Nutritional characterization of carobs and traditional carob products

Abstract Twenty traditional carob products were measured for their nutritional composition, and their results were compared with the pulp of Cypriot carob cultivars. Moisture, ash, fat, proteins, sugars, dietary fibers, minerals, caffeine‐theobromine, carbohydrates, and energy value were determined. Fluctuations of the nutritional composition values based on the ingredients’ chemical synthesis and product manufacturing process were noted. Only 60% of the products had a label indicating their nutritional value, and the majority of them (75%) were consistent with that of labeling. Chemometric analyses distinguished the carob products according to their type and the discriminator components highlighted their particular nutritional value. Carobs can be characterized as functional foods with low‐fat content, high content in dietary fibers, and high content and/or source of minerals; however, carob products partially satisfied those health and nutritional claims as expected. This pilot research contributes to the nutritional estimation of carob and highlights the traditional carob products.

Many studies have shown that carobs and their products can promote human health and help prevent specific chronic diseases. In particular, they show antiproliferative and apoptotic activity against cancer cells, they are suggested to treat diarrhea symptoms, and they present antihyperlipidemia and antidiabetic effects due to their high antioxidants, polyphenols, and high content in fibers (Theophilou, Neophytou, & Constantinou, 2017). Therefore, they are considered ideal food for people with diabetes (Youssef et al., 2013). Carob flour (from carob seeds) is used to manufacture dietetic products and products for celiac patients (gluten-free products) (Tsatsaragkou & Gounaropoulos, 2014).
Carob is an indigenous drought-and temperature-tolerant tree cultivated in Cyprus for centuries. In the past, it significantly benefited the agricultural economy of the island. It was widely known as the "black gold" of Cyprus. In recent years, carob's health benefits and nutritional value are being highlighted and therefore traditional carob-based food products end up in the market. In Cyprus, many traditional carob products are produced (Table 1); the most widely known is the carob syrup (charoupomelo) which is exported to many countries. The literature work on Cyprus carob cultivars is very limited. Currently, the geographical origin and type of Ceratonia siliqua L. material (flesh and pods), from Cyprus and other countries, were discriminated based on Fourier transform infrared (FTIR) spectroscopy and chemometrics (Christou, Agapiou, & Kokkinofta, 2018).
According to the modern market needs and progressions, the next years, carobs' importance is expected to enhance globally due to: the cocoa shortage (Loullis & Pinakoulaki, 2018), the trend toward health and nutrient supplements, the need for biological and glutenfree products, along with the necessity for natural hydrocolloids.
To our knowledge, little is known about standardization of traditional carob products. This is due to nonindustrial food processing methods, limited manufacturing practices, and lack of long-term documented research. Highlighting the importance of traditional products is a modern food industry trend (protected designation of origin, PDO and protected geographical indication, PGI), along with that of discovering or creating new functional foods (Regulation (EU) No 1151/2012; Luykx & van Ruth, 2008). Therefore, the aim of the present study is the adequate study of the nutritional value of carobs and carob products with origin from Cyprus using official and approved analytical methods and chemometrics. Toward this, the issue of insufficient labeling of traditional carob products is highlighted and data for evaluating their efficacy as functional foods are provided. The examined products are presented in detail in Table 1.

| MATERIAL S AND ME THODS
All the analyses were performed in duplicate or triplicate, under the same laboratory conditions, in an accredited laboratory using either official or accredited/validated methods. Moisture, ash, fat, proteins, sugars, dietary fibers, minerals, and caffeine-theobromine were determined in all samples, and the results were compared to that of domestic carob fruits, widely found in the island of Cyprus (cultivars: Koumpota, Kountourka, and Tylliria). Carbohydrates were calculated by difference, and energy value was calculated with the energy calculation factors.
Ash was determined by Dry Oxidation-Incineration process according to method AOAC 14-098 and (Kirk & Sawyer, 1991 Determination of caffeine and theobromine was carried out with HPLC ((HPLC -Alliance, Water 2695) and PDA detector (λ = 273 nm).

The selected mobile phase was MeOH/H 2 O 30:70 v/v, Waters
Spherical C 18 300 mm × 3.9 mm × 5 μm column was chosen, and the results were processed using Millennium software (empowered 3).
Caffeine ≥99% and theobromine ≥99% were purchased from Sigma-Aldrich, Carrez I and Carrez II solutions were in-house prepared, and Sartorius Stedim membrane filters 0.5 μm were used. The method limit of detection (LOD) was estimated at 0.085 mg/100 ml for caffeine and 0.265 mg/100 ml for theobromine.
Energy value was calculated based on the energy calculation factors, and the total carbohydrates were calculated by difference.

| Data analysis
The results of all the applied analytical methods were statistically proceeded using SIMCA 13.0 (Umetrics, Sweden) for the differentiation of the samples. Each sample was considered as an assembly of 21 variables represented by the chemical data. All the available results were used (Dataset 21 variables × 46 observations × 4 groups, shown in Supporting information Table S1), including all the samples in duplicate (46 samples). Pattern recognition tools used in this work are as follows:

| Principal component analysis (PCA)
This procedure was applied mainly to achieve a reduction of dimensionality to permit a primary estimation of the variation in the data matrix (Kokkinofta & Theocharis, 2005). The data were mean-centered with Unit Variate Scaling (UV), and the PCA model was extracted at a confidence level of 95%.
Partial least square-discriminant analysis (PLS-DA): This technique builds classification model by separating the systematic variation in X into two parts, one that is linearly related to Y (predictive information) and one that is unrelated to Y, at a confidence level of 95%. The resulting loading and contribution plots reveal the most discriminating variables (Kokkinofta et al., 2017). The efficiency of the models was described by the goodness-of-fit R 2 (0 ≤ R 2 ≤ 1) and the predictive ability Q 2 (0 ≤ Q 2 ≤ 1) values. The R 2 explains the variation (how well the data of the training set is mathematically reproduced), while the Q 2 explains the predictive ability of the model (it represents the fraction of the variation of Y that can be predicted).
The models have been validated using cross validation-analysis of variance (CV-ANOVA), with a p-value < 0.05. For validation purposes, the misclassification table was calculated.

| Uncertainty
The results of moisture, ash, and fat were given with their uncertainty (the experiments were held with triplicate samples). Proteins, dietary fibers, and sugars were analyzed in duplicate samples. The

| Nutritional analysis
In Figure 1,    The LOD for caffeine was estimated 0.085 mg/100 ml and for theobromine 0.265 mg/100 ml, c Where A, B, C, D, E, F, G = different producer Caffeine and theobromine content in carobs pulp, carob powders, and carob coffee were found below the LOD of the method.
This is in agreement with the results obtained by other researches (Craig & Nguyen, 1984;Kumazawa et al., 2002 The energy value ranges from 280.17 to 286.07 kcal/100 g in carob pods and from 280.40 to 587.59 kcal/100 g in carob products. Moreover, the results were compared with the nutritional value on product labels (Supporting information Table S3). The labels of eight products made no reference to their nutritional value and simply included their ingredients. This is a known and expectable problem in traditional products due to limited personnel and resources involved.
In the absence of research and development departments, products are frequently produced empirically. The examined products derived from small, family enterprises with limited ability to perform frequent nutritional composition analyses. However, after the transitional period of the Regulation (EU No 1169/2011), the nutritional labeling will be mandatory.
It should be stressed that the composition and nutritional quality of carobs are affected mainly by the genotype, the harvest period, the growing conditions, the climatologically conditions (e.g., sun, and water availability), the soil content, and the overall microclimate. In the same way, the postharvest conditions (e.g., storage and distribution of the food chain) should not be underestimated.

| Chemometric analysis
First, basic statistics was performed in order to extract the correlation between the original variables (nutritional components). The correlation matrix is presented in Supporting information Table S4 a. Proteins, Mg, P, and Mn are highly correlated, while Cu has a smaller but significant correlation with proteins and P. Finally, glucose shows good correlation with Ca and P. Boxplots in Supporting information Figure  syrup differentiates mainly due to its glucose content, which is significantly higher in the products from mixing carob syrup and honey (S17, S18).
Furthermore, along the second principle component, a metalbased trend was observed, as shown in the loading plot ( Figure 2b).
The creamy products of carob are strongly characterized by sodium, while calcium is responsible for distinguishing between the samples of pulp, powder, and coffee, which appear very close with great similarity.
Partial least square-discriminant analysis was applied to validate the previous results on the influence of the production process to the composition. The discrimination was satisfactorily correct, although the above mixtures of carob syrup and honey classified as "pasteli" that is not entirely wrong, considering similar production

| CON CLUS IONS
A holistic approach was followed for the nutritional analysis of carobs, from the fruit to the final commercial product. The chemical composition of Cyprus carob cultivars was examined for the first time, and the results were compared with that of commercially available carob-based traditional products using chemometrics tools. The possibility of characterized carobs as functional foods (fat, dietary fibers, and minerals) was explored, taking into consideration the presence of many antioxidant components (e.g., polyphenols and flavanols), as indicated in the previous literature studies (Stavrou, Christou, & Kapnissi-Christodoulou, 2018). Additionally, carobs contain high amounts of D-pinitol (3-O-methyl-chiroinositol), a natural bioactive and effective ingredient, with proven insulin-like function (Bates, Jones, & Bailey, 2000). According to the current results, the nutritional or chemical composition of foods changes if the amount and type of ingredients used in the formulation differed. The examined food products are usually commercially available in small local markets, and traditional products suffer from standardization, internal national control on product identity, and the requirement for strong nutritional labeling and/or packaging.
Modern diet and lifestyle are associated with severe diseases in the Southern and Eastern Mediterranean (e.g., obesity, diabetes, and cardiovascular disease), and therefore, biological food market is growing. Carobs appear to fulfill modern health criteria of consumers (i.e., gluten-and caffeine-free product, natural chocolate-like sweetener, ingredient for bread, beverages such as liqueurs and tea, nuts, tahini, and honey). Local efforts for PDO and PGI products can be supported and enhanced within Europe, especially when such fruits and products are cultivated and linked with the tradition and history of many European countries (e.g., Spain, Italy, Greece, Portugal, and Cyprus). Besides, in the old days, the consumption of fruits, nuts, and cereals was part of the Mediterranean diet.
Therefore, carob and carob powders are proposed to be included in humans' daily diet as they contain valuable nutrients, low fat and have a sweet taste. However, this is not the case for all carob products, as some of them were determined with a high-fat content, and they are neither a source of edible fibers nor minerals. Carob products partially satisfied health and nutritional claims: 60% in terms of fat, 25% in terms of dietary fibers, and 80% in terms of for minerals. Moreover, although containing enough percentage of fat, some products also contain enough nutrients. Given the nutritional value of carob, the indication that commercial products contain carob may be misleading for some consumers because they buy these products thinking they are functional foods. Nevertheless, depending on the manufacturing processing, carobs could be used as a natural ingredient for the creation of new functional foods based on the high fiber and minerals content and low fat levels. Carob, indigenous in Cyprus, is of high biological value and must be protected. Standardization of carob products is proposed in order to (a) ensure proper consumer information and protection against misleading indications in product labeling and (b) to protect producers from unfair practices in the production of carob products.
In summary, this research assesses for the first time the nutritional components in different carob products, in order to provide tools for their characterization as functional food products. This study can be considered as a pilot; more samples could be analyzed to enhance the above conclusions. Nevertheless, it contributes to nutritional estimation of carob and gives added value to Cypriot traditional carob products.

ACK N OWLED G EM ENTS
The authors would like to thank the "Black Gold" project, financially supported by the University of Cyprus, and the personnel of the "Food Composition, Food Quality and Nutritional Value" Laboratory of the State General Laboratory of Cyprus for their active contribution in the implementation of the current analyses.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing.