2. Analytical Methods for Determination of Fats in Grains
2.1. Crude Fat Determination
Official methods for fat analysis in foods have been evaluated and approved by authorized organizations that develop procedures for routine analysis in regulatory, contractual, and other laboratories. Crude fat can be extracted using published AOAC or AOCS methods. AOAC Official Method 948.22
[31][16] is a solvent extraction procedure for determining the crude and total fat contents of nuts and nut products. Samples are extracted with ether in a Soxhlet-type extractor for 16 h, after which the extracted lipid is dried at 95–100 °C and weighed. Modifications of the Soxhlet solvent extraction procedure include the Randall/Soxtec/diethyl ether (AOAC 2003.05)
[32][17] and Randall/Soxtec/hexane (AOAC 2003.06)
[33][18] extraction–submersion methods, which provide a shorter extraction time because the sample is submerged in boiling solvent. AOCS Official Method Am 5-04
[34][19] was approved for rapid determination of the total fat content of oil seeds, meats, feeds, and foods using an automated or semi-automated extraction system. Substances extracted using petroleum ether are predominantly triacylglycerides, with small amounts of other lipids and minor components.
An alternative to the solvent extraction method is a two-step hydrolysis process in which the sample is pretreated with acids, alkaline reagents, or enzymes to break down the matrix before extraction with a solvent. Hydrolytic treatment disrupts the lipid–carbohydrate bonds, proteins, polysaccharides, and plant cell walls. AOAC Official Method 983.23
[35][20] describes a hydrolytic method for determining the fat content of foods.
2.2. Fatty Acid Determination
Interest has grown in determining the content and composition of fatty acids in foods and food ingredients, prompting the development and validation of analytical methods for quantifying fatty acids using gas chromatography with flame ionization (GC–FID) or gas chromatography–mass spectrometry (GC–MS)
[36,37][21][22]. AOAC Official Method 996.01
[38][23] was approved for determination of the fat content of cereal products containing 0.5–13% total fat using a gas chromatographic method. The sample is heated while being shaken in ethanol and 8 M HCl at 80 °C for 40 min and then cooled. Subsequently, it is transferred with ethanol to a Mojonnier fat extraction flask for liquid–liquid extraction with ethyl and petroleum ether, followed by evaporation with nitrogen. The extract is saponified and methylated by the addition of sodium hydroxide (0.5 M) in a methanol solution and then esterified with 14% boron trifluoride in methanol. Fatty acids are derivatized to fatty acid methyl esters (FAMEs) and quantified using GC–FID or GC–MS. The total fat content is determined by the summation of individual fatty acids
[36,37,39,40][21][22][24][25]. Conversion factors are used to express the analyzed FAMEs as free fatty acid equivalents. It is difficult to analyze fatty acids in their free forms, as they are highly polar and will form hydrogen bonds, causing problems with adsorption
[41][26]. AOAC Official Method 996.06
[42][27] demonstrates an interlaboratory collaborative study for determining the fat content (total, saturated, and unsaturated) in food matrices, including wheat-based cereal products, with a total fat content of 1.5–46%.
3. Effects of Sprouting on the Fat Content of Grains
3.1. Lipase Activity
Lipids are abundant in the embryo, scutellum, and aleurone of whole grains as oil (triacylglycerols)
[28,43,44][28][29][30]. Lipid catabolism provides energy and carbon sources for biochemical and physicochemical changes during seed growth.
During germination, lipases release esterified fatty acids from triglycerides
[43,44][29][30]. As lipases are activated, triacylglycerols are hydrolyzed into free fatty acids, increasing the saturated/unsaturated fatty acid ratio
[28]. Free fatty acids are then degraded through β-oxidation and the glyoxylate cycle and converted to sugars
[44][30]. This may result in the development of off-flavors
[45][31].
Long sprouting times enhance undesirable flavors and odors due to increased lipase and lipoxygenase levels leading to aldehydes, free phenolic compounds, heterocyclic substances, and dimethyl sulfide
[46,47,48][32][33][34].
Lipase acts as soon as the grain structure breaks down and comes into contact with its substrates
[45][31]. Kubicka et al.
[44][30] noted that lipase activity increased 1.2- to 2.3-fold during the sprouting of cereal grains because of the synthesis of lipase in the outermost part of the endosperm. However, oats are unique and contain high lipase activity in their grains that is either maintained or reduced during sprouting
[49][35]. Aparicio-Garcia et al.
[45][31] demonstrated that lipase activity was reduced by 17% in sprouted oats compared to that in the control. It was also observed that the degradation of oil from the oat embryo occurred earlier than that of oil reserves in the scutellum
[50][36]. One to two days after imbibition, triglyceride mobilization begins in the endosperm of oats, with the accumulation of free fatty acids
[50][36]. Aparicio-Garcia et al.
[51][37] showed that lipase activity increased in a sprouted hulled oat variety but was reduced in a dehulled oat variety. Heiniö et al.
[47][33] observed that hulled oats had a lower lipid content than hull-less oats. A greater amount of triglycerides (>75%) was hydrolyzed in hulled oats steeped in water for 15 h than in dehulled oats (15%). The lipase activity was higher in oat grains than in sprouted oats. Hosseini et al.
[52][38] found greater amounts of free fatty acids in hulled than in dehulled sprouted oats, suggesting an important role in the increased lipid activity in oat hulls.
3.2. Lipid Content of Sprouted Grains
Lipid amounts in non-germinated and germinated grains were evaluated and compared using data from various studies. The lipid content was higher in sprouted oats after germination at 25 °C/60% relative humidity
[53][39]. The fat content decreased by 8–15% owing to lipase activity in sprouted millet (3 days, 20–23 °C)
[54][40]. No change was observed in the fat content of sprouted wheat compared to that of seeds when germinated for 3 days at 20 °C
[55][41]. Farooqui et al.
[56][42] reported that the fat content decreased from 2.75% in non-germinated barley to 2.10% when barley was sprouted at 25 °C for 72 h. However, Ortiz et al.
[57][43] observed the amount of fat to be 50.2% greater in the sprouted barley than in the raw barley grain when grown under light for 6 days at 20 °C. The total fat content increased from 1.62% in buckwheat grains to 2.42% in sprouted buckwheat when germinated in the dark for 3 days at 30 °C
[58][44]. Conversely, Rico et al.
[2] noticed that the fat content of sprouted barley decreased considerably compared to that of the control when grown in the dark at 12.1–19.9 °C and for 1.6–6.19 days. Jiménez et al.
[59][45] did not notice a significant change in the lipid content when sprouting quinoa for 24 h at 22–24 °C and amaranth for 48 h in the dark.
Other studies have demonstrated that higher amounts of lipids are broken down as the sprouting temperature increases. The total lipid content was reduced by 18–28% in the following: sprouted millet (2 days; 32 °C)
[60][46], sprouted wheat (2 days, 30 °C)
[61][47], and sprouted brown rice (1–5 days, 25 to 30 °C)
[62,63][48][49].
Pîrvulescu et al.
[64][50] determined that the highest lipid content from barley, wheat, and oats was found in the germinated Capo wheat genotype (11.12 g/100 g flour). The lipid content of the flours from the non-germinated grains was lower than that of the germinated grains.
In general, the fat content was lower in sprouted quinoa (0.1–3.8%)
[65,66][51][52] than in its grain (4.0–7.6%)
[67][53]. The fat content of sprouted grains generally decreases because of the use of lipids as an energy source during germination
[68][54] or increased lipolytic activity, which converts fats into fatty acids and glycerol
[69,70,71][55][56][57]. Triglycerides were the most abundant lipids in non-germinated and germinated oil seeds. Changes during sprouting depend on the crop type, genotype, and germination conditions
[59][45].
3.3. Fatty Acids in Sprouted Grains
3.3.1. Wheat
Waxy wheat (
Triticum aestivum L.) has exceptional properties for forming bread textures with soft, thick, and gelatinous breadcrumbs that prevent bread staleness. Wheat contains large amounts of essential fatty acids (linoleic and linolenic acids) that the human body cannot synthesize because humans do not have the enzymes required for their production
[61][47].
Hung et al.
[61][47] found that ungerminated and sprouted wheat contain eleven fatty acids representing 98.6–100% of total fatty acids. The major fatty acids in order of abundance are linoleic (C18:2), palmitic (C16:0), oleic (C18:1), linolenic (C18:3), and stearic (C18:0) acids. Trace amounts of other components include myristic (C14:0), palmitoleic (C16:1), arachidonic (C20:0), eicosaenoic (C20:1), eicosatrienoic (C20:3), and eicosapentaenoic (C20:5) acids. Hung et al.
[61][47] observed that the fatty acid composition of wheat remained unchanged during 48 h of germination. This study concluded that sprouted waxy wheat can enhance the texture and nutritional attributes of cereal-based products.
Ozturk et al.
[74][58] observed that the sprouting of two varieties of wheat (Demir 2000 and Konya 2002) had a significant effect on the fatty acid composition of 9-day-old wheat seeds. Although trace levels of some saturated fatty acids (C4:0, C10:0, and C12:0) were observed in the seeds, they disappeared from the sprouts. The quantity of omega-3 (18:3n-3) fatty acids increased, whereas the amounts of cis-18:1 and cis-18:2 fatty acids decreased when wheat was germinated. Márton et al.
[55][41] compared the fatty acid content of wheat seeds and wheat sprouts after 3 days of germination at 20 °C. The predominant fatty acids in seeds and sprouts were palmitic (C16:0), linoleic (C18:2 cis), and oleic (C18:1) acids. While the amounts of palmitic and linoleic acids increased in sprouted wheat, oleic acid levels decreased.
3.3.2. Buckwheat
Buckwheat (
Fagopyrum esculentum) is unrelated to wheat, unlike its name suggests. It is not a cereal or a member of the grass family but a pseudocereal because its seeds are used similarly to those of cereals for culinary and nutritional purposes. It is gluten-free
[75][59].
Kim et al.
[73][60] assessed the fatty acid composition of buckwheat and its sprouts. Oleic (C18:1cis), linoleic (C18:2), and linolenic (C18:3n-3) acids comprised 36.8%, 38.1%, and 2.7% of the total fatty acid composition of the raw ingredient, respectively. The major saturated fatty acid in buckwheat seeds, palmitic acid (C16:0), comprised approximately 17.7% of the total fatty acid content. Small amounts of myristic (C14:0), stearic (C18:0), arachidic (C20:0), and behenic (C22:0) acids were detected. The study found that linoleic acid (C18:2) was the most abundant fatty acid in the sprouts, and its content increased by up to 52.1% in 7 days, with the total amount of unsaturated fatty acids (oleic, linoleic, and linolenic acids) being greater than 83% and higher than that of the saturated fatty acids. However, the amount of saturated fatty acids rapidly decreased; myristic and stearic acids disappeared after 1 day of sprouting, and arachidic and behenic acids disappeared after 3 days of sprouting. The greatest decrease among unsaturated fatty acids was observed for oleic acid; however, the amounts of linoleic, linolenic, and arachidonic acids increased in sprouts as germination progressed
[73][60]. Linoleic, linolenic, and arachidonic acids are essential because they cannot be synthesized in the human body.
Molska et al.
[58][44] germinated buckwheat seeds in the dark for 3 days at 30 °C. Higher concentrations of unsaturated fats, constituting 78.48% of the total fatty acids in seeds and 83.04% in sprouts, were observed than saturated fatty acids, which constituted 21.2% in seeds and decreased to 16.8% in sprouts. The most abundant fatty acids in buckwheat seeds and sprouts were palmitic, oleic, and linoleic acids. However, the quantity of these fatty acids changed during sprouting. The level of palmitic acid significantly decreased in the controlled sprouts (up to 13.90%), but the amount of linoleic acid increased (up to 42.45%)
[58][44].
Conversely, Yiming et al.
[72][61] observed a different trend in the fatty acid composition of Tartary buckwheat (
Fagopyrum tataricum L.) when germinated in the dark at 37 °C for up to 7 days. The amounts of palmitic, oleic, and stearic acids increased, whereas those of linoleic and eicosenoic acids decreased.
3.3.3. Quinoa
Quinoa (
Chenopodium quinoa) is a herbaceous plant grown for its edible seeds. It is not a grass but a pseudocereal that belongs to the Amaranth family. Quinoa is the only plant with all essential amino acids, trace minerals, vitamins, and no gluten
[76][62].
Park and Morita
[77][63] studied the fatty acid composition of quinoa seeds germinated for 3 days and noticed that the levels of some fatty acids either increased or decreased. The amounts of saturated and monounsaturated fatty acids increased, whereas those of polyunsaturated fatty acids decreased, and variation was observed in the omega-6/omega-3 ratio.
Peiretti et al.
[78][64] studied the fatty acid composition of quinoa from the seed to the morphological vegetative stages from the end of June to the end of September 2010. The fatty acids in the seeds were predominantly palmitic (C16:0), oleic (C18:0), and linoleic (C18:2cis) acids. Quinoa contained the highest amount of alpha-linolenic acid (C18:3n-3; omega-3), at 47% of the total fatty acids, whereas linoleic acid (C18:2 cis, omega-6) constituted 16% of the total fatty acids in the early vegetative state. The sprouts contained high levels of linolenic acid (C18:3n-3) (385–473 g/kg of total fatty acids) and polyunsaturated fatty acids (611–691 g/kg of total fatty acids). The linoleic acid content (146–176 g/kg of total fatty acids) decreased significantly in quinoa until the shooting stage was reached and then increased, whereas the amounts of the other fatty acids, palmitic and oleic acids, did not show significant differences during growth. This showed that quinoa grown in the early and mid-vegetative stages contained higher amounts of alpha-linolenic acid and omega-3 fatty acids than linoleic acid and omega-6 fatty acids. Peiretti et al.
[78][64] showed that the omega-6/omega-3 ratio in quinoa was 0.3. Alvares-Jubete et al.
[79][65] reported an omega-6/omega-3 ratio of approximately 1:6.
Jiménez et al.
[59][45] compared the fatty acid profile between the grains and sprouted quinoa grown between 22 °C and 24 °C in the dark for 24 h. The amounts of saturated fatty acids, palmitic and behenic acids, decreased, potentially owing to lipase activity and the breakdown of triglycerides and polar lipids into simple compounds during sprouting. As the levels of saturated fatty acids decreased, those of monounsaturated and polyunsaturated fatty acids increased during germination. The omega-6/omega-3 ratio for quinoa grains was 5:24 and decreased to 4:55 when sprouted.
3.3.4. Barley
Barley (
Hordeum vulgare) is an ancient cereal from the grass family grown in mild climates worldwide and is traditionally used as animal feed and as a raw material for beer and other beverages in the malting industry
[80][66]. There has been heightened interest in its use as a food ingredient because of its nutritional benefits and superior bioactive properties
[81][67].
Rico et al.
[2] germinated two varieties of whole-grain barley flour in the temperature range of 12.1–19.9 °C for 1.6–6.19 days in the dark. The fatty acid composition was evaluated and compared with that of the control. No changes were detected in the amounts of saturated, unsaturated, monounsaturated, and polyunsaturated fatty acids in barley grown at lower temperatures and shorter times compared with those of the raw ingredient. However, after a longer germination period, sprouted barley contained approximately 37% more saturated fatty acids than barley grains, whereas the amounts of polyunsaturated acids did not show significant differences, at 59–63%. Rico et al.
[2] concluded that the most suitable condition for the sprouting of barley was 16 °C for 3.53 days.
Oritz et al.
[57][43] noticed an increase in the C18:0 (24%) and C18:3n-3 (49%) contents and a decrease in the C18:1n-9 (6%) content in barley sprouted at 20 °C for 6 days in the light. The concentration of linoleic acid (C18:2n-6), which is predominant in barley, did not vary during barley growth. Palmitic acid (C16:0) was the most abundant saturated fatty acid in the sprouted barley. Polyunsaturated fatty acids constituted most of the fatty acid composition in green shoots, the residual structure of sprouted grains plus non-sprouted grains, and root fractions ranged from 56.1% to 61.4%. Lower amounts of monounsaturated (14.3–24.6%) and saturated (15.78–21.96%) fatty acids were detected in these sprout fractions
[57][43].