Extraction of non-starch lipid from protease treated wheat flour

Siti Farhiah Abdul Manan; Jihong Li; Chao-Feng Hsieh; Jon Faubion; Yong-Cheng Shi

doi:10.1002/jsfa.11523

. Author manuscript; available in PMC: 2023 Mar 30.

Published in final edited form as: J Sci Food Agric. 2021 Sep 22;102(5):2172–2178. doi: 10.1002/jsfa.11523

Extraction of non-starch lipid from protease treated wheat flour

Siti Farhiah Abdul Manan ^a,^b, Jihong Li ^a, Chao-Feng Hsieh ^a, Jon Faubion ^a, Yong-Cheng Shi ^a,^*

PMCID: PMC8908900 NIHMSID: NIHMS1783613 PMID: 34498279

Abstract

BACKGROUND

Lipids account for 2.0-2.5% of wheat flour by dry weight and affect properties and quality of cereal foods. A new method was developed to extract non-starch lipids from wheat flour. Wheat flour was first hydrolyzed with a protease and followed by extraction of non-starch lipids by water-saturated butanol (WSB).

RESULT

Protein hydrolysis by protease followed by extraction of non-starch lipids with WSB increased yield to 1.9±0.3% from 1.0±0.1% with no protease treatment. The lipid profile showed a significant increase in phospholipid compounds extracted with protease hydrolysis (5.9±0.8 nmol/g) vs. without enzymatic treatment (2.4±1.3 nmol/g).

CONCLUSION

Improved lipid extraction yield and phospholipid compounds following protease-assisted extraction method provided additional insight towards the understanding of protein-lipid interaction in wheat flour. The new protease-assisted extraction method may be applied to analyze non-starch lipids in other types of wheat flours and other cereal flours.

Keywords: Wheat flour, non-starch lipid extraction, protease, water-saturated butanol

INTRODUCTION

Lipids account for approximately 2.0-2.5% of wheat flour by dry weight,^1-2 and can be categorized into starch lipids (0.8-1.0%) and non-starch lipids (1.2-1.5%) (Fig. 1). Starch lipids are classified as the lipids tightly bound in the internal structures of starch granules. As much as 90% of wheat starch lipids are polar lipids with lysophosphatidylcholines (LPC) as the dominant compounds. Non-starch lipids (flour lipids), on the other hand, are the lipids localized outside of starch granules, and have traditionally been subdivided into “free” lipids (extractable by non-polar solvent) and “bound” lipids (subsequently extractable by polar solvents). Starch surface lipids are considered non-starch lipids (either free or bound to proteins) in the starch-bearing tissues.³ Non-starch lipids consist of 0.7 to 0.9% free lipids based on the dry weight of wheat flour. Most free lipids are nonpolar (0.5-0.7%) while the remaining (~0.2%) is polar. Other non-starch lipids are the remaining bound lipids (0.5-0.6%).

Fig. 1 — Total lipids distribution based on the wheat flour dry weight (summarized from Chung et al., 2009; Pareyt et al., 2011).

Despite their low levels, non-starch lipids are involved in the physical, chemical and biochemical processes of dough formation and bread making and affect properties and quality of cereal foods.^{2, 4-5} The role of lipids in bread making has been reviewed by Pareyt et al..² Non-starch lipids have a major influence on the dough formation and bread loaf volume. It is of interest to study the non-starch lipids so as to better understand the roles of endogenous lipids in creating desired dough and batter properties.

The importance of protein-lipid internactions in wheat flour is well recognized, and models have been proposed to explain the protein-lipid interactions.⁶ Polar non-starch lipids (glycolipids and phospholipids) are reported to interact with starch and protein and to affect the behavior of dough and batter in bakery products.^2,7 It has been suggested that glycolipids interact with glutenin through hydrogen bonds and hydrophobic interactions, while phospholipids tend to interact with gliadin and lipid binding proteins.⁸ The principal phospholipids in wheat flour are LPC and phosphatidylcholine (PC).⁷ Higher concentrations of galactolipids and phospholipids are found in the bound lipids than the free lipids.⁸ Polar non-starch lipids (glycolipids and phospholipids) have a positive influence on gas retention and baking performance.² Alteration of flour lipids through enzymatic (lipase) modification can help to improve gas cell stability in bread dough and increase bread loaf volumn.^5,9-10

Wheat flour lipid extraction differs according to the types of solvent employed, specifically its polarity. Due to their high polarity, starch lipids need to be extracted by using highly polar solvents after the starch granules are swollen or gelatinized.^11,12 On the other hand, the non-starch lipids are extractable with solvents without undergoing starch swelling or gelatinization. The most commonly used non-starch lipid extraction solvents for cereals are petroleum ether,^11-12 benzene:ethanol:water (10:10:1),¹³ ethanol:diethyl ether:water (2:2:1),¹³ benzene,¹⁴ acetone,¹⁴ 2-propanol,¹⁴ isopropanol:water (90:10),¹¹ and hexane.^{11, 15}

The free lipid subcategory of non-starch lipid can be readily extracted by nonpolar solvents like petroleum ether^14,16and hexane.^11,15 For bound lipids subcategory, the extractions mostly require the highly polar solvents such as water-saturated butanol (WSB)^{5, 13-17} or isopropanol-water.¹¹

Since both free and bound lipids of the non-starch lipids fraction involve in the flour and dough of the bread making, it would be useful to extract the non-starch lipids fraction without separation of the free and bound lipids extraction steps. It was reported that the usage of bipolar solvents like WSB was sufficient to extract the polar and nonpolar lipids present in both free and bound lipids of the wheat flour.^4-5 In this study, we used protease to hydrolyze protein in wheat flour before the extraction of non-starch lipids by WSB. Protease has been used in the cereal starch isolation to produce higher purity starch with lower residual protein content.^18-24 Two of these studies^21-22 also analyzed the starch lipid content in addition to the residual protein content of the starch to indicate the effectiveness of the enzyme-assisted starch isolation. However, in those studies, protease has not been used to hydrolyze protein followed by extraction of non-starch lipids in cereal flours.

The objectives of this study were to use protease to hydrolyze proteins in wheat flour followed by extraction of lipids with WSB, to analyze and compare the lipid composition with that extracted without the use of protease treatment. Our hypothesis was that protease treatment would disrupt the protein matrix in wheat flour and result in the release of more lipids, specifically those bound with protein. We compared the extraction methods of the non-starch lipids with and without protease treatment. Comparison between the compounds detected in both lipid extracts would reveal which types of lipids are released after protein matrix is disrupted by protease and may provide additional insights towards understanding of the possible interaction between specific compounds within the flour components.

MATERIALS AND METHODS

Straight grade flour (9.8% protein, 12.0% moisture), which represented ~ 72% extraction rate, from Kansas hard red winter wheat (HRWW) produced by the Hal Ross Flour Mill at Kansas State University (Manhattan, KS) was used in all experiments. Danisco FoodPro® alkaline protease was obtained from DuPont Nutrition and Health (New Century, KS). All other chemicals were analytical grade and purchased from Fisher Scientific (Fair Lawn, NJ).

Non-starch lipids were extracted with WSB by direct extraction and protease-assisted methods. In addition, to examine whether the temperature, pH, and stirring affected the lipid extraction yield, a control method was performed following the protease-assisted method conditions except that no protease was added. Both direct extraction and control methods were categorized as without protease assistance extraction method. WSB was prepared by mixing butanol and distilled water at 80% to 20% (v/v) ratio in a separatory funnel. The direct extraction, protease-assisted, and control methods are described as follows.

The direct extraction procedure is summarized in Fig. 2. HRWW flour (50 g) was mixed with 115 mL WSB and placed in a shaking water bath at ambient temperature for 30 min. The mixture was then centrifuged at 3840 g for 10 min, and the supernatant was transferred to a separatory funnel (first lipid extract). The sediment was extracted twice more with 100 mL WSB creating to the second and third extracts. Extracts were combined and then carefully transferred into a round bottom flask and dried by rotary evaporation at 60 °C. Due to the large extract solvent volume, the pooled lipid extract was dried in batches. Dried lipid was then dissolved in 10 mL chloroform, filtered through 0.45 μm nylon syringe filter membrane into a pre-weighed 50 mL centrifuge tube and allowed to dry completely under a fume hood in darkness overnight. Extracted lipid was then dried in a vacuum oven (MTI Corporation, Richmond, CA) at 60 °C for 1 h to ensure complete removal of moisture. Dried lipid was stored at 4 °C in the dark before further analysis.

Protease-assisted extraction method is summarized in Fig. 3. A preliminary study determined that the optimum time for protease hydrolysis by alkaline protease was 4 h. Employing this time, the residual protein content in defatted wheat starch collected after continuous lipid extraction and centrifugation was reduced significantly (9.8% to 0.3%). HRWW flour (50 g) was mixed with distilled water at a ratio of 1:3. Protease was added at 0.5% (wheat flour basis), and the mixture was adjusted to pH 8 with 1.0 M sodium hydroxide. The protease was allowed to react in a water bath set at 45 °C, constant pH 8 and continuous stirring for 4 h. After hydrolysis, 150 mL butanol was added to the mixture, and it was placed in a shaking water bath at ambient temperature for 15 min. The mixture was then centrifuged at 3840 g for 10 min, and the supernatant was collected as the first lipid extract. The sediment was extracted two more times (second and third extracts) each with 75 mL WSB as previously mentioned. Dried lipid was stored at 4 °C in the dark before further analysis.

The control lipid method was conducted as the same as described in Fig. 3 but without protease treatment. Sediment following the continuous protease-assisted lipid extraction and centrifugation were collected and considered as defatted wheat starch. The sediment was dried in a convection oven for 48 h at 40 °C followed by grinding and passing through a 180 μm sieve. Residual protein content was analyzed with nitrogen/protein analyzer LECO Model FP628 (LECO Corporation, St. Joseph, MI) for crude protein determination by combustion method. Protein content of wheat flour without lipid extraction was also analyzed.

Non-starch lipids from direct extraction and protease-assisted extractions were analyzed for polar lipids (GL and PL). The analysis was performed by the Kansas Lipidomics Research Center (KLRC) of Kansas State University. Extracted non-starch lipids were transported to the KLRC in dried form, then dissolved in the chloroform and stored at −20 °C in vials equipped with Teflon-lined screw caps. The lipids were analyzed by direct infusion electrospray ionization triple quadrupole mass spectrometer (TQMS) following the method described by Welti et al.²⁵ and Finnie et al..¹¹ The internal standards of DGDG and MGDG were from Matreya LLC (State College, PA), and phospholipid standards were from Avanti Polar Lipids Inc. (Alabaster, AL). The GL, PL, and MAG were analyzed with a Waters Xevo TQS mass spectrometer (Waters Corporation, Milford, MA, USA); a volume corresponding to 0.009 mg dry lipid weight was taken for analysis. The capillary voltage was set at 2.8 kV, the source offset voltage at 30 V, the source temperature at 150 °C, the desolvation temperature at 250 °C, the cone gas flow at 150 L/h, the collision gas flow at 0.14 mL/min, the nebulizer gas flow at 7 Bar, the LM1 resolution at 3.2 and the HM1 resolution at 15.5. The samples were infused at 30 μL/min using an autosampler. The resolution was set so that the peak width was approximately 0.7 μ. Parameters as in Finnie et al.,¹¹ except for source voltage −4500, DP-100, EP-14, with a volume equal to 0.045 mg dry lipid weight was taken for analysis.

Extracted lipids were vacuum dried, weighed, then dissolved in 1.0 mL chloroform. Standards were present as in Finnie et al.,¹¹ with the phospholipids 1/5 as much, and the galactolipids 1/2 as much. Table 1 summarizes the concentrations (nmol/sample) of the lipid species in 5 μL of PL and GL internal standards and the scan functions used to quantify the lipid species. Data were then processed using Lipidome DB Data Calculation Environment as described by Shiva et al..²⁶ Additionally, response factors were applied to correct for the difference in response of the mass spectrometer to plant galactolipids in comparison to internal standards.

Table 1.

Concentrations (nmol/sample) of lipid species in internal standards and scan functions used to quantify the lipid species.

Internal standards	Concentrations (nmol/sample)	Lipid species quantified	Scan function
PL (5 μL):
LysoPG 14:0	0.30	LysoPG	−Prec153
LysoPG 18:0	0.30	LysoPG	−Prec153
		SQDG	−Prec225
PG (14:0/14:0)	0.30	PG	+NL189
PG (20:0/20:0)	0.30	PG	+NL189
LysoPE 14:0	0.30	LysoPE	+NL141
LysoPE 18:0	0.30	LysoPE	+NL141
PE (12:0/12:0)	0.30	PE	+NL141
PE (20:0/20:0)	0.30	PE	+NL141
PE (23:0/23:0)	0.30	PE	+NL141
LysoPC 13:0	0.60	LysoPC	+Prec184
LysoPC 19:0	0.60	LysoPC	+Prec184
PC (12:0/12:0)	0.60	PC	+Prec184
PC (24:1/24:1)	0.60	PC	+Prec184
LysoPA 14:0	0.30	LysoPA	+NL115
LysoPA 18:0	0.30	LysoPA	+NL115
PA (14:0/14:0)	0.30	PA	+NL115
PA (20:0/20:0)	0.30	PA	+NL115
PS (14:0/14:0)	0.20	PS	+NL185
Di Phy PS	0.20	PS	−NL185
PI (16:0/18:0)	0.29	PI	+NL277
PI (18:0/18:0)	0.10	PI	+NL277
GL (5μL):
DGDG (18:0/16:0)	0.44	DGDG	+NL341
DGDG (18:0/18:0)	1.48	DGDG	+NL341
MGDG (18:0/16:0)	1.67	MGDG	+NL179
		Steryl glucoside	+NL197
		GIPCer	+NL615
MGDG (18:0/18:0)	1.40	MGDG	+NL179

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Statistical analysis

Five replicates of each extraction method were carried out and analyzed by mass spectrometry. An ANOVA test of equal variance was performed for each lipid compound of extracts from protease-assisted and direct extraction methods. Then, the two-sample T-test was performed based on the assumption of either equal or non-equal variances. All the statistical analyses were performed with Minitab 18 (Minitab Inc., State College, PA).

RESULTS AND DISCUSSION

Table 2 summarizes the yield and mean values of normalized signal of the extracted lipids. By hydrolyzing flour protein prior to lipid extraction, the protease-assisted method resulted in higher yield than that of the non-enzymatic method (1.9% versus 1.0% of extracted lipid on a flour dry weight basis). The protease enzyme hydrolysis prior to total non-starch lipid extraction with WSB facilitated in the process greatly. It was reported that extraction with bipolar WSB resulted in a higher amount of polar lipid as they dissolved better in this solvent than in 2-propanol.⁵ Clements²⁷ studied the effects of various extraction solvents polarity on the wheat flour lipid yield, and reported that WSB generally gave a maximum yield of lipid especially when the wheat flour was tempered by water or moisture prior to extraction with the solvent, whereas chloroform with methanol extraction solvent gave lower lipid yield than WSB. The explanation for the difference was suggested due to the incorporation of water into the extraction solvent.

Table 2.

Yield and mean values of normalized signal per mass dry weight wheat flour non-starch lipids (polar lipids compounds) by protease-assisted and direct extraction methods.

	Extraction methods
	Protease-assisted	Direct extraction
Yield of lipid (% of 50 g flour weight)^†	1.9±0.3^a	1.0±0.1^b
Lipid class
Polar lipid
GL (nmol)^‡
DGDG	17.5±6.4^a	16.1±10.8^a
MGDG	10.5±6.5^a	7.2±4.5^a
PL (nmol)
PG	0.1±0.1^a	0.1±0.0^a
LPG	0.6±0.6^a	0.3±0.2^a
PC	0.7±0.2^a	0.6±0.5^a
LPC	3.3±1.2^a	1.2±0.6^b
PE	0.1±0.1^a	0.1±0.1^a
LPE	0.1±0.1^a	0.0±0.0^b
PI	0.7±1.2^a	0.1±0.0^a
PS	0.1±0.1^a	0.0±0.0^b
PA	0.1±0.0^a	0.0±0.0^b

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Values are means±standard deviations; n=5. Mean values with different letters within rows indicate significant differences at P<0.05.

^†

The non-starch lipid yield extracted with butanol and water-saturated butanol (WSB) after the wheat flour was held at 45 °C and pH 8 for 4 h without protease is 1.1±0.1% of 50 g flour weight.

^‡

Response factors were applied to correct for the difference in response of the mass spectrometer to GL in comparison to internal standards.

The residual protein content in the wheat starch was 0.3% after 4 h of protease hydrolysis and extraction of lipids, whereas the protein content of the wheat flour was 9.8%. This significant reduction in residual protein content agreed with previous studies using different proteases.^18-24 Tester et al.^21-22 reported that the maize starch lipid content reduced in protease-treated sample compared to the native sample without protease treatment. These findings suggest that protease may hydrolyze starch surface-bound protein and release more bound lipids.

A control set of samples was prepared to employ the protease-assisted method without the protease addition. This control extraction was to examine whether the increased in non-starch lipid yield obtained from protease-assisted method was primarily due to the protease hydrolysis or due to the extraction conditions (temperature and pH). The yield of lipid obtained from extraction at pH 8 and 45 °C for 4 h was 1.1±0.1%, which is much lower compared to the extraction at the similar condition with the addition of protease (1.9±0.3%). Based on these results, the increased in lipid yield extracted from protease-assisted method was mainly due to the enzymatic hydrolysis and not the extraction conditions (temperature and pH). The temperature used during the extraction (45 °C) was below the gelatinization of wheat starch and that avoided swelling of starch granules and extraction of starch lipid inside starch granules. Enzymes are used in lipid extraction of oilseeds like soybean as well as high oil-containing cereal such as corn. Previous studies on the extraction of oil from oilseeds with protease assistance revealed increment in lipid extraction yield compared to the non-enzymatic extraction method.^28-31

The composition of the lipid extracted from wheat flour by each method is shown in Table 2. The lipid from direct WSB extraction method (Fig. 2) was further analyzed for the polar and nonpolar lipid composition and compared with lipid extracted from protease-assisted method (Fig. 3). Enzyme-assisted lipid extraction resulted in releasing higher amounts of individual phospholipids, LPC, LPE, and PA. Indeed, LPE and PA were not even detected in the lipid directly extracted by WSB without protease treatment.

With the assistance of protease enzyme, this study shows a significant increase in specific phospholipids compared to the results of the direct extraction method. It was postulated that the enzyme hydrolysis could lead to increased release of lipid within the protein matrix. Those phospholipids (LPC, LPE, and PA) are either bound with the protein or physically trapped inside the protein matrix. Pauly et al.³² studied the effects of incubation with different enzymes, organic solvents of different polarities or media of different ionic strength on isolated soft wheat starch. Their study indicated that different peptidase and lipases removed different levels of both protein and lipids from the starch. These variations in the amount were then proposed as the basis for the close association between protein puroindoline and lipids at the starch granules. They further suggested that the removal of proteins with enzyme correlates with the reduction in polar lipids on the starch.

CONCLUSIONS

A new method (Fig. 3) was developed to extract non-starch lipids in wheat flour. Protease hydrolysis before non-starch lipid extraction with WSB resulted in higher total non-starch lipid yield. A significant reduction in residual protein indicated higher purity of defatted wheat starch following enzymatic hydrolysis of the protein and lipid extraction with WSB. Disrupting protein network attributed to the higher lipid extraction yield after proteolysis compared to the direct extraction method, as measured by weighing of the dried extract. Moreover, protease-assisted lipid extraction resulted in higher levels of phospholipids, LPC, LPE, and PA, suggesting that those lipids are tightly associated with the protein. Future studies are proposed to analyze non-polar lipids and polar lipids (e.g. DGMG, MGMG, NAPE, and NALPE) that are not measured in this study. The new protease-assisted extraction method may be applied to analyze non-starch lipids in other types of wheat flours and other cereal flours.

ACKNOWLEDGEMENTS

The lipid analyses described in this work were performed at the Kansas Lipidomics Research Center Analytical Laboratory at Kansas State University. Instrument acquisition and lipidomics method development was supported by National Science Foundation (EPS 0236913, MCB 1413036, DBI 0521587, DBI 1228622), Kansas Technology Enterprise Corporation, K-IDeA Networks of Biomedical Research Excellence (INBRE) of National Institute of Health (P20GM103418), and Kansas State University. We thank Dr. Ruth Welti and Ms. Mary Roth at the Kansas Lipidomics Research Center Analytical Laboratory at Kansas State University for their constructive discussion on the lipid results.

The authors gratefully acknowledge the Ministry of Higher Education (MOHE) in Malaysia, for supporting the doctoral scholarship, and to the Universiti Putra Malaysia, UPM Serdang, Malaysia, for giving permission to one of the authors (Siti Farhiah Abdul Manan) to study at the Kansas State University, USA. This is contribution no. 19-077-J of the Kansas Agriculture Experiment Station.

Abbreviations:

DGDG: digalatosyldiacylglycerol
DGMG: digalactosylmonoglycerol
GL: galactolipid
GIPCer: glycosylinositol-phosphoceramide
LPC: lysophosphatidylcholine
LPE: lysophosphatidylethanolamine
LPG: lysophosphatidylglycerol
MAG: monoacylglycerol
MGDG: monogalactosyldiacylglycerol
MGMG: monogalactosylmonoglycerol
NALPE: N-acyl lysophosphatidylethanolamine
NAPE: N-acyl phosphatidylethanolamine
PA: phosphatidic acid
PC: phosphatidylcholine
PE: phosphatidylethanolamine
PG: phosphatidylglycerol
PI: phosphatidylinositol
PL: phospholipid
PS: phosphatidylserine
SQDG: sulfoquinovosyldiacylglycerol
WSB: water-saturated butanol

Footnotes

CONFLICT OF INTERST

The authors declare no conflict of interest.

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[R1] 1.Chung OK, Ram MS, Park S-H, Ohm J-B, & Howitt CA (2009). Wheat lipids. In Khan K & Shewry PR (Eds.), Wheat: Chemistry and Technology (pp. 363–399). St. Paul, MN, USA: AACC International, Inc. [Google Scholar]

[R2] 2.Pareyt B, Finnie SM, Putseys JA, & Delcour JA (2011). Lipids in bread making: Sources, interactions, and impact on bread quality. Journal of Cereal Science, 54(3), 266–279. [Google Scholar]

[R3] 3.Morrison WR (1981). Starch lipids: A reappraisal. Starch - Stärke, 33(12), 408–410. [Google Scholar]

[R4] 4.Morrison WR (1978). Wheat lipids composition. Cereal Chem., 55, 548–558. [Google Scholar]

[R5] 5.Schaffarczyk M, Østdal H, & Koehler P (2014). Lipases in wheat breadmaking: Analysis and functional effects of lipid reaction products. Journal of Agricultural and Food Chemistry, 62(32), 8229–8237. [DOI] [PubMed] [Google Scholar]

[R6] 6.Chung OK (1986). Lipid-protein interactions in wheat flour, dough, gluten, and protein fractions. Cereal Foods World, 31(3), 242–256. [Google Scholar]

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PERMALINK

Extraction of non-starch lipid from protease treated wheat flour

Siti Farhiah Abdul Manan

Jihong Li

Chao-Feng Hsieh

Jon Faubion

Yong-Cheng Shi