Cashew nuts are kidney-shaped seeds obtained from apples of the cashew tree (Anacardium occidentale L) [1]. The seed has protective inner and outer shells that offer protection to the nut. Cashew is of great economic commercial value in Nigeria [2] because of the value chain products obtained from it. The nut serves as food/food ingredient in the production of other consumer products. Cashew nuts contain reasonable amounts of nutrients such as protein, fats, ash, fiber, carbohydrates, minerals, etc., needed for body metabolism [3]. Cashew nut milk is one of the important products obtained from cashew nuts. Tamuno and Monday [4] reported cashew nut milk to contain appreciable amounts of nutrients required by the body.

The presence of some anti-nutrients can prevent the bioavailability of these nutrients for metabolic activities of the body. Cashew nuts have been reported to contain anti-nutrients in them. Anti-nutrients, according to Popova and Mihaylova [5], are substances that are either natural or synthetic that can influence the absorption and utilization of nutrients and may be capable of producing adverse effects in the process. Veer et al. [6] opined that plants possess these natural substances for protection. These anti-nutrients get to the body via food/diet, and some of these toxins build up in the body to harmful levels [6]. Essack et al. [7] affirmed rashes, headache, bloating, and nutritional deficiency as some of the symptoms associated with large amounts of anti-nutrients in the body.

However, despite causing health-related challenges in large amounts, studies have reported positive health-related effects of some of these anti-nutrients acting as anti-tumor, anti-microbial, anti-inflammatory, and kidney stone inflammatory effects [8,9], among others, thus finding potential applications in the pharmaceutical industries.

Recently, the use of cashew nuts and their products has expanded in the food sector. Cashew nuts are used as a topping in ice cream. It is also blended with water to extract milk, which is used in the production of non-dairy products. The level of anti-nutrients in the nuts and/or their products depends on the processing techniques employed. A higher amount of these anti-nutrients may be detrimental to the body if ingested. To this end, this study was conducted to determine the amount of anti-nutrients in raw cashew nuts and the influence/effect of extraction variables on cashew nut milk with the view of reducing anti-nutritional factors to a minimal level.

Materials

Raw cashew nuts (Brazilian red variety) were obtained from an 8-12-year-old cashew tree from a farm in Ayedun, Kwara State, Nigeria. Cashew nut seeds were properly identified in the Department of Plant Biology, University of Ilorin, with herbarium identification number UILH/001/835/2023. All chemicals and reagents used were of analytical grade and purchased from chemicals and reagents stores in Ilorin, Kwara State, Nigeria.

Cashew Nut Processing

Raw cashew nuts were sun-dried for 14 days at ambient temperature (25 + 5°C) to relatively low moisture content until ready for use. The method of Olayinka et al. [10] was adopted for cashew nut processing. Raw cashew nuts were sorted and soaked in a 5% NaCl solution overnight. The soaked nuts were washed and steam boiled using a steam boiler (at a pressure of 0.62 Mpa) for 40 min contact time (between steam and cashew nuts). The steamed nuts were cool, shelled using a foot-pedaled shelling machine, and the nut roasted for 40 min.

Cashew Nut Milk Extraction

The nuts were dry-milled using a blender (Saisho S-748 Model), and the milk was extracted following a preliminary experiment of optimization of cashew nut milk extraction according to the experimental runs and levels in Table 1. There were 3 independent variables: extraction time (X₁), extraction temperature (X₂), and the ratio of cashew nut to water (X₃). Each variable has 5 different coded levels from negative star point (-α), low (-1), medium (0), high (+1), to positive star point (+α). The blended cashew nut was mixed at various ratios with distilled water and allowed to stand in a water bath at temperatures and times as stipulated by the central composite design (CCD) of RSM stated in Table 1. Thereafter, it was sieved using a cheesecloth to obtain the milk. The milk was pasteurized at 90°C for 30 min and allowed to cool gradually to a temperature of 37°C.

Table 1: Experimental Range and Levels of Independent Variables.

Determination of Anti-nutrients

Tannin was determined using the method described by Obum-Nnamdi et al. [11]. About 10 mL of 70% acetone was mixed with 0.2 g of the sample in a 50 mL bottle. The bottle was shaken in an ice shaker for 2 hours at 30°C and centrifuged; the supernatant was kept in ice. About 0.2 mL and 0.8 mL of the supernatant and distilled water were mixed in a test tube to obtain a test solution of 1.0 mL. A 0.5 mg/mL of the standard tannate stock was treated with 0.5 mL distilled to make a standard solution of 1.0 mL. 0.5 mL of Folin-Ciocalteu reagent and 2.5 mL of 20% Na₂CO₃ were added to test samples and standard solutions, vortexed, and incubated at room temperature for 40 min. The absorbance was read at 725 nm, and the concentration of tannin was extrapolated from the standard calibration curve.

Alkaloid determination was done with a spectrophotometer by adopting the method described by Obum-Nnamdi et al. [11]. 0.5 g of the sample was dissolved in a mixture of 96% ethanol and 20% tetraoxosulphate (vi) acid (1:1), and 1.0 mL of the filtrate was mixed with 5.0 mL of 60% tetraoxosulphate (vi) acid for 5 min. After that, 2.0 mL of 0.5% formaldehyde was added and let to sit for 3 h. The reading was taken at 565 nm absorbance, and the value extrapolated accordingly.

0.5 g of the sample was boiled in 20 mL of 1 NHCl for 4 h. After cooling, 50 mL of pet ether was added to the filtrate for the ether layer, which will then be evaporated to dryness. The residue was treated with 5.0 mL of acetone-ethanol. A 6.0 mL ferric sulfate reagent was added to three test tubes containing 0.4 mL of each, followed by 2 mL of concentrated tetraoxosulphate (vi) acid. After 10 min, it was well mixed, and the absorbance was measured at 490 nm to obtain saponin content as described by Obum-Nnamdi et al. [11].

Total phenolic content was determined according to the Folin-Ciocalteu method described by Olapade et al. [12] using gallic acid as a standard. 1.0 mL of standard solution of concentration 0.01, 0.02, 0.03, 0.04, and 0.05 mg/mL of gallic acid were prepared in methanol. Concentrations of 0.1 and 1.0 mg/mL of cashew nut milk were prepared in ethanol, and 0.5 mL of each sample was introduced into test tubes and mixed with 2.5 mL of a 10-fold dilute Folin-Ciocalteu reagent and 2 mL of 7.5% sodium carbonate. The tubes were covered with parafilm and allowed to stand for 30 min at room temperature before the absorbance was read at 760 nm using a spectrophotometer.

Flavonoid content was determined according to the method of Zhishen et al. [13]. To 100 microliters of the samples, 4 mL of distilled water was added. It was followed by the addition of 0.3 mL of 5% sodium nitrite and allowed to stand for 5 min. Thereafter, 0.3 mL of 10% aluminum chloride was added. After 6 min, 2 mL of 1 M sodium hydroxide was added to the mixture. The mixture was diluted immediately using 3.3 mL distilled water and mixed thoroughly. The absorbance was taken at 510 nm versus a blank. Catechin was used as a standard for the calibration curve.

Phytate content determination was according to the methods of Olagunju et al. [14] and Balogun et al. [15]. Approximately 8 g of sample was mixed with 200 mL of 2% HCl and vortexed for 3 h. The solution was filtered, and 10 mL of 0.3% NH₄SCN was added to 50 mL of the filtrate. The solution was titrated against 0.00195 g/mL ferric chloride solution until a persistent brownish yellow color was observed.

Where Y = response; β₀ = constant; β₁ … β₃₃ = coefficients; X₁, X₂ and X₃ = independent variables.

Anti-nutritional Contents of Raw Cashew Nut Seeds

Results of the anti-nutritional composition of raw cashew nuts are presented in Table 2. The values of tannin, alkaloid, saponin, and phenol, flavonoid, and phytate contents obtained in this study were 53.43, 49.02, 63.79, 86.88, 42.16, and 186.59 mg, respectively. These values showed that cashew nuts are high in anti-nutrients. Tannin, according to Emelike and Ebere [16], is a water-soluble polyphenolic compound that can precipitate proteins, making them not available, thus decreasing protein palatability and digestibility. In addition, 53.43 mg of tannin obtained in this study can be compared to Dakuyo et al. [17], who reported values ranging from 28.95–108.12 mg of tannin from different geographical areas in their study. The phytate content (186.59 mg) obtained in this study is higher than the range of values (49–118 mg) reported by Dakuyo et al. [17]. 

Table 2: Anti-nutritional Contents of Raw Cashew Nut Seeds.

Anti-nutritional Contents of Cashew Nut Milk

The anti-nutritional content of cashew nut milk after processing (soaking in 5% brine overnight, steaming, and roasting) and extraction (under different extraction times, temperatures, and cashew nut to water ratios) is presented in Table 3. Results indicated that tannin content ranged from 9.52 to 11.19 mg/mL. The least and highest values were obtained under experimental conditions of runs 10 and 7, respectively. These values are within the reports of N’Cho et al. [18], who reported values ranging from 0.0–19.52 mg, but higher than the reports of Akujobi et al. [19], who reported a range of values from 18.3–184.97 µg. Emelike and Ebere [16] reported a range of values between 0.07 and 2.84 mg, while Ogunwolu et al. [20] reported tannin values from 0.80–1.99% in cashew. The differences in the tannin content could be attributed to the processing methods employed in the respective studies. Table 3 showed that values of alkaloid content were between 10.23 and 12.71 mg/mL. Cashew nut milk extracted under extraction time of 15 min at 50°C using a ratio of 1.32 of cashew nut to water resulted in a low alkaloid value (10.23 mg/mL), while cashew nut milk extracted under extraction time of 10 min, extraction temperature of 40°C using a ratio of 4 of cashew nut to water resulted in high alkaloid content (12.71 mg/mL). These values are, however, higher than the values (2.73-2.77 mg) reported by Akujobi et al. [19].

Table 3: Anti-nutritional Factors of Cashew Nut Milk under Varying Process Variables.

Table 3 showed that saponin contents ranged from 30.00 to 36.90 mg/mL. The least and highest values were obtained under experimental conditions of run 13 and run 8, respectively. Also, phenol content of cashew nut milk ranged from 0.22 to 0.88 mg/mL. Experimental run 10 (15 min extraction time, 50°C extraction temperature at a 1.32 ratio of cashew nut to water) resulted in low phenol content, while the highest value (0.88 mg/mL) was obtained using experimental conditions of run 11 (20 min extraction time, 40°C extraction temperature at a 4 ratio of cashew nut to water). Akujobi et al. [19] reported a range of values between 164 and 165 µg, which are lower than the values obtained in this study. Also, the results of the phenol content obtained are lower than the range of values (137-274 mg) reported by Alasalvar and Bolling [20]. Results of flavonoid content revealed the values between 19.86 and 27.38 mg/mL. Cashew nut milk extracted at 15 min time, extraction temperature of 66.82°C, and cashew nut to water ratio of 2 recorded the highest value (27.38 mg/mL). Akujobi et al. [19] reported 3.37-3.47 mg of flavonoid content, while Alasalvar and Bolling [21] reported 2.0 mg, which are lower than what is recorded in this study. Still, this difference could be attributed to processing techniques employed in cashew nut processing. Phytate contents were between 71.89 and 102.37 mg/mL. The highest and lowest values were obtained under experimental conditions of run 8 and run 7, respectively. Results of phytate content obtained in this present study are lower than the range of values (0–37.57 mg) reported by N’Cho et al. [18]; 290 mg reported by Alasalvar and Bolling [21]; and 3.54–6.44 g/kg reported by Ogunwolu et al. [20]. In general, the differences in anti-nutrients could be attributed to sun drying method, steaming time, soaking time, roasting time, as well as extraction time and temperature. Ojo [22] opined that soaking time had an effect on the levels of anti-nutrients in plant materials.

Response Surface Modelling and Data Interpretations

Following fit statistics of response surface methodology, the model with the highest sequential p-value, coefficient of determination (R²), adjusted R², predicted R², a good coefficient of variation (CV), and adequate precision above 4 was selected as the best-fitted model for analysis of the respective response. Hence, tannin and alkaloid content of cashew nut milk were fitted to a reduced quadratic model. The choice of reduced model is to enable better efficiency of the fitted model to describe the data. Sudamalla et al. [23] reported that, to develop a regression model that is statistically significant, there are a number of insignificant terms; the insignificant terms are removed from the model, thus resulting in a model’s (reduced) efficiency in describing the response better than in its normal form. Also, saponin contents were modeled using a reduced linear model. The response surface quadratic model was used to model phenol content and flavonoid contents, while experimental data obtained for phytate content of milk samples were fitted to the response surface linear model (Table 4).

Furthermore, the effectiveness of a model is usually ascertained using R². The best R² value has been estimated between 0.8 and 1.0 [24]. R² measures the amount of variation of the observed values around the mean explained by the model. However, R² values below 0.8 have been reported to imply fair fit of the response surface model but very useful in making predictions about the experimental data [25,26]. A CV below 10% describes good precision and reliability, and the model is considered reasonably reproducible [27], while values above 10% indicate only the reliability of the experiment in making predictions. Adequate precision measures the signal-to-noise ratio, and a ratio greater than 4 implies that the model can be used to move in any direction within the experimental domain [25]. Consequently, analysis of variance showed that the fitted response surface model was predictable, reliable, and effective in describing the respective anti-nutritional contents of cashew nut milk as well as capable of navigating within the experimental domain.

Effects of Extraction Time, Temperature, and Cashew Nut to Water Ratio on Anti-Nutritional Factors of Cashew Nut Milk

The effects of extraction time, extraction temperature, and cashew nut to water ratio showed that the quadratic term of cashew nut to water ratio had a significant effect (F = 7.19; P = 0.0158) on the tannin content of cashew nut milk (Table 4). This effect could be expressed using equation (2).

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