Hemp seed oil is well-known for its health benefits because of its high polyunsaturated fatty acid (PUFA) content, especially linoleic acid (LA) ω-6 and α-linolenic (ALA) ω-3 acid [15,16,17]. The cold-pressed technique is commonly used to extract commercial hemp oil from seeds, leaving almost half of the total biomass as by-products commonly known as hemp cake or hemp meal. Hemp seed hulls are another hemp by-product obtained during the hulling process that produces hemp seed hearts, which can be consumed raw or cooked with other food. A number of studies have been conducted on hemp cake for its chemical composition and protein content [3,18,19], but no systematic study has been conducted on hemp seed hulls for their chemical characterization. The current study focuses on in-depth lipid analysis of hemp by-products, especially hemp cakes and hemp hulls, for their possible aquafeed application. Total lipid content of both hemp cake and hemp hulls was determined together with the oil extraction yield using hexane as extracting solvent. Whole hemp seeds and hemp hearts together with cold-pressed commercial hemp oils were also studied simultaneously for comparison.

The lipid content of whole hemp seeds (48.0%) was slightly lower than hemp hearts (54.7%) because of the hull’s presence, which has 17.5% total lipid. Even though the hemp cake was collected after cold-pressed oil extraction, it still contained 13.1% total lipid, similar to the previous study by Pojic et al., 2014 [3]. The hemp meal fractions reported to have oil content varied from 8.26 to 18.6% depending on the partial size. In the current study, we have used hexane as extracting solvent because it has less toxicity, is easy to evaporate and is widely used in food industries for oil extraction. The results suggested that hemp cake has the lowest hexane extractable oil, yielding only 7.4% of the biomass, which is almost half of the total lipid content (13.1%). The oil extraction yield of hemp seed hulls, hemp hearts and hemp whole seeds was also lower than the total lipid content (Table 1). Lower oil extraction yield suggested there should be polar lipids such as glycolipids or phospholipids in the residual biomass after hexane extraction; further study is needed to characterize such lipids in hemp seeds and in hemp seed by-products. Solid phase extraction (SPE) technique with silica gel cartridge was further used to determine the percentage of various classes of lipids present within the oil extracted by hexane [13]; the SPE results suggested that oils (hexane extract) extracted from hemp by-products are primarily composed of neutral lipids, which comprise >97.1% of the oil (Table 1). The proton NMR signals in their respective spectra displayed peaks only belonging to TAGs (Figure S1), i.e., either fatty acid acyl side chain or glyceride moiety [14]. The NMR spectral signals clearly demonstrated that the oil extracted either from by-products or hemp seeds is made up of mainly triglycerides (TAGs).

Hemp seed and hemp seed meal have been well-studied for their fatty acid composition; results suggested that LA, ALA, oleic acid, γ-linolenic acid and palmitic acid are the main fatty acids present in hemp seed or hemp meal oil [7,8,17,20]. LA (C18:2 n-6) is the major fatty acid that covers more than 50% of the total fatty acids and its percentage varies depending upon the extraction methods and cultivars. Oomah et al. (2002) reported 53.4–56.6% of LA in hemp seed oils extracted with petroleum ether using the Soxhlet extraction method [21]. Similarly, 58% LA was reported in hemp seed oil extracted with supercritical carbon dioxide (CO₂) [20] and 55.2–54.8% LA was reported in hemp seed meal fractions [3]. In the current study, LA content in the hemp by-products oils was 54.9–55.9% of the total fatty acids. α-linolenic acid (ALA) was the second major fatty acid in the oil extracted from hemp by-products, covering 16.7–18.4% of the total fatty acids. Longer chain fatty acids, i.e., C:20 or higher, were also detected but their concentration was significantly lower than LA and ALA. Moreover, PUFA ω-6 covers almost 60% and PUFA ω-3 covers only 18% of the total fatty acid count in the oil derived from hemp by-products. Almost identical results were observed for the oil derived from hemp hearts, hemp seeds and cold-pressed commercial hemp oil.

Dietary lipids play important roles as a source of energy and essential fatty acids necessary for fish growth and development [22]. There is urgent need for alternative sources of protein and lipid diet in the aquaculture industry because of increasing demand for aquaculture products, especially fish [9,10]. In general, freshwater fish require either LA (18:2 ω-6), ALA (18:3 ω-3) or both, whereas marine fish require long chain fatty acids, eicosapentaenoic acid (EPA, 20:5 ω-3) and/or docosahexaenoic acid (DHA, 22:6 ω-3) [23]. Oils extracted from hemp by-products, namely hemp cake and hulls, contain high levels of LA and ALA, obviously suitable for freshwater aquafeed application. Extensive research has been conducted to study the effects of alternative lipid sources, mainly vegetable oils, on the growth of both marine and freshwater aquatic animals [24,25]. Even though no growth performance advantage was observed in juvenile Tor tambroides with LA/ALA treatment against palm oil which is mainly composed of saturated fatty acids [26], significantly higher concentrations of ω-3 PUFA were found in the muscle of the LA/ALA feed diet group [27]. Studies on Atlantic salmon suggested that partial replacement of fish oil by vegetable oils (40–60%) can produce similar results as diets containing 100% fish oil during the grow-out phase of Atlantic salmon in the sea and does not significantly affect ω-3 fatty acid composition in muscle when feeding fish with a diet containing low levels of fish meal and moderate levels of fish oil [28,29], suggesting the use of vegetable oil having little or no EPA/DHA for marine aquafeed application. In another study, Xue et al. (2006) observed that the fatty acid composition of fish fillets and livers reflected the dietary fatty acid composition when studying six alternative lipid sources in Japanese sea bass (Lateolabrax japonicas) [30]. Sankian et al. (2019) demonstrated that the complete replacement of fish oil is even possible in mandarin fish diets [31]. These studies clearly demonstrate vegetable oils are already starting to replace fish oil either partially or fully in fish feed diets. Soybean is one of the well-studied vegetable oils for aquafeed, having high levels of PUFA LA and ALA, containing 55% and 13%, respectively [32]. Oil derived from hemp by-products has a slightly better ω-3 profile than soybean oil because it has higher ALA (18%). Possible use of hemp seed by-products oil for aquafeed application not only helps in managing the waste biomass, it also gives economic value for emerging hemp industries around the world. In addition, stearidonic acid (SDA), which is also present in hemp by-products, is an intermediate fatty acid in the biosynthetic pathway from α-linolenic acid to EPA/DHA, and the conversion from SDA is more efficient than from α-linolenic acid [33]. The ω-6/ω-3 fatty acid ratio is another factor which plays an important role in health benefits for both humans and aquatic animals. Human beings evolved on a diet with a ratio of ω-6/ω-3 essential fatty acids of 1 whereas in Western diets the ratio is 15/1 to 16.7/1; a lower ratio of ω-6/ω-3 is needed for the prevention and management of chronic diseases [34]. A study suggested that an increase in the dietary ω-6/ω-3 ratio was reported to influence the growth of Atlantic salmon challenged with Paramoeba perurans [35]. The ω-6/ω-3 fatty acid ratio of oils extracted from hemp cake and hemp hulls was 3.0:1 and 3.1:1, respectively. Several factors such as climatic conditions, cultivars, nutrients, etc. could affect the ω-6/ω-3 fatty acid ratios of hemp oil and their ratios were reported as 1.71:1–3.31:1 [36].

Even though fatty acids are a key component of the hemp seed oils and oils derived from hemp seed by-products, only a small amount may exist in free acid forms. The majority of fatty acids are present in ester forms, either in triacylglycerols (TAGs) or in polar lipids such as glycolipids or phospholipids. It is worth mentioning here that bioavailability of the fatty acids depends upon the structural composition of triacylglycerols. It has been reported that re-esterified triacylglycerols have superior bioavailability of EPA and DHA as compared to the natural fish oils, ethyl esters or free fatty acid forms [37]. It is crucial to know the structure composition of the intact lipids (or triacylglycerols) to understand their true health benefits for humans or as feedstock, including aquafeed application. To the best of our knowledge, no research has been conducted to date on characterization of intact TAGs in hemp seed oil or oil-derived hemp by-products. In the current study, electrospray ionization (ESI) was used to characterize TAGs in neutral lipid fractions of oil derived from hemp by-products. ESI is a soft ionization technique previously used by MacDougall at al. (2011) for profiling TAGs of microalgae [38]. ESI generates intact molecular ions, typically observed as ammonium, sodium or proton adducts (Figure 5). The ammonium adducts ion was selected for identification of TAGs using the LipidMAPS database [39]. Moreover, the molecular ion of TAG could be fragmented under low-energy collision-induced dissociation (CID) in a systematic fashion into diacylglycerol (DAG) product ions which were used for the identification of TAGs. Representative mass spectra of four TAGs having fatty acid side chains 18:3/18:2/18:1, 18:2/18:1/16:0, 24:0/18:3/18:2 and 22:0/18:2/18:1 with their fragment ions are shown in Figure 5. Based on the accurate masses of adduct and fragment ions, we have for the first time identified 47 TAGs in the oil derived from hemp by-products or hemp seeds. The MS spectrum in Figure S6 showed that the precursor ion at m/z 896.77087, 870.75549 and 844.74030 eluted at 2.35 min of hemp cake belongs to TAGs. Even though some of these TAGs were not chromatographically resolved, their identifications were incontrovertible. The major fragments observed in the MSMS spectra represent the neutral losses of fatty acids from the glyceride backbone. For example, in Figure 5, the precursor ion at m/z 896.77156 first lost an ammonium adduct and further lost major fragments at m/z 601.51970, m/z 599.50386 and m/z 597.48850, representing the neutral loss of fatty acids C18:3, C18:2 and C18:1, respectively. Unfortunately, determination of the position of individual fatty acyl chains within glyceride backbone was outside the scope of this study. Based on the intensities of the ammonium adducts ions we were able to create a hit map of the individual TAGs within the neutral lipid fractions to compare their relative concentration. The hit map diagram of individual TAGs (Table 2) strongly suggested that TAGs with fatty acyl chain 18:3/18:2/18:2 and 18:3/18:2/18:1 are major components in all the tested hemp oils derived either from hemp by-products or from hemp seeds. Fatty acid analysis data (Table 3) clearly demonstrated that linoleic acid (C18:2 n-6), α-linolenic acid (C18:3 n-3), oleic acid (C18:1 n-9), stearic acid (C18:0) and palmitic acid (C16:0) were the major fatty acids in hemp oil. This further confirmed that the TAGs having these fatty acyl chains (C18:3, C18:2, C18:1, C18:0 and C16:0) are the main TAGs in the hemp oils. TAGs with longer fatty acid acyl chains, i.e., C:20, C:22, C:23 and C:24 were also detected, but their intensities were lower as compared to the TAGs containing major fatty acid acyl chains.

Carotenoids, chlorophyll and cannabinoids, especially CBD, were also reported in hemp seed oil [36]. In the current study, we also found the presence of lutein in all oil samples extracted from either by-products or hemp seeds and cold-pressed commercial hemp oil. The light yellow color of the hemp hearts oil may be due to low levels of lutein. α-carotene and β-carotene were also detected in oils derived from hemp cake and hemp hulls plus cold-pressed hemp oil. Chlorophyll degradation products and cannabinoids, especially CBD and CBDA were observed in most of the oils except hemp hearts and whole seeds. Absence of cannabinoids and chlorophyll and only low levels of lutein presence in the hemp hearts oil clearly suggested that the cannabinoids, carotenoids and chlorophyll present only in the hemp hulls. Higher concentrations of pigments and chlorophyll in hemp cake indicated that the cold-pressed process enhances the extraction efficiency of pigments. Lower levels of lutein and absence of other carotenoids in whole seeds oil suggested that the heating process may possibly degrade the pigments. Terpenes were also present in all hemp samples, with more terpenes being detected in whole seed, cake and cold-pressed hemp oil. Hemp hearts and hemp hulls had fewer terpenes detected. Hemp seeds have been reported to have antinutrient components in them such as phytic acid or phytate [40,41]. Even though we did not analyze phytic acid or phytate in oils extracted from hemp seeds and by-products, these antinutrient components are most likely not present in the hemp seed oils based on their polarity and ability to be extracted by hexane.

Vegetables oil has been reported to have antioxidant properties [42]; thus, we further tested oils derived from both hemp cake and hemp hulls for their DPPH radical scavenging potency for comparison with other vegetable oils. Oils extracted from other hemp biomass and cold-pressed hemp oils were also tested. Cold-pressed hemp oil and oil derived from hemp cake possessed the highest DPPH radical scavenging potency compared to the oil extracted from hemp hearts and hemp hulls, which had relatively higher concentrations of α-carotene and β-carotene. The oil derived from hemp seeds showed significantly weaker DPPH radical activity as compared to other oils; most probably, the roasting process destroyed the antioxidative component. The mechanical crushing process seems to enhance extraction of antioxidative components present in the hemp oils. The IC₅₀ values of the tested oils range from 555 to 3062.5 μg/mL, weaker as compared to the positive control ascorbic acid having IC₅₀ 2.4 μg/mL. The IC₅₀ values of hemp cake and cold-pressed hemp oils are in a similar range as those of soybean oil (IC₅₀ 362 μg/mL), canola oil (IC₅₀ 400 μg/mL), sesame oil (IC₅₀ 500 μg/mL) and are better than flax seed oil (IC₅₀ 2400 μg/mL) and avocado oil (IC₅₀ 6200 μg/mL) [42].

In summary, we have studied lipid composition of oils derived from hemp seed hulls and hemp cake together with whole hemp seeds, hemp hearts and commercial cold-pressed hemp oil. Both hemp by-products, i.e., hemp hulls and hemp cakes contain significant amounts of total lipids, 17.5 and >13.1% of the biomass, respectively, having great commercial value as a source of lipid for aquafeed application. It is worth mentioning here that this is the first report of such comprehensive lipid characterization for oil derived from hemp seed hulls and hemp cake in comparison with cold-pressed commercial oil and oil extracted from hemp whole seeds and hemp hearts. The fatty acids profile suggested that, regardless of material, for either hemp seed or hemp by-products, all biomasses had almost identical fatty acid compositions. LA and ALA acids were the two dominant fatty acids present in the oil extracted from hemp by-products, similar to those of cold-pressed hemp oil. The total ω-6/ω-3 ratios of the tested hemp oils were 3.1 to 3.3. For the first time, we have characterized the triacylglycerol content of the hemp oil and 47 individual TAGs were identified by UPLC-HRMS analysis. Among them, TAGs with fatty acyl side chain 18:3/18:2/18:2 and 18:3/18:2/18:1 were the major ones. A low level of carotenoids, especially lutein, α-carotene and β-carotene was found in oils extracted from hemp by-products. Moreover, the oils derived from hemp by-products possessed dose-dependent DPPH radical scavenging properties and their potency was in a similar range as compared to other vegetable oils such as canola, soybean, avocado and sesame oils.