Clinical, nutritional, and quality assessment of flaxseeds and their supplemented muffins

ABSTRACT This study aimed to explore whether flaxseed could be used to ameliorate various CVD biomarkers. In the first phase, flaxseed’s nutritional composition was performed. In the second phase, a 32-day efficacy trial was conducted on 20 Sprague Dawley rats. All physical and biochemical tests were performed on the initial and final days. Results revealed that blood glucose, triglycerides, total cholesterol, and low-density lipoprotein cholesterol were significantly reduced, whereas high-density lipoprotein cholesterol, hematocrit, haemoglobin, red blood cells, and platelets were increased. Having significant clinical findings, a marketable flaxseed-supplemented muffin recipe was developed using flaxseed-supplemented flour at 0%, 5%, 10%, 12%, and 20%. Farinographic investigations of flaxseed-supplemented flour revealed its improved baking potential through more excellent water absorption and dough development time. The flaxseed-supplemented muffins revealed a significant rise of 15% in protein, 60% in fiber, 23% in ash, 60% in healthy vegetative fats and sensorial parameters.


Introduction
Plants have long been the primary source of food and bioactive components and have remained nature's true gift, assisting in better health.Since prehistoric times, natural foods and their products have been recognized and employed as the principal source of medicinal drugs (Anwar et al., 2022;Waseem et al., 2021).Medicinal plants have been reported to treat and cure a range of health-related concerns since ancient times, as these have minimal side effects and a wealth of advantages (Khan et al., 2021).Plant-based foods, rich in various bioactive components like omega-3 polyunsaturated fatty acids (n-3 PUFA), vitamins, phytochemicals, dietary fibers, lignans, etc., known to have anti-inflammatory, antioxidant, and immunological protection properties (Shabbir et al., 2022;Shahzad et al., 2021).Plantbased foods, particularly nuts, seeds, and legumes, were recently found to be associated with positive health outcomes even in recovering COVID-19 patients (Rabail et al., 2021).Hence, incorporating such health-protective bioactive foods into daily dietary consumption patterns can help lower the risks of communicable and non-communicable diseases (NCDs).Prevention of NCDs is the top-priority goal (World Health Organization, 2013) and cardiovascular diseases (CVDs) remain responsible for over 18.6 M global fatalities in 2019, with elevated levels of low-density lipoprotein cholesterol (LDL-C) accounting for 4.4 M deaths globally (American Heart Association, 2021).This prevalence is higher in low-and middleincome nations (World Heart Federation, 2018).Hence, dietary interventions are essential to meet health and economic goals to reduce the burden of diseases, especially CVDs, in low-and middle-income nations (Siscovick et al., 2017).
The increasing demand for n-3 PUFA as functional food has generated interest in fortifying traditional meals to improve nutrition, health promotion, and disease prevention (Konieczka et al., 2017;Manzoor et al., 2021).The American Heart Association advises consuming at least 1.4 g of n-3 PUFAs from dietary sources every day (Gowda et al., 2018), or 0.6% to 1.2% of the total calories from alpha-linolenic acid (ALA), i.e. the vegetative n-3 PUFA (McGuire, 2011).Regular consumption of flaxseeds (Linum usitatissimum L.) can be a good strategy to meet the nutritional requirements of ALA, as it is among ALA's top richest natural dietary sources.Flax name (L. usitassimum) means "very useful."It belongs to Linaceae family and is an annual plant with blue flowers and pointy flat nutty seeds of two basic varieties: brown and golden.Mostly it is termed "flaxseed" (used for dietary purposes) or "linseed (mostly for industrial purposes).Its origin dates back to 7,000 BC.It is also known as Alsi or Teesi locally.Its intake has been documented for more than 5000 years due to health advantages against cancer, autoimmune illnesses, atherosclerosis, diabetes, osteoporosis, hyperlipidemia, and neurological disorders (Das & Biswas, 2016;Morris, 2007;Prasad & Jadhav, 2016;Mohamed et al., 2020;Vinita et al., 2016).The nutritional composition of flaxseed varies due to varieties, environmental conditions, analysis methods, and seed processing (Morris, 2007). Flaxseed comprises 38-45% lipids, 20-21.23% proteins, 4-7% ash, 3.47-4.5%moisture, 18-30% fiber, of which 25% is water-soluble, and 75% is insoluble fiber.These seeds are energy dense at 4.5 kcal/gram and hold an excellent fatty acid ratio of n-6 to n-3, which is about 0.3:1 or 1:4.Among fatty acids, these contain 53-58.2%ALA is six times greater than most fish oils.(Asif, 2011;Carraro et al., 2012;Ciftci et al., 2012;Hussain & Anjum, 2008;Tolkachev & Zhuchenko, 2000;Tulsi & Darshan, 2016;Wali et al., 2017) In our contemporary living style, a wide range of foods are prepared using plain white or wheat flour baking.Most baked items are fatty and sweet, providing a delightful treat, e.g.bread, muffins, cakes, and so on.Bread and other bakery goods go friendly with home and industrial supplementation (Martinez & Gomez, 2019;Rabail et al., 2022;Usman et al., 2021;Waseem et al., 2022).In the current study, flaxseeds were selected for supplementation in bakery items as a source of n-3 PUFA (ALA), dietary fibers, lignans, and phytochemicals to ameliorate CVD-related biomarkers.Although many scientific efforts have been made until now, there is still space to overcome common people's daily dietary utilization of ALA from flaxseeds to attain its clinical, nutritional, and functional advantages.Therefore, efficacy trials were conducted first using Sprague Dawley (SD) rats to investigate these seeds' preventive and therapeutic effects.Following that, nutritional composition, supplementation, rheological, and sensory appraisals of flaxseed were evaluated.

Materials and methods
During October and November 2016, the flaxseed samples were procured from a localized merchant market in Multan, Pakistan.All raw flaxseed samples were kept at room temperature until they were used.The required flaxseed samples were pan-roasted using an electric hot plate for 3-5 min on a very low heat of roughly 50°C.These roasted seeds were finely ground in a regular kitchen blender, refrigerated in zip-lock bags, and used in triplicates for further analysis within a week.

Proximate nutritional assessment
The proximate nutritional assessment of flaxseed was determined using the Association of Official Analytical Chemists' techniques (AOAC, 2000) as were done in the last part of the study (Rabail et al., 2022) using Method # 925.08 for moisture, Method # 923.03 for ash, Method # 979.09 for protein, Method # 920.39 for crude fat, and Method # 962.09 for crude fiber.The number of carbohydrates was calculated using the following formula:

Clinical assessment
Two clinical trials, safety (S) and treatment (T), were conducted on 6-7 week-old 20 SD rats with a mean body weight of 187 g.While the T-trial intended to investigate the hypolipidemic and cardioprotective benefits of flaxseed consumption, rats in the treatment trial were fed a diet containing 2% cholesterol for 3 weeks, and the S-trial sought to determine whether flaxseed intake had any negative or positive health effects.(Rabail et al., 2022).Rats were fed a basal feed for a week to help them adjust to their new environment.Then, experimental diets were administered for the next 32 days.The diets were made by altering the control diet plan described in the last part of the study (Rabail et al., 2022) to make the control and flaxseed groups isocaloric (Table 1).The World Health Organization's (WHO) recommendation of 1 g = 1% of the total daily calories from ALA was practically met in this trial by introducing the selected amount of 5.3 g of flaxseed flour to make 100 g of the total diet.It will ultimately provide 1 g of ALA per 100 g of total weight (based on the literature described earlier in flaxseed's nutritional composition that 53-58% of flaxseed oil is ALA) (Engel, 2010;Lagiou et al., 2009).
Daily food intake and weekly weight fluctuations were monitored.At the beginning of both experiments, cardiac punctures were used to get blood samples, and rats were dissected to collect essential organs (heart, liver, kidneys, and spleen).Fat accumulation and organ weight were reported.The food efficacy ratio (FER) and daily body weight gain per day were calculated.Diagnostic tests for triacylglycerols (TAG), total cholesterol (TC), high-density lipoproteins (HDL), low-density lipoproteins (LDL), blood glucose, aspartate transaminase (AST), alanine transaminase (ALT), complete blood count (CBC), hematocrit (HCT), and serum creatinine were performed at the University Diagnostic Lab, Department of Veterinary Sciences, Bahauddin Zakariya University, Multan, Pakistan, by using commercial kits (Merck, Germany) (Sultan  et al., 2012).The Omni online calculator calculated the percentage increase or decrease using the following formula.
Results were evaluated in triplicates to determine mean values and standard deviations (Rabail et al., 2022).

Rheological assessment
These finely ground flaxseeds were combined on a dry basis with wheat flour at 0%, 5%, 10%, 15%, and 20% supplementation levels to create homogeneous flaxseed flour blends.
To ascertain the approximate composition of a given blend, the nutritional contents of white wheat flour were substituted for the nutritional contents of flaxseeds.The rheological assessment was done using a Brabender Farinograph (Electronic T150, Ohgduisburg, Germany) following the AACC, 2000: Method # 54-21 protocol.A 300 g sample of each blend was placed in the Farinograph mixing bowl; water was carefully added on Farinograph demand to produce the dough's absolute consistency.After 20 min, the computer-generated Farinograph curve was consulted to determine Farinograph properties such as water absorption (WA), dough stability time (DST), degree of softening (DoS), dough development time (DDT), Farinograph quality number (FQN), dough consistency (DC), and mixing tolerance index (MTI) (Ashraf et al., 2020;Rabail et al., 2022).

Flaxseed supplemented muffins
Flaxseed-supplemented flour blends were utilized to create a recipe for muffins that can be eaten for breakfast, brunch, or tea snacks.Table 2 describes the components in a regular control recipe, whereas flaxseed-supplemented muffin recipes were created by substituting flaxseed-supplemented flour blends with wheat flour.The wet ingredients were combined in a regular home blender for around 2-3 min to get a uniform foamy and creamy texture.In contrast, all the dry ingredients were well mixed before being combined with the liquid and swirled until a homogeneous, thick-textured batter was created.About 50 g of the resulting batter was put into individual muffin molds made of aluminum foil and baked in a preheated electric oven at 130°C for 6-8 min on the lower grill, followed by 3-5 min on both the lower and upper grills, and finally 2-3 min on the top grill.The baked muffins were taken out of the oven and allowed to cool at room temperature (Rabail et al., 2022).
The proximate nutritional composition of flaxseedsupplemented muffins was determined using the above mentioned AOAC guidelines.The color experiment using a CIELAB space colorimeter at Ayub Agricultural Research Institute in Faisalabad was conducted following the same protocols in previous works (Alshehry, 2019;Rabail et al., 2022) to analyze color values (L* lightness from 0 black to 100 white, -a* for greenness, +a* for redness, -b* for blueness, and +b* for yellowness).The following formulas were used to determine chroma and hue values: The trained sensory panel evaluated the sensory quality of flaxseed-supplemented muffins using the "15 th Centimeter Scale Sensorial Performa."Among the 10 sensory components assessed are the crust color (caramelized light brown) and crust appearance (uniformed appearance of crust firmness), the crumb color (creamy light brown) and crumb appearance (Uniformed appearance of air cells), the aroma, the mouthfeel (appealing sensation associated with mild sweetness and freshness), the texture (softness, hardness, gumminess, and springiness), the flavor, the volume (appropriate size/height gained), and the overall acceptability.The specific characteristic's strength rises progressively from 0 to 15.In addition to being assigned to various booths with fluorescent white illumination, panelists were given plain cold water to rinse their mouths between samples (Rabail et al., 2022).

Statistical design
The independent sample t-test, one-way ANOVA, and Duncan's Multiple Range Test were used to compare the means at the 95% (p0.05) confidence interval level using SPSS-16 (IBM, Chicago, IL, U.S.A).

Results for clinical assessment
This section has examined the findings for physiological and biochemical markers from both S-and T-trials.The results for physical indicators, including total body weight gain, body weight gain per day, FER, organ weights, and percent organ fats, recorded during the investigation have been presented in Table 3. Flaxseed consumption resulted in significant reductions in total body weight gain and daily weight gain in S-trial, while the weight was slightly higher during T-trial, but this increase remains non-significant for both parameters.On the other hand, daily food intake was more significant in flaxseed groups of both S-and T-trials, while this increase was non-significant in T-trial, it was highly significant in S-trial, leading to a very highly significant decrease in FER in S-trial that might have resulted due to the less weight gain and more feed intakes.Among organ weights, only liver weight showed a significantly lowered gain in S-trial, whereas, in the T-trial, heart, liver, and spleen weights showed a highly significant decrease for flaxseed groups.These reductions were highly significant for liver fats in S-trial and heart, liver, and spleen fats in T-trial.
Results for laboratory biomarkers for healthy rats of S-trial in Table 4 revealed that flaxseed consumption from D1 to D32 has significantly increased Hb levels by 10.32%, TLCs by 17.22%, and platelet counts by 10.11%.The results for HCT and RBCs were also inclining but remained nonsignificant during the trial.Similarly, flaxseed consumption showed promising amelioration in lipid profiling and LFTs at D32 when compared to D1 in healthy rats, as there was a very highly significant decrease in TG by 9.69%, TC by 11.07%, LDL by 24.13%, ALT by 13.48%, and AST levels by 19.58%.On the other hand, a notable but non-significant 4.35% increase in HDL-cholesterol and a noticeable but non-significant decrease of 3.30% in blood glucose levels were reported from S-trial.The comparison between the control and flaxseed groups on D32 has been indicated in the red font in Table 4.It revealed a significant increase in HCT, a significant increase in Hb, and a very high increase in TLCs and platelet counts in flaxseed-fed rats compared to control group rats.The decline in laboratory values for flaxseed-fed rats in blood glucose levels, ALT, AST, TG, TC, and LDL-cholesterol were also highly significant compared to the control group.
Likewise, the results of the T-trial on hyperlipidaemic rats elaborated in Table 4 indicated that the rise for HCT, Hb, and RBCs was significant in the control group, but this rise remains non-significant in flaxseed-fed rats.Surprisingly, the results indicated a very significant decrease in TLCs, whereas a very significant increase in platelet counts in both control and flaxseed groups.On the other hand, blood glucose levels indicated a significant decrease of 5.81% in flaxseed-fed rats compared to a significant rise in blood glucose in the control group.Moreover, this blood glucose ameliorative effect of flaxseed was highly significant compared to the control and flaxseed groups at D32. Flaxseed consumption resulted in a very significant reduction in AST levels, whereas the levels of ALT made a highly significant rise of 18.62% in the flaxseed group, but this rise was significantly lower than the raised values of 44.93% in the control group.The results for both LFTs are good indicators of flaxseed's hepatoprotective potential.The creatinine level in T-trial revealed a 100% rise for which the actual cause is still not understood.Flaxseed consumption brought forward strong protective results for lipid profiling by a highly significant decrease of 8.22% in TG; a very significant decrease of 20.60% in TC and 38.90% in LDL; and a highly significant rise of 16.50% in HDL.Also, the same goes for the results of flaxseed-fed rats compared with the control group at D32 (as indicated in red font in Table 2).On the other hand, the results of lipid profile parameters in the control group rats were alarming, with a highly significant rise in TC and LDL.

Results for nutritional assessment
The proximate composition and the sum of squares for flaxseed, flaxseed-supplemented blends, and flaxseedsupplemented muffins are given in Table 5. Finely ground flaxseed flour was mixed into plain wheat flour at 0%, 5%, 10%, 15%, and 20% supplementation levels.The resultant flaxseed-supplemented flour blends and muffins were examined for their proximate composition and were evaluated for rheological attributes.The increasing supplementation levels of flaxseed in blends and muffins revealed a significant decrease in their moisture and a highly significant decrease in NFE contents, whereas a highly significant increase in ash, fat, protein, and fiber contents.Improved nutritional profile in flaxseed-supplemented blends has supported the objective of developing an innovative recipe with improved functional and nutraceutical properties.Purposely, a recipe for flaxseed-supplemented muffins at various supplementation levels was developed and analyzed.The increasing supplementation levels of muffins showed a better nutritional composition of minerals, healthy fats, good-quality vegetative proteins, and dietary fiber.

Rheological assessment
The results of the Farinographic analysis have been elaborated in Table 6, and their Farinographic curves and types are depicted in Figure 1.These improvements in the results of Farinograph were significant for DC, whereas these were highly significant for DDT, DST, DoS-ICC, FQN, and MTI.Changes in the results of WA remain non-significant.

Color assessment
The color of the muffins has a considerable influence on their perceived acceptability.The results for L*, a*, and b* values *** = Very highly significant at p < .001;** = highly significant at p < .01;*= significant at p < .05;ns = non-significant at p < .05 on comparison D1 to D32.
for flaxseed-supplemented muffins revealed highly significant variations, as shown in Table 7.The intensity of redness was indicated by the "a*" value, yellowness by the "b*" value, and lightness by the "L*" value.The results showed an increase in a dark tone with a drop in L* and a decrease in yellowness with a decrease in b* both crust and crumb, although the -a* value for slight greenness was also seen.

Sensory assessment for flaxseed supplemented muffins
Sensory assessment is an important stage in product development since product acceptance at any industry, or domestic level is challenging to achieve without judgment on the product's acceptability.The sum of squares for crumb color, crumb appearance, volume, mouthfeel, and gumminess were highly significant, and those for crust color, crust appearance, taste, and hardness were significant, whereas softness and aroma remain non-significant along with supplementation levels.Figure 2 depicts how sensory rating for properties such as crust and crumb appearance, volume, softness, hardness, springiness, and gumminess declined as supplementing amount increased.

Clinical assessment
The results of the clinical assessment indicated better feed efficacy, and a similar improved feed conversion ratio and dietary intake have been reported previously (Al-Zuhairy & Taher, 2014).The reduction in body weight by flaxseed consumption may be related to its rich dietary fiber contents and gum formation or gelling ability, as investigated by (Luo et al., 2018).Its consumption can help in achieving a better body frame and prevent central obesity and its consequences by reducing body fat depositions.As indicated earlier, flaxseed consumption may enhance adiposity indicators like adiponectin levels.Consuming flaxseed might therefore be a complementary treatment to reduce central obesity (Ahmad et al., 2021;Ahmadniay Motlagh et al., 2021).Results for organ weight reductions and associated visceral fat pads are pretty conclusive.
A previous study on flaxseed consumption examined similar reductions in fats associated with the liver (Cho et al., 2009).Therefore, consuming flaxseed may aid in reducing visceral body fat deposition and relevant harmful pathophysiological consequences brought on by CVDs and reduce body weight gain tendencies without adversely impacting hunger (Ahmadniay Motlagh et al., 2021).
Results of laboratory biomarkers revealed significant improvements in various parameters investigated in both S-and T-trials.The results of the S-trial were very protective and can be compared with the improved results of blood profiles previously reported (Al-Zuhairy & Taher, 2014;Prasad, 2005).Likewise, similar health-protective and lipidameliorating results have been observed in previous studies (Cho et al., 2009;D. A. Mohamed et al., 2008;Mirfatahi et al., 2016).The t-trial investigation also revealed very conclusive and preventive roles of flaxseed consumption.The results on the hematopoietic parameters of non-significant improvements in Hb or HCT correspond with those of (Al-Zuhairy & Taher, 2014).The results for lipid profile strongly supported hypolipidaemic and cardioprotective effects of flaxseed-fed rats were highly significant compared with the control group.This study's significant lipid ameliorative results have been supported in studies (Ahmadniay Motlagh et al., 2021;Alamri, 2019;Da Silva et al., 2020).

Product development
The moisture, ash, crude fat, and protein contents of flaxseeds correspond with those of (Kulczyński et al., 2019;Mihafu et al., 2019;Sá et al., 2021).Supplementation of flaxseeds into plain wheat flour brought forward improved nutritional combinations with rich protein, and a similar improved nutritional profile has been reported in the works of (Moraes et al., 2010;Wirkijowska et al., 2020).Proximate compositional analysis of flaxseed-supplemented muffins also exhibited highly significant improvements in the nutritional profile, and these results for improved nutritional outcomes of flaxseed supplementation have been  backed up by the works of (Ganorkar & Jain, 2014;Kaur et al., 2019;Wirkijowska et al., 2020).Results for rheological assessment indicated an increase in WA.Although the increase in WA was not significant, this increase can be attributed to the increased water-holding capacity due to gelling ability and higher fibre contents (Chetana et al., 2010;Toliba & Mohamed, 2019), and mostly owing to the mucilage's strong water-binding capability (Iglesias-Puig & Haros, 2013).Moreover, any flour with a higher WA, MTI less than 30 B.U., and longer DDT is acceptable for baking or bread-making.Overall, the findings showed improved dough rheological qualities, with 10-15% flaxseed addition being the most acceptable based on maximum DDT, FQN, and MTI and minimum DoS and DST, as indicated in Figure 1.
Color assessment of muffins indicated significant variations in chroma and hue of crust as well as chroma of crumb.Overall results indicated an increase in the darker tones and the increase in supplementation, which can easily be attributed to the dark brown pigment of flaxseeds.Similar results have been obtained in previous works (Chetana et al., 2010;Wirkijowska et al., 2020).A similar decrease in L* and a* has been observed in the work of (Kaur et al., 2019;Wirkijowska et al., 2020).Similar darker tones have been reported by flaxseed supplementation (Lipilina & Ganji, 2009).Furthermore, protein concentration is inversely related to the lightness of baked goods.Higher protein percentages resulted in darker muffins (Fagundes et al., 2018).
The sensory assessment revealed that the increasing levels of supplementation had a detrimental influence on some sensory features in the consumer's opinion.However, lower levels of supplementation (5%, 10%, and 15%) remained acceptable during the study.Similar sensorial appraisal outcomes of 10-15% flaxseed supplementation have been reported previously (Kaur et al., 2019;Wirkijowska et al., 2020).

Conclusions
The study's overall findings are still pretty straightforward regarding the enhanced nutritional profile of muffins made with wheat flour enriched with flaxseed.Such cutting-edge foods can help reduce the burden of disease and nutritional deficits worldwide if consumed regularly.Practical studies have shown that flaxseed is an excellent source of vegetative n-3 PUFA (ALA), vegetative proteins, lignans, and dietary fibers for illness prevention and health promotion.They should be ingested regularly to receive these nutritional and nutraceutical advantages.Additionally, their rheological characteristics substantiated the higher baking quality of flaxseed-supplemented flour blends.The 15% supplementation dose appeared to be the most acceptable based on the sensory evaluation.In conclusion, flaxseed can be a valuable supplement for enhancing the nutritional qualities of a range of food items.Based on the current research, essential recommendations for the future might include creating new, modified recipes and examining the health advantages they provide for various clinical issues.
For financing Roshina Rabail's M.Sc.(Hons.)thesis on the topic of "Nutraceutical and functional scenario of unconventional food sources of Ώ-3 fatty acids in a cereal-based product," Muhammad Tauseef Sultan is grateful to the Higher Education Commission of Pakistan.

Table 1 .
Diet for the control, cholesterol, and flaxseed-fed rats.

Table 2 .
Ingredients in the standard muffin recipe in grams.

Table 3 .
Physiological markers of S-and T-trial.

Table 4 .
Laboratory biomarkers of S-and T-trials.

Table 5 .
Nutritional composition of flaxseed flour, its blends, and muffins.

Table 6 .
Farinographic characteristics for flaxseed supplemented blends.DDT= Dough Development Time; DST= Dough Stability Time; DoS= Degree of Softening; DoS-ICC= Degree of Softening-ICC; DC= Dough Consistency; FQN= Farinograph Quality Number; MTI= Mixing Tolerance Index.Flaxseed = acts as control; Means sharing same letters in a column are not significantly different from each other at p < .05. * = highly significant at p < .01;* = significant at p < .01;ns = non-significant at p < .05. *

Table 7 .
Colour indices of flaxseed supplemented muffins.