Vitamin B complex, amino acid profile and bioactive properties of newly developed rye variants

ABSTRACT This study was designed to investigate the amino acid profile, vitamin B complex, and bioactive properties present in recently cultivated rye variants in Pakistan. The four rye variants, named as Gp 1, Gp-2, Gp-3, and Gp-4, were procured from the Forage and Pulses section of AARI Faisalabad. The rye samples were ground into flour using a hammer-type 120-ton laboratory mill. For the analysis of amino acids, vitamin B complex, phenolic constituents, alkylresorcinols, and lignin, standard HPLC and GC-MS methods were employed following the extraction with appropriate reagents. The results revealed that among the rye variants, Gp-2 showed the highest content of vitamin B complex, with thiamin at 252.33 μg/100 g, niacin at 397 μg/100 g, and pyridoxine at 99 μg/100 g. Pantothenic acid (246.70 μg/100 g) and ascorbic acid (470 μg/100 g) were found in the highest concentrations in Gp-3. The riboflavin content of Gp-1 was 84.40 g/100 g, while the folate content of Gp-4 was 24.40 g/100 g. In terms of phenolic compounds, the range of chlorogenic acid content ranged from 86.70 to 92.43 mg/kg, p-coumaric acid from 2.70 to 5.40 mg/kg, gallic acid from 64.70 to 97.10 mg/kg, caffeic acid from 16.90 to 25.90 mg/kg, and coumaric acid from 218.10 to 228.40 mg/kg. In conclusion, Gp-2 observed as the most nutritionally rich and bioactive variant, distinguished by its high essential amino acids, vitamins, and other bioactive components.


Introduction
Rye (Secale cereal L.) is a neglected crop in terms of overall production in the world.Rye contributes to approximately 0.70% of the entire cereal crops.The global data regarding rye production is expected to be about 14.80 million tons.Rye grains are unusually winter hardy and can thrive in sandy, allowing them to be grown in regions where other cereals cannot grow. [1]It is believed to have originated from Asian wild rye types, particularly wheat flour, which spread like weeds [2] In cereals, rye contains higher total dietary fiber (24.47%) and various bioactive constituents. [3]any Nordic countries have a long-standing and rich history of including rye in their diets.All cereals, especially rye, should be baked with whole-grain flour since whole rye grains are rich in vitamins, fibers, minerals and other bioactive substances [4] Plant-derived phytochemicals are essential components used by plants for both defense systems and cell signaling processes.Aleurone cells, or the bran layer, are an essential source of various phytochemicals. [5,6]Rye has been found to contain benzoxazinoids, phenolic acids, lignans and alkylresorcinols. [7]However, rye comprises over 2000 chemicals as a result of the continuous increase in the number of phytochemicals that have been identified in the rye. [8]Therefore, rye consumption on a daily basis has found to be significantly protective against a number of diseases in recent research. [9]Rye grains contain minerals as well as carbohydrates, protein, fiber and amino acids, particularly lysine content.They also have vitamins E and B complex, which includes thiamine, folic acid, pantothenic acid and riboflavin.Rye contains less gluten than wheat on average. [10]elcour & Hoseney [11] found that the biochemical profile of rye is affected by variety, climate and farming circumstances, quality of land and other factors.Rye grain has a basic makeup that is comparable to that of other cereals. [12]The major component of rye seed is starch.Rye starch gelatinization occurs at a lower temperature as compared to wheat starch.The size of starch fraction (A-type 20-35 μm), and (B-type 5-10 μm) and shape (spherical and lenticular) of rye starch contents are comparable to wheat starch. [13]Compared to wheat starch, rye flour starch granules have more enzymatic and mechanical degradation.Rye flours have fewer proteins and starch than wheat, but more fiber. [3]The aleurone layer of rye endosperm is high in bioactive components like phenolic compounds (phenolic acids and polyphenols), vitamins (B vitamins, particularly riboflavin, thiamine, niacin as well as vitamin E), minerals (potassium, iron, zinc, magnesium and phosphorus), lignans, sterols, alkylresorcinols as well as stanols. [14]Secondary metabolites of cereal compounds that are bioactive compounds include phenolics, i.e. flavonoids, flavanones, and isoflavones. [15]This project aims to investigate the amino acid profile, vitamin B complex composition, and presence of important bioactive chemicals in different wholegrain rye variants, with a focus on lignans, alkylresorcinols, and phenolics.

Procurement of raw materials
Grains of rye were procured from Forage and pulses Section, Ayub Agricultural Research Institute AARI, Faisalabad.RJS-1001, RJS-1002, RJS-1003 and RJS-1004 were four variations that were GP-1, GP-2, GP-3 and GP-4, respectively.For the analysis, only chemicals of analytical grade were used.H 2 SO 4 and methanol are obtained from Sigma-Aldrich Chemical Co.(St.Louis, MO, USA) whereas, n-hexane and petroleum ether were purchased from Merck (Darmstadt, Germany).Rye grains were ground at Ayub Agricultural Research Institute, Faisalabad using a hammer-type 120 perton laboratory mill.The ground flour was separated using sieves that varied in size from 0.5 to 2.0 mm.This paper is a fouth part of a PhD research project.The other parts of the projects are already published [2,4,8]

Amino acid profile
Five-gram rye flour samples with 80% aqueous ethanol (10 mL) were put in a cylinder glass tube.At 110°C the glass tube containing the sample was hydrolyzed for 24 h.After adding 5 mL of distilled water to the samples, they were centrifuged for 10 min at 4000 rpm and 4°C.The supernatant was removed and filtered by a 0.45 µm syringe filter and put into a flask.Three percent of 1 mL sulfosalicylic acid (SSA) was added to the flask.The SSA samples were mixed under the vortex until homogenous and placed at 20°C.The solution was again centrifuged for 10 min at 4000 rpm and 4°C.The supernatants were withdrawn and dried at 45°C.To dissolve the solution, 1 mL of sodium acetate buffer was added.A 0.22 µm filter was used to filter the solution once more.About 300 µL of the filtrate was ready to be transferred to the Central Hi-Tech Lab, Government College University Faisalabad, Pakistan.The sample of 50 nmoL/mL was analyzed using a JEOL (JLC 500/V) automatic amino acid analyzer.The amino acids were measured in g/100 g of dry weight protein.

Sample preparation
With slight modifications, the rye samples were treated as described in Aslam et al. [16] to analyze B vitamins.Ten grams of homogenized rye flour were transferred into a conical flask.The extraction mixture was then added to the sample in a volume of 25 mL.For 40 min, samples and extraction solution were maintained at 70°C in the water bath.The samples were then cooled for 3-4 h before being filtered.

Phenolic compounds
According to the methods proposed by Irmak et al., [17] phenolic acids were calculated of rye flour.The primary phenolic acids were determined using HPLC (Perkin Elmer series 200) with a UV-visible detector.The Shim-pack GIST-HP (Octadecylsilane (C18) column was used.

Alkylresorcinols content
The procedure proposed by Mattila et al. [18] was used to determine alkylresorcinols.The sample (1.25 g) was steeped at ambient temperature in a Pyrex screw-cap tube with an equivalent weight of acetone.The extract was saved after being filtered using Whatman No. 1 paper for 24 hours.After 24 hours, the sample was again submerged in the same volume of acetone and filtered.Previous extracts were mixed with filters.Sample was dried and powdered before being re-soaked in acetone in a 1:5 ratio, filtered, and then added to the earlier extract.With the help of acetone, the volume of collected extracts was increased to 25 ml.A water bath at 850°C was used to extract the acetone from two aliquots of 2.0 ml each that had been transferred to Pyrex screw-cap tubes lined with Teflon.After being brought to room temperature, the leftover was dissolved in 0.4 cc of chloroform.The tube was sealed tightly after being filled with 0.1 ml of 75% ethanol and 0.1 ml of 75% KOH.It was then put in a shaker bath at 450 C and stirred every 2-3 minutes.
After 20 minutes, 8.4 ml of 95% ethanol and 1.0 ml of distilled water were added.10 ml in total were shaken, and they were let to stand for 30 minutes.The tube was shaken once more before to detecting the fluorescence on a Hitachi Perkin-Elmer MPF-2A spectrofluorometer (Perkin-Elmer, Norwalk, CT).The wavelengths used for excitation and emission were 420 and 520 nm, respectively.With each set of samples, a standard curve was created using 5-pentadecylresorcinol.To confirm the precision of the response factors calculated from olivetol, the standards C17:0, C19:0 and C21:0 (ReseaChem GmbH, Burgdorf, Switzerland) were used.The results are given in mg/100 g of dry weight.

Lignin contents
GC-MS (Agilent 7000E triple quadrupole GC/MS) was used to identify lignans including pinoresinol, matairesinol, isolariciresinol, secoisolariciresinol, lariciresinol and syringaresinol, with minor changes in the procedure suggested by Mazur et al. [19] The standards (50 mg) and distilled water were added to the weighed quantity of the sample.Enzymatic hydrolysis was used to prepare the samples before extraction with diethyl ether.Hydrochloric acid was used to hydrolyze in the water phase, which was then extracted using a combination of diethyl ether and ethyl acetate.Ion exchange chromatography was used to mix and purify the organic phases, as reported by Mazur et al. [19] The treated samples were analyzed by GC -MS after silylation.

Essential amino acid profile
The mean value of the isoleucine content of whole rye flour is presented in Figure 1.The present study's findings indicated that the isoleucine lysine, phenylalanine, threonine, tryptophan, valine, histidine, leucine and methionine content in the rye flour varied from 225 to 254.67, 195 to 256, 192 to 226, 178 to 201.3, 46 to 68, 265.3 to 325, 135.67 to 157, 356.33 to 395, and 43.1 to 49 g/kg N −1 respectively.The maximum isoleucine, content (254.67 g/kg N −1 ) was detected in Gp-4 while, the minimum isoleucine content was detected in Gp-3 (225 g/kg N −1 ).The highest lysine content (256 g/ kg N −1 ) was detected in Gp-3 while, the lowest lysine content was detected in Gp-1 (195 g/kg N −1 ).On the other hand, the grain protein of rye has a greater lysine concentration (15.1-28.1)than wheat (13.1-24.9). [13]Meanwhile, The maximum phenylalanine content (226 g/kg N −1 ) was detected in Gp-1 although, the minimum phenylalanine content was detected in Gp-4 (192 g/kg N −1 ).The highest threonine content (201.3 g/kg N −1 ) was detected in Gp-4 whilst, the lowest threonine content was detected in Gp-1 (178 g/kg N −1 ).In comparison, The maximum tryptophan content (68 g/kg N −1 ) was detected in Gp-1 while, the minimum tryptophan content was detected in Gp-3 (46 g/kg N −1 ).The highest valine content (325 g/kg N −1 ) was detected in Gp-1 whilst, the lowest valine content was detected in Gp-3 (265.3 g/kg N −1 ).

Non-essential amino acid
The mean value of the non-essential amino acid content of whole rye flour is presented in Figure 2.

Phenolic contents
The mean value regarding the phenolic content of whole rye flour is shown in Figure 3 The chlorogenic acid in the rye flour varied from 86.7 to 92.43 mg/kg.The maximum chlorogenic acid (92.43 mg/kg) was detected in Gp-2 then Gp-1 (91.4 mg/kg) and Gp-4 (90.76 mg/kg), While, in Gp-3 (86.7 mg/kg) minimum chlorogenic acid was present.These current results agree with the outcomes of Andreasen et al. [24] who studied ferulic acid and phenolic acids in seventeen rye varieties.Heiniö et al. [25] also mentioned similar findings while investigating quantities of phenolic compounds in the different rye varieties.Whereas the p-coumaric acid in the rye flour varied from 2.7 to 5.4 mg/kg.The maximum p-coumaric acid (5.4 mg/kg) was detected in Gp-1 then Gp-4 (3.6 mg/kg) and Gp-3 (3.5 mg/kg).While the Gp-2 (2.7 mg/kg) elucidated minimum p-coumaric acid content.Nystrom et al. [26] detected similar results and reported that p-coumaric acid in the whole rye flour is 3.3 mg/100 g.These outcomes are also in line with Bondia-Pons et al. [27] and Andreasen et al. [24] documented that p-coumaric acid in the rye flour varied from 37-65 μg/g dry mass.While the gallic acid in the rye flour ranged from 64.7 to 97.1 mg/kg.The maximum gallic acid (97.1 mg/kg) was detected in Gp-4 then Gp-1 (78.5 mg/kg) and Gp-2 (69.3 mg/kg).While the Gp-3 (64.7 mg/kg) showed minimum gallic acid content.However, the caffeic acid in the rye flour varied from 16.9 to 25.9 mg/kg.The maximum caffeic acid (25.9 mg/kg) was detected in Gp-2 then Gp-1 (21.4 mg/kg) and Gp-3 (16.9 mg/kg).In Gp-4 (20 mg/kg) minimum, caffeic acid content was detected.Mattila et al. [18] explored that caffeic acid in the whole rye flour is 1 mg/100 g.Whereas, another study carried out by Bondia-Pons et al. [27] depicted similar results.Whereas the coumaric acid in the rye flour varied from 218.1 to 228.4 mg/ kg.The maximum p-coumaric acid (228.4mg/kg) was detected in Gp-2 then Gp-3 (223 mg/kg) and Gp-1 (220 mg/kg).While the Gp-4 (218.1 mg/kg) explored minimum p-coumaric acid content.

Conclusion
The results showed that rye flour is a significant source of phenolic chemicals, alkylresorcinols, vitamin B complex, and both essential and non-essential amino acids.GP-2 is the most nutritionally robust of the four rye varieties that were evaluated (GP-1, Gp-2, Gp-3, and Gp-4).It has higher levels of vitamins, amino acids, and other bioactive components.Gp-2 is a better option than other alternatives because of its improved nutritional profile.The remarkable nutritional richness of rye flour accentuates its potential for enhancing the nutritional and functional attributes of diverse food products.Consequently, incorporating rye flour into various food formulations holds promise for elevating their overall nutritional value and functional benefits.

Figure 1 .
Figure 1.Mean values for the essential amino acid contents of four different rye flours (g/kg N −1 ).

Figure 2 .
Figure 2. Mean values for the non-essential amino acid contents of different rye flours (g/kg N −1 ).

Figure 3 .
Figure 3. Mean values for the phenolic content of different rye flours (mg/kg).

Figure 4 .
Figure 4. Mean values for the vitamin content of the different rye flour (μg/100 g).