Evaluation of the Beneficial Health Effects of an Additive-free Meat Product in Healthy Subjects (HIPOCARNE)
Phase: NA
Conditions: Inflammation, Oxidative Stress, Food Toxicity
Sponsor: Universidad de Murcia

  
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Official Title: Evaluation of the Beneficial Health 
Effects of an Additive-free Meat Product in 
Healthy Subjects (HIPOCARNE) 
 
 
 
Document Date: 04/18/2024 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

  
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1.1. Trial Design 
This study consisted of a double-blind, randomised, controlled, unicentric trial with two parallel arms 
depending on the cooked meat product consumed (control and reformulated), with a duration of 5 
weeks. The study was approved, according to the Declaration of Helsinki, by the Bioethics Committee 
of the University of Murcia (UMU) with code 550/2023. The research took place at the Department of 
Nutrition and Bromatology of the Faculty of Veterinary Medicine of the University of Murcia. 
Investigators provided a detailed explanation of the protocol to participants, and those choosing to 
participate gave their informed consent. Each participant received a unique code based on their arrival 
order. Following this, an external researcher then assigned the participants to the different groups with 
the aim of having two similar groups based on sex, age, and IMC. The specific group assignments were 
not known to either the researchers or the participants. 
Three weeks before the start of the study, a recruitment phase was carried out, in which potential 
participants were recruited and informed about the details of the study. From a total of 63 individuals 
initially recruited, fifty-eight volunteers completed the trial. Participants who abandoned the trial were 
for personal reasons unrelated to the study. Two visits were made during the experimental phase of the 
intervention: the initial and the final visit. At both visits, blood, faeces, and urine samples were obtained 
from participants, and anthropometric measurements were also taken. Two deliveries of 15 blister packs 
of 100g control or reformulated cooked meat product were made, at baseline and after 17 days. 
Individuals consumed one blister pack per day for 6 days per week. Throughout the trial, subjects 
recorded their complete diet in a collection notebook, which was given to the researchers at the end of 
the study. 
1.2. Participants 
The study comprised a total of 58 subjects divided into two groups of 29 individuals with similar 
characteristics (Table 2). To participate in the study, subjects were required to be between 18 and 65 
years of age and body mass index (BMI) between 18.5 and 30 kg/m2. Participants also had to avoid all 
of the exclusion criteria: use of antibiotics in the last 3 months; use of medication for hypertension or 
for the treatment of hyperlipaemia; consumption of pre- or probiotic supplements; consumption of 
omega-3 or omega-6 supplements; eating  , intestinal or similar problems; diabetes mellitus, chronic 
kidney disease, liver disease, infectious diseases or any type of cancer; having donated blood in the last 
month; having undergone surgery within the last three months; abusive alcohol consumption; being 
vegetarian; being pregnant; being a smoker; and participation in other clinical trials in the last 3 months. 
 
Table 1. Distribution of volunteer demographics  
 
Control 
Intervention 
p-value1 
Age (years)  
26.6 ± 11.5 
26.7 ± 11.7 
0.97 
Men  
15 
14 
1.00 
Women  
14 
15 
BMI (kg/m2)  
23.7 ± 2.7 
23.6 ± 2.8 
0.98 
Weight (kg)  
66.6 ± 11.8 
68.4 ± 12.7 
0.58 
Fat mass (%)  
26.0 ± 9.0 
26.9 ± 9.0 
0.71 
Total colesterol 
(mg/dl)  
175.8 ± 34.6 
178.0 ± 30.9 
0.80 
Oxidized LDL 
(ng/mL)  
238.6 ± 52.1 
233.6 ± 52.4 
0.71 
 
 
 
 
1 p-values between control and intervention groups were examined using a one‐way 
analysis of variance (Tukey’s test). The results are shown as mean ± SD (n=29). 

  
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1.3. Study Analyses 
1.3.1. Anthropometric measurements 
The anthropometric analysis of the participants followed the protocol of the International Society for 
the Advancement of Kinanthropometry (ISAK). The following measurements were taken with the 
corresponding equipment: height (213 measuring rod, Seca deutschland, Germany), weight (body fat 
monitor with scale BF400, OMRON Healthcare, Japan), body mass index (BMI) and percentage of fat 
mass (body fat monitor BF306, OMRON Healthcare, Japan). Abdomen circumference was also 
measured (201 antrophometric tape, Seca deutschland, Germany). In addition, hip and waist 
circumferences were measured to calculate the waist:hip ratio (WHR) (cm waist:cm hip), which is used 
to evaluate the development cardiovascular disease. 
1.3.2. Blood samples 
Subjects fasted for 12 hours prior to blood collection and were not allowed to perform moderate-
intensity exercise in the previous 24 hours. Blood samples were collected from the antecubital vein in 
gel and clot activator tubes (Vacutest Kima Srl, Arzergrande, Italy). These samples were centrifuged 
at 3500 rpm for 5 min at 4 °C to separate the serum from the cell fraction. After collection, the serum 
portions were stored at -80 °C until further analysis.  
Biochemical markers such as basal glucose, GOT, GPT, hs-CRP and lipid markers (triglycerides 
(TG), LDL-C, HDL-C, and total cholesterol (TC)) were measured using a clinical chemistry analyzer 
(BA400 Biosystems). 
Interleukin (IL) 1β, IL-6, IL-10, and tumour necrosis factor-alpha (TNFa) were analysed using 
commercially available kits (Milliplex Human High Sensitivity T Cell; Life Science, Darmstadt, 
Germany) according to manufacturer's indications. Catalase activity was assessed by an automatic 
method previously described by Slaughter et al. FRAP measurement was based on the method 
described by Benzie and Strain, with some modifications. Glutathione peroxidase (GPx) was 
determined using a commercially available method (Ransel, Randox Laboratories Limited, Crumlin, 
United Kingdom). Catalase, FRAP and GPx were measured in an automated chemistry analyzer 
(Olympus AU400, Olympus Diagnostica Europe GmbH, Ennis, Ireland). The TBARS assay was 
determined following the method by Buege and Aust. Oxidized Low Density Lipoprotein (ox-LDL) 
was determined using commercially available ELISA Kit (Human ox-LDL(Oxidized Low Density 
Lipoprotein) ELISA Kit, Texas, USA). TBARS and ox-LDL were measured using a microplate reader 
(Powerwave XS, Biotek instruments). 
1.3.3. Stool samples 
Faecal samples were collected by participants at home, using a sterile kit provided to them previously. 
Once the sample was collected, participants stored it under refrigeration (for no more than 24-48h) until 
it was delivered to the researchers. Once received, it was stored at -20°C until the corresponding 
analyses were performed. 
1.3.3.1. Sample preparation for FRAP and short chain fatty acids analysis. 
The faecal sample was previously defrosted at 4°C for 24h. Then, samples were diluted 1:10 with 0.1M 
phosphate buffer (2.33 g PO4HNa2 and 14.03 g PO4H2Na in 2L of water, pH 7.4) and sterilised. The 
mixture was centrifuged for 20 min at 5000 rpm in order to collect the supernatant and centrifuged again 
for 10 min. The supernatant was collected again and separated into 2 fractions for SCFA and FRAP 
assay analysis. The sample for AGCC was stored at -80°C until use. The FRAP assay was performed 
on the same day of sample preparation. 
1.3.3.2. FRAP assay. 
The antioxidant capacity of faeces was measured using the FRAP assay, according to the procedure 
described by Benzie and Strain. For this purpose, the supernatant portion was previously diluted with 
water at 1:10 dilution. 
1.3.3.3. Short chain fatty acids analysis 
The production of SCFAs was assessed according to the procedure described in Panzella et al. with 
some modifications. The supernatant portion was filtered through 0.20 µm PTFE filters and analysed 
by reversed-phase HPLC with diode array detector (1260 Infinity II LC System, Agilent, Santa Clara, 

  
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CA, USA). The results were obtained in ppm and converted to mmol/kg faeces, expressing the result as 
the increase of SCFAs in 24 hours. 
1.3.3.4. DNA extraction and metagenomic sequencing 
Bacterial genomic DNA was isolated from each fermentation pellet sample using NZY Soil gDNA 
Isolation kit (Nzytech, Lisboa, Portugal). Sequencing of the V3 and V4 regions of the 16S rRNA gene 
was performed on the MiSeq (Illumina, Essex, UK) instrument using 2 x 300 bp reads. The 
oligonucleotide primers targeting the 16S rRNA gene V3 and V4 regions were 5’-
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’ 
and 
5’-
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3’, 
respectively. The 16S-V4 sequencing library was first reviewed with FastQC for overall quality 
assessment, and the libraries were processed in R package DADA2 (v.1.8.0.). Reads were quality 
trimmed with the “filterAndTrim” function with “maxEE (2,5)” and reads below 165 bp were discarded. 
Forward and reverse reads are merged below to generate a table of sequences, and the resulting 
Amplicon Sequence Variants (ASVs) were subjected to de novo chimera detection, using DADA2 and 
any artifacts were removed. 
1.3.3.5. Bioinformatic analysis 
For bacteria taxonomic assignment, ASVs were queried against the Silva database v.132 [The SILVA 
ribosomal RNA gene database project: improved data processing and web-based tools] using IDTAXA  
implemented in the R package DECIPHER. Sequences identified as non-bacterial were discarded.  
The abundance matrix, the taxonomy assignment and the metadata obtained from each sample were 
merged and imported with the phyloseq v3.12 package. Alpha diversity was calculated in R using the 
phyloseq package, and several alpha indices were generated, such as ASV numbers, Chao1, ACE, 
Shannon, Simpson, InvSimpson, and Fisher and plotted using the function “plot richness”. Beta 
diversity was calculated using weighted and unweighted Unifrac distances. To test for significant 
differences in community composition among different seasons, permutational multivariate analysis of 
variance using distance matrices (PERMANOVA) was conducted using the Adonis function in the R 
package, and the results were visualized by Principal Coordinates Analysis (PCoA). 
1.3.4. Urine samples 
Participants collected the first urine sample of the morning in a sterile container given to them 
previously. Once the sample was collected, participants stored it under refrigeration (for no more than 
24-48h) until it was delivered to the researchers. Samples were stored at -20°C until use. 
1.3.4.1 Nitrates and nitrites analysis 
Urine samples were used to analyse nitrate and nitrite content. The samples were diluted 1:10, then 1 
ml of Carrez I (K4[Fe(CN6)·3H20) and 1 ml of Carrez II (ZnSO4 x 7H2O) were added and stirred for 30 
minutes at room temperature. After this time, the mixture was filtered through a 150 mm Ø filter paper. 
Finally, the samples were analysed in a SmartChem. 
1.4. Statistical analysis  
Data analysed were expressed as mean ± standard deviation (SD). Student's t-test was used to analyse 
significant differences over time (baseline and final). To analyse the differences between groups in the 
evolution of the different variables, an analysis of variance (ANOVA) was performed for repeated 
measures, with time (baseline and final) as the within-subject factor, and the product consumed (Control 
and Intervention) as the between-subject factor. Tukey´s test was carried out for post hoc analysis. 
Cross-tabulation and chi-square tests were conducted to determine the perception of respondents on 
food additives based on demographic data. 
Data processing and statistical analysis were performed using SPSS version 28.0 (IMB Corp., Armonk, 
NY, USA) with a significant level of p ≤ 0.05. 
 
 
 
 
 
 

  
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INFORMED CONSENT STATEMENT 
 
Ms. /Mr. ..................................... of .......... years of age and with ID card No. .......................... declares 
that she has been informed about the benefits that my participation could bring to cover the 
objectives of the research project entitled "RESEARCH AND DEVELOPMENT OF MINIMALLY 
PROCESSED AND HYPOALLERGENIC MEAT FOODS (HIPOCARNE)", directed by Gaspar Ros Berruezo, 
with the aim of determining the effects of meat products without synthetic additives at blood, urine 
and feces level, with certificate of the Ethical Committee of the UM, contact telephone: +34 968 
364794 and e-mail: gros@um.es.  
 
I have been informed of the possible prejudices that participation in this project may have on my well-
being and health by having read the participant information sheet about the study in question.  
 
I have been informed that my personal data will be subject to processing for scientific research 
purposes by the University of Murcia. The period of conservation of the data will be the minimum 
necessary to ensure the realization of the study or project. However, my identification data, to ensure 
optimal privacy conditions and when the study procedure allows it, could be subjected to 
anonymization or pseudo-anonymization, in any case, the identifying information that could be 
collected will be deleted when it is not necessary.  
 
I have been informed that any inquiry regarding the processing of personal data in this study or to 
request access, rectification, deletion, limitation or opposition may be directed to the address 
protecciondedatos@um.es likewise, I have been informed of my right to file a complaint with the 
Spanish Data Protection Agency.  
I have been given an information sheet and a copy of this informed consent, dated and signed.  
 
Taking this into consideration, I hereby give my consent for this data collection and sample to take 
place and to be used for the purposes specified in the project.  
 
Full name Participant                                                   Signature of Participant              Date 
 
 
Signature of Researcher: