Development of Vitamin D and Protein Rich Energy Bar with Mushroom

Vitamin D deficiency is very common due to life style changes during present days. According to various reports almost 70-100% of Indian population of different age groups is either deficient or insufficient in vitamin D (Sanwalka, 2015). Vitamin D deficiency often results in bone related disorders such as rickets and osteoporosis, cardiovascular diseases, diabetes, cancer and infections such as tuberculosis. Protein deficiency is also quite prevalent and can lead to growth problems, mental and physical development (Sharma and Devi, 2021). Natural sources of Vitamin D are very few and most of them are non-vegetarian. Among the vegetarian foods, mushrooms are fair source of vitamin D and concentrated source of ergosterol (vitamin D2 precursor) (Urbain et al., 2011). 

Mushrooms also contain good quality proteins rich in essential amino acids like leusine, methionine, tryptophan and valine (Longvah and Deosthale, 1998; Diez and Alvarez, 2001; Agrahar-Murugkar and Subbulakshimi, 2004). Being rich in good quality proteins, mushrooms can be useful infighting the protein malnutrition in the country. Low sugars and lipid levels with no cholesterol and higher proportion of polyunsaturated fatty acids is an added advantage. Mushrooms are also good for diabetic and heart patients as they don’t have any starch or cholesterol content (Wani et al., 2010). Moreover, mushrooms are also rich in dietary fiber, several other vitamins (folic acid, thiamine, riboflavin and niacin) and minerals (potassium, sodium, copper, selenium, phosphorous etc). Mushrooms are also good source of many antioxidants (Mau et al., 2004; Puttaraju et al., 2006; Ferreira et al., 2007; Oyetayo, 2007).

Keeping in mind the widespread Vitamin D and protein malnutrition in India and the demand of a nutritious, healthy and convenient snacking item, the present study was planned to develop a Vitamin D and protein rich energy bar utilizing mushrooms powder along with cereals, natural sweeteners such as honey and jaggery, peanuts and dry fruits which will help in fighting the Vitamin D and protein deficiencies prevalent among the population.

The present study was carried out at ICAR-Directorate of Mushroom Research, Solan during the year 2018.
 
Raw materials
 
Rolled oats, puffed rice, jaggery, honey, peanuts, nuts (including almond, cashew nuts and raisins) were procured from the local market of Solan, Himachal Pradesh, India. For preparing mushroom powder, fresh white button mushrooms (Agaricus biosporus) procured from ICAR-Directorate of Mushroom Research, Solan were washed properly in potable water, cut into slices and dried in a cabinet or trey dryer at 50°C for 20 hours to obtain a moisture content of less than 5%. The dried mushroom slices were then ground in a grinding mill (Retsch, Model No. SM100 Standard, Germany) followed by sieving through sieve of 0.5 mm size (ISS No. 50) to convert into a fine powder.
 
Energy bar preparation
 
Twelve formulations of mushroom fortified energy bar were prepared with varying levels of mushroom powder, sweeteners and cereals (Table 1). Honey and jaggery were used as sweeteners instead of sugar to make it healthier. Cereals (oats and puffed rice), peanuts and nuts (almonds and cashew nuts) were dry roasted in a pan for 2 min to give them a crunchy texture and good aroma and then ground coarsely for the preparation of energy bar. A syrup of 80°Brix total soluble solid (TSS) was prepared by melting sweeteners (jaggery and honey in 1:1 ratio). Energy bar was prepared by cold mixing of mushroom powder, cereals, peanuts and dry fruits with sweeteners in the mixer (Hevells, Supermix 500, India). The mixture was then put into tray measuring 18x10x2 cm pre-greased with vegetable oil and sheeting was done with the help of rolling pin. After keeping this tray in the refrigerator for an hour, the bars of approximately 50±2 g weight were cut with the help of knife. These energy bars were then wrapped in butter paper and packed individually in laminated aluminium foil pouches of suitable size and stored for quality analysis (Mridula et al., 2010).


 
Proximate composition and energy value
 
All the energy bar samples were analyzed for moisture, crude fat, ash and crude fibre content by following standard AOAC protocols (AOAC, 2010). Protein content of the bars was analyzed using Micro Kjeldahl method (Kelplus Classic Dx, Pelican Equipments). Total carbohydrate content was calculated by difference method (Total carbohydrate = 100- [Moisture + Protein + Fat + Ash content]). Energy values of the bar were obtained by multiplying protein, carbohydrates and fat content by the factor 4, 4 and 9, respectively.
 
Vitamin D
 
For determination of Vitamin D, one gram of powdered sample was extracted thrice with 25 ml of methanol/dicholoromethane (75:25 v/v) solution and the extract was evaporated to dryness. The residue was then dissolved in 2 ml of methanol/dicholoromethane, 75:25 v/v and filtered through a 0.45 μm nylon syringe filter. The absorption of filtrate was taken at 264 nm through a UV-VIS spectrophotometer. A standard curve was also plottedwith 100 to 1000 μg concentration of vitamin D₂ at A₂₆₄ (Kumar and Rajput, 2011).
 
Antioxidant activity
 
Antioxidant activity of energy bar samples was determined by 2, 2-diphenyl-2-picryl-hydrazyl (DPPH) inhibition method (Shimada et al., 1992).
 
Texture analysis
 
Texture measurements of energy bar samples were doneby using the texture analyzer (TA-XT 2, Stable Microsystems, load cell 5 kg). Three piecesof each snack bar sample of size 2.5x2.5x2 cm were then subjected to hardness testing and the results were expressed in N.
 
Color analysis
 
Hunter Colorimeter was used to determine the color values (L, a and b) of theenergy bar samples. Hue angle (h° ) and chroma (C*) are calculated by using the following formula:
 

h° = tan^-1 (b/a) 
               
C* = [a² + b²]^1/2

Where,
b= b values,
a= a values.
 
Sensory qualities
 
Energy bar samples were evaluated by a group of eleven panelists using nine point hedonic scale ratings for different organoleptic characteristics such as colourand appearance, flavor and taste, texture and mouth feel and overall acceptability. Nine point hedonic scale is as follows; like extremely - 9, like very much - 8, like moderately -7, like slightly - 6, neither like nor dislike - 5, dislike slightly 4, dislike moderately - 3, dislike very much - 2, dislike extremely - 1 (BIS, 1971).
 
Statistical analysis
 
The data obtained in the present study were subjected to one factor statistical analysis of variance (ANOVA) technique using completely randomized design (CRD). The critical difference value at 5 per cent levels of significance was calculated and used for making comparison among different formulations. Statistical ranks based on significant difference by Duncan’s multiple range tests are given in the form of superscript over corresponding data values.

Proximate composition and energy value
 
Proximate composition and energy values of bars prepared by following twelve formulations are given in Table 2. The energy bars showed a significant increase in moisture content (from 8.82 to 14.14 g/100 g) with increasing levels of mushroom powder and sweeteners. This could be due to hygroscopic nature of honey, jaggery and mushroom powder. Protein, fibre and ash content of energy bars also increased significantly (from 9.24 to 17.89 g/100 g, 0.94 to 6.57 g/100 g and 1.68 to 3.51 g/100 g, respectively) with increasing levels of mushrooms powder, which can be attributed to high protein, minerals and fiber content of mushrooms (Reis et al., 2012; Bernaś et al., 2006; Manzi et al., 2001; Manzi et al., 2004). Similar trends were obtained during energy bar formulation using flaxseed (Mridula et al., 2013) and potato extrudates (Giri and Mridula, 2016). Fat, carbohydrate and energy values of energy bars have shown a reverse trend and decreased significantly (from 10.18 to 5.29 g/100 g, 68.72 to 61.14 g/100 g and 408.38 to 357.48 kcal/100 g) with increasing incorporation of mushroom powder as mushroom is a low calorie food with low fat and carbohydrate contents (Farzana et al., 2017).


 
Per cent RDA of Vitamin D and protein from energy bars
 
During the present investigation, vitamin D content of different formulations of energy bars ranged between 92.61 to 478.04 IU/100 g (Table 3). Significant increase in vitamin D content of energy bar with increased incorporation of mushroom powder might be due to presence of provitamin D2 (ergocalciferol) in mushrooms (Phillips et al., 2011). As per the recommended dietary allowance (RDA) of Vitamin D and protein it was found that the bar incorporated with 20% mushroom powder can provide upto 53% of RDA of Vitamin D and 12.7% of RDA of protein from single snack bar (50 g) (Table 3). This mushroom fortified energy bar can be a good source of Vitamin D and protein and thus by contributing significantly to RDA of both these nutrients can help in alleviating protein and Vitamin D deficiencies prevalent in many developing countries. 


 
Antioxidant activity
 
Antioxidant activity of energy bar also varied significantly with varying concentrations of mushroom powder and sweeteners (Fig 1). The general trend was an increase in antioxidant activity (from 39.22 to 78.52% scavenging of DPPH) of the bar with increasing concentration of mushrooms powder. Higher antioxidant properties of energy bar with more mushroom powder might be due to presence of several bioactive compounds, such as polyphenols, polysaccharides, vitamins, carotenoids and minerals in mushrooms resulting in significant antioxidant properties (Kozarski et al., 2015).


 
Sensory quality
 
Sensory score for color and appearance decreased with increasing level of mushroom powder (from 7.27 to 6.09) but it remained acceptable for all the formulations (>6.0). Whereas, the sensory scores for taste and flavor, texture and mouth feel and overall acceptability were significantly higher for the bars prepared with 20% mushroom powder which might be due to a positive effect of mushroom powder on the flavor and texture of the energy bar upto this point (Table 4). In a similar study, no significant difference in sensory characteristics (color, taste, aroma, texture, flavor and overall acceptability) of cake samples was recorded up to the level of 20% replacement of refined wheat flour (maida) with mushroom powder (Arora et al., 2017).


 
Colour characteristics
 
Although, both mushroom powder and sweeteners significantly affected the color value (L, a, b, hp and C*) of energy bar (Table 5 and Fig 2), the bar samples even at the 20% level of mushroom powder were accepted well based on organoleptic scores (Table 4). Eissa et al., (2007) have also reported that bread incorporated with mushroom flour (5-15%) showed darker crust hence lower crust L values as compared to control.




 
Textural quality
 
Hardness of bar represents the force required for biting into it. A significant increase in the hardness (from 27.56 to 66.5 N) of energy bars with increasing concentration of mushroom powder was observed during the present investigation (Fig 3). This might be because of compacting effect of mushroom powder on the texture of snack bar making it more dense. In similar studies, an increase in hardness of mushroom fortified bread and cake samples with increasing level of mushroom fortification have also been reported earlier (Eissa et al., 2007; Majeed et al., 2017). Whereas, on the contrary, legume flour incorporation has reported to cause a decrease in hardness of cereal bars (Yadav and Bhatnagar, 2017). However, as per the sensory evaluation, the scores for texture and mouth-feel of energy bars were recorded to be most acceptable for 20% level of mushroom powder incorporation (Table 4).

Mushroom fortification improved the level of Vitamin D, protein, antioxidant activity and fiber in the bars while lowering the fat content and calorific value. Energy bar prepared with 20% mushroom powder, 40% sweetener, 25% cereals, 10% peanuts and a 5% dry fruits showed good sensory acceptability along with a good amount of vitamin D (423.99 IU/100 g), protein (15.18 g/100 g), antioxidant activity (78.52% scavenging of DPPH (2,2-diphenyl-1-picrylhydrazyl), crude fiber (4.76 g/100 g), ash (2.60 g/100 g) and phenols (383.45 mg Gallic acid equivalent/100 g). Single bar (50 g) of this mushroom fortified energy can provide 53% RDA of Vitamin D and 12.7% RDA of protein for an adult. This mushroom fortified energy bar can be a healthy and nutritious snacking option for people of all age groups and will help in alleviating protein-energy malnutrition as well as vitamin D deficiency prevalent in the country.  

The authors would like to acknowledge Indian Council of Agricultural Research (ICAR)-Directorate of Mushroom Research, Solan, Himachal Pradesh, India and ICAR-Indian Agricultural Research Institute, New Delhi for funding and supporting the work.

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