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Article

Commercial Hemp (Cannabis sativa Subsp. sativa) Proteins and Flours: Nutritional and Techno-Functional Properties

by
Yamina Absi
,
Isabel Revilla
* and
Ana M. Vivar-Quintana
Food Technology, Polytechnic High School of Zamora, Universidad de Salamanca, Avenida Requejo 33, 49022 Zamora, Spain
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(18), 10130; https://doi.org/10.3390/app131810130
Submission received: 16 August 2023 / Revised: 5 September 2023 / Accepted: 7 September 2023 / Published: 8 September 2023
(This article belongs to the Special Issue Chemical and Functional Properties of Food and Natural Products)
Browse Figure
Versions Notes

Abstract

:
Hemp (Cannabis sativa subsp. sativa) has been increasing in popularity in recent years owing to its nutritional composition, with an interesting combination of protein, fat, and fiber, as well as minerals. Its transformation into flours and concentrates has allowed its incorporation in different foods thanks to its techno-functional properties. In this study, four commercial brands of hemp flour and hemp protein concentrate were analyzed for their proximal, amino acid and mineral composition, and fatty acid profile. The bioactive characteristics, such as phenolic composition and antioxidant activity, and techno-functional properties, such as solubility and water-holding and oil-holding capacities, were analyzed. The results showed that the composition of the flours was characterized by a high fiber content and a high antioxidant activity due mainly to the high level of total phenolic compounds. In the case of concentrates, these showed a lower carbohydrate but higher protein content and better functional properties such as water-holding, foaming, and gel-forming capacities. Both flours and concentrates showed low fat contents with polyunsaturated fatty acids being the major fatty acids, a good amino acid profile, and high K and P concentrations. Organic products showed differences in nutritional composition but not in functional properties when compared with non-organic products.

1. Introduction

Cannabis sativa subsp. sativa, of the Cannabaceae family, is a non-conventional multipurpose crop with a wider range of adaptation to warmer and colder conditions than other plants typical of moderate climates [1]. Moreover, hemp is attracting great interest as a sustainable crop owing to its role in soil regeneration, with its roots growing deep in the soil and its short growth period of 3–4 months [2]. In addition, it has low water and input requirements [3] and traps carbon dioxide five times more effectively than forest trees on the same acreage, which helps to reduce to global warming [4]. In consequence, hemp may have an important role to play in environmental protection [5]. Moreover, its increasing legalization, together with the wide variety of products which can be obtained from a hemp plant, has attracted the interest of different industries to make it a widely cultivated and industrially important plant [2].
The increasing focus on plant-based foods for consumers has led to a growing interest in the uses of hemp seeds as food and feed [5]. For this reason, the number of non-pharmacological hemp varieties with low δ-9-tetrahydrocannabinol (THC) content is steadily increasing [6]. The term “hemp” refers to cultivars of Cannabis sativa grown for industrial purposes containing tetrahydrocannabinol (THC) in trace amounts. According to Commission Regulation (EU) 2022/1393 [7], the maximum level of delta-9-tetrahydrocannabinol in ground hemp seeds, (partially) defatted hemp seeds, and other hemp seed-derived/processed products is 3.0 mg/kg.
Hemp seeds provide significant amounts of protein (200–250 g/kg), oil (250–350 g/kg), carbohydrates (200–300 g/kg), and insoluble fiber (100–150 g/kg), together with important levels of minerals (56–80 g/kg) [8]. In fact, they contain a wide range of minerals such as iron, calcium, zinc, magnesium, and manganese [9]. In addition, several studies, both in vitro and in vivo, have demonstrated the antioxidant potential and the antihypertensive properties of hemp seed [10]. It is particularly interesting that the concentration of anti-nutritional compounds in hemp seeds is very low [11], which makes it an interesting alternative to concentrates and flours made from legumes.
The proteins present in hemp seeds are primarily albumin and edestin. These are easily digestible proteins which contain substantial levels of all essential amino acids [12] and are classified as non-allergenic [13]. The amino acid profile is similar to that found in eggs and soybeans [14], and this can help to address the issues of a sustainable alternative food. Hempseed oil consists of 80–90% polyunsaturated fatty acids (PUFA) including high levels of linoleic (LA) and α-linolenic acid (ALA) [12].
Hemp seed products, which are generally obtained after partial or total oil extraction, are positioned as a good alternative to soy for the food industry because hemp flour has high levels of fat and protein but also of fiber, essential fatty acids and amino acids, and minerals [15]. In addition, hemp flour is rich in bioactive compounds, such as polyphenols, vitamin E, and minerals such as calcium, potassium, sodium, magnesium, iron, phosphorus, sulfur, and zinc [16]. As far as hemp protein concentrate is concerned, it has very interesting functional properties such as solubility, foaming, and water-holding and fat-holding capacities According to Galves et al. [17], hemp protein concentrates showed a slightly lower oil-holding capacity (OHC) and water-holding capacity (WHC) than other protein concentrates such as safflower, sunflower, and canola. It should be taken into account that protein structure and functionality can be influenced by the method of production. Processing can, therefore, lead to modifications in protein structure, in protein folding, and in the gelation capacity [18].
This greater availability of raw materials, together with their nutritional and functional properties, has resulted in both hemp flour and hemp protein concentrates being used for the manufacture of new products. Flour has been employed in bread [16], cookies [19,20], and gluten-free biscuits [21]. Hemp flour can be applied in both confectionery and bakery products, replacing wheat flour to create clean label products with a nutritional profile, a higher protein, and micronutrient content. Meanwhile, concentrates have also been used in meat products [22], yoghurts [23], and juices [24]. Vegan and vegetarian products with a declared content of up to 10% of hemp seeds are also available on the market [25]. Zahari et al. [22] showed that it is possible to replace soy protein with hemp protein in the meat analog formulation. In addition to their industrial application, these types of products have also reached the market for direct sale to consumers. Today, it is easy to buy hemp flour and hemp protein concentrates at markets and on the Internet. As they are completely gluten-free products, both hemp flour and hemp concentrates can be used in the nutrition of those affected by intolerances such as the most common celiac disease, replacing products made from cereals (wheat, barley, or rye) containing gluten.
In the scientific literature, articles can frequently be found in which hemp seed products have been characterized, and in which the influence of variety and the growing conditions on the composition of these products have been studied. However, the products which can be purchased by consumers do not reflect this situation. Therefore, the aim of this paper is to increase knowledge of hemp protein concentrates and flours in terms of nutritional composition and physicochemical and functional properties by comparing two brands of hemp flour and two brands of hemp protein. The article presents as a novelty the fact that the samples analyzed correspond to commercial samples which can be purchased by consumers.

2. Materials and Methods

2.1. Materials

Two flours and two protein concentrates made from hemp (Cannabis sativa) seeds were purchased by e-commerce. One of each of the samples, both the flour and the concentrate, were identified as being of organic production. All four samples were packaged by EU or UK companies.

2.2. Proximate Composition Analysis

Several parameters were determined in samples of hemp flours and concentrates according to AOAC methods (AOAC, 2006). The total nitrogen was determined by the Kjeldahl method (AOAC 950.36) and the total protein content (N × 6.25) was calculated. The total fat content was analyzed by the Soxhlet method (AOAC 935.38) using petroleum ether. The ash content was calculated (AOAC 923. 03) after incinerating at 550 °C. The enzymatic method described in AOAC 996.11 was used for the determination of starch content, while the gravimetric method (AOAC 925.10) was used for analyzing the moisture content, and the total fiber content was determined using an ANKOM analyzer (ANKOM technology, New York, NY, USA) (AOAC 991.43). The total amount of carbohydrates was then calculated ((100 − (ash + protein + total fat)). Lastly, the caloric intake was calculated considering that the energy supplied by protein and carbohydrates is 4 kcal/g and 9 g/kg for fats. All determinations were carried out in triplicate, and the results were expressed in g/100 g of dry weight (dw).

2.3. Fatty Acid Analysis

Fat extraction was performed with petroleum ether from 20 g of the sample. Subsequently, the samples were methylated with KOH (methanolic) according to the method described by Lurueña-Martínez et al. [26] using the GC 6890 N chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with an FID detector. One microliter of the sample was injected using the splitless mode of the injector and analyzed using a 100 m × 0.25 mm × 0.20 μm capillary column (SP-2560, Supelco, Inc., Bellefonte, PA, USA). Fatty acids were identified taking into account their retention times using as a reference a mix of 37 commercially available standards (47885-U Supelco, Sigma–Aldrich, Darmstadt, Germany). Measurements were repeated in duplicate, and the amount was expressed as a percentage of the total fatty acid methyl esters (FAMEs).

2.4. Amino Acid Analysis

The determination was carried out by liquid chromatography (HPLC) using a Biochrom 20 analyzer (Pharmacia, London, UK), with an ion-exchange column. Detection was performed post column after a reaction with ninhydrin. Quantification was carried out using external standards. Measurements were repeated in duplicate, and the amino acid content was expressed in g/100 g of fresh weight (fw).

2.5. Mineral Analysis

Analyses of the contents of the following minerals Na, Mg, P, K, Ca, Cr, Ni, Se, Cu, Zn, Mn, Fe, Cd, and Pb were performed by ICP-MS. Samples (0.2 g) were microwave digested in Teflon vessels using HNO3 in a Milestone system. The concentration of the minerals was determined using an Agilent 7800 ICP mass spectrometer (Agilent, Santa Clara, CA, USA) and the following conditions: 1550 W of reference power, 15 L/min of plasma air flow, 0.9 L/min of auxiliary air flow, and 0.99 L/min of nebulized air flow. Certified standard solutions (1 g/L) (Panreac, Madrid, Spain) solutions were used for the quantification of the elements which were grouped into two multi-elemental standards. Two repetitions per sample were carried out, and the mineral content was expressed in mg/kg of fw.

2.6. Bioactive Components and Antioxidant Activity

2.6.1. Extraction

Extracts were prepared in duplicate according to the method described by Betances Salcedo et al. [27]. First, 20 mL of 70% ethanol was added to 1 g of the sample, and the mixture was kept in an ultrasonic bath for 8 min. The samples were centrifuged at 3000× g for 20 min (20 °C). The supernatant was transferred to a volumetric flask and made up to 25 mL with 70% ethanol.

2.6.2. Total Phenolic Content Analysis

The total phenolic content (TPC) was quantified according to the method described by Millar et al. [28]. Thus, 0.5 mL of the extracted sample, 0.5 mL of Folin–Ciocâlteu reagent, 10 mL of Na2CO3 (7.5%), and 10 mL of H2O were added and mixed in a 25 mL volumetric flask. This was then made up to 25 mL with distilled water. The TPC was determined using a Shimadzu UV 1280 spectrophotometer (Shimadzu, Kyoto, Japan) at 750 nm, and the results were expressed in mg gallic acid equivalent (GAE)/100 g fw.

2.6.3. Total Flavonoid Content Analysis

For the determination of the total flavonoid content (TFC), the method used was that described by Valencia et al. [29], based on the formation of the aluminum chloride complex with slight modifications. In a 25 mL volumetric flask, 0.5 mL of AlCl3 (5%) and 2 mL of sample extract were added and mixed, and then made up with ethanol (96%). The samples were kept in the dark for 30 min. The measurement of the absorbance was carried out at 425 nm with a Shimadzu UV 1280 (Shimadzu, Kyoto, Japan). Analyses were performed in duplicate, and the results were expressed in milligrams of rutin/100 g fw.

2.6.4. Total Flavanone and Dihydroflavonol Content Analysis

An analysis of flavanones and dihydroflavonols was carried out using the method previously reported by Popova et al. [30] with slight modifications. Briefly, a volume of 1 mL of sample extract and 2 mL of DNP (2,4 dinitrophenylhydrazine) solution was mixed, heated for 50 min at 50 °C and cooled to room temperature. Then, KOH (10% in methanol w/v) was then added up to 10 mL. Absorbance measurement was performed at 486 nm with a Shimadzu UV 1280 (Shimadzu, Kyoto, Japan). Analyses were performed in duplicate, and the results were expressed in milligrams of pinocembrin/100 g fw.

2.6.5. Total Antioxidant Capacity Analysis

The total equivalent antioxidant capacity (TEAC) was determined by the method described by Chen et al. [31]. This method is based on monitoring the decrease in absorbance at 734 nm observed upon scavenging the ABTS (2,2-azinobis (3-ethylenebenzothiazoline-6-sulphonic acid) radical cation. After preparation of the ABTS radical cation solution, an appropriate amount of this reagent was mixed with 20 µL of the extract sample and the absorbance was measured at 734 nm for 10 min (Shimadzu UV 1280, Shimadzu, Kyoto, Japan). The percentage inhibition of absorbance was calculated and plotted against Trolox (6-hydroxy-2,5,7,8-tetramethylchorman-2-carboxylic acid) concentration used as a standard. The total equivalent antioxidant capacity (TEAC) was then calculated. Each sample was analyzed in duplicate, and the results were expressed as nmol Trolox/100 mg fw.

2.7. Determination of Techno-Functional Properties

2.7.1. Water-Holding Capacity, Oil-Holding Capacity, and Water Solubility Index

The water-holding (WHC) and oil-holding (OHC) capacities of the hemp flours and protein concentrates were determined using the method described by Osen et al. [32] and Bora et al. [33], respectively. To carry out the determination of WHC, 0.5 g of the sample and 10 mL of distilled water was mixed, vortexed for 30 s, and kept for 24 h at 20 °C. Subsequently this mixture was centrifuged (20 min, 3000 rpm, Sigma-Aldrich 4K15C, Darmstadt, Germany). The WHC was expressed as the number of grams of water retained per 1 g of flour. For the determination of the OHC, 0.4 g of the sample and 5 mL of sunflower oil were mixed following the same procedure as described for determining the WHC. OHC was expressed as the number of grams of oil retained per 1 g of flour.
The water solubility index (WSI) was measured as the soluble solids present in the supernatant liquid from the WHC analysis with a Portable Refractometer SERIE 300, ZUZI (Microscopios, Barcelona, Spain) and expressed as Brix.

2.7.2. Swelling Capacity

The swelling capacity (SC) was determined by mixing 0.5 g of the sample and 5 mL of distilled water, and then stirring the mixture for 1 min with a vortex. After 24 h at room temperature, the final volume of the samples was measured. This was carried out triplicate, and the results were expressed as a volume increase after 24 h per g of sample.

2.7.3. Gel Formation

The gelation properties were assessed using the methodology proposed by Tomé et al. [34] with slight modifications. To prepare the gels, 6 g of the sample and 3 g of sodium chloride were mixed with 30 mL of distilled water. The suspensions were adjusted to pH 5. After 30 min heating of the samples in boiling water at 90 °C, the gels formed were left at 4 °C for 24 h. A penetration test with a 10 mm probe was performed in a Texture Analyzer TX-T2iplus (Stable Micro Systems, Surrey, UK) with a 5 kg load cell at 20 °C. A distance penetration of 8 mm and a cross-head speed of 1 mm/s were used. The mean maximum force from three replicates was recorded.

2.7.4. Foaming Capacity

In order to determine the foaming capacity (FC), 5 g of the samples were placed in a 600 mL beaker, and 100 mL of distilled water was added before mixing, with a blender (Ultraturrax T 25 basic, Staufen, Germany). The foam capacity (FC) was expressed as the percentage increase in foam volume measured after 2 min. The foam stability (FS) was determined by measuring the FC after standing for 5 min.

2.7.5. Color Properties

The value of flours and concentrates was measured using HunterLab MiniScan colorimeter of the XE Plus model (Hunterlab, Reston, VA, USA). The CIELab parameters determined were L* (lightness), a* (redness), and b* (yellowness) using an observer angle of 10° and the illuminant D65. The L* value represents the lightness of a sample from black to white. The a* (−green to + red) and b* (−blue to + yellow) values represent the chromatic aspects of a sample. Nine replicates were made from each sample.

2.8. Statistical Analysis

The mean values were compared using one-way ANOVA followed by Tukey’s post hoc test with α = 0.05 to determine the existence of significant differences between samples. The correlation coefficients between the different physicochemical and techno-functional parameters of the samples were calculated using Pearson’s correlation coefficient. All statistical analyses were carried out using SPSS Statistics for Windows, version 27.0 (IBM Corp., Armonk, NY, USA).

3. Results and Discussion

3.1. Proximate Composition

The proximate composition results (Table 1) showed that carbohydrates and fiber were the most important compounds in the flours analyzed. For the concentrates, the most important compounds were proteins; they had a lower carbohydrate and fiber content than the flours. The percentage of fat ranged between 9.93% and 12.31%. Although hemp seed has a fat content of 30% fat [35]; however, the production of flours and protein concentrates from hemp occurs after defatting the seed, which is why a low percentage of fat was found in the samples analyzed. The starch content was between 0.82% and 1.11%, with one of the flour samples showing contents below the detection limit. The ash content was higher in the concentrates than in the flours with mean values of 6.68% for the flours and 8.48% for the concentrates. In terms of energy (kcal/100 g), there are no significant differences between flours and concentrates. No statistically significant differences in moisture content were found between the different samples analyzed with a mean value of 6.26%.
The nutritional composition obtained in our samples was similar to that described in the literature for hemp flours [16] and for hemp protein concentrates [36]. The two commercial brands analyzed showed differences between them; in the case of the flours, the differences were significant for all the parameters analyzed. Organic flour had higher contents of carbohydrates, fiber, and ash, but lower contents of fat, protein, and starch. Furthermore, Rusu et al. [15] showed that the variety of hemp also influences the composition of the flour obtained, especially its macro- and microelement content and the content of individual carbohydrates (sucrose, fructose, and glucose). In the literature, flours with whole fat content and defatted flours can be found. Non-fat flours have lower concentrations of protein, starch, fiber, and ash [15]. This fact can lead to confusion among consumers when comparing different flours, as not all commercial brands indicate whether the flour is defatted or not.
In the case of the concentrates, although the carbohydrate, fiber, and fat contents showed no significant differences between the two brands; however, the contents of protein and starch were significantly lower in the organic concentrate. This was also observed in the organic flours. The nutritional composition of protein concentrates can be highly variable depending on the process used to extract them. Thus, the application of an isoelectric precipitation process causes a reduction in the ash content [37], a dry fractionation process affects the carbohydrate contents, and a combined treatment of alkaline extraction and isoelectric precipitation influences the protein content [36]. This highlights the great variability that consumers may find within hemp protein concentrate.

3.2. Fatty Acid Profile

According to the results (Table 2), the most abundant fatty acid in hemp flours and concentrates was linoleic acid (C18:2 n6), with levels ranging from 30.65% to 56.38%. This fatty acid was followed in hemp flours by linolenic acid (C18:3n3), reaching 15.03% to 16.69% in the same samples, while the third most important fatty acid was oleic acid (C18:1) with values between 10.68% and 13.50%. However, for the protein concentrate samples, the C18:1 fatty acid was the second most important, with levels ranging from 20.58% to 23.09%, followed by palmitic acid (C16:0) with values of between 17.23% and 21.50%.
Therefore, in terms of the C16:0, C18:0, and C18:1 fatty acids, the protein concentrates showed a significantly higher value compared with hemp flour samples. However, C18:2 n6 and C18:3 n3 had higher concentrations in the hemp flours. Owing to these differences, the concentrations of SFAs, MUFAs, and n6/n3 were higher in the protein concentrates, and this was also the case for the PUFAs in the hemp flours. The n-6/n-3 ratio is considered to be an important nutritional index. The average n-6/n-3 ratio found was 3.65 for flours and 6.21 for concentrates, which indicates a good nutritional index for the hemp by-products studied. Rusu et al. [15] similarly reported that PUFAs are the predominant fatty acids in hemp flours followed by MUFAs. For hemp protein concentrates, no studies were regarding their fatty acid composition.
Hemp seeds are rich in polyunsaturated fatty acids, which also vary among different genotypes. In addition, the fatty acid composition of hemp seed is influenced by several factors, including the origin of the seeds, as well as growth and climate conditions [38]. However, the results from this study showed no significant differences between the two samples analyzed; only the C18:3 n6 fatty acid showed a large difference between the two flours analyzed, with higher values for the conventional flour.

3.3. Amino Acid Profile

With regard to the amino acid content (Figure 1), all the essential amino acids were found in both the flours and the hemp protein concentrate. The high amounts of glutamic and arginine stand out. Significant amounts of sulfur-containing amino acids (methionine and cysteine) were also found. These results agree with those obtained by other authors reporting the presence of all essential amino acids in both hemp flour and hemp protein concentrates [39,40]. Glutamic acid, aspartic acid, and arginine have also been described as the predominant amino acids in hemp by other authors [41,42]. Tryptophan was the amino acid present in the lowest concentration in all the samples analyzed in this study, which agrees with the results obtained by Teh et al. [43] and Hadnađev et al. [44]. The amino acid profile found in both flours and concentrates confirms that these products are an excellent source of protein. Hemp protein is also classified as a source of high-quality protein comparable to that of soybean or egg white [45]. Furthermore, previous studies [46] showed that these proteins have excellent digestibility, with a very high protein digestibility corrected amino acid score (PDCAAS) value.
As far as the commercial brands are concerned, no statistically significant differences were found in the contents of any of the amino acids for hemp flours. However, the organic protein concentrates showed lower amounts of all essential amino acids (EAAs) and branched-chain amino acids (BCAAs), which is consistent with their lower protein content (Table 1). The differences found between the two commercial brands could also be linked to the manufacturing process of the concentrates. The extracting process for obtaining the protein concentrate may affect the concentration of amino acids, especially EAAs and BCAAs, as reported by Shen et al. [42].

3.4. Mineral Content

The most abundant mineral in both the flours and the protein concentrates was K, followed by P and Mg, and the microelements present in the highest concentrations were Mn and Fe (Table 3). Despite having a similar mineral profile, the protein concentrates had a higher concentration of minerals than the flours. Only sodium and chromium were present in lower amounts in the concentrates. Iron, nickel, and magnesium showed differences between commercial brands but none associated with being a flour or concentrate. The process of the production of the protein concentrates has a major influence on the mineral content. Thus, concentrates obtained by a dry fractionation process show a lower sodium content compared with wet fractionation [36].
The results obtained agree with those described by Rusu et al. [15] for hemp flours, with K being the main mineral, and with high amounts of Ca, Mg, and P also being found in all the flours analyzed, regardless of the variety of hemp. Likewise, in protein concentrates, the mineral profile described by Nasrollahzadeh et al. [36] showed high amounts of the macroelements K, P, and Mg, with a connection being noted between the mineral content and the extraction process of concentrates. The high levels of minerals such as Fe and Zn described in hemp have led to it being used for the fortification of wheat flour. Some hemp varieties, such as Delores and Joey, have Fe concentrations exceeding 50% of the recommended daily intake (RDI); the Joey variety also contributes 30.03% RDI in the case of zinc [47].
The K/Na ratio was very high in all samples, especially in the concentrates, owing to their low Na content. This ratio is very interesting from a nutritional point of view owing to its cardioprotective effect [48].
On the other hand, the microelements Ni, Cr, and Se were present in smaller quantities in all the samples, with Se being found only in protein concentrates. Similar results were described for hemp flour [15] and for protein concentrates [39], with Cr and Ni being the microelements present in lower amounts.
Regarding the two commercial brands analyzed, the flours showed statistically significant differences in the contents of all the minerals analyzed except for K. The concentrates showed no differences in K content or in the Na, Cr and Se concentrations between the two concentrates. Among all the minerals analyzed, Ni showed the greatest variability between samples. Several studies have shown that the mineral composition of hemp seeds is influenced by various parameters such as environmental and climatic conditions and soil fertilization [49]. However, Lan et al. [47] found that the contents of P, Mg, and Se were not significantly different among the varieties, which suggests that they could be variety-independent. It is noteworthy that organic hemp by-products, both flours and protein concentrates, showed significantly higher amounts of Mg, P, Ca, Cu, Zn, and Fe. This result agrees with previous studies which reported that the micronutrient content was higher for organic vegetables and legumes compared to their conventional counterparts [50]. Therefore, previous studies have shown that organic vegetables have higher contents of Mg and Fe [51] and Ca, Cu, and Zn [52].

3.5. Phenolic Composition

Significant differences between flours and protein concentrates could be observed in the total flavonoid content (TFC), whereby concentrates showed higher values (77.11 mg rutin/100 g fw) than flours (62.44 mg rutin/100 g fw). The total phenolic content (TPC) and flavanone and dihydroflavonol content (F/DF) showed no differences between flours and concentrates (Table 4).
Concentrations found in previous studies for phenolic compounds range from 79.0 to 662 mg GAE/100 g for hemp protein concentrates [53]. For flours, values from 74.4 to 468 mg GAE/100 g have been described [54]. This wide variability in concentrations can be attributed to the different composition of different hemp varieties [11]. Hemp seeds are rich in several phenolic acids, lignamides, phenolic amides, and flavonoids [55]. In hemp flour samples, the most abundant phenolic compounds found were hydroxycinnamic and protocatechuic acids [56], while flavanols (catechin and epicatechin), isoflavonoids (biochanin A), and flavonoids (quercetin and tyrosol) have also been described [57].
The total antioxidant activity (TEAC) was higher in the flours (3.6 nmols Trolox/100 g) than in the concentrates (2.44 nmols Trolox/100 g). Lanzoni et al. [58] found antioxidant activity in hemp seed products; they described an antioxidant activity similar to that of soy for protein concentrates. This activity seems to depend on the type of protease, the degree of hydrolysis, and the processing conditions of the concentrate [41]. The hydrolysis process that fragments proteins to release bioactive peptides appears to be one of the most influential factors [48].
Regarding the different commercial brands, only the TPC showed differences, with that of organic flour being higher (79.53 mg GAE/100 g). Previous studies have found that food products deriving from organic agriculture have a similar or slightly higher polyphenol content and antioxidant capacity [59]. In all the other bioactive compounds, no significant differences were observed in either flours or concentrates. Alu’datt et al. [60] suggested that phenolic compounds are closely associated with proteins, lipids, and polysaccharides via non-specific or covalent interactions. These interactions would explain why there are hardly any differences between the phenolic content of the flours and the concentrates analyzed. Potin et al. [61] showed that protein concentrates obtained by ultrafiltration had a slightly lower phenolic content than flour, despite the protein enrichment carried out, which confirms the binding of these compounds with other fractions of their composition.

3.6. Techno-Functional Properties

The water-holding capacity (WHC) showed values of between 2.25 g/g and 3.10 g/g, with values being lower in the flour samples than in the protein concentrates (Table 5). The oil-holding capacity (OHC) did not show significant differences between flours and concentrates or within commercial brands, with values ranging from 0.88 g/g to 1.26 g/g. No significant differences between brands were observed in the case of flours; however, the organic concentrate showed a lower WHC. The water-holding capacity is related to the taste and texture of foods, while the oil-holding capacity has been related to the emulsifying capacity of foods [62]. Nasrollahzadeh et al. [36] described WHC values as being slightly lower for protein concentrates between 1.3 g/g and 1.8 g/g. Protein denaturation is positively correlated with WHC [32], which could explain why flours have a lower WHC than concentrates owing to the production process. In relation to the OHC, Zayas [63] reported that native proteins show a lower capacity than their denatured counterparts owing to their structural folding, which means that flours should show a lower OHC. In our samples, the WHC showed a positive correlation with the concentration of the minerals Mg (r2 = 0.994) and K (r2 = 0.998). However, the OHC showed a negative correlation with the MUFA (r2 = −0.995) and ash (r2 = −0.992) content, and a positive correlation with the PUFA content (r2 = 0.991).
The solubility (WSI) of the hemp flours showed a large difference between the two commercial flour brands tested. However, the protein concentrates showed no differences between the two brands. Previous studies reported WSI values of 9.37 g/100 g for hemp flours [64] and 35.60% for hemp concentrates [65]. The difference from the data found in this research is due to the analysis methodology used in the different studies. The WSI is strongly related to the partial hydrolysis of proteins owing to the increase in low-molecular-weight peptides, which means that values of around 30% of protein hydrolysis give the highest values of WSI [66]. In our samples, the solubility index showed a positive correlation with the starch content (r2 = 0.982). These results agree with those previously reported by Di Cairano et al. [67], who pointed out that the WSI can be used as an indicator of starch degradation. On the other hand, WSI could also depend on the different proportion of albumin fraction in the samples, as albumins have a higher solubility than globulins owing to the lower level of some amino acids (aromatic and hydrophobic) and the less rigid conformational structure [36].
The swelling capacity of flours and concentrates is related to their ability to generate greater volume when incorporated into a food, and their value depends on temperature [68]. In this study, the SC was determined at room temperature, and the highest values were obtained from hemp flour especially from the organic sample. In relation to the foaming capacity (FC), the protein concentrates showed a higher FC than flours. As for the foaming stability, flours were capable of maintaining around 40% of the foam formed after 5 min, while, in the case of the concentrates analyzed, this value rose to 44.93% for the organic concentrate and 46.30% for the conventional one. There were no differences between commercial brands for the flours, but there were differences for the concentrates, in which the organic concentrate showed a greater capacity to form foam, but less foam stability. Shen et al. [42] found a higher foaming capacity in concentrates with a higher protein concentration, which would explain the differences between concentrates and flours in this study. This foaming capacity has also been related to factors such as the solubility and surface hydrophobicity of the proteins [42]. In our samples, the highest protein concentration was found in the non-organic concentrate, which did not show higher foam formation. This may be due to the fact that the production process also has a major influence on this property, as enzymatic hydrolysis leads to a reduction in the foaming capacity of hemp concentrates [69]. In our samples, FC showed a positive correlation with the C16:0 fatty acid concentration (r2 = 0.994), and FS showed a positive correlation with the C20:0 fatty acid concentration (r2 = 0.999).
As far as the hardness of the gels formed is concerned (Table 5), the protein concentrates were harder, and there were no statistically significant differences between the different commercial brands. The gelling capacity depends on both internal factors (protein composition or concentration) and external factors (pH, temperature, etc.) [18]. The protein extraction process has a strong influence on the gelling capacity [42]. Thus, the formation of high levels of aggregates during concentrate production reduces the gel-forming capacity, increasing the amounts of protein molecules which are necessary for the formation of a gel network [70]. In our samples, the strength of the gel formed at pH 5 showed a positive correlation with protein concentration (r2 = 0.963) and with the concentration of the amino acid aspartate (r2 = 0.995).
In relation to the color of the samples (using CIE L*a*b* color measurements) no differences were observed between flours and concentrates in the L* and a* parameters. The b* parameter showed lower values in the flours than in the concentrates. In relation to the color, organic flours were characterized by a more pronounced greenish-yellow color (lower a*, higher b*) when compared with the conventional ones. The deeper greenish hue of hemp protein concentrates may be due to the presence of chlorophyll, which is co-extracted during the production process [42]. For this reason, some concentrates are decolored after extraction to remove the green color [71]. Furthermore, oxidation of phenolic compounds gives rise to quinones, which react with the amine groups of proteins, resulting in dark-colored adducts [72]. This means that the extraction process has a major influence on the color of protein concentrates [36]. No significant correlations were found between the color parameters and any of the other parameters analyzed.

4. Conclusions

Both the flours and the protein concentrates analyzed in this study meet current European labeling regulations. Hemp flours and hemp concentrates had a similar energy intake for the consumer; however, their composition showed differences. Carbohydrates were, thus, the main component of the flours, which essentially consisted of fiber; flours also showed a high antioxidant activity with a high concentration of total phenolic compounds. In the case of concentrates, the carbohydrate content was reduced by half, and the amount of protein increased. Hemp flours and concentrates were marketed partially defatted, with a 50% reduction in the fat content of the unprocessed hemp seeds, such that both flours and concentrates had low fat values. This fat was mainly composed of polyunsaturated fatty acids (linoleic and linolenic acids). Both flours and concentrates had an excellent amino acid profile, with the presence of all essential amino acids in large amounts. Both in the flours and in the concentrates, mineralization stood out in terms of its high K and P content. On the basis of the results obtained, hemp flour could be of great interest for application in the fortification of bakery products and breads. They offer an interesting nutritional composition, in addition to bioactive compounds, while showing an excellent swelling capacity. Protein concentrates showed higher WHC, FC, FS, and GF than the flours. For this reason, protein concentrates appear to be very interesting products for application in meat products and sauces. Organic products showed an influence on the nutritional composition, with lower concentrations of proteins and starch but a higher micronutrient content and total phenolic composition. However, no differences were found in the profile of the fatty acids, amino acids, and techno-functional properties. On the basis of these results, organic flours seem to be a good alternative to be included in the diet of consumers. In those cases where a higher protein intake is necessary, especially in elderly people or athletes, organic hemp concentrate could be a very suitable product.

Author Contributions

Conceptualization and methodology, A.M.V.-Q. and I.R.; formal analysis, Y.A.; data curation, Y.A.; writing—original draft preparation, Y.A. and A.M.V.-Q.; writing—review and editing. A.M.V.-Q. and I.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be made available on request.

Acknowledgments

Y. Absi is grateful to the Algerian government for the grant supporting a long-term residential doctoral program abroad.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 13 10130 g001
Figure 1. Amino acid profile of different hemp flours and protein concentrates. EAAs: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. BCAAs: valine, isoleucine, and leucine.
Figure 1. Amino acid profile of different hemp flours and protein concentrates. EAAs: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. BCAAs: valine, isoleucine, and leucine.
Applsci 13 10130 g001
Table 1. Mean values (±standard deviation) of the proximate composition parameters of different hemp flours and protein concentrates (expressed in g/100 g dw).
Table 1. Mean values (±standard deviation) of the proximate composition parameters of different hemp flours and protein concentrates (expressed in g/100 g dw).
Hemp FloursHemp Protein Concentrates
OrganicConventionalOrganicConventional
Carbohydrates51.75 ± 0.01 c47.07 ± 0.07 b34.05 ± 0.23 a33.38 ± 0.46 a
Fiber53.56 ± 0.47 c40.90 ± 0.99 b25.31 ± 0.12 a25.50 ± 0.08 a
Proteins31.34 ± 0.13 a34.41 ± 0.41 b46.33 ± 0.40 c47.72 ± 0.15 d
Fat9.93 ± 0.01 a12.31 ± 0.03 c10.87 ± 0.12 b10.69 ± 0.13 b
Starch<0.10 *0.89 ± 0.11 a0.82 ± 0.03 a1.11 ± 0.03 b
Ash6.92 ± 0.02 b6.44 ± 0.03 a8.76 ± 0.08 d8.21 ± 0.22 c
Energy (kcal/100 g)398.32 ± 0.2 a415.40 ± 0.4 a419.33 ± 0.43 a420.63 ± 0.65 a
a–d Values in the same row followed by different letters are significantly different (p < 0.05). * Values under the detection limit of the analytical method applied.
Table 2. Mean values (±standard deviation) of the fatty acids of different hemp flours and protein concentrates (expressed in g/100 g of FAME).
Table 2. Mean values (±standard deviation) of the fatty acids of different hemp flours and protein concentrates (expressed in g/100 g of FAME).
Hemp FloursHemp Protein Concentrates
OrganicConventionalOrganicConventional
C16:06.95 ± 0.04 a8.57 ± 0.24 a21.50 ± 3.38 b17.23 ± 0.75 b
C16:10.11 ± 0.00 a0.21 ± 0.01 a0.26 ± 0.01 a0.34 ± 0.18 a
C18:03.15 ± 0.01 a2.15 ± 0.43 a7.90 ± 0.97 b6.25 ± 0.54 b
C18:1 n9t0.02 ± 0.00 a0.83 ± 0.41 ab1.90 ± 0.24 b2.36 ± 0.77 b
C18:113.50 ± 0.06 a10.68 ± 1.16 a23.09 ± 0.89 b20.58 ± 2.67 b
C18:2 n656.38 ± 0.05 b52.29 ± 2.28 b30.65 ± 6.69 a35.06 ± 3.34 a
C20:00.89 ± 0.01 a0.71 ± 0.19 a2.12 ± 0.24 a2.78 ± 1.33 a
C18:3 n60.67 ± 0.00 a6.68 ± 0.61 b0.15 ± 0.05 a0.91 ± 0.95 a
C18:3 n316.69 ± 0.06 b15.03 ± 1.53 b4.52 ± 1.10 a5.63 ± 0.15 a
SFA11.64 ± 0.08 a12.06 ± 1.19 a34.25 ± 5.34 b28.57 ± 0.73 b
MUFA14.32 ± 0.04 a12.66 ± 1.71 a27.54 ± 0.91 b25.85 ± 0.16 b
PUFA74.05 ± 0.12 b75.27 ± 2.91 b38.20 ± 6.25 a45.59 ± 0.891 a
n316.88 ± 0.06 b15.29 ± 1.44 b5.18 ± 0.51 a6.44 ± 0.10 a
n657.16 ± 0.06 b59.98 ± 1.47 b33.02 ± 5.74 a39.15 ± 0.79 a
n6/n33.38 ± 0.01 a3.93 ± 0.27 a6.34 ± 0.47 b6.08 ± 10.03 b
a,b Values in the same row followed by different letters are significantly different (p < 0.05).
Table 3. Mean values (±standard deviation) of the mineral compounds of different hemp flours and protein concentrates (expressed in mg/kg fw).
Table 3. Mean values (±standard deviation) of the mineral compounds of different hemp flours and protein concentrates (expressed in mg/kg fw).
Hemp FloursHemp Protein Concentrates
OrganicConventionalOrganicConventional
Na641.4 ± 6.6 b846.1 ± 5.6 c42.35 ± 0.5 a35.72 ± 0.5 a
Mg5255.2 ± 69.0 b4702.0 ± 149.6 a8946.1 ± 28.2 d7849.2 ± 33.4 c
P10717.6 ± 24.5 b9099.1 ± 141.1 a15814.8 ± 104.3 d13179.8 ± 109.3 c
K10686.2 ± 174.4 a10419.2 ± 144.3 a16574.9 ± 42.9 b14878.1 ± 73.8 b
Ca1776.8 ± 17.9 b1290.4 ± 0.7 a2329.4 ± 13.8 d2072.1 ± 1.45 c
Cr0.3 ± 0.01 b1.0 ± 0.02 c0.1 ± 0.00 a0.1 ± 0.01 a
Ni3.4 ± 0.03 b16.1 ± 0.08 d6.4 ± 0.02 c1.4 ± 0.02 a
SeNDND0.2 ± 0.00 a0.2 ± 0.03 a
Cu18.9 ± 0.9 b14.9 ± 1.1 a21.3 ± 0.0 b15.4 ± 0.5 a
Zn76.9 ± 0.4 c48.1 ± 0.8 a78.4 ± 0.4 c62.9 ± 0.5 b
Fe142.2 ± 0.6 b102.6 ± 1.2 a244.6 ± 1.1 d197.2 ± 0.2 c
Mn166.6 ± 0.9 a204.0 ± 2.5 b220.0 ± 6.4 c173.4 ± 4.6 a
ND: not detected. a–d Values in the same row followed by different letters are significantly different (p < 0.05).
Table 4. Mean values (±standard deviation) of the phenolic composition and antioxidant capacity of different hemp flours and protein concentrates.
Table 4. Mean values (±standard deviation) of the phenolic composition and antioxidant capacity of different hemp flours and protein concentrates.
Hemp FloursHemp Protein Concentrates
OrganicConventionalOrganicConventional
TPC (mg GAE/100 g79.53 ± 2.81 b66.42 ± 0.72 a55.32 ± 0.03 a69.14 ± 5.12 ab
TFC (mg rutin/100 g)58.24 ± 4.05 a66.65 ± 0.09 a72.12 ± 0.12 b83.10 ± 12.24 b
F/DF (mg pinocembrin/100 g)620.31 ± 6.19 a580.62 ± 37.71 a768.75 ± 6.15 b656.14 ± 4.75 ab
TEAC (nmol Trolox/100 g)3.45 ± 0.09 b3.75 ± 0.87 b2.35± 3.01 a2.54 ± 9.05 a
a,b Values in the same row followed by different letters are significantly different (p < 0.05). TPC: total phenolic content, TFC: total flavonoid content, F/DF: flavanones and dihydroflavonols, TEAC: total equivalent antioxidant activity.
Table 5. Mean values (±standard deviation) of techno-functional properties of commercial hemp flours and hemp protein concentrates.
Table 5. Mean values (±standard deviation) of techno-functional properties of commercial hemp flours and hemp protein concentrates.
Hemp FloursHemp Protein Concentrates
OrganicConventionalOrganicConventional
WHC (g/g)2.27 ± 0.12 a2.25 ± 0.07 a2.92 ± 0.01 b3.10 ± 0.06 c
OHC (g/g)1.19 ± 0.08 ab1.26 ± 0.17 ab0.95 ab ± 0.13 a0.88 ± 0.12 a
SC (%)2.49 ± 0.32 c2.11 ± 0.01 b0.92 ± 0.02 a0.95 ± 0.06 a
WSI (°Brix)4.3 ± 0.12 a9.0 ± 0.10 b9.0 ± 0.01 b8.3 ± 0.06 b
FC (%)70.35 ± 7.67 a69.81 ± 4.92 a110.14 ± 2.51 c85.71 ± 4.76 b
FS (%)29.25 ± 0.27 a27.04 ± 4.81 a44.93 ± 2.51 b39.69 ± 2.75 c
GF (N)71.64 ± 14.75 a74.32 ± 13.73 a293.13 ± 109.29 b416.49 ± 136.14 b
L*43.92 ± 2.47 c34.39 ± 3.30 a44.03 ± 0.52 c40.21 ± 0.87 b
a*1.83 ± 0.11 a3.22 ± 0.15 c2.38 ± 0.11 b3.42 ± 0.11 d
b*24.46 ± 1.21 b20.30 ± 1.36 a27.65 ± 0.34 c27.01 ± 0.36 c
WHC: water-holding capacity, OHC: oil-holding capacity SC: swelling capacity, WSI: water solubility index, FC: foaming capacity, FS: foaming stability, GF: gel formation (hardness of the gel formed at pH = 5). L*: lightness, a*: redness, and b*: yellowness. a–d Values in the same row followed by different letters are significantly different (p < 0.05).
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MDPI and ACS Style

Absi, Y.; Revilla, I.; Vivar-Quintana, A.M. Commercial Hemp (Cannabis sativa Subsp. sativa) Proteins and Flours: Nutritional and Techno-Functional Properties. Appl. Sci. 2023, 13, 10130. https://doi.org/10.3390/app131810130

AMA Style

Absi Y, Revilla I, Vivar-Quintana AM. Commercial Hemp (Cannabis sativa Subsp. sativa) Proteins and Flours: Nutritional and Techno-Functional Properties. Applied Sciences. 2023; 13(18):10130. https://doi.org/10.3390/app131810130

Chicago/Turabian Style

Absi, Yamina, Isabel Revilla, and Ana M. Vivar-Quintana. 2023. "Commercial Hemp (Cannabis sativa Subsp. sativa) Proteins and Flours: Nutritional and Techno-Functional Properties" Applied Sciences 13, no. 18: 10130. https://doi.org/10.3390/app131810130

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