extfieldsofstudy: Medicine Biology

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2019.jsonl.gz.gz

text:
Genetic Structure Analysis of a Collection of Tunisian Durum Wheat Germplasm

The Tunisian durum wheat germplasm includes modern cultivars and traditional varieties that are still cultivated in areas where elite cultivars or intensive cultivation systems are not suitable. Within the frame of a collection program of the National Gene Bank of Tunisia (NGBT), durum wheat germplasm was collected from different Tunisian agro-ecological zones. The collected samples were studied using simple sequence repeats (SSRs) markers to explore the genetic diversity and evaluate the genetic structure in Tunisian germplasm. The results demonstrated significant diversity in the Tunisian durum wheat germplasm, with clear differentiation between traditional varieties and modern cultivars. The population structure analysis allowed the identification of five subpopulations, two of which appear to be more strongly represented in germplasm collected in central and southern Tunisia, where environmental conditions at critical development phases of the plant are harsher. Moreover these subpopulations are underrepresented in modern varieties, suggesting that traits of adaptation useful for breeding more resilient varieties might be present in central and southern germplasm. Moreover, our results will support, the activity of in situ on farm conservation of Tunisian durum wheat germplasm started by the National Gene Bank of Tunisia along with the ex situ approach.

Introduction
Durum (Triticum turgidum L. subsp. durum, genome AABB, 2n = 4× = 28) and common (Triticum aestivum L. subsp. aestivum, genome AABBDD, 2n = 6× = 42) wheats are species with significant agricultural importance as cereal grains throughout much of the world. In particular, durum wheat is grown mainly in the Mediterranean basin, and is primarily used for pasta, couscous and bread. Durum wheat, which originated in the Fertile Crescent, spread west of the Mediterranean basin, reaching the Iberian Peninsula and North Africa ca. 7000 years BC [1,2]. Wheat reached this area through Egypt, which acted as the bridge to the Fertile Crescent for the diffusion of wheat in North Africa. Other sources of wheat were maritime: wheat was introduced to Libya from Greece and Crete [3]. Moreover from the south of the Italian peninsula through the Sicily region, wheat reached the coasts of Tunisia, Morocco and Algeria [3]. This migration process, combined with both natural and human selection, led to the creation of local varieties along the Mediterranean basin that were well adapted to its different agro-ecological zones. Indeed, Tunisia and Algeria have a large collection of durum local varieties with great diversity, as observed for the first time by Boeuf in 1932 [4]. Contrasting environmental conditions of North Africa triggered a wide diversification of the durum wheat genome determining the development of several local populations. Thereby, North Africa is considered one of the principal secondary centers of diversification of durum wheat [5]. Studies on the origin and migration of durum wheat based on genetic diversity of landraces have shown that Tunisian landraces and those from the Mediterranean basin have a shared allelic identity and, consequently, that they originated from related seed sources and were exposed to similar environmental pressures and accumulated distinct mutations over time [6]. Durum wheat cultivation has continued to the present day as a staple food for local populations and possesses an outstanding socio-economical value as an important basic constituent of the diet in Maghreb countries [7,8].
In Tunisia during the 2018/2019 season, 0.5 M ha of durum wheat were cultivated, representing about 85% of the area devoted to wheat (common and durum), which in turn accounted for more than half of the cereal cultivated area [9]. The durum wheat germplasm cultivated includes about 45 varieties [10] grouped in (a) traditional varieties selected from Tunisian landraces (developed up to 1930s), (b) old cultivars produced by crossing Tunisian traditional varieties and foreign materials (developed up to 1970s) and (c) modern cultivars obtained in national and international breeding centers (developed up to present). The traditional varieties still prevail in rural areas where farmers still use traditional farming systems due to the impossibility of adopting modern intensive ones, or where this germplasm ensures tolerance to stresses [11,12]. The traditional varieties can be defined as the consequence of an empiric breeding program focused on exploiting the phenotypic variability among Tunisian durum landraces, such as 'Biskri', 'Bidi', 'Mahmoudi', 'Chili' and 'Jenah Khottifa' [7,8].
Several descriptions exist to define a 'landrace' [13,14]. Villa et al. [15] remarked that a landrace is 'a dynamic population of a cultivated plant that has historical origin, distinct identity and lacks formal crop improvement, as well as often being genetically diverse, locally adapted and associated with traditional farming systems'. An autochthonous wheat landrace can be defined as a traditional variety with a high capacity to tolerate biotic and abiotic stresses, resulting in high yield stability and an intermediate yield level under a low input agricultural system [16]. Wheat landraces are valuable sources for broadening the genetic base of cultivated wheat. In fact, several studies using morphological, physiological and agronomic traits have shown that genetically diverse local germplasm well adapted to a broad range of environmental conditions can be considered an important reservoir of useful genes for exploitation in wheat breeding programs [17][18][19][20].
Molecular markers provide a satisfactory option for several genetic plant studies, since they are highly polymorphic and almost unlimited in number, do not vary among tissues or developmental stages of a plant and are not affected by environmental factors. Therefore, molecular markers can be used for genome analysis, and many of these have been applied with success for genetic mapping [21,22], cultivar identification [23] and diversity studies [19,20]. Different molecular markers have been developed (see review [24]). In particular, simple sequence repeats (SSRs) markers represent a useful alternative marker system because of their abundance in the wheat genome and easy detection as PCR-based molecular markers. Moreover, the advantages of SSRs include high information content, codominant inheritance, locus specificity and high reproducibility. Fifteen SSRs and five primer pair combinations of amplified fragment length polymorphism (AFLP) were tested by Medini et al. [10] in a wheat collection including Tunisian cultivars and wild wheat species. Cluster analysis separated the wild wheat species from the durum wheat cultivars, and these latter were further split in old and modern cultivars. The discriminant analysis carried out on 196 durum wheat lines issued from landraces collected in Tunisia and genotyped with diversity array technology sequencing (DArTseq) distinguished five groups that matched with the farmer's variety nomenclature [25].
Nowadays, thanks to the activity of the National Gene Bank of Tunisia (NGBT), a program to collect durum wheat germplasm has been started. This program includes the idea of fostering the cultivation of traditional Tunisian durum germplasm by on farm conservation [26]. Within this framework, the maintenance and sustainable management of the genetic diversity present in autochthonous durum wheats are an absolute priority in order to preserve this genetic heritage, ensure food sovereignty and help smallholders cope with climate change. In this context the main goals of this work were to: (i) explore the genetic diversity in Tunisian durum wheat germplasm collected in different agro-ecological regions; (ii) evaluate the genetic structure of the Tunisian durum wheat genotypes; and (iii) clarify synonymy cases among the Tunisian durum wheat samples collected.

Agro-Ecological Conditions of Tunisian Durum Wheat Zones
The socio-agro-ecological zones of Tunisia follow a distribution similar to the natural regions ( Figure 1). In the North, the agro-ecological zone of 'Kroumirie and Mogods' has an average yearly rainfall between 650 and 1500 mm, with severely reduced seasonal and annual variability. This area has two well-identified seasons: a wet season from October to April, and a dry season from June to September ( Figure S1). In the Center, the agro-ecological zone of 'Dorsal and Tell' is formed by two different climatic units, due to the presence of mountain ridges that intercept the humid currents from the Mediterranean (Figure 1). This is a semi-arid bio-climate with cool and temperate winters and precipitations varying with the elevations (higher in the mountain area and lower moving East). The South is characterized by an arid Mediterranean climate with a long dry season (8-9 months) and rainfall ranging from 100 to 200 mm ( Figure S1).
In these three zones, the production of durum wheat shows a strong North-to-South decline along the cline determined by the bio-climate of the areas, as reported by Latiri et al. [27]. The durum wheat germplasm utilized in the present study is representative of the three agro-ecological zones previously described.  [28]. The symbols , and are used to indicate the location of sampling from the North, Center and South Tunisia, respectively.

Genetic Diversity
A set of 16 selected simple sequence repeats (SSR) markers were used to study the genetic diversity in a durum wheat collection including 41 Tunisian samples and 13 cultivars (Table 1). A total of 95 alleles was obtained, ranging from 13 (Xgwm193) to 2 (Xgwm154). The polymorphism information content (PIC) was used to evaluate the informativeness of SSRs. The PIC mean value of the 16 SSRs was 0.57 with a range from 0.82 (Xgwm193) to 0.28 (Xwmc406) as reported in Table 1. A strong relationship (r = 0.84***; p < 0.001) was observed between the number of alleles and PIC values. The Shannon's information index (I) value ranged from 2.07 (Xgwm193) to 0.51 (Xwmc406). The expected heterozygosity (He), which corresponds to heterozygosity at a single locus, ranged from 0.28 (Xwmc406) to 0.82 (Xgwm193). The fixation index (F), which compares the expected heterozygosity (He) with observed heterozygosity (Ho) to estimate the degree of allelic fixation, ranged from -0.30 (Xgwm154) to 1.00 (Xbarc45 and Xgwm193 having all the alleles fixed) ( Table 1). The F mean value was found to be 0.82 for the whole collection, and F values were positive in 15 SSR markers, revealing that the majority of the alleles were fixed. Expressed sequence tag; PIC = polymorphism information content; Na = number of alleles, I = Shannon's information index; Ho = heterozygosity observed; He = heterozygosity expected; F = fixation index.
Based on the dissimilarity matrix, a neighbor-joining dendrogram clearly showed the presence of two main clusters ( Figure 2). The first cluster included 24 Tunisian durum wheat samples and 12 cultivars. This group included nine Tunisian durum wheat cultivars, suggesting a genetic relationship among them. Indeed, Karim and Razzak were the parental lines of several modern durum cultivars obtained from the breeding programs of the National Agronomic Research Institute of Tunisia (INRAT). The second cluster incorporated mainly Tunisian samples and the cultivar Taganrog. In this cluster, the group constituted by four Tunisian durum samples, (Chili_Lans, Chili_Msaken, Chili_Kef and Aouija_Chorb) was found to be genetically identical. This result suggests that, probably, the name 'Aouija' could have been erroneously attributed by local farmers to Chili, although morphologically different. Moreover, four Tunisian durum samples named Biskri_Saou, Biskri_OS, Biskri_Jouf and Biskri_Gafsa were found to be genetically identical and undistinguishable from three samples named Mahmoudi_OS, Mahmoudi_Jouf and Mahmoudi_Site1, thus suggesting a putative case of synonymy. The two main clusters identified in the dendrogram were used to calculate the AMOVA, revealing that 22% of the total variation was found among the clusters while the rest (78%) was within clusters (Table 2). Finally, cluster analysis distinguished the samples with white glumes and black awns from the rest of samples. The durum wheat samples with these traits are included in the second cluster, confirming that the landraces Mahmoudi, Biskri and Chili were similar in these traits [8]. Dendrogram of durum wheat collection resulting from the neighbor-joining cluster analysis based on the dissimilarity matrix obtained from SSR allelic data. The colors blue and red were used to distinguish the two main clusters. Table 2. Analysis of molecular variance (AMOVA) of durum wheat collection subdivided into the two main groups and detected using the neighbor-joining algorithm in DARwin software version 6.01.

Genetic Structure
For population genetic structure analysis, Bayesian clustering modeling was performed using STRUCTURE software with genotyping data generated according to 16 SSR markers. As the clustering model presumes the underlying existence of K clusters, an Evanno [29] test was performed and yielded K = 5 as the highest log-likelihood ( Figure S2). In this way, the durum wheat collection showed a genetic structure split into five subpopulations. A durum wheat sample was considered belonging to a subpopulation if its membership coefficient (Q) was > 0.60, as suggested by Chen et al. [30]. The five subpopulations obtained by the Bayesian method were used to calculate the AMOVA, revealing that 37% of the total variation was found among the subpopulations, while the rest of variation (63%) was within subpopulations (Table 3). The collection was divided in four groups: three included the Tunisian durum wheat samples, spilt according to the agro-ecological zones of sampling location (North, Center, and South); the fourth group included the modern durum cultivars (Figure 3). In each group, the mean Q of the five subpopulations, detected by structure analysis, was calculated and showed a strong genetic stratification. In particular, two main subpopulations (in orange and red) were relatively abundant in Tunisian durum samples, but rare (<10%) in modern durum cultivars. Two subpopulations (in orange and green) were found the main genetic components (some 80%) of durum wheat samples collected in the South of Tunisia. A subpopulation (in yellow) was present at a frequency >60% in modern durum cultivars and almost absent (<1%) in durum wheat samples collected in the South of Tunisia. The AMOVA confirmed that 9% of the total variation was found among Tunisian durum samples split according agro-ecological zones, while the rest (91%) was within groups (Table 4).

Discussion
During the second half of the last century, a significant increase in wheat yield was observed in Tunisia. This was a consequence of the introduction of modern high-yielding varieties able to optimize water and soil resources, as well as a result of improvements in agronomic techniques, such as better crop management [7,8].
In Tunisia, as in the whole Mediterranean region, rainfall is highly variable in its yearly distribution [27]. The overall yearly rainfall is differently distributed over the country, with northern regions much wetter than the Center and South (Figure 1 and Figure S1). The growing season of wheat, with sowing between November and December, extends through the wet and cool season when in central/southern regions there is a reduced level of precipitation but evaporation is relatively low, thus securing enough water for the plants' needs. More critical is the period between April and May, the period of flowering-to-grain filling, when high temperatures or drought stress can dramatically reduce kernel setting, and thus yield [22,27,31]. In this period, the difference in precipitation among the three regions of Tunisia drastically increases ( Figure S1). Therefore, it is possible to suppose that plants adapted to central and principally southern environments should possess traits of adaptation, allowing them to escape from these limiting environmental conditions. In our study, a set of 16 SSR markers with a good coverage of the A and B genomes of durum wheat were tested in the composed durum wheat collection. They allowed the identification of 95 alleles, with an average of 5,9 alleles/SSR locus. The considerable number of alleles was due to the multi-allelic nature of these markers that facilitates variability detection [32]. Similar results were reported by Chen et al. [30] in a Chinese winter wheat collection, although a large range of alleles per locus (4.5-18.1) was reported in the literature for a wider germplasm collection [33,34]. The genetic diversity of the collection (He = 0.58) was similar to the values reported by previous studies involving durum wheat collections (He values between 0.55 and 0.68) [33,35].
To define the genetic distances among samples, the matrix obtained with a simple matching coefficient was also used to produce a neighbor-joining dendrogram ( Figure 2). This dendrogram clearly divided collected germplasm into two distinct groups: the one hosting only traditional varieties, (mostly Biskri, Mahmoudi and Chili), and one with a more assorted composition (including both traditional and modern varieties). In this latter group, the distances separating the entries are much higher than in the former one, which we will refer to as the 'core traditional varieties' (CTVs) group.
To better investigate this result, an analysis of the molecular variance was performed to test different sources of variation. In the first instance, we tested the distribution of molecular variation among the CTV group and the rest of the analyzed germplasm. The results clearly indicate that the CTV group is unquestionably differentiated from the rest of the analyzed material, since 22% of the total variation is between the two groups of samples (Table 2). In a second run, three groups were tested: the CTV, the one formed by entries described by farmers as traditional varieties and, finally, modern cultivars. In this case, AMOVA demonstrated that the distribution of the molecular variance conformed to the grouping, even though at a lower extent than in the former analysis (Table S1).
Nevertheless, the above analyses did not provide any information on the distribution of genotypes along the geographic cline. To this end a Bayesian approach was utilized by analyzing genotypic data through the STRUCTURE software. Structure analysis assigns each genotype to a specific subpopulation and five different ones were identified after the Evanno test [29]. The robustness of these results was confirmed by the AMOVA used to test the distribution of variation among and within these five subpopulations. In fact, the AMOVA indicated that 37% of the total variation observed was among these five groups (Table 3). After assigning each genotype to a specific subpopulation, as described in the results section, we tested the richness of each subpopulation using the three collection zones (North, Center, and South) as discriminating factors for Tunisian samples; a fourth discriminating factor was the modern durum cultivars (see Figure 3).
The approach described above allowed us to observe that some genotypes (belonging to the subpopulations indicated with the colors red and orange in Figure 3) were increasingly frequent along the North-South cline, while at the same time the modern cultivars tended to be decreasingly represented. Conversely, in the modern cultivars, the presence of traditional germplasm was scarce.
This picture indicates that: (a) modern varieties are more fit for cultivation in the northern part of Tunisia, where the rainfall is adequate for new high-yielding varieties that develop their full potential only in the presence of moderate to high inputs; (b) there is an increased presence of traditional varieties and CTVs in the Center and South groups, which are characterized by reduced and more erratic precipitations.
These findings indicate that the traditional varieties, originally developed from North African landraces, might possess traits of resistance to unfavorable climatic conditions that are absent in the modern cultivars. This is not surprising since landraces and derived traditional varieties are the result of a non-scientific process of selection driven by farmers' cultural values and by natural adaptation to local environment [13].
The fact that some old cultivars, such as AgiliAC2, are still called by the names of traditional varieties might be the result of different causes. In fact, the improved material of the 1970s-1990s might have been distributed using the names of the traditional varieties in order to overcome the diffidence of more traditional farmers against innovations. Another reason might reside in a phenotypic resemblance of some morphological traits of the new introductions with those of the CTVs, or by the misnaming of landraces during seed exchange among farmers or during the collecting missions undertaken by the NGBT [25].
In conclusion, our results showed that the molecular markers distinguished Tunisian durum wheat germplasm (named the CTV group) and detected putative synonymy and homonymy cases. These results could be useful to increase ex situ conservation efficiency, because SSRs allow the detection of redundant duplications within durum germplasm conserved in the NGBT. In situ on-farm conservation, in addition to the ex situ approach, could ensure the long-term conservation of genetically diverse Tunisian durum wheat available in local germplasm, which is a reservoir of genes for drought, heat and salinity tolerance and quality traits. The traditional varieties could help maintain the use of local knowledge and challenge ongoing genetic erosion due to the spread of modern pure line cultivars.
The population structure analysis suggests a strong genetic stratification along the North-South cline of Tunisia. This could be the result of breeding programs from the second half of the last century that produced high-yielding varieties for the northern highly productive areas rather than tackling the agro-ecological conditions of the Center and South.
The strategy of incorporating adaptation traits found in the genetic diversity pool of landraces and traditional varieties into modern breeding programs is widely supported [36]. In this way it will also be possible to contend with the challenges imposed by climate change and the survival of agriculture in marginal lands [36][37][38]. Correct and scientifically driven programs aiming at in situ conservation of agro-biodiversity on farms are essential, particularly for those species that are the food staple of Mediterranean populations and Tunisians in particular, such as durum wheat [39]. In fact, the end-use caracteristics are among the selection drivers used by past farmers during the non-scientific selection of traditional landraces. This explains the preference of genotypes providing better couscous or more valuable tabouna (a traditional sort of bread).

Plant Material
A durum wheat collection including 41 Tunisian samples (Table 5) and 13 cultivars (Table 6) were considered in this study. The Tunisian durum samples, named as indicated by the farmers, were collected during 2012-2018 ( Figure 1) and conserved by the National Gene Bank of Tunisia (NGBT). The Tunisian durum cultivars (9), and French durum cultivar (Carioca) were obtained from NGBT, while the three Italian durum cultivars (Cappelli, Svevo and Taganrog), used as references, were obtained from the Department of Soil, Plant and Food Sciences (DiSSPA), Sect. Genetics and Plant Breeding, University of Bari Aldo Moro (Italy).
The data of monthly rainfall were obtained from the National Institute of Meteorology of Tunisia and derived from the mean of the last 30 years (1988-2018). The data were recorded at three stations in the North (Bizerte, Tunis and Tabarka), three stations in the Center (Kairouan, Monastir and Sfax) and two stations in the South (Djerba and Tozeur).   [40]; c) [41]; d) [42]; e) [43].

DNA Extraction and SSR Assays
The genomic DNA was extracted from 3 g of fresh young leaves from a single plant for each sample using the cetyltrimethyl ammonium bromide (CTAB) method [44]. The DNA quality was evaluated in agarose gel (0.8%) (Biomatik, Wilmington, DE, USA) and quantified using NanoDropTM ND2000 (Thermo Scientific, MA, USA). All samples were normalized to a standard concentration of 50 ng by adding HPLC-grade water (Sigma Aldrich, St. Louis, MO, USA). The durum wheat collection was evaluated with a set of 16 simple sequence repeats (SSRs) markers (reported in Table 1). The SSRs were selected based on the consensus map of tetraploid wheat [45] and following criteria proposed by Laidò et al. [43]: locus-specific amplification, low complexity, robust amplification and good genome coverage (nearly one marker for each chromosome arm). The sequence primers were available in the GrainGenes database [46]. The amplification reactions were performed in a final volume of 20 µL, containing the template DNA (50 ng) forward primer with the M13 (−21bp) tail at its 5 end (0.06 µM), reverse primer (0.16 µM), M13 primer labeled with 6-Carboxyfluorescein (6-FAM) (0.05 µM) or 2'-chloro-5'-fluoro-7',8'-fused phenyl-1.4-dichloro-6-carboxyfluorescein (NED) (0.10 µM), or 2 -chloro-7 phenyl-1,4-dichloro-6-carboxy-fluorescein (VIC) (0.05 µM), dNTPs (2 mM), buffer (1×) and Taq DNA polymerase (0.6 U). PCR reactions were performed in a thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) as follows: an initial denaturing step at 94 • C for 3 min, followed by 35 cycles of 94 • C for 1 min, 59 • C for 1 min, 72 • C for 2 min and a final extension step at 72 • C for 7 min. In order to verify the PCR efficiency, the PCR fragments were separated on 2.0% agarose gels containing Nancy-520 DNA Gel Stain (Sigma Genosys, St. Louis, MO, USA), and visualized under UV light. The amplification products were detected by the automatic capillary sequencer ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA), and the sample analyses were carried out with GeneMapper genotyping software version 5.0 ( Figure S3). The internal molecular weight standard was GeneScanTM 600 LIZ dye Size Standard (ThermoFisher Scientific, Waltham, MA, USA).

Data Analysis
The genetic indices as number of alleles (Na), Shannon's information index (I), heterozygosity observed (Ho), heterozygosity expected (He) and fixation index (F = He − Ho/He) were calculated using the GenAlEx program version 6.5 [47,48]. The informativeness of SSR markers was measured by the polymorphism information content (PIC) according to Weber [49] and Anderson et al. [50]. The allelic data was used to obtain the dissimilarity matrix using the simple matching coefficient by DARwin program version 6.01 [51]. This dissimilarity matrix was utilized for constructing the dendrogram using the neighbor-joining algorithm [52]. The confidence interval of the genetic relationships among the samples was determined by 1000 bootstraps.
The molecular data were processed using the STRUCTURE program version 2.3.4 [53]. The number of sub-populations (K) was estimated by 20 independent runs for each K (from 2 to 20) applying the admixture model, 100,000 Markov Chain Monte Carlo (MCMC) repetitions and a 100,000 burn-in period. The means of the log-likelihood estimates for each K were calculated. The true K was determined using the Evanno ∆K [29] using STRUCTURE HARVESTER [54,55]. Analysis of molecular variance (AMOVA) was used to partition the genetic variation into inter-and intra-gene pool diversities in the durum wheat collection using GenAlEx program version 6.5 with 1000 permutations.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/13/3362/ s1. Figure S1: Monthly rainfall (mm) in the three Tunisian macro areas: North (in green), Center (in red) and South (in yellow). Figure S2: Delta ∆K for differing numbers of subpopulations (K) estimated in durum wheat collection with 16 SSR markers. Table S1: Analysis of molecular variance (AMOVA) of durum wheat collection split in three groups: Core Traditional Varieties (CTVs), entries named as traditional varieties by farmers and durum cultivars. Figure S3: Electropherogram of Tunisian durum wheats D117 and Agili AC2 tested with Xgwm193 SSR marker labeled with fluorescent dye NED. The alleles 182 and 186 bp were found in D117 and Agili AC2 durum samples, respectively.


extfieldsofstudy: Chemistry Physics

text:
Food products with ingredients rich in ω-3 polyunsaturated fat acids

The paper shows the possibility of enriching various food products with polyunsaturated fat acids of the ω-3 group (ω-3 PUFA). A method is proposed for using a microwave-treated cod liver as the basis for a number of products, including culinary ones. Cod liver oil separated during microwave treatment is also used as a source of ω-3 PUFA. The series of products includes culinary products: salad and zrazy, as well as meat and fish sausages produced without the use of sodium nitrite as a color stabilizer. The results of experimental studies in the development of technology and the selection of the optimal composition of various products with a microwave treated cod liver (salad and zrazy, meat and fish sausages) are presented. It was found that fish oil separated during microwave treatment of cod liver can be used as an oxidizing baking improver, and glutathione from yeast - as a reducing agent. According to the results of the experiments, the most acceptable food formulations were selected.

Introduction
One of the important problems in the development of functional food products is their enrichment with fat acids (PUFAs), in particular, with ω-3 [1]. ω-3 PUFAs have a very high nutritional value, have a therapeutic effect in relation to a number of diseases, especially cardiovascular diseases [2]. Fish [3] is a traditional and very common source of ω-3 PUFAs.
However, traditional methods of processing a cod liver have several disadvantages. Firstly, it is very difficult to use frozen cod liver to produce high-quality products. Secondly, the use of very fat liver and frozen raw materials for canned foods leads to a large amount of free fat in the jar. The authors propose to use not a raw cod liver for food, but a semi-finished product from cod liver, which is obtained using microwave processing. It is also advisable to use fish oil separated during such processing. This method of treating a cod liver [4] allows for a longer storage of microwave-treated cod liver (microwave) in the frozen state and prevents the separation of fat during further processing.
The enrichment of a number of combined food products in ω-3 PUFAs by incorporating the selected cod liver oil into the microwave oven can be of particular interest. The use of fish raw materials in sausages producing technology is not a completely new method, but it seems very promising. Unlike traditional meat sausages, meat and fish products allow the use of very valuable fish raw materials, and have more traditional sensory characteristics compared to fish products [5]. Also the use of microwave semi-finished products for the preparation of various salads and zrazy is interesting. IOP  Another area of researches includes the use of cod liver oil as a bread improver. The quality of the final product greatly depends on the quality of the main raw material -wheat flour, whose properties are not always constant. Bakery improvers are necessary to provide certain characteristics (rheological, colloidal, sensory) for dough and bakery products. They allow the production of bread and bakery products of stable quality even with unstable quality of flour. One of the widespread groups of bakery agents includes oxidizing and reducing agents. Fats and oils, including extracted fish oil, are oxidative improvers. To achieve the best technological effect, the integrated use of both oxidizing and reducing agents is practiced [6].
Glutathione was selected as a reducing agent. This is a tripeptide containing a cysteine residue with a -SH group. It can break disulfide bonds in a gluten molecule, changing the structural and mechanical characteristics of the dough. Yeast can be used as a source of glutathione. It is also advisable to use accelerated kneading of the dough and reduce the time of its fermentation [7].

Materials
Microwave treated cod liver (Gadus Morhua), as well as cod liver oil isolated during cooking, were used as a source of ω-3-PUFA. The microwave oven treated cod liver was frozen and stored at a temperature of minus 18 °C. Fish oil extracted during microwave processing was purified by sedimentation and decantation methods. These semi-finished products were used to produce new types of food. Other raw materials (meat, flour, salt, spices, etc.) were obtained at the local market.

Research and processing methods
The Kjeldahl method [8] uses the SelectaBlocDigest and SelectaPro-Nitro (Spain) modules. The resulting nitrogen content was converted to raw protein (P) according to the following formula: where: N -total nitrogen content; K -conversion coefficient. The lipid content in samples of raw materials, fish protein isolate (FPI) and food products made from them (canned food) was determined using a SelectaDET / GRAS extractor (Spain) according to the Soxhlet method.
The composition of lipid fat acids was determined by high performance liquid chromatography (HPLC) with Agilent 1100 (USA) after saponification of lipids with an alcohol solution of binomial KOH and pre-column derivatization using bromophenacyl bromide and triethylamine [9].
Penetration strength was used to evaluate structural and mechanical characteristics. To obtain this characteristic, FoodChecker (Japan) was used with a spherical indenter (diameter 8 mm) immersed to a depth of 10 mm at a constant speed and at a temperature of 20 ° C.
The acid number of the oil was determined after fat extraction using a mixture of chloroform and ethyl alcohol by titration with 0.1 M sodium hydroxide and phenolphthalein. The fat peroxide number was determined by titration of iodine displaced from potassium iodide by peroxides and hydroperoxides of sodium thiosulfate [10].
The water retaining capacity and stability of the minced meat emulsion was determined using the Salavatulina method, which includes the determination of water and fat losses, as well as the characteristics of the emulsion after heating the minced meat in a jar [11].
The porosity of bakery products was determined by determining the mass and volume. The amount of raw gluten was determined using dough (made from flour and water), rinsing with cold water until the starch is completely removed. The porosity of bakery products was determined using the Zhuravlev device, the water content was determined by drying, and the amount of raw gluten was determined after washing the dough ball [12].
Organoleptic methods were carried out using rating scales [13]. A generalized sensory score (%) was calculated by this method. Experimental design methods (in particular, the central composite rotating design) were used in this study. The selection and evaluation of the regression equation and regression analysis as a whole were performed using OakdaleDatafit 9.1.

Production of culinary products based on semi-finished liver products
In order to identify the most relevant culinary products with a microwave-treated cod liver, separate marketing studies were conducted using the questionnaire method. The results of these studies showed that buyers may be interested in the following culinary products with a microwave-treated cod liver: salad (34% of the total number of respondents) and zrazy (23%). Thus, it was decided to develop salad and zrazy compositions from the microwave-treated cod liver. The central rotatable compositional plan was used to select a composition that was close to the optimal. The following factors were selected: dosage of cod liver and onion for salad, as well as the proportion of cod liver, potato and eggs for zrazy. The design and results of experiments to optimize salads and zrazy are given in tables 1 and 2, respectively. The results of table 1 do not show the high adequacy of the regression analysis due to the use of subjective sensory assessment as an answer. As a result of regression analysis, the following equation is obtained: Where a=140000; b=-12600; c=427; d=-6.40; e=0.0358; f = -9.60; g = 0.696 -regression coefficients. The Fisher criterion was 5.22, this is not enough to say both about the adequacy and inadequacy of the regression equation. Data analysis does not allow finding the optimal value of this equation in the factor space, but some results at the boundary of this factor space can be considered close to optimal. In particular, it was found that the highest sensory score belongs to salad sample No. 6 where a=4640; b = 120.5; c=-21.92; d=7673; e=4584; f=1191; g= 113.7 -regression coefficients. The Fisher test was 53.9, which confirms the adequacy of the regression equation with a confidence probability of 0.95. The response surface is shown in Figure 1. Analysis (3) showed that the optimum is achieved at X1 = 0.364; X2 = 0.0757. This corresponds to the following dosage of the ingredient per 1 kg of zrazy: cod liver -253 g; egg -53 g; potatoes -695 g. In addition, it should be noted that fairly good sensory results were found in sample No. 9 (table 2). Thus, it was managed to find the most acceptable composition of salad and the optimal composition of zrazy.

Production of baked goods with cod liver oil
A marketing survey was conducted using questionnaires to determine the most preferred types of bakery products and types of the most desirable additives. The survey results showed that consumers almost equally prefer traditional types of bread: shaped (baked in the form) and hearth. The analysis showed that seaweed (kelp) (68% of the total number of respondents) and fish lipid-based additives (27% of the total number of respondents) were selected by the respondents to be included in the bread recipe from the entire list of the proposed additives produced from marine hydrobionts. Consumers also choose the addition of grain and seeds, for example, oats, flax seeds, sunflower seeds (73% of the total number of respondents) and dietary fiber, for example, bran (56% of the total number of respondents).
Studies have been conducted on the use of cod liver oil in the production of white bread. Cod liver oil was used as an oxidizing agent for baking bread in the last stage of dough kneading. Glutathione was selected as a reducing agent for bakery products. Inactivated yeast cells were used as a source of glutathione; they were added in the first kneading step.
A series of experiments was conducted on baking bread samples with the addition of cod liver oil and glutathione. The best characteristics were obtained when using a dosage of oil -1% by weight of flour. Table 3 shows the results of studying the characteristics of gluten, and table 4 shows the properties of the samples of the finished product.  Formulations of bakery products from wheat flour with the addition of seaweed, bran and milk thistle were developed to expand the range of bakery products. Bran is a rich source of dietary fiber that helps regulate bowel function. Seaweed are a source of iodine, it also contains natural sorbents (alginates) and sterols, which can have a positive effect on the human body. Milk thistle has both dietary fiber and flavonolignans. Samples of wheat bread with such additives were prepared and evaluated; the results are shown in table 5. Thus, a sufficiently high-quality product can be achieved by adding seaweed additives. The use of bran also allows getting a high-quality product.

Implementation of FPI and microwave treated cod liver for the production of meat and sausages
The production of meat and sausages is one of the promising areas for the use of semi-finished products from cod liver. Previous studies have led to the development of technology for meat and fish sausages with microwave, which has been optimized for the main ingredients [4]. In addition to balancing the main ingredients, it is very important to find the dosage of additional components that affect the yield of the finished product, structural and mechanical characteristics, and color. So, the next stage in the development of the composition was the optimization of additional ingredients.
Guar gum (food polysaccharide-derived hydrocolloid) is used as a thickener and emulsifier to provide the most appropriate structural and mechanical characteristics. It was decided to abandon the use of tomato paste as a dye, but use fermented rice to obtain a slightly pink color.  6 An objective characteristic of the color of the finished product is its additive color model (RGB). Using this model instead of sensing color enhances the accuracy of the experiment. But the RGB model includes 3 parameters that change the range from 0 to 255, so it is reasonable to determine the total square deviation from the optimal task. A subsequent selection of colors was made, and the most acceptable colors for sausages were determined using an expert method. The results show that the optimal RGB color is 248.170.168.
Thus, a central composite design of a two-factor experiment can be developed. The generalized optimization parameter includes 5 separate parameters: Y1 -water-holding ability; Y2 -fat capacity; Y3 is the stability of the emulsion; YR4-relative structural and mechanical characteristic calculated according to (4): Where -penetration force, kPa; -optimal penetration force, kPa (36.1); ∆Y -critical change in penetration index, above which a significant effect on consistency is observed, kPa (5).
Y -deviation from the optimal color, determined by the formula Parameters Y -Y are expressed in percents, they must be maximized, therefore, their recalculation is not required, the remaining parameters were recalculated to this range. The generalized optimization parameter was calculated by the formula: where K , K , K , K , K -significance factors, respectively equal to 0.2; 0.2; 0.1; 0.1; 0.4. The plan of the two-factor experiment and the results are shown in table 6.
The Fisher criterion for this equation is 11.12, the probability of inadequacy is 0.01. All regression coefficients are significant at a confidence level of at least 0.99. The response surface is shown in Figure  2.
Using the differential method, a local maximum was found. The optimal values of the factors: X_1 = 0.33, X_2 = 0.25.
The optimal composition of meat and fish sausages are shown in

Conclusions
Technologies have been developed for the preparation of culinary products from a microwave-treated cod liver. The most acceptable salad composition with cod liver was not found. The optimal ratio of ingredients for zrazy with cod liver was determined.
It was proved that the use of a combination of cod liver oil and glutathione as bakery products improves both the structural, mechanical and sensory characteristics of bakery products. The addition of components such as seaweed, bran and milk thistle can in several ways increase the nutritional value of the product and satisfy consumer demand for baked goods with natural additives.
The technology for processing meat and fish sausages with RBI and microwave-treated cod liver has been developed, the optimal composition has been found and justified. The experimental samples are under the study.


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Problems and prospects of cheese production in Russia

The Russian cheese market is in constant dynamics. A range of products is available on the market, a wide price range makes cheese a product affordable for all segments of the population; and a steady growth in this industry can be predicted. In this regard, an analysis of the prospects for the production and sale of cheese on the Russian market is of scientific and practical interest. Analysis of statistical data of the Federal State Statistics Service, the Territorial Body of the Federal State Statistics Service for the Altai Region, as well as analytical materials of the Altai Region Administration for the development of entrepreneurship and market infrastructure are presented and critically analyzed. Trends in the production and sale of cheese products are formulated. Factors hindering their development identified; development vectors in terms of import substitution are defined. The following general scientific research methods were used: analysis and synthesis, comparison, statistical methods, modeling, expert assessment method, description, etc. The scientific novelty of the study consists in assessing the factors determining the development of cheese production in the Russian Federation, as a whole, and in the Altai Region, in particular, and in developing the institutional, industry, and local vectors for its development.

Introduction
In nutrition, cheese is recognized as a highly nutritious, a biologically complete and easily digestible product, because it contains the necessary human proteins, fats, carbohydrates, and their derivatives, mineral salts, trace elements and vitamins [1,2]. According to the Agency International Dairy Federation, France (25.9 kg/year), Iceland (25.2 kg/year), Finland (24.7 kg/year) are among the three leaders in per capita cheese consumption.
In Russia, cheese is traditionally not so popular, this is due to national traditions, low cheese involvement as an ingredient in dishes of national cuisine. However, the active expansion of foreign, mainly European national cuisines into the restaurant industry, the expansion of the product range in the food retail industry, the growth of outbound tourism has formed a steady trend towards increasing demand for this product, made it popular and even "fashionable". In the first years after food embargo, the range of cheese on the Russian market was significantly reduced. However, there is an increase in the production of cheese products by domestic producers over the past two years. Today, we can state with confidence that the fashion for cheese is unlikely to pass; it is firmly established in the diet of the modern Russian consumer. The Russian cheese market is in constant dynamics, the range of products on the market, the wide price range makes cheese a product accessible to all segments of the population; and we can predict the steady growth of the whole industry. In this regard, an analysis of the prospects for the production and sale of cheese on the Russian market is of scientific and practical interest.

Materials and Methods
An analytical review of scientific literature on the research topic and analysis of statistical data of the Federal State Statistics Service, the Territorial Body of the Federal State Statistics Service for the Altai Region, as well as of analytical materials of the Altai Region Administration for the development of entrepreneurship and market infrastructure, was conducted. The boundary conditions of the study aimed at identifying trends in the production and sale of cheese products and factors hindering their development are analyzed; development vectors in terms of import substitution are defined. To solve the set tasks, the following general scientific research methods were used: analysis and synthesis, comparison, statistical methods, modeling, expert assessment method, description, etc.
The scientific novelty of the study consists in assessing the factors determining the development of cheese production in the Russian Federation and in the Altai Region, in particular, and in developing the institutional, industry and local vectors for its development.

Results
In 2016, the Ministry of Health of the Russian Federation, taking into account changes in the production and consumption of food products, developed new recommended food consumption standards to meet modern requirements for healthy nutrition [3].
The recommended annual consumption of cheese is 7 kg per person, which is 1 kg more than it was previously recommended by the Ministry of Health of the Russian Federation [4]. The real average per capita consumption of cheese in Russia is significantly lower, both new and old, and amounts to 5.  Table 1). The reasons for this situation are the unformed culture of cheese consumption due to the acute shortage of this product during the Soviet era, the lack of national dishes with cheese. Table 1. Dynamics of consumption of cheese in Russia (according to [5,6] Before the introduction of restrictions on the import of food from a number of foreign countries [7], that is, until the autumn of 2014, imported products accounted for about 50% of the cheese consumed in Russia. According to TrendEconomy.ru [12], from 2002 to 2014, Russia occupied the leading position as an importing country on the world cheese and cottage cheese market (Table 2). Cheese was delivered to the Russian Federation from more than 30 countries of the world. Over the years, Belarus, Germany, Ukraine, Lithuania, Finland, the Netherlands, and other countries were the main importers of cheese and cottage cheese. About 70% of all imports accounted for the products of countries that fell under the food embargo in August 2014. In this regard, the volume of imports decreased significantly, and the structure of imports in the context of the counterparty countries changed. According to Cheese is also supplied from South America (Argentina, Brazil, Uruguay, Chile), the Caucasus (Armenia, Azerbaijan, Georgia), and Asia (Kyrgyzstan, Kazakhstan), Africa (Tunisia, Morocco, Egypt), and other. The share of each of the listed importing countries in the total volume of cheese imports does not exceed 4%.
Foreign manufacturers, in order not to lose their positions in the Russian market, transfer or expand their production in the Russian territory (for example, the brands "Svjalea" from Lithuania, "Valio" from Finland) or in states that are not affected by the food embargo (for example, the brand "Rokiskio" from Lithuania, because it is produced in Belarus).
The introduction of countersanctions favorably affected the development of the production of cheese and cheese products in Russia. Domestic producers began to increase production volumes. In comparison to 2010, the production of cheese and cheese products in the Russian Federation increased by 38.38% in 2016. However, this growth does not meet the requirements [8].
Traditionally, the Altai Region is a leader in the production of cheese in the Siberian Federal District (about 70% of the production of cheese and cheese products in the Siberian Federal District) and the Russian Federation (about 15% of the production of cheese and cheese products in the Russian Federation). The development trends of cheese production in the Altai Region are similar to the all-Russian.
8 cheese-producing enterprises with the status of a legal entity registered in the Altai Region. The production capacity for cheese production in Russia is increasing annually, including due to the growth of small enterprises in this area. Mini-cheese factories are opening, the production of cheese in farms is organized.
In connection with the introduction of countersanctions, at the end of 2016 and the beginning of 2017, despite the positive trends in the development of cheese production, the trend of reducing the growth rate of the industry was outlined in Russia. Since the production of cheese is "milk-rich" (10 kg of milk is necessary for the production of 1 kg of cheese), the determining factor is the shortage of raw materials provoked by the decline of agricultural production in the post-Soviet period. So, the volume of milk production in farms of all categories in Russia decreased from 31,847.3 thousand tons to 30,757.9 thousand tons for 2010-2016 [14], which amounted to -3.42%. There is a trend of decline in the production of raw milk (-6.23% and -1.58%, respectively) as well in the Siberian Federal District and in the Altai region. Therefore, experts note, milk processing production facilities in Russia are approximately loaded by 60% [15].
Compliance with the following microbiological indicators of milk is extremely important in the production of cheese: a level of bacterial contamination (in the reductase test is not lower than the first class), KMAFanM (not more than 5.0 • 105 CFU / cm 3 ), a number of spores of mesophilic anaerobic lactate digesting oil-acid microorganisms (for cheeses with a low second heating temperature, no more than 13,000, with a high second heating temperature, no more than 2,500 spores per 1 dm 3 ) [16]. However, these requirements are not being complied with in the current state of animal husbandry to the full, which reduces production efficiency and negatively affects the consumer characteristics of the finished product.

Discussion
A significant factor hindering the development of the cheese industry in Russia is the rise in average prices for raw milk sold by agricultural producers of all categories. Over an average of 7 years, prices for cow's milk rose by 83,34%. In the Siberian Federal District, an increase is about 63.14%, and it is around 83.84% in the Altai region. This directly affects the increase in the cost of production from milk, as well as the selling and retail prices for finished products, including cheese (the correlation coefficient between the prices of raw milk and prices for rennet cheese is 0.98 in the period 2011-2015). This makes it uncompetitive in price compared to imported cheese. Given the decline in real disposable income in Russia, consumer preferences of the population are focused on cheaper dairy products, which cheese a priori cannot be.
The production capacity of the Russian enterprise-producers of cheese is significantly worn out, and technical re-equipment is not available to everyone, since significant financial investments with a long payback period require for this. Industry experts note that at present, for the production of products, expensive equipment of foreign production is mainly used due to the lack of analogues of domestic. It is purchased for a currency at an unattractive rate that has been formed lately. This problem is aggravated by the fact that when using imported technological equipment for primary production, the use of imported packaging equipment for packaging the finished product of a certain configuration and parameters is necessary.
Partially, the ingredients (bacterial starter cultures, concentrates, and milk-clotting enzyme preparations) used in cheese production are of imported origin, which also leads to an increase in the production costs.
Due to the shortage of raw materials and the desire to reduce the cost of production, producers use poor quality milk with the addition of stabilizers or vegetable substitutes. Experts attribute the growth in the physical production of cheese and cheese products in Russia to the increased growth rates of palm oil imports. At the same time, unscrupulous commodity producers do not indicate the use of fats with a nondairy origin in the labeling of products, which is a violation and is recognized as counterfeit with appropriate checks.
In a hurry to occupy the vacated niche formed as a result of the introduction of the food embargo, some manufacturers use the technology of accelerated ripening of cheese. This leads to the fact that the resulting product does not meet consumer expectations.

Conclusion
Factors hindering the development of cheese production in Russia can be divided into the following: "design and development of recipes," "raw materials," "production technology," "packaging and labeling," "storage," "implementation," "transportation," and "staff" (Table 3). Table 3.
Factors constraining the development of cheese production in Russia.

Factors
Influence Design and formulation development Long innovation cycle in the development, production and marketing of new varieties of cheese; "Gap" of the innovation cycle, low commercialization of developments [9].

Raw materials
Lack of basic raw materials, both quantitative and from qualitative points of view; Seasonality; High level and increase in prices for basic raw materials; Dependence on imported ingredients (bacterial starters, concentrates and milk-clotting enzyme preparations). Production technology Limited own technologies and recipes; High depreciation of fixed assets; Dependence on foreign manufacturers of technological equipment; Limited investment in the production of cheese, their high cost and a long payback period. Packaging and labeling Dependence on foreign manufacturers of packaging equipment; Low competitiveness of domestic packaging materials; Failure to comply with the requirements of technical regulations [10] on labeling by unscrupulous manufacturers.

Storage
High capital intensity and energy intensity due to the long production cycle Implementation High share of imports; Dumping by foreign suppliers; A high level and rising prices of cheese; Low competitiveness in price and quality of domestic products; Expansion of large retail and difficult cooperation conditions for small manufacturing enterprises; Elasticity of demand for cheese; Decrease in the purchasing power of the population; Underdevelopment of cheese consumption culture. Transportation High transport rates. Staff The lack of highly qualified specialists in the production of cheese.
Despite these difficulties, many experts believe that the domestic cheese market will continue to grow. Further development of the cheese market in the medium and long term will depend on institutional, industrial, and local trends. The increase in production will be ensured by the development of domestic production, namely: strengthening the position of existing enterprises-manufacturers and the emergence of new enterprises. Thus, there is a scientific and production potential for the development of domestic cheese production. This potential needs to be raised and correctly used in the current economic conditions.


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Proximate and Amino Acid Analyses of the Rhizome of Nymphaea lotus (Water Lily)

The proximate composition and the amino acid profile analyses of the rhizome of Nymphaea lotus (water lily) were carried out based on standard methods reported by the association of official Analytical Chemists (AOAC). The results of the proximate analysis showed that Nymphaea lotus rhizome had moisture content of 48.83±3.22; crude protein 4.23±0.06; crude fibre 5.00±1.50; ash content 4.67±0.17; high available carbohydrate 35.44 ± 0.01 and energy value 742.10 kJ/100 g. The crude lipid content was low 1.83±0.60. The study detected seventeen different amino acids. The amino acid content in g/100 g protein content was: lysine = 3.11, histidine= 1.82, arginine= 4.25, aspartic acid = 7.60, threonine= 2.19, serine= 3.02, glutamic acid= 9.68, proline= 2.34, glycine= 3.06, alanine= 3.40, cycteine= 0.73, valine= 3.37, methionine= 1.09, isoleucine= 2.35, leucine= 8.24, tyrosine= 2.25 and phenylalnine= 3.72. The studied revealed that glutamic acid, aromatic acids, leucine and aspartic acid were the predominant amino acids. All essential amino acids were found to be higher than WHO/FAO/UNU requirement pattern for adults while leucine was found to be higher than the requirement pattern for preschool children. Lysine however was found to be the most limiting amino acid for preschool children. The presence of these compounds of nutritive value, as well as high energy value suggests that Nymphaea lotus rhizome could serve as a food supplement for livestock and humans.

Introduction
Currently, the world, especially developing nations is facing the problem of malnutrition. The major causes of this malnutrition are war and bad economic policies. Conventional legumes and some dicotyledonous seeds, which have been proven to be economically favourable alternative sources of proteins and energy as against animal proteins in developing countries, have played significant role in the management of these problems so far [1]. However, these are still insufficient for complete fulfillment of food and energy requirements of the fast growing population. In most parts of Nigeria, starch based food are the main staple food which supply both energy and protein. Thus, protein deficiency prevails among the populace as recognized by Food and Agricultural Organization, FAO [4]. Recent reports have revealed that quite a large number of indigenous plant species are high in nutrient value [2]. However, many of such plants have been identified but lack data on their chemical compositions [3]. Hence, there is need for exploration of non-conventional sources of nutrients supplements.
One of such non-conventional sources of nutrient is the water lily (family, nymphaeaceae). Water lilies are aquatic plants growing perennially from a sunken stem buried in the mud, where the rootlets of the plant put away for anchor with the asexual parts emerging above the water surface. It is one of the foremost aquatic macrophytes that have been identified in the Nigerian fresh water bodies [5]. The impact of some macrophytes on water bodies, have been highlighted by many authors [6][7][8][9]. However, there is dearth of information on water lily despite its presence on many fresh water bodies in Nigeria [10]. It has however been observed that people of the north eastern part of Nigeria eat and market the seeds of water lily [11]. In the light of this fact, it is clear that water lily may be a sole contributor to resolve the malnutrition problems in developing countries. The nutritional composition of the bulb of Nymphaea lotus has been reported [9]. However, nothing has been done on the rhizome of this plant. It is therefore the aim of this work to study the proximate and amino acid profile analyses of the rhizome of Nymhpaea lotus.

Sample Collection and Preparation
The Nymphaea lotus rhizomes were collected from Dan Gurbi Lake in Randa village, Tureta Local Government Area of Sokoto state, Nigeria. The sample was oven dried at 105°C, cooled in a dessicator and finely ground. The dried sample was used for the analyses, except for moisture content determination in which fresh sample was used.

Proximate Analysis
The proximate analysis (moisture content, ash content, crude protein, crude fibre, crude lipid, total carbohydrate and energy value) of Nymphaea lotus rhizome were determined using standard methods [12].

Amino Acid Profile
Amino acid determination was carried out using ionexchange chromatography with Technicon Sequential Multisample Amino Acid Analyser, TSM (Technicon Instruments Corporation, Dublin, Ireland) at Postgraduate laboratory, Zoology unit, University of Jos, Nigeria as outline in [13]. 2g sample was defatted with petroleum ether using Soxhlet extraction methods. The defatted sample was redried and milled into fine powder using porcelain pestle and mortar. 30mg sample in duplicate were weighed into a glass ampoules to which 5cm 3 6M HCl and 5µmoles norleucine were added. The ampoules were evacuated by passing nitrogen gas (to remove oxygen so as to avoid possible oxidation of some amino acids during hydrolysis), sealed with Bunsen burner flame and hydrolyzed in an oven at 110°C for 24 hours. The ampoules were cooled, broken at the tip and the contents filtered. The filtrates were evaporated to dryness at 40°C under vacuum in a rotary evaporator. The residues were dissolved to 5µL (for acid and neutral amino acids) or 10 µL (for basic amino acids) with acetate buffer, pH 2.2 and the solutions were dispensed into the cartridge of TMS. The chromatograms (amino acid peaks) obtained from automatic pen recorder corresponds to the quantity of each amino acid present. Quantification was performed by comparing the peak area of each amino acid in the sample to the area of the corresponding amino acid standard of the protein hydrolysate.

Results
The results of the analysis, involved proximate analysis and amino acid profile of the rhizome of Nymphaea lotus and they are shown in the tables 1, 2 and 3 below.

Proximate Analysis
The results of the proximate analysis of the rhizome of Nymphaea lotus were compared with that reported for the bulb of the same plant [9]. This is summarized in table 1. From the table, the moisture content for this research work is high, (48.83 ± 3.22) and is also higher than the value reported for the bulb of N. lotus (9.72 ± 0.000) in the literature which is also similar to the result obtained for the leaf sample (7.50±0.50), root sample (9.00±0.48) and seed sample (6.00±0.45) [14]. The high moisture content of the rhizome implies that it is likely to have a short storage capability [15]. The ash content showed a lower value (4.67±0.17) compared to the literature as shown in the table. The value for the ash content is also low compared to that of the leaf sample (12.67±1.26) and the root sample (26.67±1.89). It is however higher than the value reported for the seed sample (2.67±0.29) [14]. The ash content shows a reflection of the mineral contents preserved in the rhizome. Minerals are essential for the proper functioning of tissues and act as second messengers in some biochemical mechanisms [16]. The rhizome from this research, as shown in table 1 has very low crude lipid (1.83±0.60) while the reported literature for the bulb showed higher lipid content (5.07±0.014). The lipid content of most parts Nymphaea lotus is low as shown in literature, where the leaf and root samples gave values within the range for the rhizome (2.18 and 2.00 respectively). However, the seed sample showed higher crude lipid similar to the bulb in the table above (9.33±0.29) [14]. The rhizome, been a low lipid containing food can thus be recommended as part of weight reducing diet, since low fat food reduces the level of cholesterol and obesity [17]. The crude protein was 4.23 ± 0.06. This value is higher than that reported for the root and seed samples (1.02 and 1.04 respectively) [14]. The presence of crude protein in the rhizome, suggests that it can serve as a protein supplement to contribute to the daily protein needed by the body. However, the value is less than that reported for the bulb (21.66±0.014) as shown in table 1. The percentage crude fibre obtained in this work was 5.00 ± 1.50. This is low compared to the bulb (13.30 ± 0.021), and the root sample (24.33±0.58) [14]. The value however, is similar to that reported for the seed sample (5.50±0.50) [14]. The crude fibre content implies that the N. lotus rhizome could serve as a source of dietary fibre and can be employed in the treatment of diabetes, obesity and Amagbor Stella Chinelo and Umar Kabiru Jega: Proximate and Amino Acid Analyses of the Rhizome of Nymphaea lotus (Water Lily) gastrointestinal tract diseases [18]. The available carbohydrate content was very high (35.44 ±0.01), even though less than that reported for the bulb (41.92±0.056). The value is also less than that reported for the root sample (61.31±1.86) [14]. This value of available carbohydrate, suggests that N. lotus rhizome is a good source of carbohydrate which is essential for energy in livestock and man. Table 2 shows the result for the amino acid profile of N. lotus rhizome. The data are mean value of duplicate results.

Amino Acid Profile
The result indicated that non-essential amino acids (alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, proline and serine) were higher in concentration (56.53%) compared to the essential amino acids (isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine and valine) which constitute 43.48% of the total amino acids analyzed. Among the essential amino acids, leucine and aromatic (phenylalanine and tyrosine) are the predominant acids, while glutamic and aspartic acids were found to be the major non-essential amino acids in Nymphaea lotus rhizome.
To evaluate the nutritional quality of the rhizome, the percentages of the essential amino acids in the sample were compared with those of reference standard amino acid profile by WHO/FAO/UNU [19] and the result (Table 3) indicates that all the essential amino acids exceeded the reference value for adults, while isoleucine, lysine, sulphur-containing amino acids (methionine and cysteine), total aromatic essential amino acids (phenylalanine and tyrosine), threonine and valine were below the standard requirement for preschool children. However, for preschool children, lysine is the most limiting essential amino acid and the second is threonine followed by the sulphur containing essential amino acids. This implies that the rhizome will be more suitable for adults than children.

Conclusion
This research work showed the proximate analysis and amino acid profile of the rhizome of Nymphaea lotus. It therefore showed the biochemical compositions such as carbohydrates, crude protein, crude lipid, crude fibre and amino acids. From the result of this study, it has been shown that Nymphaea lotus rhizome contains relatively good amounts of nutrients. The low crude lipid imply that this rhizome can serve as a good food supplement for the treatment of obesity and the amino acid profile imply that the rhizome is suitable for adults protein supplement. All these suggest that Nymphaea lotus rhizome can actually serve as a non-conventional food supplement to help combat the problem of malnutrition amongst humans and livestock. People living around areas where this Nymphaea lotus rhizome is found, may look for processing methods to help integrate this into their food for both humans and livestock. Furthermore, the nutrient information would enhance efforts to promote wide use of plants because of their nutritional benefits and medicinal properties.