Introduction

Cereal products are staple foods in most human diets, providing a major proportion of dietary energy (more than 50%) and nutrients (approximately 75% carbohydrates and 6–15% proteins) [1]. Most cereals are milled for flour or meal that is used in dough formation. Therefore, the variety of cereal products can consist of flour, breakfast cereals, snack foods, corn meal, dough, pasta, breads, pastries, etc. [2], when various vegetable, fruit, or animal-based components are also used. For example, bakery products can consist of main ingredients (flour, fat or oil, sugar, water, chemical leavening agents) and minor ingredients (salt, egg, emulsifier, milk powder, or flavouring compounds) [3]. The dough for pasta making is traditionally prepared with durum wheat semolina flour and water, while other added materials can be eggs, vegetables or different colouring and flavouring substances [4]. Therefore, cereal products contribute to important nutrients in the diet, such as protein, lipids, dietary fibre, B vitamins (including thiamine, niacin, riboflavin), vitamin E, or minerals (calcium, magnesium, potassium, phosphorus, iron, and sodium) [1].

Cholesterol is a biomolecule that has a bidirectional (both good and bad) role in the body, therefore, some state authorities have recommended a maximum intake of 300 mg per day for adults [5, 6]. The cholesterol content in foods is variable, such as in eggs around 274 mg/egg [7] or in milk products from 104 to 2740 mg/kg, depending on the fat content [8]. Animal-based food components, which are often used in the formulation of cereal products to improve nutritional and technological characteristics, are also a source of the so-called hidden cholesterol. In addition to that, during some food processes, this compound forms its oxidised derivatives (cholesterol oxidation products), which can exhibit toxic effects on the human organism [9, 10]. Lercker and Rodriquez-Estrada [11] determined the cholesterol content in biscuits, snacks, and noodles prepared with eggs in the range of 20–370 mg/kg of lipids and the cholesterol oxidation product (7-ketocholesterol) in 1.5–54.4 mg/kg of lipids. Zardetto et al. [9] reported the cholesterol content in pasta at 146 mg/100 g of dry matter.

The most serious problem related to cholesterol is its association with the incidence of cardiovascular diseases (CVDs). CVDs are the leading cause of more than 4 million deaths in Europe each year. The key initiating event is the retention of low-density lipoprotein cholesterol (LDL-C) and other cholesterol-rich apolipoprotein B-containing lipoproteins within the arterial wall [12]. Therefore, several studies describe methods to reduce cholesterol content in animal foods. The most promising is the inclusion of the cholesterol molecule into β-cyclodextrin (β-CD) cavity to form an ´inclusion complex´ with its subsequent elimination from a matrix; it was shown that the elimination effectivity can reach up to 98% in various milk or egg products without changing their chemical and functional properties [6, 13]. According to Kolarič et al. [14], it was possible to reduce the cholesterol content by β-CD in milk about 98.1%, in butter 96.7% and in fresh cheese about 97.7%. Alonso et al. [15] reported that it was possible to reduce the cholesterol content in powdered eggs by 82.1% using β-CD treatment technology.

Up to date, there is a lack of data on the application of low-cholesterol animal-based components in cereals products. Therefore, the mail goal of this study was to prepare frequently consumed cereal products by addition of conventional as well as low cholesterol animal-based components and to study effects of this addition on technological, culinary, and texture profiles of the products. The experimental part of the work can then be divided into three parts: I. Laboratory experiment no. 1—decrease in cholesterol content in biscuit samples using low-cholesterol butter prepared by application of β-CD; II. Laboratory experiment no. 2—decrease in cholesterol content in pasta samples using low cholesterol egg melange prepared by the application of β-CD; and III. Laboratory experiment no. 3—decrease in cholesterol content in muffin samples by the use of low-cholesterol egg melange and milk prepared by the application of β-CD.

Materials and methods

Materials

For the determination of cholesterol content, 32 commercial samples were tested, including 11 biscuit samples, 6 pasta samples, 5 muffin samples, 4 salty snacks samples and 6 samples of other bakery products. All samples were purchased from local markets. Cream (40% fat content), homogenised UHT cow milk (3.5% fat content), pasteurised egg melange (93%), durum wheat semolina, fine wheat flour, semi-coarse flour, baking powder, sugar, sunflower oil, and salt were also purchased from a local market. β-CD (fine chemical) was obtained from Cyclolab (Cyclodextrin Research and Development Laboratory Ltd., Budapest, Hungary). Potasium hydroxide (KOH), chloroform, n-hexane, ethanol, and anhydrous sodium sulphate were of analytical grade and obtained from Centralchem s.r.o. (Bratislava, Slovakia). Methanol and acetonitrile were HPLC grade (Fisher Chemical, Loughborough, UK).

Determination of cholesterol content

The determination of cholesterol content was performed according to previous studies [14, 16]. Different amounts of samples (5.0 g for milk and biscuits, 2.0 g for muffins, 1.0 g for egg melange and pasta, and 0.5 g for butter) were saponified with 20 mL of 1 mol. L−1 KOH methanolic solution for 15 min. After cooling to laboratory temperature, cholesterol was extracted from the samples with 20 mL of n-hexane and chloroform mixture (1:1, v/v) in duplicate with the addition of 1 mL of ethanol (96%) and 10 mL of deionised water. The extracts collected were then filtered through anhydrous sodium sulphate and evaporated to dryness using a rotary vacuum evaporator (Witeg, Wertheim, Germany). The residue was dissolved in 5 mL of methanol and filtered using a PTFE filter with 0.2 µm particle size (Agilent Technologies, Santa Clara, CA, USA) to vial.

The HPLC system (Agilent Technologies 1260 infinity system, Santa Clara, CA, USA) consisted of a vacuum degasser, a quarterly pump, an autosampler and a UV-DAD detector. The isocratic elution was performed using a mobile phase of acetonitrile and methanol 60:40 (v/v) at a flow rate of 0.5 mL min−1. The stationary phase was C18 column (Zorbax Eclipse Plus, 2.1 × 50 mm, 5 µm particle size) with a guard column (Zorbax SB-C18, 4.6 × 12.5 mm, 5 µm particle size). The injection volume was 10 µL. The UV-DAD detector was set at 205 nm. The results were recorded using OpenLab software, ChemStation Edition for LC, and LC/MS systems (product version A.01.08.108).

Validation of analytical method

The complete validation procedure of the method for animal-based ingredients and samples was published in our previous studies [8, 16]. In addition, the linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision of method for the determination of cholesterol content in laboratory prepared biscuit samples is shown in this study. The method was validated according to Eurachem guide for the fitness for purpose of analytical methods [17]. Linearity was recorded by the correlation coefficient (R2) of the calibration curve. The calibration curve was calculated by plotting the UV signal against the known amount of cholesterol standards at 0.04, 0.06, 0.1, 0.15, 0.25, 0.35, 0.5, 0.8, 1.0, and 2.0 µg. LOD and LOQ were calculated as 3 or 10 times the standard deviation of the blanks divided by the slope of the calibration curve. The accuracy of the method was studied as a recovery test by standard addition at three concentrations (300, 500, and 700 mg/L). The precision was determined by repeatability and intermediate precision. Repeatability was recorded by analysing four replicates of samples on the same day, and intermediate precision was then evaluated on three different days. The precision was calculated from the standard deviation (SD) and relative standard deviation (RSD) [8].

Laboratory experiment no. 1

The preparation of low-cholesterol butter

Low-cholesterol butter (LCB) was prepared according to Kolarič et al. [14]. LCB samples were made from treated cream (40% fat content) with three concentrations of β-CD (2.0, 4.0, and 6.0% (w/w)). A control butter (CB) was prepared from standard untreated cream as well. 200 mL of cream was mixed (Arex-6 Connect Pro, Velp Scientifica, Usmate, Italy) with β-CD at 480 rpm, 40 °C, and for 20 min. The cream was then settled for 30 min at 4 °C and centrifuged (Hettich Zentrifugen, Tuttlingen, Germany) at 450 × g for 20 min. Then, the low-cholesterol cream was collected from the supernatant. Each cream was churched with electric blender (Hyundai Hand Mixer, Soul, Korea) until butter granules became visible. The butter was finally washed twice with cold tap water, moulded, and placed in containers for biscuit manufacturing [14].

The manufacture of biscuits

Biscuits were prepared according to the modified method by Nwakalor [18]. Sugar (45 g) and CB or LCB (55 g) were mixed well to a creamy consistency. Fine wheat flour (100 g), salt (1 g) and water (20 mL) were added to a creamy mixture. The prepared dough was rolled (3 mm) and cut into a round shape (50 mm in diameter). The biscuits were baked in a hot air oven (Mora, Czech Republic) at 170 °C for 10 min.

Qualitative characteristics of biscuits

The qualitative parameters of the biscuits were evaluated 2 h after baking. The volume of biscuits was measured using the rapeseed displacement method [19]. The specific volume (cm3 g−1) of each biscuit was calculated as the volume of the biscuit divided by the weight of the biscuit [20]. The volume index of biscuits and the spread ratio were measured using the method of Lauková et al. [21]. Weight loss (%) is determined according to the weight of the dough before baking and the weight of the product, which was detected one hour after baking according to Minarovičová et al. [22].

Texture properties of biscuits

Biscuit texture was measured using the TA.XT plus texture analyser (Godalming, Surrey, UK) using a Three Point Bend Rig (HDP/3PB) according to Lauková et al. [21]. A pre-test speed 1.0 mm s−1, test-speed 1.0 mm s−1 and post-test speed 10 mm s−1 were used. The gap distance was 26 mm.

Colour properties of biscuits

Samples for colour evaluation were ground to a fine powder and put in a cell. The colour characteristics were recorded using a UV–VIS spectrophotometer Cary 300 (Agilent Technologies, Santa Clara, CA, USA) with DRA-CA-30I (internal) sphere accessory and calculated as a CIEL*a*b* coordinates with respect to the illuminate D65 and a visual angle of 10°. The measured parameters were L* (lightness factor), a* (− a* = green, + a* = red), b* (− b* = blue, + b* = yellow), and YI (yellowness index). The spectrophotometer was standardised with a white plate supplied with the equipment. ΔE was calculated to estimate the visual differences between control samples and low-cholesterol samples according to Eq. (1) [14, 23].

$$\Delta E = \sqrt {{\left( {\Delta L^* } \right)^2 + \left( {\Delta a^* } \right)^2 + \left( {\Delta b^* } \right)^2 }}$$
(1)

The results were recorded with the Cary WinUV software (version 4.20(468)).

Laboratory experiment no. 2

The preparation of low-cholesterol egg melange

Low-cholesterol egg melange (LCE) was prepared according to the modified method of Jeong et al. [24]. Egg melange (300 g) was placed in a beaker and β-CD was added at concentrations of 5.0, 10.0, or 15.0% (w/w). The mixture was mixed (Arex-6 Connect Pro, Velp Scientifica, Usmate, Italy) at 400 rpm, 35 °C for 20 min. Then, the cholesterol-β-CD complex was separated by centrifugation (Hettich Zentrifugen, Tuttlingen, Germany) at 1600 × g for 20 min. The low-cholesterol supernatant was collected and used for pasta manufacturing.

The manufacture of pasta

Pasta samples were prepared according to the modified procedure by Alamprese et al. [25]. The dough was made from durum wheat semolina flour and fine wheat flour in a 1:1 ratio with the addition of LCE or control egg melange (CE) at the same amount. The dough was then settled for 30 min in a refrigerator and shaped into noodle sheets using pasta press (Karl Heinz Häussler GmbH, Germany), and dried at 25 °C in the air for about 24 h.

Qualitative characteristics of pasta

Pasta cooking quality was calculated according to Kolarič et al. [23]. Pasta (10 g) was cooked in 250 mL of boiling water until the opaque white core of the strand disappeared (optimum cooking time). For the determination of swelling index, 25 g of pasta samples were cooked 30 min and then calculated according to Eq. (2),

$${\text{Swelling}}\;{\text{index}} = \frac{a}{b}$$
(2)

where a is the volume of pasta after cooking and b before cooking. The volume of pasta was measured in a graduated cylinder. The pasta was then drained in a Buchner funnel for 2 min and weighted. Water absorption was calculated according to Eq. (3),

$${\text{Water}}\;{\text{absorption}} = \frac{m_1 - m_2 }{{m_2 }} \times 100$$
(3)

where m1 (g) is the weight of cooked pasta and m2 (g) is the weight of raw pasta. Cooking loss was evaluated after combining the cooking and rinse water and determined as a sediment in mL on the bottom of graduated cylinder.

Texture properties of pasta

The texture evaluation of cooked pasta samples was measured by the TA.XT plus texture analyser (Godalming, Surrey, UK) using a test for the determination of firmness (g) and work of shear (g.cm) by a light knife blade (A/LKB) with the test speed 0.17 mm/s and target distance 5 mm. Four cooked strands of pasta were used for analysis [23].

Colour properties of pasta

The colour evaluation was done with the same methodology as in Laboratory experiment no. 1.

Laboratory experiment no. 3

The preparation of low-cholesterol milk and egg melange

Low-cholesterol milk (LCM) was prepared according to Kolarič et al. [14]. LCM samples were made from treated UHT milk (3.5% fat content) with three concentrations of β-CD (1.0, 2.0, and 3.0% (w/w)). 300 mL of milk was mixed (Arex-6 Connect Pro, Velp Scientifica, Usmate, Italy) with β-CD at 840 rpm, 20 °C, and for 20 min. The milk was then settled for 120 min at 4 °C and centrifuged (Hettich Zentrifugen, Tuttlingen, Germany) at 70 × g for 20 min. Then, low cholesterol milk was collected from the supernatant and used for muffin making together with control milk (CM) without treatment with β-CD. Low-cholesterol egg melange was prepared with the same procedure as in laboratory experiment no. 2.

The manufacture of muffins

Muffins were prepared according to Gökşen and Ekiz [26]. Sugar (50 g) was mixed with LCE or CE (60 g) with an electric hand mixer. Then, sunflower oil (50 g) and CM or LCM (50 g) were added. Finally, semi-coarse flour and baking powder were added and mixed for 2 min. 50 g of batter were weighed in paper muffin cups and baked at 175 °C for 25 min in a preheated oven (Mora, Czech Republic).

Qualitative characteristics of muffins

Volume, specific volume, and weight loss evaluations were done with the same methodology as in Laboratory experiment no. 1.

Texture properties of muffins

Textural profile analysis (TPA) was evaluated in muffins using a TA.XT plus texture analyser (Godalming, Surrey, UK) according to the method described by Minarovičová et al. [27]. The test was carried out on cubes (25 mm side) taken from the centre of the muffin. The test speed was 1.7 mm s−1; the post-test speed was 10 mm s−1, and there was a 5 s interval between the two compression cycles. A trigger force of 5 g was selected. The compression of 40% was performed with a 36 mm cylindrical probe.

Colour properties of muffins

The colour evaluation was done with the same methodology as in Laboratory experiment no. 1.

Statistical evaluation

Statistical evaluation was performed using Microsoft Excel 365 (version 2012). All results are expressed as mean ± standard deviation or as a percentage and were repeated at least three times. Data were subjected to a one-way analysis of variance (ANOVA) with the Tukey comparison test and values were considered significantly different when p < 0.05. A correlation analysis was performed to determine the type of dependence between all measured parameters of the low-cholesterol and the control products. The level of association was quantified using Pearson´s correlation coefficients (r). A strong correlation was confirmed when r was between 0.8 and 1.0 [14].

Results and discussion

Cholesterol content in cereal products

The validation parameters are shown in Table 1 in the supplementary file. Linearity was proved by R2 value (R2 = 1) in the range of 0.04–2 µg of cholesterol standard. LOD and LOQ were determined at 1.82 and 6.07 mg/kg, respectively. The accuracy of the method was studied as a recovery test by standard addition in three quantities, and the results ranged from 94.2 to 97.2%. Agulló and Gelós [28] showed that the gas–liquid chromatographic method for the determination of free cholesterol in egg pasta reached a recovery of 99.5%, similar to Naviglio et al. [29], 98.7%. Oles et al. [30] reported the GC method for cholesterol determination in various food matrixes (cookie, egg powder, fresh egg, sausage, pretzel) and recoveries varied from 96.3 to 110%. The precision of the proposed method was determined by the repeatability in one day and the intermediate precision of the results obtained in 3 days and expressed as the RSD deviation. According to Murphy et al. [31], the acceptable RSD for analyte concentration 100% (e.g., 100 g/100 g) or ≥ 10% (e.g., 10 g/100 g) is ≤ 4% or ≤ 6%, respectively. In a previous study [16], this method was shown to provide RSD 0.6–3.9% by analysing the cholesterol content in the butter reference material or RSD of 0.3–2.3% by analysing the cholesterol content in milk, cream, and cheese samples [8]. Thus, from the results, it can be concluded that the proposed HPLC–UV–DAD method is suitable not only for the determination of cholesterol content in milk matrices but also in studied cereal matrices.

The cholesterol content in various cereal products is shown in Table 2 in the supplementary file. This measurement was done to confirm the theory of hidden cholesterol in 32 types of commercial cereal products. The largest analysis group was biscuits (11 samples). The cholesterol content ranged from 37.9 to 318.3 mg/kg with an average value of 131.0 mg/kg. It was expected as the biscuit formulation is very versatile. In the case of this study, the most used animal-based ingredients in biscuit formulations were dry egg yolk powder, milk powder with different fat content (10%, 7% or skimmed), milk fat, whey powder, butter, and in one case (sample no. 8) whole eggs. The cholesterol content in the egg pasta samples (6) ranged from 81.7 to 1103.6 mg/kg, depending on the number of eggs. Then, the average value was 350.9 mg/kg. In muffin samples (5), the cholesterol content was also variable, from 144.8 to 681.7 mg/kg with an average of 411.5 mg/kg. Animal-based ingredients in muffin formulations were mostly pasteurised egg melange, whey powder, and milk in different forms (as a milk powder or milk fat).

Zunin et al. [32] reported a cholesterol content in commercial egg biscuits at 300–1500 mg/kg and in commercial sweet snacks at 1100–2400 mg/kg. In other studies, the cholesterol content in egg pasta was up to 2466 mg/kg (6 eggs) [33] or 1200 mg/kg (6.3 eggs) [29]. Agulló and Gelós [28] also determined the cholesterol content in pasta samples (15) and found amounts ranging from 230 to 510 mg/kg.

During the thermal treatment of cereal products, cholesterol is also susceptible to oxidation. Oxidation products are biologically active substances with toxic effects, such as atherogenesis, carcinogenesis, neurodegenerative diseases, and other harmful effects on human health [9]. Therefore, many studies are concerned with the determination of cholesterol oxidation products in these products. Zunin et al. [32] found that the highest amounts of 7-ketocholesterol were observed in samples of biscuits containing whole milk powder and eggs. According to their research, cholesterol oxidation in products with powdered eggs occurs much faster than in products made with fresh eggs. The results in the pasta samples indicated that small amounts of cholesterol oxidation products were also formed in the egg pasta during industrial processing and the amount observed in the fresh egg pasta was much lower than in the dried egg pasta [9].

Laboratory experiment no. 1

The decrease of cholesterol content in biscuits

For products in the fast-moving consumer goods category, biscuits rank among the leading ones. The consumption of biscuits is very popular due to their pleasant flavours, long shelf life, nutritional needs, and convenience. In recent years, biscuits have been enriched with various nutrient-rich ingredients [34]. To monitor the possible way to decrease the cholesterol content in biscuits, samples were made from standard butter and butter made from cream treated with three different concentrations of β-CD (2.0, 4.0, and 6.0% (w/w)), which were chosen based on the results of previous study [14]. The results are shown in Table 1. Optimal conditions for the elimination of the cholesterol content in cream were previously published [35]. In Kolarič et al. [14], it was shown that by 5.0% of β-CD, it was possible to reduce the cholesterol content in butter by 95.6% without adverse effects on organoleptic properties. In this study, a relatively similar value was observed by 4.0% of β-CD (94.9%). The results were statistically different between 2.0% of β-CD and other concentrations at p < 0.05. Alonso et al. [36] showed a scale-up process for the manufacture of reduced-cholesterol butter by treating raw milk with even a smaller amount of β-CD (0.6% (w/v)) and reached an effectivity of cholesterol removal at around 90%.

Table 1 Cholesterol content and percentage of removal from butter and biscuits
Full size table

Thus, after the production of low-cholesterol butter, the biscuits were prepared. It was shown that with this procedure, it was possible to reduce the cholesterol content of these products by around 93%. The cholesterol content in the control group was 329.2 mg/kg and after using the low-cholesterol butter prepared with 4.0% β-CD, it decreased to 22.8 mg/kg. Up to date, there are no articles describing such an effective decrease in cholesterol content in biscuits. Moreover, the elimination of cholesterol from butter by β-CD does not affect any chemical, sensory, or organoleptic properties of the product [14, 36, 37]. Thus, it is suitable for the production of low-cholesterol biscuits.

The effect of cholesterol removal procedure on qualitative, texture, and colour properties of biscuits

The results on the effect of butter cholesterol removal procedure on the qualitative, texture, and colour properties of the biscuits are shown in Fig. 1. From the qualitative parameters of the biscuits, the spread ratio is an important property because it is a good indicator of the binding characteristics of the ingredients and the texture of the biscuits. In general, a biscuit with a larger spread ratio is more desirable [3, 38]. In this study, the spread ratio increased from 8.76 (control sample) to 9.85 (sample prepared with low-cholesterol butter using 6% of β-CD). However, the results were not statistically different at p < 0.05 between the control and low-cholesterol biscuits prepared with butter treated with 2 and 4% of β-CD. The spread ratio is mainly related to the properties of hydration of the flour but also to the fat content. Higher fat increases the mobility of the dough in melting and improves the spread rate, a lower fat content makes the flour accessible to the water and increases the consistency of the dough during baking and stops spreading [3]. However, in this case, the elimination of the cholesterol content of butter by β-CD did not significantly influence its overall fat content. Weight loss is related to the weight of the product, shape or moisture content [21]. Weight loss increased from 9.6% (control sample) to 11.5% (sample prepared with low-cholesterol butter using 6% of β-CD). The results were not statistically different at p < 0.05 between the control sample and the low cholesterol sample prepared with butter treated with 2% of β-CD. The weight loss is mainly attributed to the water activity of the dough. The higher weight loss during baking can also be related to the fact that there was no developed structure that would retain the shape of the product and its components to any external change [39]. The other qualitative parameters of the biscuit samples were also influenced by the concentration of β-CD. Volume, specific volume, as well as volume index decrease significantly, when butter was treated with 6% of β-CD. However, when 2% of β-CD was used, these parameters remained the same compared to the control. Therefore, the results showed the importance of β-CD concentration in the elimination of cholesterol from butter.

Fig. 1
figure 1

The effect of cholesterol removal to qualitative parameters (A), texture (B) and colour properties (C) of biscuit samples. +the values are significant different at p < 0.05 from control sample

Full size image

The texture parameters (hardness and fracturability) of the biscuits are shown in Fig. 1B. In the previous study [14], it was observed that the firmness of CB was statistically similar to that of LCB. However, Kim et al. [40] reported that treatment with β-CD increased some parameters of butter texture, suggesting the addition of evening primrose oil and phytosterols. The texture of the biscuits is mainly related to the mechanical properties, which can be influenced by factors, such as the proportion or replacement of fat to sugar level, flour, and the particle size of the ingredients [3]. In this study, the hardness and fracturability of the biscuits were affected by the concentration of β-CD. The hardness of the control biscuit and the low-cholesterol biscuit prepared with butter treated with 2% of β-CD was not statistically different at p < 0.05. At higher concentrations of β-CD, the hardness decreased. The same trend was also observed in a previous study [14] focused on the determination of the firmness of low-cholesterol butter. In correlation analysis, no significant relationship was found between cholesterol content and texture properties of biscuits.

The colour coordinates of biscuits are shown in Fig. 1C. The L* parameter (lightness) was found to increase from 76.33 (control group) to 80.24 (biscuits prepared with low cholesterol butter treated with 6% of β-CD). These results correlate with a previous study [14], in which the lightness factor of butter increased with higher concentrations of β-CD. However, the typical colour of the biscuits did not change, as other coordinates were not significantly different at p < 0.05 between the control and the low cholesterol biscuits, and ΔE ranged from 3.12 to 3.96. No visual differences are perceptible to consumers, when ΔE is less than five units [41].

Laboratory experiment no. 2

The decrease of cholesterol content in pasta

Egg pasta is a very popular food and is usually made of durum wheat semolina, eggs, and sometimes water. In some cases, semolina can be substituted for common wheat flour. However, within the EU, there are radically different regulations for pasta [42]. The main animal-based component in pasta formulation is egg (egg yolk or whole egg as a powder or melange). Thus, in this study, the elimination of the cholesterol content of the egg melange was examined. The results are shown in Table 2. The standard egg melange contained 3094.88 mg/kg of cholesterol. The highest percentage of cholesterol elimination was achieved by 10.0% of β-CD (90.4%). The results were statistically different at p < 0.05 between other concentrations of β-CD. According to Alonso et al. [15], the concentration of 5% of β-CD was suitable to remove 80.04% of cholesterol from yolk and powdered eggs. In their study, the highest concentration of β-CD was 7% but no differences were found compared to 5%. A higher percentage of cholesterol removal was achieved by Jeong et al. [24], 92.76%, however, they used crosslinked β-CD instead of natural powder β-CD and also quite high β-CD concentration (25%). From the results of this study, 15% of β-CD caused a significant reduction in the effectivity of cholesterol elimination. In another study [43], different forms of β-CD were examined, and the authors found that the direct addition of β-CD to the egg yolk caused the greatest decrease in cholesterol level (90%) compared to immobilised β-CD on agarose beads (37.7%) or in agar film (28.9%). The effectiveness of this process was also proved by Su et al. [44], who achieved 94.2% of the cholesterol removal rate by β-CD (β-CD:cholesterol molar ratio was 5:1).

Table 2 Cholesterol content and percentage of removal from egg melange and pasta
Full size table

The second step in this study was to use low-cholesterol egg melange in a production of pasta. The results on cholesterol content are shown in Table 2. Standard egg pasta contained a high cholesterol content, 1117.1 mg/kg, while pasta with egg melange treated with 10% of β-CD contained only 93.7 mg/kg. Therefore, the procedure exhibited a high effectivity on cholesterol decrease (91.6%). The results were statistically different at p < 0.05 between other concentrations of β-CD. In the scientific literature, there are no articles that describe the application of low-cholesterol melange in pasta production. Bonciolini et al. [45] described the effect of tannins on cholesterol content in egg pasta. Tannins (ellagitannins and gallotannins) interacted with cholesterol and affected its content in egg pasta but were significantly influenced by the shape of the pasta. The advantage of their method was also associated with the reduction of cholesterol oxidation products in the final products as a result of the antioxidant activity of tannins.

The effect of cholesterol removal procedure on qualitative, texture, and colour properties of pasta

The effect of the cholesterol removal procedure on the qualitative, texture and colour properties of pasta samples is shown in Fig. 2. In relation to the qualitative parameters of pasta cooking (Fig. 2A), no significant differences (p < 0.05) were found between the control group and the low-cholesterol group. The optimal cooking time was established between 9.05 and 9.35 min. The results are similar to other studies, Kolarič et al. [23] determined the optimal cooking time of durum semolina pasta on 9.17 and Samaan et al. [46] between 9–10.5 min. In general, the parameters of the cooking quality of pasta are associated with the properties of starch granules and the strength of the protein network [23]. Thus, it can be observed that cholesterol elimination by β-CD did not influence these important components. This was also proved by correlation analysis, as there was no strong relationship between cholesterol content and pasta cooking quality.

Fig. 2
figure 2

The effect of cholesterol removal to cooking qualitative parameters (A), texture (B), and colour properties (C) of pasta samples. +the values are significant different at p < 0.05 from control sample

Full size image

The elimination of the cholesterol content from the egg melange slightly influences the colour properties of the pasta (Fig. 2C). According to the ANOVA analysis, there were significant differences at p < 0.05 in the parameters a*, b* and YI between the control and the low-cholesterol group. L* factor was not statistically different. In a*, there were differences between all β-CD concentrations. In b* and YI, there were differences only with respect to 5% of β-CD. However, the samples were not visually different, which was calculated by ΔE parameter. There was also no strong correlation between cholesterol content and colour coordinates. The optimum β-CD concentration regarding cholesterol removal was 10% (w/w) and in this group, ΔE was only 1.07. According to Lamas et al. [43], there were no differences in colour between control egg-derived products and β-CD-added egg derived products. However, the addition of chitosan instead of β-CD decreased the amount of yellow colour of egg products. The yellowish colour of pasta is mainly related to the carotenoid content of semolina flour or egg products. From the results, it seems that the carotenoid content was not influenced by the treatment with β-CD. The intensity of pasta colour can also be improved by some natural colour components [47]. De Santis et al. [48] made low-cholesterol pasta by substituting pasteurised whole eggs with egg white and found that these pasta samples exhibited a higher L* value but were not significantly different from standard egg pasta. Low-cholesterol pasta also showed a brighter yellow colour. Moreover, ΔE value was much higher (11.4) than in this study.

In Fig. 2B, the texture properties of pasta samples are shown. No significant differences were observed at p < 0.05 between the control group and the low-cholesterol group. In egg pasta products, firmness is mainly related to protein quality (albumen vs. yolk protein) rather than quantity [42]. Alamprese et al. [42] stated that ovalbumin plays an important role in the development of the pasta protein network, but, in contrast, yolk, due to its lipid content, weakens the protein network, favouring swelling of the starch granule and increased weight in pasta during cooking. From the results of this work, it appears that treatment of egg melange with β-CD did not affect the protein properties of eggs. On the other hand, it was proved that cholesterol did not enter any reaction that influences the firmness of the pasta, as no strong correlation was found.

Laboratory experiment no. 3

The decrease of cholesterol content in muffin

A traditional muffin is an oil-in-water emulsion consisting of a mixture of eggs, water, fat, and sugar, in which the flour particles are dispersed [49]. Due to their versatile formulation, muffins can also be rich in cholesterol content. In this work, muffin samples were composed of milk (3.5% fat) and pasteurised egg melange. Therefore, the first part was related to the removal of cholesterol from these ingredients. The results are shown in Table 3. The removal procedure of cholesterol from egg melange was performed by the same methodology as in Laboratory experiment no. 2. The highest percentage of removal was achieved by 10% of β-CD (88.6%) and was statistically different at p < 0.05 compared to 5 or 15% of β-CD. Optimisation of milk cholesterol removal by β-CD was previously published [14, 35] and, based on that, similar conditions were also used in this study. The untreated milk sample contained 120.3 mg/kg cholesterol, while treatment with only 2% of β-CD caused a 94.3% elimination of cholesterol. The highest cholesterol removal effectivity was noticed by 3% of β-CD (94.7%), but with no difference to 2% of β-CD. The application of β-CD in the production of low-cholesterol milk was also published in many patents and the highest elimination of cholesterol was up to 98% [6].

Table 3 Cholesterol content and percentage of removal from egg melange, milk, and muffin samples
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The control muffin contained 487.2 mg/kg of cholesterol. Using treated egg melange with 10% of β-CD and milk with 2% of β-CD, it was possible to decrease its content to 51.08 mg/kg (by 89.5%). The results were statistically different at p < 0.05 between other concentrations of β-CD. Marcet et al. [50] published a study in which whole egg yolk was substituted with egg yolk granules in the preparation of gluten-free muffins without cholesterol. Based on their results, using 60% of the granules, it was possible to reduce the cholesterol content in the muffins by around 86%. However, egg yolk granules also decrease the amount of lipids and high-emulsifying phospholipids in final products.

The effect of cholesterol removal procedure on qualitative, texture, and colour properties of muffins

The effect of cholesterol removal on the main consumer characteristics of the muffin samples is shown in Fig. 3. First, the baking properties were examined as weight loss, volume, and specific volume (Fig. 3A). The weight loss of muffins is related to the binding capacity of the water of the ingredients and is an important parameter for the industry, because the lower the weight loss, the higher the yield and therefore the weight of the products [51]. From the results, it can be seen that the elimination of cholesterol from animal ingredients did not affect their water binding capacity as there were no statistical differences at p < 0.05 between the control and the low-cholesterol samples. The weight losses of the control muffin and muffin with treated components with optimal β-CD concentration (10% for egg melange, 2% for milk) were 11.7 and 11.6%, respectively. Marcet et al. [50] also did not observe significant weight loss changes in low-cholesterol muffin samples prepared by replacing egg yolk with egg granules. Regarding the specific volume of the muffin, significant differences were found at p < 0.05 between the control sample and the low cholesterol samples using the last two concentrations of β-CD. The specific volume increased from 151.90 to 169.25 mL/100 g. However, this parameter is mainly related to the air retention capacity of the batters during the mixing of the ingredients; therefore, it depends on the mixing time and energy rather than the cholesterol content [52].

Fig. 3
figure 3

The effect of cholesterol removal to qualitative parameters (A), texture (B) and colour properties (C) of muffin samples. +the values are significant different at p < 0.05 from control sample

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The textural parameters of the low-cholesterol and control muffins are shown in Fig. 3B. The hardness of the muffins is the most valued textural parameter and is directly related to the density of the sample [50]. No significant differences in hardness were found between the control and low-cholesterol group. According to Martinez-Cervera et al. [53], the hardness of the muffin is connected to the volume of the muffin, thus the smaller the volume, the harder the texture. In this study, only a weak negative correlation (r = − 0.52) was observed between the hardness and volume of the muffin samples. According to Kolarič et al. [14], the β-CD treatment did not have any effect on the firmness of the milk after cholesterol removal, as differences compared to standard milk were not significant at p < 0.01. In addition to the hardness value, chewiness, resilience, and cohesiveness were also recorded. Chewiness was correlated with the hardness factor (r = 0.88). Resilience is related to the degree to which the sample recovers after compression, and the value of 1 means that the sample behaves like a spring, thus returning immediately to the initial height [53]. Cohesiveness is defined according to how well the product withstands a second deformation relative to how it behaved under the first deformation [27] and has a strong correlation with resilience (r = 0.90). None of these parameters changed significantly after the removal of cholesterol from animal-based ingredients. Furthermore, no correlation was found between the texture profile of the muffins and the cholesterol content.

The colour properties of muffins are shown in Fig. 3C. Differences in L* coordinates were not significant at p < 0.05 between control and low-cholesterol samples. Statistically significant changes were found in b* and YI factor. However, ΔE values ranged only in an interval between 1.43 and 2.55. According to Marcet et al. [50], by replacing egg yolk with granules in the muffin formulation, ΔE values were higher than 3 (3.77 for 100% granules, 9.63 for 60%). The correlation analysis did not show any relationship between the colour coordinates and the cholesterol content in the muffin.

Conclusion

Many studies associate daily intake of cholesterol that exceed current average levels with elevated total or low-density lipoprotein cholesterol concentrations in serum. Therefore, dietary guidance should focus on healthy dietary pattern (e.g. Mediterranean-style and DASH (Dietary Approaches to Stop Hypertension) styles that are inherently relatively low in cholesterol content in the diet [54]. Cereal products are daily consumed throughout the world; however, their formulation often includes animal-based components, which commonly contain significant levels of cholesterol. As already mentioned, elevated cholesterol intake is associated with an elevated risk of cardiovascular disease. In addition, bakery products can contain cholesterol oxidation products, which are also associated with several harmful effects on human health. Therefore, this study describes an innovative procedure for the production of healthy products by the application of low-cholesterol animal-based components in the formulation of three types of cereal products (biscuits, pasta, and muffins). The low-cholesterol ingredients were prepared by application of β-CD treatment procedure, which is the simplest, most effective, and safe way for their preparation. When comparing the cholesterol content in original and low-cholesterol products, it was found that the decrease is equal to 93.1, 91.6, and 89.5%, respectively. Subsequently, the texture, culinary and colour properties were studied in the original or low-cholesterol products. These characteristics were influenced by the concentration of β-CD but overall, they did not change significantly. Therefore, the consumption of low-cholesterol cereal products could bring health benefits to the incidence of CVD and approach the general accepted optimal daily cholesterol intake of 220 mg, which would be in accordance with the principles of DASH. Future trends should focus on the development of animal and non-animal-based foods with a lower cholesterol content to permanently meet recommended cholesterol levels in a daily diet.