Reduction of Cyanogenic Compounds in Flaxseed (Linum usitatissimum L.) Meal Using Thermal Treatment

Abstract

Flaxseed (Linum usitatissimum L.) provides multiple nutritional benefits, including high quality protein, dietary fiber, and is the most abundant source of α-linolenic acid (C_18:3). However, the presence of anti-nutritional factors, such as cyanogenic compounds, restricts flaxseed's consumption as a food or feed. This study investigated the reduction of cyanogenic compounds, measured as hydrocyanic acid (HCN), in full-fat flaxseed using extrusion processing without a die by following the response surface methodology. The ranges of processing variables selected were: barrel exit temperature of 76.3–143.6°C; screw speed of 59.6–160.5 rpm; and feed rate of 26.4–93.6 kg/h. The experimental values of HCN reduction obtained were from 60.8 to 86.6%. Optimum extrusion conditions of barrel exit temperature, screw speed, and feed rate were found to be 143.6°C, 133.5 rpm, and 57.8 kg/h for maximum (89.1%) reduction of HCN. This effect was mainly dependent on barrel exit temperature, whereas screw speed and feed rate had no or minimal effect. The mutual interaction effect of barrel exit temperature and screw speed was found to be significant (p ≤ 0.01). The degree of correlation (R²) for HCN reduction was 97.2%, which showed the validity of applied second-order response model. The results of this study demonstrated that significant reduction of HCN in flaxseed can be achieved commercially using an extruder without a die.

Keywords:

• Flaxseed
• Anti-nutritional factor
• Hydrocyanic acid
• Heat treatment
• RSM

INTRODUCTION

Flaxseed (Linum usitatissimum L.) is one of the world's oldest domesticated crops, which is cultivated as either an oil or fiber crop. The seed of flax, known as “flaxseed,” often used interchangeably with the term “linseed,” is flat or oval (4–6 mm) with a pointed tip and varies in color from dark-brown to pale-yellow. World flax production has remained constant at about 2.5 million tonnes as compared with other oilseed crops and represents 1% of total world oilseeds supply.[1,2]

Some uses of flaxseed for human consumption include adding it to ready-to-eat (RTE), breakfast cereals, breakfast drinks, specialty breads, muffins, and other bakery products in Europe and Asia.[3] The main benefits of using flaxseed in human nutrition are its very high dietary fiber (28–30%) and abundance of α-linolenic acid (C_18:3) content.[4,5] An intake of flax omega-3 fatty acids decreases serum cholesterol, which beneficially affects blood pressure, thrombosis, atherosclerosis, arterial compliance, and hyperlipidemia response.[6,7]

The flaxseed meal remaining after oil extraction has limitations for livestock feed due to the presence of anti-nutritional compounds, primarily cyanogenic glycosides, which are an integral part of flax constituents.[8,9] Cyanogenic compounds are glycosides of aldehyde or ketone cyanohydrin. The naturally present cynanogenic compounds in flaxseed are diglycosides (linustatin and neolinustatin) and monoglycosides (linamarin and lotaustralin). When the plant tissue is damaged, cyanogenic compounds in the presence of enzyme linase produce 100–300 mg/kg hydrocyanic acid (HCN). Intake of these compounds may result in acute or chronic intoxication by complexing with ferric ions of mitochondrial cytochrome oxidase and ultimately lead towards abnormal respiration and nervousness.[10] With respect to the sensory quality, cyanogenic compounds impart an astringent taste to the diet.[11]

Increasingly, more attention is being given to reduce the toxic effect and improve the nutritional quality of flaxseed through appropriate processing. Various attempts have been made on lab-scale to reduce the cyanogenic compounds in flaxseed meal by boiling in water, microwave roasting, wet autoclaving, or acid treatment.[12–14 14 Extrusion processing, which uses high temperature and short time (HTST), has long been employed in preparation of human and pet foods. The beneficial effects of heat during extrusion to destroy anti-nutritional compounds in soybean, sunflower, beans, peas, canola, and cottonseed have been well documented but limited information is available on HCN reduction in flaxseed using extrusion technology. Past research has shown that incorporation of extruded ingredients resulted in improved palatability, digestibility, and bioavailability for different product formulations.[15,16]

The response surface methodology is a mathematical and statistical approach, which has been widely used for optimizing the response of multivariate parameters during modeling of extrusion processing.[17,18] The objective of this study was to investigate the influence of extrusion processing of full-fat flaxseed at different barrel exit temperatures, screw speeds, and feed rates on the reduction of HCN using response surface methodology.

MATERIALS AND METHODS

The flaxseed (cv. Chandni) was procured from Oilseeds Research Institute, Faisalabad, Pakistan. Seeds were cleaned to remove any debris or field dirt and stored in sealed polyethylene bags at 5 ± 1°C.

Extrusion Processing

A single-screw extruder (Extru-tech E325, Extru-tech, Sabetha, KS, USA), with a barrel length to diameter ratio of 9:1 was used for the production of full-fat flaxseed meal. This extruder is divided into six zones along the length of the barrel, with Zone-1 designated as inlet section and Zone-6 nearest the barrel discharge/exit section.

Table 1  Coded and actual levels of independent variables used for optimization of single-screw extrusion processing conditions as determined by the central composite design (CCD)

	Coded levels
Independent variable	−1.682	−1	0	1	1.682
Barrel exit temperature (°C)	76.3	90	110	130	143.6
Barrel screw speed (rpm)	59.6	80	110	140	160.5
Feed rate (kg/h)	26.4	40	60	80	93.6

The screws and steamlocks configuration were arranged in such a manner to provide a progressively tighter pitch and greater resistance from inlet section to outlet zone. The inlet section was characterized with wide flight tapered screws, Zone-2, -3, and -4 desirably with intermediate flight spacing while Zone-5 and -6 were affixed with tight flight screws to compress the raw material. A temperature probe was set at the end of the barrel section for the determination of barrel exit temperature (BET). Medium shear extruder classification was used for all experiments. The feed rate (FR) for different treatments was calculated in relation to feeder speed (FS). The FS was set at different rates (8, 12, 16, or 20 rpm) for variable processing times (1, 2, or 3 min). The average material collected during different FS was used for the development of regression equation, and FR was determined accordingly as described in Table 1.

The extruder die was removed during the processing of flaxseed under different extrusion runs. This was done to avoid potential oil percolation at the die end during processing of whole seeds having high lipid content (>16%).[19,20] The extruded samples were cooled down to room temperature and packaged in sealed polyethylene bags for analysis of HCN content.

Estimation of Hydrocyanic Acid

The HCN content of flaxseed meal was estimated by alkaline titration according to the AOAC Method 26.115.[21] Flaxseed sample (20 g) was taken in a Kjeldhal flask following the addition of 200 mL of distilled water and slowly mixed the sample. The sealed flask was held for 3 h to allow proper hydrolysis process of sample mixture. After rest, the distillation process was carried out by connecting the flask to a vapor distilling apparatus and the distillate was collected in a 250-mL flask containing 20 mL of 2.5% NaOH solution. Then, 8 mL of 6 M NH₄OH and 2 mL of 5% KI solution were transferred into the distillate solution before titration against 0.02 M AgNO₃ standard solution using a microburette. The used volume of AgNO₃ standard solution during the titration was noted for each individual sample. Titration of a blank experiment against AgNO₃ standard solution was also run for comparison under the same room conditions. The following formula was used to calculate the total contents (mg) of HCN, as given below:

In this trial, the reduction rate of HCN from flaxseed was taken as response and was calculated according to the formula mentioned by Wu et al.:[16]

Experimental Design and Statistical Analysis

This experimental study was carried out to determine the optimum values of independent variables on which maximum dependent response was obtained using the rotatable central composite design (CCD), as described by Montgomery.[22] Extrusion cooking modeling for quality changes involves multiple process control input parameters and selected product output properties. The effect of BET (76.3–143.6°C), SS (59.6–160.5 rpm), and FR (26.4–93.6 kg/h) on the response value of HCN reduction was examined. For better accuracy and simplification of result interpretation, the coded multiple regression coefficients were used and reconverted into original values at the end of the experiment using MATLAB^® (Version 7.9.0) software (Mathworks, Inc., Natick, MA, USA). The coded coefficients at five levels used in this study were −1.682 (lowest level), −1, 0 (medium level), and 1, 1.682 (highest level), respectively (Table 1).

Data was analyzed using the polynomial equation generated by BB experimental design:

(1)

where Y is the dependent variable; b₀ is the intercept; b₁ to b₃₃ are the regression coefficients; and x₁, x₂, and x₃ are the independent variables.

RESULTS AND DISCUSSION

Fitting the Proposed Model

The average HCN content in raw flaxseed was found to be 198.42 mg/kg (on wet basis). The total number of experimental runs was 20 as determined by the CCD. Details of independent variables combination for extrusion optimization and HCN reduction in flaxseed with predicted values are presented in Table 2. The HCN reduction in extruded flaxseed meal for the 20 combinations of BET, SS, and FR independent variables was found in the range of 60.8 to 86.6%. There is very limited published data that provides an information or support to the reduction of HCN content in flaxseed meal using single-screw extruder with or without die. It is to be noted from response factor values (Table 2), that there is an interaction between extruder independent parameters and HCN reduction in flaxseed.

The regression equation developed for optimization of independent variables for maximum level of HCN reduction was

(2)

where Y is the response, x ₁ = BET (°C), x ₂ = SS (rpm), and x ₃ is FR (kg/h).

Table 2  Experimental and predicted values of hydrocyanic acid (HCN) reduction in flaxseed using single-screw extrusion processing as determined by the central composite design (CCD)

	Independent variables			HCN reduction (%)
Extrusion run	BET^1 (°C)	SS^2 (rpm)	FR^3 (kg/h)	Experimental value	Predicted value
1	−1	−1	−1	68.4	66.9
2	−1	−1	−1	77.3	76.4
3	−1	−1	−1	63.8	62.6
4	−1	−1	−1	82.6	81.4
5	−1	−1	1	66.7	65.6
6	−1	−1	1	76.9	75.8
7	−1	1	1	60.8	59.4
8	1	1	1	79.6	78.8
9	−1.682	0	0	61.4	63.3
10	1.682	0	0	86.6	87.8
11	0	−1.682	0	67.4	69.0
12	0	1.682	0	66.5	68.0
13	0	0	−1.682	69.2	70.9
14	0	0	1.682	66.7	67.7
15	0	0	0	75.9	75.4
16	0	0	0	75.4	75.4
17	0	0	0	75.6	75.4
18	0	0	0	75.8	75.4
19	0	0	0	75.2	75.4
20	0	0	0	75.3	75.4
^1Barrel exit temperature; ^2screw speed; ^3feed rate.

Table 3  ANOVA of the predicted second-order polynomial model for hydrocyanic acid (HCN) reduction present in flaxseed

Source of variation	Degrees of freedom	Sum of squares	Mean squares	F-ratio
Model	9	—	—	—
Linear effects	3	731.105	243.702	96.250***
Interaction effects	3	44.894	14.965	5.910*
Quadratic effects	3	140.272	46.757	18.467**
Residual	10	25.320	2.532	—
Lack of fit	5	24.926	4.985	63.372
Pure error	5	0.393	0.079	—
Total	19	941.5895	—	—
***Significant at 0.01 level; **significant at 0.05 level; *significant at 0.10 level.

The predicted values of HCN reduction in extruded flaxseed meal were calculated using a regression model and were compared with experimental values in order to evaluate the validity of the model. Degree of correlation (R ²) computed for HCN reduction was found to be 97.2%, which indicates the proficiency of the applied second-order polynomial response model.

Table 3 shows the analysis of variance (ANOVA) obtained by fitting of the experimental data. The analytical results indicated that reduction of HCN was significantly affected by the model effects. The order of model effects observed during reduction of cyanogenic compounds in flaxseed was linear > quadratic > interaction. The significance of each coefficient was determined using the t-test of response and p value in Table 4. The corresponding coefficient was more significant when absolute t-value became larger and the p value became smaller (p ≤ 0.01). The t-values and p values are listed in Table 4. Results showed that the BET, mutual interaction term of BET and SS, and the quadratic term of SS and FR had the largest effect on HCN reduction.

Table 4  Regression coefficients of the predicted second-order polynomial model for hydrocyanic acid (HCN) reduction in flaxseed

Coefficients		Response variable Y	t-Value of Y	P value
Linear	β0	75.4425
	β1	7.2547**	16.4158	0.0001
	β2	−0.2939	−0.6650	0.5211
	β3	−0.9625*	−2.1779	0.0544
Interaction	β12	2.3125**	4.0047	0.0025
	β13	0.1625	0.2814	0.7841
	β23	−0.4875	−0.8442	0.4183
Quadratic	β11	0.0537	0.1248	0.9031
	β22	−2.4382**	−5.6683	0.0002
	β33	−2.1731**	−5.0520	0.0005
**Significant at 0.01 level; *significant at 0.10 level.

Single Factor Analysis

A closer examination of each independent factor was taken into account for response value during the experimental single-screw extrusion. For the development of single factor equations, two of three factors were set at mean values. The mean values for BET, SS, and FR were 110°C, 110 rpm, and 60 kg/h, respectively. The respective regression equations for individual factor are given below:

(3)

(4)

(5)

The extruder BET was shown to impart a strong effect on reduction of HCN in flaxseed. The reduction rate of HCN in flaxseed was slow at low temperature range during the initial point of extrusion. However, it appears that the increase in temperature may improve the HCN reduction. The action of extruder SS and FR had minimal effect on the HCN reduction. The results show that SS and FR represents a very active source of HCN reduction on low level, but at high SS and FR, it leads towards a negative decrease in reduction action (Fig. 1).

Figure 1 The effect of barrel exit temperature (BET, 76.3–143.6°C), screw speed (SS, 59.6–160.5 rpm), and feed rate (FR, 26.4–93.6 kg/h) on hydrocyanic acid (HCN) reduction during flaxseed meal production. The BET, SS, and FR were all standardized proportionally according to their coded level in the range of −1.682 to 1.682.

Analysis of Mutual Interaction Effect

The optimum extruder conditions for the maximum reduction of HCN in flaxseed were obtained by varying two independent parameters and fixing the third variable at the coded zero level. The mutual impact of BET and SS on the HCN reduction by setting the FR at 60 kg/h is shown in Fig. 2a. It can be concluded from the data shown in Table 2 that there was significant interaction between BET and SS for the reduction of HCN. The maximum predicted value of HCN reduction (89.1%) was found with a BET of 143.6°C and SS of 133.5 rpm. Increasing the BET at low SS increased the HCN reduction in flaxseed. The trend in output results can be compared to the reduction of similar anti-nutritional components in other materials. The concentration of anti-nutritional compounds reduction has been the subject of extensive investigation as reported by Mukhopadhyay et al.,[23] who predicted maximum reduction value of tannins (61.25%) in linseed meal at optimum values of BET and SS, 82.5°C and 90 rpm, respectively. The same description focusing on reduction of tannins in sesame meal was also found at optimum values of the process variables.[24]

Figure 2 Mutual interaction effect of barrel exit temperature (BET, °C) and screw speed on the hydrocyanic acid (HCN) reduction during flaxseed meal production at: (a) feed rate—FR 60 kg/h; and (b) at screw speed—SS, 110 rpm.

Setting the SS at 110 rpm, the maximum HCN reduction (87.8%) was obtained at BET 143.6°C and FR 57.8 kg/h (Fig. 2b). It can be shown that mutual influence of BET and FR is slightly significant. Increasing the BET at low FR positively influenced the HCN concentration. The high FR with increasing BET had insignificant effect on HCN reduction during flaxseed extrusion. Within this context, optimized results for barrel temperature (146.0°C) and FR (32.7 kg/h) were found for HCN removal rate (93.23%) during the twin-screw extrusion detoxification technique on flaxseed via stepwise nonlinear response surface methodology.[16]

As shown in Fig. 3, when the BET was set at a zero-coded level, i.e., 110°C, the maximum value of HCN reduction (75.5%) in flaxseed meal was found at SS 109 rpm and FR 55.5 kg/h. The mutual influence of SS and FR was found to be insignificant. The differences in HCN can be explained with the stay time of flaxseed in the cavity of the extruder barrel. Extrusion at low SS, which resulted in longer residence time in the extruder barrel, positively influenced the HCN reduction. However, at high SS, reduction of HCN content was somewhat lower, which was supported by the results of Liang et al.,[25] who reported that high SS resulted in less glucosinolate removal from rapeseed meal.

Figure 3 Mutual interaction effect of screw speed (SS, rpm) and feed rate (FR, kg/h) on the hydrocyanic acid (HCN) reduction during flaxseed meal production at BET 110°C.

CONCLUSION

The results of this study demonstrated that the full-fat flaxseed extrusion (without a die) is an effective processing technique for reducing HCN concentration. Optimum extrusion conditions of BET, SS, and FR were found to be 143.6°C, 133.5 rpm, and 57.8 kg/h for maximum (89.1%) reduction of HCN. Among the experimental conditions used in this study, BET was found to be most significant as compared to SS and FR for reduction of cyanogenic compounds. The mutual interaction effect of BET and SS was found significant. Based on the results of this study, it can be concluded that extrusion cooking can be successfully used to reduce or eliminate anti-nutritional factors in flaxseed meal.

ACKNOWLEDGMENTS

The authors wish to thank the Higher Education Commission (HEC) of Pakistan for the support of this research. Special thanks to Dr. Muhammad I. Khan (National Institute of Food Science, University of Agriculture, Faisalabad) for use of his laboratory equipment and supplies.