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Isolation and Functional Characterization of MsFTa, a FLOWERING LOCUS T Homolog from Alfalfa (Medicago sativa)

The production of hay and seeds of alfalfa, an important legume forage for the diary industry worldwide, is highly related to flowering time, which has been widely reported to be integrated by FLOWERING LOCUS T (FT). However, the function of FT(s) in alfalfa is largely unknown. Here, we identified MsFTa, an FT ortholog in alfalfa, and characterized its role in flowering regulation. MsFTa shares the conserved exon/intron structure of FTs, and the deduced MsFTa is 98% identical to MtFTa1 in Medicago trucatula. MsFTa was diurnally regulated with a peak before the dark period, and was preferentially expressed in leaves and floral buds. Transient expression of MsFTa-GFP fusion protein demonstrated its localization in the nucleus and cytoplasm. When ectopically expressed, MsFTa rescued the late-flowering phenotype of ft mutants from Arabidopsis and M. trucatula. MsFTa over-expression plants of both Arabidopsis and M. truncatula flowered significantly earlier than the non-transgenic controls under long day conditions, indicating that exogenous MsFTa strongly accelerated flowering. Hence, MsFTa functions positively in flowering promotion, suggesting that MsFTa may encode a florigen that acts as a key regulator in the flowering pathway. This study provides an effective candidate gene for optimizing alfalfa flowering time by genetically manipulating the expression of MsFTa.

Introduction
Flowering time, the switch from vegetative to reproductive growth, is a key trait for the seasonal and geographical adaptation of flowering plants. The mechanism of flowering is best understood in the annual plant Arabidopsis, in which the timely onset of flowering is regulated by an intricate network with various genetic regulators responding to endogenous (gibberellin, autonomous and aging) or environmental (vernalization and photoperiod) triggers, as reviewed in recent years [1][2][3][4]. The balance of signals from these pathways is integrated by a common set of genes, including FLOWERING LOCUS C (FLC)/MADS AFFECTING FLOWEIRNG (MAF), FLOWERING LOUCS T (FT), CONSTANS (CO) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), to determine the flowering time [5][6][7]. In Medicago, however, FLC/MAF clade flowering repressors are missing and CO-like genes do not seem to regulate flowering [8][9][10]. Hence, florigen encoding gene FT, acting as a long-distance hormone signal between leaves and shoot apical meristems, is the most important flowering integrator of the legume species.
Since the search for the flowering hormone florigen in the 1930s, an increasing number of FT-like genes have been identified in a wide range of annual plants, such as rice, soybean and barrel clover [11][12][13]. For example, in M. truncatula five FT-like genes, namely MtFTa1, MtFTa2, MtFTb1, MtFTb2 and MtFTc, have been characterized. Among them, MtFTa1, MtFTb1 and MtFTc rescued the late flowering phenotype of Arabidopsis ft-1 mutant, while the other two genes failed to act as a florigen [11]. Functional differentiation among FT paralogs derived from lineage-specific duplication has been observed in several species, such as soybean, potato and rice [13][14][15]. The diversity of the functions of FT paralogs is attributed to the expression pattern shift and the divergence in gene regulatory networks.
In recent years, studies on FT-integrated flowering regulation have focused on economically important perennial plants including grasses (e.g., switchgrass and ryegrass) [16,17] and trees (e.g., poplar, apple and cherry) [18][19][20]. In contrast to annual plants, FT is expressed in both leaves and flowers of several perennial species such as sweet cherry, kiwifruit and leafy spurge [21][22][23]. Functional coordination of perennial FT paralogs revealed the evolution of a complex perennial adaptive trait after genome duplication. For example, poplar FT1 determined the onset of reproductive stage in winter, whereas FT2 promoted vegetative growth and inhibited bud set in the growing season [18]. Ectopic over-expression of FT perennial orthologs in Arabidopsis caused early flowering in most cases, suggesting a conserved function in perennial species [16][17][18][19][20]22].
Alfalfa (M. sativa), one of the most important forages globally, exhibits seasonal flowering. Different from perennial woody plants, which are not able to form flower buds during the first several years of their life cycle, herbaceous alfalfa grows vegetatively at early developmental stages and then switches to the reproductive stage in the same year. In Northern China, a main alfalfa planting region in the country, the first cutting coincides with the rain season, which severely limits hay production. Breeding alfalfa cultivars with optimal flowering time would facilitate the improvement of the quantity and quality of alfalfa. Our study here demonstrated that MsFTa, the first isolated FT from alfalfa, not only shared the conserved functional domains of its orthologs, but also acted ectopically in accelerating flowering. These findings would supply a potential candidate for the generation of alfalfa cultivars with an optimal flowering time for the main planting regions in Northern China via genetic modulation of MsFTa expression.
To investigate flowering regulation in perennial legume alfalfa, we cloned an FT-like cDNA from the forage by combination of RT-PCR and RACE strategy based on the sequence information of MtFTs. The 1029 bp cDNA sequence contained an open reading frame (ORF) of 531 bp (GenBank: JF681135) ( Figure S1). The putative protein consists of 176 amino acids with theoretical isoelectric point (pI) of 8.23 and molecular weight of 19.7 kDa, respectively. Phylogenetic analysis of 33 FT and FT-like proteins from rice, barley, Arabidopsis, soybean, barrel clover and poplar demonstrated that the six MtFTs were grouped into three subclasses, namely Subclass A (MtFTa1, MtFTa2 & Mt6g033040), Subclass B (MtFTb1 & MtFTb2) and Subclass C (MtFTc) (Figure 1). The putative alfalfa FT protein was clustered into Subclass A sharing the same branch with MtFTa1 and MtFTa2 ( Figure  1). Hence, it was designated as MsFTa, the first FLOWERING LOCUS T gene identified in alfalfa. Compared with Subclass A members from Arabidopsis, rice and barley, MsFTa was more closely related to the FTs of legume plants such as barrel clover and soybean ( Figure 1). Sequence homology analysis showed that MsFTa shared an identity of 59-99% with MtFT members, the lowest identity with MtFTc and the highest with MtFTa1, respectively (Table S1). The sequence similarity of MsFTa with FTs from the other five species ranges from 54% to 84% (Table S1). Multiple alignment revealed that, like its orthologs, MsFTa has a ligand-binding motif with a conserved key residue Tyr (Y) at position 85, an external loop of a 14-amino acid segment (residues 128-141) and a triad (residues 150- ; Glycine max (Gm): Gm19g108100, Gm18g298900, Gm18g299000, Gm16g151000, Gm16g044200, Gm16g150700, Gm16g044100, Gm19g108200, Gm08g363100; Populus trichocarpa (Pt): Pt8g077700, Pt10g179700; Hordeum vulgare (Hv): Hv1Hr1g076430, Hv2Hr1g023180, Hv2Hr1g084540, Hv3Hr1g027590, Hv3Hr1g087100, Hv4Hr1g012200, Hv7Hr1g024610 and Oryza sativa (Os): OsHd3a (Os06g0157700), Os04g0488400, Os05g051800, Os06g0157500, Os06g0552900, Os11g0293800, Os12g0232300.
To investigate flowering regulation in perennial legume alfalfa, we cloned an FT-like cDNA from the forage by combination of RT-PCR and RACE strategy based on the sequence information of MtFTs. The 1029 bp cDNA sequence contained an open reading frame (ORF) of 531 bp (GenBank: JF681135) ( Figure S1). The putative protein consists of 176 amino acids with theoretical isoelectric point (pI) of 8.23 and molecular weight of 19.7 kDa, respectively. Phylogenetic analysis of 33 FT and FT-like proteins from rice, barley, Arabidopsis, soybean, barrel clover and poplar demonstrated that the six MtFTs were grouped into three subclasses, namely Subclass A (MtFTa1, MtFTa2 & Mt6g033040), Subclass B (MtFTb1 & MtFTb2) and Subclass C (MtFTc) (Figure 1). The putative alfalfa FT protein was clustered into Subclass A sharing the same branch with MtFTa1 and MtFTa2 ( Figure 1). Hence, it was designated as MsFTa, the first FLOWERING LOCUS T gene identified in alfalfa. Compared with Subclass A members from Arabidopsis, rice and barley, MsFTa was more closely related to the FTs of legume plants such as barrel clover and soybean ( Figure 1). Sequence homology analysis showed that MsFTa shared an identity of 59-99% with MtFT members, the lowest identity with MtFTc and the highest with MtFTa1, respectively (Table S1). The sequence similarity of MsFTa with FTs from the other five species ranges from 54% to 84% (Table S1). Multiple alignment revealed that, like its orthologs, MsFTa has a ligand-binding motif with a conserved key residue Tyr (Y) at position 85, an external loop of a 14-amino acid segment (residues 128-141) and a triad (residues 150-152) at the C-terminal end ( Figure 2) [24][25][26]. In addition, MsFTa features a large number of alpha helices, beta-folds and random coil structures ( Figure S2a). Our prediction using ScanProsite showed that most regions of MsFTa are hydrophilic ( Figure S2b), indicating it is a hydrophilic protein. 152) at the C-terminal end ( Figure 2) [24][25][26]. In addition, MsFTa features a large number of alpha helices, beta-folds and random coil structures ( Figure S2a). Our prediction using ScanProsite showed that most regions of MsFTa are hydrophilic ( Figure S2b), indicating it is a hydrophilic protein. Comparison of the exon-intron structure of FT-like genes demonstrated that like most FT-like genes from a variety of species [11,13,27,28], MsFTa is composed of four exons separated by three introns (Figure 3). Interestingly, exon 2 is universally 62 bp and exon 3 is 41 bp, except two barley FTlike genes (Hv3Hr1g0871002 and Hv7Hr1g0246102) ( Figure 3). The 2nd and 3rd exons of MsFTa encode the N-terminal of the ligand-binding motif, including the amino acids forming the ligandbinding pocket and the key residue Tyrosine (Y) at position 85 ( Figure 2). Therefore, MsFTa not only Comparison of the exon-intron structure of FT-like genes demonstrated that like most FT-like genes from a variety of species [11,13,27,28], MsFTa is composed of four exons separated by three introns

The MsFTa-GFP Recombinant Protein Resided in Both the Cytoplasm and Nucleus
To examine the subcellular localization of MsFTa, 35S::MsFTa-GFP and 35S::GFP were transformed into onion epidermal cells separately. The transient expression of the GFP control was uniformly distributed throughout the cell, whereas the MsFTa-GFP fusion protein was observed in the nucleus and cytoplasm, especially the membrane ( Figure 4). This result is consistent with the recent observations of FT-like proteins in perennial species such as switchgrass and sweet cherry [16,22].

The MsFTa-GFP Recombinant Protein Resided in Both the Cytoplasm and Nucleus
To examine the subcellular localization of MsFTa, 35S::MsFTa-GFP and 35S::GFP were transformed into onion epidermal cells separately. The transient expression of the GFP control was uniformly distributed throughout the cell, whereas the MsFTa-GFP fusion protein was observed in the nucleus and cytoplasm, especially the membrane ( Figure 4). This result is consistent with the recent observations of FT-like proteins in perennial species such as switchgrass and sweet cherry [16,22].

Diurnal and Tissue-Specific Expression Pattern of MsFTa
To monitor the temporal and spatial expression profiles of MsFTa, quantitative RT-PCR was performed using MsFTa-specific primers. Analysis of the MsFTa transcript demonstrated that the level of MsFTa progressively increased after 6 h light and reached a peak at 16 h light. The onset of darkness caused a sharp deduction in MsFTa and the descending trend continued during the night period ( Figure 5a). Spatially, four tissues from both underground and aerial organs were tested.
MsFTa was detected in all tested tissues including roots, stems, floral buds and leaves with the highest expression in leaves, which is approximately 3.4-fold the amount in roots (Figure 5b). The expression pattern of MsFTa is highly reminiscent of its legume homologs, especially MtFTa2 in M. truncatula [11]. Detailed analysis demonstrated that besides cauline leaves, a higher level of FT transcript was detected in flowers, siliques, and seeds at early stages (http://bbc.botany.utoronto.ca/efp) ( Figure S3).

Ectopic Expression of MsFTa in Arabidopsis Strongly Accelerates Flowering
To explore the biological function of MsFTa in modulating flowering, the construct of MsFTa driven by the cauliflower mosaic virus 35S promoter was introduced separately into wild type Arabidopsis and ft-10, a T-DNA insertion mutant of FT, by agrobacterium infiltration. The existence of a 35S::MsFTa construct in two independent transgenic Arabidopsis T2 lines (T2-1 & T2-4) with

Diurnal and Tissue-Specific Expression Pattern of MsFTa
To monitor the temporal and spatial expression profiles of MsFTa, quantitative RT-PCR was performed using MsFTa-specific primers. Analysis of the MsFTa transcript demonstrated that the level of MsFTa progressively increased after 6 h light and reached a peak at 16 h light. The onset of darkness caused a sharp deduction of MsFTa and the descending trend continued during the night period ( Figure 5a). Spatially, four tissues from both underground and aerial organs were tested. MsFTa was detected in all tested tissues including roots, stems, floral buds and leaves with the highest expression in leaves, which is approximately 3.4-fold of the amount in roots (Figure 5b). The expression pattern of MsFTa is highly reminiscent of its legume homologs, especially MtFTa2 in M. truncatula [11]. Detailed analysis demonstrated that besides cauline leaves, a higher level of FT transcript was detected in flowers, siliques, and seeds at early stages (http://bbc.botany.utoronto.ca/efp) ( Figure S3).

Diurnal and Tissue-Specific Expression Pattern of MsFTa
To monitor the temporal and spatial expression profiles of MsFTa, quantitative RT-PCR was performed using MsFTa-specific primers. Analysis of the MsFTa transcript demonstrated that the level of MsFTa progressively increased after 6 h light and reached a peak at 16 h light. The onset of darkness caused a sharp deduction in MsFTa and the descending trend continued during the night period ( Figure 5a). Spatially, four tissues from both underground and aerial organs were tested.
MsFTa was detected in all tested tissues including roots, stems, floral buds and leaves with the highest expression in leaves, which is approximately 3.4-fold the amount in roots (Figure 5b). The expression pattern of MsFTa is highly reminiscent of its legume homologs, especially MtFTa2 in M. truncatula [11]. Detailed analysis demonstrated that besides cauline leaves, a higher level of FT transcript was detected in flowers, siliques, and seeds at early stages (http://bbc.botany.utoronto.ca/efp) ( Figure S3).

Ectopic Expression of MsFTa in Arabidopsis Strongly Accelerates Flowering
To explore the biological function of MsFTa in modulating flowering, the construct of MsFTa driven by the cauliflower mosaic virus 35S promoter was introduced separately into wild type Arabidopsis and ft-10, a T-DNA insertion mutant of FT, by agrobacterium infiltration. The existence of a 35S::MsFTa construct in two independent transgenic Arabidopsis T2 lines (T2-1 & T2-4) with Bars represent the means ± SD of three biological replicates.

Ectopic Expression of MsFTa in Arabidopsis Strongly Accelerated Flowering
To explore the biological function of MsFTa in modulating flowering, the construct of MsFTa driven by the cauliflower mosaic virus 35S promoter was introduced separately into wild type Arabidopsis and ft-10, a T-DNA insertion mutant of FT, by agrobacterium infiltration. The existence of a 35S::MsFTa construct in two independent transgenic Arabidopsis T2 lines (T2-1 & T2-4) with kanamycin resistance was confirmed by PCR (Figure 6a). Semi-quantitative RT-PCR of the over-expression lines demonstrated that MsFTa transcript was abundantly expressed in both transgenic lines while absent in wild type Arabidopsis (Figure 6b). Phenotypically, the over-expression plants exhibited early flowering (Figure 6c). The measurement of leaf number showed that wild type plants flowered with 15.4 leaves, whereas T2-1 and T2-4 over-expression lines flowered with 12.1 and 11.4 leaves, respectively (p < 0.05) (Figure 6d). Consistently, in terms of days after germination (DAG), the first flower of line T2-1 and T2-4 appeared at 16.1 and 17.8 DAG, respectively, while the first flower of the wild type was observed at 24.3 DAG (p < 0.05) (Figure 6e). These results suggested that flowering time of the transgenic Arabidopsis was associated with MsFTa transcript level. kanamycin resistance was confirmed by PCR (Figure 6a). Semi-quantitative RT-PCR of the overexpression lines demonstrated that MsFTa transcript was abundantly expressed in both transgenic lines while absent in wild type Arabidopsis (Figure 6b). Phenotypically, the over-expression plants exhibited early flowering (Figure 6c). The measurement of leaf number showed that wild type plants flowered with 15.4 leaves, whereas T2-1 and T2-4 over-expression lines flowered with 12.1 and 11.4 leaves, respectively (p < 0.05) (Figure 6d). Consistently, in terms of days after germination (DAG), the first flower of line T2-1 and T2-4 appeared at 16.1 and 17.8 DAG, respectively, while the first flower of the wild type was observed at 24.3 DAG (p < 0.05) (Figure 6e). These results suggested that flowering time of the transgenic Arabidopsis was associated with MsFTa transcript level.

Ectopic Expression of MsFTa Promoted Flowering in Transgenic M. truncatula Plants
Given the close phylogenetic relationship and high sequence identity of MsFTa to MtFTa1, both barrel clover wild type R108 and mtfta1-1, a late-flowering mutant (NF3307) caused by the insertion of a Tnt1 retrotransposon in the first exon of MtFTa1 [11], were transformed with the construct of 35S::MsFTa mentioned above. Flowering time of the transgenic plants was measured as days to produce the first flower under LD conditions. Compared with R108 which flowered at D56, the two independent T1 lines (T1-1 & T1-2) overexpressing 35S::MsFTa produced the first flower at D35 and D32, respectively. Over-expression of MsFTa in M. truncatula resulted in statistically significant early flowering (p < 0.05) (Figure 7a). For mtft1-1 plants expressing 35S::MsFTa, the flowering time of two independent transgenic lines was analyzed. As shown in Figure 7b, the first flower of the two lines emerged at D58 and D61, while fta1-1 flowered at D83, which is significant later than R108 at D56 (p < 0.05), indicating that MsFTa successfully rescued the late flowering phenotype of fta1-1. These results suggested that similar to ectopic expression of MsFTa in Arabidopsis, introduction of MsFTa into M. truncatula caused strong flowering promotion.

Ectopic Expression of MsFTa Promotes Flowering in Transgenic M. truncatula Plants
Given the close phylogenetic relationship and high sequence identity of MsFTa to MtFTa1, both barrel clover wild type R108 and mtfta1-1, a late-flowering mutant (NF3307) caused by the insertion of a Tnt1 retrotransposon in the first exon of MtFTa1 [11], were transformed with the construct of 35S::MsFTa mentioned above. Flowering time of the transgenic plants was measured as days to produce the first flower under LD conditions. Compared with R108 which flowered at D56, the two independent T1 lines (T1-1 & T1-2) overexpressing 35S::MsFTa produced the first flower at D35 and D32, respectively. Over-expression of MsFTa in M. truncatula resulted in statistically significant early flowering (p < 0.05) (Figure 7a). For mtft1-1 plants expressing 35S::MsFTa, the flowering time of two independent transgenic lines was analyzed. As shown in Figure 7b, the first flower of the two lines emerged at D58 and D61, while fta1-1flowered at D83, which is significant later than R108 at D56 (p < 0.05), indicating that MsFTa successfully rescued the late flowering phenotype of fta1-1. These results suggested that similar to ectopic expression of MsFTa in Arabidopsis, introduction of MsFTa into M. truncatula caused strong flowering promotion. under LD conditions. Flowering time was measured as days to the emergence of the first flower under LD conditions. Two independent lines for each transformation were analyzed. Data represent average ± SD of three biological replicates. * indicates a significant difference between wild type and ft-10 (Student's t-test, p < 0.05).

Discussion
Precise flowering time ensures that the onset of flowering occurs at an ideal time to optimize plant biomass and fitness in specific environmental conditions. Although diverse upstream components of flowering pathways have been identified from plants, FLOWERING LOCUS T appears to be a universal regulator ultimately integrating flowering signaling in crops, vegetables, grass and trees [11,18,26,30]. In this study, we provided molecular and genetic evidence that MsFTa, an FT homolog from alfalfa, functions to accelerate flowering.
As a member of an ancient phosphatidylethanolamine-binding protein (PEBP) gene family, FT has a conserved exon/intron structure and highly identical protein sequence across species [13,31]. Indeed, our results demonstrated that MsFTa possessed the characteristic features of a classic FT gene. First, MsFTa, similar to FT-like genes, has four exons with conserved exon/intron boundaries [13,27,28]. Over-expression of the chimeric genes constructed by replacing individual exons of FT revealed that substitution of the fourth exon, especially the external loop caused late flowering, presumably the crystal structure was affected due to the sequence variation of the exon [24]. It has been reported recently that exons two and three were also crucial for the functional conversion of FT in Arabidopsis [13,32]. Second, MsFTa shared several well characterized amino acid residues proven to be important for flowering acceleration according to the experiments of individual residue exchange or segment replacement. For example, in Arabidopsis, alteration of Tyr 85 (Y85H) changed under LD conditions. Flowering time was measured as days to the emergence of the first flower under LD conditions. Two independent lines for each transformation were analyzed. Data represent mean ± SD of three biological replicates. * indicates a significant difference from wild type (Student's t-test, p < 0.05).

Discussion
Precise flowering time ensures that the onset of flowering occurs at an ideal time to optimize plant biomass and fitness in specific environmental conditions. Although diverse upstream components of flowering pathways have been identified from plants, FLOWERING LOCUS T appears to be a universal regulator ultimately integrating flowering signaling in crops, vegetables, grass and trees [11,18,26,30]. In this study, we provided molecular and genetic evidence that MsFTa, an FT homolog from alfalfa, functioned to accelerate flowering.
As a member of an ancient phosphatidylethanolamine-binding protein (PEBP) gene family, FT has a conserved exon/intron structure and highly identical protein sequence across species [13,31]. Indeed, our results demonstrated that MsFTa possessed the characteristic features of a classic FT gene. First, MsFTa, similar to FT-like genes, has four exons with conserved exon/intron boundaries [13,27,28]. Over-expression of the chimeric genes constructed by replacing individual exons of FT revealed that substitution of the fourth exon, especially the external loop caused late flowering, presumably the crystal structure was affected due to the sequence variation of the exon [24]. It has been reported recently that exons two and three were also crucial for the functional conversion of FT in Arabidopsis [13,32]. Second, MsFTa shared several well characterized amino acid residues proven to be important for flowering acceleration according to the experiments of individual residue exchange or segment replacement. For example, in Arabidopsis, alteration of Tyr 85 (Y85H) changed FT functions in promoting flowering, indicating that the absolutely conserved Tyr 85 is a key residue determining the protein function as an activator of flowering [25]. FTs share a triad ending with residue asparagine (N) at site 152, followed by leucine (Figure 2). The crystal structure revealed that the triplet motif contacts the adjacent external loop, which facilitates access to FT interactors, such as the bZIP transcription factor FD and 14-3-3 proteins [24,[33][34][35]. In Rosaceae species, mutations of the triad prevented the FT-like genes from promoting flowering [36]. Third, FTs have highly conserved motifs including a potential ligand-binding pocket and an external loop (Figure 2), both of which were proven to contribute to the functional specificity of FT [24,33]. Compared with other species, it seems that the external loop of legume plants is variant (Figure 2), implying varied conformation, possibly leading to flexible accessibility to a binding partner. Therefore, MsFTa, together with FT-like genes, specifies FT conformation by possessing identical exon/intron structure and highly conserved functional motifs.
FT-like orthologs from a variety of plant species have been shown to promote flowering when ectopically expressed [16,22,[37][38][39]. In the model legume M. truncatula, reverse genetics revealed that the loss-of-function mutant of MtFTa1, the closest homolog of MsFTa, caused late flowering and MtFTa1 fully complemented fta1-1 defects [11]. Consistently, an allelic series of gain-of-function mutations in MtFTa1 caused by retroelement insertions exhibited a dominant early flowering phenotype [40,41], confirming its function in flowering regulation. It appears that similar to FT, MsFTa from the perennial forage acts positively in flowering promotion. The notion is supported directly by the transgenic Arabidopsis and M. truncatula including both complementation and over-expression plants. Plants ectopically expressing MsFTa in either wild type or ft mutant of both plant species displayed a clear shift to early flowering (Figure 6d-g). Statistically, the flowering time difference between the transgenic plants and the non-transgenic ones is significant (p < 0.05). Notably, compared with wild type, complementation plants flowered slightly late, suggesting that constitutive expression of MsFTa partially rescued the flowering time of ft mutants from Arabidopsis and barrel clover. These findings imply a minute functional distinction between MsFTa and its orthologs, FT and MtFTa1, in Arabidopsis and barrel clover, respectively. Application of the endogenous promoter of MsFTa to produce transgenic alfalfa is undergoing. Moreover, generation of a loss of function mutant of MsFTa using the CRISPR-Cas9 strategy would be necessary to elucidate the endogenous function of MsFTa in alfalfa flowering regulation.
Given the detection of MsFTa in roots and stems, it is tempting to speculate that besides flowering promotion, MsFTa may participate in other development processes of alfalfa. FT orthologs have been documented to be involved in multiple functions like plant architecture [42], tuberization [15], and dormancy interruption [43]. The expression of MsFTa resembled FT orthologs in perennial plants, such as sweet cherry, loquat, leafy spurge and tea-oil tree, in which FT-like genes are expressed in diverse tissues, including mature leaves, floral buds and reproductive organs (flower organs and fruits) [21,22,27,44]. The expression divergence may be an important force in driving the functional division of FTs in perennial plants, especially in species with multiple FT paralogs [13,14,16,22]. As it is closely related to diploid legume M. truncatula, which has six identified FT-like genes, tetraploid alfalfa is more likely to have a similar number, or even more, MsFTa paralogs due to genome duplication. Long-term work of flowering regulation in alfalfa will be directed at paralogs of MsFTa.

Plant Material and Growth Conditions
Medicago sativa cv. 'Zhongmu No.1', bred by the Institute of Animal Science, the Chinese Academy of Agricultural Sciences, was used in this study. Seeds were germinated in regular soil in a 20 cm diameter pot. Medicago truncatula Tnt1 insertion mutant fta1-1 (NF3307) was obtained from the Noble Research Institute (Ardomor, OK, USA). Arabidopsis thaliana seeds of ft-10 (CS9869) were from Arabidopsis Biological Research Center (ABRC) and germinated in soil (vermiculite:soil = 1:3). All plants were grown in growth chambers at 21 • C under a 16/8 h (light/dark) photoperiod.

Cloning of MsFTa from Alfalfa
Total RNA was extracted from 2-week-old alfalfa seedlings with TRIzol reagent (Sangon, Shanghai, China). After DNase I (Fermentas-MBI, Shenzhen, China) treatment, the first-strand cDNA was synthesized using Reverse Transcriptase III (Takara, Dalian, China). A DNA fragment was amplified by using the two degenerate primers designed based on FT-like genes in M. truncatula and sequenced. The obtained partial sequence of alfalfa FT-like gene was used to design primers for amplification of the full-length MsFTa by 3 -RACE and 5 -RACE (Invitrogen, Carlsbad, CA. USA) with the specific primers according to the manufacturer's instructions. PCR amplicons were purified after separated on 1% agarose gel and cloned into pMD19-T (TaKaRa, Dalian, China) and sequenced.

Bioinformatic Analysis
For FT homologous genes, Arabidopsis FT (At1g65480) was used as query sequence to search against the database EnsemblPlants (http://plants.ensembl.org/index.html). Phylogenetic analysis was performed with bootstrap trials of 1000 using DNAMAN version 7.0 (Lynnon Corporation, Quebec, QC, Canada), and sequence alignment was analyzed using the same software with default parameters. Isoelectric point (pI) and molecular weight (MW) values were predicted using ExPaSy (https:// prosite.expasy.org). The secondary structure of the protein was predicted using LYON-GERLAND PBIL (https://prabi.ibcp.fr); and the hydrophobic structure was predicted using ScanProsite (https: //prosite.expasy.org/scanprosite).

Expression Analysis by RT-PCR
For expression analysis in different tissues, roots, stems and leaves were harvested from 6-week-old alfalfa, while flowers were from 60-day-old plants. For the diurnal analysis, leaves were collected separately from 6-week-old alfalfa at zeitgeber time (ZT) 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 time points, respectively. After extraction of total RNA, cDNA was prepared as described above. Semi-quantitative RT-PCR was carried out using gene-specific primers with MsActin serving as an internal control. The PCR product was analyzed in gel and images were captured using a Gel Image Analysis System FR-200A (Furi Science & Technology, Shanghai, China). For quantitative real-time PCR, SYBR Premix EX Taq TM qPCR Kit (Takara, Dalian, China) was used according to the instruction with BIO-RAD CFX96 TM Real-Time System (BioRad, Hercules, CA, USA). The Arabidopsis Actin gene was used as a loading control. The sequence of the primers used in this study is listed in Table S2.

Subcellular Localization Analysis
For the subcellular localization analysis of MsFTa-GFP fusion protein in onion epidermal cells, the construct of MsFTa-GFP was performed as follows: the MsFTa ORF sequence was subcloned into the pA7-GFP (35S::GFP) expression vector [29] using the primers of FT-GF and FT-GR containing XhoI and SpeI restriction sites, respectively. The plasmid of the pA7-GFP and 35S::MsFTa-GFP fusion binary vector was then transformed into separate onion epidermal cells via particle bombardment (GJ-1000, Scientz Biotechnology, Ningbo, China). The subcellular localization of the transiently expressed MsFTa-GFP fusion protein was imaged using a confocal laser-scanning microscope (Olympus FV500, Tokyo, Japan).

Plasmid construct and plant transformation
For generation of the transgenic Arabidopsis plants expressing 35S::MsFTa, first, a binary vector harboring 35S::MsFTa was constructed by amplifying MsFTa ORF with primers named pBI-FTa-f and pBI-FTa-r, and the sequence-confirmed MsFTa was introduced into the pBI121 vector (Clontech, Palo Alto, CA, USA) using Xba I and Sma I restriction sites. Secondly, the 35S::MsFTa plasmid was transformed into Agrobacterium tumefaciens strain GV3101 by electroporation, and Arabidopsis transformation was performed via the floral-dip method. T1 Arabidopsis seeds were screened on a MS medium containing 40 mg·L −1 kanamycin, and T2 lines confirmed by semi-quantitative RT-PCR were used in this study for MsFTa expression and phenotypic analysis. For Medicago transformation, explants from 4-week-old plants (R108 and fta1-1) growing under LDs were cocultivated with the agrobacterium containing the construct of 35S::MsFTa according to the Medicago handbook [45]. Kanamycin of 40 mg·L −1 was used to select transformant plants.

Conclusions
In this study, the FT homolog from alfalfa, one of the most important perennial forages, was isolated and characterized. MsFTa has highly identical sequence composition, especially exon/intron boundary and functional domains, with FTs in a variety of plant species. The diurnally regulated MsFTa is expressed preferentially in leaves and floral buds. Functionally, MsFTa accelerated flowering in Arabidopsis and the annual legume M. truncatula, indicating that the novel alfalfa FT acts as a flowering promoter.


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

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Creating functional foodstuffs from high-technological larval raw materials

The possibility of using the apiproducts of the larval origin from the drone larvae in the production of functional foodstuff, such as bakery, confectionery and honey products enriched with high-grade protein, biologically active high-functional substances and complexes that gave them therapeutic and prophylactic, and immune-modulating properties is substantiated in the paper. The recipes for the functional foodstuffs, such as the “Bilar-api” bread, “Pchelka” api-dragee, “Bilar” api-honney, “Bilar” api-honney with bee glue (propolis), enriched with apiproduct from the drone larvae have been developed. To impart an immune-modulating and functional effect to the products, the “Bilar” api-product with a high content of SH-groups, decenoic acids, vitamins, as well as flower pollen, honey, and bee glue in the form of a water-alcohol extract have been used. Preference for these substances has been given because phenolic compounds with P-vitamin activity, unsaturated compounds of isoprene nature, have antioxidant, and fungicidal impacts. They are manifested in their ability to protect the body from the destructive effect of various damaging factors (ionizing radiation, free oxidation radicals, reactive oxygen species, etc.). The “Bilar” powder has a major content of high-grade protein (50-51%), which contains all essential amino acids, and on lysine, tryptophan and histidine exceeds the level of ideal protein on the FAO/WHO scale by almost two times, on the fatty acid composition practically corresponds to the formula of balanced nutrition, vitamins, and decenoic acids (7.9%), as well as sulfhydryl compounds (1160.1 mg/%). Decenoic acid has pronounced antibiotic properties and antitumor effect, exerting an inhibitory effect on the malignant tumors. New functional food products were manufactured on the basis of traditional production technologies using sparing technological regimes and with utilization of apiproducts (the “Bilar” powder, pollen, etc.). Based on the results of the comprehensive study, the increase in the quality and quantity indicators of functional foodstuffs in comparison with control, the increase in biological and nutritional value, as well as improved organoleptic and physics and chemical parameters have been established.

Introduction
In recent years, a new direction has been formed in the science of nutrition, that is, the functional nutrition, which includes the development of theoretical foundations, manufacturing, sale and consumption of the functional foodstuffs (FFS). In the developed countries, healthy food is elevated to the level of public policy. It is not accidental. It has been proven that proper nutrition ensures the growth and development of children, contributes to the prevention of diseases, increases the capacity for work and prolongs the life of people, whereas unbalanced, on the contrary, leads to many health problems. Modern society faces a global problem of food shortages that is largely due to the growth of the population of the Earth and the diversion of a significant share of food resources for technical purposes. Along with the shortage of food raw materials, the most important issue is that the contemporary mass food industry, playing the role of a supplier of basic food substances (proteins, fats, and carbohydrates) to the population, offers modern man not only refined food deprived of a number of useful substances, but also containing substances (coloring materials, preservatives, flavors, etc.), toxic to the body. Analysis of the actual nutrition of the population in various regions of Russia indicates that it is deficient in terms of vitamins, dietary fiber (pectin and fiber), and macro-and microelements. Therefore, it is very important to enrich food products with biologically active components that can improve many physiological processes in the human body, reducing the risk of development of the nutritional-dependent diseases, that is, diseases depending on nutrition factors (allergic diseases, anemia, atherosclerosis, diseases of the digestive system, thyroid disease, hypertension, gout, obesity, osteoporosis, diabetes mellitus, some pathologies of pregnancy, etc.).
One of the ways to solve these issues is to involve environmentally friendly non-traditional raw materials into economic circulation, the use of which, in its pure form and in the production of food products, will enrich them with vital nutrients that satisfy the energy, regulatory, protective and other needs of the organism to the level, corresponding to the physiological needs of man.
According to the data of The Micronutrient Initiative (USA), the enrichment of food with biologically active substances makes it possible to prevent four out of ten child deaths, reduce maternal mortality by more than a third, increase work capacity by 40%, increase the IQ of the population by 10-15 points, increase the country's gross product by 5%. This is the direction of food biotechnology and preventive medicine, which in the 21st century will create real prerequisites for an increase in the average life expectancy, long-term preservation of physical and spiritual health, social and moral satisfaction, active life for the elderly and the birth of a healthy generation.
According to Japanese and American scientists, it is the development of the market of fortified foodstuffs that will change the general structure of nutrition for all people on the Earth in the near future, and in 25-30 years the products of this category will half-oust the existing market of medicines in the world.
Currently, in connection with the popularization of the role of nutrition among the population in the prevention of alimentary diseases, there is a steady tendency to increase the volume and expand the range of foods enriched with biologically active substances as one of the tools to increase the competitiveness of products. So far, the enriched food products account for no more than 3% of all known foodstuffs. However, judging by the forecasts of the world's leading experts in nutrition and medicine, in the next 15 to 20 years their share will reach 30% of the total food market. The need to expand the range and increase the volume of enriched products production is stipulated by the main directions of the National Concept "Healthy Nutrition Policy in Russia", approved by the Government of the Russian Federation [1].
The Russian market of ingredients offered for the enrichment of food products is represented by a large variety of substances of various origins and physicochemical and physiological effects. At present, natural functional ingredients are of special interest for both consumers and manufacturers. The life products of honey bees (Apis mellifera), which have special properties, are the source of them along with the traditional ones, such as vegetables, fruits, berries, medicinal and spicy-aromatic plant raw materials. Their uniqueness is in the fact that specific polyunsaturated fatty acids, which are produced by the bee's organism that cannot be found in nature in pure form and have special functional properties, are added to plant biologically active substances (phenolic compounds, carotenoids, vitamins, etc.).
By now, the properties of such common products of beekeeping as honey, flower pollen, and royal jelly have been thoroughly studied. However, there is practically no data on the promising new Apiproducts of the larval origin have much in common with royal jelly; they have a therapeutic and prophylactic effect (especially antioxidant, immune-modulating, antitumor, actoprotective, etc. effects). The studies conducted by Professor I.A. Prokhoda have shown that there is much more decenoic acids and sulfhydryl compounds in the drone larvae than in royal jelly. The amount of protein reaches 51%, and it contains all essential amino acids, and for lysine, tryptophan and histidine the level of ideal protein on the FAO/WHO scale is exceeded almost twice. In addition, up to 5% of fats are present in the larvae, of which 10% are low molecular weight fatty acids (essential), 28% are mono-saturated, and 45% are saturated, that is, the fat content practically corresponds to the formula of rational and balanced nutrition that increases the biological value considered in the projects of apiproducts from the drone larvae [2,8].
The foregoing allows us concluding that it is necessary and promising to carry out the claimed R&D work on the small innovative enterprise Apiproduct Ltd.
We have developed recipes for new functional bakery, confectionery and honey products and demonstrated the possibility of their manufacturing at the small innovative enterprise Apiproduct Ltd., the first Russian enterprise that manages the quality of innovative apiproduct from the drones larvae and its introduction into the food industry for the enrichment of products of mass consumption with a high-grade protein, biologically active high-functional substances and complexes.

Problem Statement
The problem of production of protein-containing food products, especially from natural raw materials, is acute in the world. In modern environmental conditions, there is a significant decrease in immunity, which is manifested in an increase in the number of people infected with dangerous diseases, for example, tuberculosis. Nutrition plays the main role in the correction of immunity. One of the ways to solve the above issues is a significant increase in the diet the proportion of foods with a high content of natural protein and biologically active substances (BAS) with immune-modulating action in the form of powders, pastes, etc.
Also, manufacturers face a huge problem of search of alternative sources of natural raw materials that can be used as mineral processing products of mass consumption and giving them highly functional properties. In modern literature, many scientists have shown that bee products such as royal jelly, honey, pollen, propolis, etc. have a medical and nutritional properties, thanks to their composition of the full-fledge proteins, phenolic compounds, biopolymers, unsaturated substances, such as decenoic acid and sulfhydryl compounds that are able to bind reactive oxygen species, oxidative free radicals and to create insoluble complexes with ions of heavy metals.
However, there is no systematic scientific information on the functional properties of the unconventional product of beekeeping of the larval origin, namely, the drone larvae. Moreover, larvae are not a traditional product of the Russian cuisine, and their use in the production of functional foods is also hampered by the emotional discomfort when used. We have previously established a high therapeutic activity of the use of the drone larvae: this nontraditional apiproduct is recognized to be more effective in comparison with the synthesized preparations of traditional medicine [2], [3], and [4].
However, our studies allow us stating that: 2.1. To date, the scientific idea of using high-functional apiproducts of the larval origin in food production has not been formed. In the Russian Federation, products of the larval origin cause emotional discomfort when consumed, and are not used for food. However, in many countries of the world, larvae are used as a food product, since they contain a rich full-fledged protein. Therefore, the solution of the issue of searching for alternative raw materials from natural food ingredients is very relevant and timely. This work is devoted to solving the actual scientific problem -to the scientific substantiation and creation of the functional food products from the larval raw materials that is an unconventional product of beekeeping, specifically, the apiproduct from the drone larvae.

Research Questions
3.1. The analysis of literature and the development of recipes of the "Bilar-api" bread, using apiproduct from the drone larvae as an enriching agent.
3.2. The development of recipes and technologies for the production of confectionery productsthe api-dragee "Pchelka".
3.3. The development of recipes and technologies for the production of confectionery products -"Bilar" api-honney, "Bilar" api-honney with propolis, enriched with apiproduct from the drone larvae, and the formation of their consumer properties.

Purpose of the Study
The purpose of the study is a scientific justification of the following: a. The possibility of using apyproduct of the larval origin for the creation of functional food products; b. The development of "Bilar-api" bread, "Pchelka" api-dragee, "Bilar" api-honney, "Bilar" apihonney with propolis, enriched with apiproduct from the drone larvae; c. Formation of consumer properties of the new functional food products.

Research Methods
Theoretical basis of this study is the scientific literature on the topic of the research, the works of domestic and foreign scientists. Also, the material-technical and laboratory base of the Apiproduct Ltd. enterprise and Internet materials have been used in the process of writing this paper.
Research methods include generic scientific methods, such as the experimental method, the analog method, as well as the organoleptic, physicochemical, microbiological and other methods.
The subject of the research is the development of recipes and the creation of functional products for the mass consumption, such as the "Bilar-api" bread, "Pchelka" api-dragee, "Bilar" api-honney, "Bilar" api-honney with bee glue, enriched with apiproduct from the drone larvae, and the formation of their consumer properties.

Findings
6.1. In the course of the research, recipes and technologies for the production of new products -"Bilar-api" bread have been developed, its commodity quality assessment has been given that allows expanding the production range, increasing nutritional value, and improving the organoleptic, physicochemical indicators of the finished product.
In the development of recipes and the selection of the chemical composition of the new products, we followed the recommendations of the Institute of Nutrition of the Academy of Medical Sciences of Russia on the nutrition of people living in areas with increased radiation, as well as on the nutrition of To make the products immune-modulating and provide it with functional effects, powdered apiproduct "Bilar", and propolis in the form of an extract with a high content of SH-groups, decenoic acids, phenolic compounds, tannins, vitamins, as well as honey and flower pollen have been used. Preference for these substances has been given because phenolic compounds with P-vitamin activity, unsaturated compounds of isoprene nature and tannins, which are part of propolis, possess antioxidant, bactericidal and fungicidal activity. They are manifested in their ability to protect the body from the destructive effect of various damaging factors (ionizing radiation, free oxidation radicals, reactive oxygen species, etc.).
The "Bilar" powder has a high content of high-grade protein (50-51%), which contains all essential amino acids, and on the lysine, tryptophan and histidine exceeds the level of ideal protein on the FAO/WHO scale by almost twice, on the fatty acid composition practically correspond to the formula of balanced nutrition, vitamins, decenoic acids (7.9%), and the same is for sulfhydryl compounds (1160.1 mg/%). Decenoic acid has pronounced antibiotic properties and antitumor effect, having an inhibitory effect on malignant neoplasms [2].
The "Bilar-api" bread has been produced according to the traditional technology of bakery products by a sputtered method using sparing technological production modes. The recipe for the production of the "Bilar-api" bread is characterized by the use of apiproducts (Bilar powder and flower pollen) as a biologically active additive for giving bread prophylactic and functional properties (Table 1). Table 1. Consumption of components for the "Bilar-api" bread (summary formulation) per 100 kg of flour. Powdered apiproduct "Bilar" was introduced in an amount of 2%, flower pollen -3% of the total amount of flour. The flower pollen is recommended to grind or dissolve in a small amount of water immediately before mixing the dough.

Name of raw materials and process indicators
"Bilar" is used in the form of a powder without additional treatment. These doses were introduced according to the recommendations of dieticians when using royal jelly for enriching food products, including bakery products and they are allowed by the Ministry of Health (MOH) of Russia. The doses are selected based on the organoleptic evaluation of the final product and economic feasibility. It was found that the addition of additives in these dosages slows the process of staling, since an increase in the amount of hydrophilic components contributes to stabilization of the forms of moisture binding in the crumb, an increase in the share of capillary and adsorption-bound moisture.
The products with api-supplements are characterized by high organoleptic indicators: the crumb gets a uniform thin-walled porous structure without voids and has a light yellow color, and a peculiar tar floral smell and floral odor. Flower pollen, which contains natural coloring pigments, phenolic compounds and carotenes, imparts the yellowish color and floral aroma to the "Bilar-api" bread. Based on the results of the comprehensive study, an increase in the qualitative and quantitative indices of the "Bilar-api" bread was established in comparison with the control ("Rozhdestvensky" bread, enriched with bran and produced according to a similar recipe). The biological value of the new "Bilar-api" bread increased at a constant humidity of 44.0%, acidity of 3.9º and fat and carbohydrate content; protein content was 11.5%; the bread is enriched with vitamins.
Technical documentation has been developed for the new "Bilar-api" bread with the use of the "Bilar" powder and pollen. Recipes and technologies of the enriched products are approved and introduced on "The Komarichsky Bakery".
6.2. The recipes and technologies for the production of confectionery products -the api-drageehave been developed. As raw materials, in the preparation of the api-dragee have been used sugar, molasses, nuts, honey, fats, food acids, dyes, essences, vegetable oils, etc. Starch has been used as a forming material. Paraffin and beeswax have been used to gloss the surface. The preparation of raw materials for production and the manufacturing technology is the same as in the production of other types of confectionery products, except that apiproducts have been introduced in the process of coating. From traditional production technologies, the api-dragee differs in using apiproducts. To strengthen the protective effect, ascorbic acid has been added, which is not only an antioxidant, but also a synergist. Flower pollen and honey have been injected into the formulations in an amount of 4.5-5%. The dose is selected based on the organoleptic evaluation of the final product and economic feasibility. Apiproduct Bilar was used in an amount of 0.1%. This dose is used for the preparation of food products and their enrichment with royal jelly and is authorized by the MOH of Russia. The apiaddition from propolis was added in an amount of 0.06% per dry matter, by analogy with the use of natural preservatives. The introduction of apiproducts provides a high level of P-active substances in the finished products that have the ability to accept free radicals and thus reduce the intensity of oxidative processes that initiate a decrease in human immunity.
The study of the quality of the api-dragee showed that they have a pleasant taste and a peculiar tar honey aroma with a shiny surface, round or oval in their organoleptic characteristics. Humidity of the api-dragee ranges from 2.5-6.5%. Studies of the content of BAS in the api-dragee showed that the mass fraction of β-carotene was 1.1-1.5 mg per 100 g, and they are characterized by a high content of ascorbic acid (315-321 mg per 100 g). The recommended preventive dose of api-dragee is 25 g, and it contains 78.7-80.3 mg of vitamin C that corresponds to the daily requirement of a person in vitamin C. It is also shown that the new api-dragee have a high content of phenolic compounds, such as chlorogenic acid -843-898 mg per 100 g, flavonol glycosides -215-221 mg per 100 g, free catechins -104-112 mg per 100 grams, and tannins -348-375 mg per 100 g.
Studies of the microbiological parameters of the new api-dragee showed that the total number of mesophilic aerobic and facultative anaerobic microorganisms was 2.8-3.5 x 103 units per gram that does not exceed the parameters provided by the state standards (GOST). It is shown that the quality of api-dragee "Pchelka" is practically unchanged for 6 months: the mass fraction of β-carotene decreased by 13-18%. Medico-biological studies of the api-dragee carried out at Kharkov National Pharmaceutical University have shown that they possess therapeutic and prophylactic properties and are recommended for regular use.
6.3. "Bilar" api-honney is a mixture of natural honey with a powdery apiproduct "Bilar" (100:1), "Bilar" api-honney with propolis is a mixture of the same components with the addition of the propolis api additives in the form of water-alcohol extract (100:1:2).
Preparation of api-honney was made with the help of a mixer according to the generally accepted technology. When using crystallized honey, it was previously "dissolved" in a heated water bath at a temperature of plus 40°C to evenly mix the components. Packing of the api-honney "Bilar" was carried out in glass jars, with a volume of no more than 0.5 dm3, covered with sterile metal lacquer lids. In the room where the packing process was taking place, the relative humidity of the air should not be more than 75%, and the temperature not higher than 25°C.
According to the organoleptic indicators, api-honney "Bilar" has a sweet taste with a peculiar floral aroma, and api-honney "Bilar" with propolis has a pleasant sweet taste and a peculiar tar floral aroma peculiar to propolis. The addition of apiproducts did not affect the appearance of the api-honney -a thick syrupy mass; the color is rich yellow due to propolis, which contains phenolic compounds that impart an additional intensity to the staling. The addition of lipid additives to honey made it possible to enrich its BAS and was directed to form a quality: the mass fraction of vitamin C was 12.2-12.5 mg per 100 g, phenolic compounds, such as chlorogenic acid, 185-198 mg in 100 g, flanolol glycosides 42.0-50.0 mg per 100 grams, free catechins 10.0-12.0 mg per 100 grams, tannins 380-420 mg per 100 grams. The main component of honey is carbohydrates (95%), so api-honney "Bilar" and "Bilar" with propolis are enriched with biologically active substances, which give them enhanced immunemodulatory and functional qualities in comparison with natural honey.
The study of the microbiological indices of the api-honney "Bilar" showed that the total amount of mesophilic aerobic and facultative anaerobic microorganisms was 2.0x100 units per gram that does not exceed the standard specified in the GOST for natural honey.
The results of the conducted tastings showed that the developed products have high organoleptic and structural and mechanical characteristics and enriched chemical composition compared to traditional ones, due to the introduction of apiproduct from the drone larvae and other apiproducts.
According to clinical trials, apiproduct from the drone larvae has a multifaceted biological effect: its use is recommended for stabilization of the immune and nervous (with vegetative-vascular dystonia, cerebral blood flow disorders, etc.) systems, hematopoietic system of children with anemia; it normalizes appetite, increases the body's resistance to infections, and is the source of essential amino acids, vitamins, macro-and microelements and other essential biologically active complexes. Apiproduct promotes increased growth, normalization of the hormonal background, especially during puberty and the adverse effects of menopause, and in the treatment of sexual infertility. It is recommended in physical exhaustion and during the reconvalescence of patients, for improving memory and vision, mental and physical performance, and for rejuvenating the body.

Conclusion
Possibility of using apiproducts of the larval origin from the drone larvae for the creation of functional foodstuffs -bakery, confectionery and honey products enriched with high-grade protein, biologically active high-functional substances and complexes, and giving them therapeutic-prophylactic and immune-modulating properties is scientifically substantiated.
The "Bilar-api" bread has been produced according to the traditional technology of bakery products using sparing technological production modes.
The "Bilar-api" bread is characterized by high organoleptic characteristics: the crumb acquires a uniform thin-walled porous structure without voids and has a light yellow color, a peculiar tar floral smell and floral scent. The yellowish color and floral aroma are imparted to the "Bilar-api" bread by flower pollen, which contains natural coloring pigments, phenolic compounds and carotenes.
Based on the results of the comprehensive study, an increase in the qualitative and quantitative indices of the "Bilar-api" bread has been established in comparison with the control ("Rozhdestvensky" bread, enriched with bran and produced according to a similar recipe). The biological value of the new "Bilar-api" bread increased at a constant humidity of 44.0%, acidity 3.9º and fat and carbohydrate content; protein content was 11.5% and bread is enriched with vitamins.
Technical documentation has been developed for the new the "Bilar-api" bread with the use of the Bilar powder and pollen. Recipes and technologies of the enriched products are approved and introduced on "The Komarichsky Bakery". The developed recipes and technologies for the production of confectionery products, namely the api-dragee "Pchelka". As raw materials, sugar, molasses, nuts, honey, fats, food acids, dyes, essences, vegetable oils, etc. have been used in the preparation of the api-dragee. Starch has been utilized as a forming material. The api-dragee differs from traditional production technologies with using apiproducts. Flower pollen and honey were injected into the formulations in an amount of 4.5-5%. The apiproduct Bilar was used in an amount of 0.1%, the additives from propolis -in an amount of 0.06% on a dry matter. According to the organoleptic indices, the api-dragee "Pchelka" has a pleasant taste and a peculiar tar honey aroma with a shiny surface, round or oval. Humidity of the api-dragee ranges from 2.5-6.5%. Studies of the content of BAS in the api-dragee showed that the mass fraction of βcarotene was 1.1-1.5 mg per 100 g; they are characterized by a high content of ascorbic acid (315-321 mg per 100 g). The recommended preventive dose of api-dragee 25 g per dose contains 78.7-80.3 mg of vitamin C, which corresponds to the daily requirement of a person in this vitamin. It is also shown that the new api-dragee have a high content of phenolic compounds such as chlorogenic acid -843-898 mg per 100 g, flavonol glycosides -215-221 mg per 100 g, free catechins -104-112 mg per 100 grams, and tannins -348-375 mg per 100 g.
Studies of the microbiological parameters of the new api-dragee have shown that the total number of mesophilic aerobic and facultative anaerobic microorganisms is 2.8-3.5 x 103 units per gram that does not exceed the standard provided by the GOST. It is shown that the quality of the api-dragee "Pchelka" is practically unchanged for 6 months: the mass fraction of β-carotene decreased by 13-18%. Medical and biological studies of the api-dragee "Pchelka" carried out at Kharkov National Pharmaceutical University have shown that they possess therapeutic and prophylactic properties and are recommended for regular use.
The api-honney "Bilar" is a mixture of natural honey with a powdery apiproduct "Bilar" (100:1), the api-honney "Bilar" with propolis is a mixture of the same components with the addition of propolis in the form of water-alcoholic extract (100:1:2).
On its organoleptic indicators, the api-honney "Bilar" has a sweet taste with a peculiar floral aroma; the api-honney "Bilar" with propolis has a pleasant sweet taste and a peculiar tar floral aroma peculiar to propolis. The addition of apiproducts does not affect the appearance of the api-honney -a thick syrupy mass; the color is rich yellow due to the apiproduct of propolis, which contains phenolic compounds that impart an additional intensity to the staling. The addition of lipid additives to honey made it possible to enrich its BAS and is directed to form a quality: the mass fraction of vitamin C was 12.2-12.5 mg per 100 g, phenolic compounds such as chlorogenic acid -185-198 mg in 100 g, flanolol glycosides -42.0 -50.0 mg per 100 grams, free catechins -10.0-12.0 mg per 100 grams, and tannins -380-420 mg per 100 grams. The main component of honey is carbohydrates (95%), so the api-honney "Bilar" and "Bilar" with propolis are enriched with biologically active substances, which give them enhanced immune-modulatory and functional properties in comparison with natural honey.
The study of the microbiological indices of the api-honney "Bilar" showed that the total amount of mesophilic aerobic and facultative anaerobic microorganisms is 2.0x100 units per gram that does not exceed the standard specified in the GOST for natural honey.
The results of the conducted tastings showed that the developed products have high organoleptic and structural and mechanical characteristics and enriched chemical composition compared to traditional ones, due to the introduction of apiproduct from the drone larvae and other apiproducts.


extfieldsofstudy: Medicine Chemistry

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Protein-Based Fingerprint Analysis for the Identification of Ranae Oviductus Using RP-HPLC

This work demonstrated a method combining reversed-phase high-performance liquid chromatography (RP-HPLC) with chemometrics analysis to identify the authenticity of Ranae Oviductus. The fingerprint chromatograms of the Ranae Oviductus protein were established through an Agilent Zorbax 300SB-C8 column and diode array detection at 215 nm, using 0.085% TFA (v/v) in acetonitrile (A) and 0.1% TFA in ultrapure water (B) as mobile phase. The similarity was in the range of 0.779–0.980. The fingerprint chromatogram of Ranae Oviductus showed a significant difference with counterfeit products. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) successfully identified Ranae Oviductus from the samples. These results indicated that the method established in this work was reliable.

Introduction
Rana chensinensis is mainly distributed in the Changbai Mountain area, China. Ranae Oviductus is the dried oviduct of female Rana temporaria chensinensis David. The Ranae Oviductus is a potent traditional Chinese medicine that has been used in clinical studies for thousands of years. Today it is widely used as a nutrient food. It has been reported that Ranae Oviductus has significant effects in enhancing immunity, anti-fatigue, anti-aging, and lowering blood fat [1][2][3][4]. As a precious traditional Chinese medicine, Ranae Oviductus has been in short supply because of its limited production [5]. Its high price and lucrative profits have tempted many counterfeit products, such as bullfrog oviduct, toad oviduct, or frog oviduct, to inundate the market, resulting in the uneven quality of Ranae Oviductus in the market [6,7]. Those counterfeits have a similar appearance but have less efficacy. To guarantee the quality of Ranae Oviductus, its authenticity identification has attracted more and more attention from the pharmacists, doctors, and medicinal scientists. The identification method of Ranae Oviductus is still under development. In the 2005 China Pharmacopoeia, the appearance and expansion degree were employed as discriminating items of Ranae Oviductus [8]. Our group has reported using UV spectra to identify Ranae Oviductus [9]. According to a previous study, it is difficult to identify the Ranae Oviductus and counterfeit products using traditional methods [10]. Therefore, it is essential to establish a highly reliable method for the identification of Ranae Oviductus.
More than 40% of the components in Ranae Oviductus are proteins and the proteins are the major bioactive components of Ranae Oviductus [11,12]. However, the identification of Ranae Oviductus and

Chemicals and Samples
The petroleum ether, guanidine hydrochloride, and ammonium sulfate analytical grade were purchased from Beijing Chemical Factory (Beijing, China). The dithiothreitol (DTT) and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The HPLC-grade acetonitrile (MeCN) and HPLC-grade methanol were purchased from Fisher (Fisher Scientific, USA). The ultrapure water was obtained from a gradient water purification system (Water Purifier, Sichuan, China).
Ranae Oviductus, bullfrog oviduct, toad oviduct and frog oviduct were provided by Jilin Province Rana Industry Association which were collected from the Changbai Mountain area in the Jilin province of China. The specific location is shown on the map in Figure 1. Ten batches of Ranae Oviductus samples were collected from different regions from the main producing area of the Changbai Mountain range. The specific collection information is shown in Table 1.

Protein Extraction
The dried Ranae Oviductus was pulverized into a powder (passing through a 20-mesh sieve) and degreased with petroleum ether at room temperature. After filtration, the powder was placed in an oven at 55 °C for 1 h. Afterward, 0.50 g of the sample was added to PBS buffer (50 mL, 0.1 M pH 7.4). After continuously stirring for 8 h, the mixture was centrifuged at 5000 r/min for 15 min. The supernatant was collected and the precipitate was extracted again. The two centrifugal supernatants were combined. To the supernatant, an ammonium sulfate solid was slowly added until to 60% saturation [21,22]. The mixture was centrifuged at 8000 r/min for 20 min after standing at 4 °C for 1 h. The precipitate was dissolved in 6 M guanidine hydrochloride (containing 10 mM DTT) [23,24], and dialyzed in distilled water in a dialysis bag (molecular weight cutoff: 8000 Da) for 12 h [25]. The sample solution was finally scaled to 5 mL with 6 M guanidine hydrochloride (containing 10 mM

Protein Extraction
The dried Ranae Oviductus was pulverized into a powder (passing through a 20-mesh sieve) and degreased with petroleum ether at room temperature. After filtration, the powder was placed in an oven at 55 • C for 1 h. Afterward, 0.50 g of the sample was added to PBS buffer (50 mL, 0.1 M pH 7.4). After continuously stirring for 8 h, the mixture was centrifuged at 5000 r/min for 15 min. The supernatant was collected and the precipitate was extracted again. The two centrifugal supernatants were combined. To the supernatant, an ammonium sulfate solid was slowly added until to 60% saturation [21,22]. The mixture was centrifuged at 8000 r/min for 20 min after standing at 4 • C for 1 h. The precipitate was dissolved in 6 M guanidine hydrochloride (containing 10 mM DTT) [23,24], and dialyzed in distilled water in a dialysis bag (molecular weight cutoff: 8000 Da) for 12 h [25]. The sample solution was finally scaled to 5 mL with 6 M guanidine hydrochloride (containing 10 mM DTT) in a volumetric flask and filtrated with a 0.45 µm filter membrane prior HPLC injection [26]. The preparations of bullfrog oviduct, toad oviduct and frog oviduct were the same as that for Ranae Oviductus.

RP-HPLC Chromatography Analysis
The samples were separated using an Agilent Technologies 1200 Series liquid chromatograph (Agilent Technologies, Pittsburgh, PA, USA) equipped with a quaternary pump, autosampler, thermostatted column compartment, diode array detector (DAD), and UV detector. The columns used were the Agilent Zorbax 300SB-C8 column (250 × 4.6 mm, 5 µm) and Agilent Zorbax SB-C18 column (250 × 4.6 mm, 5 µm) with mobile phase A (0.085% TFA in v/v with acetonitrile) and mobile phase B (0.1% TFA in v/v with ultrapure water) [27,28]. Gradient elution was adopted as follows, from 12-30% A in the first 52 min, and from 30-44% A in the next 28 min. The injection volume was 20 µL.
The optimized separation conditions were tested under the different detection wavelengths, flow rates and temperatures [29]. The data were recorded and processed using the Agilent Chemstation software.

Validation of the RP-HPLC Method
Ranae Oviductus sample (S1) was used to verify the RP-HPLC method. A precision analysis was carried out by repeatedly injecting the same solution 5 times on the same day. The repeatability was assessed by injecting 5 separate solutions obtained from the same Ranae Oviductus sample. The stability was evaluated by analyzing the same sample solution at different time periods of 0, 2, 4, 8, 16 and 24 h at room temperature.

Establishment of the HPLC Fingerprint
The common characteristic peaks and similarities of fingerprint data of 10 batches of Ranae Oviductus were investigated using the professional software Similarity Evaluation System for the Chromatographic Fingerprint, according to the recommendations of the State Food and Drug Administration (SFDA). The HPLC fingerprint data of the samples were imported to the evaluation system (the solvent peaks in the first 4 min were removed and the time window was set at 0.2 s). The calibration method was multi-point calibration. The significant common peaks were labeled as mark peaks and the reference chromatogram fingerprint was generated with a mean value method. The similarity of the fingerprint data was represented by a correlation coefficient (similarity) and the higher similarity between the two samples resulted in a correlation coefficient value close to 1. The correlation coefficients of all chromatograms of 10 batches of Ranae Oviductus samples were calculated throughout the study and a correlation analysis was performed.

Data Analysis
Hierarchical clustering analysis (HCA) is a cluster analysis technique that reflects the similarities and differences between samples in the form of a hierarchical tree diagram [30,31]. This method is easier to observe than the complex raw data. Based on the clustering method between different groups and the Pearson correlation intervals, SPSS (version 25.0; SPSS Inc., Chicago, IL, USA) was used to group the different samples in this study.
Principal component analysis (PCA) is a classification method that uses dimensionality reduction techniques to simplify numerous original variables into several representative composite indicators [32,33]. According to the contribution rate of each comprehensive indicator, the information of the original data could be reflected when using appropriate numbers of principal components (PCs) [34]. In this study, PCA was performed using SPSS (version 25.0; SPSS Inc., Chicago, IL, USA) and the fractional scatter plot was interpreted by the relationship between PC1, PC2, and PC3 for visual analysis of the data matrix.

Optimization of the RP-HPLC Conditions
In order to improve the separation rate of the proteins in Ranae Oviductus, the Ranae Oviductus (S1) collected from the China Changbai mountain area were systematically investigated. The RP-HPLC chromatography method was optimized through the detection wavelength, separation column, flow rate and temperature. Three classical UV detection conditions were previously reported: 215 nm corresponding to the maximum absorption of peptide bonds; 254 nm corresponding to the maximum absorption of phenylalanine residues; and 280 nm corresponding to tyrosine and maximum absorption of tyrosine residues and tryptophan residues [35]. Figure 2a shows the UV absorption diagram of Ranae Oviductus using a diode array detector (DAD) with a wavelength range of 195-300 nm. The red region in the diagram indicated a larger absorption value. Although obvious solvent peaks around 215 nm were observed, the analysis of the core substance was not affected. The UV absorption diagram suggested that the separation effect at 215 nm was better than 254 nm and 280 nm.
Two types of columns (Agilent Zorbax SB-C18 column 250 × 4.6 mm, 5 µm, 80 Å and Agilent Zorbax 300SB-C8 column 250 × 4.6 mm, 5 µm, 300 Å) were used to examine the column effect on the protein separation of Ranae Oviductus. The results showed that the C8 column had a higher separation rate than the C18 column, which could be attributed to the large molecular weight of the proteins ( Figure 2b). Therefore, the C8 column with a 300 Å pore diameter was selected for this study.

Optimization of the RP-HPLC Conditions
In order to improve the separation rate of the proteins in Ranae Oviductus, the Ranae Oviductus (S1) collected from the China Changbai mountain area were systematically investigated. The RP-HPLC chromatography method was optimized through the detection wavelength, separation column, flow rate and temperature. Three classical UV detection conditions were previously reported: 215 nm corresponding to the maximum absorption of peptide bonds; 254 nm corresponding to the maximum absorption of phenylalanine residues; and 280 nm corresponding to tyrosine and maximum absorption of tyrosine residues and tryptophan residues [35]. Figure 2a shows the UV absorption diagram of Ranae Oviductus using a diode array detector (DAD) with a wavelength range of 195-300 nm. The red region in the diagram indicated a larger absorption value. Although obvious solvent peaks around 215 nm were observed, the analysis of the core substance was not affected. The UV absorption diagram suggested that the separation effect at 215 nm was better than 254 nm and 280 nm.
Two types of columns (Agilent Zorbax SB-C18 column 250 × 4.6 mm, 5 μm, 80 Å and Agilent Zorbax 300SB-C8 column 250 × 4.6 mm, 5 μm, 300 Å) were used to examine the column effect on the protein separation of Ranae Oviductus. The results showed that the C8 column had a higher separation rate than the C18 column, which could be attributed to the large molecular weight of the proteins (Figure 2b). Therefore, the C8 column with a 300 Å pore diameter was selected for this study.  Since the flow rate of the mobile phase can affect the isolation efficiency, three flow rates (1.0, 1.5, 2.0 mL/min) were tested in this study. High flow rates showed that peaks overlapped (Figure 2c). The flow rate of 1.0 mL/min showed the highest separation effect and this, therefore, was chosen for the study.
On the other hand, the temperature played an important role in the RP-HPLC separation. Theoretically, high temperatures can increase the motion rate of proteins. In this study, three different temperatures (40, 45, and 50 • C) were investigated (Figure 2d). From the results, we could see that only one peak (t = 74.8 min) at 40 • C was observed, but two shoulder by shoulder peaks appeared at 45 and 50 • C. More proteins separated at 45 and 50 • C. Excessive temperature may damage the column's sorbent, therefore, 45 • C was selected as the optimum temperature.

RP-HPLC Methodology Validation
The accuracy of the RP-HPLC method was investigated through consecutive tests five times, using the same sample solution (Ranae Oviductus sample S1) within one day. The relative standard deviations (RSD) of the retention times and peak areas of the 12 common peaks were smaller than 2.02% and 4.23%, respectively. The repeatability was determined by injecting five separate sample solutions of the Ranae Oviductus sample. The results showed that the RSD of the retention time and peak area of the 12 common peaks were smaller than 2.96% and 5.62%, which suggested that the RP-HPLC method had good repeatability. The stability test was carried out at room temperature for 0, 2, 4, 8, 16 and 24 h. The RSD of the retention times and peak area were smaller than 2.62% and 5.22%. All tests indicated that the RP-HPLC method established in this work satisfied the requirements of protein fingerprinting analysis of Ranae Oviductus.

HPLC Fingerprint of Ranae Oviductus Protein
The protein chromatographic spectra of Ranae Oviductus collected from 10 sampling sites in Changbai Mountain area showed a similar profile using the optimized RP-HPLC method (Figure 3a). Based on the retention time, the 12 significant common-peaks were labeled with number 1 to 12. The 12 significant common-peaks in the Ranae Oviductus protein spectra were labeled as mark peaks according to the Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) (Beijing, China). A reference fingerprint chromatographic spectrum of 10 batches of Ranae Oviductus was created (Figure 3b). The similarity was in the range of 0.779-0.980 ( Table 2). The RSD value of the retention time of each common-peak was smaller than 4.70% and the RSD value of the relative peak area was smaller than 5.47%. This result pointed out that the common-peaks appearing in the chromatographic spectra were reliable in the analysis of Ranae Oviductus.

Fingerprint Spectra Analysis
The fingerprint spectra analysis of Ranae Oviductus and counterfeit products (bullfrog oviduct, toad oviduct and frog oviduct) were performed depending on the aforementioned optimized RP-HPLC method. The results showed a significant difference. By comparing Figure 4a,b, we could see that the significant common-peaks appeared at around 30 min in the reference fingerprint of Ranae Oviductus. In contrary, the counterfeit products, including the bullfrog oviduct, showed four common-peaks (peak A, peak B, peak C and peak D) in 0-30 min and the toad oviduct, showed three common-peaks (peak J, peak K, peak L) in the same time period. Ranae Oviductus showed 12 commonpeaks (peak1-peak12) in 30-80 min, whereas, the bullfrog oviduct and toad oviduct only showed five common-peaks. The frog oviduct only showed four tiny common-peaks (Figure 4c), which was a finding consistent with a previous report. Huang, et al. [36] reported that the protein types in frog oviduct were less than that of other species by using the SDS-PAGE method. Both the protein extraction method and the RP-HPLC conditions were optimized according to the Ranae Oviductus sample, which may have not been adequate for frog oviduct. Through the comparison, we noticed that even the three counterfeit products had a significant difference (Figure 4d). The bullfrog oviduct (nine peaks) and toad oviduct (eight peaks) had more peaks than the frog oviduct (four peaks), but the retention time was different. Therefore, although Rana chensinensis, bullfrog, toad, and frog are similar amphibians, they are not the same species. Their genetic differences cause the expression of different types of proteins in the fallopian tubes, so that in RP-HPLC chromatographic spectra, they showed significant differences. Those differences can be used to identify Ranae Oviductus and counterfeit products.

Fingerprint Spectra Analysis
The fingerprint spectra analysis of Ranae Oviductus and counterfeit products (bullfrog oviduct, toad oviduct and frog oviduct) were performed depending on the aforementioned optimized RP-HPLC method. The results showed a significant difference. By comparing Figure 4a,b, we could see that the significant common-peaks appeared at around 30 min in the reference fingerprint of Ranae Oviductus. In contrary, the counterfeit products, including the bullfrog oviduct, showed four common-peaks (peak A, peak B, peak C and peak D) in 0-30 min and the toad oviduct, showed three common-peaks (peak J, peak K, peak L) in the same time period. Ranae Oviductus showed 12 common-peaks (peak1-peak12) in 30-80 min, whereas, the bullfrog oviduct and toad oviduct only showed five common-peaks. The frog oviduct only showed four tiny common-peaks (Figure 4c), which was a finding consistent with a previous report. Huang, et al. [36] reported that the protein types in frog oviduct were less than that of other species by using the SDS-PAGE method. Both the protein extraction method and the RP-HPLC conditions were optimized according to the Ranae Oviductus sample, which may have not been adequate for frog oviduct. Through the comparison, we noticed that even the three counterfeit products had a significant difference (Figure 4d). The bullfrog oviduct (nine peaks) and toad oviduct (eight peaks) had more peaks than the frog oviduct (four peaks), but the retention time was different. Therefore, although Rana chensinensis, bullfrog, toad, and frog are similar amphibians, they are not the same species. Their genetic differences cause the expression of different types of proteins in the fallopian tubes, so that in RP-HPLC chromatographic spectra, they showed significant differences. Those differences can be used to identify Ranae Oviductus and counterfeit products.

Hierarchical Cluster Analysis (HCA)
Hierarchical cluster analysis was carried out using the relative peak areas of the characteristic peaks of Ranae Oviductus and counterfeit products. The 16 samples were analyzed using SPSS 25.0 software and the results are shown in Figure 5a. Obviously, there were four clusters when the interval of abscissa was 10. Cluster I, Cluster II and Cluster III were composed of the bullfrog oviduct sample, frog oviduct sample and toad oviduct sample, respectively. Cluster IV referred to the 10 samples of Ranae Oviductus used in the establishment of the fingerprint. The sample S1 with low similarity to Ranae Oviductus also showed a low correlation in Cluster IV. When the interval of abscissa was 25, the sample was divided into two clusters, one authentic and another one counterfeit.

Hierarchical Cluster Analysis (HCA)
Hierarchical cluster analysis was carried out using the relative peak areas of the characteristic peaks of Ranae Oviductus and counterfeit products. The 16 samples were analyzed using SPSS 25.0 software and the results are shown in Figure 5a. Obviously, there were four clusters when the interval of abscissa was 10. Cluster I, Cluster II and Cluster III were composed of the bullfrog oviduct sample, frog oviduct sample and toad oviduct sample, respectively. Cluster IV referred to the 10 samples of Ranae Oviductus used in the establishment of the fingerprint. The sample S1 with low similarity to Ranae Oviductus also showed a low correlation in Cluster IV. When the interval of abscissa was 25, the sample was divided into two clusters, one authentic and another one counterfeit.

Principal Component Analysis(PCA)
As an effective data analysis technique, PCA has been used to study the classification of samples [37]. To directly reflect the difference between authentic and counterfeit products, 16 samples were used to perform the PCA analysis, based on the relative peak areas of the characteristic peaks of the samples. The variance contribution rates of the three main components (PC1, PC2, and PC3) were 31.34%, 27.61%, and 26.73%, respectively. The cumulative variance contribution rate of the three PCs was 85.68% and those variables reflected the majority of total information. To visualize the analysis results, the score charts were drawn using the three main components of PC1, PC2 and PC3 ( Figure  5b). Four aggregation states are showed in Figure 5b. Ranae Oviductus, bullfrog oviduct, toad oviduct, and frog oviduct samples were classified in the a, b, c, and d regions, respectively. The Ranae Oviductus samples S1-10 could be classified in the same area (the a region), the bullfrog oviduct was classified in the b region, the toad oviduct was classified in the c region, and the frog oviduct was classified in the d region. The results were consistent with the HCA analysis, that both Ranae Oviductus and the counterfeit products were correctly classified. Comparing the similarity analysis with the HCA, PCA can provide a more visual comparison of the chromatograms.

Conclusions
This study used the RP-HPLC method and fingerprint technique to establish a chromatographic fingerprint of the proteins from Ranae Oviductus. Ten batches of Ranae Oviductus collected from the Changbai mountain area were used to analyze the protein components. The results showed 12 common-peaks in the reference fingerprint chromatographic spectrum. In combination with stoichiometry HCA and PCA, the results suggested that the method established in this work can satisfy the identification of Ranae Oviductus and counterfeit products. The method established in this work provides a promising approach for the identification of Ranae Oviductus and counterfeit products.

Principal Component Analysis(PCA)
As an effective data analysis technique, PCA has been used to study the classification of samples [37]. To directly reflect the difference between authentic and counterfeit products, 16 samples were used to perform the PCA analysis, based on the relative peak areas of the characteristic peaks of the samples. The variance contribution rates of the three main components (PC1, PC2, and PC3) were 31.34%, 27.61%, and 26.73%, respectively. The cumulative variance contribution rate of the three PCs was 85.68% and those variables reflected the majority of total information. To visualize the analysis results, the score charts were drawn using the three main components of PC1, PC2 and PC3 (Figure 5b). Four aggregation states are showed in Figure 5b. Ranae Oviductus, bullfrog oviduct, toad oviduct, and frog oviduct samples were classified in the a, b, c, and d regions, respectively. The Ranae Oviductus samples S1-10 could be classified in the same area (the a region), the bullfrog oviduct was classified in the b region, the toad oviduct was classified in the c region, and the frog oviduct was classified in the d region. The results were consistent with the HCA analysis, that both Ranae Oviductus and the counterfeit products were correctly classified. Comparing the similarity analysis with the HCA, PCA can provide a more visual comparison of the chromatograms.

Conclusions
This study used the RP-HPLC method and fingerprint technique to establish a chromatographic fingerprint of the proteins from Ranae Oviductus. Ten batches of Ranae Oviductus collected from the Changbai mountain area were used to analyze the protein components. The results showed 12 common-peaks in the reference fingerprint chromatographic spectrum. In combination with stoichiometry HCA and PCA, the results suggested that the method established in this work can satisfy the identification of Ranae Oviductus and counterfeit products. The method established in this work provides a promising approach for the identification of Ranae Oviductus and counterfeit products.

Conflicts of Interest:
The authors declare no conflict of interest.