Project title: Taxonomy, Ecology and Utilization of Carob Tree (Ceratonia siliqua L.) and Bay Laurel (Laurus nobilis L.) in Croatia

 

Acronym: TEUCLIC

 

Agency: Croatian Science Foundation

 

Grant number: 3304

 

Duration: 48 months (start: 7/7/2014; end: 7/7/2018)

 

Total budget: 719,945.59 HRK

 

Budget in first year: 249,222.50 HRK

 

Principal investigator: Dr Siniša Srečec, scientific counsellor

 

Institution of principal investigator: Križevci College of Agriculture

 

Collaborating institution (by alphabetical order): Faculty of Agriculture University of Zagreb, Faculty of Pharmacy and Biochemistry University of Zagreb, Faculty of Food Technology and Biotechnology, Faculty of Science Dept. of Biology University of Split, Faculty of Veterinary University of Zagreb 

 

Number of researchers: 22

 

 

Extended Synopsis of the project:

 

Carob tree (Ceratonia siliqua L.) is nutritionally and protectively very valuable plant. The most important part of carob are seeds, containing polysaccharides, which are widely used in the food industry. They are, therefore, a cheap source of natural polyphenolic phytochemicals, whose nature and importance is, as yet, poorly investigated. Antioxidant behaviour of carob pod extract is a rather complicated issue, considering that the antioxidant characteristics examined represent, in essence, the integration of actions of more than one polyphenolic classes. It can be claimed that extracts containing the same total polyphenol content as aged red wines appear significantly more potent (Makris and Kefalas, 2004). Using of carob is also known in feeding of ewes and weaning kids, because feeding with carob-based diet with PEG (polyethylene glycol) increase feed intake, crude protein digestibility and growth to the rate obtained with the control diet (Silanikove et al., 2006; Silanikove et al., 1994). Goats have inhabited Mediterranean scrubland areas for 7000 years. One of the most important abilities of goats is to consume large amounts of tannin-rich plant material without exhibiting toxic effects. Goats are well adapted to the nutritional environment and may consume large amounts of tannins (1.1-2.7 g kg-1 BW per day condensed tannins and 0.4-0.9 g kg-1 BW day-1 soluble phenolics) without suffering any ill effects (Silanikove et al., 1996). Generally in all the countries of Mediterranean basin, Carob trees provide shelter and nutritious fruit for livestock. Its pods and seeds are a good source for protein, carbohydrates, and fibres. Both pods and seeds contain enough crude protein and energy to meet the maintenance and lactation requirements of ewes. Livestock break the seed-coat dormancy and disperse seed to new sites. However, goats may be more efficient transporters of carob seeds because of their ability to disperse viable carob seeds over a longer period (Moh’d Khair J. El-Shatnawi and Khalil I. Ereifej, 2001). According to some research, extracts of carob (Ceratonia siliqua L.) (i.e. ethanol extract, n-hexane extract, methanol extract and water extract) showed also a good antimicrobial activity against some of the most important pathogenic microorganisms, such as: Escherichia coli ATCC 29998, Escherichia coli ATCC 25922, Escherichia coli ATCC 11230, Salmonella typhimurium CCM 5445, Enterobacter cloacae ATCC 13047, Enterococcus faecalis ATCC 29212, Staphylococcus epidermidis ATCC 12228, Candida albicans ATCC 10239, Pseudomonas aeruginosa ATCC 27853 and even against Staphylococus aureus ATCC 6538P, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228 (Kivçak, 2001; Alzorekya and Nakahara, 2003).

In spite of the historical fact that the value of carob tree was recognized by the ancient Greeks, who brought it from its native Middle East to Greece and Italy, and by the Arabs, who disseminated it along the North African coast and north into Spain and Portugal, Carob has been neglected with respect to both cultural practices and research and development. It was spread in recent times to other Mediterranean-like regions such as California, Arizona, Mexico, Chile and Argentina by Spaniards, to parts of Australia by Mediterranean emigrants and to South Africa and India by the English. Even nowadays, carob pods and juice has a very important role in diet of Mediterranean countries (Akbulut and Bayramoglu, 2013; Rababah et al., 2013). During the nineties years of the last century, world production of carobs is estimated at about 310 000 t/year produced from some 200 000 ha with very variable yields depending on cultivar, region and farming practice (Batlle and Tous, 1997). According to FAOSTAT, a total world harvested area of carobs in 2011 was 84 168 ha, with total production of some 187 080 tonnes. In spite of that fact, carob tree is important component of the Mediterranean vegetation and its cultivation in marginal and prevailing calcareous soils of the Mediterranean region is important environmentally and economically. According to FAOSTAT harvested area of carobs in Croatia in 2011 was 550 ha (or 0.65 % of total world harvested area) with total production of 553 tonnes (or 0.29 % of total world production). Traditionally, grafted carob trees have been interplanted with olives, grapes, almonds and barley in low intensity farming systems in most producing countries (Batlle and Tous, 1997). Thus, development of production of carob in Croatia might have a positive impact on economical and consequently demographical revitalization of Croatian islands. Unfortunately, in last decade, because of aggressive urbanization of Croatian islands, a lots of carb trees has been reclaimed. So, one of the specific goals of this project is to stop that tendency, because the carob tree is one of the important economic resources of Croatian islands.

          Bay Laurel (Laurus nobilis L.) is also like carob tree (Ceratonia siliqua L.) nutritionally and protectively very valuable plant, which grows in similar ecological conditions. It is also overspread on the Mediterranean basin and bay laurel is also an important component of the Mediterranean vegetation and its cultivation in marginal and prevailing calcareous soils of the Mediterranean region. The most important parts of the bay laurel plant are leaves which contain considerable quantity of essential oil.

The major chemical constituent of essential oils in leafs of bay laurel is linalool, which presented certain lower antibacterial activity against foodborne pathogenic bacteria such as Escherichia coli ATCC 25922 (Millezi et al., 2013). The yield of free volatile aglycones in fresh plant material of bay laurel leaves is among 0.14 mg g-1. Also, the yield of essential oils in the same plant tissue is among 6.20 mg g-1. Because of that phytochemical compounds, bay laurel has a valuable antioxidant potential.  However, during maturation, storage, industrial pretreatment or processing, volatile compounds can be released from these non-volatile precursors by enzymatic or chemical reactions. Consequently, glycosidically bound volatile compounds can be considered a hidden antioxidant potential of plant materials and may contribute to total antioxidant activity of bay laurel, which is very important for potential use of herbs and spices in the food and pharmaceutical industries to increase the shelf life of foodstuffs, as well as to prevent cellular damage, a cause of human diseases and ageing (Politeo et al., 2007) However, ethanol extracts of bay laurel shows great potential as a source of microbial growth and quorum sensing inhibitors, particularly against Chromobacterium violaceum (Al-Hussaini and Mahasneh, 2009). Leaves of bay laurel are traditionally used to treat some symptoms of gastrointestinal problems, such as epigastric bloating, impaired digestion, eructing and flatulence. According to results of modern research high amounts of phenolic compounds can be extracted from leaves of bay laurel by ultrasound-assisted extraction technology (Muñiz-Márquez et al., 2013) and also by supercritical fluid extraction (Santoyo et al., 2006). It is very important to point out that cultivated laurel had a high content of terpenes such as linool, α-terpinol, α-terpinyl acetate, thymol, caryophyllene, aromandrene, selinene, farnesene, and cadinene, while wild laurel had a high content of eugenol and methyl eugenol, vitamin E, and sterols in the comparison with bay laurel from spontaneous vegetation. On the other hand, significant antioxidant activity measured in bovine brain was observed in wild laurel (Conforti et al., 2006). According to Tabassum and Vidyasagar (2013), plant essential oils are potential source of antimicrobials of natural origin. Essential oils and extracts obtained from many plants have recently gained a great popularity and scientific interest. Consumer demand for natural preservatives has increased, whereas the safety aspect of chemical additives has been questioned. The plant oil has been reported to have antibacterial, antifungal, antiviral, antiparasitic and antidermatophytic properties. It is now considered as a valuable source of natural products for development of medicines against various diseases and also for the development of industrial products. Which means that bay laurel, as almost forgotten plant, has very important and even increasing role in future agriculture, as well as in modern food preservation and pharmacology. Respecting the small level of pollution of Croatian islands and some coastal areas, out of main roads and urban areas, growing, and processing of bay laurel is completely complementary with growing of carob trees and both of them, as alternative or even main crops, might have a positive impact on reconstructing of agriculture and economy of Croatian islands. Additionally, bay laurel in the southe Dalmation (in colloquially language know as ‘Dubrovački lovor’) posses the oil of unusually good quality and it is worth to be investigated.

In order to realize the three main research goals; to investigate taxonomical and ecological characteristics, as well as utilization of carob tree and bay laurel (as stays in project tittle), the 21 members of the research team are divided into 3 research divisions, such as:

 

1.   Division for taxonomical characterizations of plants

Aim: to determine morphological, genetic and phytochemical traits of Croatian populations of carob tree and bay laurel. Carob tree and bay laurel contain many phytochemicals and because of that the considerable biological activity could be expected. With regard to isolation of some island' populations, significant morphometric, genetic and phytochemical traits some differences among populations could be expected. Through this investigation we would like to help the revitalization of carob tree in Croatia. Additionally, we would like to stimulate cultivation of best populations of bay laurel as well as carob.

Research: Morphometric and phytochemical investigations of ten to fifteen indigenous populations of carob tree and bay laurel will be made on leaf and fruit (carob tree) samples (gathered in situ), while the genetic variability will be determined using AFLP analysis of genomic DNA isolated from the leaf. Plant material of both species will be collected along the Adriatic coast and Croatian islands. From each of ten to fifteen population samples of leaves and fruits will be collected from 10 to 20 individuals, depending of individuals’ number, for morphometric and genetic investigations. Samples of leaves will be dried with silica gel and stored in plastic bags and fridge on –80 °C. For phytochemical investigations leaves and fruits samples will be dried on air in dark room and stored in paper bags. For determination differences in essential oil contents leaves of bay laurel will be collected several times during the years. On each carob tree leaves following traits will be measured: number of leaflets, petiole length, length and width of leaflets. Also, the weight, length and width of pod and seed will be measured. In bay laurel the length and width of leaves will be measured. Plant extracts will be prepared from leaves and fruits and content of phenolic compound will be determinate. Content of total polyphenol and tannin will be determined using Folin-Ciocalteu spectrophotometric method while the content of total flavonoids (quercetin type) will be determined using the spectrophotometric method (Christ and Müller, 1960) as well as HPLC-method. Antioxidant activity will be determinate using several in vitro metods. Free radical scavenging activity (RSA) will be evaluated by the scavenging of DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2-azinobis 3-etilbenzotiazolin-6sulfonat) methods. Additionally, the FRAP (Ferric reducing power) assay will be used to estimate the antioxidant potential of tested extracts. Analysis of essential oil will be done using gas chromatography and mass spectrometry (GC-MS).

 

2.   Division for ecological research of plant habitats and statistics

Aim: to determinate the ecological characteristics of plant habitats, particularly soil characteristics in order to find the interactions between ecological factors (i.e. concentration of plant nutrients in soil, organic matter in soil and concentration of heavy metals in soil, altitude of habitats, annual rainfalls and annual sum of temperatures and their distribution etc.) on some chemical characteristics of carob and bay laurel. This is particularly important for possible explaining the differences in chemical characteristics of plants between and inside the population(s) in case of weak genetic variability between populations. This division is in charge for statistical data processing.

Research: pH of soil in H2O and 1 M solution of KCl will be analysed according to ISO 10390: 1994 method. Content of organic matter (%) will be analysed according to Tjurin’s method. Total nitrogen (%) will be analysed according to ISO 11261:1995 method. Total content of CaCO3 will be analysed according to Scheibler method and content of physiological active phosphorus and potash, calculated in mg of P2O5 and K2O per 100 grams of soil, will be analysed according to Egner-Riehm-Domingo’s ammonium lactate method. Analyses of heavy metals will be conducted using the atomic absorbance spectrophotometry. All gathered data (both of taxonomical as well as ecological research) will be statistically analysed using the suitable statistic methods. Descriptive statistics (average, standard deviation, coefficient variability) will be computed. For small samples t-test, F-test, Wilcoxn mathed pair test and Mann-Whitney U test will be used. For larger samples ANOVA and MANOVA will be used, as well as different post-hoc tests (Tukey, Duncan, Scheffé). Data will be also analysed using the different methods of multivariate analysis (cluster analysis, principal component analysis, linear as well as multiple correlations, etc.). Statistical analysis of obtained results will be made with Statistica v. 10.0. (StatSoft, Tulsa, SAD). The researchers will, depending of situation and “grouping” of analytical data, also use a non-parametric statistical methods such as Spearman’s Rank Order Correlations and Kendall t. According to nature of analytical data and their distribution, the researchers will use some of data transformations such as log(x) and ln(x) transformation, and arcsin transformation. The minimal level of pronouncing significance achieved differences between analytical data is p<0.05.

 

3.   Division for research of carob and bay laurel utilization

3.a Team for research of carob and bay laurel utilization in veterinary and animal husbandry. Aim: In modern intensive farming of animals (porcine, poultry, small ruminants) for human consumption considerable efforts are being made in the understanding of intestinal infectious diseases, their diagnosis, including the biology of pathogens, host resistance and treatment, where little is known about the prevention of these diseases uses using immunomodulatory and nutritional strategies. In fact, all these problems so far have been solved by adding a suboptimal dose of antibiotic growth promoters in animal feed. Fears from a possible risk to human health because of use and / or misuse of antibiotic growth promoters in food for animals, has led to a ban on their use in the EU (Regulation EC No. 1831/2003). In order to adjust to the withdrawal of antibiotic growth promoters in food for animals (in the EU since 2006), it is obligated for Croatia, as an EU member state since July 1 of 2013, to comply with the legislation of the EU and to join the European scientific trends in veterinary medicine in order to determine the relevant health criteria, as well as science-based recommendations for the use of alternative strategies for antibiotic growth promoters in food for animals. The major problem in modern pig production, especially in weaned pigs, are infections caused by enterotoxigenic Escherichia coli (ETEC) strains. So today, when usage of antibiotic growth promoters in farm animal production is abandoned, one of the possible alternative strategies for the control and prevention of E. coli infection could be a blending of carob tree, Ceratonia siliqua L. in animal feed, where it is necessary to verify and define its potential probiotic and immunomodulatory effects on the health and productivity of weaned pigs. Specifically, flavonoid components of carob tree, as natural substitute for antibiotic growth promotors in food for animals, and it has shown positive effects on the health status of animals and production indicators. Therefore, the aim of proposed research is to validate possible positive effects of addition supplement of carob tree in standard food for animals on health and production parameters in weaned pigs as the natural replacement of antibiotic growth promoters.

Research: According to available literature, positive effect of addition of carob tree on the digestive system of domestic pigs, was described after addition of supplement of carob tree in concetration of 10 % in standard daily mixture. In modern intensive pig production, coli diarrhea and coli enterotoxemia usually appear 5-10 days after weaning, i.e. in the period when the piglets in a short time must be adapted to a numerous changes in their organism, the environment and nutrition. The experiment will be carried on according to principles of good practice in animal husbandry and good veterinary practice respecting the Animal Welfare Law of the Republic of Croatia and EU legislation.

 

3.b Team for research of utilization of carob and bay laurel in food technology.

Aim: Fruit of the Carob has a high nutritive value.  To make the best use of the fruit it should be dried, and grinned. Carob powder, made by grinding the roasted pod, is used for the human food industry (with cocoa products and syrups). Due to increasing consumer demand for functional foods it is our goal to   develop a new, enriched product that could substitute different less healthy fillings of backed products as well as bases for soft drinks.  Physical as well as chemical characteristics of Carob powder are important to determine its behaviour in different filling mixtures or their solubility characteristics if they are a part of a drink.

Ripe pods also have potential as a high energy stockfeed. The gum is used as a thickening or binding agent, particularly for canned pet food products.

Research: It is known from prior research that the effect of different granulation of milled powders is essential to the quality of products, therefore after milling a granulometric analysis will be made  on Malvern 2000 (Malvern Instruments Ltd., (Worcestershire, Great Brittan) so that further experiments can be done with particles of different size. These fractions will be mixed in Turbula shaker mixer with other ingredients according to recopies with the aim to obtain filings for drinks, pastries and cakes, and animal feed. Flow characteristics of these mixtures will be analysed with Powder Flow Analyser  (Stable MicroSystems, Godalming, Great Britain) while their behaviour as a bulk are of importance for further production and its behaviour in it. These products will be analysed on texture analyser to see how they behave under different production systems which is parameter important to the industry. All the obtained new products (Carob based fillings, Carob based soft drinks, Carob based animal feed) will be sensory evaluated to see how consumers and animals will accept them. The interdependence of all the physical characteristics of the same sample will be estimated by Spearman correlation coefficients as a method of nonparametric statistics with a significance level of p <0.05.

 

Research work in these three divisions will be coordinated by Project Headquarter:

1.    Dr Siniša Srečec, scientific counsellor – principal investigator (PI) and coordinator of Division for ecological research of plant habitats and statistics

2.    Dr Dario Kremer, scientific counsellor – executive deputy of principal investigator and coordinator of Division for taxonomical characterizations of plants

3.    Prof Dr Maja Popović – coordinator of Team for research of carob and bay laurel utilization in veterinary and animal husbandry

4.    Prof Dr Ingrid Bauman - coordinator of Team for research of carob and bay laurel utilization in in food technology