HYDROLYZED HEMP SEED PROTEINS AS BIOACTIVE PEPTIDES

Hemp seed cake, a by-product of cold oil processing, represents a food waste material of high nutritional value. Therefore, in this research, it was used as starting raw material for bioactive peptides production. Alkali extraction followed by isoelectric precipitation was employed for hemp protein isolation.Subsequently, the influence of different enzymes (alcalase and pancreatin), as well as the degree of hydrolysis on the kinetics of different molecular weights peptides production and their antioxidant potential was investigated. The DPPH• scavenging activity of hemp protein hydrolysates and hemp protein isolate and their reducing power was determined.The obtained results showed that the strongest antioxidant activity exhibited peptides characterized by the highest degree of hydrolysis. In general, it was concluded that the properties of the obtained hydrolysates were dependent on the type and specificity of the employed protease, as well as the hydrolysistime.


INTRODUCTION
During the food processing, the occurrence of biologicalspecific waste is inevitable.The disposal of food waste is difficult due to its biological instability, potentially pathogenic nature, high water content, potentially rapid autoxidation and very high enzymatic activity.High risk of environmental pollution caused by the uncontrolled release of food by-products has led to the necessity of reducing the amount of waste through development of various bioprocesses toconvert the starting material tonovel food products.Numerous recent studies have shown that food industry by-products represent rich source of proteins and other nutraceuticals (Hadnađev et al., 2017;Rodrigues et al., 2012).
The oil industry represents an important resource of large quantities of underutilized by-products.The extraction of oil results in oilcake or meal, which quantity has a continuous tendency of growth all around the world (Moure et al., 2006b;Ramachandran et al., 2007).The most common use of these cakes and meals is in animal nutrition or as organic fertilizers (Ramachandran et al., 2007).Oils cakes and meals generally contain high amount of protein, ranging from 15% to most commonly 50 %.Despite the fact that these by-products contain other numerous biologically active compounds (vitamins, minerals, phenols), proteins due to possibility of being modified and, consequently, manifest various physiological functions, remain in the focus of current scientific research (Kreft et al., 2002;Moure et al., 2006a;Popović et al., 2015;Šimurina et al., 2015).
Hemp proteins used in food and feed industry originate from speciesCannabis sativa L., which unlike marijuana (Cannabis indica), containsonly traces of δ-9-tetrahydrocannabinol (THC).Hemp seed is composed of over 30% of oil and 25% of high quality protein.Hemp seed proteins are generally comprised of two major fractions -edestin (globulin) and albumin (Patel et al., 1994;Tang et al., 2006).These proteins contain most of the essential amino acids (Wang et al., 2008).Comparedto soybean seed, hemp seed contains lower amount of trypsin inhibitors that limit protein digestion and subsequent protein absorption (Odani and Odani, 1998).
In the last few years, the number of scientific researches in the field of bioactive peptides has increased considerably.Biopeptides are natural chemical compounds found in plants and animals that have the ability to improve certain health functions when found in the human organism (Hernandez-Ledesma et al., 2007).They represent short amino acid sequences (2-20 amino acid residues) found in the primary protein structure.They are derived from food proteins by the action of enzymes and when they enter the body they can act as modulators of certain physiological processes, similar to endogenous peptides with hormonal activity (Tang et al., 2009).Their physiological effects are related to: regulation of elevated blood pressure (inhibition of angiotensin and converting enzyme), antioxidant activity, prevention of platelet aggregation, modulation of the immune system, lowering of cholesterol and triglycerides in plasma, stimulation of the functions of the nervous system, antimicrobial effect, improving the transport and absorption of minerals (Hartmann and Meisel, 2007;Korhonen and Pihlanto, 2006).
Assuming that the proteins obtained from hemp seed cakecan be converted to bioactive peptides with the action of different proteases, the aim of this research was to study the effect of enzyme type (alcalase and pancreatin) and the enzymatic hydrolysis degree on the functional properties of hemp proteinhydrolysates, with particular reference to their antioxidant potential.
Hemp protein isolate preparation Hemp protein isolate (HPI) was prepared according to the method described by Tang et al. (2006).Hemp cake was ground in a laboratory mill Foss Knifetec 1095 (Foss, Hillerød, Denmark) equipped with cooling system in order to prevent overheating during grinding procedure.Obtained hemp flour was fractioned using a universal laboratory sifter (Bühler AG, Uzwil, Switzerland) and a particle size fraction of <250 μm was used as a starting raw material for protein isolation.Subsequently, obtained hemp flour fraction was defatted using hexane in double hexane extraction at a 1:5 meal to hexane ratio (w/v) during 24h, with constant stirring at room temperature for the first 2h, followed by hexane decantation.The defatting process was repeated twice.Defatted hemp flour was then suspended in water in a ratio of 1:20, and the pH was adjusted to 10.0 using 2M NaOH solution.Suspension was constantly mixed at 37 °C during 75 min and the pH was kept at 10.0.After the extraction procedure, suspension was centrifuged for 20 min at 8000 g and temperature of 20 °C (Sorvall® RC-5B Refrigerated Suprespeed Centrifuge, Du Pont Instruments, USA).Supernatant was then collected; pH was adjusted to 5.0 using 1M HCl which resulted in protein precipitation.Protein precipitate was finally centrifuged for 20 min at 8000 g and temperature of 20 °C and dried in a vacuum drier (Binder, Tuttlinger, Nemačka) at 37 °C.

Hemp protein hydrolizates preparation
Six grams of HPI was suspended in 150 mL of distilled water at room temperature.The suspension was heated to the temperature needed to achieve the optimum activity of each enzyme and pH was adjusted to the required values.Six samples were prepared: three using alcalase (A3 -degree of hydrolysis 3 %, A6 -degree of hydrolysis 6 %, A9 -degree of hydrolysis 9 %) and three with pancreatin treatment (P3 -degree of hydrolysis 3 %, P6 -degree of hydrolysis 6 %, P9 -degree of hydrolysis 9 %).Enzymatic hydrolysis conditions were pH 8.5 and temperature of 55 °C for alcalase and pH 7.5 and temperature of 37 °C for pancreatin hydrolysis, while enzyme/substrate ratio was 3 % calculated on theprotein content of the sample.Protein and enzyme mixture was heated in an enzyme digestion apparatus (VELP Scientifica, Italy).pH value was kept constant during the hydrolysis process by adding 0.5 M NaOH solution.Hydrolysis was stopped by heating the mixture in boiled water for 10 minutes followed by cooling to room temperature.Obtained protein hydrolysates were centrifuged for 20 min at 8000 g in order to remove insoluble compounds.Supernatant was collected and dried in a vacuum drier.
The degree of hydrolysis (DH), can be defined as the ratio of the number of cleaved peptide bonds relative to the number of peptide bonds prior to hydrolysis -htot (the number of free amino groups formed during hydrolysis).The value of htot is equivalent to the protein amino acid composition calculated from the analysis by adding mmolof individual amino acids per gram.Degree of hydrolysis was determined using pH-stat method (Adler-Nissen, 1986) and calculated using the following equation: where B represents the consumption of alkali expressed in mL, Nb-alkali molarity, Mp -protein mass used for hydrolysis (g), htot -total number of peptide bonds in substrate (meqv/g protein).In previous analyzes of the amino acid composition, it was calculated that htotfor hemp protein isolates is 7.39 meqv/g (Yin et al., 2008).

2400
(3 where T is the temperature of the reaction.

Protein yield and content determination
Protein yield was calculated relative to the defatted hemp flour content, while protein content in HPI was determined according to Kjeldahl method (AOAC, 2011).

DPPH radical scavenging activity
Effect of the HPI and hydrolysatessolutions on the content of 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH•) was determined according to the method described by Tang et al. (2009).Three millilitres of hydrolysatesand protein isolate solutions of different concentrations were mixed with 3 mL of 2.0 x 10-4 M freshly prepared DPPH•methanol solution.After that, mixture was left for 30 min in the dark, followed by absorbance measurements at 517 nm (Specord M40, Carl Zeiss, Jena, Germany) against the blank (instead HPI and protein hydrolysates3 mL of distilled water was used).Lower absorbance of the reaction mixture indicates higher DPPH antiradical activity.
The antiradical activities of tested samples were expressed as IC50 value (mg/mL), which represents the concentration of a sample needed to quench 50 % of the initial amount of DPPH•.Antiradical activity of the investigated samples was compared to activity of L-glutathione reduced (GSH).

Reducing power
Reducing power of the HPI and hydrolysates solutions was measured according to the method of Tang et al. (2009) and Jaisan et al. (2015).The quantity of 2.5 mL of sample solutionwas mixed with 2.5 mL of phosphate buffer (0.2 M, pH=6.6) and 2.5 mL of potassium ferricyanide (1%).Subsequently, the mixture was heated for 20 min at 50 °C, cooled down and mixed with 2.5 mL of 10 % trichloroacetic acid and then centrifuged for 3 min at 3000 g.Finally, 2.5 mL of the obtained supernatant was mixed with 2.5 mL of distilled water and 1 mL of 0.1% ferric chloride.Absorbance was measured at 700nm.Higher absorbance indicates higher reducing power, as well.Ability of protein hydrolysates and isolate to reduce Fe3+ was demonstrated as effective concentration, IC50 i.e. the required concentration of investigated solutions at which the absorbance of reaction mixture reaches 0.5 of reducing power.Reducing power of the investigated samples was compared to the one of L-glutathione reduced (GSH).

Statistical analysis
Data were analyzed by one-way analysis of variance with LSD test, which was performed using Statistica 8.0 (Statsoft, Tulsa, USA).The significance of differences among the mean values was indicated at the 95 % confidence level.

RESULTS AND DISCUSSION
Hemp protein isolate obtained in this study was characterized with protein content of 89.95 %, and the yield relative to defatted hemp flour of 25.81 %.These values are in agreement with Tang et al. (2006) and Isinguzo (2011) who obtained 86-91 % protein content of HPI and the yield in the range between 21 -26 % relative to defatted hemp flour.The colour of the product was dark green due to the presence of bound phenolic compounds and chlorophyll (Pojić et al., 2014).
Unlike numerous research papers comparing the action of different enzymes on theantioxidant activity of the protein hydrolysates bykeeping the time of hydrolysis constant, in this paper, the degree of hydrolysis (DH) for both enzymes was kept constant.This approach enabled comparison of enzymes specificity to certain protein sites rather than hydrolysis degrees on peptides antioxidant capacity.For both alcalase and pancreatin,hydrolysis was performed until a degree of hydrolysis of 3, 6 and 9 % was achieved.It can be seen in Figure 1that with the use of alcalase the desired degree of hydrolysis was reached for a shorter period of time than with the use of pancreatin.

Fig.1. Change in the degree of hydrolysis with the hydrolysis time
Alcalase,as a serine type endoprotease, has a wide specificity for substrates and can hydrolyse most of the peptide bonds in the protein molecule.It represents the product of Bacillus licheniformis strain.Although the use of alcalase is widespread, its specificity has not yet been fully characterized.On the contrary, pancreatin is a mixture of several digestive enzymes derived from exocrine pork pancreas cells, and represents a blend of endo-and exopeptidases.It is a wide-spectrum protease comprised of amylase, trypsin, lipase, and ribonuclease (Sigma Aldrich, 2017).Although pancreatin is consisted of several enzymes, at a temperature and pH conditions which represent a catalytic optimum for this enzyme, the pancreatin solution is highly unstable due to autolytic and proteolytic degradation of the enzyme itself (Biozym, 2017).Using the same amount of protein substrate, as well as the same amount of enzyme, the required degree of hydrolysis (3, 6 and 9 %) was achieved for the shorter period of time using alcalase enzyme than pancreatin (Table 1).This can be explained by the fact that alcalase has a greater affinity for peptide cleavage.Therefore, alcalase could be observed as a more effective choice for the hemp proteins hydrolysis than pancreatin.In general, alkaline proteases such as alcalases show better activity in peptide hydrolysis in comparison to acid and neutral proteases (Klompong et al., 2007).
The ability of hemp protein hydrolysates, HPI and GSH, which served as the control sample,to scavenge DPPH• was shown in Figure 2. Comparison of the antioxidant activity of the samples was done according to the IC50 value.It can be seen that the ability of the sample to scavenge DPPH•is closely related to protein hydrolysis degree.The strongest antioxidant activity was exhibited in samples characterized by the highest degree of hydrolysis (9 %) regardless of the enzyme employed.Comparing the IC50 values,it can be concluded that hydrolysates obtained by pancreatinshowed a stronger antioxidant activity than hydrolysatesderived by alcalase.Previous studies showed that the ability of protein isolates to scavenge DPPH•was associated with the high hydrophobicity of the proteins themselves.It was also indicated that smaller protein fractions possessed a higher proportion of hydrophobic amino acids (Li et al., 2008;Pownall et al., 2010).Moreover, it is well known that the antioxidant effect of the proteins is related to amino acid composition (Chen et al., 1995).Since pancreatinrepresents a mixture of enzymes with different specificities, results of scavenging activity on DPPH•indicated that in the case of pancreatin hydrolysis, bioactive peptides with a higher proportion of hydrophobic amino acids have been released.The results presented in this paper further confirmed that protein hydrolysates exhibited greater scavenging activity on DPPH• than the isolate.Moreover, it can be assumed that certain DPPH antiradical activity of HPI was probably due to polyphenol compounds and pigments that have been co-extracted with the isolate, as indicated by the green colour of the isolate itself.Namely, phenolic compounds are the reason of the dark colour and undesirable taste of protein isolates.According to Xu and Diosady (2002), phenolic compounds bind to proteins in aqueous solution through the following mechanisms: hydrogen bonds, covalent bonds, hydrophobic interactions and ionic bonds, and their removal represents a major challenge.
As shown in Figure 3, IC50 values for reducing power of the protein hydrolysates were in the range of 1.98-4.70mg/mL, depending on the enzyme used, as well as the hydrolysis time or degree.

Fig.3. Reducing power of hemp protein hydrolysates and hemp protein isolate relative to L-glutathione
These results are in agreement with the results obtained by Tang et al. (2009)who investigated the effect of variousproteases on the antioxidant properties of the resulting hemp protein hydrolysates.Glutathione exhibited the greatest ability in reducing Fe3+compared to hemp protein isolate and hydrolysates.It is a tripeptide containingcysteine -amino acid with sulfhydryl group.Sulfhydryl groups are considered to be responsible for the expression of the reducing activity (Battin and Brumaghim, 2009).The hydrolysates obtained by using pancreatinshoweda higher reducing power relative to those obtained by alcalase treatment.Battin and Brumghim (2009) stated that the type of amino acids, as well as peptide type plays an important role in reducing power activity.It can be noticed that hydrolysates obtained using pancreatin and alcalase treatment have a lower proportion of sulfhydryl groups in their composition than glutathione, which contributed to their poorer ability to reduce Fe3+ (Isinguzo, 2011).The relatively high ability to reduce Fe3+ in the case of hemp protein isolates was probably the consequence of the presence of phenolic compounds bound to the protein molecule.

CONCLUSION
Hydrolysis of hemp protein,isolated from hemp seed meal, with the aid of various enzymes (alcalase and pancreatin) revealed that the same degree of hydrolysis can be achieved for a shorter period by using the alcalase in comparison to pancreatin treatment.The strongest antioxidant activity was shown for hydrolysates characterized by the highest degree of hydrolysis (DH 9 %) in the case of both enzymes.The hydrolysates obtained using pancreatin exhibited stronger DPPH antiradical activity as well as reducing power than the ones derived by alcalase treatment.It can be explained by the fact that pancreatin,a mixture of enzymes of varying specificity, released bioactive peptides with a higher proportion of hydrophobic amino acids as well as sulfhydryl groups,which are responsible for antioxidative activity of proteins.Hemp protein hydrolysates expressed stronger antioxidative potential thanthe starting raw materialhemp protein isolate.

Fig. 2 .
Fig. 2. Scavenging activity on DPPH • of hemp protein hydrolysates and hemp protein isolate relative to L-glutathione

Table 1 .
Time required for hydrolysis of HPI using different proteases and degree of hydrolysis