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Development and Validation of a Food Frequency Questionnaire for Population of Adolescents in Croatia

Ana Močić Pavić1*#orcid tiny, Sara Sila1#orcid tiny, Tena Niseteo1orcid tiny, Iva Hojsak1,2,3orcid tiny and Sanja Kolaček1,2orcid tiny

1Children’s Hospital Zagreb, Klaićeva 16, 10000 Zagreb, Croatia

2University of Zagreb, School of Medicine, Šalata street 2, 10000 Zagreb, Croatia

3J.J. Strossmayer University of Osijek, Faculty of Medicine in Osijek, Josipa Huttlera 4, 31000 Osijek, Croatia

Article history:

Received: 10 January 2020

Accepted: 11 March 2021

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Key words:

food frequency questionnaire, 3-day food record, questionnaire validation, adolescents


Research background. Food frequency questionnaire (FFQ) is an important method for estimation of dietary intake in epidemiologic studies. The aim of the study is to develop a FFQ and evaluate its relative validity for adolescents 12 to 18 years old.

Experimental approach. FFQ was developed from previously validated youth/adolescent diet questionnaire (YAQ) by modifying it in order to include Croatian national foods. The final version of the FFQ (FFQ-m) comprised 87 food items. The reference method was a set of two 3-day food records (3DFR) administered twice during the 3 non-consecutive days, one month apart. The FFQ-m was administered approximately on the last day of the second applied dietary food record. Adolescents were recruited from randomly selected elementary and high schools in urban and rural areas of Croatia. FFQ-m was validated on a sample of 84 adolescents (70.2 % female). Nutritional intake from FFQ-m and 3DFR was analysed for each participant. Spearman correlation coefficients (r) and Bland-Altman method were used to assess the validity of the FFQ-m compared to 3DFR. Anthropometric parameters were assessed in 78 adolescents.

Results and conclusions. The mean nutrient intake estimated by the FFQ-m were higher than that of the 3DFR. The average correlation coefficient for energy and nutrients in our validation study was 0.40. On average, 76.5 % of adolescents were classified in the same or adjacent quartile of the nutrient intake. Bland-Altman analysis showed good agreement with all macronutrients and some micronutrients (sodium, phosphorus, potassium, calcium, magnesium and iron). A simple self-administrated questionnaire completed by adolescents is a valid tool for measuring energy and nutrient intake among adolescent population. 

Novelty and scientific contribution. This is the first FFQ developed and validated for population of adolescents in Croatia. It will contribute to further research of nutritional intake in the population of adolescents, especially those from the region.

*Corresponding author: +385958272367


#Both authors contributed equally and share first authorship

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Physicochemical Characterization, Stability and Cytotoxicity of a Blue Dye Obtained from Genipap Fruit (Genipa americana L.)

Camila Verly de Miranda Sabino1orcid tiny, Bárbara Janaína Paula da Silva1orcid tiny, Danielle Lima Bezerra de Menezes2orcid tiny, Felipe Moura Araújo da Silva3orcid tiny, Tatiane Pereira de Souza1orcid tiny, Hector Henrique Ferreira Koolen4orcid tiny, Ádley Antonini Neves de Lima2orcid tiny and Emerson Silva Lima1*orcid tiny

1Faculty of Pharmaceutical Sciences, Federal University of Amazonas, Av. Gen. Rodrigo Otavio, 6200, CEP 69077-000, Manaus, AM, Brazil

2Faculty of Pharmacy, Federal University of Rio Grande do Norte, Av. Sen. Salgado Filho, 3000, CEP 59078-970, Natal, RN, Brazil

3Institute of Exact Sciences, Federal University of Amazonas, Av. Gen. Rodrigo Otavio, 6200, CEP 69077-000, Manaus, AM, Brazil

4Metabolomics and Mass Spectrometry Research Group, University of Amazon State, Av. Carvalho Leal, 1777, CEP 69050-010, Manaus, AM, Brazil

Article history:

Received: 20 May 2020

Accepted: 4 March 2021

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Key words:

Genipa americana, genipap fruit, natural dye, geniposide


Research background. The current commercial scenario indicates an increase in the demand for natural dyes. Compared to synthetic dyes, natural ones have the advantage of being sustainable, making them of great interest for the food and cosmetic industries. The development of new natural dyes is necessary, as well as the carrying out of complementary research regarding the existing ones. 

Experimental approach. The present study aims to characterize the physicochemical and biological characteristics of the dye obtained from dehydrated endocarp of the genipap (Genipa americana) fruit, as well as perform the relevant stability and cytotoxicity tests. The chemical characterization was performed by HPLC-MS/MS analyses. The stability studies were carried out by spectrophotometry and cytotoxicity assays using cell culture and fluorometric methods.

Results and conclusions. After dehydration and milling of the fruit endocarp, water was added to the obtained powder (in the ratio 4:1) to extract the dye. HPLC-MS/MS elucidated five compounds and confirmed the presence of the geniposide as its main compound. With the X-ray diffraction and electron microscopy analysis, we characterised the obtained powder as being amorphous and of porous structure with a variable size. The thermogravimetric analysis indicated a maximum loss of 61 % mass after exposure to a temperature range from 240 to 760 °C. The obtained blue dye was stable in the absence of light, at room temperature and had neutral pH. In the cytotoxicity assay, (95.0±1.3) % of viable human fibroblast were observed after exposure to this dye. The genipap fruit can be a viable alternative to produce a natural blue dye, since it is easy to obtain and has very low toxicity in food, pharmaceutical or cosmetic products.

Novelty and scientific contribution. This study demonstrates for the first time the physicochemical and biological properties of a natural blue dye from G. americana fruit.

*Corresponding author: +5592988177360

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A Molecular Approach for the Detection and Quantification of Tribolium castaneum (Herbst) Infestation in Stored Wheat Flour

Aditi Negi1,2orcid tiny, Arunkumar Anandharaj3*orcid tiny, Sureshkumar Kalakandan3orcid tiny and Meenatchi Rajaman1*orcid tiny

1Department of Primary Processing, Storage and Handling, Indian Institute of Food Processing Technology, Thanjavur-613005, Tamil Nadu, India

2Bharathidasan University, Tiruchirappalli - 620024, Tamil Nadu, India

3Department of Food Safety and Quality Testing, Indian Institute of Food Processing Technology, Thanjavur-613005, Tamil Nadu, India

Article history:

Received: 20 July 2020

Accepted: 4 March 2021

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Key words:

insect detection, wheat flour, T. castaneum, qRT-PCR, DNA, red flour beetle


Research background. The presence of insect fragments is one of the major constrains in stored food commodities and cause considerable loss in the quality of the produce. The management of the pest is viewed as a huge challenge in food processing industry. Conventionally, the detection of Tribolium castaneum in the food processing industry is carried out by acid hydrolysis and staining methods that are time consuming and lack precision.

Experimental approach. Considering the importance of a quick and effective method, a quantitative real-time polymerase chain reaction (qRT-PCR)-based approach was developed and elucidated in this study. The mitochondrial cytochrome oxidase I (mtCOI) gene was identified as a target due to the abundance in the pest. Specific primers were designed against the target gene by Primer Premier software for amplification in qPCR.

Results and conclusions. This method is capable of detecting all the ontogenic stages of T. castaneum in stored wheat flour. Earlier experiments had demonstrated that about 20 µg of DNA can be obtained from 2.2 mg of insects. To quantify the infestation levels, the cycle threshold (Ct) values obtained from known samples were subjected to regression analysis and expressed as adult equivalents. In the unknown samples, the infestation was calculated as 1.74 and 0.046 adult insects in 5 g of wheat flour. The maximum permissible limit of insect fragments in flour is 75 insect fragments or approx. 3 adults per 50 g of flour as per the US Food and Drug Administration (FDA). Hence, by adopting this new method, it is possible for the warehouse operators to arrive at a decision to proceed with efficient management practices were wheat flour is stored. Also, this method can be ratified by government agencies associated with international business to ascertain whether the wheat flour meets the standards set by the respective country before subjecting to foreign trade.

Novelty and scientific contribution. This study is the first of its kind in the detection and quantification for T. castaneum in milled products. So far only conventional methods have been employed to assess the presence of the pest and manual counting of fragments is practiced to quantify the infestation levels. The developed qRT-PCR method is faster, more reliable and can be employed in milling industries, bakery industries, food processing plants and foreign trade units for critical detection and quantification T. castaneum pest infestation.

*Corresponding author: +919750968420; +919940058889

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Proteolytic Lactococcus lactis and Lipolytic Enterococcus durans of Dairy Origin as Meat Functional Starter Cultures

Mirna Mrkonjic Fuka1*orcid tiny, Ivica Kos2orcid tiny, Ana Zgomba Maksimovic1orcid tiny, Melita Bačić1orcid tiny and Irina Tanuwidjaja1orcid tiny

1Department of Microbiology, University of Zagreb Faculty of Agriculture, Svetosimunska cesta 25, Zagreb, Croatia

2Department of Animal Technology, University of Zagreb Faculty of Agriculture, Svetosimunska cesta 25, Zagreb, Croatia

Article history:

Received: 1 July 2020

Accepted: 22 February 2021

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Key words:

fermented sausages, native starter cultures, dairy origin, rep-PCR, sensory properties


Research background. As fermentation is an integral feature of both, dry sausage and cheese production this has led to the evaluation of bacterial cultures Lactococcus lactis ssp. cremoris (LL8307) and Enterococcus durans (ED0207) originally isolated from artisanal Croatian hard type cheese to diversify the range of flavours of dry fermented sausages and to increase their microbiological safety. Both strains were chosen for their high or medium acidifying, proteolytic and/or lipolytic activity and bioprotective potential after step-by-step selection of wild isolates. Therefore, this study aims to evaluate the survival rate of selected starter cultures in wild boar meat sausages during the ripening period of 40 days at a local small-scale facility under artisanal conditions as well as their influence on sausage quality parameters. 

Experimental approach. Safety, biotechnological and probiotic properties of twenty-three enterococcal and lactococcal isolates of dairy origin were studied. Based on the results, two best candidates were selected and added to the meat batter during the artisanal wild boar meat sausage preparation where their survival rate, the effect on physicochemical, microbiological and sensorial properties and histamine content were evaluated.

Results and conclusions. As revealed by repetitive element-polymerase chain reaction (rep-PCR), native starter cultures survived up to 15 days of ripening and were either absent from (LL8307) or reduced by 80 % (ED0207) in final products. The application of native starter cultures rapidly decreased pH (p<0.05) leading to the significantly lower load of E. coli, coliforms and Enterobacteriaceae in ready-to-eat sausages prepared by the addition of starter cultures (3.04-3.94 log CFU/g) than the control (3.88-5.00 log CFU/g). Analysis of hedonic test data revealed that some of the sensory traits (odour, flavour, juiciness) of treatments with starter cultures were highly liked by the higher percentage of consumers. The results suggest that these starter cultures would represent a valuable tool to improve the homogeneity of artisanal manufacture and hygienic quality of fermented sausages and can be safely used for food application.

Novelty and scientific contribution. This is the first study to explore in depth the biotechnological potential of bacterial cultures isolated from artisanal Croatian cheese as functional starter cultures for high-quality game meat sausages production.

*Corresponding author: +38512394034

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Mozzarella Cheese Stretching: A Review

Mônica Correia Gonçalves1*orcid tiny and Haíssa Roberta Cardarelli2orcid tiny

1Federal University of Paraíba, Department of Food Engineering, Center of Technology, Campus I, Cidade Universitária, Av. Castelo Branco s/n, João Pessoa, PB, 58051-900, Brazil

2Federal University of Paraíba, Center of Technology and Regional Development, Campus I, Rua dos Escoteiros, s/n, Mangabeira, João Pessoa, PB, 58058-600, Brazil

Article history:

Received: 15 March 2020

Accepted: 3 March 2021

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Key words:

pasta filata cheese, functional properties, stretching temperature, calcium


Mozzarella cheese stretching is a thermomechanical treatment influenced by factors such as pH, acidity, stretching time and temperature. The aim of this minireview is to provide information about the stretching step and the effect of the main factors on the functional properties of mozzarella. The presented studies show that stretching under higher temperatures promotes more interactions in the protein matrix, and changes occur in the calcium balance throughout the storage period that influence water mobility, proteolysis and lead to changes in mozzarella properties. Therefore, the information presented in this minireview may facilitate the production of mozzarella cheese with specific functional properties.


*Corresponding author:

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