, T., Magkoutis, A., Biagki, T., Dimou, C., Karantonis, C., Koutelidakis, A. 
(2021). Postprandial bioactivity of spread cheese, enhanced with mountain tea and orange 
peel extract, in healthy volunteers. A pilot study. Proceedings, 70(1), pp. 19. 
https://creativecommons.org/liscences/by/4.0/. 
 
Fidelix, M., Milenkovic, D., Sivieri, K., Cesar, T. (2020). Microbiota modulation and effects on 
metabolic biomarkers by orange juice: a controlled clinical trial. Food & Function, 11(2), pp. 
1599-1610. doi: 10.1039/c9fo02623a. 
 
Drouin-Chartier, J. P.; Côté, J. A.; Labonté, M. É.; Brassard, D.; Tessier-Grenier, M.; Desroches, 
S.; Couture, P.; Lamarche, B. Comprehensive Review of the Impact of Dairy Foods and Dairy 
Fat 
on 
Cardiometabolic 
Risk. 
Adv. 
Nutr., 
2016, 
7 
(6), 
1041–1051. 
https://doi.org/10.3945/an.115.011619. 
 
Jędrusek-Golińska, A.; Górecka, D.; Buchowski, M.; Wieczorowska-Tobis, K.; Gramza- 
Michałowska, A.; Szymandera-Buszka, K. Recent Progress in the Use of Functional Foods for 
Older Adults: A Narrative Review. Compr. Rev. Food Sci. Food Saf., 2020, 19 (2), 835–856. 
https://doi.org/10.1111/1541-4337.12530. 
 
Lamothe, S.; Langlois, A.; Bazinet, L.; Couillard, C.; Britten, M. Antioxidant Activity and Nutrient 
Release from Polyphenol-Enriched Cheese in a Simulated Gastrointestinal Environment. Food 
Funct., 2016, 7 (3), 1634–1644. https://doi.org/10.1039/c5fo01287b. 
Magrone, T.; Heredia, F. P. De; Jirillo, E.; Morabito, G.; Marcos, A.; Serafini, M. Treatment of 
Diet-Related Diseases 1. Can. J. Physiol. Pharmacol. Vol., 2013, 396 (April), 387–396. 
 
 
 
 
 
 
CONSENT FORM 
 
for the nutritional intervention with the title: " Postprandial Inflammatory and Metabolic 
Responses Induced by the Authentic Ladotyri Mytilinis PDO Cheese and Italian Parmesan 
PDO Cheese" 
I hereby declare that I agree to participate as a volunteer in the dietary intervention - clinical 
study with the aim to investigate the postprandial inflammatory and metabolic response 
induced by the consumption of authentic Mytilini cheese, in biomarkers of healthy volunteers. 
 
The study will last 7 days and I will need to visit the premises of the University of the Aegean 
two (2) times after consultation with the researchers. During my visits to the premises of the 
University of the Aegean and in the context of the clinical study-nutritional intervention, I will 

receive a meal that will include either Authentic Ladotyri Mytilinis PDO cheese or Italian 
Parmesan PDO cheese, amounting to 100 grams (g), together with two slices of bread of 80 
grams (g ) and a glass of 250 ml water. 
 
Before the administration of the above meals, but also 30 minutes (min), 1.5 and 3 hours (h) 
after their completion, blood will be taken from me by a cooperating doctor, with the aim of 
using it in measuring biomarkers (antioxidant capacity, lipid profile, glucose, etc.). I will also 
need to complete questionnaires to assess my clinical condition and nutritional habits. 
 
My participation in the study is voluntary, solely my choice, and no remuneration or other 
compensation is provided. I can withdraw from the study at any time, without giving any 
reason and without any consequences. 
 
By signing this document and filling in the boxes below, I agree and give my consent to the 
following: 
 
1. As a participant I have first been informed about the study, had the 
opportunity to review the information, decide about my participation, ask 
questions and receive satisfactory answers. 
2. The purposes of the research are fully understood and have been 
adequately explained to me. 
3. I understand what is proposed and the procedures in which I am to 
participate. 
4. I understand that my participation in this study and in particular the data 
from this research will remain strictly confidential. Only the researchers 
involved in the study will have access to the data which will not be given 
to others. 
5. I know how the results will be used upon completion of the research 
program. Personal data will not be included in the results and 
publications. 
 
 
 
For any additional information and assistance, the possibility of a planned visit to a special 
area of the University of the Aegean, where the study will take place, is provided. 
 
 
 
The volunteer 
The researcher 

Name & Surname 
Signature 
Date